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Summarize: Charlotte officials say they are preparing for more protests today following a night of violence over a police officer’s fatal shooting of an African-American man Tuesday in the University City area. The dead man was identified as Keith Lamont Scott, 43. Sixteen police officers were injured overnight in a series of clashes, and there were reports early Wednesday of motorists on Interstate 85 being hurt and their vehicles damaged when protesters threw rocks, bottles and traffic cones off interstate overpasses onto traffic below. The officers hurt suffered mostly minor injuries, though one was hit in the face with a rock, officials said. $20 for 365 Days of Unlimited Digital Access Last chance to take advantage of our best offer of the year! Act now! At a Wednesday news conference, city leaders appealed for calm and promised a thorough investigation. Charlotte Mayor Jennifer Roberts held a Wednesday press conference and urged the community to remain calm and wait for the facts in the case to be released before jumping to conclusions. Her comments came just an hour before one activist group held its own news conference, urging the black community to start an economic boycott of white-owned businesses Charlotte. Roberts says she has been in contact with the governor’s office, the White House and the NAACP, and said the city would work to get out information as quickly as possible, while also dispelling rumors being spread on social media. Charlotte-Mecklenburg Police Chief Kerr Putney said the department is still viewing video from the scene, though the officer involved in the shooting himself was not wearing a camera. Putney said the officer who fired the shots was in plain clothes, wearing a vest and was accompanied by uniformed officers when they approached the victim. It remains unclear whether Scott was pointing a gun at the officer when he was shot, Putney said. The officer was also African-American. Putney added that officers have not found a book at the scene of the shooting, contrary to social media claims that Scott was holding a book. “I can tell you we did not find a book that has been referenced to,” Putney said. “We did find a weapon. The weapon was there and witnesses have corroborated it, beyond just the officers.” Putney said the department would be staffed Wednesday in expectation of more protests, which he believes will be peaceful. “We’ll be prepared for whatever we see...We’re hoping for best but will be prepared for the worst,” he said. SHARE COPY LINK Several hundred people protested a CMPD officer-involved fatal shooting in the University Area that extended from Tuesday evening into Wednesday morning. A dozen police officers were injured in the clashes. The destruction late Tuesday and early Wednesday included blocking all lanes of Interstate 85 and looting a Walmart on North Tryon Street at about 3:30 a.m. The store was closed early Wednesday, with wooden pallets piled in front of the doors and shopping carts blocking the driveway into the lot. Three or more tractor trailer trucks were stopped and looted on Interstate 85, and at least two fires were started on the interstate, as the protesters burned items removed from the trucks. Motorists were reportedly stuck on Interstate 85 for hours at the height of the protests, which ignited at a time when the nation has seen a spate of police shootings of black men, which has led to protests from Ferguson, Mo., to Tulsa to Chicago and started the Black Lives Matter movement. Only one person has been arrested so far, police said. The neighborhood where the incident occurred was quiet Wednesday, aside from a large media presence. SHARE COPY LINK A dozen police officers were injured Tuesday night in a series of clashes. Charlotte’s Tuesday night protests began on Old Concord Road at Bonnie Lane, where a Charlotte-Mecklenburg police officer fatally shot a man in the parking lot of a University City apartment complex Tuesday afternoon. The officer who fired the fatal shot was CMPD Officer Brentley Vinson, a police statement said. Police said they had been searching for someone who had an outstanding warrant at The Village at College Downs complex on Old Concord Road when they saw Scott leave his car holding a gun. Officers approached Scott after he got back into the car. He emerged from the car again armed with a firearm “and posed an imminent deadly threat to the officers, who subsequently fired their weapon striking the subject,” police said in a statement. “The officers immediately requested Medic and began performing CPR.” Medic took Scott to Carolinas Medical Center, where he was pronounced dead. Scott was not the person officers were searching for to arrest on the outstanding warrants, CMPD Chief Kerr Putney told reporters later. Police said they recovered the firearm Scott was holding. But a woman who said she is Scott’s daughter claimed on a live-streamed video on Facebook that Scott was unarmed when he was shot. The video went viral, with more than 521,000 views by 9:30 p.m. In the video, the woman said her father was sitting in his car reading a book and waiting for the school bus to drop off his son. She claimed that her father was Tasered and then shot four times, and that he was disabled. “IT WAS A BOOK” one protester’s sign read. Police declined to respond directly to the woman’s accusations. A public records search shows that Scott was convicted in April 2004 of a misdemeanor assault with a deadly weapon charge in Mecklenburg County. Other charges stemming from that date were dismissed: felony assault with a deadly weapon with intent to kill, and misdemeanors assault on a child under 12, assault on a female and communicating threats. His mother, Vernita Walker of Charleston, SC, said Wednesday her son had seven children. “He was a family man,” she said. “And he was a likeable person. And he loved his wife and his children.” She said she had just talked with her son on the phone that day. In April 2015 in Gaston County Court, Scott was found guilty of driving while intoxicated. Roberts tweeted early Wednesday: “The community deserves answers and full investigation will ensue. Will be reaching out to community leaders to work together.” The protesters began to gather as night fell, hours after the shooting. They held signs that said “Stop Killing Us” and “Black Lives Matter,” and they chanted “No justice, no peace.” The scene was sometimes chaotic and tense, with water bottles and stones chucked at police lines, but many protesters called for peace and implored their fellow demonstrators not to act violently. A CMPD helicopter circled low over the crowd, shining a bright searchlight on the protesters. Old Concord Road was shut down. Some protesters began to throw water bottles and rocks. Shortly before 11 p.m., police donned gas masks. Soon, clouds of tear gas bloomed in front of their lines. Protesters damaged at least two CMPD vehicles, one cruiser and one SUV, which were removed from the scene. One officer was hit in the face with a rock, CMPD said. Observer news partner WBTV said three of its reporters were hit during the protest, and at least one went to the hospital after a blow to the head. At one point, the crowd began pushing down the ramp from Old Concord to Harris Boulevard West, blocking the road. Police deployed tear gas on that road as well. Not all the interactions were so tense. Around 1 a.m. Wednesday, police were seen handing bottles of water to the several dozen people who were still protesting. Live Blog Protests erupt in Charlotte following fatal officer-involved shooting As is standard procedure with any fatal police shooting, CMPD’s Internal Affairs Bureau will conduct a separate but parallel investigation to determine whether CMPD policies and procedures were followed. Per department protocol, Vinson will be placed on administrative leave. Anyone with information about the shooting is asked to call police at 704-432-TIPS (8477) or Crime Stoppers at 704-334-1600. CHARLOTTE, N.C. — Family members speak to WCCB, Charlotte’s CW reporter Courtney Francisco following the fatal shooting of Keith Lamont Scott. Scott was shot and killed by a Charlotte-Mecklenburg Police officer on Tuesday, September 20th in northeast Charlotte. Police say officers with the Metro Division Crime Reduction Unit were searching for a suspect with an outstanding warrant on him at The Village at College Downs just before 4pm. Officers say they saw a Scott inside a vehicle in the apartment complex. Scott exited the vehicle armed with a firearm, according to police. Officers say Scott got back into the vehicle and then the officers started to approach Scott. That’s when police say Scott got back out of the vehicle armed with a firearm and posed an imminent deadly threat to the officers who subsequently fired their weapon striking him. The officers immediately requested MEDIC and began performing CPR, according to a news release. Scott was pronounced dead on the scene. Police say the officer involved in the shooting is Brentley Vinson. As is standard procedure with any officer involved shooting, Officer Vinson has been placed on paid Administrative Leave. Officer Vinson has been employed with the CMPD since July 21, 2014 and is currently assigned to the Metro Division. Family members say Scott was sitting in a vehicle reading a book while he waited for his son to get off a school bus. Family members claim the book was mistaken as a gun. Protests broke out shortly after the shooting. At 10pm protesters were seen throwing water bottles at police. Around 11pm, police cruisers were being damaged by protesters. Police started deploying tear gas and smoke bombs into the crowd, according to a source. Several officers have been injured in the protests. Charlotte Police Chief Kerr Putney said on Sept. 22 that he has no plan to release publicly a video of the deadly encounter with Keith Lamont Scott. (Reuters) For the latest updates from Charlotte, head here. CHARLOTTE — Hours after North Carolina Gov. Pat McCrory (R) declared a state of emergency and the National Guard and state troopers moved in, Charlotte-Mecklenburg Police Chief Kerr Putney resisted calls to release video footage of a police shooting that sparked intense protests in this city. Family members of Keith Lamont Scott have asked to view video of his shooting and that authorities are trying to accommodate them, Putney said. Attorneys for the relatives said they planned to watch it later in the day. One of the attorneys, Justin Bamberg, said during an afternoon news conference that Scott’s wife, Rakeyia Scott, witnessed her husband’s death. “It’s my understanding that his wife saw him get shot and killed,” Bamberg said. “That’s something that she will never, ever forget.” Putney suggested that his department has no imminent plans to release the video footage to the public, and the Scott family’s attorneys made no promises to reveal its contents once they had seen it. “Transparency is in the eye of the beholder,” Putney told reporters. “If you think I’m saying we should display a victim’s worst day for public consumption, that is not the transparency I’m speaking of.” Putney said his department would release the video only “when we believe it is a compelling reason,” but the footage — which, he noted, doesn’t definitively show Scott pointing a gun — probably would not do much to calm the city anyway. “I can tell you this: There’s your truth, my truth and the truth,” Putney said. “Some people have already made up their minds.” Charlotte officials spoke as the city tried to recover from a second night of demonstrations that left several businesses damaged and one man clinging to life. Mayor Jennifer Roberts (D) noted that it had been “a difficult couple of days” for the city, adding: “This is not the Charlotte we know and love.” Although city leaders said Charlotte was open for business, Uptown was more of a ghost town than bustling city center, with some businesses cleaning up and others closing shop. Workers moved quickly to repair damage from the previous night’s protests and were seen mending windows at the Ritz-Carlton Hotel and a nearby bank building. Duke Energy, Wells Fargo and Bank of America, which is headquartered here, had told employees to stay home. During the lunch rush, restaurants and coffee shops had few customers. “Today is dead,” said hot-dog vendor Kidane Engida. “It’s like a bank holiday: If the bankers are not working, there’s nobody.” City officials stressed that the business district was safe and secure after a second spasm of overnight unrest. On Wednesday, peaceful protests turned into chaos when demonstrators attempted to follow police in riot gear into a hotel lobby. Officers used tear gas, and then a reporter heard a gunshot and saw a man lying in the street near the hotel entrance. The man, who was not identified, was taken to a hospital with wounds that medics said were “life-threatening.” Officials announced on Twitter that the man had died, then later tweeted that he was on “life support.” Putney said the man was in critical condition Thursday morning. Investigators are reviewing video to determine who shot the man, the chief said, noting that an allegation was made “that one of our officers was involved.” City officials say a man was shot during the second night of demonstrations in Charlotte after police fatally shot a black man outside an apartment complex on Sept. 20. (Cleve Wootson/The Washington Post) The protests stemmed from Tuesday’s fatal police shooting of Scott — putting Charlotte on a growing list of communities across the country that have erupted amid a growing debate on racial bias in policing. Some protesters ignited small fires and shattered hotel windows. Businesses were damaged and looted. Nine civilians were injured, and two police officers suffered “minor” eye injuries and three were treated for “heat issues,” the chief said. There were 44 overnight arrests on charges such as failure to disperse, assault, and breaking and entering, Putney said. More arrests are likely after investigators review surveillance video, he said. Officials weighed the possibility of implementing a curfew Thursday night — though the police chief noted at the morning news conference that “right now, we don’t see the need to shut the city down at a specific hour.” McCrory, the governor, said National Guardsmen were mobilized to help protect buildings and structures, and that state troopers were deployed to control traffic and help local police do their jobs. “As governor,” he said, “I firmly believe that we cannot tolerate any type of violence.” Michael Smith, chief executive of the downtown development corporation Charlotte Center City Partners, said the influx of law enforcement personnel gave him “much greater confidence that we will respond the way we need to” after two nights of chaos. But Corinne Mack, who heads the Charlotte chapter of the NAACP, said the increased police presence could prove problematic. “More police presence is never going to help,” she said. Instead, she said: “More transparency helps.” [Blame, rumor and blood run in Charlotte as protests surge. 1 critically wounded, 4 cops injured.] Law enforcement officials have fatally shot 706 people this year, 163 of them black men, according to a Washington Post database tracking fatal police shootings. A growing divide in public rhetoric over that toll has been fed by a summer of high-profile deaths captured on social media and deadly assaults on police officers in Dallas and Baton Rouge. The latest encounters — in Charlotte, and Tulsa, where protesters called for the arrest of the officer involved in the fatal shooting of a black man there Friday — come as the presidential race has tightened, and both candidates have offered positions and solutions. Attorney General Loretta E. Lynch pleaded again Thursday for protesters to remain peaceful. “For the second day in a row, protests in response to Mr. Scott’s death took place in Charlotte last night,” Lynch, a North Carolina native, said during a news conference. “And for the second day in a row, those protests were marred by violence — this time leaving one person on life support and several individuals injured — an awful reminder that violence often only begets violence.” Lynch called for “those responsible for bringing violence to these demonstrations to stop,” adding, “you’re drowning out the voices of commitment and change and ushering in more tragedy and grief in our communities.” The FBI and the Justice Department are monitoring the situation involving Scott’s death, Lynch said, but federal officials have not launched an investigation. McCrory said Thursday that the North Carolina State Bureau of Investigation is leading an independent probe into the shooting. Charlotte police have insisted that Scott had a gun and was posing an “imminent deadly threat” when officers shot him outside an apartment complex near the campus of the University of North Carolina at Charlotte. Scott’s family, however, said he was unarmed when he was killed and was instead reading a book in his car while waiting for his child to get out of school — a detail that quickly went viral on social media and was seized upon by protesters here. Putney said police recovered a gun and found no book at the scene. The police chief said the officer who shot Scott was in plainclothes, wearing a vest with a police logo, and was accompanied by other officers in full uniform. The plainclothes officer wasn’t wearing a body camera, but the other officers were. [Charlotte mayor: ‘I understand the anger’] Whether authorities can defuse the anger on the streets could hinge on that body-camera footage. The shooting has thrust Charlotte to the forefront of a national debate about access to police body cameras. During an occasionally testy exchange with reporters on Thursday, Putney was asked when the public could expect the release of video showing the fatal shooting. “You shouldn’t expect it to be released,” he said, noting that he did not want to “jeopardize the investigation.” Having watched the footage, Putney said, “the video does not give me absolute definitive visual evidence that would confirm that a person is pointing a gun. I did not see that in the videos that I’ve reviewed.” Still, he said, “When taken in totality of other evidence, it supports what we’ve heard and the version of the truth about the circumstances that happened that led to the death of Mr. Scott.” A new state law effective Oct. 1 forbids police agencies from making body-camera footage public without a court order. “At a time when you’re seeing other states becoming more transparent, North Carolina is taking this tremendous step backward,” said Mike Meno, a spokesman for the American Civil Liberties Union of North Carolina. The violent protests and conflicting accounts in Charlotte prove “just how misguided this new law is,” Meno said, and show exactly why public access to such footage is crucial. [Fatal Force: 706 people have been shot and killed by police in 2016] In a Facebook Live video widely circulated before Tuesday’s protest, a woman who identified herself as Scott’s daughter said officers used a stun gun on him, then shot him four times with their service weapons. She added that Scott was disabled. “My daddy didn’t do nothing; they just pulled up undercover,” she said in the video. By Wednesday afternoon, the video had been taken down. Hours later, Scott’s wife, Rakeyia, released a statement saying the family was “devastated.” “Keith was a loving husband, father, brother and friend who will be deeply missed every day,” she wrote. “As a family, we respect the rights of those who wish to protest, but we ask that people protest peacefully. Please do not hurt people or members of law enforcement, damage property or take things that do not belong to you in the name of protesting.” The family, she said, had “more questions than answers about Keith’s death. Rest assured, we will work diligently to get answers to our questions as quickly as possible.” Bamberg, one of the family’s attorneys, confirmed Thursday that Scott, who had been married for two decades and had seven children, had a disability due to injuries suffered in an accident. He said there were different accounts of the shooting, including some people who say Scott was holding a book and others who said his hands were empty. Bamberg did not agree to release any information after viewing the video of Scott’s death, saying, “My priority is the greater good of this family.” Authorities said the officer who shot Scott is black, and they identified him as Brentley Vinson, who has worked for the Charlotte-Mecklenburg police force since July 2014. He was placed on paid administrative leave pending an investigation. This city also was the scene of another high-profile police shooting, when officers killed Jonathan Ferrell, a 24-year-old black man who had crashed his car in a residential neighborhood several miles from the complex where Scott died, in September 2013. Officer Randall Kerrick fired 12 rounds at Ferrell, who was unarmed, striking him 10 times. Police said Ferrell ignored officers’ instructions. Last year, the jury deadlocked during Kerrick’s trial. While most jurors voted to acquit the officer, four voted to convict him. After a judge declared a mistrial, the state said it would not seek another trial. Ferrell’s family and the city of Charlotte settled a lawsuit stemming from the shooting for a reported $2.25 million. But anger from the 2013 shooting never went away, lurking beneath the surface until Tuesday night, when it exploded again into the open. Jibril Hough, a local activist who organized protests during Kerrick’s trial, said the recent demonstrations stem from lingering frustrations over Ferrell’s shooting. “What you’re seeing is people have been put in that situation for so long and they’re tired of talking,” he said. “They’re tired of talking and talking and candlelight vigils and dialogue and nothing getting done.” Hough said he did not agree with the violent turn the protests have taken. But, he said, there’s a “boiling point” — and some people in Charlotte have reached it. Bever and Berman reported from Washington and Adam Rhew and Wesley Lowery from Charlotte. William Wan, Derek Hawkins and Julie Tate contributed to this report, which has been updated multiple times. Story highlights Charlotte police say the man had a gun Protesters gather in apartment complex (CNN) [Breaking news alert, posted at 12:37 a.m. ET Wednesday] The Charlotte-Mecklenburg Police Department said "approximately 12 officers" have been injured during protests following the fatal shooting of Keith Lamont Scott by a police officer. The department also tweeted that one of its officers was hit in the face with a rock. [Previous story, posted at 11:30 p.m. ET Tuesday] Charlotte, North Carolina, police looking to serve a warrant Tuesday shot and killed a man in the parking lot of an apartment complex. The man was armed but not the man police were looking for, the Charlotte-Mecklenburg Police Department said in a news release. Read More
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Summary: Yet another American city has erupted in protests after a fatal police shooting. Police in Charlotte, NC, say around a dozen officers were injured late Tuesday and early Wednesday after a man was shot and killed in the parking lot of an apartment complex Tuesday afternoon, CNN reports. Police say they were searching for somebody at the complex when they saw Keith Lamont Scott, 43, leave his car holding a gun. Police say Scott-who was not the man they were looking for-was approached by officers after he got back in the car and was shot dead when he emerged from the car with a gun again. Police say Scott "posed an imminent deadly threat," but family members give a very different account of the shooting to WCCB. They say Scott was sitting in his car reading a book while he waited for his son to get off a school bus, and police mistook the book for a weapon. Brentley Vinson, the officer involved in the shooting, has been with the force since 2014 and is on paid administrative leave while the incident is investigated. Sources tell the Charlotte Observer that Vinson, like Scott, is African American. The Washington Post reports that tear gas and rubber bullets were fired during Tuesday night's protests and protesters shut down traffic on I-85 early Wednesday.
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Summarize: Published on Aug 3, 2015 The license of this video is Creative Commons Attribution (CC-BY). Feel free to share it. If you have doubts about this license, you can see the details here: http://creativecommons.org/licenses/b... SUNNYVALE (KPIX 5) — A deputy patrolling the Caltrain tracks in Sunnyvale pulled a driver to safety after his car ended up on the tracks Monday evening, seconds before a train slammed into the vehicle. Authorities told KPIX 5 that two deputies were patrolling the Caltrain rails at Mary Avenue in Sunyvale, when a driver crashed into the crossing arm and pole around 6:30 p.m. Authorities said the man appeared drunk. One of the deputies pulled the man out of the car, moments before it was hit by a northbound train. ALSO READ: Dead Frog Reportedly Found In Bag Of Organic Spinach Leaves “These deputies did not hesitate, they acted. Had they not acted, who knows what would have happened, I believe because of their actions, this person is alive today,” said Detective Salvador Zuno of the San Mateo County Sheriff’s Department, the agency that handles law enforcement on Caltrain. While the deputy pulled the man to safety, Deputy Erick Rueppel ran onto the tracks toward the oncoming train to slow it down. “I’m just happy at the result…Nobody died, that would be the worst outcome of this situation,” Rueppel said. “And I’m glad we were here.” “These kinds of incidents aren’t just hard for the families of the victims, they’re hard for our engineers and crews on board the train so we’re so grateful that tragedy was averted because these two deputies were in the right place at the right time,” said Caltrain spokesperson Jayme Ackemann. MORE: Lone Gray Wolf Spotted In Northern California The deputy had been responding to a call nearby prior to the crash, Ackemann told Bay City News Service. The crash could be seen in two YouTube videos. The driver of the car can be seen being dragged away from the tracks by transit police before the first train struck the car. The incident resulted in delays of up to 45 minutes as trains single-tracked through the area. Both tracks had reopened for service by 7:45 p.m., according to Ackemann. Crews were repairing the broken crossing arm and pole as of late Monday night. The sheriff’s department did not give the driver’s name, but said he was in his 20s and suffered minor injuries. TM and © Copyright 2015 CBS Radio Inc. and its relevant subsidiaries. CBS RADIO and EYE Logo TM and Copyright 2015 CBS Broadcasting Inc. Used under license. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed. Bay City News Service contributed to this report. Alleged drunk driver pulled from car seconds before it is hit by Caltrain Photo: Caltrain Image 1 of / 1 Caption Close Image 1 of 1 San Mateo County sheriff's deputies Erik Rueppel (left) and Lance Whitted helped save the life of a man whose car was found on the tracks in Sunnyvale San Mateo County sheriff's deputies Erik Rueppel (left) and Lance Whitted helped save the life of a man whose car was found on the tracks in Sunnyvale Photo: Caltrain Alleged drunk driver pulled from car seconds before it is hit by Caltrain 1 / 1 Back to Gallery A San Mateo County sheriff’s deputy dragged a suspected drunken driver from a car stuck on Caltrain tracks in Sunnyvale just seconds before a train plowed into the vehicle, a dramatic rescue that was captured on video, officials said. The collision happened on the tracks at South Mary Avenue near West Evelyn Avenue about 6:40 p.m. Monday. Police later determined that the driver, Nelson Gomez, 20, had been involved in two hit-and-run collisions before he drove a black Volkswagen Jetta through the crossing-guard arms and became stuck, Sunnyvale Department of Public Safety officials said Tuesday. San Mateo County sheriff’s deputies Lance Whitted and Erik Rueppel were conducting a traffic stop nearby when they spotted the Jetta on the tracks, authorities said. The deputies are part of the transit police bureau, which provides law-enforcement services along Caltrain property between San Francisco and Gilroy. Rueppel ran down the tracks in hopes of getting the train to slow down. Whitted grabbed Gomez from the car. In video of the incident caught by a bystander, the two can be seen walking away from the car, but then Gomez falls on the ground. The footage then captures Whitted dragging Gomez away from the tracks, just seconds before the northbound train comes barreling though the intersection, destroying the front end of the Jetta. The deputies told reporters that they simply did what they are trained to do and rejected assertions that they were heroes. “I don’t think there was time to be scared,” Rueppel said. “You just kind of do things as safe as you can and respond and that’s it. It kind of just happens.” Rueppel said, “This unfolded in front of us. So we had to do something. So you don’t think about whether it’s heroic or not. You just have to be safe, for one, because we don’t want to become a casualty in the situation.” Whitted said, “I can’t say why I did it or why someone else wouldn’t do it. At that point, it’s just instincts that kick in. You don’t have time to analyze the situation. I knew that my job, working in law enforcement is to preserve life, and that’s what I did.” Police later determined that Gomez had hit a parked car in a parking lot on the 100 block of South Bernardo Avenue and fled the scene, said Sunnyvale police Capt. Jeff Hunter. Gomez then hit an unoccupied bicycle at Bernardo and Evelyn Avenue and again fled before driving onto the tracks, police said. Gomez was treated for minor injuries at a hospital and then given a citation for drunken driving, hit-and-run, driving without a license and a probation violation. Caltrain spokeswoman Jayme Ackemann, said she it was fortunate that everyone escaped major injury. “These incidents aren’t just hard for the families of the victims, they’re hard for our engineers and crew on board the train, so we’re grateful that tragedy was averted because these two deputies were in the right place at the right time.” Henry K. Lee and Kale Williams are San Francisco Chronicle staff writers. E-mail: [email protected] and [email protected] Twitter: @henryklee @sfkale
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Summary: A California driver is very lucky that two deputies happened to be nearby after he crashed and ended up on railroad tracks yesterday. Because, yes, a train was bearing down on him. San Mateo County Sheriff's deputy Erik Rueppel ran toward the train to try to slow it down as deputy Lance Whitted worked on the getting the driver out, reports the San Francisco Chronicle. Whitted got him out, but the unidentified man fell down, and video shows the deputy dragging him to safety with only seconds to spare. The driver suffered minor injuries, reports CBS San Francisco.
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Summarize: People celebrate the capture in Tripoli of Moammar Gadhafi's son and one-time heir apparent, Seif al-Islam, at the rebel-held town of Benghazi, Libya, early Monday, Aug. 22, 2011. Libyan rebels raced... (Associated Press) Heavy clashes have broken out near Moammar Gadhafi's compound in the Libyan capital. Rebel spokesman Mohammed Abdel-Rahman says that tanks emerged from the complex, known as Bab al-Aziziya, early Monday and began firing. An Associated Press reporter at the nearby Rixos Hotel where foreign journalists are staying could hear gunfire and loud explosions that have been going on for more than 30 minutes. No further details were immediately available on the fighting. Abdel-Rahman says that Gadhafi troops remain a threat to rebels advanced into the city Sunday, and that as long as Gadhafi remains on the run the "danger is still there." THIS IS A BREAKING NEWS UPDATE. Check back soon for further information. AP's earlier story is below. TRIPOLI, Libya (AP) _ Euphoric Libyan rebels took control of most of Tripoli in a lightning advance Sunday, celebrating the victory in Green Square, the symbolic heart of Moammar Gadhafi's regime. Gadhafi's defenders quickly melted away as his 42-year rule crumbled, but the leader's whereabouts were unknown and pockets of resistance remained. State TV broadcast Gadhafi's bitter pleas for Libyans to defend his regime. Opposition fighters captured his son and one-time heir apparent, Seif al-Islam, who along with his father faces charges of crimes against humanity at the International Criminal Court in the Netherlands. Another son was under house arrest. "It's over, frizz-head," chanted hundreds of jubilant men and women massed in Green Square, using a mocking nickname of the curly-haired Gadhafi. The revelers fired shots in the air, clapped and waved the rebels' tricolor flag. Some set fire to the green flag of Gadhafi's regime and shot holes in a poster with the leader's image. The startling rebel breakthrough, after a long deadlock in Libya's 6-month-old civil war, was the culmination of a closely coordinated plan by rebels, NATO and anti-Gadhafi residents inside Tripoli, rebel leaders said. Rebel fighters from the west swept over 20 miles (30 kilometers) in a matter of hours Sunday, taking town after town and overwhelming a major military base as residents poured out to cheer them. At the same time, Tripoli residents secretly armed by rebels rose up. When rebels reached the gates of Tripoli, the special battalion entrusted by Gadhafi with guarding the capital promptly surrendered. The reason: Its commander, whose brother had been executed by Gadhafi years ago, was secretly loyal to the rebellion, a senior rebel official Fathi al-Baja told The Associated Press. Al-Baja, the head of the rebels' political committee, said the opposition's National Transitional Council had been working on the offensive for the past three months, coordinating with NATO and rebels within Tripoli. Sleeper cells were set up in the capital, armed by rebel smugglers. On Thursday and Friday, NATO intensified strikes inside the capital, and on Saturday, the sleeper cells began to rise up. President Barack Obama said Libya is "slipping from the grasp of a tyrant" and urged Gadhafi to relinquish power to prevent more bloodshed. "The future of Libya is now in the hands of the Libyan people," Obama said in a statement from Martha's Vineyard, where he's vacationing. He promised to work closely with rebels. By the early hours of Monday, opposition fighters controlled most of the capital. The seizure of Green Square held profound symbolic value _ the plaza was the scene of pro-Gadhafi rallies organized by the regime almost every night, and Gadhafi delivered speeches to his loyalists from the historic Red Fort that overlooks the square. Rebels and Tripoli residents set up checkpoints around the city, though pockets of pro-Gadhafi fighters remained. In one area, AP reporters with the rebels were stopped and told to take a different route because of regime snipers nearby. Abdel-Hakim Shugafa, a 26-year-old rebel fighter, said he was stunned by how easy it was. He saw only about 20 minutes of gunbattles as he and his fellow fighters pushed into the capital at nightfall. "I expect Libya to be better," said Shugafa, part of a team guarding the National Bank near Green Square. "He (Gadhafi) oppressed everything in the country _ health and education. Now we can build a better Libya." In a series of angry and defiant audio messages broadcast on state television, Gadhafi called on his supporters to march in the streets of the capital and "purify it" of "the rats." He was not shown in the messages. His defiance raised the possibility of a last-ditch fight over the capital, home to 2 million people. Government spokesman Moussa Ibrahim claimed the regime has "thousands and thousands of fighters" and vowed: "We will fight. We have whole cities on our sides. They are coming en masse to protect Tripoli to join the fight." But it seemed that significant parts of Gadhafi's regime and military were abandoning him. His prime minister, Al-Baghdadi Al-Mahmoudi, fled to a hotel in the Tunisian city of Djerba, said Guma el-Gamaty, a London-based rebel spokesman. NATO Secretary-General Anders Fogh Rasmussen said Gadhafi's regime was "clearly crumbling" and that the time to create a new democratic Libya has arrived. It was a stunning reversal for Gadhafi, who earlier this month had seemed to have a firm grip on his stronghold in the western part of Libya, despite months of NATO airstrikes on his military. Rebels had been unable to make any advances for weeks, bogged down on the main fronts with regime troops in the east and center of the country. Gadhafi is the Arab world's longest-ruling, most erratic, most grimly fascinating leader _ presiding for 42 years over this North African desert nation with vast oil reserves and just 6 million people. For years, he was an international pariah blamed for the 1988 bombing of a Pan Am jumbo jet over Lockerbie, Scotland, that killed 270 people. After years of denial, Gadhafi's Libya acknowledged responsibility, agreed to pay up to $10 million to relatives of each victim, and the Libyan rule declared he would dismantle his weapons of mass destruction program. That eased him back into the international community. But on February 22, days after the uprising against him began, Gadhafi gave a televised speech vowing to hunt down protesters "inch by inch, room by room, home by home, alleyway by alleyway." The speech caused a furor that helped fuel the armed rebellion against him and it has been since mocked in songs and spoofs across the Arab world. As the rebel force advanced on Tripoli on Sunday, taking town after town, thousands of jubilant civilians rushed out of their homes to cheer the long convoys of pickup trucks packed with fighters shooting in the air. One man grabbed a rebel flag that had been draped over the hood of a slow-moving car and kissed it, overcome with emotion. Akram Ammar, 26, fled his hometown of Tripoli in March and on Sunday was among the rebel fighters pouring back in. "It is a happiness you can't describe but also some fear. It will take us time to clear the entire city. I expect a long time for Libyans to get used to the new system and the new democracy," he said, dressed in camouflage pants and black shirt and sporting the long beard of a conservative Muslim. "But in the end it will be better." The rebels' leadership council, based in the eastern city of Benghazi, sent out mobile text messages to Tripoli residents, proclaiming, "Long live Free Libya" and urging them to protect public property. Internet service returned to the capital for the first time in six months. The day's first breakthrough came when hundreds of rebels fought their way into a major symbol of the Gadhafi regime _ the base of the elite 32nd Brigade commanded by Gadhafi's son, Khamis. Fighters said they met little resistance. They were 16 miles from the big prize, Tripoli. Hundreds of rebels cheered wildly and danced as they took over the compound filled with eucalyptus trees, raising their tricolor from the front gate and tearing down a large billboard of Gadhafi. From a huge warehouse, they loaded their trucks with hundreds of crates of rockets, artillery shells and large-caliber ammunition. One group started up a tank, drove it out of the gate, crushing the median of the main highway and driving off toward Tripoli. The rebels also freed more than 300 prisoners from a regime lockup, most of them arrested during the heavy crackdown on the uprising in towns west of Tripoli. The fighters and the prisoners _ many looking weak and dazed and showing scars and bruises from beatings _ embraced and wept with joy. "We were sitting in our cells when all of a sudden we heard lots of gunfire and people yelling 'God is great.' We didn't know what was happening, and then we saw rebels running in and saying 'We're on your side.' And they let us out," said 23-year-old Majid al-Hodeiri. He said he was captured four months ago by Gadhafi's forces crushing the uprising in his home city of Zawiya. He said he was beaten and tortured while under detention. From the military base, the convoy sped toward the capital. Mahmoud al-Ghwei, 20 and unarmed, said he had just came along with a friend for the ride. "It's a great feeling. For all these years, we wanted freedom and Gadhafi kept it from us. Now we're going to get rid of Gadhafi and get our freedom," he said. The uprising against Gadhafi broke out in mid-February, and anti-regime protests quickly spread. A brutal regime crackdown quickly transformed the protests into an armed rebellion. Rebels seized Libya's east, setting up an internationally recognized transitional government there, and two pockets in the west, the port city of Misrata and the Nafusa mountain range. Gadhafi clung to the remaining territory, and for months neither side had been able to break the other. In early August, however, rebels launched an offensive from the Nafusa Mountains, intending to open a new, western front to break the deadlock. They fought their way down to the Mediterranean coastal plain, backed by NATO airstrikes, and captured the strategic city of Zawiya. Rebel fighters who spoke to relatives in Tripoli by phone said hundreds rushed into the streets in anti-regime protests in several neighborhoods on Sunday. "We received weapons by sea from Benghazi. They sent us weapons in boats," said Ibrahim Turki, a rebel in the Tripoli neighborhood of Tajoura, which saw heavy fighting the past two days. "Without their weapons, we would not have been able to stand in the face of the mighty power of Gadhafi forces." Thousands celebrated in the streets of Benghazi, the rebels' de facto capital hundreds of miles to the east. Firing guns into the air and shooting fireworks, they cheered and waved the rebel tricolor flags, dancing and singing in the city's main square. When rebels moved in, the regime unit guarding the capital, known as the Mohammed Megrayef battalion, surrendered and its commander ordered its troops to put down their arms. Al-Baja, the rebel official, said that the commander, Barani Eshkal, had secretly defected earlier to the rebels, embittered by the 1986 execution of his brother, who had joined a coup attempt against Gadhafi. Eshkal also pointed out to the rebels the hiding place of Gadhafi's son Seif al-Islam in a hotel, al-Baja said. Rebel chief Mustafa Abdel-Jalil in Benghazi confirmed to the AP that the rebels captured Seif but refused to give details. In the Netherlands, the prosecutor at the International Criminal Court, Luis Moreno-Ocampo, said his office would talk to the rebels on Monday about Seif al-Islam's transfer for trial. "It is time for justice, not revenge," Moreno-Ocampo told the AP. Seif al-Islam, his father and Libya's intelligence chief were indicted earlier this year for allegedly ordering, planning and participating in illegal attacks on civilians in the early days of the violent crackdown on anti-regime protesters. Another son, Mohammed, was under house arrest. Mohammed, who is in charge of Libyan telecommunications, appeared on the Arabic satellite channel Al-Jazeera, saying his house was surrounded by armed rebels. "They have guaranteed my safety. I have always wanted good for all Libyans and was always on the side of God," he said. Close to the end of the interview, there was the sound of heavy gunfire and Mohammed said rebels had entered his house before the phone line cut off. ___ Hadeel Al-Shalchi in Cairo contributed to this report. Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more Seif al-Islam, his father and Libya's intelligence chief were indicted earlier this year for allegedly ordering, planning and participating in illegal attacks on civilians in the early days of the violent crackdown on anti-regime protesters. In the Netherlands, the prosecutor at the International Criminal Court, Luis Moreno-Ocampo, said his office would talk to the rebels on Monday about Seif al-Islam's transfer for trial. "It is time for justice, not revenge," Moreno-Ocampo told the AP. 'Time for justice, not revenge' Citing a source close to Gadhafi's inner circle, NBC News reported that Seif al-Islam had tried to flee Tripoli while disguised as a women. "Whether or not Gadhafi reads the tea leaves the same way is the big question," the official said. Referring to news that Gadhafi's sons Seif al-Islam, his onetime heir apparent, and Mohammed had been arrested and were in rebel custody, the U.S. official said, "We could be watching the game changer unfolding." As international news agencies broadcast video of huge crowds celebrating in Benghazi, the rebels' eastern headquarters, a U.S. official told NBC News that "the battle for Tripoli is clearly under way, and what has often seemed impossible — the fall of Gadhafi — may now be attainable." NBC News was not able to independently confirm the reports. Other top officials were reported to be fleeing, led by Prime Minister Al Baghdadi AlMahmoudi, who was spotted Monday morning in Jaraba, Tunisia, Al-Jazeera reported. Another major Arabic news agency, Al Arabiya, reported that indications were that Gadhafi may be in Tajoraa Hospital. Al-Jazeera's Arabic-language service reported that officers who had defected to the rebels found Gadhafi overnight near Tajoraa Hospital east of Tripoli. Gadhafi resisted, and two of the officers who tried to arrest him were killed, said the news agency, quoting a military source. "The traitors are paving the way for the occupation forces to be deployed in Tripoli," he said, calling on his supporters to march in the streets of the capital and "purify it" from "the rats." 'Traitors' Gadhafi delivered a series of defiant audio messages on state television Sunday night. He acknowledged that the opposition forces were moving into Tripoli and warned that the city would be turned into another Baghdad. "The future of Libya is now in the hands of the Libyan people," the president said. President Barack Obama said in a statement late Sunday from vacation in Martha's Vineyard, Mass., that "Tripoli is slipping from the grasp of a tyrant" and that Gadhafi's regime "is showing signs of collapsing." Rebel fighters from the west swept over 20 miles in a matter of hours Sunday, taking town after town and overwhelming a major military base as residents poured out to cheer them. At the same time, Tripoli residents secretly armed by rebels rose up. The startling rebel breakthrough, after a long deadlock in Libya's 6-month-old civil war, was the culmination of a closely coordinated plan by rebels, NATO and anti-Gadhafi residents inside Tripoli, rebel leaders said. Hundreds of people remained in the streets of the capital Monday morning. Many of them were armed, and they fired repeatedly at propaganda posters of Gadhafi. Men celebrate the arrival of rebel fighters in Tripoli in the streets of Misrata, Libya, on Monday. Earlier, rebels waving opposition flags and firing into the air drove into Green Square, a symbolic showcase of the government until it was recently used for mass demonstrations in support of Gadhafi. Rebels immediately began calling it Martyrs' Square. Two of Gadhafi's sons were captured by the rebels but the whereabouts of Gadhafi himself, one of the world's longest ruling leaders, were unknown. A rebel spokesman also told Al-Jazeera that some pro-Gadhafi forces were still fighting and controlled 15 to 20 percent of Tripoli. Bab al-Aziziya, a sprawling compound that long served as the command center for the regime, has been heavily damaged by repeated NATO airstrikes over the past five months. Tripoli resident Moammar al-Warfali, whose family home is next to Bab al-Aziziya, said tanks rolled out from the compound after a group of rebels tried to get in. He said there appeared to be only a few tanks belonging to the remaining Gadhafi forces that have not fled or surrendered. Nouri Echtiwi, another rebel spokesman in Tripoli, told Reuters: "Four hours of calm followed the street celebrations. Then tanks and pick-up trucks with heavy machine guns mounted on the back came out of Bab al-Aziziya... and started firing and shelling. They fired randomly in all directions whenever they heard gunfire." Rebel spokesman Mohammed Abdel-Rahman said tanks emerged from the complex, known as Bab al-Aziziya, and began firing shortly after dawn. Heavy fighting was reported near Moammar Gadhafi's compound Monday as government forces launched a fightback after rebels swept into the heart of the Libyan capital and crowds took to the streets to celebrate what they saw as the end of the dictator's four decades in power. P.J. Crowley, former chief spokesman for the State Department, said in an interview with msnbc TV that the swift takeover of the capital was "simply remarkable." Crowley cautioned that the world community would need to act just as swiftly to ensure stability. The National Transitional Council, a committee formed in March by anti-Gadhafi forces, has been "preparing for this day," but it would have been surprised by the overnight takeover, he said. "They will have to move swiftly," Crowley said, asking: "What will happen to their security services?... What kind of justice system will we have?" Obama keeps full vacation day after Libya briefing Earlier Sunday, the rebels overran a major military base defending the capital, carted away truckloads of weapons and raced to Tripoli with virtually no resistance. The rebels' surprising and speedy leap forward was packed into just a few dramatic hours. By nightfall, they had advanced more than 20 miles to Gadhafi's stronghold. Thousands of jubilant civilians rushed out of their homes to cheer the long convoys of pickup trucks packed with rebel fighters shooting into the air. Some were hoarse, shouting, "We are coming for you, frizz-head," a mocking nickname for Gadhafi. In villages along the way that fell to the rebels one after another, mosque loudspeakers blared, "Allahu Akbar," or "God is great." "We are going to sacrifice our lives for freedom," Nabil al-Ghowail, 30, a dentist, said as he held a rifle in the streets of Janzour, a suburb just 6 miles west of Tripoli. Heavy gunfire erupted nearby. As town after town fell and Gadhafi forces melted away, the mood turned euphoric. Some shouted: "We are getting to Tripoli tonight." Others were shooting into the air, honking horns and yelling "Allahu Akbar." Once the rebels reached Tripoli, a convoy of more than 10 trucks entered the neighborhood of Ghot Shaal on the western edge of the city and set up checkpoints. The rebels moved on to the neighborhood of Girgash, about a mile and a half from Martyrs Square. They said they came under fire from a sniper on a rooftop in the neighborhood. Sunday's first breakthrough came when hundreds of rebels fought their way into a major symbol of the Gadhafi regime — the base of the elite 32nd Brigade, commanded by Gadhafi's son Khamis. Fighters said they met little resistance. Hundreds of rebels cheered wildly and danced as they took over the compound filled with eucalyptus trees, raising their tricolor from the front gate and tearing down a large billboard of Gadhafi. Inside, they cracked open wooden crates labeled "Libyan Armed Forces" and loaded their trucks with huge quantities of munitions. One of the rebels carried off a tube of grenades, while another carted off two mortars. Across the street, rebels raided a huge warehouse, making off with hundreds of crates of rockets, artillery shells and large-caliber ammunition. The warehouse had once been used to store packaged foods, and in the back, cans of beans were still stacked toward the ceiling. The prisoners had been held in the walled compound, and when the rebels rushed in, they freed more than 300 of them. "We were sitting in our cells when, all of a sudden, we heard lots of gunfire and people yelling 'Allahu Akbar.' We didn't know what was happening, and then we saw rebels running in and saying, 'We're on your side.' And they let us out," said Majid al-Hodeiri, 23, of Zawiya. He said he was captured four months ago by Gadhafi's forces and taken to the base, where he said he was beaten and tortured. Many of the prisoners looked disoriented as they stopped at a gathering place for fighters several miles away from the base. Some had signs of severe beatings. Others were dressed in tattered T-shirts or were barefoot. Crackdown fuels rebellion The uprising against Gadhafi broke out in mid-February, and anti-regime protests quickly spread across the vast desert nation of 6 million people. A brutal regime crackdown quickly transformed the protests into an armed rebellion. Rebels seized Libya's east, setting up an internationally recognized transitional government there, and two pockets in the west, the port city of Misrata and the Nafusa mountain range. Gadhafi clung to the remaining territory, and his forces failed to subdue the rebellion in Misrata, Libya's third-largest city, and in the Nafusa mountains. Since the start of August, thousands of rebel fighters, including many who had fled Gadhafi-held cities, joined an offensive that was launched from the mountains toward the coast. The fighters who had set out from the mountains three weeks ago rushed toward Tripoli on Sunday, starting out at dawn from a village just east of the coastal city of Zawiya. Only a day earlier, the rebels had claimed full control of Zawiya, an anti-regime stronghold of 200,000 people and home to Libya's last functioning oil refinery. Rebels said Saturday that they had launched their first attack on Tripoli in coordination with NATO, and gunbattles and mortar rounds rocked the city. NATO aircraft also made heavier-than-usual bombing runs after nightfall, with loud explosions booming across the city. NATO has conducted airstrikes in Libya since the end of March to fulfill a U.N. mandate calling for military action to protect civilians. Oana Lungescu, a NATO spokeswoman, said the Western alliance would continue to enforce its mandate.
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Summary: Automatic gunfire and explosions could be heard early today as tanks rolled out of Moammar Gadhafi's complex in Tripoli and troops loyal to the Libyan strongman continued a last-ditch battle against rebels. Gadhafi's troops remain a threat to rebels who surged into the city yesterday, and "danger is still there," rebel spokesman Mohammed Abdel-Rahman told AP. Earlier, rebels were waving flags and shooting into the air to celebrate their takeover of Tripoli's central Green Square. The war seemed all but over, and President Obama has called on Gadhafi to "relinquish power once and for all." But Gadhafi has not surrendered and his whereabouts are unknown. Gadhafi called on supporters to "kill the rats" in a series of desperate audio messages on state TV last night. Though much of Gadhafi's security and palace guard has reportedly surrendered, forces loyal to him still control some 20% of the city, a rebel leader has told Al-Jazeera. The International Criminal Court issued arrest warrants for Gadhafi, his son Saif al-Islam, and the head of Libya's intelligence service for crimes against humanity, reports the BBC. Gadhafi's eldest son has surrendered and Saif al-Islam was captured, according to reports.
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Summarize: A mother has been supplying police with information about her Muslim convert son, who travelled to Somalia in hope of becoming a suicide bomber. Sally Evans took action because she fears that 24-year-old Thomas will kill innocent people after joining the Islamist Al Shabaab group behind atrocities in neighbouring Kenya. The 56-year-old said she would rather see her son in a British prison than dying for his ‘warped ideology’ in a foreign country. Thomas Evans, now known as Abdul Hakim, who has converted to Islam and is now fighting in Somalia after joining the terrorist group al-Shabaab. ‘I did tell police what happened because I have nothing to hide,’ said Mrs Evans, of Wooburn Green in Buckinghamshire. ‘I. would rather have Thomas alive behind bars than dead in the middle of. nowhere because he wouldn’t just die innocently but die doing something. that he shouldn’t be doing. Mother: Sally Evans, whose son Thomas (black shirt), has converted to Islam and is fighting with Al Shabab in Somalia. Her other son Micheal is in red. ‘And if he took other lives, how do you live with this? If he was a suicide bomber, how would I live with that?’ In. a phone conversation with her son that police were made aware of, Mrs. Evans asked if he was thinking of becoming a suicide bomber. She told. the Sunday Times he replied: ‘If asked, I would, Inshallah [God. willing].’ About 50 British. jihadists are believed to have gone to Somalia to join Al Shabaab. Hundreds have also travelled to Syria to take part in the civil war,. prompting police to urge women to report male relatives planning to go. abroad to become terrorists. Mrs. Evans told the Daily Mail that her son left school at 16 and had been a. ‘normal’ boy until he became depressed after splitting with a long-term. girlfriend and slipped into ‘troublemaking’. Evans. converted to Islam when he was 19 and adopted the Islamic name Abdul. Hakim. His beliefs became increasingly fundamental after he started. going to a small prayer centre. Now. with a beard and shaved head, he tried to convince his mother and. younger brother Micheal to become Muslims, saying: ‘I want you to. convert to Islam, otherwise you will burn in hell.’ Divorcee Mrs Evans said one of the few friends he brought to her house was Donald Stewart-Whyte. Another. convert, Stewart-Whyte was arrested in 2006 in connection with the. liquid bomb plot to blow up planes but was later cleared of any. involvement. 'Normal boy': Thomas at his home in Wooburn Green, Buckinghamshire, when he was about 18 years old. Evans attempted. to fly to Kenya in February 2011 but was blocked by anti-terrorism. police at Heathrow. A few months later he went to Egypt, saying he was. planning to study Arabic. Police. later informed his mother he had been trying to enter Kenya. It wasn’t. until January 2012 that he phoned and told her he was in Somalia and was. prepared to die for his Islamist beliefs. He. said she wouldn’t see him again. In August that year he claimed he had. been shot in the leg and treated at a ‘field hospital’. Mrs. Evans added: ‘I am heartbroken at his decision to leave me and his. brother to follow a warped ideology but I still love him. I wish they. had a magic wand to bring him back.’ The Metropolitan Police, who have oversight on counter-terrorism issues, declined to comment
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Summary: Thomas Evans from Buckinghamshire converted to Islam at aged of 19. Changed his name to Abdul Hakim and lost all interest in his hobbies. Travelled to Somalia in 2011 to fight alongside terror group al-Shabaab. Mother Sally Evans, 56, fears he could become a suicide bomber. She keeps police informed of his activities when he gets in contact. Says she'd rather see him behind bars than fighting with terror group.
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Summarize: Background LSC was established in 1974 as a private, nonprofit, federally funded corporation to provide legal assistance to low-income people in civil matters. LSC provides the assistance indirectly through grants to competitively selected local programs. LSC distributes funds to the grantees on the basis of the number of low-income persons living within a service area. Grantees may receive additional funding from non-LSC sources. During 1998, LSC funded 262 local grantees that operated through approximately 900 neighborhood law offices employing about 3,600 attorneys and 1,400 paralegals. Each local program is governed by its own board of directors and is required to spend at least 12.5 percent of its LSC grant to encourage private attorney involvement (PAI) in delivering legal services to low-income clients. In fiscal years 1998 and 1999, LSC received appropriations of $283 million and $300 million, respectively. LSC’s authorizing legislation restricts it from engaging in lobbying; political activities; class actions except under certain conditions; and cases involving abortion, school desegregation, and draft registration or desertion from the military. Annual appropriations laws have placed additional restrictions on the activities in which LSC grantees can engage, even with non-LSC funds. In 1996, for example, grantees were prohibited from engaging in challenges to welfare reform, litigation on behalf of prisoners, representation in drug-related public housing evictions, and representation of certain categories of aliens. Grantees must serve only those clients who meet financial and citizenship/alien eligibility requirements. With respect to client financial eligibility, local programs are to establish their own criteria, which, in general, require that clients’ income not exceed 125 percent of the federal poverty guidelines. With appropriate documentation of the grantee’s decision, clients who are between 125 percent and 187.5 percent of the federal poverty level may be found eligible. LSC regulations require that grantees (1) adopt a form and procedure to obtain eligibility information and (2) preserve that information for audit by LSC. With regard to citizenship/alien eligibility, only citizens and certain categories of aliens are eligible for services. For clients who are provided services in person, a citizen attestation form or documentation of eligible alien status is required. For clients who are provided services on the telephone, grantees must document that they made inquiries regarding the individuals’ citizenship/alien eligibility. LSC uses a Case Service Reporting (CSR) system to gather quantifiable information from grantees on the services they provide that meet LSC’s definition of a case. The CSR Handbook is LSC’s primary official guidance to grantees on how to record and report cases. According to the 1999 CSR Handbook, which revised and expanded the guidance in LSC’s 1993 Handbook, information about cases is an important indicator of the number of legal problems that programs address each year, and LSC relies on such case information in its annual request for federal funding for legal services. Audit reports on five grantees issued by LSC’s OIG between October 1998 and July 1999 reported that all five grantees misreported the number of cases they had closed during calendar year 1997 and the number of cases that remained open at the end of that year. The OIG found that all five grantees overstated the number of closed cases, while four overstated and one understated open cases. The OIG attributed the overreporting to such factors as (1) counting as cases telephone calls in which individuals were not provided any legal assistance and only partial eligibility determinations were made; (2) counting wholly non-LSC funded cases as LSC cases; (3) double counting of the same cases; (4) reporting cases as closed during 1997, or as still open at the end of 1997, when service ceased in prior years; and (5) counting as cases the provision of services to over-income clients. In June 1999, in response to Congress’ request for information on whether the 1997 case data of other LSC programs had problems similar to those reported by LSC’s OIG, we issued a report on our audit of five of LSC’s largest grantees: Baltimore, Chicago, Los Angeles, New York City, and Puerto Rico. We found similar types of reporting errors at the five grantees and estimated that, overall, 75,000 of the 221,000 open and closed cases that the five grantees reported to LSC were questionable. Interviews that we conducted with LSC officials and executive directors of the 5 audited grantees indicated that they had taken or were planning to take steps to correct the causes of these case-reporting problems. Objectives, Scope, and Methodology Our objectives were to determine (1) what efforts LSC and its grantees have made to correct problems with case service reporting, and (2) whether these efforts are likely to resolve the case reporting problems that occurred in 1997. To address the objectives, we reviewed documents that contained LSC case reporting guidance, interviewed cognizant officials at LSC headquarters, and conducted structured telephone interviews with a random sample of grantee executive directors. Specifically, we reviewed LSC regulations and LSC’s 1993 and 1999 CSR Handbook, as well as supplemental guidance that LSC distributed to grantees in the form of program letters and frequently asked questions and answers about case reporting. We also collected documentation and interviewed LSC officials and grantee executive directors to gather information about a self- inspection process that LSC required all grantees to undertake in order to determine the accuracy of their 1998 case data. In our telephone interviews with executive directors, we asked if the directors viewed LSC’s case reporting requirements as being clear, what changes they have made or planned to make as a result of the requirements, and the results of their self-inspection of 1998 case data. We also interviewed LSC officials and grantee executive directors about areas of case reporting that they felt needed further clarification. To identify the universe of LSC grantees, an LSC official referred us to the list of LSC programs on the corporation’s Internet Web site. As of July 15, 1999, the Internet site listed 256 programs. From this list, we randomly selected 80 programs for our sample. We developed the interview instrument, pretested it with executive directors from two grantees, revised the instrument based on the pretest results, and completed approximately 1-hour-long structured interviews by telephone with the executive directors of 79 of the 80 programs, for a 99 percent response rate. (After numerous attempts over several days, we were unable to contact the executive director of one LSC grantee.) Our sample was designed so that we could generalize our findings with 95-percent confidence and a maximum 10-percent margin of error to the universe of LSC grantees. We performed our work from July through September 1999 in accordance with generally accepted government auditing standards. We requested comments on a draft of this report from the President of LSC. LSC’s comments are discussed at the end of this letter and included as appendix I. LSC Clarified CSR Requirements and Grantees Have Been Making Program Changes to Comply With the Requirements, but Some Grantees Remain Unclear About Certain Requirements LSC issued a new CSR Handbook and distributed other written communications intended to clarify reporting requirements to its grantees. Most grantees indicated that the new guidance helped clarify LSC’s reporting requirements, and virtually all of them indicated that they had or planned to make program changes as a result of the requirements. Many grantees, however, identified areas of case reporting that remained unclear to them. LSC Issued New CSR Guidance To address specific problems identified by the OIG and LSC’s own internal program reviews, LSC has issued revised reporting guidance. In November 1998, LSC issued the 1999 CSR Handbook, which replaced the 1993 CSR Handbook. The 1999 handbook instituted changes to some of LSC’s reporting requirements and provided more detailed information on other requirements than previously existed. LSC also distributed program letters and a list of frequently asked CSR questions and answers that further elaborated on points made in the 1999 handbook. The 1999 CSR Handbook instituted several notable changes to case reporting requirements. These included (1) procedures for timely closing of cases; (2) procedures for management review of case service reports; (3) procedures for ensuring single recording of cases; (4) requirements to report LSC-eligible cases, regardless of funding source; and (5) requirements for reporting PAI cases separately. In addition, grantees were required to use automated case management systems and procedures that would ensure that program managers had timely access to accurate information on cases and the capacity to meet their reporting requirements. On November 24, 1998, LSC informed its grantees that two of the changes in the 1999 CSR Handbook were to be applied to the 1998 case data. The two changes pertained to timely closing of cases and management review of case service reports. The timely closing provision required grantees to ensure that cases in which legal assistance had ceased in 1998, and was not likely to resume, would be closed prior to grantees’ submission of case service reports to LSC in March 1999. To the extent practicable, cases in which the only assistance provided to the client was counsel and advice, brief service, or referral after legal assessment were to be closed in the year in which these types of service were provided. Cases involving other types of service were to be closed in the year in which program staff determined that further legal assistance was unnecessary, not possible, or inadvisable and a closing memorandum or other case-closing notation was prepared. The management review provision required the executive director, or a designee, to review the program’s case service reports prior to their submission to LSC in order to ensure their accuracy and completeness. The remaining new provisions of the 1999 CSR Handbook were not applicable to 1998 cases. For example, for 1998, there was no requirement for grantees to ensure that cases were not double counted. For 1999, LSC is requiring the use of automated case management systems and procedures to ensure that cases involving the same client and specific legal problem are not reported to LSC more than once. For 1998, grantees could report only those cases that were at least partially supported by LSC funds. For 1999, LSC is requiring grantees to report all LSC-eligible cases, regardless of funding source. LSC intends to estimate the percentage of activity spent on LSC service by applying a formula that incorporates the amount of funds grantees receive from other funding sources compared with the amount they receive from LSC. For 1998, grantees were required to report their LSC-funded PAI cases together with non-PAI cases. For 1999, PAI cases are to be reported separately. In addition to changing certain reporting requirements, the 1999 handbook also provides more detailed guidance to grantees than the 1993 handbook. For example, the 1999 handbook provides more specific definitions of what constitutes a “case” and a “client” for CSR purposes. The 1999 handbook also addresses documentation requirements that were not discussed in the 1993 handbook. For example, the 1999 handbook indicates that, except for telephone service cases, the client’s file must contain an attestation of citizenship or documentation of alien eligibility in order for the case to be reported to LSC. The 1999 handbook also imposes requirements for documentation of information on client income and assets. The handbook states that, for all cases reported to LSC, the eligibility documentation must include specific information about income and assets. The 1999 handbook also contains a requirement that legal assistance, to be counted as a case, must be provided by an attorney or paralegal. Grantee Directors Reported That They Are Implementing Changes to Comply With Reporting Requirements On the basis of our survey, we estimate that over 90 percent of grantee executive directors viewed the changes in the 1999 CSR Handbook as being clear overall, and virtually all of them planned to or had made at least one change to their program operations as a result of the revised case reporting requirements. Program changes that were cited included revising policies and procedures, providing staff training, modifying forms and/or procedures used during client intake, implementing computer hardware and software changes, and increasing review of cases. Nearly half of the grantees indicated that they planned to or had revised their policies and/or procedures to comply with the 1999 handbook requirements, and slightly less than a third planned to or had conducted staff training on the requirements. Respondents told us that the focus of their training was on such issues as the current definition of a case, how to determine and document clients’ financial eligibility, how to determine and document citizenship/alien eligibility, timely closing of cases, and prevention of duplicate case reporting. More than half of the grantees indicated that they planned to or had changed their intake forms and/or procedures. Of the 41 respondents who made this comment, 26 said they were making these changes in order to document client income and assets. Slightly over 40 percent of the grantees reported that they planned to or had made computer changes. The computer changes included such actions as adding new fields to their automated case management (e.g., to ensure that client eligibility and acceptance information is recorded), making programming changes (e.g., to identify duplicate cases, ensure that cases are not assigned separate numbers for the same client with the same legal problem, and better document client assets), and installing new software (e.g., to generate reports so that they can track how long cases are open). About 10 percent of grantee executive directors indicated that they could better comply with CSR requirements if they had uniform case management software. LSC, too, believes that grantees’ case management systems should provide for more consistency in the collection and processing of CSR information. According to an LSC official, LSC is developing a strategy for modifying grantees’ case management systems so that data errors can be detected and prevented. As part of the strategy, LSC has hired an expert in case management systems and is working to develop standard input requirements for these systems. LSC intends to work with case management system vendors to modify the systems so that they prevent cases from being accepted if grantees do not record the required compliance information. LSC expects modifications to the case management systems to be implemented in calendar year 2000 and available for application to grantees’ 2000 CSR data. In the nearer term, LSC plans to work with a contractor to develop customized case management queries that would enable grantees to detect and remedy errors in their 1999 case data. In 1999, LSC plans to pilot test the computer queries at five programs that have the most commonly used case management systems. Nearly three-fourths of the grantees indicated that they planned to or had increased their review of cases to comply with LSC requirements. Respondents cited such review activities as more intense monitoring of open cases, to ensure that they are closed in a timely manner, and more thorough monitoring of PAI cases. Some grantees said they have directed increased attention to reporting requirements during routine reviews of cases, while others said they instituted more frequent reviews of cases. One executive director reported to LSC that managing attorneys would meet with all case-handling staff and reemphasize the importance of keeping accurate activity records. These managing attorneys are to monitor compliance with the requirement initially on a weekly basis and then on a random basis. If deficiencies are discovered, the managing attorney is to direct the case handler to correct them immediately, and the managing attorney is to recheck the file within 48 hours to ensure compliance with the directive. Another executive director told us that his program had not previously reviewed CSR data. He said that they now are reviewing reports, along with listings of open and closed cases, to ensure their compliance with LSC guidelines. Some executive directors said that they planned to become more involved in reviewing case files and case management reports. Many grantees reported making other efforts to comply with reporting requirements, such as disseminating the new handbook to case handlers, developing compliance checklists, emphasizing the importance of compliance to staff, sending written instructions or memos to staff, holding meetings and discussions, providing more feedback to case handlers, and more stringently enforcing requirements. Many Grantees Remain Unclear About Certain Reporting Requirements Although most of the grantee executive directors reported that the new LSC guidance helped clarify requirements, many of them also indicated that they were still unclear about certain requirements and that additional clarification was needed. Among the areas of confusion or uncertainty that executive directors identified were requirements pertaining to asset and citizenship/alien eligibility documentation, single recording of cases, and who can provide legal services. Asset documentation: The 1999 CSR Handbook states that, for all cases to be reported to LSC, the eligibility documentation should include specific information about income and assets. About 30 percent of the executive directors indicated that LSC’s requirements for documenting client assets were clear only to some, to little, or to no extent. Of the 24 respondents who gave this response, 23 made comments to the effect that LSC should clarify what it means by assets and asset limits and/or clarify its documentation requirements for client assets. In a July 14, 1999, program letter to grantees, LSC noted that although many grantees inquire about applicants’ assets, they do not consistently document either the inquiries or the applicants’ responses. In its program letter, LSC sought to clarify its requirements for documenting and preserving the asset information obtained from each applicant. Because we conducted our telephone survey in late July and early August, we do not know how many of the executive directors with whom we spoke had reviewed LSC’s July 14 guidance and felt that it sufficiently clarified their questions concerning asset documentation. However, two respondents told us that LSC’s July 14 guidance was still unclear. One respondent said, for example, that the program letter left unclear the meaning of the term “household goods,” whether exempted assets had to be documented, who was to value the goods, and who was to determine what to count and what not to count as part of assets. Our own analysis of LSC guidance on the asset issue indicated that LSC has not been consistent in its directives to grantees about the specificity of asset information they must have in order to comply with CSR reporting requirements. For example, in its March 24, 1999, communication on frequently asked CSR questions and answers, LSC instructed grantees to document, at minimum, the total amount of household assets accessible to the client. In its July 14, 1999, program letter, LSC instructed grantees to identify and document all of the liquid and nonliquid assets of all persons who are resident members of a family unit. Citizenship/alien eligibility documentation requirements for telephone cases: The 1999 CSR Handbook does not explicitly address the citizenship or eligible alien status documentation requirements for situations where assistance is provided only over the telephone. Nearly one-fourth of the executive directors indicated that LSC’s documentation requirements in this area were clear only to some, to little, or to no extent. Of 19 respondents who gave this response in our survey, 15 said that more clarification was needed on the grantees’ documentation responsibilities. Respondents said, for example, that they were confused about exactly when they needed to obtain a written attestation, whether it was sufficient simply to record that questions about citizenship/alien eligibility had been asked, whether certain types of service required documentation while others did not, and whether the requirement changed if an individual receiving assistance over the telephone came into the office to drop off documents. In its July 14, 1999, program letter to grantees, LSC sought to clarify its requirements for documenting citizenship/alien eligibility information in telephone cases. LSC stated that it requires recipients to make appropriate inquiry of every telephone applicant and record the inquiry and response. All such documentation is to be maintained in the client file. We do not know how many of the respondents to our survey were familiar with LSC’s July 14 guidance when we interviewed them in late July and early August. Single recording of cases: The 1999 CSR Handbook requires that programs ensure that cases involving the same client and specific legal problem are not recorded and reported to LSC more than once. Over one-fourth of the executive directors indicated that the requirement for preventing duplicate case reporting was clear only to some, to little, or to no extent. Of the 22 respondents who gave this response in our survey, 17 said that the distinction between specific and related legal problems is difficult to determine. Several respondents cited examples in family law cases where more than one problem can arise within one family case. Depending on the situation, it was not clear to them at what point legally related issues become separate enough to count as separate cases. One respondent indicated that attorneys in his program have a hard time interpreting this requirement, and that he got calls every week about this issue. Provider of legal assistance: The CSR handbook requires that legal assistance be provided by an attorney or paralegal in order for a service to be called a case. Slightly over 40 percent of executive directors indicated that LSC was clear only to some, to little, or to no extent about who can provide such legal assistance. Respondents noted in our telephone survey, for example, that the terms “case handler,” and “paralegal” are not clearly defined, and that they did not know whether nonlawyers (e.g., intake workers) who are supervised by lawyers can provide legal assistance. Comments made to us by the executive directors revealed that grantees held varying views about who can provide legal assistance to clients. Several respondents specifically stated that this is an important issue that LSC should address in its handbook. Our own analysis of LSC guidance on this issue indicates that LSC has not been consistent in its advice to grantees. For example, in its communication with grantees on frequently asked CSR questions and answers, LSC stated that a telephone conversation between an intake specialist and a caller who was accepted for service can be counted as a case if the caller received some advice that addressed a specific legal problem. LSC officials told us that LSC’s current position on this issue is that the person giving legal advice must be someone who is authorized to practice law or is under the supervision of an attorney in accordance with local rules of practice. Therefore, legal advice can be given by a (1) lawyer, (2) paralegal, or (3) intake specialist or law student under the supervision of a lawyer, as long as the assistance does not violate local rules of practice. Most Grantees Certified to the Accuracy of Their 1998 CSR Data, but Questions About Data Accuracy and Interpretation Remain LSC sought to determine the accuracy of grantees’ case data by requiring that grantees do self-inspections of their open and closed caseload data for 1998. Grantees were required to determine whether the error rate in their data exceeded 5 percent. If the error rate was 5 percent or less, they could certify that their data were substantially correct. If the error rate was higher than 5 percent, they were to determine how the problems identified could be addressed. LSC found that about three-fourths of the grantees were able to certify to the substantial accuracy of their data. LSC used the results of the self-inspections to estimate the total number of case closings in 1998. Our review of LSC’s self-inspection process raised concerns about the accuracy and interpretation of the results, and what the correct number of certifying programs should be. LSC Grantees Conducted Self-Inspections of 1998 CSR Data On May 14, 1999, LSC issued a memo to all grantees instructing them to complete a self-inspection procedure by July 1, 1999. The purpose of the self-inspection was to ensure that (1) grantees were properly applying instructions in the 1999 edition of the CSR Handbook that were applicable to the 1998 data, and (2) LSC had accurate case statistical information to report to Congress for calendar year 1998. LSC provided detailed guidance to grantees on the procedures for the self- inspection. Each grantee was to select and separately test random samples of open and closed cases to determine whether the number of cases it reported to LSC earlier in the year was correct. Grantees were to verify that the case file contained a notation of the type of assistance provided, the date on which the assistance was provided, and the name of the case handler providing the assistance. Grantees were also to determine whether assistance had ceased prior to January 1, 1998; was within certain service categories as defined by the 1999 handbook; was provided by an attorney or paralegal; and was not prohibited or restricted. Finally, grantees were to verify that each case had eligibility information on household income, household size, household assets, citizenship attestation for in-person cases, and indication of citizenship/alien status for telephone-only cases. According to LSC officials, the self-inspection was a single procedure that was undertaken within a limited time period, and LSC did not expect the self-inspection to resolve all case-reporting problems. In requiring grantees to verify that their 1998 case files contained information on client assets and an indication of citizenship/alien status for telephone-only cases, LSC imposed stringent criteria on the self- inspections. The criteria were stringent in that LSC had not promulgated explicit documentation requirements related to these issues until it released the 1999 CSR Handbook and July 14 program letter. Grantees were not required to apply these new documentation requirements to their 1998 case data. Recognizing that in 1998 many grantees did not keep sufficient documentation on assets or citizenship/alien eligibility to comply with the stringent self-inspection requirements, LSC allowed grantees some latitude in determining whether they were in fundamental compliance with the requirement that legal assistance only be provided to eligible individuals. In conversations with grantee executive directors during the self-inspection, LSC officials advised them that if they had a level of certainty that their program staff had asked questions about assets, and if their certainty was sufficient for them to sign a form attesting to this, that was an acceptable basis for asserting compliance with the reporting requirement for assets. Similarly, if grantees were sufficiently certain that, for telephone cases, their program staff had asked questions about citizenship/alien status but had not documented the inquiry, that too was acceptable. Finally, LSC allowed grantees some latitude with respect to the requirement that an attorney or paralegal must be the provider of legal assistance. LSC advised grantees during the self-inspection that it was acceptable for them to count as valid cases instances in which legal advice was given by a (1) lawyer, (2) paralegal, or (3) intake specialist or law student under the supervision of a lawyer, as long as the assistance did not violate local rules of practice. If any single aspect of a case failed to meet LSC’s requirements, the case was to be classified as an error for reporting purposes. If the grantees found that their CSR case sampling had an error rate of 5 percent or less, the program directors and policy board chairs were to sign a certification form and return it to LSC. Grantees who could not certify to the correctness of their 1998 CSR data were to submit a letter to LSC describing (1) the problems they had identified during the self-inspection process and (2) the corrective actions they had instituted to address the problems. Grantees could resubmit their 1998 CSR data to LSC if they identified one or more problems in the random sample and corrected their entire 1998 database so that the problems no longer appeared. If, by correcting the problems, the error rate in the data was reduced to 5 percent or less, the grantees could resubmit their 1998 data along with a signed certification attesting to the substantial accuracy of the resubmitted data. In this way, grantees who were unable to certify at one point in time could certify at a later point in time. As of July 29, 1999, 26 grantees had resubmitted their 1998 CSR data after having made corrections to the data, and 20 of them had certified their data. Most Grantees Certified Their 1998 CSR Data According to LSC officials, about three-fourths of the grantees certified the accuracy of their 1998 case data. As of August 26, 1999, LSC documents indicated that 199 of 261 grantees (76 percent) reported substantially correct CSR data to LSC. The remaining 62 grantees (24 percent) did not certify to LSC that their CSR data were substantially correct. On the basis of the self-inspection results, LSC estimated that grantees closed 1.1 million cases in 1998. LSC officials told us that they were surprised that such a large number of grantees were able to certify their 1998 CSR data. They attributed the lower-than-expected error rates to the following factors: The self-inspection did not attempt to identify duplicate cases. LSC officials explained that, during most of 1998, LSC did not have a standard regarding duplicate cases, and that they believed it would have been too burdensome on grantees for LSC to require them to apply the new standard retroactively. According to an LSC official, duplicate cases are best caught at the time of intake. The official also noted that duplicate cases had not in the past been found to be a major problem in comparison with other identified problems. Grantees received the new 1999 CSR Handbook in November1998, and a number of them had implemented the requirements that applied to their 1998 data by the time they submitted their 1998 case statistics. Grantees were aware of the problem that the OIG had identified with cases coded as referral after legal assessment. That is, some programs had inappropriately reported as CSR cases numerous instances of making a telephone referral without providing legal advice and/or without documenting an individual’s eligibility. Because grantees had been sensitized to this issue, LSC officials believed that they were less likely to count these referrals as cases in 1998. According to LSC officials, CSR problems were more common at larger grantees that had heavier case loads and multiple branches. They noted that approximately 30 of the 50 grantees with the largest caseloads were unable to certify their 1998 data. They hypothesized that larger programs may have difficulty certifying if such programs have (1) numerous branch offices, one or more of which are out of compliance with regulations and causing reporting problems for the entire program, and/or (2) numerous sources of funding and more compliance requirements, which could add to the complexity of their reporting process. The officials acknowledged that these hypotheses needed further exploration. LSC officials also reported that noncertifying grantees identified two principal problems. One pertained to the lack of citizenship attestations in case files. The second pertained to the lack of information on client assets. LSC officials also noted that some grantees had reported matters, such as referrals, as cases, and some had reported cases as open when they did not meet the timely closing requirement. In our telephone interviews with 79 executive directors, 24 reported that they did not certify their 1998 data to LSC. Factors that the executive directors of noncertifying programs believed greatly affected the errors in their 1998 data included lack of clear guidance from LSC (cited by 11 respondents), computer problems (cited by 7 respondents), and insufficient attention by their programs to administrative matters (cited by 5 respondents). Computer problems that were noted included difficulties merging databases and using several different software packages, and problems with upgrading the computer system. LSC Used Self-Inspection Results to Estimate Number of Cases Closed Based on the self-inspection results, LSC estimated that its grantees closed about 1.1 million cases in 1998. LSC arrived at this figure by subtracting the total number of closed cases estimated to be in error (135,498) from the total number of cases that grantees reported to LSC (1,260,351). As of July 29, 1999, LSC estimated that its grantees’ total closed caseload for 1998 was 1,124,853. LSC intends to report only the estimated number of cases closed in 1998, even though the self-inspections were to encompass both open and closed cases. According to LSC officials, LSC has less confidence in the open-case numbers than the closed-case numbers. One reason for this is that they believe open cases are more likely than closed cases to have timely closing problems. A second reason is that some grantees experienced problems when converting to a new computer system. System conversions sometimes caused dates to be lost or cases to be mistakenly coded as being open. Self-Inspection Results Raised Concerns Our review of LSC’s self-inspection results raised some concerns about LSC’s interpretation of the results and about the accuracy of the data provided to LSC by grantees. As a result, we could not assess whether the number of certified programs and case closures that LSC estimated for 1998 is correct, lower, or higher than it should be. Although LSC provided instructions to grantees on how they should select test samples and what case information they should verify, LSC did not issue standardized procedures for grantees to use in reporting the results of their self-inspections. Grantees that could not certify their data wrote letters to LSC in which they described the errors they uncovered. The letters contained varying degrees of detail about the errors. Some programs provided an overall error rate and did not separately report how much error they found in open and closed cases. Others provided detailed information on both the number of errors found in open and closed cases, respectively, as well as the number of errors broken out by type. Since LSC did not have a standard protocol for collecting the results of the self- inspections, in some cases LSC officials had to rely on interpreting grantee letters that described the problems that were discovered. LSC officials believe that their numerous contacts with grantees who called them to ask questions about the self-inspections, combined with their analysis of the grantee letters, enabled them to correctly determine the number of certifying programs and estimate the number of closed cases. We are uncertain how many programs should have been counted as certified because we are uncertain if LSC applied a consistent definition of “certification.” Most programs that were on LSC’s certification list determined that they had error rates of 5 percent or less for both open and closed cases. However, LSC placed some programs on the certified list if the program’s overall error rate for closed cases was 5 percent or less, even if the overall error rate actually was higher than 5 percent. We encountered this situation in two instances in which executive directors told us in telephone interviews that they did not certify their CSR data because their overall error rate exceeded 5 percent. However, these programs appeared on LSC’s list of certified programs. When we asked an LSC official about this, he told us that they advised grantees that if their closed case error rate did not exceed 5 percent, they should “partially certify” their data. In response to our inquiry, the official reviewed the certification letters submitted by nearly 200 grantees, and he identified 5 certified programs whose error rates for open cases exceeded 5 percent. Given that some grantees submitted only an overall estimate of data error, we do not know how many programs qualified to be certified overall, just for closed cases, or just for open cases. In another instance, an executive director told us that she did not certify her program because 8 of the 14 case-handling offices had error rates exceeding 5 percent. Nonetheless, this program appeared on LSC’s certified list. An LSC official explained that, after reviewing the information provided by this program, the official agreed that the program should not have been classified as certified. In a fourth instance, in which an executive director reported to us that he did not certify his program, an LSC official said that, although they thought the grantee’s data had been corrected, the program had not yet certified its data and might need to do additional sampling. We are also concerned that LSC’s instructions to grantees on how to conduct the self-inspections may have led some of the smaller grantees to select too few test cases to make a reliable assessment of the proportion of error in their case data. For example, LSC instructed grantees to select every tenth case for review if the program handled less than 1,000 cases in 1998. This was to be done separately for open and closed cases. Based on 1998 case statistics that grantees submitted to LSC between January and March 1999, several programs would have based their certification determinations on reviews of a relatively small number of cases. Seven grantees had fewer than 300 closed cases, and 43 grantees had fewer than 300 open cases. Three programs had fewer than 300 cases combined. The smallest program would have based its self-inspection on 3 closed cases and 1 open case. We believe that, in general, samples of 30 or fewer cases are too small to provide reliable estimates of the total number of case data errors. Because these were smaller grantees, this limitation would have had little effect on LSC’s estimate of total closed caseload. However, it could have affected—by either overstating or understating—LSC’s count of the number of certified programs. LSC does not know how well grantees conducted the self-inspection process, nor how accurate the results are. We spoke with several executive directors who did not correctly follow LSC’s reporting requirements. In one case, the executive director sought clarification from LSC headquarters about the use of a CSR closure code and was given incorrect verbal guidance. The situation concerned whether applicant files could be closed as “client withdrew” cases when individuals who were accepted for service and assigned to staff attorneys did not subsequently appear for a meeting with the attorney. According to both the executive director and an LSC official, the executive director was told that if the program attempted to contact these individuals, by telephone or letter, to determine whether they were still interested in obtaining legal assistance, they could be counted as cases. The executive director told us that she would review her entire database for this type of error and make corrections. This program was included on LSC’s certified list, but we do not know whether the program would stay on that list if all the errors were identified. Another executive director told us that he was concerned that LSC did not want grantees to count assistance over the telephone as a case. This is not an entirely correct interpretation of LSC guidance since, under certain conditions, LSC permits legal assistance over the telephone to be counted as a case. Although this program was included on LSC’s certified list, any valid telephone cases that were not counted would have erroneously increased its error rate. A third executive director told us that the timely closing rule required him to close new cases in December. If the person sought assistance with a similar problem in January, it would be treated as a new case because a new reporting year had begun. In a March 1999 written communication intended to supplement the 1999 handbook, LSC advised grantees to exercise discretion about when to close cases that were opened near the end of the year. LSC did not require grantees to close all cases at year-end. These examples illustrate the possibility that incorrect interpretations of LSC guidance may have resulted in some programs certifying their 1998 data when they shouldn’t have, and other programs not certifying their 1998 data when they should have. An LSC official told us that, although they have conducted CSR training sessions for grantee executive directors, there are thousands of case handlers in grantee offices who have not received similar training. The official acknowledged that written guidance and telephone contacts with grantees may not be sufficient to ensure correct and consistent understanding of reporting requirements, and that LSC may consider alternative ways of providing training to staff, such as through videos. Incorrect interpretations of LSC guidance could also have affected the accuracy of LSC’s estimate of closed cases. The Inspector General told us that his office has completed audits of six grantees’ 1998 case data. One additional audit of a grantee’s CSR data had not yet been completed. The six completed audits included both certifying and noncertifying grantees. According to the Inspector General, the results of the six completed audits would be provided to Congress by September 29, 1999. LSC officials told us that the self-inspection was valuable and that LSC plans to have grantees complete self-inspections again early next year as part of the 1999 CSR reporting process. We agree that a self-inspection process can be valuable, provided that grantees have clear, consistent, and appropriate guidance on the procedures for reviewing their case data, determining whether to certify the data, correcting errors, and providing their results to LSC in a standardized way that facilitates validation of the results. Conclusions LSC’s 1999 CSR Handbook and other written communications have improved the clarity of reporting requirements for its grantees. However, many grantees remain unclear about and/or misunderstood certain aspects of the reporting requirements. LSC’s practice of disseminating guidance primarily by written or telephone communications may not be sufficient to ensure that grantees correctly and consistently interpret the requirements. LSC sought to determine the accuracy of grantees’ 1998 case statistics by requiring grantees to conduct self-inspections. However, we do not know the extent to which the results of the self-inspection process are accurate. The validity of the results are difficult to determine because LSC did not standardize the way that grantees were to report their results, some of the grantees used samples that were too small to assess the proportion of error in their data, some grantees did not correctly follow LSC’s reporting guidance, and LSC had done no verification of the grantees’ self-inspection procedures. We do not believe that LSC’s efforts, to date, have been sufficient to fully resolve the case reporting problems that occurred in 1997. Recommendations We recommend that the President of LSC clarify and disseminate information on the specific information on client assets that grantees must obtain, record, and maintain; clarify and disseminate information on the types of citizenship/alien eligibility information grantees must obtain, record, and maintain for clients who receive legal assistance only over the telephone; clarify and disseminate LSC’s criteria for single recording of cases; clarify and disseminate LSC’s policy concerning who can provide legal assistance to clients for the service to be counted as a case; explore options for facilitating correct and consistent understanding of reporting requirements, such as developing and disseminating a training video for grantee staff; develop a standard protocol for future self-inspections to ensure that grantees systematically and consistently report their results for open and closed cases; direct grantees to select samples for future self-inspections that are sufficient to draw reliable conclusions about magnitude of case data errors; and finally, ensure that procedures are in place to validate the results of LSC’s 1998 self-inspection, as well as of any future self-inspections. Agency Comments The President of LSC provided written comments on a draft of this report, which are printed in full in appendix I. LSC generally agreed with our findings and stated that it intends to implement our recommendations. Concerning the issue of the clarity of case reporting guidance, LSC’s letter stated that LSC is troubled by our finding that some grantees are continuing to have difficulty in this area. LSC’s letter reiterated a point that we made in the report; namely, that the overlap in time between our data collection effort and LSC’s distribution to grantees of a July 1999 program letter intended to clarify client eligibility documentation requirements may have caused some of the executive directors not to factor in the new guidance when they responded to our telephone interview questions. Although, as we state on pages 9 and 10 of the report, some respondents may not have been familiar with LSC’s July 1999 guidance, two respondents told us that they were familiar with the new guidance and that they were still unclear about the requirements dealing with assets. We also state on page 10 of the report that our own analysis indicated that LSC was not consistent in its guidance to grantees on what asset information they needed to obtain from clients. Therefore, we do not believe that grantees’ lack of clarity concerning reporting requirements on client eligibility was due solely to the overlap in the time period of our data collection and LSC’s distribution of the July 1999 program letter. With respect to grantees’ lack of clarity about how to determine duplicate case counting, LSC indicated that it will consider providing additional guidance to grantees through further revisions to the CSR handbook. LSC also indicated that it will revise the handbook to clarify the issue of when legal assistance by nonlawyers can be reported as a case. LSC also reiterated that it is developing additional methods to supplement written guidance, including (1) conducting training and technical assistance, (2) developing case management standards that would detect and prevent cases from being accepted and reported if the required eligibility documentation was not obtained, and (3) developing database queries for grantees to apply to their case management systems to identify instances where cases lacked required compliance documentation. Concerning the self-inspection results, LSC noted that its principal purpose in requiring grantees to self-inspect their 1998 case data was to ensure that case-reporting guidance was being properly applied. LSC stated that assessing the accuracy of the 1998 case statistics that grantees submitted to LSC in March 1999 was a secondary purpose of the self-inspection. LSC believes that the self-inspection increased grantees’ awareness of reporting requirements and prompted them to make changes in their case reporting practices. Indeed, in LSC’s view, the self-inspection requirement resulted in more program improvements than did the 1999 CSR handbook and other written guidance issued by LSC because grantees that identified significant problems were required to take corrective actions. LSC notes correctly that, in relation to the self-inspection, our report focuses on the accuracy of LSC’s determination of the number of grantees that certified their data, and the factors that affected the accuracy of LSC’s estimate of the total number of closed cases in 1998. Our review focused on the stated goals of the self-assessment. Although we did not assess whether, relative to LSC’s other efforts, the self-inspection had a greater effect on grantees’ awareness of and compliance with CSR reporting, we do report that 26 grantees made corrections to their 1998 data as a result of their self- inspections. As arranged with your offices, unless you publicly announce the contents of this letter earlier, we plan no further distribution until 7 days after the date of this letter. At that time, we will send a copy of the report to the Chairmen and Ranking Minority Members of LSC’s appropriations and legislative committees and to Mr. John McKay, the President of LSC. The major contributors to this report are acknowledged in appendix II. If you or your staff have any questions concerning this report, please contact me or Evi L. Rezmovic on 202-512-8777. Comments From the Legal Services Corporation GAO Contacts and Staff Acknowledgments GAO Contacts Acknowledgments In addition to those named above, Mark Tremba, Kristeen McLain, Jan Montgomery, David Alexander, Barry Seltser, Lemuel Jackson, Brian Lipman, and Walter Vance made key contributions to this report. The first copy of each GAO report and testimony is free. Additional copies are $2 each. Orders should be sent to the following address, accompanied by a check or money order made out to the Superintendent of Documents, when necessary. VISA and MasterCard credit cards are accepted, also. Orders for 100 or more copies to be mailed to a single address are discounted 25 percent. U.S. General Accounting Office P.O. Box 37050 Washington, DC 20013 Room 1100 700 4th St. NW (corner of 4th and G Sts. NW) U.S. General Accounting Office Washington, DC Orders may also be placed by calling (202) 512-6000 or by using fax number (202) 512-6061, or TDD (202) 512-2537. 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Summary: Pursuant to a congressional request, GAO determined: (1) what efforts the Legal Services Corporation (LSC) and its grantees have made to correct problems with case service reporting; and (2) whether these efforts are likely to resolve the case reporting problems that occurred in 1997. GAO noted that: (1) LSC revised its written guidance and issued a new handbook to its grantees to clarify case reporting requirements; (2) based on telephone interviews with a sample of 79 LSC grantee executive directors, GAO estimates that 90 percent of grantees viewed the new guidance as having clarified reporting requirements, overall; (3) virtually all grantees said they responded to the new requirements by making or planning to make one or more changes to their program operations; (4) however, many grantees indicated that they were unclear about certain aspects of LSC's reporting requirements, particularly regarding: (a) the specific information required on client assets; (b) the information required for documenting citizenship/alien eligibility for services provided over the telephone; (c) the criteria for avoiding duplicate counts of cases; and (d) who can provide legal assistance to clients in order for the service to be counted as a case; (5) LSC initiated a self-inspection procedure in which grantees were required to review their 1998 case data and submit certification letters to LSC if they found that the extent of error in their data was 5 percent or less; (6) grantees who could not certify their 1998 data were required to develop corrective actions that would address the problems identified; (7) about 75 percent of the grantees submitted letters to LSC certifying that the error rate in their 1998 data was 5 percent or less, while about 25 percent of the grantees submitted letters to LSC indicating that they could not certify their 1998 data; (8) according to LSC, about 30 of the 50 grantees with the largest caseloads were unable to certify their 1998 case data; (9) GAO could not assess whether the number of certified and uncertified programs that LSC obtained for 1998 was correct, lower, or higher than it should be; (10) this is because LSC did not provide grantees with a standardized way of reporting their self-inspection results, and LSC's instructions on how to conduct the self-inspections may have led some of the smaller grantees to select too few cases to reliably assess the amount of error in their case data; (11) some grantees did not correctly interpret LSC's case reporting requirements; and (12) for these reasons, GAO does not believe that LSC's efforts to date have been sufficient to fully resolve the case reporting problems that occurred in 1997.
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Summarize: Eight Canadians posted to Cuba have experienced mysterious symptoms such as headaches, dizziness and nose bleeds in recent months, but Global Affairs officials say they are still at a loss to explain what is causing them. The most recent incident affecting Canadian diplomats and members of their families took place in December. The person affected said they suddenly felt a wave of pressure. Symptoms reported by others have included headaches, dizziness, nose bleeds, loss of balance and sleeplessness. The symptoms reported by the Canadians have not been as severe as those reported by some Americans. While one person affected continues to experience headaches, Canadian officials said Wednesday there is no indication following medical testing that anyone has experienced permanent damage. Officials told reporters during a background briefing in Ottawa Wednesday they are in uncharted territory and have never seen anything like it anywhere else in the world. At this point, they said, they haven't ruled out anything, including foul play. The mysterious ailments, which have triggered speculation worthy of a spy thriller, appear to only be hitting Canadian and American diplomats posted to Havana, Cuba. U.S. media reported last year that U.S. authorities were investigating the possibility the diplomats and their families had been targeted by some kind of "sonic attack." However, The Associated Press on Monday cited a new FBI report that said the U.S. has found no evidence sonic waves were used. U.S. officials told a congressional hearing Tuesday they are still working on several theories — including the possibility a virus was used. Canadian officials said there have been no reports of similar symptoms among staff of other embassies or among Cubans. Nor have there been any reports of unusual symptoms among the thousands of Canadian tourists who visit Cuba every year. The United States reduced the number of people posted to its embassy in response to the problems reported by 24 U.S. personnel and members of their families. However, Canadian officials said they have no plans to reduce staff at its embassy. While a small number of diplomats and their families have opted to return to Canada, others have been sent to replace them after being fully briefed. At the same time, the government has increased security measures around both the embassy and the residences of staff members. Among the aspects officials have been studying are infrasound, ultrasound and environmental factors. While doctors have determined that some of the symptoms could be produced by auditory trauma, they said there is no known sonic weapon. The U.S. government warns travellers to Cuba that embassy employees have been targeted by attacks and U.S citizens could be as well — particularly around the Hotel Nacional and Hotel Capri in Havana. The Canadian government is not planning to add a similar warning to its travel advisory. While the officials did not say it, adding a warning to a travel advisory can have significant repercussions for businesses like insurance companies and airlines. The first reported symptoms occurred in March 2017 but there was a cluster around May 2017 after the Canadian Embassy was approached by the American Embassy and it began to ask staff if they had experienced anything. Overall, 27 staff members or their family members went through a medical examination and eight received further followup medical treatment. The government is also doing baseline testing of those being posted to the embassy in Cuba, including those sent there temporarily, to allow them to determine if there are any future changes. While a number of American staff said they heard a high-pitched sound before they began experiencing symptoms, only one Canadian reported that kind of sound. The Canadians reported feeling the symptoms at their homes, rather than at the embassy. Officials said Cuban officials appear to be as puzzled by the strange symptoms as the Canadians and Americans. Breaking News Emails Get breaking news alerts and special reports. The news and stories that matter, delivered weekday mornings. / Updated By Abigail Williams and Tracy Connor It's not just the U.S. State Department that is baffled by the health problems of diplomats who've worked in Cuba. The Canadian government said Wednesday it also has no idea who or what is behind the mysterious ailments. At a briefing in Ottawa, a Canadian official said eight of their citizens stationed in Havana, out of 27 tested, required medical care after reporting symptoms that ranged from dizziness and headaches to nosebleeds. Three diplomatic families left Cuba as a result, the official said. The United States says 24 Americans affiliated with the embassy in Cuba have reported similar symptoms and signs consistent with traumatic brain injury. The State Department recalled all nonemergency personnel in late September and has no plans to return them, straining ties with Cuba. Cuba has welcomed FBI agents to Havana to investigate. But officials told NBC News that the probe found no evidence of attacks. Additionally, some U.S. officials have questioned whether there's a single cause for the symptoms. The State Department says the phenomenon is real. "Is there any thought given to the fact that this is a case of mass hysteria? That a bunch of people are just being hypochondriacs and making it up?” Sen. Marco Rubio, R-Fla., asked during a Senate Foreign Relations subcommittee hearing on Tuesday. "Twenty-four people have had symptoms and findings consistent with what looks like a mild traumatic brain injury. The objective tests that were done were not ones that could basically be easily faked,” State Department Medical Director Dr. Charles Rosenfarb testified. "The findings suggest that this is not a case of mass hysteria," he said. While officials in Washington initially theorized that the staffers and their family members were sickened by some kind of sonic attack, other possible culprits are under investigation. "I think there’s viral, there’s ultrasound, there is a range of things that technical experts are looking at," Todd Brown, diplomatic security assistant director at the State Department, said during the hearing. "When you say viral, you are talking about somebody intentionally implanting a virus?" Sen. Tom Udall, D-N.M., asked. "That could be a possibility," Brown said. Francisco Palmieri of the State Department told a Senate subcommittee it's "incomprehensible" that Cuba doesn't know who is behind alleged attacks that harmed U.S. and Canadian diplomats. KEVIN LAMARQUE / Reuters Secretary of State Rex Tillerson has now decided to ratchet up the inquiry and convene an independent panel known as an Accountability Review Board. At the hearing, Rubio grilled officials on why the board had not been set up earlier, in accordance with law. "The law very clearly requires that within 60 days of an incident, and 120 days at most, that an accountability review board be stood up and they did not do that until almost a year after the first reports of the attacks,” Rubio told reporters. "That’s unacceptable." The Cuban government has vociferously denied any involvement in or knowledge of attacks on American and Canadian diplomats. "There is no evidence of any kind, not of the cause, not of the perpetrators, not of the origin, not of the motivations," a Cuban foreign ministry official, Josefina Vidal Ferreiro, said Tuesday evening. "There is no evidence at all, not one piece of evidence." Despite the lack of evidence, the State Department said it's "incomprehensible" that leaders in Havana don't know what made the Americans and Canadians sick. Rubio, an ardent critic of the island's Communist government, agreed. "We can say we don't know how it happened, we can even say we can't know for sure who did it," he said. "But two things we know for sure: People were hurt, and the Cuban government knows who did it."
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Summary: Fresh off the news that an FBI report based on months of investigation found no evidence that sound waves could have damaged the health of American diplomats in Cuba, a new twist in the case: Canada on Wednesday said the inexplicable problems haven't let up. The CBC reports that eight Canadians have now suffered headaches, dizziness, and nose bleeds after time in Cuba, with the latest incident occurring just last month. In that case, the afflicted person reported feeling a "wave of pressure." Unlike the Americans, none of the affected have had lasting symptoms or damage. The CBC notes no other countries have reported similar incidents. NBC News reports that the eight Canadians needing medical care came from a pool of 27 who were tested. The US has found 24 Americans to have been mysteriously afflicted, and while the FBI report undermines the idea that a sonic weapon was used, other theories were discussed during a Senate hearing Tuesday. A State Department official said "there's viral, there's ultrasound, there is a range of things that technical experts are looking at." When asked by Sen. Tom Udall if viral meant someone "intentionally implanting a virus," the official answered that it was a possibility.
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Summarize: The images of European cities left smouldering and in ruins at the end of the Second World War have been starkly echoed in new pictures revealing wholesale devastation across eastern Ukraine. Heavily shelled tower blocks, abandoned hotels and airplane noses that look to have dropped from the sky are among the sights depicting the destruction in Donetsk, which in parts equals that seen after the Second World War in cities such as Stalingrad and Dresden. It comes as heavy weaponry was today towed away from the front line at the village of Paraskoviyvka, north of the government stronghold of Artemivsk, in a move that signified a France and Germany-brokered ceasefire may be beginning to take hold 11 days after it was agreed. Scroll down for video. Destroyed: A shell of a car lies among dead trees in front of heavily shelled tower blocks in Donetsk. Ruins: A heavily damaged hotel stands in ruins near to Donetsk airport in Donetsk, Ukraine. Crushed: A tank can be seen among the shattered buildings in the industrial city of Donetsk that was at the centre of the fighting. A direction sign at Donetsk airport is left riddled with bullet holes, while huge blast craters can be seen on a nearby building. On guard: A separatist soldier stands close to a ruined hotel in Donetsk as weapons were moved away from the front line. A partially collapsed building sits amid the barren landscape after the area surrounding the airport was left ravaged by months of shelling. The move to withdrawn heavy weaponry was Kiev's most direct step to acknowledge that the ceasefire was finally holding, a week after suffering one of the worst defeats of the war at the hands of rebels who initially ignored the ceasefire to launch a major advance. The pro-Russian rebels, who committed to the truce after their successful offensive, have been pulling back heavy weapons for two days, but Kiev had until now held back from implementing the withdrawal, arguing that fighting had not yet ceased. However, the army today reported no combat fatalities at the front for a second straight day - the first time no troops have been killed since long before the French and German-brokered truce was meant to take effect. The withdrawal of artillery is 'point two' of the peace agreement reached in the Belarus capital Minsk, so it amounts to an acknowledgement that 'point one' - the ceasefire itself - is being observed. 'Today Ukraine has begun the withdrawal of 100 millimetre guns from the line of confrontation,' the military said in a statement, saying the step would be monitored by the Organization for Security and Cooperation in Europe. It said it reserved the right to alter the schedule of withdrawal 'in the event of any attempted offensive'. Barren trees and a bullet ridden stop sign are all that is left standing in a section of Donetsk airport. A partially destroyed church remains standing despite the obvious damage it has received during months of warfare. One of the main buildings of Donetsk airport is left in ruins after troops withdraw from the bitterly disputed area. Ukrainian troops and rebel forces both began withdrawing artillery from the frontline today in a sign the peace plan may be taking hold. Pictured is the battle worn Donetsk airport. The roof of this abandoned administrative building in Donetsk was completley destroyed during the heavy bombing. A gutted bus sits in the middle of the road between the towns of Debaltseve and Artyomovsk in eastern Ukraine. A part of the airport passengers once used to board flights is left a wreck, with only the frame of the building remaining upright. A rebel soldier wanders through part of Donetsk airport as artillery began withdrawing from the area. A rebel walks through the remains of the airport amid reports both sides have begun withdrawing artillery from the frontline. Rebel soldiers force Ukrainian prisoners of war to search through the wreckage of Donetsk airport to remove dead bodies and weaponry. The airport has been left in ruins, with collapsed roofs and walls burying soldiers after months of shelling and fighting. Witnesses in rebel-held Donetsk said they had heard no artillery in the night although the occasional distant blast or gunshot could be heard during the day. Rebels brought Ukrainian war prisoners to the ruins of the airport on the north of the town to recover the dead bodies of their fellow Ukrainian troops, left buried in the wreckage since the terminal was captured in January. Rebels also carried out controlled explosions to blast holes through walls inside the ruined terminal and sent the prisoners down a ladder where the floor had collapsed. Three dead bodies still lay at the site out of five that had been recovered from the debris yesterday. Prisoners said they were searching for three more they believed were still buried. The commander of the separatist 'Sparta' battalion, going by the nom de guerre 'Motorola', said the prisoners had been assigned the task because 'it's not our job to recover dead bodies, it's our job to make them. 'They take their comrades out to return them to their mums and dads. Did they think we would feed them for free?' Airplane noses sit partially damaged near Donetsk airport. The site has been one of the most heavily fought over pieces of land. Damage: A commercial aircraft lies destroyed at the region's airport, which came under heavy bombardment during months of fighting. Bullet-ridden: A destroyed commercial airplanes sit scattered at the airport, revealing the extent of damage caused by months of fighting. Shells: Burnt out vehicles lie strewn next to a destroyed building in Pisky village, in the eastern Donetsk region. Obliterated: An armed soldier of the separatist self-proclaimed Donetsk People's Republic army stands inside the damaged Donetsk airport. Rubble: A pro-Russian rebel stands guard while Ukrainian prisoners of war are forced to search through the wreckage. A flimsy building remains standing but covered in shelling damage and bullet holes amid the rubble of Donetsk. The twisted remains of a tank lie near Donetsk airport. On the left is its base, while metres to the right sits the turret. The battle of Stalingrad (pictured), which took place during the Second World War, was a prolonged and entrenched battle which left much of the Russian city in ruins. Donetsk airport has been a totemic battlefield for both sides. Ukrainian troops had held out there for months until the rebels assaulted it after abandoning a previous ceasefire agreed in September. The separatist rebels initially ignored the new truce last week to launch an advance that led to one of the biggest battles of a war that has killed more than 5,600 people. But since capturing the strategic town of Debaltseve, where the rebels said the truce did not apply, they have taken pains to emphasise that they now intend to abide by it. Western countries denounced the rebels and their presumed sponsor, Russian President Vladimir Putin, for advancing on Debaltseve after the truce was meant to take effect. But they have since held out hope that the ceasefire will now hold, with the rebels having achieved that objective. In the days after its troops were driven from Debaltseve, Kiev maintained that it believed the rebels were reinforcing for another advance, particularly expressing fear for the city of Mariupol, a port of 500,000 people. Western countries have threatened to impose new economic sanctions on Moscow if the rebels advance further into territory the Kremlin calls 'New Russia'. Moscow, which denies aiding its sympathisers in Ukraine, said today the threats of more sanctions were cover for Western efforts to undermine the truce. 'It's an attempt to... distract attention from the necessity to fulfil the conditions of the Minsk agreements,' Foreign Minister Sergei Lavrov said. Vladimir Putin. and his Cypriot counterpart Nicos Anastasiades signed an. agreement to give Russian military ships access to. Cypriot ports. Ties between Russia and the West have plummeted in the wake. of the Ukraine crisis, but Putin said the ships allowed to dock. at Cypriot ports would mostly be used in international. anti-terrorism and piracy efforts. 'I don't think this should worry anyone,' he said. Cyprus, which is heavily dependent on Russian investment,. played down Wednesday's deal, saying Russian ships had always. had access to its ports. A government source said it was simply. the first time access had been spelled out in a separate accord. Russia has sought to forge stronger ties with individual. members of the European Union, including Cyprus, Hungary and. Greece, after the 28-nation bloc, along with the United States,. imposed cumulative sanctions on Moscow for its role in Ukraine. Officials in Brussels fear this policy is aimed at weakening. EU resolve and preventing a further tightening of sanctions. Moving away: Members of the Ukrainian armed forces ride armoured personnel carriers as they pull back from Debaltseve region, near Artemivsk. A rebel soldier makes his way through the debris which litters the ground of Donetsk airport. A pro-Russian rebel smokes a cigarette while making his way through the ruins of Donetsk airport, which has been left completely destroyed. Ukrainian prisoners of war are lined up by rebels before they are ordered to begin sifting through the rubble. Withdrawal: Pro-Russian rebels move tanks and heavy weaponry away from the front line of fighting in accordance with the Minsk II agreement. A pro-Russian rebel stands guard while Ukrainian prisoners of war are forced to search through the wreckage for weaponry and dead bodies of comrades. Pro-Russia rebels are pictured moving tanks and heavy artillery away from the frontline as agreed upon in the recent ceasefire. A rebel soldier looks on from the comfort of his tank after it appeared the France and Germany-brokered ceasefire today began to take hold. A rebel brandishes his assault rifle while tanks withdraw in the distance. The withdrawal of heavy weaponry constitutes the second phase of the peace agreement. A tank travels along a road near Olenivka village, Donetsk, after rebels appeared to adhere to the ceasefire following their defiance of the peace plan when they launched an attack on Kiev troops a week ago. Ukrainian soldiers also started withdrawing heavy weapons. Pictured are a group of soldiers riding an armoured personnel carrier as it tows a cannon away from the frontline. Ukrainian heavy artillery is withdrawn as officials claim 100 millimetre guns are being removed from the line of confrontation
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Summary: The devastation left across eastern Ukraine echoes that seen in European cities at the end of the Second World War. Donetsk airport and its surrounding regions are abandoned with only partially destroyed buildings left standing. New pictures reveal the airport, once used as a hub for Euro 2012, is a scene of wholesale devastation. Ukrainian troops have towed artillery away from the conflict's front line in a sign the ceasefire is finally holding. Both the Government troops and rebels have reported no combat fatalities at the frontl ine for a second straight day. The artillery withdrawal is 'point two' of the France and Germany-brokered peace deal agreed upon 11 days ago.
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Summarize: A volunteer firefighter is suspected of igniting a string of fires around the town, including a blaze that destroyed a wooden CN Rail bridge in Alberta Tuesday. Lawson Michael Schalm, 19, is charged with 18 counts of arson in connection with a recent rash of fires, RCMP said Saturday night. “The community’s in shock right now,” Mayerthorpe Mayor Kate Patrick said. “They’re happy to know that someone has been apprehended, but they’re certainly in shock as to who it is.” Schalm has worked as a volunteer firefighter with the Mayerthorpe Fire Department, Patrick said. His father Albert was mayor of the town for three years, and now serves as a school trustee with the Northern Gateway school district, Patrick said. The Tuesday CN trestle bridge fire was so intense that Lac Ste. Anne County firefighters called in provincial forest firefighters, an air tanker, and a helicopter to prevent the blaze from spreading to the surrounding trees. The resulting thick plume of black smoke prompted the evacuation of two Mayerthorpe schools and a nearby trailer park. The broken rail link has caused logistical headaches for some local businesses. RCMP spokeswoman Cpl. Sharon Franks said there have been more than 20 fires in the Mayerthorpe area during the last 30 days. More charges may be pending, Franks said. Most of the recent blazes have been grass fires, Patrick said. On Friday, firefighters responded to five fires, she said. Four were in the wee hours of the morning, including two brush fires on sports grounds, one in a river valley, and another on the town’s main street. That afternoon, there were flames again on the river bank, Patrick said. A paramedic found, and extinguished, another small fire near the local legion hall. Firefighters also responded to spot fires near the trestle bridge before Tuesday’s spectacular blaze. Schalm was involved with the cadets, school theatre productions, and teen drug use prevention programs in his community, Patrick said. A Mayerthorpe Freelancer article from 2014 says cadets can volunteer with the fire department as young as age 15, and can begin responding to fire calls when they’re 17. Mayerthorpe’s fire chief couldn’t be reached Saturday evening. Franks said Schalm was not previously known to police. In custody on Saturday night, Schalm is due to appear in Stony Plain Provincial Court on Wednesday. A former Mayerthorpe mayor says he remains supportive of his firefighter son, who faces 18 counts of arson for a spree of major fires he allegedly started in the Alberta town. The charges against 19-year-old Lawson Schalm are related to numerous suspicious fires in the area that began on April 19, including a massive one that destroyed the CN trestle bridge. On Sunday, Mayerthorpe fire Chief Randy Schroeder confirmed Schalm was one of the volunteer firefighters called to actively battle that fire, as well as four other fires during the course of the spree. "There's an unconditional love for my son," Albert Schalm said, his voice trembling. "No matter how this ends up, there will always be a dinner plate at my table for my son. He is always welcome in my home." Albert Schalm said his son began working for the Mayerthorpe fire department when he was 15 years old and was considering a possible future with the department. "We were shocked," he said. "He was planning out his future and where it would go from there, and obviously some of that will be on hold for a while." Schalm is in custody and is scheduled to make his first court appearance in Stony Plain provincial court on Wednesday. Fire department shocked, chief says The CN bridge fire forced the evacuation of nearby schools and a trailer park with 38 mobile homes. Some Lac St. Anne County residents living nearby were also told to be ready to leave on one-hour notice. Fire chief Schroeder said last week almost three dozen firefighters from four different fire departments in the area were called to help douse the flames, alongside agriculture and forestry services members, helicopters and a water bomber. The loss of the bridge will impact the lumber and oil industries "extensively," Schroeder said. He said the fire department is struggling to come to terms with the charges against one of their own. "We're certainly very shocked, bewildered, wounded and a little hurt on the discovery of this," he said. "The discovery that one of our own potentially lit 18 fires is definitely affecting our station, for sure." Schroeder and Mayerthorpe Mayor Kate Patrick said in a news release that members of the fire department will undergo "critical incident stress debriefing," and that individual counselling will be available to all members over word of the charges. Schroeder said the department is still functioning normally and providing service as usual for the community, and also extending their support to the Schalm family. "We understand that this has got to be a tremendously trying time for his family members," he said. 'There will always be a dinner plate at my table for my son' Albert Schalm said he hasn't yet been able to speak with his son, but that the community has been supportive of the family. Schalm was mayor of Mayerthorpe in 2005, when four RCMP officers were shot and killed near the town. "This is a community that I've poured my heart and soul into. Moving this community forward, that's still going to continue." - Albert Schalm, father of accused in Mayerthorpe, Alta., arsons "We're thankful that this is over for the community, and for ourselves and for everybody, and that nobody got hurt, there were no serious injuries. You have to be really thankful for that. "This is a community that I've poured my heart and soul into. Moving this community forward, that's still going to continue." Schalm's son was expected to graduate high school in two months, and the family is now focusing on their son's future, Schalm said. Despite what he calls a "dark period," he said his family's focus will remain on his son, and their Christian faith. "We refuse to be devastated by this. I refuse to throw my son under the bus. I'm not that kind of dad. My goal is to get him back on the road, get his future going again." Mayerthorpe’s fire chief said there’s a sense of betrayal following the arrest of a volunteer firefighter in connection with a slew of suspicious fires in the area. “We didn’t see this coming,” Randy Schroeder said. “The aspect of what we do to protect the community, the lengths at which we strive in order to train…It’s devastating.” READ MORE: Mayerthorpe junior firefighter charged with 18 counts of arson On Saturday, Lawson Michael Schalm, 19, of Mayerthorpe, was charged with 18 counts of arson in relation to a recent rash of fires in the area, including the blaze which destroyed a CN trestle bridge. READ MORE: Massive Mayerthorpe trestle bridge fire being investigated as arson Schalm is a junior firefighter with the Mayerthorpe Fire Department and was standing alongside firefighters battling the trestle bridge blaze. “There’s a sense, on behalf of the members, a sense of betrayal, because the fact is we work very hard,” Schroeder said. Schroeder said Schalm was a great firefighter and had good prospects of becoming a lifelong firefighter. “It’s heartbreaking,” the fire chief added. The fact that Schalm served in a volunteer capacity makes this even more upsetting, Schroeder said. Schalm signed on to be part of the volunteer department when he turned 18. “This member started as a junior at 15 and…our cadet program emphasizes great community service.” Schroeder said potential firefighters submit a job application, must pass a physical test and character references are checked. If an applicant begins the process after the age of 18, a criminal record check is requested by the department. He said there is also a sense of shock over the arrest. “[Schalm] was on five of the scenes – as an active firefighter – that are in question and at least four as a bystander,” Schroeder said. “We didn’t see it coming.” He said Schalm showed no signs of being involved whatsoever. “He was part of the attack to a limited basis with his training,” Schroeder said. “He wasn’t qualified for interior attack, but certainly on support lines and on exterior.” The fire chief wonders what the department could have missed. “I try not to take it personally. I’m struggling like any of our members with questions of ‘why?’ and ‘how?’ and ‘what did we miss?’ I would like to be able to roll the tape back metaphorically speaking and get a better understanding,” the fire chief said. #Mayerthorpe fire chief Randy Schroeder and mayor Kate Patrick address the media. #YEG pic.twitter.com/VLF33Uoc02 — Julia Wong (@JWongGlobalNews) May 1, 2016 The mayor called the whole situation “heartbreaking and sad.” “The community is saddened to hear of the arrest of this person but are happy to know it’s been successfully completed,” Kate Patrick said. She confirmed Schalm was a member of the Mayerthorpe Fire Department and Lac Ste. Anne Fire Services. Global News has learned Schalm is the son of former Mayerthorpe mayor Albert Schalm. “We respectfully wish the community supports their family,” Patrick said. “They’re well-respected, a very nice family. We want the community to move forward and honestly respect them.” Theresa Wallace, 16, has known Schalm for several years. The Grade 11 student calls him a “super nice kid.” “I don’t think he’s capable of this,” Wallace said. “He’s… super laid back. I don’t know, he’s a good kid. He’s shy, totally shy, but once you get to know him he’s super nice,” she said. Wallace said Schalm, who was set to graduate Grade 12 this year, was involved with school plays and other programs. “It’s kind of appalling when I found out he got arrested,” she said. “Everybody is just focusing on if he’s OK. We’re more worried about his well-being than anything else.” READ MORE: Citizen patrol group launched after string of suspicious fires in Mayerthorpe The community launched a citizens patrol group after the 17 fires in the area were sparked over a period of just six days. Then on Friday, five more suspicious fires were set. Barry Fulford, a citizens patrol member, said the community is relieved following the arrest. “Last night was kind of quiet in town. There were no fire trucks, no fires – it was kind of relaxing.” “It’s a little more comforting,” he said. “It’s a little more settling. A sense of relief I’m sure will take place today.” The patrol group is still going ahead despite the arrest. Mayerthorpe, Alta. is located 120 kilometres north of Edmonton. RCMP are not seeking any other suspects in the fires. They said further charges against Schalm could be laid, but did not elaborate. With files from Julia Wong, Global News Follow @Emily_Mertz
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Summary: After more than 20 suspicious fires in the space of a few weeks, it didn't take Sherlock Holmes to figure out that an arsonist was at work in the small town of Mayerthorpe, Alberta-but the real shock was the suspect's identity. On Saturday, volunteer firefighter Lawson Michael Schalm, 19, was arrested and charged with 18 counts of arson, including one relating to a spectacular fire that destroyed a rail trestle bridge last week, the National Post reports. Authorities say Schalm, the son of an ex-mayor, helped fight at least five of the fires and was a bystander at several others. "We're certainly very shocked, bewildered, wounded, and a little hurt," fire Chief Randy Schroeder says. "The discovery that one of our own potentially lit 18 fires is definitely affecting our station." Schroder tells Global News that the situation is "heartbreaking" and firefighters are struggling to deal with the apparent betrayal by Schalm, who started out as a cadet at 15 and had good prospects of becoming a career firefighter. "I would like to be able to roll the tape back metaphorically speaking and get a better understanding," the chief says. Former mayor Albert Schalm says that while he is shocked by the allegations, his love for his son is "unconditional" and there "will always be a dinner plate at my table" for him. "I refuse to throw my son under the bus. I'm not that kind of dad," he tells the CBC. "My goal is to get him back on the road, get his future going again."
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Summarize: Cinema, teatri e sale da concerto ripartono. Dopo mesi di silenzio e di buio in sala, si potranno riaccendere riflettori, proiettori e amplificatori. Un altro comparto della cultura può ricominciare, ma solo dal prossimo 27 marzo, tra un mese. Lo aveva preannunciato anche il ministro dei Beni Culturali, Dario Franceschini, oggi pomeriggio. Si tratta solo di una bozza, per ora. È quella del nuovo Dpcm, il primo del governo Draghi, che al momento è al vaglio delle Regioni e che punta ad essere approvato e firmato dal presidente del Consiglio all'inizio della prossima settimana. Tante le novità, anche per i musei, che apriranno in zona gialla anche il weekend, ma con biglietto prenotato online. Per quanto riguarda cinema, teatri e sale da concerto, la riapertura verrà accordata a determinate regole. Innanzitutto si parla, chiaramente, solo di zona gialla. Quindi niente da fare in zona arancione e zona rossa. Secondo quanto scritto nella bozza del Dpcm serviranno "posti a sedere preassegnati e distanziati e a condizione che sia comunque assicurato il rispetto della distanza interpersonale di almeno un metro sia per il personale, sia per gli spettatori che non siano abitualmente conviventi". Resta da capire a che ora si potrà andare al cinema o a teatro, visto che il coprifuoco è stato confermato dalle 22 alle 5.
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Summary: Cinema, teatri e sale da concerto riapriranno (in zona gialla) il 27 marzo, secondo quanto scritto nella bozza del primo Dpcm del governo Draghi, che al momento è al vaglio delle Regioni. Distanziamento di almeno un metro e nuovo protocollo del Mibac validato dal Cts, del quale però ancora non si sa nulla. Resta da capire a che ora si andrà al cinema o a teatro, visto che il coprifuoco dalle 22 alle 5 è confermato.
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Summarize: The seed for Wide00014 was: - Slash pages from every domain on the web: -- a ranking of all URLs that have more than one incoming inter-domain link (rank was determined by number of incoming links using Wide00012 inter domain links) -- up to a maximum of 100 most highly ranked URLs per domain - Top ranked pages (up to a max of 100) from every linked-to domain using the Wide00012 inter-domain navigational link graph UK-based online gambling holding Gamesys has launched a new Facebook app called ‘Bingo Friendzy’, the first to hit the social network’s App Center that lets UK users aged 18 and over to play an online game for real cash prizes. We knew this was coming, but now the wait is over. Gamesys, which operates the UK’s leading Bingo and Slots website Jackpotjoy.com, says the new Friendzy App comes with a series of 90 Ball Bingo and Slots games that enable adult Facebook users in the UK to play for real cash. Comments Julien Codorniou, Facebook’s Head of EMEA Gaming Partnerships: “People love playing new types of games with their friends on Facebook and real money gaming is a popular leisure activity in the UK. We’re delighted to be working with Gamesys to enable them to offer their games on Facebook so that people can enjoy playing more games with their friends.” Facebook has effectively partnered with Gamesys to fuel the distribution of the new game on the social networking service, and plans to team up with more gambling companies in the future. Gamesys says a series of controls should protect users under 18 years of age, as well as “vulnerable people”. Also, in line with gambling legislation, all users will have access to a number of self-help tools to limit their spending and exclude themselves from playing at anytime. Zynga, Facebook’s largest gaming partner by a margin, is also planning to venture into the real-money online gambling space in the near future. And who knows, maybe some day this will become legal in the US (it will, eventually). Also read: What happens when professional gambling meets social gaming Zynga is “all in” to acquire Bwin.party-owned poker business Ongame: Report Read next: Calling on steroids: Korea's SK Telecom to launch world's first HD voice over LTE service Facebook could find better growth for its slow-going Facebook Payments revenue line, after it debuts paid gambling games on its platform from Tuesday. UK online bingo game maker Gamesys‘ new game Bingo & Slots Friendzy is the first on Facebook App Centre to pay real cash prizes; several more are likely to follow. Release. While U.S. regulators only recently began allowing some kinds of online gambling on a state-by-state basis, it is commonplace in the UK – a nation usually marked by greater regulatory control. Furthermore, recent deregulation of gambling advertising has prompted news publishers, bookmakers and various other brands to launch their own bingo and betting games, promoted through day-time TV ad campaigns. Bingo & Slots Friendzy will be accessible only to those aged 18 and over. Gamesys promises: “A series of controls will be in place to protect under 18s and vulnerable people. In line with industry standard gambling legislation, all users will have access to a number of self-help tools to limit their spending and exclude themselves from playing at anytime.” Facebook’s alternative to its core advertising earnings is its Facebook Payments stream, which has relied heavily on payments from Zynga games that are becoming less popular. Facebook Payments revenue grew a disappointing three percent through Q2 this year… So Facebook Payments could stand to benefit from online bingo fans who pay money to play on the platform, which will also allow users to play along with friends. There is another category of operator which could also benefit from the move. Gamesys also powers the bingo platforms for News International’s Sun Bingo and Fabulous Bingo sites – two of the largest in the UK. The market has become fiercely competitive, with Trinity Mirror’s rival Mirro Bingo losing players over the last year for example. If Gamesys or other vendors take their clients’ games to Facebook, the publishers could find new audiences of players.
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Summary: Because Facebook isn't already addictive enough... UK users will now be able to gamble, in addition to posting photos of the excellent pork tenderloin they made for dinner last night. The Next Web reports that Gamesys' new app Bingo Friendzy is the first to allow those over 18 to play in hopes of winning cash. The app reportedly pulls together 90 Ball Bingo and Slots games, and also features "controls" that Gamesys says will keep those who are "vulnerable" or under 18 from playing. Look for Zynga to make a foray into this domain, too, reports Next Web, which predicts that gambling games will eventually become legal on US shores. Smells like a smart move for Facebook, writes Robert Andrews for paidContent. The company's supplement to its ad revenues is the Facebook Payments stream, "which has relied heavily on payments from Zynga games that are becoming less popular." Facebook Payments revenue growth has been sluggish so far this year, but users eager to try their luck could change that.
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Summarize: By. Damien Gayle. and Sian Boyle. If you packed a cardigan for a day out yesterday, you might be able to leave it behind today. Temperatures are forecast to peak at a rather warm 20C (68F), right on cue for Bank Holiday Monday. Yesterday glorious sunshine sent the average UK temperature creeping up from a cool 12C (54F) earlier in the week to 18C (64F) in many parts of the country, including Aberdeen, while Bristol enjoyed highs of 16C (61F). Families flocked to the coast in Brighton and Dorset, while roads were gridlocked in Llandudno, North Wales, as day trippers made their way home from the seaside resort. Scroll down for video. Two-and-a-half-year-old Beck Colman, cools off from the May Bank Holiday Sunshine with an ice cream on Great Yarmouth Beach in Norfolk. People punting on the River Cam in Cambridge, enjoying the Bank Holiday sunshine. Holly Lush joined thousands across the UK, flocking to the British coast. The 27-year-old lapped up the sunshine on Great Yarmouth Beach in Norfolk. Figures from Asda suggested the. sunshine inspired a wave of impromptu barbecues, with sales of sausages. and equipment expected to triple those of April. The fine weather was not evenly spread, however, according to a forecaster from the Met Office. 'Large. swathes of England, Wales and southern parts of Scotland had good. spells of sunshine but further north and west there was thicker cloud. producing outbreaks of rain,' he said. 'Today. there is likely to be an east/west split with the best sunshine in. the south-eastern parts of England from London up to Lincoln, and. temperatures will reach 20 or 21C (68-70F).' Outbreaks of rain from the west to the east are expected to arrive in the evening and could carry on into Tuesday. Yesterday,. with no work till Tuesday and clear blue skies and temperatures nudging. the mid teens in the South and East, children and adults were out. playing in the parks and flocking to the coasts. A cavalcade of narrow boats descended on the Little Venice Canal in Paddington, London to make the most of the weather. Dozens of boats lined the waterways, bearing bright bunting as passers-by stopped to enjoy the spectacle. But. it wasn't quite yet warm enough for serious sunbathing and, although. there were ice creams aplenty, many were still taking precautions with. hats and jackets. In. Bournemouth, East Sussex, children were out playing by the sea, building. sandcastles and dipping their toes into the still chilly waters despite. a stiff breeze. Bursts of. colour blanketed Kent, meanwhile, as powdery blue carpets of bluebells. shone in almost celestial light of the glorious early morning sun. Photographer. John Turp snapped the plants, which have been voted Britain's. favourite, in two locations near his home in the county. 'I. was fortunate to stumble across some bluebells outside of Woodchurch by. accident,' he said. 'They inspired me to find more and I visited the. Hucking Estate a week late and found this beautiful carpet of flowers. 'I love how the almost unnatural blue stands out against the woodland green.' Boaters enjoyed winding their way along the Canal and through north London. Sheffield's Peace Gardens bask in the sunshine, as dozens flock to the pretty city centre spot to make the most of the warm weather. 'I love how the almost unnatural blue stands out against the woodland green.': John Turp photographed these bluebells in bloom near his home in Kent. A couple take a ride in a kayak, enjoying meandering along the River Cam in Cambridge. But the change in weather, while almost universally welcome, has come with warnings. Nature lovers have begged holidaymakers not to get too close to huge but harmless basking sharks which are moving close to beaches on the West Country coastline. The docile monsters of the deep, which can reach 30ft in length and weigh up to eight tonnes, drift round just below the surface with their vast 4ft wide jaws wide open as a net to catch millions of microscopic plankton, their staple food. Shark fins have been spotted cleaving the water, with this spring's first sightings close to the shore at Porthcurno and near Mount's Bay in Penzance, Cornwall. Today wildlife tour operator Marine Discovery, based in Penzance, urged excited and curious holidaymakers to take great care when they spot basking shark cruising lazily in shallow water. 'We're now getting into the time of year when basking sharks can be found feeding off the Cornish coast and it's fantastic to see them,' said a spokesman. 'But it's important to remember that they need to be approached carefully so as not to disturb their natural behaviour. 'If a shark thrashes its tail and dives or stops feeding and dives, then it is likely you have disturbed it. If this happens learn from the mistake and try not to repeat it.' Carla Pietersen, three, from Wiltshire, enjoys a paddle on Bournemouth beach. She was joined there Lily and Holly Bell, two and one respectively, from Herefordshire. Sand between the toes: Jude Adams, three, and his sister Seren, eight months, enjoy the sun on Bournemouth beach. In Bristol people enjoyed an interactive temporary art installation, the Park and Slide project in the city centre. Meanwhile, in Cambridge, university officials have urged students to boycott a notorious annual booze-up today in the city's Jesus Green park. 'Caesarian Sunday' attracts thousands of students every year who dress up and play drinking games. But locals and visitors have been upset as the historic riverside park has been left littered with rubbish, empty bottles and vomit. After years of complaints university officials are calling on students to shun today's event. A University spokesman said: 'The Colleges will be asking students preferably not to attend but if they do to be considerate of others and avoid any behaviour which may cause offence.' The three-day forecast: Temperatures will increase tomorrow, but a band of rain moving in from the west will gradually cover the country and sit there for the rest of the week. But with a fine afternoon and evening expected, it may be difficult to dissuade Britons from making the most of the springtime sun. Calum McColl, a forecaster with the Met Office, told MailOnline that for much of England and Wales 'there's some good weather to be had.' Although cloud is still overshadowing central and northern parts of the country, many inland areas will enjoy temperatures of up to 15C even as far north as Sheffield. The best of the sun will be in the South and the East, particularly around coastal areas, which will nevertheless be slighter cooler under the influence of the sea breeze. Overnight the clear skies over these areas will lead to a frost and a chilly morning, but May Day bank holiday temperatures tomorrow will then increase up to 18C in London and surrounding areas, with much of the rest of the country also enjoying a 'pretty good day'. However, rain will start moving into western parts over the course of the day, bringing with it colder temperatures and heavier cloud cover. 'There's a little bit of a change coming into western areas,' said Mr McColl. 'There is a band of rain coming through.' By Tuesday, rain will advance across much of the country, with summery weather holding out only in the far west, in an outlook that looks likely to persist for the rest of the week
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Summary: Britain basks in temperatures reaching the mid-teens and clear skies across the South and East. Families hit the beaches on bank holiday outings, even as stiff breezes make for lower temperatures by the sea. Temperatures are forecast to peak at a rather warm 20C (68F), right on cue for Bank Holiday Monday. However rain is forecast to return in the West and will spread across the country for the rest of the week.
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Write a title and summarize: Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor' s response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development. The striatum is the main input structure of the basal ganglia, which is necessary for proper motor function and habit formation. The medium spiny projection neurons (MSPNs), which comprise ∼95% of striatal neurons, undergo changes in synaptic strength during the learning of a motor task [1]. This synaptic plasticity is thought to be the cellular basis of motor learning and habit formation, and it is disrupted in animal models of Parkinson' s Disease [2] and Huntington' s Disease [3]. One of the critical mechanisms for inducing synaptic plasticity in neurons is calcium elevation in the spine. The sources of calcium are quite diverse, and depend on brain region and direction of plasticity. In particular, LTD often requires release of calcium from internal stores [4] or voltage dependent calcium channels [4], [5]. In contrast, the source of spine calcium that contributes to long-term potentiation (LTP) is the NMDA receptor (NMDAR) in the hippocampus [6], cortex [7], and striatum [8]. Because NMDARs permit calcium influx in response to the coincidence of pre-synaptic glutamate release and post-synaptic depolarization, they are well situated to modulate spike timing dependent plasticity (STDP). In STDP protocols, an action potential (AP) is caused by depolarizing the soma of a neuron and is paired in time with a pre-synaptic stimulation. However, NMDARs differ in several properties that may be critical for timing-dependent synaptic plasticity. They contain various combinations of GluN1,2, and 3 subunits which can change their maximal conductance, current decay time, and sensitivity to magnesium block [9]. While the GluN1 splice variant has some control over the kinetic properties of the NMDAR, the four GluN2 subunits (A, B, C, and D) strongly control them when the GluN1 splice variant is kept the same [9]. The GluN2 subunit can thereby alter the calcium influx through the NMDAR. Because the specific differences between GluN2 subunits are the ones that would affect the NMDARs dependence on AP timing, and because calcium through the NMDAR plays an essential role in striatal timing-dependent long term potentiation (tLTP) [10]–[12] we hypothesized that changes in GluN2 subunit would modulate STDP in the striatum. The MSPNs of the striatum contain both GluN2A and GluN2B subunits in abundance [13], and it has been suggested that GluN2D subunits may be present in low concentrations [14]. In animal models of Parkinson' s disease, the NMDAR subunit composition is altered in the striatum [2] and subunit-specific NMDAR antagonists have been shown to alleviate Parkinson' s like symptoms [15]. However, the intracellular consequences of such altered NMDAR subunit composition has not yet been made clear. In this study, we investigate the effects of altering NMDAR subunit composition on tLTP in the striatum. Using a multi-compartmental model of a MSPN, we examine NMDAR-mediated calcium influx through receptors containing different GluN2 subunits and under different STDP conditions. We find that calcium elevation depends on which GluN2 subunit the NMDAR contains, the relative timing of the AP, the duration of somatic depolarization, and the number of consecutive APs. More significantly, model predictions about the effect of GluN2 subunit on the shape of the STDP curve are confirmed experimentally. To evaluate the role of the GluN2 subunit in STDP, we used a model of a dorsal striatal MSPN, which was modified from a nucleus accumbens neuron model [16], [17]. The model of the dorsal striatum MSPN had the same general morphology as the nucleus accumbens model (Figure 1A), with explicit spines that include synaptic glutamate receptors (AMPA and NMDA) and voltage dependent calcium channels. Kinetics and maximal conductances of voltage dependent channels (See Tables 1 and 2) were tuned to match the characteristics of neurons in the dorsal striatum, such as smallest current that evokes a single AP, long latency to first spike during somatic depolarization (Figure 1B), AP width of ∼1 ms, and distinct inward rectification (Figures 1C and see Text S1). Once the model was tuned to match these parameters, it also closely matched the average current-voltage relationship of 25 MSPNs from the mouse dorsal striatum undergoing a series of hyperpolarizing and depolarizing currents incrementing by 50 pA from −500 pA to 200 pA (Figure 1D and see Text S1). This model did not contain GABAergic synapses and represents experimental conditions in which GABAergic channels are blocked [10], [11], [18]. Because NMDAR channel opening requires both glutamate to activate the channel and membrane depolarization to relieve the voltage-dependent magnesium block, the occurrence of a somatic AP close in time to glutamatergic stimulation can drastically increase the calcium elevation due to the NMDAR. Previous studies have shown that different somatic depolarization patterns back-propagate into the dendrites with varying strength [19]–[21]. Consequently, STDP protocols that have been used on striatal cells vary in AP number and length of somatic depolarization. To investigate the influence that different protocols have on the NMDAR-mediated calcium elevation in the spine, simulations were performed for each STDP protocol that has been used in the striatum: a 30 ms depolarization to 1 AP [10], [22], a 5 ms depolarization to 1 AP [11], and an AP triplet at a frequency of 50 Hz [11], [18] (Figure 2A). Each simulation involved the stimulation of two adjacent spines on the secondary dendrite of the model cell and a somatic depolarization to AP initiation, with the two events separated by a specific temporal interval (Δt). For each STDP protocol, the peak calcium elevation due to the NMDAR in a single spine was recorded for both positive and negative Δt and was compared to the peak NMDAR-mediated calcium elevation due to control stimulation with no somatic AP (Figure 2B). When repeated over a range of Δt, simulations reveal that a 30 ms depolarization to 1 AP significantly increased the NMDAR-mediated calcium in the spine when it occurred +2 to +20 ms after pre-synaptic stimulation (∼250% increase), while a 5 ms depolarization to AP caused much reduced elevations in NMDAR-mediated calcium at these intervals (∼175% increase). The NMDAR-mediated calcium elevation observed with negative Δt was reduced compared to positive Δt for all three protocols, consistent with the experimental observation that in the presence of GABA inhibitors, negative Δt does not produce tLTP [10]. Because in reality, the AP does not always occur at the exact same time within the depolarization, STDP simulations were repeated with added random synaptic input to cause noise and jitter to the timing of the AP within the 30 ms depolarization. Simulations were repeated 6 times using different input frequencies and random seed values. The averages of these simulations overlay the control conditions, demonstrating that this model and protocol are robust to noise (Figure S1). Because calcium through the NMDAR is essential for tLTP [10], [12], the model predicts that the 30 ms depolarization protocol, which causes a strong increase in NMDAR-mediated calcium, will more readily induce tLTP than the 5 ms depolarization protocol which causes a weak increase in NMDAR-mediated calcium. This model prediction was confirmed experimentally with whole-cell patch-clamp recordings in rat striatal slices. STDP experiments show that using Δt of +5 to +30 ms, robust tLTP is induced with the 30 ms depolarization protocol, but tLTP is not induced with the 5 ms depolarization protocol (Figures 2D &E). Indeed, with the 30 ms depolarization protocol, the mean EPSC amplitude value, recorded 60 minutes after the induction protocol, was 186. 0±11. 6%, (Δt = +18. 6±1. 5 ms, n = 22), a value significantly different (p<0. 01) from baseline (Figure 2E). However, 5 ms postsynaptic suprathreshold depolarizations paired with presynaptic stimulations (Δt = +10. 3±0. 4 ms, n = 5) were not able to induce significant plasticity. The mean EPSC amplitude recorded 60 minutes after the induction protocol (97. 5±11. 0%) did not significantly differ from baseline (Figure 2E). The type of GluN2 subunit (A, B, C, and D) strongly determines the maximal conductance, decay time, and sensitivity to magnesium block for the NMDAR [9]. Therefore, differences in GluN2 subunit are predicted to modulate synaptic plasticity by controlling the calcium influx through the NMDAR. To evaluate this hypothesis, simulations were repeated using the 30 ms depolarization STDP protocol that caused tLTP for four types of NMDAR each representing a receptor containing two GluN1 subunits and two GluN2 subunits of the same type (either A, B, C, or D). The current decay time, maximal conductance, and magnesium block parameters of these NMDARs were adjusted to reflect experimental measurements [23], [24] (Table 3, Figures S2A & B). Calcium elevation due to NMDAR was evaluated for each GluN2 subunit condition over all Δt (from −100 to +100 ms). The resulting calcium curves (Figure 3) show clear differences in shape depending on GluN2 subunit. GluN2A and GluN2B conditions show the highest normalized calcium peaks, reaching greater than 250% of control, while GluN2C demonstrates a much reduced increase in calcium due to AP timing, just reaching 200% at the narrowest intervals. Similarly, GluN2D exhibits reduced calcium peaks and shows very little change in calcium concentration based on the AP timing (Figure 3). These simulations suggest that tLTP would be readily induced in GluN2A and GluN2B-containing synapses, but would not be as easily induced in GluN2C and GluN2D-containing synapses. Although GluN2A and GluN2B-containing receptors both reach calcium peaks greater than 250%, the shapes of the calcium curves are strikingly different. Because of the faster decay time of GluN2A, the range of Δt that causes strong calcium elevation is much narrower than for the more slowly decaying GluN2B-containing receptors. Calcium elevation due to influx through GluN2A only reaches levels >250% for narrow Δt. However, the GluN2B-containing receptors maintain elevations in calcium >250% even at wide Δt (Figure 3). Because the predicted intervals that allow significant amounts of calcium through NMDARs differ between GluN2A and GluN2B and because striatal MSPNs contain both GluN2B and GluN2A subunits [25], the model predicts that blocking the GluN2B subunit in an MSPN would narrow the range of Δt that produces tLTP. To test this prediction, STDP experiments were conducted in either normal aCSF, or in the presence of 10 µM Ifenprodil, a potent and selective GluN2B antagonist. In the control condition, Δt between +5 ms and +30 ms elicits tLTP (186. 0±11. 6%, n = 22) (Figure 4A–D). This Δt range includes both narrow timing intervals (5 ms<Δt<12 ms), and wide timing intervals (12 ms<Δt<30 ms). Under control aCSF conditions, the tLTP induced using narrow Δt (207±26%, n = 5) does not significantly differ from the tLTP induced using wide Δt (179. 9±13. 4%, n = 17) (P = 0. 72). In contrast, when GluN2B-containing NMDARs are blocked with Ifenprodil, tLTP at intervals wider than +12 ms is abolished (97. 7±6%, n = 7), while tLTP between +5 ms and +12 ms was maintained (142±12%, n = 12). This difference between wide and narrow timing intervals is significant for the Ifenprodil condition (p<0. 05; Figure 4D). These results demonstrate that GluN2 subunits not only control whether potentiation occurs, as previous studies have shown [26]–[28], but also that they hone plasticity, making it sensitive to wider or narrower time intervals between pre-synaptic neurotransmitter release and post-synaptic firing. To reflect these experiments in the model cell, control aCSF simulations were run using a combined GluN2A and B decay time constant (25% GluN2B, 75% GluN2A [25]), and compared to the GluN2A only condition. For both cases the calcium response was normalized by the control aCSF no AP condition. The calcium curves generated by the model MSPN reflect the experimental STDP curves. When GluN2A and GluN2B are present, calcium elevation due to NMDAR stays above 250% of control for both narrow (2 ms<Δt<12 ms) and wide (12 ms<Δt<30 ms) Δt, whereas when GluN2A is present alone, calcium elevation due to NMDAR drops below 250% of control for wide timing intervals (Figures 4E&F). Interestingly, when GluN2A and GluN2B are present, the interval at which NMDAR-mediated calcium drops below 250% in the model (+40 ms Δt) is the same interval that no longer gives tLTP in the experiments (Figures 4C &E). During the narrow timing intervals (5 ms<Δt<12 ms), Ifenprodil significantly reduced the amount of tLTP induced for the experiments, and reduced the NMDAR mediated calcium in the model simulations. This result is not surprising considering that the synapses in the Ifenprodil condition have fewer total NMDARs available than those in the control condition. To test whether a general 75% reduction in NMDAR conductance would produce the same calcium effects as the GluN2A only condition, simulations were run using 75% of the NMDAR conductance and the combined GluN2A+B kinetics (Figures 4E&F). This general reduction reduced the effect of AP pairing, but did not reproduce the timing difference seen with the GluN2A only case. Our model predicts that if NMDAR conductance is reduced by 75%, LTP would be reduced for both narrow and wide Δt. Previous studies in other neuronal types have shown that distance from the soma alters the strength [29] and direction of STDP [30]. Because the AP decays as it back-propagates in MSPNs [31], we hypothesized that the timing dependence seen in secondary dendrites of these neurons would shift with distance from the soma. To investigate these interactions, we evaluated the peak calcium elevation during the 30 ms depolarization STDP protocol while stimulating spines located on the third segment of the tertiary dendrite, 150 µm away from the soma, compared to the secondary dendrites at 40 µm. Because a smaller branch diameter increases the input resistance at the tertiary dendrite, stimulating two adjacent tertiary spines resulted in a larger post-synaptic potential (PSP) than stimulating the two adjacent secondary spines, when seen at the spine head (Figures 5A & B). However, the greater electrotonic distance for the tertiary stimulation causes the same stimulation to be smaller when seen at the soma (Figures 5A&C). As predicted by cable theory [32], the depolarization seen in the spine due to the back propagating AP is smaller in the tertiary dendrites than in the secondary dendrites (Figure 5D). A reduced sensitivity to AP timing for positive Δt is observed under every GluN2 subunit condition (Figure 5E), due to both the decrement in back-propagating AP, which decreases the amplitude of the NMDAR-mediated calcium elevation, and the larger PSP, which decreases the need for depolarization by the AP. In contrast, virtually no change in calcium elevation or timing dependence appears for negative Δt (Figure 5E). The most drastic decreases in NMDAR-mediated calcium elevation occurs in the subunit conditions most sensitive to the magnesium block, i. e. GluN2A and GluN2B, while the GluN2C and GluN2D subunit conditions showed a smaller decrease in NMDAR-mediated calcium elevation due to distance. Because there is no change for negative Δt and a decrease in peak calcium for positive Δt, the difference between positive and negative Δt is strongly reduced when synapses are on the tertiary dendrite. For example, when GluN2A is stimulated on the tertiary spine, positive Δt causes nearly the same elevation in calcium as negative Δt (Figure 5E inset). Under these distal dendrite conditions, positive Δt is unlikely to produce tLTP, and the dependence on AP timing is drastically reduced. The MSPNs of the striatum receive inputs from almost all areas of the cortex [33] and from the thalamus [34]. Using a recently developed slice preparation which preserves both cortico-striatal and cortico-thalamic fibers [35], Ding et al. [25] and Smeal et al. [36] have characterized the two major glutamatergic inputs to the striatum in mouse and rat respectively. Ding et al. found that thalamo-striatal synapses had a lower NMDAR/AMPAR ratio than cortico-striatal synapses in the mouse, while Smeal et al. found the opposite, that the NMDAR/AMPAR ratio was higher for thalamo-striatal synapses in the rat. Measuring the decay time constant, Smeal et al. found that thalamo-striatal synapses had lower GluN2B/NMDAR ratios than cortico-striatal synapses, while using bath application of Ifenprodil Ding et al. found the opposite, that thalamo-striatal synapses had a higher GluN2B/NMDAR ratio. Smeal et al. also found that the thalamo-striatal synapses were electrotonically more distant from the soma than the cortico-striatal synapses, Ding et al. did not measure this (Table 4). To investigate the implications of differences in the NMDAR/AMPAR ratio, the GluN2B/NMDAR ratio, and the electrotonic distance from the soma, we modeled synapses with specific cortico-striatal characteristics and specific thalamo-striatal characteristics. Because the two studies report contrasting results [25], [36], we ran separate simulations using each set of data. We found that, based on different electrotonic properties, thalamo-striatal and cortico-striatal synapses differentially affect the NMDAR mediated calcium elevations for positive Δt, but not negative Δt. The distal thalamo-striatal synapses had lower peak calcium, and a weaker dependence on AP timing than did the cortico-striatal synapses for both mouse and rat (Figure 6A). This result suggests that NMDAR-dependent tLTP would be more readily induced in cortico-striatal synapses than in thalamo-striatal. However, this result assumes that thalamo-striatal synapses are more distal than the cortico-striatal as has been experimentally suggested [36]. To distinguish the effect of distance from the effect of NMDAR/AMPAR and GluN2B/NMDAR ratios, simulations were repeated with thalamo-striatal synapses in the same location (secondary dendrite) as the cortico-striatal synapses. When the synapses were located at the same distance from the soma, the effect of Δt on peak calcium concentration did not differ strongly between thalamo-striatal and cortico-striatal synapses (Figure 6A). Therefore, the lower calcium peaks in the thalamo-striatal simulations are entirely due to its location on the tertiary dendrite. Interestingly, this result was independent of which dataset (Ding et al. or Smeal et al.) was used. In conclusion, we predict that the cortico-striatal and thalamo-striatal synapses onto an MSPN will respond similarly to STDP protocols if they are located a similar distance away from the soma. However, if as Smeal et al. [36] have shown, the thalamo-striatal synapses are more distal, then we predict it will be more difficult to induce NMDAR dependent tLTP in those synapses than in cortico-striatal synapses. In the model, NMDAR-mediated calcium elevations show strong sensitivity to AP timing in proximal dendrites, but this sensitivity as well as the maximum change in calcium is diminished at distal synapses, independent of STDP protocols and GluN2 subunit. Given the diminished effect of AP timing in tertiary dendrites, LTP at distal synapses might be achieved through entirely different plasticity mechanisms not requiring a somatic AP. A recent study suggests that a limited number of inputs on distal dendritic branches can induce upstates in MSPNs due to the high input resistance of the branch and non-linearity of the NMDA receptor [43]. Thus, the conjunction of many synaptic inputs may be more important than somatic depolarization for distal synaptic plasticity. However, further work is needed to investigate whether this upstate induction can induce LTP at synapses distal to the soma. We have used the recently characterized thalamo-striatal and cortico-striatal inputs to MSPNs to model synapses of each type. Simulations show that distance from the soma was the only factor that created a differential response to STDP protocols between these two types of synapse. Thus, our results predict that any disparity in plasticity expression between these synapses will not be NMDAR related, and will come from other neuromodulatory mechanisms, such as differences in pre-synaptic endocannabinoid receptor expression [44]. Our model shows that the time course and voltage dependence of GluN2 subunit can influence the way synapses respond to STDP protocols. GluN2A and GluN2B produce a stronger sensitivity to AP timing than GluN2C and GluN2D. Although the striatum contains mostly GluN2A and GluN2B subunits, our findings predict that neuron types with predominantly GluN2C or GluN2D will not exhibit NMDAR dependent tLTP. In addition, GluN2A, because of its fast decay time, results in a narrowing of the STDP curve (compared to GluN2B) by decreasing the Δt that permits sufficient calcium influx. While previous studies have focused on whether a particular GluN2 subunit is necessary for plasticity [26]–[28], we have shown that the relationship between GluN2 subunit and plasticity is more complex than simply allowing or preventing LTP. In addition to the basic subunit characteristics modeled here, there are other, less well understood parameters that differ between the four GluN2 subunits. For example, differing amino acid patterns in the c-terminal tail allow differential phosphorylation by kinases and differential binding to scaffolding proteins [45]. Some of these differences are known to change the calcium permeability of the channel [46], and could further influence the receptor' s response to STDP. Similarly, the intracellular location of the GluN2 subunits is not well known in the striatum. A few studies have looked at the synaptic versus extrasynaptic location of GluN2A and GluN2B [47], [48] in the striatum, but the techniques used were comparative rather than quantitative; thus we cannot be certain that we are not stimulating extrasynaptic NMDARs, but the low frequency stimulation (always 1 Hz or lower) used here is unlikely to cause significant glutamate spillover [49]. It is possible that NMDA triheteromers (containing both GluN2A and B) are present in the striatum ([50], but see [25]). Our experimental protocol does not distinguish between di or triheteromers and therefore we cannot specifically determine whether the effect is due to Ifenprodil' s full blockade of GluN2B diheteromers or its weak blockade of GluN2A/B triheteromers [51]. The balance between GluN2A and GluN2B, and thus the shape of the STDP curve may be modulated dynamically. Studies in the hippocampus have demonstrated a widening of the STDP curve in response to dopamine D1 receptor stimulation [52], and β-adrenergic receptor stimulation [53]. Interestingly, both D1 and β-adrenergic receptors activate the cAMP-dependent protein kinase (PKA) which is essential for striatal LTP [54]. PKA phosphorylation is known to increase the calcium permeability of NMDARs and in particular, those containing GluN2B subunits [46]. Therefore, PKA activation may widen the STDP curve by increasing the calcium permeability of GluN2B-containing NMDARs. The role for NMDAR subunits in neurological disorders has recently been suggested. Studies conducted in rodent models of Parkinson' s Disease have shown that dopamine depletion results in the reconfiguration of the NMDARs, specifically in a reduction of GluN2B subunits [47]. Other studies have found that the administration of GluN2B antagonists reduces dyskinesia in animal models of Parkinson' s Disease while GluN2A antagonists may increase it [55]. Similarly, GluN2B containing receptors are selectively potentiated by mutant huntingtin [56], suggesting abnormal GluN2B subunit activity in Huntington' s Disease. An imbalance of GluN2B containing NMDARs may allow non-specific potentiation that could lead to excessive and uncontrolled movement. Our results contribute to the emerging picture of GluN2 subunit antagonists as treatments for neurological disorders of the striatum by elucidating a possible mechanism for GluN2 subunit alterations to alter striatal plasticity and therefore motor behavior. Our findings suggest a novel role for the GluN2A NMDAR subunit in striatal synaptic plasticity. Instead of allowing or preventing LTP, this subunit hones plasticity, narrowing the STDP curve and allowing for the fine-tuning of neuronal pathway strength. Previous work has shown that the medial striatum undergoes plastic changes during the early, more coarse, phase of skill learning, while the lateral striatum undergoes plasticity during the late, fine-tuning, phase of skill learning [1]. Interestingly, the lateral striatum contains a higher ratio of GluN2A to GluN2B subunits than the medial striatum [13]. A high density of the GluN2A subunit may functionally underlie the fine-tuning phase of skill learning, allowing potentiation of only the most closely-timed connections. While the higher ratio of GluN2B subunits in the medial striatum would be useful for less specific, but possibly faster acquisition of a skill. This role for GluN2A may also underlie the experience-dependent developmental shift from GluN2B to GluN2A in the visual cortex [57], and may be responsible for the increase in spatial learning ability that coincides with the developmental switch from GluN2B to GluN2A at hippocampal synapses [58]. All plasticity experiments were performed in accordance with local animal welfare committee (Center for Interdisciplinary Research in Biology and College de France) and EU guidelines (directive 86/609/EEC). For electrophysiology used to tune the computational model, animal handling and procedures were approved by the George Mason University IACUC committee (Text S1). Every precaution was taken to minimize stress and the number of animals used in each series of experiments. Animals, OFA rats (Charles River, L' Arbresle, France) (postnatal days 17–25) were sacrificed by decapitation and brains were immediately removed. Patch-clamp recordings of MSPNs were performed in horizontal brain slices (330 µm) from OFA rats. These horizontal slices included the somatosensory cortical area and the corresponding cortico-striatal projection field [22] and were prepared with a vibrating blade microtome (VT1000S and VT1200S, Leica Micosystems, Nussloch, Germany). Patch-clamp whole-cell recordings were performed in the somatosensory area of the dorsal striatum and made as previously described [10], [22]. Briefly, borosilicate glass pipettes of 5–7 MΩ resistance contained (mM): 105 K-gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0. 3 GTP-Na, 0. 3 EGTA (adjusted to pH 7. 35 with KOH). The composition of the extracellular solution was (mM): 125 NaCl, 2. 5 KCl, 25 glucose, 25 NaHCO3,1. 25 NaH2PO4,2 CaCl2,1 MgCl2,10 µM pyruvic acid bubbled with 95% O2 and 5% CO2. Picrotoxin (50 µM) (Sigma, Saint Quentin, France) was dissolved in ethanol and then added in the external solution for a final ethanol concentration of 0. 01%. All recordings were performed at 34°C using a temperature control system (Bioptechs ΔTC3, Butler, PA, USA and Bath-controller, Luigs&Neumann, Ratingen, Germany) and slices were continuously superfused at 2–3 ml/min with the extracellular solution. Individual neurons were identified using infrared-differential interference contrast microscopy with CCD camera (Hamamatsu C2400-07; Hamamatsu, Japan). Signals were amplified using EPC10-2 amplifiers (HEKA Elektronik, Lambrecht, Germany). Current-clamp recordings were filtered at 2. 5 kHz and sampled at 5 kHz, and voltage-clamp recordings were filtered at 5 kHz and sampled at 10 kHz using the program Patchmaster v2x32 (HEKA Elektronik). The series resistance was compensated at 75–80%. Electrical stimulation of the cerebral cortex was performed with a bipolar electrode (Phymep, Paris, France) placed in the layer 5 of the somatosensory cortex [22]. Electrical stimulations were monophasic at constant current (Stimulator WPI, Stevenage, UK or ISO-Flex stimulator controlled by a Master-8, A. M. P. I., Jerusalem, Israel). Currents were adjusted in order to evoke striatal excitatory postsynaptic currents (EPSCs) ranging in amplitude from 50 to 200 pA. Repetitive control stimuli were applied at 0. 1 Hz, a frequency for which neither short- nor long-term synaptic efficacy changes in EPSC amplitudes were induced [22]. STDP protocols consisted in pairings of pre- and post-synaptic stimulations with the two events separated by a specific temporal interval (Δt) repeated 100 times at 1 Hz. Pre-synaptic stimulations correspond to cortical stimulations and the post-synaptic stimulation to an AP evoked by a direct application of a depolarizing current step (5 or 30 ms duration) in the MSPN. Neurons were recorded for 10 minutes during baseline and for at least 60 minutes after the cellular conditioning protocol; long-term synaptic efficacy changes were measured after approximately 60 minutes. Series resistance was monitored and calculated from the response to a hyperpolarizing potential (−5 mV) step during each sweep throughout the experiments and a variation above 20% led to the rejection of the experiment. Repetitive control stimuli were applied at a frequency of 0. 1 Hz for 60 minutes. Drugs were applied in the bath, after recording 10 minutes of baseline and 10 minutes before cellular conditioning protocol, and were present continuously until the end of the recording. Ifenprodil was dissolved in water at 15 mM and then added to extracellular solution for a final concentration of 10 µM (Tocris, Ellisville, MO, USA). Off-line analysis was performed using Igor-Pro 6. 0. 3 (Wavemetrics, Lake Oswego, OR, USA). All results are expressed as mean±SEM and statistical significance was assessed using the Student' s t-test or the non-parametric Wilcoxon signed-rank test when appropriate at the significance level (p) indicated. Statistical analysis was performed using Prism 5. 0 software (San Diego, CA, USA). A dorsal striatum MSPN model cell was created based on the nucleus accumbens neuron model by Wolf et al. (2005) [16]. The Wolf model was translated from NEURON into Genesis simulation software, and channel concentrations and kinetics were adjusted to closely match those of a mature (>3 weeks old) MSPN in the mouse dorsal striatum (Text S1). The MSPN morphology is the same as in Wolf et al. [16], but with the addition of individual spine compartments on the primary dendrites to the third segment of the tertiary dendrites (Figure 1A). Primary dendrites are 20 µm long, secondary dendrites 24 µm, and each of the 11 tertiary segments is 36 µm long. For all simulations, two adjacent spines were stimulated (on the third segment of the tertiary dendrite, or on the secondary dendrite), and the NMDAR-mediated calcium was recorded from one of the two spines. This model is available on Model DB: http: //senselab. med. yale. edu/modeldb/ All channels kinetics (Table 1) were taken from published data, using dorsal striatum MSPNs when possible. The Na+ kinetics were obtained from dissociated dorsal striatum MSPNs in the guinea pig [59]. The fast A-type potassium channel (Kv4. 2) data was obtained from slice dorsal striatum MSPNs in rat [60]. The slow A-type potassium channel (Kv1. 2) data was obtained from dissociated and slice dorsal striatum MSPNs in rat [61]. The inwardly rectifying potassium channel (Kir) kinetics were extracted from the computational studies of Wolf et al. [16] and Steephen and Manchanda [62]. The KDr channel was from Migliore et al. [63] The BK channel [64] and SK channel [65] were activated by a specific pool of calcium from the N and R type calcium channels, but not the T or L type calcium channels [16], [66]. L (Cav1. 2 and Cav1. 3), N, R, and T type voltage sensitive calcium channels use the same parameters as Wolf et al. [16]. Calcium channels were added to the soma, and dendritic shafts. L, R, and T-type channels were also added to spines [20]. MSPNs of the dorsal striatum display different characteristics from those of the ventral striatum [67]. Both hand tuning and the simulated annealing parameter optimization routine in Genesis were used to adjust channel maximal conductances (Table 2), and channel activation and inactivation time constants (Table 1, scaling factor). These parameters were adjusted to match spike frequency, spike width, and latency to first spike extracted from current clamp data obtained at 30–32°C from mouse dorsal striatum MSPN (Figures 1B–D). The change in channel time constants of NaF, KDr, and KAf (scale in table 1) was required to produce the correct spike width as faster time constants produced spikes that were too narrow. In contrast, varying the maximal conductances by ±10% did not significantly change spike width (data not shown). Δt for both experiments and the model MSPN is defined as the time from pre-synaptic stimulation (stimulus artifact in the experimental case) to the peak of the AP. AMPA and NMDAR channels were added using the synchan object in Genesis to all spines in the model. The synchan object uses equation 1 to calculate the conductance of the channel from the activation and inactivation time constants (τ1 and τ2 respectively), time t relative to the action potential, and the maximal conductance (gmax). K is a normalization constant which is calculated from the time constants and allows Gsyn to reach a peak value of gmax. (1) GABA channels were not added to this model, thus all simulations should be interpreted as occurring in the presence of GABA receptor antagonists. The default AMPA maximal conductance is 342 pS, which agrees with data from Carter and Sabatini [20]. Default NMDA maximal conductance was 940 pS to maintain the NMDA/AMPA ratio of 2. 75/1 in cortico-striatal terminals [25]. AMPARs have an activation time constant (τ1) of 1. 1 ms and an inactivation time constant (τ2) of 5. 75 ms [16], [68]. NMDARs have an activation time constant (τ1) 2. 25 ms [13], but inactivation (τ2) depends on subunit. Magnesium sensitivity to the NMDAR was implemented by using the “Mg_block” object in Genesis which utilizes equation 2: (2) In which, parameter B = 1/62, while parameter A depends on subunit (Table 3). Specific GluN2-subunit containing NMDARs were modeled by adjusting the decay time constant, the maximal conductance, and the sensitivity to magnesium block according to published data [23], [24]. GluN2 subunits differ in open probability [69] and affinity for glutamate [70]. These differences, though not modeled explicitly, contribute to the maximal conductance and the decay time which are taken into account in this model. Single decay time constants, averaged from the fast and slow time constants in Vicini et al. [24], were used for each GluN2 subunit (Table 3). All NMDA decay time constants are adjusted for temperature by dividing by a scaling factor of 2. Tau decay in tables 3 and 4 are the scaled values. Maximal conductances were calculated from the slope of the magnesium-free data [23] The ratio between the maximal conductance of GluN2A+B, C, and D was maintained, but the conductances were universally reduced such that the value for GluN2A+B (the predominate subunits in striatal MSPNs) matched cortico-striatal NMDAR/AMPAR ratios [25]. Current-voltage curves for each subunit in the presence of 1 mM magnesium from Monyer et al. [23] were matched by adjusting parameter “A” in this equation (Table 3, Figure S2B).
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Title: The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons Summary: The striatum of the basal ganglia plays a key role in fluent motor control; pathology in this structure causes the motor symptoms of Parkinson' s Disease and Huntington' s Chorea. A putative cellular mechanism underlying learning of motor control is synaptic plasticity, which is an activity dependent change in synaptic strength. A known mediator of synaptic potentiation is calcium influx through the NMDA-type glutamate receptor. The NMDA receptor is sensitive to the timing of neuronal activity, allowing calcium influx only when glutamate release and a post-synaptic depolarization coincide temporally. The NMDA receptor is comprised of specific subunits that modify its sensitivity to neuronal activity and these subunits are altered in animal models of Parkinson' s disease. Here we use a multi-compartmental model of a striatal neuron to investigate the effect of different NMDA subunits on calcium influx through the NMDA receptor. Simulations show that the subunit composition changes the temporal intervals that allow coincidence detection and strong calcium influx. Our experiments manipulating the dominate subunit in brain slices show that the subunit effect on calcium influx predicted by our computational model is mirrored by a change in the amount of potentiation that occurs in our experimental preparation.
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Summarize: BACKGROUND OF THE INVENTION When performing dental procedures, using a nose hood to administer inhalation gases, there is a possibility of foreign materials reaching the patient's eyes. The nose hood has a flexible gasket that seals the hood around the nose. The areas covered by the hood would be interfered with by conventional goggles. Access to the oral cavity is required for dental surgery, which renders prior art face shields, which cover the mouth, as unsuitable for providing eye protection during dental surgery. In addition to materials that might reach the eyes from above, such as things dropped or spilled while working in the oral cavity, there is the risk to the eyes from matter projected from the oral cavity. Such matter might include particles dislodged during drilling or other surgical procedures, matter atomized by drilling or fluid pressure, and matter projected from the mouth by the patient. These materials would arise below a conventional eye shield and present a danger to the eyes even with the conventional shield in place due to the nose hood interfering with a proper fit of the shield. OBJECTS It is therefore an object of this invention to provide a protective eye shield for use on patients when the patient is wearing an inhalation-gas dispensing nose hood. It is further an object of this invention to provide the shield described above wherein the shield provides access to the oral cavity and shields the eyes from matter arising from the oral cavity. It is further an object of this invention to provide the shield described above wherein the shield is indexed to the nose hood and is adjustable so as to be securely and comfortably accommodating to the patients head shape. It is further an object of this invention to provide the shield described above wherein the shield is of substantially one piece construction and of a semi-flexible transparent material that is sufficiently low in cost as to permit economical disposal of the shield after use. Other objects of the invention will become apparent from the following descriptions, drawings and claims. BRIEF DISCUSSION OF THE PRIOR ART The prior art abounds in face shields that protect the eyes of the wearer. U.S. Pat. No. 4,986,282 to Stackhouse et al. teaches a shield that is typical of such devices. The art also abounds in head gear that has protection for the eyes and a means of introducing inhalation gases to the wearer, and for expelling exhalation gases from the head gear. U.S. Pat. No. 789,145 to Derx is an early example of such a piece of head gear. U.S. Pat. No. 3,241,155 to Phillips teaches a cushioning pad at the edge of a face shield. U.S. Pat. No. 4,945,574 to Dagher teaches adjustable side contacts for a protective mask. If broad language is used, it can be said that the prior art provides a number of the utilities and functions that are the same as those of the instant invention. However, the prior art does not provide an eye shield that protects the eyes of the wearer and provides access to the oral cavity when the user is wearing an inhalation gas providing nose hood. BRIEF DESCRIPTION OF THE INVENTION The invention, in its simplest form, is an eye shield, constructed of semi-flexible clear or tinted plastic material, and having a broad, eye protecting central span having a face side surface and an outside surface. The central span has a perimeter having a forehead edge, a nose edge, opposite the forehead edge, and a right side edge and a left side edge. The right and left side edges span the distances between the forehead edge and the nose edge, one to each side of the shield, to form a closed perimeter for the shield. A nose hood index is formed as a part of the central span near the nose edge of the shield. The nose hood index is configured so as to receive and index to an inhalation-gas dispensing hood. The forehead edge is provided with a forehead contacting surface which is configured so as to rest comfortably on a human forehead. The side edges have projecting therefrom, temple pieces which are configured to contact with and index to the temple area of a human head. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a pictorial view of the shield of this invention taken from the nose edge of the shield. FIG. 2 is a pictorial view of the shield of FIG. 1 taken from the forehead edge of the shield. FIG. 3 is an elevational view of the shield of FIGS. 1 and 2 in place on a patient. FIG. 4 is a sectioned fragmentary view of a temple piece and a cushioning sleeve having an insert. FIG. 5 is a partially sectioned elevational view of the nose edge of the shield showing a Velcro (TM) joinder means. FIG. 6 is a partially sectioned elevational view of the nose edge of the shield of this invention showing a tape joinder means. FIG. 7 is a sectioned elevational view of the nose edge of the shield of this invention showing a clip means of joinder. FIG. 8 is a plan view of the shield of this invention showing a threaded fastener means of joinder. DETAILED DESCRIPTION OF THE INVENTION In the figures, like numbers refer to like objects. In the figures some sections have been thickened for ease of illustration. Referring now to FIGS. 1 through 4. Protective eye shield 1 has its principle elements formed of a semi flexible clear or tinted plastic material and is characterized by having a broad eye shielding central span 2 having a face side surface 32 and an outside surface 33. Eye shielding central span 2 is bounded on one side by a forehead edge 3, and on the opposite side by nose edge 4. Right side edge 5 and left side edge 6 span the distance between forehead edge 3 and nose edge 4. Edges 3,4,5, and 6 form a closed perimeter for central span 2. Nose hood 20 is provided with flexible seal 21 which contacts the patient's nose, cheeks and upper lip to form a seal against the entry or loss of gases. Hood 20 is also provided with hoses 22 which carry inhalation gases to hood 20 and carry exhalation gases away from hood 20. Hoses 22 lie closely along the face of the patient and form an effective barrier to materials passing between the patients face and nose hood 20 or hoses 22. Nose edge 4 of shield 1 is provided with hood index 7 which serves to index shield 1 to nose hood 20. Hood index 7 is hereshown as an integral part of shield 1 and index 7 is in the form of an inverted cup which approximates the shape of an inhalation gas providing nose hood 20 with which it cooperates and to which it indexes. Forehead edge 3 has as a part thereof, forehead contacting surface 8 which is configured so as to rest comfortably against the patient's forehead 34. Forehead contacting surface 8 is in the form of a curved bar which projects from forehead edge 3 of shield 1 and the curve of the bar approximates the curve of the forehead 34 of a human head and the curve of the bar presents a smooth surface 35 that will rest comfortably upon the forehead of the patient. Right side edge 5 and left side edge 6 are provided with right temple piece 9 and left temple piece 10 respectively. Temple pieces 9 and 10 serve to provide positional security to shield 1 relative to the face of the patient. The comfort of temple pieces 9 and 10 and the closeness of their fit is enhanced by the use of cushioning sleeves 11. As shown in the sectioned view of FIG. 4, cushioning sleeve 11 is constructed of a compressible resilient material such as elastic cotton or the like. Cushioning sleeves 11 engages the free ends 36 of temple pieces 9 and 10. Adjustment inserts 12, of folded cotton gauze or the like, are insertable into sleeve 11 on the face side of temple pieces 9 and 10. Inserts 12 serve to cushion the pressure of temple pieces 9 and 10 against the head of the patient, and also serve to insure a firm and secure fit of shield 1 with the head of the patient. Shield 1 as disclosed above indexes to and cooperates with nose hood 20 to form an eye protection system for use during dental surgery when a patient is wearing an inhalation gas dispensing nose hood and access to the oral cavity is required. Shield 1 can be joined to or combined with nose hood 20 by a number of convenient means. Referring now to FIG. 5 wherein shield 1 at nose edge 4 and adjacent to hood index 7, is provided with a first Velcro (TM) fastening tape 19 and wherein nose hood 20 is provided with a second, mating Velcro (TM) fastening tape 19' at the intersection 14 of hoses 22 and nose hood 20. A similar set of tapes is provided to each side of index 7 and hood 20. Referring now to FIG. 6 wherein shield 1 is provided at its nose edge 4 with adhesive tapes 15, one to each side of index 7, and which hingeably join shield 1 to intersection 14 of hoses 22 of nose hood 20. Tapes 15 serve to maintain the joinder of shield 1 with hoses 22 while permitting shield 1 to be raised to permit access to hood 20, or seal 21 or to blot perspiration from the patient's brow. Tapes 15 have first end segments 37 which are secured to intersection 14 of hoses 22 of nose hood 20 and second end segments 38 which are secured to shield 1 near nose edge 4. Referring now to FIG. 7 wherein a mechanical clip 16 is secured near nose edge 4 of shield 1, one at each side of index 7, and the clip mechanically engages intersection 14 of hoses 22 of hood 20. Shield 1 has as a part of nose edge 4, a curved segment 17 which, in combination with mechanical clip 16, encompasses more than half of the perimeter of intersection 14 of hoses 22. To join shield 1 with nose hood 20 at intersection 14 of hoses 22, curved segment 17 and clip 16 are urged apart, in the maner that is customary in applying spring clips, and are passed, one to each side of intersection 14 of hoses 22 and allowed to return to their normal unstressed position, thereby mechanically joining shield 1 with intersection 14 of hoses 22. Clip 16 may be positioned so as to engage hoses 22 of nose hood 20 when it is seen as desirable to do so. Referring now to FIG. 8 wherein shield 1 is shown as being joined with nose hood 20 by means of threaded fasteners 18 to form a substantially unitary structure. In some situations it will be desirable to have nose hood 20 and shield 1 combined to form a substantially unitary structure. Threaded fasteners 18 provide that utility while at the same time permitting the separation of shield 1 from hood 20 should it be desirable to do so. Threaded fasteners 18, which may be conventional screws, pass through shield 1 and are threadably engaged into intersection 14 of nose hood 20. Preferred embodiments of this invention have been disclosed and discussed above. Many variants and combinations of the elements of the invention will suggest themselves to one skilled in the art. Such variants might include those necessary for the adaption of the shield of this invention to the specific geometries of a specific manufacture of nose hood. Disclosure and discussion of all possible combinations and variants of the elements of this invention would greatly multiply the drawings and cause the specifications to become prolix. Therefore, the invention should not be limited to the embodiments disclosed above, but be limited only by the appended claims and all equivalents thereto that would become obvious to one skilled in the art.
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Summary: This invention is a protective eye shield that indexes to an inhalation gas providing nose hood and the hood's hoses to provide secure eye protection to a patient wearing such a hood during the performance of dental procedures. The shield further indexes to the temples and forehead of the wearer to provide secure and comfortable positioning of the shield. The shield cooperates with the inhalation hood in providing a shield against materials reaching the patients eyes.
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Write a title and summarize: RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. Viruses are among the most important plant pathogens that cause huge economic losses in many agriculturally important crops worldwide. The invasion of the host by viruses deeply alters the physiology of the plants at cellular and tissue levels due to the interaction of the virus with the cellular pathways, which ultimately leads to viral symptom development. During evolution, plants have developed diverse strategies to combat virus infections. Amongst others, RNA silencing is one of the most important mechanisms that serve to fight against viruses [1,2]. RNA silencing is a conserved eukaryotic pathway involved in almost all cellular processes like development, stress responses and antiviral defense. RNA silencing relays on the 21–24 nt short interfering RNAs (siRNAs) or micro RNAs (miRNAs) the hallmark molecules of silencing [3]. The siRNAs and the miRNAs (collectively named small RNAs, sRNAs) are processed by RNase III-type ribonucleases, the DICER (in plants Dicer-Like, DCL) enzymes [4,5] in collaboration with their partner DOUBLE-STRANDED RNA BINDING (DRB) proteins [6–9]. sRNAs are 2’-O-methylated by HUA ENHANCER1 (HEN1) at their 3’ protruding ends [10], a reaction that serves to protect them against poly-uridylation and subsequent degradation [11]. sRNAs then associate with ARGONAUTE (AGO) proteins [12–14] the central effectors of RNA-induced silencing complex (RISC) [15,16]. Based on the sequence complementarity, sRNAs guide RISC to silence cognate RNAs through cleavage or translational repression (post-transcriptional gene silencing, PTGS) or induce chromatin/DNA modifications of the specific genomic locus (transcriptional gene silencing, TGS) [17–19]. In some specific cases, amplification of silencing occurs through double-stranded RNA synthesis by RNA-dependent RNA polymerases (RDRs) and secondary siRNA production [20–22]. sRNAs are non-cell autonomous, they can move within the plant to transmit gene silencing from cell-to-cell or systemically on long distance as mobile silencing signals [23–25]. Players of the antiviral silencing overlap with those of the endogenous silencing pathway [1,26]. Antiviral RNA silencing is triggered by the presence of viral dsRNA structures such as replication intermediates or intra—molecular fold—back structures of the invading virus. These dsRNA structures are processed by DCL4 or DCL2, to produce viral short interfering RNAs (vsiRNAs) [2,3, 27–32]. The vsiRNAs guide self-silencing of the parental viral genomic RNA as part of the antiviral response through the action of AGO effectors [1,13]. Among the AGOs implicated in antiviral defense, AGO1 and AGO2 were identified as the most important players. AGO1 was shown to have antiviral roles against a number of viruses in A. thaliana [33–36], N. benthamiana [26,37,38] and in rice [39]. AGO2 was found to be important in the antiviral silencing response in A. thaliana [35,36,40–45]. In N. benthamiana, the important model organism for plant virology studies, AGO2 was proposed to protect against Potato virus X [46] and the suppressor-deficient Tomato bushy stunt virus (TBSV) [47,48]. However, recent observation suggested that AGO1 constitutes a solid layer of defense against tombusvirus infections [49]. As AGO1 is the negative regulator of AGO2, it is believed that AGO2 represents a second layer of antiviral defense [40]. Viruses, to counteract host defense, have evolved viral suppressors of RNA silencing (VSRs) providing strong evidence for the antiviral nature of silencing [1,50,51]. Most viruses studied so far were found to encode at least one VSR. VSRs were shown to block silencing at multiple steps like initiation, effector complex assembly, silencing amplification but also through transcriptional control of endogenous factors, hormone signal modulation or interaction with protein-based immunity [51,52]. The absence or inactivation of VSRs leads to the recovery of plants from viral infections, demonstrating the effect of plant antiviral silencing response [53–55]. Although several VSRs have been identified in the past, our knowledge about the precise molecular basis of their action and their multifunctional roles have only been resolved in a few cases [1]. The p19 protein of tombusviruses is one of the best-known VSR. Crystallographic studies have shown that p19 tail-to-tail homodimer acts as a molecular caliper to size-select and sequester siRNA duplexes in a sequence-independent manner [56–58] preventing the loading of siRNAs into AGO effector proteins [59,60]. Based on p19 expressing transgenic plants it was proposed that during virus infection p19 efficiently prevents miRNAs loading into RISC deeply compromising the endogenous miRNA pathways of the plants [61–63]. In particular, it was reported that three distinct VSRs (HcPro, p19 and P15) compromised the regulation of the miR167 target AUXIN RESPONSE FACTOR 8 (ARF8) when constitutively expressed in transgenic plants [64]. It was also proposed that misregulation of miR167 is the major cause for the developmental aberrations exhibited by VSR transgenic plants and for the phenotypes induced during viral infections [64]. Contradictory to these, other data suggests that during genuine Cymbidium ringspot virus (CymRSV) infections miRNA sequestration by p19 is very poor and may depend on spatial and temporal co-expression of miRNA duplex and the VSR [65]. vsiRNAs but not miR159 were shown to be sequestered efficiently into p19-homodimer: siRNA nucleoprotein complex, whereas miR159 was efficiently incorporated into RISC complex [66,67]. These findings suggest that, in virus-infected plants, p19 potently affects vsiRNA-pathway and at the much lesser extent the miRNA one. Besides, independently of its siRNA binding capacity, p19 similarly to other VSRs can promote miR168 transcriptional induction that results in miR168-guided AGO1 down-regulation [66,68]. Thus, the interaction of p19 with endogenous silencing pathways and its contribution to viral symptom development is far from being fully uncovered. To better understand the impact of p19 on silencing and its role in viral symptom development we employed a synthetic p19-expressing transgenic N. benthamiana plant line (p19syn) or wild type plant as control in combination with its wild-type (CymRSV) or suppressor deficient virus (Cym19stop) infection: (i) wt virus infection of wt N. benthamiana (p19 in “cis”), (ii) Cym19stop-infection in wt N. benthamiana (virus present, no suppressor), (iii) Cym19stop-infection in p19syn plants (p19 in “trans”) and (iv) p19syn plants (p19 only). In this way we were able to compare the impact of p19 on its own or in the genuine virus-infected background. We analyzed p19 ability to sequester vsiRNAs and plant endogenous sRNAs, with and without viral background. Consistently with our previous results, we have found that p19 can bind vsiRNAs when expressed either “in cis” (from the CymRSV wild-type virus, in wild-type N. benthamiana) or “in trans” (transgenically expressed in p19syn plants infected with the suppressor-deficient Cym19stop virus). In line with our previous findings, p19 efficiently bound endogenous sRNA duplexes only in the absence of the virus infection, suggesting that p19 impact on endogenous pathways is restricted. Analyzing the siRNA pool immunoprecipitated by p19 through high-throughput sequencing, we found that p19 changes the bias of positive vsiRNAs towards a more equilibrated positive/negative strand ratio, suggesting a preference for perfect ds-vsiRNAs. We also showed that p19 prevents mainly AGO1 but not AGO2 loading with vsiRNAs. This finding suggests a key role of AGO1 opposed to AGO2 during the antiviral response. To uncouple p19 effects elicited by virus infection on RNA silencing and host plant symptom development we prepared p19-expressing N. benthamiana plants (Fig 1A–1C). To avoid the interference between the p19 transgene and the challenging virus (p19-deficient, Cym19stop), we modified the p19 transgene introducing all possible silent nucleotide changes. In this way, we reduced the nucleotide sequence similarity between the transgene and the challenging virus to 68% while keeping the amino acid identity at 100% (S1 Fig). These plants were named synthetic-p19 expressing plants (p19syn). The p19syn plants showed strong phenotype characterized with elongated stem internodes and typical leaf distortions (Fig 1A, 1C and 1D and S2A Fig) suggesting that the expressed p19 protein retained it suppressor activity, thus potentially compromising the endogenous silencing pathways. Importantly, this phenotype was clearly different from that of virus-infected stunted dwarf plant (S2A Fig). Transgenic line 1–57 was selected for further studies (Fig 1A and 1B). First we tested the silencing suppressor activity of transgenically expressed p19 in a GFP transient assay (see Materials and Methods). When GFP sense transgene was transiently expressed in wild-type plant leaves, spontaneously triggered silencing almost completely diminished GFP expression at four days post infiltration. In contrary when GFP was expressed in p19syn plants its expression was still strong as visualized under UV light (Fig 1D). The lack of GFP silencing in p19syn plants confirmed the suppressor activity of the p19 transgene. Next we tested p19 suppressor activity in an authentic virus infection context: we challenged the p19syn plants by the infection with Cucumber mosaic virus + yellow satellite RNA (CMV + Y-satRNA). CMV + Y-satRNA was reported to induce bright yellow symptoms on N. benthamiana through targeting the tobacco magnesium protoporphyrin chelatase subunit I (ChlI) gene involved in chlorophyll biosynthesis by Y-satRNA derived siRNA [69]. The CMV-Y-satRNA infected wt N. benthamiana plants developed the bright yellow symptoms while the infected p19syn plants failed to show the typical yellowing (Fig 1E). All these confirmed that the transgenically expressed p19 works as a silencing suppressor in vivo. It is generally assumed that virus encoded suppressors strongly interfere with the endogenous silencing pathway and are central players in the development of viral symptoms [1,61–64]. However, this notion mostly comes from studies that used VSR-expressing transgenic plants without analyzing the effect of the VSRs in an authentic virus infection background. To reinvestigate this dogma we set up an experiment in which we could compare p19 effects (vsiRNA and endogenous sRNA binding) with or without its parental virus infection background. We compared the sRNA binding capability of p19 both in mock- and Cym19stop-inoculated p19syn plants. This setup allowed us to analyze the impact of p19 when provided “in trans” during virus infection. It is worth noting that the suppressor mutant Cym19stop virus in infected p19syn plant was able to invade whole leaves similarly to the CymRSV in wt plants (S2B Fig). In contrast, in the absence of p19, the Cym19stop virus is restricted to the veins [70] (S2B Fig). Besides this, we also inoculated wt plants with CymRSV to study p19 activity “in cis“. Based on previous studies [61,62] we expected p19 to bind ds-sRNAs of both plant and viral origin. p19 immunoprecipitations (IP) were prepared from pooled systemically-infected leaves of virus-infected plants and the corresponding mock-inoculated leaves of p19syn plants. cDNA libraries of sRNAs were generated using RNA samples isolated from inputs and p19 IPs. After quality control filtering and processing steps (see Materials and Methods), sequences flanked by the 3’ and 5’ Solexa adaptors, and ranging in length from 16 to 28 nt, were aligned to the N. benthamiana and the CymRSV genomes, respectively [71,72]. Analysis of p19-bound sRNAs from mock-inoculated p19syn plants revealed that p19 binds efficiently endogenous sRNAs (Fig 2A and 2B), including members of several miRNA families (S3A Fig) [73]. Surprisingly, the analysis of p19-bound sRNA libraries derived from both CymRSV-infected wt N. benthamiana (“cis“-p19) and Cym19stop-infected p19syn plants (“trans-p19“) have shown a different picture: p19 bound almost exclusively vsiRNAs but not endogenous sRNAs (Fig 2A and 2C–2F). This suggests that the abundantly produced vsiRNAs may outcompete the plant sRNAs from p19 binding during virus infection. Specific enrichment of vsiRNAs versus endogenous miR159, one of the most abundant miRNA, was quantified by Northern blot analysis. p19 had a much weaker affinity for miR159 during virus infection: the IP/input ratio of p19 bound miR159 was 1. 2 mock-inoculated samples, while during virus infection (Cym19stop-infected p19syn plants) it dropped to 0. 29 (Fig 3A). We also quantified the percentage of enrichments in case of vsiRNAs and endogenous sRNAs within p19 IPs compared to inputs from our deep seq data (Fig 2A). The input library of Cym19stop-infected p19syn plants contained 28% N. benthamiana reads while in the p19-IP they represented only 2% (p19 specifically enriched vsiRNAs from 72% in the input to 98% in the p19-IP). Similarly, in the CymRSV-infected wt plant 12% N. benthamiana reads in the input sample has dropped to 1% (p19 enriched the 88% vsiRNAs of the input to 99% in the p19-IP). We concluded therefore that p19 ability to sequester endogenous sRNAs is strongly decreased by the virus infection and p19 preference to vsiRNAs does not depend on the expressional origin of p19 protein (viral vs transgenic expression). To better understand the biological relevance of vsiRNA-mediated endogenous sRNA binding and out-competition/release from p19 sequestration we analyzed the behavior of known miRNA-target mRNA pairs [73]. We compared RNAseq data obtained from mock-inoculated p19syn plant samples (when p19 binds to miRNAs) and from Cym19stop virus-infected p19syn plant samples (when p19 binds preferentially vsiRNAs while miRNAs are outcompeted/released). In the absence of the virus, p19 efficiently bound miRNA duplexes (S3A Fig) and this correlated with elevation of most of the miRNA-target mRNAs as the consequence of miRNA duplex sequestration by p19 and inability to program miRISC for cleavage (p19syn compared to wt N. benthamiana, S3B Fig). Upon Cym19stop virus infection however, the levels of most miRNA target RNAs were downregulated (compared to mock-infected p19syn) as the consequence of miRNA out-competition/release from p19 (S3B Fig). We went further and specifically looked to accumulation of trans-acting RNAs derived from TAS3 precursor, the target of miR390 [74] in a Northern blot assay (S3C Fig). In p19syn plants the level of miR390 was slightly elevated while the TAS3-derived D7 tasiRNA dropped below the detection level (compared to wt N. benthamiana). This was likely the consequence of the inhibition of the cleavage of TAS3 transcripts by p19-captured miR390. Indeed, miR390 is efficiently enriched in p19 IP (p19syn mock-infection, S3A Fig). When p19syn plants were infected with Cym19stop virus, miR390 binding by p19 decreased (S3A Fig), and consequently the activity of miR390 was restored that lead to D7 tasiRNA accumulation (to a similar level as detected in wt N. benthamiana, S3C Fig). Altogether our findings support the hypothesis that during virus infection p19 preferentially binds vsiRNAs while endogenous sRNAs are outcompeted/released from binding. High-throughput sequencing analysis showed that CymRSV-derived vsiRNAs produced during infection have a strong bias towards positive strand polarity (95% positive, 5% negative polarity) (Fig 3B and S4 Fig). These data, which are in line with our and other previous observations, suggest that the majority of vsiRNAs are produced from fold-back structures of the positive strand of the viral RNAs [27,30,31,75]. Hot spots of vsiRNA generation were observed (S4 Fig) as earlier [31,75]. The polarity analysis of p19-immunoprecipitated vsiRNAs revealed a more equilibrated positive/negative strand ratio (significant enrichment in negative strand derived vsiRNAs with 65% positive, 35% negative strands) in the CymRSV-infected plants (Fig 3B). In Cym19stop-infected p19syn plants 79% of vsiRNAs produced were positive-stranded (21% negative), while p19syn—immunoprecipitated the ratio changed to 62% positive, 38% negative (Fig 3B). Based on these we conclude that p19 preferentially enriches positive-negative ds-vsiRNA pairs possessing perfect duplex structure. To formally test the impact of mismatches within the duplex sRNAs on the affinity of p19 we compared the affinity of p19 protein towards the miR171 duplex miRNA family (Arabidopsis miR171a, miR171b, miR171c all containing mismatches) and a perfect artificial siR171 duplex (for structures see Fig 4A–4D) using in vitro electro-mobility shift assay [57]. The presence of mismatches within the stem of sRNAs strongly reduced p19 binding affinity towards duplex sRNAs (Fig 4A–4F). Consistent with our findings, it has been also shown previously that p19 preferentially binds to perfect sRNAs duplexes but not imperfect miRNAs duplexes [76]. We have also analyzed the 5’-nucleotide preference of p19 binding. No 5’-nucleotide sorting of vsiRNAs in the p19 complex was observed regardless of p19 expressional context (from the virus or the transgene) (Fig 2C and 2E). The relative abundance of sRNAs possessing different 5’-nucleotides closely followed the ratio of the input samples. The distribution of p19 bound vsiRNAs along the viral genome was found to be similar to that in the input (S4 Fig) showing that there is no sequence preference in p19 vsiRNA binding. To better understand the p19 protein effects on vsiRNAs we analyzed the size distribution of these during infection. Upon CymRSV infection vsiRNAs produced are predominantly of 21nt and 22nt in length (Fig 3C). In addition to these, the 20nt long vsiRNAs are still present although at much lower level. In Cym19stop virus-infected wt plants we observed a shift towards slightly longer forms: most of the vsiRNAs were 22nt long, the abundance of the 21nt and 20nt long forms being reduced (Fig 3C). These results are in line with our previous findings [31]. miRNAs having enhanced electrophoretic mobility were also detected earlier in the presence of p19 [62,77]. To test if shortening is indeed an effect of p19 protein itself we analyzed parental virus- (Cym19stop) derived vsiRNA when p19 was provided “in trans“. The length shift to 1- or 2-nucleotide shorter vsiRNA forms was confirmed (Fig 3A–3C). Shortening of endogenous miRNAs was also observed in the absence of virus infection (Fig 3D). Analysis of selected endogenous miRNAs, where the precise sequence and biogenesis/maturation are known, allowed us to establish that the truncation occurred at the 3’ end but not 5’ end. The truncation of miRNAs happened mainly in p19-sRNA complexes as was observed in p19-IP, however not all p19 bound miRNAs are truncated and the reason for this has not been clarified yet (Fig 3D). Nuclease treatments on in vitro bound p19: siRNA complexes further confirmed that the p19 protein can protect the double-stranded stretch of sRNA duplexes (S5 Fig). The exonuclease (RNaseA) -mediated digestion occurred in discrete 1- and 2-nucleotide steps while the dsRNA region (19nt length) was protected by the p19 protein. Shortening of sequestered sRNAs, therefore, is not dependent on the virus infection, occurs on 3’ end, involves both vsiRNAs and miRNAs and is likely the direct consequence of p19-binding and exonuclease activity. Multiple AGOs were shown to have antiviral functions. In A. thaliana and N. benthamiana AGO1 and AGO2 were described as the most important effectors while others such as AGO5,7 and 10 to have minor roles during antiviral silencing [13,34,36,44]. The current model of the inhibitory effect of p19 suggests that sequestering vsiRNAs prevents AGO loading. The inhibitory effect of p19 protein on RNA silencing during infection was quite evident. In fact, wt virus infection showed strong viral symptoms that culminated in complete necrosis and collapse of the plants while the Cym19stop-infected wt plants recovered from viral infection [70], the virus accumulation was restricted to the vascular tissues and a few cell layers around the veins ([70] and S2 Fig). To get a better insight into the detailed mechanism of p19 actions we analyzed the AGO1- and AGO2-bound vsiRNAs in CymRSV- and Cym19stop-infected wt N. benthamiana by co-immunoprecipitations (Fig 5) followed by deep sequencing analysis (Fig 6 and S6–S10 Figs). Loading of siRNAs into a particular AGO is preferentially directed by their 5’-terminal nucleotide: AGO1 prefers sRNAs having 5’U while AGO2 preferentially binds 5’A sRNAs [78,79]. As expected, the AGO1 co-immunoprecipitated plant sRNAs possessed predominantly 5’U while AGO2 immunoprecipitated sRNAs mainly 5’A, with a relatively high amount of 5’U species (Fig 6). We have also found 5’U endogenous sRNA binding by AGO2 when we processed the raw data obtained from previous report [36]. We expected that p19 would drastically reduce the loading of vsiRNAs into AGO1 and AGO2. Surprisingly, vsiRNA loading into AGO1 was compromised in the presence of p19 (during CymRSV-infection compared to Cym19stop-infection): we observed relatively high “background” of vsiRNAs without 5’ sorting preference in AGO1 (compare Fig 6D with 6F). Conversely, the amount of vsiRNAs and their 5’ sorting into AGO2 was very similar during CymRSV- and Cym19stop- infections (Fig 6D–6F). This suggests that the presence of p19 preferentially impact vsiRNAs’ AGO1 but not AGO2 effector loading. In the same time (the same sample set) endogenous sRNAs were efficiently precipitated as 5' U by AGO1-IP proving that the IP worked correctly (Fig 6C–6E). Note that the reads of endogenous sRNAs in CymRSV and Cym19stop are lower compared to mock-infected sample due to the high amount of vsiRNA presence (that impacts the bias during deep sequencing). The AGO1 IP derived from CymRSV-infected plants contained a similar miRNA profile as the mock inoculated plants, in contrast to AGO2 IP in which the levels of analyzed miRNAs were reduced (Fig 6C and 6D and S6 and S7 Figs). Importantly, this occurred only in wt CymRSV infection when a high level of p19 is expressed. Efficient incorporation of vsiRNAs into AGO2 but not AGO1 may cause the out-titration of AGO2-bound endogenous sRNA species (during CymRSV infection). In contrast, during Cym19stop-infection AGO1-loading occurred as expected predominantly by 5’U-sorting of vsiRNA and endogenous sRNAs (Fig 6E and 6F). The obtained results were confirmed with a second AGO1 and AGO2 IP that gave very similar results although had slightly higher background of contaminating 24nt species (S9 Fig). These findings suggest that p19 protein itself compromises AGO1- but not AGO2-loading during viral infection. The specific impact of p19 on vsiRNA AGO1-loading found in the deep sequencing analysis was also confirmed by Northern blot analysis. vsiRNAs loading into AGO1 was less efficient than AGO2 in CymRSV-infected plants when the p19 was provided in “cis” (Fig 5A) or in “trans” when two independent p19syn lines were infected with Cym19stop virus (Fig 5B). We also analyzed the distribution of AGO1- and AGO2-bound vsiRNA along the viral genome. This generally followed the biogenesis of vsiRNAs and we could not define any sequence preference or specific hotspots of AGO1- or AGO2-loading (S8 Fig). The strong spikes of certain vsiRNAs may arise due to the sequencing bias, therefore, do not necessarily represent vsiRNAs preferred for binding [80]. During virus infection, high amounts of vsiRNAs are produced. These vsiRNAs are efficiently sequestered by p19 suppressor inhibiting their incorporation into RISC [67]. The consequence of p19 vsiRNA binding is that the strong positive strand bias of vsiRNA biogenesis in the input sample (95: 5 positive/negative) is changed to a more equilibrated positive/negative stand ratio (65: 35 positive/negative). This result suggests that there are qualitative structural differences between vsiRNAs and that p19 preferentially binds vsiRNAs derived from perfect dsRNA or highly structured RNA species. The preference of p19 towards even structured ds-vsiRNAs is in agreement with p19 crystal structure: p19 homodimer leans on the ds-sRNA backbone. If the backbone structure is distorted by mismatches, the sRNA could become less accessible to p19 sequestration. Indeed, p19 bound siR171a perfect duplex with higher affinity than natural miR171a, miR171b or miR171c duplexes (Fig 4). This may likely be one of the reasons why ds-vsiRNAs are preferred by p19 instead of mismatches containing endogenous miRNA duplexes during viral infection (Figs 2 and 3A). The excess of vsiRNAs over endogenous miRNAs in virus-infected plant may also contribute to the preferential binding of vsiRNAs by p19. Moreover, the difference in the biogenesis of miRNAs versus vsiRNAs could also be a further important factor in the mechanism of the sRNA sequestration by p19. In addition to the previous findings, we have also shown that sRNA binding by p19 happens without 5’-end nucleotide selection including vsiRNAs or endogenous miRNAs. Previous studies [62,77] have reported the truncation of p19-bound sRNAs by 1 or 2 nucleotides. In the case of vsiRNAs, the site of truncation (5’ vs. 3’ end) cannot be defined since the generated vsiRNAs started from almost every single nucleotide of viral genome (S4 and S8 Figs). During Cym19stop-infection on wild-type plants truncation does not occur while in p19syn plants, which provide p19 in trans, it can be observed. Shortening also happens on miRNAs in p19syn plants without virus infection. In summary, the truncation is likely induced directly by p19-binding and is not due to the virus infection or restricted to a specific class of sRNAs. Why shortening does not happen in the absence of p19, on the free vsiRNAs, which theoretically would be more accessible? In cells endogenous free sRNA duplexes (e. g., miR168/miR168star) [54] and free vsiRNAs in Cym19stop infection can be observed [32,67]. The stability of these sRNAs (miRNAs and vsiRNAs) is conferred by HEN1-mediated methylation [11]. The crystal structure of p19: siRNA complex shows that the last two single-stranded nucleotides at 3’ terminus of siRNAs are protruding from the complex [57,58]. Furthermore, the p19 bound vsiRNAs are not methylated at 3’ terminus [65] therefore may be sensitive to exonucleases. We propose therefore that p19 binding inhibits sRNAs methylation and as a consequence of this the protruding unprotected two nucleotides at the 3’-end of sRNAs are trimmed by cellular 3’-exonucleases. Whether the truncation of sRNAs is a simple byproduct of binding or has a definite biological importance remains to be seen. Trimming of sRNAs may inactivate and render them incompetent for AGO-loading. Contradictory to this, we find efficient binding of 19nt and 20nt vsiRNAs by AGO2 (Fig 6D, S9D Fig). This observation supports the “catch and release” of vsiRNAs by p19 proposed earlier [81]. It has been long suggested that VSRs interfere with endogenous silencing pathways, and this may contribute to the viral symptom development [1,61–64]. Constitutive expression of p19 in N. benthamiana leads to the development of a strong phenotype that is quite different from symptoms observed during parental viral infection (Fig 1 and S2 Fig). The strong phenotype of p19syn plants may arise, at least in part, due to the sequestration of endogenous sRNAs by p19. Indeed, we could immunoprecipitate endogenous miRNAs with p19 from transgenic plants (Fig 2B). Importantly, however, miRNA sequestration by p19 provided either in cis or in trans was drastically reduced when the virus was present (Fig 2C–2F). Importantly, miRNA out-competition/release correlated with downregulation of miRNA targets (S3B Fig) and reestablishment of tasiRNA biogenesis (S3C Fig). Out-competition/release of p19-bound endogenous sRNAs/miRNAs upon virus infection seems to be biologically relevant and could have an important role in moderating the virus impact on plant. This needs to be further investigated. In conclusion therefore, our findings deny the model in which miRNA binding by p19 is the key step for the development of virus-induced symptoms [1,61–64]. It is more likely that p19 has an indirect effect through the specific inhibition of antiviral plant response and the viral symptoms are the outcome of a complex virus-host interaction during the viral invasion of plant cells. What could be the criteria for vsiRNA selection by AGO-loading machinery? We previously observed that in wt tombusvirus infection, p19 protein prevents vsiRNA loading to AGO/RISC complexes, however, even in the absence of p19 only a small fraction of the vsiRNAs is loaded into effector complexes (Fig 5) the rest remaining in a free, probably double-stranded form [32,67]. This suggests that a big part of the abundantly produced vsiRNAs is AGO-incompetent, or there is no free AGO protein present to be loaded into. The structure of the ds-sRNA stem could be an important feature for vsiRNA selection into AGOs (as we have shown for p19). A similar analysis of sRNA duplexes as in the case of p19 cannot be done, since in p19 binding both strands of ds-sRNAs are retained, while in AGOs, after loading, one strand is eliminated. The other possibility of the inability of vsiRNAs to load into effector complexes could be the shortage of silencing proteins like DCL/DRB or AGOs during the assembly of these effectors. It was shown that specific regulatory mechanisms are induced by the virus to dampen silencing: translation of AGO1 protein is decreased by the suppressor-mediated miR168 over-accumulation [54,66]. However, the down-regulation of AGO1 protein was not observed in p19syn plants (Fig 1B). The reason for this could be the relatively low level of p19 produced from transgene compared to virus infection (Fig 5). Another important observation is that vsiRNAs loading is selectively prevented mainly into AGO1 but not AGO2 in the presence of p19 in both CymRSV infected wt or Cym19stop infected p19syn plants (Fig 5). During virus infection, the decrease in the translation of AGO1 protein leads to accumulation of AGO2, due to the absence of AGO1-miR403-mediated posttranscriptional down-regulation of AGO2 [54,66]. One possibility, therefore, is that the vsiRNAs will be loaded into the available AGO2 while AGO1-loading will be decreased. We could not observe a significant increase of AGO2 protein in the presence of p19 (Fig 1B). The other possibility is that AGO1 and p19 compete for the same set of vsiRNAs while AGO2 requirement for vsiRNA features is different. p19 therefore, would selectively impact AGO1 but not AGO2-loading. Interestingly, we observed relatively high p19-depending “background” of vsiRNAs without 5’ sorting in AGO1 IP, unlikely to be AGO1-incorporated sRNAs. p19 could affect the connection between biogenesis/loading complexes DCLs/DRBs with AGO1 effector [7,9]. In line with this hypothesis, it was demonstrated that p19 can compromise the transfer of siRNA from DICER-R2D2 into RISC complex using Drosophila embryo extracts based in vitro system [59]. Regardless of the reason of how AGO1-loading is compromised by p19, it seems that AGO2 is not enough to fight off the virus and help the plant to recover in the absence of AGO1-loading/activity. It has been suggested previously that AGO2 but not AGO1 plays role in the antiviral response against tombusvirus infections, including Tomato bushy stunt virus (TBSV) [47,48]. We have done TBSV-VIGS (Virus Induced Gene Silencing) experiment, using p19 inactivated virus vector (TBSVp19stop), which carried Nb-PDS and Nb-AGO1 sequence (S11 Fig). When NbAGO1 was silenced by VIGS the virus accumulated at higher level and plants have shown stronger phenotype (S11 Fig). The obtained results further support the idea that AGO1 has a major role in antiviral response against tombusvirus infection. However, the additional role of other plant AGOs in antiviral response remained to be explored and it likely depends on specific features of the highly diverse plant viruses. The availability of CRISPR/Cas9 system for plant research will also help to clarify the specific roles of plant effectors in antiviral silencing response. The synthetic CymRSV ORF5 (p19) was essentially constructed following the previously described antivirus-induced transgene silencing strategy [82]. As the first step, we introduced, in CymRSV ORF5, all possible silent point mutations by selecting those most compatible with the N. tabacum codon usage. The resulting nucleotide sequence was further modified to avoid the presence of cryptic splicing and polyadenylation signals using Net2gene splicing prediction (http: //www. cbs. dtu. dk/services/NetGene2/) and HCpolyA (http: //bioinfo4. itb. cnr. it/~webgene/wwwHC_polya_ex. html) software, respectively. The synthetic ORF5 (S1A Fig) was synthesized by Life Technologies and cloned in pJIT61 [83] between the CaMV 35S promoter and 35S terminator. The gene cassette was excised with KpnI and BglII and cloned in KpnI-BamHI of pBin19 (pBinCymRSVp19syn) N. benthamiana was transformed with the recombinant Agrobacterium tumefaciens strain C58C1 (pGV2260) harboring the plasmid pBinCymRSVp19syn, and kanamycin-resistant plants were regenerated as previously described [83]. The primary transformants were checked for the presence of p19syn transgene by PCR and for the expression of the p19 protein by Western blotting with the anti-CymRSV-p19 antibody as previously described [56]. N. benthamiana plants were grown at 22°C. At six-leaves stage plants were infiltrated with A. tumefaciens C58C1 harboring the appropriate constructs in the pBIN61 plasmid. pBIN61-Cymp19 and pBIN61-GFP were grown on selective media overnight, resuspended in the infiltration buffer (10 mM MES, 0. 15 mM acetosyringone, 10 mM MgCl2) kept on 25°C for 4h, and subsequently infiltrated into wild-type or p19syn plant leaves at OD600 = 0. 4. In vitro transcription of CymRSV, Cym19stop, TBSV-PDS-GFP and TBSV-PDS-AGO1-1 RNAs from linearized template plasmids and inoculation of RNA transcripts onto N. benthamiana plants were performed as described previously [84]. CMV Y-sat infection was performed as described earlier [69]. Total RNA was extracted from 100 mg of leaf tissue. The homogenized plant materials were resuspended in 600 μl of extraction buffer (0. 1 M glycine-NaOH, pH 9. 0,100 mM NaCl, 10 mM EDTA, 2% SDS) and mixed with an equal volume of phenol. The aqueous phase was treated with equal volumes of phenol-chloroform and chloroform, precipitated with ethanol, and finally resuspended in sterile water. RNA gel blot analysis of higher molecular weight RNAs was performed as described previously [84]. RNA gel blot analysis of 21–24 nt RNAs was performed as follows. Approximately 5 μg of total RNA was separated by 15% PAGE with 8. 6 M urea and 1xTris-borate-EDTA. RNA was electro-blotted onto Hybond-NX membranes and fixed by chemical crosslinking at 60°C for 1 hr [85]. Small RNA Northern blot hybridization and analysis were performed using complementary locked nucleic acid (LNA) oligonucleotides (Exiqon, http: //www. exiqon. com). Mock- or virus-infected systemical leaf tissues were homogenized in extraction buffer (150 mM Tris-HCl, pH 7. 5,6 M urea, 2% SDS, and 5% μ-mercaptoethanol). Samples were boiled, and cell debris was removed by centrifugation at 18,000 x g at 4°C for 10 min. The supernatants were resolved on 12% SDS-PAGE, transferred to Hybond PVDF membranes (GE Healthcare) and subjected to Western blot analysis. For detection anti-p19 [70], NbAGO1 [86] and NbAGO2 custom antibody were used. NbAGO2 antibody was generated by immunization of rabbits with the synthetic peptide (CLEDPEGKDPPRDVF) (GenScript, http: //www. genscript. com/). The proteins were visualized by chemiluminescence (ECL kit; GE Healthcare) according to the manufacturer’s instructions. For immunoprecipitation, 1–5 grams of mock-, CymRSV- or Cym19stop-infected N. benthamiana leaves showing systemic symptoms (or leaves at the same stage and positions from mock-inoculated plants) were collected, ground in 1: 3 (w/v) amount of immunoprecipitation buffer (40 mM HEPES/KOH 7. 4,100 mM KOAc, 5 mM MgOAc, 5% glycerol, freshly added 4 mM DTT), and cleared by centrifugation (twice at 15,000 x g for 5 min). Cleared lysates were kept on ice until immunoprecipitation with antibody-coated protein A-Sepharose (GE Healthcare). Beads were washed before adding the antibodies (described earlier). For mock immunoprecipitation preimmune serum was used. Antibodies coated beads were incubated with the relevant cleared lysates for 4h at 4°C. After immunoprecipitations the beads were washed five times with ice-cold immunoprecipitation buffer for 2 min each. Input extracts and eluates of immunoprecipitations were used for Western and Northern blot analysis. The library preparation was described previously [31], shortly: RNA samples were purified by cutting the sRNA region from 8% denaturing polyacrylamide gels (acrylamide: bisacrylamide (19: 1) 1xTBE, 8. 6 M urea). After gel electrophoresis, the gels were stained by SYBRGold (Thermo Fisher Scientific). Bands at the small RNA range were cut out and crushed. Gel particles were shaken overnight in RNase free water at 4°C, followed by RNA isolation (described above). TruSeq Small RNA Sample preparation kit (Illumina) was used for library preparation; we followed the manufacturer’s protocol. In the case of the AGO1- and AGO2- immunoprecipitation 9 libraries were pooled together. In the case of the p19 immunoprecipitations, 4–4 libraries were pooled together. The libraries were sequenced on Illumina HiScanSQ platform (UD-GenomMed Medical Genomic Technologies Ltd., Debrecen, Hungary) that yielded approximately 100 M reads per lane (50bp, single end) (S10 Fig). RNA-seq library preparation was done according to the manufacturer protocol (TruSeqStranded mRNA Library Prep Kit). The libraries were sequenced on Illumina HiScanSQ. 135 M 100 bp paired-end reads were produced per lane. 3 samples were pooled together on a lane. The libraries were submitted to GEO and can be accessed through series accession number GSE77070. One hundred nanograms of synthetic sRNA (5’-UGAUUGAGCCGCGCCAAUAUC-3’) was 5’ end labeled by T4 polynucleotide kinase (Fermentas) with 32P isotope. After the reaction was stopped, 10 ng was saved for further process and the rest was mixed with 500 ng of unlabeled synthetic RNA with the sequence of 5’-UAUUGGCGCGGCUCAAUCAGA-3’. The mixture was heated to 95°C for 2 minutes in a thermocycler and was cooled to 5°C (2°/2 min) to gain 19 nt perfectly matched double strand with 2 nt protruding at the 3’ end. The sufficient amount of DNA loading dye was added to the mixture and to the saved labeled single stranded RNA. Both samples were run on a 8% acrylamide: bis-acrylamide 19: 1 1xTBE gel. Gel was directly exposed. The dsRNA region was cut from the gel. The gel piece was shredded using a 0. 5 ml tube with several holes in the bottom in a 2 ml tube via centrifugation. The shredded gel pieces were shaken overnight in 500 μl of 300 mM NaCl at 4°C. Gel pieces were filtered out by using Spin-X column (Corning). 400 μl of dsRNA solution was precipitated by adding 20 μg glycogen (Fermentas) and 1 ml ethanol. The precipitated ds-RNA was resolved in IP buffer described before. Purified p19 described earlier was used for the assay [56]. A dilution series of p19 (~1 μg) was made in 1x IP buffer. An equal amount of gel-purified ds-siRNA was added to each p19 dilution and incubated at room temperature for 10 min. Then 10 ng of RNase-A (Sigma) was added and incubated for 10 and 30 min at room temperature. After incubation the samples were mixed with DNA dye and ran on a 16% acrylamide: bis-acrylamide (19: 1) 1xTBE gel. Decade marker (Ambion) and synthetic RNAs were used as size markers. Gels were dried and were directly exposed. For band shift assays wild-type p19 protein was purified from E. coli as described previously [56,67]. Custom RNAs used were ordered from Dharmacon, (http: //dharmacon. gelifesciences. com) for sequence see Fig 4. Labeling and annealing of si/miRNA duplexes was carried out as described previously [67]. Purified p19 protein and labeled si/miRNAs were incubated for 30 min at room temperature in band-shift buffer [67]. Complexes were resolved on 8% polyacrylamide 0. 5xTBE gels. Gels were dried and exposed to a storage phosphor screen and bands quantified (Molecular Dynamics Typhoon Phosphorimager, GE Heathcare). In situ hybridization was performed as previously described in [87]. Detection of viral RNA expression patterns were made by using nonradioactive in situ hybridization on histological sections of leaf tissues. Digoxigenin labeled antisense RNA probe was synthesized by in vitro transcription from the linearized CymRSV construct. After demultiplexing of the raw data we used the UEA workbench version 3. 0 [88] for adapter removal. Quality control consisted of filtering out reads with less than 14 nt (without the adapter sequences) and reads showing low complexity. We used PatMaN v1. 2. 2. [89] to align reads allowing 0 mismatches. Reads not matching either genome were removed. Reads passing quality control is referred as “total” in this article. In Fig 3B reads were normalized to 1 million viral reads. In S7 Fig reads were normalized to 1 million N. benthamiana genome matching reads. In all other cases reads were normalized to 1 million total reads. After demultiplexing we used FastQC 0. 10. 1 to check general attributes. Trim_galore 0. 4. 1 and FASTX Toolkit 0. 0. 13 were used to remove adaptor sequences, low quality bases, reads under 20 nt and unpaired reads. Bowtie2 [90] was used to align reads to Nbv5 [91] transcriptome database. Reads were counted for homologs of known miRNA targets. NCBI-blast+ 2. 2. 28 [92] was used to validate miRNA targets. Samtools 0. 1. 19-96b5f2294a was used during alignment evaluation. Read counts were normalised to 1 million total reads.
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Title: Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants Summary: To better understand the specific effect of p19 viral suppressor of RNA silencing (VSR) on antiviral silencing and endogenous small RNA pathways, we generated a N. benthamiana plant (p19syn) capable of sustaining the ectopic expression of the Cymbidium ringspot virus (CymRSV) p19 upon infection with a suppressor-deficient CymRSV (Cym19stop). By using wt and p19syn plants in combination with CymRSV and Cym19stop, we were able to analyze the effects of p19 provided "in trans" and "in cis" during the viral invasion of the plant. We have shown that p19 can efficiently sequester endogenous small RNAs (sRNAs) in mock-inoculated p19syn plants while it does not bind these sRNAs upon Cym19stop infection. Also, the presence of p19 in virus infection did not alter the expression of miRNAs significantly. These findings do not support the widely accepted assumption that viral symptoms are the direct consequence of the impact of VSRs on endogenous silencing pathways. We demonstrated that p19 preferentially sequesters positive: negative viral short interfering RNAs (vsiRNAs) pairs and that the binding by p19 is independent of vsiRNA sequence or the type of the 5'-end nucleotide. We have also found that 3' truncation is induced on p19 bound sRNAs. Finally using AGO1- and AGO2- immunoprecipitation experiments we observed that p19 specifically compromises vsiRNAs' loading into AGO1 but not AGO2. Since antiviral silencing is strongly inhibited by p19, this suggests that AGO1 is the main effector protein against CymRSV tombusvirus.
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Summarize: The society out of which Cecil proposed to rescue Lucy was perhaps no very splendid affair, yet it was more splendid than her antecedents entitled her to. Her father, a prosperous local solicitor, had built Windy Corner, as a speculation at the time the district was opening up, and, falling in love with his own creation, had ended by living there himself. Soon after his marriage the social atmosphere began to alter. Other houses were built on the brow of that steep southern slope and others, again, among the pine-trees behind, and northward on the chalk barrier of the downs. Most of these houses were larger than Windy Corner, and were filled by people who came, not from the district, but from London, and who mistook the Honeychurches for the remnants of an indigenous aristocracy. He was inclined to be frightened, but his wife accepted the situation without either pride or humility. "I cannot think what people are doing," she would say, "but it is extremely fortunate for the children." She called everywhere; her calls were returned with enthusiasm, and by the time people found out that she was not exactly of their milieu, they liked her, and it did not seem to matter. When Mr. Honeychurch died, he had the satisfaction--which few honest solicitors despise--of leaving his family rooted in the best society obtainable. The best obtainable. Certainly many of the immigrants were rather dull, and Lucy realized this more vividly since her return from Italy. Hitherto she had accepted their ideals without questioning--their kindly affluence, their inexplosive religion, their dislike of paper-bags, orange-peel, and broken bottles. A Radical out and out, she learnt to speak with horror of Suburbia. Life, so far as she troubled to conceive it, was a circle of rich, pleasant people, with identical interests and identical foes. In this circle, one thought, married, and died. Outside it were poverty and vulgarity for ever trying to enter, just as the London fog tries to enter the pine-woods pouring through the gaps in the northern hills. But, in Italy, where any one who chooses may warm himself in equality, as in the sun, this conception of life vanished. Her senses expanded; she felt that there was no one whom she might not get to like, that social barriers were irremovable, doubtless, but not particularly high. You jump over them just as you jump into a peasant's olive-yard in the Apennines, and he is glad to see you. She returned with new eyes. So did Cecil; but Italy had quickened Cecil, not to tolerance, but to irritation. He saw that the local society was narrow, but, instead of saying, "Does that very much matter?" he rebelled, and tried to substitute for it the society he called broad. He did not realize that Lucy had consecrated her environment by the thousand little civilities that create a tenderness in time, and that though her eyes saw its defects, her heart refused to despise it entirely. Nor did he realize a more important point--that if she was too great for this society, she was too great for all society, and had reached the stage where personal intercourse would alone satisfy her. A rebel she was, but not of the kind he understood--a rebel who desired, not a wider dwelling-room, but equality beside the man she loved. For Italy was offering her the most priceless of all possessions--her own soul. Playing bumble-puppy with Minnie Beebe, niece to the rector, and aged thirteen--an ancient and most honourable game, which consists in striking tennis-balls high into the air, so that they fall over the net and immoderately bounce; some hit Mrs. Honeychurch; others are lost. The sentence is confused, but the better illustrates Lucy's state of mind, for she was trying to talk to Mr. Beebe at the same time. "Oh, it has been such a nuisance--first he, then they--no one knowing what they wanted, and everyone so tiresome." "But they really are coming now," said Mr. Beebe. "I wrote to Miss Teresa a few days ago--she was wondering how often the butcher called, and my reply of once a month must have impressed her favourably. They are coming. I heard from them this morning. "I shall hate those Miss Alans!" Mrs. Honeychurch cried. "Just because they're old and silly one's expected to say 'How sweet!' I hate their 'if'-ing and 'but'-ing and 'and'-ing. And poor Lucy--serve her right--worn to a shadow." Mr. Beebe watched the shadow springing and shouting over the tennis-court. Cecil was absent--one did not play bumble-puppy when he was there. "Well, if they are coming--No, Minnie, not Saturn." Saturn was a tennis-ball whose skin was partially unsewn. When in motion his orb was encircled by a ring. "If they are coming, Sir Harry will let them move in before the twenty-ninth, and he will cross out the clause about whitewashing the ceilings, because it made them nervous, and put in the fair wear and tear one.--That doesn't count. I told you not Saturn." "Saturn's all right for bumble-puppy," cried Freddy, joining them. "Minnie, don't you listen to her." "Saturn doesn't bounce." "Saturn bounces enough." "No, he doesn't." "Well; he bounces better than the Beautiful White Devil." "Hush, dear," said Mrs. Honeychurch. "But look at Lucy--complaining of Saturn, and all the time's got the Beautiful White Devil in her hand, ready to plug it in. That's right, Minnie, go for her--get her over the shins with the racquet--get her over the shins!" Lucy fell, the Beautiful White Devil rolled from her hand. Mr. Beebe picked it up, and said: "The name of this ball is Vittoria Corombona, please." But his correction passed unheeded. Freddy possessed to a high degree the power of lashing little girls to fury, and in half a minute he had transformed Minnie from a well-mannered child into a howling wilderness. Up in the house Cecil heard them, and, though he was full of entertaining news, he did not come down to impart it, in case he got hurt. He was not a coward and bore necessary pain as well as any man. But he hated the physical violence of the young. How right it was! Sure enough it ended in a cry. "I wish the Miss Alans could see this," observed Mr. Beebe, just as Lucy, who was nursing the injured Minnie, was in turn lifted off her feet by her brother. "Who are the Miss Alans?" Freddy panted. "They have taken Cissie Villa." "That wasn't the name--" Here his foot slipped, and they all fell most agreeably on to the grass. An interval elapses. "Wasn't what name?" asked Lucy, with her brother's head in her lap. "Alan wasn't the name of the people Sir Harry's let to." "Nonsense, Freddy! You know nothing about it." "Nonsense yourself! I've this minute seen him. He said to me: 'Ahem! Honeychurch,'"--Freddy was an indifferent mimic--"'ahem! ahem! I have at last procured really dee-sire-rebel tenants.' I said, 'ooray, old boy!' and slapped him on the back." "Exactly. The Miss Alans?" "Rather not. More like Anderson." "Oh, good gracious, there isn't going to be another muddle!" Mrs. Honeychurch exclaimed. "Do you notice, Lucy, I'm always right? I said don't interfere with Cissie Villa. I'm always right. I'm quite uneasy at being always right so often." "It's only another muddle of Freddy's. Freddy doesn't even know the name of the people he pretends have taken it instead." "Yes, I do. I've got it. Emerson." "What name?" "Emerson. I'll bet you anything you like." "What a weathercock Sir Harry is," said Lucy quietly. "I wish I had never bothered over it at all." Then she lay on her back and gazed at the cloudless sky. Mr. Beebe, whose opinion of her rose daily, whispered to his niece that THAT was the proper way to behave if any little thing went wrong. Meanwhile the name of the new tenants had diverted Mrs. Honeychurch from the contemplation of her own abilities. "Emerson, Freddy? Do you know what Emersons they are?" "I don't know whether they're any Emersons," retorted Freddy, who was democratic. Like his sister and like most young people, he was naturally attracted by the idea of equality, and the undeniable fact that there are different kinds of Emersons annoyed him beyond measure. "I trust they are the right sort of person. All right, Lucy"--she was sitting up again--"I see you looking down your nose and thinking your mother's a snob. But there is a right sort and a wrong sort, and it's affectation to pretend there isn't." "Emerson's a common enough name," Lucy remarked. She was gazing sideways. Seated on a promontory herself, she could see the pine-clad promontories descending one beyond another into the Weald. The further one descended the garden, the more glorious was this lateral view. "I was merely going to remark, Freddy, that I trusted they were no relations of Emerson the philosopher, a most trying man. Pray, does that satisfy you?" "Oh, yes," he grumbled. "And you will be satisfied, too, for they're friends of Cecil; so"--elaborate irony--"you and the other country families will be able to call in perfect safety." "CECIL?" exclaimed Lucy. "Don't be rude, dear," said his mother placidly. "Lucy, don't screech. It's a new bad habit you're getting into." "But has Cecil--" "Friends of Cecil's," he repeated, "'and so really dee-sire-rebel. Ahem! Honeychurch, I have just telegraphed to them.'" She got up from the grass. It was hard on Lucy. Mr. Beebe sympathized with her very much. While she believed that her snub about the Miss Alans came from Sir Harry Otway, she had borne it like a good girl. She might well "screech" when she heard that it came partly from her lover. Mr. Vyse was a tease--something worse than a tease: he took a malicious pleasure in thwarting people. The clergyman, knowing this, looked at Miss Honeychurch with more than his usual kindness. When she exclaimed, "But Cecil's Emersons--they can't possibly be the same ones--there is that--" he did not consider that the exclamation was strange, but saw in it an opportunity of diverting the conversation while she recovered her composure. He diverted it as follows: "The Emersons who were at Florence, do you mean? No, I don't suppose it will prove to be them. It is probably a long cry from them to friends of Mr. Vyse's. Oh, Mrs. Honeychurch, the oddest people! The queerest people! For our part we liked them, didn't we?" He appealed to Lucy. "There was a great scene over some violets. They picked violets and filled all the vases in the room of these very Miss Alans who have failed to come to Cissie Villa. Poor little ladies! So shocked and so pleased. It used to be one of Miss Catharine's great stories. 'My dear sister loves flowers,' it began. They found the whole room a mass of blue--vases and jugs--and the story ends with 'So ungentlemanly and yet so beautiful.' It is all very difficult. Yes, I always connect those Florentine Emersons with violets." "Fiasco's done you this time," remarked Freddy, not seeing that his sister's face was very red. She could not recover herself. Mr. Beebe saw it, and continued to divert the conversation. "These particular Emersons consisted of a father and a son--the son a goodly, if not a good young man; not a fool, I fancy, but very immature--pessimism, et cetera. Our special joy was the father--such a sentimental darling, and people declared he had murdered his wife." In his normal state Mr. Beebe would never have repeated such gossip, but he was trying to shelter Lucy in her little trouble. He repeated any rubbish that came into his head. "Murdered his wife?" said Mrs. Honeychurch. "Lucy, don't desert us--go on playing bumble-puppy. Really, the Pension Bertolini must have been the oddest place. That's the second murderer I've heard of as being there. Whatever was Charlotte doing to stop? By-the-by, we really must ask Charlotte here some time." Mr. Beebe could recall no second murderer. He suggested that his hostess was mistaken. At the hint of opposition she warmed. She was perfectly sure that there had been a second tourist of whom the same story had been told. The name escaped her. What was the name? Oh, what was the name? She clasped her knees for the name. Something in Thackeray. She struck her matronly forehead. Lucy asked her brother whether Cecil was in. "Oh, don't go!" he cried, and tried to catch her by the ankles. "I must go," she said gravely. "Don't be silly. You always overdo it when you play." As she left them her mother's shout of "Harris!" shivered the tranquil air, and reminded her that she had told a lie and had never put it right. Such a senseless lie, too, yet it shattered her nerves and made her connect these Emersons, friends of Cecil's, with a pair of nondescript tourists. Hitherto truth had come to her naturally. She saw that for the future she must be more vigilant, and be--absolutely truthful? Well, at all events, she must not tell lies. She hurried up the garden, still flushed with shame. A word from Cecil would soothe her, she was sure. "Cecil!" "Hullo!" he called, and leant out of the smoking-room window. He seemed in high spirits. "I was hoping you'd come. I heard you all bear-gardening, but there's better fun up here. I, even I, have won a great victory for the Comic Muse. George Meredith's right--the cause of Comedy and the cause of Truth are really the same; and I, even I, have found tenants for the distressful Cissie Villa. Don't be angry! Don't be angry! You'll forgive me when you hear it all." He looked very attractive when his face was bright, and he dispelled her ridiculous forebodings at once. "I have heard," she said. "Freddy has told us. Naughty Cecil! I suppose I must forgive you. Just think of all the trouble I took for nothing! Certainly the Miss Alans are a little tiresome, and I'd rather have nice friends of yours. But you oughtn't to tease one so." "Friends of mine?" he laughed. "But, Lucy, the whole joke is to come! Come here." But she remained standing where she was. "Do you know where I met these desirable tenants? In the National Gallery, when I was up to see my mother last week." "What an odd place to meet people!" she said nervously. "I don't quite understand." "In the Umbrian Room. Absolute strangers. They were admiring Luca Signorelli--of course, quite stupidly. However, we got talking, and they refreshed me not a little. They had been to Italy." "But, Cecil--" proceeded hilariously. "In the course of conversation they said that they wanted a country cottage--the father to live there, the son to run down for week-ends. I thought, 'What a chance of scoring off Sir Harry!' and I took their address and a London reference, found they weren't actual blackguards--it was great sport--and wrote to him, making out--" "Cecil! No, it's not fair. I've probably met them before--" He bore her down. "Perfectly fair. Anything is fair that punishes a snob. That old man will do the neighbourhood a world of good. Sir Harry is too disgusting with his 'decayed gentlewomen.' I meant to read him a lesson some time. No, Lucy, the classes ought to mix, and before long you'll agree with me. There ought to be intermarriage--all sorts of things. I believe in democracy--" "No, you don't," she snapped. "You don't know what the word means." He stared at her, and felt again that she had failed to be Leonardesque. "No, you don't!" Her face was inartistic--that of a peevish virago. "It isn't fair, Cecil. I blame you--I blame you very much indeed. You had no business to undo my work about the Miss Alans, and make me look ridiculous. You call it scoring off Sir Harry, but do you realize that it is all at my expense? I consider it most disloyal of you." She left him. "Temper!" he thought, raising his eyebrows. No, it was worse than temper--snobbishness. As long as Lucy thought that his own smart friends were supplanting the Miss Alans, she had not minded. He perceived that these new tenants might be of value educationally. He would tolerate the father and draw out the son, who was silent. In the interests of the Comic Muse and of Truth, he would bring them to Windy Corner.
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Summary: Chapter 10 explains some of the history of the Honeychurch family. Lucy's father had been a prosperous local lawyer who built Windy Corner before the area had been developed. When wealthy Londoners moved to the area and built their mansions, they mistook the Honeychurches for country aristocrats instead of the nouveau riche they really were, and therefore, Lucy had been accepted into the higher society of the area. Now after having been to Italy, Lucy sees that this society is rather narrow, but she still has a great affection for it, and cannot despise it. Cecil wants to introduce her to broader society, but fails to see that Lucy is beyond that; all she really wants to find is a soul mate, an equal partner. Lucy and Minnie Beebe play bumble-puppy, a silly game with tennis balls, while talking to Mr. Beebe and Mrs. Honeychurch about the Miss Alans, who are planning to move into Cissie Villa. Meanwhile, Cecil stays inside, to avoid being hurt as the game deteriorates into roughhousing. Freddy announces that it is not the Miss Alans, but a family called Emerson, who are going to live in the villa. Lucy is shocked to find that the Emersons are none other than George and his father, who have been invited by Cecil as a joke on Sir Harry, as Cecil thought them very vulgar. Cecil claims that he believes in democracy and wants to punish a snob, but Lucy is angry that he has spoiled her work in inviting the Miss Alans and made her look bad.
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Summarize: Il 13 aprile 1967, a Londra si teneva la premiere della parodia più riuscita della storia relativa alla saga di 007. “James Bond 007 – Casino Royale”, diretto dai registi John Huston, Kenneth Hughes, Val Guest, Robert Parrish e Joseph McGrath è basato sul primo romanzo di Ian Fleming e vede Davdi Niven nei panni di Sir James Bond, ormai ritiratosi dall’attività di spia al servizio di Sua Maestà, fino a quando alcuni rappresentanti dell’M16, della CIA e del KGB gli fanno visita per pregarlo di tornare, vista la minaccia rappresentata dall’organizzazione SMERSH. Bond non ci pensa proprio, anche perchè si è ritirato in campagna per amore della sua Mata Hari, dalla quale ha avuto la figlia Mata Bond (Joanna Pettet). Ma quando la sua residenza sarà distrutta, si recherà in Scozia, diventando il capo dell’M16 e, facendosi aiutare proprio da sua figlia e da Vesper Lynd (Ursula Andress) e dal giocatore esperto Evelyn Tremble (Peter Sellers), riuscirà a sventare i piani di Le Chiffre (Orson Welles), battuto anche al tavolo verde del Casino Royale. Man mano, si saprà che a capo della SMERSH c’è il Dr. Noah, ossia Jimmy Bond (Woody Allen), nipote smidollato di 007 che ha sgangherati e diabolici piani. Basato su una sceneggiatura scritta da Wolf Mankowitz, John Law, Michael Sayers, la pellicola è diventata un cult, soprattutto, per la presenza del cast davvero stellare, che presenta ingredienti ben congegnati, mescolando alla perfezione l’eleganza di David Niven, l’ironia di Peter Sellers e di un inedito e giovanissimo Woody Allen e le originali performance di icone come Orson Welles, John Huston (M), Deborah Kerr (Agente Mimì), William Holden (Ransome), Charles Boyer (le Grand), per finire con la partecipazione di Barbara Bouchet (Moneypenny). Pur trattandosi di una palese satira o parodia di uno dei franchise più longevi e famosi al mondo, il film è estremamente curato sotto il profilo scenografico, musicale (la colonna sonora è stata composta da Burt Bacharach e il pezzo “The Look of Love” è stato candidato agli Oscar del 1968), degli effetti speciali e delle scene d’azione, anche se un tantino danneggiato dalla regia a più mani che ne da un aspetto frammentario.
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Summary: La premiere della parodia più riuscita della storia della saga di 007, si teneva a Londra, esattamente il 13 aprile del 1967. Con un cast fatto di star del calibro di David Niven, Peter Sellers, Woody Allen, Orson Welles, il film è pieno di scene surreali, ingegnose e divertenti, ma estremamente curato sotto il profilo tecnico e musicale, e ciò ne fa un cult assoluto.
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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to athletic boots and more particularly it concerns a novel, molded athletic boot construction wherein an insert of cloth-like material is embedded or encapsulated in the material of the boot to provide strength and rigidity in selected regions without adding significantly to the weight or thickness of the boot material. The novel boot construction of the present invention is especially suited for ski boots and skate boots, such as boots for hockey skates. 2. Description of the Prior Art Athletic boots have previously been molded of synthetic materials such as polyurethane rubber. These molded boots require additional support in certain regions of the boot to provide sufficient stiffness and rigidity. Accordingly, some prior art molded boots had regions that were built up in thickness to provide this needed extra support. Such construction made the boot excessively heavy and bulky. Other prior art molded boots used a separate insert element made from a rigid plastic, mechanically fastened within a molded outer shell. One arrangement for fastening the insert element to the outer shell is shown in U.S. Pat. No. 3,807,062. According to that patent apertures are formed at selected locations in the insert element and the outer shell is molded around the insert element so that the material of the outer shell flows into the apertures to lock the insert and shell together. This arrangement suffers from the disadvantage that the reinforcing insert applies maximum stiffness adjacent the foot of the wearer and therefore it is uncomfortable to wear. Also the mechanical interlocks between the boot and the insert are subject to very high stresses. Moreover, the strain produced in the boot shell is not evenly transmitted to the insert. SUMMARY OF THE INVENTION The present invention avoids the disadvantages of the prior art and provides easily manufactured molded athletic boots having great tensile strength and rigidity in precisely predetermined regions thereof without localized stress concentrations or the tendency to pull apart. According to one aspect of the present invention, there is provided a novel athletic boot comprising a molded outer shell shaped to encase a human foot, with a porous insert of a cloth-like material embedded in and impregnated by the material of the outer shell and interlocked therewith. The material of the insert has a greater elastic modulus and higher strength than the outer shell; and it cooperates with the outer shell to provide increased rigidity and strength in the regions where the insert is located. According to another aspect of the invention, there is provided a novel boot molding process in which a porous sock-like insert assembly is slipped over a last which in turn is encased within a mold. A castable material, such as polyurethane is then injected into the mold to fill the mold interior about the last and at the same time to impregnate and encapsulate the insert assembly. The insert assembly shifts slightly off the last and thus becomes embedded in and integrated into the material of the molded boot shell to provide strength and rigidity. According to a further aspect of the present invention, there is provided a novel insert assembly for use in molded athletic boots. This insert assembly comprises a highly porous elastic sock-like holding and positioning member within which is contained one or more insert elements of porous material of high elastic modulus. These insert elements are secured to predetermined locations within the holding and positioning member by sewing, gluing, tack welding or the like. The holding and positioning member slips over the last and holds the insert elements in proper location during the mold filling operation; and yet it permits the insert elements to drift away, slightly, from the mold so that the insert elements become fully embedded in the boot material. There has thus been outlined rather broadly the more important features of the invention in order that the detailed description thereof that follows may be better understood and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject of the claims appended hereto. Those skilled in the art will appreciate that this invention may be utilized as a basis for designing other structures or methods for carrying out the several purposes of this invention. It is therefore important that the claims be regarded as including such equilvalent constructions and methods as do not depart from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Two embodiments of the invention have been chosen for purposes of illustration and description and are shown in the accompanying drawings forming a part of the specification, wherein: FIG. 1 is a perspective view of a molded boot shell in which the present invention is embodied; FIG. 2 is a section view, taken along line 2--2 of FIG. 1; FIG. 3 is a further section view, taken along line 3--3 of FIG. 2; FIG. 4 is an exploded perspective view showing the positioning of an insert assembly on a last as a preliminary step in the molding of the boot shell of FIG. 1; FIG. 5 is an exploded perspective view showing the components of the insert assembly of FIG. 4; FIG. 6 is an exploded perspective view showing the positioning of a last, with insert assembly attached, within a mold, as a further step in the molding of the boot shell of FIG. 1; FIG. 7 is a side elevation section view of the mold of FIG. 6 closed about the last with insert assembly secured thereto; FIG. 8 is a view similar to FIG. 7 but showing the mold filled with a castable boot forming material; and FIG. 9 is a fragmentary section view showing an alternate construction of the molded boot shell of FIG. 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As shown in FIG. 1 there is provided a hockey skate boot shell 10 of generally shoe shaped configuration. The shell 10 is formed with a generally flat sole 20 to which a skate blade (not shown) is later attached. The boot shell 10 is also formed with a hardened toe region 22 which provides protection against impact from sticks, skates and pucks during hockey play. An open instep region 24 is formed on the boot shell 10 and eyelets 26 are provided along each side for lacing the boot tightly to the foot. The boot shell 10 is also provided with a top opening 28 which surrounds the ankle of the wearer. In most cases a tendon and ankle guard (not shown) is attached to the sides of the boot shell just below the top opening 28 to extend up and protect the tendons and ankles of the wearer. The boot shell 10 is cast or molded of a synthetic material such as polyurethane rubber. One suitable polyurethane rubber is available from E. I. du Pont de Nemours & Co. Inc., Wilmington, Del., under the trademark Adiprene. The urethane material has sufficient elasticity to conform closely to the foot of the wearer when the boot 10 is tightly laced. Also this material provides a high degree of scuff and wear resistance. In skating, as in other sporting activities, the footwear used becomes subjected to especially high stresses at various portions of the footwear; and these stresses act in various directions, depending upon the particular sport involved. In hockey, for example, the skate boot must resist stresses which are caused on the one hand by forces imposed by the foot, and on the other hand by reactive forces imposed by the skate blade. These forces produce tensile stresses in the plane of the sole of the boot as well as torque stresses about axes perpendicular to this plane. In order to resist these stresses, and to avoid undue stretching as a result of them, the boot sole must have a high elastic modulus, i.e. a high rigidity in its own plane. Although rigidity can be increased to a certain extent by increasing the thickness of the sole, this results in a great expense and an undesirable increase in overall weight of the boot. As will be seen herein, the present invention makes it possible to increase the elastic modulus and rigidity along the plane of the boot sole to a substantial degree without appreciably increasing the weight or bulk of the boot. The toe region of a hockey skate encounters a different type of stress, namely, that produced by impact from sticks, pucks and other skates. Consequently the toe region must be reinforced against bending stresses in localized regions. Again, the present invention, as explained below, provides localized reinforcement against bending and denting without undue increase in weight or thickness. Turning now to FIGS. 2 and 3 it will be seen that the skate boot shell 10 is composed primarily of an outer molded portion 30 within the material of which is embedded a multiple layer insert assembly made up of an outer sock-like holding and positioning layer 32 a woven layer 34 and a nonwoven layer 36. As indicated above, the outer portion 30 is preferably of a polyurethane rubber such as that available under the trademark Adiprene. The outer portion 30 may also be of a polyether, polyester or polycaprolactam type polyurethane. In order to achieve variations in tensile strength, rigidity and elastic elongation up to a certain degree in various regions, the thickness of the outer portion 30 is varied. However, in the sole region 20 and in the toe region 22 additional reinforcing is provided according to the present invention by means of the cooperative effect of the woven and nonwoven layers 34 and 36. Both the woven and nonwoven layers 34 and 36 are of a polyamide fiber such as nylon, preferably the material sold under the trademark Kevlar, by E. I. du Pont de Nemours & Co. Inc., Wilmington, Del. Other materials which may be used include nylon-11, nylon-12 and nylon-13. The woven layer 34 extends over and around the toe region 22 and along the length of the sole region 20. This woven layer is made up of fibers which extend along the plane of the toe and sole regions. Because of the porosity of the woven layer 34, the layer is impregnated with and is encapsulated within the material of the outer shell 30; and so it becomes an integral part of the outer shell. As a result, the elastic modulus of the material of the woven layer 34, which is substantially higher than the elastic modulus of the outer shell material 30, provides a high overall elastic modulus in the plane of the sole region 20 and in the plane of the toe region 22 which enables these regions to withstand high tensile stresses without appreciable stretching. It has been found, in fact, that the stretching of the polyurethane to its ultimate strength can be reduced from 200 percent to less than 5 percent by the use of the layers 34 and 36. This provides resistance to shearing forces and torque forces imposed by the skate blade during use. The nonwoven layer 36 may be of the same material as the woven layer 34. However, the nonwoven layer is felted or matted with the fibers thereof being oriented randomly. It will also be noted that the nonwoven layer is much thicker than the woven layer 34. This thickness serves to provide resistance to bending or denting when it is impregnated with the material of the outer portion 30. Thus, a cooperative effect is produced between the outer shell material 30 which impregnates and maintains the spacing of the fibers of the nonwoven layer 30 and the material of the individual fibers themselves whose tensile strength in their spaced arrangement produces a resistance to bending. The degree of this bending resistance depends upon the thickness or bulk of the nonwoven layer. This bulk should be such that the nonwoven layer occupies 40-60 percent, preferably 45-55 percent of the overall volume of the boot region containing it. As can be seen in FIG. 3, the layers 34 and 36 extend up over the top of the toe region 22 of the boot to provide strength and rigidity over this entire portion of the boot. However, the layers 34 and 36 extend back from the toe region only along the sole region 20 to strengthen it. The remainder of the boot contains no reinforcing material since the particular requirements for strength and rigidity in those other regions do not call for such reinforcement. Moreover, it is important that those other regions remain flexible to accommodate normal foot movements during skating. Of course, either or both the layers 34 and 36 may be extended or repositioned to different boot regions depending upon the particular requirements of the boot. Thus, ski boots would have reinforcing layers arranged at different locations than those shown herein for skate boots. The outer sock-like holding and positioning layer 32 performs no appreciable reinforcing or strengthening function. Its purpose instead is to hold the other layers in place during the molding of the boot shell. This outer layer is of highly porous elastic material such as stretch nylon. It should not include any material which is incapable of withstanding the high temperature encountered during the molding of the material of the outer shell, nor should it include any material, such as latex which would weaken the outer shell. It will be noted from FIG. 2 that the layer 32 includes a band-like side portion 32a which extends around the sides of toe and heel of the boot 10 and extends up about an inch 20 the sole region 20. although this may vary according to type and size of boots. The layer 32 also includes a bottom portion 32b which extends under the woven layer 34. The side portion 32a is preferably formed of a material comprising a polyurethane core wrapped with stretch nylon. One such material is sold under the trademark Lycra by E. I. du Pont de Nemours & Co. Inc., Wilmington, Del. The bottom portion 32b may be entirely of stretch nylon. In order to allow free flow of polyurethane material during a casting or molding operation, the layer 32 should have a porosity of about 95 percent. FIG. 4 shows an insert assembly 38 about to be positioned on a last 40 as a preliminary step to the molding of the skate boot shell 10 of FIG. 1. The last 40 is shaped in accordance with the human foot; and, of course, its size will depend upon the size of the boot shell to be formed. The insert assembly 38 appears in FIG. 4 as a fabric sock somewhat smaller than the bottom of the last 40. However, the outer holding and positioning layer 32 is of stretch material so that the assembly can be stretch fitted over the last 40 and it will remain in position thereon during subsequent steps. As shown in the exploded view of FIG. 5, the insert assembly 38 comprises the outer holding and positioning member 32 with the woven and nonwoven layers 34 and 36 affixed therein. While the shape of the outer holding and positioning member 32 is such as to fit over the last 40, the shape of the woven and nonwoven layers 34 and 36 depends only upon the particular location in the boot shell where reinforcing is to be provided and upon the type of reinforcing required. It will be appreciated that since the reinforcing material does limit the flexibility of the outer shell material to a certain extent, it is not advantageous to provide the reinforcing throughout the entire shell. The inner layers 34 and 36 may be tack stitched, glued or otherwise held in position to the outer holding and positioning layer 32. Turning now to FIG. 6 it will be seen that the last 40, after being fitted with the insert assembly 38, is positioned to be encased by outer mold parts 42 and 44 which come together about the last to define the extend configuration of the boot shell. As can be seen in FIGS. 6 and 7, the mold parts 42 and 44 are formed with port of entry 46 and sprue and vent passages 48 in the vicinity of the heel of the boot so that moldable material may be poured under pressure into the interior regions of the mold surfaces to displace the air therein. In order to insure complete evacuation of air and penetration of moldable material, a bottom filling mold is desirable. It is also preferable, in some instances, to form the mold with additional secondary ports (not shown) to the mold cavity leading from a common port of entry 46 along the sole of the boot. It will further be noted in FIG. 7 that the insert assembly 38, although elastically held to the last 40 is somewhat loose so that the woven and nonwoven layers 34 and 36 may pull away slightly from the last to allow the moldable material to encapsulate these layers without, however, allowing them to shift laterally from their desired locations. Turning now to FIG. 8 it will be seen that when moldable polyurethane or similar material 50 is injected into the mold, it impregnates the entire insert assembly 38 including each of the layers 32, 34 and 36. While the initial inflow of the material 50 may tend to push the insert assembly 38 against the last 40, it so happens that the continued flow of the material 50 through the mold eventually causes the insert assembly 38 to pull away slightly from the last 40. As a result only the outer holding and positioning layer 32 contacts the last 40; and this contact occurs only along the outermost edge of the layer 32. As a result the woven and nonwoven reinforcing layers become fully encapsulated and therefore integrated into the finally molded boot. After the injected material 50 solidifes, the mold parts are taken apart and the molded boot shell 10, with the insert assembly 38 encapsulated therein, is removed from the last 40. It will be appreciated that the preformed insert assembly 38, with different woven and nonwoven layers positioned therein, makes it possible to provide various types and amounts of reinforcement at any desired location within a molded boot without complex manual operations. One variation made possible by the present invention is illustrated in FIG. 9 wherein a boot 52 is formed with a nonwoven reinforcing layer 54 which extends only throughout its toe region 56 and a woven type reinforcing layer 58 extending throughout both its toe region 56 and its sole region 60. The nonwoven layer 54 and the woven layer 58 are both held in their proper locations by means of an elastic, porous outer holding and positioning layer 62 to which they are sewn or glued. Having thus described the invention with particular reference to the preferred forms thereof, it will be obvious to those skilled in the art to which the invention pertains, after understanding the invention, that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the claims appended hereto.
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Summary: A molded athletic boot, a reinforcing insert assembly for a molded athletic boot and a method of making the boot are described. A cloth-like insert is mounted on a last and a mold is then closed about the last. A castable material, such as polyurethane is injected into the mold so that it impregnates and encapsulates the insert prior to solidifying to form an athletic boot having great tensile strength and rigidity. The described insert assembly comprises a porous, elastic, sock-like element which fits over the last. A layer of a woven or knitted polyamide and a layer of a nonwoven polyamide are secured inside the sock-like element at selected locations.
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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to international application PCT/DE2006/001039, filed Jun. 20, 2006 and German application DE 10 2005 045 006 filed on Sep. 21, 2005. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention generally relates to the area of medicine in the surgical field. Containers are used in surgery for application tools for applying objects. Some containers are especially suitable for holding special objects, in particular hemostatic clamps. Hemostatic clamps are also referred to as ligature clamps. Ligature clamps are used to tie or clamp blood vessels during surgical procedures, and kept in containers. These containers usually consist of holders, cassettes or magazines. The invention relates to such a magazine. [0004] 2. Related Art [0005] Ligature clamps have been used in surgery for many years. They serve two basic functions, specifically tying vessels, nerves or fluid-carrying vessels of the human body, or marking a special surgical site, typically the periphery of a tumor, for example, which can later be seen in an X-ray. The ligature clamps (hereinafter referred to as clamps) are available in three basic sizes-small, medium and large, as well as in two special sizes combination small/medium and impervious to X-rays. Clamps used early on were V-shaped. However, the disadvantage to clamps shaped like this is that they pressed the vessel away from the clamp or cut into the vessel instead of tying it off. This problem was resolved through the use of pre-shaped, horseshoe-shaped clamps that were described in U.S. Pat. Nos. 3,323,216 and 3,363,628 and are currently in general use. The horseshoe-shaped clamp consists of two essentially parallel legs, which initially fix the vessel between the ends when closed, thereby holding it between the actual ligature between the ends. [0006] In a typical magazine for holding and providing clamps in the prior art, a plurality of side-by-side clamps, e.g., ten, are pressed onto a rigid middle beam or pillow block. The outer shape of the pillow block corresponds to the inner shape of the horseshoe-shaped clamps. However, the clamps are somewhat smaller than the pillow block, so that the contact pressure between the parallel legs of the clamp and the pillow blocks holds the clamps during transport and prevents the clamps from falling out of the magazine if the magazine is turned or dropped. But this contact pressure is associated with several disadvantages. First of all, the contact pressure makes it hard to grip the clamp via the clamping jaws or branches of an application tool, in particular the special forceps. Secondly, it makes removal a very unsafe process, even though the application tool has two branches that can move relative to each other, the free ends of which each carry longitudinal grooves on the facing sides that run parallel to the branches and are open relative to the opposing branch. Application tools with a narrow clamping jaw width make it more difficult to grip the clamp, especially since the clamp already tightly abuts the pillow block, and therefore cannot be compressed. This significantly increases the friction between the application tool and the clamp, and metal might be shaved off the clamp. If the clamping jaws of the application tool are too wide, the clamp is sometimes not reliably gripped by the clamping jaws after removed from the magazine. As a result, the clamp might slip from the operating position inside the application tool or fall out of the application tool. Another disadvantage is that the contact tension makes it difficult to remove the clamp from the holding device of the magazine. The tight abutment between the clamp and pillow block generates significant friction between the pillow block and clamp, and the relatively small magazine must be held to enable removal. The required handling of the clamp and magazine is associated with additional disadvantages. [0007] Further developments in prior art are intended to eliminate the above disadvantages. For example, U.S. Pat. No. 4,936,447 discloses a cassette for holding ligature clamps, in which the downwardly open end of the ligature clamps is plugged into a free compartment via a nose saddle, and both sides of its outer contour are held by a respective elastic holding device provided with a notch at the end. The holding device is designed with a special insert, which must additionally be connected with the basic body of the cassette. The notches on the holding device must reliably grip the ligature clamps, but easily release them again during removal by means of an application tool provided for this purpose. In the known cassette, the holding devices must be manufactured to exacting tolerances with respect to dimensions and resilience. They are also only suitable for mounting a single type of ligature clamp. Even if this magazine embodiment makes it possible to eliminate the aforementioned friction disadvantages, there remains the problem that the application tool and retaining straps disrupt each other, since these abut the arms of the clamps, which happen to form the areas into which the forceps reaches. This is the cause of disruptions. Another serious disadvantage to this cassette is that it has no guide for the application tool for removing the ligature clamps. Therefore, disadvantages are associated with handling when gripping the ligature clamps, i.e., the ligature clamp is not optimally situated in the application tool, so that mistakes can be encountered when tying off the vessels. [0008] In the magazine described in WO 98/05260 A1 and the publications cited therein, each clamp sits astride a fixed element (reverse position with “U” turned upside down) with a complementary shape, which is seamed by a pair of massive transverse walls used to hold the clamps. The clamps incorporated in such a magazine are in part very small, e.g., the dimensions measure on the order of 1.5 mm, making it extremely difficult to handle the clamps. In the known magazines, the clamps are held in such a way that a forceps-like application tool can grasp the clamps when inserted into a magazine, and then pull them out of the magazine. The disadvantage to known magazines here is that the clamps are fixed laterally by elastically resilient clamping elements, which only abut the middle area of the web, and are lifted during the insertion of an application tool. Therefore, the clamps might become tilted or even jammed in the magazine while inserting the application tool, then making it impossible to introduce the clamp into the application tool in the manner envisaged. [0009] Another problem is encountered in holding devices in clamp magazines according to prior art. In order to ensure that the application tool encompasses the clamps, the clamping jaws of the application tool must be stronger than the clamps. To allow the application tool to reach into the clamp compartment, this compartment must therefore be wider than the clamp. As a result of the wider clamp, the clamp can be varyingly positioned in the compartment as the application tool enters, and not be centered, so that the clamp is not gripped precisely by the middle of the application tool. A clamp not correctly gripped by the clamping jaws of the application tool can cause the clamp to close unevenly. The principle for removing the clamps from a magazine is identical for all known clamp sizes. The magazines for the varying clamp sizes are color-labeled for differentiation purposes. For example, the magazine accompanying the small clamp size is yellow, while it is blue for medium and orange for large. [0010] There is a need therefore, for an inexpensive container with a simple functional geometry to hold hemostatic clamps, with elevated demands on safe storage and removal, and hence on tying off vessels, exhibiting a structural shape suitable for reliably removing clamps via a surgical instrument. SUMMARY OF THE INVENTION [0011] In order to create a container exhibiting these features of the invention for holding hemostatic clamps, in particular ligature clamps for surgery, the invention proposes that a container be designed in such a way that the clamps in the container are completely and clearly held without tilting, in particular when introducing a surgical instrument (hereinafter referred to as application tool) into the container. The invention is a container that consists of two components for holding a plurality of storable and retrievable objects, in particular ligature clamps, of which each is formed in a specific mold and can be gripped by an application tool. The two components consist of a lower and upper part. The lower part is fitted with a plurality of clamp compartments, of which each contains at least one lateral wall for bordering the clamping elements and a pillow block for loosely storing the clamp. The upper part is provided with a plurality of clamping elements for holding the clamp in each of the clamp compartments, wherein a clamping element consists of two movable retaining means, which envelope the pillow block in each of the clamp compartments, and of which each retaining means has a contact section that holds the respective clamp on the pillow block. [0012] The device according to the invention is used in practice as described below. This description is not to be construed as a limitation, but rather serves to describe the general principle underlying the invention. [0013] In general, this invention has a container consisting of a lower and upper part, wherein the lower part represents the retaining device. The basic part is provided with a plurality of clamp compartments, advantageously with six to ten compartments, each of which can hold a prefabricated ligature clamp. Therefore, each compartment has a pillow block that is dimensioned in such a way as to roughly correspond to the interior dimensions of a ligature clamp, and holds the clamp loosely in the clamp compartment. In order to prevent the clamp from being held by friction, the pillow block has a channel according to the invention. The channel is arranged in the transverse direction to the pillow block and formed on the outer contour of the pillow block, or on the middle line of the pillow block, wherein the middle line of the pillow block is simultaneously the middle line of the clamp compartment. Another advantage to the channel is that the clamp can be centered when fitting the basic part onto the pillow block. This is also necessary because the clamp compartment with pillow block contained therein is larger than the ligature clamp. The size of the clamp compartment is determined by the size of the application tool to be used, which has to be larger in order to grasp the ligature clamp. [0014] Therefore, the application tool determines the size of the clamp compartment, and the channel determines the location of the clamp on the pillow block, and hence in the clamp compartment. If the basic part of the container is fitted with one clamp per clamp compartment in the clamp compartment, which may also occur automatically as a result of the simple and advantageous construction of the basic part, e.g., with a automatic pick and place machine known from SMD technology, the basic part is fabricated with the retaining device. [0015] During fabrication, the webs and separating walls arranged on the basic part according to the invention pass through openings situated in the retaining device, wherein the retaining device can be secured to the basic part in various sways, e.g., via bonding or latching on connecting pins projecting at the corners of the upper side of the basic part, wherein the retaining device has the correspondingly necessary holes at the corners. As an alternative, the pins may also be arranged on the lower side of the retaining device, and the holes in the basic part. Other fastening modes or connections between the basic part and retaining device are within the scope of the invention. [0016] As evident from one embodiment of the container, the clamp centered in the chamber of the pillow block of the basic part is held by a clamping element, wherein the clamping element is formed out of two retaining means per clamp compartment, which envelop the pillow block in the fabricated state. To this end, the retaining means is U-shaped. The clamping element is hence formed out of a pair of retaining means, and holds the clamp between the latter two. As a result, the clamp is clamped from both sides. Clamping takes place via the retaining means in the contact area that are fitted with a latching nose. The latching nose is located at the vertex of the retaining means, and points in the direction of the clamp. In order for the clamp attached in the channel of the pillow block to yield a detachable clamping, the U-shaped retaining means are flexibly arranged on the retaining device. The flexibility stems from the fact that the legs of the retaining means are relatively thin, and secured to the retaining device only at their ends, yielding a certain mobility of the retaining means. Because the U-shaped strap of the retaining means is inclined, the material causes a specific tension (spring force) to be exerted on the clamp inserted in the channel. [0017] To advantageously remove a clamp from a clamp compartment of a container, the basic part according to the invention is fitted with webs arranged on both longitudinal sides of the basic part on the one hand, and the pillow blocks located in the clamp compartment are provided with a pedestal on the other. The webs are used by the application tool to guide the clamping jaws, while the pedestals are used by the front surfaces of the application tool as a solid stop. [0018] A clamp is removed with an application tool by inserting the application tool from above between the webs of a clamp compartment on the one hand, and between the two retaining means of a clamping element on the other. The inclined guiding surface on the webs according to the invention leads the application tool against the U-shaped straps of the retaining means, during which the retaining means are forced apart, and the application tool is fixed against the clamp. The application tool reaches its end position once the front surfaces of the two clamping jaws hit the stop of the pedestal, i.e., the insertion depth of the application tool is limited by the stop formed by the pedestal on the pillow block. Once the application tool has been inserted, the clamping jaws of the application tool are optimally situated relative to the clamp. The two legs of the clamps are defined in this position of the application tool in the channels of the two clamping jaws. The leg ends of the clamp are now flush with the ends of the clamping jaws of the application tool. The webs of the U-shaped retaining means according to the invention and the pedestal stop on the pillow bearing ensures a frictionless removal of the clamp from the pillow block, and no friction comes about when introducing the application tool between the chamber and clamping jaws either. [0019] An exemplary embodiment of the invention is depicted in purely a schematic form in the drawings, and will be described in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS [0020] FIG. 1 is a perspective view of a container for holding a plurality of hemostatic clamps, and [0021] FIG. 2 is a perspective view of the basic part, and [0022] FIG. 3 is a perspective view of the retaining device, and [0023] FIG. 4 is a sectional view A-A through the container on FIG. 1 DETAILED DESCRIPTION [0024] FIG. 1 shows a perspective view of a container 1 for ligament clamps 12, consisting of a lower base 2 and upper 3 part, wherein the lower base part 2 represents the basic part and the upper part 3 represents the retaining device. The basic part 2 is fitted with a plurality of clamp compartments 4 (see also FIG. 2 ), of which each has at least one lateral wall 5 for limiting the clamping element 7 and a pillow block 10 for loosely storing the clamps 12, wherein the pillow block 10 matches the contour 11 of the clamp 12. In addition, the basic part 2 is provided with a plurality of webs 14, wherein each clamp compartment 4 accommodates two webs 14, 14 ′, and the webs 14, 14 ′ are used to guide the application tool 15 (see FIG. 4 ) for reliably removing the clamp 12. The webs 14, 14 ′ are arranged on the longitudinal sides 16, 16 ′ of the basic part 2 at a specific distance X in the longitudinal direction 17 relative to each other, and at a specific distance Y in the transverse direction 18 of the transverse side 19 relative to the pillow block 10. This arrangement of the webs 14, 14 ′ in a longitudinal 17 and transverse 18 direction yields two webs 14, 14 ′ per clamp compartment 4, which are centrally arranged relative to the clamp compartment 4, and located on the middle line Z of the transverse direction 18 relative to the pillow block 10. The middle line Z in the area of the pillow block 10 incorporates a channel 13 in its outer contour 11. The channel 13 is used to center the clamp 12 on the pillow block 10, and is adjusted to the inner contour 9 of the clamp 12 (not shown). The clamp 12 is loosely accommodated in the channel 13. The height of the pillow block 10 and height of the lateral wall 5 may correspond to the height of the webs 14, 14 ′. The basic part 2 may be one-piece, and can be made out of various materials, preferably out of a plastic that serves medical purposes and resists sterilization. The sectional view A-A through the container 1 is evident from FIG. 4. [0025] As the basic part 2 on FIG. 2 shows, the pillow block 10 has another function according to the invention. The function involves equipping the pillow block 10 with a pedestal 20. The pedestal 20 runs in the longitudinal direction 17 of the basic part 2 on either side of the pillow block 10. The upper side 21 of the pedestal 20 serves as a stop for the front faces 23 of the clamping jaws 22 of the application tool 15 (see FIG. 4 ). In addition, the surface 24 of the webs 14, 14 ′ is inclined on the side facing the pillow block 10. Therefore, the base surface 25 of the webs 14, 14 ′ narrows toward the top end. As a result, the distance of the webs 14, 14 ′ in a clamp compartment 4 viewed in the transverse direction 18 is smaller in the base area than in the top area due to the inclined surface 24. The shortened distance between the two webs 14, 14 ′ of a clamp compartment 4 automatically presses the application tool 15 against the retainers 8 in a first step in order to open the latter, and guides it against the clamp 12 in a second step, which is situated between the two retainers 8 to remove them. [0026] As evident from FIG. 3, the retaining device or base cover 3 has openings 30 through which the webs 14, 14 ′ of the basic part 2 can extend, and openings 31 that accommodate separating walls 6. The openings are situated at both longitudinal sides of the retaining device 3 at a certain distance X in the longitudinal direction relative to each other, and at a certain distance Y in the transverse direction similarly to the webs 14, 14 ′ in the basic part 2, wherein the shape of the openings 30 for accommodating the webs 14, 14 ′ matches their shape. Other openings 31 in the retaining device 3 are used to accommodate the separating walls 6 of the basic part 2 during fabrication. The openings 31 are located between the adjacent clamping elements 7, and a separating wall 6 has two lateral walls 5, 5 ′, which serve as a lateral stop for the retainers 8, 8 ′ (see FIG. 2 ). In addition, the retaining device 3 is fitted with a plurality of clamping elements 7, wherein one clamping element 7 is allocated to each clamp compartment 4. A clamping element 7 here consists of two paired retainers 8, 8 ′, with there being one pair per clamp compartment 4. The retaining means 8, 8 ′ are U-shaped, wherein the legs 32 of the U-shaped strap 33 project from the retaining device 3. The vertex area 34 or contact area of the U-shaped strap 33 has a latching nose 35, which assumes the function of holding the clamp 12 on the pillow block 10. The retainers 8, 8 ′ are inclined, and meet at the vertex 34. The distance “U” of the paired retainers 8, 8 ′ in the base area 36 of the straps 23 is selected in such a way that the clamping jaws 22 of the application tool 15 can be easily introduced between them. The clamping jaws 22 reach between the retainers 8, 8 ′ in a horizontal direction according to the invention, such that no forces act on the container in a tangential direction. The U-shaped strap 33 is biased towards a retaining position. [0027] In the depicted embodiment the retaining position holds latch 35 over a top surface of the clamp. As the clamping jaws 22 are introduced further, the retaining means 8, 8 ′ are bent apart in the vertex area 34, causing the latching noses 35 to release the clamp 12. The maximum path that a retaining element 8, 8 ′ can traverse from the closed setting to the open setting is limited by the lateral walls 5, 5 ′ of the separating wall 6. This precludes any excess bending of the retaining means 8, 8. If the retainers 8, 8 ′ abut the lateral walls 5, 5 ′, the clamp 12 is free, and can be removed from the pillow block 10 by the application tool 15 without friction. [0028] FIG. 4 shows a sectional view A-A of the container 1 fitted with a clamp 12 according to FIG. 1, consisting of a basic part 2 and the retaining device 3, wherein the upper part 3 is advantageously provided with a bordering 38. The bordering 38 can improve incorporation of the basic part 2 on the one hand, and ease handling by the user on the other. A fastening means 41 (not shown) can be provided on the lower side 39 of the basic part 2 or on the lower side 40 of the bordering 38. The fastening means 41 consists of a carrier provided on either side with a fixation, which may be double-sided adhesive tape, or alternatively a rigid foam product. As also evident form the sectional view A-A, the clamp compartment 4 is limited in the transverse direction 18 by the webs 14, 14 ′ and in the longitudinal direction 17 by a lateral wall 5. Visible in the middle of the clamp compartment 4 is the pillow block 10 on the pedestal 20, wherein the pillow block 10 carries a clamp 12. The clamp 12 is clamped by the latching nose 35 of a retaining means 8. Schematically depicted on the inclined surface 24 of the webs 14, 14 ′ are clamping jaws 22 of an application tool 15 that have not yet reached the end position for removing the clamp 12. At the end position, the front faces 23 of the clamping jaws 22 would be on the top side 21 of the pedestal 20, and the clamp 12 would be accommodated by the channels in the clamping jaws 22, so that the loose clamp can be removed without friction. REFERENCE LIST [0000] 1 Container 25 Base surface 2 Lower part (base) 26 Top surface 3 Upper part (device) 27 Free 4 Clamp compartment 28 Free 5 Lateral wall 29 Free 5 ′ Lateral wall 30 openings (webs) 6 Separating wall 31 Openings (lateral wall) 7 Clamping element 32 Leg 8 Retaining means 33 Strap 8 ′ Retaining means 34 Vertex 9 Inner contour 35 Latching nose ′ 10 Pillow block 36 Base area 11 Contour 37 Free 12 Clamp 38 Bordering 13 Channel 39 Lower side (for 2 ) 14 Web 40 Lower side (for 3 ) 14 ′ Web 41 Fastening means 15 Application tool 16 Longitudinal side 16 ′ Longitudinal side 17 Longitudinal direction 18 Transverse direction 19 Transverse side 20 Pedestal 21 Upper side 22 Clamping jaw 23 Front face 24 Inclined surface′
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Summary: This invention generally relates to the area of medicine in the surgical field. Containers are used in surgery for application tools for applying objects. Containers especially suitable for holding special objects, in particular hemostatic clamps. Hemostatic clamps are used to tie or clamp blood vessels during surgical procedures. Special containers are used to store and advantageously provide hemostatic clamps. These containers completely and clearly hold clamps in each phase without tilting, and enable reliable removal by a surgeon using a surgical instrument. This is achieved with the container according to the invention, which avoids friction between the clamp and the surgical instrument.
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Summarize: Strange. I remember Ron Howard's 1989 ode to suburban child-rearing, "Parenthood," being a lot funnier than the new NBC television series with the same name. The new version, which premieres March 2 at 9 p.m. on KSL Ch. 5, takes the same parenting-is-hell playbook from the movie, but it's stripped of much of the laughter that helped offset the tears. The new series changes the names of the characters but retains the archetypal personalities from the movie. Peter Krause ("Six Feet Under") is Adam Braverman (Steve Martin's role), an anxious but caring father who only wants the best for his son, who he later learns has a mild form of autism called Asperger's syndrome. He also has two sisters and a brother. Ericka Christensen ("Traffic") is Julia, a lawyer caught up in her career instead of her daughter. Lauren Graham ("Gilmore Girls") is Sarah, a recently divorced mother of two who moves back in with their parents (Graham replaced "ER's" Maura Tierney after Tierney was diagnosed with cancer). And "Punk'd" co-star Dax Shepard is Crosby, the younger free-floating brother who craves his independence. The patriarch of the Braverman family is Zeek (Craig T. Nelson), a rough father figure who believes parenting is like preparing his kids for war. In the pilot episode, much of the same ground is covered as in the film. Adam coaches his son's baseball team but doesn't know why the boy acts differently. Sarah has a rough post-divorce time juggling her Advertisement kids' problems with her fear of dating (her character is based on the one played in the movie by Dianne Wiest). "Parenthood's" strongest suit is an able, well-cast ensemble of actors who so far show off a fair amount of chemistry. For anyone who has kids -- or just remembers being one -- raising children is full of quirky, sometimes knee-slapping moments that are permanently burned in our memories. ABC's offbeat comedy, "Modern Family," shows off those moments well. "Parenthood" -- at least in the episode available for review -- exhibited more drama than comedy, seemingly more interested in conveying the more noble gestures of parenting than what we saw in Howard's blockbuster movie. But this is early, and the seeds of a good comedy -drama are there. Perhaps, like a good parent, the network can nurture this series into something it can be proud of. Vince Horiuchi's column appears Fridays. He can be reached at [email protected] or 801-257-8607. For more television insights, visit Horiuchi's blog, "The Village Vidiot," at blogs.sltrib.com/tv/. Send comments to [email protected]. Martin dressed up as a cowboy for his son's birthday party. Moranis tried to create a prodigy, showing his daughter flashcards of chemical symbols. And Hulce learned that he had a son. A black son. Named Cool. Ron Howard's 1989 movie was sprawling and ambitious, with a cast including Steve Martin, Tom Hulce, Rick Moranis, Jason Robards, Mary Steenburgen, Dianne Wiest, and a sulking Leaf Phoenix who had not yet changed his name to Joaquin. Whenever I watch a remake, I wonder: Why now? And sometimes, just "Why?" I think that's what we were all thinking when NBC tried to gas up a new version of "Knight Rider" in 2008. So why "Parenthood"? Now? Since I was 19 when I saw it, the storyline that resonated most with me was when the teenage daughter (Plimpton) took some naughty photos to get developed with her boyfriend (Keanu Reeves!) - that were accidentally picked up by her mother. Yikes. That's the kind of lesson that sticks with a teenager. Today, of course, any nude photos would be taken by phone and sexted out to everybody on your buddy list within seconds. So I guess times have changed. As for the "Why now?" question... the truth is, they've never stopped trying to remake "Parenthood." A 1990 version with Ed Begley Jr. was critically well received, but didn't last long. Most notably involved: *a pre-"Buffy the Vampire Slayer" Joss Whedon as a writer on the show; *a pre-screaming-teenagers Leonardo DiCaprio as one of the kids, and *a pre-Courteney-Cox David Arquette in the Keanu role. Over and over, TV has tried to capture both the comedy and agony of family. "The Parent 'Hood" (1995-99), "Life Goes On" (1989-93), "Seventh Heaven" (1996-2007), and most recently "Brothers and Sisters," "The Middle" and "Modern Family." Now Ron Howard's getting back into the game again with partner Brian Grazer, as well as big names from "Friday Night Lights," "Arrested Development" and "The West Wing." The exquisite Maura Tierney had to bow out due to breast cancer (don't worry - she's well enough now to star in a Los Angeles play), but she's been gamely replaced by Lauren Graham. Basically, it's her "Gilmore Girls" role, except her daughter is a goth rebel instead of a perky overachiever. The remarkably close Braverman family lives in Berkeley, Calif., in a series of funkily charming homes. The solid cast also includes: *Craig T. Nelson as Zeek, the gruff patriarch who may be having an affair. *Bonnie Bedelia as Camille, his wife. We know from "Presumed Innocent" that she *knows how to end an affair. *Peter Krause as Adam, who learns that his son has Asperger's Syndrome. *Monica Potter, as his freaking-out wife. *Erika Christensen as Julia, the lawyer bread-winner in her family, who's worried that her daughter prefers her dad. *Dax Shepard as Crosby - and how could he be anything but a laid-back music producer who lives on a houseboat with a name like Crosby? *Joy Bryant as Jasmine, Crosby's ex, who shows up one day to tell him he has a son. A black son. Named Jabar. *Mae Whitman (who's giving "Juno's" Ellen Page some competition as America's slacker sweetheart) as a teenager who hasn't learned to run from a boyfriend named Damien. "Parenthood" strikes all the right notes, although it's not yet can't-miss television. It's sweet and funny and heartbreaking, and relatable to the point of cliché. Doesn't it seem like the only way you can show your son that you love him these days is to argue with the ump and get thrown out of his Little League game? The series drives home the undeniable fact that it takes a village to raise a child. You can pay the village, I suppose, or depend on the kindness of strangers. But family can be one-stop shopping for day care, therapy, advice, criticism and casseroles. And though you may save financially, boy will you pay for it emotionally.
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Summary: Can the new Parenthood, a remake of the Ron Howard flick of the same name, help repair NBC's 10pm timeslot? Maybe, critics say, but it's got some problems. Here's what they're saying about the show, which premieres tomorrow: It's a "credible dramedy with the misfortune of arriving after the similar-and better-Modern Family," writes Brian Lowry of Variety. There are so many plot threads that it "risks becoming Brothers and Sisters-Lite (which is already pretty lite, come to think of it)." "It's sweet and funny and heartbreaking, and relatable to the point of cliché," says Paige Wiser of the Chicago Sun-Times. "Parenthood strikes all the right notes, although it's not yet can't-miss television." "Where are the laughs?" asks Vince Horiuchi of the Salt Lake City Tribune. There's potential here, but the show needs to strike a better blend of comedy and drama. "Perhaps, like a good parent, the network can nurture this series into something it can be proud of."
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Summarize: CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to French Application No. 0513368 filed Dec. 27, 2005, which is incorporated herein by reference. BACKGROUND OF THE INVENTION a) Field of the Invention The invention concerns the field of applicators for fluid or pasty products, typically applicators of cosmetic products such as mascaras. b) Description of the Prior Art A large number of mascara applicators are already known. These applicators, which are designed to cooperate with a container forming a reservoir for mascara, typically comprise: a) a cap adapted to seal said container and to serve as means for gripping said applicator, b) an axial rod, c) and a brush, said rod being fixedly connected to said cap at one of its ends, and with said brush at the other one of said ends, said brush comprising a metallic twist binding a plurality of bristles. With respect to said brush, a large number of models of brushes are already known. Thus, the brushes described in the following French Patents are known: FR 2 505 633, FR 2 605 505, FR 2 607 372, FR 2 607 373, FR 2 627 068, FR 2 627 363, FR 2 637 471, FR 2 637 472, FR 2 650 162, FR 2 663 826, FR 2 668 905, FR 2 675 355, FR 2 685 859, FR 2 690 318, FR 2 701 198, FR 2 706 749, FR 2 715 038, FR 2 745 481, FR 2 748 913, FR 2 749 489, FR 2 749 490, FR 2 753 614, FR 2 755 693, FR 2 774 269, FR 2 796 531, FR 2 796 532, FR 2 800 586. The brushes described in the following U.S. Patents are also known: U.S. Pat. Nos. 4,733,425, 4,861,179, 5,357,987, 5,595,198, 6,241,411, 6,427,700. On the one hand, in view of the permanent evolution of mascara formulations, there is a need for developing new applicators which can be used to apply these formulations, and which are typically adapted to each now formulation. On the other hand, for the persons who use these formulae, there is the requirement that they must rely on a wide range of applicators to obtain different effects, for example different loading levels of product to be applied for the applicator, and/or different effects in combing eyelashes. In addition, there is an increasing requirement to incorporate new technical functions in the applicators, in order to respond to the needs of the persons using them, as well as to be distinct from the “bottom-of-the-line” products for which the production may be shifted to cheap labor countries. Finally, in the field of cosmetic products, there is a permanent demand for new products, in order for example to customize the products, whether we are concerned with the formulations, the applicators or also wrappings in general, to the extent that replacement of the products becomes an absolute commercial requirement, to the risk of disappearing from the market place. SUMMARY OF THE INVENTION According to the invention, the applicator for a fluid or pasty cosmetic product, has an axial direction and comprises an axial rod that is fixedly connected, at its so-called lower end, with an application means provided with means for retention of said cosmetic product, and at its so-called upper end, with means for manually grabbing said applicator. It is characterized in that: a) said axial rod is a co-axial rod comprising a central rod and an external sheath, said central rod comprising a rod portion that is coaxial with said external sheath and a so-called free rod portion which is not covered with said central rod, b) said application means comprises a plurality of N axial members providing an inner space there between, wherein N varies from 2 to 8, said axial elements being fixedly connected, at their upper ends, with said external sheath at the bottom end thereof, and at their lower ends, with said rod portion at the bottom end thereof, c) said grabbing means comprises a so-called fixed grabbing portion and a knob which is rotatably fixedly connected to said central rod, a manual rotation of said knob according to an angle α with respect to said fixed portion causing a relative rotation of said lower ends with respect to said upper ends and thus a twist of said axial elements, in such a manner that it is possible for example to modify at will the volume of said inner space and thereby the quantity of said cosmetic product held back by said application means. Following rotation of said knob, the applicator according to the invention makes it possible to more or less tighten said axial members, and to modify the inner space so as to operate on the quantity of cosmetic product held back in said application means, as well as on the external volume of said application means. Since said applicator is typically a part of a dispenser comprising a reservoir containing said cosmetic product, for example a mascara, the person using the applicator can operate the knob, either when the applicator acts together with the reservoir, said application means dipping into said cosmetic product, before being removed from the reservoir, said application means then passing through wringer, or once the applicator has been separated from the container, and possibly during make-up. Thus, the person using the applicator disposes of a means for modifying the applicator all along the make-up, depending on the type of effect it is desired to be obtained. BRIEF DESCRIPTION OF THE DRAWINGS All the figures relate to the invention. For reasons of clearness of the figures, all the elements or members represented in the figures have not been hatched. FIGS. 1 a to 1 h relate to a first embodiment of the invention. FIG. 1 a is a side view in perspective of the applicator. FIG. 1 b is a side view in perspective of the reservoir which is adapted to operate jointly with the applicator in order to constitute the dispenser. FIG. 1 c is a side view in perspective of the dispenser in closed position. FIG. 1 d is a cross-section of the dispenser along line D-D of FIG. 1 c. FIG. 1 e is an axial cross-section of the dispenser along line E-E of FIG. 1 c. FIG. 1 f is an enlarged view of the part of FIG. 1 e that is surrounded by a rectangle illustrated in dotted line, indicated by “f”. FIG. 1 g is an enlarged view of the part of FIG. 1 e that is surrounded by a rectangle illustrated in dotted line, indicated by “g”. On this figure, the upper end of the application means is an upper end that is ratchetly engaged in the lower end of the external sheath. FIG. 1 h is a cross-section view in the transverse plane H-H of FIG. 1 g, which is a plane that is perpendicular to an axial direction, Illustrating the case where the application means comprises three axial members oriented at 120° with respect to the axial direction. FIGS. 2 a to 2 e illustrate various embodiments and representations of axial members formed of a support that fixedly connects a plurality of radial members having a space there between, allowing for a sampling of the product and its later application. FIG. 2 a illustrates the case where the radial members are parallel to one another and are offset with respect to the support. FIG. 2 b illustrates the case where all the radial members are not parallel to one another. FIG. 2 c illustrates the case where the radial elements are parallel to one another and are centrally aligned with respect to the support. On the right portion of FIGS. 2 b and 2 c, by way of illustration, two types of projections or sections of radial members, of circular shape or in the shape of an oval, are represented. FIG. 2 d corresponds to FIG. 2 c and is a simplified representation thereof. FIG. 2 e is another simplified representation of an axial member showing a curve, as in the case of FIG. 1 g. FIGS. 3 a to 4 b relate to another embodiment of the invention. FIG. 3 a is an axial section of the applicator. FIG. 3 b is a side view in perspective which illustrates the torsion of the axial members, two only having been illustrated, one in the front in full line, one in the back in dotted line. FIG. 3 c is an enlarged view of the lower part of FIG. 3 a, surrounded by a rectangle in dotted lines. FIG. 3 d is a cross-section along line D-D of FIG. 3 c showing the presence of four axial members spaced 90° from one another. FIG. 4 a is a cross-section view of the dispenser comprising the applicator of FIG. 3 a and the reservoir represented in axial section of FIG. 4 b. FIGS. 5 a to 7 c illustrate various embodiments of application means or of members of applications means, in axial section. FIG. 5 a and 5 b illustrate an embodiment in which said application means constitute a one-piece member comprising said upper collar at its upper end, and said lower collar at its lower end. FIG. 5 a illustrates the cooperation of the bottom end of the external sheath with said upper collar through a ratchet engagement and rotary joint operation means and the joint operation of said bottom end of said free rod portion with said lower collar, through a ratchet engagement and rotary cooperation means. FIGS. 5 c and 5 d are respectively similar to FIGS. 5 a and 5 b. They differ in that said upper collar is a collar that is distinct from said axial members. In this embodiment, he application means constitute a one-piece member comprising a lower collar at its lower end. FIGS. 6 a and 6 b are respectively similar to FIGS. 5 a and 5 b. They differ in that the lower collar is a collar that is distinct from he axial members. In this embodiment, the application means constitutes a one-piece member comprising an upper collar at its upper end. FIGS. 6 c and 6 d are respectively similar to FIGS. 6 a and 6 b. They differ in that the upper collar is a collar that is distinct from the axial members. In this embodiment, the application means constitutes a one-piece member with the external sheath. FIGS. 7 a and 7 b are respectively similar to FIGS. 5 a and 5 b. They substantially correspond to the embodiment of FIG. 1 g, the free rod portion having a bottom end defining a male part that is ratchetly engaged in the collar constituting a female part. In this embodiment, the application means defines a one-piece member comprising a lower collar at its lower end. FIG. 7 c illustrates the bottom part of the co-axial rods, the external sheath comprising an annular part at its bottom end, which is intended to operate jointly with the upper ends. FIGS. 8 a and 8 b are respectively similar to FIGS. 5 a and 5 b. They differ in that the application means and said central rod constitute a one-piece member. FIGS. 8 c and 8 d are respectively similar to FIGS. 5 c and 5 d. They differ in that the application means and the central rod constitute a one-piece member. FIGS. 9 a to 9 c illustrate an embodiment of application means comprising a plurality of annular members which are fixed to the free rod portion, each annular member comprising an annular part surrounding a portion of the free rod, and a radial part defining a comb tooth. FIG. 9 a, which corresponds to FIG. 3 c, is a front lateral view In perspective. FIG. 9 b is an axial cross-section B-B, along the axial direction, of FIG. 9 a. FIG. 9 c is a cross-section C-C, which is perpendicular to the axial direction of FIG. 9 a. DESCRIPTION OF PREFERRED EMBODIMENT According to the invention, and as illustrated on FIG. 3 c, the free rod portion 221 can extend to an axial height H which substantially corresponds to the axial height of the application means 3. As illustrated for example on FIG. 3 c, the axial members 30 may have at their upper ends 300 a so-called upper connecting means 5. The upper connecting means 5 may be a so-called upper ring 50 possibly rotatably connected to external sheath 23, at its bottom end 230. As illustrated for example on FIGS. 5 a and 5 b, upper collar 50 may be a collar 50 ′ which constitutes a one-piece molded member of plastic material 7, 7 a with the plurality of axial members 30, upper collar 50 ′ comprising an axial ratchet engagement means 51 with external sheath 23. As illustrated in FIGS. 5 c and 5 d, the upper collar 50 may be a collar 50 ″ which operates jointly with external sheath 23 at its bottom end 230, in a manner to block the upper ends 300 of the axial members 30 and to rotatably connect the upper ends 300 to the external sheath 23. As illustrated for example on FIG. 3 c, the axial members 30 may provide at theirs lower ends 301 a so-called lower connection means 6, so as to rotatably cooperate with the free rod portion 221 at its bottom end 222. The lower connection means 6 may be a so-called lower collar 60, connected, possibly rotatably, to the free rod portion 221, typically through an axial ratchet engagement means 61. As illustrated on FIGS. 5 a and 5 b, the lower collar 60 may be a collar 60 ′ defining a one-piece molded member of plastic material 7 b with the plurality of axial members 30. As illustrated on FIGS. 6 a and 6 b, the lower collar 60 may be a collar 60 ″ that acts together with the free rod portion 221 at its bottom end 222, in a manner to block the lower ends 301 of the axial members 30 and to rotatably connect lower ends 301 to the free rod portion 221. On FIGS. 6 a to 6 d, the collar 60 ″ acts together with the lower ends 301, 301 ′ through ratchet engagement means. However, according to another variant, the lower collar 60 could also constitute a crown that surrounds the ends 301, in a similar manner to the left part of FIG. 7 a in which the annular portion 231 operates jointly with the upper ends 300. As illustrated on FIG. 5 b, the plurality of axial members 30 may define with upper 50 and lower 60 collars, a one-piece molded member of plastic material 7 c. As illustrated on FIGS. 8 a to 8 d, the plurality of axial members 30 may constitute a one-piece molded member 7 d, 7 d ′ of plastic material with the free rod portion 221. As illustrated on FIGS. 6 c and 6 d, the plurality of axial members 30 may, through upper ends 300, define with external sheath 23 a one-piece molded member 7, 7 e of plastic material. According to the invention, the rod portion 220 which is coaxial with the external sheath 23, and said free rod portion 221 may typically have a same diameter. However, the rod portion 220 which is coaxial with the external sheath 23 and the free rod portion 221, may not have the same diameter, only rod portion 220 which is coaxial with external sheath 23 being cylindrical, in a manner to allow for said manual rotation. As illustrated in FIG. 1 f, the fixed part 40 and the external sheath 23 constitute a one-piece member 11 which is molded and is made of plastic material and comprising a horizontal part 110 and a peripheral skirt 111 comprising a lower interiorly threaded part 112 and an upper part 113 that is capable of operating jointly with knob 41 typically by means of a head 42 defining a one-piece member 12 with the central rod 22, the head typically comprising an annular skirt 420 that is coaxial with upper part 113. According to the invention, knob 4 can operate jointly with fixed part 40 by joint action of a helical ramp and a lug that moves in ramp, said manual rotation of knob 41 by an angle α with respect to the fixed part 40 leading, in addition to the relative rotation of upper ends 300 with respect to lower ends 301, to an axial movement of the central rod 22 with respect to external sheath 23, an angular rotation by an angle+α leading to a forward axial movement which has a tendency to exert an axial tension on the axial members 30, a reverse angular rotation by an angle−α leading to a rearward axial movement tending to exert a compression on the axial members 30. Since this embodiment can easily be deducted from the embodiment represented on FIG. 1 f, it has not been illustrated on a distinct figure. As illustrated on FIGS. 2 a to 2 e, axial member 30 may comprise an axial support 31 and a plurality of n radial members 32 fixedly connected to axial support 31, wherein n varies from 5 to 30. The n radial members 32 may be regularly spaced along axial height H. According to the invention, N may vary from 3 to 5. As illustrated on FIGS. 3 a and 3 c, the plurality of axial members 30 may, before the rotation, constitute a substantially cylindrical cluster 33 of members that are parallel to one another and substantially parallel to the free rod portion 221 which is not covered with external sheath 23. As illustrated on FIGS. 5 a to 8 d, the plurality of axial members 30 may, before the rotation, constitute a cluster 33 ′ which bulges out at the level of its central part, in the manner of a spindle. As illustrated on FIGS. 9 a to 9 c, the application means 3 may comprise a plurality of annular members 8 which are fixedly connected to the free rod portion 221, the annular members 8 comprising an annular part 80 and a radial part 81 typically defining at least one comb tooth. Another object of the invention consists of a dispenser 9 for a cosmetic product comprising a rerservoir 90 which is capable of containing the cosmetic product, typically a mascara, and provided with a threaded neck 91 that operates jointly with the manual gripping means 4 of applicator 1 according to any one of claims 1 to 21, the fixed part 40 of the manual gripping means 4 comprising a thread capable of operating jointly with the threaded neck 91 by screwing/unscrewing, the neck possibly comprising a wringer 92 for the application means 3. Such a dispenser 9 has been illustrated on FIG. 4 a. EXAMPLES OF EXECUTION All the figures constitute examples of execution. LIST OF REFERENCE MARKS Applicator... 1 Axial direction... 10 One-piece member 40 + 23... 11 Horizontal part... 110 Peripheral skirt... 111 Inwardly threaded part... 112 Upper part... 113 Axial rod of 1... 2 Co-axial rod... 2 ′ Bottom end... 20 Top end... 21 Central rod... 22 Rod portion coaxial to 23... 220 Free rod portion... 221 Bottom end of 221... 222 External sheath... 23 Bottom end... 230 Application means... 3 Axial member... 30 Upper end... 300, 300 ′ Lower end... 301 Axial support of 30... 31 Radial member... 32 Cluster of 30... 33, 33 ′ Manual gripping means... 4 Fixed part of 4... 40 Knob connected to 22... 41 Head connected to 22... 42 Annular skirt connected to 41... 420 Upper connection means of 300 to 23... 5 Upper collar... 50, 50 ′, 50 ″ Catch means of 50 & 23... 51 Lower connection means of 301 to 221... 6 Lower collar.... 60, 60 ′ Catch means of 60 & 221... 61 One-piece molded member of 3... 7 Upper collar 50 and plurality of 30... 7 a Lower collar 60 and plurality of 30... 7 b Collars 50 & 60 and plurality of 30... 7 c Plurality of 30 & 22... 7 d, 7 d ″ Annular member fixedly connected to 221.... 8 Annular part... 80 Radial part defining a comb tooth... 81 Dispenser... 9 Container... 90 Neck... 91 Wringer... 92
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Summary: In an embodiment, an applicator includes an external sheath and an internal rod extending through and beyond a first end of the external sheath. A first end of the internal rod may be coupled to a plurality of axial members that extend between the first end of the internal rod and the first end of the external sheath. By actuating a knob coupled to the internal rod, the distance between the first end of the internal rod and the first end of the external sheath may be adjusted. By adjusting the distance, the configuration of the axial members may be adjusted and this adjustment may modify the amount of product that may be stored between the axial members.
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Write a title and summarize: Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit. An estimated one third of the cellular proteome is reversibly phosphorylated on specific serine/threonine residues, indicating an involvement of phosphorylation in many, if not all, cellular processes. The rapid reversibility and the selective functional effects of phosphorylation on substrate functions contribute to its critical importance in signaling cascades. The net phosphorylation of any given regulatory site is determined both by the enzymes catalyzing the phosphorylation reaction—the protein serine/threonine kinases (PSTKs) —and by the enzymes catalyzing the dephosphorylation reaction—the protein serine/threonine phosphatases (PSTPs). The molecular architecture that allows these two opposing enzyme classes to achieve substrate specificity has evolved very differently in each class. This is reflected in the number of genes coding for PSTKs (~430) versus only about 30 genes encoding catalytic subunits of the PSTPs [1]. Most PSTKs are multidomain, single-chain enzymes containing a catalytic and a substrate regulatory domain. PSTPs, however, are multisubunit enzymes in which the catalytic and regulatory subunits are encoded by different genes. Thus the assembly of different combinations of subunits to produce phosphatase holoenzymes with different substrate specificities compensates for the low number of PSTP catalytic subunits. A prime example of the PSTP multisubunit architecture is protein phosphatase 2A (PP2A). PP2A in vivo comprises a large number of heterodimeric and -trimeric complexes, all of which contain a catalytic C subunit. In the majority of PP2A holoenzymes, the C subunit is bound to the regulatory A subunit, which serves as a scaffold for the interaction with one of several B-type subunits. The B-type subunits are responsible for the substrate specificity and intracellular localization of the PP2A holoenzymes. Four B-type subunit families have been identified in mammalian cells, each family consisting of several highly related isoforms. In theory, more than 72 heterotrimeric holoenzymes can be assembled from the large pool of PP2A subunit isoforms [2]. A major determinant of holoenzyme composition in vivo is the availability of specific regulatory subunits, which in turn depends on their spatially and temporally regulated expression. Whereas some holoenzymes only form in certain developmental stages, others are present at all times and constitute “housekeeping” PP2A complexes. Another important determinant seems to be the stoichiometric balance of C, A, and B-type subunits. Down-regulation of the PP2A C or A subunit by RNA interference (RNAi) in vivo leads to the rapid degradation of the non-targeted subunits, suggesting that PP2A subunits are stabilized in the holoenzyme, but are instable as monomeric subunits [3,4]. Consequently, native PP2A isolated from various tissues consists of dimeric and trimeric complexes, but does not contain the free monomeric C subunit [5]. How the absolute levels of PP2A subunits and, therefore, of holoenzymes are adjusted to the cell' s respective requirements is unknown, but the catalytic PP2A subunit with its strictly controlled expression levels seems to play a central role in this regulatory circuit. Monomeric C subunits isolated from in vivo–assembled PP2A holoenzymes possess promiscuous and high activity in vitro [5,6]. Complex formation with the regulatory A and B-type subunits suppresses nonspecific C subunit activity, but increases the substrate affinity, as reflected by the decreased maximum velocity (Vmax) and Km [7]. Tight control of C subunit levels provides one explanation for how cells prevent the consequences of having free and unregulated C subunit [8]. However, it leaves open the question of how the catalytic activity of the newly translated C subunit (or transiently free C subunit, if recycling occurs) is kept in check until holoenzyme assembly can occur. Holoenzyme assembly is promoted by reversible methylation of the C subunit carboxyterminal leucine, which increases its affinity for the A and B-type subunits [9,10]. Deletion of protein phosphatase methyltransferase 1 (PPM1), encoding the yeast protein phosphatase methyltransferase solely responsible for PP2A methylation, not only negatively affects the interaction between the yeast C subunit homologs, PPH21 and PPH22, and the scaffold subunit, TPD3, but also affects the interaction with the B subunits, CDC55 and RTS1 [9,10]. The recently solved structures of a heterotrimeric PP2A complex suggest charge neutralization of the C subunit C-terminal carboxyl group as a potential cause for the affinity increase by methylation [11,12]. Although carboxymethylation is required for stable complex formation of PP2A holoenzymes in vivo [9,10], this does not seem to be the case in vitro [12], indicating a more complex role for C subunit methylation in vivo. Interestingly, deletion of PPM1 also reduces the catalytic activity of C subunit [13]. The discrepancy between the high catalytic activity of the free C subunit isolated in vitro from pre-existing holoenzymes and the decreased catalytic activity of the C subunit from a ppm1Δ strain with a hampered holoenzyme formation suggests that holoenzyme assembly might be coupled to the generation of the catalytically active C subunit and that methylation might play an important role in both processes. Recently, we obtained experimental evidence for a conserved mechanism to generate active and specific C subunit in vivo [13]. Deletion of rapamycin-resistant deletion 1 and 2 (RRD1 and RRD2), encoding the yeast homologs of the PP2A phosphatase activator (PTPA), produces a catalytic subunit with a conformationally relaxed active site, as indicated by its low catalytic activity towards phospho-serine/phospho-threonine (P-Ser/P-Thr) substrates, an altered substrate specificity, and its metal dependence. RRD2 stably interacts with the PP2A C subunit and, when expressed ectopically in the rrd1Δ/rrd2Δ strain, partially rescues the catalytic activity of the PP2A C subunit. RRD1, on the other hand, seems to be required for the generation of active SIT4, a PP2A-related phosphatase. Our data suggested a mechanism by which cells may avoid the risk of free and unregulated catalytic phosphatase subunits, namely the synthesis of a low-activity form that requires a functional interaction with an activator protein for full activity. However, this leaves open the question how the biogenesis of the catalytic subunit is coupled to the methylation-dependent PP2A holoenzyme assembly. Here we present evidence that the scaffolding PP2A subunit, TPD3, interacts functionally and physically with RRD/PTPA in the generation of active and specific PP2A in vivo. Further, the PP2A methylesterase, PPE1, plays a multifaceted role, since deletion of PPE1 in an rrd1Δ/rrd2Δ strain increases holoenzyme assembly with the B-type subunit CDC55 without generation of the active C subunit, whereas deletion of PPE1 in a tpd3Δ strain restores C subunit activity despite a failure of holoenzyme formation. Considered in detail, our results support a model in which C subunit biogenesis and PP2A holoenzyme assembly depend on a series of interlocking steps that proceed under surveillance of the PP2A methylesterase PPE1 by keeping in check the untimely methylation of the C subunit. The scaffolding A subunit is a prerequisite for the assembly of most PP2A holoenzymes. In the absence of the A subunit, TPD3, yeast proliferate poorly and have defects in tRNA transcription at high temperatures and in cytokinesis at low temperatures [14]. This latter phenotype is similar to the one caused by overexpression of the PP2A catalytic subunit [15]. It has therefore been assumed, that the unrestricted and untargeted catalytic activity of the C subunit is responsible for the observed phenotype in the absence of TPD3. Our recent work indicated, however, that the PP2A C subunit can exist in a low-activity form that requires the functional interaction with RRD/PTPA for the switch into an active P-Ser/P-Thr–specific enzyme [13]. Thus we asked whether the observed phenotype of the tpd3Δ strain, a strain impaired in holoenzyme assembly, is indeed caused by the unrestricted activity of the free C subunit. To analyze PP2A activity under these diverse conditions, we expressed hemagglutinin (HA) -tagged PPH21 or HA-tagged PPH22 in the wild-type, tpd3Δ, and rrd1Δ/rrd2Δ strains, and in a cdc55Δ/rts1Δ strain, which lacks the known B-type subunits of yeast. Anti–HA-tag immunoprecipitates were tested for catalytic activity towards the P-Ser/P-Thr substrate phosphorylase a (Figures 1A and S1A). Contrary to the prediction, the catalytic activity of PPH21 or PPH22 from a strain lacking the TPD3 subunit was decreased to approximately 25% or approximately 18%, respectively, of the wild-type activity. The extent of the decrease was similar for substrate concentrations ranging from 1 μM phosphorylase a to 100 μM, a concentration 20-fold above the reported Km value for phosphorylase a (at which concentration the velocity converges to the Vmax of the enzyme) (Figure S2) [16]. A decreased catalytic activity, however, was not a general feature of strains lacking PP2A regulatory subunits. Deletion of the two known B-type subunits in yeast, CDC55 and RTS1, did not impair C subunit activity towards phosphorylase a. In addition, we analyzed the catalytic activity of endogenous C subunit immunoprecipitates from lysates of a wild-type or a tpd3Δ strain that did not express ectopically HA-tagged PPH21 or PPH22 (Figure S3B). The endogenous C subunit activity was also severely impaired towards phosphorylase a in the tpd3Δ strain. Thus, TPD3 seems to fulfill functions in addition to serving as the scaffold for the interaction with B-type subunits. The C subunit in the rrd1Δ/rrd2Δ strain shows a decreased P-Ser/P-Thr–specific activity. Strikingly, however, it gains high activity towards para-nitrophenyl phosphate (pNPP), a small-molecule substrate with a bulky phospho-tyrosine–like group [13] (Figure 1B). Both C subunits, PPH21 as well as PPH22, displayed several-fold higher activities towards pNPP in the tpd3Δ strain than in the wild-type strain (Figures 1B and S1B). Thus, loss of TPD3 led to the generation of C subunits with altered catalytic properties, indicating a role for TPD3 in C subunit biogenesis. The decreased C subunit activity and altered substrate specificity in the tpd3Δ strain were reminiscent of the hampered PP2A activity in a strain lacking RRD1 and RRD2 [13]. RRD2 is known to form stable interactions with the yeast C subunits and to be significantly more active in vivo on the PP2A catalytic subunit than RRD1 is. We asked whether the interaction of the C subunit with RRD2 was affected by deletion of TPD3. Anti–HA-tag immunoprecipitates from lysates of the wild-type, tpd3Δ, rrd1Δ/rrd2Δ, cdc55Δ/rts1Δ, and ppm1Δ strains were analyzed for the presence of RRD2 (Figure 2A). The interaction of HA-tagged PPH21 with RRD2 was only slightly reduced in the tpd3Δ strain (64% ± 26% of wild-type strain level, n = 10), but the levels of associated TAP42, a highly conserved and essential subunit of the PP2A family, increased several-fold, as has been shown by others [17]. Competency for TAP42 binding indicated that deletion of TPD3 did not lead to formation of denatured C subunit. The increased levels of TAP42: C subunit complexes in a tpd3Δ strain have been attributed to the lack of PP2A heterotrimers, which may promote the dissociation of the TAP42: C subunit complex by dephosphorylation of TAP42 [18]. However, in contrast to the tpd3Δ strain, the level of TAP42: C subunit complexes remained unchanged in the cdc55Δ/rts1Δ strain, indicating that the presence or absence of heterotrimeric PP2A does not alter the stability of the TAP42: C subunit complexes. Genetic evidence indicates an inhibitory role for TAP42 on phosphatase activity [19]. To exclude the possibility that the decreased catalytic activity and altered substrate specificity of the C subunit isolated from the tpd3Δ strain is simply due to formation of the TAP42: C subunit complex or binding of an unknown inhibitor, we measured the catalytic activity of immunoprecipitated HA-tagged PPH21 that was stripped of associated proteins, including TAP42, at alkaline pH (Figure 2B and 2C). To verify the monomeric nature of the C subunit, we analyzed pH-treated lysates from a wild-type or tpd3Δ strain by sucrose gradient sedimentation/centrifugation (Figure S4). The pH treatment shifted HA-tagged PPH21 to lower molecular weight fractions, indicating dissociation of C subunit complexes and the presence of monomeric C subunits. Compared with the wild-type strain, the “free” C subunit from the tpd3Δ strain was still impaired in catalytic activity (Figure 2C), indicating that the defect is intrinsic to the C subunit and not caused by the increased binding of TAP42 or any other inhibitor. C subunit activity was decreased even at high substrate concentrations (Figure S5), confirming the results obtained with the complexed C subunit and indicating a defect in the catalytic mechanism. In summary, loss of TPD3 led to alterations of C subunit activity and specificity, which are most probably due to changes at the active site that are reminiscent of the alterations observed in the absence of RRD1/RRD2. The similarity in the defect of the C subunit in the tpd3Δ and rrd1Δ/rrd2Δ strains raised the possibility of a physical and/or functional interaction between RRD2 and TPD3: PPH21 dimers. We therefore tested whether RRD2, TPD3, and PPH21 can form a trimeric complex in a wild-type strain, a possibility that has been suggested recently, but not proven [20]. First we analyzed myc-tagged RRD2 immunoprecipitates from lysates of the wild-type strain co-expressing HA-tagged PPH21, for associated PP2A subunits (Figure 3A) and compared it with the composition of HA-PPH21 immunoprecipitates from lysates of the same strain. The analysis revealed that approximately 5%–10% of PPH21 was associated with RRD2 in a wild-type cell. Moreover, the RRD2-bound PPH21 was unmethylated, indicating that methylation probably was not a requirement for complex formation with RRD2. This was in agreement with the association between PPH21 and RRD2 in the ppm1Δ strain (Figure 2A). RRD2 also associated with TPD3, but not with the regulatory subunits TAP42, CDC55, RTS1, and PPE1 (unpublished data). The lack of interaction with TAP42 was in contrast to the recent identification of TAP42: RRD1 and TAP42: RRD2 complexes and might be due to differences in experimental conditions and/or the strain background [20]. To test whether RRD2, TPD3, and PPH21 co-exist in the same complex, we immunoprecipitated myc-tagged RRD2 from lysates of a wild-type strain co-expressing HA-tagged PPH21, eluted the RRD2 complexes with the myc-epitope peptide, and re-immunoprecipitated the complexes via HA-tagged PPH21 (Figure 3B). Analysis of the HA-PPH21 immunoprecipitates with antibodies to the A subunit, TPD3, revealed that these proteins indeed are in the same multimeric complex. These findings are consistent with TPD3 and the RRD proteins playing a common role in C subunit biogenesis. To further test the functional interaction between these proteins, we deleted TPD3 and both RRD genes and examined the strain viability by a proliferation assay. In line with our hypothesis, deletion of TPD3 and both RRD genes was synthetically lethal (Figure 3C), providing further evidence for the essential functional and/or physical interaction of these proteins. The 36% decreased interaction between RRD2 and the C subunit (Figure 2A) cannot explain the 75% decreased catalytic activity towards phosphorylase a in a tpd3Δ strain (Figure 1A), because deletion of RRD2 reduces catalytic activity towards phosphorylase a and other P-Ser/P-Thr substrates only by about 45%–50% [13]. We therefore investigated whether the reversible methylation of the C subunit, which is required for stable holoenzyme formation [9,10], but probably also for the activation of PP2A activity [13], was also altered in the tpd3Δ strain (Figure 4A). Anti–HA-tag immunoprecipitates from lysates of the wild-type, tpd3Δ, rrd1Δ/rrd2Δ, and ppm1Δ strains were analyzed with the methylation-specific monoclonal antibody 2A10. No C subunit methylation was detected in the strain deleted for PPM1 (Figure 4A, lane 5), proving the specificity of the 2A10 antibody. Surprisingly, PPH21 was almost completely unmethylated in the tpd3Δ strain and markedly hypomethylated in the rrd1Δ/rrd2Δ strain compared to the wild-type strain, suggesting that in the absence of TPD3 or RRD/PTPA, the catalytically impaired C subunit became either a preferred substrate for PPE1 or an unfavored substrate for PPM1. To identify the potential cause for the decreased methylation levels in these strains, we analyzed HA-tagged PPH21 immunoprecipitates for associated PPE1 or PPM1. The yeast methyltransferase, PPM1, did not form stable complexes with the C subunit in either the wild-type or the deletion strains, suggesting that methylation occurs during a transient interaction between substrate and enzyme (unpublished data). However, deletion of either TPD3 or RRD1/RRD2 caused an increase in the abundance of a PPE1: PPH21 complex (Figure 4A), suggesting that the reduced methylation levels of PPH21 resulted from increased PPE1 binding (Figure 4A, lower panel). The complex formation between PPE1 and PPH21 in the ppm1Δ strain, however, also showed that methylation was not a prerequisite for the interaction of PPE1 with the C subunit. In summary, these data indicated that the PPE1: C subunit complex formation represents an early step in PP2A biogenesis and that interruption of the cascade causes accumulation of these complexes. An increase in the abundance of PPE1: PPH21 complexes and demethylation of the C subunit correlated with an impaired catalytic activity in the tpd3Δ or rrd1Δ/rrd2Δ strains. We therefore asked whether overexpression of PPE1 in a wild-type strain would also negatively affect C subunit activity (Figure 4B and 4C). Loss of C subunit methylation by high-level overexpression of PPE1 correlated with a large increase in the amount of PPE1: PPH21 complexes, a profound reduction in the binding of PPH21 to RRD2 and TPD3 (Figure 4B), and a severely reduced PPH21 phosphatase activity towards phosphorylase a (Figure 4C). These data suggest that the impaired catalytic activity of the C subunit in a tpd3Δ strain was due to the increased PPE1 binding and demethylation of the C subunit, which prevented the next critical step in C subunit biogenesis. If demethylation blocks C subunit maturation, then deletion of the PPE1 gene in a tpd3Δ strain might allow the biogenesis to proceed and restore the impaired catalytic activity of the C subunit. Deletion of PPE1 in the absence of RRD1/RRD2, the essential factors for the generation of active PP2A [13], however, should, if anything, worsen the defective C subunit biogenesis. Thus, we analyzed the activity and complex composition of HA-PPH21 from a series of deletion strains (Figure 5A and 5B). As before, tpd3Δ and rrd1Δ/rrd2Δ strains had marked hypomethylation of PPH21. As predicted, deletion of PPE1 not only restored C subunit methylation in these strains, but even increased it, indicating that PPE1 is responsible for the reduced amounts of methylated C subunits in these strains (Figure 5A). Importantly, PPE1 deletion affected C subunit biogenesis in distinctly different ways in the tpd3Δ versus the rrd1Δ/rrd2Δ strain. In the tpd3Δ strain, PPE1 deletion restored C subunit activity (n = 7, p < 0. 001) towards phosphorylase a almost to the level of the wild-type strain (Figure 5B). Thus, TPD3 is not required for formation of the catalytically active C subunit. In contrast, the RRD1/RRD2 proteins were required for the restoration of phosphatase activity: in the rrd1Δ/rrd2Δ strain, the impaired C subunit activity not only could not be rescued by PPE1 deletion, but was further reduced (n = 7, p < 0. 05) (Figure 5B). The same was true for the activity towards cdc2-phosphorylated histone H1, a substrate known to be specifically dephosphorylated by heterotrimeric complexes containing the B subunit [21] (unpublished data). The restoration of C subunit methylation in this strain, however, led to a huge increase in formation of inactive holoenzymes with TPD3 and CDC55. Of note, in these inactive complexes, a subset of CDC55 migrated with decreased electrophoretic mobility (Figure 5A). Treatment with λ-phosphatase shifted the slower form to the faster migrating form of CDC55, indicating that the altered electrophoretic mobility of CDC55 was probably due to phosphorylation (unpublished data). Interestingly, our previous analysis had revealed that in the absence of RRD1/RRD2, the level of a post-translationally modified form of CDC55 is also increased [13], raising the intriguing possibility that RRD/PTPA function regulates the modification/phosphorylation state of CDC55 and probably also of the other B-type subunit, RTS1 (unpublished data). The exacerbation of the impaired C subunit activity in the rrd1Δ/rrd2Δ strain upon deletion of PPE1 indicated a functional and/or physical interaction between these proteins. Since we did not detect a physical interaction between PPE1 and RRD1 or RRD2 (unpublished data), the data are consistent with RRD1/RRD2 and PPE1 being in the same functional pathway without being in a complex. The impaired C subunit activity in the tpd3Δ strain seems to be responsible for the severe proliferation phenotype of this strain. If the impaired activity is indeed the cause for the phenotype, then reactivation of C subunit activity upon PPE1 deletion might alleviate the severity of the phenotype. Therefore, we measured the proliferation of these strains at different temperatures (Figure 5C). However, the tpd3Δ/ppe1Δ strain with its restored PP2A activity grew only slightly better at all tested temperatures than the tpd3Δ strain. We suspect that this minor improvement is due to the lack of properly targeted and regulated PP2A holoenzymes. In the rrd1Δ/rrd2Δ strain, the proliferation phenotype was aggravated upon PPE1 deletion, despite the increase in trimeric holoenzymes, most likely due to the severe defect in catalytic activity. The increase of C subunit methylation in the tpd3Δ/ppe1Δ strain correlated with a restoration of the impaired catalytic activity, indicating a potential requirement for methylation in the C subunit activation process, a role to which our previous data had already pointed [13]. To test this hypothesis, we deleted the PPM1 gene in the tpd3Δ/ppe1Δ strain and analyzed the methylation state and catalytic activity towards phosphorylase a in this strain (Figure 6A and 6B). Loss of C subunit methylation in this strain caused a huge decrease in catalytic activity (n = 7, p < 0. 001) (Figure 6B), underscoring the importance of methylation for C subunit activation. Thus, PPE1 probably delays progression of the biogenesis cascade in the tpd3Δ strain by keeping the C subunit in an unmethylated state. Here we identify a critically important functional and physical interaction between RRD2 and the structural subunit TPD3 in C subunit maturation. RRD2/TPD3 interacts preferentially with the demethylated and inactive form of the C subunit by targeting the PPH21: PPE1 complex. PPE1, therefore, may fulfill a surveillance function of TPD3/RRD2-dependent C subunit maturation by preventing untimely C subunit methylation and hence the premature generation of active C subunit and holoenzyme assembly (see model, Figure 7). The catalytic subunit of a multisubunit enzyme like PP2A achieves substrate specificity by complex formation with regulatory subunits [22]. The combinatorial assembly of holoenzymes from a large pool of distinct regulatory subunits generates a great number of different substrate-specific complexes and explains how a single catalytic subunit is able to perform so many functions. Inherent to the multisubunit enzyme architecture, however, is the problem of how the promiscuous catalytic activity of the free catalytic subunit is kept in check until holoenzymes form. Recently, we obtained experimental evidence for a potential control mechanism, which involves synthesis of the PP2A catalytic subunit in a low-activity form that requires the functional interaction with RRD/PTPA for the switch into an active and specific enzyme [13]. Here we show that the scaffolding subunit TPD3 functionally and physically interacts with RRD2 in the generation of the active and specific PP2A C subunit, indicating that C subunit maturation is coupled and coordinated with holoenzyme assembly. Biochemical analyses indicated that in vitro, binding of the A subunit to the C subunit decreases the phosphatase catalytic rate and increases substrate specificity [7]. However, the effects of PP2A A subunit binding on C subunit catalytic properties were determined with C subunits isolated from catalytically active PP2A holoenzymes, because the free C subunit does not seem to exist in substantial amounts in vivo. On the basis of these in vitro observations, it has been assumed that loss of the A subunit in vivo will leave behind the monomeric C subunit with high and promiscuous catalytic activity. The data presented here demonstrate that this assumption is incorrect. Our analysis of the tpd3Δ strain indicates that the A subunit provides an additional function besides narrowing the substrate specificity of an otherwise promiscuous C subunit. As evidenced by genetic and physical interactions, the A subunit also cooperates with RRD/PTPA in the generation of the active C subunit in vivo. What is the regulatory role of TPD3 in this process? TPD3 formed a multimeric complex with RRD2 and PP2A C subunits. This complex appears to be essential for PP2A biogenesis and differs in several aspects from B-type subunit-containing holoenzymes. In contrast to B-type subunits, RRD2 interacted preferentially with the unmethylated C subunit, suggesting methylation state-dependent regulation of RRD2: C subunit interaction. We found, consistent with this hypothesis, that deletion of PPE1 (as well as PPM1 overexpression; unpublished data) decreased the levels of RRD2: TPD3: C subunit complexes. RRD2: TPD3: PPH21 holoenzyme assembly constitutes the first example of a PP2A complex whose stability is regulated in an opposite manner to other PP2A holoenzymes, namely by demethylation rather than methylation. The binding preference of RRD2 for the demethylated C subunit suggests an important role for PPE1, the enzyme catalyzing the demethylation, as well as for the carboxyl terminus itself in the interaction with RRD2. In accordance with our prediction, a C subunit mutant lacking the carboxy-terminal nine amino acids including the site of methylation did not bind RRD2, and the monoclonal antibody 7A6 directed to the C subunit carboxyl terminus did not co-precipitate RRD2 (unpublished data), indicating that the C subunit carboxyl terminus seems to be essential for the interaction (directly or indirectly) with RRD2. The impaired catalytic activity of the deletion mutant towards phosphorylase a (unpublished data) provided further evidence for the functional cooperation between the C subunit carboxyl terminus and RRD2. So far, no obvious phenotype has been identified following deletion of PPE1 in yeast [10], raising questions about PPE1′s role and importance in vivo as the opposing enzyme of the PP2A methyltransferase, PPM1. Whereas PPM1 function (C subunit methylation) is a prerequisite for stable complex formation with B-type subunits [9,10], the PPE1 function in the regulation of trimeric PP2A holoenzymes remained obscure because conflicting data exist on its ability to demethylate the C subunit once it is bound to B-type subunits [23,24]. Thus, it is currently thought that PPE1 regulates PP2A holoenzyme assembly with B-type subunits by shifting the equilibrium between the demethylated and the methylated AC core dimer. Our data indicate a role for PPE1 earlier in the biogenesis of PP2A, namely in the regulation of complex assembly with RRD2 rather than with B-type subunits. PPE1 appears to be required for stable complex formation between the C subunit and TPD3: RRD2, and counteracts the premature methylation of the C subunit that has not yet been activated by TPD3: RRD/PTPA. Defects in C subunit biogenesis, for example by deletion of either the TPD3 or RRD genes, caused the accumulation of complexes of PPE1 and the C subunit in its low-activity conformation. Thus, PPE1 seems to constitute a surveillance mechanism that prevents the premature generation of the active C subunit in the absence of the scaffolding subunit TPD3, the major constituent of PP2A holoenzymes, or premature holoenzyme assembly in the absence of the essential activator RRD/PTPA. PPE1 seems to achieve this function in two ways, by competing with RRD2 for the binding to the C subunit and, more importantly, by keeping the C subunit in its demethylated state and thereby preventing untimely methylation that would otherwise complete the C subunit activation and promote complex formation with B-type subunits. In line with this prediction, deletion of PPE1 in a tpd3Δ strain overcame the PPE1-dependent block of C subunit maturation despite the inability for holoenzyme formation, and deletion of PPE1 in the rrd1Δ/rrd2Δ strain increased dramatically the levels of trimeric holoenzymes with CDC55 despite the fact that the C subunit was in its inactive conformation. How do defects in C subunit biogenesis lead to the increased interaction between PPE1 and the C subunit? At the moment, our data do not allow us to distinguish between several potential scenarios of PPE1 function. In the first scenario, PPE1 constitutes one of the first steps of C subunit biogenesis, interacting with the newly synthesized C subunit in its low-activity conformation. Interruptions of the biogenesis cascade would then cause the accumulation of complexes from earlier steps because dissociation of these complexes is interlocked with formation of the subsequent complex, e. g., the TPD3: RRD2 complex. In the second scenario, PPE1 plays a more active role by interacting with and demethylating C subunits that have not yet been activated by RRD/PTPA. PPE1 could achieve this by a substrate preference for any C subunit in its low-activity conformation, including those that are newly synthesized and inactive. Our results from PPE1 overexpression experiments (but also the finding of increased PPE1 binding levels in the tpd3Δ and rrd1Δ/rrd2Δ strains) are also consistent with a third mechanism in which PPE1 targets the catalytically active C subunit, demethylates it, and through its binding to catalytic site residues, converts the C subunit into the low-activity conformation, which subsequently requires RRD/PTPA as well as C subunit methylation for recycling in the biogenesis cascade. This biogenesis mechanism appears to function in mammals as well as yeast. Consistent with an affinity of PPE1 for the inactive C subunit, we were able to identify and isolate the mammalian PPE1 ortholog, PME1, by its increased interaction with catalytically defective C subunit mutants [25], and PME-1 was recently co-purified with inactive PP2A [24]. Interestingly, several of the human C subunit active-site mutant proteins also bind increased levels of PTPA (the mammalian ortholog of RRD2) as well as α4/TAP42 [13], suggesting a binding preference of both PTPA and α4/TAP42 for the inactive conformation of the C subunit. The increased binding of these molecules to C subunit mutants that are unable to fold into the catalytically active conformation and complete the biogenesis suggests that they might play key roles in the early steps of C subunit biogenesis. TAP42 is an essential protein [17] known to interact with the catalytic subunits of the PP2A family of phosphatases, including PPH21, PPH22, SIT4, and PPH3 [26]. A recent study identified RRD1 and RRD2 as part of the TAP42: phosphatase complexes [20] in the W303 strain, and confirmed earlier findings that RRD/PTPA proteins are required for phosphatase activity [13,27]. In addition, these authors detected a complex between the RRD proteins and TPD3, but the significance of this complex remained unclear. Our results now identify the functional and physical interaction between RRD2 and TPD3 in C subunit biogenesis. In contrast to their findings, we were unable to detect significant complex formation between RRD2 and TAP42 in the BY strain. Thus, the observed differences may be due to the different genetic backgrounds of the W303 and BY strains. It will be interesting to test whether and how the differences in TAP42: RRD complexes affect PP2A biogenesis in these strains. By what mechanism might RRD/PTPA regulate C subunit biogenesis? We showed recently that loss of RRD proteins results in the generation of PP2A and SIT4 catalytic subunits with impaired catalytic activity, indicating alteration of the active site. Thus, we hypothesized that RRD/PTPA might play a role in the generation of the native conformation of the PP2A and SIT4 active sites. In line with our assumption, preliminary evidence has been provided for a potential peptidyl-prolyl cis/trans isomerase (PPIase) activity of RRD/PTPA [28] that is stimulated by ATP/Mg2+ and, which could be responsible for switching the C subunit into the active conformation. Evidence for a potential PPIase activity was further provided by the recent structure determination of RRD/PTPA in complex with a proline-containing PPIase peptide substrate [29]. No ATP binding site, however, could be identified in the RRD/PTPA structure [29,30]. Interestingly, SSB2, a ribosome-associated chaperone that possesses ATPase activity and plays a role in the folding of newly synthesized proteins has been found just recently in a complex with TPD3 [31]. It is tempting to speculate that the energy necessary for the PTPA-catalyzed conformational change in the C subunit might be provided by ATP hydrolysis through the TPD3-bound SSB2. It would also provide a potential explanation for our finding of the functional and physical interaction between RRD/PTPA and TPD3. Chao et al. [32], however, identified an ATP binding pocket in their structure of PTPA and showed that PTPA and the PP2A A-C dimer together constitute a composite ATPase, whose activity is required for the transient change of PP2A into a phosphotyrosine (P-Tyr) -specific phosphatase, at least in vitro. How the loss of this transient P-Tyr–specific activity would lead to the huge decrease of the P-Ser/P-Thr–specific activity of trimeric PP2A holoenzymes that is observed in the rrd1Δ/rrd2Δ strain [13], or now in the rrd1Δ/rrd2Δ/ppe1Δ strain, is unclear. The requirement for an activator protein, however, is not unique to the PP2A family of phosphatases. Inhibitor-2 (I-2), a highly conserved protein, interacts with the catalytic subunit of protein phosphatase 1 (PP1) and inhibits its catalytic activity in vitro [33]. Deletion of GLC8, encoding the yeast I-2 ortholog, however, leads to a reduction and not to an increase of GLC7 catalytic activity, as would have been expected for the loss of an inhibitor [34]. The catalytically inactive PP1 in complex with I-2 is activated upon phosphorylation of I-2, which probably induces a conformational change in PP1, suggesting a function of inhibitor-2 as a PP1 chaperone [35,36]. Whether the I-2 chaperone function, and thus the generation of active PP1, is also coupled to and coordinated with PP1 holoenzyme assembly is currently unknown, but—given our present results—this is a likely possibility. Deletion of RRD/PTPA affected the post-translational modification state of the yeast B-type subunits CDC55 [13] and RTS1 (unpublished data), suggesting the intriguing possibility that phosphorylated B-type subunits are substrates of RRD/PTPA complexes in vivo. RRD/PTPA may represent a unique species of PP2A regulatory subunits, with a very restricted number of (potentially even P-Tyr) phosphorylated substrates. In this way, the generation of the active C subunit would be tightly coupled to the formation of substrate-specific holoenzymes. Cells could avoid potential damage from the highly active but untargeted C subunit by coupling the RRD/PTPA-dependent maturation process to the interaction of the C subunit with the scaffolding subunit TPD3, which in turn constitutes a prerequisite for holoenzyme assembly with B-type subunits (model, Figure 7). The order of events seems to be under the surveillance of the PP2A methylesterase that preferentially binds to the C subunit in its low-activity conformation, consistent with PPE1′s increased affinity for inactive C subunit mutants. Demethylation of the C subunit by PPE1 is required for the efficient interaction with RRD2, and at the same time, demethylation prevents interaction with B-type subunits. TPD3 and RRD2 are both required for the release of PPE1 and the generation of the active and specific C subunit. The RRD2-dependent activation of the phosphatase catalytic activity is coupled to the assembly with B-type subunits by targeting the RRD2: TPD3: C subunit complexes to their potential substrates, phosphorylated B-type subunits. The process is completed by C subunit methylation destabilizing the interaction with RRD2 and, at the same time, stabilizing the one with B-type subunits. This model provides an explanation for how cells deal with the inherent danger of the multisubunit enzyme architecture, namely the untargeted and unrestricted catalytic activity of the free C subunit. Saccharomyces cerevisiae strains used in this study are listed in Table 1. The double-deletion strains cdc55Δ/rts1Δ and tpd3Δ/ppe1Δ were generated by PCR-based gene targeting [37] from the single-deletion strains rts1Δ and tpd3Δ, respectively. The rrdΔ/rrd2Δ/ppe1Δ strain was obtained by mating the strains rrd1Δ/rrd2Δ (MATa) and ppe1Δ (MATα, EUROpean Saccharomyces Cerevisiae ARchive for Functional analysis [Euroscarf], http: //web. uni-frankfurt. de/fb15/mikro/euroscarf/index. html), followed by sporulation and tetrad dissection. The tpd3Δ/ppe1/Δppm1Δ strain was obtained in the same way, by mating the strains tpd3Δ/ppe1Δ (MATa) and ppm1Δ (MATα; Euroscarf). After phenotypic analysis, gene deletions were confirmed by PCR and by immunoblotting analysis of yeast lysates with specific antibodies. The strains were grown at 30 °C in rich medium (YPD) or complete minimal (CM) dropout medium supplemented with 2% glucose or 2% galactose and 0. 1% raffinose. Plasmids used for this study are summarized in Table 2. Detailed cloning strategies and information of the individual constructs can be obtained upon request. Analysis of 74 tetrads obtained by mating rrd1Δ/rrd2Δ (MATa [13]) with the tpd3Δ (MATα; Euroscarf) strain revealed the absence of any viable rrd1Δ/rrd2Δ/tpd3Δ triple-deletion mutant. To corroborate these data, the heterozygous diploid strain was transformed with the plasmid pYES2[myc-TPD3] (2 μm, URA3) and sporulated. The spores were dissected, analyzed phenotypically for the presence of resistance and auxotrophic markers (KanR, His+, and Ura+), and tested for protein expression by immunoblotting. The Ura+ rrd1Δ/rrd2Δ/tpd3Δ mutants were spotted on synthetic complete (SC) medium plates ± 1 g/l 5-fluoroorotic acid (5-FOA). The preparation of whole-cell yeast extracts was carried out as previously described [13]. HA- or myc-tagged proteins were immunoprecipitated by using anti-HA (12CA5) or anti-myc (4A6; Upstate, http: //www. upstate. com) antibodies cross-linked to BSA-coated protein A sepharose beads (Pharmacia Biotech [GE Healthcare], http: //www. gehealthcare. com/usen/index. html) as described previously [38]. To avoid stripping of the immunoblots after antibody incubation, immunoprecipitates were split into equivalent aliquots after boiling in sample buffer and analyzed by 10% SDS-PAGE. Detection of the co-immunoprecipitated proteins was performed using the monoclonal antibodies anti-HA (clone 16B12; Covance Research Products, http: //www. crpinc. com), anti-methyl-PP2A (clone 2A10; Upstate, http: //www. upstate. com), anti-RRD2 [13], and anti-TPD3 (raised against residues 65–597, clone 5G2), and the rabbit polyclonal antibodies anti-CDC55 [9], anti-PPE1 (raised against full-length PPE1), anti-PPH21 [13], anti-TAP42 (raised against full-length TAP42), and anti-TPD3 [9]. For the experiment in Figure 3B, peptide competition and re-immunoprecipitation was carried out as follows: 200 optical density at 600 nm (OD600) logarithmically growing yeast cells were used for myc-RRD2 immunoprecipitation. Immunoprecipitates were washed and eluted by two sequential incubations for 5 min at 30 °C in the presence of 100 or 50 μg/ml (first and second elution, respectively) of the myc peptide (MEQKLISEEDL) in TBS buffer. The eluates were pooled and used for the second immunoprecipitation, carried out in the same way as before, except using anti-HA antibody cross-linked to protein A sepharose beads. Monomeric HA-PPH21 was isolated by immunoprecipitation of yeast lysates, which had been treated with a basic pH shift to disrupt protein complexes, followed by neutralization. The lysates were generated as described before by [13] except that the yeast lysis buffer contained 10 mM instead of 50 mM Tris-Cl. The basic pH shift (pH 11. 5) was obtained by adding 6. 25 μl of triethylamine to 500 μl of yeast cell lysate. After 5 min incubation at room temperature, the reaction was neutralized by adding 330 μl of 0. 1 N HCl and was used for immunoprecipitation experiments/phosphatase assays as described in the next paragraph. Phosphatase activity of PPH21 and PPH22 immunoprecipitates was assayed towards 32P-labeled phosphorylase a, 32P-labeled histone H1, and para-nitrophenyl phosphate (pNPP; Sigma, http: //www. sigmaaldrich. com). Details about 32P-labeled phosphorylase a and 32P-labeled histone H1 preparation were described previously (see [38]). To determine the phosphatase catalytic activity of HA-PPH21 or HA-PPH22 towards phosphorylase a, histone HI, or pNPP, the equivalent of 50 OD600 logarithmically growing cells were used for native whole-cell lysate preparation as previously described [13]. The lysates were used for immunoprecipitation (1 h at 4 °C, under rocking) with 40 μl of anti-HA (12CA5) antibody cross-linked to BSA-coated protein A sepharose beads (1: 1 suspension in PBS). For PPH21 endogenous activity (Figure S3), the lysate was incubated with 2 μl of rabbit polyclonal anti-PPH21 serum (or preimmune serum for control immunoprecipitation) for 30 min followed by another 30 min in the presence of 40 μl of BSA-coated protein A sepharose beads (1: 1 suspension in PBS) at 4 °C. The immunoprecipitates were washed one time with yeast lysis buffer and three times with TBS. To test the catalytic activity of PPH21/PPH22 complexes (or the monomeric C subunit) towards 32P-phosphorylase a or pNPP, one fourth of the immunoprecipitate (resuspended in 400 μl of TBS) was used for activity determination. For 32P-histone HI activity analysis, 1/32 of the immunoprecipitate was used. In addition, one fourth of each of the immunoprecipitates was used to normalize the PPH21 amounts (see below). Further steps were performed as previously reported in detail for PP2A isolated from mammalian cells [38]. The unspecific activity (background) was determined by using a wild-type strain containing an empty vector (or for endogenous PPH21, a wild-type strain incubated with PPH21 preimmune serum) and was subtracted from each point determination. The assay values (average of at least three independent experiments) are presented as a percentage of the wild-type strain activity, which was set as 100%. For this, the values were normalized to the amount of immunoprecipitated PPH21/PPH22 as determined by immunoblot and densitometer analysis using the Personal Densitometer SI (Molecular Dynamics [GE Healthcare], http: //www. gehealthcare. com/usen/index. html) or the Odyssey Infrared Imaging System (LI-COR, http: //www. licor. com). We included for the assay calculations only quantifications that revealed levels of HA-PPH21/PPH22 that did not differ by more than 5. 5-fold from the wild-type reference (only the immunoblots for Figure 4C were analyzed with conventional HRP detection). Moreover, we repeated the quantification if C subunit levels differed more than 5. 5-fold, as follows: the remaining aliquots of the immunoprecipitates were reanalyzed after adjustment based on the rough quantification of the first immunoblot and analyzed by SDS-PAGE/immunoblot analysis as described above. Data are presented as mean values ± standard deviation (SD), and were analyzed using the Student t-test. Differences with p-values less than 0. 05 were considered statistically significant.
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Title: Generation of Active Protein Phosphatase 2A Is Coupled to Holoenzyme Assembly Summary: Multisubunit enzymes, such as protein phosphatase 2A, consist of a catalytic subunit and one of several regulatory subunits that are responsible for substrate specificity. Whereas this molecular architecture enables the assembly of a few components into many different substrate-specific enzymes, it possesses an inherent danger in the form of the uncomplexed catalytic subunit with its unspecific phosphatase activity. Until substrate-specific complexes assemble, the catalytic subunit would constitute a risk to the cell if no control mechanisms existed. We recently obtained evidence for the existence of the catalytic subunit in a low-activity conformation that requires an activator for the switch into the active conformation. This requirement suggested that the existing model of protein phosphatase 2A biogenesis was incomplete, because it could not explain how the activity of the catalytic subunit is kept in check until it is assembled with the substrate-targeting subunits. In this study, we provide evidence that the generation of the active catalytic subunit is coupled with and regulated by holoenzyme assembly. We propose a novel model of protein phosphatase biogenesis in which a tightly controlled activation cascade protects cells from the potential risk of unspecific dephosphorylation events.
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Summarize: Rep. Tom Price was confirmed as the next Department of Health and Human Services secretary early Friday, despite Democratic outrage over the Georgia Republican's alleged business conflicts. Price, a 62-year-old orthopedic surgeon from Atlanta, was confirmed in a 52-47 vote shortly after 2 a.m. The middle of the night vote places Price in charge of carrying out President Trump’s campaign promise to repeal and replace Obamacare. Price has been a staunch critic of the former President’s signature legislation, and has advocated for drastically cutting low-income health care provisions such as Medicare and Medicaid. Price didn’t get a single vote from Democrats, who sharply opposed his nomination after learning that he actively traded stocks in pharma companies while simultaneously pushing conservative health care policies in Congress. Trump signs executive orders he says will crack down on crime “Tom Price wants to repeal the ACA w no replacement, throwing the entire health care system into chaos,” Sen. Chris Murphy (D-Conn.) tweeted after the vote. “What a terrible choice for HHS.” The longtime GOP congressman is an unapologetic free market advocate, and has proposed replacing the Affordable Care Act with tax credits, personal health savings accounts and high-risk pools for consumers with preexisting conditions. In a statement, Speaker of the House Paul Ryan said that "for Americans struggling under Obamacare, today is a big step forward." Supporters of the Affordable Care Act in Colorado were one of many groups rallying earlier in the week in support of Obamacare, of which Price is a known opponent. (Brennan Linsley/AP) "Having Dr. Tom Price at the helm of HHS gives us a committed ally in our work to repeal and replace Obamacare and finally provide Americans with a better system," he wrote. Undocumented Arizona woman detained in display of Trump order Democrats have argued that such a replacement plan would leave millions of Americans uninsured or unable to cover significant expenses. The odd-hour vote came after Democrats prolonged a deeply divided confirmation debate. Senior GOP Sen. John Cornyn of Texas accused Democrats of threatening "the stability of the government" by blocking President Trump's nominees. Democrats responded by ripping Price's preference for restricting women's access to birth control, engaging in questionable stock trading and deriding any type of government-subsidized health care. "He seems to have no higher priority than to terminate health coverage for millions of people," said Sen. Jeanne Shaheen (D-N.H.). Rep. Tom Price, a seven-term congressman and an orthopedic surgeon by trade, had solid backing from Senate Republicans. AP Photo Senate confirms Obamacare opponent Price to lead health department The Senate early Friday morning installed Rep. Tom Price as the nation’s top health care official, putting him in charge of HHS and Republicans’ effort to make good on their pledge to repeal and replace the sweeping Affordable Care Act. Price’s confirmation came by a slim 52-47 margin, ending weeks of partisan rancor marked by ethics allegations, boycotts and sharp breaks with Senate tradition and decorum. The seven-term congressman maintained full Republican support throughout the process, ensuring his path to HHS secretary was never in real danger. No Democrats voted for him. Story Continued Below But the bitter opposition to Price’s nomination — which culminated with Democrats delaying a final vote for nearly 30 hours — served as a preview of the fight to come over the future of American health care. And Price could well start that fight immediately by using his authority as secretary to roll back or not enforce select pieces of Obamacare — the mandated benefit package, perhaps, or the hot-button birth control coverage rules. “I feel passionately about the fact that this is the wrong person to serve in that job,” Sen. Jeanne Shaheen (D-N.H.) said during a Thursday floor speech. “He seems to have no higher priority than to terminate health coverage for millions of people across this country.” The Trump administration has hailed Price, a former orthopedic surgeon, as the quarterback of its plan to tear down Obamacare and construct a replacement package by year-end. The Georgia Republican, who has served as House Budget chairman, favors a far more conservative approach to health care. He authored a 2015 replacement bill that would have eliminated many of Obamacare’s broad health benefits, and he supports drastically rolling back funding for both Medicare and Medicaid. At HHS, Price is expected to start immediately reshaping the health care system, using his administrative powers to ease Obamacare’s regulations and smooth the path for congressional Republicans to repeal and ultimately replace the law. That will likely start with relaxing rules for insurers, in hopes of soothing the industry’s nerves over participating in Obamacare’s insurance markets in the short term. “He’ll be a very thoughtful leader and will help us hopefully, from an administrative standpoint, look at the essential health benefits,” Sen. Tim Scott (R-S.C.) said, referring to requirements that insurers cover certain health services such as maternity care. Scott said the goal is “to make them less prescriptive so there’s more flexibility, and hopefully that means there’ll be more competition in the marketplace.” At the same time, Price, along with congressional leadership and the White House, will face the tough task of rallying the GOP around a single repeal and replacement strategy. Republican leadership wants to finish all that by the end of the year — a tight timeline, given the lack of consensus among Republican lawmakers — and are looking to Price to make it happen. He’ll get no help from Democrats, who vowed to fight Obamacare’s replacement every step of the way. In the run-up to Price’s confirmation, they painted his health care beliefs as extreme and regressive, and they hammered him for potential conflicts of interest tied to his more than $300,000 in personal health-related stock investments. “It’s hard to imagine who in America would be better off under Congressman Price’s leadership at HHS,” said Sen. Patty Murray (D-Wash.), ranking member of the HELP Committee and one of Price’s chief critics. “What makes his nomination even more troubling are the serious ethics questions that haven’t been resolved as it has been jammed through the Senate.” The scrutiny over Price’s investments produced one of the most heated moments in the new Congress, when Democrats refused to show up for a committee vote on Price’s nomination. The impromptu boycott, which was in response to a Wall Street Journal report suggesting Price misled lawmakers about his stake in an Australian biotech firm, delayed the committee vote. The following day, Republicans suspended committee rules to force through Price’s nomination without any Democrats present. “What a situation that our colleagues tried to put this good man in. It’s disreputable,” Finance Committee Chairman Orrin Hatch (R-Utah) said Thursday, still fuming over the boycott. “There ought to be some courtesy here, and I’m kind of shocked that there isn’t.” Price has committed to divesting from his health care investments within 90 days of joining the Trump administration. But Democrats, who repeatedly called for investigations into Price’s financial holdings, warned that scrutiny will follow Price into his new job. Price’s confirmation represents the last major piece Republicans say they need to start dismantling Obamacare. After more than seven years of waiting, the GOP must now figure out how exactly they will go about repealing and replacing a law that provides coverage to 20 million people — a question Republican lawmakers hope Price, one of the few to author a health care plan, will be able to answer. “I think you have to admire that he was willing to make a serious proposal,” said Sen. Tom Cotton (R-Ark.). “That’s the kind of leadership we need.” The Senate confirmed Rep. Tom Price (R-Ga.) to serve as secretary of Health and Human Services during a 2 a.m. vote Friday. No Democratic senator supported Price in the 52-47 vote. He overcame an initial procedural hurdle on Wednesday evening along party lines. ADVERTISEMENT With the GOP's 52-seat hold on the Senate and only a simple majority required, Democrats don't have the manpower to block any nominees on their own. But they signaled early on that Price would be a top target and spent hours on the Senate floor ahead of the vote protesting his confirmation because of his support for nixing the Affordable Care Act and overhauling Medicare. “Make no mistake: In the dark hours of the early morning, with the confirmation of Secretary Price, the Republicans launch the first assault in the war on seniors,” said Senate Minority Leader Charles Schumer Charles (Chuck) Ellis SchumerRSC Chairman: Harvey aid could be jeopardized if linked with debt ceiling Dems prep for major fight over Trump USDA science pick Ex-Medicare chief promotes ObamaCare enrollment on Twitter after Trump cuts outreach funding MORE (D-N.Y.). Democrats argue Price’s positions are out of line with Trump's campaign promises, among them that he wouldn’t cut entitlement programs. Sen. Bernie Sanders Bernard (Bernie) SandersWeek ahead: Congress returns to take up bipartisan health care effort Sanders urges support for progressive policies on Labor Day Top Dem: Trump ending DACA would be his administration hitting 'rock bottom' MORE (I-Vt.) called Price — who has previously backed privatizing Medicare — the "exact opposite" of what Trump campaigned on. "My opposition to Congressman Price has less to do with his well-known extreme right-wing economic views than it has to do with the hypocrisy and dishonesty of President Trump," Sanders said. Price, who was chairman of the House Budget Committee when nominated, said late last year that Republicans would move to overhaul Medicare within six to eight months of Trump’s inauguration. He also voiced support in 2015 for a proposal to reduce Medicare costs by delivering benefits through a voucher program. Democrats are all but guaranteed to use the vote on Price, and his position on Medicare, to target Sens. Dean Heller Dean Arthur HellerWeek ahead: Congress returns to take up bipartisan health care effort Trump, Congress face packed September agenda Heller leads primary by 22 points in internal poll MORE (Nev.) and Jeff Flake Jeffrey (Jeff) Lane FlakeThe Memo: Trump faces critical fall North Korea fears demand attention despite domestic concerns Flake: ‘Harsh rhetoric’ not slowing down North Korea MORE (Ariz.) — two Republicans up for reelection in 2018. Price faced a rocky confirmation battle, also coming under fire for a growing number of reports about his stock trading as a member of Congress. CNN and Time reported last month that he had invested in health-related companies shortly before introducing or supporting legislation that benefited them. Price denied wrongdoing, adding that he made the investments through a broker. But Sen. Elizabeth Warren Elizabeth Ann WarrenGoogle faces blowback after think tank fires critic Judd Gregg: The left's theater of the absurd Kamala Harris and Kirsten Gillibrand will lead Democrats to 2020 victory MORE (D-Mass.) — in her first floor speech since she was temporarily banned from speaking on Tuesday night — said Price should be disqualified for “basic ethics.” "He should have withdrawn his nomination weeks ago, and if he didn't go voluntarily, the president and his friends in Congress should have quietly but forcefully pushed him out," she said. The Wall Street Journal also reported last month that he received a 12 percent discount on his purchase of biomedical stock. But Republicans never publicly signaled that they were wavering on Price. They believe getting him confirmed is key to syncing up with the White House on the plan to repeal and replace ObamaCare. Trump and congressional Republicans — as well as House and Senate lawmakers — have at times struggled to get on the same page about a timeline for repealing ObamaCare or how to replace it. They included a Jan. 27 date for repeal proposals in a budget bill passed earlier this year, but lawmakers acknowledged at the time that they wouldn't meet the deadline. Senate Majority Whip John Cornyn John CornynSenate panel expected to hold hearing on immigration visas Conservative leader: Don't tie Harvey relief to debt ceiling Cruz rips Christie after Harvey criticism: He is 'desperate' for media spotlight MORE (R-Texas) said Thursday that he hopes confirming Price will speed up the process. “After seven years we’ve got all kinds of great ideas, but we have to come together behind one,” he told reporters. Senate Majority Leader Mitch McConnell Addison (Mitch) Mitchell McConnellThis week: Harvey aid at top of long to-do list as Congress returns The Memo: Trump faces critical fall Week ahead in finance: Lawmakers brace for high-stakes September MORE (R-Ky.) praised Price separately as the “right man” for to lead the department. “Price knows more about health care policy than just about anyone. He doesn’t just understand health care policy as a policy maker … he also understands it as a practicing physician,” he said. “He gets the real-world impact.” Georgia Republican Tom Price, the conservative lawmaker who has been one of Congress’s most vehement opponents of the Affordable Care Act, was sworn in Friday morning as health and human services secretary after a starkly divided Senate vote to confirm him. Price won confirmation on a party-line vote of 52-47, which came around 2 a.m. Senate Democrats, united in their opposition to him as the government’s top health official, argued that the intersection of his personal investments and legislative behavior warranted deeper scrutiny of his ethics. But without the numbers to defeat his confirmation, Democrats instead marshaled a protracted war of words, reading testimonials from Americans with severe, expensive-to-treat illnesses and gratitude to the ACA, Medicare or Medicaid — cornerstones of federal health policy that the Democrats accused the nominee of wanting to undermine. The new secretary offered no remarks following his swearing-in hours later by Vice President Pence. But the biggest challenge he faces — one on which progress in Congress seems uncertain for now, despite GOP lawmakers’ pledges for immediate action — was the first subject Pence mentioned in his introduction. “President Trump has made it the top priority of this new Congress to repeal and replace the Affordable Care Act with health-care reform that will lower the cost of health insurance without growing the size of government,” Pence said. Vice President Pence swears in Tom Price as the new health and human services secretary as his wife, Betty Price, holds a bible. (Mark Wilson/Getty Images) Price’s confirmation to lead the Department of Health and Human Services raises anew the question of what role the White House will play as Republicans try to come up with an approach to abolishing the 2010 law and creating a more conservative set of health-care policies. [Republican town halls are getting very, very nasty] At his first news conference after the election in November, Trump said that his administration would submit a repeal-and-replace plan “almost simultaneously” with Price becoming secretary. He said the plan “will most likely be on the same day or the same week, but probably the same day. Could be the same hour.” In an interview with The Washington Post the following weekend, Trump said that a plan with the goal of “insurance for everybody” was “very much formulated down to the final strokes.” He reiterated that he was just waiting for Price to take office. Yet when Price was asked at his confirmation hearing whether he was involved with writing a plan, he drew laughs by partly deflecting the question. Trump has since seemed to retreat from his earlier timetable. In an interview with Fox News host Bill O’Reilly that aired Sunday, he described the process of crafting a new plan as “very complicated” and acknowledged that undoing a law and putting something in its place “takes a while to get.” “Maybe it’ll take till sometime into next year,” Trump said, although “the rudiments” could be released in late 2017. His comments were ambiguous about whether the White House was preparing its own plan. “We’re gonna be putting it in fairly soon,” he said, without elaborating. In their home districts, some Republican lawmakers are hearing from constituents increasingly worried that their health coverage could be interrupted or lost if the GOP-controlled Congress rescinds the law, as long promised, without a clear sequel. Leaders for the health insurance industry and state insurance commissioners warned last week that more health plans almost certainly will defect from ACA marketplaces for 2018 unless Congress and the Trump administration provide some concrete assurances within the next two months. Price, a 62-year-old orthopedic surgeon from a wealthy Atlanta suburb, chaired the House Budget Committee and led Republican opposition to the ACA, including helping to draft several comprehensive bills to replace it. The debate over his nomination had opened in the Senate on Wednesday night with Democrats casting the prospect of him leading HHS in apocalyptic terms. [Tom Price belongs to a doctors group with unorthodox views on government and health] It began with Sen. Ron Wyden (Ore.), the ranking Democrat on the Senate Finance Committee, predicting that “this is about whether the United States will go back to the dark days when health care worked only for the healthy and wealthy.” Sen. Bernie Sanders (I-Vt.), who as a 2016 presidential candidate energized many progressive voters, accused the White House of hypocrisy. The president, he contended, had campaigned on promises not to cut the nation’s main entitlement programs but then chose as his HHS secretary a congressman who has long sought to weaken them. “The American people are still waiting for that one tweet that says, ‘I will keep my promise,’” Sanders said. However, Senate Majority Leader Mitch McConnell (R-Ky.) delivered a sharply different portrayal, saying that Price “doesn’t just understand health-care policy as a policymaker, though he does deeply. He also understands it as a practicing physician.” As debate was ending, Wyden returned with a stinging denunciation. The Democrat focused on what he called “a coverup” by Price about his purchase last year of discounted stock in a small Australian biotechnology company in which a fellow GOP congressman is the largest shareholder. Alleging that the nominee “failed to come clean” about whether he had special access to buy the stock because of his position, Wyden said, “It ought to shake this body’s confidence in Dr. Price.” The starkly split vote that followed mirrored the polarized outcome of several of Trump’s Cabinet-level appointments — which moved forward with little of the comity that has traditionally defined the Senate’s culture. Friday morning, House Speaker Paul D. Ryan (R-Wis.), who has been closely aligned with Price for years, praised his confirmation. “For Americans struggling under Obamacare, today is a big step forward,” Ryan said in a statement. “Having Dr. Tom Price at the helm of HHS gives us a committed ally in our work to repeal and replace Obamacare and finally provide Americans with a better system.”
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Summary: A man who wants to do things to ObamaCare that the Hippocratic oath might forbid him doing to a patient is now the top health official in America. Tom Price, a longtime Republican congressman from Georgia, was confirmed as the next Department of Health and Human Services secretary early Friday, the New York Daily News reports. He was confirmed in a 52-47 vote at around 2am without a single Democrat voting in favor. Price, a 62-year-old former orthopedic surgeon, has long been one of ObamaCare's fiercest critics and also supports slashing Medicare and Medicaid funding, Politico reports. During confirmation hearings, Price said he was "offended" by questions from Democrats about his trading in medical stocks. Democrats had little hope of blocking the nomination, though they protested with hours of Senate speeches, the Hill reports. "Make no mistake: In the dark hours of the early morning, with the confirmation of Secretary Price, the Republicans launch the first assault in the war on seniors," Sen. Chuck Schumer said. Sen. Bernie Sanders called Price's policies the "exact opposite" of what Trump promised on the campaign trail. "My opposition to Congressman Price has less to do with his well-known extreme right-wing economic views than it has to do with the hypocrisy and dishonesty of President Trump," Sanders said. Majority Leader Mitch McConnell, one of the few Republicans to speak, praised Price as somebody who understood health care both as a policymaker and a physician, the Washington Post reports.
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Summarize: CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of Ser. No. 09/757,863 filed Jan. 11, 2001 now abandoned. BACKGROUND OF THE INTENTION 1. Field of the Invention The present invention generally relates to food substances, and in particular to beverages. More particularly, the present invention relates to ultra heat treated (UHT) milk drinks which have been carbonated or aerated. Preferred embodiments of the present invention include an ultra heat treated, (UHT) sparkling skim milk including combinations of all ultra heat treated, (UHT) milk and all flavors of ultra beat treated, (UHT) milk, all ultra heat treated, (UHT) fruit juice combinations, ultra heat treated, (UHT) fermented yogurts, ultra heat treated, (UHT) non-dairy products, e.g.: soya, field milk, artificial milk and the like. In addition, the present invention relates to a method of carbonating ultra heat treated, (UHT) milk in aseptic and non-aseptic packaging. Although the present invention will be described with particular reference to an ultra heat treated, (UHT) milk drink aerated with sterilized and purified carbon dioxide and to methods of carbonating the treated milk, it is to be noted that the present invention is not necessarily limited to this embodiment and the scope of the invention may be more extensive by including within it other beverages aerated with other gases and gas mixtures of varying ratios, such as substitute milk products, for example soya substitutes. It is to be noted that the use of the general term, ultra heat treated (UHT) milk products include all kinds of ultra heat treated (UHT) milk products whether natural or artificial milk or substitute milk materials, such as for example, whole milk, low fat milk, modified milk, yogurt, and milk products in general including combinations with other beverages such as fruit juices, soy and the like. 2. Description of Related Art Soft drink sales and sales of aerated and or carbonated beverages in general have over a period of time increased so that not only are more beverages in general being sold but the market share of soft drinks and other carbonated drinks have been increasing at the expense of other types of drinks, such as milk product drinks. In order to increase the appeal of milk as a drink and hopefully to gain an increased market share for milk drinks, particularly in situations away from a refrigerator such as during out-of-home activities, it has been thought that if carbonated or aerated milk was available, this product, because of its similarities to soft drink in appeal, would be successful in view of it being perceived as a more healthy alternative to soft drinks. The use of ultra heat treated (UHT) milk is well established in the art. It is well known that preheating milk to a temperature of 80 degrees centigrade then heating the preheated milk intensively for a short period of time, i.e. for approximately 2-5 seconds, to approximately 130 degrees-150 degrees centigrade followed by instant cooling to a temperature below 80 degrees-90 degrees centigrade will allow milk to become shelf stable. This heat sterilization process which also uses pressure during the ultra heat process kills micro organisms and allows one to keep milk shelf stable without refrigeration. Typical ultra heat treated (UHT) milk formulation processes are set forth in the U.S. Pat. No. 3,973,048 to Tetra Pak Development SA (1976), U.S. Pat. No. 6,163,532 to Parmalat S.p.A. (2000) and in U.S. Pat. No. 4,921,717 to Milk Marketing Board (1990). Unfortunately, these patents do not relate to the process of carbonating or aerating the ultra heat treated (UHT) milk. It is to be noted that the carbonation of milk is also well established in the art. Typical milk carbonation formulas arc set forth in U.S. Pat. No. 607,362 to FWH Graeff (1898), in U.S. Pat. No. 1,570,975 to D. Sweeney (1926), in U.S. Pat. No. 2,394,303 to H. Griesbeck (1940), in U.S. Pat. No. 4,804,552 to Dam' Research (1989) and U.S. Pat. No. 4,919,960 to Dairy Research (1990). Unfortunately, these patents do not relate to carbonating ultra heat treated milk products using a pre-heat and pressure process. In addition, these patents relate to the need of refrigerating the finished milk product and are not shelf stable. Thus, a need exists for a carbonated or aerated ultra heat treated (UHT) milk beverage which uses a pre-heat/pressure process and does not need refrigeration for the finished product and is shelf stable. OBJECTS AND ADVANTAGES Accordingly, several objects and advantages of my invention are: (A) to provide a method for carbonation of pre-heated and pressurized ultra heat treated (UHT) milk products and combinations of preheated and pressurized ultra heat treated (UHT) milk products and to provide a pre-heated and pressurized ultra heat treated (UHT) carbonated milk product that may also be flavored. (B) to provide a method to aerate or carbonate pre-heated pressurized ultra heat treated (UHT) milk with suitable gas or mixture of gases in a suitable package whereby the preheated and pressurized ultra heat treated (UHT) milk products remains carbonated in the package for a reasonably long shelf life until opened and no refrigeration is required. (C) to provide a method of aerating or carbonating pre-heated and pressurized ultra heat treated (UHT) milk comprising of introducing a gas or a mixture of gases such as carbon dioxide into an pre-heated and pressurized ultra heat treated (UHT) milk product under pressure, maintaining the pre-heated and pressurized ultra heat treated (UHT) milk product in an aerated or carbonated state and packaging the aerated or carbonated preheated and pressurized ultra heat treated (UHT) milk product into a package. Typically, the package includes bottles, aluminum cans, cartons of paperboard, aseptic or non aseptic packaging, plastics including PET, polypropylene and mixtures thereof or the like. (D) to provide a method of aerating or carbonating pre-heated and pressurized ultra beat treated (UHT) milk to include whole milk, low fat milk, total milk solids, non-fat milk, soy or any other product conventionally referred to as milk or dairy product or non-dairy product or artificial milk product or milk substitute product where solid or liquid of any viscosity. (E) to provide a method of carbonating pre-heated and pressurized ultra heat treated (UHT) milk without the excessive foaming and destabilization caused by sparging with gases at lightly elevated temperatures. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a general flow chart of a typical embodiment of the invention. FIG. 2 is a flow chart of the preferred embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION In the present invention, the foregoing difficulties are obviated in that there is provided a method to carbonate or aerate preheated and pressurized ultra heat treated (UHT) milk. In a preferred embodiment the carbonated or aerated pre-heated ultra heat treated (UHT) milk is flavored and packaged in sterile bottles, cans or aseptic packaging. In accordance with the invention, the un-carbonated milk can be processed into preheated and pressurized ultra heat treated (UHT) milk in the usual well know in method in the art. To carbonate and package, the following formulations can be utilized: EXAMPLE 1 A. (Ultra Heat Treatment) The milk sample, optionally containing any suitable, conventional or desirable additives is pumped from a stainless steel mix tank to an ultra heat treatment (UHT) plant where in the first stage which is the UHT pre-heater being a plate heat exchange, the milk is indirectly heated from a temperature of from 4 to 8 degrees centigrade to a temperature of about 138 degrees centigrade. After attaining a temperature of 138 degrees centigrade the milk is ultra heated from 138 to 150 degrees centigrade in the UHT heater where it is held at a pressure of 700 KPA or an appropriate pressure for about 2.25 seconds within limits as required to kill microorganisms, pathogens, bacteria and the like. It is noted the precise conditions can change such as at a temperature of 153 degrees centigrade or similar. Thereupon it is cooled, preferably to below 10 degrees centigrade more preferably to 2 degrees centigrade immediately. At this point a flavoring agent may be introduced preferably from about 0.1% to 3% or as desired. Thus, at this stage in the regeneration part of the UHT plant, there is now provided a pre-heated and pressurized ultra heat treated optionally flavored milk product which is well known in the art. B. (Milk Product Carbonating) The pre-heated and pressurized ultra heat treated (UHT) milk product may be carbonated in the following ways by direct injection of sterilized, purified carbon dioxide gas into the coding chamber section of the plate heat exchange to the stainless steel holding receptacles, refrigerated in aseptic modified pressurized maturing tanks. One form of the carbonator is substantially integral with the UHT plant. Another form of the carbonator may be located remote from the UHT plant. Briefly, the carbonator which may be especially designed or modified for the present UHT plant or may be a conventional carbonator either as is or modified as required comprises a carbon dioxide inlet for receiving carbon dioxide or a mixture of gases, a filter and an injection nozzle (atomizer). Typically, the supply of carbon dioxide is from 11 to 56 kg per hour and is typically delivered under pressure range of from 50 KPA to 200 KPA. The carbon dioxide then passes through a filter sterilization to ensure that the carbon dioxide which is used to carbonate the pre-heated ultra heat treated milk is pure, sterilized and aseptic. Typically, the carbon dioxide undergoes gas filtration and sterilization to produce a substantially purified carbon dioxide. After passing through the filter the carbon dioxide is passed to a carbon dioxide injector for actual injecting gaseous carbon dioxide into the pre-heated ultra heat treated milk. Typically, the injector is an adjustable or otherwise controllable carbon dioxide used in the beer brewing or bottling industry or a modified sparger. Typically, the amount of carbonation is about 3 vols./8 vols. and the carbon dioxide is supplied at a pressure of 60 p.s.i to the preheated ultra heat treated milk. The injection of the carbon dioxide takes place by sparging the pre-heated ultra heat treated milk. An inlet for the carbonator is provided to admit the pre-heated ultra heat treated milk thereto from the remainder of the UHT plant. A further inlet in the form of a conduit is provided for admitting carbon dioxide for atomization. Another example is a sparge pipe provided with an injector for dispersion, in one embodiment the pre-heated ultra heat treated milk product is passed through the injector. Once the pre-heated ultra heat treated milk has been carbonated at 2 degrees centigrade +/−1 degrees centigrade it is pumped to a pressurized carbonator holding tank where the carbonated product is held under pressure in order to maintain its carbonation to fully mature and saturate. Typically, the carbonator holding tank is maintained at a pressure of 450 KPA and a temperature of 2 to 6 degrees centigrade. The holding tank is provided with a carbon dioxide sensor probe for measuring the amount of the carbonation of the pre-heated ultra heat treated milk product. In another embodiment, carbon dioxide from the carbonator is used to carbonate the pre-heated ultra heat treated milk product in line in a manner similar to that described using an injector but with the difference that in this case it is in-line sparger. In this embodiment, the carbon dioxide is delivered at a pressure which is compatible with the filling operations, typically of about 450 KPA. The carbon dioxide is cooled to about 0.2 centigrade or thereabouts and is filtered by two sterile micron filters, typically each being a 3 stage filter arrangement. The amount, quality and other characteristics of the carbon dioxide is maintained by an in-line gas analyzer set up for carbon dioxide gas. Control of the distribution of carbon dioxide to the in line type modified sparger for pre-heated ultra heat treated milk is by means of a carbon dioxide control panel which controls the amount of carbon dioxide being delivered to various locations for carbonating the ultra heat treated milk product. Conventional gas control means are employed. In the event that the amount of carbon dioxide levels or the amount of the carbonation of the pre-heated ultra heat treated milk product is insufficient, suitable sensors connected to the gas analyzer detect this condition so that by means of the control panel, the insufficiently carbonated product can be diverted to be reprocessed through the carbonator in a return loop to a holding tank to be re-pasteurized to be within the specification. C. (Packaging) After the pre-heated ultra heat treated milk is carbonated by the process described above either using a carbonator more or less integral with the UHT plant, a separate carbonator or an in line sparging pipe, it is conveyed to an aseptic pressure balanced tank which is specifically designed to settle/pacify the pre-heated carbonated ultra heat treated milk. It is then conveyed to the packaging station where the pre-heated carbonated ultra heat treated milk product is packaged into sterile containers. Prior to packing the milk, suitable containers such as for example cans or bottles, are sterilized by dry heat such as for example using hot blasts of air heated to a temperature of 150 degrees centigrade directed onto the cans or bottles for about 58 seconds or a chemical (cooled refrigerated) twist rinse sterilizing bath suitable for bottles, including plastic bottles using ultra violet sterilization. The bottles or cans may be additionally sterilized by the injection of nitrogen. The milk product is maintained at a suitable pH depending on the product, preferably 4.0 to 5.7 during the packaging operations. This suitable pH levels keep the pre-heated ultra heat treated milk from approaching or going below the isoelectric point of the milk and thus avoiding the unwanted destabilization and or foaming experienced by the prior art. Just prior to filling, the sterilized containers are purged with sterilized purified carbon dioxide and or nitrogen. Typically, the packages into which the carbonated milk is packed are bottles, cans, plastic containers, either rigid or flexible. Additionally, the caps, tops, lids, closure means or the like are either separately or simultaneously sterilized by any suitable or convenient method. After packaging the milk product into individual containers, the milk may be further sterilized by non-toxic radiation or pasteurization. The filling operations is by means of a modified counter pressure aseptic filler conveniently housed in a clean environment, The finished packages are then pressure checked, product coded, boxed into cartons and the cartons are warehoused accordingly. A general flow chart for the typical embodiment of the invention described herein is illustrated in FIG. 1 at 100. As shown in the chart, the milk 110 is preheated 120 before it undergoes UHT treatment 130. The milk is then cooled 140. In the next step, flavor is added 150, followed by carbonization 160. The product is then placed in sterile packaging 170. A general flow chart for the preferred embodiment is shown in FIG. 2 at 200. First, a solution of non-fat dry milk is created 210 and introduced to the process 220 in the main tank. Then there is a first microfiltering step 230 of the raw milk solution. This pretreatment filtering step removes microorganisms and proteases before the UHT treatment. This step improves the flavor and the texture of the final product. The solution is then pre-heated and given UHT treatment at a high temperature for a short time 240. The solution 220 is then put in a blending tank 250. As desired, flavorings, added vitamins, preservatives and other added ingredients found in one or more storage tanks 260 are added to the solution in the blending tank 250. As described below, the additions are preferably added sequentially. Next, the product is homogenized 270 and put through a final blending process 280. The product is then cooled 290 and carbonated 300. Next the product is put through a filling process 310 into a container. In the preferred embodiment, the product is placed in cans 320 after they have been placed through a rinser 330. One inside the container, the product is cooled 360. The cooling is preferably accelerated by an air blowing step 370. The containers are then coded 380. Coding 380 may include date coding, plant coding and batch coding. The product is preferably then tested 390, especially to determine if it has the proper pH. The product is then cased 400 and sent to storage 410 if desired. In the blending tank, ingredients are preferably added in sequence and are each mixed at a low shear speed. Sugar and corn syrup are added and mixed for a minimum of five minutes. Flavorings and colors are added next and mixed for a minimum of five minutes. Calcium supplementation and vitamin supplementation are added next and mixed for a minimum of two minutes. Phosphoric acid and citric acid are added next and mixed for a minimum of two minutes. Preservatives and stabilizers are added next and mixed for a minimum of five minutes. The blended mixture is then homogenized under high pressure of approximately 2,000 psi to 5,000 psi for approximately 5 minutes. After homogenization, the mixture is blended again for a minimum of five minutes to ensure even viscosity and stabilization of the milk proteins. The mixture is then cooled, followed by carbonization. The carbonated mixture is then sent to the filling station, sealed, pasteurized and cooled. Calcium benzoate is the preferred calcium supplementing agent, although other agents such as calcium picolinate, calcium aspartate, calcium gluconate, calcium ascorbate, calcium glutamate and calcium palimate may be used. The preferred sugar is commercially available granulated extra fine sugar; however, other sweeteners such as crystalline fructose and fructose polymers, glucose, saccharine, sucrose, sucralose and aspartme may be used. It is also preferred that vitamin supplementation is made with Vitamin A, Vitamin C and Vitamin D. The vitamins may also be added sequentially. The preferred preservative is benzoic acid, although equivalent preservatives such as sodium benzoate, potassium benzoate and calcium benzoate may be used. The preferred stabilizer is kappa-carageenan. Using the above described steps, the product made is of a flavor and consistency which has never been known before. Children have found this nutritious product to be tastier than any known similar product. Thus, the subject product is more fun, healthy, and tasty for children than any product of the prior art. While the process can be made for unflavored product, or any flavor at all, three flavors have had heretofore previously unknown success in flavorfulness: lemon-vanilla, strawberry and orange. The lemon vanilla flavor preferably has a sweetness of approximately 12.6° on the Brix scale. The added flavoring preferably has a total acidity of approximately 26.6 gm/L. Also, the preferred amount of carbonation in the final product is 2.2% volume of CO2, with a total acidity of approximately pH 3.5 +/−0.05. Also for a 250 ml can of the final lemon vanilla flavored product, the contents generally comprise those listed in Table 1, in the general proportions stated. TABLE 1 LEMON VANILLA FLAVORED BEVERAGE RAW MATERIAL CONTENTS (G) RATIO (%) Skimmed Milk Powder 2.39080 0.91000 Sugar 15.10004 5.74747 Corn Syrup 20.13340 7.66330 Citric Acid 0.34732 0.13220 Phosphoric Acid 0.07882 0.03000 Vitamin A 0.01540 0.00586 Vitamin D 0.00402 0.00153 Flavor 0.26887 0.10234 Liquified Carbon Dioxide 2.16073 0.82243 Water 222.22560 84.58486 TOTAL 262.72500 100.00000 The strawberry flavor preferably has a sweetness of approximately 12.0° on the Brix scale. The added flavoring preferably has a total acidity of approximately 21.3 gm/L. Also, the preferred amount of carbonation in the final product is approximately 2.2% volume of CO2, with a total acidity of approximately pH 3.5 +/−0.05. Also for a 250 ml can of the final strawberry flavored product, the contents generally comprise those listed in Table 2, in the general proportions stated. TABLE 2 STRAWBERRY FLAVORED BEVERAGE RAW MATERIAL CONTENTS (G) RATIO (%) Skimmed Milk Powder 1.83470 0.70000 Sugar 14.59394 5.56808 Corn Syrup 19.45859 7.42411 Citric Acid 0.28126 0.10731 Phosphoric Acid 0.05242 0.02000 Vitamin C 0.02621 0.01000 Vitamin A 0.01541 0.00588 Vitamin D 0.00401 0.00153 Natural Food Color 0.03932 0.01500 Flavor 0.44806 0.17095 Liquified Carbon Dioxide 2.16073 0.82439 Water 223.18535 85.15275 TOTAL 262.10000 100.00000 The orange flavor preferably has a sweetness of approximately 12.0° on the Brix scale. The added flavoring preferably has a total acidity of approximately 21.0 gm/L. Also, the preferred amount of carbonation in the final product is approximately 2.2% volume of CO2, with a total acidity of approximately pH 3.5 +/−0.05. Also for a 250 ml can of the final orange flavored product, the contents generally comprise those listed in Table 3, in the general proportions stated. TABLE 3 ORANGE FLAVORED BEVERAGE RAW MATERIAL CONTENTS (G) RATIO (%) Skimmed Milk Powder 2.38511 0.91000 Sugar 14.30432 5.45758 Corn Syrup 19.0724 7.27677 Citric Acid 0.24425 0.09319 Phosphoric Acid 0.07863 0.03000 Vitamin C 0.02621 0.01000 Vitamin A 0.01541 0.00588 Vitamin D 0.00401 0.00153 Natural Food Color 0.02359 0.00900 Flavor 0.29059 0.11087 Liquified Carbon Dioxide 2.16075 0.82440 Water 223.49472 85.27078 TOTAL 262.10000 100.00000 Also, for the optimum flavor profile, the lemon vanilla flavoring is preferred to be a combination of the following flavor ingredients available from the Lotte Chilsung Beverage Company in Korea: Soda Flavoring No. 228, Soda Flavoring No. 87A-341, Givavdan NK-1200 and Hyvong-Base-T. The strawberry flavoring is preferred to be a combination of Soda Flavoring No. 228, Givavdan NK-1300, Hyvong-Base-T, Nocke 96-0985-D and Q61129. Conclusion, Ramification and Scope of the Invention Accordingly, the reader will see that a carbonated pre-heated and pressurized ultra heat treated (UHT) milk beverage of the inventor provides a solution to: aerating or carbonating a pre-heated and pressurized ultra heat treated milk product comprising introducing a carbon dioxide˜as or mixture of gases under pressure into a milk product. maintaining the pre-heated ultra heat treated milk product in an aerated or carbonated state and packaging the aerated or carbonated milk product into a shelf stable non-refrigerated package. the destabilization effect (frothing and foaming) which occurs when milk is carbonated. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within its spirit and scope.
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Summary: An aerated or carbonated milk product drink having a shelf stable pre-heated and pressurized ultra-heat treated milk product which has been carbonated with a gas or gases under pressure and packaged into a container. The milk product may be natural or artificial milk product including dairy products and non-dairy milk products and includes combinations of milk products with other beverages such as fruit juices. The method of producing the shelf-stable carbonated milk product of the present invention comprises injecting under pressure carbon dioxide gas or a mixture of gases into the milk product at low temperature of less than 10 degrees centigrade and high pressure of from 50 kpa to 200 kpa. The carbonated milk product remains carbonated and shelf stable in the package until opened.
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Summarize: 2 Killed In 1-Car Crash In Collin CountyThe Department of Public Safety said preliminary information indicates a Chevrolet Camaro was traveling north on SH 121 north of Anna when the driver lost control. Search Party Finds Body In Creek Matching Description Of Missing Ennis MotherSearch volunteers found the body of a female matching the description of missing Ennis mother Emily Wade in a creek bed. 2 Former Plano High Players Will Face Off In Super Bowl LIIIRex Burkhead, running back for the New England Patriots and Los Angeles Rams rookie offensive lineman Joseph Noteboom are the Plano Wildcat alumni going against each other on Super Bowl Sunday. NFL On CBS Color Analyst Tony Romo Garners Praise For AFC Championship CallNFL on CBS color analyst Tony Romo is collecting MVP votes for his call of the AFC Championship game between the New England Patriots and the Kansas City Chiefs on Sunday. JPS Hospital Worker Ends Up In ER After Elevator AccidentAn employee at John Peter Smith Hospital was injured Sunday in an elevator accident, according to a spokesperson for the trauma center. Dallas Officer Arrested For DWI With His 2 Children In CarCedar Hill Police said around 12:40 a.m. a driver traveling behind Kevin Masters reported Masters' vehicle "almost hit a wall and was swerving in and out of its lane." Body Found After Apartment Fire In North Richland HillsIt happened at the Hilltop Apartments in the 6400 block of Iron Horse Blvd. The roof of one of the apartment buildings collapsed. Former Cowboys Running Back Darren McFadden Arrested At McKinney Whataburger For DWIFormer Dallas Cowboys running back and Arkansas Razorback standout Darren McFadden was arrested Monday in Collin County for driving while intoxicated. Some trouble today for a couple of Sulphur Springs Middle School teachers after they handed out classroom awards to each student bearing the moniker, "The Ghetto Classroom" awards. One student received the "Huh?" award. His grandmother, Debra Jose, was not amused. "I had to take a second look. I was like, really? How could a teacher put this on there?...Did she just say ghetto on a certificate that she was giving my grandson?” Jose wondered. A picture of the award was shared on Facebook. Turned out, other parents didn't ‘get the joke' either. It did not take long for Sulphur Springs Superintendent Michael Lamb to hear about it. Lamb describes his initial reaction, "Shocked. Shocked. Truly, it goes in layers... You kind of ask yourself, had anything else been used, the 'teacher's name' award, would it start to seem more acceptable. The “huh?” award just begs questions. And then the 8th annual brings questions too." Two teachers' names, Mrs. Garner and Mr. Couch, are on the certificate, as well as the principal's. Lamb says the principal did not, in fact, sign them, and was completely unaware of the awards. Mrs. Garner is a second year faculty member. "I believe she'd done the same in previous district," Lamb says. But not just in other districts; here, as well. "It's my understanding the same award was given last year to up to 60 kids." The school has begun an investigation into these awards and are in the process of meeting with teachers involved as well as students' families. Lamb wants to offer the benefit of the doubt, saying, "You'd like to think the best, and, as bad as it may seem, you'd hope there's some logical explanation and it's not that bad, and that's part of what were looking into today." For Jose, what may seem as innocent as a classroom end-of-year celebration brings up a slew of past issues with the use of one word. "Back in the day, when I was growing up, they segregated us. They put us in a part where they said we were 'ghetto.' If she knew what ghetto meant, she would have never approached that, because, being an African-American, we were always thrown that.” Jose says she's waiting for an apology and is looking forward to seeing what steps the school district takes. Copyright KLTV 2015. All rights reserved.
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Summary: Coming home with an award from school is one thing-bringing home a "ghetto classroom award" signed by your teacher is quite another. "Tears just started falling out of my eyes," Texas resident Debra Jose tells CBS DFW of her grandson's dubious honor. "I was like, 'What did they just do to him again?' I just lay in bed and thought about it all night long." Officially labeled "8th Annual Ghetto Classroom Awards," the certificates were handed out in a class of learning-challenged students at Sulphur Springs Middle School in Sulphur Springs, Texas. They bear signatures over the names of two teachers in the class and "SSMS Principal," but Superintendent Michael Lamb says it's news to him: "Shocked," he says. "Shocked. Truly, it goes in layers." The school is investigating after the award was posted on Facebook and "other parents didn't 'get the joke' either," WGCL Atlanta reports. Lamb says one of the faculty members named on the award, second-year teacher Mrs. Garner, handed out such awards "for six years in a prior district. It went unnoticed." The teacher had been a special-ed teacher in the Carrollton Farmers-Branch school district in Carrollton, Texas, which serves the Dallas area. Lamb says Garner also gave such awards to as many as 60 kids last year at her current school. So what's the point? "It's like you're putting them in a 'place,'" says Jose. "If [the teacher] knew what ghetto meant, she would have never approached that, because, being an African-American, we were always thrown that."
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Summarize: Sweatpants with stilettos? Taking casual chic to its extreme, a number of established designers including Michael Kors, Thakoon and Jean Paul Gaultier are touting versions of the slouchy pant usually associated with stay-at-home sick days. Gussied up with expensive materials, some tailoring details and price tags as high as $1,000, this new breed of sweatpant is intended to be worn proudly in public with blazers, dressy tops and high heels. Designers say their customers are looking for fashion that's less flashy, more practical. Plus, in shaky economic times, swanky sweatpants are viewed as a safe bet. "Look, whenever you can combine fashion, style and comfort in one item, it's going to work at retail," says Mr. Kors, whose cashmere versions will sell for $995. Sally Lohan, a trend expert at fashion-consulting service WGSN, also says the sweatpant trend will have legs. "We really balked at wearing leggings but eventually we all did—they were a good comfortable item to wear," she says. Sweatpants Go Luxe A look at various ways luxury sweatpants are being worn View Slideshow Alexander Wang So far, the dressy sweatpants seem to be selling. Stores have already sold out of Rag & Bone's $255 "long johns" for spring, says a label spokesperson, who called the response "incredible." Bottega Veneta boasts that its runway version priced at $290 to $360 is selling "very well." Topshop quickly sold out of $160 high-waisted sweatpants with mirrored grommets designed by Christopher Kane, and already has sold out of spring versions with metal-studded sides by London-based designer Ashish, according to Karen Bonser, Topshop's head of design. Sweatpants date back to the 1920s, first appearing on college athletes, according to Patricia Campbell Warner, author of a 2006 book about American sportswear. The broader public quickly adopted them, but sweatpants mostly floated near the bottom of the clothing hierarchy until the late-1990s, when brand Juicy Couture struck it big pitching a $200 velour track suit. The look became so iconic that a pink Juicy track suit is part of the permanent fashion collection at London's Victoria & Albert Museum. Two years ago sweatpants made it to the runways, with the likes of Isabel Marant and Alexander Wang presenting sweats and heels. But unlike Juicy sweats, which are usually worn to the mall or the movies, these dressy sweats are intended for more formal occasions, which some say could lead to embarrassing encounters. "You should be aware people perceive sweatpants as workout gear," cautions Daniel Post Senning, moderator of the question-and-answer blog of etiquette experts Emily Post Institute. "Sweatpants will communicate workout wear to the vast majority of people." Intrigued after seeing a model wearing the Rag & Bone version, Loren Ridinger, a senior vice president for an Internet marketing company in Miami, purchased a few pairs of Alexander Wang sweatpants in December. "It's all about the heels," she says, saying she paired her black sweats with a form-fitting white sweater and black high-heeled ankle booties. "It takes it to a whole new level." Her husband, James, initially wasn't as sure. "Where are you going with this? You're going to change, right?" he asked when he spied his wife donning her new $265 army-green sweatpants to meet friends at a restaurant. (Mr. Ridinger says he likes the look now.) Ms. Ridinger, who has since taken her new look to other dinners, parties and work, confesses that people sometimes ask her if she's really wearing sweatpants. While some friends and strangers have offered compliments, "I wonder if [others] think it looks weird," she says. Paulina Petkoski, a 23-year-old fashion-school student in New York who says she never wore sweatpants before, says she's donned her new fashion-forward sweatpants for work, typically with a "boyfriend" blazer and high heels. "Most of the time the reaction is positive," she says, though she adds that her guy friends have told her they are not crazy about the look, finding it unsexy. Some fashion followers vow to sit this one out. "Maybe there is an irony there I'm missing, like taking the sweatpant and subverting it, but sweatpants do not become OK outside of the house just because there is a designer name slapped on them," says Fiona Taylor, who works for an interactive marketing firm in New York. "I think people who buy them will look back at pictures of themselves in a few years with regret." Write to Ray A. Smith at [email protected] Sweatpants Trend to Give Those Who ‘Get It’ Feelings of Superiority Designer sweatpants hit the runways of Alexander Wang and Isabel Marant a couple of years ago. They've since appeared on the runways of Bottega Veneta ($290 to $360), Michael Kors ($995, but they're cashmere), and rag & bone ($255), and made a strong showing for both the spring and fall 2010 seasons. The Wall Street Journal, which has just gotten wind of this trend, is dubious, asking, "Sweatpants with stilettos?" Yes, sweatpants with stilettos. It's not a perfect trend, but so few are in their incarnation. One trend analyst pointed to leggings. When they came out, everyone thought only fashion riffraff would wear those, and then they became absolutely huge, and prices easily soared to four figures for the best buttery-leather pairs. In the past five years, the paper might have asked: "Leggings with mesh panels?" "Booties with open toes?" "Florals for fall?" Now we can definitively say yes, yes, and yes. However, the problem with sweatpants is that not everyone gets them. Leggings, open-toed booties, and florals in the snow may look odd at first, but not necessarily inappropriate and ill-mannered. "You should be aware people perceive sweatpants as workout gear," cautions Daniel Post Senning, moderator of the question-and-answer blog of etiquette experts Emily Post Institute. "Sweatpants will communicate workout wear to the vast majority of people." This is something we personally experienced the first time we wore sweatpants and heels out almost a year ago. The pants, we later learned, were deemed rude by one or two of our dining companions. Shows what they know! we thought. Just wait till next spring when everyone's wearing them! Intrigued after seeing a model wearing the Rag & Bone version, Loren Ridinger, a senior vice president for an Internet marketing company in Miami, purchased a few pairs of Alexander Wang sweatpants in December. "It's all about the heels," she says, saying she paired her black sweats with a form-fitting white sweater and black high-heeled ankle booties. "It takes it to a whole new level." Her husband, James, initially wasn't as sure. "Where are you going with this? You're going to change, right?" he asked when he spied his wife donning her new $265 army-green sweatpants to meet friends at a restaurant. (Mr. Ridinger says he likes the look now.) Ms. Ridinger, who has since taken her new look to other dinners, parties and work, confesses that people sometimes ask her if she's really wearing sweatpants. While some friends and strangers have offered compliments, "I wonder if [others] think it looks weird," she says. They do, but that's the beauty of the sweatpant: Those who "get it" know they strut on the forefront of style, while those who don't are so behind. A fashionable person loves nothing more than knowing this is what separates them from the masses. And that is why sweats are here to stay. The $1,000 Sweatpants [WSJ]
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Summary: Designer sweatpants have arrived, with some versions costing nearly $1,000-but not everyone is convinced the notoriously informal attire is a great sartorial choice for anywhere but the gym. The Wall Street Journal is especially skeptical, opening a recent article with the question, "Sweatpants with stilettos?" Um, "yes, sweatpants with stilettos," counters Amy Odell in New York. The only problem with sweatpants "is that not everyone gets them." Other questionable fashion trends that eventually made good, like leggings, "may look odd at first, but not necessarily inappropriate and ill-mannered." Sweatpants, on the other hand, can be "deemed rude"-even if you are wearing them with heels. The trick is to remember that you "strut on the forefront of style, while those who don't are so behind. A fashionable person loves nothing more than knowing this is what separates them from the masses. And that is why sweats are here to stay."
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Summarize: RELATED APPLICATION [0001] This application is a divisional application of U.S. patent application Ser. No. 11/359,942 filed Feb. 22, 2006. FIELD OF THE INVENTION [0002] The present invention is directed to a method and apparatus for physical exercise using air spring technology. BACKGROUND OF THE INVENTION [0003] Exercise equipment, in general, is known in the art. Air springs are also known in the art. An air spring is an elastomeric bellows having end closures that allow for mounting on vehicles and industrial equipment. The bellows contains a fluid such as air, some other gas or a liquid, usually under pressure. BRIEF SUMMARY OF THE INVENTION [0004] The present invention is directed to physical therapy equipment based upon an air spring acting as a flexure joint, and methods of exercise utilizing the air spring. Using such a flexure joint will allow deflections in the range of nearly 90 degrees and a rotational deflection direction of a full 360 degrees. [0005] In one embodiment the exercise device is constructed from an air spring or referred to herein as a flexure joint device, a lever arm attached to an upper portion of the flexure joint device, a rigid unsprung frame member attached to a portion of the outer surface (lower side) of the flexure joint device to prevent motion of the portion of the flexure joint device attached to the rigid member when the lever arm is moved, and a human body engaging member attached to the lever arm, whereby the lever arm can be moved with resistance by the human body engaging member in any direction away from an in rest position of the lever arm. The resistance is adjustable and can be selectively controlled by the user by adjusting the pressure within the flexure joint device. [0006] Air springs or flexure joint devices suitable for use in this invention are commercially available from Companies such as the Firestone Industrial Products Company. This company calls such devices AIRSTROKE® actuators and AIRMOUNT® isolators. Although such devices are adapted to be used in pneumatic systems, they can be adapted to be used in hydraulic and aero-hydraulic systems. The elastomeric bladder is typically sandwiched between an upper and lower covers, which make the elastomeric bellows or bladder airtight. These covers are sometimes called by the industry bead plates. They can be designed with flanges as means for mounting to brackets or solid surfaces. In the present invention, it is anticipated that the air springs assembly be adapted with means to attach its bottom to a solid platform, floor or other surface such as a table (rigid unsprung frame member described above); and the upper part be adapted with means for engaging a lever arm as described above. Typically, the upper part includes an inlet port for the air or hydraulic fluid medium. [0007] The human body engaging member can be a hand grip, a foot-engaging member, such as a foot pad or any other body engaging member. The hand grip can be a T-bar configuration or cross-shaped along the lever arm containing two hand grips. The body-engaging member can be secured/strapped to work out specific body parts and allow work-out while pushing or pulling against the air spring assembly. [0008] The human body-engaging member can be resilient or gyroscopic. The gyroscopic hand or foot feature interfaces to reduce strain and transmit force with natural body movements. [0009] The exercise device preferably contains pressure-regulating means for adjusting pressure in the flexure joint device. This is usually done by those skilled in the art by the use of a regulating valve and relief valve. The fluid medium filling the flexure joint device can be air for pneumatic control or hydraulic fluid for hydraulic control or other similar fluid media suitable for using under pressure and suitable for varying by the use of regulators/valves, including the combination of air and hydraulic components/features. [0010] The base member of the flexure joint device can be mounted on a vertical surface. In this embodiment, the mounting can be on a wall, a post or on a frame system that allows for height (vertical) adjustment or lateral adjustment and/or combinations of such adjustment to suit the height of the person using the equipment and/or the standing position on a floor or platform of the user, as well as the part of the body engaging member being exercised. Such adjustments can also accommodate persons in wheelchairs, sitting, standing, lying down on side or back. The apparatus can be mounted to hospital beds, home beds, wheelchairs and home furniture. [0011] In the above description and as further described below, the flexure joint device provides a great advantage in allowing for exercise motion in any direction, including performing rotational actions, side to front, front to side, up and down, inward, etc. For example, the apparatus could incorporate both linear and flexure joint movement to simulate human movement. This would help with occupational therapy such as lifting a box over your head. [0012] Such adjustable means are known in the art. For example, the flexure joint device and its lever arm can moved along a track and tightened when moved to a desired location or located with a quick-release pin type system where pins are inserted in apertures or friction lock tubing. It is preferred that the equipment's flexure joint device be designed such that a user can adjust the resistance of the apparatus by adjusting the pressure. A gauge can be provided and connected to the pressurizing lines, with a user friendly interface indicating levels of resistance as is the case with many aerobic exercise machines today. The gauge can be located so to be in plain view of the user. The resistance adjustment controls should also be located so as to be readily adjusted by the user of the equipment. The levels can be visualized digitally or by a needle scale type of arrangement. User interface can include a computer interface so a physical therapist or other attendant, for example, can control the workout program. For example, air springs could have pressure transducers, electronic pressure regulator, and rotational flexure transducers to interface with a computer. This would allow real-time monitoring of the air spring pressure, deflection direction, and amount of deflection. Amount of force exerted on the apparatus could also be determined by the air spring pressure differential produced when the air spring deflects and compresses the air. Mechanical stops could be used to limit the travel of the flexure member so that the patient's range of motion would be controlled so as not to re-injure or aggravate an injury. A computer rehabilitation program could be used by the physical therapist or other attendant to monitor and/or modify the characteristics of the flexure member. [0013] The exercise device can have a bicycle type pedal mounted on the lever arm. In this embodiment, the lever arm can be resistance adjusted through the flexure joint device and the pedals can also have means for adjusting the peddling resistance. In this embodiment, the present invention becomes two exercising devices for exercising both arms and/or both legs. Seating means are provided either independent of the invention so the bicycle embodiment can be used or the bottom of the flexure joint device can be mounted on a surface common to the mounting of the seat means. Effectively, the user sits on a bicycle-like seat and is able to exercise by peddling while at the same time moving the arm with typically two hand grips inward and outward. Of course, the individual features can be used separately. That is, the legs can be exercised by the peddling action and the arms can be exercised by the flexure of the flexure joint device, combination or individually. In addition, in the bicycle-like seat embodiment or when a person is in a wheelchair, the person can pull up and lift his body of the seat/chair using the flexure apparatus, lean forward, left and right, against the flexure apparatus, and push and pull on the apparatus for exercising. [0014] In another embodiment, legs and hips could be exercised by a stair stepping action provided by the invention. [0015] In another embodiment, the T-bar or generally T-shaped hand grips can themselves be adapted to include a flexure joint device. In this case the rigid frame member may be a relative long frame member extending from a wall or floor or it may be a very short base member attached to a table top surface or the like or the wall. This embodiment is great for exercising the shoulders, forearms, wrists and hands. [0016] Another embodiment is the use of two side by side flexure joint devices, each having on its top a generally planar and elongate foot standing surface that extends a pre-determined lever arm distance (for example, 12 to 24 inches away from the flexure joint device), and each generally parallel to each other. A person can stand on each lever arm and use the apparatus as a step exerciser. It is preferable if handrails for balance be available to the user. [0017] The combination can have a foot positioning surface for exercise of the feet and legs and to ensure that a person's foot does not slide off the surface. These surface forms are typical of stair stepping exercise equipment used in gyms. [0018] In other embodiments, the flexure apparatus can be configured so that the bottom of an air spring is mounted to the floor or stationary base and a small platform is mounted to the top of the air spring. This would be used to help with balancing and will strengthen hip flexors. Air pressure would be regulated so that higher air pressure stiffens the platform and limits range-of-motion and the opposite for lower air pressures. A safety hand rail should preferably be built around the balancing plate. [0019] Further, specific flexure devices can be configured for activities such as: arm wrestling, strengthening baseball and football throwing arms, strengthening all joints, recovery from operations such as knee or elbow surgery, tendonitis, tennis elbow, and similar conditions. BRIEF DESCRIPTION OF THE DRAWINGS [0020] FIG. 1 is a perspective conceptual view of one embodiment of the present invention with the lever arm in use; [0021] FIG. 2 is a conceptual depiction of the embodiment of FIG. 1 with a peddle assembly being used; [0022] FIG. 3 is a conceptual depiction of another embodiment of the invention where the lever arm is relatively short for exercise of wrist and forearms; [0023] FIG. 4 is a conceptual depiction of an embodiment similar to that of FIG. 3, except that a T-shaped handle is provided for gripping by both hands; [0024] FIG. 5 is a conceptual partial cross-section depiction of a bellows system where the fluid medium is hydraulic fluid filled from a hydraulic pump source; [0025] FIG. 6 is a conceptual partial cross-section depiction of a bellows system where the fluid medium is air filled from an air compressor source; [0026] FIG. 7 is a conceptual depiction of an embodiment similar to FIG. 1, except the lever arm is shorter and the invention is mounted on a table or elevated surface; [0027] FIG. 8 is a conceptual depiction of an embodiment similar to FIG. 1, further depicting an example of how resistance can be applied to the peddling action; [0028] FIGS. 9 a and 9 b are conceptual depiction of the invention where the rigid member to which the flexure joint assembly is attached is a T-shaped structural member to facilitate exercising with both hands at the same time; [0029] FIG. 10 is a conceptual depiction of an example of mounting the invention on a wall surface with elevation adjustment possibilities; [0030] FIG. 11 is a conceptual depiction of various embodiments of the invention being applied to a universal gym type frame system; [0031] FIG. 12 is a conceptual depiction of another embodiment of the invention where two flexure joint assemblies are used with parallel lever arms adapted to support the feet of the person exercising with a common resistance regulating system and fluid source so that the invention can be used as a stair stepping exercise machine; and [0032] FIG. 13 is a conceptual depiction of another embodiment of the invention configured to be used as a balance exercise machine. DETAILED DESCRIPTION OF THE INVENTION [0033] Turning now to FIGS. 1-11, one or more embodiments of the invention 10 are generally depicted. There is shown a flexure joint assembly 12 (also referred to herein by its industrial generic name as an air spring or air spring assembly), having an inflatable bellows or bladder 14, filled with a fluid 16 such as air or hydraulic fluid. To the upper part of the assembly 12, is attached a lever arm 18 having a hand grip 20. The lever arm 18 is attached to the assembly's 12 upper plate 22, which covers the bellows 14. Pressure is introduced into bellows 14 via conduit means 24 for supplying the fluid 16 to and for pressurizing the flexure joint assembly 12. [0034] The lower portion or plate 26 of the flexure joint assembly 12 is attached to a rigid unsprung base member 28. By “unsprung” is meant that the air spring assembly 12 does not impart any springing action to base member 28. Base member 28 merely holds air spring assembly in place when lever arm 18 is moved to exercise a user. The base member 28 attached to the flexure joint assembly 12 can be mounted on a vertical surface 30. In this embodiment, the mounting can be on a wall 30 a or on a frame system 30 b (examples only) or other working/anchoring member, including a post, that allows for height (vertical) adjustment or lateral adjustment and/or combinations of such adjustment to suit the height of the person using the equipment and/or the standing position on a floor or platform of the user, as well as the part of the body engaging member being exercised. Of course, frame system 30 b could be independent or itself mounted to a wall surface 30 a or other working/anchoring member. [0035] The flexure joint assembly 12 has a fluid inlet port 32, which is in fluid communication with the bellows 14, the conduit means and a fluid supply source 34, which typically an air compressor system or a hydraulic pump system. [0036] FIGS. 3, 4 and 7 depict examples where the invention 10 is shown on an elevated surface or table surface 36. Means for regulating the pressure to adjust resistance is conceptually depicted as 38 in FIGS. 5 and 6. Conceptually, the means 38 for regulating the pressure is a control device that can be located so as to be used by an attendant, such as a therapist, either adjacent to the invention or remotely from an attendant working station. This includes setting up means 38 to be computer controlled. This conceptual regulating means applies to all embodiments shown in all the drawings, although not shown in FIGS. 3 and 4 and only partially shown in FIG. 7. The amount of pressure, which should be presented on the gauge face in user friendly terms such as resistance levels 1 - 10, is shown conceptually as gauge 40. Of course, the gauge face could also be presented in an actual pounds per square inch (psi), foot-pounds, or a similar analogous scale that imparts some meaning to the person using the equipment according to the embodiments of the invention, depending on the preference of the manufacture and the intended use of the invention. The levels can be visualized digitally or by a needle scale type of arrangement. [0037] Handle means 42 at the upper end of the lever arm 18 are provided. The handle means 42 can provide for different types of hand grips 20. Typically hand grips 20 can be designed to be integral to the lever arm 18, or designed to cover a portion of the lever arm 18 such as a rubber or foam base hand grip. The lever arm's 18 opposite end is attached to flexure joint assembly 12, which in turn is mounted on table surface 36. [0038] When mounted to a frame system 30 b, the air spring assembly 12 and the frame system 30 b (as well as the wall surface 30 a ) can be adapted so that assembly 12 is engaged to allow vertical and/or horizontal movement to suit the height or position of the user. This can be done by a number of ways known in the art, including the use of a track or channel with position tightening means such as bolts or quick release pins through mating apertures in the assembly lower portion 26 and the frame system 30 b or even the wall surface 30 a. Of course, another alternative is to have the bolts or release pins or other tightening means engage a channel in the wall or frame system under pressure. This allows the invention 10 to be moved up and down on a vertical wall surface 30, thereby allowing the height of lever arm 18 to be adjusted by the user. [0039] The air spring assembly or flexure joint assembly 12 can be mounted or attached on a floor or other planar or generally horizontal platform 44. In an embodiment from the basic invention 10, a peddle assembly 46 is added to lever arm 18 to provide for exercise of the legs while exercising the arms with handle means 42 at the upper end of the lever arm 18. The resistance of pedal assembly 46 can also be adjustable using means known in the art, such as by using tension friction belt-type of systems as often used with exercise bikes or by using tubing with a smooth bore and a friction (phenolic) block inserted around the peddle shaft with an air inlet for pressurizing the back side of the block to obtain the desired resistance, or by using disc pads 46 a with adjustable friction faces. [0040] Various air spring assemblies can be employed in the practice of the present invention. Various lengths are available, as are assemblies having one or more convolutions. The style used will typically be a matter of design choice and aesthetic. For example, a more tubular or cylindrical shaped bellows may be desirable for the hand/wrist exercise embodiment, such as the bellows 14 shown in FIGS. 3, 4, 9 a and 9 b. [0041] In another embodiment depicted in FIG. 12, two parallel air spring assemblies 12 are attached to a floor or horizontal platform 44. Generally flat lever arm 18 a, 18 b, each attached to one of the two air spring assemblies 12, extend relatively parallel to the plane of the floor a pre-determined distance from the flexure joint assembly 12. Typically, the lever arms 18 a, 18 b will be about 12 to 18 inches, but can be more or less. The user can exercise his or her feet and legs by moving them up and down while standing on lever arms 18 a, 18 b and using the embodiment as a stair stepper. A support frame for maintaining balance 48 should be used in conjunction with this embodiment of the invention 10, so that a user has something to hold on to while exercising. [0042] In another embodiment similar to that depicted in FIG. 12, FIG. 13 depicts a single air spring assembly 12, which is attached to a floor or platform 44 and the lever arm 18 is configured to serve as a human body engaging member so that a person exercising to enhance his or her balance can stand on the platform/lever arm portion. That is, the human body engaging member is in an overlying relationship to the closed upper portion of the flexure joint assembly and is configured so that a person exercising can stand on said human body engaging member for performing a balance exercise. [0043] It should be understood that in the above mentioned embodiments, a vacuum cup with a 12 VDC portable air and vacuum compressor may be utilized. [0044] It should also be understood that the preceding is merely a detailed description of one or more embodiments of this invention and that numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit and scope of the invention. The preceding description, therefore, is not meant to limit the scope of the invention. Rather, the scope of the invention is to be determined only by the appended claims and their equivalents.
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Summary: An air spring is used as a resistance device in exercise equipment having a lever arm for exercising arms and legs. In one embodiment, the equipment can be adapted to be used as a stair stepper. In another, it can be adapted to be used on a table surface for exercising hands, wrists and forearms. In still another embodiment, it can incorporate a bike pedal assembly to simultaneously aerobically exercise the legs and arms. The equipment includes resistance level regulating components and a visual indicator using a gauge or similar device to monitor resistance levels.
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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a non provisional patent application of U.S. Provisional Patent Application Ser. No. 61/690,704, filed Jul. 2, 2012 Priority of U.S. Provisional Patent Application Ser. No. 61/690,704, filed Jul. 2, 2012, hereby incorporated herein by reference, is hereby claimed STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable REFERENCE TO A “MICROFICHE APPENDIX” [0003] Not applicable BACKGROUND OF THE INVENTION [0004] 1. Field of the Invention [0005] The present invention relates to artificial lures for fishing. More particularly, the present invention relates to an improved spinner or lure that utilizes a uniquely configured jointed lure central structure with weighted hook and a unique adjustable line attachment piece. [0006] 2. General Background of the Invention [0007] Fishing with artificial lures is the most popular fishing method in the U.S. In very general terms, a lure, carrying a hook and attached to a fishing line, is cast into the water and pulled toward the angler. Visual appearance, motion pattern, and vibratory signals of the lure entice predatory fish to chase and swallow it, which, in turn, allows for the capture of the fish. [0008] Spinner-type lures are one of the most popular artificial baits on the market. The two basic forms are spinners and spinner baits. Spinners (in Britain sometimes referred to as ‘bar-spoons’), feature a rotating blade mounted on a wire shaft which has a proximal loop for the attachment of fishing line, a distal loop for the attachment of a fishing hook, and usually a lure body, a weight element around the shaft located between the hook and the spinner blade. Spinner baits have two wire shafts, joined at one end by a looped connection, which serves as line attachment point and which positions the two wire shafts in the shape of a V relative to each other. One wire shaft carries one or more rotating elements for the attraction of fish, while the other wire element carries a weighted fishing hook. [0009] Both spinner-type lures, spinners and spinner baits, primarily attract fish through the visual and vibratory signals emitted by the rotating blade, yet neither of them will catch unless the fish swallows the hook, located at some distance from the fish-attracting blade. In order to make the hook more appealing to fish, manufacturers often add decorative ‘dressing’ to its shank, such as buck tail hair or tinsel material. [0010] While both spinners and spinner baits attract fish by the rotational movement of the spinning blade, this rotational movement is symmetric and even for spinners whose wire shaft is moving coaxially with the travel direction of the lure, whereby it is asymmetric and uneven for spinner baits, whose blades are rotating around a wire shaft which moves at an angle to the travel direction of the lure. Such uneven rotational movements introduce additional, fish attracting thumping vibrations, in theory an advantage for the spinner bait type lure over the even and less pulsating rotational signals emitted by the spinner. This advantage of spinner baits is a disadvantage at the same time, though, since the enhanced attractiveness of the blade-carrying arm of the spinner bait detracts the fish's attention from the object intended for its affection—the hook-carrying opposite arm of the lure. This problem could be tackled if it were possible to combine the advantage of the spinner (fish-catching hook and fish-attracting blade mounted in close spatial proximity on a single wire element) with the advantage of the spinner bait (emission of both rotational and pulsating vibrations owing to an angulated position of the rotational axis of the spinning blade in relation to the travel direction of the lure). It is an objective of this invention to present a spinner that fulfills these conditions. [0011] In addition, before it can attract and catch fish, a good lure has to be cast into the right spot by the angler. Spinners, with a weighted lure body in the middle of the lure, and often bulky but light-weight dressing around a treble hook at the end, are notorious for the hook fowling the line during the casting process, subsequently preventing the lure from working properly when in the water. It is therefore an additional objective of this invention to present changes which greatly reduce the chance that the hooks fowls the line during the casting process. [0012] Further, due to the rapid rotation of the blade around the axis of the lure, spinners often present another vexing problem: line twist. The rotating blade imparts a rotating movement on the spinner itself, which is transmitted onto the fishing line, causing its eventual entanglement and damage. It is an objective of this invention to present changes which will reduce this problem. [0013] Lastly, all artificial lures, as opposed to natural baits, pose yet another problem to the fisherman: fish tend to come off after striking the lure by ‘throwing the hook’. Natural baits, usually presented to the fish in a static way, are often swallowed whole and deeply, with no chance for the fish to come off. Matters are different with artificial baits, which are often large, and which have to be chased by the fish. In most cases, the lure itself will remain outside the mouth of the fish, with only one of the hooks embedded lightly in its lip. Twisting and turning movements of the fish, and shaking of its head, will often lead to dislodgement of the hook and loss of the fish, with the lure serving as a lever. In general, the longer and the more rigid the lure, the easier for the fish to translate rotational and angular movements into effective leverage to ‘throw the hook’—crankbaits and spoons, but also larger spinners and spinner baits such as those designed for muskie fishing are infamous in that regard. It is therefore an objective of this invention to present changes which greatly reduce the ability of the fish to use the lure as a lever to dislodge the hook. BRIEF SUMMARY OF THE INVENTION [0014] The apparatus of the present invention solves the problems confronted in the art in a simple and straightforward manner. [0015] The apparatus has a single long axis, as opposed to the two long axes arranged in the ‘safety pin’ fashion that are typical for many spinner baits. The presented invention breaks down the single long axis into a plurality of elements. The proximal portion of this long axis is formed by a stiff wire shaft element, kept as short as possible with the minimum length dependent on the size and shape of the attached rotating blade, and ending in a proximal and a distal loop. The proximal loop can be used to attach the lure to fishing line, the distal loop is used to attach a subsequent element forming the distal portion of the lure's long axis, either a second stiff wire shaft element, or a piece of flexible material, preferably wire, or one or more split rings. [0016] There is a rotating blade mounted on the proximal stiff wire shaft (either and preferably by way of a clevis, or by direct passing of the wire shaft through a portion of the blade), however, deviating from the traditional model, the stiff wire shaft ends right below the attachment site of the spinning blade. Traditional spinners have a lure body, a weighted element usually located distally to the spinning blade on the stiff wire shaft. This novel lure shortens the stiff wire shaft as much as possible, and features a second, possibly flexible, wire or split ring element, forming the lure's distal long axis portion, which may carry one or more lure body elements that are, in this point also deviating from the traditional model, not heavily weighted in the preferred embodiment of the invention. [0017] The current invention features a rigid line attachment head, mounted on the proximally located wire shaft towards the proximal end thereof. This rigid line attachment element has one or more apertures which allow for the attachment of fishing line at increasing distances from the wire shaft. If the lure is attached to the line in the traditional way at the proximal end of the proximal stiff wire shaft, the shaft will follow the fishing line, and the direction of the pull, in a coaxial fashion, and the blade, rotating around the stiff wire shaft, will stay at the same angle in relation to the wire shaft throughout the 360° turn, thereby emitting regular and constant pressure signals. If the lure is attached to the line at one of the apertures located on the rigid line attachment head, the wire shaft will follow the fishing line at an angle in relation to the line and the direction of the pull, with the degree of deviation from a straight coaxial position increasing commensurate with the distance of the chosen aperture from the wire shaft. Owing to the now tilted position of the wire shaft in relation to the direction of the pull, the rotating blade will find uneven water resistance during its turn around the shaft, with maximum resistance encountered at its closest position towards the point of line attachment, and minimum resistance at its maximal distance from said point of line attachment. This varying resistance, in turn, will lead to an inverse change in the angle of rotation of the blade, with a maximum angle of rotation in relation to the shaft at the time point of minimal water resistance, and a minimum angle of rotation in relation to the shaft at the point of maximal water resistance. Now, in addition to emitting an even and constant rotational pressure signal, the lure also emits a superimposed thumping signal with each rotation, most appealing to a predatory fish's appetite and aggressive instincts. Unlike a spinner bait type lure, however, this spinner lure features the thumping signal emitting blade and the hook in close spatial proximity, on the same wire element, eliminating for the fish any and all feelings of uncertainty as to what lure part to swallow—the rotating blade or the hook—since both can be devoured with ease at the same time. [0018] Next, this invention features at least one weight element mounted either around the shank of a hook or adjacent to its distal bends (in the case of the hook type most commonly used, the treble hook, this area is hereafter referred to as the ‘trifurcation’), and further, also departing from the design of traditional spinner lures, the current invention minimizes the weight of the lure body elements. In doing so, the current invention shifts the center of mass of the lure from its mid-portion toward its distal-most portion, the hook. Casting the lure will now inevitably put the heaviest element, the hook, ahead of all lighter elements, most notably the fishing line located on the opposing end of the wire shaft, thereby all but eliminating any possibility of the hook fowling the line. [0019] Next, and again unlike traditional spinner lures, the long axis of the lure is formed of a plurality of segments, either two rigid but short wire elements, or a rigid wire element and a flexible wire element, mounted one after the other, or a single wire element and one or more split rings attached thereto, thereby significantly shortening the portions of the lure's total long axis which are rigid. This arrangement not only limits possible bending damage to the rigid wire elements due to non-coaxial force that might be exerted on the lure by pulling, as might occur due to snagging or due to a large fish being played, but also, by keeping the rigid portions of the long axis of the lure short, the ability of a loosely hooked fish to use the lure as a lever to free itself is significantly reduced. [0020] Finally, the proximal portion of this novel lure, consisting of the initial wire element carrying the attachment head and the rotating blade, can be used separately from the other features described in this invention, in that the proximal portion can be linked to, and set ahead of, many currently commercially available fishing lures, thereby given them the added attractiveness and fish-catching potential caused by the proximal portion's off-axis rotating blade. [0021] There are therefore numerous advantages of the present invention, as listed below: [0022] 1. The invention's novel weight distribution, shifting the center of gravity towards the hook and away from the point of line attachment, ensures that lure-entangling and line-fowling during the casting process are minimized. [0023] 2. Further, the weight distribution of this lure increases the precision with which it can be cast to the suspected fish-holding location. [0024] 3. The invention's off axis rotation of the spinning blade adds a thumping pressure signal to the standard constant rotational signal of a spinner type lure, thereby increasing its attractiveness to fish. [0025] 4. Also owing to the described off-axis rotation pattern, the total resistance encountered during the retrieval of the lure is maximized, leading to an increase of the amplitude of all pressure signals emitted, thereby further increasing the lure's attractiveness to fish. [0026] 5. In the preferred embodiment, with more than one alternative line attachment aperture located at the rigid line attachment head mounted on the wire shaft, the angler can chose the angle at which the wire shaft moves in relation to the direction of travel of the lure, and thereby adjust the intensity of the pressure signals exactly to the fish's liking. [0027] 6. Owing to the fact that the hook is attached to a plurality of elements, rather than to a single, elongated and inflexible wire element as in traditional spinners, the lure cannot be misused by fish as a levering instrument to unhook themselves. [0028] 7. In the preferred embodiment, the rigid line attachment head mounted on the wire shaft offers space for decorative enhancements, an option not otherwise available for spinner type lures, such as decorative elements resembling eyes, known to attract fish to lures. [0029] 8. Given the non-coaxial relationship of the stiff wire shaft and the direction of pull, and given the surface area presented by the rigid wire attachment head, the rotational inertia of the lure is greatly increased, thereby preventing the rotational movements of the blade from rotating the wire shaft itself. This reduces line-twist, a vexing problem otherwise encountered with standard spinners, often forcing the angler to add a vane or weighted element to the lure assembly. [0030] 9. The front portion of this novel lure can be used separately, by linking it to lures already in the possession of the fisherman, thereby immediately increasing the fish catching potential of previously unsuccessful lures. [0031] 10. In summary, therefore, this novel lure is easier to cast precisely to its intended location, there will be reduced tangling and line-fowling, the lure looks better to fish, its thumping signals will prove virtually irresistible, and, once hooked, its flexible hook attachment will prevent fish from unhooking themselves. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0032] For a further understanding of the nature, objects, and advantages of the present invention, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein: [0033] FIG. 1A is a perspective view of a traditional spinner; [0034] FIG. 1B is a perspective view of a traditional spinner, whereby the rotational movement of the spinner blade is depicted, with the position of the blade shown at two points of its rotation, 180° apart; [0035] FIG. 2A is a side view of a spinner bait; [0036] FIG. 2B is a side view of a spinner bait, whereby the rotational movement of the spinner blades are depicted, with the position of the blades shown at two points of their rotation, 180° apart; [0037] FIG. 3A is a perspective view of a traditional spinner with an elongated, triangular wire-shaft eye; [0038] FIG. 3B is a perspective view of a traditional spinner with an elongated, triangular wire-shaft eye, caught on submerged roots, with attempts under way to free the lure from its entanglement; [0039] FIG. 4 is a perspective view of a first embodiment of the current invention; [0040] FIG. 5 is a perspective view of a second embodiment of the current invention; [0041] FIG. 6 is a perspective view of a third embodiment of the current invention; [0042] FIG. 7 is a perspective view of a fourth embodiment of the current invention; [0043] FIGS. 8A-8D is an exploded perspective view of the parts needed to assemble the invention in its various preferred embodiments; and [0044] FIG. 9-12 show side views of the preferred embodiment of the apparatus of the present invention illustrating fishing line attached to the lure at various attachment openings, further showing the effects of individual attachment positions on the rotational positions of the blade. DETAILED DESCRIPTION OF THE INVENTION [0045] FIG. 1A shows a traditional spinner 11. A rotating blade 12 is mounted on a single wire shaft 13 by a clevis 14. Below clevis 14, a small metal or plastic bead 15, also mounted on wire axis 13, facilitates the rotational movements of clevis 14 and blade 12 around axis 13. Below bead 15, one or more lure body components 16 provide weight to allow for better casting of the lure. At the proximal and distal ends of single wire shaft 13, proximal wire shaft eye 17 and distal wire shaft eye 18 allow for the attachment of fishing line 19 (proximally) and of a treble hook 20 (distally). Most spinners also feature decorative dressing material 21 (often consisting of colored animal hair such as buck-tail material), attached with yarn 22 to hook shank 23 below hook eye 24. [0046] FIG. 1B shows traditional spinner 11 in motion. Fishing line 19 is attached to proximal wire shaft eye 17, and spinner 11 is pulled through the water (not depicted) in a direction generally coaxial to fishing line 19 and axis 13, as indicated by arrow 25. The motion of spinner 11 against the resistance of the water leads to rotational movements of clevis 14 and blade 12 around wire 13. Ignoring the forces of gravity and water current, and assuming a constant velocity pull in a direction that is coaxial with wire 13, then blade 12 will rotate around wire 13 at an angle α that is dependent mainly upon the shape of blade 12 and, importantly, which is constant around the 360° of rotation. FIG. 1B demonstrates this by depicting blade 12, shown in broken lines, in an upper position 26 and in a lower position 27. Rotational movement direction of blade 12 is indicated by arrows 28, and blade/axle angle α, identical for upper 26 and lower 27 positions is indicated by arrows 29. Spinner type lures, exhibiting this constant angle rotation, emit a monotonous and ‘artificial’ pressure signal when retrieved. [0047] FIG. 2A depicts a typical spinner bait type lure 30. In its most common form, a V-shaped wire 31 has two arms, a straight arm 32 and an offset arm 33, joined by line attachment loop or bend 34. Straight arm 32, at its distal end, is joined to the proximal end of lure head-weight 35 which, in turn, is joined at its opposing end to single hook 36. Decorative dressing 21 is usually applied in a manner similar to traditional spinner 11. Offset arm 33 carries an upper blade 37, mounted rotatably to arm 33 by means of standard clevis 14. Distal to clevis 14, one or more beads 15 are mounted on arm 33, which then ends in distal offset arm eye 38. A standard swivel 39 is attached with one end to distal offset arm eye 38 and with its opposing end to lower blade 40. In its typical form, spinner bait 30 can feature a decorative eye painting 41 on both sides of lure head-weight 35. [0048] FIG. 2B shows spinner bait 30 in motion. Fishing line 19 is attached to attachment loop 34, and spinner bait 30 is pulled through the water (not depicted) in a direction coaxial to fishing line 19 and relatively coaxial to straight arm 32, as indicated by arrow 25. The motion of spinner bait 30 against the resistance of the water leads to rotational movements of clevis 14 and upper blade 37 around offset arm 33, as well as to rotational movements of lower blade 40 and swivel 39 around their own long axes. Again ignoring the forces of gravity and water current, and assuming a constant velocity pull in a direction that is fairly coaxial with straight arm 32, but at an angle to offset arm 33, then upper blade 37 will rotate around offset arm 33 at a varying angle (indicated by arrows 29 ) that constantly shifts between narrow angle 13, located at the outside of spinner bait 30, and wide angle γ, located at the inside of spinner bait 30. [0049] Direction of rotation of upper blade 37 is indicated by arrows 42, direction of rotation of lower blade 40 is indicated by arrows 43. In a manner similar to FIG. 1B, both moving blades are depicted as broken lines, whereby broken lines 44 show upper blade 37 in its uppermost position, forming narrow angle β with offset arm 33, and broken line 45 show upper blade 37 in its lowermost position, forming wide angle γ with offset arm 33. Lower blade 40 is shown in dotted or broken lines in two positions, in a plane parallel to the view at 47 and in a plane orthogonal to the view at 46. While rotating lower blade 40 offers virtually no resistance to the water and emits a minimal pressure signal, rotating upper blade 37 emits a strong rotational signal that constantly varies in intensity, leading to a ‘thumping’ pressure signal upon retrieval of the lure. As discussed above, however, this feature attracts the fish to the wrong wire arm: the arm without the hook. [0050] FIG. 3A shows a common modification 51 of the traditional spinner 11, featuring an elongated and triangular proximal wire shaft eye 52. Examples of such elongated and triangular proximal wire shaft eye are the above quoted fishing lure of Driesel (U.S. Pat. No. 4,998,371) or the currently commercially available Shyster® (Luhr-Jensen Co) spinner. Other elements of the lure, such as clevis 14, blade 12, lure body 16, treble hook 20 and dressing 21 are similar to the standard spinner model 11 featured in FIG. 1. While the purpose of this wire eye design appears to be to keep the lure body off center, thereby reducing line twist, that design, however, also imparts, nolens volens, a small degree of off-axis rotation to the movement of the blade. Regrettably, a larger degree of off-axis rotation that would lead to a significant degree of “thumping” in the lure's pressure signal could not be obtained if one were to exaggerate the size and angulation of the proximal wire shaft eye, owing to the inherent instability of the wire material, as further illustrated below. [0051] FIG. 3B demonstrates the instability of the wire axis 13 and bent proximal wire shaft eye 52 of common spinner modification 51 when under pressure, a common scenario while fishing: Modified spinner 51 is caught, with treble hook 20, in the roots of submerged vegetation 53, and pull, indicated by arrow 54, is exerted to free the lure. Similar pressure will be exerted each time a larger fish is played. This pulling pressure damages and straightens triangular wire eye 52, reducing its angulation at point 55, while at the same time deforming single wire shaft axle 13 between bent wire eye 52 and clevis 14 at point 56, impeding the ability of clevis 14 to freely rotate. The problem is that, between the attachment points of fishing line 19 and treble hook 20, a single elongated rigid piece of wire (formed by wire axis 13 and bent wire shaft eye 52 ) cannot possibly withstand even those only modestly deformational forces routinely encountered while fishing. [0052] FIG. 4 illustrates a first embodiment of the apparatus of the present invention, designated generally by the numeral 87. A proximal wire part 57 (shown in more detail in a subsequent illustration) runs twice through rigid attachment head 58, forming upper loop 60 at the upper end or forward of attachment head 58, and lower loop 61 at the lower or rear end of attachment head 58, thereby securely attaching parts 57 and 58 to each other. Blade 12 attaches to wire 57 with clevis 14. Head 58 provides multiple openings 59, each opening providing an attachment position for a fishing line 19 and swivel 49. Loop 62 is provided at the distal end of proximal wire part 57. Distal loop 62 of proximal wire part 57 attaches to proximal loop 64 of distal wire part 63 which, in turn, by way of its distal loop 65 attaches to hook eye 24 of hook 71. Proximal wire part 57, below rigid attachment head 58, rotatably mounts clevis 14 attached to blade 12. Below clevis 14, a pearl or bead 15 is mounted on proximal wire part 57, ensuring clevis 14 's free movement. Between its proximal loop 64 and its distal loop 65, distal wire part 63 carries lure body 66, made of light weight material (e.g., plastic). Treble hook 71 has a weight or lead element 72 mounted distally on its hook shaft 23. Mounted proximally on hook shaft 23 is attachment portion 74 of dressing 73. [0053] FIG. 5 shows an alternative embodiment 88 of the present invention, showing its most versatile application. Similar to the first embodiment 87 are proximal wire part 57 and rigid attachment head 58 and their various features (e.g., blade 12, clevis 14 ). In FIG. 5, the ensemble of proximal wire part 57, attachment head 58, blade 12, clevis 14, and pearl 15 is referred to as upper lure assembly 98. This upper lure assembly 98 connects to a lower lure assembly 97, in FIG. 5 consisting of bait mounting wire 92 at bait mounting wire eye 93. Distally, bait mounting wire 92 carries internal weight 94, and, further distally yet, it connects to double hook 95. Mounted on this system consisting of elements 92, 93, 94, 95 can be a soft plastic bait 96, as a typical example, in fish form. The unit of elements formed by parts 92 - 96 is given as an example of how lower lure assembly 97 can look. While lower lure assembly 97 is illustrated here as an internal mounting system carrying a soft plastic bait in fish form, any other standard soft plastic bait could be used or mounted on elements 92 - 95, or, alternatively, a natural dead bait could be mounted just as easily. Similarly, instead of elements 92 - 95, any standard and commercially available mounting system or rig for natural dead-baits or soft plastic baits could be used, in conjunction with a natural dead-bait or with a conventional soft plastic bait to form lower lure assembly 97. The versatility of this invention is due to the fact that upper lure assembly 98 can be used by itself as an independent fish-attracting element, to be combined with multiple currently commercially available products serving as lower lure assembly 97, including but not confined to standard dead-bait spinning rigs, standard soft plastic baits, crank-baits, or even standard spinners. [0054] FIG. 6 illustrates another alternative embodiment 89 of the present invention. Similar to the first embodiment 87 are proximal wire part 57 and rigid attachment head 58 and their various features as seen in FIGS. 4-5 (e.g., blade 12, clevis 14 ). Proximal wire part 57 again connects to distal wire part 63 in a similar fashion, but distal wire part 63 carries a plurality of round body parts 67, again made of light weight material in the preferred embodiment. [0055] Distal wire part 63, with its distal loop 65, connects to hook eye 24 of upper treble hook 75 which carries, parallel to hook shaft 23, wire brace 76 which, in turn, on its distal end carries weight element 78. In its distal-most portion, wire brace 76 forms loop 77 which, in turn, connects to hook eye(s) 24 of one or more lower treble hooks 79, each of which carry lead element or weight 80 around the distal aspects of their hook shafts 23. In this embodiment, long dressing 81 attaches with portion 82 to upper treble hook 75, covering upper hook 75 and lower hook(s) 79. [0056] FIG. 7 shows a fourth embodiment 90 of the present invention. Rigid attachment head 58 and blade 12, as depicted in this example, are smaller, and the overall length of the lure is significantly shorter than in embodiments 87 and 89. Therefore, instead of distal wire part 63 or flexible wire material 68, proximal wire part 57 and hook eye 24 are joined by split ring 86 ; a single split ring 86 is used in the illustration, although a plurality of split rings can be used if needed. In this example, both lead element 72, mounted around hook shank 23, and weight element 78 attached with wire brace 76, provide a backward distribution of weight facilitating casting of embodiment 90, and preventing line entanglement. Distal loop 77 of wire brace 76 allows for the attachment of stinger hooks (not depicted) if desired. [0057] FIG. 8 illustrates the various component parts of the four suggested embodiments of FIGS. 4-7 in more detail. For ease of overview, the various parts are grouped in four different levels A, B, C, and D, in order of their location towards the proximal or distal end of the lure apparatus 87, 88, 89, 90. [0058] In FIG. 8, level A shows attachment head 58 with the two longitudinal openings 70, whose path through attachment head 58 is indicated by broken lines 91. The parallel wire portions of proximal wire part 57 fit in these longitudinal openings 70 and form upper loop 60 and lower loop 61, thereby securely connecting proximal wire part 57 to attachment head 58. Fishing line 19 can be attached upper loop 60, or directly to attachment head 58 at any one of attachment holes 59 (e.g., see FIGS. 9-12 ). Also shown at this level are clevis 14 and bead or pearl 15, which can be mounted on the lower portion of proximal wire part 57 in between lower loop 61 and distal loop 62. The ensemble of the parts depicted in level A, in conjunction with blade 12, form upper lure assembly 98. Upper lure assembly 98, in and by itself, without any of the other parts here depicted, can easily be combined with currently commercially available standard artificial lures or rigs, whereby upper lure assembly 98 would be attached proximally to fishing line, and distally, with loop 62, to whatever element is selected to serve as lower lure assembly 97, including but not confined to mounted natural baits, crank-baits, spinners, soft-plastic baits, or even spoons. [0059] Level B shows distal wire part 63 with its proximal 64 and distal 65 attachment loops. As an alternative to distal wire part 63, flexible wire material 68 can be used, forming an elongated loop closed by way of a standard crimp sleeve 69. A plurality of loops formed of flexible wire material 68 could be used, for added safety. The advantage of using flexible wire material at this level is that it cannot be used by fish as leverage to unhook themselves. As another alternative, one or more split ring(s) 86 could be placed in between proximal wire part 57 and a hook eye 24. Further shown in FIG. 8, level B are oval single lure body part 66 or, as an alternative, rounded multiple lure body parts 67, made preferably of light weight material (e.g., plastic). No lure body parts are needed if wire part 57 and hook eye 24 are connected by one or more split ring(s) 86. Shown also is spinner blade 12 which can be chosen in a variety of shapes. Lastly, a standard system for the mounting of soft plastic baits or natural deadbaits is depicted, with bait mounting wire 92, proximal bait mounting wire eye 93, internal weight 94, twin hook 95, and, as a broken line silhouette, bait 96, shown in fish form, which could be a soft plastic artificial bait or a natural dead-bait. [0060] In FIG. 8 at level C, most differences between the two embodiments 87 and 89, shown in FIGS. 4 and 6, respectively, are apparent. FIG. 4 embodiment 87 presented a single treble hook 71, carrying lead element 72 around its shank 23. Lead elements 72 can be oval shaped elongated lead pieces with a central longitudinal opening, slit open at the side, commercially available as ‘rubber core sinkers’, with the rubber core removed and the lead element firmly affixed to the distal portion of treble hook shank 23. In the single treble hook embodiment 87 of FIG. 4, short dressing 73 is fixed with its attachment portion 74 to the proximal portion of treble hook shank 23. To the left at level C are the elements pertaining to the embodiment 89 shown in FIG. 6. Wire brace 76 wraps around upper treble hook 75, whereby upper loop 83 of wire brace 76 fits through hook eye 24 of upper treble 75, and lower loop 84 of wire brace 76 cradles trifurcation 85 of upper treble 75. Weight element 78 is mounted on wire brace 76 between lower loop 84 and distal loop 77, thereby placing it directly below trifurcation 85. [0061] In FIG. 8 at level D, one or more distal treble hook(s) 79 are shown which, in the assembled lure, are attached with their hook eye(s) 24 to distal loop 77 of wire brace 76. Also shown at level D, for added clarity, and given the numeral 48, is the hook 75, brace 76 and weight 78 assembly of those parts individually depicted at level C of FIG. 8. [0062] FIGS. 9-12 show embodiments of the present invention in motion. In FIG. 9, fishing line 19 is attached to attachment head 58 by way of a snap swivel 49 (commercially available), and the effects of various attachment positions on the rotational angle of the blade are illustrated. In FIG. 9, swivel 49 attaches to upper loop 60 of proximal wire part 57, leading to a direction of travel of the lure essentially coaxial with fishing line 19 and proximal wire part 57. Similar to a traditional spinner 11 as depicted in FIG. 1, the motion of the lure against the resistance of the water leads to rotational movements of clevis 14 and blade 12 around proximal wire part 57. Blade 12 is shown in hard lines in an upper position 26 and in broken lines in a lower position 27. The blade/axle angle α (formed by blade 12 and underlying distal wire element 63 ) is identical for upper 26 and lower 27 positions. With this line attachment leading to constant angle rotation, the lure will emit a monotonous and ‘artificial’ pressure signal when retrieved, similar to a traditional spinner. Of note, two oval shaped lure body parts 50, hook 71, lead or weighted element 72 and dressing 73 are used in this illustration. [0063] In FIG. 10, swivel 49 is attached to an attachment hole 59 in attachment head 58, thereby positioning proximal wire part 57 at a slight angle in relation to fishing line 19. This, in turn, results in blade 12 forming, in its upper position 26, a slightly larger blade/axle angle γ and, in its lower position 27, a slightly smaller blade/axle angle β. This asymmetry of rotation induces the ‘thumping’ signal referred to above. [0064] In FIG. 11, an attachment hole 59 one position further away from upper loop 60 is chosen for line attachment, thereby further increasing the degree of asymmetry of blade 12 rotation. FIG. 12 shows the point of line attachment in the attachment hole 59 the furthest away from loop 60, thereby maximizing blade/axle angle γ and minimizing blade/axle angle β, and, consequently, maximizing the intensity of the ‘thumping’ signal emitted by the lure. [0065] FIG. 11 further illustrates another important point, highlighting the significance of breaking up the lure's long axis into a plurality of segments for the functioning of this invention: if one were to attach head 58 to a single long wire axis as in traditional spinners, represented in FIGS. 11 and 12 as ‘hypothetical’ broken line 99, blade 12 in its lower position 27 would interfere with that axis and therefore be unable to rotate. Therefore, in order to allow blade 12 to rotate significantly off axis in relation to wire part 57, this part 57 has to be shortened, with the connection to treble hook 71 being formed by one or more parts 63 or 68 or 86 whose location puts them inside the rotational orbit of blade 12. It is therefore the combined effects of head 58 's various attachment positions, and of the breaking down of the lure's long axis into a plurality of elements 57 and 63 (or 68 or 86 ) that enables blade 12 to engage in significant off axis rotation. PARTS LIST [0000] Number Description 11 traditional in-line spinner 12 blade 13 single wire shaft axle 14 clevis 15 bead 16 lure body component 17 proximal wire shaft eye 18 distal wire shaft eye 19 fishing line 20 treble hook 21 dressing 22 yarn 23 hook shank 24 hook eye 25 arrow indicating direction of pull 26 upper blade position 27 lower blade position 28 arrows indicating rotational direction of blade 29 arrows indicating angle formed by rotating blade and wire shaft axle 30 spinner bait 31 V-shaped wire 32 straight arm 33 offset arm 34 attachment loop 35 head weight 36 single hook 37 upper blade 38 offset arm eye 39 standard swivel 40 lower blade 41 head weight decoration 42 rotational arrows for upper blade 43 rotational arrows for lower blade 44 upper blade in upper position 45 upper blade in lower position 46 lower blade in orthogonal position 47 lower blade in parallel position 48 hook, brace, and weight assembly 49 snap swivel 50 oval shaped multiple lure body parts 51 modified traditional inline spinner 52 bent proximal wire shaft eye 53 submerged vegetation 54 arrow indicating direction of pull 55 point indicating reduced angulation 56 point indicating wire shaft damage 57 proximal wire part 58 attachment head 59 attachment hole 60 upper loop 61 lower loop 62 distal loop of proximal wire part 57 63 distal wire part 64 proximal loop of distal wire part 63 65 distal loop of distal wire part 63 66 oval shaped single lure body 67 round shaped multiple lure body parts 68 flexible wire material 69 crimping sleeve 70 longitudinal openings of attachment head 71 treble hook 72 lead element of treble hook/weighted element 73 short dressing 74 attachment of short dressing 75 upper treble hook 76 wire brace 77 distal loop of wire brace 76 78 weight element of upper treble hook 75 79 lower treble hook 80 lead weight element of lower treble hook 81 long dressing 82 attachment of long dressing 83 upper loop of wire brace 76 84 lower loop of wire brace 76 85 trifurcation of upper treble 75 86 split ring 87 artificial spinner apparatus 88 artificial spinner apparatus 89 artificial spinner apparatus 90 artificial spinner apparatus 91 broken lines 92 bait mounting wire 93 proximal bait mounting wire eye 94 internal weight 95 double hook 96 bait 97 lower lure assembly 98 upper lure assembly 99 dotted line [0156] The foregoing embodiments are presented by way of example only; the scope of the present invention is to be limited only by the following claims.
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Summary: A fishing lure having a long axis provided at its proximal portion, an elongated stiff wire shaft having a bent or looped portion on its proximal end for attaching fishing line or a swivel thereto, and a bent or looped portion on its distal end. The distal portion of said long axis is formed by either a flexible element, or by a second elongated stiff wire shaft, or by one or more split rings. At the distal end one or more hooks are attached, at least one carrying a weight element. The fishing lure includes at least one spinner blade assembly connected to a span of the proximally located stiff wire shaft, that it is free to rotate 360°. A rigid element is attached to the wire shaft between its proximal end and the spinner blade assembly, containing one or more openings representing attachment locations for fishing lines or swivels.
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en
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Summarize: Diners have been left spitting feathers after a second branch of Nando's ran out of chicken this week. Customers in Wimbledon, London were left astounded when staff told them the restaurant had run out of its famous Peri Peri dish. It comes just days after the Leicester branch of the popular Portuguese-themed restaurant chain also ran out of poultry. Customers at the Russell Road branch of Nando's in Wimbledon, London were left astounded when staff told them the restaurant had run out of its famous Peri Peri chicken this week. It is thought the shortcomings have been caused by a new online takeaway service launched by the chain resulting in an increase in orders, as well as customers trying to redeem points on their paper loyalty cards before they expire. However, the Wimbledon branch may also have experienced a surge in diners this week as thousands descended on the town for the annual tennis championships. One customer, Susan French, 30, tried to dine at the Wimbledon branch after watching the action at the All England Club this week but was told by staff there was no chicken left. She told the Standard: ‘When I went in they told me they had run out of chicken. It was absolutely ridiculous. ‘They’re a chicken restaurant that can’t serve chicken. I could not believe it. It was a complete joke.’ It came just days after customers took to Twitter to vent their despair after the Leicester restaurant was temporarily unable to serve some of its poultry dishes after running out of the meat. One user called NestaMcG wrote on Wednesday night: ‘I don’t want to cause mass hysteria but @NandosUK in Leicester has run out of chicken. ‘I’m still coming to terms with what happen [sic]. One colleague is inconsolable.’ Diners in Leicester took to Twitter to vent their despair earlier this week after the branch was temporarily unable to serve chicken dishes after running out of poultry. Another customer, Steffan Powell, posted: 'So I'm in @NandosUK in Leicester...and there is no chicken... I repeat NO Chicken...' Customers have been desperately trying to redeem the final ‘chillies’ on their paper loyalty cards which were set to be made redundant from the beginning of July and replaced with plastic loyalty cards. Nando's said this, along with the launch of its online takeaway service, had contributed to the chaos. A spokesman said: ‘The launch of Online Takeaway has been incredibly popular, and with many customers also wanting to use their final remaining chillies on their old paper loyalty cards, unfortunately some restaurants ran out of certain options for a short period of time. ‘We would like to apologise to everyone who couldn’t get their meal of choice, and assure our customers that our restaurants are fully stocked with Per-Peri chicken.’ Nando’s specialises in flame-grilled chicken basted in Peri-Peri sauce and has around 300 restaurants across the UK. Its popularity has soared over the last few years, partly thanks to its celebrity devotees who often boast about eating at the restaurant
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Summary: Diners left in a flap after Nando's branch in Wimbledon runs out of poultry. Customers astounded after being told the restaurant had no chicken left. Comes just days after Leicester branch of chain ran out of Peri Peri dishes. Nando's has blamed popularity of new takeaway service on shortcoming. Also blamed drought on diners trying to redeem rewards on paper loyalty cards which are being replaced by plastic cards.
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Summarize: BACKGROUND OF THE INVENTION [0001] The present invention relates to digital radiography (X-ray) sensors designed for use in a filmless radiography system and, more particularly, to rotatable and/or detachable digital X-ray sensors. [0002] While the present invention is considered to be applicable to both the medical and dental professions, for example, for ease in description, the following discussion will focus on an intraoral embodiment of the present invention. [0003] Dentists and oral surgeons typically use x-radiation (“X-rays”) to obtain images of their patients' teeth, mouths and gums to aid in diagnosis and treatment. In traditional oral and dental radiography, a cartridge containing a piece of photographic film is placed in the patient's mouth, for example, behind a patient's tooth, and an X-ray beam is projected through the tooth and onto the film. The film, after being exposed in this manner, is developed in a dark room or a closed processor using special chemicals to obtain a photographic image of the tooth. [0004] More recently, the field of filmless dental radiography has emerged. In filmless dental radiography, an X-ray beam is still projected through the target area such as a patient's tooth, but no photographic film is used. Instead, an electronic sensor is placed in the patient's mouth behind the tooth to be examined. The electronic sensor may include a charge-coupled device (CCD) or any other filmless radiation sensor. The X-rays pass through the tooth and impinge on the electronic sensor, which converts the X-rays into an electrical signal. The electrical signal is transmitted over a wire to a computer, either directly or through a module containing intermediate processing circuitry. The computer then processes the signal to produce an image on an associated output device, such as a monitor or a printer. [0005] Filmless dental radiography offers several advantages over traditional film-based radiography. Most importantly, the electronic sensor is much more sensitive to X-rays than is film, allowing the dosage of X-rays to the patient to be lowered by as much as 90%. Also, the image of the tooth is generated by the computer almost instantaneously, thus eliminating the entire developing process, including the use of potentially harmful chemicals. In addition, because the images are generated electronically, they can be stored electronically in a computer database. Examples of filmless dental radiography systems include those described in U.S. Pat. No. 4,160,997 to Robert Schwartz and U.S. Pat. No. 5,434,418 to David Schick. Filmless dental radiograph systems typically utilize a standard desktop computer, such as an IBM or IBM compatible type personal computer. [0006] Recently, various sensors have been developed which are directed to improve the fit and/or performance of sensors within the oral cavity. However, even with the more advanced sensors, typically at least two different sized sensors are necessary to obtain a complete X-ray set of a single patient. Bitewing X-rays are a vital part of any diagnosis and are often the most widely taken X-ray. Periapical X-rays are generally taken for specific diagnosis of dental pathology, particularly for an issue with the tooth root and bone structure supporting the tooth. [0007] Even using different sized sensors, the known sensors are often inadequate at taking full tooth X-rays including the root. Furthermore, often the fixed cable extending from the sensor makes it difficult to properly position the sensor within the oral cavity. Thus, the present invention allows for better sensor placement within the oral cavity which will allow the dental staff to capture better X-ray images. SUMMARY OF THE INVENTION [0008] The present invention provides an intraoral sensor which is rotatable for proper positioning within a dental patient's oral cavity without significant interference from the cable which is connected to a computer or module containing intermediate processing circuitry. As a result, the intraoral sensor is configured to fit comfortably and in closer proximity to a target area in an intraoral cavity than currently known static sensor and cable corrections. By allowing for rotation of the sensor, not only does the sensor fit more comfortably, but the sensor can be positioned to capture a greater image of the teeth, including the roots or other targeted areas, such as bone structure. These features are believed to reduce refractive error in the image received by the sensor, thereby improving the image data transmitted by the sensor. Preferably, the sensor is selectively detachable from the cable leading to the computer and/or image display unit. Thus, unlike presently known filmless X-ray sensors, if either the cable or the sensor becomes damaged, they can be readily replaced. [0009] Still further, the preferred configuration of the sensor is relatively thin, which also contributes to the improved ergonomics of the sensor, and enables the sensor to get closer to the target area of an oral cavity, thereby improving the image data transmitted by the sensor. [0010] Further features of the present invention will become apparent from the following detailed description taken with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0011] The present invention will become more fully understood from the detailed description and the accompanying drawings, wherein: [0012] FIG. 1 is an exploded perspective views of a sensor assembly according to a first embodiment of the present invention; [0013] FIG. 2 is an exploded perspective view of a sensor assembly according to a second embodiment of the present invention; [0014] FIGS. 3A-3C are top views of the sensor assembly shown in an installation position as well as first and second installed positions; [0015] FIG. 4A is a cut-away view showing the sensor head and cable in an installation position; [0016] FIG. 4B is a cut-away view showing the sensor head and cable in an installed position; [0017] FIG. 5 is a perspective view of a sensor assembly according to a third embodiment of the present invention; [0018] FIG. 6 is a perspective view of a sensor assembly according to a fourth embodiment of the present invention; [0019] FIG. 7 is a perspective view of a sensor assembly according to a fifth embodiment of the present invention; [0020] FIGS. 8A-8C demonstrate the rotatable nature of the sensor assemblies for repositioning of a sensor for elongated or wide angle views; [0021] FIG. 9 is a cut away side view of a sensor assembly according to a fifth embodiment of the present invention; and [0022] FIG. 10 is a schematic representation of a filmless radiography system using a sensor according to the teachings of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0023] A sensor according to the present invention is designed to be used with a filmless radiography system. As an example, a sensor 10 according to the present invention can be used as part of a filmless radiography system 12 which is designed according to the principles of Schwartz U.S. Pat. No. 4,160,997 (Schwartz patent), which is incorporated hereby by reference. As illustrated in FIG. 5, the sensor 10 transmits digital image data to a preprocessor 14, and the preprocessor transmits the image data either directly to a display device 16, or to a computer 18 which is connected to the display device 16. The preprocessor 14 is configured to normalize the image data transmitted by the sensor 10, to improve the contrast (color and/or grayscale) of such data in relation to the raw image data produced by the sensor. The image data is then transmitted directly to the display device 16, or the image data is transmitted to the computer 18 which can manipulate the image data on the display device. The manner in which the image is processed, transmitted and/or displayed can be in accordance with the principles of the Schwartz patent, or in accordance with other well known devices and systems for processing and displaying the image data. Such devices and systems are well known to those in the art and should not require further explanation. [0024] In addition, the sensor 10 has internal structure, which is designed in accordance with the principles of the Schwartz patent. The internal structure includes components which receive radiated energy image data from a target area of an intraoral cavity (e.g. X-ray images of a patient's teeth, gums, etc), convert the resulting X-ray image data into a visible light image, and transmit the visible light image to a charge coupled device (CCD) or any other visible light sensor forming part of the structure. [0025] As noted in U.S. Pat. No. 6,652,141 B1 to Cianciosi, which is hereby incorporated by reference, the internal structure is suggested to include an array of CCD detectors, a printed circuit board associated with the CCD detectors, and a radiant energy screen with the appropriate coating to convert a radiant energy image to a visible image that impinges on the CCD detectors. The printed circuit board is coupled to a cable 30, as shown in FIG. 1 for example, which transmits the image data from the CCD array to the preprocessor 14 which preprocesses and transmits the image data to the display device 16 and/or to the computer 18. Again, the internal structure of the sensor can be constructed in various known ways, and should not require further explanation to those in the art. [0026] The basic configuration for a sensor assembly 10 according to the present invention as disclosed with reference to FIGS. 1-4B. In FIG. 5, the sensor assembly 10 comprises a sensor body (or housing) 20 configured to fit in a patient's oral cavity proximate a target area of the oral cavity. The sensor body 20 houses the CCD detectors, circuit board and radiant energy screen examples of which are described in U.S. Pat. No. 6,652,141 which is hereby incorporated by reference by way of non-limiting example. The side of the sensor body which is disposed adjacent to the target area of the teeth and/or jaw line, is generally formed from a biocompatible material that is transparent to X-ray energy. The side of the sensor body 24 which hosts the rotatable connector assembly 26 can be formed, for example, from a biocompatible thermoplastic material which is not transparent to X-ray energy. The side sections at the sensor body are bonded or otherwise fastened together, and enclose the internal structure components of the sensor. [0027] The rotatable connector assembly 26 is in the form of either a male or female component 30 which mates with a complementary male or female component 40 disposed along the terminal portion 52 of the electrical cable 50. More particularly, according to the embodiment depicted in FIG. 1, the component 30 is a male component including a well 32 having an upwardly projecting post 34 which seats within a central recess (not shown) contained on the component 40. The component 40, as shown, includes a radially extending flange 42 having cut-out portions 44 (holes) for assembling the components 30 and 40 to complete the necessary circuit for conveying the digital image data. The cut-out portions 44 are fit over the inwardly projecting tabs 36 and the sensor is rotated such that the face 46 of the flange 42 is held in place under the tabs 36, which extend radially inwardly over the well thereby holding the components 30 and 40 in contact to allow for image data communication. As shown in the embodiment of FIGS. 2 and 4B, the flange 42 may include spaced apart recesses 46 into which the tabs 36 insert to better secure the body 20 and cable 50 together. Additionally, to enhance contact between the sensor body 20 and the cable 50, either a portion of the post 34 or the receiving end 52 of the cable 50 into which the post is inserted may be magnetized to facilitate magnetic coupling in addition to the mechanical attachment. [0028] FIG. 3A-3C show the connection between the sensor body 20 and the connecting cable at various stages of the installation process. For example, FIG. 3A shows the flange 42 positioned such that the cut out portions 44 pass over the tabs 36. FIG. 3B shows the sensor body rotated 45° such that the sensor body 20 is fixed in a position whereby the cable 50 extends beyond one end of the generally rectangular sensor body. FIG. 3C, in contrast, shows the sensor body 20 rotated 45° in the opposite direction such that the cable 50 extends beyond one side of the upper body. Further reference can be made to FIGS. 4A-4B which show the flange 12 installed into the well 32 in both the insertion position and the installed positions, respectively. [0029] As shall be understood by those skilled in the art, the embodiments of FIGS. 1 and 2 (along with the assorted detailed figures) can be formed such that the male and female connections are alternatively disposed on the sensor body and cable as shown in FIG. 5. [0030] Referring to FIG. 6, an alternative rotatable intraoral sensor 120 is illustrated as including connector assembly 126 having a male portion 130 located along the side 124 of the sensor body 120 and a female portion 140 associated with the cable 150. Hereto, the male portion includes a post 134 which seats within a female receptacle 146 to complete the image data circuit upon sufficient connection. Again, the opposing male/female relationship is contemplated according to FIG. 7. In addition to the male/female coupling of the embodiments of FIGS. 6 and 7, an added feature includes detents 150 and mating protrusions 152 which seat within the detents when the sensor body is apportionately rotated relative to the cable. As can be appreciated, the embodiments of FIGS. 6 and 7 allow for incremental rotation of the sensor body relative to the cable position and thus the cable can extend via the connection at various positions from the sensor body. The sensor assembly embodiments of FIGS. 6 and 7 are believed to be particularly suited for magnetic coupling between the flange 140 and the well 132, over and above the mechanical coupling occurring at the post 146 and receptacle 134 interface. [0031] A major problem with prior art X-ray sensor embodiments which is addressed by the present invention is illustrated with reference to FIGS. 8A-8C. For example, when currently known X-ray sensors are employed in the posterior region of an intraoral cavity, particularly when a full tooth X-ray is desired, the connector cable 50 interferes with the oral cavity due to the rigid connection between the cable and the sensor body making it difficult to obtain an accurate X-ray. By rotating the sensor relative to the cable as shown in FIG. 8C, not only is there sufficient clearance for the cable, but a full tooth X-ray is also possible. [0032] As shown, in FIG. 9, yet another embodiment of the present invention is depicted. According to this embodiment, the coupling between the sensor body and the cable are accomplished via a ball and socket design which is intended to allow for a free swivel-type rotation of the sensor body relative to the cable. This should allow for clearance of the cable without the need for removing the sensor from the mouth and rotating the body prior to re-insertion to an X-ray target area. As shown, a flange similar to that shown in FIGS. 1-5 has a socket for receiving a ball bearing disposed along the terminal end of the cable. Alternatively, the sensor body may include a projecting ball bearing assembly which seats within a socket provided along the terminal end of the sensor cable. [0033] Preferably, the sensing bodies of the various embodiments are positioned so that when the sensor is properly positioned relative to a target area, the sensing structure will receive X-ray energy at about 90° to the direction of the X-ray energy. By providing a comfortable fit relative to the target areas, the sensors are ergonomically improved, in terms of its comfort and feel to a dental patient. In addition, the configuration of the sensor is designed to allow the sensor to be placed closer to a target area in an oral cavity than prior sensors (i.e. closer to target teeth, gum, etc). This feature, and the fact that when the sensor is properly positioned relative to a target area the X-ray energy will by received by the sensing structure at about 90° to the direction of the X-rays is believed to reduce refractive error in the image received by the sensor, thereby improving the image data transmitted by the sensor. Still further, preferred embodiments of the sensor body are relatively thin (e.g. with a thickness of about 5 mm), which also contributes to the improved ergonomics of the sensor, and enables the sensor to get closer to the target area of an oral cavity, thereby improving the image data transmitted by the sensor. [0034] In addition to the advantages of rotation of the sensor body relative to the sensor cable, it should be noted that the numerous embodiments of the present invention allow for quick disconnect-type coupling of the sensor body and cable. This feature allows for the replacement of either the cable or the sensor body if they are dysfunctional, without having to discard the entire assembly which is currently the case in the industry and is unduly expensive. [0035] Accordingly, the foregoing disclosure provides sensor configurations which are particularly useful as a filmless radiography sensor. With the foregoing disclosure in mind, the manner in which the principles of the invention can be used to form various types of intraoral sensors for various uses comparable to filmless radiography will be apparent to those in the art.
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Summary: A new and improved digital sensor assembly for use in a filmless radiography system is disclosed. The sensor is configured to be universal in that it can be rotated about a cable connection thereby allowing the sensor to be used both vertically and horizontally in the oral cavity. The rotatability of the sensor allows it to fit comfortably and close to a target area in an intraoral cavity. By providing a comfortable relative fit to the target area, the sensor is ergonomically improved, in terms of its comfort and feel to a dental patient. Alternative sensor assembly embodiments disclosed herein relate to quick disconnect-type coupling arrangements between the sensor body and the sensor cable.
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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Renewable Energy Alternative Production Act'' or the ``REAP Act''. SEC. 2. CREDIT FOR PRODUCTION OF RENEWABLE ENERGY. (a) In General.--Section 45 of the Internal Revenue Code of 1986 is amended by adding at the end the following new subsection: ``(f) Credit Allowed for Production of Non-Electric Energy.-- ``(1) In general.--The credit allowed under subsection (a) shall be increased by an amount equal to the product of-- ``(A) the dollar amount determined under paragraph (2), and ``(B) each million British thermal units (mmBtu) of qualified fuel which is-- ``(i) produced by the taxpayer-- ``(I) from qualified energy resources, and ``(II) at any facility during the 10-year period beginning on the date such facility was placed in service, ``(ii) not used for the production of electricity, and ``(iii) sold by the taxpayer to an unrelated person during the taxable year. ``(2) Dollar amount.--The dollar amount determined under this paragraph shall be the amount determined by the Secretary to be the equivalent, expressed in British thermal units, of the credit allowed under subsection (a) for 1 kilowatt hour of electricity. ``(3) Reduction for grants, tax exempt bonds, subsidized energy financing, and other credits.--Rules similar to the rules of subsection (b)(3) shall apply for purposes of paragraph (1). ``(4) Definitions and special rules.--For purposes of this subsection-- ``(A) Qualified fuel.--The term `qualified fuel' means an energy product which is produced, extracted, converted, or synthesized from a qualified energy resource through a controlled process, including pyrolysis, electrolysis, and anaerobic digestion, which results in a product consisting of methane, synthesis gas, hydrogen, steam, manufactured cellulosic fuels, or any other form of energy provided under regulations by the Secretary and which is used solely as a source of energy. ``(B) Allocation of credit to patrons of agricultural cooperatives.--Rules similar to the rules of subsection (e)(11) shall apply for purposes of paragraph (1).''. (b) Conforming Amendments.-- (1) The heading for section 45 of the Internal Revenue Code of 1986 is amended by striking ``electricity'' and inserting ``energy''. (2) The table of sections for subpart D of part IV of subchapter A of chapter 1 of such Code is amended by striking ``Electricity'' in the item relating to section 45 and inserting ``Energy''. (c) Effective Date.--The amendments made by this section shall apply to taxable years beginning after the date of the enactment of this Act. SEC. 3. ENERGY CREDIT FOR ONSITE RENEWABLE NON-ELECTRIC ENERGY PRODUCTION FACILITIES. (a) Credit Allowed.--Clause (i) of section 48(a)(2)(A) of the Internal Revenue Code of 1986 is amended-- (1) by striking ``and'' at the end of subclause (III), and (2) by adding at the end the following new subclause: ``(V) qualified onsite renewable non-electric energy production property,''. (b) Qualified Onsite Renewable Non-Electric Energy Production Property.--Subsection (c) of section 48 of the Internal Revenue Code of 1986 is amended by adding at the end the following new paragraph: ``(5) Qualified onsite renewable non-electric energy production property.-- ``(A) In general.--The term `qualified onsite renewable non-electric energy production property' means property which produces qualified fuel-- ``(i) from qualified energy resources, ``(ii) not used for the production of electricity, and ``(iii) used primarily on the same site where the production is located to replace an equivalent amount of non-renewable fuel (determined based on the number of British thermal units of non-renewable fuel consumed by the taxpayer in the prior taxable year) or to provide energy primarily on such site for a use that did not exist prior to the later of the date of the enactment of this paragraph or the date such property was placed in service. ``(B) Definitions.--For purposes of this paragraph-- ``(i) Qualified fuel.--The term `qualified fuel' means an energy product which is produced, extracted, converted, or synthesized from a qualified energy resource through a controlled process, including pyrolysis, electrolysis, and anaerobic digestion, which results in a product consisting of methane, synthesis gas, hydrogen, steam, manufactured cellulosic fuels, or any other form of energy provided under regulations by the Secretary and which is used solely as a source of energy. ``(ii) Qualified energy resources.--The term `qualified energy resources' has the meaning given such term by paragraph (1) of section 45(c). ``(iii) Termination.--The term `qualified onsite renewable non-electric energy production property' shall not include any property for any period after the date which is 10 years after the date of the enactment of the Renewable Energy Alternative Production Act.''. (c) Effective Date.--The amendments made by this section shall apply to periods after the date of the enactment of this Act, under rules similar to the rules of section 48(m) of the Internal Revenue Code of 1986 (as in effect on the day before the date of the enactment of the Revenue Reconciliation Act of 1990). SEC. 4. RENEWABLE NON-ELECTRIC ENERGY PRODUCTION FACILITIES ELIGIBLE FOR NEW CLEAN RENEWABLE ENERGY BONDS. (a) In General.--Paragraph (1) of section 54C(d) of the Internal Revenue Code of 1986 is amended to read as follows: ``(1) Qualified renewable energy facility.--The term `qualified renewable energy facility' means a facility which is-- ``(A)(i) a qualified facility (as determined under section 45(d) without regard to paragraphs (8) and (10) thereof and to any placed in service date), or ``(ii) a facility which produces qualified fuel (as defined in section 45(f)(4)(A)) which is derived from qualified energy resources (within the meaning of section 45(f)(4)(B)) and not used for the production of electricity, and ``(B) owned by a public power provider, a governmental body, or a cooperative electric company.''. (b) Effective Date.--The amendment made by this section shall apply to obligations issued after the date of the enactment of this Act.
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Title: A bill to amend the Internal Revenue Code of 1986 to provide for an energy carrier production tax credit, and for other purposes Summary: Renewable Energy Alternative Production Act or the REAP Act - Amends the Internal Revenue Code to allow: (1) a new tax credit for the production of non-electric energy from renewable resources; (2) an energy tax credit for investment in property used to replace non-electric energy at the production site for such energy; and (3) new clean renewable energy bond financing for renewable non-electiric energy production property.
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Summarize: Lady Bird is obsessed with place. Her place in the world, in her Catholic high school, in her school play, in her own family and in her unglamorous hometown of Sacramento ("the Midwest of California") and the even more unglamorous part she lives in. She craves sophistication in a way that she can't quite put into words or actions, beyond a vague desire to go to an East Coast college, but is certain that whatever she has in her middle-class existence in 2002 isn't it. Part Angela Chase, Lindsay Weir, Jo March and Anne Shirley, she is selfish and self-centered in that very particular way that teenage girls, who can't yet comprehend that this is a phase that might pass, can be. And she is, quite simply, one of the more achingly realistic teenage characters that we've had the pleasure of meeting in a movie. Played by the Irish-American actress Saoirse Ronan ("Brooklyn," ''Atonement"), Lady Bird and the film bearing her name is the semi-autobiographical creation of actress and writer Greta Gerwig in her solo directing debut. "Lady Bird " chronicles one year in the life of its titular character, whose real name is Christine McPherson, from the start of her senior year of high school to freshman year of college and all of its beautiful banalities — sex, prom, money, grades, boys, nuns and that gnawing dissatisfaction that has plagued every modern 17-year-old who'd rather die than admit that things might be OK. The film begins with a quote from Joan Didion: "Anybody who talks about California hedonism has never spent a Christmas in Sacramento." It's the kind of quote Lady Bird would love to think she's the only one who understands, but the truth is, she has likely not yet discovered that patron saint of California girls. She hasn't discovered a lot of things — clove cigarettes, Jim Morrison, what "the deuce" is, how to drink liquor, or that her school has an annual musical — and doesn't yet know how to look out for both herself and others too, whether it's her nurse mother, Marion (Laurie Metcalf), or her best friend, Julie (Beanie Feldstein). All she can see is what she doesn't have, so she carelessly skips over the tiny triumphs of her friend, and is blind to the fact that her mom might actually have her best interests in mind, or that her father Larry (Tracy Letts) might be struggling, financially and mentally. It's these tiny and painfully honest details that make up Gerwig's rich and lovingly composed film, which is bursting with wit, humanity, joy and truth. Ronan adds another superb performance to her already stunning resume as this somewhat unlikable yet empathetic character in flux, who will make you cry, laugh and cringe. But it's the wonderfully drawn supporting characters who truly bring this world to life and make this film such an undeniable pleasure to live in for a too-brief 93 minutes. Metcalf is operating at the top of her game as Marion, and there are too many delightful side characters to do justice here: Lucas Hedges and Timothee Chalamet as two very different kinds of high school boyfriends; Lois Smith as a wise and funny nun; Jake McDorman as an impossibly charming teacher; and Stephen McKinley Henderson as a melodramatic drama coach are among the standouts. But it is Feldstein's performance as Julie is its own kind of sleeper triumph, and one that makes you sit up and take notice of an excellent actress who had up until this point basically only been used as a punchline (see: "Neighbors 2: Sorority Rising"). "Lady Bird" feels like a companion piece to both "Mistress America" and "Frances Ha," both of which Gerwig co-wrote with Noah Baumbach and which he directed. Left to her own devices, Gerwig has arrived and solidified her place as one of the most invigorating, observant and authentic voices in movies today with a director's acumen to match. There are a lot of things rotting right now in the world and in Hollywood, and, basically, we should be especially grateful when something as lovely as "Lady Bird" comes along. "Lady Bird," an A24 release, is rated R by the Motion Picture Association of America for "language, sexual content, brief graphic nudity and teen partying." Running time: 93 minutes. Four stars out of four. ___ MPAA Definition of R: Restricted. Under 17 requires accompanying parent or adult guardian. ___ Follow AP Film Writer Lindsey Bahr on Twitter: www.twitter.com/ldbahr The French philosopher Simone Weil wrote often of attention as a kind of spiritual discipline. “Attention is the rarest and purest form of generosity,” she wrote in her notebooks, an idea she later would continue to develop, eventually concluding that attention “presupposes faith and love.” In a Q&A following a festival screening of her masterful solo directorial debut Lady Bird, Greta Gerwig quoted Weil, and it’s clear from the film that this spirit of faith, love, generosity, and attention animates the whole endeavor. Lady Bird is a coming-of-age film starring the great Saoirse Ronan as Christine — or “Lady Bird,” as she’s re-christened herself — and it’s as funny, smart, and filled with yearning as its heroine. Lady Bird is an act of attention, and thus love, from Gerwig, not just toward her hometown of Sacramento but also toward girlhood, and toward the feeling of always being on the outside of wherever real life is happening. Rating vox-mark vox-mark vox-mark vox-mark vox-mark The movie also confirms what movie lovers have long suspected: Gerwig is by far one of the most talented filmmakers working today, both onscreen and behind the camera, confident and winsome in a way nearly unparalleled by her contemporaries. Lady Bird is the rare movie that manages to be affectionate, entertaining, hilarious, witty, and confident; it’s one of the best films of 2017, and certainly my favorite. Lady Bird chronicles one girl’s senior year of high school Lady Bird opens with an epigraph, something Joan Didion told Michiko Kakutani in a 1979 New York Times profile: “Anybody who talks about California hedonism,” Didion said, leaning to look out an airplane window as they flew from Los Angeles to her hometown of Sacramento, “has never spent a Christmas in Sacramento.” Didion and Gerwig are both from Sacramento, and so is Lady Bird — the name, she says, is her given name, because “it’s given to me, by me.” She starts her senior year at a Catholic girls’ high school in 2002, with pink-dyed hair, a deep love for Dave Matthews Band’s song “Crash Into Me,” and dreams of getting out of Sacramento to go to college in New York City. On her bedroom wall, she’s scrawled a line from Anna Karenina — “Boredom: The Desire for Desires.” She wants, more than anything, to lead a life that’s the opposite of Sacramento. She wants to be extraordinary. Lady Bird lives on what she jokingly calls “the wrong side of the tracks,” along with her father Larry (Tracy Letts), her mother Marion (an extraordinary Laurie Metcalf), and her brother Miguel (Jordan Rodrigues) and his girlfriend Shelly (Marielle Scott). They’re all crammed into a neat but small house; her parents had hoped they’d be able to upgrade long ago, but tight finances kept getting in the way. Lady Bird bickers with Marion, whose fears about finances and her daughter’s future often tilt her into passive aggression, and who’s insistent that a California college is the best option. Lady Bird’s escape from home is at school, where she hangs out with her best friend Julie (the absolutely fantastic Beanie Feldstein) and joins the drama club in an attempt to pad out her college applications. Drama is conducted jointly with the nearby Catholic boys’ school and led by Father Leviatch (Stephen Henderson). During rehearsals for the fall musical production of Merrily We Roll Along, Lady Bird develops a crush on Danny (Lucas Hedges), the sweet redheaded boy from a big Irish Catholic family who becomes her first real love, though she can’t help eyeing too-cool-for-school Kyle (Timothée Chalamet) at a party too. The film’s arc is deceptively simple, following Lady Bird through her senior year (which is helped along by some excellent editing and a joyful, buoyant score from Jon Brion). She struggles with math and kisses Danny and fights with Marion. She loses her virginity. She and Julie lie on the floor at school and giggle about sex and eat unconsecrated communion wafers. They go to mass and celebrate holidays. They dream about the future. Lady Bird harbors remarkably deep ideas beneath its surface But there’s a remarkable amount of depth beneath this apparently conventional arc. In Lady Bird, Gerwig has taken the familiar teenage girl coming-of-age plot and turned it into something rather extraordinary and carefully observed, the kind of movie that actually reveals layers on repeated viewing. Inside Lady Bird are meditations on parenting, on struggling with money, and on the ways the future is never quite what we wanted it to be. But its strongest and subtlest theme is that closely attending to the world around us, and to the people around us, is a kind of grace that makes space for us to love. The film is mostly told from Lady Bird’s perspective, and like most high school seniors, her attention is all focused on herself and her own troubles. But through the year, a series of small conversations, captured in vignettes and glimpses, punctures her self-involvement. Everything that happens — heartbreak, embarrassment, disappointment, elation — all of it is evidence that other people struggle too, whether it’s Julie’s sadness at being left out of Lady Bird’s expanding social circle, or Lady Bird’s father’s growing realization that his best efforts to bolster his career are never going to be good enough. The biggest troubles in Lady Bird’s world are in her family; they’re struggling with finances in a way that a teenager usually can’t fully comprehend, and when Larry loses his job it ratchets up Marion’s anxieties too. Lady Bird’s yearning to leave Sacramento hurts her mother; her shame about her family’s humble house — especially compared to those of some of the affluent kids at school — hurts her father. The movie’s central pairing is Lady Bird and Marion, in a mother-daughter relationship that’s drawn with rare sensitivity. Marion’s awareness of her family’s money struggles spills over into almost every conversation with her daughter. She refers to Lady Bird’s “wealthy friends” with a kind of bitterness that paves over insecurity, and she’s clearly terrified that Lady Bird will look down on her parents. But Lady Bird doesn’t care all that much about the money beyond how it affects her college plans; she’s worried that her mom simply doesn’t like her very much. The two of them are in deep need of actually seeing each other, instead of peering through glasses they’ve tinted themselves over 18 years of living in the same house. Lady Bird’s arc moves toward this point — helped along by the inevitable changes that come with high school graduation and the start of college — and when the realization finally hits mother and daughter that they truly love each other, it’s a rare and grace-filled moment to see in a teen-focused comedy. Lady Bird is beautifully realized and filled with wisdom To get all this depth into a film is hard enough; to get it into such a purely enjoyable movie is a small miracle, especially one that’s so loaded with delights. Lady Bird gets a lot about being a teenager right, but it’s especially good on being a teenager during the 2002-’03 school year, right down to the fashions (Lady Bird’s wealthier friends wear the ubiquitous Abercrombie rugby shirts, while Lady Bird’s clothing clearly comes from a thrift store and is a season or two off) and the music — contemporary songs like Justin Timberlake’s “Cry Me a River” butt up against the sort of mid-’90s tracks that teenagers of the era grew up listening to, hits by Dave Matthews Band, Alanis Morissette, and Bone Thugs-N-Harmony. Lady Bird is about a year younger than Gerwig (and me, incidentally), and though Gerwig’s insisted in interviews that the film isn’t a roman à clef, she renders Lady Bird’s senior year — and the texture of the city where she lives — with such loving detail that it’s obvious how much of it is rooted in a shared reality with her character. Much of that love is directed at the Catholic girls’ school that Lady Bird attends (Gerwig attended one as well), and in particular, headmistress Sister Sarah Joan (Lois Smith), who is a voice of wisdom (and good humor) for the otherwise religiously uninterested Lady Bird. But the movie sustains a kind of grace and faith, with the school year’s rhythms fitting into the religious holidays that can’t help but seep into Lady Bird’s bones. And it’s Sister Sarah Joan, ultimately, who opens Lady Bird’s eyes to her real feelings about Sacramento. After reading her college entrance essay, she remarks that Lady Bird clearly loves the city. “You write about Sacramento so affectionately, and with such care,” she tells her. When Lady Bird replies that she just pays attention, Sister Sarah Joan says, “Don’t you think they are the same thing? Love and attention?” Lady Bird seems only half-convinced, but by the end of the movie it’s clear that the lesson has sunk in. She has, by fits and starts, started to really look at the people around her, and thereby to grow into who she is becoming. Growing up means learning to love the world for what it is — and in making Lady Bird, Gerwig has done the same. Simone Weil would be proud. Lady Bird opens in limited theaters on November 3 and will roll out in the weeks following. Go to the Legal Help page to request content changes for legal reasons. The opening moments of “Lady Bird” accomplish so much so quickly, it takes your breath away. A mother and daughter are engaged in the time-honored tradition of the senior-year road trip to check out college campuses. It is 2002, and they are intently listening to a book on tape—in this case, The Grapes of Wrath. As it concludes, the two smile at one another, sigh and wipe the tears from their eyes. Tom Joad and Ma would approve. Enjoy the lack of familial tension while you can. This is just about the last time parent and child will agree on anything as 17-year-old Christine, aka the self-proclaimed Lady Bird (Saoirse Ronan, the very picture of adolescent pique), impatiently expresses her post-graduation intention to flee from her staid Sacramento and take off to the East Coast “where the culture is.” Later, she will deride her hometown as the “Midwest of California” and not bother to correct someone who thinks she is from San Francisco. Advertisement Meanwhile, Marion (Laurie Metcalf of “Rosanne” TV fame, staking her claim in the movie mom hall of fame) takes maternal passive-aggressiveness to new levels as a persnickety psychiatric nurse. Forced to work double shifts after her husband is laid off, she attempts to sell Lady Bird on a cheaper in-state institution, to no avail. The push-pull of their conflicting points of view, informed by fear of the unknown and begrudging expressions of affection, is the energy that drives one of the more accomplished female-led coming-of-age tales since the Golden Age of John Hughes. Little wonder there are subtle echoes of “Pretty in Pink” throughout, from not one but two pink-hued frocks and a pair of unsuitable suitors to a dad who is down on his luck (playwright Tracy Letts) and a batch of pop oldies on the soundtrack (Alanis Morissette and Dave Matthews Band loom large). It is also one of the better solo directing debuts by an actor in recent memory. Hardly a false step is taken by Greta Gerwig in her semi-autobiographical script that centers on Lady Bird’s final year at her rather progressive Catholic high school. While she might have poured parts of herself into her co-written screenplays for "Frances Ha" and "Mistress America," there is a special authenticity here that can’t be denied. Her film manages to feel like an indie dramedy but powers along like a mainstream film. Part of that comes from how Gerwig has colored in her supporting characters with marvelous quirks and telling details while showing exceptional taste in casting. Somehow, she has recruited last year’s Oscar It Boy, “Manchester by the Sea’s” Lucas Hedges and this year’s potential Oscar It boy, “Call Me by Your Name’s” Timothee Chalamet as Lady Bird’s crushes. But nothing beats Lady Bird’s BFF Julie (bubbly Beanie Feldstein, a true find), a numbers whiz who moons over their blandly handsome male math teacher. I don’t think there is another scene this year that I have enjoyed more than watching Lady Bird and her chum burst into giggle fits while chowing down on a box of communion wafers as if they were potato chips. Doing their heavenly best to do justice to Gerwig’s words are such veterans as Lois Smith as an understanding nun and August Wilson specialist Stephen McKinley Henderson as a depressed priest. Of course, the MVP here is Ronan, whose Lady Bird is as far from her sweet Irish lass in “Brooklyn” as she can be. Bedecked with a messy blood-red dye job, a smattering of acne and thrift-shop chic sensibilities, she is thoughtful and impulsive, sharp and naïve in equal measure. Lady Bird at one point declares that “the learning part of high school is over.” And, yet, there is still much to be absorbed when it comes to losing one’s virginity, cheating on quizzes, settling for being in the chorus of a drama club production, ditching a guy who clearly has no special feelings for you, breaking up and making up with your bosom buddy and finding out that smoking and drinking are not all that they’re cracked up to be. Yes, “Lady Bird” is occasionally too episodic and obvious—such as when our heroine decides to buy cigarettes, a lottery scratch-off ticket and a Playgirl magazine on her 18th birthday just because now she legally can. But then there are such displays of tenderness as a prom reunion and a missed goodbye that feel genuine. “Lady Bird” might not be perfect, but it touched many of the right emotional buttons for me. As for Gerwig, I can’t wait to see what she cooks up next time behind the camera. By the time you read this, you’ll probably have a pretty good idea of what the movie “Lady Bird” is about, seeing as how it’s the kind of modest, miraculous low-budget gem that takes on a life of its own. But sight unseen, the title sounded a lot like a sequel to last year’s “Jackie,” seeing as how LBJ succeeded JFK in office, and Lady Bird was the name of his wife. Turns out Lady Bird was also the name restless high school senior Christine McPherson picked for herself — a first act of defiance in a battle to assert her own identity as separate from her parents. “Lady Bird” is indie darling (and one-time mumblecore muse) Greta Gerwig’s directorial debut, and the character is basically her, 15 years younger and played by Saoirse Ronan, sporting a vampire-red rinse and a face full of acne (both signs of its attention to detail). Early Telluride reactions seem particularly impressed that such an accomplished film could be anybody’s first, although those who’ve been following Gerwig’s career surely saw it coming: She’s been starring in, co-writing and even co-directing smart, soul-baring dramedies for more than a decade. The real surprise is just how honest and personal this film proves to be — again, par for the course with Gerwig, and yet, fairly rare among first-time directors, who haven’t had nearly so much practice simply being real. Just think, it took Mike Mills 11 years to work up to examining that chapter of his life in “20th Century Women,” a movie in which Gerwig played a key supporting role, and a spiritual model for this richly complicated homage to her own mother (a once-in-a-lifetime part for Laurie Metcalf). But the more appropriate comparison might be to “Frances Ha,” which also turns on a young woman’s what’s-in-a-name coming-of-age concerns. The only thing missing from “Lady Bird” is Gerwig herself, although one could argue that she inhabits every frame of this movie, and especially those in which Ronan appears — that’s how thoroughly the young Irish actress channels her Sacramento-raised writer-director. And wouldn’t it be great if this were but the first instance of a career-long series of stand-ins, the way Woody Allen casts stammering act-alikes to basically play him on screen? Not that you have to be familiar with any of Gerwig’s previous roles to appreciate “Lady Bird,” and considering the college-age demographic to which this A24 release is most likely to appeal, the vast majority probably won’t have seen a single one. Absorbing Gerwig’s mannerisms and way of speaking, Ronan comes across as a bright young woman who feels as if she’s only experienced a fraction of what life has to offer. She also looks less like a Greta than she does a Lady Bird. The self-selected nickname not only suits her character — fancifully girlish, yet eagerly anticipating womanhood — but is a lot less eccentric than the affectations other screenwriters invent to distinguish their oddball teen characters (say, Bud Cort’s faux suicides in “Harold and Maude” or Paul Dano’s notebook writing in “Little Miss Sunshine”). Gerwig isn’t being quirky merely for the sake of being quirky, but to make a point, and that point is: How’s a middle-class gal with average grades and no hobbies supposed to distinguish herself from her parents, much less her peers? There’s the Manic Panic red hair, of course, but beyond that, “Lady Bird” is about that universal late-teen need to reject our parents’ advice and to pave our own way, even at the cost of making a few easily avoidable mistakes. Only then do most of us come to appreciate how right our parents may have been. In the opening scene, Lady Bird’s mom (Metcalf, in her meatiest role since “Roseanne”) is chaperoning her on a college tour, basically any school within driving radius of Sacramento, where the McPhersons live modestly (unemployed dad Tracy Letts and adopted brother Jordan Rodrigues round out the family) and send their daughter to a private school on scholarship. Like so many movie teens, Lady Bird really wants to get as far away from home as possible. She believes that she has outgrown Sacramento — as Gerwig must have felt when she herself went off to college — but in truth, the movie doubles as a sweet homage to a city seldom depicted on screen (coincidentally, it’s also where Ben Stiller lives in college-application comedy “Brad’s Status”). Loosely structured and jauntily paced, the events of “Lady Bird” span the 2002 school year — although the film inexplicably skips from prom to its freshman-year coda at the end, breezing by the last summer at home when so many coming-of-age films unfold. It’s a genre that’s already been explored from practically every angle, though as a writer, Gerwig shows a gift for specificity that turns her first audition, first kiss and first heartbreak into singular events. Treating each of its characters with respect no how small their parts, Gerwig’s script derives much of its comedy from the nun-run Eternal Flame school (where Lady Bird’s grades aren’t that good, and she has no lasting hobbies to speak of), and to a lesser degree, her friendship woes and romantic disappointments. While nowhere near as sassy as Diablo Cody nor as self-assured as Lena Dunham, “Lady Bird” comes from a fundamentally genuine place — as evidenced by the character’s betrayal of lifelong bestie Jules (charismatic newcomer Beanie Feldstein). The movie is also frank — and funny — about the awkwardness of first-time sexual experiences, and casts realistic actors as Lady Bird’s two senior crushes: theater geek Danny (“Manchester by the Sea” breakout Lucas Hedges) and aloof rocker Kyle (Timothée Chalamet). But the relationship that matters most is the one between Lady Bird and her mother, based in love but increasingly combative as young Christine asserts her own identity — felt from the first scene, when Lady Bird throws herself out of the moving car when her mom’s nagging becomes too much, with the consequence that she wears a bright pink cast for at least half the movie. Metcalf has never had a big-screen role as rich as this, and she makes the most of it, playing a character who doesn’t hold back in criticizing her daughter’s faults (from academics to future job prospects), but works double shifts in the psych ward to provide for her future. On the surface, she’s tough on Lady Bird, but Metcalf manages to communicate between each line how it all comes from a place of caring. The big showdown between them involves Lady Bird’s choice of college, and what will become of the family if she goes off to the east coast. Gerwig may have flown the nest, but “Lady Bird” is proof that she hasn’t forgotten where she came from.
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Summary: Actress Greta Gerwig makes her solo directorial debut in Lady Bird, a film she wrote based on her experience growing up in Sacramento. But don't expect it to feel like a first attempt. The film following a year in the life of a teenage girl played by Saoirse Ronan, whom you may remember from The Lovely Bones and Hanna, with Laurie Metcalf as her mother, had a 100% rating from 65 critics on Rotten Tomatoes. Here's what they're saying: With this collection of "tiny and painfully honest details" of teenage life, "Gerwig has arrived and solidified her place as one of the most invigorating, observant and authentic voices in movies today with a director's acumen to match," writes Lindsey Bahr at the AP. "Bursting with wit, humanity, joy and truth," Lady Bird is "an undeniable pleasure to live in for a too-brief 93 minutes," she writes. Ronan is "supurb," while Metcalf is "at the top of her game." "Lady Bird is the rare movie that manages to be affectionate, entertaining, hilarious, witty, and confident; it's one of the best films of 2017, and certainly my favorite," writes Alissa Wilkinson at Vox. Achieving such "a remarkable amount of depth" in "such a purely enjoyable movie is a small miracle," she adds. But then, "Gerwig is by far one of the most talented filmmakers working today." There's "hardly a false step" in Lady Bird, "one of the more accomplished female-led coming-of-age tales since the Golden Age of John Hughes," writes Susan Wloszczyna at RogerEbert.com. She credits the "energy" of Metcalf and "MVP" Ronan but also "a special authenticity" provided by Gerwig, who delivers "one of the better solo directing debuts by an actor in recent memory." Lady Bird is the rare bird that feels "like an indie dramedy but powers along like a mainstream film." "It's the kind of modest, miraculous low-budget gem that takes on a life of its own," but "the real surprise is just how honest and personal this film proves to be," writes Peter Debruge at Variety. Ronan is terrific, but not all of his praise is reserved for her. "Metcalf has never had a big-screen role as rich as this, and she makes the most of it."
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Summarize: Il Green Pass sarà obbligatorio per tutti i lavoratori da metà ottobre. La decisione del governo è attesa per oggi, dopo l'incontro con i capidelegazione di maggioranza e il successivo Consiglio dei ministri che, nel pomeriggio, dovrebbe approvare il testo del decreto unico con cui l'obbligo di certificazione verde sarà esteso ai dipendenti del settore pubblico e privato. "Se c'è un'emergenza sanitaria non capiamo perché non venga messo un obbligo vaccinale", ha detto il segretario generale della Uil Pierpaolo Bombardieri intervenendo alla trasmissione "L'Italia s'è desta" di Radio Cusano Campus. Durante l'incontro di ieri con il governo, i sindacati non hanno opposto resistenza alla certificazione verde nei luoghi di lavoro, ma hanno suggerito di percorrere la strada dell'obbligatorietà. "Il governo – ha aggiunto Bombardieri – preferisce arrivare al vaccino attraverso il Green Pass perché non possono verificare che una persona sia vaccinata per strada, ma possono farlo nei luoghi di lavoro". La road map del governo, quindi, sembra essere quella di estendere il più possibile il Green Pass per raggiungere una percentuale di vaccinati tra l'80 e il 90%. Il decreto che sarà varato questo pomeriggio dovrebbe entrare in vigore nella seconda settimana di ottobre e questo potrebbe essere un invito a vaccinarsi per tutti quei lavoratori che non hanno ancora ricevuto la prima dose e che, in questo modo, potrebbero ottenere in tempo la certificazione verde per entrare a lavoro, senza doversi sottoporre a tampone. È quella che in molti, durante i dibattiti parlamentari degli ultimi giorni, hanno definito una "spinta gentile alla vaccinazione". Il tema è proprio questo: il governo non esclude di introdurre l'obbligo vaccinale, ma vi ricorrerà solo se la percentuale di immunizzati non dovesse raggiungere la soglia fissata.
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Summary: In vista dell'estensione del Green Pass ai lavoratori pubblici e privati, i sindacati suggeriscono al governo la strada dell'obbligatorietà dei vaccini. "Se c'è un'emergenza sanitaria non capiamo perché non venga messo un obbligo vaccinale", ha detto il segretario generale della Uil Pierpaolo Bombardieri. Per il momento, invece, l'esecutivo preferirebbe incentivare le vaccinazioni con lo strumento del Green Pass.
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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Rebuilding Local Business Act of 2010''. SEC. 2. REBUILDING COUNTIES. (a) In General.--Section 3(p) of the Small Business Act (15 U.S.C. 632(p)) is amended-- (1) in paragraph (1)-- (A) in subparagraph (D), by striking ``or'' at the end; (B) in subparagraph (E), by striking the period at the end and inserting ``; or''; and (C) by adding at the end the following: ``(F) rebuilding counties.''; and (2) in paragraph (4), by adding at the end the following: ``(E) Rebuilding counties.-- ``(i) In general.--The term `rebuilding county' means an initial period rebuilding county or an extension period rebuilding county. ``(ii) Initial period rebuilding county.-- The term `initial period rebuilding county' means a county, parish, or similar political subdivision-- ``(I) for which the Administrator determines that the 1-year unemployment rate average is not less than 110 percent of the 1-year average unadjusted unemployment rate for the United States, based on the most recent data available from the Secretary of Labor; ``(II) that-- ``(aa) as of the date of the determination under subclause (I), is not a HUBZone; or ``(bb) will cease to qualify as a HUBZone not later than 2 years after the date of the determination under subclause (I); and ``(III) during the 3-year period beginning on the date on which the Administrator makes the determination under subclause (I). ``(iii) Extension period rebuilding county.--The term `extension period rebuilding county' means a county, parish, or similar political subdivision-- ``(I) for which the Administrator has made a determination under clause (ii)(I); ``(II) for which the 3-year period described in clause (ii)(III) has ended; ``(III) for which the Administrator determines that the average unemployment rate for the 1-year period ending on the date on which the 3-year period described in clause (ii)(III) ends is not less than 140 percent of the 1-year average unadjusted unemployment rate for the United States, based on the most recent data available from the Secretary of Labor; and ``(IV) during the period beginning on the date on which the Administrator makes the determination under subclause (III) and ending on the earlier of-- ``(aa) the date that is 3 years after the date of the determination under subclause (III); and ``(bb) the date on which the Bureau of the Census publicly releases the initial results of the first decennial census occurring after the date of the determination under subclause (III). ``(iv) 1-year unemployment rate average.-- The term `1-year unemployment rate average' means the average unemployment rate, based on the most recent data available from the Secretary of Labor, during any 1-year period during the period-- ``(I) beginning on the date on which a recession begins, as determined by the National Bureau of Economic Research; and ``(II) ending on the date that is 180 days after the date on which the National Bureau of Economic Research publicly releases the determination under subclause (I).''. (b) Recession of 2007.--For purposes of applying section 3(p)(4) of the Small Business Act, as added by subsection (a), in relation to the recession announced by the National Bureau of Economic Research on December 1, 2008, the term ``1-year unemployment rate average'' means the average unemployment rate during the 1-year period ending on the date of enactment of this Act, based on the most recent data available from the Secretary of Labor.
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Title: To amend the Small Business Act to temporarily designate as a HUBZone counties that are most affected by a recession Summary: Rebuilding Local Business Act of 2010 - Amends the Small Business Act to designate as a HUBZone (historically underutilized business zone), for purposes of assistance under such Act: (1) an initial period rebuilding county; and (2) an extension period rebuilding county. Defines an initial period rebuilding county as a county, parish, or similar political subdivision: (1) for which the Administrator of the Small Business Administration (SBA) determines that the one-year unemployment rate is at least 110% of the one-year average unadjusted unemployment rate for the United States; (2) that is not a HUBZone or will cease to qualify as a HUBZone two years after such unemployment determination; and (3) during the three-year period beginning on the date of such unemployment determination. Defines an extension rebuilding county as a county, parish, or political subdivision: (1) in which the Administrator has made the above unemployment determination; (2) for which the above three-year period has ended; (3) for which the Administrator determines that the unemployment rate as of a specified date is at least 140% of the average unadjusted unemployment rate; and (4) for which such designation extends for an additional specified period after the three-year period.
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Summarize: CROSS-REFERENCE TO RELATED APPLICATION This application claims priority from German patent Application No. 201 07 778.7, titled PUNCTURE CANNULA, filed in Germany on May 8, 2001, the entire contents of which is incorporated by reference herein as though set forth in full. FIELD OF THE INVENTION The instant invention relates to a puncture cannula, particularly for nerve stimulation, comprising a steel cannula attached to a cannula hub and a cable extending through an insertion channel of the cannula hub and having a core connected to the steel cannula. BACKGROUND OF THE INVENTION European Patent 0 102 538 B1 discloses a puncturing and catheterizing device which is suited for the puncturing and catheterizing of nerve strings. This device comprises a steel cannula with a cannula hub provided at the distal end of the cannula. The steel cannula is connected to a cable by which an electric potential can be applied to the steel cannula. When the steel cannula, which has an exposed tip, is brought into a position close to a nerve, electrical pulses, which are applied to the cable by a suitable stimulation device, will cause a nerve stimulation, resulting in corresponding reflexes of the patient. In this manner, it can be verified whether the tip of the cannula has been guided sufficiently close to the selected nerve. Thereafter, an anesthetic agent can be injected either through the cannula or through a catheter which has been set by use of the cannula in order to perform local anesthesia. In the known device, the electrical connection of the core of the cable to the steel cannula is performed by winding the core around the cannula, or by soldering the core to the cannula and subsequently enmolding the connection. Such a connection technique is complicated and overly expensive. It is an object of the invention to provide a puncture cannula which is easily manufactured and guarantees a safe contact between the cable and the puncture cannula. SUMMARY OF THE INVENTION According to the instant invention, the hub of the cannula comprises a metallic clamping element which is formed with a first clamping slot for engaging the steel cannula and with a second clamping slot for engaging the core of the cable. By insertion of the clamping element into the hub of the cannula, the steel cannula and the core of the cable will be automatically positioned in their respective clamping slot to be tightly surrounded therein. In this manner, these two components are clamped into their desired positions at the same time, while the clamping of one component will not be affected by the other component. The clamping element is provided as a one-piece member, with its clamping slots arranged in such a configuration that the first clamping slot clamps the steel cannula when the second clamping slot clamps the core of the cable. Thus, the clamping slots have the same mutual distance as the steel cannula and the core of the cable and will be activated by displacing the clamping element. A considerable advantage of the invention resides in the simple and safe mounting process. By displacement of the clamping member, the steel cannula and the cable are fixed relative to each other and are also fixed relative to the hub of the cannula. It is also possible to perform the clamping prior to the attaching of the steel cannula in the hub of the cannula. In this case, an adhesive is inserted into a recess of the cannula hub after insertion of the clamping element. This adhesive will enter into all gaps to thus fasten the steel cannula in the hub of the cannula. Further, the adhesive serves as a surrounding electrical shielding for the cable and lends further stability the fixation of the cable. Further, the adhesive fulfills the function to cover all metallic parts which exist on or in the hub of the cannula, thus precluding the possibility that a person might inadvertently come into contact with any one of the current-carrying parts. According to a preferred embodiments of the invention, it is provided that the second clamping slot is formed with cutting edges adapted to penetrate an insulation of the cable. This obviates the need to first strip the cable and expose the core of the cable. When the clamping element is inserted into the hub of the cannula, the cutting of the cable insulation and the clamping of the core of the cable are performed automatically in the process. Preferably, the clamping slots are arranged behind each other in the clamping element, with a converging opening provided between the clamping slots so as to decouple the two clamping slots from each other. The puncture cannula of the invention is particularly suited for nerve stimulation. The instant puncture cannula is compatible with different techniques for using a cannula. Thus, for instance, an anesthetic agent can be injected directly through the steel cannula, or the steel cannula can be connected to a short catheter or a capillary tube. It is also possible to set a catheter via the puncture channel generated by the steel cannula, either with or without a guide wire. The clamping element can be of a design adapted to various diameters of steel cannulae and/or of cores of cables. A preferred embodiment of the invention will be described in greater detail hereunder with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a longitudinal sectional view of an embodiment of the puncture cannula; FIG. 2 is a cross-sectional view along the line II—II of FIG. 1; and FIG. 3 is a view from the direction of the arrow III in FIG. 1. DETAILED DESCRIPTION OF THE INVENTION The puncture cannula comprises an elongate hollow steel cannula 10 attached to a cannula hub 11 of a plastic material. Steel cannula 10 consists of a tube provided with a nonconductive coating 12. The tip of the cannula 10 (not shown) is exposed. Seated on a tubular connecting piece 13 of cannula hub 11 is a protective hose 9 which surrounds steel cannula 10 and extends beyond the length of cannula 10 to protect users from accidental injuries which might be caused by the tip of the cannula. The protective hose 9 can be withdrawn from the connecting piece 13. The distal portion 10 a extends through a cavity or a recess 14 of the hub 11 of the cannula and ends in a cavity 15 which is closed by a hose connector 16. A hose 17, starting from hose connector 16, is provided for the administration of an anesthetic agent which will then be injected into the patient's body through steel cannula 10. An introduction channel 18 is arranged to enter the recess 14 of hub 11 and has a cable 19 extending therethrough. Introduction channel 18 is formed along a part 18 a of its length as a circumferentially closed bore, while another part 18 b of the length of channel 18 is formed as an open channel. The channel part 18 b terminates at an end wall 20 forming a stop face for the end of cable 19. Cable 19 comprises a cable core 21 made of copper and an insulation 22. Introduction channel 18 is oriented under an acute angle relative to the longitudinal axis of steel cannula 10. A guideway 23, formed for movement of a clamping element 24 therein, is arranged in a transverse direction relative to steel cannula 10 and cable 19. Clamping element 24 comprises an elastic plate 25 made from spring steel. As illustrated in FIG. 2, this plate is formed with a first clamping slot 26 for steel cannula 10 and with a second clamping slot 27 for the core 21 of cable 19. Both clamping slots 26, 27 are arranged behind each other along a common axis and have a widened opening 28 arranged therebetween. Each of the clamping slots 26, 27 has a tapering shape in the direction of insertion, i.e. from top to bottom in FIG. 2, so that a progressing insertion of the clamping element 24 into the cannula hub 11 will cause an increasingly stronger clamping action on the steel cannula 10 and the cable core 21, respectively. The edges of second clamping slot 27 are cutting edges provided to cut through the insulation 22 of cable 19, thus generating a safe electrical contact to the core 21 of cable 19. Clamping slot 26 is delimited by wings 29, 30 which can be bent about bending regions 31 for adaptation to different diameters of steel cannulae 10. In FIG. 1, one of these wings, 29, is shown as bent in outward direction. Clamping element 24 comprises a plate which, however, does not necessarily have to be flat. The plate is guided in a linear guideway 23 for displacement between a clamping position and a release position. When the steel cannula 10 and the cable 19 are to be mounted, the steel cannula 10 is inserted into the channel of cannula hub 11 which is provided for this purpose, and the cable 19 along with its insulation 22 is introduced into insertion channel 18. Subsequently, clamping element 24 is inserted into guideway 23 and pushed into the same until reaching the end stop, while the clamping slot 26 exerts a clamping grip on steel cannula 10 and the clamping slot 27 cuts through the insulation 22 into the core 21 of cable 19. The hub 11 of cannula 10 is then positioned in such an orientation that the recess 14 is facing upwards. In this condition, FIG. 3 represents a plan view from above. Next, a liquid adhesive 33 is filled into recess 14. This adhesive will occupy the recess 14 completely. The adhesive enters into the annular gap between steel cannula 10 and cannula hub 11 and into the annular gap between introduction channel 18 and the cannula hub 11 and will after hardening also keep the clamping element 24 fixed in position. Thus, a sole adhesion process is sufficient to fix all of the components relative to each other and to close them in a tightly sealed manner. Also the entrance orifice 34 of guideway 23 is closed by adhesive and sealed so that the whole clamping element 24 is embedded in adhesive material. Thus, no electrically conductive components are accessible from the outside.
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Summary: A puncture cannula, particularly for nerve stimulation, comprises a steel cannula ( 10 ) attached to a cannula hub ( 11 ), and a cable ( 19 ) extending through an insertion channel ( 18 ) and having its core ( 21 ) connected to the steel cannula ( 10 ). The hub ( 11 ) of the cannula ( 10 ) comprises a metallic clamping element ( 24 ) formed with a first clamping slot engaging the steel cannula ( 10 ) and with a second clamping slot engaging the core ( 21 ) of the cable ( 19 ).
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Summarize: WALNUT GROVE — The search for 55-year-old Prasad Moparti, who went missing during his daughter’s wedding reception in Walnut Grove, has been suspended indefinitely, the Sacramento County Sheriff’s Department said Tuesday. The sheriff’s department and other agencies have been searching the area surrounding the historic Grand Island Mansionin Walnut Grove, where the wedding reception was held, since Feb. 13. There has been no sign of Moparti since family says he walked away from the reception on foot about 4 p.m. on Feb. 13. Moparti arrived in the U.S. from India last month and did not know the area well. Some family members said that Moparti was suffering from depression. There is no evidence of suspicious circumstances of foul play. Moparti was considered a voluntary missing person, but since he wasn’t familiar with the area, the sheriff’s department tried to locate him. The sheriff’s department is still asking anyone who may have seen Moparti to call (916) 874-5115. Starting in 1996, Alexa Internet has been donating their crawl data to the Internet Archive. Flowing in every day, these data are added to the Wayback Machine after an embargo period. The search for 55 year old Prasad Moparti has been suspended indefinitely. The Sacramento Sheriff’s Department, with assistance of other outside agencies, searched the areas around Grand Island Mansion in Walnut Grove since the evening of February 13, 2016. All resources were utilized to search for Moparti on the ground, water, and from the air with no results. The Sheriff’s Department was seeking the public’s assistance in locating Moparti, who went missing from Grand Island Mansion around 4:00 p.m on February 13, 2016. He was reported missing by family members after he left on foot from a wedding reception and never returned. Moparti arrived in the United States from India in January of 2016, and did not know the area well. Family members further indicated Moparti suffered from depression, and that he showed indicators of his depression leading up to the wedding. He was last seen by family members walking away from the wedding reception alone. There is no indication at this time of any suspicious circumstances or foul play leading to his disappearance. Moparti was considered a voluntary missing person, but because of Moparti’s lack of knowledge of the area and his depression, the Sheriff’s Department utilized all resources available in attempts to locate him. Moparti is described as a dark skinned East Indian male with short black/gray hair and black eyes. He is 5’10” tall, weighs approximately 180 pounds, and was last seen wearing traditional dress in brown/gold colored top with a cream-colored undergarment. The Sheriff’s department is still asking anyone knowing the whereabouts of this missing person to call the Sheriff’s Department at (916) 874-5115. Sergeant Tony Turnbull Sheriff’s Spokesman Please enable Javascript to watch this video WALNUT GROVE-- It's a picturesque setting for a wedding; the historic Grand Island Mansion sits between the Sacramento River and rows of orchards in Walnut Grove. Saturday, Durga Moparti got married at the mansion. Two days later she's still in the area, stressing instead of celebrating. "I think he just went for a casual walk because one of my family members actually saw him walking down the road," Moparti said. Her father, Prasad Moparti, hasn't been seen since wandering off during the reception. Relatives tell FOX40 the 55 year-old came to California from India about a month ago to see his daughter walk down the aisle. "I believe that he was just walking and he fell unconscious, and he just tripped or something like that, but I'm really not sure what happened, and this seems like a total mystery," Moparti said. Many wedding guests spent the day trying to solve that mystery by scouring the area alongside search crews. The Sacramento Sheriff's Department had a boat out in the river with a cadaver dog. Family and friends spread missing person's posters throughout the area. Because he walked away from the party, the sheriff's department says Moparti is considered a "voluntary missing person" and that some relatives told investigators he was recently depressed. The bride paints a different picture. "He was a very happy person, and he enjoyed the entire wedding. He was actively participating in all the events," Moparti said. For now, all plans are on hold until the family figures out what happened to the father of the bride. "Our only focus is to get our father back, and our efforts will continue until we get our father back," Moparti said. The 55-year-old was last seen wearing the traditional brown and gold dress seen in the photograph.
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Summary: The night before her wedding ceremony in Walnut Grove, Calif., Durga Moparti's father danced. What followed the next day "seems like a total mystery," the 29-year-old tells Fox40. Prasad Moparti apparently told a relative he was going for a brief walk. The 55-year-old wandered off around 4pm Saturday and never returned to Grand Island Mansion, where the reception had been held. Family members and friends spread fliers over the past few days, while local police and the Coast Guard searched along the shore and the California Highway Patrol looked for clues from the air, all to no avail. On Tuesday, the Sacramento County Sheriff's Department called off the search, reports the Sacramento Bee. A sheriff's rep says there is no sign of "suspicious circumstances or foul play" and that family members indicated the native of India suffered from depression. Although Prasad was considered to be missing of his own accord, "all resources were utilized" to look for him, per a sheriff's department statement, because of his possible mental health issues and the fact he wasn't familiar with the area. Durga says her dad-who she thinks possibly tripped or fell unconscious-doesn't have his passport or cash on him. The only sign of discontent she had noticed: his idleness since coming to the States about a month prior. "He was a workaholic back in India and was managing several projects," she says, per the Bee. "He was bored."
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Summarize: The U.S. Department of Defense announced Wednesday that the remains of 17 service members who died in a 1952 Globemaster crash on the side of an Alaskan glacier were recovered and are being sent home to their families after being lost for more than six decades. An Alaska National Guard Blackhawk helicopter spotted the wreckage two years ago emerging from the side of the Colony Glacier, which is about 50 miles east of Anchorage. Days later, a military operation was conducted to recover items from the site, and possible future recovery expeditions may continue. The plane, which was carrying 52 people, crashed on Nov. 22, 1952, during a flight from McChord Air Force Base in Washington state. It became buried in snow and likely churned beneath the surface of the glacier for decades, the Army said. An Associated Press report days after the crash said the Globemaster was the third big Air Force transport plane to crash in Alaska that month and the sixth around the Pacific Rim. Soon after the crash, a military team tried three times to make it to the site, but was thwarted by bad weather, Tonja Anderson, whose grandfather was on board the flight, told the Associated Press in 2012. After returning from the site, Terris Morris, a civil air patrol member and president of the University of Alaska, told reporters that the plane "obviously was flying at full speed" when it hit Mount Gannett, sliding down the snow-covered cliffs, exploding and disintegrating over two or three acres. Only the tail and flippers of the craft were intact, but the tail numbers were enough for an identification. Moore said blood was found on a piece of blanket and there was a "sickly-sweet smell of death." Reuters reported that beside the human remains collected at the site in 2012, searchers found a mini-box of Camel cigarettes, a survival suit and a hockey puck. In all, military personnel removed about 1,800 pounds of debris. Scientists from the Armed Forces DNA Identification Laboratory used forensic tools to identify the service members. The department identified the recovered remains belonging to Army Lt. Col. Lawrence S. Singleton; Army Pvts. James Green Jr. and Leonard A. Kittle; Marine Corps Maj. Earl J. Stearns; Navy Cmdr. Albert J. Seeboth; Air Force Cols. Noel E. Hoblit and Eugene Smith; Air Force Capt. Robert W. Turnbull; Air Force 1st Lts. Donald Sheda and William L. Turner; Air Force Tech. Sgt. Engolf W. Hagen; Air Force Staff Sgt. James H. Ray; Air Force Airman 1st Class Marion E. Hooton; Air Force Airmen 2nd Class Carroll R. Dyer, Thomas S. Lyons and Thomas C. Thigpen; and Air Force Airman 3rd Class Howard E. Martin. They will receive a burial with full military honors, the department said. Fox News' Edmund DeMarche and The Associated Press contributed to this report For more than six decades, a tombstone at the Sunnyside Cemetery in Caney, Kan., has marked Pvt. Leonard Kittle’s final resting place. On Saturday, he will at last be buried there as his family looks on. Private Kittle was one of 17 people aboard a military plane that crashed in Alaska in 1952 whose remains have been identified after being recovered from a melting glacier where the wreckage was found, the Department of Defense said Wednesday. The service members’ remains will be returned to their families for burial with full military honors, Defense Department officials said. There were 52 crew members and service members from various military branches onboard when the plane crashed near Anchorage on Nov. 22, 1952. A week after the crash, the military declared Private Kittle and everyone onboard dead, said his younger sister, Beatrice Crawford, 82, of Bartlesville, Okla. “They told us that they were no longer with us, that they’d given up the hunting,” she said Wednesday. ANCHORAGE, Alaska (AP) — The remains of about a third of the service members who died when their military transport plane crashed into an Alaska mountain and then was buried for decades in glacier ice have been identified, military officials said Wednesday. FILE - This Thursday, July 12, 2012, file photograph shows the Joint Task Force-Alaska Team from Joint Base Elmendorf-Richardson and Fort Wainwright recovering debris on Colony Glacier near Anchorage,... (Associated Press) FILE - This Thursday, July 12, 2012, file photograph shows the Joint Task Force-Alaska Team from Joint Base Elmendorf-Richardson and Fort Wainwright recovering debris on Colony Glacier near Anchorage,... (Associated Press) FILE - This Thursday, July 12, 2012, file photograph shows the Joint Task Force-Alaska Team from Joint Base Elmendorf-Richardson and Fort Wainwright recovering debris on Colony Glacier near Anchorage,... (Associated Press) The Department of Defense released the identities of 17 people onboard the C-124 Globemaster, which crashed in 1952, and said the remains will be returned to their families for burial with full military honors. The first believed burial is planned Saturday in Caney, Kansas, for Army Pvt. Leonard A. Kittle, said Tonja Anderson-Dell, a Tampa, Florida, woman who has researched the crash for years. Her interest was stoked about the crash when her grandmother, now deceased, told her details of her grandfather, Isaac Anderson, 21, who died when the plane hit the mountain on Nov. 22, 1952. Anderson-Dell also maintains a Facebook page about the crash and recovery efforts. The identifications were bittersweet for her because her grandfather wasn't among them. "He wasn't part of the ones that will be coming home," she told The Associated Press by telephone Wednesday. "However, I am still happy for the families that are bringing their servicemen home." The plane from McChord Air Force Base, Washington, was headed to Elmendorf Air Force Base in Anchorage when it went down with 52 crew members and passengers aboard. Efforts to reach the crash site immediately after the crash were halted by bad weather. Days later, a member of the Fairbanks Civil Air Patrol, along with a member of the 10th Air Rescue Squadron, landed at a glacier and positively identified the wreckage as the Globemaster. The civil air patrol member was Terris Moore, president of the University of Alaska. He later told reporters the plane "obviously was flying at full speed" when it hit Mount Gannett, sliding down the snow-covered cliffs, exploding and disintegrating over 2 or 3 acres, according to an Associated Press story from the time. The heavy transport plane with 41 passengers and 11 crew members became buried in snow and likely churned beneath the surface of the glacier for decades. The Alaska National Guard discovered the wreckage in June 2012 on Colony Glacier, about 40 miles east of Anchorage. An eight-man Joint POW/MIA Accounting Command recovered materials such as a life-support system from the wreckage and possible bones from the glacier two summers ago, and took the evidence to the command's lab in Hawaii for analysis. Military officials said in a release that the remaining 35 service members have not yet been recovered from the wreckage, and the site will be monitored for possible future recovery efforts. Further details were not immediately available from a military spokeswoman who didn't return a message to The Associated Press. Anderson-Dell said it is her understanding the military will continue to fly over the area to see if the glacier has given up any additional material from the wreckage.
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Summary: The families of 17 servicemen killed when an Air Force transport plane crashed into an Alaskan mountain in 1952 can now finally bury their loved ones. Wreckage from the Globemaster aircraft was found near Mount Gannett in 2012 after having spent decades buried under the ice of Colony Glacier, and POW/MIA teams have scoured the area for remains of the 52 people who were on board. The remains that investigators have managed to identify will be returned to their families for burial with full military honors, reports Fox News. The first burial will be that of Pvt. Leonard Kittle, who will be laid to rest in his hometown in Kansas on Saturday. His younger sister, 82-year-old Beatrice Crawford, says the military declared everybody on board dead a week after the crash. "They told us that they were no longer with us, that they'd given up the hunting," she tells the New York Times; bad weather stymied immediate recover efforts, and the wreckage was later IDed as the C-124 Globemaster. Military officials say that 35 service members have yet to be recovered, and the site will be monitored to see if the glacier gives up any more remains, the AP reports. At the time of the crash, the AP reported that the plane exploded and disintegrated over as many as three acres; a compass, a survival kit, and a hockey puck were among the other items recovered at the scene.
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Summarize: A woman left heartbroken by seven miscarriages has finally achieved her dream of becoming a mother - thanks to a stitch in her cervix and daily doses of aspirin. Samantha Bourner had spent a traumatic 12 years trying to start a family since meeting her husband, David, in 2003. The 33-year-old initially became pregnant after two years, but was devastated when she lost the baby - whom she had named after her grandfather Alfie - at just 22 weeks. Over the next decade she suffered another five miscarriages and almost gave up hope of ever having a child. Samantha Bourner, 33, suffered seven miscarriages and she and her husband David, 35, nearly gave up on ever having a child. On December 3 2014 she gave birth to Noel Arthur Paul - thanks to a stitch in her cervix. Mrs Bourner said Noel had a personality straight away. She said: 'He likes lots and lots of cuddles and attention and doesn't like going down in his bed' She was diagnosed with Antiphospholipid syndrome, which causes an increased risk of blood clots and raises the risk of miscarriage in pregnant women. Doctors said the condition – which causes the blood to be 'too sticky' - could be causing her recurring miscarriages. They prescribed aspirin, which helps thin the blood, and another drug called Clexane which stops blood clots forming. Last March Mrs Bourner became pregnant for a seventh time and was delighted when doctors told her she would be having twins. But she lost one of the babies at just nine weeks, and she and her husband David, 35, feared the other child would not survive. However, doctors inserted a stitch into her cervix, which stops it opening too early, reducing the chance of a miscarriage. On December 3 Mrs Bourner gave birth to Noel Arthur Paul four weeks early by emergency Caesarean section. Mrs Bourner, from Hamstreet, Kent, said: 'It's been a long time coming but nothing prepares you for being a mum. 'It's a lot harder than I thought it would be and I'm always tired. 'But it's really exciting, watching every stage he goes through. Just seeing him every day is amazing. 'I never felt safe during the pregnancy. 'Everyone was getting really excited but right up until the point I was holding him I thought it was going to go wrong.' Mrs Bourner, who runs a craft company, added: 'Noel had a personality pretty much straight away. A cervical stitch is also known as a cervical suture or cervical cerclage. It involves'stitching' a tape around the cervix to support it. This aims to keep the cervix closed and therefore to help prevent a baby being born too early. Doctors might advise a cervical stitch because they believe the woman has a weakness in the cervix – they call this ‘cervical incompetence’. This means the cervix runs the risk of opening before the end of a 40-week pregnancy, and maybe even very early on in pregnancy. If the cervix opens, there is an increased risk of the baby being born early and not surviving, or surviving with a serious risk of long-term problems. Source: Miscarriage Association. 'He likes lots and lots of cuddles and attention and doesn't like going down in his bed. 'He can be fast asleep in your arms and as soon as you put him down he's wide awake. He also loves being in the bath.' Noel was born at St Mary's Hospital in London weighing 5lb14oz - but Mrs Bourner still had worries right to the last minute. During her pregnancy, Mrs Bourner had suffered from gestational diabetes – a condition affecting pregnant women where there is too much glucose in the blood - and had to go on a strict low sugar diet. Then, one morning she woke up to discover she had been bleeding overnight and dialled 999 in tears. She told the operator she thought she had lost another baby and was rushed to hospital, where doctors performed an emergency Caesarean immediately. When Noel was born, Mrs Bourner said his first cry sounded 'amazing'. She believes her son's twin, whom she lost at nine weeks old, may actually have helped Noel to survive. The twin was absorbed into the womb rather than being miscarried in the normal way, a condition known as a 'vanishing twin' which sometimes occurs when more than one baby develops in the womb. Mrs Bourner said: 'Noel was always on one side of my tummy to begin with but when his twin died he moved over to where they had been. 'I think he was looking for him or her. The sibling was a vanishing twin, I never miscarried, it was reabsorbed into the womb. 'I believe that second twin meant there were extra hormones from that brief little life to help keep the other going.' Research suggests that vanishing twin syndrome occurs in roughly a third of pregnancies in which an early ultrasound detects two or more developing babies. After years of heartbreak, Mrs Bourner and her husband are delighted to be parents. Mrs Bourner said: 'We are over the moon with Noel, he really is a miracle and I adore him.' While Mrs Bourner says she is thrilled to finally be a mother, she currently has no plans to try for another baby. 'People keep saying now we've done it once we could do it again. 'But I would have to have to have that exact combination of hormones, supplements and diets - and even then it might not happen,' she said. 'It puts a strain on your body and doctors have said it might be dangerous for me to have another child. 'We are over the moon with Noel, he really is a miracle and I adore him.' Experts say just one per cent of couples suffer three or more miscarriages, so Mrs Bourner's success after seven was remarkable. Ruth Bender Atik, national director of the Miscarriage Association, said: 'Random miscarriages are horribly common and one in four women will have at least one. 'It is a far smaller group that have one after another, after another. It is only one per cent of couples trying to conceive who will have three or more.' She added: 'When it happens again and again it becomes really hard to bear - for more than half of those people they will never find out why they are miscarrying. 'Some couples will keep trying and some of them just feel like they need to stop because they can't cope anymore. 'I am really happy to hear Samantha has had success but I'm sure that it won't stop her feeling the grief for the babies that she has lost.'
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Summary: Samantha Bourner suffered six miscarriages and was left heartbroken. Doctors said her miscarriages were caused by Antiphospholipid syndrome. This condition causes an increase risk of blood clots and miscarriage. They prescribed an anti-clotting drug and aspirin to thin the blood. Doctors put a stitch in her cervix to prevent it opening too early. She was delighted to become pregnant again in March - and with twins. Lost one of the babies early on, and it was absorbed into the womb. Was thrilled to give birth to Noel Arthur Paul by emergency Caesarean.
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Summarize: Le estrazioni del Lotto, SuperEnalotto e 10eLotto di giovedì 5 novembre in diretta su Fanpage.it: sono stati appena comunicati i numeri vincenti delle tre lotterie italiane. Sale a 60,5 milioni di euro il jackpot in palio per il SuperEnalotto, in attesa di un fortunato vincitore. Vediamo tutti i numeri estratti per il Lotto e il 10eLotto serale, insieme ai simboli vincenti del Simbolotto di oggi. Ecco i numeri del Lotto estratti oggi su tutte le ruote: Bari: 66 – 85 – 12 – 38 – 67 Cagliari: 30 – 13 – 58 – 43 – 79 Firenze: 18 – 67 – 52 – 42 – 63 Genova: 61 – 12 – 66 – 74 – 89 Milano: 90 – 68 – 73 – 26 – 33 Napoli: 72 – 38 – 43 – 70 – 42 Palermo: 73 – 22 – 67 – 24 – 69 Roma: 39 – 38 – 42 – 61 – 4 Torino: 83 – 12 – 45 – 61 – 55 Venezia: 64 – 83 – 52 – 28 – 68 Nazionale: 83 – 58 – 76 – 38 – 5 Seconda estrazione della settimana per la lotteria più antica d'Italia, che prevede l'estrazione di 5 numeri da 1 a 90 su 11 ruote, 10 delle quali prendono il nome di altrettanti capoluoghi italiani. Su Lottomatica la verifica della vincita della schedina per l'estrazione di oggi. Combinazione vincente SuperEnalotto: 5 – 10 – 18 – 69 – 24 – 85 Numero Jolly: 65 Numero Superstar: 50 Jackpot: 60.500.000€ Sale a 60,5 milioni di euro il jackpot del SuperEnalotto in palio per chi centra i 6 numeri vincenti. Al pari di Eurojackpot, anche il SuperEnalotto mette in palio un montepremi milionario, che cresce di estrazione in estrazione. Quote del SuperEnalotto e relative vincite consultabili su Sisal e su superenalotto.com.
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Summary: I numeri vincenti delle estrazioni di oggi per il Lotto, SuperEnalotto e 10eLotto di giovedì 5 novembre 2020: 60,5 milioni di euro il jackpot del SuperEnalotto, attesa per conoscere le quote e sapere se qualche fortunato giocatore ha vinto il montepremi. Vediamo tutti i numeri estratti sulle ruote del Lotto e la combinazione del 10eLotto di oggi.
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Summarize: Overview of the Foreign Sovereign Immunities Act The Foreign Sovereign Immunities Act (the FSIA) applies to all foreign states and their "agencies and instrumentalities." Immunity for sovereign nations against suits in U.S. courts has a long history and is based on the principle that conflicts with foreign nations are more effectively addressed through diplomatic efforts than through judicial proceedings. Congress passed the FSIA to codify these long-standing principles and to clarify limitations on the scope of immunity that had emerged in international practice. The FSIA contains both a general, presumptive rule against litigation in U.S. courts and a number of exceptions permitting suits. As a general rule, foreign states, together with their agencies and instrumentalities, are "immune from the jurisdiction of the courts of the United States and from the states." However, the FSIA authorizes jurisdiction over foreign nations in several exceptions. Namely, a foreign state is not immune from U.S. courts' jurisdiction where (1) the foreign state has waived its immunity; (2) the claim is a specific type of admiralty claim; (3) the claim involves commercial activities; (4) the claim implicates property rights connected with the United States; (5) the claim arises from tortious conduct that occurred in the United States; (6) the claim is made pursuant to an arbitration agreement; or (7) the claim seeks money damages against a designated state sponsor of terrorism for injuries arising from a terrorist act. The exception for designated state sponsors of terrorism provides jurisdiction over cases involving designated "state sponsor[s] of terrorism" in suits involving "personal injury or death that was caused by an act of torture, extrajudicial killing, aircraft sabotage, hostage taking, or the provision of material support or resources for such an act if such act or provision of material support or resources is engaged in by an official, employee, or agent of such foreign state while acting within the scope of his or her office, employment, or agency." However, the exception seems to apply only to countries designated by the U.S. Department of State as state sponsors of terrorism. This list currently includes Iran, Sudan, and Syria. At the time suit was brought in the In re Terrorist Attacks litigation, the previous terrorism exception remained in force. Jurisdiction in Cases Against Foreign Defendants Before asserting jurisdiction to accept a case, a federal court must establish its authority over the dispute involved and the parties to the litigation. In other words, courts must assert both subject matter jurisdiction over each claim and personal jurisdiction over each defendant in a case. For cases involving foreign defendants, the analyses for subject matter and personal jurisdiction differ according to whether the FSIA applies. Subject Matter Jurisdiction For claims by U.S. plaintiffs against foreign non-state defendants to whom the FSIA does not apply—for example, claims against individuals or non-government owned corporations—federal law authorizes subject matter jurisdiction as long as the "amount in controversy" exceeds $75,000. In contrast, for claims against foreign states and their instrumentalities, the FSIA is a jurisdictional gatekeeper. The FSIA denies subject matter jurisdiction over claims against foreign defendants entitled to immunity. Conversely, the FSIA authorizes subject matter jurisdiction over claims in which a foreign state would be entitled to immunity under the FSIA but for the application of an exception. Individual foreign officials are not covered by the FSIA if they are sued in their capacity as individuals, but may be immune from suit under the common law of foreign sovereign immunity. One such defendant, the former president of two of the Saudi-run charities sued for alleged involvement in funding the 9/11 terrorist attacks, prevailed in a motion to dismiss the lawsuit against him on the basis of common-law foreign sovereign immunity. Personal Jurisdiction Personal jurisdiction is the second threshold hurdle for assertion of judicial authority in cases involving foreign defendants. Whereas subject matter jurisdiction governs courts' power over particular claims, personal jurisdiction governs courts' power over particular defendants. Thus, even if a court establishes jurisdiction over the subject matter of a claim, it cannot exercise its authority over a defendant for whom it lacks personal jurisdiction. Personal jurisdiction requires both statutory authority and satisfaction of Fifth Amendment due process standards. As with subject matter jurisdiction, statutory authority for personal jurisdiction over foreign defendants follows one of two distinct routes according to the FSIA's application. If the defendant is a foreign state or its agency or instrumentality, personal jurisdiction is statutorily authorized under the FSIA if subject matter jurisdiction is established. Alternatively, for a defendant who is not a foreign state or its agency or instrumentality, the ordinary procedure for obtaining statutory authority for personal jurisdiction applies; typically, a federal court must find statutory authority for personal jurisdiction in the laws of the state in which it sits. However, constitutional limits apply regardless of a statutory basis for personal jurisdiction. Under the due process clause, personal jurisdiction is constitutional if (1) defendants have had "certain minimum contacts with" the judicial forum attempting to assert jurisdiction, and (2) asserting such jurisdiction "does not offend traditional notions of fair play and substantial justice." The type and quantity of contacts necessary to constitute "minimum contacts" differ according to the type of personal jurisdiction—general or specific—that applies. General jurisdiction, which allows a court to exercise jurisdiction over a foreign defendant for any claim, does not require contacts related to the specific claim in the case but instead requires "continuous and systematic" contacts with a forum. Conversely, specific jurisdiction, which limits a court's jurisdiction over a defendant to claims in a particular case, involves no "continuous and systematic" requirement; instead, it requires that a defendant's contacts with the forum "relate to" or "arise out of" the claim at issue in the case. U.S. Court of Appeals Decision in In Re Terrorist Attacks on September 11, 2001 ("Terrorist Attacks III") In August 2008, the U.S. Court of Appeals for the Second Circuit (Second Circuit) affirmed dismissals of claims against the Kingdom of Saudi Arabia, a Saudi charity, Saudi princes, and a Saudi banker in In r e Terrorist Attacks on September 11, 2001. Plaintiffs in the case were victims of the September 11 terrorist attacks. They alleged that the Saudi defendants had supported Al Qaeda's financial backers prior to the attacks and were therefore civilly liable for plaintiffs' injuries. However, the court of appeals did not reach the merits of these allegations. Instead, the court held that U.S. courts lack jurisdiction over the claims against the Saudi defendants. The legal bases for this holding were lack of subject matter jurisdiction under the FSIA and lack of personal jurisdiction. The most significant aspects of the court of appeals' opinion were interpretations of the FSIA, namely (1) its interpretation of "agency or instrumentality" under the FSIA as extending both to the Saudi charity and to individuals sued in their official capacities, and (2) its interpretation of the commercial activities and tort exceptions under the FSIA as having a narrower scope than plaintiffs had advocated. The Supreme Court later abrogated the first of these holdings, and the Second Circuit reversed its own position with respect to the tort exception to foreign sovereign immunity. In December 2013, the Second Circuit ordered these claims against Saudi Arabia and its agencies or instrumentalities be reinstated in the interest of justice to determine whether the tort exception applies. The district court again dismissed the claims based on foreign sovereign immunity, and the case is on appeal at the U.S. Court of Appeals for the Second Circuit. Background In r e Terrorist Attacks of September 11, 2001 ( In re Terrorist Attacks ) is a case consolidated for pre-trial purposes in the U.S. District Court for the Southern District of New York. The Second Circuit Court of Appeals' opinion reviewed dismissals of only a subset of the claims at issue in the case. Plaintiffs in In r e Terrorist Attacks are individuals and businesses injured by the September 11 terrorist attacks. They brought claims based on state and federal tort law and various federal laws, including the Torture Victim Protection Act, for injuries suffered as a result of the attacks. The dismissed claims fall into four categories: (1) claims against the Kingdom of Saudi Arabia; (2) claims against four Saudi princes in their official capacities; (3) claims against the Saudi High Commission for Relief to Bosnia and Herzegovina (the SHC), a charitable organization operated in connection with the Saudi government; and (4) claims against a banker and Saudi princes in their personal capacities. Underlying all of the claims was the allegation that defendants had "played a critical role in the September 11 attacks by funding Muslim charities that, in turn, funded al Qaeda." The appeals court in 2008 affirmed dismissals of the first three sets of claims for lack of subject matter jurisdiction under the FSIA. Because the FSIA precludes courts from asserting jurisdiction over claims against foreign states, one of the FSIA exceptions must apply before a U.S. court may assert jurisdiction over the Kingdom of Saudi Arabia or any of its "agencies or instrumentalities." As discussed below, the Second Circuit held that none of the FSIA exceptions applied. The fourth set of claims (those brought against princes in their personal capacities) fell outside of the scope of the FSIA. Nonetheless, as discussed below, the court dismissed those claims for lack of personal jurisdiction. Charity and Princes as "Agencies and Instrumentalities" of the Kingdom Because a foreign state's "agency or instrumentality" is entitled to the same immunity to which the state itself is entitled under the FSIA, a key threshold question was whether the SHC and the princes sued in their official capacities qualified as agencies or instrumentalities under the FSIA. The FSIA defines "agency or instrumentality" as any entity which is (1) a "separate legal person, corporate or otherwise"; (2) "an organ of a foreign state or political subdivision thereof, or a majority of whose shares or other ownership interest is owned by a foreign state or political subdivision thereof"; and (3) not a U.S. citizen or created under the laws of a third country. The SHC Charity Whether the SHC was an agency or instrumentality turned on whether it was an "organ" of the Kingdom of Saudi Arabia. The court applied a multi-factor test, derived from a previous Second Circuit decision and from decisions from other circuits, to determine whether SHC was such an "organ." Specifically, the court applied the following five criteria: "(1) whether the foreign state created the entity for a national purpose; (2) whether the foreign state actively supervises the entity, (3) whether the foreign state requires the hiring of public employees and pays their salaries, (4) whether the entity holds exclusive rights to some right in the [foreign] country; and (5) how the entity is treated under foreign state law." Emphasizing that the Saudi government had formed SHC and paid its employees, the court held that the SHC was an organ, and thus was an agency or instrumentality of the Kingdom. Officials The plaintiffs sued four Saudi princes for actions taken within their official capacities. All four princes held positions of power in the SHC; three of the princes were members of the country's "Supreme Council of Islamic Affairs," the body responsible for monitoring and approving "Islamic charitable giving both within and outside the Kingdom"; and the fourth prince was the SHC's president, in addition to his roles as a provincial governor and crown prince. Although several other federal courts of appeals had ruled on the extension of foreign sovereign immunity to foreign officials, treatment of officials under the FSIA was a question of first impression for the Second Circuit. Raising a number of textual arguments and referencing the FSIA's legislative history, the court held that individuals acting within their official capacities were indeed "agencies" of their states and were therefore entitled to immunity under the FSIA to the same extent as their states. The court noted that at the time the FSIA was enacted, Congress expressed a desire to codify common law principles, one of which was that immunity extends to a state's officials. The court also emphasized the potential erosion of immunity for foreign states if immunity extended only to government actions distinct from the actions of officials as individuals, noting that "the state cannot act except through individuals." The Second Circuit's holding was consistent with the conclusions of five of the six other federal courts of appeals that had considered whether an individual may be protected as an agent or instrumentality. Only the U.S. Court of Appeals for the Seventh Circuit (Seventh Circuit) had reached the opposite conclusion. In Terrorist Attacks III, the Second Circuit characterized the Seventh Circuit as an "outlier" on this issue. However, after the Fourth Circuit also adopted the minority position, the Supreme Court granted review and established the minority position as the correct one. Consequently, jurisdiction over remaining Saudi officials is subject to the same inquiry that applies to other individuals and possibly a determination as to whether common law immunity applies. Because the officials dismissed from this case were not part of the motion to vacate, plaintiffs did not have an opportunity to pursue their lawsuit against them on remand. Claims against them in their official capacity would likely be deemed to be claims against the state or its instrumentality in any event. Relevant FSIA Exceptions After holding that the FSIA applied not only to the Kingdom of Saudi Arabia but also to Saudi officials and the SHC as an agency or instrumentality of the Kingdom, the court of appeals next examined whether any FSIA exception applied. First, the court held that the terrorist state exception did not apply because the U.S. State Department has not designated the Kingdom of Saudi Arabia as a state sponsor of terrorism. Next, although the court found two other exceptions—the commercial activity and tort exceptions—"potentially relevant," neither exception applied to the Saudi defendants. Commercial Activities Exception To support their argument that the commercial activities exception should apply to the Saudi defendants, the In r e Terrorist Attacks plaintiffs characterized defendants' charitable contributions to Muslim groups as a form of money laundering. The court rejected this characterization as incompatible with the Supreme Court's interpretation of the commercial activities exception. The FSIA defines "commercial activity" as "a regular course of commercial conduct or a particular commercial transaction or act." The court noted the "circularity" of this definition and relied upon the Supreme Court's definition of "commercial activity" (for the context of the FSIA exception) as "the type of actions by which a private party engages in 'trade and traffic or commerce.'" Under this definition, the court noted that the appropriate focus in determining whether an action constitutes "commercial activity" is on an action's nature rather than its purpose. With this framework, the court upheld the district court's finding that defendants' "charitable contributions" fell outside the scope of the commercial activities exception by reason of their non-commercial nature, regardless of the contributions' alleged money laundering purpose. This portion of the decision remains undisturbed. Tort Exception Finally, the court of appeals rejected the tort exception as inapplicable to claims against the Saudi defendants. Specifically, the court noted that Congress's purpose in enacting the tort exception was to create liability for incidents such as traffic accidents that occur in the United States. Furthermore, the court was concerned about the effect that an expanded tort exception would have on the other FSIA exceptions. It emphasized that if the exception were expanded to include all conduct conceivably characterized as tortious, the tort exception would "vitiate" the terrorist state exception's limitation to designated terrorist states. In a separate case, a later panel of the appellate court disagreed with this aspect of the decision, however, effectively overturning it for the Second Circuit. In December 2013, the appellate court granted the plaintiffs relief from judgment in the interests of justice, sending the case back to the district court to determine whether the tort exception applies or whether the defendants were entitled to immunity based on the discretionary nature of their actions. The lower court was also asked to determine whether the "entire tort" rule would apply, in which case the fact that the relevant Saudi government activity took place outside the United States would make the tort exception inapplicable. Princes Sued in Their Personal Capacities For claims made against a Saudi banker and against several Saudi princes for actions taken in their personal capacities, subject matter jurisdiction was not precluded by the FSIA. However, the appeals court upheld the district court's determination that it lacked personal jurisdiction over the Saudi defendants sued in their personal capacities. Specifically, the court concurred with the district court's finding that the princes sued in their personal capacities lacked sufficient contacts with the forum to permit personal jurisdiction under the constitutional "minimum contacts" standard. Plaintiffs argued that the minimum contacts test was satisfied because the defendants had purposefully directed activity at the judicial forum by supporting the attacks. The court rejected this argument, acknowledging that it had been a successful argument in cases where defendants were "primary participants" in the terrorist acts but holding that the banker and princes' activities were too attenuated from the actual attacks to satisfy due process requirements. Similarly, the court rejected the plaintiff's argument that potential foreseeability of the terrorist attacks was a sufficient basis for establishing minimum contacts. It noted that foreseeability alone is insufficient to pass constitutional muster for personal jurisdiction; instead, the constitutional standard requires "intentional" conduct, "expressly aimed" at residents in the forum. In Re Terrorist Attacks on Remand to the District Court On remand to the district court, Judge Daniels of the U.S. District Court for the Southern District of New York dismissed the claims against the Kingdom of Saudi Arabia and the SHC, holding the FSIA tort exception does not apply because of the "entire tort" rule, and suggested that the discretionary act exception to the tort exception might otherwise apply to preclude jurisdiction. The "Entire Tort" Rule The Second Circuit applies the so-called "entire tort" rule to determine whether the conduct of a foreign government or its instrumentality is subject to the FSIA tort exception. The Supreme Court has described the tort exception as "cover[ing] only torts occurring within the territorial jurisdiction of the United States." Consequently, it is insufficient to overcome immunity to allege that an injury or damage occurred within the United States; it must also be demonstrated that the defendant's tortious conduct occurred within the United States. In affirming the dismissal with respect to the Saudi Joint Relief Committee (SJRC) and the Saudi Red Crescent Society (SRC), the appellate court did not appear to foreclose a finding of jurisdiction unless all conduct associated with the injury occurs wholly within the United States, but rather only that some tortious act must be committed by the defendant in the United States. In the case of the Saudi charities, the appellate court stated that all of the defendants' conduct had occurred abroad, and noted in a footnote that Although the September 11, 2001 attacks constitute a "tort," the SJRC and the SRC are not alleged to have participated in that "tort." Instead, the "torts" allegedly committed by the SJRC and the SRC only involve giving money and aid to purported charities that supported al Qaeda. … The September 11, 2001 attacks thus are distinct and separate from the "torts" allegedly committed by the SJRC and the SRC. Consequently, it is possible that the indirect nature of the defendants' alleged support for the terrorist attacks has some bearing on the application of the "entire tort" rule, so that a more direct role in orchestrating from abroad a terrorist attack that takes place in the United States would nevertheless qualify for jurisdiction under the tort exception. The district court found that the same considerations that applied to the charities also commanded immunity for the Kingdom and the SHC. The plaintiffs, recognizing that their allegations about indirect funding for the attacks would not satisfy the tort exception, sought to allege that the activities of certain "operational level agents and alter-egos" of Saudi Arabia within the United States in connection with the attacks are attributable to Saudi Arabia. The court found that there was an insufficient factual basis to conclude that any of these individuals were employees of Saudi Arabia acting within the scope of their employment when they carried out the allegedly tortious activities in the United States. The Discretionary Act Exclusion Neither the appellate court nor the district judge had occasion to determine whether the discretionary function exclusion to the FSIA tort exception would apply to the Saudi government and the SHC, since immunity was already protected under the "entire tort" rule. However, the district judge set forth the applicable two-part rule for determining whether immunity is retained for otherwise tortious conduct: (1) the acts alleged to be negligent must be discretionary, in that they involve an element of judgment or choice and are not compelled by statute or regulation, and (2) the judgment or choice in question must be grounded in considerations of public policy or susceptible to policy analysis. He noted the 2005 district court decision in Terrorist Attacks I granting the defendants immunity for decisions involving which charities to fund, specifically, the holding that "Saudi Arabia's treatment of and decisions to support Islamic charities are purely planning level 'decisions grounded in social, economic, and political policy." With respect to the SHC, the previous judge had explained that "SHC's alleged misuse of funds and/or inadequate record-keeping—even if it resulted in the funds going to terrorists—was the result of a discretionary function and cannot be the basis for overcoming SHC's immunity." Courts have regarded the FSIA discretionary act exclusion as analogous to the similar exclusion for U.S. sovereign immunity in the Federal Tort Claims Act. Accordingly, some courts have found the discretionary act exclusion inapplicable to tortious acts in the United States that amounted to illegal conduct. A district court in the D.C. Circuit found in Letelier v. Republic of Chile that an assassination occurring on U.S. soil under the direction of a foreign government was not a discretionary act within the meaning of the FSIA tort exception, stating the following: While it seems apparent that a decision calculated to result in injury or death to a particular individual or individuals, made for whatever reason, would be one most assuredly involving policy judgment and decision and thus exempt as a discretionary act under section 1605(a)(5)(A), that exception is not applicable to bar this suit. As it has been recognized, there is no discretion to commit, or to have one's officers or agents commit, an illegal act.... Whatever policy options may exist for a foreign country, it has no "discretion" to perpetrate conduct designed to result in the assassination of an individual or individuals, action that is clearly contrary to the precepts of humanity as recognized in both national and international law. Accordingly there would be no "discretion" within the meaning of section 1605(a)(5)(A) to order or to aid in an assassination and were it to be demonstrated that a foreign state has undertaken any such act in this country, that foreign state could not be accorded sovereign immunity … for any tort claims resulting from its conduct. Similarly, the Ninth Circuit has held that the discretionary act exclusion does not apply in the case of a foreign official who, while acting in his home country, orders a killing in the United States that would be a violation of the domestic law of that country. Both of these cases applied the tort exception to injuries in the United States orchestrated from abroad, suggesting that the "entire tort" rule either is not in use in these appellate circuits or that it is not to be taken literally. That is, courts may tend to view direct involvement on the part of a foreign official overseas in tortious activity that takes place on U.S. territory as included in the same tort, while indirect contribution by means of lawful conduct could be viewed as at most a separate tort or as a discretionary act. If the appellate court reverses the district court's dismissal of the suit on "entire tort" grounds, the case will likely return to the district court for a determination of whether the suit is precluded on grounds that the alleged tortious acts retain immunity as discretionary functions. U.S. Court of Appeals' Dismissal of Claims Against Private Banks In a separate decision in In re Terrorist Attacks on September 11, 2001, the U.S. Court of Appeals for the Second Circuit affirmed the dismissal of claims against five defendants whose cases had been dismissed by the district court for failure to state a claim on which relief could be granted. These defendants were (1) Al Rajhi Bank, (2) Saudi American Bank, (3) Saleh Abdullah Kamel, (4) Dallah al Baraka Group LLC, and (5) Dar Al–Maal Al–Islami ("DMI") Trust. Plaintiffs brought claims against these defendants under a common-law tort theory as well as the Anti-Terrorism Act (ATA), the Alien Tort Statute (ATS), and the Torture Victims' Protection Act (TVPA). The Anti-Terrorism Act The ATA provides in 18 U.S.C. §2333 that U.S. nationals injured in their person, property, or business "by reason of an act of international terrorism" can recover treble damages, plus costs, but does not describe who can be held liable. The plaintiffs in this case alleged that the defendants were liable for knowingly providing financial support and services to charities that supported Al Qaeda, but did not identify any actions on the part of the defendants that proximately caused their injuries. Citing its recent holding in Rothstein v. UBS AG, the appellate court held that the ATA does not encompass liability on an aiding-and-abetting theory, but, rather, based on the Supreme Court's interpretation of the phrase "by reason of," requires a showing of proximate cause. The Alien Tort Statute and Other Causes of Action The Alien Tort Statute provides federal jurisdiction for tort claims brought by non-U.S. citizens seeking damages for conduct that violates the "law of nations or a treaty of the United States." While the Supreme Court in 2004 held that the statute does not provide a cause of action for such lawsuits, it permitted courts to give effect to the statute for civil actions arising from the violation of a very narrow class of international norms "defined with a specificity comparable to the features of the 18 th century paradigms" that were understood to govern at the time of the ATS's drafting in 1789. The Second Circuit concluded that international terrorism did not meet this standard as of September 11, 2001. Quoting its 2003 opinion in United States v. Yousef, the court stated the following: We regrettably are no closer now... to an international consensus on the definition of terrorism or even its proscription; the mere existence of the phrase "state-sponsored terrorism" proves the absence of agreement on basic terms among a large number of States that terrorism violates [customary] international law. Moreover, there continues to be strenuous disagreement among States about what actions do or do not constitute terrorism, nor have we shaken ourselves free of the cliché that "one man's terrorist is another man's freedom fighter." We thus conclude... that terrorism—unlike piracy, war crimes, and crimes against humanity—does not provide a basis for universal jurisdiction [under customary international law]. The court also rejected the plaintiffs' reliance on the Torture Victim Protection Act for a cause of action, following the Supreme Court's interpretation of that law as providing for a right of action only against natural persons who act under color of foreign law. Finally, the court rejected the plaintiffs' complaints based on common law tort theories, holding that they did not demonstrate that the defendants breached any duty of care owed to the plaintiffs or that their actions proximately caused the plaintiffs' injuries. Legislative Developments In Terrorist Attacks III, the U.S. Court of Appeals for the Second Circuit adopted narrow interpretations of the commercial activities and tort exceptions under the FSIA, appearing to preclude efforts by September 11 victims and other plaintiffs seeking recovery in U.S. courts against foreign officials and government-controlled entities like the Saudi charity. Plaintiffs' efforts to hold non-government financial institutions liable for providing material to terrorist organizations have also seemingly been stymied by the courts' interpretations of the ATA as precluding secondary liability for terrorist acts and requiring that plaintiffs demonstrate that the support alleged was a proximate cause of their injuries or losses. Several bills have been introduced in Congress that would broaden the FSIA tort exception explicitly to cover terrorist acts that occur within the United States and make other changes that could boost plaintiffs' efforts against some of the Saudi defendants. Previous Legislative Activity The 111 th Congress held a hearing to consider S. 2930, the Justice Against Sponsors of Terrorism Act, which, among other measures, would have amended the tort exception to the FSIA specifically to cover terrorist attacks within the United States. In the 112 th Congress, new legislation was introduced to reduce some of the burdens faced by victims of state-sponsored terrorism in the United States who seek to bring lawsuits against foreign officials. S. 1894, the Justice Against Sponsors of Terrorism Act, was ordered to be reported favorably out of the Senate Judiciary Committee in September 2012. A companion bill, H.R. 5904, did not receive further action. Identical versions of the Justice Against Sponsors of Terrorism Act were introduced in the 113 th Congress as S. 1535 and H.R. 3143. S. 1535 was reported favorably out of the Senate Judiciary Committee with some amendments to the findings and provisions addressing aiding and abetting liability under the Anti-Terrorism Act, and was passed by the Senate in December 2014. The House did not vote on either version of the bill. The bills would have amended the tort exception to the FSIA expressly to include "any statutory or common law tort claim arising out of an act of extrajudicial killing, aircraft sabotage, hostage taking, terrorism, or the provision of material support or resources for such an act.... " Although the aspect of the Terrorist Attacks III decision interpreting the tort exception as inapplicable to terrorist acts occurring in the United States was effectively overruled by another panel of judges, it is possible that other courts could read the terrorism exception as foreclosing suits against states not designated as sponsors of terrorism. The tort exception would also have been amended to clarify that there is no rule holding that the "entire tort" must occur within the United States, but rather that such claims are covered "regardless of where the underlying tortious act or omission occurs." Additionally, the bills would have expanded liability for foreign government officials in civil actions for terrorist acts no matter where they occur by amending 18 U.S.C. Section 2337, which currently exempts all government officials. The amended version of Section 2337 would have exempted only U.S. officials. H.R. 3143 would have included the provision of material support for terrorism in the jurisdiction provision, although its revision of the ATA cause of action did not expressly include liability for material support. 114th Congress, Justice Against Sponsors of Terrorism Act The Justice Against Sponsors of Terrorism Act (JASTA) has returned to the 114 th Congress as S. 2040, which the Senate passed by voice vote on May 17, 2016, after accepting an amendment in the nature of a substitute offered by Senator Cornyn, and H.R. 3815, which was introduced late in 2015 and referred to the House Judiciary Subcommittee on Constitution and Civil Justice. Amendments to the FSIA H.R. 3815 would amend the FSIA tort exception in 28 U.S.C. §1605(a)(5) by limiting the type of injury to "physical injury" rather than "personal injury," and specifically excluding "any claim for emotional distress or derivative injury suffered as a result of an event or injury to another person that occurs outside of the United States." It would include "any statutory or common law tort claim arising out of an act of extrajudicial killing, aircraft sabotage, hostage taking, terrorism, or the provision of material support or resources for such an act, or any claim for contribution or indemnity relating to a claim arising out of such an act" to the tortious acts or omissions for which foreign sovereign immunity may be abrogated. It would also eliminate the "entire tort" rule by providing that the non-commercial tort exception covers physical injuries, deaths, and property damage or loss occurring in the United States "regardless of where the underlying tortious act or omission occurs." The discretionary function exclusion would remain unchanged; however, the bill would include among congressional findings that "no country has the discretion to engage knowingly in the financing or sponsorship of terrorism, whether directly or indirectly." S. 2040 (as passed by the Senate) would create a new exception to the FSIA for acts of international terrorism that occur on U.S. territory, to be codified as 28 U.S.C. §1605B. "International terrorism" is defined with reference to the ATA, including the exclusion for "acts of war." The amendment would dispense with the "entire tort" rule and the statutory discretionary act exclusion for such cases only, leaving current law intact with respect to the FSIA non-commercial tort exception in other cases. Under the bill, a plaintiff could seek money damages from a foreign state for physical injury to person or property or death caused by both (1) an act of international terrorism in the United States, and (2) a tortious act or acts on the part of the foreign state or official, employee, or agent of that foreign state acting within the scope of his or her employment, regardless of where the tortious conduct took place. The bill would also clarify that a tortious act or omission would not give rise to jurisdiction where it constitutes "mere negligence." Finally, it would clarify the relationship between the FSIA exception and the ATA's civil liability exception precluding suits against foreign officials. If the new FSIA exception applies, a U.S. national could pursue an ATA lawsuit against the foreign state. S. 2040 would provide for exclusive federal court jurisdiction over foreign states under the new FSIA provision, and would authorize the Attorney General to intervene in such cases to seek a stay of the civil action if the Secretary of State certifies that the United States is engaged in good faith discussions with the defendant state to resolve the claims. The court would be authorized to issue a stay for 180 days, which could be renewed for additional 180-day periods if the Secretary of State certifies that such negotiations remain ongoing. Civil Liability Under the Anti-Terrorism Act All current versions of JASTA include a finding by Congress that expresses approval in the context of civil liability under the ATA for the test established by the D.C. Circuit for aiding and abetting and conspiracy liability. The D.C. Circuit defined aiding and abetting as including the following elements: (1) the party whom the defendant aids must perform a wrongful act that causes an injury; (2) the defendant must be generally aware of his role as part of an overall illegal or tortious activity at the time that he provides the assistance; (3) the defendant must knowingly and substantially assist the principal violation. Conspiracy, according to the D.C. Circuit, gives rise to civil liability when it consists of (1) an agreement between two or more persons; (2) to participate in an unlawful act, or a lawful act in an unlawful manner; (3) an injury caused by an unlawful overt act performed by one of the parties to the agreement; (4) which overt act was done pursuant to and in furtherance of the common scheme. The bills would implement secondary liability in somewhat different ways. S. 2040 (as passed by the Senate) and H.R. 3815 would amend the ATA to provide for secondary liability in the case of an act of international terrorism planned, committed, or authorized by an entity designated as a foreign terrorist organization at the time of the planning, commission, or authorization of the attack. The House bill would also apply liability in the case of an entity designated as a foreign terrorist organization as a result of the attack that gives rise to the lawsuit. Secondary liability would attach under the Senate bill to any person who "aids and abets, by knowingly providing substantial assistance, or who conspires" with the perpetrator; it also expressly adopts the definition for "person" from Section 1 of Title 1 to define who may be held secondarily liable. The House bill would authorize secondary liability against anyone who aided, abetted, or conspired with the perpetrator, without clarifying what type of intent or knowledge would be necessary to establish liability. Neither bill would explicitly elucidate whether criminal liability under the material support provisions of the ATA would also establish civil liability, as some courts have found, but language in the findings section suggest the intent that the knowing or reckless provision of material support or resources to a foreign terrorist organization, whether directly or indirectly, should give rise to liability. H.R. 3815 would amend the ATA to remove the immunity of foreign officials from lawsuits as currently provided in 18 U.S.C. §2337(2). It would also add a provision to the ATA to address issues plaintiffs have confronted in establishing personal jurisdiction over foreign nationals. H.R. 3815 would declare that The district courts shall have personal jurisdiction, to the maximum extent permissible under the 5 th Amendment to the Constitution of the United States, over any person who commits or aids and abets an act of international terrorism or otherwise sponsors such act or the person who committed such act, for acts of international terrorism in which any national of the United States suffers injury in his or her person, property, or business by reason of such an act in violation of section 2333. S. 2040 (as passed by the Senate) would leave the foreign official exclusion under the ATA intact, but, as noted above, would permit lawsuits by U.S. nationals against the foreign state itself if the new FSIA exception is triggered. Aliens as well as U.S. nationals would likely be able to bring lawsuits under causes of action other than the ATA if jurisdiction is available under the new FSIA exception.
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Summary: Practical and legal hurdles, including the difficulty of locating hidden Al Qaeda members and the infeasibility of enforcing judgments in terrorism cases, hinder victims' attempts to establish liability in U.S. courts against, and recover financially from, those they argue are directly responsible for the September 11 terrorist attacks. Instead, victims have sued numerous individuals and entities with only indirect ties to the attacks, including defendants who allegedly provided monetary support to Al Qaeda prior to September 11, 2001. Within the consolidated case In re Terrorist Attacks of September 11, 2001, one such group of defendants was the Kingdom of Saudi Arabia, several Saudi princes, a Saudi banker, and a Saudi charity. Plaintiffs argued that these Saudi defendants funded groups that, in turn, assisted the attackers. A threshold question in In re Terrorist Attacks was whether U.S. courts have the power to try these Saudi defendants. In August 2008, the U.S. Court of Appeals for the Second Circuit affirmed dismissals of all claims against the Saudi defendants, holding that U.S. courts lack jurisdiction over the claims. Specifically, the court of appeals held that in this case, U.S. courts lack (1) subject matter jurisdiction over the Kingdom of Saudi Arabia, because the Kingdom is entitled to immunity under the Foreign Sovereign Immunities Act (the FSIA) and no statutory exception to immunity applies; (2) subject matter jurisdiction over the Saudi charity and Saudi princes acting in their official capacities, because they are "agents or instrumentalities" of the Kingdom and thus, under the FSIA, are entitled to immunity to the same extent as the Kingdom itself; and (3) personal jurisdiction over Saudi princes sued in their personal capacities, because the princes had insufficient interactions with the forum to satisfy the "minimum contacts" standard for personal jurisdiction under the Fifth Amendment due process clause. In 2011, the Second Circuit reversed itself with respect to the immunity of non-terrorist states, finding that the tort exception under the FSIA does not exclude terrorist acts that take place within the United States. In 2013, the court ordered these claims against Saudi Arabia and its agencies or instrumentalities be reinstated in the interest of justice to determine whether the tort exception applies. The district court again dismissed the claims in 2015 on the basis that the "entire tort" defendants were alleged to have committed did not occur within the United States. This decision has been appealed to the U.S. Court of Appeals for the Second Circuit. In a separate decision, the court of appeals affirmed the dismissal of claims against private Saudi banks on the basis that the Anti-Terrorism Act (ATA) does not permit secondary liability under an aiding-and-abetting theory. It also affirmed the dismissal of claims against these defendants brought under the Alien Tort Statute and the Torture Victims Protection Act, as well as claims brought as common law torts. To address some issues related to the interpretation of the FSIA, among other related matters, Congress is considering the Justice Against Sponsors of Terrorism Act (JASTA; S. 2040, H.R. 3815).This report summarizes the FSIA and jurisdiction in cases against foreign defendants, analyzes the court decisions, and reviews legislative efforts to revise the FSIA and the ATA.
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Summarize: Starting in 1996, Alexa Internet has been donating their crawl data to the Internet Archive. Flowing in every day, these data are added to the Wayback Machine after an embargo period. Beyonce’s Baby to be Bathed in Baubles Thanks to ‘Auntie Kelly’ www.thediamondbathtub.com What baby gift do you buy your best friend when she is one-half of the highest-earning couple in show business? And what baby gift do you buy for that friend after you reveal to the world her most closely guarded secret, the gender of her unborn child? If you are Kelly Rowland, you buy a bathtub. Not just any bathtub, but a very expensive, Swarovski-crystal studded Baby Diamond Bathtub worth $5,200. And you give it to your best friend, Beyonce. The “Best Thing I Never Had” singer reportedly received the gift from Rowland, her former Destiny’s Child bandmate, in anticipation of the bundle-of-joy she’s expecting this February with husband Jay-Z. Rowland famously let the gender secret the couple had been closely guarding slip when talking to reporters at the Cosmopolitan Awards in London earlier this month. “I have no idea what I’m going to buy Beyonce at the baby shower because Jay is going to buy that little girl every single thing possible,” the singer, 30, told the UK’s BANG Showbiz. “She won’t be spoiled but she will be very well looked-after.” Well looked-after by both her parents and, evidently, her “Auntie Kelly,” as Beyonce told Closer magazine Rowland will be known to her child. Crafted by California-based interior designer Lori Gardner, the baby-sized, footed, porcelain tub is covered in 44,928 imported crystals that Gardner reportedly spends more than two months applying by hand. While Beyonce was gifted the pink version, the bling bathtub, weighing in at more than 3,000 pounds, is available in all the Swarovski colors. Not to be impractical, the bathtub, according to the designer, can also be used for pets or as a beverage chiller for parties. Just don’t expect to see any Cristal champagne chilling in any Baby Bathtub belonging Beyonce and Jay-Z’s baby offspring, of course.
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Summary: If there ever was an infant truly impossible to shop for, it's the child who will be born to Beyonce and Jay-Z-what baby shower gift can you possibly buy for a couple who already has a $1 million "mom van" and a 2,200-square-foot nursery? Fortunately for Kelly Rowland, who faced just such a dilemma, the $5,200 pink-Swarovski-crystal-covered Baby Diamond Bathtub exists. Rowland-who Beyonce has said will be known as "Auntie Kelly"-also had one other little issue to contend with when deciding upon her pricey gift. You see, Rowland accidentally let slip earlier this month that Bey and Jay are having a girl. "I have no idea what I'm going to buy Beyonce at the baby shower because Jay is going to buy that little girl every single thing possible," said the former Destiny's Child singer. "She won't be spoiled but she will be very well looked-after." Hopefully the 3,000-pound tub, bedazzled with 44,928 crystals, makes up for that little oops. ABC News notes that, for those who don't have a baby but simply must have the tub anyway, it can also be used as a beverage chiller.
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Write a title and summarize: Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. Le. infantum and Le. tropica typically cause visceral or cutaneous leishmaniasis, respectively, but Le. infantum strains transmitted by P. tobbi can cause cutaneous disease. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared. cDNA libraries of P. tobbi and P. sergenti female SGs were constructed, sequenced, and analyzed. Clones (1,152) were randomly picked from each library, producing 1,142 high-quality sequences from P. tobbi and 1,090 from P. sergenti. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41. 9-kDa superfamily of proteins. Phylogenetic analyses and multiple sequence alignments of putative proteins were used to elucidate molecular evolution and describe conserved domains, active sites, and catalytic residues. Proteomic analyses of P. tobbi and P. sergenti SGs were used to confirm the identification of 35 full-length sequences (18 in P. tobbi and 17 in P. sergenti). To bridge transcriptomics with biology P. tobbi antigens, glycoproteins, and hyaluronidase activity was characterized. This analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research. Sand flies are bloodsucking nematoceran Diptera that transmit the protozoan parasites of the genus Leishmania. Similar to that of other bloodsucking arthropods, sand fly saliva comprises antihemostatic, immunomodulatory, and antigenic components. The saliva is deposited into the host skin every time the sand fly ingests a blood meal to facilitate feeding. Also during the bite by an infected sand fly, Leishmania parasites are egested into the wound with the saliva. Sand fly saliva can enhance Leishmania infection in naive mice [1], [2]. Conversely, pre-exposure of mice to sand fly saliva conferred a protective effect against Leishmania infection [3], [4]. Even single salivary proteins have been characterized as potential Leishmania vaccine candidates in mouse, hamster, and dog models of cutaneous or visceral leishmaniasis [5]–[9]. The potent effects of sand fly saliva stimulate a protective host cellular immune response [3]–[9], and the antigenic nature of saliva also provides a humoral immunity measurement of host exposure to sand fly bites already used in several human epidemiological studies [10]–[18]. Identifying markers of vector exposure based on anti-saliva antibodies are essential in epidemiologic and vector control surveillance [15], [16], [18], [19]–[21]. However, anti-saliva antibodies are highly specific [12], [16], [22] and with over 80 species of sand flies implicated in Leishmania transmission, it is vital to continue describing the salivary proteins in the search for markers of exposure as well as vaccine candidates. Sand fly salivary gland proteins have been well studied in Lutzomyia longipalpis [23], [24] and Phlebotomus papatasi [6]. Recently, transcriptomic and proteomic data have been published for several other sand fly species, vectors of visceral (P. ariasi, P. argentipes, and P. perniciosus) and cutaneous (P. arabicus, P. duboscqi) forms of leishmaniasis [25]–[28]. To broaden the repertoire of subgenus Larroussius salivary proteins and provide the first report from a subgenus Paraphlebotomus sand fly, we prepared and analyzed the transcriptomes and proteomes of P. tobbi and P. sergenti, both proven vectors in the Old World. Phlebotomus sergenti, subgenus Paraphlebotomus, is the main vector of Le. tropica, principally an agent of cutaneous leishmaniasis [29]–[31]. Phlebotomus tobbi, on the other hand, is an important vector of Le. infantum [32] together with the taxonomically related P. ariasi and P. perniciosus, sand flies of the subgenus Larroussius [29], [33], [34]. In contrast to other members of the subgenus, P. tobbi transmits the cutaneous form of the Le. infantum [32]. Additionaly, we characterized P. tobbi antigens, glycoproteins, and hyaluronidase activity; the later one compared with 6 sand fly species belonging to vectors of cutaneous or visceral leishmaniases. Colonies of P. tobbi (originating from Turkey), P. papatasi (Turkey), P. sergenti (Israel), P. argentipes (India), P. arabicus (Israel), P. perniciosus (Spain), and L. longipalpis (Brazil) were kept in the insectary of Charles University in Prague as described in [35]. The P. sergenti colony, originating from Turkey, was reared in similar conditions at the Laboratory of Malaria and Vector Research, National Institutes of Health (Rockville, MD, USA). For mRNA extraction, salivary glands (SGs) from non-bloodfed 1- to 2-day-old female sand flies were dissected and stored in RNA Later (Ambion, Inc., Austin, TX, USA). For other assays and analysis, SGs of non-bloodfed 5- to 7-day-old females were stored at −70°C; SGs were stored in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA, USA) for proteome analysis and in Tris buffer (20 mM Tris, 150 mM NaCl, pH 7. 8) for hyaluronidase assays, affinity blot, and immunoblot. Before use, samples were homogenized by three freeze-thaw cycles in liquid nitrogen. Protein concentration in resulting SG homogenate (SGH) was measured on Qubit Fluorometer (Invitrogen) following manufacturer' s guidelines. An SG cDNA library was constructed from P. sergenti (Turkey) and P. tobbi. MicroFastTrack mRNA isolation kit (Invitrogen) was used to isolate SG mRNA from 40 SG pairs dissected into 20 µl of RNA Later (Ambion). A cDNA library was constructed using SMART™ cDNA Library Construction Kit (BD Clontech, Palo Alto, CA, USA) following the manufacturer' s protocol, with some modifications as described in [36]. For each species, three cDNA libraries were constructed according to PCR product size – large, medium, and small. PCR amplicons were washed and concentrated to 4–7 µl on Microcon YM-100 columns (Millipore, Billerica, MA, USA). Concentrated samples (3 µl) were ligated into the λTripleEx2 vector and packed into the phage particles with Gigapack III Gold Packaging Extract (Stratagene, La Jolla, CA, USA). Phage libraries were used to infect the log-phase XL-1 Blue Escherichia coli (Clontech) plated onto four LB agar plates per each library size. Transfected plaques were randomly selected and transferred into 96-well V-shape plates with 75 µl of ultrapure water per well. Four 96-well plates of phage were picked per each library size, resulting in 12 plates (1,152 clones) per sand fly species. Phages (3 µl) were subjected to PCR using FastStart PCR Master Mix (Roche, Molecular Biochemicals, Indianopolis, IN, USA) and vector-specific primers (PT2F1 5′-AAGTACTCTAGCAATTGTGAGC-3′ and PT2R1 5′-CTCTTCGCTATTACGCCAGCTG-3′). Amplification conditions were as follows: 1 hold of 75°C for 3 min, 1 hold of 94°C for 2 min, 34 cycles of 94°C for 1 min, 49°C for 1 min, and 72°C for 2 min. The final elongation step lasted for 10 min at 72°C. Products were cleaned using ExcelaPure 96-well UF PCR Purification Plates (Edge Biosystems, Gaithersburg, MD, USA) and cleaned PCR products were used as a template for cycle-sequencing reaction using BigDye Terminator v3. 1 cycle sequencing kit (Applied Biosystems, Fullerton, CA, USA) and a vector-specific forward primer (PT2F3 5′-CTCGGGAAGCGCGCCATTGT-3′). Products of cycle-sequencing reaction were cleaned using Sephadex and MultiScreen HV Plates (Millipore), dried, resuspended in formamide, and stored at −20°C until sequenced on an ABI 3730XL 96-Capillary DNA Sequencer (Applied Biosystems). For mass spectrometric (MS) analysis, SGH samples of P. sergenti (Turkey) and P. tobbi were dissolved in Laemmli sample buffer in parallel with or without 2-mercaptoethanol and electrophoretically separated on 12% polyacrylamide SDS minigel with initial voltage 80 V and 120 V upon entry of sample to the gel. Gels were stained for total proteins with Coomassie Brilliant Blue R-250. Individual bands were cut, destained and digested as was described in [37]. Samples (0. 5 µl) were transferred to a 384 spot stainless steel MALDI target (AB Sciex, Framingham, MA, USA) and let to dry. Dried droplets were covered with a 0. 5 µl drop of alpha-cyano-hydroxycinnamic acid (Fluka, Switzerland) solution (2 mg/ml in 80% acetonitrile) and allowed to dry. Spectra were acquired with 4800 Plus MALDI TOF/TOF analyzer (AB Sciex) equipped with a Nd∶YAG laser (355 nm; firing rate 200 Hz). Voltages were set as follows: source1 20 kV, grid1 16 kV, source1 lens 10 kV, lens1 5 kV, mirror1 14. 085 kV, mirror2 20. 3 kV and reflector detector 1. 905 kV. Digitizer bin size was set to 0. 5 ns, vertical scale 0. 5 V, vertical offset 0. 0, input bandwith 500 MHz. Spectra were externally calibrated using ProteoMass peptide MALDI calibration kit (Sigma-Aldrich). Spectra were recorded in the range 700 to 4000 Da, focus mass 2100 Da. Spectra were summed from 40 positions per 50 shots, 2000 shots in total. Spectra were processed by 4000 Series Explorer version 3. 5. 3 (AB Sciex) without smoothing; baseline subtraction was performed with peak width set to 50. Spectra were deisotoped and peaks with a local signal-to-noise ratio greater than 5 were picked and searched by local Mascot v. 2. 1 (Matrix Science, Boston, MA, USA) against a database of protein sequences derived from the cDNA library. Database search criteria were as follows: enzyme: trypsin; taxonomy: none; fixed modification: carbamidomethylation; variable modification: methionine oxidation; peptide mass tolerance: 80 ppm; one missed cleavage allowed. Only hits that were scored as significant (P<0. 05) were included. The data associated with this manuscript may be downloaded from ProteomeCommons. org Tranche using the following hash: mCZfFsOLaBtSfR+Jh6o8OwEgjrqDp4m3VntpJdAPqPGFNzNpTPry8IhEuGeLw9 TmpHcTRMSiiuiNNRL/6xP65TLvyNwAAAAAAAADGQ = =. The hash may be used to prove exactly what files were published as part of this manuscript' s data set, and the hash may also be used to check that the data has not changed since publication. Expression sequence tags (ESTs) were analyzed using the dCAS software (Desktop cDNA Annotation System, version 1. 4. 3) [38] with all third-party components recommended: CAP3 assembler program [39], Phred [40], [41], and BLAST programs [42]. Sequences with Phred quality scores lower than 25 were removed, as well as primers and vector sequences. Resulting sequences were grouped based on nucleotide homology of 90% identity over 100 residues and aligned into consensus transcript sequences (contigs) using the CAP3 sequence assembly program. BLAST programs were used to compare contigs and singletons (contigs with a single sequence) to the non-redundant protein database of the NCBI, the Gene Ontology database (GO) [43], to COG conserved domains database [44], Protein Family database (Pfam) [45], Simple Modular Architecture Tool database (SMART) [46], and to rRNA Nucleotide Sequences, and Mitochondrial and Plasmid Sequence (MIT-PLA) databases available from NCBI. The three frame translations of each dataset were submitted to the SignalP server [47] to detect signal peptides. The grouped and assembled sequences, BLAST results, and SignalP results combined by dCAS software in an Excel spreadsheet were manually verified and annotated. Additionally, glycosylation sites were determined in selected sequences using NetNGlyc prediction server [48]. For phylogenetic analysis, protein sequences without signal peptide were aligned using ClustalX (version 2. 0) [49] with related sequences obtained from GenBank and manually refined in BioEdit 7. 0 editing software. For each alignment, best substitution matrix was determined by ProtTest software, version 2. 0 [50]. This matrix was then used by TREE-PUZZLE 5. 2 [51] to reconstruct maximum likelihood phylogenetic trees from the protein alignments using quartet puzzling with 1000 puzzling steps. Resulting trees were visualized in MEGA 4 [52]. All protein and nucleotide accession numbers mentioned in the text, tables and figures are listed in Text S1. Hyaluronidase activity was compared between seven sand fly species: P. tobbi, P. sergenti (Israel), P. papatasi, P. argentipes, P. arabicus, P. perniciosus, and L. longipalpis. Hyaluronidase activity in SGs was quantified using a sensitive assay on microtitration plates coupled with biotinylated HA (bHA). bHA, prepared as described in [53], was immobilized onto Covalink NH microtiter plates (Nunc, Placerville, NJ, USA) using the method in [54] at a final concentration of 1 µg bHA per well. The plates were incubated overnight at 4°C and washed three times in PBS (containing 2 M NaCl and 50 mM MgSO4, pH 7. 2). Plates with immobilized bHA were blocked with 1% BSA in PBS for 45 min, washed and equilibrated to pH 5. 0 (0. 1 M acetate, 0. 1 M NaCl, 0. 1% Triton X-100, pH 5. 0), the pH optimum for sand fly salivary hyaluronidase [53]. SGHs were incubated for 45 min at 37°C in triplicate at a final concentration of 0. 5 gland per well. As a standard, bovine hyaluronidase (Sigma) at a concentration of 0. 01 Turbidity Reducing Units (TRU) /µl was serially diluted in 0. 1 M acetate buffer (0. 1 M NaCl, 0. 1% Triton X-100, pH 4. 5). Wells without bHA or enzyme were used as controls. The reaction was terminated by 6 M guanidine 200 µl/well. Plates were washed in PBS (containing 2 M NaCl, 50 mM MgSO4,0. 05% Tween 20, pH 7. 2) and then equilibrated with PBS, 0. 1% Tween 20, pH 7. 2. Avidin-peroxidase (Sigma) was added at a final concentration of 0. 2 µg/well and incubated for 30 min at room temperature. Color reaction was developed with o-phenylenediamine substrate in 0. 1 M citrate-phosphate buffer (pH 5. 5). After 10 min in dark, plates were read at 492 nm (Tecan-Infinite M 200 Fluorometer; Schoeller Instruments, Prague, Czech Republic). The obtained results were expressed as relative TRU (rTRU). Three independent experiments were performed with a different set of SGH samples in each experiment. For hyaluronidase zymography, 8% polyacrylamide gels (0. 75 mm thick) were copolymerized with 0. 002% hyaluronic acid (HA). As the hyaluronidase activities and band patterns varied among sand fly species, different loads were used per lane to obtain bands of equal intensity. The equivalent of 1/2 gland (L. longipalpis and P. sergenti) or 1/20 gland (other tested species) was loaded for zymography under non-reducing conditions, and the equivalent of 2. 5 glands (L. longipalpis and P. sergenti) or 1/4 gland (other tested species) was loaded for zymography under reducing conditions. The total protein content per lane was as follows (non-reducing/reducing conditions): L. longipalpis = 110/550 ng; P. papatasi = 12. 5/62. 5 ng; P. sergenti = 140/700 ng; P. argentipes = 14/70 ng; P. arabicus = 10. 5/52. 5 ng; P. tobbi = 10/50 ng; P. perniciosus = 10. 5/52. 5 ng. For reducing conditions, samples were treated with 3% 2-mercaptoethanol for 40 min at 45°C. SDS-PAGE electrophoresis was carried out using Mini-Protean II apparatus (Bio-Rad, Hercules, CA, USA) and constant voltage at 150 V. After electrophoresis, gels were rinsed 2×20 min in 0. 1 M Tris, pH 7. 8, and 20 min in 0. 1 M acetate buffer, pH 5. 5 (both with 1% Triton X-100 to wash out SDS) and then incubated in 0. 1 M acetate buffer (without detergent) for 120 min at 37°C. The gels were then washed in water, soaked in 50% formamide for 30 min and stained in Stains-all (Sigma, St. Louis, MO, USA) solution (100 mg/ml in 50% formamide) for 24 h in the dark. Hyaluronidase activity was visible as a pink band on a dark blue background. Immunoblot was performed using P. tobbi SGH separated by SDS-PAGE on 10% polyacrylamide gel under non-reducing conditions using the Mini-Protean III apparatus (Bio-Rad). Separated proteins were electrotransferred onto nitrocellulose (NC) membrane by iBlot Dry Blotting System (Invitrogen). After transfer, the NC membrane was cut into strips with the equivalent of four glands/strip and free binding sites were blocked by 5% low-fat dried milk in 20 mM Tris buffer with 0. 05% Tween (Tris-Tw) overnight at 4°C. The strips were then incubated with serum obtained from rabbit repeatedly exposed to P. tobbi females. Serum was diluted 1∶250 in Tris-Tw and incubated with P. tobbi proteins for 1 h, followed by 1 h incubation with peroxidase-conjugated swine anti-rabbit IgG (Sevapharma, Prague, Czech Republic) diluted 1∶1,000 in Tris-Tw. Substrate solution contained Tris buffer, diaminobenzidine, and H2O2. Affinity blot was performed using P. tobbi SGH separated and electrotransferred as described for Immunoblot. After transfer, free binding sites on NC membrane were blocked by 5% BSA in 20 mM Tris-Tw overnight at 4°C. The strip was then incubated for 1 h at room temperature with biotinylated lectin from Canavalia ensiformis (Concanavalin A, Sigma) diluted 0. 2 µg/ml in Tris-Tw. To control the reaction specificity, another strip was incubated with lectin preincubated for 30 min with the ligand, 0. 5 M methyl-α-D-mannopyranoside. Avidin-peroxidase (Sigma) was added at a final concentration of 2. 5 µg/ml and incubated for 1 h at room temperature. Substrate solution contained Tris buffer, diaminobenzidine, and H2O2. All animals used in this study were maintained and handled strictly in accordance with institutional guidelines and legislation for the care and use of animals for research purpose Czech Act No. 246/1992 coll. on Protection Animals against Cruelty in present statues at large that complies with all relevant European Union and international guidelines for experimental animals. The experiments were approved by the Committee on the Ethics of Animal Experiments of the Charles University in Prague (Permit Number: 24773/2008-10001) and were performed under the Certificate of Competency (Registration Number: CZU 934/05) in accordance with the Examination Order approved by Central Commission for Animal Welfare of the Czech Republic. All efforts were made to minimize suffering of experimental animals within the study. Phlebotomus tobbi and P. sergenti cDNA libraries were constructed from SGs of female sand flies dissected one day after emergence. From each cDNA library, 1,152 randomly selected clones were sequenced. Obtained ESTs were deposited in the NCBI dbEST database under accession numbers GW814275–GW815416 (1,142 sequences) for P. tobbi and GW813185–GW814274 (1,090 sequences) for P. sergenti. High-quality sequences were grouped together based on sequence homology, and resulting assembled sequences were analyzed using the dCAS cDNA annotation software [38] and verified by manual annotation. In the P. tobbi cDNA library, 997 high-quality sequences were grouped into 68 contigs and 125 singletons (one sequence in cluster); in P. sergenti, 853 high-quality sequences were grouped into 56 contigs and 196 singletons. Similar to other sand flies studied so far, the most abundant transcripts in both libraries were those coding for putative secretory proteins. BLAST comparison of translated nucleotide sequences with the non-redundant (NR) protein database showed high similarity with other sand fly secreted salivary proteins. In P. tobbi, 81 clusters containing 863 sequences (average 10. 7 sequences per cluster) matched to sand fly salivary proteins. Of them, we found 62 clusters (796 sequences) with predicted signal peptide sequence. In P. sergenti, 50 clusters containing 553 sequences (average 11. 1 sequences per cluster) matched to sand fly salivary proteins. Of them, 32 clusters (482 sequences) with predicted signal protein sequence were found. Tables 1 and 2 list representative secreted salivary proteins from P. tobbi and P. sergenti, respectively, deposited into NCBI GenBank database. The tables show GenBank accession numbers, putative mature protein features, best match to NR protein database, and presence in the proteome analysis as confirmed by MS (Figure 1). Additionally, Figure 1A and 1B show detailed analysis of MS results for P. tobbi and P. sergenti, respectively, including cluster name, Gen Bank accession number, and molecular weight of mature proteins under reducing and non-reducing conditions. The putative secreted salivary proteins of P. tobbi and P. sergenti could be divided into ten main protein families (Figure S1): antigen 5-related protein, apyrase, hyaluronidase, D7-related and PpSP15-like protein (odorant-binding proteins superfamily), ParSP25-like protein, PpSP32-like protein, yellow-related protein, the 33-kDa salivary proteins, and the 41. 9-kDa superfamily. The following paragraphs describe these families in detail, focusing on protein family characteristics, possible function, biochemical, immunomodulatory, and antigenic properties, and phylogenetic analysis in context with related proteins from other sand flies. Antigen 5-related proteins (Ag5r) are present in saliva of all sand fly species studied so far [55], [56], including P. tobbi (PtSP77/HM140620, PtSP78/HM140621, PtSP79/HM140622) and P. sergenti (PsSP52/HM537134). Sand fly Ag5r proteins are members of CAP superfamily consisting of mammalian Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5) originally described from wasp venom, and plant Pathogenesis-related 1 proteins (PR-1) [55]. Proteins with CAP domain occur across all living organisms, including prokaryotes [57], and are mostly extracellular/secreted. All sand fly Ag5r proteins have similar predicted molecular mass (ranging from 28. 8 to 31. 2 kDa) and are alkaline (Table S1). In P. tobbi and P. sergenti, the predicted molecular mass corresponded well with the one measured in proteomic analysis (Figure 1, Tables 1 and 2) suggesting single-domain protein and negligible post-translational modifications. We identified 14 highly conserved cysteine residues proportionally distributed through the whole sequence length (Figure 2), possibly involved in disulfide bonding. Although the members of this family were described in sialotranscriptomes of all bloodsucking arthropods characterized [55], [56], their role is mostly unknown with a few exceptions. In Stomoxys calcitrans, Ag5r protein possesses immunoglobulin Fc binding activity [58]. In Tabanus yao, members of the Ag5r protein family can probably serve as an inhibitors of angiogenesis (RTS disintegrin motif) [59] or a potent platelet inhibitor (RGD motif) [60]. The Ag5r proteins are not specific for salivary glands thus they may possess other functions not associated with feeding [23], [56]. Several studies showed antigenic properties associated with Ag5r proteins. Plasmid coding for Ag5r protein from P. ariasi (ParSP05/AAX44092) induced a cell-mediated immune response in Swiss Webster mice [27], showing that sand fly Ag5r proteins might modulate cell-mediated host immune response. This presumption is also supported by several T cell epitopes predicted for P. duboscqi Ag5r proteins [26] that include regions highly conserved among sand flies (Figure 2). Antibody response to sand fly Ag5r proteins was demonstrated in P. perniciosus; Ag5r protein (PpeSP07/ABA43055) reacted with IgG antibodies from sera of P. perniciosus bitten dogs [21]. In other bloodsucking diptera, Ag5r proteins are mostly associated with IgE antibody response. Ag5r protein of Simulium vittatum seems to be the major allergen for insect bite hypersensitivity sharing common IgE-binding epitopes with Ag5r protein from Culicoides nubeculosus [61], [62]. Specific anti-Ag5r IgE antibodies were also observed in Ugandan individuals bitten by Glossina morsitans [63]. Phylogenetic analysis of Ag5r proteins from sand flies and other insects showed a strongly supported distinct clade of sand fly Ag5r proteins (Figure 3) similar to a previous analysis by [28]. The relationship within the sand fly clade reflected phylogenetic relationship within phlebotomine sand flies [33], showing three distinct branches: clade I with species belonging to subgenera Euphlebotomus, Larroussius, and Adlerius; clade II with Phlebotomus and Paraphlebotomus species (P. papatasi, P. duboscqi, and P. sergenti); and Lutzomyia in clade III (Figure 3). Apyrase (EC 3. 6. 1. 5) appears to be a universal enzyme used to prevent blood coagulation by diverse hematophagous animals such as bloodsucking leeches, ticks, triatomine bugs, fleas, and mosquitoes. This enzyme hydrolyses both ATP and ADP to AMP, thus destroying an important physiologic stimulus of platelet aggregation released from damaged tissues and blood cells. Apyrases of bloodsucking insects are divided into three families: CD-39 (the actin/heat shock 70/sugar kinase superfamily); 5′-nucleotidase; and Cimex-type [55], [56]. Sand flies are not an exception; transcripts coding for apyrases have been found in the saliva of all tested species [6], [23]–[28], including P. tobbi (PtSP4/HM135951, PtSP10/HM135952) and P. sergenti (PsSP40/HM560860, PsSP41/HM560862, PsSP42/HM560861) (Tables 1 and 2). The predicted molecular mass of the translated molecules is uniform for all sand fly species, varying between 35 and 36 kDa (Table S1). All sand fly apyrases deposited in GenBank have also been found in the proteomic analysis (Table S1). In P. tobbi and P. sergenti, the predicted molecular mass corresponds well with the molecular weight measured under non-reducing conditions (33. 0–37. 6 kDa) (Figure 1; Tables 1 and 2). Sand fly apyrases belong to the Cimex-type apyrase family. They hydrolyze ADP at a faster rate than ATP [64] and, similar to Cimex lectularius, the activity strictly depends on Ca2+ but not Mg2+ ions [6], [23], [64]–[66]. Apyrase activity has been demonstrated in the saliva of L. longipalpis [23], [64], P. argentipes [65], P. colabaensis [65], P. duboscqi [66] P. papatasi [65], [67], P. perniciosus [65], and as well as in recombinant apyrases of P. papatasi (PpApy/AF261768) [67] and P. duboscqi (PduApy2/DQ834331) [66]. Bacterially expressed P. duboscqi apyrase inhibited ADP- as well as collagen-induced platelet aggregation [66], indicating that post-translational modifications such as glycosylation are not necessary for apyrase activity. Orthologs of the Cimex apyrase family have also been identified in vertebrates and termed calcium-activated nucleotidases (CANs) [68]. In contrast to sand flies, human soluble CAN-1 (SCAN-1) preferentially hydrolyses UDP and GDP; however, the engineered SCAN-1 mutant Glu92Tyr shows five times and seven times higher hydrolysis activity for ADP and ATP, respectively [69]. This mutated tyrosine is conserved among species of the genus Phlebotomus (Figure 4), supporting its key role in substrate specificity for phlebotomine apyrases [69]. In human SCAN-1, other amino acid residues essential for binding nucleotide and Ca2+ were identified [70], some of them being absolutely conserved among the analyzed apyrase proteins (Asp44, Ser100, Asp114, Glu216, Arg232, Ser277), while others were uniformly mutated within sand fly apyrases (Asp101Glu, Gly160Ser, Ile214Trp) (Figure 4). Besides hydrolyzing activity, sand fly apyrases also possess antigenic properties. Antibodies from dogs experimentally or naturally exposed to P. perniciosus strongly recognized PpeSP01 (ABB00906) and PpeSP01B (ABB00907) apyrases [21]. In humans naturally exposed to sand flies, anti-sand fly saliva IgG antibodies recognized a protein band corresponding, in molecular weight, to apyrase [11], [12]. Moreover, antibodies elicited by P. duboscqi saliva also recognized bacterially expressed P. duboscqi apyrase [66], indicating that not all antibodies are specific for possible glycan modifications of sand fly apyrases. Phylogenetic analysis of sand fly apyrases reflects the same taxonomic relationship as Ag5r proteins. Figure 5 shows three distinct clades separating species in clade I (P. arabicus, P. argentipes, P. ariasi, P. perniciosus, P. tobbi) from Phlebotomus and Paraphlebotomus subgenera in clade II (P. papatasi, P. duboscqi, and P. sergenti), and genus Lutzomyia in clade III. This analysis showed a very close relationship within the Larroussius species, P. tobbi and P. perniciosus (Figure 5). Hyaluronidase is an enzyme that catalyzes the hydrolysis of hyaluronic acid, a major component of the extracellular matrix in vertebrates. It is an ubiquitous enzyme found in mammals, bacteria and in the venom of bees, wasps, spiders, and snakes [71]. In bloodsucking Diptera, hyaluronidase activity has been found primarily in the saliva of telmophagic insects: horse flies, black flies, biting midges, and sand flies [72]. Thus, hyaluronidase is believed to decreases host skin tissue viscosity, assisting other salivary components to diffuse and create a pool of blood [60], [72], [73]. Sand fly hyaluronidase belongs to the same family as mammalian and Hymenopteran hyaluronidases (endo-β-N-acetyl-hexosaminidases, E. C. 3. 2. 1. 35) and is different from that of bloodsucking leeches and nematodes (endo-β-glucuronidases, E. C. 3. 2. 1. 36) [71], [74]. Hyaluronidase activity has been detected in all eight sand fly species studied to date ([23], [28], [53], [73], Figure 6). Our zymographic analyses of P. tobbi (Figure 6) and P. sergenti originating from Israel (Figure 6) and Turkey [53] showed the potent activity of sand fly hyaluronidase. Based on the microplate method, P. tobbi hyaluronidase activity is one of the highest measured (Figure 6A). In contrast, hyaluronidase of P. sergenti had the lowest activity among the species of the genus Phlebotomus (Figure 6A). Under non-reducing conditions, P. tobbi and P. sergenti hyaluronidase revealed diffuse bands with the molecular weight of around 110 and 135 kDa, respectively (Figure 6B). Hyaluronidase of P. sergenti is probably a homodimer, because under reducing conditions, the activity was observed at about half of the molecular weight, both in the Israeli (Figure 6B) and Turkish strains [53], while hyaluronidase of P. tobbi was monomeric with similar molecular weight under non-reducing and reducing conditions and the activity reduced to minimum when denaturated and treated with β-mercaptoethanol (Figure 6B). Similar features were observed for the hyaluronidase of P. perniciosus, the other Larroussius species ([53], Figure 6), which suggests common biochemical characteristics of this enzyme between closely related species. In general, the remarkably high activity of salivary hyaluronidase may aid the spread of other salivary components as well as transmitted pathogens. Indeed, hyaluronidase coinjected with Le. major promotes infection in BALB/c mice [72]; however, no association was found between hyaluronidase activity and the sand fly capacity to vector either cutaneous or visceral leishmaniasis (Figure 6A). Although sand fly hyaluronidase is a very potent enzyme, it is scarcely found in transcriptomic and proteomic approaches probably due to the low abundance of transcripts combined with the large size of the protein. Hyaluronidase transcripts have been reported in only two of seven salivary cDNA libraries, namely in L. longipalpis and P. arabicus [23], [24], [28]. In P. sergenti, no transcript was found, and in P. tobbi, only one 3′-truncated transcript was identified (PtSP125/JN192442). Amino acid residues that constitute the catalytic site (Asp111, Glu113, and Glu247) and form disulfide bridges (Cys22–Cys313 and Cys189–Cys201) in bee hyaluronidase [75] are conserved among the sand fly hyaluronidase sequences (Figure 7). Based on the NetNGlyc prediction server [48], several putative glycosylation sites were predicted in sand fly hyaluronidases, including one highly conserved among aligned sequences (Figure 7). Allergenic properties of sand fly hyaluronidase are not known, although it has been identified or suspected as the main allergen in the saliva of other bloodsucking Diptera, namely biting midges and horseflies [59], [76]. However, there is no record of typical IgE-mediated allergic reaction to sand fly saliva; only negligible amount of anti-saliva IgE was measured in hosts repeatedly bitten by sand flies [11], [19], [77]. Two sand fly salivary protein families, D7-related proteins and PpSP15-like proteins, are related to the arthropod pheromone/odorant binding protein superfamily (OBP, protein domain PBP-GOBP, pfam01395) [56], [78]. D7-related (D7r) proteins are named after the D7 protein, originally described in Aedes aegypti as a major salivary protein exclusively synthesized in bloodsucking females [79], [80]. Salivary proteins related to D7 have also been found in black flies and biting midges [56], [78] and all sand fly species studied to date. In the P. tobbi SG cDNA library we found seven clusters homologous to D7r sequences (HM164145–HM164151) and three clusters in the P. sergenti cDNA library (PsSP4/HM560863, PsSP5/HM569360, PsSP7/HM560864) (Tables 1 and 2). Within all sand flies, D7r proteins have similar predicted molecular mass (25. 3–28. 1 kDa) and wide range of pI (4. 82–9. 5) (Table S1). Based on the results from NetNGlyc prediction server [48], we found a mixture of putative glycosylated and non-glycosylated D7r sequences in most of the sand fly species studied with the exception of L. longipalpis, P. sergenti, and P. papatasi, where no N-glycosylation sites were found (Figure 8). All sand fly D7r predicted proteins contain nine highly conserved cysteine residues (Figure 8), implying there is a single non-disulphide-bond-forming cysteine. The other family related to OBPs, PpSP15-like proteins, is closely related to larger D7-related proteins [25], [56] and are named after 15-kDa salivary protein of P. papatasi (PpSP15/AF335487) [6]. They have not been identified in any Diptera other than sand flies [25], [56]. It is the most abundant family among sand fly salivary proteins, and P. tobbi and P. sergenti are not exceptions; six and seven members of this family were found in each cDNA library, respectively (Tables 1 and Table 2). Several members were also detected by proteomic analysis, having similar molecular mass as predicted based on the amino acid sequences (Tables 1 and Table 2; Figure 1). Within the sand flies, PpSP15-like proteins have a similar predicted molecular mass (12. 2–17. 1 kDa) and surprisingly wide range of pI (6. 33–9. 44) (Table S1). In accordance with previous reports [25], [28], all sand fly PpSP15-like proteins show high degree of variability of around six conserved cystine residues (Figure S2). In mosquitoes, some salivary D7 strongly bind biogenic amines and leukotrienes as well as components of the coagulation cascade, thus promptly antagonizing the host defense system [81]–[83]. D7r and PpSP15-like sand fly salivary proteins have not yet been characterized functionally; however, the motif [ED]-[EQ]-x (7) -C-x (12,17) -W-x (2) -W-x (7,9) -[TS]-x-C-[YF]-x-[KR]-C-x (8,22) -Q-x (22,32) -C-x (2) -[VLI], found in mosquito D7 salivary proteins that bind cysteinyl leukotrienes [83], is also found in the sand fly D7r proteins (Figure 8). Sand fly PpSP15-like proteins and D7r proteins possess antigenic properties. PpSP15-like proteins were reported as promising anti-Leishmania vaccine candidates [6], [27], [84]. Phlebotomus papatasi SP15 protein is able to protect mice against Le. major challenge, and a DNA vaccine containing the PpSP15 cDNA provided the same protection [6]. ParSP03 (AAX56359), a PpSP15-like protein from P. ariasi, elicited similar delayed-type hypersensitivity and humoral immune responses upon DNA vaccination [27]. D7r could serve as a marker of exposure to sand fly bites. In humans, all tested serum samples from individuals naturally exposed to P. papatasi strongly bound to a P. papatasi protein band with a molecular mass corresponding to PpSP30 D7r protein (AAL11049) [12], [18]. As an ideal marker of exposure, this protein was recognized by both IgE and IgG antibodies, including all tested IgG subclasses [18]. D7r proteins seem to be applicable also for measurement of dog exposure, the main reservoir host for visceral leishmaniasis, since IgG antibodies from animals bitten by P. perniciosus [21] or L. longipalpis [16], [85] recognized D7r proteins of the respective species (PpeSP4/DQ150623, PpeSP04B/DQ150624, PpeSP10/DQ153104, LJL13/AF420274). Moreover, L. longipalpis-bitten dogs bind also to the LJL13 D7r recombinant form [16]. Phylogenetic analysis of D7r proteins showed several major clades (Figure 9). Phlebotomus sergenti sequences clustered together forming a distinct subclade within clade III that contains P. papatasi and P. duboscqi. In contrast, P. tobbi D7r protein sequences are divided among clades I and II, which contain sequences from P. arabicus, P. ariasi, P. argentipes, P. perniciosus, and L. longipalpis. Interestingly, clade II only contained sequences with predicted N-glycosylation sites, which may suggest a unique functional characteristic of D7 molecules within this clade that have arisen after gene duplication. Similarly, phylogenetic analysis of PpSP15-like proteins (Figure 10) revealed several separated groups, consistently clustering P. sergenti sequences with P. duboscqi and P. papatasi, and P. tobbi sequences with those from P. perniciosus and other sand flies studied to date, including a single member from L. longipalpis. PpSP15 could be a multicopy gene, as more than two alleles were found in several P. papatasi individuals, some of them unique to the population origin [86]. This family is named PpSP32 from the original identification in P. papatasi (AAL11050) [24] and due to the lack of homology to a conserved protein domain. PpSP32-like proteins have been described solely in sand flies and are found in all species studied so far; we identified homologous sequences also in P. tobbi (PtSP27/HM173642, PtSP28/HM173643, PtSP29/HM173644) and P. sergenti (PsSP44/HM569368). The predicted molecular mass of P. tobbi PpSP32-like proteins (24. 5 kDa) is slightly lower than what was measured in proteomic analysis (Figure 1, Tables 1 and 2). All sequences have a wide range of predicted molecular mass (ranging from 22. 5 to 34. 9 kDa), no protein domain match, and are alkalic (pI ranging from 9. 3 to 10. 6) (Table S1). An interesting common feature of this protein family is that it possesses highly conserved N- and C- terminal regions with extremely variable internal sequence (Figure 11). Within the genus Phlebotomus there are predicted N-glycosylation sites in the variable and C-terminal regions (Figure 11). To date, no function has been associated with sand fly PpSP32-like proteins, although L. longipalpis and P. perniciosus proteins have been hypothesized to possess collagen binding activity [24], [25] and in P. papatasi, PpSP32 transcripts are expressed independently of either diet or age [87], indicating a vital role for these molecules in feeding. Phylogenetic analysis of PpSP32-like proteins reflects again the taxonomic relationship within Phlebotomine sand flies [33]. True to form, phylogenetic position of P. tobbi PpSP32-like proteins are within a subclade I with P. perniciosus and the P. sergenti PpSP32-like protein is within the Phlebotomus and Paraphlebotomus clade II (Figure 12). Phlebotomine yellow-related proteins are characterized by the presence of major royal jelly protein domain (MRJP; pfam03022). Originally, MRJP proteins were described from honeybee larval jelly, making up to 90% of the protein content [88]. Sequences related to MRJP proteins were described in Drosophila, where it is related to cuticle pigmentation and, when mutated, it produced a yellow phenotype and thus named Yellow proteins [89], [90]. In bloodsucking Diptera, salivary yellow-related proteins have only been described in sand flies [55], [56] and black flies [91]. Yellow-related proteins are found in all sand fly species studied to date. In the P. sergenti cDNA library, five different clusters were found (PsSP18/HM569361, PsSP19/HM560865, PsSP20/HM560866, PsSP22/HM560867, PsSP26/HM569362) compared with P. tobbi, where only two clusters were found (PtSP37/HM140618 and PtSP38/HM140619) (Tables 1 and 2). Sand fly yellow-related proteins have a similar predicted molecular mass (41. 5–45. 2 kDa), wide range of pI (4. 75–9. 8), and contain four conserved cysteine residues shown to form two disulfide bonds in LJM11 (AAS05318) [9] (Table S1, Figure 13). Yellow-related proteins are modulated on a transcriptional level [87] and are likely post-translationally modified, as variants with different mobility have been detected on SDS-PAGE [6], [25] (Figure 13). Ribeiro and Arca [55] proposed that in Phlebotomines, salivary yellow-related proteins work as kratagonists, the binders of biogenic amines. Indeed, Xu et al. [9] proved that the bacterially expressed L. longipalpis yellow-related proteins (LJM11, LJM17/AAD32198, and LJM111/ABB00904) bind biogenic amines, namely serotonin, catecholamines, and histamine. The proteins differed in affinity to the particular ligand, suggesting functional divergence within the family [9]. The midgut yellow protein in Aedes aegypti is involved in the melanization pathway as a dopachrome conversion enzyme [92]; however, in sand flies the yellow-related proteins found in the midgut lumen probably originating from swallowed saliva [93] and researchers failed to detect dopachrome convertase activity in salivary yellow-related proteins [28], [56]. In Glossina morsitans, the ubiquitous tissue expression of the protein suggests also a housekeeping role for yellow-related proteins [91]. Sand fly salivary yellow proteins possess antigenic properties as they are recognized by serum antibodies of experimentally bitten mice [12] and dogs [19], [21], as well as naturally exposed dogs, humans, and foxes [11], [16], [18], [21], [77], [85]. Additionally, a combination of recombinant LJM17 and LJM11 successfully substituted L. longipalpis whole SG sonicate in probing sera of individuals for vector exposure [16], [20]. Yellow proteins are also under consideration for anti-Leishmania vector-based vaccines. LJM17 from L. longipalpis elicited leishmanicidal Th1 cytokines in immunized dogs [8], and LJM11 protected laboratory animals against both Le. major and Le. infantum [7], [9]. In contrast, mice immunized with P. papatasi yellow-related proteins PpSP42 or PpSP44 (AAL11052 and AAL11051, respectively) elicited Th2 cytokines and exacerbated Le. major infection [84]. It remains to be elucidated whether the protection induced by yellow-related proteins is related to particular protein immunogenicity, to sand fly species, or to the vector-Leishmania-host combination, as all of these factors can contribute to vaccine efficacy. Recently, Xu et al. [9] showed that L. longipalpis LJM11 but not LJM111 produces a DTH response in mice challenged by SGH. The authors related this immunogenicity to electrostatic potential on the protein surface, which is positive in LJM11; thus the protein is probably more attractive to antigen-presenting cells [9]. Yellow-related proteins are highly conserved among sand flies. Phylogenetic analysis produced three major clades combining Larroussius, Adlerius and Euphlebotomus (clade I); Phlebotomus and Paraphlebotomus (clade II); and Lutzomyia (clade III), while subclades discerned each subgenus (Figure 14). Interestingly, P. sergenti illustrates a gene duplication event that preceded speciation and was followed by a clear gene duplication expansion that is seen in one of the subclades. Gene duplication in bloodsucking arthropod salivary molecules is fundamental for the functional diversification of proteins, as can be seen with the range of substrates bound by the L. longipalpis yellow-related proteins [9]. Within clade II, two subclades can be seen distinguishable by the presence of putative N-glycosylation sites. Moreover, sequences in clade IIa have a slightly higher predicted isoelectric point than the glycosylated sequences in clade IIb (Figure 14, Table S1), indicating another feature that might be responsible for functional diversification. ParSP25-like transcripts were found in P. tobbi but not in P. sergenti SG library. Phlebotomus tobbi ParSP25-like molecules (PtSP73/HM173639, PtSP75/HM173640, and PtSP76/HM173641) have predicted molecular mass ranging from 27. 8 to 38. 8 kDa and contain a large proportion of acidic residues resulting in a pI of 4. 5±0. 1. The sequences share similarity with eight other sand fly salivary proteins from three sand fly species [25], [27], [28] (Figure 15), all of them with predicted pI between 4. 4 and 5. 0 (Table S1). Analysis of the putative protein sequences revealed highly conserved regions rich in amino acid residues such as Asp, Tyr, Glu, and Ser and no predicted N-glycosylation sites (Figure 15). Though the function is not known, some members of this family were shown to be highly antigenic. Mice immunized with a plasmid coding for ParSP25 (AAX55664) elicited high levels of anti-P. ariasi IgG1 and a strong DTH reaction when challenged with P. ariasi saliva [27]. Moreover, dogs exposed to P. perniciosus bites strongly bind to protein band characterized as PpeSP08 (ABA43056) [21]. Sand fly ParSP25-like proteins are most likely genus-specific because, so far, the sequences have been found only in Adlerius (P. arabicus) and Larroussius species (P. ariasi, P. perniciosus, P. tobbi) and not in representatives of the other subgenera (Figure 15). These proteins, named by Anderson et al. [25] as members of the 33-kDa family, have not yet been found in any Diptera other than sand flies. PsSP49 (HM569369) and PtSP66 (HM173645) share sequence similarity with seven other sand fly salivary proteins from six sand fly species both from both New and Old World sand flies [24]–[28] (Figure 16). All sand fly 33-kDa family proteins have similar predicted molecular weight (32. 3–34. 5 kDa) and alkalic pI (8. 2–9. 1) (Table S1). PsSP49 and PtSP66 were both identified in the proteomic analysis (Figure 1). Two highly conserved N-glycosylation sites were predicted among all sand fly sequences (Figure 16) and both PsSP49 and PtSP66 were found above the predicted molecular weight in the proteomic analysis (Figure 1, Tables 1 and 2), indicating a post-translational modification. Indeed, the two proteins from P. arabicus (PabSP32/ACS93510 and PabSP34/ACS93511) showed glycosylation by ProQ Emerald staining [28]. The function is unknown; however, P. perniciosus PpeSP06 (ABA43054) and the L. longipalpis LJL143 (AAS05319) were identified as antigens for dogs living in endemic areas of Le. infantum [16], [21], the later one also shown to be a candidate for vaccine against canine leishmaniasis [8]. 41. 9-kDa protein superfamily is specific to bloodsucking Nematocera encompassing members of mosquitoes, biting midges, black flies, and sand flies [56]. The P. sergenti and P. tobbi members of this superfamily, PsSP82 (HM569371) and PtSP49 (HM173648), share sequence similarity with five other sand fly salivary proteins from five sand fly species (Figure 17). These sand fly proteins have a wide range of predicted molecular weight (27. 5–56. 6 kDa) and pI (4. 3–8. 5) (Table S1) but only one of them, P. perniciosus PpeSP19 (ABA43063), has been found by proteomic analysis [25]. All sequences are rich in putative N-glycosylation sites (Figure 17) and the function is not known. Several other putative salivary proteins were identified in the transcriptomes of P. tobbi and P. sergenti SGs. They are smaller than 15 kDa, their function is not known, and are, thus far, unique to sand flies. Additionally, none of these small proteins have been found in the proteomic analysis (Figure 1, Tables 1 and 2). PsSP28 (HM569370), PtSP8 (HM173646), and PtSP81 (HM173647) share sequence similarity with P. ariasi ParSP23 (AAX55663) and P. perniciosus PpeSP15 (ABB00905) (Figure 18). The proteins have a low predicted molecular weight (2. 4–5. 0 kDa) and an alkalic pI (9. 2–10. 7). PsSP98 (HM569366) has a predicted molecular weight similar to PpSP15-like proteins (14. 3 kDa) but is highly acidic (pI = 4. 73). The protein sequence is related to 16-kDa proteins from P. arabicus (PabSP64/ACS93507, PabSP63/ACS93506) and P. argentipes (PagSP73/ABA12153) (Figure 19). PsSP73 (HM569367) has a predicted molecular weight 12. 2 kDa and is highly acidic (pI = 4. 51). The predicted protein sequence is related to proteins found in P. arabicus (PabSP75/ACS93508) and P. ariasi (ParSP13/AAX55657) (Figure 20). PtSP71 (HM173638) has a low predicted molecular weight (4. 5 kDa) and an alkalic pI (10. 6). The protein sequence is related to molecules identified in P. perniciosus (PpeSP12/ABA43060, PpeSP13/ABA43061) and P. ariasi (ParSP15/AAX55658) (Figure 21), indicating these sequences might be unique to Larroussius species. To identify antigens and glycoproteins in P. tobbi SGH, electrophoretically separated proteins were incubated with anti-P. tobbi rabbit serum and a lectin Concanavalin A (ConA), respectively (Figure 22). When compared with the proteome analysis in Figure 1, the protein bands visible by silver staining are most likely yellow-related proteins (PtSP37 and PtSP38), apyrases (PtSP4 and PtSP10), antigen 5-related proteins (PtSP77 and PtSP79), PpSP32-like proteins (PtSP28 and PtSP29), D7-related proteins (PtSP58 and PtSP60), and PpSP15-like proteins (PtSP9, PtSP23, and PtSP32). Anti-P. tobbi antibodies recognized all identified bands as well as other six high molecular weight proteins not visible by silver staining (Figure 22, lane 2). Most of the P. tobbi proteins reacted with ConA, indicating they are N-glycosylated. The lectin binding was specific, as the reactivity was totally inhibited when ConA was preincubated with specific monosaccharide methyl-α-D-mannopyranoside. The most intense reaction was observed with the high molecular weight band not visible by silver staining, and with the bands of molecular weight similar to one yellow-related protein (PtSP38) and both apyrases. Among the nine silver-stained bands, three did not react with ConA, namely bands with molecular weight similar to D7-related proteins, PpSP15-like proteins, and one yellow-related protein (PtSP37) (Figure 22, lane 4). The reactivity with ConA is in agreement with N-glycosylation as predicted by NetNGlyc server [48], with the exception of PtSP10 apyrase (Table 3). We can speculate that the most glycosylated band with the highest molecular weight might be hyaluronidase. Although producing a minor unstainable band, it is predicted to be highly glycosylated (Figure 7) and its activity is clearly visible around 135 kDa in zymography analyses (Figure 6). Within sand fly yellow-related proteins, it is common that glycosylated and non-glycosylated forms occur in the same species. As proved for P. papatasi [93] and predicted for protein sequences of Phlebotomus (P. papatasi and P. duboscqi) and Paraphlebotomus (P. sergenti) species, at least one form is glycosylated, forming a well supported subclade with glycosylated sequences from other species (Figure 14). Glycosylated and non-glycosylated forms are also present in P. tobbi, as proven by blot analysis (Figure 22), although the closely related P. perniciosus possesses only glycosylated forms. Interestingly, in sand fly species within the clades I and III (Figure 14), all published sequences are glycosylated with an exception of P. tobbi and P. ariasi, which has at least one non-glycosylated form. Further research is needed to investigate whether the presence of sugar side chains may contribute to the antigenicity of the yellow-related proteins. With over 80 species of sand flies implicated in Leishmania transmission, it is vital to continue describing their salivary proteins in the search for vaccine candidates and markers of exposure. In this study, we prepared and analyzed the transcriptome and proteome data of P. tobbi and P. sergenti to broaden our knowledge on the repertoire of Larroussius salivary proteins and provide the first report from a Paraphlebotomus sand fly, respectively. P. tobbi has been reported to transmit Le. infantum that causes cutaneous leishmaniasis [32]. Interestingly, the salivary proteins of P. tobbi are highly homologous to those of P. perniciosus, a vector of Le. infantum that causes visceral disease. It is likely that, in this instance, the salivary proteins of P. tobbi are not the determining factor for these different disease manifestations. However, in general, it is possible that the divergence, diversity or amount of sand fly salivary proteins or non proteinaceous components of the saliva correlate with different disease manifestations of the same species of Leishmania. The transcriptome data can be utilized to prepare recombinant proteins that can be used to test their potential as anti-Leishmania vaccines or in epidemiologic studies to develop more specific and efficient methods for measurement of vector exposure. Finally, recombinant salivary proteins may also help us to understand the mechanism of blood sucking or find biological activities of many of these novel sequences.
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Title: Salivary Gland Transcriptomes and Proteomes of Phlebotomus tobbi and Phlebotomus sergenti, Vectors of Leishmaniasis Summary: Phlebotomine female sand flies require a blood meal for egg development, and it is during the blood feeding that pathogens can be transmitted to a host. Leishmania parasites are among these pathogens and can cause disfiguring cutaneous or even possibly fatal visceral disease. The Leishmania parasites are deposited into the bite wound along with the sand fly saliva. The components of the saliva have many pharmacologic and immune functions important in blood feeding and disease establishment. In this article, the authors identify and investigate the protein components of saliva of two important vectors of leishmaniasis, Phlebotomus tobbi and P. sergenti, by sequencing the transcriptomes of the salivary glands. We then compared the predicted protein sequences of these salivary proteins to those of other bloodsucking insects to elucidate the similarity in composition, structure, and enzymatic activity. Finally, this descriptive analysis of P. tobbi and P. sergenti transcriptomes can aid future research in identifying molecules for epidemiologic assays and in investigating sand fly-host interactions.
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Summarize: Background Tantalum is a hard, gray metal with a high melting point that is highly resistant to corrosion. It is a good conductor of heat and electricity. These attributes make it a valuable material for use in numerous military applications such as capacitors used in electronics equipment including computers, turbine engines for aircraft, and the linings of missile warheads. It is also used in numerous commercial consumer products such as mobile phones, personal computers, chemical processing equipment, heat exchangers, anti-lock brake systems, and airbag activation systems. It is possible to substitute other materials for tantalum, but substitutions usually result in a loss of performance. Worldwide tantalum supply begins with mining tantalum ore—a concentration of tantalum-containing minerals, such as tantalite, microlite, and wodginite—that is economically feasible to mine. Ore is the principal raw material from which tantalum is derived, and can be mined through industrial operations which may be mechanized or through artisanal mining operations, which are characterized by a lack of mechanization. The ore is further processed into tantalum concentrate by physically removing unwanted materials. Tantalum is also derived from synthetic concentrates, where tantalum is extracted from slags, for example, from processing tin concentrates or other forms of industrial processing. These concentrates can be chemically processed into tantalum salts, tantalum oxides, or other marketable tantalum materials such as tantalum powder, metal, and alloys, which are then further manufactured into end- products. Figure 1 shows details of the tantalum supply chain. The USGS collects, monitors, and analyzes information about natural resource conditions, issues, and problems, including those related to tantalum, to provide information to various government agencies, including DOD. Within USGS, the National Minerals Information Center develops and provides statistics and information on the worldwide production, consumption, and flow of minerals and materials essential to the United States economy and national security. To communicate this information, the USGS produces two publications that include information on tantalum, among other minerals. The Minerals Yearbook is an annual publication that provides statistical data on over 90 commodities over a 5-year period. It also includes data from over 175 countries on mineral production and trade, among other things. The Mineral Commodity Summaries is based on the data reported in the Yearbook and published each January. The annual summary includes similar historical data as reported in the Minerals Yearbook, as well as production estimates from the current reporting year. According to the USGS’ 2015 Mineral Commodity Summaries, the United States relies solely on imports to meet needs for tantalum; there has been no significant tantalum mined in the United States since 1959 because the tantalum cannot be economically mined at prevailing prices. Countries for which the USGS reported tantalum mine production in 2014 are Brazil, Burundi, China, Democratic Republic of the Congo, Ethiopia, Mozambique, Nigeria, and Rwanda. Although Australia has some of the largest reserves of tantalum, USGS reported no production for the country in 2014. The USGS estimated in its 2015 Mineral Commodity Summaries that the two largest producers of mined tantalum in 2014 were Rwanda and the Democratic Republic of the Congo, producing approximately 600 and 200 metric tons, respectively. This amount represented approximately two-thirds of USGS’ total 2014 estimated global mine production. The National Defense Stockpile maintains a domestically held inventory of strategic and critical materials. DOD uses USGS information on materials, in part, to help address its National Defense Stockpile program objectives. One objective of the National Defense Stockpile program is to decrease the risk of dependence on foreign suppliers or single suppliers for strategic and critical materials, such as tantalum, that are used in defense and essential civilian applications. Authority for stockpiling strategic and critical materials, which can include tantalum, is contained in the Strategic and Critical Materials Stockpiling Act (Stockpile Act) that lays out the purpose and responsibilities for the National Defense Stockpile. DOD generates national emergency scenarios for the Stockpile Act and related National Defense Stockpile Report. These scenarios cover four- year timeframes where the first year represents a period of conflict, and years two through four represent a period of recovery. The one-year period of conflict takes into consideration the following conditions: (1) a catastrophic attack on a United States city by a foreign terrorist organization or rogue state, (2) two near simultaneous major combat operations, (3) war damage from a highly capable enemy, and (4) ongoing military activities such as a military presence in a foreign country. On behalf of DOD, DLA-Strategic Materials serves as the National Defense Stockpile program manager, whose functions include: determining materials deemed strategic and critical, precluding, when possible, a dangerous and costly dependence by the United States upon foreign sources for supplies of such materials in times of national emergency, and submitting a biennial report to Congress detailing stockpiling requirements and recommendations for the National Defense Stockpile based on certain national emergency planning assumptions. USGS and DOD have an interagency support agreement to facilitate data sharing in support of DOD’s biennial Stockpile Report. According to the agreement, USGS provides DOD with data on United States production, consumption, imports, exports, and world production by country for materials in the National Defense Stockpile and other materials critical for defense applications. USGS data on worldwide tantalum production is the primary production data source used by DOD to support its stockpile analyses. As the program manager for the National Defense Stockpile, DLA-Strategic Materials is responsible for compiling the data it receives from a number of entities, including the USGS and Department of Commerce, and performing analyses to determine if shortfalls for materials will occur during potential conflict scenarios. Table 1 provides a list of the sources of data and examples of the data used by DLA- Strategic Materials. The analyses focus on the sources of materials and the likelihood these materials will be available in the conflict scenario based on forecasted demand for materials in critical defense and civilian uses. In the case of a material shortfall, the analyses also consider mitigation strategies such as increased production or substitutions that could be made for these materials. The results of these analyses are published biennially in DOD’s Stockpile Report and are the basis for DOD’s recommendations to stockpile certain materials. Tantalum Data from Government and Industry Sources Vary Due to Differences in Forms of Tantalum Reported and Data Collection Methods Government and industry tantalum information differ because the government reports on tantalum ore from mining operations, whereas industry data include not only mining but also data on additional forms of processed tantalum, such as synthetic concentrates and scrap. In addition, government and industry have different data collection methods to address their specific needs. Differences include which forms of tantalum are measured, the frequency of data collection, and data validation practices. Having reliable tantalum data is important because DOD uses the information to assess the availability of tantalum supply by country during specific planning scenarios, among other efforts. Government and Industry Report Tantalum Data Differently Government and industry data differ on the forms of tantalum reported. DOD’s primary government source for mineral production data, the USGS, captures data on tantalum ore from mining operations but does not include or estimate synthetic concentrates such as tantalum derived from tin slags, other means of industrial processing, or secondary types of tantalum such as scrap or recycled tantalum. USGS officials told us that they have limited visibility into the supply from secondary types of tantalum, such as scrap or recycled materials, and thus for reliability reasons, only report mine production. However, USGS officials also said their data includes some artisanal mining reported from countries like Rwanda or the Democratic Republic of the Congo, although it is difficult to quantify how much of the tantalum production they report is from industrial versus artisanal mining. Industry data, which, for the purpose of our review, are largely publically available data from the TIC, include tantalum supply from sources other than mined ore. For example, the TIC reports tantalum data that includes supply from mining as well as synthetic concentrates and slags from processing tin concentrates and other materials. Further, the TIC reports data on purchases of tantalum ore, synthetic concentrates, and some recycled material from member companies. These data may also include artisanal mined ore and processed materials of unknown origin. As a result, TIC data on tantalum purchased by processors—companies that modify the tantalum and change it into different forms for use—show higher supply figures than USGS reports on tantalum ore from mining operations. Government and Industry Have Different Data Collection Methods Our review also identified differences in data collection methods that affect USGS and industry tantalum reporting, including differences in how and when data are collected and validated. The USGS collects data on tantalum and other minerals to provide information to various government agencies, including DOD. Table 2 summarizes a comparison of government and industry data reporting and collection practices. To facilitate its data collection, the USGS employs country and mineral commodity specialists to help estimate tantalum production by country of origin. USGS officials said they use the term commodity to refer to ore, minerals, and materials, and their mineral commodity specialists have expertise in mineral and material markets and industries. According to USGS and DLA-Strategic Materials officials, providing production information by specific country is important to DLA-Strategic Materials’ ability to assess tantalum supply risks and how much tantalum the United States may be able to acquire during conflict scenarios. USGS country and mineral commodity specialists annually survey foreign governments on the production of tantalum from mining, gather data and information from in-country visits, and review mineral reporting provided by embassies and industry sources to estimate tantalum production. USGS officials told us that while the quality of the data provided by foreign governments can vary widely, its specialists use their expertise with a mineral commodity or country to estimate production even if the data are incomplete or questionable. For example, in cases where the amount of tantalum reported is significantly different than previous information, specialists can use mining permits, trade analysis, and mine capacities to confirm estimates. In contrast, TIC data are provided as a service to its member companies rather than in support of the federal government, and are based on tantalum supply data voluntarily reported by those members. The TIC employs an independent company to compile surveys from its member mining and processing companies on a quarterly basis. Supply data are compiled and aggregated into categories such as raw material reported by mining, sales of tantalum products, and material purchased by companies that further process tantalum. These data are not reported by company or country of origin. USGS and industry also vary in their procedures for validating tantalum data. USGS data are required to meet USGS Fundamental Science Practices, a set of consistent practices, philosophical premises, and operational principles that serve as the foundation for research and monitoring activities. These practices describe how USGS work is carried out and how the resulting information products, such as maps, imagery, and publications, are developed, reviewed, and approved. The USGS also examines the mineral commodity data it collects by using a set of 14 statistical standards, most recently revised in November 2014. These 14 standards include definitions of statistical terms, conversion factors, and guidelines for graphic display of statistical information, among other things. In addition, USGS mineral commodity or country specialists compile and review survey data. In cases where these experts have different estimates, USGS officials said that managers resolve the differences based on discussion with the analysts, their own expertise, and contacts within other governments or industry to arrive at a final USGS position. Further, USGS guidelines provide that at least 75 percent of surveys should be returned in order to report data in its publications. In comparison, TIC officials stated that they do not have processes in place to validate the data received from their member companies, but they do have some guidelines for compiling the data. For example, the TIC guidelines request that member companies not report sales and purchases from other member companies to avoid double-counting of primary tantalum production. Further, the guidelines require that two- thirds of member companies, and certain major companies, provide data before the publications are released. However, industry officials we spoke with told us the TIC is not in a position to check or validate information provided by its members and that it is uncertain how much of the tantalum market the TIC’s members encompass, making it difficult to ascertain how much of the worldwide tantalum market is reflected in its data. DOD Has a Detailed Process in Place to Evaluate the Availability of Tantalum and Is Taking Steps to Mitigate Risk Posed by Existing Data DOD has established a process for evaluating materials’ availability, including tantalum, as part of the National Defense Stockpile’s biennial requirements assessment process as required under the Stockpiling Act. To inform DOD’s stockpile analysis, USGS provides DLA-Strategic Materials with the data it collects and validates, and DLA-Strategic Materials takes steps to ensure that the data are reliable. Further, as the program manager for the National Defense Stockpile, DLA-Strategic Materials assesses the availability of tantalum and other materials for national defense emergency planning scenarios as required under the Stockpiling Act. For example, in DOD’s 2015 Strategic and Critical Materials Report on Stockpile Requirements, tantalum was among over 90 materials assessed for a potential shortfall. DLA-Strategic Materials is also taking additional steps to review and analyze industry data sources to address tantalum data challenges and better inform its stockpile analysis. DOD Has a Detailed Process to Assess Tantalum Availability for National Emergency Planning Scenarios DOD has a process for assessing the available supply of tantalum in selected planning scenarios as part of the National Defense Stockpile’s biennial requirements assessment process, as required under the Stockpiling Act. Prior to evaluating tantalum and other materials’ availability, DOD officials take steps to ensure that supply data used in its stockpile analysis are reliable. As discussed previously, USGS has documented procedures to validate its data and DLA-Strategic Materials officials identified additional steps they take to ensure that data are reliable, consistent with Standards for Internal Control in the Federal Government and other guidelines. For example, DLA-Strategic Materials officials said that their material specialists coordinate closely with USGS commodity and country specialists to verify data sources, such as whether production data are from USGS surveys or mining publications, and to determine how missing or incomplete data have been accounted for in its reports. Moreover, DLA-Strategic Materials officials told us they check USGS calculations and unit conversions to ensure accuracy prior to sending the data to the Institute for Defense Analyses, the federally funded research center responsible for the model used to perform the stockpile analysis. DLA-Strategic Materials officials also noted that they rely heavily on their own subject matter expertise, as well as interviews with knowledgeable industry and government officials and complementary data sources to inform their review of USGS production data and make decisions about whether to adjust tantalum figures. While DLA-Strategic Materials officials told us they are aware of limitations to USGS data, they told us they have generally relied on USGS mine data as the primary source for production data because its estimates are more conservative. Nevertheless, representatives stated that they believe the information USGS is able to collect and confirm is a basis for DOD’s stockpile recommendations. Further, DLA-Strategic Materials officials told us that USGS has the knowledge-base and resources to effectively gather and validate reliable and unbiased mine production data as part of its stated mission and responsibilities. The Stockpiling Act is intended to provide a process to mitigate dangerous and costly reliance on foreign sources of materials during national emergencies. In accordance with the Stockpiling Act, DLA- Strategic Materials reports its findings of potential shortfalls of materials in the Stockpile Report every two years. In its 2015 report, tantalum was among over 90 materials assessed for a potential shortfall. Additionally, from 2011 through 2015 DLA-Strategic Materials identified potential shortfalls for tantalum. Since 2013, it has recommended stockpiling various amounts of tantalum. For example, in its 2013 Stockpile Report, DLA-Strategic Materials reported a shortfall for tantalum of 623,307 pounds, and DLA-Strategic Materials subsequently recommended stockpiling 187,000 pounds of tantalum divided evenly over a four-year period along with other mitigation strategies to address the estimated shortfall. In May 2015, subsequent to issuing the 2015 Stockpile Report, DLA-Strategic Materials was provided with updated primary tantalum production data from USGS that revised the supply of tantalum upward, reducing the estimated shortfall. However, based on historical and consistent shortfalls as well as the United States’ reliance on foreign sources, DLA-Strategic Materials officials told us they plan to continue to seek legislative authority to stockpile tantalum. Table 3 identifies the reported tantalum shortfalls in DOD’s Stockpile Report from 2011 to 2015 and recommended stockpiling actions. DLA-Strategic Materials uses a repeatable three-step process to identify potential shortfalls for materials during national emergency scenarios. In the first step, DLA-Strategic Materials identifies materials to assess and establishes data requirements needed for the shortfall assessments. DLA-Strategic Materials monitors a “watch list” of over 160 materials, which includes tantalum, that are of interest to the defense community. DLA-Strategic Materials then narrows the watch list to a smaller number of materials using an internal ranking system where additional weight is given to risks associated with heavy reliance on foreign sources of supply and single points of failure along the supply chain, among others. In the second step, DLA-Strategic Materials determines whether material shortfalls exist in the context of national emergency scenarios. A component of this step is to estimate the global supply of tantalum available for United States civilian and defense needs. To estimate available supply, DLA-Strategic Materials and the Institute for Defense Analyses rely on country reliability assessments, as well as the specific assumptions of the national emergency scenarios, to decrement foreign production during a conflict. DLA-Strategic Materials receives inputs from the intelligence community for the country reliability assessments and officials told us they may “zero out” a country’s supply as part of their analyses if that country is deemed to be unreliable under the assumptions of the national emergency scenario. Because USGS production data is country- based, DLA-Strategic Materials can apply the assumptions of the national emergency scenario and country reliability assessments to decrement those amounts. In the third step of the process, DLA-Strategic Materials decides what mitigation strategies are appropriate to reduce any identified shortfalls, which may include recommendations to stockpile the material. For example, when a shortfall estimate is initially generated, DLA-Strategic Materials will determine whether mitigating options, such as substitution, reducing exports, or additional purchases of a material are sufficient to eliminate the shortfall. If mitigating options are not sufficient to eliminate the shortfall, the result is a net shortfall and DLA-Strategic Materials may decide to recommend a stockpiling action for that material. Figure 2 illustrates the shortfall analysis processes. DOD is Taking Steps to Mitigate Risks from Existing Data While DOD has a detailed process for evaluating the availability of tantalum and other materials, DOD, USGS, and industry officials identified several challenges that affect their ability to gather complete and reliable tantalum data. For example, the USGS reported that unlike other materials and precious metals, tantalum concentrates are not publicly traded through commodities exchanges. Rather the material is bought and sold through networks of dealers and on contract between producers and consumers, some of whom may not provide accurate statistical data. Further, according to industry officials, artisanal mining, which is difficult to quantify in USGS reporting, may account for a significant portion of global tantalum production. In addition, industry officials commented that some artisanal mining production may enter the supply chain labeled as scrap to disguise its origin. While DLA-Strategic Materials officials told us that some of these concerns are not unique to tantalum, they said the lack of transparent tantalum supply data affects their ability to collect complete information to inform the stockpile analysis. Given the above challenges, officials from DOD, USGS, and industry acknowledged that tantalum has an “opaque” supply chain, making it difficult to collect more accurate data. Recognizing these challenges, DLA-Strategic Materials is taking additional steps to inform its analysis of tantalum availability. For example, DLA-Strategic Materials has requested that the Institute for Defense Analyses perform additional work on materials of interest. In 2013, the Institute for Defense Analyses completed a review that identified weaknesses in the supply chain for processed tantalum used in some defense applications. Further the Stockpiling Act permits DOD to identify materials requiring further study. According to the 2015 Stockpile Report, significant reliance on foreign sources or a single point of failure is the main basis for additional study, but other reasons, such as a net shortfall or the potential for supply chain disruption can also result in additional research into a material. Under this authority, DLA-Strategic Materials said they are conducting research into whether certain materials, such as tantalum, could be recovered through recycling of manufactured materials. Moreover, citing concerns about differences in tantalum data reported by USGS and industry sources, DOD’s 2015 Stockpile Report noted that DLA-Strategic Materials will be comparing data and performing additional analyses on these two sources in advance of their 2017 report. DOD’s efforts to review industry data were ongoing during our review, but officials noted several limitations to TIC data. For example, as discussed above, the TIC’s data is not reported by country, which limits its use in support of the National Defense Stockpile analyses. Further, DLA- Strategic Materials officials told us they are uncertain about what portion of the industry is covered by TIC’s reporting members, or how accurate the data are since there may be incentives for members to over- or under- report. Given these concerns, DLA-Strategic Materials officials identified several potential next steps they are considering upon completing their review of the TIC data. For example, officials said they could use the data as a complementary source to inform their review of USGS mine production data, similar to DLA-Strategic Materials’ use of Roskill Information Services market report data. Additionally, officials told us that given the challenges of collecting accurate and reliable tantalum data, they may consider running two sets of analyses for tantalum in future stockpile reports—one using only USGS data and another that includes industry data estimates. In addition to their review of TIC’s data, DLA-Strategic Materials is also in the process of compiling its own estimates for the supply of tantalum derived from slag, scrap, and recycled tantalum based on data and interviews with various sources, including USGS, Roskill Information Services, and tantalum processing companies. DLA-Strategic Materials officials told us that they plan to use these estimates to inform their 2017 analysis since the United States mostly imports these secondary types of tantalum, which are not included in USGS figures. Agency Comments We are not making recommendations in this report. DOD and the Department of the Interior were provided copies of the draft report. Neither DOD nor the Department of the Interior provided written comments. The Department of the Interior provided technical comments that were incorporated, as appropriate. We are sending copies of this report to the appropriate congressional committees, the Secretary of Defense, the Under Secretary of Defense for Acquisition, Technology, and Logistics, the Secretary of the Interior, the Director of the United States Geological Survey, the Secretary of Commerce, the Chairman of the Securities and Exchange Commission, and other interested parties. In addition, the report is available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at (202) 512-4841 or at [email protected]. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made key contributions to this report are listed in appendix II. Appendix I: Objectives, Scope and Methodology The House Armed Services Committee Report on a bill for the National Defense Authorization Act for Fiscal Year 2016 included a provision for GAO to examine the global tantalum supply chain. This report addresses (1) how data on tantalum supply reported by government sources differ from data reported by industry and (2) the extent to which the Department of Defense (DOD) has assessed the availability of tantalum during national defense emergency planning scenarios. To compare available tantalum data reported by government sources to industry sources, we met with United States Geological Survey (USGS) officials, Defense Logistics Agency Strategic Materials (DLA-Strategic Materials) and other DOD officials, and tantalum industry officials from the Tantalum-Niobium International Study Center (TIC) to identify tantalum data sources and collection methods, and to discuss the availability of tantalum supply data. For the purpose of our review we focused on USGS tantalum production estimates, which are DOD’s primary source for estimated tantalum production in the 2015 Strategic and Critical Materials Report on Stockpile Requirements (Stockpile Report). We also reviewed industry data collected by the TIC, an international, non-profit association founded in 1974 with about 90 member companies worldwide. Some of the TIC’s data are publically available, and the TIC is one of several industry data sources used by DOD to inform their analysis in the Stockpile Report. We obtained and analyzed the USGS tantalum production data that DOD used in developing the 2015 Strategic and Critical Materials Report on Stockpile Requirements as well as industry data to identify similarities and differences between the data sources and the methods of compiling estimates. We assessed the reliability of USGS data by (1) reviewing existing information about the data and the system that produced them, and (2) interviewing agency officials knowledgeable about the data. USGS takes steps to collect and confirm production estimates for use in their publications such as the Mineral Commodity Summaries and the Minerals Yearbook. Based on our review of the data and interviews with USGS and DOD we determined that USGS data were sufficiently reliable for our purposes. To assess the processes used by USGS to ensure the reliability of its data, we obtained USGS Fundamental Science Practices and Statistical Standards and discussed how those practices are implemented in the reviews of data to be published in USGS publications. In addition, due to differences in data collection and reporting methods, USGS and industry tantalum data are not directly comparable and, therefore, we do not compare supply estimates from these sources for the purpose of this report. We use the information to demonstrate differences between industry and USGS data and collection methods. For industry, we met with officials from the TIC, Roskill Information Services Ltd., Commerce Resources Corporation, and Global Advanced Metals. To the extent possible we confirmed types of information provided by the TIC by comparing it to published reports. We examined quarterly reports showing tantalum supply and discussed the types of data collected and reported and the processes used to assess reliability. However, based on our review of the process for collecting the data and assessing the reliability of the data provided by the members and on interviews with industry officials, we determined that the industry supply numbers were self-reported, but not verified, and, therefore, not sufficiently reliable for direct comparison to USGS data. Therefore, we did not compare USGS and industry figures for this report. To assess the extent to which DOD has evaluated the availability of tantalum during national defense emergency planning scenarios, we obtained and analyzed information in reports prepared by DLA-Strategic Materials for DOD’s Office of the Undersecretary of Defense for Acquisition, Technology, and Logistics including the Stockpile Reports from 2011 through 2015 and the Annual Industrial Capabilities Reports to Congress. We met with officials from DLA-Strategic Materials and the Institute for Defense Analyses, the federally funded research center that assists with the stockpile analysis, to determine what steps were taken to assess the reliability of data used in the assessment process and mitigate risk. We obtained and examined DLA-Strategic Materials’ proposed Annual Materials Plans for fiscal years 2015 through 2017. We reviewed relevant legislation, regulations, and policy including: the Strategic and Critical Materials Stock Piling Act (50 U.S.C. § 98 et seq.); provisions of the Dodd-Frank Wall Street Reform and Consumer Protection Act (Pub. L. No. 111-203 §1502; policy documents from the Office of the Under Secretary of Defense for Acquisition, Technology, and Logistics concerning determining potential shortfalls and the organizational structure for executing the provisions of the Stockpiling Act. We reviewed DLA-Strategic Materials data used in the assessments and documentation of the steps they took to assure data reliability. To determine the extent to which DOD has assessed the availability of tantalum during specific national defense emergency scenarios as part of the National Defense Stockpile’s biennial requirements assessment process, we reviewed the interagency support agreement between USGS and DLA-Strategic Materials. To understand the data and support USGS provides DLA-Strategic Materials, we reviewed the worldwide USGS production data provided to DLA-Strategic Materials for its assessment. We reviewed DOD reports to understand the analyses DOD had conducted to identify risks related to the availability of tantalum. Further, we met with officials from USGS, DLA-Strategic Materials, and with analysts from the Institute for Defense Analyses. We discussed sources of tantalum supply with industry representatives to determine what other sources of tantalum supply data are available and the general reliability of such data. We obtained documentation on the model DLA-Strategic Materials uses to conduct its stockpile analysis including the categories of information considered by the model to determine potential shortfalls and provide support for recommending stockpiling amounts. In addition, we interviewed DOD officials from the Under Secretary of Defense for Acquisition, Technology, and Logistics and DLA-Strategic Materials about actions underway to increase the accuracy of the data reported in the Stockpile Report. We obtained and analyzed such documentation as was available about proposed and ongoing actions to improve the reliability and accuracy of tantalum data. We assessed DOD’s policies, procedures, and practices against criteria in applicable statutes, Standards for Internal Control in the Federal Government, and GAO’s Framework for Assessing the Acquisition Functions at Federal Agencies. We determined whether or not documentation for the determination of reliability of the data used in the DLA-Strategic Materials model was provided but did not assess the adequacy of the information provided. We interviewed officials from USGS and DLA-Strategic Materials to clarify the issues. We concluded that the data had been collected and used in accordance with principles developed by USGS and DLA-Strategic Materials and were sufficiently reliable for the purpose of obtaining an understanding of the process used to collect the data, the model for determining potential shortfalls of material, and the reports produced by DLA-Strategic Materials. We conducted this performance audit from August 2015 to March 2016 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. Appendix II: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the contact named above, Penny Berrier, Assistant Director; Marie Ahearn; Pedro Almoguera; John Beauchamp; Stephanie Gustafson; Jeffrey Harner; Mathew Jacobs; Carol Mebane; Jean McSween; Meghan Perez; Katrina Pekar-Carpenter; and Roxanna Sun made significant contributions to this review.
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Summary: The United States relies on foreign mine production of tantalum, a corrosion-resistant metal that is used in commercial and defense applications. Having reliable information on the global supply of tantalum is important for defense planning, particularly in determining if it is necessary to stockpile in case of future shortages. The House Armed Services Committee Report on a bill for the National Defense Authorization Act for Fiscal Year 2016 included a provision for GAO to examine the global tantalum supply chain, with a focus on why data reported by the government and by industry vary. This report addresses (1) how tantalum supply data reported by government sources differ from industry data, and (2) the extent to which DOD has assessed the availability of tantalum during emergency planning scenarios. GAO reviewed data compiled by the USGS-DOD's primary source for tantalum production data-and by a tantalum industry organization that makes its information publicly available. GAO interviewed DOD and industry officials about the reporting and collection methods for the data; examined the data DOD uses to determine potential shortfalls of materials, including data for its biennial Strategic and Critical Materials Reports on Stockpile Requirements; and discussed with DOD officials steps they have taken to assess the reliability of the data used in the analyses. Data published by government and industry on the global supply of tantalum vary due to differences in forms of tantalum reported and data collection methods. For example, government data prepared by the United States Geological Survey (USGS) on tantalum production reports information on tantalum ore from mining. Industry data GAO obtained from the Tantalum-Niobium International Study Center includes additional forms of processed tantalum, such as synthetic concentrates and slags and recycled tantalum materials. In collecting data, the USGS employs specialists to estimate production data by country of origin for government agencies, including the Department of Defense (DOD), by conducting annual surveys of foreign governments on mine production and relying on country specialists. In contrast, industry data compiled by the Tantalum-Niobium International Study Center is based on aggregated data voluntarily reported by its member companies as a service to those members rather than in support of the federal government. The table below summarizes the differences in USGS and industry data. Source: GAO analysis of United States Geological Survey and tantalum industry data and collection methods. I GAO-16-335 DOD assesses the availability of tantalum, among other materials, for selected planning scenarios for the National Defense Stockpile's biennial assessment process. Further, DOD takes steps to help ensure that tantalum supply data used in its stockpile analyses are reliable. For example, USGS provides the Defense Logistics Agency-Strategic Materials (DLA-Strategic Materials)-the stockpile program manager-with the data it collects and validates. Consistent with internal control standards, DLA-Strategic Materials officials said they then verify sources and check calculations to ensure that data are reliable before conducting their stockpile analyses. Since 2013, DLA-Strategic Materials has identified potential shortfalls for tantalum and recommended stockpiling. Given DLA-Strategic Materials' interest in using the most accurate information available, it is taking additional steps to review and analyze existing industry tantalum data sources to better inform its stockpile analyses.
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Summarize: A youthful yet chilling voice belonging to a nine-year-old boy calls for the reclamation of Islamic authority around the world. His father, who proudly shared the video on social media, is a devout supporter of Hizb-ut-Tahrir - the controversial Muslim group which organised protests in western Sydney last week against 'government aggression'. In the footage, seen by Daily Mail Australia, the child stands before a Sydney gathering to deliver a sermon about sacrifice and victory of the Muslims. The boy addresses his fellow 'brothers and sisters' as he chants passages from the Koran and preaches in his Australian accent. A video of the nine-year-old preaching to his 'brothers and sisters' in Sydney has been posted by his father. Another video shows the boy, 7, in 2012 addressing protesters at a rally in Sydney. 'Today we see the same sacrifices in the Muslims and the mujaheens in Syria and Afghanistan and in Palestine and all around the Muslim lands who know that only Islam can bring mercy to mankind,' the boy preaches. 'Let us take this opportunity, in this month (Ramadan) … by sacrificing everything for the sake of Allah. 'Let us pray that Allah will release the calamities to this Ummah and brings back Islam as an authority and as life to be an example to the humanity and send mercy to mankind.' His father, who Daily Mail Australia has chosen not to identify, posted the footage on Facebook in July - during Ramadan - which was then shared a number of times. More videos of the boy delivering a sermon were posted by his father on Facebook in 2013. The father of the boy is a supporter of controversial Muslim group Hizb-ut-Tahrir. His father, who also shows his support for Hizb-ut-Tahrir online, has also posted similar videos of the boy in previous years. The child stood before a Sydney protest in support of the Syrian uprisings in 2012, at the age of seven, where he delivered another speech. 'We witness that our brothers and sisters in Syria, in Afghanistan, in Palestine and other countries has been oppressed by corrupt tyrant rulers,' the boy said. 'They sacrifice their lives and they sacrifice their families for the sake of Allah. May Allah help our Ummah to work day and night to establish the (caliphate) that will protect and free all Muslims from oppression.' On the father's Facebook page, disturbing images of soldiers brandishing Islamic flags and children holding machine guns can be seen. He also posted an image which declares: 'Say no to democracy.' It also appears he attended a Sydney rally organised by Hizb-ut-Tahrir last Thursday night, following a series of pre-dawn anti-terrorism raids - the largest counter-terrorism operation in Australian history. Daily Mail Australia has contacted the nine-year-old boy's father for comment. The operation last week saw 800 police officers carry out 25 search warrants across Sydney's north-west and in three Brisbane suburbs. About 200 protesters took to the streets to defend their rights as Islamic Australians in Lakemba, western Sydney, with signs reading 'terror raids cannot break the spirit of Muslims' and'stop terrorising Muslims'. In August, a shocking photo of a young boy holding a head of a decapitated soldier in Syria emerged on social media when it was posted by his father and convicted Australian terrorist, Khaled Sharrouf. It was accompanied by a photo of Sharrouf himself holding the same head, written with the words: 'What a head', and another of him and three young boys brandishing rifles. Since departing Australia, Sharrouf has been using his social media pages to boast about the Islamic State group. Sharrouf is wanted by the Australian Federal Police for alleged terrorist activities in the Middle East after he fled the country in December on his brother's passport
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Summary: The boy stood before a Sydney gathering as he speaks about sacrifice and victory of the Muslims. He preaches in his Australian accent and chants passages from the Koran. The video was posted online by the boy's father - a supporter of controversial Muslim group Hizb-ut-Tahrir. When the boy was seven-years-old, he also addressed protesters in Sydney who gathered in support of the uprisings in Syria. Hizb-ut-Tahrir is known to have organised a protest in western Sydney last week following a series of pre-dawn anti-terrorism raids by police.
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Summarize: Nay, if the gentle spirit of moving words Can no way change you to a milder form, I'll woo you, like a soldier, at arms' end, And love you 'gainst the nature of love, force you. --Two Gentlemen of Verona The apartment to which the Lady Rowena had been introduced was fitted up with some rude attempts at ornament and magnificence, and her being placed there might be considered as a peculiar mark of respect not offered to the other prisoners. But the wife of Front-de-Boeuf, for whom it had been originally furnished, was long dead, and decay and neglect had impaired the few ornaments with which her taste had adorned it. The tapestry hung down from the walls in many places, and in others was tarnished and faded under the effects of the sun, or tattered and decayed by age. Desolate, however, as it was, this was the apartment of the castle which had been judged most fitting for the accommodation of the Saxon heiress; and here she was left to meditate upon her fate, until the actors in this nefarious drama had arranged the several parts which each of them was to perform. This had been settled in a council held by Front-de-Boeuf, De Bracy, and the Templar, in which, after a long and warm debate concerning the several advantages which each insisted upon deriving from his peculiar share in this audacious enterprise, they had at length determined the fate of their unhappy prisoners. It was about the hour of noon, therefore, when De Bracy, for whose advantage the expedition had been first planned, appeared to prosecute his views upon the hand and possessions of the Lady Rowena. The interval had not entirely been bestowed in holding council with his confederates, for De Bracy had found leisure to decorate his person with all the foppery of the times. His green cassock and vizard were now flung aside. His long luxuriant hair was trained to flow in quaint tresses down his richly furred cloak. His beard was closely shaved, his doublet reached to the middle of his leg, and the girdle which secured it, and at the same time supported his ponderous sword, was embroidered and embossed with gold work. We have already noticed the extravagant fashion of the shoes at this period, and the points of Maurice de Bracy's might have challenged the prize of extravagance with the gayest, being turned up and twisted like the horns of a ram. Such was the dress of a gallant of the period; and, in the present instance, that effect was aided by the handsome person and good demeanour of the wearer, whose manners partook alike of the grace of a courtier, and the frankness of a soldier. He saluted Rowena by doffing his velvet bonnet, garnished with a golden broach, representing St Michael trampling down the Prince of Evil. With this, he gently motioned the lady to a seat; and, as she still retained her standing posture, the knight ungloved his right hand, and motioned to conduct her thither. But Rowena declined, by her gesture, the proffered compliment, and replied, "If I be in the presence of my jailor, Sir Knight--nor will circumstances allow me to think otherwise--it best becomes his prisoner to remain standing till she learns her doom." "Alas! fair Rowena," returned De Bracy, "you are in presence of your captive, not your jailor; and it is from your fair eyes that De Bracy must receive that doom which you fondly expect from him." "I know you not, sir," said the lady, drawing herself up with all the pride of offended rank and beauty; "I know you not--and the insolent familiarity with which you apply to me the jargon of a troubadour, forms no apology for the violence of a robber." "To thyself, fair maid," answered De Bracy, in his former tone--"to thine own charms be ascribed whate'er I have done which passed the respect due to her, whom I have chosen queen of my heart, and lodestar of my eyes." "I repeat to you, Sir Knight, that I know you not, and that no man wearing chain and spurs ought thus to intrude himself upon the presence of an unprotected lady." "That I am unknown to you," said De Bracy, "is indeed my misfortune; yet let me hope that De Bracy's name has not been always unspoken, when minstrels or heralds have praised deeds of chivalry, whether in the lists or in the battle-field." "To heralds and to minstrels, then, leave thy praise, Sir Knight," replied Rowena, "more suiting for their mouths than for thine own; and tell me which of them shall record in song, or in book of tourney, the memorable conquest of this night, a conquest obtained over an old man, followed by a few timid hinds; and its booty, an unfortunate maiden, transported against her will to the castle of a robber?" "You are unjust, Lady Rowena," said the knight, biting his lips in some confusion, and speaking in a tone more natural to him than that of affected gallantry, which he had at first adopted; "yourself free from passion, you can allow no excuse for the frenzy of another, although caused by your own beauty." "I pray you, Sir Knight," said Rowena, "to cease a language so commonly used by strolling minstrels, that it becomes not the mouth of knights or nobles. Certes, you constrain me to sit down, since you enter upon such commonplace terms, of which each vile crowder hath a stock that might last from hence to Christmas." "Proud damsel," said De Bracy, incensed at finding his gallant style procured him nothing but contempt--"proud damsel, thou shalt be as proudly encountered. Know then, that I have supported my pretensions to your hand in the way that best suited thy character. It is meeter for thy humour to be wooed with bow and bill, than in set terms, and in courtly language." "Courtesy of tongue," said Rowena, "when it is used to veil churlishness of deed, is but a knight's girdle around the breast of a base clown. I wonder not that the restraint appears to gall you--more it were for your honour to have retained the dress and language of an outlaw, than to veil the deeds of one under an affectation of gentle language and demeanour." "You counsel well, lady," said the Norman; "and in the bold language which best justifies bold action I tell thee, thou shalt never leave this castle, or thou shalt leave it as Maurice de Bracy's wife. I am not wont to be baffled in my enterprises, nor needs a Norman noble scrupulously to vindicate his conduct to the Saxon maiden whom he distinguishes by the offer of his hand. Thou art proud, Rowena, and thou art the fitter to be my wife. By what other means couldst thou be raised to high honour and to princely place, saving by my alliance? How else wouldst thou escape from the mean precincts of a country grange, where Saxons herd with the swine which form their wealth, to take thy seat, honoured as thou shouldst be, and shalt be, amid all in England that is distinguished by beauty, or dignified by power?" "Sir Knight," replied Rowena, "the grange which you contemn hath been my shelter from infancy; and, trust me, when I leave it--should that day ever arrive--it shall be with one who has not learnt to despise the dwelling and manners in which I have been brought up." "I guess your meaning, lady," said De Bracy, "though you may think it lies too obscure for my apprehension. But dream not, that Richard Coeur de Lion will ever resume his throne, far less that Wilfred of Ivanhoe, his minion, will ever lead thee to his footstool, to be there welcomed as the bride of a favourite. Another suitor might feel jealousy while he touched this string; but my firm purpose cannot be changed by a passion so childish and so hopeless. Know, lady, that this rival is in my power, and that it rests but with me to betray the secret of his being within the castle to Front-de-Boeuf, whose jealousy will be more fatal than mine." "Wilfred here?" said Rowena, in disdain; "that is as true as that Front-de-Boeuf is his rival." De Bracy looked at her steadily for an instant. "Wert thou really ignorant of this?" said he; "didst thou not know that Wilfred of Ivanhoe travelled in the litter of the Jew?--a meet conveyance for the crusader, whose doughty arm was to reconquer the Holy Sepulchre!" And he laughed scornfully. "And if he is here," said Rowena, compelling herself to a tone of indifference, though trembling with an agony of apprehension which she could not suppress, "in what is he the rival of Front-de-Boeuf? or what has he to fear beyond a short imprisonment, and an honourable ransom, according to the use of chivalry?" "Rowena," said De Bracy, "art thou, too, deceived by the common error of thy sex, who think there can be no rivalry but that respecting their own charms? Knowest thou not there is a jealousy of ambition and of wealth, as well as of love; and that this our host, Front-de-Boeuf, will push from his road him who opposes his claim to the fair barony of Ivanhoe, as readily, eagerly, and unscrupulously, as if he were preferred to him by some blue-eyed damsel? But smile on my suit, lady, and the wounded champion shall have nothing to fear from Front-de-Boeuf, whom else thou mayst mourn for, as in the hands of one who has never shown compassion." "Save him, for the love of Heaven!" said Rowena, her firmness giving way under terror for her lover's impending fate. "I can--I will--it is my purpose," said De Bracy; "for, when Rowena consents to be the bride of De Bracy, who is it shall dare to put forth a violent hand upon her kinsman--the son of her guardian--the companion of her youth? But it is thy love must buy his protection. I am not romantic fool enough to further the fortune, or avert the fate, of one who is likely to be a successful obstacle between me and my wishes. Use thine influence with me in his behalf, and he is safe,--refuse to employ it, Wilfred dies, and thou thyself art not the nearer to freedom." "Thy language," answered Rowena, "hath in its indifferent bluntness something which cannot be reconciled with the horrors it seems to express. I believe not that thy purpose is so wicked, or thy power so great." "Flatter thyself, then, with that belief," said De Bracy, "until time shall prove it false. Thy lover lies wounded in this castle--thy preferred lover. He is a bar betwixt Front-de-Boeuf and that which Front-de-Boeuf loves better than either ambition or beauty. What will it cost beyond the blow of a poniard, or the thrust of a javelin, to silence his opposition for ever? Nay, were Front-de-Boeuf afraid to justify a deed so open, let the leech but give his patient a wrong draught--let the chamberlain, or the nurse who tends him, but pluck the pillow from his head, and Wilfred in his present condition, is sped without the effusion of blood. Cedric also--" "And Cedric also," said Rowena, repeating his words; "my noble--my generous guardian! I deserved the evil I have encountered, for forgetting his fate even in that of his son!" "Cedric's fate also depends upon thy determination," said De Bracy; "and I leave thee to form it." Hitherto, Rowena had sustained her part in this trying scene with undismayed courage, but it was because she had not considered the danger as serious and imminent. Her disposition was naturally that which physiognomists consider as proper to fair complexions, mild, timid, and gentle; but it had been tempered, and, as it were, hardened, by the circumstances of her education. Accustomed to see the will of all, even of Cedric himself, (sufficiently arbitrary with others,) give way before her wishes, she had acquired that sort of courage and self-confidence which arises from the habitual and constant deference of the circle in which we move. She could scarce conceive the possibility of her will being opposed, far less that of its being treated with total disregard. Her haughtiness and habit of domination was, therefore, a fictitious character, induced over that which was natural to her, and it deserted her when her eyes were opened to the extent of her own danger, as well as that of her lover and her guardian; and when she found her will, the slightest expression of which was wont to command respect and attention, now placed in opposition to that of a man of a strong, fierce, and determined mind, who possessed the advantage over her, and was resolved to use it, she quailed before him. After casting her eyes around, as if to look for the aid which was nowhere to be found, and after a few broken interjections, she raised her hands to heaven, and burst into a passion of uncontrolled vexation and sorrow. It was impossible to see so beautiful a creature in such extremity without feeling for her, and De Bracy was not unmoved, though he was yet more embarrassed than touched. He had, in truth, gone too far to recede; and yet, in Rowena's present condition, she could not be acted on either by argument or threats. He paced the apartment to and fro, now vainly exhorting the terrified maiden to compose herself, now hesitating concerning his own line of conduct. If, thought he, I should be moved by the tears and sorrow of this disconsolate damsel, what should I reap but the loss of these fair hopes for which I have encountered so much risk, and the ridicule of Prince John and his jovial comrades? "And yet," he said to himself, "I feel myself ill framed for the part which I am playing. I cannot look on so fair a face while it is disturbed with agony, or on those eyes when they are drowned in tears. I would she had retained her original haughtiness of disposition, or that I had a larger share of Front-de-Boeuf's thrice-tempered hardness of heart!" Agitated by these thoughts, he could only bid the unfortunate Rowena be comforted, and assure her, that as yet she had no reason for the excess of despair to which she was now giving way. But in this task of consolation De Bracy was interrupted by the horn, "hoarse-winded blowing far and keen," which had at the same time alarmed the other inmates of the castle, and interrupted their several plans of avarice and of license. Of them all, perhaps, De Bracy least regretted the interruption; for his conference with the Lady Rowena had arrived at a point, where he found it equally difficult to prosecute or to resign his enterprise. And here we cannot but think it necessary to offer some better proof than the incidents of an idle tale, to vindicate the melancholy representation of manners which has been just laid before the reader. It is grievous to think that those valiant barons, to whose stand against the crown the liberties of England were indebted for their existence, should themselves have been such dreadful oppressors, and capable of excesses contrary not only to the laws of England, but to those of nature and humanity. But, alas! we have only to extract from the industrious Henry one of those numerous passages which he has collected from contemporary historians, to prove that fiction itself can hardly reach the dark reality of the horrors of the period. The description given by the author of the Saxon Chronicle of the cruelties exercised in the reign of King Stephen by the great barons and lords of castles, who were all Normans, affords a strong proof of the excesses of which they were capable when their passions were inflamed. "They grievously oppressed the poor people by building castles; and when they were built, they filled them with wicked men, or rather devils, who seized both men and women who they imagined had any money, threw them into prison, and put them to more cruel tortures than the martyrs ever endured. They suffocated some in mud, and suspended others by the feet, or the head, or the thumbs, kindling fires below them. They squeezed the heads of some with knotted cords till they pierced their brains, while they threw others into dungeons swarming with serpents, snakes, and toads." But it would be cruel to put the reader to the pain of perusing the remainder of this description. [29] As another instance of these bitter fruits of conquest, and perhaps the strongest that can be quoted, we may mention, that the Princess Matilda, though a daughter of the King of Scotland, and afterwards both Queen of England, niece to Edgar Atheling, and mother to the Empress of Germany, the daughter, the wife, and the mother of monarchs, was obliged, during her early residence for education in England, to assume the veil of a nun, as the only means of escaping the licentious pursuit of the Norman nobles. This excuse she stated before a great council of the clergy of England, as the sole reason for her having taken the religious habit. The assembled clergy admitted the validity of the plea, and the notoriety of the circumstances upon which it was founded; giving thus an indubitable and most remarkable testimony to the existence of that disgraceful license by which that age was stained. It was a matter of public knowledge, they said, that after the conquest of King William, his Norman followers, elated by so great a victory, acknowledged no law but their own wicked pleasure, and not only despoiled the conquered Saxons of their lands and their goods, but invaded the honour of their wives and of their daughters with the most unbridled license; and hence it was then common for matrons and maidens of noble families to assume the veil, and take shelter in convents, not as called thither by the vocation of God, but solely to preserve their honour from the unbridled wickedness of man. Such and so licentious were the times, as announced by the public declaration of the assembled clergy, recorded by Eadmer; and we need add nothing more to vindicate the probability of the scenes which we have detailed, and are about to detail, upon the more apocryphal authority of the Wardour MS.
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Summary: This chapter's quote is from Shakespeare's Two Gentlemen of Verona, which Scott also uses at the beginning of Chapter 11. These lines are from Act V, Scene 4, when Proteus threatens the female lead, Silvia, with rape.At around noon the following day, De Bracy appears in Rowena's rooms.He has taken the time to get all dressed up to impress her.De Bracy tries to woo Rowena, but she isn't having it. He has taken her and her father captive, after all.De Bracy loses his temper. He tells Rowena that she will either leave Torquilstone as his wife or not at all. De Bracy has no idea how she can refuse him - to his mind he's a Norman and rich, while she's a just a lowly Saxon.De Bracy assures her that Wilfred of Ivanhoe will always be penniless because his friend King Richard will never come back to England. If Rowena will marry De Bracy, he'll make sure that Front-de-Boeuf doesn't kill Ivanhoe.After all, Ivanhoe is Front-de-Boeuf's competitor for the castle of Torquilstone, so Front-de-Boeuf has good reason to assassinate him.If Rowena does what De Bracy tells her, then De Bracy will rescue both Ivanhoe and Cedric. How can she refuse? Rowena starts to cry. De Bracy feels embarrassed. He can't go back on his plan now, but he can't talk to her while she's crying.As De Bracy tries to calm Rowena down, he hears the three horn blasts at the gate.De Bracy is relieved to have an excuse to leave the room.The narrator jumps in to say that it's weird that the same people who went on to help protect English liberty started out so oppressive.He lists many other examples of the cruelties of the Normans. Basically, the 11th and 12th centuries were not a great time in English history.
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Summarize: Story highlights Naps on the weekend don't really make up for pulling droopy-eyed all-nighters The widely held notion that you can pay back a "sleep debt" may be a myth Mice forced to sleep like shift workers had heavy damage in a key group of nerve cells Scientists want to study deceased shift workers to see if they suffered similar damage Are you a truck driver or shift worker planning to catch up on some sleep this weekend? Cramming in extra hours of shut-eye may not make up for those lost pulling all-nighters, new research indicates. The damage may already be done -- brain damage, that is, said neuroscientist Sigrid Veasey from the University of Pennsylvania. The widely held idea that you can pay back a sizeable "sleep debt" with long naps later on seems to be a myth, she said in a study published this week in the Journal of Neuroscience. Long-term sleep deprivation saps the brain of power even after days of recovery sleep, Veasey said. And that could be a sign of lasting brain injury. Veasey and her colleagues at the University of Pennsylvania medical school wanted to find out, so, they put laboratory mice on a wonky sleep schedule that mirrors that of shift workers. They let them snooze, then woke them up for short periods and for long ones. Then the scientists looked at their brains -- more specifically, at a bundle of nerve cells they say is associated with alertness and cognitive function, the locus coeruleus. They found damage and lots of it. "The mice lose 25% of these neurons," Veasey said. This is how the scientists think it happened. When the mice lost a little sleep, nerve cells reacted by making more of a protein, called sirtuin type 3, to energize and protect them. But when losing sleep became a habit, that reaction shut down. After just a few days of "shift work" sleep, the cells start dying off at an accelerated pace. The discovery that long-term sleep loss can result in a loss of brain cells is a first, Veasey said. "No one really thought that the brain could be irreversibly injured from sleep loss," she said. That has now changed. More work needs to be done on humans, she said. And her group is planning to study deceased shift workers to see if they have the same kind of nerve damage. They hope their research will result in medicines that will help people working odd hours cope with the consequences of irregular sleep. Image copyright Thinkstock Image caption Sleep loss is bad for mood and mental processing Sleep loss may be more serious than previously thought, causing a permanent loss of brain cells, research suggests. In mice, prolonged lack of sleep led to 25% of certain brain cells dying, according to a study in The Journal of Neuroscience. If the same is true in humans, it may be futile to try to catch up on missed sleep, say US scientists. They think it may one day be possible to develop a drug to protect the brain from the side-effects of lost sleep. The study, published in The Journal of Neuroscience, looked at lab mice that were kept awake to replicate the kind of sleep loss common in modern life, through night shifts or long hours in the office. We now have evidence that sleep loss can lead to irreversible injury Prof Sigrid Veasey, University of Pennsylvania School of Medicine A team at the University of Pennsylvania School of Medicine studied certain brain cells which are involved in keeping the brain alert. After several days of sleep patterns similar to those followed by night workers - three days of night shifts with only four to five hours sleep in 24 hours - the mice lost 25% of the brain cells, in part of the brain stem. The researchers say this is the first evidence that sleep loss can lead to a loss of brain cells. But they add that more work needs to be done to find out if people who miss out on sleep might also be at risk of permanent damage. Prof Sigrid Veasey of the Center for Sleep and Circadian Neurobiology, told BBC News: "We now have evidence that sleep loss can lead to irreversible injury. "This might be in a simple animal but this suggests to us that we are going to have to look very carefully in humans." She said the next step was to examine the brains of shift workers after death for evidence of any loss of brain cells. Image copyright Getty Images Image caption Shift work may have permanent health implications In the long-term, they think it might be possible to develop a medicine that protects brain cells, by boosting a natural chemical involved in sleep recovery. Prof Hugh Piggins of the University of Manchester said the experiment indicated in a mouse model of sleep deprivation what might go wrong in the human brain. "The authors draw parallels with night shift work in humans and suggest how chronic sleep deprivation could adversely affect not only our physical, but also our mental health," he said. "This possibility will need to be tested by a lot more research. Nonetheless, it is consistent with many recent reports of importance of circadian clocks and sleep cycles for optimal well-being. "
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Summary: A lack of shut-eye may not just leave you feeling groggy-it could seriously injure your brain. A new study of mice has found 25% of brain cells died off after prolonged sleep loss, meant to duplicate night shifts or long hours at the office-the first evidence of its kind and a scary find if it applies to humans. "We now have evidence that sleep loss can lead to irreversible injury," a researcher told the BBC, noting that when the mice lost a little sleep, they were protected by an energizing protein, but brain cells began to die off when sleep loss became a habit. "This suggests to us that we are going to have to look very carefully in humans." If you don't get the recommended six to eight hours of sleep per night but substitute extra hours of snores later to catch up-like on weekends-that may not make up the difference either, researchers say, as the damage may already be done, CNN notes. While more research is needed-the group plans to study the brains of shift workers after death to look for nerve damage-there may also be a silver lining to look forward to: The team notes its research may eventually lead to medicines that help people deal with irregular sleep patterns caused by odd work hours.
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Summarize: ****Update, Jan. 5, 2010: The reports and unconfirmed rumors in the post below have since been updated in this confirmed, updated report on Beyoncé’s performance. Earlier reports which suggested that Saif al-Islam Gaddafi, one of the sons of Libyan leader Muammar Gaddafi, had hosted the event were incorrect — according to the New York Post, this year’s party was hosted by his brother Hannibal Gaddafi. As noted below, Saif Gaddafi was in New Zealand over New Year’s. Reports cited below saying he hosted last year’s party, where Mariah Carey performed, were also incorrect; New York Daily News columnists Rush & Molloy subsequently reported that Saif Gaddafi was in Asia at the time of the event, and that the party was actually hosted by his brother, Moatessem-Billah Khadafy, which is not the brother who hosted this year’s event. The full, confirmed, updated version of this story may be found here; the rest of this post (below) has been left as it was published on Jan. 3, 2010 to keep the record intact. Did Beyoncé perform on New Year’s Eve for the family of Libyan leader Colonel Muammar Gaddafi? The mega-star and her mega-star husband, Jay-Z, are confirmed to have spent their New Year’s holiday in St. Barth thanks to numerous sightings, tweets/twitpics and paparazzi photographs over the last few days of 2009. The UK’s Daily Mail ran pictures of Beyoncé, including the one above, performing at the exclusive Nikki Beach club, as well as after in a group that included her husband Jay-Z, Usher, and Jon Bon Jovi (Usher did the countdown to New Year’s). Confirmations of a show-stopping New Year’s Eve performance came via Twitter as well, from Asylum Records CEO Todd Moscowitz and Swedish supermodel Victoria Silvstedt, who also snapped a picture. So where do the Gaddafis come in? Well, last year they made waves with a similar party: Mariah Carey was reportedly paid $1 million to sing just four songs at the Nikki Beach Club for a party reportedly thrown by the family of the Libyan leader. Jay-Z and Timbaland also reportedly performed. (NB: Those reports were widespread, but not confirmed anywhere that I found.) Saif Gaddafi, who acts diplomatically on behalf of Libya and runs with playboys and powerbrokers, was in the news earlier this year regarding the release of Libyan Abdel Basset Ali al-Megrahi, better known as the Lockerbie bomber, who had been convicted of placing the bomb that brought down Pan Am 103 in December 1988. Gaddafi fils facilitated the release and flew the bomber home to Libya on his father’s private jet, and caused a stir after with comments that the deal may have been linked to trade talks (he pulled back from those comments in a NYT op-ed, in which he also pushed back against reports that Megrahi had received a “hero’s welcome” in Libya; this photo of him with Megrahi with their hands held high as they emerged from the plane indicates otherwise. It’s from the album, “Another success for Libya“). Saif Gaddafi has hosted a swank New Year’s Eve party on Nikki Beach for the past few years, according to sources familiar with the St. Barth’s social scene, including last year’s event headlined by Carey, where Jay-Z also performed and Beyoncé was also in attendance. Though there were reports that he himself was in New Zealand through the New Year, the party seemed to go on as scheduled, according to this tweet from DJ Sam Young: (The other big New Year’s Eve party on St. Barth was thrown by Russian billionaire Roman Abramovich.) So was Gaddafi there? An article by Brazilian celebrity/gossip columnist Vivi Mascaro, in Portuguese, reported on the event, mentioning “Kadafi” and noting that Lindsay Lohan and George Michael. (Moscowitz also noted Lohan “melting down and dropping numerous f** bombs on the promoter.”) According to Mascaro, 300 people were at the party, drinking magnums of Champagne and seated at tables that each cost a cool $12,000. (According to a source who was there, reservations were required in advance and it was pay-your-own-way, but the question remains about who paid for Beyonce. An email to Nikki Beach asking for clarification has not been returned). Beyoncé, for her part, reportedly sang the five songs mandated by her contract, previously reported by Mascaro — and, according to iPhone translator, “leaving the audience vip blown away with her curves and booty nearly albums.” (Audience video here and here.) Reports are now emerging — unconfirmed — that Beyoncé doubled Carey’s last-year take with a cool $2 million for the performance. Influential urban blogger Necole Bitchie wrote “I am hearing they were at a party for Libyan dictator Moammar Khadafy’s son and Beyonce was reportedly paid 2 million to perform.” UK celebrity and entertainment columnist Dean Piper wrote in the Daily Mirror about the Abramovitch party, noting “[m]eanwhile at another party at nearby Nikki Beach, Beyonce Knowles (watched by husband Jay-Z) was being paid a rumoured six-figure sum by none other than the family of Libyan dictator Col Gaddafi!” Emails and a phone call to Beyonce’s publicist have not been returned. So: Why does this matter? Well, as respectable as Saif Gaddafi is with his charitable endeavors, pedigreed friends and PhD studies, his father is not exactly Mr. Popular — especially after his super-crazy speech at the U.N. in September. Gaddafi’s Libya has long been seen as a foe by the U.S., particularly in the 80s when Gaddafi was held to have been a major sponsor of international terrorism, and directly responsible for ordering the 1986 bombing of a Berlin nightclub which killed two U.S. marines — for which President Ronald Reagan ordered airstrikes on military targets in Tripoli, including the Gaddafi compound, where Gaddafi’s baby adopted daughter Hannah was killed. Gaddafi was also thought to have been a sponsor of Black September, which perpetrated the ‘Munich Massacre’ at the 1972 Olympics — and, of course, there was Pan Am 103. The shorthand for the flight is Lockerbie, from the town in Scotland where the it fell, but the flight was en route from Heathrow to JFK. Of the 243 passengers and 16 crew members killed in that bombing — plus 11 people on the ground — 190 were Americans, including many students returning home from studying abroad. Don’t read the “Victims” section of the Pan Am 103 Wikipedia page if you don’t want to choke up. It’s tough reading. Performing for Gaddafi may not have seemed a big deal for Carey and Jay-Z last year — 20 days later, Carey was in Washington serenading newly-inaugurated President Barack Obama — but this year, the wounds of Pan Am 103 have been ripped open anew with the release of Megrahi, and Gaddafi’s first visit to the U.S., both of which angered the victims’ families greatly and catalyzed a wave of backlash against Libya’s role in the bombing 21 years later. And while President Obama made history this summer by becoming the first U.S. president to shake hands with the Libyan dictator — after all, Libya renounced WMDs and is trying to re-enter the international community, and the U.S. finally dropped its sanctions against the country — Gaddafi was not invited to Obama’s September reception for world leaders attending the U.N. Assembly because of the Megrahi release and that so-called “hero’s welcome.” So: should Beyoncé care about any of this? Shouldn’t she be entitled to play a fun show in a glamorous locale with her husband and friends? The U.S. no longer has sanctions against Gaddafi, after all. Should it fall to Beyoncé to uphold a higher standard? Or, in the wake of the Pan Am bomber release — and the scary terrorism near-miss of Northwest 253 on Christmas — is the timing just a little off? (And more bad timing: Gaddafi’s brother, Moutassim Bilal Gaddafi, known as Hannibal, was reportedly involved in a violent domestic dispute with his wife in London last week.) It’s early yet — as I said this story is still unconfirmed, and just starting to trickle out — but there are signs of backlash on Twitter (@DarlingJadey: “Beyonce performed for Gaddafi’s son. She needs help ASAP.” @LolaCheri: “no but seriously tho why is bey performing for a Libyan dictator neway? &last yr they booked mariah? how U doin Moatessem al-Gaddafi? baha!” @MomVsWild “WTF!Is this true,was Beyonce per4ming 4 the Gaddafi Family last night?Did she 4get about the Lockerbie Bombing & 200+ ppl died?”) This may be unfairly singling out Beyoncé for something that Carey and her own husband did just last year, within a rarefied group of high-powered people for whom it’s clearly not an issue. But still — she’s an all-American superstar with multiple endorsement deals across the board, who just recorded a version of “Proud To Be An American” — and the Gaddafis are a family with a pretty chequered history. A $2 million payday is pretty sweet, but depending on who paid it, it may leave a bitter aftertaste. ****** Translation of Portuguese article by Vivi Mascaro above, translated by Maisa Fonseca: New Year’s eve in St. Barths: Beyoncé’s show and Lindsay Lohan in the audience in a party at Nikki Beach Beyoncé and Usher’s performances. Only three hundred people, like the Libyan Kadafi and Linday Lohan. Magnum Champagne. In short, that was the New Year’s eve party at Nikki Beach, in St. Barths, one of the most exclusive parties in the planet. Who attended the party had to pay the minimum amount of US$ 12 thousand each table, which didn’t scare away names like George Michael, Nizan Guanaes and Paul Allen, one of Microsoft’s founders. As the blog said before, Beyoncé fulfilled her contract: sang only five songs, leaving the audience speechless with her curves and her near-Brazilian sway. The Brazilian stylist Márcio Vicentini got excited and went to the stage to dance with the singers. Her husband Jay-Z was also there. (Translation notes: Mascaro used the expression ‘rebolado quase tupiniquim’ to describe part of Beyonce’s performance. ‘Rebolado’ is the hip/butt circular/side to side movements that compose a lot of dances, including Samba. ‘Tupiniquim’ is an adjective we use to describe native Brazilian things. The journalist was saying her moves could be almost considered Brazilian-like.) Update: Mascaro’s initial report that Microsoft Founder Paul Allen was at the party was incorrect. According to a source with direct knowledge of the situation, Allen was in the area (as evidenced by Russell Simmons’ photo of his yacht) but was not at the Beyoncé-Gaddafi event. Video from the audience at Nikki Beach (warning, TERRIBLE audio. Combined from here and here): (NB: For those of you watching NBC’s New Year’s Eve special hosted by Carson Daly — the part with Jay-Z and Rihanna was pre-taped.) Tonight we received an urgent email from the PR firm representing shithead Libyan Dictator Muammar al-Gaddafi's son, Saif al-Islam: We incorrectly reported Beyonce performed for him this New Year's! She actually performed for Col. Gaddafi's other shithead son, Hannibal. This weekend, Foster wrote an admirably sprawling analysis of speculation by Mediaite's Rachel Sklar that Saif al-Islam paid Beyonce to perform at his New Year's Eve party in St. Barth last week. Turns out it wasn't him. And Ethan Wagner, of the PR firm Brown Lloyd and James, sent us an email letting us know that... I wasn't pleased to read the accusations in your piece. I'm quite familiar with Gawker's style of journalism and you are entitled to print your opinions about Saif, however inflammatory they may be; in using the obscene language that you do to describe him, I am assuming you're not familiar with the direct, personal role he has played in securing the release of western hostages, distributing aid among Africa's poor, and fighting for human rights in the region; rather, you've opted to smear him with profanities because of your perceptions of his father and his country. Pretty much spot on, except for one quibble: Foster smeared Saif al-Islam not because of his "perceptions" of Col. Gadaffi, but because Col. Gadaffi is in fact a ranting, terrorist-backing shithead of truly staggering proportions. It is as if God literally took a pile of shit, fashioned it into the shape of a head, placed it gingerly on the neck of Muammar al-Gaddafi, then let him run a good-sized country using only the worm-addled brain contained therein. Not a "perception": A cold, hard, metaphorical fact. But the main point of Wagner's email was that Saif al-Islam was not serenaded by the hottest woman on the planet this past New Year's. He was in fact being a shithead in New Zealand, on the opposite side of the globe, when we placed him in the audience of a the banging St. Barth Beyonce blowout. Wagner included a few helpful links proving it and demanded that: you print a correction, on the same page where your original story ran, clarifying that your original story about Saif al-Islam Gaddafi was incorrect; I see that you've done something similar already for Paul Allen, and while you may have a higher opinion of Allen than you do my client, responsible journalism and objective fairness demand that you at least take similar action to clarify that Saif al-Islam Gaddafi was not involved in any way. So, consider it corrected: Saif al-Islam Gadaffi was not the shithead who threw the infamous Beyonce bash. It was his younger brother, Hannibal! And if you thought Saif was a shithead, just check out the amazing amount of shitheaded things 33 year-old Hannibal (nee Moutassim) Gadaffi has done in his short, useless life: 2009: Involved in domestic disturbance incident with his wife Christmas day that looks suspiciously like he punched her in the face. A witness claims he saw his wife "bleeding heavily from her nose and face. Her nose was clearly broken and it looked like she would need surgery." When police arrive at his hotel room, Hannibal runs the Libyan embassy to demand diplomatic immunity. Involved in domestic disturbance incident with his wife Christmas day that looks suspiciously like he punched her in the face. A witness claims he saw his wife "bleeding heavily from her nose and face. Her nose was clearly broken and it looked like she would need surgery." When police arrive at his hotel room, Hannibal runs the Libyan embassy to demand diplomatic immunity. 2008: Arrested by Swiss police for allegedly beating his servants with a belt and clothes hanger. Arrested by Swiss police for allegedly beating his servants with a belt and clothes hanger. 2005: Arrested for allegedly punching his wife, model Aline Skaf, in a Paris hotel while she was 8 months pregnant and brandishing a 9mm handgun at her. Arrested for allegedly punching his wife, model Aline Skaf, in a Paris hotel while she was 8 months pregnant and brandishing a 9mm handgun at her. 2004: Leads police on a chase down Paris' Champs Elysees in his Porsche. Avoids charges after Libyan diplomats apologize. Leads police on a chase down Paris' Champs Elysees in his Porsche. Avoids charges after Libyan diplomats apologize. 2003: Involved in a brawl in Rome that leaves six photographers hospitalized Involved in a brawl in Rome that leaves six photographers hospitalized 2001: Attacks three Italian policemen with a fire extinguisher, then demands dilomatic immunity. So, hopefully that's all cleared up now.
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Summary: Beyoncé spent her New Year's Eve performing for Libyan dictator Moammar Gadhafi's son-although Seif al-Islam's PR firm will have you know it wasn't him, as originally reported. "She actually performed for Col. Gadhafi's other shithead son, Hannibal," writes Adrian Chen on Gawker, before running down a list of Hannibal's, er, achievements. Chen "corrects" Gawker's original story after an indignant email from aforementioned PR firm insisting that al-Islam was in New Zealand and not Beyoncé's show in St. Barts. The rep also accused original writer Foster Kamer of "smearing" al-Islam simply because of incorrect "perceptions," which Chen is quick to refute: "Foster smeared Seif al-Islam not because of his 'perceptions' of Col. Gadhafi, but because Col. Gadhafi is in fact a ranting, terrorist-backing shithead of truly staggering proportions. Not a 'perception': A cold, hard, metaphorical fact." For more on the Beyoncé concert, see Mediaite and the New York Post.
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Summarize: Background To protect its critical assets, DOD has established several protection measures for weapon systems. These measures include information assurance to protect information and information systems, software protection to prevent the unauthorized distribution and exploitation of critical software, and anti-tamper techniques to help delay exploitation of technologies through means such as reverse engineering when U.S. weapons are exported or lost on the battlefield. Examples of anti-tamper techniques include software encryption, which scrambles software instructions to make them unintelligible without first being reprocessed through a deciphering technique, and hardware protective coatings designed to make it difficult to extract or dissect components without damaging them. In 1999, the Under Secretary of Defense for Acquisition, Technology, and Logistics (AT&L) issued a policy memorandum for implementing anti- tamper protection in acquisition programs. In the following year, AT&L issued a policy memorandum stating that technologies should be routinely assessed during the acquisition process to determine if they are critical and if anti-tamper techniques are needed to protect these technologies. In 2001, an AT&L policy memorandum designated the Air Force as the Anti- Tamper Executive Agent. The executive agent’s office, which currently has four staff, is responsible for implementing DOD’s anti-tamper policy and managing anti-tamper technology development through the Air Force Research Laboratory. The executive agent also holds periodic information sessions to educate the acquisition community about anti-tamper policy, initiatives, and technology developments. To coordinate activities, military services and defense agencies, such as the Missile Defense Agency, have an anti-tamper point of contact. Program managers are responsible for ensuring anti-tamper protection is incorporated on any weapon system with critical technologies that need protection. Since it is not feasible to protect every technology, program managers are to conduct an assessment to determine if anti-tamper protection is needed. When assessing if anti-tamper protection is needed, program managers make several key decisions regarding the identification of critical technologies, assessment of threats and vulnerabilities, and determination of anti-tamper techniques or solutions. The process begins with determining whether or not their system’s critical program information includes any critical technologies. If it is determined that the system has no critical technologies, program managers are to document the decision and request concurrence from either the office within their component that is designated with anti-tamper responsibilities or the Anti-Tamper Executive Agent. For systems that are determined to have critical technologies, the next key steps are to identify potential threats and vulnerabilities and select anti-tamper techniques to protect those technologies. Techniques are ultimately verified and validated by a team composed of representatives from the DOD components. The program manager documents decisions in an annex of the program protection plan. In 2004, we reported that program managers had difficulty in carrying out DOD’s anti-tamper policy on individual weapons, such as identifying critical technologies and experiencing cost increases or schedule delays when applying anti-tamper techniques—particularly when the techniques are not fully developed or when the systems are already in design or production. We made several recommendations, including increasing oversight over the identification of critical technologies across programs, improving tools and resources for program managers in identifying critical technologies, ensuring early identification of anti-tamper costs and solutions, monitoring the development of generic anti-tamper solutions and evaluating their effectiveness, and developing a business case to determine whether the current organizational structure and resources are adequate. DOD concurred or partially concurred with these recommendations. DOD has taken some steps to implement our recommendations including identifying available anti-tamper technical resources and developing a searchable spreadsheet of critical technologies, incorporating information in the Defense Acquisition Guidebook on the need for early identification of anti-tamper solutions in a weapon system, and sponsoring a study on anti-tamper techniques and their general effectiveness. While DOD has taken these steps to address parts of the recommendations, all remain open. DOD Lacks Departmentwide Direction for Implementing its Anti- tamper Policy DOD has recently taken several actions aimed at raising awareness about its anti-tamper policy and assisting program managers in implementing anti-tamper protection on a weapon system. Despite these actions, DOD still lacks departmentwide direction to implement its anti-tamper policy. Without such direction, DOD components are left to develop their own initiatives to assist program managers in implementing anti-tamper protection. While individual efforts are important, such as a database to track critical program information DOD-wide, their effectiveness may be limited because they have yet to be accepted and adopted across all DOD components. DOD Has Developed Some Resources for Program Managers to Use in Implementing Anti-tamper Protection Since our 2004 report, DOD, through the Anti-Tamper Executive Agent, has developed some resources aimed at assisting program managers as they go through the anti-tamper decision process. DOD’s resources range from providing general information about the anti-tamper policy to research on anti-tamper solutions. Specifically, DOD has developed a guidebook that includes a checklist to assist program managers in identifying security, management, and technical responsibilities when incorporating anti-tamper protection on a weapons system; developed a searchable spreadsheet to assist program managers in developed a Web site for program managers to provide general anti- tamper information, policy resources, conference briefings, implementation resources, and current events; coordinated with Defense Acquisition University to design and launch an online learning module on anti-tamper protection; funded Sandia National Laboratories to study anti-tamper techniques and their general effectiveness; and sponsored research to develop generic anti-tamper techniques through Small Business Innovation Research, a research program that funds early-stage research and development projects at small technology companies. DOD has also updated two acquisition documents with general anti- tamper information. The first document—DOD Instruction 5000.2, Operation of a Defense Acquisition System—currently states that one of the purposes of the System Development and Demonstration phase of a weapon system is to ensure affordability and protection of critical program information by implementing appropriate solutions such as anti- tamper protection. The second document—the Defense Acquisition Guidebook—has been updated to include some basic information on the importance of implementing anti-tamper protection early in the development of a weapon system and describes program managers’ overall responsibilities for implementing the anti-tamper policy. DOD Has Not Provided Direction to Implement Its Anti-tamper Policy While DOD has issued broad policy memorandums that reflect the department’s desire for routinely assessing weapon systems to determine if anti-tamper protection is needed, the department has not fully incorporated the anti-tamper policy into its formal acquisition guidance. Specifically, DOD Instruction 5000.2 mentions anti-tamper protection, but the department has not provided direction for implementation of anti- tamper in a formal directive or instruction. Currently, the department is coordinating comments on a draft instruction (DOD Instruction 5200.39) on protection of critical program information that includes anti-tamper implementation. However, in commenting on the draft instruction, several DOD components have raised concerns about when and how to define critical program information that warrants protection, which have contributed to long delays in finalizing the instruction. In addition, the department has not provided specific guidance for program managers on how to implement anti-tamper protection in a DOD manual because DOD officials said this process cannot begin until the instruction is finalized. The date for finalizing the instruction has not yet been determined. Officials from the executive agent’s office stated that departmentwide direction would give credence to the anti-tamper policy in practice. Anti- tamper points of contact told us that the policy memorandums are not sufficient to ensure that program managers are implementing anti-tamper protection on weapon systems when necessary. One service anti-tamper point of contact stated that program managers might disregard the policy memorandums because they are high-level and broad. Another service anti-tamper point of contact said that implementation is ultimately left up to the individual program manager. While a program manager’s decision should be approved by the milestone decision authority and documented in the program protection plan, some service and program officials said that programs are not always asked about anti-tamper protection during the review. Absent Departmentwide Direction, Components Have Been Left to Develop Their Own Anti-tamper Initiatives Lacking departmentwide direction for the anti-tamper policy, DOD components have been left to develop their own initiatives to assist program managers in anti-tamper implementation. However, the usefulness of these initiatives depends on the extent to which other components participate in these efforts. For example, the Missile Defense Agency developed a risk assessment model to help program managers identify how much anti-tamper is needed to protect critical technologies. Specifically, the model helps program managers assess the criticality of the technology relative to the risk of exploitation. However, when the Missile Defense Agency sought comments on the initiative, the executive agent and services indicated that it was too lengthy and complex to use. The executive agent, in coordination with anti-tamper points of contact from the Missile Defense Agency and services, has taken over this effort, and it is still in development. The Navy is also implementing an initiative: a database intended to capture the information that programs across DOD components have identified as critical. Many officials we spoke with pointed to this database as a potential tool to improve identification of critical program information across DOD components. To date, the Navy and the Army are submitting information for the database, but the Missile Defense Agency and Air Force are not. The Missile Defense Agency anti-tamper point of contact stated that its information is classified at a level above what the database can support and its program managers will not submit information for the database unless DOD requires submissions by all DOD components. However, the Missile Defense Agency does have access to the database and uses it as a cross-check to determine if it is identifying similar critical program information. The Air Force has been briefed on the initiative but does not yet have consent from all of the commands to participate. Without full participation across all DOD components, the usefulness of this database as a tool to identify critical technologies that may need anti- tamper protection will be limited. Program Managers Face Several Challenges in Identifying Critical Technologies, Threats, and Sufficient Anti-tamper Solutions To determine whether anti-tamper protection is needed, program managers must identify which technologies are deemed critical, determine the potential threats and vulnerabilities to these technologies, and identify sufficient anti-tamper solutions to protect the technologies. Such decisions involve a certain level of subjectivity. However, program managers lack the information or tools needed to make informed assessments at these key decision points. As a result, some technologies that need protection may not be identified or may not have sufficient protection. Limited Information and Coordination on What Is Critical Increase the Risk That Some Technologies May Not Be Identified Determining technologies that are critical is largely left to the discretion of the program managers. While DOD has some resources available to program managers to help identify critical technologies, they may be of limited use. For example, the executive agent’s searchable spreadsheet of critical technologies may not be comprehensive because it relies on DOD’s Militarily Critical Technologies List, which we reported in 2006 was largely out of date. Also, some program offices have used a series of questions established in a 1994 DOD manual on acquisition systems protection to help guide their discussions on what is critical. However, these questions are broad and subject to interpretation, and can result in different conclusions, depending on who is involved in the decision-making process. In addition, identifying what is critical varies by DOD component and sometimes by program office. For example, one Air Force program office tried various approaches, including teams of subject matter experts, over 2 years to identify its list of critical program information. In contrast, the Army took the initiative to establish a research center to assist program managers in identifying critical program information, but Army officials stated that the approach used by the center has led to an underestimating of critical program information and critical technologies in programs. At the same time, there has been limited coordination across programs on technologies that have been identified as critical—creating a stove piped process—which could result in one technology being protected under one program and not protected under another. While informal coordination can occur, programs did not have a formal mechanism for coordinating with other programs, including those within their service. For example, officials from one program office stated they had little interaction from programs within their service or other services to ensure protection of similar technologies. A program under one joint program executive office had not coordinated with other programs to identify similar technologies as critical. In addition, according to an Army official, contractors who have worked on programs across services have questioned why one service is applying anti-tamper solutions to a technology that another service has not identified as critical. Finally, one program office we spoke with identified critical program information on its system but indicated that a similar system in another service had not identified any critical program information and, therefore, had no plans to implement anti- tamper protection. Despite the risk that some technologies that need protection may not be identified or may not be protected across programs, no formal mechanism exists within DOD to provide a horizontal view of what is critical. However, any effort to do so could be undermined by the programs’ and services’ different definitions and interpretations of “critical program information” and “critical technologies.” The Anti-Tamper Executive Agent defines critical program information as capturing all critical technologies. In contrast, the Army’s interpretation is that critical program information only includes critical technologies that are state-of-the-art. For the Navy, critical program information includes software, while hardware is part of what the Navy defines as critical technologies. One program that is part of a joint program office identified critical program information as including company proprietary information. As a result, tracking critical program information may not provide a horizontal view of all technologies services and programs have identified as needing anti-tamper protection. Contradictory and Insufficient Intelligence Information Has Hindered Some Programs in Identifying Potential Threats Once a program office identifies critical technologies, the next step in the anti-tamper decision process is to identify threats to those technologies. DOD’s Program Manager’s Guidebook and Checklist for Anti-tamper states that multiple threat assessments should be requested from either the service intelligence organization or counterintelligence organization. One program office we visited stated that it has requested and received multiple threat assessments from the intelligence community, which have sometimes contradicted one another, leaving the program office to decipher the information and determine the threat. According to an anti- tamper point of contact, other programs have received contradictory information—typically relating to foreign countries’ capabilities to reverse engineer. The potential impact of contradictory intelligence reports is twofold: If the threat is deemed to be low but is actually high, the technology is susceptible to reverse engineering; conversely, if the threat is deemed to be high and is actually low, the anti-tamper solution is more robust than needed. To assist with the process of identifying threats, program offices may request threat assessments from a group within the Defense Intelligence Agency. However, this group was not able to complete assessments for approximately 6 months during 2006. While the group has resumed completing assessments, an agency official stated that it is not able to produce as many assessments as before due to limited resources. The Defense Intelligence Agency does not turn down program offices that may request assessments, but does have to put them in a queue and provide them with previous assessments, if they exist, until it can complete a full assessment for the program office. One program office indicated that it took 6 to 9 months for the agency to complete its assessment. Program Managers Need Tools to Assist in Designing Effective Anti- tamper Solutions and Estimating Related Costs Program managers also lack the tools needed to identify the optimal anti- tamper solutions for those critical technologies that are vulnerable to threats. Most notably, program managers lack a risk model to assess the relative strengths of different anti-tamper solutions and a tool to help estimate their costs. According to National Security Agency officials, who are available to provide support to program managers considering or implementing anti- tamper protection, program managers and contractors sometimes have difficulty determining appropriate solutions. Four of five programs we spoke with that had experience in this area of the anti-tamper decision process had difficulty identifying how much anti-tamper protection was enough to protect a critical technology. For example, one program official told us that an anti-tamper solution developed for one of the program’s critical technologies may not be sufficient to prevent reverse engineering. Another program office stated that it is difficult to choose between competing contractors without knowing how to determine the appropriate level of anti-tamper protection needed. An anti-tamper point of contact said that program managers need a tool to help them assess the criticality of a technology versus the types of threats to that technology. Implementing a suboptimal anti-tamper solution can have cost and performance implications for the program. Specifically, if the solution provides less anti-tamper protection than is needed, the program may have to retrofit additional anti-tamper protection to allow for a more robust solution. Not only can such retrofitting add to a program’s costs, it can compromise performance. Given limited resources and tools for determining anti-tamper solutions, some program office officials told us that to satisfy anti-tamper solutions they relied on other protection measures. For example, officials in one program office stated that anti-tamper protection and information assurance were interchangeable and indicated that following the National Security Agency’s information assurance requirements—which number in the hundreds—should be sufficient as an anti-tamper solution for this system. This same program was not aware of anti-tamper resources and did not coordinate with an anti-tamper validation and verification team on its solutions. Also, an official from another program office indicated that anti-tamper protection and information assurance are similarly defined. While DOD and service officials agreed that some information assurance and anti-tamper measures may overlap, fulfilling information assurance requirements does not guarantee a sufficient anti-tamper solution. Conclusions In establishing various policies to protect its critical assets, DOD saw anti- tamper as a key way to preserve U.S. investment in critical technologies while operating in an environment of coalition warfare and a globalized industry. Program managers are ultimately responsible for implementing DOD’s anti-tamper policy. However, a lack of direction, information, and tools from DOD to implement its policy has created significant challenges for program managers. Further, this policy can compete with the demands of meeting program cost and schedule objectives, particularly when the optimal anti-tamper solution is identified late in the schedule. Until DOD establishes a formal directive or instruction for implementing its policy departmentwide and equips program managers with adequate implementation tools, program managers will continue to face difficulties in identifying critical technologies and implementing anti-tamper protection. Recommendations for Executive Action As DOD examines its policies for protecting critical assets, we are recommending that the Secretary of Defense direct the Under Secretary of Acquisition, Technology, and Logistics, in coordination with the Anti- Tamper Executive Agent and the Under Secretary of Defense for Intelligence, to issue or be involved in developing and providing departmentwide direction for application of its anti-tamper policy that prescribes how to carry out the policy and establishes definitions for critical program information and critical technologies. To help ensure the effectiveness of anti-tamper implementation, we also recommend that the Secretary of Defense direct the Anti-Tamper Executive Agent to identify and provide additional tools to assist program managers in the anti-tamper decision process. Agency Comments and Our Evaluation In written comments on a draft of this report, DOD concurred with our recommendation that the Secretary of Defense direct the Anti-Tamper Executive Agent to identify additional tools to assist program managers in the anti-tamper decision process. DOD stated that the Anti-Tamper Executive Agent is drafting Anti-Tamper Standard Guidelines to facilitate proper implementation of anti-tamper protection across the department. DOD did not concur with our recommendation that the Secretary of Defense direct the Under Secretary of Defense (AT&L) in coordination with the Anti-Tamper Executive Agent and the Under Secretary of Defense (Intelligence) to issue departmentwide direction for application of its anti- tamper policy that prescribes how to carry out the policy and establishes definitions for critical program information and critical technologies. DOD stated that the Under Secretary of Defense (Intelligence) has primary responsibility for DOD Directive 5200.39, a security and counterintelligence support directive to acquisition programs, and its successor, DOD Instruction 5200.39 regarding protection of critical program information. The Under Secretary of Defense (Intelligence) is currently coordinating an update to this directive. Once it is issued, the department plans to update DOD 5200.1-M, which provides the execution standards and guidelines to meet the DOD Instruction 5200.39 policy. While DOD has issued broad policy memorandums beginning in 1999 that reflect the department’s desire for routinely assessing weapon systems to determine if anti-tamper protection is needed, the department has not fully incorporated anti-tamper policy into its formal acquisition guidance. As we have reported, service officials indicated collectively that these policy memorandums are high-level, broad, and leave implementation ultimately up to the individual program manager. DOD did not indicate when the update of DOD Directive 5200.39 might be complete and guidance on anti- tamper implementation issued. We continue to believe that such direction is currently needed and that the Under Secretary of Defense for Acquisition, Technology, and Logistics, who issued the policy memorandums and is responsible for anti-tamper policy, should be involved in developing and providing the appropriate direction whether it be the update to DOD Directive 5200.39 or another vehicle. That direction should include how to implement the anti-tamper policy and how critical program information and critical technologies are defined. We continue to believe that the direction, which has been lacking since the policy was initiated in 1999, should not be further delayed. If DOD continues to experience delays in updating DOD Directive 5200.39, it should consider interim measures to meet the immediate need for anti-tamper direction. DOD’s letter is reprinted in appendix II. We are sending copies of this report to interested congressional committees, as well as the Secretary of Defense; the Director, Office of Management and Budget; and the Assistant to the President for National Security Affairs. In addition, this report will be made available at no charge on the GAO Web site at http://www.gao.gov. Please contact me at (202) 512-4841 or [email protected] if you or your staff have any questions concerning this report. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. Others making key contributions to this report are listed in appendix III. Appendix I: Scope and Methodology To identify actions the Department of Defense (DOD) has taken to implement its anti-tamper policy since 2004, we reviewed DOD policies and guidance governing anti-tamper protection on weapon systems and obtained documents on various initiatives. We interviewed officials from the Anti-Tamper Executive Agent, military services, and other DOD components such as the Missile Defense Agency; Acquisition, Technology and Logistics; Defense Intelligence Agency; National Security Agency; and the Air Force Research Laboratory about initiatives or actions taken regarding anti-tamper. Through these interviews and documents, we also determined the status of our 2004 anti-tamper report recommendations. We interviewed DOD officials from Networks and Information Integration, Science and Technology, and Counterintelligence to discuss anti-tamper protection and how it relates to other program protection measures. To determine how program managers implemented DOD’s anti-tamper policy, we interviewed officials from 14 program offices. We are not identifying the names of the programs due to classification concerns. We conducted structured interviews with 7 of the 14 program offices to discuss and obtain documents about their experiences with implementing the anti-tamper decision process and identify any challenges they faced. We selected 6 of these programs from a list of weapon systems identified in Anti-Tamper Executive Agent, services, and component documents as considering and/or implementing anti-tamper protection and a seventh program considering anti-tamper that we identified during the course of our fieldwork. Systems we selected represented a cross section of acquisition programs and various types of systems in different phases of development. For the remaining programs, we interviewed 7 not identified by the Anti-Tamper Executive Agent or the services as considering and/or implementing anti-tamper to obtain their viewpoints on DOD’s anti-tamper policy and implementation. We selected these programs by identifying lists of DOD acquisition programs and comparing them to the Anti-Tamper Executive Agent’s, services’, and components’ lists of program considering and/or implementing anti-tamper. We did not evaluate whether programs had implemented sufficient anti-tamper protection. Appendix II: Comments from the Department of Defense Appendix III: GAO Contact and Staff Acknowledgments GAO Contact Acknowledgments In addition to the contact named above, Anne-Marie Lasowski (Assistant Director), Gregory Harmon, Molly Whipple, Karen Sloan, John C. Martin, and Alyssa Weir made major contributions to this report. Related GAO Products High-Risk Series: An Update. GAO-07-310. Washington, D.C.: January 2007. Export Controls: Challenges Exist in Enforcement of an Inherently Complex System. GAO-07-265. Washington, D.C.: December 20, 2006. Defense Technologies: DOD’s Critical Technologies Lists Rarely Inform Export Control and Other Policy Decisions. GAO-06-793. Washington, D.C.: July 28, 2006. President’s Justification of the High Performance Computer Control Threshold Does Not Fully Address National Defense Authorization Act of 1998 Requirements. GAO-06-754R. Washington, D.C.: June 30, 2006. Export Controls: Improvements to Commerce’s Dual-Use System Needed to Ensure Protection of U.S. Interests in the Post-9/11 Environment. GAO-06-638. Washington, D.C.: June 26, 2006. Defense Trade: Enhancements to the Implementation of Exon-Florio Could Strengthen the Law’s Effectiveness. GAO-05-686. Washington, D.C.: September 28, 2005. Industrial Security: DOD Cannot Ensure Its Oversight of Contractors under Foreign Influence Is Sufficient. GAO-05-681. Washington, D.C.: July 15, 2005. Defense Trade: Arms Export Control Vulnerabilities and Inefficiencies in the Post-9/11 Security Environment. GAO-05-468R. Washington, D.C.: April 7, 2005. Defense Trade: Arms Export Control System in the Post-9/11 Environment. GAO-05-234. Washington, D.C.: February 16, 2005. Defense Acquisitions: DOD Needs to Better Support Program Managers’ Implementation of Anti-Tamper Protection. GAO-04-302. Washington, D.C.: March 31, 2004. Defense Trade: Better Information Needed to Support Decisions Affecting Proposed Weapons Transfers. GAO-03-694. Washington, D.C.: July 11, 2003.
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Summary: The Department of Defense (DOD) invests billions of dollars on sophisticated weapon systems and technologies. These may be at risk of exploitation when exported, stolen, or lost during combat or routine missions. In an effort to minimize this risk, DOD developed an anti-tamper policy in 1999, calling for DOD components to implement anti-tamper techniques for critical technologies. In March 2004, GAO reported that program managers had difficulties implementing this policy, including identifying critical technologies. This follow-up report (1) describes recent actions DOD has taken to implement its anti-tamper policy and (2) identifies challenges facing program managers. GAO reviewed documentation on actions DOD has taken since 2004 to implement its anti-tamper policy, and interviewed officials from the Anti-Tamper Executive Agent's Office, the military services, other DOD components, and a cross-section of program offices. Since 2004, DOD has taken several actions to raise awareness about anti-tamper protection and develop resources that provide program managers with general information on its anti-tamper policy. These actions include developing a Web site with anti-tamper information and events, establishing an online learning module on anti-tamper protection, and sponsoring research on generic anti-tamper techniques. However, DOD lacks departmentwide direction for implementation of its anti-tamper policy. Without such direction, individual DOD components are left on their own to develop initiatives. For example, the Navy is developing a database that is intended to provide a horizontal view of what DOD components have identified as critical program information. While many officials we spoke with pointed to this database as a potential tool for identifying critical technologies that may need anti-tamper protection, the database is currently incomplete. Specifically, the Missile Defense Agency is not providing information because its information is classified at a level above what the database can support. Also, the Air Force is not currently providing information because not all commands have provided consent to participate. At the same time, program managers face challenges implementing DOD's anti-tamper policy--due largely to a lack of information or tools needed to make informed assessments at key decision points. First, program managers have limited information for defining what is critical or insight into what technologies other programs have deemed critical to ensure similar protection across programs. Determining whether technologies are critical is largely left to the discretion of the individual program manager, resulting in an uncoordinated and stove piped process. Therefore, the same technology can be identified as critical in one program office but not another. Second, program managers have not always had sufficient or consistent information from the intelligence community to identify threats and vulnerabilities to technologies that have been identified as critical. The potential impact of inconsistent threat assessments is twofold: If the threat is deemed to be low but is actually high, the technology is susceptible to tampering; conversely, if the threat is deemed to be high and is actually low, an anti-tamper solution is more robust than needed. Finally, program managers have had difficulty selecting sufficient anti-tamper solutions--in part because they lack information and tools, such as risk and cost-estimating models, to determine how much anti-tamper protection is needed. As a result, program managers may select a suboptimal solution. Given these combined challenges, there is an increased risk that some technologies that need protection may not be identified or may not have sufficient protection.
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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Florida Coast Protection Act of 1999''. SEC. 2. ENVIRONMENTAL IMPACT STATEMENT REQUIREMENTS. Section 307(c)(3) of the Coastal Zone Management Act of 1972 (16 U.S.C. 1456(c)(3)) is amended by adding at the end the following: ``(C) Necessary data and information.--For purposes of subparagraph (B), a State shall not be considered to receive all necessary data and information with respect to a plan for exploration, development, or production before the date on which the State receives a copy of an environmental impact statement under section 102(2)(C) of the National Environmental Policy Act of 1969 (42 U.S.C. 4332(2)(C)) that applies to that exploration, development, or production.''. SEC. 3. UNIFORM DOCUMENTATION REQUIREMENTS. Section 25 of the Outer Continental Shelf Lands Act (43 U.S.C. 1351(a) is amended-- (1) in paragraph (a)(1), by striking ``other than the Gulf of Mexico,'' each place it appears; and (2) by striking subsection (l). SEC. 4. OIL AND GAS DEVELOPMENT AND PRODUCTION. Section 25(e) of the Outer Continental Shelf Lands Act of 1972 (43 U.S.C. 1351(e)) is amended-- (1) by striking ``(e)(1) At least'' and inserting the following: ``(e) Major Federal Action.-- ``(1) Outside the gulf of mexico.-- ``(A) In general.--At least''; (2) by striking ``(2) The Secretary'' and inserting the following: ``(B) Preliminary and final plans.--The Secretary''; and (3) by adding at the end the following: ``(2) In the gulf of mexico.-- ``(A) In general.--The approval of a development and production plan in a covered area (as defined in section 8(p)(1)) shall be considered to be a major Federal action for the purposes of the National Environmental Policy Act of 1969 (42 U.S.C. 4321 et seq.). ``(B) Time for review following receipt of environmental impact statement.--In the case of a development and production plan in a covered area, the Secretary shall ensure that each affected State for which a development and production plan affects any land use or water use in the coastal zone of the State with a coastal zone management program approved under section 306 of the Coastal Zone Management Act of 1972 (16 U.S.C. 1455), receives the final environmental impact statement not less than 180 days before determining concurrence or objection to the coastal zone consistency certification that is required to accompany the environmental impact statement under section 307(c)(3)(B) of the Coastal Zone Management Act of 1972 (16 U.S.C. 1456(c)(3)(B)).''. SEC. 5. LEASING ACTIVITY OFF THE COAST OF FLORIDA. Section 8 of the Outer Continental Shelf Lands Act (43 U.S.C. 1337) is amended-- (1) in subsection (a)(1), by striking ``The Secretary'' and inserting ``Except as provided in subsection (p), the Secretary''; and (2) by adding at the end the following: ``(p) Leasing Activity Off the Coast of Florida.-- ``(1) Definitions.--In this subsection: ``(A) Covered area.--The term `covered area' means-- ``(i) the Eastern Gulf of Mexico Planning Area (as established by the Secretary) which is adjacent to the State of Florida as defined by 43 U.S.C. 1333(a)(2)(A); ``(ii) the Straits of Florida Planning Area (as established by the Secretary); and ``(iii) the South Atlantic Planning Area (as established by the Secretary) which is adjacent to the State of Florida as defined by 43 U.S.C. 1333(a)(2)(A); within 100 miles off the coast of Florida. ``(B) Preleasing activity.-- ``(i) In general.--The term `preleasing activity' means an activity relating to a lease that is conducted before a lease sale is held. ``(ii) Inclusions.--The term `preleasing activity' includes-- ``(I) the scheduling of a lease sale; ``(II) the issuance of a request for industry interest; ``(III) the issuance of a call for information or a nomination; ``(IV) the identification of an area for prospective leasing; ``(V) the publication of a draft or final environmental impact statement or a notice of sale; and ``(VI) the performance of any form of rotary drilling in a prospective lease area. ``(iii) Exclusions.--The term `preleasing activity' does not include an environmental, geologic, geophysical, economic, engineering, or other scientific analysis, study, or evaluation. ``(2) Prohibition of preleasing activities and lease sales.--The Secretary shall not conduct any preleasing activity or hold a lease sale under this Act in a covered area.''.
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Title: Florida Coast Protection Act of 1999 Summary: (Sec. 3) Amends the Outer Continental Shelf Lands Act to remove provisions: (1) exempting any area of the outer Continental Shelf in the Gulf of Mexico from a requirement to submit a development and production plan before development and production under an oil and gas lease; and (2) allowing the Secretary of the Interior to apply provisions relating to oil and gas development and production to a lease located in the Gulf adjacent to the State of Florida. (Sec. 4) Declares that, in the Gulf, the approval of such a plan is a major Federal action for purposes of NEPA. Directs the Secretary to ensure that each affected State receive the final environmental impact statement at least 180 days before the Secretary makes a determination on whether each plan activity complies with the those States' enforceable policies of approved management programs. (Sec. 5) Prohibits the Secretary from conducting any preleasing activity or holding a lease sale under this Act in an area within 100 miles off the coast of Florida in: (1) the Eastern Gulf of Mexico Planning Area adjacent to Florida; (2) the Straits of Florida Planning Area; or (3) the South Atlantic Planning Area.
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Write a title and summarize: Lymphatic filariasis (LF) is a neglected tropical disease for which more than a billion people in 73 countries are thought to be at-risk. At a global level, the efforts against LF are designed as an elimination program. However, current efforts appear to aim for elimination in some but not all endemic areas. With the 2020 goal of elimination looming, we set out to develop plausible scale-up scenarios to reach global elimination and eradication. We predict the duration of mass drug administration (MDA) necessary to reach local elimination for a variety of transmission archetypes using an existing model of LF transmission, estimate the number of treatments required for each scenario, and consider implications of rapid scale-up. We have defined four scenarios that differ in their geographic coverage and rate of scale-up. For each scenario, country-specific simulations and calculations were performed that took into account the pre-intervention transmission intensity, the different vector genera, drug regimen, achieved level of population coverage, previous progress toward elimination, and potential programmatic delays due to mapping, operations, and administration. Our results indicate that eliminating LF by 2020 is unlikely. If MDA programs are drastically scaled up and expanded, the final round of MDA for LF eradication could be delivered in 2028 after 4,159 million treatments. However, if the current rate of scale-up is maintained, the final round of MDA to eradicate LF may not occur until 2050. Rapid scale-up of MDA will decrease the amount of time and treatments required to reach LF eradication. It may also propel the program towards success, as the risk of failure is likely to increase with extended program duration. Lymphatic filariasis (LF) is a neglected tropical disease (NTD) primarily prevalent in poor populations in 73 countries [1]. LF is caused by infection with Wuchereria bancrofti, Brugia malayi, or B. timori transmitted by a variety of mosquito genera [2]. Infection with the filarial nematodes can damage the lymphatic vessels, the main clinical manifestations being lymphedema, hydrocele, and elephantiasis [3]. In addition to disfigurement and disability, people affected by LF face stigma, social adversity, and economic hardship [4–6]. LF is spread by mosquitoes that take up circulating microfilarae (mf) in the peripheral blood of infected humans [7]. Administration of albendazole with ivermectin or diethylcarbamazine citrate (DEC) has been shown to reduce circulating mf to such low levels that transmission cannot be sustained [8]. For this reason, LF is one of six diseases considered to be potentially eradicable [9]. Accordingly, in 1997 the World Health Assembly (WHA) adopted resolution WHA 50. 29, which calls for the elimination of LF as a public health problem and, in 2000, the World Health Organization (WHO) established the Global Program to Eliminate Lymphatic Filariasis (GPELF). The GPELF aims to eliminate LF in all endemic countries by 2020 through annual mass drug administration (MDA) maintained over multiple years [8]. The program benefits through donations from Merck & Co. and GlaxoSmithKline (GSK), which have pledged to provide enough ivermectin and albendazole, respectively, to achieve elimination, as well as from Eisai, which in 2010, pledged 2. 2 billion DEC tablets [10,11]. The GPELF has scaled up rapidly and is among the fastest growing disease elimination programs in the world [12]. By the end of 2013,56 LF-endemic countries had carried out MDA, of which 15 are now undertaking post-MDA surveillance. In 2013 alone, more than 410 million anti-filarial treatments were distributed under the GPELF. However, the program is not without its challenges: mapping is incomplete in 12 countries, 14 countries requiring MDA are yet to begin, and many of the other endemic countries are targeting relatively small proportions of their at-risk populations [13]. Issues with compliance, contraindications of ivermectin and DEC in areas with hyper Loa loa-endemicity, and interruptions in funding also plague the program [14,15]. At a global level, the efforts against LF could be considered a global elimination program (elimination of infection in some but not all countries) as the name suggests, or an eradication program (permanent reduction to zero of the worldwide incidence of infection) as implied by the stated aims of the program [13,16,17]. In order to assist decision makers in determining whether efforts for LF should be scaled up to try to achieve eradication, it has been proposed to use an analytic and deliberate methodology to produce evidence-based guidance on the rationale for investing [18,19]. As part of this endeavor, we herein predict the duration of MDA necessary to reach local elimination for a variety of transmission archetypes using an existing model of LF transmission, outline plausible scale-up scenarios leading to global elimination and eradication, and estimate the number of treatments required under each scenario. Potential delays in implementation, previous progress, and different intensities of infection and transmission are also taken into account. Studies on the economic and financial costs, the impact on disease burden, and cost-effectiveness of these scenarios are to be published as companion papers. Scenarios were developed by first reviewing the WHO preventive chemotherapy (PCT) databank to assess progress made towards LF elimination as of 2012 [13]. The scenarios were further refined, with key assumptions agreed upon in a series of technical advisory group meetings, including stakeholders from WHO, Centers for Disease Control and Prevention (CDC), funders, pharmaceutical companies, and program managers from endemic countries. In the global elimination scenario, countries that have not yet started will not start, and countries that have started continue according to their assigned level of scale-up (see: Rate of scale-up). In the eradication I scenario, countries that have already started MDA continue as in the global elimination scenario and countries that have not yet started implement MDA following an ‘average’ level of scale-up. The eradication II scenario represents the case in which all countries scale-up MDA more quickly (fast). Eradication III serves as the ‘best case’ scenario, whereby all endemic countries provide MDA to their entire at-risk populations immediately. Thus, this analysis provides insight into the differences in the amount of time and treatments required to extend elimination efforts to all endemic countries (eradication I), increase MDA intensity (eradication II) and, most ideally, scale-up instantaneously (eradication III). An important assumption underlying this study is that annual MDA using DEC with albendazole, or, in onchocerciasis-endemic countries, ivermectin and albendazole, will be sufficient to reduce circulating mf enough to interrupt the transmission cycle of LF if maintained for an appropriate number of years. Therefore, hardly predictable features that could undermine success, including systematic non-compliance with MDA, but particularly events such as civil unrest and humanitarian emergencies (e. g. earthquakes in Haiti and Nepal; Ebola epidemic in West Africa) that could compromise the health system’s capacity, could not be accounted for. We also assume that countries undertake MDA without interruption. Administration of ivermectin to communities with high prevalence (>40%) of L. loa is contraindicated, as the microfilaracidal actions of the drug poses an unjustifiably high risk of causing severe adverse events. As such, the WHO provisionally recommends the LF program to instead treat these areas with albendazole monotherapy distributed bi-annually and vector control [20]. Here we assume that this strategy will be equally efficacious as annual albendazole-ivermectin, and thereby assume the number of years of MDA required in areas co-endemic with L. loa to be equivalent to the number of years required with albendazole-ivermectin. The GPELF advises LF endemic countries to conduct MDA for 4–6 years [8]. This duration only holds at a country level if all endemic areas are treated simultaneously. To incorporate scaling-up of geographic coverage for each scenario, we divided each country’s at-risk population into deciles, and assumed MDA to start in subsequent deciles after varying durations according to four schedules of scale-up. In schedule I (fast), 20% of the at-risk population is added to the MDA schedule annually. In schedule II (average), one decile is added each year, in schedule III (slow) one decile is added every two years and in schedule IV (very slow) this period is three years. In the global elimination scenario, scale-up is based upon the proportion of the at-risk population each country previously targeted. In order to be allocated to schedule I, the at-risk population targeted in the most recent round of MDA had to exceed 50%. Schedule II has been assigned to countries previously targeting 30–50%, schedule III to those targeting 20–29. 9%, and schedule IV to those targeting <20%. Rather than attempting to recreate the progress of each country exactly, we used these categories to incorporate a range of scale-up levels encountered. Previous progress made towards local elimination was further taken into account by counting the number of previously effective years of MDA, which was considered as any year in which program coverage within the targeted area (regardless of the at-risk population targeted) exceeded 65%. We then subtracted the number of effective years previously achieved from the number of years of MDA deemed necessary (see below: Transmission Archetypes; Table 4) in order to determine the number of years of MDA remaining. The number of rounds corresponds to the minimum at which at least 97. 5% of simulations went to elimination. For all scenarios, we assume that countries that have finished mapping but not begun MDA have a 1-year delay, whereas countries that have not completed mapping nor begun MDA have a 4-year delay. While countries face challenges of different magnitudes and require different durations to map, the 4-year delay assumed corresponds to the average number of years that mapping took in countries with available data to support the calculation [13]. To account for heterogeneity in transmission intensity within countries, we obtained paired baseline circulating filarial antigenaemia prevalence, measured through immunochromatographic tests (ICTs), and mf prevalence data from sentinel site surveys from program countries across the AFRO region. As specified by the WHO, these surveys involve collecting fingertip blood, between 10 p. m. and 2 am. from at least 300 participants aged five years and above [21]. We gained additional access to ICT prevalence data from mapping studies in 17 African countries. The relationship between mf and antigenaemia prevalence was estimated using the non-parametric regression proposed by Passing and Bablock, which assumes linearity and uncertainties in both variables [22]. The regression equation calculated from the paired prevalence data was then used to infer mf prevalence from the ICT mapping data. We determined the percentage of the at-risk population that fell into prevalence quartiles: <5%, 5–10%, 10. 1–15%, >15%, for each country that provided district level prevalence data. To account for uncertainties in this approach, we took 500 random draws from a multinomial distribution with probabilities based on weighted averages from the dataset and assumed these to be the possible ranges of pre-intervention prevalence distributions for all countries in our analysis. It has been theoretically demonstrated that the required duration of MDA is region-specific and dependent on various factors, including drug regimen and level of coverage, vector species, and pre-intervention transmission intensity [23–25]. In order to broadly capture the heterogeneous transmission patterns of LF, we defined transmission archetypes (Table 4). In addition to prevalence levels and drug regimens, we accounted for differences in transmission between Anopheles spp. and Culex spp., which notably differ in their mf-density dependent likelihood of becoming infected [26]. Predicting regional anopheline- or culicine-mediated LF transmission has been shown to require different model formulations and parameterizations [27]. For our analysis we made several simplifications: we assumed transmission of W. bancrofti by Aedes spp. was similar to transmission efficacy by Culex spp., while transmission of Brugia spp. was assumed to be comparable to W. bancrofti transmission by Anopheles spp. Where the primary vector was unclear, infection by Culex spp. was assumed in order to avoid underestimating the number of MDA rounds required. The duration of MDA required to eliminate LF was predicted for the transmission archetypes using a deterministic model of LF transmission, EpiFil [28]. The model used for the current analysis has been described in detail, validated against multiple data sets for both transmission settings with Anopheles spp. and Culex spp., and used extensively to predict LF intervention outcomes [28–31]. Details on model structure, equations, and the approach to obtaining parameter estimates are provided in Supporting Text 1: LF model description. For all transmission archetypes, we ran 500 simulations of once-yearly MDA of varying total durations, drawing from a range of parameter estimates. The lowest number of rounds at which the 95th percentile range of the simulations resulted in an mf prevalence below 1% 50 years after the start of the MDA program was taken as a conservative measure of the number of rounds required to ensure elimination. Population at-risk figures were taken from the WHO PCT database for 2012 and adjusted for population growth using country-specific 2012 United Nations estimates [13,32]. MDA coverage rates were assumed to be 85% for all countries. Except for areas co-endemic with L. loa, treatments are assumed to occur annually. Based on the pre-intervention prevalence distributions, we developed 500 estimates of the number of treatments needed for each country and scenario. Results are reported as the mean number of treatments by region and scenario, along with 95% credible intervals (CI). Our results indicate that interrupting LF transmission in all countries by 2020 is unlikely, though if MDA is drastically scaled-up and expanded, the final round of MDA to eradicate LF could be carried out by 2028 (eradication III; Fig 1). If scale-up continues at the current rate, as modeled in our global elimination and eradication I scenarios, the last round of MDA will not be given until 2050, largely due to slow scale-up in areas where transmission occurs through Culex spp. The eradication II scenario reaches the last round of MDA by 2032. As this scenario assumes that all countries add 20% of their at-risk populations to MDA annually, the last countries to reach local elimination are those that were delayed due to mapping, and whose vector and treatment combination included Anopheles spp. and ivermectin or Culex spp. and DEC, including: Angola, Chad, the Democratic Republic of Congo, South Sudan, Sudan, Zambia, and Zimbabwe. Fig 2 provides a visual representation of the impact different intensities of scale-up and expansion have on time to reach local elimination for each country. Since the scenarios take into account population growth, rapid scale-up of MDA also decreases the number of treatments required. As depicted in Fig 3, the eradication III scenario initially requires substantially more treatments, but by 2024, the treatments under this scenario are projected to be less than that required under all other scenarios. The global elimination scenario is projected to require approximately 3,409 million treatments (95% CI: 3,185m–3,538 million). Expanding the program to all endemic countries will increase the number of treatments to 4,666 million (95% CI: 4,419m–4,904 million). Scaling up MDA more rapidly, as under the eradication II scenario, results in savings of nearly 300 million treatments compared to the eradication I scenario. Under the most optimistic scenario (eradication III), eradication could be achieved with 4,159 million treatments (95% CI: 3,924m–4,382 million). As shown in Fig 1, this represents nearly 750 million treatments more than the global elimination scenario but 210 million treatments less than the intensified eradication scenario (eradication II). Owing to the largest burden, the AFRO region requires the majority of treatments, followed by Southeast Asia. With the shift from global elimination to eradication, the number of treatments required in the Eastern Mediterranean region increases by more than 380 fold due to treatments required for Sudan, which is not considered under the elimination scenario (Table 5). As not all LF endemic countries are considered under the global elimination (comparator) scenario, any eradication campaign will require a massive increase in treatments. However, if LF is to be eliminated in all endemic countries, then rapid scale-up as soon as possible will lead to increased savings—both in terms of time and treatments. Accelerated MDA may also propel the program towards success, as the risk of failure (due to lapses in funding, donor fatigue, or occurrence of calamitous events) potentially increases with extended program duration [33]. It is conceivable that a decrease in program duration may also decrease the likelihood of drug resistance evolution [34]. Noticeably missing from our analysis is India. While India has the greatest burden of LF [35], it has made substantial progress against the disease, having distributed nearly 3. 5 billion antifilarial treatments since 2001 [13]. As such, our model suggests that further rounds may not be necessary for India. However, previous studies have found pockets of systematic non-compliance in India, leading to MDA coverage in those areas to fall below effective coverage [36]. It is therefore possible that transmission of LF may still occur in India. However, in order to remain consistent in our approach, and in recognizing that to provide global estimates we cannot take into account all eventualities, additional treatments for India have not been considered. We sought data from a number of diverse sources. Due to the inherent structure of the LF program, however, our analysis relies heavily on data that have been collected and reported directly by each country. While this arrangement raises a number of issues, discrepancies in the data could also decrease the validity of our estimates. Inconsistencies in coverage data may affect the number of years required to interrupt transmission, while inaccuracies in at-risk estimations would directly impact the number of treatments projected to reach our scenario endpoints. Whether these issues would result in underestimates or overestimates is dependent upon the direction and magnitude of the error. While we avoided underestimating scale-up potential through our eradication III scenario, it is possible that we overestimated the capacity of some countries to scale-up. It is possible that we also overestimated the effectiveness and ability to proceed with rapid scale-up in areas co-endemic with L. loa. While WHO has provisional guidelines for dealing with LF and L. loa co-endemicity, no such areas have been broadly targeted for LF elimination as yet, and thus the effectiveness and feasibility of the strategy remains unclear. At the same time, the mass distribution of long-lasting insecticidal nets (LLINs) in many malaria endemic sites is likely to have a large impact on LF transmission by anophelines [37,38]. Because the impact remains difficult to quantify, and uncertainty remains regarding the duration LLINs have to remain in place, we have not included this here. The time and treatment estimates in this study are based on data and model formulations and parameterizations currently available to the authors. Many of the assumptions and simplifications inherent to our scenarios are in need of closer investigation. Ideally, models would be fit to specific transmission settings within and between countries, as parameter values have been shown to differ by region [29]. Other aspects equally deserving of more attention, but likewise beyond the scope of this project, are the effectiveness of twice-yearly albendazole in concert with vector control for areas co-endemic with L. loa, and the consequences of mid-program delays, [39,40]. Care should thus be taken when interpreting these results, particularly at a country-specific level. Our duration estimates are considerably longer than those proposed under the GPELF, which envisages all endemic countries to reach full geographic coverage by 2016, with post-MDA surveillance in all countries anticipated by 2020 [17]. While this level of scale-up is similar to that proposed under our eradication III scenario, we project the last round of MDA to occur nearly a decade later, in 2028. This divergence arises from differences in the assumed number of rounds of MDA required to interrupt transmission. Depending on baseline prevalence and vector-treatment combinations, our model estimates interruption in transmission to occur after 6–15 rounds of MDA (Table 4). In contrast, the GPELF assumes five years of MDA in all areas [17]. It is worth noting that the durations in this study represent a potentially conservative measure, as they were based on the 95th percentile range of simulations leading to elimination, accounting for the uncertainty in our parameter estimates. This measure was taken to represent the time that could guarantee elimination with a reasonable level of certainty, but does not preclude that shorter durations may be sufficient in many areas. However, the discrepancy between predicted MDA durations and those advocated by GPELF was also evident in previous estimates with both deterministic and stochastic LF transmission models [41]. While aggressive goals for disease elimination and eradication potentially propel campaigns forward, overly optimistic projections could stifle innovations and further investment, ultimately hindering the initiative. This study adds to the growing body of evidence on the feasibility of eradicating LF. While our estimates suggest more time may be needed to reach LF elimination than what is currently projected, the treatment estimates for our scenarios represent 66–89% of that which has already been distributed under the GPELF. Thus, our analysis indicates that with continued commitment, eradicating LF is within reach.
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Title: What Is Needed to Eradicate Lymphatic Filariasis? A Model-Based Assessment on the Impact of Scaling Up Mass Drug Administration Programs Summary: Lymphatic filariasis (LF) is a disease caused by filarial worms transmitted by different types of mosquitos that can lead to massive disability, including elephantiasis and hydrocele. LF has no significant zoonotic reservoir and is thought to be a potentially eradicable disease through once yearly treatment distributed by mass drug administration (MDA). In this study, we set out to determine how many treatments and over how much time it might take to globally eliminate and eradicate LF under different levels of treatment intensities. We created a model that took into account country-specific and disease-specific variables, and found that if the current intensity of MDA is maintained, 3,409 million treatments distributed over the next 37 years will be required. However, if treatment is rapidly expanded to the entire at-risk population in all endemic countries, eradication could be achieved with 4,159 million treatments and in less than half the time. While our estimates suggest more time may be needed to reach LF elimination than what is currently projected, with continued commitment, eradicating LF is within reach.
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Summarize: John Terry has praised Chelsea team-mate Gary Cahill for his 'immense' match-defining block to deny Josip Drmic in England 2-0 win over Switzerland. The former England captain, working as a pundit on Sky Sports, identified Cahill's 70th-minute intervention, with England leading 1-0 at the time, as the crucial moment in a morale-boosting win for Roy Hodgson's men. Terry, who won 78 caps for England before retiring in 2012, said: 'At 1-0, he's got himself back in there. That recovery speed is something people tend to overlook. VIDEO Scroll down to watch No more negativity - Cahill. Praise: John Terry, working as a pundit on Sky Sports, picked out Gary Cahill's block during the second half of England 2-0 win over Switzerland as the crucial moment. Crucial: Gary Cahill denied Switzerland's Josip Drmic a certain goal when England were just a goal ahead. Strong display: Cahill, seen here receiving the captain's armband after Wayne Rooney is subbed off late on, was one of several key performers for England as their Euro 2016 campaign began with a win. Warm welcome: Terry said he'd had a 'great night' after posting this image to his official Instagram account. 'I think Harty [Joe Hart] does well here initially, he delays him [Drmic], pushes him wide and Gaz comes back with a great block there. 'It's very important and an immense challenge. Over five yards, he's very quick. He does that for Chelsea an awful lot as well.' England got their Euro 2016 qualifying campaign off to the perfect start and lifted the post-World Cup gloom as Danny Welbeck's double secured victory in Basel. And Terry was full of praise for the front three of Welbeck, captain Wayne Rooney and Raheem Sterling, who caused endless problems for the Swiss defence. Opinions: Terry made an appearance in the Sky Sports studio to talk about Monday's international action. At the double: Danny Welbeck scored twice as England secured an important victory in Basel. He said: 'They've done very well. The manager and the team will be delighted with this performance. 'The pace up front from Sterling, Welbeck and Wazza [Rooney] stretched the opponents and that definitely benefited us.' On Sterling's contribution, Welbeck added: 'He's been brilliant all night, he's been the difference. Every time he got the ball, he wanted to do something. 'He's very versatile, he can play behind, on the left or on the right. He can open up defences.' Morale-boost: Roy Hodgson celebrates during the 2-0 win, which lifted the post-World Cup gloom. Lionheart: Terry in action as England captain in a World Cup qualifier with Ukraine back in 2009. Before the match, Terry had expressed his support for Rooney's recent appointment as captain following Steven Gerrard's retirement from international football following the World Cup. He said: 'He's still one of the best players in the world and the right choice for the captain. 'He's a leader in the dressing room and the training field, he sets an example on daily basis. I've been on a the receiving end of a few of the shouts from him. 'When you're not performing well in training, he's the first one to let you know.' VIDEO No more negativity - Cahill
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Summary: John Terry was working as a pundit on Sky Sports as England beat Switzerland 2-0 to start their Euro 2016 qualifying campaign. Chelsea defender praised club-mate Gary Cahill for vital block to deny Josip Drmic during the second half. Two goals from Danny Welbeck lifted post-World Cup gloom. Terry was impressed by front three of Welbeck, Wayne Rooney and Raheem Sterling in Basel victory.
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Summarize: LinkedIn has filed a registration statement with the Securities and Exchange Commission, setting the stage for the professional-focused social networking site to go public. Though the company does not reveal a price, number of shares, or date for its offering, it does offer insight into its financials. LinkedIn generated $120 million in revenue for 2009, and $161 million through the first nine months of 2010, where it also turned a profit with a net income of about $10 million. That revenue comes from three sources — advertising, job listings and premium subscriptions. The latter has declined significantly as a revenue source since 2007 (from 53% of sales then to 27% through the first nine months of 2010) while job listings and advertising have become the company's primary growth areas (41% and 32% of revenue, respectively). In its filing, LinkedIn notes that Facebook, Twitter, Google and Microsoft are all potential competitors, writing that they "could develop competing solutions or partner with third parties to offer such products." That said, the company finished 2010 with more than 90 million members, and claims to be adding 1 per second in its filing. Recently, private trading of LinkedIn shares valued the company at nearly $3 billion. That would make the company's debut bigger than that of Demand Media, which went public on Wednedsay at a valuation of around $1.5 billion. With Skype, Groupon and potentially Facebook also in the mix of IPO candidates this year, LinkedIn's offering will be another big barometer for the public's appetite for Internet IPOs. A name change, not making any money, a lot of debt: If that description sounds like a recipe for a dicey online date, then investors might want to apply the same principles to the coming week's IPO of Web-site operator FriendFinder Networks Inc. View Full Image none needed A FriendFinder page:'social networking' but not exactly Twitter. The page includes a link at top-center of the page to IPO information. FriendFinder, which operates more than a dozen Web sites aimed primarily at people looking for relationships or flings, changed its name 18 months ago from Penthouse Media Group. It isn't profitable, and until recently was in default on its debt covenants. It struck a deal in October with its creditors to waive its defaults in exchange for all the money it raises in its initial public offering. It is hoping for $200 million in net proceeds from the sale of 20 million shares through RenCap Securities and Ledgemont Capital Markets LLC, according to its prospectus, with an estimated price range of $10 to $12, and a listing on the New York Stock Exchange with symbol FFN. The company is portraying itself as a potential catch in the social-networking industry, a sector that has few stand-alone stocks and a lot of buzz. It describes itself as a leading "social networking and multimedia entertainment company," and in prerecorded roadshows compares itself with privately held Facebook Inc. and MySpace, owned by News Corp., publisher of The Wall Street Journal. But FriendFinder differs from both: Members on Facebook and MySpace tend to keep their clothes on, while several of FriendFinder's sites encourage the opposite. Though the firm does operate several general-interest dating sites—FriendFinder, BigChurch.com and SeniorFriendFinder.com—its largest is AdultFriendFinder.com, which targets swingers and people who want to meet, according to the Web site, partners "tonight." Executives at FriendFinder are somewhat circumspect when it comes to describing their Web sites in the company's prerecorded roadshow, available at RetailRoadshow.com. The company's dark-suited, bespectacled chief executive, Mark H. Bell, describes one of the Web sites, GetItOn.com, as a fast-growing new site that allows people to text search and "type in a sentence like, 'I'm looking for people who ride motorcycles.' " Mr. Bell didn't return a call seeking comment on the IPO. FriendFinder isn't exactly the kind of social networking that most investors have in mind when they talk about a site like Twitter or LinkedIn someday going public, says Josef Schuster, founder of IPOX Schuster LLC, a Chicago financial-services firm specializing in IPO products. Aside from some of the racy content on FriendFinders' sites, the $471 million of debt the company carries makes it look more like a leveraged-buyout turnaround than a high-growth new-media prospect, Mr. Schuster says. FriendFinder also differs from MySpace and Facebook in its business model: Most of its revenue is generated by converting casual viewers into paying subscribers, not advertising. While subscription-based revenue is more predictable than advertising, it has been affected by global economic conditions, and net revenue remained flat at $244 million in the first nine months of 2009, compared to the same period of 2008. The company's net loss narrowed to $27 million from a loss of $32 million a year earlier. The company plans to increase its focus on driving more advertising revenue in the future, according to its prospectus. Because many of its sites focus on particular tastes—be it a spiritual Christian wife or a casual fling with a sadomasochist—the subscriber model does make sense, says Michael Gartenberg, vice president of strategy and analysis at market research firm Interpret LLC. "If you have more eclectic tastes that go beyond what might come up in polite conversation, it could be hard to find people with similar interests using traditional social sites," Mr. Gartenberg said. "These are the folks who would be the people most likely to pay for a subscription service in order to match with someone who shares their particular interests and needs." FriendFinder isn't being shy about marketing its IPO, either. Besides doing a roadshow for investors, it has sent out blast emails to its employees, affiliates and the various sites' members telling them how they can participate in the stock offering. Write to Lynn Cowan at [email protected]
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Summary: After weeks of speculation, LinkedIn has confirmed what many suspected-it will go public later this year. The career-focused social networking site filed a registration statement with the SEC yesterday, setting the stage for the company to become the biggest social network yet to land on Wall Street, reports Mashable. With revenues of $164 million in the first nine months of 2010 and 90 million users, LinkedIn is one of the biggest social networks this side of Facebook, and financial observers are keenly watching its IPO for a sense of broader market demand. Experts say 2011 could be a big year for social networking websites going public. "Facebook has definitely escalated people's interest in the sector and I think there's a lot of demand (for more Internet IPOs)," said one analyst. Friend Finder Networks, the company behind the smutty hookup site Adult Friend Finder, filed its intention to go public last week. However, with the company's subscription-heavy revenue model, $471 million in debt, and risque content, FFN is considered a more unusual case.
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Summarize: Colonna d'aria su un fondo o su una costruzione La colonna d'aria è definita come la proiezione verso l'alto dello spazio aereo sovrastante un suolo o una costruzione. La colonna d'aria diventa giuridicamente rilevante (ed economicamente importante) in quanto è lo spazio che andrebbe ad occupare una futura elevazione di un edificio già esistente oppure una costruzione realizzata su un suolo. Di fatto la colonna d'aria sopra un suolo permette la realizzazione di una futura costruzione sul suolo (o la sopraelevazione di una costruzione già esistente). La titolarità della colonna d'aria sovrastante il suolo (o una costruzione) spetta al proprietario del suolo; la titolarità della colonna d'aria è strettamente connessa con il diritto di costruire sul suolo o di sopraelevare su un edificio già esistente Di conseguenza, il diritto di costruire sul suolo o di sopraelevare una costruzione già esistente spetta al medesimo soggetto titolare della colonna d'aria sul suolo o sulla costruzione (salvo le ipotesi in cui è possibile scindere tali facoltà cone nel diritto di superfice ex art. 952 cc o nel diritto di sopraelevare nel condominio ex art. 1127 cc condominio ex art. 1127 cc). Come ogni diritto anche la costruzione nello spazio compreso nella colonna d'aria può essere limitato o escluso, questi limiti possono derivare da norme di legge (divieti di costruire ecc.) oppure da preesistenti diritti di terzi (come balconi, finestre realizzati dall'originario proprietario dell'edificio al momento della costruzione). Infatti, può capitare che edifici limitrofi abbiano il diritto di veduta sul fondo e, da un lato, tale diritto di veduta non deve essere limitato dalla realizzazione di altre sopraelevazioni del suolo, oppure, dall'altro alto, tale diritto di veduta deve essere esercitato in appiombodiritto di veduta deve essere esercitato in appiombo fino alla base dell’edificio senza ostacoli, potendosi opporsi alla costruzione (come una veranda o un pergolato realizzato a copertura del terrazzo di un appartamento sottostante) che pregiudichi l’esercizio di tale suo diritto. Quando si pensa alla lesione del diritto (esclusivo) sulla colonna d'aria del proprietario del suolo (o della costruzione) il problema non è ipotizzare in modo astratto se il diritto sulla colonna d'aria può essere violato (e di conseguenza ipotizzare se il diritto sulla colonna d'aria può essere tutelato), ma il problema reale è ipotizzare in modo concreto come è possibile ledere (o occupare) la colonna d'aria. Per rendere chiara la vicenda si potrebbe pensare ad una costruzione di un edificio i cui balconi aggettanti "occupano" la colonna d'aria di un fondo limitrofo.
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Summary: La Cassazione del 20.4.2018 n. 9877 ha affermato che in presenza di una invasione di colonna d'aria di un fondo altrui, nessun rilievo ha la deduzione per la quale sul fondo altrui si aprirebbero finestre del condominio, quindi si tratterebbe di uso meramente più intenso della medesima veduta ex art. 1102 cc. In realtà, non si tratta di uso di cosa comune ex art. 1102 cc., ma di servitù su cosa aliena e nelle servitù è sancito il divieto di innovazioni che rendono più gravosa la condizione del fondo servente art. 1067 cc.
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Summarize: By. Daily Mail Reporter. PUBLISHED:. 14:48 EST, 29 April 2013. |. UPDATED:. 08:28 EST, 30 April 2013. Guilty? Teresa Moses, 23 at the time of this 2006 mugshot, was found not guilty by reason of insanity in 2010 of murder, torture and child abuse after her son, Raijon Daniels, 8, was found dead next to a pool of his own vomit. A San Francisco woman who tortured, abused and murdered her eight-year-old son is a free woman today, seven years after her little boy was found dead in a pool of his own vomit in 2006 and three years after she was sentenced. Teresa Moses, 30, from Richmond, was declared not guilty by reason of insanity in 2010 for killing her son Raijon Daniels and has spent the last three years in a mental hospital receiving treatment for a condition that doctors have called 'fleeting'. No longer deemed a risk to society, the rehabilitated mother has been placed in Contra Costa County's Forensic Conditional Release Program, known as Con-REP under which her continued recovery will be monitored closely. Ms Moses was mother to Raijon and a younger daughter who was placed in foster care while she awaited trial. The news of Ms Moses' premature release will no doubt come as a shock to many who followed the story in 2006. Ms Moses was charged with torture and child abuse after Raijon's body was discovered in her house on October 27th. Autopsy reports showed that the boy had been beaten, scratched and starved and further investigation revealed that he had been locked in a bedroom in Ms Moses' house where he was strapped to the bed and watched by surveillance cameras. Ms Moses was said to have used a high pressure water hose to wash feces off his body and forced her son to take baths in his own waste mixed with cleaning detergent Pine Sol to deter him from relieving himself. On the day of his death, she admitted to having poured the highly chemical product on his genitals. Responding to news of her client's release on Saturday, deputy public defender, Stephanie Regular told Contra Costa Times: 'The incident which led to Ms. Moses' commitment is a horrible tragedy - a tragedy which Ms Moses will regret for the rest of her life. 'However, I also think it is important to remember that Ms Moses was legally insane at the time she committed the crime. The person she was then is not the person who she is now.' Victim: Raijon Daniels, 8, was strapped to a bed by his mother who poured Pine Sol on his genitals, made him wash in his own waste and starved him. Ms Regular's claims were backed up by three doctors during the trial who all testified that Ms Moses was legally insane at the time of the torture. A friend of the defendant who went to see her shortly after she was taken to hospital told the Contra Costa Times that Ms Moses was genuinely'shocked' to hear of her son's death. She told her friend that she had learned her parenting skills from watching ants while growing up in the projects of Richmond. 'Of course, I thought she meant "aunts"', said the woman who preferred to remain anonymous. 'No, she meant the insects.' Though Ms Moses' medical records are kept strictly private, she was described by one attorney as having paranoid delusions. Mr Butts quoted doctors in Napa as describing her condition as 'a mental illness than can be fleeting.' Explaining the way the law treats insanity cases such as Ms Moses', deputy district attorney Derek Butts said: 'As unfortunate as it may be, a person can suffer a short period of insanity and not be held accountable for the crimes they commit, however heinous they may be. 'It is a difficult part of our justice system to accept, especially under circumstances such as these.' He also added that a not guilty by reason of insanity verdict in a burglary case would be given the same punishment as a murder case. The District Attorney's office raised objections to Ms Moses' release in February but since all the defendant's doctors at Napa State Hospital supported it and called her a'model patient', Mr Butts did not pursue a trial. For Ms Regular, the reversal has shown the remarkable way in which the mentally ill can be given a second chance provided they receive the right treatment and support. She referred to the agreement made between doctors, lawyers and judges, 'an insurmountable task.' Sentenced: Ms Moses has served only three years in a mental hospital and has been released on the advice of doctors who called her mental condition at the time of the torture 'fleeting' and her transformation, remarkable. While Ms Moses' attorney and doctors. have applauded her recovery and have pointed to her work with 'New. Start', a program she co-facilitated at the hospital, her nameless. friend has called her transformation'remarkable'. 'For. her, being in Napa has been a real gift, and she took it as that,' she. said. 'One of her graces is she really wanted to take responsibility and. wanted help and took advantage of every opportunity that she could.' But one person struggling to understand the transformation so readily is Raijon's father, Desmond Daniels. 'How can three years pass and she be OK after the acts that she did? How could someone who put Pine-Sol in a child's food, allow a child to jump from a second-story building, how could they be a model patient?' he wonders. 'I still have so many questions.' Still, even despite the way in which his young son's life ended, Mr Daniels has astonishingly found a way to forgive Ms Moses. 'In the beginning, I really wanted to see her suffer because she had made him suffer,' he admitted. "But I can't pass judgment, I wasn't in the home. I forgive her, as crazy as that may sound, because of the faith that I have. Who am I to beat her down and dog her?'
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Summary: Teresa Moses' son Raijon Daniels was found dead at her home in 2006. Autopsy reports and an investigation found the boy had been strapped to his bed, beaten, starved and tortured with Pine Sol and cold water hoses. Ms Moses was found not guilty by reason of insanity in 2010 and sentenced to a mental hospital. Doctors treating her have called her a'model patient' who has been transformed.
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Summarize: By. Amy Oliver. PUBLISHED:. 11:29 EST, 29 May 2012. |. UPDATED:. 13:07 EST, 30 May 2012. A senior nurse was told by her boss that she was 'the wrong colour' to get a top job in nursing, a misconduct hearing was told. British-born Sarina Saiger, who has Indian heritage and a Phd, was advised to 'go back to where she came from' rather than try to become a director of nursing in Cumbria. The Nursing and Midwifery Council heard how Dr Saiger's boss, Bruce Skilbeck, made the remarks during a 2005 appraisal into her performance. Dr Saiger, who has already been awarded £115,000 in damages at an earlier tribunal after being sacked in 2008, said: ‘After we did a brief resume of the work I had been doing, I remember clearly he said technically I was the most brilliant nurse he had ever worked with. Victim: British, Dr Sarina Saiger, who has Indian heritage and a PhD, was labelled the 'wrong colour' to be a director of nursing in Cumbria. ‘But I would never be a director of nursing in Cumbria because I was the wrong colour and wrong culture for the organisation. ‘He said I needed to go back to where I came from to get a director of nursing position.’ Skilbeck, who was the director of nursing for North Cumbria Acute Hospitals NHS Trust but has now retired, has been hauled before the NMC charged with two counts of misconduct against Dr Saiger which was allegedly ‘racially motivated’ Halifax-born Dr Saiger had been parachuted into the trust in 2004 to raise standards as assistant director of nursing. She had excelled academically and harboured ambitions of going on to the top job, but was shot down by Skilbeck in the appraisal meeting on November 3, 2005, shortly after he joined the trust. Dr Saiger added: ‘I was just stunned, I have been a nurse since 1986 and I’ve faced many things in nursing. ‘It’s a difficult profession, but I never expected to hear that, not from someone who is supposed to be of an intellectually senior standing.’ A tearful Dr Saiger told the hearing how she was studying for her PhD and raising her son Michael while working in an increasingly hostile environment. She said: ‘I was finding things out after the event, excluded from meetings. My PA Maria was absolutely loyal and would get me information because I wasn’t kept informed. ‘I didn’t have an office; I was working out of the back of my car. Other nurses and senior managers who were appointed after me were given an office and a desk, and access to equipment. ‘I was actually quite lonely.’ Eventually, Dr Saiger launched a grievance against the trust, including the comments made by Skilbeck. But a prolonged investigation delivered a ‘whitewash’ which absolved senior managers of blame, she said. She said: ‘It was just a farce really. It completely and utterly absolved everyone and anything. ‘They would find reasons, excuses, policies - everything I was saying was completely untrue.’ Skilbeck is also accused of violently grabbing Dr Saiger by the arm and dragging her along a corridor, on May 31, 2007. Dr Saiger reported the incident, which left her with a badly bruised arm, to the police, but no charges were brought. She was sacked from her £42,000-a-year post in 2008, but launched an employment tribunal case which ruled she had been a victim of a campaign of racially-motivated discrimination and harassment. The tribunal found she had been subjected to an ‘intimidating, hostile, degrading, humiliating or offensive environment’. The tribunal upheld 16 of her 27 complaints and awarded her £115,000 in damages. Skilbeck, who is now retired, did not attend the hearing in central London today, but emailed papers to the NMC to support his case. The hearing into his alleged misconduct continues. Sorry we are unable to accept comments for legal reasons
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Summary: British-born Sarina Saiger has Indian heritage. Told she was the 'wrong colour' to get top nursing job. Former top boss Bruce Skilbeck charged with two counts of misconduct against Saiger. Has already been awarded £115,000 in an employment tribunal after being sacked from her post in 2008. That tribunal ruled she had been subjected to an 'intimidating, hostile environment'
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Summarize: Sally Anne Bowman's mother today spoke of her anger at Spanish police after discovering that they had a chance to catch her daughter's killer years before he murdered her. Indisputable DNA evidence has emerged that links Mark Dixie, currently serving a life sentence for the 2005 murder of 18-year-old Sally in Croydon, to a rape and two sex assaults in the Costa Del Sol in 2003. A Dutchman was jailed for the crimes in 2005 without forensic evidence. His lawyer strongly believes he was wrongly convicted. Tragic: Linda Bowman (right) has expressed her anger at the Spanish police, who she believes could have apprehended Mark Dixie before he murdered her daughter, Sally (left), if they'd used DNA evidence. Linda Bowman says she is dismayed at the thought that if the Spanish authorities had investigated the case more thoroughly, her daughter might still be alive. She told MailOnline: 'I am absolutely appalled. An innocent man has spent 11 years of a 16 year sentence in prison for a crime he did not do. 'I feel very strongly that if the Spanish authorities had done their jobs properly, my Sally Anne would be alive. 'He did not assault those three women in Spain - Sally Anne's killer did. He is someone's son and he is locked up for something he didn't do.' The man serving time for the attacks in Spain is 41-year-old Romano Van der Dussen. He's served 11 years of a 16-year sentence for the crimes, despite forensic evidence linking Dixie to the rape emerging almost eight years ago, after Interpol sent his DNA to police forces across Europe. Official documents – seen by MailOnline – show that DNA found on the Spanish rape victim in an examination straight after the attack, match a DNA profile for Dixie on Interpol's Veritas database. DNA evidence has emerged linking Mark Dixie (left), serving life for the 2005 murder of Sally Anne Bowman, to a rape and two sex assaults in the Costa Del Sol in 2003 that Romano Van der Dussen (right) was jailed for. Yet Spanish authorities refuse to accept the miniscule 54 million-to-one chance that a DNA profile from the sex attack did not match one of Dixie supplied to Interpol by Scotland Yard. They have requested a fresh DNA sample from Dixie so more tests can be carried out - before they release Romano. In April this year a Spanish court sent an international letter of request to the Home office requesting a saliva sample from Dixie to confirm the DNA match as 'a matter of urgency.' In the event of Dixie refusing to supply a profile, the court has asked that the Spanish rape DNA sample is run through the UK National DNA Database to compare with genetic codes held on Dixie. But Romano's lawyers say they have heard nothing since then and Romano is now facing the prospect of another five years in a Spanish jail for a crime he did not commit. Desperate Romano is now urging the British government to intervene and hand over the DNA so his conviction can be quashed. Linda said: 'When the DNA was found on Sally Anne it was sent to Holland, Australia, Spain and all over. 'If the Spanish police and judges had done the same, they would have got him. 'If they had done their work properly, they would have taken proper DNA from the three victims in 2003 and done what we did. It gets me choked thinking about it... even saying that thing's name.' She added: 'I have campaigned so much about DNA and the national database. To me, this is all about the DNA. Dixie was caught nine months after 18-year-old Sally Ann's murder when DNA taken from him after a minor pub scuffle was matched to the crime. 'It can incriminate people but it can also eliminate people. 'We are born with a birth certificate, we have a national insurance number and a passport number - why can't we all have our DNA taken at birth too? 'This would not have happened. My child would still be here. I am so angry.' Dixie was living in Fuengirola, on the Costa Del Sol, when the Spanish victim was raped in August 2003 in startling similar circumstances to Sally's murder in Croydon, South London. Spanish court documents, obtained by MailOnline, describe how in the early hours of the 10 August 2003, two women were robbed and sexually assaulted in the street in Fuengirola, southern Spain. The attacker then carried on his crime spree – targeting a third female victim who was walking along the street in he Costa del Sol town at around 4:30am. He leapt on her, hit her several times before throwing her on the floor and raping her. The case bears chilling similarities to Dixie's rampage on September 25, 2005 - the night stunning blonde model Sally Anne was stabbed to death and raped. After downing drinks and snorting cocaine with friends in Croydon, Dixie is believed to have attacked a female motorist and stolen her mobile phone. He then watched Sally Anne being dropped home by her boyfriend Lewis Sprotson at around 4.10am before stabbing her in the neck and chest and having sex with her as she lay dead on her doorstep. Romano's lawyers say their client is innocent of the Spanish crimes and was mistakenly convicted solely on 'inconsistent' statements from the victims who identified him from photos. He was convicted solely on eyewitness testimony - no DNA or fingerprints linked Romano to the crimes. It is believed Romano may have been the victim of mistaken identity as he bears a striking resemblance to the Dixie – with the same brown curly hair and thick eyebrows. In 2007, his lawyer in Spain discovered that DNA found on the rape victim 'was compatible' with that of Dixie's DNA profile on Interpol database. But despite years of legal wrangling, the Spanish courts said they would not order a retrial or acquit Romano until they receive a saliva sample from Dixie – so that his DNA can be tested more throughly alongside the rape victim's. Sally Anne Bowman on her 18th birthday in Kos with boyfriend Lewis Sprotson. So far Dixie has not given this sample so Romano's last hope is that the Home Office agrees to share Dixie's DNA profile from the UK's national database with the Spanish authorities. Romano's lawyer in Spain Silvio Garcia Sierra, told MailOnline: 'We are waiting for the British authorities to answer to this request, meanwhile Romano is in prison for a crime that all the evidence shows he has not committed. I requested the suspension of the sentence to the Supreme Court, which was not granted because the court did not accept my request. 'The court in Fuengirola, which is leading the case of DNA and other proceedings, is working flawlessly, but we are in the hands of the British authorities, which are in no hurry to forward any DNA testing of Dixie to the Spanish courts.' He added: 'This conviction was based solely on the statements of victims through photographic reconnaissance and recognition in the trial, without any evidence to DNA or fingerprints on the scene. 'Despite the inconsistencies in the statements of victims and witnesses who claimed to have witnessed one of the assaults from a balcony, the court sentenced for these crimes.' Romano, meanwhile, said he has had to endure 'years of torment' while serving his sentence in Palma prison, Majorca. A Dutch charity named the Prison Law Foundation has been campaigning for Romano's release. Dixie was caught nine months after 18-year-old Sally Anne's murder when DNA taken from him after a minor pub scuffle was matched to the crime. He was jailed for life and told he must serve at least 34 years behind bars. The chef was a serial sex offender and detectives have long believed he is responsible for more sex attacks and even murders. At age 16 he was charged with indecent assault after holding a woman a knifepoint and fondling her breasts. Just a year later he was jailed for six months after attacking a mum-of-three. At his murder trial, a Thai woman gave evidence about how Dixie had stabbed and raped her at her home in Subiaco, Western Australia in 1998. Campaign: Linda Bowman wants the DNA of every British citizen to be added to a national database as soon as they're born. The killer's DNA matches that left at the scene of a sexual assault in 2001, where he is believed to have masturbated in front of a woman in a telephone booth. Dixie has also been a prime suspect in one of the notorious unsolved murder cases in Australia - the Claremont killings between 1996 and 1997, as it is believed he was in the area at the time of the killings. The Home Office has so far declined to comment on the case
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Summary: Linda Bowman has spoken of her anger over the work of Spanish police. She said they missed a chance to arrest the man who killed her daughter in Croydon in 2005. DNA evidence emerged linking Mark Dixie to the sex assaults carried out in Spain in 2003. However, a Dutchman has spent 11 years behind bars for the attacks after he was jailed on eye witness testimony. Bowman says forensic evidence would have led to Dixie's arrest.
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Summarize: CROSS REFERENCE TO RELATED APPLICATIONS This application claims benefit under 35 U.S.C. 119(e) of Provisional Application No. 62/243,837, filed Oct. 20, 2015 and Provisional Application No. 62/299,634, filed Feb. 25, 2016, and claims benefit under 35 U.S.C. 119(a) of Canadian Patent Application filed Jul. 19, 2016 under the same Applicant and title as the present application. Each of the forgoing applications is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The present invention relates generally to orthodontics. More specifically, the present invention relates to a direct bonding appliance system that increases the accuracy and efficiency of bonding attachments to teeth for orthodontic purposes. DESCRIPTION OF THE RELATED ART Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. By convention, orthodontic attachments (such as brackets) may be bonded directly to the patient's teeth. To facilitate bonding, the tooth surface is prepared (etched), and a bonding agent is placed. Adhesive is then placed on the base of each attachment, which in turn is then placed on the selected tooth surface (facial or lingual). Prior to curing (completing the bond), the attachment may be repositioned (moved along the tooth surface) to a preferred location on the tooth. Once the location of the attachment is determined to be satisfactory, the adhesive is cured (bonding the attachment to the tooth). Many challenges exist whilst utilizing the aforementioned technique. Assessing the optimal position for attachment placement intra-orally (where views are obstructed) is difficult. Contamination of the bonding surface by the contents of the patient's oral cavity must also be controlled. Therefore employing “moisture control” appliances further restricts the operator's access/view. This restricted access renders optimal placement of attachments near impossible to achieve. Ergonomics are also not favourable, as the operator must often contort their body to access (view) tight/crowded areas. Ultimately, these restrictions impair the accuracy of the attachment placement, and also extend the time required to satisfactorily bond attachments on the teeth. Attempts have been made to mitigate these challenges. For example, Align Technology Inc. has proposed a template having openings or windows therein for guided placement of orthodontic brackets in family of U.S. patents including U.S. Pat. No. 7,056,115, U.S. Pat. No. 7,578,673, U.S. Pat. No. 7,600,999, U.S. Pat. No. 7,658,610, U.S. Pat. No. 8,496,473 and U.S. Pat. No. 8,734,149. However, several limitations of the prior art prevent optimal (most accurate and efficient) direct bonding of orthodontic attachments to teeth from being achieved. The ‘template’ designs of the prior art use thermal forming dental material, which may fail to produce an intimate cohesive seal at the template to tooth interface due to the rigidity of the final template product. The resulting space between the template and teeth allows excess adhesive on the orthodontic attachment to “splay” under the template tray upon seating. This presents an issue with removing the excess adhesive prior to curing, or worse it creates more laborious work to remove the excess adhesive after curing (and after template removal). The ‘template’ designs of the prior art may also fail to produce an intimate cohesive seal at the template-to-attachment interface. The “attachment support edge” will not adapt to provide a clear positive “vertical stop” when placing the orthodontic attachments. That is, the potential space left between the tooth and the template at the perimeter of the window at the closed end thereof may allow the attachment to partially slip into this space, i.e. slide slightly past the proper target position. The ‘template’ designs of the prior art extend past the attachment base gingivally. Assessment of “attachment seating” is more challenging when the template extends gingivally past the attachment base. That is, once the attachment has been inserted into the respective window, access to the perimeter of the attachment to test the interface between the tooth and the attachment is prevented or severely restricted, at least until all of the attachments have been placed and the template removed. Also, potential issues arise in this removal of the template after bonding. It has been proposed in the prior art, that increasing the ‘thickness’ of the template will provide better guidance to the operator when placing attachments. However, the ‘template removal issues’ are further exacerbated if the ‘template’ is made thicker/more rigid. Therefore, with the template spanning gingivally past the attachment, the operator must either sacrifice ‘flexibility’ in the template removal (with a thicker template), or sacrifice the ‘guidance’ (ease of placement), of the individual attachment (with a thinner template) The ‘template’ designs of the prior art do not address the utility in creating “repositioning appliances”. Orthodontists by convention will often reposition multiple attachments over time to improve/achieve desired tooth movement, or place attachments on surfaces where access was previously restricted by overlapping teeth. The ‘template’ designs of the prior art do not address ‘lingual placement’ of attachments. In fact, prior art fabrication indicates removing significant portion of the lingual surface on the ‘template’ (aligner) to help facilitate removal. The lingual surfaces of anterior teeth (being concave in nature), and posterior teeth (below the crest of curvature), pose an issue in removal with gingival extensions reaching past the attachments. The ‘template’ designs of the prior art do not address bi-maxillary or inter-arch (both top and bottom arches connected in one piece) unit advantages. The efficiency of placing attachments is substantially increased if the patient is held in biting position during bonding. Inter-arch (bi-maxillary) designs also offer significant ‘moisture control’ advantages that may be incorporated. Controlling contaminants (moisture) present in the oral cavity is paramount in the bonding process. In these respects, the direct bonding system according to the present invention substantially departs from the conventional concepts and designs of the prior art, and in doing so provides an apparatus primarily developed for the purpose of increasing the accuracy, efficiency and comfort of the orthodontic direct bonding procedure. SUMMARY OF THE INVENTION In view of the foregoing disadvantages inherent in the known types of apparatuses in the prior art, the present invention provides a new orthodontic direct bonding system construction wherein the same can be utilized for increasing the accuracy, efficiency, and comfort of the direct bonding procedure. The general purpose of the present invention, which will be described subsequently in greater detail, is to provide a new orthodontic direct bonding system that has many of the advantages of the prior art mentioned heretofore and many novel features that result in a new orthodontic direct bonding system which is not anticipated, rendered obvious, suggested, or even implied by any of the prior art either alone or in any combination thereof. An orthodontic ‘direct bonding stent’ is disclosed to support ideal positioning of physical (real) attachments on a patient's teeth in order to facilitate orthodontic tooth movement. Novel designs of direct bonding stents are disclosed. Also, methods are disclosed for fabricating the direct bonding stents. The methods include digitizing the patient's teeth; adding virtual attachments to predetermined locations on the digitized virtual model; 3-D printing the virtual model (with virtual attachments attached) to create a physical model (with printed attachments attached), and then lab fabrication of the direct bonding stent utilizing the physical 3-D printed model. Another method discussed involves creation of the stent virtually and fabrication utilizing additive manufacturing technologies (such as stereolithography or 3D printing) to produce the direct bonding stent. The direct bonding stent is designed to provide negative impressions, or partial footprints or window frames of the orthodontic attachments. These window frames act as guides for placement of real attachments at a predetermined location on the patient's tooth. Advantages of the stent may include one or more of the following: The methods, steps, and algorithms described above that are used to form the requisite digital representation of an orthodontic direct bonding stent. Additional features, bodies, or component data files can be used in addition to the tooth files used to form said stent. The elements, or concepts of the creation methods described above can be mixed or matched. That is, a file may also be created by using some steps from one method and other steps from one or more other methods. The stent allows orthodontic brackets to be ideally positioned on teeth more efficiently. The ideal placement may be determined physically or virtually, and the placement of the brackets can be done using a stent that is, in essence, a removable guide. According to one aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows for placement of the attachments into abutment with the patient's teeth through said windows, said plurality of windows each passing through said attachment support wall in a facial-lingual direction, each having an open end at a terminal end of the attachment support wall that is situated oppositely of the arc-shaped occlusal portion in the gingival direction, and each terminating at a closed end that lies opposite to said open end and adjacent to the occlusal arch-shaped portion; wherein a depth of each window is less than a height of the respective attachment such that the attachment support wall of the stent stops short of a gingival end of the attachment when the respective attachment is seated against the closed end of the window. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows each passing through said attachment support wall in a facial-lingual direction for placement of the attachments into abutment with the patient's teeth through said windows; wherein a contact surface of the attachment support wall that is positioned for placement against the patient's teeth consists of semi-rigid material of elastomeric properties so as to provide an intimate-cohesive seal between said attachment support wall and the patient's teeth. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows each passing through said attachment support wall in a facial-lingual direction for placement of the attachments into abutment with the patient's teeth through said windows; wherein the attachment support wall extends gingivally from the occlusal arch-shaped portion in two opposing directions, and the plurality of windows comprises a first set of windows situated superiorly of the occlusal arch-shaped portion for placement of respective attachments on the patient's maxillary teeth and a second set of windows situated inferiorly of the occlusal arch-shaped portion for placement of respective attachments on the patient's mandibular teeth. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; a plurality of windows each passing through said attachment support wall in a facial-lingual direction for placement of the attachments into abutment with the patient's teeth through said windows; and a second wall extending in the gingival direction from the arc-shaped occlusal portion along a second side thereof opposite to the attachment supporting wall, wherein the second wall is of lesser height than the attachment supporting wall in the gingival direction. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; a plurality of windows each passing through said attachment support wall in a facial-lingual direction for placement of the attachments into abutment with the patient's teeth through said windows; and a second wall extending in the gingival direction from the arch-shaped occlusal portion along a second side thereof opposite to the attachment supporting wall; wherein the attachment supporting wall extends from the arch-shaped occlusal portion along a facial side thereof, the second wall extends from the arch-shaped occlusal portion along a lingual side thereof, and the second wall is shaped to terminate short of a cresting point of a lingual side curvature of each tooth in a facial-lingual plane thereof. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows each passing through said attachment support wall in a facial-lingual direction for placement of the attachments into abutment with the patient's teeth through said windows; wherein the attachment support wall extends from the occlusal arch-shaped portion at a lingual side thereof. According to another aspect of the invention, there is provided a method of producing a stent for guiding placement of attachments on a patient's teeth, the method comprising, on a working model that includes physical representations of both the patient's teeth and said attachments to be placed thereon, applying a flowable material over the physical representations of the patient's teeth in a manner conforming closely against the physical representations of the attachments, without facially covering said physical representations of the attachments, whereby setting or curing of the flowable material creates a stent in which negative impressions of the physical representations of the attachments create windows passing fully through the stent in lingual-facial directions to accommodate placement of the attachments against the patient's teeth at prescribed positions thereon through the windows, whose positions on the stent are dictated by positions of the physical representations of the attachments in the working model. According to another aspect of the invention, there is provided a method of placing attachments on a patient's teeth, the method comprising: obtaining a stent that has been custom-fabricated for said patient's teeth and, that comprises semi-rigid material of elastomeric properties, and that comprises windows therein for guided placement of said attachments to the patient's teeth through said stent; positioning the stent on the patient's teeth in a manner placing the semi-rigid material in cohesively and intimately sealed contact with the patient's teeth at a perimeter of each window; adhering each attachment to the patient's teeth through a respective one of said windows, during which splaying of the adhesive out from beneath the attachments is prevented by the cohesively and intimately sealed contact between the stent and the patient's teeth. According to another aspect of the invention, there is provided a stent for use in repositioning of select attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows each passing through said attachment support wall in a facial-lingual direction for placement of the select attachments into abutment with the patient's teeth through said windows; and bypass openings in the support wall that pass fully therethrough in the facial-lingual direction and that open into the support wall from a terminal end of the attachment support wall that is situated oppositely the occlusal arch-shaped portion in the gingival direction, each bypass opening being larger than the windows so as to space the stent away from other existing attachments on the patient's teeth that are not being repositioned during repositioning of the select attachments. According to another aspect of the invention, there is provided a method of producing a stent for guiding placement of attachments on a patient's teeth, the method comprising using computerized means to: producing a virtual model that includes a virtual dentition that is representative of the patient's teeth and virtual objects that are representative of the attachments to be placed on the patient's teeth and have been virtually positioned on the virtual dentition at desired target locations for the attachments; adding a virtual stent to the virtual model, including customizing a shape of a support wall the virtual stent to include virtual windows therein that reside at the target locations occupied by the virtual objects and that open through the support wall of the stent in a facial-lingual direction; and from the virtual stent, producing a physical stent in an automated additive manufacturing process, whereby the physical stent features actual windows corresponding to the virtual windows of the virtual stent for use in guiding placement of the attachments on the patient's teeth via said windows. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows for placement of the attachments into abutment with the patient's teeth through said windows, said plurality of windows each passing through said attachment support wall in a facial-lingual direction, each window having an open end at an originating end of the attachment support wall where said attachment support wall joins with the arc-shaped occlusal portion, and each window terminating at a closed end that lies opposite to said open end in an occlusal-gingival direction. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows for placement of the attachments into abutment with the patient's teeth through said windows, said plurality of windows each passing through said attachment support wall in a facial-lingual direction and each having a depth measured between an open end of the window and an opposing closed end thereof in a gingival-occlusal direction; wherein at least one posteriorly located window is deeper in the gingival-occlusal direction at a distal side of said posteriorly located window than at an opposing mesial side thereof to improve access to said posteriorly located window from the mesial side during placement of the respective attachment in said posteriorly located window. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows for placement of the attachments into abutment with the patient's teeth through said windows, said plurality of windows each passing through said attachment support wall in a facial-lingual direction; wherein the support wall comprises at least one inter-dental area of reduced lingual-facial thickness that is located between a respective pair of adjacent windows and slopes distally and gingivally toward a more posterior one of said respective pair of adjacent windows for improved mesial access to said more posterior one of said pair of adjacent windows. According to another aspect of the invention, there is provided a stent for guiding placement of attachments on a patient's teeth, the stent comprising: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of voids in said attachment support wall, each void passing through said attachment support wall in a facial-lingual direction; a respective bridge portion of the attachment support wall spanning across each void at a gingival side thereof opposite the occlusal arch-shaped portion for seating of a respective attachment against said bridge portion at a gingival side thereof opposite the void; wherein a tooth-facing side of each respective bridge portion is spaced outwardly from a tooth-facing side of an occlusal portion of the attachment support wall that lies across said void from the respective bridge portion and joins with the occlusal arch-shaped portion, whereby in a worn position of the stent, each bridge portion is spaced outward from a respective tooth against which the tooth-facing side of the respective occlusal portion of the attachment support wall is abutted. According to another aspect of the invention, there is provided a method of placing one or more attachments on a patient's teeth, the method comprising: obtaining a stent having: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; a support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of voids in said attachment support wall, each void passing through said attachment support wall in a facial-lingual direction; and a respective bridge portion of the attachment support wall spanning across each void for seating of a respective attachment against said bridge portion at a side thereof opposite the void; positioning the stent in a worn position on the patient's teeth, and thereby placing each bridge portion at a location spaced outward from a respective tooth to leave a gap between said bridge portion and said respective tooth; placing a base of a selected attachment against the respective tooth in a position seating the attachment against the respective bridge portion at said location spaced outwardly from the respective tooth, from which the attachment reaches inward to the respective tooth across said gap. According to another aspect of the invention, there is provided a method of placing one or more attachments on a patient's teeth, the method comprising: obtaining a stent having: an occlusal arch-shaped portion for fitting against occlusal surfaces of the patient's teeth; and an attachment support wall extending in a gingival direction from the arc-shaped occlusal portion along one side thereof; and a plurality of windows for placement of the attachments into abutment with the patient's teeth through said windows, said plurality of windows each passing through said attachment support wall in a facial-lingual direction, each having an open end and an opposing closed end; positioning the stent in a worn position on the patient's teeth, and thereby placing the closed end of each window at a location spaced outward from the respective tooth to leave a gap between said closed end of the window and said respective tooth; placing a base of a selected attachment against the respective tooth in a target position seating the attachment against the closed end of the respective window at said location spaced outwardly from the respective tooth, from which the attachment reaches inwardly to the respective tooth across said gap. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A-C shows frontal, sagittal, and transverse views of an Intra-arch (Facial) direct bonding stent designed for placement of attachments on the facial surface of teeth of a single arch. FIG. 2A-C shows frontal, sagittal, and transverse views of an Intra-arch (Lingual) direct bonding stent designed for placement of attachments on the lingual surface of teeth of a single arch. FIG. 3A-C shows frontal, sagittal, and transverse views of an Inter-arch (Facial) direct bonding stent designed for placement of attachments on the facial surface of teeth of both maxillary and mandibular arches. FIGS. 4A and 4B show exemplary methods or processes to fabricate a direct bonding stent to position an attachment on a patient's tooth. FIG. 5 shows an exemplary method or process for placing an orthodontic attachment on a patient's tooth. FIG. 6A shows a frontal view of an Intra-arch (Facial) direct bonding repositioning stent designed for placement of attachments on the facial surface of teeth at an intermediate attachment-repositioning phase of an orthodontic treatment timeline. FIG. 6B shows a transverse view of an Intra-arch (Facial) direct bonding repositioning stent similar to that of FIG. 6A, but with enlarged by-pass openings in the stent to stay clear of existing attachments that are to be left in place during the repositioning of other attachments. FIG. 7A shows a sagittal view of an Intra-arch (lingual) direct bonding stent which differs from that of FIG. 2 in that the windows of the stent in which the attachments are to be placed during use are inversed relative to the embodiment of FIG. 2. FIG. 7B shows a cross-sectional view of the Intra-arch (lingual) direct bonding stent of FIG. 7A, as viewed along line B-B thereof. FIG. 8 shows a close-up sagittal view of an Intra-ach direct bonding stent in which the windows for posterior teeth have reduced mesial sides for improved mesial access to the windows during placement of attachments therein. FIG. 9A shows a frontal view of an Intra-arch (Facial) direct bonding stent designed for placement of attachments on the facial surface of teeth in which the incisal support wall portion of the stent window frame is reduced to a facially elevated bridge. FIG. 9B shows a close-up incisal view of an Intra-ach direct bonding stent in which the incisal support wall portion of the stent window frame is reduced to a facially elevated bridge for improved incisal access during placement of attachments therein. FIG. 9C shows a close-up lateral view of an Intra-ach direct bonding stent in which the incisal support wall portion of the stent window frame is reduced a facially elevated bridge. DESCRIPTION FIG. 1 shows one embodiment of a direct bonding stent. The stent 10 has the appearance of a removable appliance; however, it has geometrical features resembling partial ‘window frames’ or ‘partial footprints’ (negative impressions) to guide the placement of an attachment on a given tooth surface. This intra-arch (Facial) direct bonding stent 10 is designed to place attachments on the facial (cheek side) of teeth. Therefore, a significant portion of the lingual surface of the tooth is not covered by the stent to help facilitate removal. The lingual surfaces of anterior teeth (being concave in nature), and posterior teeth (below the crest of curvature), pose an issue in removal with gingival extensions reaching past the attachments. More specifically, the first embodiment stent 10 features an occlusal arch-shaped portion 12 for fitting conformingly against occlusal surfaces and incisal edges of the patient's maxillary or mandibular teeth in an occlusal plane. Along an outer facial side of the occlusal arch-shaped portion 12, an attachment support wall 14 extends therefrom in a gingival direction, either superiorly or inferiorly depending on whether the stent 10 is intended for the maxillary of mandibular arch of the patient. At discrete positions along the arch-shaped path followed by the support 14 wall, a plurality of window-like openings 16 pass through the support wall 14 in a facial-lingual direction, each being bordered on three sides by a respective one of the aforementioned ‘window frames’. Each window 16 has an open end 16 a at a terminal edge of the support wall 14 that lies opposite to the occlusal arch-shaped portion 12 in the gingival direction. Each window 16 terminates at a closed end 16 b that lies opposite of the open end at a location adjacent to the occlusal plane in which the arch-shaped portion 12 resides. By adjacent, it is meant that the closed end 16 b of the window resides nearer to the occlusal plane than the open end of the window. The closed end 16 b of the window denotes one of the three sides of the respective window frame that delimits the area of the window. At this closed end of the window, a thickness of the support wall 14 defines an ‘attachment support edge’ against which a respective attachment is abutted during use of the stent to define a properly attained position at which the attachment is to be adhered to the respective tooth. Two opposing sides 16 c, 16 d of the window extend perpendicularly from opposite ends of the ‘attachment support edge’ to join up with the terminal end of the support wall 14 at the open end 16 a of the window, thereby completing the respective three-sided window frame. The width of each window 16 between its two sides closely matches the width of a respective attachment whose placement on the patient's tooth is to be guided by said window during use of the stent. On the other hand, a depth of each window 16 measured from the open end 16 a thereof at the terminal edge of the support wall 14 to the opposing closed end 16 b is less than the corresponding height measurement of the attachment from a gingival end thereof to an occlusal/incisal end thereof. The gingival end refers to an end of the attachment that resides nearest to the patient's gingiva in the final adhered position of the attachment on the patient's tooth, while the occlusal/incisal end of the attachment refers to the end of the attachment nearest to the biting edge (incisal edge) or biting surface (occlusal surface) of the tooth. In the preferred embodiment, the depth of the window 16 is approximately equal to one-third of the attachment height. Placement of the respective attachment into the window 16 with the sides and incisal/occlusal end of the attachment closely adjacent the closed end and sides of the window sets the attachment in the target position on the patient's tooth. In this properly seated position of the attachment, with the gingival two-thirds of the attachment reach out of the window into an exposed outside the confines of the stent. That is, the respective window surrounds only the incisal/occlusal third of the attachment. Window frames may also feature contrasting coloration or distinctive markings on the stent surface. The purpose of the contrasting coloration or markings is to further illuminate the stent position thereby increasing the clinician's ability to visualize of the stent window frame intra-orally. The contrasting coloration may also aid in assessment and determination of full seating of the attachment. Both of the aforementioned advantages increase the speed and efficiency of attachment placement. With reference to FIG. 8, the stent window frame may also have a portion of the mesial or distal aspect of the support wall removed, resulting in a shorter window depth at one side of the window than in the other embodiments. In FIG. 8 the mesial support wall 16 c of the window is shorter in order to facilitate placement of the attachment. For example, when placing attachments on posterior teeth, removal of a portion of support wall at the mesial (anterior) side of the window in a manner reducing the support wall height at this mesial side of the window allots the clinician more access to position the attachment into the appropriate position. This design modification may also involve increasing the occlusal-gingival height of the support wall at the opposing distal side 16 d of the window relative to the earlier embodiments in order to allow a more positive horizontal stop when positioning the attachment. Accordingly, the support wall and related window depth at the distal side of the window may be greater those at the mesial side of the window, as shown in FIG. 8. In the fully seated position of the attachment, it abuts against the taller distal side 16 d of the window and the closed end 16 b of the window. The depth of the window at the taller distal-side 16 d is again preferably about one third of the height of the expected attachment, while the reduced mesial side 16 c of the window is shallower to provide increased mesial access to the window and attachment during placement of the attachment into the properly seated position. In FIG. 8, the most-posterior window is shown unoccupied to illustrate the shallower/shorter mesial side 16 c of the window versus the deeper/taller distal side 16 d of the window, while the other two illustrated windows are shown with respective attachments fully seated therein. Another advantage of this modification is improved visual access to the window at the mesial side thereof to assess positive attachment seating. Concurrent to the reduction of the support wall at the mesial side of the windows, removal or scalloping of the stent material inter-dentally (i.e. at the area between adjacent teeth in the same arch) to improve access even further may also be incorporated into stent designs. Such scalloping or removal reduces the lingual-facial thickness of the stent's support wall at these inter-dental areas. The scalloping or removal is performed in manner creating a respective sloped channel that slopes distally and gingivally toward the mesial side of a more-posterior one of the two windows between which this inter-dental area resides. The attachment can be inserted into the more posteriorly situated window via this channel in a sloped gingival/distal direction to pass the attachment over the reduced-height mesial area of the support wall area at the mesial side of the window, whereby the channel of reduced lingual-facial thickness improves mesial access to the window. The smoothly and concavely curved cross-sectional profile of a scallop-like channel that widens toward the outer surfaces of the surrounding thicker areas of the support wall minimizes impedance to smooth passage of the attachment through the channel toward the more posteriorly situated window. The direct bonding stents of the preferred embodiments are fabricated from semi-rigid materials with elastomeric properties (such as the addition cured silicones, ‘Tango™’ family or other SLA elastomeric resins) to produce an intimate cohesive seal at the stent-to-tooth interface. This seal prevents excess adhesive on the orthodontic attachment from “splaying” under the stent upon seating of attachment against the tooth. An intimate cohesive seal is also produced at the stent-to-attachment interface. The “attachment support edge” is able to more closely adapt to the attachment to provide a clear positive “vertical stop” when placing the orthodontic attachments. That is, the intimate cohesive seal between the support wall 14 at the boundaries or window frame of each window 16 maintains the attachment support edge at the closed end 16 b of the window in directly adjacent relation to the facial surface of the tooth, whereby the inserted attachment cannot slip between the stent and tooth out of the targeted alignment. Therefore the accuracy and efficiency in placing the attachments with the stent is increased, as the provider can also tactilely “feel” the correct attachment position in the stent window frame (as the attachment fits in the negative impression). Once the attachment is seated, attachment placement/seating forces cannot displace the stent, as the sealed condition of the stent against the patient's dentition resists flexing or displacing of the stent out of position, thereby maintaining the positional integrity of the window during placement of the attachment. This reinforces the positive “vertical stop” to aid in placement. There is also a more accurate transfer of the attachment to the tooth, as there is no displacement when seating the attachment. As described above, the stent ‘window frames’ terminate prior to the gingival two-thirds of the attachment. This early termination ensures ‘passive removal’ of the stent from the dentition. No lifting of the gingival end of the support wall 14 away from the facial surfaces of the teeth is required to clear the adhered attachments during removal of the stent. Therefore, no concerns exist regarding inadvertent “debonding” (bond failure) of the attachments when removing the stent. Also, assessment of “attachment positive seating” on the tooth is more efficient with the partial height ‘stent window.’ Each side of the attachment is readily accessible over a majority of its length in order to assess the seating of the attachment and/or test the bonding of the attachment to the tooth prior to removal of the stent, whereby any necessary repositioning and re-seating of the attachment can be performed without removal of the stent from the patient. A second wall 18 of the stent extends gingivally from the occlusal arch-shaped portion 12, just like the attachment support wall 14. However, unlike the attachment support wall 14, the second wall 18 extends along the inner or lingual side of the occlusal arch-shaped portion 12, and has a reduced height in the gingival direction relative to the height of the attachment support wall 14. Lying opposite to the support wall 14 that conforms against the facial surface of the patient's teeth, the second wall 18 thus conforms against the lingual surface of the patient's teeth. Being of lesser height, the second wall 18 spans less of each tooth in the gingival direction than the support wall 14, and terminates prior to a point at which the concave curvature of the lingual surface of each tooth crests in a facial-lingual plane. FIG. 2 shows another embodiment of a direct bonding stent 10 ′. Whereas the stent 10 of the first embodiment has the attachment support wall on the facial side of the arch to enable placement of attachments on the facial surface of the patient's teeth, the intra-arch (Lingual) direct bonding stent 10 ′ of the second embodiment is designed to place attachments on the lingual (tongue side) of teeth. The second embodiment stent 10 ′ shares many of the same properties as the intra-arch (Facial) version of the stent; however, a significant portion of the facial surface of the tooth is not covered by the stent in order to help facilitate removal. That is, in the second embodiment, the shorter second wall 18 ′ resides on the outer/facial side of the arch for mating with the facial surfaces of the teeth in a position lying opposite to the lingually-located support wall 14 ′. As with the first embodiment, the stent ‘window frames’ terminate prior to the gingival two-thirds of the attachment, which also helps facilitate removal as the lingual surfaces of teeth (being concave in nature) would pose an issue in removal of the stent if the support wall 14 ′ were to extend further in the gingival direction and span the full attachment height in the gingival direction. With reference to FIG. 7, a modification to the intra-arch lingual stent's window orientation may useful for maxillary or mandibular lingual stents when placing anterior attachments. In this embodiment, the stent window 16 ′″ is oriented with the open end 16 a of the window directed towards the incisal or occlusal surface instead of toward the gingiva. The open end 16 a of the window 16 ′″ thus resides at the originating end of the support wall 14 ′ that joins to the arc-shaped occlusal portion 12 of the stent, and the closed end 16 b of the window 16 ′″ resides intermediately between the originating and terminal ends of the support wall so that the window 16 ′″ terminates short of the opposing terminal end of the support wall 14 ′. The attachment is thus inserted into the window in a gingival direction through the occlusally-facing open end 16 a of the window. These occlusally-open lingual-side windows 16 ′″ are deeper than the gingivally-open windows of the preceding embodiment since the full height of the attachment must be receivable in the window so that the seated attachment resides fully past the occlusal plane of the tooth in the gingival direction. This inverted orientation of the windows on the lingual side of the intra-arch lingual bonding stent aids in positioning the attachments using indirect vision, and increases ease of placement. Removal of the stent remains passive as the posterior occlusal aspect of the stent may be unseated, moved posteriorly, and then rotated (superiorly or inferiorly) without placing strain on the bonded attachments. FIG. 3 shows another embodiment of a direct bonding stent 10 ″. This inter-arch (Facial) direct bonding stent is designed to place attachments on the facial (cheek side) of teeth. The stent shares many of the same properties as the preceding embodiments, however, both top-arch (maxillary) and bottom-arch (mandibular) stents are connected in a one-piece unit. In this case, the support wall 14 ″ and the shorter second wall 18 ″ each extend both superiorly and inferiorly from a shared occlusal arch-shaped portion 12 ″ in the gingival directions of the respective dental arches. The upper surface 12 a of the shared occlusal arch-shaped portion 12 ″ is shaped to conform to the occlusal surfaces and incisal edges of the patient's maxillary teeth, while the lower surface of the shared occlusal arch-shaped portion 12 ″ is shaped to conform to the occlusal surfaces and incisal edges of the patient's mandibular teeth. A first set of windows 16 ′ is defined at a superior terminal end of the support wall 14 ″ for use in placement of attachments to the patient's maxillary teeth, while a second a second set of windows 16 ″ is defined at an inferior end of the support wall 14 ″ for use in placement of attachments to the patient's mandibular teeth. This design of the third embodiment offers several advantages. The efficiency of placing attachments is substantially increased if the patient is held in biting position during bonding (reduced patient jaw movement). Inter-arch (bi-maxillary) designs also offer significant ‘moisture control’ advantages that may be incorporated. An example of these moisture control features is shown in the form of a length of suction tubing 20 that perforates the shared occlusal arch-shaped portion 12 ″ of the stent (inter-arch) 10 ″ to enter the lingual portion of the oral cavity (tongue side). A connection portion 20 a of the tubing 20 a extends from an anterior apex of the shared occlusal arch-shaped portion 12 ″ to enable coupling of a suitable vacuum source to the tubing 20 a outside the oral cavity. An internal passage of the tubing extends anteriorly through the connection portion 20 a, and onward through the shared occlusal arch-shaped portion 12 ″ of the stent in the facial-lingual direction, where the passage continues through an internal cavity portion 20 b of the tubing that reaches posteriorly into the oral cavity at the center of the arch-shaped stent. This tubing may be configured in a variety of formations (both in number and position), and may also be attached/fixed to the lingual portion of the stent. Controlling contaminants (moisture) present in the oral cavity is paramount in the bonding process, and the tubing 20 enables suction of excess moisture from the oral cavity through the stent during bonding. Other inter-arch perforations may also be present in stent design to help facilitate patient respiration, for example as shown by the presence of two respiration passages 22 perforating the shared occlusal arch-shaped portion 12 ″ in the facial-lingual direction on either side of the centrally located suction tube 20. Inter-arch designs may also incorporate buccal mucosa isolation (‘cheek spreading arms/wings’) to further segregate the intra-oral tissues from the bonding surfaces (not shown in figures). With the ‘inter-arch’ stent in place, the only major source of saliva in the bonding zone of the oral cavity is the parotid gland duct (Stensen's duct). Stensen's duct is located laterally to the second maxillary molar, and may pose an issue in bonding without isolation. Therefore, buccal mucosa isolation may be employed to combat potential saliva contaminant during bonding. Likewise, other embodiments, including intra arch designs, may also incorporate buccal mucosa isolation (‘cheek spreading arms/wings’) to further segregate the intra-oral tissues from the bonding surfaces. FIG. 4A shows an exemplary method or process to fabricate a stent for positioning of an attachment on a patient's tooth. First, the process requires production of models of the patient's dentition. This may be accomplished utilizing physical (non-digital), or virtual (digital) means. Historically, models have been produced by non-computer-aided techniques. Typically, impressions of the patient's dental arches are made using hydrocolloid or elastomeric materials. Replica plaster or “stone” casts (full arch models) are made from each impression and sealed. Currently, models may be produced utilizing computer-aided techniques. In this method teeth are digitized to produce virtual models of the patient's dentition. This digitization process may be accomplished utilizing a variety of methods. For example, the patient's dentition may be scanned or imaged using well-known technology. There are a variety of reflective systems in use. Some of these reflective systems utilize non-optical incident energy sources (such as sonar or microwave radar). Others systems available utilize optical energy. Those ‘non-contact-type’ systems utilizing reflected optical energy further contain special instrumentation configured to allow for precise measurement recording (e.g., triangulation, imaging radar, and interferometry). The second step in the stent fabrication process involves addition of objects/attachments (orthodontic brackets for example) to the models in preferred locations on the teeth. If the physical cast method is utilized, the preferred positions for the objects are first determined on the stone model. Once the location has been determined, the objects are bonded to the sealed stone models using a temporary adhesive. If the virtual model method is utilized, the preferred positions for the objects are first located on the digitized teeth (3D virtual model). The virtual models may be a computer aided design (CAD) model, or may be scanned using scanners described above. Once the location has been determined on the virtual model, the “virtual objects” are added. The virtual objects are in essence 3D models of actual physical attachments (orthodontic brackets for example) that are to be placed on the patient's teeth. The objects may be positioned on a digitized tooth using a computer or workstation having a suitable graphical user interface (GUI) and software appropriate for viewing and modifying the images. Following positioning of the objects, the virtual model (complete with objects) is converted (fabricated) into a physical model. Fabrication utilizes well-known technology (3-D printing technology) to create a non-scaled (1:1) identical physical version of the virtual model. In either of the aforementioned methods listed, the result is production of “working models” used for laboratory fabrication of dental appliances. These working models are used to fabricate the “direct bonding stent”. The working model is fabricated using “identical”, “non-scaled”, “non-cavitated”, models and object projections. Stent fabrication involves a working model, which includes physical representations of the patient's teeth and “physical” objects attached to the modeled teeth to serve as physical representations of the actual attachments that are to be attached to the patient's actual teeth. Stent design (i.e. intra-arch vs. inter-arch, facial vs. lingual placement, etc.) must also be selected prior to fabrication of the direct bonding stent, for example from among the three embodiments specifically described above and illustrated in FIGS. 1-3. Finally, once the stent design has been selected, fabrication of the stent takes place. A lab technician, or automated machine process (e.g. assembly line), utilizes the working model to fabricate the direct bonding stents. Variation in fabrication process may exist, however, all embodiments encompass the following fabrication principles. Direct Bonding Stents utilize materials with elastomeric properties (addition silicones for example) to provide an intimate/accurate fit to teeth. The stent window frames are in effect negative impressions (partial footprints) of the object projections, not a removal or “cut out” of vacuum formed plastics. The stent cannot be formed by pressure or vacuum forming techniques as the stent material transforms (polymerizes) from a reversible (surface wetting) state to irreversible (cohesive seal) state. One fabrication process involves flowing polymerizable stent material (addition silicone for example) over the working model dentition (occlusal, buccal and lingual surfaces), and intimately between the teeth and object at the areas around the interface shared between the object and the modeled tooth, without fully covering the objects facially on the model dentition. Air-driven techniques may be utilized to coerce the flowable material into tight spaces, and encourage a more intimate cohesive seal. Once the material has suitably encased the objects, the stent material is cured (or allowed to set). The stent is then modified by removal of the over-extended material (now polymerized into more rigid irreversible state). During this step, around each object, any set/cured material is removed from adjacent the gingival two-thirds of the object, creating a partial window frame or footprint for accommodating the incisal/occlusal one third of the actual attachment during use of the stent. Also depending on the modeled dentition surface the object is placed on (buccal or lingual), the cured/set material from the opposite surface of the modeled dentition is removed (at the crest of the tooth curvature for example) such that only partial coverage is provided by the stent on the surface of the modeled tooth opposite that at which the objects are located. These modifications allow easier placement of attachments, and also aid in the removal of the stent following bonding. During the fabrication process, the shared occlusal arch-shaped portion 12 ″ of the stent may also be reinforced (with acrylic for example) to form a “bite block”, or increase the dimensional stability of the direct bonding stent. One modification to this fabrication process exists with the fabrication of the inter-arch (bi-maxillary) stent 10 ″ of the third embodiment. This version of the direct bonding stent places the patient into a constructed bite whereby the patient's maxillary and mandibular teeth are disoccluded (opening approximately 10-15 mm). Upper and lower arches of the stent may be connected (bridged) by the same material as the stent (addition silicone), resulting in a thicker shared occlusal arch-shaped portion 12 ″ that has the same uniform material composition as the rest of the stent. Alternatively, the bridge may be a different material (acrylic for example). In the case of the latter, the resulting shared occlusal arch-shaped portion 12 ″ may have a multi-layered configuration of materially distinct layers, where upper and lower layers that abut the occlusal surfaces and incisal edges of the maxillary and mandibular teeth, respectively, comprise the same elastomeric material as the attachment support walls 14 of the stent, while an intermediate layer between the upper and lower layers is a more rigid material, such as acrylic. Whether the shared occlusal arch-shaped portion 12 ″ is of singular material or multi-material construction, the bridge between the arches must be rigid, or dimensionally stable. For example, in the instance of a singular material construction, the greater thickness of the bridge, which may for example be 10-15 mm thick, compared to the thinner arch-shaped portion of the intra-arch embodiments, imparts a greater rigidity to this area of the stent, despite having the same material composition, in order to provide the required dimensional stability. The material forming the bridge may also be perforated to allow for moisture control applications (suction tubing), and improved patient oral respiration (if nasal respiration is impossible). In the case of the multi-material construction with a more rigid core or intermediate layer, the aforementioned moisture control and/or respiration passages may be provided within the more rigid intermediate layer in order to best resist closure of these passages by compressive action between the patient's maxilla and mandible. Another modification exists in fabrication of a mid-treatment or repositioning stent (see FIGS. 6A and 6B ). At any given point in a treatment timeline, an orthodontist may wish to alter the position of some (but not all) of the attachments, and a stent may be fabricated to aid in the repositioning process. Any of the novel direct bonding stent designs may used or modified to allow for midcourse correction of attachment placement. The main advantage of the direct bonding stent design exists in the improved passive removal (due to early stent termination prior to the gingival two-thirds of the attachment). Placing the repositioning stent would be exceedingly more difficult, and the risk of accidental debonding of existing attachments would be increased, with full-length gingival extensions of the support wall. Alternatively, or additionally, the stent may be fabricated to completely by-pass the existing attachments that are present (and correctly positioned) on the patient's dentition. The direct bonding stent may be terminated occlusally to the ideally placed existing attachments, thus ensuring a completely passive removal of the direct bonding stent. This option is shown in FIG. 6B, where at areas of the stent that correspond to the positions of the existing attachments 24 that are not to be repositioned, large bypass openings 26 of greater size (in both width and depth) than the attachment-guiding windows 16 are provided so as to leave the existing attachments and surrounding areas of the tooth uncovered by the stent. When fabricating such a stent from a physical model, the bypass openings (like the windows 16 ) may be created either by controlling the application of the flowable material to leave voids at the intended areas of said openings, or by later trimming away the set or cured material at these areas. FIG. 4B shows an alternative exemplary method or process to fabricate a stent to position an attachment on a patient's tooth. In this method teeth are digitized to produce virtual models of the patient's dentition (as described in FIG. 4A ). The preferred positions for the placement of objects are located on the digitized teeth (3D virtual model). Once the location has been determined on the virtual model, the “virtual objects” are added. Following addition of the objects, the virtual model (complete with objects) is used to create a “virtual stent”. This virtual stent is then fabricated utilizing well-known technology such as a rapid prototyping device (stereolithography machine) to create a non-scaled (1:1) identical physical version of the virtual stent. Recent advances in stereolithographic materials (such as ‘Tango™’ family or other SLA elastomeric resins) that are able to simulate properties of elastomerics (rubber like properties) allow this fabrication process to be possible. FIG. 5 shows an exemplary method or process for placing orthodontic attachments on a patient's teeth. The process utilizes the direct bonding stent fabricated in the process of FIG. 4A or 4B. To facilitate bonding, the tooth surface is prepared (etched), and a bonding agent is placed (with or without the stent seated). Adhesive is then placed on the base of each attachment, which in turn is then placed on the selected tooth surface (facial or lingual). Following initial seating, any excess adhesive (if not flash free) is removed by the operator. Once the attachment is determined to have been seated satisfactory, the adhesive is cured (bonding the attachment to the tooth). The adhesive may be light or chemical curing. Light curable adhesives employ a photo-initiator that initiates the curing reaction upon exposure to light. Conversely, chemical curing adhesives utilize two or more components that when mixed together initiate curing reaction. FIGS. 9A-9C show another embodiment of a direct bonding stent 10 ″. Whereas the stents of the earlier embodiments have the attachment support wall fully intact over the entire incisal portion of the attachment, i.e. spanning continuously from the occlusal arch-shaped portion 12 to the terminal edge of the support wall 14 over the full-length of the arch, this embodiment instead features a respective void 28 in the support wall 14 at each tooth. With reference to FIG. 9A, each void 28 is located intermediately between the originating end of the support wall 14 that joins to the occlusal arch-shaped portion 12 and the terminal end of the support wall at which the respective attachment-receiving window is defined. An occlusal/incisal portion 14 a of the support wall 14 remains intact at the originating end thereof to connect the support wall to the arch-shaped portion 12, and defines an occlusal/incisal side of the void. Across the void 28 from the occlusal/incisal portion 14 a of the support wall 14, the opposing gingival side of the void is defined by a bridge portion 30 of the support wall that spans across the void 28. For all teeth except the rearmost molars, the remaining two sides of each void 28 are defined by intact inter-dental areas 14 b of the support walls that overlie the areas between adjacent teeth and span fully from the originating end of the support wall to the opposing bridge portions thereof. On the gingival side of this bridge portion 30 that faces away from the void 28, the respective window 16 is defined for use in guiding the placement of the respective attachment 24 ′ on the facial surface FS of the respective tooth T during use of the stent, as described herein above for the other illustrated embodiments. The gingival side of the bridge portion 30 is thus shaped to define the two opposing sides 16 c, 16 d of the respective window 16 and the closed end thereof against which the respective attachment 24 ′ is to be placed. Like the windows 16, the voids 28 pass fully through the support wall in the lingual-buccal direction, but unlike the open-ended windows 16, the voids 28 are closed on all sides thereof. Turning to FIG. 9B, the tooth-facing lingual side 30 a of the bridge portion 30 of the support wall 14 is spaced facially outward from the tooth-facing lingual side 31 of the occlusal/incisal portion 14 a of the support wall 14. The distance of this facially outward offsetting of the bridge portion 14 a relative to the occlusal/incisal portion 14 a exceeds that which is dictated by the curvature of the tooth's facial surface FS, thus resulting in a gap G between the tooth-facing lingual side 30 a of the bridge portion 30 of the support wall 14 and the facial surface FS of the tooth when the stent is placed in the worn position. When the respective attachment 24 ′ is seated in the target position against the bridge portion 30, only a facially outer portion of the attachment is directly supported by the narrow bridge portion 30, while an inner lingual portion of the attachment reaches inwardly across the gap G from the bridge portion 30 of the support wall 14 to the facial surface FS of the tooth T. Accordingly, access to the base 24 b of the attachment is available via the respective void 28 and the adjoining gap G. The width of the void 28 between the inter-dental areas 14 b of the support wall 32, and the corresponding width of the gap G measured in the same direction, both exceed the corresponding width of the respective window 16 and attachment 24 ′, whereby access is available to three of the four sides of the attachment's base 24 a through the gap G. This embodiment thus facilitates removal of excess adhesive that may accumulate around the incisal portion of the attachment base, and provides improved visibility to ensure positive seating of attachments on the teeth. As described for other embodiments, the depth of the window at each bridge may be approximately one-third of the attachment height, leaving the gingival two-thirds of the attachment height free of the stent for easy gripping and manipulation thereof, which also helps facilitate passive removal. While FIG. 9 illustrates a stent whose support wall is on the facial side of the arch-shaped occlusal portion 12 for placement of attachments on the facial surfaces FS of the patient's teeth T, the voids, windows and offset bridge portions could alternatively be used in a lingually positioned support wall for placement of attachments on the lingual surfaces of the teeth. In such instance, the bridge portions will be outwardly offset from the incisal/occlusal portions of the support wall in the lingual, rather than facial direction, likewise creating a gap between the teeth and bridge portions in the lingual-facial direction to enable improved access to the base of the attachment via the respective void. While FIG. 9 shows an intra-arch version of the stent, it will be appreciated that similar use of voids and gaps for improved access and visibility to placed attachments could likewise be employed one or both maxillary and mandibular halves of an inter-arch stent. While the described embodiments feature a window depth of approximately one-third of the attachment height, the ratio between the window and attachment sizes may be varied while still retaining the advantage of leaving a gingival portion of the attachment exposed outside the constraints of the stent. The illustrated embodiments with a 1:3 ratio of window depth to attachment height is one example of preferred embodiments in which the window depth is 50% or less of the attachment height, while the window depth other embodiments may vary inside or outside the preferred range, for example with window depths between 25% and 50% of the attachment height, between 25% and 60% of the attachment height, or between 25% and 75% of the attachment height. Any stents disclosed herein configured for placement on the maxillary arch may incorporate palatal coverage/extension to increase retention of the stent intraorally. Since various modifications can be made in my invention as herein above described, and many apparently widely different embodiments of same made within the scope of the claims without departure from such scope, it is intended that all matter contained in the accompanying specification shall be interpreted as illustrative only and not in a limiting sense.
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Summary: An orthodontic direct bonding stent facilitates ideal positioning of physical attachments on a patient's teeth. Novel designs of direct bonding stents include uniquely configured windows for guiding placement of the attachments. Windows of reduced depth leave a majority of each attachment exposed beyond the stent for easy grip and manipulation, and use of a reduced height wall opposite the window-equipped wall eases removal of the stent without disruption of the adhered attachments. Use of elastomeric material at the window boundaries provides an improved seal with the tool for less adhesive splay, and inter-arch stents enable maxillary and mandibular placement with a single appliance. Lingual windows enable lingual placement of attachments for reduced visibility, and reduced wall thickness between adjacent windows provides improved mesial access to attachments. Enlarged bypass openings accommodate previously placed attachments, and bridged voids provide improved access and visibility for adhesive cleanup and accurate placement.
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Summarize: Introduction Recent and planned nuclear reactor closures have raised questions about the future of nuclear power in the United States and its potential contribution to the U.S. electricity mix. The nuclear power industry and its supporters have proposed that Congress take action to prevent the shutdown of currently operating U.S. reactors before the expiration of their operating license. Supporters contend that nuclear power should be valued as a domestic source of highly reliable, low-carbon electricity. However, opponents contend that nuclear power suffers from too many drawbacks (e.g., safety risks) and that federal incentives should focus instead on renewable energy and efficiency. There are currently 60 operating nuclear power plants in the United States comprising 99 reactors and representing 99,316 Megawatts of generating capacity—nearly 10% of existing utility-scale generating capacity, and approximately 20% of total power generation. Generally, U.S. nuclear plants are located in one of two market types: (1) competitive—where the value of electricity fluctuates based on supply-side price offers that are generally a function of fuel (e.g., natural gas) costs and demand-side price bids, and (2) cost-of-service—where the value of electricity is set at a rate based on regulator-approved costs, operating expenses, and a reasonable investment return. Some nuclear plants located in competitive power markets are subject to full or partial rate regulation, which reduces financial risk associated with fluctuating electricity prices. Access to competitive wholesale power market prices can result in regulatory bodies deciding that early nuclear plant retirement is in the best interest of rate payers based on projected electricity prices. Other plants located in competitive power markets operate on a "merchant" basis, where electricity prices and revenues are subject to commodity price fluctuations and supply/demand dynamics. Merchant nuclear power plants are the focus of this report. Competitive power markets are managed by either a Regional Transmission Organization (RTO) or an Independent System Operator (ISO), which operates electricity and capacity markets that determine revenues for nuclear, and other, power generators. Unlike cost-of-service market areas—where generators have electricity price rates that are approved and periodically revised by a state utility commission—competitive power markets are subject to electricity price volatility that results from supply and demand fundamentals as well as fuel (e.g., natural gas and coal) costs for the marginal price-setting generators. While generators operating in cost-of-service market areas are not immune to fuel competition, to date nuclear power plants that have announced early closure or have been identified by financial consultants and ratings agencies as "at-risk" of closure are located in competitive market areas. U.S. nuclear power plants operating in competitive power markets are in an era of challenging economic conditions—increasing operating costs and low wholesale power prices in some locations—that create uncertainty regarding the future of some plants. Six reactors have permanently shut down during the past five years; 10 reactors—at eight plant sites—have announced, since 2010, their intent to close—nine in the past two years (see Figure 1 ); and 10 have been identified by consultants and ratings agencies as "at-risk" of closing prior to the expiration of their operating licenses. In total, these plants represent 22,078 Gigawatts of generating capacity, which is roughly 20% of the current nuclear power fleet. Of the 10 power reactors that have announced their intent to close, some operate under full or partial rate regulation—they receive a set price for electricity sales—and some operate solely as merchant power plants—they are subject to the price dynamics of competitive power markets. However, the one common link among the reactors that have announced their intent to close is that they are located in competitive power markets ( Figure 1 ). Because they are subject to wholesale power price dynamics, merchant generators operating in competitive power markets are the focus of the economic discussion included in this report. This report provides background on the evolution of nuclear power in the United States, discusses recent and announced plant closures, explains the economic conditions creating financial challenges for merchant nuclear power plants, and presents policy considerations associated with the future of existing nuclear power plants. Caveats and Limitations The financial situation for each nuclear power plant is unique. Operating costs, expected capital expenditures, wholesale power prices, capacity payments, hedging exposure, power purchase agreements (PPAs), and full or partial regulated rate structures are financial components that are plant and location specific. Nevertheless, much of the economic and financial information presented in this report is generalized in order to illustrate the cost and pricing trends across the entire industry. While some plant-by-plant analysis is referenced and contained in this report, there are important data assumptions that should be noted. To date, CRS has not been able to locate or aggregate a comprehensive data set that would allow for a plant-by-plant analysis that accurately reflects all financial variables. Overview of U.S. Nuclear Power Generation Commercial-scale nuclear power generation in the United States was inaugurated in 1957 by the startup of the Shippingport reactor in western Pennsylvania. Built by the U.S. Atomic Energy Commission (AEC) and operated by Duquesne Light Company, the relatively small (60 Megawatt) power plant was not considered economically viable. However, Shippingport paved the way for additional demonstration reactors that were rapidly scaled up in generating capacity to provide a viable alternative to power plants fired by fossil fuels. Harnessing the tremendous energy potential of nuclear fission had been a major federal goal since the development of nuclear weapons during World War II. Post-war policymakers widely anticipated that nuclear power plants could generate much or most of the power that would be needed to satisfy the nation's rapidly escalating demand for electricity, particularly if plutonium and uranium from spent nuclear fuel were recycled for use in advanced fast-neutron reactors. It was also hoped that the widespread adoption of nuclear power would mitigate volatile and rising fossil fuel prices, growing dependence on foreign oil, and air pollution. The legal framework for the U.S. nuclear power industry was established by the Atomic Energy Act of 1954 (AEA), which authorized the private sector to own and operate nuclear facilities under AEC licensing. To encourage private-sector adoption of the new and potentially hazardous technology, Congress enacted the Price-Anderson Act as an amendment to the AEA in 1957, establishing a special public liability system for reactor operators and suppliers. Shippingport and other small, early commercial nuclear plants were constructed with subsidies under the AEC's Power Reactor Demonstration Program. Beginning in 1963, reactor suppliers began selling substantially larger units—around 500 megawatts and above—on a "turnkey" basis to electric utilities. By 1965, plants as large as 1,100 megawatts were being ordered by utilities under a non-turnkey basis, with contract terms similar to those of non-nuclear power plants. Rising coal prices and the apparently improved economics of larger reactors prompted electric utilities to order 20 new nuclear units in 1966 and 31 in 1967—only a decade after the startup of Shippingport. Reactor orders continued at a strong pace through 1971 and then jumped to 38 in 1972 and 41 in 1973. However, the wave of reactor orders then receded as dramatically as it had begun. Surging energy prices resulting from the Arab oil embargo in 1973-1974 dramatically slowed the growth of U.S. electricity demand, and it became apparent that electric utilities had ordered too much generating capacity of all types. In addition, nuclear power plant construction costs were sharply escalating, interest rates were rising, and greater safety expenditures were required after the 1979 core-melt accident at Three Mile Island 2. All reactors ordered from 1974 through 1978 were subsequently canceled, and no further reactor orders were placed in the United States until 2008, when contracts were signed for four new units now being built in Georgia and South Carolina. Most of the reactors ordered from 1971 through 1973 were also canceled, for a total of 126 cancellations. In addition to the cancellations, the lower-than-expected growth in electricity demand led electric utilities to slow the construction of many reactors that had not been canceled, stretching their completion into the 1980s and 1990s, and to 2016 in the case of one reactor, Watts Bar 2. Of the 256 reactor orders placed through the 1970s, 134 reactors, including the early AEC demonstration plants, ultimately were completed and placed into service. Throughout the history of the U.S. nuclear power program, reactors have permanently shut down for a variety of reasons. Many of the small, early AEC reactors were not necessarily expected to operate for a long time, and nine of them had been permanently closed by the late 1970s. Three Mile Island 2, a nearly new reactor, closed in 1979 after its accident. Four reactors were permanently closed during the 1980s, including the Shoreham plant on Long Island, NY, which had never begun full-power operation. During the 1990s, 10 reactors shut down, including the large, two-unit Zion plant in Illinois. Rising natural gas prices in the early 2000s significantly improved the economics of nuclear power, because they made gas-fired electric generation more expensive while nuclear generation costs remained comparatively low (discussed in detail later in this report). Existing reactors were no longer being closed, and the first U.S. license applications for new reactors since 1978 were filed with the Nuclear Regulatory Commission (NRC, the regulatory successor to the AEC) beginning in 2007. However, gas prices fell sharply after 2009 and have remained low, primarily because of strong production of domestic shale gas. Six reactors have permanently shut down since the beginning of 2013, and additional closures have been announced. The current economic situation has clouded the outlook for new U.S. reactors. NRC has issued construction and operating licenses to seven new reactors and is currently considering applications for seven more. However, except for the four units in Georgia and South Carolina that are now under construction, no commitments have been made to build any others. As shown in Table 1, of the 134 commercial reactors that have started operating since the beginning of the U.S. nuclear power program, 35 have permanently closed, leaving 99 currently operating and 4 under construction. The 99 operating U.S. commercial power reactors are located in 30 states at 60 plant sites. Nuclear power plants annually provide about 20% of total U.S. electricity generation, most recently 19.5% in 2015. Nuclear power accounted for 61.6% of U.S. "zero carbon" electricity generation in 2015. Under the Atomic Energy Act, nuclear power reactors are licensed to operate for 40 years and are eligible for 20-year license renewals thereafter. Because about half of U.S. reactors started operating in the 1970s or earlier, the licenses of much of the reactor fleet would have expired by now without renewal. As of July 2016, according to NRC, 80 of the 99 currently operating reactors have received 20-year license renewals, and 12 additional renewal applications are under review. The 20-year renewals, allowing 60 years of operation, would extend the licenses of most current reactors through the 2030s and of almost all the remainder through the 2040s. Further 20-year renewals are also possible under the AEA, extending plant lives to 80 years or longer. Actual, Planned, and Possible Reactor Closures After experiencing no commercial reactor closures during the first decade of the 21 st century, the U.S. nuclear power industry has seen six reactors retire during the past five years, with at least 19 more considered to be at risk of closure by their owners or major credit rating agencies (see Table 2 ). However, New York and Illinois have recently taken action to keep seven of the at-risk reactors operating. These actual, announced, and potential nuclear plant shutdowns fall into several broad and sometimes overlapping categories. The fundamental problem for most of the at-risk plants is that their revenues depend almost entirely on regional wholesale electricity markets, where prices have been insufficient to cover their costs. Some plants, such as Quad Cities, have relatively low operating and maintenance costs, but still cannot bring in sufficient revenue in their regional markets, because of a combination of oversupply, transmission congestion, and other local factors. Several single-unit nuclear plants, which generally have higher costs per megawatt-hour, are having trouble competing even in markets with relatively high power prices. For example, the Pilgrim plant in the New England market is reportedly receiving revenues that are insufficient to cover fuel, operating and maintenance, and capital expenditures, as discussed below. The chronic inability to cover costs during routine operations is not the only source of nuclear plant retirement risk: Marginally economic plants can be vulnerable to closure because of major repairs, capital expenditures, prolonged shutdowns, and other non-routine events. Merchant plants may be especially vulnerable, but such occurrences can even prompt the retirement of rate-regulated plants, as seen at San Onofre and Crystal River. Non-revenue factors, such as concerns about seismic safety at Diablo Canyon, can also contribute to nuclear plant closures. Recent Reactor Retirements The first nuclear shutdown resulting from the recently changing competitive environment was the 566 megawatt single-unit Kewaunee plant in Wisconsin. In its October 2012 announcement that Kewaunee would be closed, plant owner Dominion said the small plant could not cover its costs in the midst of low regional power prices. An industry consultant commented after the announcement, "This might be the beginning of a new wave of shutdowns." Kewaunee closed in May 2013. Duke Energy decided on February 5, 2013, to retire the Crystal River 3 reactor in Florida. Crystal River's previous owner, Progress Energy, had severely damaged the reactor's massive concrete containment structure while attempting to replace the plant's steam generators. Duke Energy, which acquired Crystal River in 2012, said it decided to close the plant because of unacceptable uncertainty about the scope, cost, and duration of repairing or replacing the damaged containment structure. About two-thirds of U.S. nuclear power plants use steam generators to transfer heat from reactor cooling water to a secondary piping loop in which hot water is converted to steam to drive the turbine generators that produce electricity. Most steam generators have required replacement after 20-30 years of service, a capital expenditure of several hundred million dollars per reactor. Steam generators are such large components that temporary holes must be cut in the reactor containment to remove the old ones and bring in new ones. Some nuclear plants have been closed and decommissioned to avoid the cost of steam generator replacement, such as Oregon's Trojan reactor in 1992. However, most nuclear plant owners have opted to replace the steam generators when necessary, and these projects, while requiring complex planning and execution, have become fairly routine in the nuclear power industry. No other steam generator replacement project caused containment damage similar to that suffered by Crystal River 3. Nevertheless, unrelated problems with another steam generator replacement project, at California's San Onofre station, also led to that plant's permanent shutdown just a month after Crystal River 3. Southern California Edison (SCE), the plant's owner, replaced the steam generators in units 2 and 3 in 2010 and 2011, respectively (unit 1 had been retired previously). One of the new steam generators experienced internal leakage about a year later; inspections showed vibration-related damage to all the new steam generators, later attributed by SCE to design flaws. After considering options to repair or replace the damaged steam generators, SCE announced in June 2013 that it would retire San Onofre 2 and 3. The company cited uncertainty about whether and when the units would be allowed to restart at lower power by NRC, pending steam generator replacement or repair, as a major factor in the shutdown decision. Other considerations were the projected price of replacement power while repairs were being implemented and uncertainty about renewal of the two reactors' operating licenses in 2022, according to SCE. The Vermont Yankee plant was permanently closed in December 2014 because, according to plant owner Entergy, low power prices in New England were causing chronic losses. The single-reactor plant had received a 20-year license renewal from NRC in 2011, despite vigorous opposition from the state of Vermont that focused on leaks of radioactive tritium. The license renewal allowed the plant to operate until 2032, but Entergy decided two years later to begin decommissioning the unit at the end of 2014. The company cited competition from shale gas-fired generation, the relatively high cost of operating a single-unit nuclear plant, and "artificially low energy and capacity prices in the region." Another single-unit nuclear plant, Fort Calhoun in Nebraska, was the most recent U.S. reactor to permanently close, on October 24, 2016. Fort Calhoun had been the smallest operating power reactor in the United States. Because it was owned by the Omaha Public Power District (OPPD), which sets its own rates, the nuclear plant had not been directly subject to the relatively low power prices in the region. However, OPPD determined that it could save up to $994 million for its customers over the next 20 years by closing Fort Calhoun and purchasing low-cost replacement power in the wholesale market. "The economic analysis clearly shows that continued operation of Fort Calhoun Nuclear Station is not financially sustainable," according to OPPD's chief executive. Announced Retirements and Potential Shutdowns Ten operating reactors are currently facing permanent shutdown dates imposed by their owners or reached through negotiated agreements, as listed in the "announced shutdowns" section of Table 2. FitzPatrick and Ginna Facing the most imminent closure on this list are two single-unit New York plants, FitzPatrick, owned by Entergy, and Ginna, owned by Exelon. In announcing plans to close FitzPatrick, Entergy cited the relatively high operating costs of single-unit nuclear plants and "excess power supply and low demand" in the plant's upstate New York market. "Current and forecast power prices have fallen by about $10 per megawatt-hour, which equates to a projected annual loss of more than $60 million in revenues for FitzPatrick," said an Entergy statement. Ginna faces a similar economic situation, according to Exelon. The company said the plant's total market-based revenues would be well below the $55-$60 per megawatt-hour required for profitability. In an effort to keep FitzPatrick and Ginna operating, along with Exelon's two-unit Nine Mile Point plant next to FitzPatrick, the State of New York Public Service Commission approved a system of Zero Emission Credits (ZECs) that would provide additional revenue for the four reactors. The ZEC program would require Exelon to purchase FitzPatrick from Entergy and operate all four of the upstate New York reactors through 2029. Exelon has agreed to the purchase and to keep the four reactors running if the program is implemented as planned. This ZEC funding is being provided in the wake of more than 10 years of experience with a market price on carbon in New York State through the Regional Greenhouse Gas Initiative (RGGI). Given the stated economics of the FitzPatrick and Ginna plants, this price advantage under the market price set through RGGI (approximately $5/ton of carbon dioxide or $2.70 per MWh) for low-carbon power units has not been sufficient to make these plants profitable. Clinton and Quad Cities The next two shutdowns currently scheduled are Exelon's single-unit Clinton plant and the two-unit Quad Cities plant, both in Illinois. "Quad Cities and Clinton have lost a combined $800 million in the past seven years, despite being two of Exelon's best-performing plants," the company said in announcing the shutdowns. The two plants must sell their power into wholesale electricity hubs at consistently lower prices than at adjacent hubs, a situation that has been attributed to chronic transmission congestion. The Illinois General Assembly passed a bill (S.B. 2814) on December 1, 2016, that would provide ZECs to the Clinton and Quad Cities plants, along the lines of the New York ZEC program. Exelon had previously indicated that such assistance could keep the plants operating beyond their planned shutdown dates. Palisades Entergy announced on December 8, 2016, that it would close its single-unit Palisades plant in Michigan on October 1, 2018. Palisades is in a competitive wholesale power market but earns nearly all its revenue under a PPA with Michigan utility Consumers Energy. The PPA, scheduled to continue until 2022, was signed in 2007, when power prices were relatively high and expected to continue rising. In announcing the plant's retirement, Entergy said that, since 2007, "market conditions have changed substantially, and more economic alternatives are now available to provide reliable power to the region." As a result, prices under the PPA, which started at $43.50 per MWh in 2007 and were to rise to $63 per MWh by 2022, have risen substantially above the market level. Entergy's announcement said the early termination of the PPA would reduce electricity costs to Consumers Energy by $344 million from 2018 to 2022, and that the savings would be evenly divided between Entergy and Consumers Energy. Pilgrim Entergy plans to close its single-unit Pilgrim plant in Massachusetts in 2019. "The company is retiring the Pilgrim plant because of continued and projected low energy prices, with no expectation of market structure improvements, along with increased costs," according to Entergy's website. A recent analysis by the R Street Institute said the plant's operation and maintenance costs had risen in recent years and that it needed significant capital expenditures to address safety-related equipment problems that had prompted heightened NRC scrutiny. Although wholesale power prices in the New England market are high enough to cover Pilgrim's operation and maintenance costs, according to the analysis, revenue is not sufficient to justify the capital expenditures needed for the long term. Oyster Creek Exelon's single-unit Oyster Creek plant in New Jersey, the nation's longest-operating power reactor, is also scheduled to be retired in 2019. When the plant's initial 40-year NRC license was renewed for 20 years in 2009, the State of New Jersey required it to comply with water discharge requirements by building closed-circulation cooling towers to reduce warm water discharges into Barnegat Bay. Exelon said the cooling towers would have cost $700-$800 million and that it would retire the plant if required to build them. Exelon and the New Jersey Department of Environmental Protection reached an agreement in 2010 to close Oyster Creek in 2019, a decade before its license expiration, without building the closed-circulation cooling system. Diablo Canyon In contrast to the above closures, the planned shutdown of the two-unit Diablo Canyon nuclear power plant in California is not explicitly based on economic factors. Diablo Canyon is a rate-regulated plant, and therefore its owner, Pacific Gas and Electric (PG&E), is allowed to recover the plant's regulator-approved costs from ratepayers. PG&E contends that the plant "provides low-cost, carbon-free energy" and is a "vital energy resource for California." However, plant opponents have argued for decades that Diablo Canyon's location in a seismically active region poses unacceptable safety risks. The environmental group Friends of the Earth calls the plant "dangerous, destructive and expensive." Friends of the Earth and other groups strongly opposed PG&E's 20-year license renewal applications to NRC, which would allow Diablo Canyon 1 and 2 to operate after 2024 and 2025, respectively. They also opposed the extension of a state land lease for the Diablo Canyon site that was to expire in 2018. To resolve the controversy, PG&E reached an agreement with a coalition of environmental and labor groups on June 21, 2016, to abandon the applications to NRC for Diablo Canyon license extensions in return for support from plant opponents for the state land lease extension (which was granted a week later). As a result, the Diablo Canyon reactors are now scheduled to operate until their current NRC licenses expire in 2024 and 2025. By that time, according to PG&E, sufficient alternative electricity generation will be available to meet regional market demand. Other Potential Closures Several nuclear power units have been identified by their owners as being at risk of permanent shutdown but without any specific dates or deadlines. An Exelon official was reported in March 2016 to have said the company's Nine Mile Point plant was "losing a lot of money," despite being a dual-unit plant with relatively large total generating capacity, and that the company might have to retire it in the future. As mentioned above, Nine Mile Point may continue operating because of Zero Emission Credits being implemented by the state of New York. A FirstEnergy executive reportedly told financial analysts in November 2016 that his company would close or sell its merchant nuclear and coal plants within 18 months unless they could return to cost-based rate regulation or receive financial assistance. FirstEnergy's nuclear plants are the single-unit Perry and Davis-Besse plants in Ohio and the two-unit Beaver Valley plant in Pennsylvania. The analysts were reportedly told that the plants could not compete in regional wholesale power markets with low-cost electricity from natural gas plants and wind turbines. In addition to nuclear plants identified by their owners, several other reactors have been singled out by at least one of the major financial ratings agencies as being at risk of early retirement, generally because of market conditions. Byron 1 and 2 in Illinois were listed by UBS, and the single-unit Palisades plant in Michigan and Three Mile Island plant in Pennsylvania were listed by Fitch Ratings. Merchant Nuclear Power Plant Economics At the most basic level, existing nuclear power plants need to generate revenues from electricity sales, capacity markets (where available), and other potential sources that exceed the average total cost (ATC)—fuel, capital, and operating—of producing electricity in order to economically justify continuing operations. Generally, prices for wholesale electricity—the largest source of revenue for merchant nuclear power plants participating in competitive markets—have decreased in recent years while nuclear generation ATCs have increased. As a result, the financial condition of some U.S. nuclear power plants has been stressed and in some cases ATCs have exceeded revenues, resulting in actual and planned closures. However, the degree of financial pressure across the nuclear fleet is not uniform. Each power plant is subject to a unique mix of market and cost variables, including (1) plant site and locational pricing, (2) the Regional Transmission Organization (RTO) market, (3) non-electricity revenue sources (e.g., capacity payments), (4) plant size, (5) transmission constraints, (6) generation mix within the transmission area, and (7) the marginal cost of electricity from price-setting fuels (e.g., coal and natural gas). A limited plant-level assessment of electricity revenues and fuel and operations and maintenance (O&M) costs is included in this report (see the Appendix ). However, a detailed plant-by-plant financial analysis that includes all revenue and cost variables is beyond the scope of this report, as are the economics and levelized cost of electricity (LCOE) considerations associated with constructing new nuclear plants. The following sections provide an overview of concepts and trends that impact the financial and economic conditions considered by existing U.S. nuclear power plants. Nuclear Power Generation Costs Much of the discussion surrounding nuclear power economics has focused on revenue items such as low natural gas prices, the depressed wholesale market clearing prices that result, and low capacity revenues in some organized markets. These items certainly impact the economics of nuclear power, and this report includes discussion of these revenue-related issues. However, the cost portion of the financial equation is also important to understand due to its impact on economic viability. At an industry-wide level, the Nuclear Energy Institute (NEI) reports that average total generating costs—which include fuel, capital, and operating costs—for nuclear electricity increased from $28.27 per megawatthour (MWh) to a peak of $39.70 per MWh in 2012 and in 2015 were reported to be $35.50 per MWh. The NEI analysis indicates that while all cost categories have increased, capital expenditures were the largest contributor to average total costs, having more than doubled over the same time period. NEI also reports that total average costs can vary depending on the number of reactor units at each power plant. Total average costs for multi-unit plants were $32.90 per MWh in 2015 versus $44.52 for single unit power plants. The majority of this difference is in the "operating" cost category. In response to these cost trends, NEI has an active initiative called "Delivering the Nuclear Promise" which aims to reduce fleet-wide total generation costs by 30% by 2018 from the 2012 high of $39.70 per MWh. For the purpose of comparing revenues and costs for the industry as a whole, average total costs of generation are used in order to illustrate the relationship between electricity sales revenue and all cash costs that are incurred. In addition to the NEI industry-wide average total cost information, CRS has access to 2015 plant-level fuel and O&M costs for 33 nuclear power plants that operate in competitive power markets. This plant-level fuel and O&M cost information is compared with nodal electricity prices paid to each generator in order to determine whether revenue from electricity sales exceeds fuel and O&M costs (for additional information see the Appendix ). How Are Competitive Wholesale Electricity Prices Determined? In a competitive power market, the per-unit wholesale price of electricity (e.g., dollars per Megawatthour) paid to generators is their primary revenue source and is based on the economic concepts of supply and demand curves. Price determination in competitive markets is subject to Security Constrained Economic Dispatch (SCED), which takes into account costs, transmission limitations, and reliability requirements. In simple terms, all generators within a competitive market offer electricity at prices and volumes that are organized by the RTO from lowest to highest price in order to construct a supply curve (see Figure 2 ). Once expected demand levels and price bids for this time period (e.g., hourly) are determined, the price at which demand and supply intersect, when taking into account SCED considerations, is referred to as the "market clearing price." This is the price received by all generators that offered electricity at a price equal to or below the market clearing price level. As an example, a nuclear plant might offer to sell a certain volume of electricity at $7 per MWh (approximate value for fuel costs) and a wind generator may offer electricity for $0 per MWh. However, if the clearing price is $40 per MWh (because the marginal generator submitted a $40 per MWh offer) then the nuclear plant and wind generator will be dispatched (i.e., ordered to run) and both will receive $40 per MWh for electricity provided during this time interval. Figure 2 provides a hypothetical illustration of how wholesale electricity prices are generally determined. Nuclear power being offered at less than the market clearing price does not necessarily mean that it is profitable. Merchant nuclear plants are generally viewed as "price takers" in a wholesale power market since the plants generally prefer to operate on a continuous basis, regardless of the market price. However, indications are that some nuclear generators may start following demand load patterns in order to improve electricity sales revenues in some locations. Typically, merchant nuclear plants want to offer prices low enough to clear the market and will participate in the competitive power markets in a way that ensures market clearance and dispatch. As a result, nuclear plant profitability is generally a function of the market clearing price level that is set by price-setting units. In practice, price formation in wholesale power markets can be quite complex, with the RTO/ISO having to manage various system constraints (e.g., transmission, scarcity, reliability) that can challenge the simple application of supply and demand curves. Nevertheless, supply and demand fundamentals are the underlying premise by which wholesale electricity prices are determined. Generally, price offers are a function of the fuel cost needed to generate a unit of electricity, and do not include capital costs. The fuel cost for wind is zero and, according to the Nuclear Energy Institute, the fuel cost for nuclear was approximately $7 per MWh in 2015. Also, nuclear generators—because plants generally prefer to operate on a consistent and near-continuous basis—will offer power at low prices. Wind generators—because many projects receive out-of-market revenue/value sources (e.g., power purchase agreements, tax credits, renewable energy credits)—also want to clear the market and may be motivated to submit zero or even negative price offers, if allowed by the respective RTO, to ensure that they clear and are dispatched (for more information see text box below titled Wind Power and Negative Electricity Prices: What Is the Relationship? ). Coal and natural gas generators, on the other hand, will typically offer electricity prices that are a function of fuel acquisition costs, which can vary based on benchmark fuel prices, the location at which the generator purchases the fuel, and the conversion efficiency (heat rate) of the power plant. How Natural Gas and Coal Impact Wholesale Electricity Prices Natural gas and coal play an important price-setting role in competitive power markets. For the purpose of this discussion, focus is placed on the role of natural gas and how the price of this fuel can affect the wholesale price of electricity paid to generators. Natural gas affects wholesale electricity prices in two primary ways. First, because natural gas power generators are generally able to follow demand/load patterns and can be dispatched with relative ease, they often set prices during peak demand hours—when electricity demand and prices are typically the highest each day. Second, natural gas power generation is a primary price-setting fuel for wholesale electricity prices in competitive power markets. Table 3 indicates the percentage of time intervals that various fuels set the real-time price in three different RTOs during different years. Data contained in Table 3 suggest that natural gas is a primary price-setting fuel within the three RTOs included in the table. The price-setting contribution of natural gas in MISO and PJM has increased over the respective periods included in the table. While the ERCOT price-setting information suggests that natural gas may have declined in terms of its price-setting role within that RTO, the Market Monitor report indicates that the 34% real-time energy price decline observed in this market from 2014 to 2015 was "primarily driven by lower natural gas prices." Commodity fuel prices for natural gas and coal generally have the largest impact on wholesale electricity prices. With natural gas increasing its price-setting role in some competitive markets, a general analysis of fuel costs for natural gas-derived electricity provides an indication about the downward pressure natural gas has exerted on wholesale electricity prices, and therefore revenue for electric power generators. See Figure 3. The fuel cost range for generating electricity from natural gas has declined since peaking in 2008, while the ATC (fuel, operations and maintenance, and capital) for nuclear power generation has increased, relative to 2008 levels. As discussed above, lower natural gas costs generally translate into lower wholesale power prices. The combination of rising nuclear generation costs and declining natural gas prices is contributing to the financial challenges encountered by nuclear power plants in competitive power markets. Natural gas fuel costs per MWh ranges in Figure 3 reflect U.S.-wide averages, and it should be noted that each individual nuclear plant will be subject to a unique set of market and location-based price variables that can impact revenue from electricity sales. Locational Marginal Prices In competitive power markets, there is not a single wholesale electricity price paid to all electricity generators operating in an RTO region. Rather, electricity revenues received by generators are a function of locational marginal prices (LMPs), which vary within each RTO. LMPs generally include three primary price components: (1) energy, (2) transmission congestion, and (3) energy losses. LMPs can be affected by market variables such as fuel prices, generation mix, and transmission constraints at a specific location. In some locations, the presence of renewable electricity generation from wind and solar projects can affect LMPs and in some instances can cause real-time LMPs to drop below zero for limited periods of time. (See text box below: Wind Power and Negative Electricity Prices: What I s the Relationship? ) As discussed above, the cost of fuel (i.e., natural gas and coal) affects clearing prices, and fuel costs can also vary by location. For example, due to potential transportation cost and infrastructure limitations, the price of natural gas paid by a power generator in Pennsylvania might be different from that paid by a generator in New York. All else being equal, the electricity market clearing price at these respective locations might also be different. Figure 4 shows the range of LMPs for nuclear power plants operating in competitive power markets along with average total costs for nuclear power generation. LMP and ATC projected estimates for calendar year 2016 indicate that the entire expected electricity price range for nuclear power plants in competitive markets may be less than the ATC of nuclear electricity generation. While this projection does not include other revenue sources and does not suggest that all nuclear power plants will realize revenue that is less than cost, it does indicate that the location and cost structure of each nuclear power facility is an important consideration when assessing the financial challenges of a specific plant. Other Revenue Sources In addition to electricity sales, which are the largest revenue source for nuclear power plants, some RTO/ISO markets provide other revenue opportunities for power generators. Capacity, uplift, and ancillary services—all defined in the following text—are some areas where generators can be compensated for providing certain services other than selling electricity. Figure 5 provides average all-in electricity prices for various RTO/ISO markets by revenue source. The largest source of non-energy revenue in some competitive power markets is in the form of capacity payments. To ensure resource adequacy, most RTO/ISOs have developed and evolved capacity markets that provide an economic signal that would provide incentives for adequate generation for peak power demands and to ensure that generation resources are available to satisfy future demand. Depending on the RTO, capacity payments can be made through either a bilateral contract between a load serving entity (LSE) and a generator or through participation in a capacity auction that is organized and administered by the RTO/ISO. However, not all RTO/ISOs have a capacity market (e.g., ERCOT does not have a capacity market and uses scarcity pricing as an incentive for motivating new generation sources). For those that do, each has a unique market design that determines the value of and time frame for capacity payments. As indicated in Figure 5, capacity payments can vary by RTO, and some RTOs have changed their capacity market designs in such a way that may benefit nuclear power generators. Additionally, power plant location can impact the value of capacity payments received by nuclear and other power generators. For example, PJM's 2019/2020 Reliability Pricing Model (RPM) capacity market resulted in clearing prices that ranged from $100 per MW-day to $202 per MW-day across different zones. RPM and similar forward capacity markets guarantee payments for new capacity that performs as projected. Nuclear power plants are eligible for capacity payments, although not all merchant nuclear generators clear capacity auctions. Power Purchase Agreements (PPAs) Some nuclear power plants that operate in competitive power markets have separate power purchase agreements (PPAs) outside the RTO market that provide a specified value for electricity generation. The existence of a PPA reduces the price risk to nuclear power producers associated with participating in competitive power markets. It is not clear how many nuclear plants in competitive power markets have PPAs. However, an industry source suggests that at least six nuclear plants have PPAs for either all or a portion of their generating capacity. Depending on the contractual terms—not made available to CRS—included in each agreement, these PPAs could potentially provide some financial stability to plants with such agreements. Case Study: Quad Cities Nuclear Generating Station Located in Cordova, IL, the Quad Cities Nuclear Generating Station consists of two nuclear reactors with a combined net summer electric capacity of approximately 1,820 Megawatts. The plant is partially rate regulated, with 25% of the plant under Iowa rate regulation and the other 75% participating in the PJM power market. On June 2, 2016, Quad Cities owner-and-operator Exelon announced that it was moving forward to shut down and retire the plant before the end of its operating license. Available price information, capacity payments, and production costs (fuel, operations and maintenance) make it apparent that the Quad Cities power plant is under a degree of financial stress. Generally, the nodal electricity price that determines the value of electricity sold by the Quad Cities power plant has been volatile in the years 2013 to 2016 and is expected to be below fuel and O&M costs in 2016. PJM capacity payments for Quad Cities provide some additional revenue. However, the combination of capacity payments and electricity sales is estimated—assuming constant production costs at 2015 levels—to be less than production costs in 2016, not considering any capital expenditures. Current electricity price projections and capacity auction results indicate that electricity sales will not be large enough to pay for production costs, although capacity payments in 2017 may provide supplemental revenue that cover production costs. The Quad Cities plant did not clear the 2018/2019 or 2019/2020 capacity auctions; therefore the plant is not eligible to receive capacity payments during those years. Faced with low electricity price projections and revenues that are expected to be less than fuel and O&M costs, not to mention any capital expenditures, Exelon management decided that it is in the financial best interest of the company to shut down the power plant. The Illinois legislature passed S.B. 2814 on December 1, 2016, which includes a Zero Emissions Credit (ZEC) incentive program for existing nuclear plants in the state. This may result in continued operations of the Quad Cities nuclear plant. Plant-Level Analysis of Electricity Sales Revenue and Fuel and O&M Costs Plant-specific cost and price information available to CRS for 33 nuclear power plants that operate in competitive power markets was used to assess whether electricity revenue did, or is expected to, exceed 2015 fuel and O&M costs for each plant for the years 2015 to 2019. The analysis is unique, compared to materials reviewed for this report, in that it compares plant-level nodal electricity prices with plant-level fuel and O&M costs. Nevertheless, this analysis is inherently limited and is included in this report as an initial financial condition assessment of merchant nuclear power plants. Additional revenue sources (e.g., capacity payments) and other costs (e.g., capital) at the plant level were not available to CRS and were not included in the estimates presented. As a result, CRS is not making an assessment of which reactors are at risk of closure. The estimates provide an incomplete view of nuclear power economic viability. Figure 7 summarizes the results of this analysis. Table A-1 in the Appendix provides plant-specific estimates for each of the 33 plants on a per MWh basis. Generally, from the perspective of electricity revenues versus fuel and O&M costs, calendar year 2016 is projected to have been quite challenging for many existing nuclear power plants. Based on data sources available to CRS, and assuming that all electricity is sold in the wholesale market, 19 of the 33 power plants are estimated to have fuel and O&M costs that exceed electricity sales revenue in 2016. Due to forward electricity prices being projected to increase in the near term, this number is projected to drop to seven for the years 2017 to 2019. While this analysis is limited by available plant-level data for all revenues and costs, it does indicate that some nuclear power plants are encountering financial challenges that may be difficult to manage in the near term. Policy Options and Considerations The increasing number of recent nuclear power plant closures and announced early retirements has motivated discussion about whether the federal government should provide financial support mechanisms for existing nuclear power facilities. The nuclear industry and its supporters contend that such support is in the national interest because nuclear power plants provide large amounts of highly reliable, low-carbon electricity. However, opponents contend that nuclear power suffers from too many drawbacks (such as accident risk and waste management problems) and that federal incentives should focus instead on renewable energy and efficiency. Furthermore, federal financial incentives for existing nuclear power would likely be opposed by owners of other generating assets that could potentially benefit financially as a result of early nuclear plant retirements. Nevertheless, if federal support for nuclear power were pursued, Congress would have a variety of options to consider, each with its own set of policy questions. One challenge associated with federal-level nuclear incentives is that financial conditions across the entire fleet are not uniform. Each nuclear power facility is subject to a unique set of cost, price, and financial performance variables. To date, much of the policy action has taken place at the state level, with several states considering, passing, or implementing legislation/regulations to support nuclear power (see text box below, State-L evel Policy Action to Support Existing Nuclear Power Plants ). Secretary of Energy Ernest Moniz has reportedly indicated that the federal government has limited existing authority to provide financial support for operating nuclear power plants and that doing so is currently a state issue. A recent report by the Secretary of Energy Advisory Board Task Force on the Future of Nuclear Power included this recommendation for the Department of Energy (DOE): For existing nuclear plants, the Task Force endorses DOE's efforts to work with the Federal Energy Regulatory Commission (FERC), State regulatory authorities, and regional and independent system operators to encourage arrangements that will preserve the U.S. fleet until the end of their useful life, subject to continued compliance with prevailing safety and environmental regulations. The Task Force believes this is essential if U.S. carbon goals are to be achieved. A range of policy options are available to Congress, should it choose to act, that could potentially provide some degree of financial support for operating U.S. nuclear power plants. Some of these potential options are discussed in the following sections. Power Market Price Formation Changes In March 2015 the Nuclear Energy Institute (NEI), along with other industry groups, announced a joint effort to pursue market and price reforms in competitive electric power markets. The groups sent a letter to the Federal Energy Regulatory Commission outlining a set of principles for price formation reforms. Generally, the primary market reform argument is that clearing prices do not include all costs (e.g., start-up and uplift) associated with operating an electric power system. As a result, LMPs may be lower than they otherwise would be if all costs were included. FERC has an ongoing Energy Price Formation initiative to evaluate electricity price formation in RTO and ISO markets. In June 2016 FERC issued a rule requiring RTOs/ISOs to change their settlement procedures and shortage pricing triggers in order to more accurately compensate generation resources. FERC is also evaluating other price formation issues (e.g., uplift payments); future rules could potentially impact electricity price formation in wholesale markets. Congressional action in this area could include oversight of how FERC's Energy Price Formation initiative might impact nuclear power generators. This effort is consistent with the Future of Nuclear Power task force recommendation referenced above. Tax Incentives A federal production tax credit (PTC) is currently available for the generation of electricity by new, advanced nuclear power facilities during their first eight years of operation. However, no federal tax incentives are available for existing nuclear power plants. Generally, currently available energy tax credit incentives are designed to encourage construction and operation of new energy production facilities and are based on either energy/electricity production (i.e., production tax credit or PTC) or investment (i.e., investment tax credit or ITC). In addition to the nuclear PTC for new plants mentioned above, two specific energy tax credits currently available to renewable energy technologies are (1) the Renewable Electricity Production Tax Credit, and (2) the Investment Tax Credit for renewable energy. Legislation making existing nuclear power generation eligible for either of these existing tax credit incentives would provide some financial value and could potentially support continued operation of at-risk plants. However, since the financial condition across the entire U.S. nuclear fleet is not uniform there would likely be plants that capture the tax credit value as a windfall, without actually needing it to justify continued operations. Carbon Price Because of the low-carbon attributes of nuclear-generated electricity, a price on carbon emissions could potentially benefit existing nuclear power plants if the carbon price were reflected in wholesale electricity prices. However, the design of such a carbon price policy could determine whether or not existing nuclear power would be financially advantaged. A carbon price policy could take many forms, including a carbon tax, a cap-and-trade approach, or an environmental regulatory approach such as the Clean Power Plan (CPP). A carbon tax would place a value on carbon emissions that, all else being equal, would increase the cost of producing electricity from carbon emitting fuels. To the extent that such costs are included in marginal-unit price offers from coal and natural gas generators, a carbon tax could effectively increase wholesale market clearing prices that determine electricity sales revenue for existing nuclear power plants. However, a regulatory approach such as the CPP, which would likely result in an effective carbon price in many states, may have varied effects for existing nuclear facilities, depending to some degree on whether states achieve CPP compliance with a rate-based or mass-based target. A CPP rate-based standard (metric tons of carbon per megawatt-hour of electricity generation) does not include existing nuclear in the calculation of megawatt-hours. Therefore, if an existing nuclear plant closes, there will be no change in a state's carbon emissions rate under the rate-based approach. Under the rate-based standard, replacement of an existing nuclear plant with a gas-fired plant could reduce a state's calculated emissions rate, if the emissions rate from the new gas-fired capacity were lower than the state average. By comparison, states using a mass-based approach would likely have a stronger incentive to maintain existing nuclear power. Under a mass-based approach, if an existing nuclear plant shut down and was replaced by gas-fired generation, the total carbon emissions in the state would rise, making it more difficult for a state to achieve its CPP reduction goals. According to EPA's analysis of the CPP, "existing nuclear generation could be slightly more competitive under a mass-based implementation than under a rate-based implementation, because the former tends to create more wholesale price support for those generators." Federal Power Purchase Agreements (PPAs) Power Purchase Agreements (PPAs) established between at-risk nuclear power plants and federal agencies are a policy option that could potentially provide a degree of electricity price certainty for both parties. PPAs have been used by the federal government to purchase power from renewable electricity projects over multiple years. A federal agency could potentially contract with a nuclear plant to purchase power at an agreed-upon price over a multi-year period. One possible contractual mechanism might be a Contracts for Difference (CfD) contract model. A CfD contract essentially sets a price level for electricity and requires/entitles the parties to make up the difference between the actual market price and CfD contract price. This contract structure provides price certainty to both the buyer and the seller while eliminating the requirement to physically deliver nuclear electricity to a federal facility. Appendix. Plant-level Analysis of Electricity Sales Revenue and Fuel and O&M Costs Table A-1 below contains analysis of electricity revenues and fuel and O&M costs for 33 nuclear power plants operating in competitive power markets within the United States. For each plant, 2015 fuel and O&M costs are subtracted from electricity sales revenue. Both revenue and cost data used to perform this analysis are at the plant level, with revenues represented by the nodal electricity price paid to each power plant and plant-level fuel and O&M cost information as reported by third-party sources. The analysis in Table A-1 is unique in that it compares plant-level electricity sales revenue with plant-level costs. Published material reviewed for this report included nodal electricity prices compared with industry average costs as well as plant-level costs compared with regional hub prices. Although this combination of plant-level data sets is a unique attribute of this analysis, nevertheless it provides a limited view of the financial state of U.S. nuclear power plants and does not consider other cash expenses such as capital expenditures or additional revenue sources such as capacity payments—where available—and PPA contracts. Additionally, CRS is not assessing whether or not plants are at risk of closure. Estimates included in the table indicate only whether or not electricity sales generate enough revenue to cover fuel and O&M costs. However, total plant-level profitability, and therefore the economic viability of each plant, can be influenced by other cost and revenue considerations that are not included in the estimates presented. Data-access limitations prevented CRS from performing such analysis for this report.
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Summary: Some of the 60 operating nuclear power plants (comprising 99 nuclear reactors) in the United States have experienced financial stress in recent years due to a combination of low wholesale electricity prices and escalating costs. Six nuclear reactors have permanently shut down during the past five years, and 19 others have announced their intention to close or have been identified as "at-risk" of closure by financial consultants and ratings agencies. Generally, U.S. nuclear plants are located in one of two market areas: (1) competitive-where the value of electricity fluctuates based on supply-side price offers that are generally a function of fuel (e.g., natural gas) costs and demand-side price bids, and (2) cost-of-service-where the value of electricity is set at a rate based on regulator-approved costs, operating expenses, and a reasonable investment return. Most of the U.S. plants considered vulnerable to shut down before expiration of their operating licenses are "merchant plants" that sell all or most of their power into competitive wholesale power markets. The price paid to merchant plants for electric power varies by location and is influenced by the price-setting fuel (usually natural gas and coal), transmission congestion, and other factors. Wholesale electricity prices in certain locations have fallen and electricity sales revenue may be below the fuel and operating and maintenance (O&M) costs of some plants, not considering capital expenditures that may also be incurred. CRS analysis of third-party data indicates that 19 of 33 power plants operating in competitive power markets may incur fuel and O&M costs that exceed electricity revenues for each plant in 2016. However, this number declines to seven in 2017 due to rising forward electricity prices as reported by Bloomberg. While merchant generators do have other revenue sources (i.e., capacity payments where available, power purchase agreements, and hedging positions) and additional costs (e.g., capital), CRS was not able to locate plant-specific information about these revenues and costs that would allow for a holistic financial assessment at the plant level. The nuclear power industry and its supporters have proposed that Congress take action to prevent currently operating U.S. reactors from shutting down before their licenses expire. Supporters contend that nuclear power should be valued as a domestic source of highly reliable, low-carbon electricity. However, opponents contend that nuclear power suffers from too many drawbacks and that federal incentives should focus instead on renewable energy and efficiency. Nuclear power plants annually provide about 20% of total U.S. electricity generation. To date, all of the policy action related to financial support for existing nuclear plants has been at the state level. New York has implemented a Clean Energy Standard (CES) that includes payments to qualified nuclear power plants in the state starting at approximately $17 per megawatt-hour in 2017. The CES has been challenged on legal grounds. A similar program was recently approved by the Illinois legislature, and Ohio has also considered nuclear support. Since each nuclear power plant is subject to a unique combination of financial variables, federal-level incentives are challenged because some nuclear plants are expected to continue operating without federal financial support. Should Congress choose to debate financial incentives for existing nuclear plants, several options may be considered. Tax incentives based on capital investment or electricity production could potentially provide financial support for existing nuclear plants. Establishing a carbon price-carbon tax, cap-and-trade, emissions regulations-could also provide some financial assistance to nuclear power, depending on how a carbon price mechanism was designed and implemented. Finally, Congress could authorize and require the federal government to enter into power purchase agreements with nuclear power plants that would provide a guaranteed price for nuclear-generated electricity. Additionally, the nuclear industry has been advocating that the Federal Energy Regulatory Commission (FERC) institute changes to electricity price formation in competitive power markets.
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Summarize: Keith Urban Thought Rehab Would "Destroy" Nicole Kidman Marriage Keith Urban and Nicole Kidman were still newlyweds --- married for about four months -- when he checked into rehab for drug and alcohol addiction in October 2006. Urban, 43, worried that the time away would wreak havoc on their new union. PHOTOS: Hollywood's most romantic couples "[I thought], 'Surely let's give it a few years so we've got some solidity, because this kind of thing could tear us apart,'" the country singer said Monday on The Oprah Winfrey Show. "'This could just destroy us.'" But it was Kidman herself, plus a few close pals, who staged an intervention for Urban, who was hooked on cocaine and other substances. "It was really a profound moment in so many ways," the crooner said of the intervention. "The way in which Nic handled that moment was just perfect...Everything was just designed, I believe, for that moment to fuse us together." PHOTOS: Nicole and Keith's personal wedding pix It was, in fact, Urban's third time in rehab in 8 years. "I had to make a decision which road I was going to take, once and for all," he said. "I'd been at that crossroads before and always taken the wrong road." What was different this time? "Love," he told Oprah Winfrey. PHOTOS: How Nicole slammed her ex Tom Cruise Urban left for rehab the same night as that intervention, he said. "I didn't care about anything else." He gushed of his wife (and mother to daughter Sunday Rose, nearly 2): "I look back now and realize Nic has taught me so much and brought so much into my life and opened my eyes in so many ways...[This is] the best place I've ever been." Actress Kidman also opened up on Oprah about their enduring romance. "I think marriage is about, every day, trying to make it better and working through things and when things are tough, saying, 'I'm going to come to you and talk,'" the Rabbit Hole star mused. "We're just very tight, and we stay really tight." PHOTOS: Sunday and other adorable A-list babies Australian like her husband, Kidman has really taken to their new life in Nashville, Tenn. "Some people like the much higher sort of more glamorous lifestyle," she said. For us, we just prefer a much quieter, private [life]." Her favorite things to do with Sunday Rose, who they sometimes call "Sunny": Sharing her cappuccino foam -- and taking baths. "I love her little shoulders, you know?" Kidman cooed. "She's in the bath, and I see those tiny little shoulders. Oprah, don't get me started!" Keith Urban on Nicole Kidman: I Thought Rehab Would 'Destroy Us' Email This Sitting down with "[I thought], 'Surely let's give it a few years so we've got some solidity, because this kind of thing could tear us apart,'" he told Oprah, But it was the Oscar-winning actress who ended up staging an intervention, which Urban believes help foster their bond. "It was really a profound moment in so many ways. The way in which Nic handled that moment was just perfect... Everything was just designed, I believe, for that moment to fuse us together." Sitting down with Oprah Winfrey Keith Urban spoke candidly about entering rehab just four months after he married Nicole Kidman "[I thought], 'Surely let's give it a few years so we've got some solidity, because this kind of thing could tear us apart,'" he told Oprah, Usmagazine.com reports. "This could just destroy us."But it was the Oscar-winning actress who ended up staging an intervention, which Urban believes help foster their bond."It was really a profound moment in so many ways. The way in which Nic handled that moment was just perfect... Everything was just designed, I believe, for that moment to fuse us together." http://xml.channel.aol.com/xmlpublisher/fetch.v2.xml?option=expand_relative_urls&dataUrlNodes=uiConfig,feedConfig,entry&id=753791&pid=753790&uts=1291122677 http://www.popeater.com/mm_track/popeater/music/?s_channel=us.musicpop&s_account=aolpopeater,aolsvc&omni=1&ke=1 http://cdn.channel.aol.com/cs_feed_v1_6/csfeedwrapper.swf Nicole Kidman Photos Actress Nicole Kidman smiles while on the sidelines before the NFL football game between the New Orleans Saints and Dallas Cowboys, Thursday, Nov. 25, 2010, in Arlington, Texas. (AP Photo/Mike Fuentes) AP FR103746-AP His 2006 stint in treatment was the third time in eight years that the country singer went to rehab. "I had to make a decision which road I was going to take, once and for all. I'd been at that crossroads before and always taken the wrong road.""I look back now and realize Nic has taught me so much and brought so much into my life and opened my eyes in so many ways... [This is] the best place I've ever been," he added.Urban and Kidman married in June of 2006. They have one daughter, Sunday Rose. Kidman has two children with her ex-husband Tom Cruise. The actress is enjoying early Oscar buzz for her role in the family drama 'Rabbit Hole,' which earned her raves at the Toronto Film Festival.
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Summary: Keith Urban and Nicole Kidman had been married just four months when he checked into rehab for drug and alcohol addiction-and the country star thought it would ruin them. Yesterday he told Oprah Winfrey his thoughts at the time were, "Surely let's give it a few years so we've got some solidity, because this kind of thing could tear us apart. This could just destroy us," Us reports. Even so, it ended up being Kidman herself who staged his intervention. It was his third rehab stint in eight years, but it stuck-because of "love," he says. For more from the interview, click here.
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Summarize: "They were praising their God," Pastor de Vega said. Tunggal Panaluan, the firing range on Nusakambangan where Myuran Sukumaran and Andrew Chan were executed. Credit:James Brickwood "It was breathtaking. This was the first time I witnessed someone so excited to meet their God." Pastor de Vega said it was the most beautiful experience she had ever had. "They bonded together," she said. "Brotherhood. They sang one song after another. Praising God. They sang a few songs together, like in a choir. "The non-Christian, I believe, also sang from his heart. It was such an experience." One of the ambulances carrying a coffin of one of the executed leaving Wijaya Pura in Cilacap. Credit:James Brickwood At 15 minutes to midnight, the families of the condemned lit a candle as they watched the grim procession of cars taking the prisoners to the execution site. One of the people in the group led a recitation of the Lord's Prayer. Immediately afterwards they were startled by loud gunshots. Coffins leaving Wijaya Pura in Cilacap after the executions on Nusakambangan. Credit:James Brickwood Many became hysterical while others, including the spiritual advisers, offered consolation. But by the time the coffins arrived for identification and official handover, a measure of calm had returned and the process went smoothly. Father Charles Burrows, who provided spiritual guidance to condemned Brazilian man Rodrigo Gularte, said the men met their fate without blindfolds, staring straight ahead. A midnight candlelight vigil held at Wijaya Pura in Cilacap on the day of the execution of Andrew Chan and Myuran Sukumaran. Credit:James Brickwood It was breathtaking. This was the first time I witnessed someone so excited to meet their God. Pastor Karina de Vega "Everyone was looking forward, it seems everyone accepted their fate," Father Burrows said. He said it was difficult because Gularte, who was diagnosed with schizophrenia when he was a teenager, was mentally ill. Gularte talked to animals and was afraid of electromagnetic waves from satellites watching him above his prison on the island of Nusakambangan. In his deluded state, he believed Indonesia had abolished capital punishment and established a prisoner extradition agreement with Brazil, which meant he could go home next year. "We didn't think [the execution] would happen," Father Burrows said. "It is finished. It's all done." Pastor Tuhu Santoso led the prison church Mass in Besi prison on the island before the condemned prisoners were put in isolation on Saturday. Nigerian Raheem Agbaje Salami, who was known as Stefanus, described how Jesus had saved him. Chan played the guitar and read a verse from the Bible stating everything that happened was God's will. Not a single feather would fall off a sparrow, without God permitting it, Chan read. Chan and Sukumaran told Pastor Tuhu they still believed in miracles and that God would save them but, if they were executed, it was part of God's plan. The pair were initially told they could not be accompanied by their choice of religious counsellor in their final hours. In an SMS, an angry Michael Chan, Andrew's brother, told Fairfax Media: "Last bit of dignity denied." However, Indonesian authorities changed their minds at the last moment and Christie Buckingham, a senior pastor at Bayside Church, and David Soper, an old friend of the Chan family, were able to be with the men until the final minutes. Fairfax Media understands both men died quickly. A source said all eight prisoners died after being shot in the heart. It was not necessary for the commander to shoot anyone in the head, as is the case if prisoners don't die after 10 minutes. One of the condemned men, Nigerian gospel singer Okwudili Ayotanze, was confident right until the end that he was going to be taken off the execution list due to his pending case in the Administrative Court. Friends and those who knew him for more than 10 years in prison were dismayed his case had been left to the 11h hour. They believed that, if the court process had started earlier, Ayotanze's life could have been saved. A friend close to Ayotanze said he never stopped helping other prisoners. He would make sure everyone would come out of their cells and attend Mass. "He was basically the Nigerian version of Andrew Chan," he said. "He was a good person. I will really feel the loss." The Chan and Sukumaran families released a statement on Wednesday morning. "Today we lost Myuran and Andrew," the families said. "Our sons, our brothers. In the 10 years since they were arrested, they did all they could to make amends, helping many others." The family said they asked for mercy, but there was none. "They were immensely grateful for all the support they received. We too, will be forever grateful." Brin Sukumaran, Myuran's sister, posted a tribute on Facebook at midnight. "Bless the lord o my soul. Myu likes this song. He sang it today. Please sing it for him," she wrote. Ambulances containing eight coffins left Nusakambangan and are on the 12-hour journey to Jakarta. Australia's consul-general is required to identify the bodies of the Australians, Foreign Affairs Minister Julie Bishop said. The bodies of Chan and Sukumaran will then be returned to Australia, most likely on Friday. Media playback is unsupported on your device Media caption Tony Abbott: "Australia deeply, deeply regrets these executions in Indonesia" Australia has recalled its ambassador from Indonesia after two Australian men were executed for drug smuggling. Andrew Chan and Myuran Sukumaran were among eight people from several countries executed by firing squad in the early hours of Wednesday on the prison island of Nusakambangan. Brazil's government also expressed its "deep dismay" at the execution of one of its citizens, Rodrigo Gularte. But the execution of a Philippine woman was called off at the last minute. Mary Jane Fiesta Veloso was spared after Philippines officials said a woman she had accused of planting drugs on her was in police custody. How was Mary Jane Veloso spared? The families of the condemned prisoners paid them final, emotional visits on Tuesday afternoon before the sentences were carried out. All eight refused offers of blindfolds and were singing together in their final moments, a pastor present at the execution told the Sydney Morning Herald. Hours after the executions, ambulances left the prison island, taking the prisoners' bodies to their chosen funeral sites in Indonesia or their home countries. Image copyright AFP Image caption Andrew Chan (left) and Myuran Sukumaran were sentenced to death in 2006 Image copyright AP Image caption Australia said Sukumaran and Chan were reformed characters - Sukumaran painted while in prison, creating this self portrait Australia had mounted a lengthy diplomatic campaign to save Chan and Sukumaran, convicted in 2006 of being the ringleaders of a group of Australian heroin traffickers known as the Bali Nine. Australian Prime Minister Tony Abbott said on Tuesday that the executions were "cruel and unnecessary", adding that Chan and Sukumaran had been "fully rehabilitated" while in prison. "We respect Indonesia's sovereignty but we do deplore what's been done and this cannot be simply business as usual," he said. Media playback is unsupported on your device Media caption Footage shows ambulances carrying coffins and candlelit vigils held by activists This is the first time Australia has recalled an ambassador from Indonesia, and its first-ever recall over the execution of one of its citizens abroad. Indonesian Attorney General Muhammad Prasetyo defended the executions, saying his country was fighting a "war" on drugs. Indonesia is an important country to Australia, the BBC's Jon Donnison says, with the two working closely together on asylum seekers and terrorism issues. Analysis: Karishma Vaswani, BBC Indonesia editor Joko Widodo, Indonesia's president, has publicly declared a war on drugs and said he will refuse all clemency applications from convicted drug traffickers - a stance that could prove legally problematic given that the very point of a presidential clemency is to evaluate each individual on a case-by-case basis. But that will be little comfort to the families of the eight people who were executed. Indonesia risks souring relations with other nations over its hardline approach, as it has already with Australia, home of two of the smugglers. But Indonesia's new president appears willing to take the risk, even if it means tarnishing his own reputation as a liberal, reform-minded leader. Joko Widodo stands firm Viewpoints: Are Australia-Indonesia ties at risk? Brazil's government later issued a statement saying the execution of Gularte marked "a serious event in the relations between the two countries". Gularte's family had said he had schizophrenia and should not have been on death row. Gularte was the second Brazilian to be executed for drug smuggling in Indonesia in four months. Marco Archer Cardoso Moreira was killed by firing squad at Nusakambangan in January, prompting Brazil to recall its ambassador. Image copyright Reuters Image caption Some of those executed have already been buried British drug smuggler Lindsay Sandiford, who is on death row in Indonesia, called the execution of fellow inmates "senseless and brutal". Her lawyer said she could be among the next to be executed. But there was delight on Monday for the family of Philippine woman Veloso, after her execution was called off at the last minute following a request by the Philippine president. Her mother described the reprieve as a "miracle". "We are so happy, I can't believe it. I can't believe my child will live," Celia Veloso told Philippine radio station DZMM. Sonny Coloma, a spokesman for the Philippine president, told the BBC he was "relieved and thankful" but stressed "the death sentence on her has not been lifted". Image copyright AFP Image caption Veloso's family celebrated the news of her reprieve in Cilacap, close to the prison Who were the executed prisoners? Australians Myuran Sukumaran and Andrew Chan Ghanaian Martin Anderson Indonesian Zainal Abidin bin Mgs Mahmud Badarudin Nigerians Raheem Agbaje Salami, Sylvester Obiekwe Nwolise, Okwudili Oyatanze Brazilian Rodrigo Gularte Frenchman Serge Areski Atlaoui and Filipina Mary Jane Fiesta Veloso were spared as they have ongoing appeals Jon Donnison: Double standards over Bali Nine A Frenchman also convicted of drug offences, Serge Areski Atlaoui, was originally due to be executed with the group but has an appeal outstanding. The French government said it "reiterates its opposition to the death sentence, in all cases and all circumstances". Indonesia has some of the toughest drug laws in the world and ended a four-year moratorium on executions in 2013. It says it takes a hard line because of the country's own drugs problem - 33 Indonesians die every day as a result of drugs, according to Indonesia's National Narcotics Agency.
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Summary: "Breathtaking" is probably not a word you'd usually associate with the executions of eight people, but that's exactly how a pastor describes the scene in Indonesia just after midnight local time. Seven foreigners and a local man refused blindfolds as they stood in front of a firing squad over drug-smuggling convictions and broke out into a chorus of "Amazing Grace" while staring down their executioners. A second song, "Bless the Lord O My Soul," began but was cut short by gunfire, the Sydney Morning Herald reports. "They were praising their God," says a pastor who witnessed the executions, adding it was the most beautiful experience of her life: "It was breathtaking. This was the first time I witnessed someone so excited to meet their God." Outside, family members, many hoping for a last-minute stay, lit candles and recited the Lord's Prayer as they watched cars carry the inmates to the execution site. At the sound of gunshots, many became inconsolable, though spiritual advisers were on hand. A source tells the Herald all eight prisoners were shot in the heart and none in the head, which is required if an inmate isn't dead after 10 minutes. Their bodies have been transferred to Jakarta, and those of Myuran Sukumaran and Andrew Chan, members of the so-called Bali Nine, will be returned to Australia. "We respect Indonesia's sovereignty but we do deplore what's been done, and this cannot be simply business as usual," Prime Minister Tony Abbott says, per the BBC, which notes Australia has yanked its ambassador from Indonesia.
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Summarize: BACKGROUND OF THE INVENTION The human hand has a bony framework consisting of eight carpal or wrist bones arranged in two rows, five metacarpal or palm bones, and fourteen phalanxes or finger bones, all of which are held together by a complex system of ligaments that allow an amazing degree of movement to achieve grasping or comprehension. The hand includes highly coordinated muscular action, and complex nervous receptors, that senses touch, pressure, temperature and pain and further enables an object to be grasped with great force. On the other hand, the complex arrangement of the nerves, ligaments, and bones occassionally do not function properly due to injury or disease and this disfunction often is curable by placing one's injured hand in the care of an expert who has spent a lifetime studying the human hand. It is therefore desirable to be able to evaluate the extent of injury to one's hand in order to determine the seriousness and character of an injury. Often, for one reason or another, a person will not display the true extent of his injured hand by not making his maximum effort when the force of his grasp is being measured and thus the true extent of injury to the hand is obscured to the evaluator. Method and apparatus by which the extent that the patient making his maximum effort when his grasp is being measured is the subject of the present invention. SUMMARY OF THE INVENTION This invention comprehends a grasp analysis apparatus by which the power of grasp in the human hand can be measured by the provision of a hand held transducer having collapsible walls on opposed sides thereof for engagement of the fingers and the palms, whereupon increased force of grasp forces the opposed walls toward one another and the transducer generates a signal that is proportional to the magnitude of the force of the grasp. More specifically, the grasp analysis apparatus by which the power of the grasp of the human hand can be measured includes a palm engaging part spaced from a finger engaging part and adjustably connected together whereby different selected portions of the fingers are engaged by the finger engaging part to thereby enable the force exerted by various different areas of the fingers to be accurately measured. Still more specifically, the before mentioned grasp analysis apparatus further includes a transducer for generating a signal that is proportional to the force of the grasp and changing the signal into data which is representative of the magnitude thereof. The above grasp analysis apparatus enables a method of evaluating the force of grasp to be carried out wherein the true force that one can exert with a hand is determined. More particularly, the method evaluates whether the true force of grasp is being exerted with the hand when one is exerting less than the maximum force he is actually capable of exerting. The method of the present invention is carried out by measuring the force of grasp exerted between the fingers and palm of the hand at a first location near the end of the fingers, at another location near the palm, and at another location between the first and second location. The measured force of grasp at the first, second and third locations are compared to determine the configuration a curve plotted from the results of the measured force. The shape of the curve determines if the patient is truly exerting a maximum effort while his grasping force is being measured, or whether the patient is not making his maximum effort and therefore the weakness of the grasp can be attributed to this lack of effort rather than to other causes such as muscular weakness and the like. Accordingly, a primary object of the present invention is the provision of method and apparatus for measuring the power of the grasp of one's hand at a plurality of locations. Another objection of the invention is to provide a hand held apparatus having movable wall sections that compress a transducer which measures the force of one's grasp. A further object of this invention is to disclose and provide a transducer device having adjustable movable wall sections arranged to engage predetermined areas of the hand and measure the force of grasp therein. A still further object of this invention is the provision of a method of evaluating the potential force of grasp exerted by one's hand when the person fails to make his maximum effort during the measurement. Another and still further object of this invention is the provision of a transducer compressed between the palm and selected areas of the fingers for measuring the force of one's grasp. An additional object of the present invention is the provision of a system by which a transducer generated signal from a grasp measuring device is treated to provide a method of evaluating the potential force of grasp exerted by one's hand when the person fails to make his maximum effort during the measurement. These and various other objects and advantages of the invention will become readily apparent to those skilled in the art upon reading the following detailed description and claims and by referring to the accompanying drawings. The above objects are attained in accordance with the present invention by the provision of a method for use with apparatus fabricated in a manner substantially as described herein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective, part cross-sectional view of a grasp analysis apparatus made in accordance with the present invention, with some parts being cut away therefrom in order to disclose the interior thereof; FIG. 2 is a reduced, part schematical, part diagrammatical, top plan view of the apparatus of FIG. 1 shown grasped within one's hand; FIG. 3 is similar to FIG. 2 and shows the apparatus in an alternate configuration; FIG. 4 is a longitudinal, cross-sectional view of the apparatus disclose in FIGS. 1-3; FIG. 5 is a fragmentary, representation of a plot illustrating the method of the present invention; FIG. 6 is diagrammatical, part schematical representation of a human finger; FIG. 7 shows the finger of FIG. 6 in a different configuration; FIG. 8 shows the finger of FIGS. 6 and 7 in a different configuration; FIG. 9 shows the configuration of the finger of FIGS. 6-8 in another configuration; and, FIG. 10 is a diagrammatical, top, part cross-sectional view of the finger disclosed in FIGS. 6-9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 of the drawings disclose a grasp analysis apparatus 10 made in accordance with the present invention. The apparatus 10 includes a palm engaging part 12 spaced from a finger engaging part 14 with the palm and finger engaging parts being adjustably held together by a cylindrical shaft 16. The shaft 16 permits the palm engaging part 12 to be moved towards the finger engaging part 14 in direct proportion to the compressive force applied by the hand thereto. As best seen in FIG. 4, together with FIGS. 1-3, the cylindrical shaft 16 is reciprocatingly received within the illustrated aperture 17. A plurality of drilled holes 16A are formed laterally through shaft 16. Stop member 18, in the form of a pin, is attached to a medial portion of shaft 16 and bears against the finger engaging part 14. Numeral 19 indicates a cylindrical counterbore, preferably open to ambient, for reciprocatingly receiving one marginal end of shaft 16; while the opposed end of shaft 16 is affixed to a movable wall 20. The wall 20 is mounted for movement within the interior of the palm engaging part 12. Strain gauge or transducer 21 can take on any number of different forms so long as it generates a signal within conductor 22 which is proportional to the grasp force exerted to move the finger engaging part 14 toward the palm engaging part 12. Computer 23, which can take on a number of different forms, treats the signal at conductor 22 and converts the signal into a measurement that is indicative of the magnitude of the force applied to compress the finger engaging part 14 toward the palm engaging part 12 of the grasp analysis apparatus 10. The converted signal is treated by suitable electronics at 23A to provide a curve at 23B. The apparatus 21, 22, 23, 23A, and 23B therefore is any device that converts the resultant force of the grasp into stored knowledge that is equivalent to the information presented by the curve of FIG. 5. In FIGS. 1 and 4, the palm engaging part 12 includes the back or rear wall 24, a lower wall 26, and a confronting face 28 that is opposed to the confronting face 30 of the finger engaging part 14. A finger grip 32 is provided for each of the four fingers of the hand. Numeral 34 indicates the top end wall and numeral 35 indicates the bottom end wall of the finger engaging part 14 while numeral 36 indicates a top end wall of the palm engaging part 12. In FIGS. 6-10, numeral 40 indicates the joinder of the finger to the palm 42. Numeral 44 indicates the end section of the finger. The middle or second section of the finger is indicated by numeral 46, while the first section of the finger is indicated by numeral 48. There is a finger joint between sections 44 and 46, 46 and 48, 48 and 42. In FIG. 10 the legend FDS is intended to illustrate a flexor digitorum superficialis while the legend FDP indicates a flexor digitorum profundus. Numeral 50 indicates the intrinsic musculature which exerts pressure through the knuckle joint of the finger. Accordingly, there are three groups of muscles brought into play for exerting maximum effect on the power of one's grasp, with the FDP being the first group of muscles, the FDS being the second group of muscles, and the flexor digitorum superficialis tendon being the third group of muscles. Numeral 52 of FIG. 10 generally indicates the before mentioned joint located below the location 40 illustrated in FIG. 6; numeral 54 indicates the FDS; numeral 56 indicates the insert of the FDP; numeral 58 indicates the joint between the end and second sections 44 and 46; and numeral 60 indicates a medial part of the FDP. The palm engaging part 12 of the grasp analysis apparatus 10 of FIGS. 1-4 includes two members, the movable wall 20 and tranducer 21, as shown in FIGS. 1 and 4. The transducer 21 is support by the rear wall 24 of the palm engaging part 12. The transducer 21 is activated by the movable wall 20 that moves longitudinally across the chamber as pressure is applied at finger grip 32. The finger engaging part 14 opposite to the palm engaging part 12 comprises a formed member that is contoured as indicated at finger grip 32 to fit the individual fingers so that suitable grasp can be obtained. The cylindrical shaft 16 is adjustably connected thereto which passes out of the housing formed within the finger engaging part 14 and through aperture 17 which freely receives the shaft in a slidable manner. The distance between members 12 and 14 can be easily established by selecting the appropriate hole, of a plurality of holes provided at 16A in shaft 16 through which the stop member 18, in the form of a small pin, is passed. The selected location of this pin respective to shaft 16 will result in a different width between the two members 12 and 14. Thus the appropriate width of the apparatus for each of the five positions along the fingers, as illustrated in the drawings, can be obtained, and the appropriate amount of grip in each of these positions measured by the movable wall 20 compressing and activating the pressure responsive transducer. The results of the measurement obtained with the transducer 21 are then conveyed at conductor 22 into a computer 23 of FIG. 4 which is programmed to create the equivalent of the graph as shown in FIG. 5. Hence the resultant printout 23B in the form of the illustrated curve is the final reading. The grasp analysis apparatus 10 can be used in a single position in a manner similar to a known or prior art manometer; and, the graph need not be actually drawn so long as data is compiled and presented to achieve the equivalent of stored knowledge, as illustrated by the curve of FIG. 5, for example. However, the grasp analysis apparatus 10 has the capability of creating the grasp analysis curve from which the strength of grasp can be better evaluated in a new and unobvious manner. This capability should be of great advantage to the hand surgeon or the rehabilitationist who must do evaluations of patients with proported impaired hand conditions, and in doing these evaluations one must ascertain whether or not the patient is making maximum effort when his grasp is measured. Heretofore, there has been no systemized procedure for achieving this desirable goal. The present invention provides a unique method and apparatus for providing this accomplishment. The grasp analysis apparatus 10 of the present invention is to be used for the measurement of the power of grasp in the human hand, and at the same time evaluate the amount of effort being utilized by the patient for creating the curve as seen in FIG. 5, thus indicating the magnitude of the force of the grasp that is present in certain locations relative to the width of the grasp. When a very wide object, as seen in FIG. 2, for example, is grasped with the hand, only the flexor tendon that attaches distally (the flexor digitorum profundus tendon) acts and this results in a certain amount of pressure being applied to the pressure transducer 21 in the grasp analysis apparatus 10. This is indicated at P on the curve of FIG. 5. With the next smaller grasp width, the flexor digitorum superficialis tendon comes into play since its function is primarily on the middle phalanx of the finger. This is indicated at PS on the curve of FIG. 5. In position three, the function of the profundus, the superficialis is aided by the action of the intrinsic musculature so that all three muscle groups are exerting maximum effect and jointly represent the magnitude of the power of grasp exerted between the palm and fingers. This is indicated at PSI on the curve of FIG. 5. In the next smaller width, or position four, the profundus can mechanically no longer function on this small a width so that the magnitude of the power of graph is produced only by the superficialis tendon 60 and the intrinsics 50. This is indicated at SI on the curve of FIG. 5. Finally, in the smallest width possible, the superficialis and profundus tendons both have become mechanically ineffective because of the smallness of the width between members 12 and 14, and only the intrinsic musculature which exerts pressure through the proximal or knuckle joint 40 of the finger is functional and the reading at 23B will so indicate. This is indicated at I on the curve of FIG. 5. In analyzing these five locations on the curve of FIG. 5, it will be seen that a bell shaped curve is achieved by plotting normal activity. This configuration of the curve will be evidenced by the data at P, PS, PSI, SI, and I; no matter what strength musculature motivates the hand; and, in most circumstances will be present when the patient is exerting maximum effort at each of the five recited measuring locations. Should the patient, for one reason or another, fail to exert maximum effort, a more straight configuration of curve will result, or a curve that has a downward slant will occur, so that the Examiner realizes that the patient is not making his maximum effort, and therefore the weakness of grasp can be attributed to this lack of effort, rather than to other causes such as muscular weakness and the like. Stated differently, data that results in a curve other than the bell curve of FIG. 5 is a strong indication that the patient is deliberately faking the extent of his injury. The greater the departure of the curve from the bell shaped curve of FIG. 5 is proportional to the degree of the false effort made by the patient.
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Summary: A grasp analysis apparatus and method by which the power of grasp exerted by the human hand can be measured, and by which a determination can be made related to the maximum effort of grasp that is made during the measurement. The apparatus includes a housing having opposed walls which can be forced towards one another in proportion to the magnitude of the compressive strength exerted by the person that grasps the housing. A transducer enclosed within the housing is compressed between the movable opposed walls and provides a signal that is proportional to the magnitude of the compressive force of the grasp. The signal is converted into a measurement indicative of the magnitude of the force. A plurality of measurements taken at different locations along the fingers are plotted to reveal the true effort being made while grasping the apparatus.
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Write a title and summarize: 500 empresas en el mundo fueron víctimas de la falsificación de informes de calidad sobre los materiales de Kobe Steel. La trama comenzó la pasada semana, cuando el tercer mayor fabricante de acero del mundo admitió que había falsificado los datos sobre la calidad de sus productos. Al comienzo la empresa dijo que las pruebas de calidad adulteradas afectaron a 200 clientes de la industria del transporte que le habían comprado aluminio y cobre. Pero las alarmas se prendieron definitivamente cuando saltaron a la mesa los nombres algunos de los clientes afectados: el de los gigantes General Motors, Boeing y Toyota. A pesar del escándalo, en ese momento pocos sabían que se venía una avalancha. Y lo que se sabe hasta hora es que una investigación interna realizada por la compañía arrojó que en realidad son 500 las empresas afectadas. Kobe Steel le vendió productos adulterados a empresas como General Motors, Boeing y Toyota. Y no solo eso. Las falsificaciones se remontan diez años atrás. Y como si eso fuera poco, no solo adulteraron la calidad del aluminio y del cobre, también lo hicieron con el acero. Para rematar el asunto, la firma ha reconocido finalmente que se trata de 16 subproductos -los cuales forman parte esencial de industrias como el transporte, las maquinarias, el sector eléctrico o la construcción- que no se ajustan a las exigencias de calidad en términos de resistencia y duración. Por ahora ninguna empresa ha planteado la posibilidad de retirar productos del mercado, pero como hay una investigación abierta que revela nuevos detalles cada día que pasa, no se sabe qué esconden realmente las paredes de Kobe Steel. Se desploma en la bolsa Apenas se enteró el mercado de lo que estaba ocurriendo, las acciones de Kobe Steel cayeron casi 40% en dos días, y el valor de mercado de la empresa en una semana disminuyó en cerca de US$1.800 millones. "La credibilidad de Kobe Steel cayó a cero", dijo el presidente de la firma, Hiroya Kawasaki. Y se ha sabido también que, además de Toyota o General Motors, Honda y Mazda también han sido afectados. Las acciones de Kobe Steel se fueron al suelo. Daimler y Airbus también estarían en la lista, según el medio japonés Nikkei, el mayor diario de información financiera del mundo. Frente a un escándalo de esta magnitud, el gobierno japonés se vio obligado a intervenir, pidiéndole a la compañía que entregue un informe sobre la seguridad de sus productos dentro de las próximas semanas. ¿Cómo nadie se dio cuenta? De inmediato surgieron las interrogantes sobre cómo fue posible que por tantos años los controles de calidad fallaran. Gary Ysuchida, un portavoz de la empresa, le dijo a la BBC que la compañía se arrepiente de la falsificación. "Hubo decenas de empleados involucrados. Parece que en algunos casos el control de calidad estuvo en manos de trabajadores con años de experiencia que gozaban de la confianza de la organización y, de algún modo, hubo discrepancias que fueron deslizadas por ellos", dijo Ysuchida. Esa explicación deja la responsabilidad en manos de los empleados. Pero Karishma Vaswani, corresponsal de Negocios de BBC en Asia, puso en cuestión la explicación corporativa. La información falsa se remonta a una década. "¿Sabían los directivos que los informes eran falsificados? ¿Hay fallas sistémicas en la compañía que llevaron a este escándalo? Las explicaciones que entregue la empresa en las próximas semanas revelarán qué tan profunda puede ser la crisis", dijo Vaswani. Varias de las empresas afectadas dijeron que hasta ahora encontraron ningún problema en la seguridad de los productos que fueron fabricados con aluminio, cobre o acero de la firma japonesa. Sin embargo, el alcance del escándalo es aún desconocido y las investigaciones -tanto dentro de Kobel Steel, como las que están llevando a cabo los propios clientes afectados- están en desarrollo. Esta crisis corporativa -que también salpica la imagen de Japón- se suma a otros problemas de compañías, como por ejemplo Nissan Motors, que anunció hace unas semanas el retiro de más de un millón de vehículos tras admitir que empleados sin la calificación suficiente habían hecho las inspecciones finales de automóviles que serían vendidos en el mercado local. Y trae a la memoria el escándalo de Takata, cuando declaró la bancarrota luego que sus airbags defectuosos fueran vinculados a 16 muertes y decenas de heridos en accidentes en todo el mundo.
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Title: El escándalo de Kobe Steel, la empresa japonesa que le vendió metales falsificados a General Motors, Boeing, Toyota (y otras 500 empresas Summary: ¿Podrían decenas de autos, trenes y aviones estar a punto de ser retirados del mercado por razones de seguridad? No se sabe, pero lo que sí está claro es que las grandes multinacionales que le compraron aluminio, cobre y acero falsificado a la empresa japonesa Kobe Steel están investigando las consecuencias de un escándalo con impacto internacional.
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Summarize: Background Overview of FEMA’s Workforce FEMA’s workforce consists of employees hired under both Title 5 and the Stafford Act, as well as individuals from two other workforce components who are not FEMA employees, but who can be deployed for disaster response: the DHS Surge Capacity Force and FEMA Corps. Title 5 employees are permanent and temporary employees who form FEMA’s day-to-day workforce, and can be deployed as needed during a disaster. Stafford Act employees include CORE and Reservists hired specifically to support disaster-related activities on a temporary or intermittent basis. COREs are temporary employees with 2- to 4-year appointments, while Reservists work on an intermittent basis and are deployed as needed. Surge Capacity Force volunteers include employees of other DHS components who augment FEMA’s workforce in the event of a catastrophic disaster. Their first and only deployment as of May 2017 was to assist in response and recovery efforts after Hurricane Sandy in 2012. FEMA manages and coordinates the Surge Capacity Force program. FEMA Corps is a national service program managed by AmeriCorps National Civilian Community Corps (NCCC). FEMA Corps members support disaster response and recovery efforts and work under the direction of FEMA staff. As shown in table 1 below, FEMA’s available disaster response workforce, including Surge Capacity Force and FEMA Corps totaled over 22,600 in March 2017. FEMA’s workforce categories are subject to different employee rights and legal protections. For example, Title 5 employees are generally afforded notice and appeal rights, while Reservists serve in temporary intermittent positions and can be terminated at any time with or without cause. FEMA Misconduct Directive and Manuals In 2012, FEMA issued a policy directive outlining procedures for reporting misconduct within the agency, conducting administrative investigations, and reporting actions taken in cases where the misconduct allegation was sustained. The FEMA Administrative Investigations Policy directive (FD 123-19) lays out misconduct responsibilities for employees, managers, and supervisors, and for the personnel management offices involved in managing misconduct. The directive also describes three types of administrative investigations: Managerial Inquiry: managers or supervisors may be assigned to conduct an inquiry when the allegation is not complex (e.g., tardiness, absence without leave) and involves a minimal number of witnesses. Office of the Chief Security Officer (OCSO) Investigation: OCSO personnel are assigned to conduct investigations of allegations that are criminal in nature, or have the potential to be criminal in nature, for example travel card fraud or a physical altercation. Independent Investigation: senior FEMA or other government officials may be assigned to conduct investigations into complex allegations that involve multiple offices and witnesses, senior employees, or prohibited personnel practices such as political coercion. The process for conducting investigations is further discussed in FEMA’s Administrative Investigations Policy manual (FEMA Manual 123-19-1). FEMA’s Employee Discipline Manual (FEMA Manual 255-3-1) describes the policies, procedures, and responsibilities for taking conduct-related discipline against permanent Title 5 employees. The manual discusses options for disciplinary actions, which include reprimands and suspensions of 14 days or less, and adverse actions, which include suspensions for 15 days or more, demotions, and removals. The manual also lays out employee grievance and appeal rights. The policies and procedures described in the manual apply solely to permanent Title 5 employees. As discussed later in this report, the discipline and appeals process varies for other workforce categories. FEMA Offices Involved in Managing Misconduct Within FEMA, three primary offices are involved in reviewing, investigating, and adjudicating employee misconduct allegations. OCSO Internal Investigations Branch (IIB): conducts investigations related to more serious allegations, such as those that may involve potential criminal misconduct. The Office of the Chief Component Human Capital Officer (OCCHCO) Labor and Employee Relations Branch (LER): advises supervisors who conduct lower level investigations and inquiries, such as time and attendance violations, and provides recommendations on any counseling or any disciplinary or adverse action for all cases. The Office of the Chief Counsel (OCC) Personnel Law Branch (PLB): provides legal advice during investigations and conducts legal reviews of certain reports of investigation and all disciplinary and adverse actions. Representatives from these three offices form FEMA’s Administrative Investigations Directive (AID) Committee, which reviews misconduct allegations, assigns investigators, and tracks the status of open cases. DHS OIG Role in Reviewing and Investigating Misconduct DHS OIG also plays a role in reviewing and investigating certain misconduct allegations. DHS Management Directive 0801.1 requires officials from all DHS components, including FEMA, to refer certain categories of misconduct to DHS OIG for review, such as allegations of criminal misconduct against a DHS employee and any allegations of misconduct against senior employees. After reviewing the allegation, DHS OIG may elect to initiate an investigation or refer the case back to the component, such as FEMA, for review. DHS OIG receives complaints (from employees, supervisors, the public, and agency referrals) against employees in all DHS components through the DHS OIG Hotline, which is a resource for reporting corruption, fraud, waste, abuse, mismanagement, or misconduct. Table 2 below shows the average number of misconduct complaints received through the DHS OIG Hotline from fiscal year 2014 through 2016 by selected DHS component. These complaints represent alleged offenses only, and not final actions or adjudication results. General Misconduct Process FEMA receives allegations of employee misconduct from individuals both within and outside the agency. Individuals (including members of the public) can report these allegations through a number of different mechanisms, including, but not limited to, FEMA’s OCSO Tipline, LER specialists, and the DHS OIG Hotline. Employees can also report misconduct to their supervisors and union representatives. The AID Committee reviews most allegations, including those declined by DHS OIG and returned to FEMA. LER reviews complaints received directly from managers or employees to determine if they need to be discussed at the AID Committee meetings. Allegations may then be assigned to one of the three types of administrative investigations discussed earlier. Once completed, reports of investigation are forwarded to LER for review. LER recommends appropriate disciplinary or adverse action, if warranted. Figure 1 below outlines the general steps in FEMA’s misconduct process, including DHS OIG’s role in reviewing and investigating allegations. Variations in potential case outcomes and the appeals process by workforce category are discussed later in this report. FEMA Has Developed and Documented Misconduct Policies and Procedures for Most Employees, but Not its Entire Workforce FEMA Has Documented Misconduct Policies and Procedures for Most of its Employees, and Is Taking Steps to Offer Misconduct Training to Supervisors FEMA has developed a policy outlining procedures regarding investigations of misconduct as documented in FEMA’s Administrative Investigations Policy directive. The directive applies to all FEMA personnel. While FEMA Corps and Surge Capacity Force members are not FEMA employees, OCSO officials stated that the investigations process is the same regardless of the workforce category. FEMA has also documented misconduct policies and procedures regarding options to address misconduct and appeals for Title 5 and CORE employees. For Title 5 employees the options to address misconduct include both disciplinary and adverse actions. FEMA Corps disciplinary policies and procedures are the responsibility of AmeriCorps NCCC and are documented in their member handbook. Figure 2 outlines the options to address misconduct and appeal rights for Title 5, CORE, and FEMA Corps as documented in their respective employee discipline and program manuals. According to LER officials, as of February 2017, most supervisors had been offered training on FEMA’s 2015 Employee Discipline Manual, which applies to Title 5 employees. Three more training sessions are planned for 2017. Additionally, PLB partnered with LER in 2016 to offer misconduct and documentation training for all supervisors. In 2016, PLB and LER completed 21 joint trainings. They held two more joint trainings in March 2017. FEMA Has Not Documented Misconduct Policies and Procedures for Surge Capacity Force FEMA has not documented misconduct policies and procedures for Surge Capacity Force members. DHS issued the Surge Capacity Force Concept of Operations in 2010, which outlines FEMA’s base implementation plan for the Surge Capacity Force. However, the document does not address any elements pertaining to Surge Capacity Force human capital management, specifically misconduct and disciplinary policies and procedures. According to the FEMA Surge Capacity Force Coordinator, despite the lack of documentation, any incidents of misconduct would likely be investigated by FEMA’s OCSO, which would then refer the completed report of investigation to the employee’s home component for adjudication and potential disciplinary action. Additionally, OCSO officials said that while they were unaware of any misconduct investigations involving Surge Capacity Force members to date, if an incident were to occur they would follow the procedures in the Administrative Investigations Policy directive and also notify the member’s home component of the incident. However, although no allegations of misconduct were made at the time, the FCO in charge of one of the Hurricane Sandy Joint Field Offices said he had not seen anything in writing or any formal guidance that documents or explains how the process would work and stated that he would have had to contact FEMA headquarters for assistance in determining how to address any misconduct. Furthermore, he noted that taking the time to figure out the Surge Capacity Force misconduct process would have detracted from time spent on the Joint Field Office’s mission. LER officials stated that they would be able to walk through the procedures with supervisors and managers if asked. However, because these procedures are not documented, the process is not transparent and information is not readily available. LER officials noted that while LER specialists can quickly answer questions, it would be beneficial for the procedures to be documented. A PLB official also told us that FEMA should have a written policy for potential Surge Capacity Force misconduct. Standards for Internal Control in the Federal Government advises management to develop and maintain documentation of its internal control system. This documentation can assist in management’s ability to communicate controls to personnel, and is a means to retain organizational knowledge as well as communicating that knowledge to external parties. Without documented guidance, FEMA cannot ensure that Surge Capacity Force misconduct is addressed adequately in a timely and comprehensive manner, which could negatively affect the extent to which Joint Field Offices can accomplish their mission after a disaster. Furthermore, FEMA’s 2015 update to the Surge Capacity Force Concept of Operations calls for increasing the size of the Surge Capacity Force workforce from its current size of approximately 6,000 volunteers to 36,000 volunteers and also does not address any human capital management issues, including misconduct. This planned expansion further underscores the importance of documenting policies and procedures in order to address potential misconduct. Reservist Policies and Procedures Do Not Outline Disciplinary Actions or Address the Appeals Process FEMA’s Reservist Program Manual lacks documented policies and procedures on disciplinary options to address misconduct and appeal rights for Reservists. Both LER and PLB officials told us that, in practice, disciplinary actions for Reservists are limited to reprimands and termination. According to these officials, FEMA does not suspend Reservists because they are an intermittent, at-will workforce deployed as needed to respond to disasters. FCOs and cadre managers have the authority to demobilize Reservists and remove them from a Joint Field Office if misconduct occurs, which may be done in lieu of suspension. Furthermore, LER and PLB officials also told us that, in practice, FEMA grants Reservists the right to appeal a reprimand or termination to their second-level supervisor. Although officials stated that FEMA is carrying out these policies and procedures in practice, the actions are not documented in the Reservist Program Manual. Standards for Internal Control in the Federal Government advises management to document policies for each unit in its area of responsibility. Each unit, with guidance from management, determines the policies necessary to operate, as well as documents policies in the appropriate level of detail to allow management to effectively monitor activity. According to FEMA officials, because of the at-will nature of their employment, Reservists are not subject to the same policies, procedures, and appeal rights as Title 5 employees, such as a suspension as an option to address misconduct. However, the procedures that are executed in practice are not reflected in the Reservist Program Manual. Without documented Reservist disciplinary options and appeals policies, supervisors and Reservist employees may not be aware of all aspects of the disciplinary and appeals process. Additionally, seven supervisors and managers in two of the three regions and one of the three cadres we spoke with noted that there is a perception of inconsistency and unfairness in the discipline process. For example, one manager told us that if cadre management provided more information on outcomes in addressing misconduct, it would help improve the perception that misconduct is being seriously addressed at FEMA. Clear documentation of the Reservist disciplinary options and appeals policies and procedures currently in practice would help to address the concerns of inconsistency. FEMA Does Not Communicate Range of Offenses and Penalties to its Entire Workforce FEMA revised its employee disciplinary manual for Title 5 employees in 2015, and in doing so, eliminated the agency’s table of offenses and penalties. Tables of offenses and penalties are used by agencies to provide guidance on the range of penalties available when formal discipline is taken. They also provide awareness and inform employees of the penalties which may be imposed for misconduct. Since revising the manual and removing the table, FEMA no longer communicates possible punishable offenses to its entire workforce. Instead, information is now communicated to supervisors and employees on an individual basis. Specifically, LER specialists currently use a “comparators” spreadsheet with historical data on previous misconduct cases to determine a range of disciplinary or adverse actions for each specific misconduct case. The information used to determine the range of penalties is shared with the supervisor on a case-by-case basis; however LER specialists noted that due to privacy protections they are the only FEMA officials who have access to the comparators spreadsheet. PLB and LER officials stated that the new comparators spreadsheet is an improvement over the old table, which contained overly broad categories and had not been updated since 1981. According to officials, the comparators spreadsheet is easier to use and thus it is easier to ensure cases are consistent across the agency. Supervisors and managers we spoke with shared their perspectives on how offenses and penalties are communicated both through the prior table and the new comparators spreadsheet. Specifically, 11 supervisors and managers in all three regions and cadres we spoke with, as well as both union representatives we interviewed, cited the benefits of a table in communicating punishable offenses and the range of penalties. These benefits included transparency, consistency, and possible deterrence against engaging in misconduct. For example, one supervisor noted that a table of offenses and penalties is beneficial for both employees and supervisors because it removes ambiguity and makes the disciplinary process more transparent. Additionally, as discussed earlier, other supervisors and managers told us that there is a perception that misconduct cases are handled inconsistently. However, management officials in one region, and one supervisor in another region, noted they preferred the new case-by-case comparators system and did not see benefits to having a table of offenses and penalties. Another manager noted that a table was useful in certain cases with specific rules, such as travel card misuse; however, many cases are more complicated and in those situations a strict table is less effective. The remaining supervisors and managers did not offer their perspective on this topic. Standards for Internal Control in the Federal Government advises management to consider standards of conduct, assigned responsibility, and delegated authority when establishing expectations. Management establishes expectations of competence for key roles as well as for all personnel through policies within internal control systems. Because information about offenses and penalties is not universally shared with supervisors and employees, FEMA management are limited in their ability to set expectations about appropriate conduct in the workplace and to communicate consequences of inappropriate conduct. Additionally, a FEMA OCCHCO official noted that FEMA is considering re-introducing a table which would inform employees and supervisors of misconduct offenses and penalties. Communicating information about offenses and common ranges of penalties, such as in a summary table that does not include individual case information, could help to provide transparency for employees and their supervisors on the range of penalties to expect for different types of misconduct and mitigate the perception that misconduct is handled inconsistently across FEMA’s workforce. FEMA Records Data on Employee Misconduct Cases and Their Outcomes, but Could Improve the Quality and Usefulness of These Data Multiple FEMA Offices Collect and Store Data on Employee Misconduct The three offices on the AID Committee involved in investigating and adjudicating employee misconduct complaints each maintain separate case tracking spreadsheets with data on employee misconduct to facilitate their respective roles in the process. OCSO collects data in a case tracking spreadsheet about employee misconduct complaints and investigations. For example, the spreadsheet contains fields with narrative descriptions of alleged offenses and investigation updates, the locations and FEMA regions where alleged offenses occurred, and the number of days an investigation was open. According to agency officials, all of the cases discussed during weekly AID Committee meetings are in the OCSO case tracking spreadsheet. LER also collects data on employee misconduct complaints in a case tracking spreadsheet. Specifically, LER records information on lower- level allegations that do not rise to the level of AID Committee review as well as adjudication information for allegations which were investigated by OCSO. In addition to employee misconduct complaints, LER specialists record all inquiries from supervisors in the LER case tracking spreadsheet, including questions about performance, grievances, and employee counseling. Fields in the LER case tracking spreadsheet include, for example, narrative descriptions of alleged offenses and case summaries, comments with case status updates, and the disciplinary or adverse actions taken, if any. PLB collects data on misconduct-related disciplinary or adverse actions it reviews in a case tracking spreadsheet. Fields in the PLB spreadsheet include narrative descriptions of the charge or issue, the employee position and type, and the disciplinary or adverse action taken. According to PLB officials, the data are based on information provided by LER. FEMA OCSO Recorded Approximately 600 Misconduct Complaints from January 2014 through September 30, 2016 We analyzed data provided by OCSO in its case tracking spreadsheet and found that there were 595 complaints from January 2014 through September 30, 2016. The complaints involved alleged offenses of employee misconduct which may or may not have been substantiated over the course of an investigation. Some complaints involved multiple allegations of various offenses against multiple subjects. In order to better summarize the number and type of alleged offenses, we developed eight general categories (see table 3 below). Based on our analysis, the 595 complaints contained approximately 799 alleged offenses from January 2014 through September 30, 2016. As shown in figure 3 below, the most common type of alleged offenses were integrity and ethics violations (278), inappropriate comments and conduct (140), and misuse of government property or funds (119). For example, one complaint categorized as integrity and ethics involved allegations that a FEMA employee at a Joint Field Office was accepting illegal gifts from a FEMA contractor and a state contractor. Another complaint categorized as inappropriate comments and conduct involved allegations that a FEMA employee’s supervisor and other employees had bullied and cursed at them, creating an unhealthy work environment. Finally, a complaint categorized as misuse of government property or funds involved allegations that a former FEMA employee was terminated but did not return a FEMA-owned laptop. LER officials provided summary data on employee misconduct outcomes separately from the LER case tracking spreadsheet. According to the summary data, there were 546 disciplinary or adverse actions related to employee misconduct taken during the last three calendar years — from January 2014 through December 2016. Of those actions, the most common were removals or terminations (354), reprimands (115), suspensions of 14 days or less (64), and suspensions of 15 days or more (6). The most common employee types affected by actions were Reservist (235) and Title 5 (110). We also analyzed data provided by PLB in its case tracking spreadsheet and found that PLB reviewed 454 final disciplinary or adverse actions related to employee misconduct from January 2014 through September 30, 2016. The most common types of action were reprimands (144), suspensions ranging from 1 to 45 days (121), and terminations (118). The most common employee types affected by actions were Title 5 (265), CORE (180), and Reservist (6). There are several potential explanations for the differences between the LER and PLB data on employee misconduct outcomes. A PLB official stated that PLB does not review all disciplinary actions against Reservists; therefore, LER Reservist actions may not appear in the PLB spreadsheet. Further, LER officials stated that they recently started adjusting their recordkeeping and it was possible that some actions were not input by LER specialists into the case tracking spreadsheet. Differences in the data may also be related to limitations we identified in the spreadsheets, as discussed below. Aspects of FEMA’s Data Limit Their Usefulness for Identifying and Addressing Trends in Employee Misconduct OCSO, LER, and PLB collect data on employee misconduct and outcomes, but limited standardization of fields and entries within fields, limited use of unique case identifiers, and a lack of documented guidance on data entry restricts their usefulness for identifying and addressing trends in employee misconduct. Limited Standardization of Fields and Entries We found that there was limited standardization of fields based on our review of OCSO, LER, and PLB case tracking spreadsheets. For example, we attempted to summarize misconduct allegations by employee type using the OCSO spreadsheet. Out of approximately 704 subjects named in complaints from January 2014 through September 30, 2016, the most common employee type was unknown because either the information was not consistently available, or not enough information was available in the case tracking spreadsheet. In 2014 and 2016, the OCSO spreadsheet sometimes included information on the subject’s employee type embedded within narrative summary of allegation fields. In 2015, that information was sometimes included in the narrative fields and sometimes listed in a subject type field unique to that year. There was also limited standardization of entries within fields. For example, in all years the offense field in the OCSO spreadsheet consisted of text entries that described similar offenses in different ways, such as “Travel Card Violation” and “Travel Policy Violation”, or multiple offenses related to a specific complaint, such as “Fraud / Travel Card Violation”. The LER spreadsheet also contained examples of fields with limited standardization of entries. For example, the fields which described a subject’s pay grade consisted of varied text entries, such as “9”, “GS-09”, and “GS-9”. The PLB spreadsheet had similar issues with other fields. Limited standardization of fields and entries within fields restricts the usefulness of the data for identifying and addressing trends in employee misconduct because it makes timely evaluation, summarization, and verification of the data more difficult. Limited Use of Unique Case Identifiers We found that there was limited use of unique case identifiers in the OCSO, PLB, and LER case tracking spreadsheets. The OCSO spreadsheet contained several unique case identifier fields, including an IIB case number, OIG case number, and incident number. However, the LER and PLB spreadsheets provided to GAO did not contain these unique case identifier fields. A PLB official confirmed that the LER and PLB case tracking spreadsheets do not include case numbers as identifiers, only subject names. OCSO officials also stated that LER does not use the OCSO IIB case number or the OIG case number, although LER does have access to the OCSO IIB case number from AID Committee meetings. As a result, the spreadsheets do not share the same unique identifier, which makes it more difficult to track the status and outcome of cases across the three case tracking spreadsheets. Lack of Documented Guidance on Data Entry OCSO, LER, and PLB officials stated that they do not have data dictionaries or documented guidance on data entry for their respective case tracking spreadsheets. A PLB official reported that data are entered into the PLB spreadsheet by a paralegal and spot-checked by a senior PLB official. LER officials noted that they did not provide data dictionaries or guidance because the original case tracker involved a simple Excel spreadsheet rather than a more complicated database system. However, a lack of documented guidance on data entry may make it more difficult to maintain and verify the completeness and accuracy of the data. For example, we identified variations in data entry into the LER case tracking spreadsheet across LER specialists. In 2015, of the 9 LER specialists who entered information into the case tracking spreadsheet, 6 specialists did not enter information into the employee pay grade field, 2 specialists sometimes entered information, and 1 specialist always entered information. Standards for Internal Control in the Federal Government advises management to process data into quality information which is appropriate, current, complete, accurate, accessible, and provided on a timely basis. Additionally, management should evaluate processed information, make revisions when necessary so that the information is quality information, and use the information to make informed decisions. As described above, FEMA employee misconduct data are not readily accessible and cannot be verified as accurate and complete on a timely basis. These limitations restrict management’s ability to process the data into quality information which can be used to identify and address trends in employee misconduct. For example, an OCSO official stated that senior OCSO officials recently requested employee misconduct information based on employee type, such as the number of Reservists. However, the data are largely captured in narrative fields, making it difficult to extract without manual review. LER officials stated that LER specialists do not regularly conduct trend analysis on employee misconduct cases, but if they notice an apparent trend such as a number of cases involving misuse and non- payment of travel credit cards they will discuss it during quarterly meetings with component management. Five supervisors and management officials from each of the three regions we spoke with said that information on trends and patterns in employee misconduct would be useful. For example, supervisors could use trend analysis to identify specific types of employee misconduct which have become more common, allowing them to send out policy guidance or schedule targeted trainings to help address the issue. LER and OCSO are taking steps to improve their case tracking spreadsheets. Specifically, LER officials reported that they began using Microsoft SharePoint software in January 2017 which includes drop-down selections for all fields except the subject name and comments. The officials stated that they switched to SharePoint in response to DHS requests for specific misconduct information as well as our requests, which raised their awareness of the LER spreadsheet’s limitations. Similarly, an OCSO official stated that OCSO planned to add new fields to their spreadsheet which will make it easier to generate reports and show trends. These are positive steps towards addressing the issues with employee misconduct data quality identified above. However, it is not clear that they will be sufficient to address each of the data limitations we identified and improve the ability to conduct trend analysis. For example, although the LER SharePoint spreadsheet does include an entry number field, it was unclear whether that would allow officials to track the status and outcomes of specific cases across all three case tracking spreadsheets. New fields and increased use of drop-down selections in the LER SharePoint spreadsheet may help improve standardization of fields and entries within fields, but LER specialists may differ in how they interpret fields without documented guidance on data entry. Additionally, it is unclear whether OCSO’s modifications will include increased use of drop-down selections or other means to standardize entries. An OCSO official stated that they are also exploring DHS OIG’s database software, and noted that, if it could be adapted for FEMA’s purposes, it would be a substantial improvement. There are a number of possible quality control measures that could be implemented to help improve the usefulness of FEMA’s data for use in identifying and addressing trends in employee misconduct. At a minimum, based on our analysis of FEMA’s misconduct spreadsheets, the offices could: add additional drop-down fields with standardized entries to make fields easier to summarize; add unique case identifier fields to improve the ability to track cases across the three program offices’ case tracking spreadsheets; develop guidance documents to ensure standardized data entry within each office, including a procedure for quality control checks; or consider adopting the use of database software, which could improve standardization and case tracking across offices. These actions, combined with routine reporting on misconduct trends, could improve upon the initial steps already underway at LER and OCSO and better enable FEMA to manage and report on misconduct information. FEMA Shares Misconduct Case Information Among Personnel Management Offices and with DHS OIG, but Does Not Accurately Track DHS OIG Referred Misconduct Complaints FEMA Personnel Management Offices Meet Regularly to Discuss Misconduct Allegations and Ongoing Investigations and Send Monthly Status Updates to DHS OIG In accordance with FEMA’s Administrative Investigations Policy directive, officials from OCSO, LER, and PLB conduct weekly AID Committee meetings to coordinate information on misconduct allegations and investigations. The committee reviews allegations, refers cases for investigation or inquiry, and discusses the status of investigations. According to PLB officials, FEMA’s process for addressing employee misconduct was ad hoc and informal prior to the release of the directive in 2012. For example, officials from key offices did not always meet and share information on a regular basis, as they do now. PLB officials noted that a new FEMA Chief Counsel in 2010 drove changes calling for a formalized directive and clear instructions for managing misconduct. In addition to the weekly AID Committee meetings, LER and PLB officials stated that they meet on a regular basis to discuss disciplinary and adverse actions and ensure that any penalties are consistent and defensible in court. Employee misconduct information is also shared directly with FEMA’s Chief Security Officer and Chief Counsel. For example, OCSO provides the Chief Security Officer with a monthly report of all open investigations. Additionally, PLB provides the Chief Counsel with a monthly report of significant employee litigation, and includes in that report a list of all significant investigations, such as those involving high-level employees as the subject or witness in a case. Within FEMA, these regular meetings and status reports provide officials from key personnel management offices opportunities to communicate and share information about employee misconduct. FEMA also provides DHS OIG with information on employee misconduct cases on a regular basis. DHS Management Directive 0810.1 requires that all DHS organizational elements, including FEMA, provide monthly reports to DHS OIG on all open investigations. OCSO complies with this requirement and sends a monthly report that includes updates on investigative activity to DHS OIG. OCSO officials stated that they also provide limited outcome information on a case-by-case basis if DHS OIG specifically requests this information. According to OCSO and DHS OIG officials, DHS OIG does not regularly provide FEMA with updates on ongoing investigations it conducts. However, OCSO officials stated they ask DHS OIG for status updates on these cases frequently, especially if the employee was placed on administrative leave during the investigation. FEMA’s Procedures for Tracking DHS OIG Referred Cases Need Improvement Our review indicates that OCSO has not established effective procedures to ensure that all cases referred to FEMA by DHS OIG are accounted for and subsequently reviewed and addressed. As discussed earlier, FEMA is required to refer certain misconduct allegations to DHS OIG for review before taking any action. OCSO officials told us they follow the guidelines closely and refer all serious misconduct allegations to DHS OIG for initial review. They also noted that DHS OIG declines to investigate and refers most cases back to FEMA for action. As also discussed above, OCSO sends a monthly report of open investigations to DHS OIG. However, while these reports provide awareness of specific investigations, according to OCSO officials, neither office reconciles the reports to a list of referred cases to ensure that all cases are accounted for. We reviewed a non-generalizable random sample of employee misconduct complaints DHS OIG referred to FEMA for review and found that FEMA did not adequately track all referred complaints and therefore could not ensure that all complaints in the sample we selected were reviewed and addressed at the time of our inquiry. Specifically, we tracked a random sample of 20 fiscal year 2016 employee misconduct complaints DHS OIG declined to investigate and referred to FEMA for action. We found that FEMA missed 6 of the 20 complaints during the referral process and had not reviewed them at the time of our inquiry. DHS OIG referred 3 of the 6 complaints to FEMA a year or more prior to our review. As a result of our review, FEMA subsequently took action to review the complaints. The AID Committee recommended that OCSO open inquiries in 3 of the 6 cases to determine whether the allegations were against FEMA employees, assigned 2 cases to LER for further review, and closed 1 case for lack of information. According to an OCSO official, OCSO subsequently determined that none of the allegations in the 3 cases they opened involved FEMA employees and the cases were closed. The remaining 2 cases were open as of April 2017. The results from our sample cannot be generalized to the entire population of referrals from DHS OIG to FEMA; however, they raise questions as to whether there could be additional instances of misconduct complaints that FEMA has not reviewed or addressed. FEMA OCSO officials offered several possible explanations for why the complaints we identified were missed. According to these officials, they sometimes receive large batches of complaints through the FEMA Tipline – some of which may be duplicative. DHS OIG referrals also come in through the Tipline and officials said that it is possible that they may not always accurately identify and record all of the referrals for the AID Committee since some of them are misclassified and some do not involve FEMA employees. Standards for Internal Control in the Federal Government advises management to perform ongoing monitoring – including comparisons and reconciliations or other routine actions. Such activities, either undertaken internally within OCSO or in coordination with DHS OIG, could improve the process. A senior OCSO official agreed that reconciliation procedures would help ensure that all complaints referred by DHS OIG to FEMA are accounted for and noted that FEMA is working with DHS OIG to improve reporting processes and case reconciliation. Conclusions Employee misconduct can detract from FEMA’s mission and negatively impact public perceptions of the agency, particularly when associated with disaster response efforts. Given the broad scope of FEMA’s mission and the growth and different categories that make up its workforce, an effective process is key to mitigating any negative employee misconduct effects. FEMA has taken actions to manage the employee misconduct investigation and adjudication process. Specifically, FEMA has developed and documented misconduct policies and procedures for most of its employees, and has established procedures for regular internal communication and coordination, as well as information sharing with DHS OIG. However, misconduct policies and procedures for Surge Capacity Force members and outcome options and appeals policies and procedures for Reservists are not documented, and FEMA does not communicate the range of penalties for offenses to all employees. Clear documentation establishing who is responsible for investigating and adjudicating misconduct is especially important given FEMA’s goal of significantly expanding the Surge Capacity Force. Because the Reservist disciplinary action options and appeals currently in practice are not documented, FEMA supervisors and Reservist employees may not be aware of all aspects of the process. Similarly, the lack of communication on the range of penalties for specific offenses to FEMA’s workforce limits management’s ability to set expectations about appropriate conduct in the workplace and to communicate consequences of inappropriate conduct. Clearly documented policies and procedures for all workforce categories and communication about offenses and penalties could help to better prepare management to address misconduct and to mitigate any perceptions that misconduct is handled inconsistently across FEMA’s workforce. In addition, while several FEMA offices collect data on employee misconduct allegations, investigations, and outcomes, limitations related to how the data are collected and managed restrict their usefulness for identifying and addressing trends in employee misconduct. Addressing these limitations by implementing quality control measures could improve FEMA’s ability to track misconduct cases and to identify potential problem areas and opportunities for targeted training. Moreover, developing reconciliation procedures to track cases referred from DHS OIG to FEMA could help reduce the risk that FEMA does not address all misconduct complaints. Recommendations for Executive Action In order to improve employee misconduct policies and procedures, the Secretary of Homeland Security should direct the FEMA Administrator to take the following three actions: document policies and procedures to address potential Surge document Reservist disciplinary options and appeals policies and procedures that are currently in practice at the agency; and communicate the range of penalties for specific misconduct offenses to all employees and supervisors. In order to better identify and address trends in employee misconduct, the Secretary of Homeland Security should direct the FEMA Administrator to take the following actions: improve the quality and usefulness of the misconduct data it collects by implementing quality control measures, such as adding additional drop-down fields with standardized entries, adding unique case identifier fields, developing documented guidance for data entry, or considering the adoption of database software; and once the quality of the data is improved, conduct routine reporting on employee misconduct trends. In order to ensure that all allegations of employee misconduct referred by DHS OIG are reviewed and addressed, the Secretary of Homeland Security should direct the FEMA Administrator to develop reconciliation procedures to consistently track referred cases. Agency Comments and Our Evaluation We provided a draft of this report to DHS and FEMA for review and comment. DHS provided written comments which are reproduced in appendix II. In its comments, DHS concurred with our recommendations and described actions planned to address them. FEMA and DHS OIG also provided technical comments, which we incorporated as appropriate. With regard to our first recommendation, that FEMA document policies and procedures to address potential Surge Capacity Force misconduct, DHS stated that FEMA is developing a Human Capital plan for the Surge Capacity Force and will include policies and procedures relating to potential misconduct. DHS noted that these policies and procedures will take into account FEMA’s limited authorities over Surge Capacity personnel who are not FEMA employees. DHS estimated that this effort would be completed by June 30, 2018. This action, if fully implemented, should address the intent of the recommendation. With regard to our second recommendation, that FEMA document Reservist policies and procedures related to disciplinary options and appeals currently in practice at the agency, DHS stated that FEMA will update FEMA Directive 010-06, FEMA Reservist Program, to include procedures for disciplinary actions and appeals current in practice at the agency. DHS estimated that this effort would be completed by December 31, 2017. This action, if fully implemented, should address the intent of the recommendation. With regard to our third recommendation, that FEMA communicate the range of penalties for specific misconduct offenses to all employees and supervisors, DHS stated that FEMA’s OCCHCO is currently drafting a table of offenses and penalties and will take steps to communicate those penalties to employees throughout the agency once the table is finalized. DHS estimated that this effort would be completed by December 31, 2017. This action, if fully implemented, should address the intent of the recommendation. With regard to our fourth and fifth recommendations, that FEMA improve the quality and usefulness of its misconduct data by implementing quality control measures, and, once the quality of the data is improved, conduct routine reporting on employee misconduct trends, DHS stated that FEMA’s OCCHCO is working with the DHS OIG to develop a new case management system. The system will use drop-down fields with standardized entries and provide tools for trend analysis. Once the new system is implemented, DHS stated that FEMA will be able to routinely identify and address emerging trends of misconduct. DHS estimated that these efforts would be completed by March 31, 2018. These actions, if fully implemented, should address the intent of the recommendations. With regard to our sixth recommendation, for FEMA to develop reconciliation procedures to consistently track referred cases, DHS stated that once the new case management system described above is established and fully operational, FEMA will be able to upload all DHS OIG referrals into a single, agency-wide database. Additionally, FEMA will work with DHS OIG to establish processes and procedures that will improve reconciliation of case data. DHS estimated that these efforts would be completed by March 31, 2018. These actions, if fully implemented, should address the intent of the recommendation. We are sending copies of this report to the Secretary of Homeland Security and interested congressional committees. If you or your staff have any questions about this report, please contact me at (404) 679-1875 or [email protected]. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made key contributions to this report are listed in appendix III. Appendix I: Objectives, Scope, and Methodology The objectives of this report were to determine (1) the extent to which the Federal Emergency Management Agency (FEMA) has developed policies and procedures for addressing employee misconduct; (2) what data are available on FEMA employee misconduct cases and their outcomes, and the extent to which FEMA uses these data to identify and address trends in employee misconduct; and (3) the extent to which information regarding misconduct cases is shared within FEMA’s personnel management offices and with the Department of Homeland Security Office of the Inspector General (DHS OIG). To address objective one, we reviewed, where available, FEMA’s policies and procedures for reporting, investigating, and adjudicating allegations of misconduct across all of the agency’s workforce categories, including: Title 5 employees, Cadre of On-Call Response/Recovery Employees (CORE), Reservists, Surge Capacity Force members, and FEMA Corps members. Specifically, we reviewed investigation and discipline directives and manuals, as well as program directives and manuals, and interagency agreements related to FEMA Corps, to determine the extent to which FEMA has developed misconduct policies and procedures. At FEMA headquarters we interviewed senior officials from the Office of the Chief Security Officer (OCSO), Office of the Chief Component Human Capital Officer, Labor and Employee Relations Branch (LER), and Office of the Chief Counsel, Personnel Law Branch (PLB). Additionally, we interviewed the Surge Capacity Force and FEMA Corps Coordinators, as well as AmeriCorps officials, to discuss Surge Capacity Force and FEMA Corps misconduct policies and procedures. We interviewed cadre management officials from FEMA’s three largest cadres - Public Assistance, Individual Assistance, and Logistics - to discuss Reservist misconduct policies and procedures. We also spoke with union representatives from two FEMA bargaining units to gain their perspective on how misconduct policies and procedures are implemented for Title 5 and CORE employees. Additionally, we interviewed a non-probability sample of supervisors and managers in three FEMA regions. Specifically, we interviewed three FEMA Regional Administrators or their designated representatives; five Federal Coordinating Officers (FCO), and 11 Public Assistance, Individual Assistance, and Logistics Branch Chiefs from FEMA Regions 2,4, and 6. We selected these three regions based on factors such as geographic dispersion and regions that typically respond to different types of disasters, as well as those with the highest number of misconduct allegations reported to OCSO from January 2014 through September 30, 2016. The results of these interviews are not generalizable to all 10 of FEMA’s regions or all cadres; however, they provided us with both regional and Joint Field Office perspectives on policies and procedures for addressing employee misconduct and supervisory misconduct training. To address objective two, we reviewed and analyzed misconduct case tracking spreadsheets maintained by OCSO, LER, and PLB. We were unable to identify and account for possible duplication of the same complaints in the different spreadsheets. As such, we focused our analysis on individual spreadsheets rather than on aggregating information across all spreadsheets. Specifically, in order to summarize alleged misconduct offenses recorded in the OCSO case tracking spreadsheet, we first reviewed all of the data available on complaints which were received from January 1, 2014 through September 30, 2016. Next, we created eight offense category definitions based on prior GAO reports, discussions with stakeholders, and our review of the summary of allegations and offense fields. Finally, we assigned alleged offenses from the OCSO spreadsheet to the categories. We also attempted to review and summarize the subjects included in each complaint by employee type. However, our ability to do so was limited because that information was not consistently included in the OCSO spreadsheet. In order to summarize final disciplinary and adverse actions reviewed by PLB and recorded in the PLB case tracking spreadsheet, we reviewed all of the data available from January 2, 2014 through September 28, 2016. Next, we selected actions which were categorized as related to employee misconduct and excluded proposed actions. Finally, we summarized final actions by the action type and employee type using existing spreadsheet fields. We also reviewed information on past employee misconduct outcomes and disciplinary actions provided by LER. To assess the reliability of these data, we reviewed the three spreadsheets for any gaps and inconsistencies. We also interviewed agency officials from each office about how data are entered into the spreadsheets, who enters the data, whether they have guidance documents for data entry, and the process of assigning complaints to the offices. We identified limitations which we discuss in the report, but overall found the data in the spreadsheets sufficiently reliable to provide some general information on the nature and characteristics of employee misconduct complaints. In order to determine the extent to which FEMA currently uses these data to identify and address trends in employee misconduct, we interviewed OCSO, LER, and PLB officials. Additionally, we included related questions during the interviews with supervisors and managers in FEMA Regions 2, 4, and 6. Although information obtained from these interviews, as mentioned above, is not generalizable to all 10 regions or all FEMA cadres, it provided insights into how employee misconduct information is shared with FEMA field supervisors. To address objective three, we first reviewed FEMA and DHS OIG employee misconduct directives and manuals to identify any requirements for coordination among internal FEMA offices and with DHS OIG. Next, we interviewed senior OCSO, LER, PLB, and DHS OIG officials to determine the extent to which they coordinate and communicate misconduct information and to obtain their perspectives on information sharing related to misconduct investigations and outcomes. We analyzed documents, including case tracking spreadsheets and reports, to determine what and how frequently employee misconduct information is shared within FEMA and with DHS OIG. Finally, we used fiscal year 2016 data from DHS OIG’s Enforcement Data System to randomly select a non-generalizable sample of 20 FEMA employee misconduct complaints which were referred from DHS OIG to FEMA. We compared these complaints to the OSCO case tracking spreadsheet provided by FEMA. For complaints we were unable to locate, we requested that OCSO provide us with their statuses, including whether or not OCSO had taken action to review or investigate the complaints. Additionally, we asked OCSO to coordinate with LER and PLB and obtain information on the outcomes of all 20 complaints. While not generalizable to all complaints referred from DHS OIG to FEMA, the results of our review provided insight about FEMA’s procedures for tracking referred complaints and ensuring that all allegations of misconduct are addressed. For all three objectives, we reviewed the documents and information we gathered and evaluated them against Standards for Internal Control in the Federal Government. We conducted this performance audit from June 2016 to July 2017 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. Appendix II: Comments from the Department of Homeland Security Appendix III: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the contact named above, Ben Atwater (Assistant Director), Sarah Turpin (Analyst-in-Charge), David Alexander, Dominick Dale, Eric Hauswirth, Rianna Jansen, Stephen Komadina, Kristiana D. Moore, and Heidi Nielson made key contributions to this report.
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Summary: FEMA is responsible for coordinating government-wide efforts in preparing for, responding to, and recovering from natural or man-made disasters, including acts of terror. The agency relies on permanent and disaster-related temporary employees and has a total workforce of over 22,000. Employee misconduct incidents can detract from FEMA's mission, damage the agency's reputation, and hamper its ability to respond to disasters and maintain public trust. GAO was asked to review employee misconduct at FEMA. This report examines: (1) the extent to which FEMA developed policies and procedures for addressing misconduct; (2) available data on FEMA misconduct cases and the extent to which FEMA uses the data to identify and address trends; and (3) the extent that misconduct cases are shared within FEMA and with DHS OIG. GAO reviewed FEMA procedures, analyzed misconduct data, and interviewed officials from FEMA HQ and three regions (selected based on geographic dispersion and number of misconduct allegations). GAO also analyzed a random, non-generalizable sample of 20 complaints referred from DHS OIG to FEMA to determine whether they were addressed. The Federal Emergency Management Agency (FEMA) has developed and documented misconduct policies and procedures for most employees, but not its entire workforce. Specifically, FEMA has developed policies and procedures regarding misconduct investigations that apply to all FEMA personnel and has also documented options to address misconduct and appeal rights for Title 5 (generally permanent employees) and Cadre of On-Call Response/Recovery Employees (temporary employees who support disaster related activities). However, FEMA has not documented misconduct policies and procedures for Surge Capacity Force members, who may augment FEMA's workforce in the event of a catastrophic disaster. Additionally, FEMA's Reservist (intermittent disaster employees) policies and procedures do not outline disciplinary actions or the appeals process currently in practice at the agency. As a result, supervisors and Reservist employees may not be aware of all aspects of the process. Clearly documented policies and procedures for all workforce categories could help to better prepare management to address misconduct and mitigate perceptions that misconduct is handled inconsistently. FEMA records data on misconduct cases and their outcomes; however, aspects of this data limit their usefulness for identifying and addressing trends. GAO reviewed misconduct complaints recorded by FEMA's Office of the Chief Security Officer (OCSO) from January 2014 through September 30, 2016, and identified 595 complaints involving 799 alleged offenses, the most common of which were integrity and ethics violations. FEMA reported 546 disciplinary actions related to misconduct from calendar year 2014 through 2016. In addition to OCSO, two other FEMA offices involved in investigating and adjudicating misconduct also record data. However, limited standardization of data fields and entries within fields, limited use of unique case identifiers, and a lack of documented guidance on data entry across all three offices restricts the data's usefulness for identifying and addressing trends in employee misconduct. Improved quality control measures could help the agency use the data to better identify potential problem areas and opportunities for training. FEMA shares misconduct case information internally and with the Department of Homeland Security Office of Inspector General (DHS OIG) on a regular basis; however, FEMA does not have reconciliation procedures in place to track DHS OIG referred cases to ensure that they are reviewed and addressed. GAO reviewed a random sample of 20 cases DHS OIG referred to FEMA in fiscal year 2016 and found that FEMA missed 6 of the 20 complaints during the referral process and had not reviewed them at the time of GAO's inquiry. As a result of GAO's review, FEMA took action to review the complaints and opened inquiries in 5 of the 6 cases (1 case was closed for lack of information). In 3 of these cases, officials determined that the complaints did not involve FEMA employees. The 2 remaining cases were open as of April 2017. While the results from this review are not generalizable to the entire population of referrals from DHS OIG to FEMA, they raise questions as to whether there could be additional instances of misconduct complaints that FEMA has not reviewed or addressed. Procedures to ensure reconciliation of referred cases across FEMA and DHS OIG records could help ensure that FEMA accounts for all complaints.
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Summarize: The National Enquirer has uncovered a 2006 police report about an alleged incident of Al Gore making unwanted sexual contact with a Portland masseuse. According to the District Attorney's office it's legit. Is Al Gore a dirty old man? According to the National Enquirer, in 2006: No criminal charges were brought against Gore, but the Portland police prepared a document marked "Confidential Special Report" - which records the explosive allegations of "unwanted sexual contact" by Al Gore "at a local upscale hotel." The Oregonian reports that the Multnomah County District Attorney's office has confirmed that "a woman who alleged unwanted sexual contact by Al Gore reported it to police in 2006,". The initial report was filed December 16, 2006, "several weeks" after the alleged incident occurred in a fancy downtown Portland hotel. But when police tried to follow up with the unidentified woman, she cancelled meetings three times before her attorney told them the case would be handled civilly (i.e. for money) and the investigation was closed in early 2007. The police briefly reopened the case in 2009, according to The Oregonian, though it's unclear what, if anything, they found. (The police report is sadly sparse on details. Update: Actually, a 2009 report is very, very detailed. See below.) Maybe this masseuse just trying to get a piece of Al Gore's sweet global warming cash. (Her reluctance to meet with police is odd.) But, still: Crazy! Al Gore!? This hints that Al and Tipper's split was not the amiable drifting apart they've sold it as, and it adds to speculation that Al Gore had an affair with Larry David's ex-wife, Inconvenient Truth producer Laurie David. Were Bill Clinton's trysts obscuring the sexual whirlwind hidden in Al Gore—strong enough to power an entire farm's worth of wind turbines? Only time, and a Tiger Woods-style parade of mistresses, will tell. Update: So, we just finished reading the police interview with the masseuse from 2009 and, Wow. That is 45 pages of (alleged) scary, sex-crazed Al Gore. Here's a very, very brief outline of what she alleges occurred in a hotel room in downtown Portland on October 24, 2006: The woman, a Licensed Massage Therapist, showed up and was told her client was Al Gore. She was happy! But when Al Gore answered the door, he gave her a hug that was just a tad too long. Then, he asked for massages in weird places: Like, the inside of his thigh. Kind of creepy, but still within the purview of legitimate massage. But things went downhill during the massage, as Al Gore growled at her to "go lower": I decided to clarify things... I said well, why don't you show me... he grabbed my right hand hard, shoved it down under the sheet to his pubic area, my fingers brushing against his penis and firmly planted my hand on his pubic crest region and said to me, "There!!" in a very sharp, loud, angry-sounding tone. She refused, and he growled some more. When that didn't work, he started pleading with her pathetically "for release of his second chakra there. 'There's so much tension being held…'" Still, she refused! Then told him it was time to go. But while she was packing up he grabbed her in "an inescapable embrace" and "caressed my back and buttocks and breasts." She distracted him with a box of chocolates (really!) which backfired when he tried to get her to eat them out of his hand. Al Gore then forced her to take a sip of minibar Grand Marnier. Then Al Gore "forced an open mouth kiss on me," and when she protested he "laughed, clearly enjoying himself at the expense of my discomfort." This groping and flailing and horrible seduction goes on for a while, and such things allegedly happened as: He had been pressing his body up against mine, holding me tightly, holding me tightly and I was not certain if his robe was open or closed... It seemed partway open like the robes are small. He's rotund, you know? And I did not want to look down to see. I could feel what seemed to be an erection from him. and: he turned to me and he immediately flipped me flat on my back and threw his whole body face down over atop me, pinning me down and outweighing me by quite a bit. Get off me, you big lummox! I loudly protested to him and I struggled with my whole body to shove him as hard as I could to roll him off me... and: He insisted I look at his ipod… the song was 'Dear Mr. President' by Pink Eventually she makes her way to the door. There, Al Gore made his final gambit: He pleaded, grabbed me, engulfed me in embrace, tongue kissed me, massaged me, groped my breasts and painfully squeezed my nipples through my clothing, pressed his pelvis against mine, rubbed my buttocks with his hands and fingers and rubbed himself against my crotch… She finally escapes, having somehow avoided being raped by Al Gore. But at home she examines her black slacks: I took 'em off and I said what's those stains on the front of those? And I wondered if it was soap or bodily fluids from Gore pushing up against me repeatedly? If you want to read the whole 45-page epic, read it at KGW's website. But you really don't want to read it, trust us. Here's the 2006 police report (Click to enlarge): Starting in 1996, Alexa Internet has been donating their crawl data to the Internet Archive. Flowing in every day, these data are added to the Wayback Machine after an embargo period. The seed for this crawl was a list of every host in the Wayback Machine This crawl was run at a level 1 (URLs including their embeds, plus the URLs of all outbound links including their embeds) The WARC files associated with this crawl are not currently available to the general public.
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Summary: Thanks to the uncovering of a 2009 police report, you're about to know more-much more-about Al Gore than you ever wanted to. The report, obtained by KGW, details a Portland masseuse's allegations of sexual assault at Gore's hands, beginning with an inappropriately long embrace when she arrived at his hotel room. Things start getting weird when he specifically asks for massage work on his abdomen and inner thigh, but that's just the beginning. A possibly-drunk Gore began "moaning, groaning, moving and acting in a very suggestive way" and asking the masseuse to "go lower," she claims. When she asked him to clarify, he shoved her hand onto his "pubic crest region," and got angry when she didn't comply-then changed tactics and pleaded "for release of his second chakra there." You cannot make this stuff up. To read more-including how she tried to distract Gore with chocolate and the mysterious stains she found on her pants when she got home-click here, or read Gawker's recap.
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Summarize: By. Anna Hodgekiss and Claire Bates. PUBLISHED:. 08:04 EST, 13 November 2012. |. UPDATED:. 04:57 EST, 14 November 2012. A man has miraculously cheated death after being hit by a 15 tonne lorry thanks to a dedicated team of 34 doctors and nurses. Lewis Godfrey, 23, suffered such horrendous injuries that doctors pronounced him dead twice. The smash had left him so badly injured his insides were left strewn across the road and he was rushed to hospital with his body parts held together in a blanket. Dr Nicholas Foster, who was working as a. volunteer GP with paramedics was the first doctor to treat Mr Godfrey. Dr Foster said: 'He had already died at. the scene. When I got there, I saw the most horrific injuries - he had. been ripped open. 'The level of trauma was so massive he shouldn’t have made it. I knew I had to stop the bleeding, so we put him back together and wrapped him up to keep him in one piece.' TOP ROW (left-right): Holly Stirland, staff nurse; Hannah Freeman, staff nurse; Dr Nicholas Foster, the first person on the scene of Lewis's accident; Lynn Brindley, healthcare assistant, major trauma ward; Joanne Critchley, staff nurse; Bob Winter, consultant, intensive care; Lindsay Warhurst, staff nurseBOTTOM ROW (left-right): Beth MacAlindin, staff nurse; Joanne Cooper, senior physiotherapist; Elaine Stevenson, auxiliary nurse; Shane MacSweeney, consultant vascular surgeon who operated on Lewis to repair his veins and arteries; June Pindard, scrub sister in operating theatres; Ian Nash, senior pain nurse specialist who advised and supervised pain relief for Lewis; Annalea Severn, staff nurse. TOP ROW (left-right): Annamma Giboy, staff nurse; Rebecca Thorpe, staff nurse in the emergency department; Hayley Queen, senior operating department practitioner in theatres; Miriam Duffy, clinical programme manager for the major trauma centre; Adam Brooks, consultant surgeon and head of the major trauma centre; Lucy Jennings, deputy sister on the major trauma ward; Katherine Knowle, staff nurse, intensive care MIDDLE ROW (left-right): Ian Woodruff staff nurse, intensive care; Pamela Weaver, staff nurse; Sharon Sanderson, case manager, major trauma centre; Hayley Conroy, physiotherapist; Justy Baby, staff nurse, major trauma ward; Nuala Hughes, healthcare assistant, major trauma ward; Daren Forward, consultant surgeon in orthopaedics and major trauma who operated on Lewis when he first arrived at hospitalBOTTOM ROW (left-right): Rohan Revill, case manager in the major trauma centre; Sarah Tolman, staff nurse in the emergency department; Tina Talbot, auxiliary nurse; Sam Rossiter, senior occupational therapist; Claire Chapman, emergency department assistant; Tim Mitchell, senior occupational therapist who worked with Lewis on his return home, ensuring he had the right equipment such as splints and braces. Mr Godfrey had a substantial head injury and his skin had been ripped. off his body in a band about a foot wide. His bowel had been damaged. and his bladder ripped off. The young man from Loughborough, Leicestershire, went on to spend 16 days in. a coma. He was expected to have brain damage and never to walk again. But five months after the horror smash he has made a remarkable recovery thanks to the team of medical staff at Nottingham Queens Medical Centre. This included six doctors, 18 nurses, three health care assistants, two physiotherapists, two occupational therapists and three healthcare managers. Each member of staff played their. part from Shane MacSweeney, the consultant vascular surgeon who repaired. Lewis' veins and arteries, to Joanne Cooper, the senior physiotherapist. who helped him walk again. Mr Godfrey said: 'I’m lucky. and my heart goes out to everyone single person that helped look after. me. Without them I wouldn’t be here today.' Lewis Godfrey has miraculously cheated death. after being hit by a 15 tonne lorry - leaving him so badly injured his. insides were left strewn across the road. Mr Godfrey points to where the skin was ripped from his body. Lewis reveals a foot-long scar after his life-saving operation. He added: 'It has taken a while for everything to sink in. Sometimes I can’t believe I’m here. I can’t remember anything from the fight or the accident, but I am really shocked when my dad tells me bits about what happened. 'I think it’s probably just as well I can’t remember anything. I was gutted when the doctors told me I would never walk again, but I was just glad to be here. 'I was determined to get my life back on track now, and with the help of a lot of people, I’m now able to walk around again.' Mr Godfrey, a cable technician, was. on a night out on June 2 when he was attacked by a group of men - who. punched him in the head and pushed him into the road, where he was. dragged under the wheels of a lorry. He continued to be dragged along as the truck as the driver was unaware he had hit someone,. A doctor pronounced Mr Godfrey dead at the scene after his heart had stopped beating - but incredibly, he came back to life. In the ambulance - as his body was held together by a blanket - his heart stopped again, but paramedics battled to restart it. He suffered from huge blood loss, severe head injuries, a broken pelvis and ripped all of the muscle off his upper right leg. A titanium plate was inserted into his skull to keep the shape of the head relatively normal. Mr Godfrey, a cable technician, also had his bladder ripped off and suffered bowel damage and a broken pelvis. He had skin removed from his leg for a graft (pictured) to cover a band about a foot wide where his skin had been ripped away. He has since taught himself to walk again. On the night he was admitted to the Queens Medical Centre he was operated on by surgeon, Mr Daren Forward. Mr Forward said: 'When he came in it was clear he’d been crushed under a lorry. He had a bleed in his head, with a substantial head injury. Five months after the horrific smash Mr Godfrey has made a full recovery, thanks to staff at the Queens Medical Centre in Nottingham. As well as undergoing emergency surgery, the 23-year-old has had a skin graft to repair his thigh. He also had a titanium plate inserted into his skull to keep the shape of his head relatively normal. Prior to going home, he also required extensive physiotherapy to walk again - and succeeded, despite initially being warned he may be paralysed. 'The skin had essentially been ripped off his body in a band about a foot wide. His bowel had been damaged and his bladder ripped off.' Mr Godfrey said: 'When they told me I. wouldn’t be able to walk again it was overwhelming. But with some help I. managed to learn to walk again in six months. 'It was a strain at first - I couldn’t even feed myself but it’s a lot better now.' Mr Godfrey's father Graham, 48, said: 'When we first arrived at the hospital, we were only allowed to see Lewis very briefly. 'They draped a sheet over him to cover his injuries, and I held his hand for a bit, and whispered in his ear to keep fighting. 'There were at least 12 tubes in him, and there was a huge bandage wrapped around his head.' Five months on, Mr Godfrey is well on the road to recovery. Although he faces further surgery and physiotherapy he hopes to be back at work by January
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Summary: Lewis Godfrey, 23, was attacked on a night out and pushed into the road, where he was dragged under the wheels of a lorry. Suffered such horrendous injuries that doctors on the scene pronounced him dead TWICE. So badly injured that his organs were strewn over the road. Spent 16 days in a coma, was expected never to walk again and to have brain damage. But five months on he has made a remarkable recovery.
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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Marine Debris Act Amendments of 2016''. SEC. 2. NOAA MARINE DEBRIS PROGRAM. Subsection (b) of section 3 of the Marine Debris Act (33 U.S.C. 1952(b)) is amended-- (1) in paragraph (4), by striking ``and'' at the end; (2) in paragraph (5)(C), by striking the period at the end and inserting a semicolon; and (3) by adding at the end the following: ``(6) work with other Federal agencies to develop outreach and education strategies to address both land- and sea-based sources of marine debris; and ``(7) work with the Department of State and other Federal agencies to promote international action to reduce the incidence of marine debris.''. SEC. 3. ASSISTANCE FOR SEVERE MARINE DEBRIS EVENTS. Section 3 of the Marine Debris Act (33 U.S.C. 1952) is amended by adding at the end the following new subsection: ``(d) Assistance for Severe Marine Debris Events.-- ``(1) In general.--At the discretion of the Administrator or at the request of the Governor of an affected State, the Administrator shall determine whether there is a severe marine debris event. ``(2) Assistance.--If the Administrator makes a determination under paragraph (1) that there is a severe marine debris event, the Administrator is authorized to make sums available to be used by the affected State or by the Administrator in cooperation with the affected State-- ``(A) to assist in the cleanup and response required by the severe marine debris event; or ``(B) such other activity as the Administrator determines is appropriate in response to the severe marine debris event. ``(3) Federal share.--The Federal share of the cost of any activity carried out under the authority of this subsection shall not exceed 75 percent of the cost of that activity.''. SEC. 4. SENSE OF CONGRESS ON INTERNATIONAL ENGAGEMENT TO RESPOND TO MARINE DEBRIS. It is the sense of Congress that the President should-- (1) work with representatives of foreign countries that produce the largest amounts of unmanaged municipal solid waste that reaches the ocean to learn about, and find solutions to, the contributions of such countries to marine debris in the world's oceans; (2) carry out studies to determine-- (A) the primary means by which solid waste enters the oceans; (B) the manner in which waste management infrastructure can be most effective in preventing debris from reaching the oceans; (C) the long-term economic impacts of marine debris on the national economies of each country set out in paragraph (1) and on the global economy; and (D) the economic benefits of decreasing the amount of marine debris in the oceans; (3) work with representatives of foreign countries that produce the largest amounts of unmanaged municipal solid waste that reaches the ocean to conclude one or more new international agreements-- (A) to mitigate the risk of land-based marine debris contributed by such countries reaching an ocean; and (B) to increase technical assistance and investment in waste management infrastructure, if the President determines appropriate; and (4) consider the benefits and appropriateness of having a senior official of the Department of State serve as a permanent member of the Interagency Marine Debris Coordinating Committee established under section 5 of the Marine Debris Act (33 U.S.C. 1954). SEC. 5. INCLUSION OF DEPARTMENT OF STATE ON THE INTERAGENCY MARINE DEBRIS COORDINATING COMMITTEE. Section 5(b) of the Marine Debris Act (33 U.S.C. 1954(b)) is amended-- (1) in paragraph (4), by striking ``; and'' and inserting a semicolon; (2) by redesignating paragraph (5) as paragraph (6); and (3) by inserting after paragraph (4) the following: ``(5) the Department of State; and''. SEC. 6. AUTHORIZATION OF APPROPRIATIONS. Section 9 of the Marine Debris Act (33 U.S.C. 1958) is amended to read as follows: ``SEC. 9. AUTHORIZATION OF APPROPRIATIONS. ``There are authorized to be appropriated for each fiscal year 2017 through 2021-- ``(1) to the Administrator for carrying out sections 3, 5, and 6, $10,000,000, of which no more than 10 percent may be for administrative costs; and ``(2) to the Secretary of the Department in which the Coast Guard is operating, for the use of the Commandant of the Coast Guard in carrying out section 4, $2,000,000, of which no more than 10 percent may be used for administrative costs.''. Passed the Senate December 10 (legislative day, December 9), 2016. Attest: Secretary. 114th CONGRESS 2d Session S. 3086 _______________________________________________________________________ AN ACT To reauthorize and amend the Marine Debris Act to promote international action to reduce marine debris and for other purposes.
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Title: Marine Debris Act Amendments of 2016 Summary: Marine Debris Act Amendments of 2016 (Sec. 2) This bill amends the Marine Debris Act to revise the Marine Debris Program by requiring the National Oceanic and Atmospheric Administration (NOAA) to work with: (1) other agencies to address both land- and sea-based sources of marine debris, and (2) the Department of State and other agencies to promote international action to reduce the incidence of marine debris. (Sec. 3) The bill also revises the program by allowing NOAA to make sums available for assisting in the cleanup and response required by severe marine debris events. (Sec. 4) The bill urges the President to: (1) work with foreign countries that produce the largest amounts of unmanaged municipal solid waste that reaches the ocean in order to find solution to the marine debris; and (2) study issues related to marine debris, including the economic impacts of marine debris. (Sec. 5) The Interagency Marine Debris Coordinating Committee must expand to include a senior official from the Department of State. (Sec. 6) This bill reauthorizes through FY2021: (1) the Marine Debris Program, (2) an information clearinghouse on marine debris, and (3) enforcement of laws about discarded marine debris from ships.
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Summarize: BURBANK, Calif. — For the past few months, Jay Leno’s decided President Barack Obama makes for a good joke. That’s a problem for the White House — one that the president came here Tuesday trying to fix. Text Size - + reset Obama gives Leno car for collection Obama: 'No patience' for Russia's anti-gay laws Throughout his career, Leno’s demonstrated show business’s most accurate sense of what the greatest denominator of people find funny. Leno may make his fellow comedians and the Amtrak corridor groan, but he’s spent 20 years on top of the ratings, and is finishing his run at the “Tonight Show” with them on the rise. (PHOTOS: Obama’s second term) Obama and Leno covered a lot of serious ground — about the threat to U.S. embassies, the troubled relationship with Russia over Edward Snowden and the country’s anti-gay laws, and the NSA’s surveillance activities. And they also covered Obama’s relationships with former rivals John McCain and Hillary Clinton, how the president really does like broccoli, and even added the obligatory joke about getting so old that he and his friends needed ibuprofen after they played basketball and golf together on his birthday. But even in an interview that was the most gracious and substantive he’s conducted in months, none of what Obama said mattered nearly as much as the underlying message of his sober, charming, occasionally smiling display as he sat cross-legged at Leno’s side for most of an hour: He’s campaigning to get back to being Barack Obama again. One Jay Leno interview is unlikely to change perceptions overnight, but as Obama works to reframe himself ahead of the fall budget negotiations with Republicans, he’s got to win back the people and the places where the squeaky-voiced king of late night plays the strongest — those that most readers of Obama’s interview with The New York Times last week might tend to lump together as “middle America.” (Also on POLITICO: Transcript of Jay Leno's interview with President Obama) Hopeful conservatives have eagerly drawn a direct connection between Leno’s Obama jokes and his recent ratings surge, but the jokes seem to demonstrate Leno’s sense of his audience that’s deeper, more poisonous and potentially irreversible for any politician: They’ve heard it all before. And for a man who was propelled by the rhetoric of change, it turns out that Obama’s just like all the rest — a detached politician who makes them only shake their heads and laugh. “The White House announced in the coming days President Obama will be reaching out to Americans who’ve lost their jobs,” Leno said to open the show. “In fact, that’s why he’s here with me tonight; he’s here to talk with me personally — that’s right, we’re going to work something out.” Then Leno said he was excited for a face-to-face conversation, since that would save Obama “the trouble of going through my emails and phone calls.” (Also on POLITICO: Obama: 'No patience' for anti-gay laws) There’s no missing the numbers for Obama, whether that’s Gallup’s 44 to 48 percent approval-disapproval rating, or state-by-state polls. In two swing states he won last year, the Democratic firm Public Policy Polling shows Obama approval and disapproval even at 48 percent in Virginia, and dropped to 46 approval compared with 50 percent disapproval in Iowa as of a month ago. Part of this is the natural attrition of a president working his way through his second term. Part is the unhealed wounds from the spring scandals. Part is the great national distaste for all things Washington. (Also on POLITICO: Obama: Putin still a Cold Warrior) Late-night comedians struggled to make jokes about Obama for five years — for all the time spent on the 2008 and 2012 elections, they never nailed the all-important caricature of him the way Al Gore was wooden or George W. Bush was dumb or John McCain was old (still a Leno joke as of Tuesday) or John Kerry and Mitt Romney were Thurston Howell IV and V. Those days are gone. A George Mason University study released ahead of the Leno taping showed Obama was the most popular subject of late-night comedy of any public figure, prompting more than twice as many jokes as Anthony Weiner, the runner-up. President Barack Obama, left, talks with Jay Leno during a commercial break during the taping of his appearance on The Tonight Show with Jay Leno. advertisement President Barack Obama gave an unexpectedly serious and wide-ranging interview to Jay Leno on Tuesday night, weighing in on a terror threat, U.S. tensions with Russia and even his recent lunch with Hillary Clinton on "The Tonight Show" — a venue where he was more accustomed to light-hearted joking. Obama used his appearance on the show – his sixth – to give his first public comments on recent warnings of a possible terrorist attack on U.S. interests in the Middle East. The warnings have prompted the State Department to shutter 19 diplomatic posts across the Middle East and North Africa until Saturday. "Well, it's significant enough that we're taking every precaution," Obama said to Leno, adding that radical violet extremism "is still out there, we've got to stay on top of it." The president also reiterated the White House's warning that the threat was significant and urged Americans to act "prudently" when planning travel and checking in with the State Department and embassies to see what precautions they should be taking. "The general rule is show some common sense and some caution," Obama said, as the first sitting president ever to go on the show, making his fourth appearance since he took office. He also said that Americans have shined in times of danger and peril, pointing out that people kept going to ball games and went on business as usual after the Boston Marathon bombings in April. "That's the right reaction. Terrorists depend on the idea that we're going to be terrorized," Obama said. The president also commented on the case of National Security Agency leaker Edward Snowden, plus the secret government data surveillance programs his leaks to the press uncovered. "We don't have a domestic spying program," Obama said, defending some surveillance as a "critical component to counterterrorism" and saying the information it gathered was "useful." Photos and Videos Audience Members React to President’s "Tonight Show" Appearance President Obama made his sixth appearance on the “Tonight Show” Tuesday. The President spoke to Jay Leno about the economy, his recent lunch with Hillary Clinton, and gay rights in Russia. Beverly White reports from Burbank for the NBC4 News at 6 p.m. on August 6, 2013. WATCH Audience Members React to... "Tonight Show" Guests Surprised Obama Making Appearance Guests of the "Tonight Show With Jay Leno" were surprised to find out a secret guests was scheduled to be President Barack obama, who was in town for his sixth taping of the show. Toni Guinyard has the report for the NBC4 News at noon on Tuesday, Aug. 6, 2013. WATCH "Tonight Show" Guests... More Photos and Videos But NBC News' correspondent Andrea Mitchell told "The Rachel Maddow Show" Tuesday said that he also appeared to express some caution about the surveillance, suggesting some level of discomfort with the NSA's programs. Obama didn't comment on the legality of Snowden's leaks. Snowden faces espionage charges for them and has been granted temporary asylum in Russia in the face of them. "We don't know exactly yet what he did," Obama said. "It's important for me not to prejudge something." The president said he was disappointed by Russia's decision to grant Snowden asylum, but maintained that the U.S relationship with Russia is still intact. "There's still a lot of business that we can do with them, but there are times when they slip back into Cold War thinking," Obama said. He confirmed to Leno that he will attend the G-20 summit in St. Petersburg with Russian president President Vladimir Putin — despite some protests that he should not, due to Russia's granting Snowden asylum, and others over Russia's new spate of laws cracking down on gay Russians' civil rights. On Wednesday, however, Obama canceled a meeting with Putin that had been scheduled on the sidelines of the summit. Leno himself told MSNBC host Lawrence O'Donnell in an interview on his show "The Last Word" Tuesday night, after the Obama taping, that the question of Russia — and of its crackdown on gay rights — was one he had most looked forward to asking Obama. The president said he had no patience for countries that try intimidate or harm gay, lesbian or transgender people and said it was his duty to speak out about basic freedoms. Another topic Leno had most wanted to ask Obama about: The high-profile case of Trayvon Martin and his fatal shooting by George Zimmerman, who was acquitted of second-degree murder and manslaughter last month. Leno praised the president's public comments on the case and on the experiences of black men and boys in America and asked him about his remarks. "I think all of us were troubled by what happened," Obama told Leno. "It doesn't mean Trayvon was a perfect kid. None of us were." Obama said he thought the attention paid to the Martin shooting was indicative of how badly Americans want a fair and just criminal justice system. "What I wanted to explain was why this was a particularly sensitive topic for the African-American community," he continued. "The system should work for everyone, and what I'm trying to do is just make sure that we have a conversation." On "The Last Word" Tuesday night, after taping, Leno said he had been particularly interested in hearing about the comments on the shooting's impact. "He put every American in the shoes of the average black teenage boy," Leno said. Leno also asked the president about the economy and the constant partisan battles in Congress over whether to boost the economy with infrastructure projects and other spending. And it was also pointed out that the president's health care law goes into full effect on Oct. 1. The late-night host also took a few swings at the president for becoming a bit closer to his 2008 presidential rival, John McCain, who was recently instrumental in pushing a comprehensive immigration bill through the Senate. Obama's appearance on Leno's show wasn't all serious, however, and he managed to have at least some fun with the late night host, with the help of some chat about former Secretary of State Hillary Clinton, whom he lunched with recently. "Who invited who to lunch?" Leno asked. "I invited her, and we had a great time," Obama said. "She had that post-administration glow — you know, when folks leave the White House, and two weeks later they look great." So was Clinton, a speculated 2016 presidential contender, measuring the Oval Office drapes for a possible future stint there? "She's been there," Obama said of the former first lady. "She doesn't have to measure them."
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Summary: President Obama covered a lot of bases-most of them serious-in his fourth Tonight Show appearance as president. Some highlights from the Jay Leno interview, which Politico sees as part of the president's push to "get back to being Barack Obama again" and stop being the butt of late-night jokes: On the global travel warning for US citizens: "The general rule is show some common sense and some caution," Obama said, praising the reactions of Americans who went about life as usual after the Boston Marathon bombing. "Terrorists depend on the idea that we're going to be terrorized," he said. On Trayvon Martin: "All of us were troubled by what happened, and any of us who are parents can imagine the heartache that those parents went through," Obama said after Leno praised his "eloquent" remarks after George Zimmerman's acquittal. "It doesn't mean that Trayvon was a perfect kid, none of us were," Obama said, adding that the issue of having a fair justice system for everybody is a "particularly sensitive topic for the African-American community." On Russia's anti-gay laws and the Sochi Olympics: "I think Putin and Russia have a big stake in making sure that the Olympics work," the president said, "and I think that they understand that for most of the countries that participate in the Olympics we wouldn't tolerate gays and lesbians being treated differently." On NSA surveillance and Edward Snowden: "We don't have a domestic spying program," claimed Obama, calling the controversial NSA programs "mechanisms that can track a phone number or an email address that is connected to a terrorist attack." He confirmed that he would attend the G20 summit in St. Petersburg and that the US-Russia relationship is intact, despite disappointment with Moscow's decision to grant asylum to Snowden. There is "still a lot of business that we can do with them, but there are times when they slip back into Cold War thinking," he said. The interview had a few lighter moments, including discussion of Obama's recent lunch with Hillary Clinton, NBC notes. Asked whether Clinton was measuring the Oval Office for drapes, Obama quipped, "Keep in mind she's been there. She doesn't have to measure them."
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Write a title and summarize: Brucellosis and leptospirosis are among neglected tropical zoonotic diseases particularly in the resource limited countries. Despite being endemic in these countries, there is paucity of information on its magnitude. This study investigated seropositivity of Brucella spp. and Leptospira spp., and associated factors among abattoir workers and meat vendors in the city of Mwanza, Tanzania. A community based cross-sectional study was conducted in Mwanza city from May to July 2017. Socio-demographic and other relevant information were collected. Detection of Brucella spp. and Leptospira spp. antibodies were done using slide agglutination test and microscopic agglutination test, respectively. Data were analyzed using STATA version 13 Software. A total of 250 participants (146 abattoir workers and 104 meat vendors) were enrolled with median age of 31 (IQR: 25–38) years. The overall, seropositivity of Brucella spp. antibodies was 48. 4% (95% Cl: 42–54). Seropositivity of B. abortus was significantly higher than that of B. melitensis (46. 0%, 95%Cl: 39–52 vs. 23. 6%, 95% Cl: 18–28, P<0. 001) while seropositivity of both species was 21. 2% (95%Cl: 16–26). The seropositivity of Leptospira spp. was 10. 0% (95% CI: 6–13) with predominance of Leptospira kirschneri serovar Sokoine which was detected in 7. 2% of the participants. Being abattoir worker (OR: 2. 19,95% CI 1. 06–4. 54, p = 0. 035) and long work duration (OR: 1. 06,95%CI: 1. 01–1. 11, p = 0. 014) predicted presence of both B. abortus and B. melitensis antibodies. Only being married (p = 0. 041) was significantly associated with seropositivity of Leptospira spp. Primary education was the only factor independently predicted presence of Brucella spp. antibodies among abattoir workers on sub-analysis of occupational exposure. None of factors were found to be associated with presence of Brucella spp. antibodies among meat vendors on sub-analysis. Seropositivity of B. abortus antibodies among abattoir workers and meat vendors is high and seem to be a function of being abattoir worker, having worked for long duration in the abattoir and having primary education. In addition, a significant proportion of abattoir workers and meat vendors in the city was seropositive for Leptospira kirschneri serovar Sokoine. There is a need to consider ‘one health approach’ in devising appropriate strategies to control these diseases in the developing countries. Brucellosis and Leptospirosis are among neglected tropical diseases which are endemic in resource limited countries including those in the sub-Saharan African region [1–3]. They are major public health concern due to their epidemiological patterns which involves animal-human interfaces resulting into economic losses and sub-clinical infections among human population. In human, these infections present with nonspecific symptoms, as a result they are misdiagnosed with other febrile illnesses such malaria, typhoid fever and rheumatic fever [4]. Leptospirosis is worldwide distributed particularly in tropical and some temperate regions. It is an occupational disease affecting individuals working close with animals. Leptospirosis outbreaks often occur after floods whereby the infected urine from animals such as rodents, dogs and cattle easily contaminate the water and environment hence spread the infection to humans [5,6]. The annual incidence of human Leptospirosis is estimated to be 1. 03 million cases worldwide with 58,000 deaths being attributed to the disease [7]. In East African region the annual incidence is estimated to be 25. 6 cases per 100,000 population [7]. Brucellosis is a contagious bacterial zoonotic disease of public health importance. Abattoir workers and others that work closely with animals or animal products have a high risk of contracting the disease[8,9]. The disease is endemic in the south and the Central America, Mediterranean, Africa, Indian subcontinent, Asia, Arab peninsula and Middle East. The annual incidence is estimated to range from 214. 4 to 1603. 4 cases per 100,000 population [10–12]. In Tanzania, the prevalence has been reported to range from 0. 7 to 23. 9% among the high risk groups [13–18]. Livestock brucellosis and leptospirosis [3,19–21] are endemic in the lake zone that supply animals destined for slaughter in Mwanza city. Abattoir workers and meat sellers may be at high risk if biosafety measures are not in place. Despite livestock brucellosis and leptospirosis being common in Tanzania especially in the Lake Victoria zone, there is paucity of data regarding the seropositivity of these pathogens among human population in the city of Mwanza, Tanzania. This study was designed to provide baseline information regarding the seropositivity of these pathogens, the information that may be useful for designing control interventions and provide insights for future research in this area. This was a community based cross-sectional study (S1 File) that was conducted between May and June 2017 in the city of Mwanza, Tanzania. The study was conducted among Igoma abattoir workers and meat vendors in the city. The abattoir has 250 workers, and about 200 cows and 50 goats are slaughtered per day. The city of Mwanza is the second largest in Tanzania with a total population of 706,453 according to 2012 National census [22]. The city possesses two districts namely; Ilemela and Nyamagana with a total population of 343,001 and 363,452, respectively. The city depends on Igoma abattoir to supply meat to more than 90 meat selling shops in the city. The sample size was estimated by using Kish Leslie formula (1965), using the Brucella spp. seropositivity of 14. 1% from a previous study by Mngumi et al., [17]. The Minimum sample size obtained was 186, however a total of 250 participants were enrolled. After obtaining a written informed consent, participants working in abattoir and meat retail shops were serially enrolled. Socio-demographic and other relevant information were collected using interview standard questions (S2 File). Data collected included: age, sex, occupation (abattoir worker, retail meat seller), residence, education and work duration in years. About 4 to 5 mls of venous blood was collected in a plain vacutainer tubes (Becton, Dickinson and Company, USA) and transported to the Catholic University of Health and Allied sciences (CUHAS) multipurpose laboratory within 4 hours of collection. The sera were extracted by centrifugation at 2500 rpm for 10 minutes, decanted into cryovials in duplicates and stored at -40°C until processing. One set of the samples was transported to the Pest management centre at the Sokoine University of Agriculture (SUA) whereby the detection of Leptospira spp. antibodies was made. The detection of specific Brucella spp. antibodies was done at Catholic University of Health and Allied Sciences (CUHAS). Detection of specific Brucella antibodies for B. abortus and B. melitensis was done using commercial rapid agglutination test according to the manufacturer’s instructions. The Eurocell A was used for B. abortus and Eurocell M for B. melitensis (Euromedi equip LTD. UK). In each run the positive and negative control were used. Results were interpreted as positive if the agglutination reaction was similar to that of positive control. The test has been found to have sensitivity and specificity of 95% and 100%, respectively [23]. Regarding the detection of Leptospira spp. antibodies, local Leptospira serovars previously isolated from animals (domestic animals and rodents) in Tanzania namely: Leptospira kirschneri serovar Sokoine, L. interrogans serovar Lora, L. kirschneri serovar Grippotyphosa, L. borgpetersenii serovar Kenya and L. interrogans serovar Hebdomadis were selected and used in microscopic agglutination test (MAT) [24,25]. The selected serovars were cultured into fresh Leptospira Ellinghausen and McCullough, modified by Johnson and Harris (EMJH) culture medium incubated at 30°C for 4 to 7 days before using as live antigen in (MAT). Culture purity and density was checked using dark-field microscope whereby a density of 300×108 Leptospires/ml was considered adequate for MAT. Serum samples were serially diluted with phosphate buffered saline (pH 7) in a ratio of 1: 10–1: 80 in U–bottomed microtiter plate and 50 μl was used in MAT. Prepared live Leptospires antigen (50 μl) was added to the diluted serum to give final dilutions of 1: 20–1: 160 (100 μl total volume) of serum-antigen mixture in each microtiter well. The first row was used for negative control while positive control was added in the same row as the sample. The plates with serum–antigen mixture were incubated at 30°C for 2–4 hours before being examined for agglutination under dark field microscope. A sample was considered positive for a specific serovar if more than 50% of the microorganisms in the microtiter well were agglutinated at the titer of ≥ 1: 80. Data collected was entered into a Microsoft excel sheet then analyzed using STATA version 13 software. The categorical variables were presented as proportions while continuous variables (age and work duration) were summarized as median with interquartile ranges. Cross-tabulation was done to determine factors with collinearity using Pearson Chi squared test. The median age and median work duration of Brucella spp. seropositive and seronegative participants were compared by Wilcoxon Mann-Whitney / ranksum tests. Logistic regression model was used to determine factors associated with the presence of specific Brucella spp. and Leptospira spp. antibodies. Factors with p value of less than 0. 2 on univariate analysis were subjected to multivariable regression analysis. Odds ratio (OR) and 95% Confidence interval (Cl) were noted. A P value of < 0. 05 was considered statistically significant. The protocol for conducting this study was approved by the joint CUHAS/BMC research ethics and review committee with ethical clearance number CREC/336/2017. The permission was further granted by the city council director, village leaders and abattoir manager. Written informed consent was obtained from each participant prior recruitment to the study. All participants in the current study aged 18 years and above All 250 participants were available for analysis. The median age of the study participants was 31 (inter quartile range (IQR): 25–38) years. One participant was female (0. 4%) and the majority 212 (84. 8%) of the participants were from urban areas. The majority of the participants 192/250 (76. 80%) were married. Out of 250 participants, only 51 (20. 40%) attended secondary education as shown in Table 1. The median work duration (years) of those with low education was 7 (IQR) 3–12 and that of those with high education was 3 (IQR) 2–5, p<0. 001. Overall seropositivity of Brucella spp. antibodies was found to be 48. 4% (121/250,95% Cl: 42–54). Seropositivity of B. abortus and B. melitensis was found to be 46. 0% (115/250, Cl: 39–52) and 23. 6% (59/250,95% Cl: 18–28), respectively while seropositivity of co-infection of B. abortus and B. melitensis was 21. 2% (53/250,95% Cl: 16–26) On univariate analysis, age (p = 0. 029), residing in rural areas (p = 0. 021), having primary education (p = 0. 001), being abattoir worker (p<0. 001) and work duration (p = 0. 002) were significantly associated with the presence of specific B. abortus antibodies. The median work duration of B. abortus seropositive participants was 7 (IQR4-15) years compared to 5 (IQR 3–10) of those who were seronegative (p = 0. 0016), Fig 1. By multivariable logistic regression analysis, having primary education (OR: 2. 64,95% CI: 1. 25–5. 55, p = 0. 011), being abattoir worker (OR: 2. 66,95% CI: 1. 49–4. 77, p = 0. 001) and having long work duration (OR: 1. 05,95% CI: 1. 015–1. 09, p = 0. 041) were found to predict B. abortus seropositivity (Table 2). By univariate analysis, residing in rural areas (p = 0. 037), being abattoir worker (p = 0. 023) and long work duration (p = 0. 014) were significantly associated with B. melitensis seropositivity. However, only long work duration (OR: 1. 05,95% CI: 1. 00–1. 10, p = 0. 024) was found to predict B. melitensis seropositivity on multivariable logistic regression analysis (Table 3). On univariate analysis residing in rural areas (p = 0. 030), being abattoir worker (p = 0. 012) and long work duration (p = 0. 004) were significantly associated presence antibodies for both species. By multivariable logistic regression analysis; being abattoir worker (OR: 2. 19,95% CI: 1. 06–4. 54, p = 0. 035) and having long work duration (OR: 1. 06,95% CI: 1. 01–1. 11, p = 0. 014) were found to predict presence of antibodies for both species (B. abortus and B. melitensis) (Table 4). When sub analysis was done among the two groups (abattoir workers and meat vendors) the following was observed. Among the variables tested in the abattoir workers group for B. abortus; increased in age (p = 0. 001), long work duration (p = 0. 004), being married (p = 0. 007) and having primary education (p<0. 001) were significantly associated with B. abortus seropositivity. By multivariable logistic regression analysis only having a primary education (OR: 3. 77,95% CI: 1. 45–9. 76, p = 0. 006) was found to predict B. abortus seropositivity. Regarding B. melitensis. only long work duration (p = 0. 013) was significantly associated with B. melitensis seropositivity. By multivariable logistic regression analysis none of the factors was found to predict B. melitensis seropositivity. Primary education (OR: 2. 93,95% CI: 1. 19–7. 23, p = 0. 019) independently predicted Brucella spp. seropositivity. None of the factors tested was found to be associated with Brucella spp. seropositivity among meat vendors. (S3 File) Overall seropositivity of Leptospira spp. antibodies was found to be 26/250 (10. 0%, 95% CI: 6–13). When categorized by occupation, there was no significant difference on seropositivity among abattoir workers and meat vendors (11/46 (7. 7%) vs. 14/104 (13. 5%), p = 0. 124). Among the five Leptospira serovars tested, the most prevalent was Leptospira kirschneri serovar Sokoine (7. 2%), followed by L. interrogans serovar Lora (2. 0%) and L. kirschneri serovar Grippotyphosa (1. 2%) (Fig 2). Other serovars tested were L. borgpetersenii serovar Kenya and L. interrogans serovar Hebdomadis in which none of the samples were found to be seropositive. Among the factors tested, only being married was significantly associated with seropositivity of Leptospira spp. (p = 0. 041). Despite high specificity of serological test used in the current study, B. abortus and B. melitensis antigens are not specific when it comes to antigen-antibody assays because these two spp are more than 95% in structural homology. It should be noted that this study was done in the specific groups therefore results cannot be generalized to the general population of Mwanza city and being a cross-sectional study the trend of the outcome by time could not be established. In addition, the high seropositivity of Brucella spp. might be due to cross-reactivity of Brucella antigens with varieties Enterobactericeae antibodies. Brucella species identification through serology is markedly affected by cross reactions[41] and this is associated with high false positive. B. abortus seropositivity among abattoir workers in Mwanza city is alarmingly high and is predicted having long work duration and having primary level of education. In addition, a significant proportion of this population is seropositive to Leptospira kirschneri serovar Sokoine. Being important zoonoses and neglected tropical diseases, there is a need to emphasize on biosafety measures during slaughtering, surveillance strategies, and treatment across the country particularly in high risk groups. Moreover, this calls for the need to adopt “One Health Approach” in controlling these diseases across the country. Further studies focusing on molecular detection of the pathogens to provide opportunities for understanding the infection patterns and the epidemiological implications of the pathogens to the high risk communities are highly warranted.
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Title: Seropositivity of Brucella spp. and Leptospira spp. antibodies among abattoir workers and meat vendors in the city of Mwanza, Tanzania: A call for one health approach control strategies Summary: Brucellosis and leptospirosis are among neglected diseases in many low-income countries affecting both animals and human populations. Despite being common, the information on their distribution are scarce. In a view of that, this study investigated the proportion of participants with positive antibody test specifically for the two diseases among slaughter house workers and meat sellers in Mwanza city. The study involved 250 participants aged between 25 and 38 years. Overall, 48. 4% of participants were Brucella spp. seropositive. Proportion of participants who were positive for B. abortus specific antibodies was higher than that of B. melitensis while 21. 2% of them found to have antibodies for both species studied. Furthermore, about 10% of the participants had Leptospira antibodies. Being abattoir worker, long work duration and having primary education were associated with the presence of Brucella antibodies while only being married was associated with the presence of Leptospira antibodies. The findings from this study emphasize the need for multisectoral approach in devising control strategies for these pathogens.
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Summarize: Picture this. It's 7.50pm, and you're walking out of the tunnel at the Abbey Stadium to the welcome of a 7,987-strong attendance. To your left is Radamel Falcao – a man who earns £280,000 per week, or £40,000 a day, while you earn pennies by comparison. Cambridge United were in that very situation a little over a week ago, when a Manchester United XI – total cost, £276million – drew 0-0 with their League Two counterparts – total cost, zilch. Radamel Falcao walks out for Manchester United in their FA Cup tie with Cambridge at the Abbey Stadium. As in so many matches this season, Falcao was ineffective as United were held 0-0 by the League Two side. Signed on: September 1. Wages per week: £280,000. Wages per day: £40,000. Goals in all competitions: 3. Pay per goal as of January 29: £2million. Yet forget that FA Cup hiccup because this week marks an anniversary, and sometimes that calls for celebration. Jorge Mendes can only have been tempted to use the good China on Thursday, when his client, Falcao, passed 150 days at Old Trafford. The milestone for the man with three Barclays Premier League goals marks the day his wages hit a cool £6m since moving to Manchester from Monaco. Tax aside. Bonuses not included. It was on August 31 when Falcao was photographed peeking out of the window of a private jet. No stop off in Madrid or London, despite apparent interest from Carlo Ancelotti and Jose Mourinho. The moment Falcao touched down in a private jet to sign for Manchester United on deadline day last summer. Louis van Gaal got him. A club desperate to become European heavyweights once more added the world-renowned striker named after Paulo Roberto Falcao – the Brazilian that starred for the greatest side not to win the 1982 World Cup. In a £6m season-long swoop plus wages they nabbed the natural-born killer from 12 yards or less, coached in the art of terrorising defenders by one himself: his father, Radamel Garcia. It was Falcao's father who arranged his first contract – £50 per week – before his debut on August 28, 1999, in the Colombian second division for Lanceros Boyaca at the age of 13 years, 199 days. Fast forward 15 years and it was Mendes, the most powerful agent in football, who orchestrated his latest contract – leaving behind a club that average less than 15,000 fans at Stade Louis II. Falcao poses with the Monaco No 9 shirt after joining the Ligue 1 side from Atletico Madrid in 2013. Falcao score 13 goals in 22 appearances for Monaco over two injury-affected seasons. How Falcao scored his last goal against Stoke City. Yet his move from Monaco to Manchester has not matched expectations. Not by a long shot. Not enough to warrant the Glazer family spending £2m per goal, nor enough to feel obligated to relinquish £43m to make his loan arrangement permanent. Falcao has not scored or provided an assist since New Year's Day against Stoke City. Between then and now, he's faced Yeovil Town (60 minutes), Queens Park Rangers (90) and Cambridge (90). Against QPR, to his credit, Falcao's positioning was second to none. Yet every chance – the point-blank header from five yards out, for example – was squandered. One miss in particular was ridiculed, mainly because it was captured from an unkind angle where you could not see Steven Caulker get his studs to it beforehand. Twitter reacted to the air shot appropriately: 'Even Fernando Torres...' You can guess the rest. Manchester United created more chances (16) against QPR than any other team this year. The closest after that being against Crystal Palace, Chelsea and Aston Villa (all 15). Had it not been for Rob Green, Falcao would have his fourth, perhaps fifth or even sixth goal in all competitions. The pesky QPR goalkeeper was seemingly hell-bent on not allowing him relief. Falcao fails to make contact with the ball as he passes up a sitter of a chance in United's match with QPR. The missed chance at Loftus Road has been one of a number of moments of disappointment this season. Falcao's heat map from United's 2-0 win at Queens Park Rangers a fortnight ago. And so to Cambridge, where Falcao managed 24 touches and one shot on target against a team loitering some 76 places below them in the Football League basement. The appropriately-named Richard Money, meanwhile, is rubbing his hands. His club are prepared for a £1.7m windfall in their return trip to Old Trafford on February 3, a smidgen more than their annual income of £1.6m. Bang for the buck, indeed. All this frustrates Mendes, who accordingly stuck his nose in. The not-so-average Mr 10 Percent took aim at his client's new manager, insisting Falcao would be better off under the guidance of Manchester United's previous leader. No, not David Moyes. 'I would have liked it if Falcao was with Sir Alex Ferguson,' he told La Sexta last week. 'He is a player who needs to play 90 minutes and I don't understand [why he isn't].' Falcao pictured with his agent Jorge Mendes, who has leapt to his defence in recent weeks. Minutes: 90. Goals: 0. Assists: 0. Shots: 3. Shots on target: 3. Total passes: 33. Accurate passes: 22 (66.7%) Duels: 17. Duel success: 58.8%. Offsides: 1. Minutes: 90. Goals: 0. Assists: 0. Shots: 3. Shots on target: 1. Total passes: 18. Accurate passes: 18 (100%) Duels: 9. Duel success: 33.3%. Offsides: 1. Be careful what you wish for, Jorge. Falcao's 90 minutes apiece against QPR and Cambridge saw six shots, four on target, 40 accurate passes, two offside calls, no goals and no assists. All this, shown above. This doesn't fit the reputation of a man voted for by Cristiano Ronaldo to win the 2013 Ballon d'Or. No petty, political, Eurovision voting by the Madridista that year, it seems. Is it a lack of service? An injury hangover? Louis van Gaal's formation? Plain, old rotten luck? Well, against QPR, Falcao (No 9) played a supporting role behind Angel di Maria (No 7), as the average positions below demonstrate. A conundrum in itself. Against Cambridge, he averaged a role further up the pitch, more central, slightly ahead of Di Maria. Next up, Leicester City, the club bottom of the Premier League, and minnows Cambridge – both at Old Trafford. Score against them, and what were we worried about? Fail, and uh oh. Falcao (No 9) among Manchester United's average positions against QPR (left) and Cambridge (right) When Falcao signed in the early hours of September 1, the headlines wrote themselves. Take, for example, The Daily Telegraph's 'Striking it rich', Daily Star's '£350,000 a week', or Daily Mail's 'Wow Falcao'. That buzz, however, has left. The hype, barely existent. Patience wearing thin. This was always a gamble. They hired a man that suffered anterior cruciate damage in January – an injury which stole the 2014 World Cup and eight months from him. It was the third severe knee injury of his career. Falcao, still to this day, is understood to ice the delicate area following every training session. A rumour strengthened when he uploaded a photo to Instagram, inadvertently showing what looked to be an ice pack strapped to his left knee. There are still concerns about Falcao's knee, which caused him to miss the World Cup last year. United must make a decision on whether or not to sign the Colombian striker on a permanent basis. Not so reassuring, either, is Falcao's diminishing records at Manchester United when compared to Monaco and Atletico Madrid. His shot conversion rate (15 per cent) is the 64th best in the Premier League. It is by far his lowest at every club he's been with since 2011, as the table below shows. His average shots per game (1.5) is down, a far cry from his record with Atletico in 2011/12 (3.2) and 2012/13 (2.9). Yet not all is lost. His shooting accuracy (70 per cent) is better than ever, and he now has as many assists (3) as he has managed in any season since 2011. Falcao greets Lionel Messi at the Ballon d'Or awards ceremony in Zurich back in 2012. Perhaps this is a storm in a teacup, then. Maybe his so-called 'adjustment period' is just longer than others. But Louis van Gaal is losing patience, no matter what he says. Dropping a man being paid £280,000 per week against Southampton – your Champions League qualification rivals – hardly screams faith. A goal against Leicester or Cambridge would be a start because, so far, there's no value for money to be had here. And if it persists, Manchester United would be better washing their hands of Falcao
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Summary: Radamel Falcao has scored just three goals since arriving at Old Trafford. The striker earns £280,000 per week on his season-long loan from Monaco. His agent, Jorge Mendes, said last week about Falcao: 'He is a player who needs to play 90 minutes and I don't understand [why he isn't]' Falcao played 90 minutes against QPR and Cambridge but couldn't score. The 28-year-old has struggled with injury throughout his career. Louis van Gaal must wash his hands of Falcao if he form does not improve.
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Summarize: This application is a continuation of application Ser. No. 08/490,306 filed Jun. 14, 1995. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is broadly concerned with improved sleeve assemblies useful for orthopaedic or prosthetic suspension purposes. More particularly, it is concerned with sleeve assemblies of this type which include anti-slip material applied to a portion of the inner surfaces thereof in order to minimize migration or slippage of the sleeve assemblies in use. 2. Description of the Prior Art Tubular orthopaedic supports of various types have long been used by individuals to support a weak or injured limb during rehabilitation or as a general protective measure. To give but one example, tubular knee supports are commonly used by athletes and include an elongated, sleeve-like tubular body adapted to be placed over the knee, lower thigh and upper calf regions of the leg. Such devices are typically fabricated of closed cell neoprene rubber material which may be lined with a soft terry or nylon fabric. Prosthesis suspension devices used by amputees to support a limb prosthesis are also well known. Generally speaking, these devices include a tubular sleeve member adapted to fit around the limb at the region of the amputation, for the purpose of engaging and at least partially holding the prosthesis in position adjacent the limb stump. In the case of leg prosthesis supports, a belt is typically provided as a part of the overall device which extends around the waist of the wearer to provide additional support and strength. Prosthesis suspensions units of this character are also commonly made of faced neoprene rubber material. A very significant problem with prior orthopaedic sleeves or prosthesis suspension devices is the tendency of these units to migrate or shift, particularly during walking other exercise. In the case of orthopaedic supports, these can fall or migrate a significant distance, particularly during athletic activity. By the same token, prosthesis suspension devices can slip or allow the prosthesis to move and shift to an unacceptable degree. In response to these problems, it has been known to form the sleeve assemblies using neoprene rubber material having only the outer surface thereof faced with fabric, leaving an inner skin-engaging neoprene rubber surface. This has been done in an attempt to increase the resistance of these devices to migration or slippage. However, this expedient causes problems in its own right. First of all, unfaced neoprene rubber tends to erode and wear out quickly. Moreover, neoprene rubber causes skin irritation in a significant number of patients. There is accordingly a real and unsatisfied need for improved sleeve assemblies useful in the context of orthopaedic supports or prosthesis suspension devices which largely eliminates the problems associated with sleeve migration, slippage and/or inadequate holding power. SUMMARY OF THE INVENTION The present invention overcomes the problems outlined above, and provides improved sleeve assemblies which are especially designed to give significantly enhanced resistance to slippage and migration. Broadly speaking, the sleeve assemblies of the invention are adapted to be worn on a limb and are in the form of tubular bodies formed of resilient, stretchable material. An anti-slip material is applied to a portion of the inner surfaces of the bodies and has a coefficient of static friction which is greater than that of the corresponding inner surfaces. In preferred forms, the anti-slip material is applied as an essentially continuous band adjacent at least one end of the sleeve assembly, or as a plurality of thin, spaced apart bands. The anti-slip material is advantageously a synthetic resin which can be bonded adequately to the inner surfaces of the sleeve assemblies. Most preferably, a silicon-based synthetic resin adhesive is used for this purpose. BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is an essentially schematic view illustrating a tubular knee support in accordance with the invention, shown applied to the knee of a user; FIG. 2 is an isometric view depicting the inner surface of a blank used to form the knee support illustrated in FIG. 1; FIG. 3 is a view illustrating the initial step of the manufacture of a knee support from the blank of FIG. 2, wherein the side margins of the blank are placed in opposition prior to taping of the resultant seam; FIG. 4 is an elevational view of the completed knee support; FIG. 5 is a fragmentary view of a blank in accordance with the invention, used in the fabrication of a leg prosthesis suspension device; FIG. 6 is a view similar to that of FIG. 5, but depicting the completed suspension device; FIG. 7 is a view similar to that of FIG. 2, but illustrating the use of a plurality of thin spaced apart bands of anti-slip material; and FIG. 8 is an enlarged sectional view taken along line 8--8 of FIG. 2 and illustrating the construction of the knee support. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Turning now to the drawings, and particularly FIGS. 2-4, a tubular knee support 10 (FIG. 2) is illustrated. The support 10 is in the form of an elongated, tubular body 12 presenting upper and lower opposed, open ends 14, 16. The body 12 includes an uppermost, diverging, somewhat frustoconical section 18 adapted to receive and conform with the lower thigh; a lower, arcuate in side elevation section 20 for receiving and conforming with the upper calf; and a central knee section 22. In addition, the support 10 includes a pair of anti-slip bands 24, 26 applied to the inner surface thereof adjacent each open end 14, 16. The tubular body 10 is preferably formed of an inner layer 28 (see FIG. 8) of closed cell neoprene rubber synthetic resin material, which has sufficient softness, resiliency and strength for this purpose. As shown, the layer 28 is essentially continuous throughout its thickness and is substantially air-impervious. Normally, inner and outer surfaces of the neoprene rubber layer 28 are faced with fabric. In practice, use has been made of G-231-N and R-1400-N closed cell neoprene rubber sheeting commercialized by the Rubatex Corporation of Bedford, Va. The neoprene rubber is normally faced with stretched nylon 30 on the surface thereof which forms the exterior of the support 10; the opposed surface of layer 28 presenting the interior surface of the support 10 can be faced with stretch nylon or stretch Jersey fabric 32 (e.g., 900 standard light stretch Jersey commercialized by Rubatex Corporation). Rubatex brochures entitled "Closed Cell Rubber and Plastic Sheets", and "Neoprene/Stretch Jersey Laminates" which describe and characterize the neoprene rubber material and fabric facings are incorporated by reference herein. The anti-slip bands 24, 26 are, in the present embodiment, continuous bands having a width of about 3/4 to 11/2". Advantageously, these bands are formed of IPOCON adhesive commercialized by IPOS N.A., Inc. of Niagara Falls, N.Y. This adhesive is a silicon-based synthetic resin material described as polydimethylsiloxane interlaced with fillers and acetoxysilan. This adhesive has been found to properly adhere to the inner surface of the support 10 and will not delaminate or erode during normal use. The bands 24, 26 can be applied by brush application of the viscous IPOCON adhesive or by any other acceptable technique. Normally, after application a cure time of at least 24 hours, and most preferably about 72 hours, is provided to insure adequate drying of the adhesive to form the anti-slip bands 24, 26. It is also important that the anti-slip material of the invention be applied as a relatively thin layer, so as to not unduly limit the stretchability of the faced neoprene rubber material. Adequate stretchability is required, especially in the case of prosthesis suspension devices, in order to prevent a blood flow-restricting "tourniquet effect" which is especially troublesome for amputees because of restricted blood circulation at the area of the amputation. In practice, the anti-slip material should have a thickness of from about 0.0050-0.1 inches, and most preferably from about 0.01-0.05 inches. In the case of the bands 24, 26, a thickness of about 0.02 inches has been found to give very satisfactory results. FIG. 2 illustrates a unitary one-piece blank 34 used in the fabrication of knee support 10. As illustrated, the bands 24, 26 are applied to the inner Jersey fabric 32 adjacent the opposed ends of the blank. In order to complete the construction, the transverse side margins of the blank 34 are brought together as illustrated in FIG. 3 to create an axially extending seam. This seam is completed by application of a length of seam tape. In particular, an adhesive material such as Rubatex R-27706 natural color adhesive is applied to the outer side margins of the blank 34. When these margins are brought into essentially abutting relationship as shown in FIG. 3, the seam tape is applied along the length of the seam in order to create the finished tubular support 10. The preferred details of the method of construction in seaming of the support 10 are set forth in U.S. Pat. No. 5,382,223 entitled "Contoured Orthopaedic Support Having Reduced Skin Irritation Properties"; such patent is incorporated by reference herein. The use of support 10 is illustrated in FIG. 1, where it will be seen that the anatomically contoured support, together with anti-slip bands 24, 26, serve to maintain the position of the support even during strenuous exercise. FIG. 7 illustrates a blank 34a which is identical with blank 34 except for the provision of differently configured anti-slip marginal bands 24a, 26a. In particular, each of these bands is formed of a plurality (here three) of thin strips of adhesive material with spacing between respective strips. Such a construction gives adequate anti-slip properties, but lessens the material requirements, as compared with the embodiment of FIGS. 1-4. All other construction techniques and material details are as described with reference to the first embodiment. FIGS. 5-6 illustrate the construction of a leg prosthesis suspension device 36. Referring first to FIG. 5, it will be observed that a blank 38 is provided having a lower section 40 adapted to be interconnected to form a lower sleeve assembly 42. In addition, the upper portion of the blank 38 includes an elongated belt section 44 which is adapted to encircle the waist of a user. The outer end of belt 44 is provided with Velcro 46 which mates with an opposing Velcro section (not shown) on tab 48. The blank 38 is formed of the same faced neoprene rubber material described with reference to support 10. Similarly, the lower margin of blank section 40 is provided with an anti-slip band 50 which is made from the described IPOCON adhesive. Finally, the sleeve portion 42 of the device 36 is formed in the same manner as support 10, i.e., an appropriate adhesive is applied to the side margins of the lower section 40 of the blank, and these are brought into abutment; a length of seam tape 52 is then applied along the exterior surface of the blank to complete the sleeve section 42. In the use of device 36, the sleeve section 42 receives the stump of the user, and belt 44 is tightened around the user's waist. Thereafter, a leg prosthesis (not shown) is positioned adjacent the stump, with the anti-slip band 50 engaging the prosthesis. In this fashion, the prothesis is prevented from undesired shifting or slippage during use.
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Summary: Improved orthopaedic supports (10) and prosthesis suspension devices (36) are provided in the form of tubular bodies (12, 42) formed of resilient, stretchable material and configured to fit about a human limb. The inner surfaces (32) of the tubular bodies (12, 42) have anti-slip material (24, 26, 50) applied thereto. The anti-slip material (24, 26, 50) is preferably a synthetic resin material having a coefficient of static friction greater than that of the inner surfaces (32).
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Summarize: I ricercatori della Riserva nazionale “Big Cypress” sita nella Florida meridionale hanno catturato e ucciso una gigantesca femmina di pitone birmano (Python bivittatus) lunga più di 5 metri, pesante 63,5 chilogrammi e con 73 uova in via di sviluppo nel corpo. Si tratta del più grande esemplare rinvenuto in questa area protetta di 3mila chilometri quadrati, che si trova a circa 70 chilometri da Miami e confina con il più famoso Parco nazionale delle Everglades. L'annuncio della cattura e dell'uccisione dell'enorme rettile è stato dato sulla pagina ufficiale del parco, corredato da una foto con i sorridenti protagonisti dell'impresa. Sacrificio. Ma perché quattro ricercatori dovrebbero posare soddisfatti con in braccio la carcassa di un animale così maestoso? Il motivo risiede nel fatto che i pitoni birmani, originari del Sud-Est asiatico, sono annoverati tra le specie più invasive in assoluto, e sono capaci di devastare – letteralmente – un intero ecosistema. Per fare un esempio pratico, nel parco delle Everglades, come indicato dagli scienziati dello United States Geological Survey (USGS), i pitoni birmani sono stati responsabili della scomparsa del 99,3 percento dei procioni; dell'87,5 percento delle linci rosse (Lynx rufus) – i cosiddetti “bobcat” – e del 98,9 percento degli opossum. I conigli dalla coda di cotone o silvilaghi, le volpi e i conigli di palude sono invece stati completamente annientati. A rendere ancor più agghiacciante l'impatto di questi serpenti, il fatto che tale ecatombe è stata registrata in soli 5 anni, tra il 1997 e il 2012.
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Summary: La gigantesca femmina di pitone birmano, lunga oltre 5 metri e pesante 63,5 chilogrammi, è stata catturata e uccisa nella Riserva nazionale "Big Cypress" della Florida. Gli scienziati l'hanno eliminata poiché la sua specie (aliena negli USA) è una delle più invasive in assoluto: i pitoni birmani hanno fatto sparire il 99,3% dei procioni e l'87,5% delle linci rosse nel parco delle Everglades.
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Write a title and summarize: Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry. The seven botulinum neurotoxin serotypes (BoNT/A–G) produced by Clostridium botulinum are the causative agents of the neuroparalytic syndrome of botulism and pose a serious threat for bioterrorism [1]. Conversely, BoNT/A is a highly effective therapy for treating neurological disorders [2]. The naturally occurring BoNTs are released together with up to four non-toxic neurotoxin-associated proteins (NAPs) (also called associated non-toxic proteins, ANTPs) in the form of progenitor toxin complexes (PTCs) with different molecular compositions [3]. Such PTCs are highly potent food poisons, e. g., the PTC of BoNT/A displays an oral LD50 of ∼35 µg/kg body weight [4]. While BoNT is sensitive to denaturation by the acidic environment and digestive proteases present in the gastrointestinal (GI) tract [5], the PTCs of different serotypes exhibit ∼360–16,000-fold greater oral toxicity than free BoNT [4], [6], [7], [8]. The NAPs are encoded together with the bont gene in one of two different gene clusters, the HA cluster or the orfX cluster [9]. Both clusters encode the non-toxic non-hemagglutinin (NTNHA) protein, which adopts a BoNT-like structure despite its lack of neurotoxicity [5]. The HA gene cluster also encodes three hemagglutinins (HA70, HA17, and HA33; also called HA3, HA2, and HA1, respectively), which together with BoNT and NTNHA constitute the large PTC (L-PTC) [10]. The structure and function of the corresponding orfX proteins are largely unknown [11]. Structural information of HAs is available for serotypes C and D, such as the crystal structures of HA33 of serotype C (HA33-C) [12], [13], a complex composed of HA17 and HA33 of serotype D [14], and HA70 of serotype C (HA70-C) [15], [16]. However, BoNT/C and D rarely cause human botulism but are known to cause the syndrome in cattle, poultry, and wild birds. For BoNT/A, the major cause of human botulism, only the structure of HA33 (HA33-A), which displays an amino-acid identity of ∼38% to HA33-C and D, has been solved [17]. We have recently determined the crystal structure of the BoNT/A–NTNHA complex [5]. However, it remains largely unclear how the HAs assemble with one another and how they interact with BoNT and NTNHA. Various structural models have been proposed for the L-PTC. One recent paper suggested a complex composed of BoNT∶NTNHA∶HA70∶HA17∶HA33 in a 1∶1∶2∶2∶3 ratio for L-PTC/A [18], whereas earlier studies suggested a stoichiometry of 1∶1∶3–5∶5–6∶8–9 or 1∶1∶3∶3∶4 for L-PTC/A, or 1∶1∶2∶4∶4 for L-PTC/D [19], [20], [21]. In comparison, electron microscopy (EM) studies on L-PTC/A, B and D supported a stoichiometry of 1∶1∶3∶3∶6 [14], [22]. The functional roles of NAPs are also not well defined. We have recently shown that NTNHA shields BoNT against low-pH denaturation and proteolytic attack in the GI tract by forming the minimally functional PTC (M-PTC), and releases it during entry into the general circulation [5], [23]. However, it is not clear whether HAs further protect the toxin. At the same time, the L-PTC may contribute to BoNT internalization into the host bloodstream through interactions with intestinal cell surface glycans [24], [25], [26]. The HAs of BoNT/A and B could disrupt the human epithelial intercellular junction through species-specific interaction with E-cadherin, presumably facilitating BoNT transport via the paracellular route [27], [28], [29]. Defining the L-PTC structure would permit a more complete understanding of the complex' s role in toxin shielding and delivery, and would help to describe the molecular mechanism underlying these important actions. Here, we report the structure of a ∼760 kDa L-PTC/A using a combination of X-ray crystallography, single-particle EM and three-dimensional reconstruction (3D-EM). We found that L-PTC/A consists of two structurally and functionally independent sub-complexes, the M-PTC and the HA complex. The HA complex is composed of HA70, HA17, and HA33 in a 3∶3∶6 stoichiometry and adopts an extended three-blade architecture, whereas the M-PTC is situated on top of the HA complex platform. BoNT/A absorption is mainly mediated by nine glycan-binding sites on the HA complex that together form multivalent interactions with host carbohydrate receptors on intestinal epithelial cells. HA complex-mediated toxin absorption can be blocked in vitro by carbohydrate receptor mimics. The monosaccharide IPTG also inhibits oral BoNT/A intoxication in mice, providing the first approach for a possible preventive treatment prior to deliberate BoNT poisoning. The high toxicity of BoNT/A prevents imaging of the fully active toxin by cryo-EM. So, we began our analysis with negative-staining EM and determined the 3D molecular envelope of L-PTC/A at ∼31 Å resolution (Fig. 1A–C and Fig. S1 in Text S1). The M-PTC moiety was clearly identified in the EM density map based on its crystal structure [5]. Beneath the M-PTC, the HAs adopt a symmetric three-blade architecture that is ∼100 Å tall and ∼260 Å wide between the tips of neighboring blades. Surprisingly, the M-PTC and the HA complex are relatively independent of each other and associate only through two small interfaces (Fig. 1D and Fig. S2 in Text S1). This arrangement contrasts with the extensive interactions between BoNT/A and NTNHA that are required for mutual protection in the GI tract [5], suggesting that the HA complex might play a minimal role in BoNT protection. We did not observe the LL-PTC under EM, which has been proposed to be a dimer of the L-PTC with a molecular weight of ∼900 kDa that might only occur at high concentrations [20], [30]. To determine the molecular architecture of the HA complex, we produced highly homogeneous recombinant proteins of HA70, HA33, HA70–HA17, HA17–HA33, HA70D3 (residues Pro378–Asn626) –HA17–HA33 (termed the mini-HA complex), and the complete HA70–HA17–HA33 (the HA complex). HA17 formed inclusion bodies and heterogeneous soluble aggregates when expressed and purified alone. This is probably due to the large hydrophobic patches on its surface, which are protected by its binding partners within the HA complex. We then systematically analyzed the solution association of these individual proteins and their complexes using analytical ultracentrifugation (AUC), which was performed at pH 2. 3 and 7. 6 to mimic the physiological conditions in the GI tract (Table S1 in Text S1). Our data indicate that the HA complex assembles at both pHs as a hetero-dodecamer consisting of HA70, HA17, and HA33 in a 3∶3∶6 ratio to yield a ∼470 kDa complex. Specifically, homo-trimeric HA70 forms the core of the complex with each C-terminal HA70D3 domain binding to one HA17, which in turn simultaneously coordinates two HA33s. We next separated the HA complex into two major components: the central hub composed of homo-trimeric HA70 and the blade composed of HA70D3–HA17–HA33. Their crystal structures were determined at 2. 9 Å and 3. 7 Å, respectively (Fig. 2C–D, Table S2 in Text S1, Fig. S3–S4 in Text S1). We also obtained a high-resolution structure of the blade by combining the structures of HA70D3–HA17 and HA17–HA33, which were determined at 2. 4 Å and 2. 1 Å, respectively (Fig. 2A–B and Fig. S5–S6 in Text S1). Each HA adopts an almost identical conformation in the independently solved structures, despite differences in crystal packing, suggesting that they represent physiologically relevant conformations. HA70 consists of three domains (D1–3) (Fig. S3 in Text S1). The D1 and D2 domains, which adopt similar structures, mediate the trimerization of HA70 with each protomer contributing ∼3,100 Å2 of solvent-accessible area for interactions. The D3 domain, sitting at the tip of the trimer, is composed of two similar jelly-roll-like β-sandwich structures. The linker between D1 and D2 (residues Thr190–Ser205) is degraded and not visible in the crystal structure, which is reminiscent of the post-translational nicking of HA70 into ∼25 and ∼45 kDa fragments that occurs physiologically [20]. HA17 has a compact β-trefoil fold and connects HA70 and HA33. Based on the crystal structure of the HA70D3–HA17 complex, the interactions between HA70 and HA17 bury a solvent-accessible area of ∼795 Å2 (per molecule) (Fig. 3A and Fig. S5 in Text S1). The structure of HA70D3 is almost identical to its equivalent domain in the full-length HA70 with a root-mean-square deviation (rmsd) of ∼0. 93 Å over 232 Cα atoms. The major HA70–HA17 interactions are composed of 13 pairs of hydrogen bonds and salt bridges. In addition, HA70-Phe547 is buried in a hydrophobic region in HA17 composed of Ile18, Ile92, Ala93, Thr96, and Met140 (Fig. 3A and Fig. S5 in Text S1). HA17 simultaneously binds to two HA33 molecules that form a dumbbell-like shape composed of two β-trefoil domains linked by an α-helix. The two pairs of HA17–HA33 interfaces bury a solvent-accessible area of ∼666 Å2 and ∼410 Å2 (per molecule), respectively (Fig. 3B and Fig. S6 in Text S1). The two HA33-binding interfaces on HA17 are adjacent but non-overlapping. HA17 contributes seven and four pairs of hydrogen bonds and salt bridges to bind the two HA33 molecules, respectively. Complementing these hydrophilic interactions, the two HA33s contain a hydrophobic surface (Trp75/Leu116/Leu129) that interacts with two neighboring hydrophobic patches on the HA17 surface (Phe75/Pro125/L127 and Leu108/Pro130/Phe132) (Fig. 3B). The two molecules of HA33 in each blade of the HA complex are almost identical (rmsd of ∼0. 35 Å over 286 Cα atoms) and bury a solvent-accessible area of ∼961 Å2 (per molecule) between them (Fig. 3C). All the interacting residues are in the N-terminal domain of HA33, whereas the interface consists of hydrophilic interactions on the periphery and a hydrophobic core in the center (Fig. S6C in Text S1). Due to the two-fold symmetry between the two molecules, intra-HA33 interactions are generally symmetric. Finally, we assembled the subunit crystal structures to create a complete structure of the HA complex (Fig. 2E). The 12-subunit HA complex is stabilized by numerous protein–protein interactions that include interactions among the HA70s of the central trimer, between HA70 and HA17, between HA17 and the two HA33 molecules, and between the two HA33s in each blade. The assembled HA complex structure could be unambiguously docked into the 3D-EM density of the native L-PTC/A (correlation coefficient, CC∼87. 7%) (Fig. 1), whereas a small difference was observed in the C-terminal domain of HA33 due to its structural flexibility. We also performed an independent 3D-EM reconstruction of our recombinant, in vitro-reconstituted HA complex at ∼31 Å resolution (CC∼93. 1%) (Fig. 2E), and found it to be almost identical to the HA complex present in the L-PTC. The crystal structure of the M-PTC was unambiguously docked into the 3D-EM density of the native L-PTC (CC∼87. 3%), which is situated on top of the HA complex, yielding a ∼760 kDa 14-subunit complex (Fig. 1 and Fig. S2 in Text S1). BoNT/A interacts with the HAs only through its receptor-binding domain (HC domain). The interface is likely composed of Gly399 and Ile400 in HA70 and Val1128, Gly1129, Glu1210, Pro1212, and Asp1213 in HC (pairwise Cα–Cα distance within 15 Å) (Fig. 1D and Fig. S7A in Text S1). Gly399 and Ile400 of HA70 are located in a loop that has weak electron density in the crystal structures, suggesting high flexibility. Moreover, the potentially interacting residues in HC are located in two flexible loops and not conserved among various BoNT serotypes (Fig. S7B in Text S1). Thus, the BoNT/A–HA70 interface in the L-PTC may be formed by induced fit. The major interface between the M-PTC and the HAs is mediated by NTNHA. The potential interface residues in NTNHA, which are within 12 Å Cα–Cα distance of the HAs, are located in loop Gly308–Gly313 and the residues flanking loop Gly116–Ala148 (nLoop) [5]. The corresponding interface residues in the HA complex are located around the center of the HA70 trimer (Fig. 1D). The nLoop displays no visible electron density in the structure of the M-PTC and is spontaneously nicked in the free NTNHA or the M-PTC [5], [31], [32], [33], [34]. However, the nLoop remains intact in the L-PTC, suggesting it may be buried by the HA complex [30], [31], [35]. We found that the synthetic nLoop peptide binds to HA70 with high affinity (Kd∼340 nM) at a stoichiometry of one nLoop to one HA70 trimer (Table S3 in Text S1). This finding unambiguously established the orientation of the pseudo 2-fold symmetric M-PTC on top of the HA complex. The nLoop of NTNHA binds strongly to HA70 at pH 7. 6, which is in contrast to the M-PTC that dissembles and releases BoNT/A at neutral or basic pH [5], [30]. This suggests that BoNT may be the only component released from the L-PTC in response to the pH change encountered upon entering the circulation [30]. The HA complex and the M-PTC are stable at low pH (e. g., pH 2. 3) and are resistant to digestive proteases, as shown by in vitro cleavage by trypsin and pepsin (Fig. S8 in Text S1) [5]. The loose association between these two complexes suggests that they may have distinct functions during oral intoxication. The penetration of BoNT through an epithelial cell barrier to reach the general circulation is the first essential step of oral BoNT intoxication, which prompted us to investigate the role of HAs in BoNT/A absorption from the GI tract. For this study, we used the well-characterized intestinal epithelial cell line Caco-2. Although derived from a human colon adenocarcinoma, Caco-2 cells differentiate to form a polarized epithelial cell monolayer that provides a physical and biochemical barrier to the passage of ions and small molecules, resembling the uptake and barrier properties of the small intestinal epithelial layer [36], [37], [38], [39]. Caco-2 cells have been extensively used to investigate their permeability upon infection, e. g. by rotavirus [40] or enteropathogenic E. coli [41], and transcytosis upon intoxication with cholera toxin [42] or BoNT [43], [44], [45]. Furthermore, it was demonstrated that the transepithelial electrical resistance (TER) of Caco-2 cell monolayers was reduced by the L-PTC of BoNT/A and B. Although the mechanism by which this may occur is unclear, BoNT absorption has been proposed to occur via the paracellular route [27], [28], [29]. We found that treatment of Caco-2 cells with the in vitro-reconstituted HA complex markedly reduced the TER. This effect was more marked when the HA complex was applied to the cell monolayer from the basolateral side than from the apical side, which needed ∼17 nM and ∼58 nM to achieve a 90% and 70% decrease in TER after 12 hours, respectively (Fig. S9A–B in Text S1). Remarkably, the potency of the isolated HA complex was similar to that of the intact L-PTC (Fig. 4A–B). In contrast, there was no effect on Caco-2 TER by BoNT/A, NTNHA, the M-PTC, or by the subunits of the HA complex, including HA70, HA33, the HA17–HA33 complex, and the mini-HA complex (Fig. 4C–D). Taken together, these data suggest that the fully assembled HA complex is the functional unit of the L-PTC that facilitates intestinal absorption of BoNT, and acts by compromising the integrity of the epithelial cell layer. Many human receptors for microbial pathogens or toxins are glycoconjugates. The L-PTC is known to initiate toxin absorption by binding to intestinal cell surface glycans [24], [25], [26]. We therefore performed a comprehensive thermodynamic analysis to characterize the interactions between HAs and several common monosaccharides and oligosaccharides (Fig. S10 and Table S3 in Text S1). We found that HA33 bound to lactose (Lac), N-acetyllactosamine (LacNAc), and galactose (Gal) with dissociation constants (Kd) of ∼1. 0 mM, ∼1. 0 mM, and ∼1. 8 mM, respectively, and that it also bound to isopropyl β-D-1-thiogalactopyranoside (IPTG) [46], a non-metabolizable galactose analog, with a Kd of ∼0. 8 mM. HA70 bound to α2,3- and α2,6-sialyllactose (SiaLac), both with Kd of ∼0. 5 mM, and displayed a lower affinity for N-acetylneuraminic acid (Neu5Ac) (Kd∼7. 8 mM). There was no overlap between the carbohydrate selectivity of HA70 and HA33. To determine the physiological relevance of these HA–glycan interactions during toxin absorption, we examined their ability to interfere with the HA complex-mediated disruption of Caco-2 TER. Lac, Gal, and IPTG markedly inhibited the TER reduction induced by the HA complex, and showed higher potencies when applied to the apical than to the basolateral compartment (Fig. 5A–B and Fig. S11A–D in Text S1). In contrast, α2,3- and α2,6-SiaLac, and to a lesser extent Neu5Ac, inhibited the decrease in TER only when applied apically, albeit more weakly than Lac (Fig. 5A–B and Fig. S11E–F in Text S1). We then examined the transport of the HA complex across the Caco-2 monolayer using a fluorescence-labeled HA complex (HA*) (Fig. 5C). Lac and IPTG efficiently inhibited the transport of HA* when it was applied to the apical or basolateral chamber. Blocking the transport of HA* via α2,3- and α2,6-SiaLac was more potent toward the basolateral compartment than toward the apical side. Neu5Ac at 50 mM did not inhibit transport of HA* from either side of the Caco-2 cell monolayer. These data are consistent with the ability of these carbohydrates to inhibit TER reduction induced by the HA complex. Collectively, these results suggest that the binding of HAs to Neu5Ac- and Gal-containing glycans on epithelial cells is essential for the transport of BoNT across the intestinal wall. Moreover, the carbohydrate receptors may play a more important role in the initial L-PTC absorption in the intestinal lumen, whereas other host receptors (e. g., E-cadherin) are involved once it gains access to the basolateral side. To fully understand the binding specificity, we determined the crystal structures of HA70 in a complex with α2,3- or α2,6-SiaLac (Fig. 6 and Table S4 in Text S1). We found that α2,3- and α2,6-SiaLac bound to the same region in the D3 domain of HA70, where the terminal Neu5Ac in both glycans mediates the majority of the HA70–glycan interactions through six pairs of hydrogen bonds (Fig. 6A and Fig. S12 in Text S1). Mutating the Neu5Ac-binding residues (e. g. T527P, R528A, or T527P/R528A) completely abolished the binding (Table S3 in Text S1). The Neu5Ac-binding mode in HA70-A is also conserved in HA70-C (Fig. S13 in Text S1) [15], [16], suggesting HA70 is unlikely to be a major determinant of the host tropism of various BoNT serotypes. In contrast to the well-defined conformation of Neu5Ac, the Gal–Glc moiety seems to have a larger structural flexibility and is not essential to HA70–glycan recognition. Specifically, α2,3-SiaLac adopts a linear conformation, which is likely stabilized by a Glc-mediated crystal contact with its symmetry mate. However, α2,6-SiaLac binding to the same site adopts a folded conformation in which there is no crystal contact and Glc has no visible electron density (Fig. S12B in Text S1). Furthermore, these conformations are also different than that observed in the structures of α2,3- and α2,6-SiaLac when they bound to HA70-C, despite the conserved Neu5Ac-binding mode [16]. The different glycan conformations and the weak electron densities for Gal–Glc observed here are probably due to the intrinsic flexibility of SiaLac in solution [47]. The ability of HA70 to bind SiaLac with different glycosidic linkages contrasts with the binding profile of influenza virus HA. Neu5Ac binds to a deep pocket in influenza HA, which restricts the composition and topology of glycans that can bind to influenza HA [48], [49], [50]. In contrast, the Neu5Ac-binding site in HA70 is on a flat surface, allowing more freedom for additional glycan binding beyond the terminal Neu5Ac. We also determined the crystal structures of the HA17–HA33 complex bound with Gal, Lac, or LacNAc (Table S4 in Text S1). All three bind to an identical site in HA33, where the HA33–glycan interactions are mediated only by the Gal moiety through extensive hydrogen bonding and a crucial stacking interaction between Phe278 and the hexose ring of Gal (Fig. 6B). The HA33–Gal interaction is well-defined and identical for the two HA33 molecules in one asymmetric unit (AU). The Glc or GlcNAc moiety does not directly interact with HA33. One Glc/GlcNAc in the AU is involved in a crystal packing and shows clear electron density, while the density for the other copy is weakly defined; the latter is likely caused by the weak HA33–glycan binding affinity and intrinsic structural flexibility of HA33 that will be discussed later (Fig. S12D–F in Text S1). To further confirm the structural findings, we mutated the Gal-binding residues in HA33 (e. g., D263A or F278A) and found that these mutations almost completely abrogated the Lac binding (Table S3 in Text S1). Gal binds at an equivalent site in HA33 of L-PTC/C (HA33-C) (Fig. S14 in Text S1) but with ∼15-fold lower binding affinity than with HA33-A [13], which is likely caused by the replacement of Phe278 in HA33-A with Asp271 in HA33-C. In addition, HA33-C binds Neu5Ac in an adjacent binding site [51]. However, HA33-A does not bind Neu5Ac-containing sugars because the key Neu5Ac-binding residues in HA33-C, Trp176 and Arg183, are replaced in HA33-A with Tyr180 and Asn187, respectively (Table S3 in Text S1). These differences between HA33-A and HA33-C indicate that the known host susceptibility to different BoNT serotypes may be determined in part by the interaction between HA33 and host glycan receptors. To further analyze the functional role of BoNT' s glycan receptors, we “knocked-down” specific glycan binding to the HA complex using structure-based mutagenesis. The HA33-DAFA complex (composed of the wild-type (WT) -HA70, WT-HA17, and HA33-D263A/F278A) did not bind to Gal, whereas the HA70-TPRA complex (composed of the HA70-T527P/R528A, WT-HA17, and WT-HA33) failed to bind to Neu5Ac (Table S3 in Text S1). We found that the HA33-DAFA complex did not reduce TER when applied from either side of the Caco-2 cell monolayer. Furthermore, the loss of the Gal-binding site prevented the transport of HA33-DAFA through the Caco-2 monolayer (Fig. 5C), indicating the crucial role of the carbohydrate interaction during transcytosis. The HA70-TPRA complex maintained activity only when applied from the basolateral side, which was inhibited by Lac (Fig. 6D–E). These data suggest that there are at least two steps at which HA–glycan interactions play an important role in toxin absorption. Both Neu5Ac- and Gal-containing glycans are important for the initial L-PTC absorption in the intestinal lumen, but Gal-containing receptors on the basolateral surface of the epithelial cells may also participate, presumably in facilitating transport via the paracellular route [27], [28], [29]. To determine whether the glycans could interfere with BoNT absorption in vivo, we examined the effect of the monosaccharides Neu5Ac, Gal, and IPTG on the oral toxicity of L-PTC/A in mice [4]. Concomitant oral administration of L-PTC/A and IPTG at 500 mM significantly extended the median survival time (MST) of animals to ∼91 hours compared with ∼39 hours for the control group. Furthermore, IPTG was effective when it was administered one hour prior to treatment with L-PTC/A with an increase of MST to ∼62 hours. Some improvement in survival was also evident with IPTG treatment one hour after intoxication with L-PTC/A, with an increase of MST to ∼55 hours (Fig. 6F). Since IPTG does not affect the neurotoxicity of BoNT/A based on the mice phrenic nerve hemidiaphragm assay, this finding suggests that receptor mimics could block BoNT/A intestinal absorption at an early point of oral intoxication. Gal and Neu5Ac (up to ∼500 mM) did not confer significant protection, most likely due to their low binding affinity and/or metabolism (Fig. S15 in Text S1). Here, we report the complete structure of a 14-subunit ∼760 kDa L-PTC/A, which is achieved by building novel crystal structures of each subunit into 3D-EM reconstruction. To our knowledge, this is the largest bacterial toxin complex known to date. The L-PTC/A adopts a unique bimodular architecture, whereas BoNT/A and NTNHA form a compact M-PTC and three HA proteins adopt an extended three-arm shape. Our results conclude the same stoichiometry and a similar overall architecture as suggested by recent EM studies of L-PTC/A, B, and D [14], [22]. Furthermore, our complementary crystallographic, EM, and biochemical studies have revealed for the first time that both BoNT/A and NTNHA are involved in interactions with the HA complex, and that the two modules associate through two small interfaces, in contrast to numerous protein–protein interactions within each module. Aside from a small interface involving the BoNT/A receptor-binding domain, the majority of the interactions between the M-PTC and the HAs are mediated by the NTNHA nLoop. In spite of the overall structural similarity between BoNT/A and NTNHA, the nLoop is a unique feature of NTNHA, which is fully exposed on the M-PTC surface [5]. The nLoop is conserved in the NTNHAs that shield BoNT/A1, B, C, D, and G, and assemble with HAs into the L-PTC. However, the nLoop is missing in NTNHAs that assemble with BoNT/A2, E, and F, which do not have accompanying HA proteins and only form the HA-negative M-PTC [11], [52], [53]. We have found that one molecule of the synthetic nLoop peptide binds to the trimeric HA70 with a high affinity, clearly suggesting that the nLoop is bridging the M-PTC and the HA complex. This is consistent with previous reports that the nLoop is intact in the context of the L-PTC but spontaneously nicked in the free NTNHA or the M-PTC [5], [30], [31], [32], [33], [34]. Structural and sequence analyses suggest that the 12-subunit architecture of the HA complex is likely conserved across different BoNT serotypes [14], [22]. For example, pairwise structural comparisons yield rmsd of ∼1. 28 Å (582 Cα atoms) and ∼1. 20 Å (137 Cα atoms) for HA70-A/HA70-C and HA17-A/HA17-D, respectively; they are ∼0. 87 Å (129 Cα atoms) and ∼1. 23 Å (134 Cα atoms) for the two domains of HA33 between serotypes A and D and similarly between HA33-A and HA33-C [13], [14], [15], [16]. Moreover, the protein–protein interactions within the HA70 trimer and between HA17 and HA33 are largely conserved among our crystal structures of serotype A and the known crystal structures of serotypes C and D. Despite the largely rigid structure of the HA complex, HA33 seems to have an intrinsic structural flexibility. The N-terminal domain of HA33 is fixed in the HA complex through extensive inter-HA33 and HA17–HA33 interactions, but its C-terminal domain is largely unrestricted. When comparing two HA33-A structures that were determined in different crystal forms, we found that the N- and C-terminal domains of HA33-A twist against each other by ∼14° (Fig. S16 in Text S1) [17]. A more significant conformational change is observed between HA33-A and C (∼61°) and HA33-A and D (∼65°) (Fig. S16 in Text S1) [13], [14]. In the context of the assembled HA complex, such a conformational change leads to a shift up to ∼23 Å for the C-terminal Gal-binding site in HA33. We suggest that HA33 could require such structural flexibility to achieve its multivalent host-receptor binding in the intestine. The loose linkage between the M-PTC and the HA complex clearly suggests divided functions. We previously reported that the M-PTC' s compact structure protects BoNT against digestive enzymes and the extreme acidic environment of the GI tract [5], [23]. We now show that the HA complex is mainly responsible for BoNT absorption in the small intestine, through binding to specific host carbohydrate receptors. This new finding permitted the identification of IPTG as a prototypical oral inhibitor that extends survival following lethal oral BoNT/A intoxication of mice. Multivalent interactions involving nine binding sites for Neu5Ac- and Gal-containing glycans increase the overall avidity of binding between the L-PTC and glycans on the epithelial cell surface, and thus compensate for the modest glycan-binding affinities at individual binding sites (Fig. 6C). Similarly, the potency of carbohydrate receptor mimics could be improved by optimizing the HA–glycan interactions as revealed here or by introducing new HA–inhibitor interactions at individual binding sites based on rational design, as well as by designing multivalent inhibitors. Although such inhibitors cannot be used to treat fully developed food-borne botulism, they could provide temporary protection upon pre-treatment and could also be useful for cases of intestinal colonization with C. botulinum spores such as in cases of infant or adult intestinal botulism. Our results also suggest that the L-PTC could be exploited for alternative applications. For example, protein-based therapeutics could be coupled to the modified non-toxic L-PTC to allow oral delivery by improving drug stability, absorption efficiency, and bioavailability. The Institutional Animal Care and Use Committee of the United States Department of Agriculture, Western Regional Research Center approved the experimental and husbandry procedures used in these studies (protocol # 12-2). All animal experiments were conducted under the guidelines of the U. S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training. The sequences corresponding to full-length HA70 (residues M1–N626), HA70D3 (residues P378–N626), full-length HA17 (residues M1–I146), and full-length HA33 (residues M1–P293) from BoNT/A1-producing C. botulinum strain 62A were cloned into expression vectors pQE30, pGEX-6p-1, pRSFDuet-1, and pET28a, respectively. In addition, HA17 and HA33 were cloned into the bicistronic pRSFDuet-1 for co-expression. To facilitate protein purification, a 6×His tag followed by a thrombin cleavage site was introduced to the N-termini of HA70, HA17, and HA33. HA70D3 was cloned into pGEX-6p-1 following the N-terminal GST and a PreScission cleavage site. For HA17 and HA33 in the pRSFDuet-1 vector, HA17 was produced with an N-terminal 6×His tag followed by a PreScission cleavage site, while HA33 had no affinity tag. All HA33 or HA70 mutations were generated by QuikChange site-directed mutagenesis (Stratagene). Four different protein expression schemes were used to produce the individual HAs or HA complexes. (1) HA70 (pQE30), HA70D3 (pGEX-6p-1), and HA33 (pET28a) were expressed alone; (2) HA70 (pQE30) and HA17 (pRSFDuet-1) were co-transformed into bacteria and co-expressed; (3) HA70D3 (pGEX-6p-1) and HA17 (pRSFDuet-1) were co-transformed into bacteria and co-expressed; and (4) HA17 and HA33 were co-expressed using the bicistronic pRSFDuet-1 vector. All recombinant proteins were expressed in the E. coli strain BL21-RIL (DE3) (Novagen). Bacteria were grown at 37°C in LB medium in the presence of the appropriate selecting antibiotics. Expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when OD600 had reached 0. 7. The temperature was then decreased to 18°C and expression was continued for ∼16 hours. The cells were harvested by centrifugation and stored at −20°C until use. For purification of His-tagged proteins (HA70, HA33, the HA70–HA17 complex, and the HA17–HA33 complex), proteins were bound to a Ni-NTA (nitrilotriacetic acid, Qiagen) affinity column in a buffer containing 50 mM Tris (pH 8. 0) and 400 mM NaCl, and subsequently eluted in the same buffer containing 300 mM imidazole. The eluted fractions of each protein were pooled and dialyzed overnight at 4°C against a buffer composed of 20 mM Tris (pH 8. 0) and 50 mM NaCl, then the His-tag was removed with thrombin (for HA70, HA33, and the HA70–HA17 complex) or PreScission protease (for the HA17–HA33 complex). GST-tagged HA70D3 and the HA70D3–HA17 complex were purified using Glutathione Sepharose 4B resins (GE Healthcare) in phosphate-buffered saline, and eluted from the resins after on-column cleavage using PreScission protease. The following three schemes were used to further purify the proteins. (1) HA70 and the HA70–HA17 complex was purified by MonoQ ion-exchange chromatography (GE Healthcare) in a buffer containing 20 mM Tris (pH 8. 0) and eluted with a NaCl gradient, followed by Superdex 200 size-exclusion chromatography (GE Healthcare) in 20 mM Tris (pH 8. 0) and 50 mM NaCl. (2) HA33 and the HA17–HA33 complex were purified by MonoS ion-exchange chromatography in a buffer containing 20 mM sodium acetate (pH 5. 0) and eluted with a NaCl gradient, followed by Superdex 200 chromatography in 20 mM Tris (pH 8. 0) and 50 mM NaCl. (3) HA70D3 and the HA70D3–HA17 complex were purified by MonoQ ion-exchange chromatography in 20 mM Tris (pH 8. 0) followed by Superdex 200 chromatography in 20 mM Tris (pH 8. 0) and 50 mM NaCl for HA70D3 or 20 mM sodium citrate (pH 5. 0) and 100 mM NaCl for the HA70D3–HA17 complex. Each protein or protein complex was concentrated to ∼3–6 mg/ml using Amicon Ultra centrifugal filters (Millipore) and stored at −80°C until used for further characterization or crystallization. The purified HA70 was labeled with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Life Technologies) according to the manufacturer' s instructions. The labeled HA70 was further purified by Superdex 200 chromatography in 20 mM Tris (pH 8. 0) and 50 mM NaCl. The calculated dye to protein ratio was ∼2 moles of dye per mole of monomeric HA70. The HA17–HA33, the HA70–HA17, and the HA70D3–HA17 complexes were produced by co-expression and co-purification as described above. To assemble the mini-HA complex (HA70D3–HA17–HA33), the purified HA33 and the HA70D3–HA17 complex were mixed at a molar ratio of ∼2. 5∶1 and incubated at 4°C overnight. The excess HA33 was removed by Superdex 200 chromatography with 20 mM Tris (pH 7. 6) and 50 mM NaCl. The fully assembled HA complex was reconstituted by mixing the purified HA70 and the HA17–HA33 complex at a molar ratio of ∼1∶1. 3. The mixture was incubated at 4°C overnight and the excess HA17–HA33 complex was removed from the mature HA complex by Superdex 200 chromatography with 20 mM Tris (pH 7. 6) and 50 mM NaCl. The fluorescence-labeled HA complex was prepared with Alexa Fluor® 488-labeled HA70 and unlabeled HA17–HA33 complex (HA*) or HA17–HA33DAFA complex (HA33DAFA*) using a similar protocol. Sedimentation equilibrium (SE) experiments were performed in a ProteomeLab XL-I (BeckmanCoulter) analytical ultracentrifuge. Purified HA samples were dialyzed extensively against a buffer containing 50 mM Tris (pH 7. 6) and various NaCl concentrations, or 50 mM citric acid (pH 2. 3) and various NaCl concentrations. Protein samples at concentrations of 0. 4,0. 2, and 0. 1 unit of OD280 were loaded in 6-channel equilibrium cells and centrifuged at 20°C in an An-50 Ti 8-place rotor at the first speed indicated until equilibrium was achieved and thereafter at the second speed. HA33 was analyzed at rotor speeds of 19,000 and 22,000 rpm. The HA17–HA33 and the HA70D3–HA17–HA33 complexes were analyzed at 12,000 and 14,000 rpm. The HA70–HA17 and the HA70–HA17–HA33 complexes were run at speeds of 6,000 and 8,000 rpm. For each sample, data sets for the two different speeds were analyzed independently using HeteroAnalysis software (by J. L. Cole and J. W. Lary, University of Connecticut). Three independent experiments were performed for each sample. The AUC data showed that HA33 is predominantly monomeric in solution at pH 2. 3 or pH 7. 6. HA17–HA33 forms a tight complex at pH 2. 3 or pH 7. 6, and the data were best fit to a model composed of one HA17 and two HA33 molecules. The HA70–HA17 complex precipitated at pH 2. 3 and was therefore analyzed only at pH 7. 6. The best fits for HA70–HA17 clearly suggested a complex composed of three HA70 and three HA17 molecules. The data for the HA70D3–HA17–HA33 complex were best fit to a model composed of one HA70D3, one HA17, and two HA33 molecules. HA70–HA17–HA33 forms a tight complex containing three HA70, three HA17, and six HA33. Weak dimerization was observed for the mini-HA complex (Kd of ∼23. 1 µM) and the full HA complex (Kd of ∼10. 9 µM) at pH 7. 6 in the presence of 100 mM NaCl, but was not observed at higher ionic strength. The weak oligomerization Kd suggests that the mini-HA and the full HA complex are monomeric under physiological conditions. The L-PTC of BoNT/A was obtained from List Biological Laboratories, Inc. (Campbell, California) and Miprolab GmbH (Göttingen, Germany). The recombinant HA complex was reconstituted in vitro as described above. Negatively stained EM specimens were prepared following a previously described protocol [54]. Briefly, 3 µl of the L-PTC (∼0. 02 mg/ml in 20 mM MES, pH 6. 2, and 100 mM NaCl) or the HA complex (∼0. 01 mg/ml in 20 mM Tris, pH 7. 6, and 50 mM NaCl) was placed on a freshly glow-discharged carbon-coated EM grid, blotted with filter paper after 40 seconds, washed with two drops of deionized water, and then stained with two drops of freshly prepared 1% uranyl formate, which also served to fix the proteins. Particle images were acquired using a 4k×4k TVIPS CCD camera on a Tecnai F20 electron microscope (FEI) equipped with a field emission electron source operated at 200 kV, at a nominal magnification of ∼70,000, resulting in a calibrated pixel size of 4. 28 Å/pixel on the object scale after binning. The defocus values were set in the range of 1. 5–3. 2 µm. The electron dosage was ∼40 electrons/Å2. Image quality was monitored on the basis of power spectra quality. Particle boxing, CTF correction, initial model generation, 3D refinement, and resolution assessment were all carried out with the EMAN2 package [55]. Particles were semi-automatically boxed out and subjected to reference-free class-averaging using EMAN2. The standard EMAN2 initial model generation program (e2initialmodel. py) was used to obtain initial templates for refinement. With the use of this methodology, models were constructed from a series of randomly generated Gaussian blobs and refined against reference-free-generated 2D class averages. The resulting models were ranked on the basis of the agreement of the projection with the class average. The top initial templates were used as starting models for the subsequent refinement with EMAN2. For the L-PTC, no symmetry was imposed throughout the 3D reconstruction and refinement, while for the HA complex, a C3 symmetry was imposed. Refinement was terminated when no significant changes could be visually detected. A data set of 15,140 particles was used for the final reconstructed map of the L-PTC, for which the resolution was estimated to be ∼30. 8 Å based on the resolution criteria of Fourier shell correlation (FSC) at 0. 5 cutoff. A data set of 3,746 particles was used for the final reconstructed map of the HA complex, for which the resolution was estimated to be ∼30. 6 Å. The density maps were filtered to 30 Å with the low-pass filter in EMAN2. Handedness of the maps was determined on the basis of the HA complex structure derived from crystal structures. Visualization and rigid-body docking of atomic models into the 3D-EM density maps were performed using UCSF Chimera [56]. The Chimera Fit in Map utility, which maximizes the cross-correlation coefficient between the 3D-EM density map and the calculated density map (filtered to 30 Å) of the atomic structures, was used to optimize the docking of atomic structures into 3D-EM maps. After fitting refinement, the positions with highest correlation coefficient (cc) values were chosen. For the HA complex, the atomic structure derived from crystal structures of HA70 and the HA70D3–HA17–HA33 complex fitted the 3D-EM map very well (cc = 93. 1%). There was a slight deviation at the C-terminal domain of HA33 that is located at the tip of the complex, which may be due to the structural flexibility of HA33. For the L-PTC, the densities for the M-PTC and the HA complex could be clearly identified in the 3D-EM reconstruction map, and the density for the HA complex was manually 3-fold averaged using Chimera and EMAN2. Atomic structures of the M-PTC and the HA complex were then docked separately into their 3D-EM densities, with highest cc values of 87. 3% and 87. 7%, respectively. The docked M-PTC and the HA complex were then merged to generate the complete pseudo-atomic model for the L-PTC. Initial crystallization screens were performed using a Phoenix crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits (Hampton Research, Qiagen, or Emerald BioSystems), followed by extensive manual optimization. The best single crystals were grown at 18°C by the hanging-drop vapor-diffusion method in a 1∶1 (v/v) ratio of protein and reservoir, as follows. (1) HA70 was crystallized with a reservoir solution composed of 0. 1 M sodium acetate (pH 4. 4) and 1. 5 M ammonium chloride. Carbohydrate complexes were obtained when HA70 crystals were soaked in the mother liquor supplemented with 100 mM α2,3-SiaLac, α2,6-SiaLac, or Neu5Ac at 18°C overnight. (2) The HA17–HA33 complex was crystallized using a reservoir of 0. 1 M MES (pH 6. 2), 0. 1 M MgCl2, and 5% (w/v) PEG [poly (ethylene glycol) ] 8K. Micro-seeding was used to improve crystal quality. Carbohydrate complexes were obtained when crystals of the HA17–HA33 complex were soaked with 100 mM Gal, Lac, or LacNAc at 18°C overnight. (3) The HA70D3–HA17 complex was crystallized using 0. 1 M sodium acetate (pH 4. 8), 12% (w/v) PEG MME 2K, and 0. 1 M CsCl. (4) The HA70D3–HA17–HA33 complex was crystallized using 0. 1 M Tris (pH 8. 2), 0. 1 M NaCl, and 6% (w/v) PEG 20K. The crystals of HA70 and its carbohydrate complexes were cryoprotected in their original mother liquor supplemented with 20% (v/v) ethylene glycerol and flash-frozen in liquid nitrogen. Crystals for all the other samples were cryoprotected in 22% (v/v) glycerol with their mother liquors and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) or Advanced Photon Source (APS). The data were processed with HKL2000 [57] or iMOSFLM [58]. Data collection statistics are summarized in Tables S2 and S4 in Text S1. The structure of HA70 of BoNT/A was determined by molecular replacement software Phaser [59] using the HA70 of BoNT/C (PDB code 2ZS6) [15] as the search model. The D3 domain of HA70 of BoNT/A, together with HA17 of BoNT/D (PDB code 2E4M) [14] and HA33 of BoNT/A (PDB code 1YBI) [17], were used as the search models to solve the structure of the HA70D3–HA17–HA33 complex by Phaser. The structures of the HA17–HA33 and the HA70D3–HA17 complexes were determined by Phaser using partial structures of the HA70D3–HA17–HA33 complex as search models. The manual model building and refinements were performed in COOT [60] and PHENIX [61] in an iterative manner. The carbohydrates were modeled into the corresponding structure during the refinement based on the Fo-Fc electron density maps. The refinement progress was monitored with the free R value using a 5% randomly selected test set [62]. The structures were validated through the MolProbity web server [63] and showed excellent stereochemistry. Structural refinement statistics are listed in Tables S2 and S4 in Text S1. The coordinate and diffraction data for all the structures reported here will be deposited in the Protein Data Bank. The conformational change of HA33 was measured by DynDom [64]. All structure figures were prepared with PyMol (http: //www. pymol. org). The calorimetry titration experiments were performed at 23°C on an ITC200 calorimeter from Microcal/GE Life Sciences (Northampton, MA). The HA samples were used as the titrand in the cell and the carbohydrates were used as titrants in the syringe. To control for heat of dilution effects, protein samples were dialyzed extensively against the titration buffer (50 mM Tris, pH 7. 6, and 100 mM NaCl) prior to each titration. Carbohydrates and nLoop peptide were dissolved in the same buffer. The pH of the acidic Neu5Ac solution was carefully adjusted to pH 7. 6. The following concentrations were used for pair-wise titrations: HA33 (200 µM) vs. carbohydrates (Gal, Lac, LacNAc, IPTG, or α2,6-SiaLac) (50 mM); HA70D3 (200 µM) vs. α2,3-, or α2,6-SiaLac (40 mM); HA70D3 (160 µM) vs. Neu5Ac (80 mM); and HA70 (30 µM) vs. nLoop (400 µM). The data were analyzed using the Origin software package provided by the ITC manufacturer. The thermodynamic values reported are the average of three independent experiments (Table S3 in Text S1). The recombinant HA70–HA17–HA33 complex, HA70, the HA17–HA33 complex, and the M-PTC were subjected to limited proteolysis with trypsin and pepsin overnight at room temperature. The trypsin digestions were performed at two different pHs in buffers containing 50 mM sodium phosphate (pH 6. 0 or 7. 5) and 300 mM NaCl, or in the Krebs-Ringer' s solution (119 mM NaCl, 2. 5 mM KCl, 1. 0 mM NaH2PO4,2. 5 mM CaCl2,1. 3 mM MgCl2,20 mM Hepes, and 11 mM D-glucose). The trypsin∶sample ratios (w/w) were 1∶10 (pH 6. 0) or 1∶20 (pH 7. 5). The digestions were stopped by adding 1 mM PMSF and boiling the samples in reducing SDS-loading buffer for 10 minutes. The pepsin digestions were performed at a 1∶100 ratio (w/w) of pepsin∶sample in a buffer containing 50 mM citrate acid (pH 2. 6, an optimal pH for the pepsin reaction) and 300 mM NaCl. Pepsin digestions were terminated by addition of a 1 M Tris-HCl (pH 8. 0) stock solution to give a final concentration of 200 mM and samples were then boiled in the reducing SDS-loading buffer. All samples were subjected to SDS-PAGE. Cell culture: Caco-2 cells were obtained from the German Cancer Research Center (Heidelberg, Germany). Cells were cultured in Dulbecco modified Eagle medium (DMEM, Gibco® | Life Technologies, Darmstadt) supplemented with 10% fetal bovine serum, 100 U of penicillin per ml, and 100 mg of streptomycin per ml for up to six months. The cells were subcultured twice a week and seeded on BD Falcon Cell Culture Inserts (#353494, growth area 0. 9 cm2, pore size 0. 4 µm) at a density of approximately 105 cells cm−2 for flux studies and determination of transepithelial electrical resistance (TER). Measurement of TER: All TER experiments were conducted in 0. 5 ml and 1. 5 ml of Iscoves Modified Dulbeccos Medium without phenol red (IMDM, Gibco® | Life Technologies, Darmstadt) in the apical and basolateral reservoir, respectively. TER was determined with an epithelial volt-ohm meter (World Precision Instruments, Berlin, Germany) equipped with an Endohm 12 chamber for filter inserts. Filters with cell monolayers were used at day 11 after seeding which is seven days of post confluency. Only filters with an initial resistance of ≥300 Ω cm−2 were used. For analysis of independent experiments subsequent results were expressed as percentages of the corresponding resistance of each data set determined immediately after administration of samples. Values are expressed as means of ≥3 independent experiments with duplicate samples ± standard deviations. Carbohydrate inhibition assays: Lac, Gal, IPTG, Neu5Ac, α2,6- and α2,3-SiaLac were dissolved in IMDM, sterile filtered and stored at −20°C. Neu5Ac stock solution was adjusted to pH 7. 4. The wild type HA complex (HA wt), fluorescence-labeled HA complex (HA*), or the HA70-TPRA complex were pre-incubated with the corresponding carbohydrate over night at 4°C in IMDM and diluted to the final concentration with IMDM prior to administration. The TER upon administration of each carbohydrate in the highest concentrations used was checked in the absence of HA and was virtually identical to that of the control without sugars. Transport measurement: For paracellular transport studies, filters were incubated in IMDM added to the apical (0. 5 ml) and basolateral (1. 5 ml) reservoir. As marker substance Alexa Fluor® 488 labeled HA* or HA33-DAFA* was administered to the apical or basolateral reservoirs at final concentrations of 58 nM and 17 nM, respectively. After 24 hour of incubation, 200 µl of samples were taken from the apical and the basolateral reservoir. The marker substance was measured in a BioTek Synergy 4 fluorescence spectrophotometer at 495 nm excitation and 519 nm emission wavelengths. The mouse protection assay was performed following a previously described protocol [4]. Briefly, random sets of 10–20 female Swiss Webster mice (20–23 g) were used per dose. Mice were treated by oral gavage with 100 µl containing 1. 9 µg of L-PTC/A (Metabiologics) in phosphate–gelatin buffer (10 mM phosphate buffer, pH 6. 2, and 2% gelatin), with or without the indicated concentrations of IPTG, Neu5Ac, or Gal. Mice were also administered 100 µl of 500 mM IPTG by gavage 1 hour prior or after treatment with 100 µl containing 1. 9 µg of L-PTC/A in phosphate gelatin buffer. The acidic Neu5Ac was adjusted to pH 6. 2 for administration. Mice were monitored for botulism symptoms for up to 14 days post-intoxication. Median survival and p-values were determined with the GraphPad Prism 5 program (San Diego, CA). Atomic coordinates and structure factors for HA70, HA70D3–HA17–HA33, HA70D3–HA17, HA17–HA33, HA17–HA33–Lac, HA17–HA33–Gal, HA17–HA33–LacNAc, HA70–α2,3-SiaLac, and HA70–α2,6-SiaLac have been deposited with the Protein Data Bank under accession codes 4LO4,4LO7,4LO8,4LO0,4LO2,4LO1,4LO3,4LO5,4LO6, respectively. EM 3D reconstructions for the L-PTC and the HA complex have been deposited with the Electron Microscopy Data Bank (EMDB) under accession codes EMD-2417 and EMD-2416, respectively.
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Title: Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity Summary: Food-borne botulinum neurotoxin (BoNT) poisoning results in fatal muscle paralysis. But how can BoNT-a large protein released by the bacteria clostridia-survive the hostile gastrointestinal (GI) tract to gain access to neurons that control muscle contraction? Here, we report the complete structure of a bimodular ∼760 kDa BoNT/A large progenitor toxin complex (L-PTC), which is composed of BoNT and four non-toxic bacterial proteins. The architecture of this bacterial machinery mimics an Apollo lunar module, whereby the "ascent stage" (a ∼290 kDa module) protects BoNT from destruction in the GI tract and the 3-arm "descent stage" (a ∼470 kDa module) mediates absorption of BoNT by binding to host carbohydrate receptors in the small intestine. This new finding has helped us identify the carbohydrate-binding sites and the monosaccharide IPTG as a prototypical oral inhibitor, which extends survival following lethal BoNT/A intoxication of mice. Hence, pre-treatment with small molecule inhibitors based on carbohydrate receptor mimicry can provide temporary protection against BoNT entry into the circulation.
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Summarize: Gaza’s streets on Tuesday slowly filled with cars, donkey carts and trucks, many of them piled with the belongings of displaced families moving from one spot to another toting mattresses, kitchen supplies and bags of clothes. Gaza faces a major challenge in reconstruction, with its infrastructure, always shaky, badly damaged. Electrical cables are down, the only power plant is out of action, the water and sewage systems are damaged and hospitals urgently need resupplying. About 260,000 of Gaza’s 1.8 million residents have been displaced by the fighting, according to the United Nations, and many thousands of them remain huddled in schools or with friends and relatives. Many have no homes to return to. “We lost in one instant all we had worked for 40 years to build,” said Fouad Harara, 55, who had worked for decades as a laborer in Israel. “The only thing we gained is destruction.” Video In the Shejaiya neighborhood in eastern Gaza City, where many Israeli soldiers were killed and where entire streets looked as if they had been through an earthquake, Hani Harazeen, 42, searched the rubble of his collapsed home for anything he could find. “We used to praise God that we all had work and lived together,” he said of himself and his brothers, who lived with their families in adjacent buildings. “Now we are scattered all over and looking for places to sleep.” He said he had not seen any Palestinian fighters before he fled the area and had no idea how they had fought. Advertisement Continue reading the main story “The war started, and the resistance responded,” he said. “But does Hamas have fighter jets? Can its rockets do this to a home?” After being nearly invisible to most Gazans throughout the war, Hamas police officers emerged in some areas, patrolling in blue and white trucks and inspecting damaged neighborhoods. New billboards had been put up recently in Gaza City, one of them showing a group of fighters and a tunnel with the words “The Tunnels of Glory” and “Passages to Arrive in Jerusalem.” Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Hamas’s Al Aqsa radio station alternated between triumphant jihadi anthems and talk shows about how “the resistance” had vanquished the “Zionist enemy” with its rockets, forcing it to withdraw from Gaza. Lt. Col. Peter Lerner, the Israeli military spokesman, said that Israel had destroyed about 32 tunnels built by Hamas and leading into Israel, and that Israeli forces had killed about 900 militants, a figure that is bound to be challenged by Hamas. He said that Israel had destroyed more than 3,000 rockets belonging to Hamas and Islamic Jihad and that those groups had launched more than 3,300 rockets toward Israel. Israel said it suspected that they had 3,000 rockets left. But Hamas and Islamic Jihad appeared ready for the conflict to come to a halt. The cease-fire could break down over negotiations for a more durable arrangement, or it could be extended beyond the initial 72 hours. “It’s clear now that the interest of all parties is to have a cease-fire,” said Bassam Salhi, a Palestinian delegate in Cairo. “It’s going to be tough negotiations because Israel has demands, too. We don’t have any guarantees the siege will be removed.” Israel is demanding security, a durable end to attacks from Gaza and strong control over what comes in and out of Gaza, aided by the Egyptians, to prevent Hamas and Islamic Jihad from easily rearming or building new networks of tunnels with diverted or smuggled cement. Yossi Kuperwasser, director general of the Israeli Ministry of Strategic Affairs, said that in Cairo, Israel wanted “to come out with arrangements that assure us that this cease-fire will be different from previous ones, that it lasts a long time and Hamas won’t rearm itself.” He added, “Fortunately, now we have an Egypt that seems willing to contribute to this outcome,” with a president, Abdel Fattah el-Sisi, who opposes Hamas. Israel has not succeeded in destroying Hamas, which was said to be a goal of the campaign, or in dislodging it from control over Gaza. Hamas is expected to call the monthlong conflict a victory. Hamas killed more Israeli soldiers, 64, than in the last major conflict, in 2008-9, when 10 soldiers were killed, four of them by friendly fire. Hamas commanders largely survived the war; its rockets reached nearly all of Israel, forcing thousands into shelters, and even led to the cancellation of many international flights to Israel’s airport near Tel Aviv for two days after a rocket landed nearby. Advertisement Continue reading the main story Hamas has been hurt, but it has survived and has earned respect among many Palestinians and other Arabs. That has infuriated some on the Israeli right, who blame Prime Minister Benjamin Netanyahu for lacking gumption. Uzi Landau, for example, the tourism minister from the party Yisrael Beiteinu, told Israel Radio, “The operation ended with no achievement that ensures quiet.” Yaakov Peri, the minister of science from the centrist Yesh Atid party but also a former head of Shin Bet, Israel’s internal security service, said the army’s achievements were significant. “But I think we have to measure the current confrontation and the current suffering by the yardstick of the political achievements that Israel has to be very, very determined about,” he said. Israel has also suffered a significant blow to its international standing, with the United Nations and Western nations, including the United States, Britain, France and Germany, lamenting the human cost of the war and criticizing some of Israel’s attacks on or near United Nations schools that were harboring thousands of displaced civilians. There will also be new debates in Israel about the preparedness of the army for urban combat and even an investigation into why the extent of the Hamas tunnel system into Israel was either not known or not prepared for better. The Iron Dome air defense system, which destroys or diverts missiles, most likely saved many Israeli lives, but the Hamas tunnel system was a psychological and tactical surprise. Egypt is also eager to bolster the status of Mahmoud Abbas, president of the Palestinian Authority, and Fatah, its political wing, which govern the West Bank. Even Mr. Netanyahu and his team, who have often dismissed Mr. Abbas, are now looking to him and Fatah as potential partners. But Mr. Abbas is 79 and has health problems, raising questions about who will succeed him. One possible candidate, Mohammed Dahlan, 52, who was born in Gaza, is favored by some Egyptian officials. But he is disliked by Mr. Abbas and hated by Hamas, which Mr. Dahlan tried to suppress when he effectively ran Gaza for the Palestinian Authority. Just before the cease-fire went into effect at 8 a.m., a last salvo of rockets was fired toward Israel from Gaza, causing warning sirens to sound in Tel Aviv and Jerusalem, while the Israeli Air Force carried out at least five strikes in Gaza, a customary last word before another in a series of conflicts between Israel and Hamas came to an end. A definitive end to the four-week conflict in Gaza appeared possible on Tuesday morning as an agreed 72-hour ceasefire between Hamas and Israel came into effect and the Israeli military said it had withdrawn from the Palestinian territory. The suspension of hostilities came into force at 8am on Tuesday and is due to be followed up with further discussions in Egypt about ending the four-week war. The Israeli military said it had withdrawn all forces from Gaza by 8am. A volley of last-minute rockets was fired by militants towards Israel. The likelihood of Israel agreeing to a longer-term ceasefire appeared to increase on Tuesday as Israeli radio stations reported that ground forces had completed their main Gaza war mission of destroying cross-border tunnels. At least 32 of the underground passages and dozens of access shafts had been located and blown up, Israel Radio and Army Radio said. A member of the Hamas delegation in Cairo, Bassam Salhi, said it was "clear now that the interest of all parties is to have a ceasefire. It's going to be tough negotiations because Israel has demands too." Mark Regev, an Israeli government spokesman, said: "Israel will honour the ceasefire and will be watching to see if Hamas does too." An hour into the ceasefire, no rockets had been fired from Gaza and fisherman in the coastal territory ventured back into the sea. The month of conflict has however left significant damage. The Palestinian deputy finance minister, Taysir Amro, estimated at least $4-6 billion in damages. Representatives of Palestinian factions had been in Cairo since Sunday to agree a set of demands and a possible end to hostilities. More than 1,800 Palestinians have died, health officials in Gaza say. Israeli casualties include 64 soldiers and three civilians killed by rocket fire. The new proposal was communicated late on Monday night to the Israelis, who accepted the ceasefire plan around midnight. An Israeli official confirmed a delegation would be heading to Cairo for talks. Ziad al-Nakhala, deputy secretary general of Palestinian Islamic Jihad, had earlier told the Guardian that he believed a deal would be reached. At least one key issue for Israel – of cross-border tunnels that allow infiltration by militants – had not been discussed, al-Nakhala said, but Egyptian officials accepted the need to ease the siege of Gaza. US State Department spokeswoman Jen Psaki said late on Monday that Washington strongly supported the latest ceasefire proposal and urged "both parties to respect it completely". The Palestinian Authority's envoy to Egypt, Gamal Shobky, who was also present at the talks in Cairo, said "many people" in the Egyptian capital were working to reach a ceasefire agreement. Egyptian officials said they were hopeful an Israeli delegation would arrive on Tuesday. Both Hamas and the Israeli government will have to convince supporters and the more general population that the war has brought them victory. A final deal could see Egypt loosen tight restrictions on passage through its border into the Gaza strip, providing a major boost to the local economy and to the popularity of Hamas, which has been isolated diplomatically and weakened financially in recent years. Israel has consistently underlined its desire to see the Islamist organisation, which took de facto control of Gaza in 2007 after winning Palestinian elections the previous year, disarmed. On a visit to the Israeli army's southern command on Monday afternoon, the Israeli prime minister, Binyamin Netanyahu, said that the Israeli Defence Forces (IDF) were concluding their action to destroy Hamas tunnels but pledged the military operation would end "only when quiet and security are restored to the citizens of Israel for a lengthy period". Israel has previously said that it would not agree to ceasefires which it fears will be broken but will declare an end to the conflict unilaterally when it believes the right moment has come. However, public statements by officials that the Israeli military has achieved at least some of its objectives in the war and has inflicted significant damage on Hamas has been interpreted by some analysts as preparing public opinion for a declaration that the conflict is now over. The Israeli military said it had resumed its attacks on the Gaza Strip on Monday, ending a self-declared unilateral ceasefire that had been in effect for much of the day. Israel had declared a seven-hour "humanitarian window" in Gaza amid international outrage after a blast outside a UN school sheltering displaced Palestinians, which killed nine, and mounting pressure for the bloodshed to end. The blast has been blamed on an Israeli airstrike and military officials acknowledged that they had been targeting militants on a motorbike in the vicinity. Monday's unilateral ceasefire was the eighth temporary pause in fighting, nearly all of which have broken down amid mutual accusations of violations. The ceasefire slowed violence, though two Israeli missiles hit a house in the Shati refugee camp in northern Gaza around the time it started. That strike killed an eight-year-old girl and wounded 29 people, medics said. At least 18 Gazans were killed on Monday, Palestinian officials said, most before the ceasefire came into force. Dozens of bodies were also retrieved from ruins. Another Israeli strike killed a commander in the Islamic Jihad group, a close ally of Gaza's militant Palestinian Hamas rulers, the group said. Israel says almost half of those killed in Gaza in recent weeks were combatants. The UN say two thirds were civilians. The British Foreign Office said it was "urgently investigating" claims that a British aid worker had been killed in the southern Gaza town of Rafah, which has seen some of the heaviest fighting in recent days. The violence in Gaza appeared to be spilling over on Monday, with Jerusalem the scene of what police said were two suspected terrorist attacks, amid clashes between Palestinian youths protesting over the Gaza conflict and Israeli security forces. A bus was rammed by an industrial digger in an ultra-Orthodox neighbourhood close to the main thoroughfare through the city. The driver, reported to be a Palestinian from east Jerusalem, was shot dead by police. A 25-year-old Israeli man died after being hit by the vehicle before it ploughed into the bus, overturning it. Five people were lightly injured. Less than three hours later, an Israeli soldier was shot in the stomach in a tunnel near the main campus of Hebrew University. "Multiple shots were fired. One man was hit in the stomach and rushed to the hospital in serious condition," said police spokesman Micky Rosenfeld. Police were searching for the assailant. Police detained 12 Palestinians overnight who it said were involved in "rioting" near the Old City. Protests in east Jerusalem and the West Bank over the war in Gaza in recent weeks have led to at least 10 Palestinians being killed by Israeli security forces. The Israeli military reported that 53 rockets had been fired at Israel on Monday. There were no reports of injuries. The Israeli ceasefire in Gaza had exempted the area around Rafah, where the UN school was struck on Sunday, and fighting continued there. Troops were working on destroying a cross-border tunnel in the area. Israeli army spokesman Peter Lerner said the IDF were close to completing their mission to destroy the network of tunnels leading into Israel. "We've caused substantial damage to this network to an extent where we've basically taken this huge threat and made it minimal," he said. Netanyahu said Israel had "no intention of attacking the residents of Gaza" during a visit to the military's southern command headquarters, according to a statement released by the government press office. "We struck a very severe blow at Hamas and the other terrorist organisations. We have no intention of attacking the residents of Gaza. In practice, it is Hamas that is attacking them and denying them humanitarian aid. I think that the international community needs to strongly condemn Hamas and also demand, just as we are demanding, that the rehabilitation of Gaza be linked to its demilitarisation." Egypt, a traditional broker in Hamas-Israeli conflicts, had proposed last month, soon after the latest conflict erupted, an unconditional ceasefire followed by talks between the two sides. Israel accepted that plan but Hamas rejected it, accusing Cairo of bypassing the Palestinian movement. Last week Cairo invited the two sides again to send their delegations for talks to work on a durable, long-term ceasefire. But Israel refused to send its negotiators, accusing Hamas of breaching a UN-backed 72-hour humanitarian truce that began on Friday but collapsed within hours. Earlier on Monday, Hamas accused Israel of breaching that truce and of trying to scuttle the Cairo talks. The Palestinian demands agreed on Sunday include "a ceasefire; Israeli troop withdrawal from Gaza; the end of the siege of Gaza and opening its border crossings". They have also demanded fishing rights up to 12 nautical miles off Gaza's coast and the release of Palestinian prisoners demanded by Hamas and the Palestinian president, Mahmoud Abbas. Gaza City (CNN) -- With negotiations for a longer-lasting cease-fire set to begin soon, many Gazans returned to their neighborhoods Tuesday while some remained at shelters not trusting the break in the violence. For now, a 72-hour humanitarian pause is in place, allowing delegations to gather in Cairo, Egypt, to talk about how to make the truce last. For Gaza's 1.8 million residents it also offered a chance to go to a street market for food and goods, or to check on abandoned homes. For Israel, where more than 2,300 rockets have landed since early July, there was, for a change, no sirens blaring. In Gaza City, one man told CNN he was happy the shooting had stopped, but his problems were just beginning. The man said he couldn't understand what he was seeing -- the home his family invested $100,000 in, now destroyed. No insurance company will give him money to rebuild, he told CNN. There are no winners in this war, he said. But on Twitter, the Israel Defense Forces declared: "Mission accomplished." The IDF said it had destroyed 32 tunnels -- many of which ran under the border into Israel -- during the four-week conflict. Israel says Hamas militants used the tunnels to sneak into Israel for terror attacks. Nearly 1,900 Palestinians have been killed in Gaza during the conflict, according to the Palestinian Health Ministry. It's unclear how many were militants. The United Nations estimates that about 70% of the dead were civilians. But the IDF says it estimates about 900 militants were killed. IDF spokesman Lt. Col. Peter Lerner said it was a preliminary figure based on field reports from troops returning from battle. Israeli officials have said 64 Israeli soldiers and three civilians in Israel died in violence. 'Peace? What peace?' After the cease-fire began, residents trickled into Shujaya, an area near Gaza City that experienced some of the most destructive violence of the conflict. They found craters and ruins where homes and shops once stood. People scaled crumbled concrete and twisted metal to rummage for any belongings left in the rubble. Nal Mohammed, a Ph.D. student whose family home was demolished, lamented the situation. "Peace? What peace? We have no home, no water, no power," he said. "There is no peace here." Residents were stunned as they returned to their neighborhoods for the first time since being displaced by the fighting. "After the cease-fire agreement took effect, the world will now see the level of destruction which should serve as an evidence of the level of crimes of the enemy," Ismail Haniyeh, a senior leader of Hamas, said in a statement aired on Hamas-run Al-Aqsa television, according to a CNN translation. The conflict has displaced more than 200,000 people across the densely populated territory. Residents are coming back to similar sights -- rubble, ruins, buildings pockmarked by shrapnel. A white-haired man, Hany Mahmoud el Harezen, stood on the roof of his collapsed two-story home. "I am a wedding photographer, I have nothing to do with this war," he said. "Maybe if we had gotten some concessions, it would be worth it. But we got nothing." For the first time Tuesday, the number of people packed into U.N. shelters decreased, said U.N. official Chris Gunness in Gaza. But not everyone left the U.N. facilities. Others were more pragmatic, thinking it best to wait and see. "They said there was a truce before and we left," one man told CNN. "But five minutes after we got home, the airstrikes started." Israel released a map noting numerous sites it targeted in Shujaya, which it said "Hamas used for military purposes." The IDF said the map showed locations of tunnels, hideouts, rocket firings and launchings, and more. Troop withdrawal "We have no forces within Gaza," IDF spokesman Lerner told CNN Tuesday. With its goal of demolishing the tunnels achieved, Israel announced the withdrawal of ground forces for what is supposed to be a three-day cease-fire with Hamas. Israel is implementing the Egyptian-brokered truce, which took effect Tuesday morning, while maintaining "defensive positions" outside Gaza, the IDF said. Officials from the United Nations and United States, who have been pushing for a cease-fire for weeks, hope that the 72-hour pause will allow negotiations to take place for a more lasting peace. Why are so many civilians dying in Hamas-Israel war? Meetings in Cairo An Israeli delegation arrived Tuesday evening in Cairo for negotiations, two senior Egyptian government officials told CNN. The officials, who asked not to be named, said they expected indirect negotiations to begin Wednesday, with the Egyptian officials acting as intermediaries. The two sides are not expected to talk directly to each other. Egyptian and Palestinian negotiators had arrived over the weekend. Key to any talks, Israeli government spokesman Mark Regev said, is that Hamas must disarm. Hamas leaders say that they want to negotiate an end to the Israeli blockade of Gaza, or at least a body other than the Israelis controlling the borders. Haniyeh, the senior Hamas leader, said his televised statement that Hamas members will work with the Palestinian delegation to end the blockade. Israel: 'The onus is on Hamas' Israel repeatedly accepted an Egyptian cease-fire proposal during the conflict, which Hamas rejected. Temporary humanitarian cease-fires repeatedly crumbled, with each side blaming the other. "The onus is on Hamas," Regev told CNN of the 72-hour truce. "We are entering this with our eyes open," he said. "We have been burnt more than once." The sentiment was similar from Hamas spokesman Osama Hamdan, who told CNN that as long as Israel honors the agreement, so will the Palestinians. "We hope they can take it and be committed to a cease-fire," he said. While Egypt has not released details of the truce, Regev suggested it was the same agreement that Israel has accepted all along. The United States urged both sides to honor the cease-fire agreement. "The United States has been steadfast in our insistence on an end to rocket and tunnel attacks against Israel and an end to the suffering of the people of Gaza," State Department spokeswoman Jen Psaki said. U.N. Secretary-General Ban Ki-moon "urges the parties to commence, as soon as possible, talks in Cairo on a durable ceasefire and the underlying issues," his office said in a statement. British minister resigns over Gaza In the latest example of the ripples the conflict has sent around the globe, a British government minister said she was resigning over her country's policy on Gaza. "With deep regret I have this morning written to the Prime Minister & tendered my resignation. I can no longer support Govt policy on Gaza," Sayeeda Warsi, senior minister of state at the Foreign and Commonwealth Office, wrote on Twitter. Warsi, a member of the House of Lords and the first Muslim in a British Cabinet, posted a photo of her resignation letter on Twitter. READ: CNN exclusive: Inside the mind of Hamas' political leader READ: Nobel laureate Wiesel: Hamas must stop using children as human shields READ: Gaza conflict: Can economic isolation ever be reversed? READ: Life in Gaza: Misery heightened by war CNN's Salma Abdelaziz, Karl Penhaul and Martin Savidge reported from Gaza, Matthew Chance reported from Jerusalem, Reza Sayah reported from Cairo and Steve Almasy reported from Atlanta. CNN's Jethro Mullen, Josh Levs, Claudia Rebaza, Phil O'Sullivan, Chelsea J. Carter and Elaine Ly contributed to this report.
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Summary: Just before an Egypt-brokered ceasefire took hold in Gaza this morning, both sides exchanged fire. Several hours into the truce, however, things have been calm, the Washington Post reports, while the Guardian sees a chance at a "definitive end" to the latest conflict. Israel says it has withdrawn all its troops from Gaza, pointing to support for the Egyptian effort, the New York Times notes. The latest from the region: Israel has finished destroying border tunnels, local media report, boosting the odds of a more lasting ceasefire, the Guardian notes. But both sides are wary, spokespeople tell CNN. "We are entering this with our eyes open," says an Israeli government rep. "We have been burnt more than once." A British government minister has resigned over the UK's Gaza policy, calling it "morally indefensible." Sayeeda Warsi, a senior minister at the Foreign and Commonwealth Office, tweeted her fears of a "detrimental impact on our reputation internationally and domestically," CNN reports. The AP sees signs that Hamas could let Palestinian President Mahmoud Abbas take charge of rebuilding operations, despite former divisions. And there's plenty of rebuilding to be done. "Peace? What peace?" a student near Gaza City asks CNN. "We have no home, no water, no power... There is no peace here." The death toll now stands at 1,865 Palestinians-about 70% civilians, the UN says-and 67 Israelis, including three civilians, both sides report, per CNN. The next step: Talks in Egypt to forge a longer-term agreement, the Times reports.
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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Budget Enforcement Legislative Tool Act of 2008''. SEC. 2. EXPEDITED CONSIDERATION OF CERTAIN PROPOSED RESCISSIONS. (a) In General.--Part B of title X of the Congressional Budget and Impoundment Control Act of 1974 (2 U.S.C. 681 et seq.) is amended by redesignating sections 1013 through 1017 as sections 1014 through 1018, respectively, and inserting after section 1012 the following new section: ``expedited consideration of certain proposed rescissions ``Sec. 1013. (a) Proposed Rescission of Discretionary Budget Authority.--In addition to the method of rescinding discretionary budget authority specified in section 1012, the President may propose, at the time and in the manner provided in subsection (b), the rescission of any discretionary budget authority provided in an appropriations Act. Funds made available for obligation under this procedure may not be proposed for rescission again under this section or section 1012. ``(b) Transmittal of Special Message.-- ``(1) Not later than 3 days after the date of enactment of an appropriations Act subject to rescission under this section, the President may transmit to Congress a special message proposing to rescind amounts of discretionary budget authority provided in that Act and include with that special message a draft bill or joint resolution that, if enacted, would only rescind that discretionary budget authority. ``(2) In the case of an Act that includes accounts within the jurisdiction of more than one subcommittee of the Committee on Appropriations, the President in proposing to rescind discretionary budget authority under this section shall send a separate special message and accompanying draft bill or joint resolution for accounts within the jurisdiction of each such subcommittee. ``(3) Each special message shall specify, with respect to the discretionary budget authority proposed to be rescinded, the matters referred to in paragraphs (1) through (5) of section 1012(a). ``(c) Limitation on Amounts Subject to Rescission.-- ``(1) The amount of discretionary budget authority which the President may propose to rescind in a special message under this section for a particular program, project, or activity for a fiscal year may not exceed 25 percent of the amount appropriated for that program, project, or activity in that Act. ``(2) The limitation contained in paragraph (1) shall only apply to a program, project, or activity that is authorized by law. ``(d) Procedures for Expedited Consideration.-- ``(1)(A) Before the close of the second day of continuous session of the applicable House after the date of receipt of a special message transmitted to Congress under subsection (b), the majority leader or minority leader of the House of Congress in which the Act involved originated shall introduce (by request) the draft bill or joint resolution accompanying that special message. If the bill or joint resolution is not introduced as provided in the preceding sentence, then, on the third day of continuous session of that House after the date of receipt of that special message, any Member of that House may introduce the bill or joint resolution. ``(B) The bill or joint resolution shall be referred to the Committee on Appropriations of that House. The committee shall report the bill or joint resolution without substantive revision and with or without recommendation. The bill or joint resolution shall be reported not later than the seventh day of continuous session of that House after the date of receipt of that special message. If the Committee on Appropriations fails to report the bill or joint resolution within that period, that committee shall be automatically discharged from consideration of the bill or joint resolution, and the bill or joint resolution shall be placed on the appropriate calendar. ``(C) A vote on final passage of the bill or joint resolution shall be taken in that House on or before the close of the 10th calendar day of continuous session of that House after the date of the introduction of the bill or joint resolution in that House. If the bill or joint resolution is agreed to, the Clerk of the House of Representatives (in the case of a bill or joint resolution agreed to in the House of Representatives) or the Secretary of the Senate (in the case of a bill or joint resolution agreed to in the Senate) shall cause the bill or joint resolution to be engrossed, certified, and transmitted to the other House of Congress on the same calendar day on which the bill or joint resolution is agreed to. ``(2)(A) A bill or joint resolution transmitted to the House of Representatives or the Senate pursuant to paragraph (1)(C) shall be referred to the Committee on Appropriations of that House. The committee shall report the bill or joint resolution without substantive revision and with or without recommendation. The bill or joint resolution shall be reported not later than the seventh day of continuous session of that House after it receives the bill or joint resolution. A committee failing to report the bill or joint resolution within such period shall be automatically discharged from consideration of the bill or joint resolution, and the bill or joint resolution shall be placed upon the appropriate calendar. ``(B) A vote on final passage of a bill or joint resolution transmitted to that House shall be taken on or before the close of the 10th calendar day of continuous session of that House after the date on which the bill or joint resolution is transmitted. If the bill or joint resolution is agreed to in that House, the Clerk of the House of Representatives (in the case of a bill or joint resolution agreed to in the House of Representatives) or the Secretary of the Senate (in the case of a bill or joint resolution agreed to in the Senate) shall cause the engrossed bill or joint resolution to be returned to the House in which the bill or joint resolution originated. ``(3)(A) A motion in the House of Representatives to proceed to the consideration of a bill or joint resolution under this section shall be highly privileged and not debatable. An amendment to the motion shall not be in order, nor shall it be in order to move to reconsider the vote by which the motion is agreed to or disagreed to. ``(B) Debate in the House of Representatives on a bill or joint resolution under this section shall not exceed 4 hours, which shall be divided equally between those favoring and those opposing the bill or joint resolution. A motion further to limit debate shall not be debatable. It shall not be in order to move to recommit a bill or joint resolution under this section or to move to reconsider the vote by which the bill or joint resolution is agreed to or disagreed to. ``(C) Appeals from decisions of the Chair relating to the application of the Rules of the House of Representatives to the procedure relating to a bill or joint resolution under this section shall be decided without debate. ``(D) Except to the extent specifically provided in the preceding provisions of this subsection, consideration of a bill or joint resolution under this section shall be governed by the Rules of the House of Representatives. ``(4)(A) A motion in the Senate to proceed to the consideration of a bill or joint resolution under this section shall be privileged and not debatable. An amendment to the motion shall not be in order, nor shall it be in order to move to reconsider the vote by which the motion is agreed to or disagreed to. ``(B) Debate in the Senate on a bill or joint resolution under this section, and all debatable motions and appeals in connection therewith, shall not exceed 10 hours. The time shall be equally divided between, and controlled by, the majority leader and the minority leader or their designees. ``(C) Debate in the Senate on any debatable motion or appeal in connection with a bill or joint resolution under this section shall be limited to not more than 1 hour, to be equally divided between, and controlled by, the mover and the manager of the bill or joint resolution, except that in the event the manager of the bill or joint resolution is in favor of any such motion or appeal, the time in opposition thereto, shall be controlled by the minority leader or his designee. Such leaders, or either of them, may, from time under their control on the passage of a bill or joint resolution, allot additional time to any Senator during the consideration of any debatable motion or appeal. ``(D) A motion in the Senate to further limit debate on a bill or joint resolution under this section is not debatable. A motion to recommit a bill or joint resolution under this section is not in order. ``(e) Amendments Prohibited.--No amendment to a bill or joint resolution considered under this section shall be in order in either the House of Representatives or the Senate. No motion to suspend the application of this subsection shall be in order in either House, nor shall it be in order in either House to suspend the application of this subsection by unanimous consent. ``(f) Requirement To Make Available for Obligation.--Any amount of discretionary budget authority proposed to be rescinded in a special message transmitted to Congress under subsection (b) shall be made available for obligation on the day after the date on which either House defeats the bill or joint resolution transmitted with that special message. ``(g) Definitions.--For purposes of this section-- ``(1) continuity of a session of either House of Congress shall be considered as broken only by an adjournment of that House sine die, and the days on which that House is not in session because of an adjournment of more than 3 days to a date certain shall be excluded in the computation of any period; and ``(2) the term `discretionary budget authority' means the dollar amount of discretionary budget authority and obligation limitations-- ``(A) specified in an appropriation law, or the dollar amount of budget authority required to be allocated by a specific proviso in an appropriation law for which a specific dollar figure was not included; ``(B) represented separately in any table, chart, or explanatory text included in the statement of managers or the governing committee report accompanying such law; ``(C) required to be allocated for a specific program, project, or activity in a law (other than an appropriation law) that mandates obligations from or within accounts, programs, projects, or activities for which budget authority or an obligation limitation is provided in an appropriation law; ``(D) represented by the product of the estimated procurement cost and the total quantity of items specified in an appropriation law or included in the statement of managers or the governing committee report accompanying such law; or ``(E) represented by the product of the estimated procurement cost and the total quantity of items required to be provided in a law (other than an appropriation law) that mandates obligations from accounts, programs, projects, or activities for which dollar amount of discretionary budget authority or an obligation limitation is provided in an appropriation law.''. (b) Exercise of Rulemaking Powers.--Section 904 of such Act (2 U.S.C. 621 note) is amended-- (1) by striking ``and 1017'' in subsection (a) and inserting ``1013, and 1018''; and (2) by striking ``section 1017'' in subsection (d) and inserting ``sections 1013 and 1018''. (c) Conforming Amendments.-- (1) Section 1011 of such Act (2 U.S.C. 682(5)) is amended-- (A) in paragraph (4), by striking ``1013'' and inserting ``1014''; and (B) in paragraph (5)-- (i) by striking ``1016'' and inserting ``1017''; and (ii) by striking ``1017(b)(1)'' and inserting ``1018(b)(1)''. (2) Section 1015 of such Act (2 U.S.C. 685) (as redesignated by subsection (a)) is amended-- (A) by striking ``1012 or 1013'' each place it appears and inserting ``1012, 1013, or 1014''; (B) in subsection (b)(1), by striking ``1012'' and inserting ``1012 or 1013''; (C) in subsection (b)(2), by striking ``1013'' and inserting ``1014''; and (D) in subsection (e)(2)-- (i) by striking ``and'' at the end of subparagraph (A); (ii) by redesignating subparagraph (B) as subparagraph (C); (iii) by striking ``1013'' in subparagraph (C) (as so redesignated) and inserting ``1014''; and (iv) by inserting after subparagraph (A) the following new subparagraph: ``(B) he has transmitted a special message under section 1013 with respect to a proposed rescission; and''. (3) Section 1016 of such Act (2 U.S.C. 686) (as redesignated by subsection (a)) is amended by striking ``1012 or 1013'' each place it appears and inserting ``1012, 1013, or 1014''. (d) Clerical Amendments.--The table of sections for subpart B of title X of such Act is amended-- (1) by redesignating the items relating to sections 1013 through 1017 as items relating to sections 1014 through 1018; and (2) by inserting after the item relating to section 1012 the following new item: ``Sec. 1013. Expedited consideration of certain proposed rescissions.''. (e) Application.--Section 1013 of the Congressional Budget and Impoundment Control Act of 1974 (as added by subsection (a)) shall apply to amounts of discretionary budget authority provided by appropriation Acts (as defined in subsection (g)(2) of such section) that are enacted after the date of the enactment of this Act. SEC. 3. TERMINATION. The authority provided by section 1013 of the Congressional Budget and Impoundment Control Act of 1974 (as added by section 2) shall terminate effective on the date in 2012 on which the Congress adjourns sine die.
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Title: A bill to establish procedures for the expedited consideration by Congress of certain proposals by the President to rescind amounts of budget authority Summary: Budget Enforcement Legislative Tool Act of 2008 - Amends the Congressional Budget and Impoundment Control Act of 1974 to authorize the President to propose, at any time and in any manner provided in this Act, the rescission of any discretionary budget authority in an appropriation Act. Prohibits funds made available for obligation under this procedure from being proposed for rescission again. Sets forth requirements for: (1) the President's transmittal to Congress of a special message regarding a proposed rescission; and (2) expedited consideration of such proposal.
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Summarize: Toys “R” Us Inc. told employees Wednesday the struggling big-box retailer will sell or close all its U.S. stores, a collapse that threatens up to 33,000 American jobs in the coming months. The 70-year-old chain, which filed for bankruptcy protection in September, has more than 700 remaining U.S. locations, including Babies “R” Us stores. It would be one of the biggest retail liquidations since Sports Authority filed for bankruptcy in 2016 with 14,500 workers and closed more than 460 stores.... Game Over For Toys R Us: Chain Going Out Of Business Enlarge this image toggle caption Jack Taylor/Getty Images Jack Taylor/Getty Images Updated at 7:10 p.m. ET After decades of being the go-to toy store for many Americans, Toys R Us is officially going out of business. Unable to get its finances in order through a months-long bankruptcy process, the retail chain has reached the end of the line. Toys R Us has informed employees that the company plans to sell or close all of its U.S. stores, according to a person familiar with the plans who spoke anonymously because the official liquidation filing has not been publicly disclosed yet. The person did not say how long the wind-down will take but said the shutdown will not be immediate. The chain employs more than 30,000 people in the U.S. and includes Babies R Us stores. Toys R Us has struggled with a heavy load of debt inherited from a 2005 buyout, as well as intense competition from Walmart, Amazon and Target — made worse by disappointing holiday sales. The chain — whose history traces back to a post-World War II baby furniture store — has spent many decades as the country's largest dedicated toy emporium. In 2017, Toys R Us accounted for roughly one-fifth of toy sales in the U.S., according to Jefferies analyst Stephanie Wissink. But in recent years, Toys R Us has found itself amid a trifecta of demographic and social changes, Wissink says. Today's parents are the millennial generation who grew up with the Internet and approach purchasing decisions and time they spend with children differently from baby boomers. Generally, foot traffic is falling at brick-and-mortar stores. And children are playing differently than they used to decades ago. "Kids are generally happily scheduled many nights of the week," Wissink says, "so the ability to devote dedicated time to play is being constrained." They spend more time on activities and with electronic screens, she says. Wissink also points out that the toy business is seasonal — three-quarters of sales take place during the holidays. This has weighed on Toys R Us, especially in the face of massive pressure from Amazon and Walmart. Without Toys R Us, Wissink estimates that 85 percent to 90 percent of the shopping that would normally happen at the toy chain would shift to other retailers. "What I worry about are many, many small companies," she says — toymakers not big enough to negotiate deals with the giant retailers, but which have relied on being discovered by Toys R Us shoppers. "Many of those companies are likely going to be up for sale," Wissink says. At a Toys R Us store in Bailey's Crossroads, a Virginia suburb of Washington, D.C., numerous shoppers were turning up this week to spend their gift cards, worried about the impending closings. Mary Henely brought her son Thomas to buy some Lego toys. "I recognize that it's a sign of the times with online shopping, perhaps. But I think it's disappointing," said Henely, who said she shops at Toys R Us about once a month. Eunice Turner came to the store to buy in-line skates for her daughter. "It's sad," she said about the decline of Toys R Us. "In wintertime I look forward to going to Toys R Us, because this is the only place where kids can actually see toys and play — more than Kmart, more than Walmart and all those places. This is just meant for kids." Business Eyeing Toys R Us' Future, Analyst Suggests Hands-On Displays Eyeing Toys R Us' Future, Analyst Suggests Hands-On Displays Listen · 4:13 4:13 The company has been laden with $5 billion in debt — a relic of a 2005 leveraged buyout. Private equity firms Bain Capital and KKR, together with real estate investor Vornado Realty Trust, took over Toys R Us and loaded the company with debt. For years, Toys R Us paid a hefty interest fee. Meanwhile, the chain lagged further behind in technology and online sales strategy, while missing the mark on some major investments, such as licensed Star Wars toys and Lego movies. When Toys R Us declared bankruptcy in September, the company said it would keep its stores open while it tried to regain financial footing. In January, Toys R Us announced plans to close almost 200 stores. While most retailers saw a boon from a perky holiday shopping season, Toys R Us faced what it called "operational missteps." According to The Wall Street Journal, Toys R Us owes a lot to the biggest toymakers: "Mattel and Hasbro are among Toys 'R' Us's biggest unsecured creditors. Mattel is owed more than $135 million, while Hasbro is owed $59 million, according to court papers." NPR's Ian Wren contributed to this report.
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Summary: "We're putting a for sale sign on everything," the Wall Street Journal quotes CEO David Brandon as telling Toys R Us employees Wednesday. The company, which started as a baby furniture store following WWII, will be closing or selling its over 700 US stores, putting up to 33,000 jobs at risk. NPR reports the writing has been on the wall for Toys R Us for some time, despite accounting for around 20% of all US toy sales in 2017. The toy-seller has been unable to put much of a dent in the nearly $8 billion of debt it largely acquired in a 2005 buyout, according to the Washington Post. It filed for bankruptcy in September and announced it was closing 182 stores in January. "The last six months have been pure hell," the Journal quotes Brandon as saying. Toys R Us has been hurt by competitors with better deals like Amazon and Walmart, and Brandon says sales during the most recent holiday season were "no short of devastating." Brandon says shoppers who failed to support Toys R Us in the past year "will all live to regret what's happening here." A group of toy makers-to which Toys R Us owed over $21 million when it filed for bankruptcy-is seeking to buy up to 400 stores to continue operating under the Toys R Us name. Brandon says "all anyone has to do is offer one dollar more" than what liquidation firms are offering. Employers were told store closures would be spread out over months. Toys R Us will reportedly pay employees at least 60 days of salary and benefits.
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Write a title and summarize: Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. Partition, the elementary form of mitosis that segregates sibling replicons in bacteria, depends critically on replicon-specific NTPases. These proteins, generically termed ParA, contact their replicons by interacting with the cognate ParB protein bound to a set of short sequence repeats (parS) that serves as the centromere. The interaction provokes dynamic behaviour of ParA which somehow directs replicon copies to the new cells-to-be. By far the most common type of partition NTPase is characterized by a variant ATP-binding Walker A box motif [1], [2] (reviewed by Vecchiarelli et al. [3]). These ParAs are specified by most low copy number plasmids and are the only type encoded by sequenced chromosomes. Mutation of conserved ATP interaction motifs in ParA proteins leads to frequent mis-localization and segregation failure, particularly of low copy-number plasmids [4]–[8], leaving no doubt that ATP is essential to partition. Just how ATP is involved is harder to define. The ATP hydrolysis activity of Walker-box ParA proteins is intrinsically very weak but is synergistically stimulated in vitro by cognate ParB proteins and by DNA [9]–[11], particularly when the latter includes the centromere [12]. Likewise, the ability of these ParA proteins to migrate en masse from end to end of the nucleoid a few times per cell cycle, presumed to be relevant to plasmid segregation [6], [13], is seen only in the presence of the cognate ParB and parS. These observations, together with the parallel effects of ATPase mutations on partition and ParA dynamics and the correlated movements of ParA and its plasmid [6], [13]–[16], argue for the involvement of ATP hydrolysis in the partition mechanism. On the other hand, binding of ATP without hydrolysis changes the conformation of ParA proteins [7], [17] and alters their properties: it facilitates dimerization of ParA [18]–20 and it enables dimers to bind to non-specific DNA [20]–[23] as well as to each other to form polymeric assemblies [4], [14], [15], [21], [22], [24], [25]. Thus, even in the case of partition-defective mutants that demonstrably bind but fail to hydrolyse ATP, it has not been possible to exclude conformational effects as the key reason for the functional deficiency. We have recently reported that in the case of the Sop partition system of the Escherichia coli plasmid F, mutation of two arginine residues in the SopB N-terminus, neither of which significantly affects affinity for SopA, reduces stimulation of SopA-catalyzed ATP hydrolysis in vitro [12]. The mutants are interesting for two reasons. One is mechanistic: the characteristics of R36 and R42 mutants are consistent with the former acting as an arginine-finger [26], inserting itself into the ATP-binding pocket of SopA to facilitate processing of the transition state, and the latter serving to optimize positioning of the finger, as previously suggested for analogous mutations in the ParG partition protein of plasmid TP228 [4]. The other reason is the delegation of responsibility for modulating ATP hydrolysis to a physically distinct partner protein. It should be possible to use the SopB arginine-finger mutants to reduce ATP hydrolysis by wild type SopA protein whose conformational responses to ATP binding are normal, and thus to distinguish the substrate and cofactor roles of ATP for any activity in which SopA is involved. We report here that SopB-mediated stimulation of ATP hydrolysis by SopA is needed to restrain oscillatory movement of SopA, to stabilize plasmid positioning and to limit plasmid clustering. We substituted the R36 and R42 alleles for wild type sopB in two normally stable mini-F plasmids. In one, pDAG218, a promoterless lacZ gene is inserted between sopB and the sopC centromere, allowing direct monitoring of plasmid loss and of transcription from the natural, autoregulated sop promoter. Assays of plasmid retention during growth in non-selective minimal-glycerol medium (Figure 1A) showed the mutants to be surprisingly stable. The R36A mutation caused about a six-fold increase in mini-F loss, the other mutations almost none, within measurement error. Assays of β-galactosidase showed that only the R36A mutant caused a significant rise in transcription, 1. 8-fold (data not shown), a result confirmed by the 1. 6-fold increase in SopA protein determined by immunoblot assay of cell extracts (Figure S1A). To determine whether higher sopAB expression accounted for the instability of the R36A mutant relative to the others, we assayed stability of mutant derivatives of the other mini-F, pDAG173, in which sopAB is transcribed constitutively from pL-tetO [27]. With Sop protein levels thus rendered uniform (though 3. 4-fold higher than with pDAG218; Figure S1A), the sopB mutant derivatives of pDAG173 showed much the same degree of modest instability as their pDAG218 equivalents (Figure 1A); the increased loss rate of the R36K mutant is offset to some extent by the variability inherent in low loss rate estimates, such that we cannot assess its significance. The essential result is that the R36A, and possibly R36K, mutation results in slight destabilization of mini-F. Attempting to increase the sensitivity of the stability assay, we reconfigured it by placing the sopAB operon under plac on an oligocopy (pSC101) vector, in trans to a mini-F ΔsopAB derivative. We considered that SopB protein radiating from the point of its production near the sopAB 3′ end might well be less plentiful at sopC owing to free diffusion and increased competition from non-specific DNA, resulting in formation of smaller, less effective SopB assemblies that amplify the handicap imposed by the mutant SopB defects. While wt sopAB still fully stabilized the mini-F (0. 005% loss per generation) in this trans test, the mutations provoked much higher mini-F instability than when in cis (Figure 1B). The R36A mutation had the strongest effect, causing ∼1000-fold destabilization, about half that seen with no active partition, while the R36K and R42A mutations destabilized less, ∼360- and 240-fold respectively, and the R42K mutation not at all. Whether or not our rationale for applying the trans test is correct, by amplifying loss rates the test serves as an indicator of Sop system performance. All succeeding experiments were carried out using this trans configuration. Alteration in SopB affinity for DNA, for SopA or for itself is unlikely to explain the partition phenotypes seen. We have shown that the mutant SopB proteins are unchanged with respect to specific and non-specific DNA binding, and exhibit affinity for SopA similar to that of wt SopB [12]. They also behave as wt SopB in destabilizing mini-F when in excess and in silencing transcription from a nearby promoter, two properties which depend on interaction of SopB dimers via their N-terminal domains (Figure S4). More importantly, the deficiency of each SopB in ATPase stimulation is closely correlated with the rate of mini-F loss (Figure 1B). Thus we can be confident that the restricted ability of the SopB R36A, R36K and R42A mutant proteins to stimulate SopA-mediated ATP hydrolysis is the major or exclusive cause of the defective partition seen. Because ATP hydrolysis is thought to be necessary for the segregation event that localizes plasmids in cell-halves, we next sought clues to the role of SopB-stimulated ATP hydrolysis by directly visualizing mini-F molecules. To observe mini-F, we inserted an array of Tet repressor binding sites into a mini-F equivalent to that used in the trans stability tests, and introduced the resulting plasmid (pDAG848) into a TetR: : Gfp-producing strain for visualization by fluorescence microscopy. The concentration of Sop proteins made from the pSC101-based plasmids is only about twice the wt mini-F level (Figure S1B; Table S1), owing to regulation of sopAB expression by Lac repressor in this MG1655 (lacI+) -based strain. Figure 2A shows that cells growing in MGC at 1. 2 generations per hour, with an average of 3 copies of pDAG848 per cell, contained 1. 2-fold more foci in the presence of wt SopB than of SopB-R36A. The difference was significant (see legend) but not strong, and was not increased by addition of the R42A mutation (Figure 2A). It was accentuated in cells growing in LB at 2. 4 generations per hour with an average of 4. 6 copies per cell [28], as shown together with foci/cell for the other sopB alleles in Figure 2B. An average of 3 foci per cell were seen in cells with wt SopB, 2. 2 with the R42K mutant, 1. 5 with R36K or R42A, and 1. 2 with R36A. Consistent with this observation, R36A foci were on average brighter than wt foci (Figures 2C, S3A). Copy number per mini-F-carrying cell was not affected by the nature of the SopB protein (Table S2). Hence, whereas in the presence of wt SopB at least half the foci consist of a single plasmid molecule, the number of molecules per focus rises progressively as the ability of SopB to stimulate ATP hydrolysis declines, up to nearly four in the case of R36A. The frequency of cells with no focus also rises, consistent with destabilization by the R36A allele in LB-grown cells (Table S1). SopB-stimulated ATP hydrolysis apparently contributes to splitting mini-F clusters or preventing their formation. To determine whether the R36/42 mutations might influence plasmid clustering independently of SopA we observed pDAG848 supplied with the SopB proteins only. Strong destabilization of mini-F by removal of SopA has been attributed to failure to resolve clusters [29], but inability to direct plasmid movement could also contribute [18], [22]. pDAG848 foci were indeed not seen in half the cells and seldom numbered more than one when present (Figure 2A). In addition, the preferred mid-cell positioning of single mini-F foci gave way to a more polar localization (Figures 2D, S3B). The same reduction in focus number and change in focus distribution occurred whether cells carried the wt or the R36A allele of sopB. Likewise, in the presence of SopA, fluorescent derivatives of wt and R36A SopBs both formed foci distributed over the long cell axis like those of labelled mini-F (Figure S3C). We thus saw no evidence for an effect of the R36/42 residues other than stimulation of ATP hydrolysis by SopA. The elements that most effectively stimulate SopA to hydrolyse ATP, SopB and sopC, are also needed to activate the migration of SopA between nucleoid poles, implying that SopB mutants with significant deficiencies in ATPase stimulation, R36A, R36K and R42A, would reduce or eliminate SopA relocation. In order to examine dynamic behaviour of SopA in the presence of wild type and mutant SopB proteins, we introduced into the chromosome of both DLT1900 and DLT2583 a sopA: : xfp fusion whose product fully stabilizes mini-F [18]; the construction restored lacI and lacY to DLT1900, putting sopA: : xfp expression in this strain under LacI control and enabling the two strains to be used interchangeably. The DLT1900 derivative (DLT2687) was transformed with pYAS47 (pSC101-plac: sopAB) and pDAG209 (mini-F-Δ (sopAB) sopC+), and the movement of SopA: : Xfp was followed by fluorescence microscopy. As seen elsewhere [13], fluorescent SopA moved from end to end of the nucleoid 3–5 times per cell cycle provided sopC and wild type SopB were present (data not shown). We then measured the frequency of SopA relocation after substituting the mutant R36 and R42 codons in sopB. The time-lapse sequences shown in Figure 3A illustrate the surprising result depicted in Figure 3C - a major increase in oscillation frequency for all mutants except R42K, which behaves as wt. The average period of migration to the far nucleoid pole and back was 3. 2 minutes for the R36A mutant and 3. 6 minutes for R36K and R42A, respectively 2. 7- and 2. 4-fold less than the return trip with wt SopB (8. 6 minutes). The increase in SopA relocation frequency was only a little higher for the R36A mutant than for R36K and R42A, rather than proportional to ATPase stimulation deficiency as with mini-F in stability and clustering (Figures 1C, 2B); a factor other than reduced ATP hydrolysis may be rate-limiting at this high oscillation frequency. Total SopA levels in these experiments were 3–4 times higher than that in mini-F-carrying cells, but relocation frequencies were comparable at SopA concentrations ∼ten-fold higher still (Figure S2); the high frequencies are thus not a result of SopA excess. The superimposed kymographs of Figure 3B suggested that the more rapid oscillation of SopA in the presence of mutant SopBs arose from virtual elimination of the normal pause between SopA' s arrival at one end of the nucleoid and its setting out for the other. Accordingly, we repeated the experiment with shorter image-capture intervals. Because of the shorter viewing period the progressive decline of oscillation rate, owing to decelerating growth or other factors, has less effect, so that the average SopA return trip was shorter in this experiment, 1. 8 minutes for R36A; nevertheless, the 3-fold increase relative to wild type was comparable to the 2. 7-fold of 30-minute acquisition experiments, as shown in Figure 4. These kymographs confirm the initial indications that the difference in oscillation rates arises mainly from the short time that SopA spends at the nucleoid ends. The average speeds of SopA movement along the nucleoid were similar whether SopB was wt or mutant, whereas the average dwell-time decreased five-fold with the mutant SopB. Indeed the relative intensities of nucleoid-end signal in wt and mutant SopB cells suggest that in the latter, SopA arriving at the ends does not wait for following molecules before setting out again, although we have not ruled out the possibility that in this case much of the SopA is dispersed and relatively immobile. Western blotting showed similar levels of SopA: : Xfp protein in both cases (Figure S1C); the difference is thus not due to blockage by piled up or aggregated protein. These observations imply that stimulation of ATP hydrolysis by SopB slows the process by which SopA reinitiates its migration but is not required for the migration itself. Partition ATPase dynamics is generally thought to determine the trajectory and destination of segregating plasmid copies. Accordingly, we next investigated how the R36 and R42 mutations affect mini-F movement. The average positioning of mini-F plasmids was little altered by substitution of SopB-R36A for wild type. Figure 5A shows the distances from pole to pDAG848 focus in the one- and two-focus classes of cells grown in MGC medium (Figure 2A); although R36A foci predominate at the margins of the distributions, the widening of the spread in R36A cells is slight. Figure 5B summarizes these data as plasmid focus position relative to cell length. Regardless of whether the cells were producing wt SopB or the most defective mutant, R36A, about 60% of single foci were within the central fifth of the cell and about 80% of double foci were grouped around the one-third and two-thirds positions. Similar positioning was seen in cells grown in LB medium (Figure 5B), despite the high degree of plasmid clustering observed in such cells (Figure 2B). Plasmid clusters appear to be as readily positioned as single molecules by the Sop partition apparatus. The most straightforward interpretation of this pattern is that the capacity of SopB to stimulate ATP hydrolysis does not strongly affect the average positions of segregation units once these have been defined by initial separation events. Nevertheless, abnormal positioning was evident in certain R36A cells whose two foci were clearly on the same side of mid-cell (blue symbols in Figure 5A); unless rectified before the next cell division this mis-positioning would result in partition failure. To examine mini-F movement, we observed pDAG848 foci in MGC-grown cells after labelling the plasmid by brief induction of tetR: : gfp. Cumulative plasmid trajectories (Figure 6A) show mini-F movement to be less constrained in the presence of the R36A mutant than with wt SopB. By imaging pDAG848 in thirty single-focus cells at 5-second intervals over 5 minutes, we could estimate that on average it moves about 1. 5 fold faster (from cumulative distance, Figure 6B). We discerned no consistent relationship between SopA and mini-F movements when these were monitored simultaneously but we could estimate from the oscillation data (above) that in the 5 min viewing time a SopA wave would pass on average once for wt SopB and three times for the mutant. The illustrative kymographs in rows 1 and 2 of Figure 6D reveal that compared to wt SopB, which generally maintains mini-F steady near the centre or quarter positions (row 1 - a–e; h and i show the two exceptions), the R36A mutant provoked a jumpy, exaggerated movement (row 2 - e, f are exceptions) and a frequent approach to the cell poles (c, g, h, i) not seen with wt. The frequencies of detectable direction reversal were similar, 13. 6+/−5. 5 (s. d.) for wt and 14. 6+/−7. 1 for R36A. Kymographs of 23 two-focus cells (Figure 6E) showed the maintenance of focus position to be, again, fairly constant with wt SopB and erratic with the R36A mutant (d in each row shows exceptions). Nevertheless, the time-averaged locations of the foci corresponded to the quarter positions of the long cell axis, as determined earlier (Figure 5). Correlated deviations in the positions of both foci were infrequent - 3 for wt (e. g. h, i), 4 for R36A (e. g. e, f, i) - but instances of temporary occupation of a single cell-half by both foci were more common for the mutant SopB (7 - e. g. c, e, g, h) than for the wt (1 - d). In R36A mutant cells the two foci often approached each other closely, presumably a simple result of their wider ambit, and in one of the 50 cells examined in this experiment were seen to fuse (row 2 - h). The one close approach seen in 50 wt SopB cells (row 1 - d) did not result in fusion. This frequent and occasionally protracted positioning of both mini-F copies on one side of the eventual division septum in SopB-R36A cells, as seen in the four examples of Figure 5A, is consistent with the higher plasmid loss rate associated with this mutant (Figure 1B). Our approach to defining the role of ATP hydrolysis differs from previous attempts, in that it targets the activator, SopB, rather than the instrument, SopA. The assumption that the behaviour of a mutant ATPase which binds but does not hydrolyse ATP is due to a defect in hydrolysis per se can be misleading; inability to hydrolyze ATP may be a secondary consequence of the mutation or be relevant only at a later step in the pathway. For example, the partition disabilities initially attributed to defective ATP hydrolysis by the K122Q mutant of P1 ParA [30] and the K120R mutant of SopA [7] are now known to be caused by impairment of conformational transitions, seen by changes in tryptophan fluorescence [23] and in CD spectrum (helicity) [7] respectively; these conformational shifts allow the wild type proteins to bind to DNA [23] and to polymerize (our unpublished data), activities which require ATP binding but not hydrolysis. Other observations of reduced ParA dynamics associated with mutations that diminish ATPase activity [6], [13], [15], [16] might require reinterpretation. We find that when hydrolysis itself is unambiguously targeted, the consequences for plasmid stability are surprisingly modest. Even the SopB-36A mutant, which generates only 5% of normal ATPase activity, allows relocation of SopA along the nucleoid at normal speed, movement of plasmids and a high level of plasmid stability. Indeed we could obtain accurate estimates of partition deficiency only by distancing the sopAB operon from the mini-F test plasmid. Our initial presumption was that reduction of SopB concentration near sopC aggravates the partition deficiencies of the SopB mutants, but other explanations are possible; for example the ten-fold higher average level of Sop proteins in this experiment (Figure S1A) might alter SopA adherence to the nucleoid, as seen in vitro [31]. The possibility that the weak partition phenotype results from a smaller deficit in ATPase stimulation in vivo than that measured in vitro must be put aside provisionally; the lack of a specific in vivo ATPase assay prevents its being tested. The further possibility that the R36 and R42 mutations also affect SopA activity in a way that compensates for loss of ATPase stimulation is not formally excluded but is rendered unlikely by the strong correlation of increased plasmid instability and clustering with in vitro ATPase reduction (Figures 1B, 2). Such instability as there is appears to stem mostly from plasmid clustering or failure to split clusters (Figure 2). This finding does not sit comfortably with current views of the partition mechanism. The primordial model of Leonard et al [20] and those evolved from it [15], [16], [22], [23], [32], [33] posit hydrolysis of ATP as the primary activator of ParA relocation and hence of plasmid segregation. Binding of ATP induces SopA, like other ParA proteins, to undergo a conformational change that enables it to associate with non-specific DNA [21]–[23] and thereby to coat the nucleoid [13], [14], [18], [34]. It also promotes interaction of SopA with SopB [12]. According to these models, the mini-F partition complex thus encounters SopA-ATP primed by contact with the nucleoid to hydrolyse ATP, and prompts it to do so. After hydrolysis SopA can no longer bind to DNA and departs for the cytoplasm where it reacquires ATP and binds to DNA further along the nucleoid, setting up a wave-like relocation between the nucleoid ends. By acting as a mobile attractor, the SopA wave creates and maintains distance between plasmid copies [35]–[38]. All type I plasmid partition systems studied conform to this general description, regardless of differences in detail. Thus the main role of ATP hydrolysis is currently thought to be dislodgement of SopA from the nucleoid. But our results (Figures 3,4) show that SopA relocation, which requires detachment from the nucleoid, is not impeded by severely weakened hydrolysis. The bulk of SopB-stimulated ATP hydrolysis thus appears to be unneeded for SopA detachment from the nucleoid and relocation. On the other hand, basal hydrolysis stimulated two-fold by DNA or residual SopB-mediated stimulation (5% for SopB-R36A) may well suffice to activate or maintain a largely auto-propagating mode of SopA relocation. The observation that pB171 ParA: : Gfp continues to retract polewards after disconnection from ParB [16] is in line with this possibility. But if so, a role other than SopA detachment from the nucleoid must be sought for most of the ATP hydrolysis that SopB stimulates. Furthermore, other mechanisms of SopA detachment must be considered. One possibility is competition by the SopB molecules massed in the extended partition complex for the nucleoid surface occupied in meta-stable fashion by SopA-ATP, which could be examined using the approach of Havey et al. [39] for observing behaviour of dynamic complexes of the P1 Par system components in vitro. An alternative is suggested by the recent finding that in an in vitro analogue of the Sop system SopB enhances dissociation of SopA-ATP from a DNA surface [31]. The authors suggested that SopB induces a conformational change in SopA that weakens its affinity for DNA and potentiates subsequent ATP hydrolysis. Specific removal of hydrolysis from this process would generate SopA-ATP unable to immediately rebind the nucleoid but primed to relocate along it. Indeed an even stronger effect of SopB revealed in this study was the inhibition of SopA-ATP association with DNA, implying that once removed from the nucleoid SopA-ATP would be unable to reassociate with it until outside the extended partition complex zone. Figure 7 illustrates the three partition processes in which we propose ATP hydrolysis to play a direct role. Hydrolysis-independent dissociation from DNA, as discussed above, would help account for one of them, the high frequency of SopA: : Xfp oscillation seen in response to the SopB R-finger mutants (Figures 3,4). In the presence of wt SopB, SopA waves were interrupted by a waiting period at the nucleoid poles (as also seen by Hatano et al. [13]), whereas with the R36A mutant this period was very brief, perhaps non-existent at the level of the individual SopA molecule. The difference implies that SopA molecules can arrive at the nucleoid tip in distinct forms that determine their ability to reverse direction. SopA arriving in response to the R36A mutant is already competent to relocate and does so immediately, whereas SopA stimulated by wild type SopB requires adaptation before setting out again, as illustrated in Figure 7B. This conditioning could consist of a time-consuming conformational change in the ATP-bound form of SopA, such as that culminating in ability of P1 ParA to bind DNA [23]. A similar case of apparent reconditioning at nucleoid ends was reported by Autret & Errington [40], who observed that the MinD division topology factor was implicated in relocation of the SopA homologue, Soj, from nucleoid ends in Bacillus subtilis. In a prior study [41] these authors had isolated a mutant of the cognate SopB homologue, Spo0J, (spo0J19) that increased the frequency, but not the speed, of Soj relocation. Modelling of Soj dynamics led Doubrovinski & Howard [36] to propose that Spo0J19 might increase relocation frequency by expelling Soj from the nucleoid in a form that rebinds the nucleoid ends without involving MinD, clearly analogous to bypassing the SopA conditioning that we suggest is a normal prelude to relocation. Our observations suggest that ATP hydrolysis might function as a means of restricting the rate of SopA relocation. Such regulation could be needed to maintain stable positioning of the plasmid. The effects of the mutant SopBs on mini-F positioning are intuitively easier to relate to plasmid stability. As well as driving segregation of mini-F copies, SopA restricts their subsequent movement [35]. This restriction broke down when the partition complexes contained SopB-R36A instead of wild type. Most of the traces shown in Figure 6 are presumably of positioned mini-F molecules not in the first phase of active segregation. ATPase stimulation is thus necessary for positional stability. If ATP hydrolysis normally repositions plasmids by removing SopA from the nucleoid, we would expect R36A partition complexes to move more sluggishly than wild type, not faster as seen. It is possible that the duration of contact between SopA-ATP and the partition complex is the key determinant of plasmid movement in this context, such that the longer contact expected from weak hydrolysis stimulation by an R36A partition complex might result in the more prolonged pulling of the plasmid by retracting SopA, accounting for the greater displacements seen (Figure 6; depicted in Figure 7C). Regardless of mechanism, the wider ambit of mini-F carrying SopB-R36A could lead to instability by increasing the chance of all plasmid copies finding themselves in the same cell half or in clusters (Figure 6D) at division. There is reason to question, however, whether temporary mis-positioning and cluster formation are the major causes of instability - fluorescent foci of various wild type plasmids have been seen to fuse [16], [42], [43] and fusion/splitting may be part of normal positioning [44]. The greater threat to stability might be failure to break clusters formed immediately after plasmid replication. The inter-plasmid links that form clusters are not based on pairing of partition complexes [45], but active partition breaks them [46]. We suggest that segregation of recent replicas requires ATP hydrolysis to play a different role from the triggering of a conformational switch that releases SopA from the nucleoid at later stages of positioning [31], namely provision of energy for the initial segregation event. We proposed previously that following replication SopB spreads from the partition complex onto nucleoid DNA, creating a DNA-free zone that permits SopA to polymerize and thus initiate partition [21]. The recent observation that in an in vitro reconstituted system SopA-ATP does not form filaments on a DNA carpet is consistent with this proposal [31]. Our evidence that clustering is accentuated in proportion to the deficit in ATPase stimulation (Figure 2) encourages a speculation on the role of SopA polymerization - the forced separation of clustered plasmids powered by hydrolysis of ATP (portrayed in Figure 7A), as an initial step that enables partition complexes to then be drawn apart by the retreating SopA wave. While we have no direct evidence for this proposal, it is compatible with the two-phase segregation of F plasmids observed by Gordon et al. [47], in which a very rapid initial separation was followed by gradual progress to the destination. Biochemical and microscopical study focused on this aspect of SopA/B behaviour is now needed to test the proposal In summary, our results reveal that SopB-mediated stimulation of SopA-catalyzed ATP hydrolysis serves mainly to split F plasmid clusters following replication and to prevent them forming later. SopA relocation and plasmid movement might depend on basal ATP hydrolysis but need little or no further stimulation by SopB. SopB-stimulated hydrolysis appears rather to regulate SopA relocation frequency. The contribution of basal hydrolysis could be approached using SopA mutated at residues directly involved in catalysis whose minimal conformational effects can be demonstrated. This altered perspective should be taken into account in attempts to determine how the partition of other replicons works. The strains are derivatives of E. coli K12 and are listed in Table 1. DH10B was used as a transformation recipient for routine plasmid construction. The base strains for mini-F loss rate determinations were MC1061 and DH10B. For microscopic observation, the following mutations were introduced by recombineering, or by P1 transduction from the recombineerants: Δ (araFGH) and Ωpcp18: : araE533 into MG1655 as in the construction of DLT1900 [9] to moderate arabinose induction, Ω (para-tetR: : egfp) to follow movement of mini-F (pDAG848; see below), and ΔlacZΩ (plac-sopA: : xfp) to monitor SopA relocation; details of the two latter constructions are given in Supplementary Information (Text S1). Cultures were grown at 37°C with aeration in Luria-Bertani (LB) broth, or in M9 salts-thiamine (1 µg/ml) supplemented with 0. 4% glycerol and either 0. 2% Casamino acids (MGC medium) or 20 µg/ml leucine, as noted in the text. When needed for selecting plasmid maintenance, antibiotics (Sigma) were added (µg/ml): kanamycin (30), spectinomycin (30), chloramphenicol (10). Plasmids are listed in Table 1. Construction outlines are given here, with details available on request. pDAG218: lacZ with its Shine-Dalgarno sequence was incorporated in the sopAB operon by insertion into the 75 bp AccI-ApaI interval between sopB and sopC in pDAG114. pDAG848: deletion of sopAB from pDAG114 and insertion 195 bp downstream from sopC of 2. 36 kb of the tetO array of pFX240. pYAS47: insertion of a PCR fragment containing sopAB with its Shine-Dalgarno sequence downstream from the lac promoter in pAM238. sopB alleles encoding the R36A-, R36K-, R42A- and R42K mutant SopBs were introduced into pDAG173, pDAG218 and pYAS47 by restriction fragment replacement to yield the derivatives listed in Table 1. pYAS70, -71: in-phase deletion of sopA codons 115–358 by cleavage with BstXI and SapI, Klenow end-filling and ligation. pYAS81: introduction of R36A (sopB nt106-7 CG>GC) to pYAS66 by quick-change mutagenesis. pJYB259, -260: insertion of a PCR fragment containing a fusion of sopB to mVenus [48], a derivative of Venus rendered monomeric by mutation A207K (this laboratory). Cells grown with selection for mini-F plasmids were diluted into antibiotic-free medium and the resulting cultures sampled at intervals for assay of the plasmid-free fraction as follows. pDAG173 and derivatives, and pDAG209: cells were spread on LB agar and the resulting colonies tested by replica plating to selective medium [49] or by the three-layer method described [50]. pDAG218 and derivatives: cells were spread on MGC agar containing X-gal (40 µg/ml) and white and blue colonies scored after incubation. Cells of strains expressing sopA: : xfp from the chromosomal lac promoter, DLT2687 and DLT2740, or tetR: : gfp from the tet promoter, DLT2583 and DLT2740, were transformed with the mini-Fs pDAG848 or pDAG209 and pSC101-based vectors expressing sopAB. Transformants grown overnight in MGC medium were diluted 1/400 into MGC (except as noted), supplemented in the case of DLT2740 derivatives with IPTG at 0–10 µM (see Results), and grown at 30°C to OD600 ∼0. 25, then sampled for observation; for visualization of pDAG848, tetO: : gfp was induced by addition of arabinose to 0. 05% 30 min beforehand. Samples were processed for microscopy as described [18]. Slides were maintained at 30°C in a thermostated chamber and examined by phase contrast and fluorescence microscopy using inverted microscopes: (i) Olympus IX81, images captured with a Coolsnap 2 (Roper) camera and processed using Metamorph software; (ii) Nikon Ti-E, acquisition with a EMCCD (Hamamatsu) camera (gain 144), and processing with NIS- element software. Metamorph software was used to generate kymographs, and to measure absolute distances (x, y) of mini-F displacement between images with the “Track Object” tool.
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Title: Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids Summary: Genes enabling bacteria to survive and thrive in challenging environments are very often found on small, non-essential DNA molecules called plasmids. Many plasmids are naturally present in the cell in very few copies and so risk being lost from one of the daughter cells upon division. These plasmids elaborate a partition system, functionally similar to mitosis, which assures their faithful inheritance. Chromosomes also generally possess such systems. We know that partition systems involve two proteins, that one (B) stimulates the other (A) to hydrolyse ATP, and that upon binding to A protein ATP confers properties needed for partition. ATP' s double action, as hydrolysis substrate and cofactor, complicates definition of its role in the mechanism. The novelty of our approach lies in use of B protein mutants that do not stimulate hydrolysis. Our results reveal that the major function of ATP hydrolysis is not to displace plasmid molecules to their positions in each cell half, as generally thought, but to split initial sibling plasmid pairs and prevent their reforming. This study is the first to dissect ATPase activity in vivo using normal A-protein ATPase, and so opens a new avenue to exploration of the mechanisms that ensure plasmid and chromosome inheritance.
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Summarize: A few miles away, a fresh barrier had been erected at a gap in the border fence where thousands of migrants had walked into Hungary in recent weeks, and new arrivals encountered only razor wire and a line of police officers pointing them back toward Serbia. Image Migrants in Horgos, Serbia, on Tuesday, by the newly built fence on the A5 highway that is stopping people from entering Hungary. Credit Sergey Ponomarev for The New York Times The migrant crisis that has rattled Europe and fractured its already shaky unity — producing heart-rending images of squalor, death and the joyful celebration of those who made it through — has now found fresh focus on this flat, forested border at the edge of the European Union. Just one day after European leaders failed to find a substantial collective approach to the crisis, and two days after Germany, Austria, Slovakia and the Netherlands instituted new border controls, Chancellor Angela Merkel of Germany and Chancellor Werner Faymann of Austria called for an urgent summit meeting next week in Brussels to again try to come up with a unified strategy. With refugees, fleeing from war, and migrants, weary of poverty, pouring in from the Middle East, Afghanistan, Pakistan and Africa, any obstruction quickly creates growing pools of the desperate. That is what happened last week at Budapest’s Keleti train station when Hungary temporarily cut off access for migrants to westbound trains. And it is what is beginning to happen now in northern Serbia. “We hope that the messages we have been sending migrants for a long time have reached them,” said Gyorgy Bakondi, an aide to Prime Minister Viktor Orban of Hungary. “Don’t come. Because this route doesn’t lead where you want to go.” Image A newly built fence stopped migrants in Horgos, Serbia, from entering Hungary on Tuesday. Credit Sergey Ponomarev for The New York Times Indeed, Hungarian officials said, after several days that saw record numbers of migrants crossing the border — 9,000 on Monday alone — the numbers were much lower on Tuesday, proof that people were heeding the warning, or at least waiting for the picture to clear. Media playback is unsupported on your device Media caption James Reynolds reports on protests by migrants at the Hungary-Serbia border Hungary has declared a state of emergency in two southern counties as tough new laws to stop migrants entering illegally came into force. Police said they had arrested 60 people accused of trying to breach a razor-wire fence on the border with Serbia. The state of emergency gives police extra powers and would allow troop deployments if parliament approves. The EU is facing a huge influx of migrants, many fleeing conflict and poverty in countries including Syria. Meanwhile, Germany and Austria are calling for a special meeting of EU leaders next week to discuss the crisis. German Chancellor Angela Merkel told a news conference that "this problem can only be solved together. It is a responsibility for the entire European Union". The EU's border agency said more than 500,000 migrants had arrived at the EU's borders this year, compared with 280,000 in 2014. The vast majority have come by boat across the Mediterranean. A boat following the most popular recent route, between Turkey and Greece, sank on Tuesday leaving 22 people dead, Turkish media reported. Starting on Tuesday, the EU has agreed to relocate 40,000 migrants from Greece and Italy to other EU states. But it has yet to agree on mandatory quotas for a further 120,000 asylum seekers. After the new Hungarian laws came into effect at midnight (22:00 GMT Monday), police sealed a railway crossing point that had been used by tens of thousands of migrants. Around midday there were tense scenes as hundreds streamed towards the fence, some searching for a way through and others starting a sit-down strike, throwing down food and water in protest at not being granted passage. Media playback is unsupported on your device Media caption Fergal Keane at the Hungarian border: "Police have been given extra powers under a state of emergency" Follow Fergal Keane on the Hungary-Serbia border Image copyright AP Image caption Hundreds have been waiting at the newly closed border fence Image copyright Reuters Image caption Hungarian police have started to make arrests Police buses will now take asylum applicants to registration centres, but if their applications are refused they will now be returned to Serbia rather than being given passage through Hungary, the BBC's Nick Thorpe reports from the border. Hungarian authorities said more than 9,000 - a new record - crossed into the country before the border was closed on Monday. Some 20,000 crossed into Austria. From Tuesday, anyone who crosses the border illegally will face criminal charges, and 30 judges have been put on standby to try offenders. The laws also make it a criminal offence - punishable by prison or deportation - to damage the newly-built four-metre (13ft) fence along Hungary's 175km (110 mile) border with Serbia. Hungarian government spokesman Zoltan Kovacs said "the official and legal ways to come to Hungary and therefore to the European Union remain open. That's all we ask from all migrants - that they should comply with international and European law." Will Hungary's clampdown work? Afghan boy's lonely start in Germany What next for Germany's asylum seekers? Caring for solo child refugees Crisis explained in graphics But the Serbian minister responsible for the migrant crisis, Aleksandar Vulin, argued the closure was unsustainable. "They will have to open the border," he said. The European Commission said it was seeking clarification of parts of the new legislation, to check whether it is in line with EU asylum rules. At talks in Brussels on Monday, a majority of states had agreed in principle to the idea of relocating a further 120,000 through mandatory quotas, and there was hope the proposal could be finally approved at a meeting on 8 October. Media playback is unsupported on your device Media caption Vice-President of the European Commission Frans Timmermans said closing all borders to asylum seekers was ''unrealistic, populist and simply impossible'' However, the Czech Republic, Slovakia and Hungary have opposed the quotas. "There was no consensus, several countries disagreed," Slovak Interior Minister Robert Kalinak said after the talks. German Interior Minister Thomas de Maiziere said on Tuesday there should be ways of "exerting pressure" on states that refused binding quotas, possibly by reducing the amount of EU funding they receive. But the Czech state secretary for the EU, Toma Prouza, said such threats were "empty but very damaging to all" A spokeswoman from UN refugee agency, Melissa Fleming, said she expected migrant "chaos" to continue in the absence of more decisive action by the EU, with migrants seeking a new route. Germany introduced temporary border controls on Monday. That slowed down the passage of migrants from Austria, where about 2,000 people slept in railway stations overnight. Austria - one of several EU countries to say it would tighten border controls - is starting to deploy hundreds of troops to help the police deal with migrant arrivals. The moves are a challenge to the EU's Schengen agreement on free movement, although the rules do allow for temporary controls in emergencies. Hungarian police officers guard the border at the Horgos border crossing into Hungary, near Horgos, Serbia, Tuesday, Sept. 15, 2015. Hungary has declared a state of emergency in two of its southern counties... (Associated Press) BUDAPEST, Hungary (AP) — The latest developments as European governments rush to cope with the huge number of people moving across Europe. All times local (CET): ___ 12:50 p.m. Hungarian authorities say they detained 60 migrants for attempting to illegally enter the country's southern border with Serbia by breaching a razor-wire fence. The detentions on Tuesday came as a set of new laws took effect that are meant to stop the huge flow of migrants through the country. According to the new legislation, it is a felony to cross or damage to the 4-meter (13-foot) fence that the country has built along its southern border with Serbia. Gyorgy Bakondi, homeland security adviser to Prime Minister Viktor Orban, said authorities caught 45 people trying to cross at the southern border and 15 deeper in the country. They got across by damaging the fence and are now in police custody and are being charged with committing offenses under the new laws. Authorities are repairing the spots where the fence was damaged. ___ 12 p.m. The International Organization for Migration is predicting more deaths in the Aegean Sea and increased strains on Greece because of EU "indecision" about how to handle an influx of migrants and refugees to Europe. IOM spokesman Leonard Doyle said Tuesday that decisions by some European countries to clamp down travel across their borders might have a "domino effect" and affect countries like Greece, where it's harder to stop people — mostly Syrian refugees — from crossing from the Turkish coast. Some migrants have called into an anti-human trafficking phone hotline set up by IOM while they are treading water in the Aegean. ___ 11:50 a.m. Turkish media say 22 people, including four children, have drowned after a 20-meter wooden boat sank off Turkey's coast. The private Dogan agency said 211 people were rescued from Tuesday's sinking in international waters between Datca in Turkey and the Greek island of Kos. It wasn't immediately clear where the people were from, or what caused the sinking. The incident comes two days after 34 people, including 15 children, drowned off the Greek island of Farmakonissi when the wooden boat they were on sank. Nearly 100 other people were rescued. More than 250,000 people have entered Greece so far this year in an attempt to reach the more prosperous northern European countries. The vast majority are Syrian refugees making the perilous sea crossing from the nearby Turkish coast. ___ 11:35 a.m. Hungary has declared a state of emergency in two of its southern counties bordering Serbia because of the migration crisis, giving special powers to police and other authorities. Government spokesman Zoltan Kovacs said the decision was made at Tuesday's Cabinet meeting. The measure also paves the way for the deployment of the army to assist police with border patrol and migrant-related duties — pending approval from parliament which is expected next week. On Tuesday, Hungary began enforcing tougher rules on people entering the country illegally and also increased prison terms for convicted human traffickers. ___ 11:25 a.m. Germany's vice chancellor says Europe has again made a fool of itself in the migration crisis and is calling for a summit soon of the 28-nation bloc's leaders. EU interior ministers meeting Monday fell short of agreeing on the redistribution of 120,000 newcomers around the bloc — something that Germany, which has seen the biggest number of migrants, is keen to see. Vice Chancellor Sigmar Gabriel said Tuesday that "Europe made a fool of itself again yesterday." He added: "Europe's heads of state and government must meet quickly, because this cannot peter out in such ministerial meetings so that we don't move one step further." Gabriel said that "what we are experiencing now threatens Europe much more than the Greek crisis." ___ 10:20 a.m. Germany's interior minister says he doesn't plan to increase his official forecast that 800,000 migrants will arrive in the country this year, after the vice chancellor said 1 million newcomers were likely. Thomas de Maiziere told ZDF television Tuesday: "I don't want to change the figures." He added that "anything you say here leads to a strange psychological effect." De Maiziere said in mid-August that Germany could see as many as 800,000 migrants arriving this year, four times last year's number. He said Tuesday that, in Afghanistan, some took that to mean that Germany was introducing a quota of 800,000 and they had to rush to get in. Vice Chancellor Sigmar Gabriel said in a letter to members of his center-left party Monday that there could be a million arrivals. ___ 10:10 a.m. Germany's interior minister is backing the idea of cutting European Union funding to countries that refuse to share the burden of hosting refugees. Several EU countries, particularly in the former Eastern bloc, have rejected calls from Germany and the EU's executive Commission for mandatory quotas to spread refugees out among the 28-nation bloc. German Interior Minister Thomas de Maiziere said Tuesday there needs to be discussion of how to exert pressure. He told ZDF television that the countries rejecting quotas often receive significant amounts of EU funding. He said that Commission chief Jean-Claude Juncker has suggested — "and I find that right" — that "we should talk about (them) getting less money from the structural funds." ___ 9:50 a.m. Hungarian police say two crossings on the border with Serbia have been closed to all traffic as stricter rules about the entry of migrants are applied. Col. Laszlo Balazs said Tuesday that the measure affects the smaller of two border crossings near the village of Roszke and one checkpoint at the village of Asotthalom. Balazs said he had "no information" about where migrants seeking to apply for asylum in Hungary would be let in. Prime Minister Viktor Orban has indicated that asylum requests from migrants trying to enter Hungary from Serbia will be rejected because Serbia is a safe country where migrants do not risk war or persecution. Some 200,000 migrants have reached Hungary so far in 2015, nearly all by walking across the southern border with Serbia. ___ 9:10 a.m. The Hungarian government is expected to declare a state of emergency due to the large number of refugees and others who have been entering the country. The state of emergency would allow the government to mobilize the army — pending parliamentary approval next week — to help police with border control, force courts to prioritize cases involving people caught entering Hungary illegally and give police the power to search homes without a warrant if they suspect migrants may be hiding there. The Cabinet will make a decision about declaring the state of emergency at its meeting on Tuesday. Prime Minister Viktor Orban said in an interview on Hungarian broadcaster TV2 that he expects the Cabinet to do so. ___ 9:05 a.m. Hundreds of migrants have spent a night in tents or out in the open on the Serbian side of the border with Hungary awaiting the opening of the crossing at Roszke, which was closed shortly after midnight. Hundreds continue to arrive, though Serbian border guards say that the crossing into Hungary will no longer be open. Two official crossings will allow migrants to cross, but will only allow in those who have applied for asylum in Serbia. Those who have not will be returned to Serbia. Serbian officials have said they will not accept migrants back from Hungary.
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Summary: Hungary declared a state of emergency and police arrested 60 migrants at or near the country's 110-mile southern border with Serbia after new laws took effect at midnight making it a criminal offense to cross the border illegally or mess with the 13-foot razor-wire fence, the BBC reports. With the state of emergency, police have been given more power to detain migrants and search homes they believe are housing migrants, and courts are able to expedite migrant cases, the AP reports. Troop deployment is also on the table if the Hungarian parliament approves. "We will start a new era," government rep Zoltan Kovacs said. "We will stop the inflow of illegal migrants over our green borders," though he added that "official and legal ways to come to Hungary, and therefore to the European Union, remain open." Several countries, including Hungary, have opposed the EU Commission's call for migrant quotas to spread the refugees more equitably throughout its 28 member countries, the AP reports. Hungarian authorities say more than 200,000 migrants have crossed into their nation this year, notes the BBC. Police will be stationed every 115 feet or so along the Serbian border, per the New York Times, and those trying to breach could be jailed or deported. Meanwhile, a UN rep in Serbia tells the paper that refugees, especially those from Syria, should be offered protection. "More than 70% of the population escaping Syria are refugees, running away from a situation that is for them a matter of life and death, and it is [up] to us to do all that we can to help them." Kovacs' counter, per the Times: "The [migrants have] arrived... from 100 different countries, proving that this is a migration crisis and not a refugee crisis."
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Write a title and summarize: Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis. Obesity, characterized by an elevation in circulating lipids, has been associated with increased incidence of cancer [1]. A hallmark of cancer cells is an increase in de novo fatty acid synthesis. This occurs as a result of induction in the expression and function of lipogenic enzymes including fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) [2], [3]. Elevated expression of these lipogenic enzymes is observed in the early premalignant stage of a variety of cancers and is thought to play a critical role in tumorigenesis and cancer progression. Enhanced expression of these genes is correlated with poor survival. Consistent with these findings, inhibiting expression of these genes in cancer cell lines promotes growth arrest and apoptosis, prompting efforts to design therapeutic agents to target FASN [4]. However, the side effects of existing FASN inhibitors have so far precluded their clinical use in cancer. Thus, a better understanding of how lipid homeostasis is regulated will provide important new therapeutic opportunities. PTEN is a critical tumor suppressor and is the second most frequently mutated gene in human cancer, behind only p53 [5]. Loss of PTEN results in the activation of the PI3 kinase (PI3K) signaling pathway, which plays a pivotal role in the proliferation and survival of cancer cells. PI3K/AKT signaling induces de novo lipogenesis through enhanced expression and activation of the transcription factor sterol regulatory element-binding protein-1 (SREBP1) [6]. Insulin treatment or constitutive AKT activation promotes the nuclear accumulation of SREBP1 and its recruitment to the lipogenic enzyme gene promoters, resulting in their coordinate induction. That SREBP activates lipogenic genes is well established; whether there are processes that can oppose SREPB-mediated activation is not known. Maf1 is a transcriptional repressor initially characterized in S. cerevisiae where it is a central node for the repression of RNA polymerase (pol) III-dependent gene expression [7]. Maf1 inhibits transcription by its direct recruitment to tRNA and 5S rRNA gene promoters through its interaction with RNA pol III [8]. In contrast to its role in yeast, in humans, Maf1 negatively regulates transcription resulting from all three nuclear RNA polymerases [9]. In addition to its function in repressing RNA pol III-dependent transcription, Maf1 directly represses RNA pol II-dependent transcription of the central transcription initiation factor, TATA-binding protein (TBP). As TBP is can be a limiting component for RNA pol I-dependent rRNA synthesis, Maf1-mediated regulation of TBP expression also modulates rRNA gene transcription. Cellular levels of TBP determine the proliferative [10] and transforming properties of cells [11]. Elevated synthesis of tRNAs and rRNAs, a hallmark of neoplasia, is required for cellular transformation and tumorigenesis [12], [13]. The ability of Maf1 to repress the transcription of genes encoding TBP, tRNAs and rRNAs supports the idea that it plays a key role in suppressing oncogenesis. Consistent with this concept, expression of Maf1 in PTEN-deficient human glioblastoma cells inhibits anchorage-independent growth [9]. To explore the biological role of Maf1 and to investigate how it is regulated, we asked whether the tumor suppressor PTEN modulates Maf1 expression. Pten-deficient mouse liver and prostate tissues exhibited marked decreases in Maf1 protein, whereas re-expression of PTEN in human PTEN-deficient cells increased Maf1 expression. Consistent with these results, a decrease in nuclear Maf1 expression is observed in both human hepatocellular carcinoma and prostate cancers that have lost PTEN. Furthermore, increased Maf1 expression represses cellular transformation. PTEN represses Maf1 expression through its ability to regulate the PI3K/AKT2/FoxO1 signaling pathway. Consistent with this idea, diet-induced insulin signaling reduces Maf1 expression in mouse liver. Changes in FoxO1 signaling alter Maf1-targeted RNA pol III-dependent gene transcription, defining a new pathway for the regulation of these genes. In addition, we identify a new class of Maf1-regulated genes. Maf1 negatively regulates the expression of the lipogenic enzymes, FASN and ACC1. Maf1 occupies the FASN promoter to oppose SREBP1c-mediated activation. Consistent with these findings, decreased Maf1 expression in human hepatoma cells promotes the accumulation of intracellular lipids whereas overexpression of Maf1 in mouse liver inhibits diet-induced lipogenesis. Together these studies identify a new mechanism for Maf1 regulation through PTEN/PI3K signaling and a new target of PTEN that plays an important role in the ability of this pathway to negatively regulate both tumorigenesis and lipid metabolism. To begin to explore how Maf1 might be regulated, we asked whether the tumor suppressor, PTEN, might control Maf1 levels. In MEFs, loss of Pten resulted in a substantial decrease in Maf1 protein levels (Fig. 1A). In tissue lysates derived from mouse models where Pten is selectively deleted in the liver or prostate, Maf1 expression was similarly decreased (Fig. 1A). The reduction in Maf1 is observed before the onset of tumors in these tissues [14], [15] indicating that the changes in Maf1 are concurrent with the loss of Pten. Consistent with a reduction in Maf1 upon loss of PTEN, induction of PTEN expression in PTEN-deficient human glioblastoma U87 cells resulted in an increase in Maf1 expression and required the phosphatase activity of PTEN (Fig. 1B). Together, these results demonstrate that PTEN regulates Maf1 expression in a variety of cell types. Given that liver and prostate tissues from Pten conditional mutant mice exhibit reductions in Maf1 expression, we tested whether Maf1 expression might be similarly deregulated in PTEN-deficient human cancers. Three matched normal tissues and hepatocellular carcinomas that were PTEN negative were examined by immunostaining for potential changes in Maf1 expression. These tissues were also hepatitis B and C negative. A representative case is shown (Fig. 2A). Maf1 was predominantly localized in the nuclei of normal liver tissue. In contrast, matched tumor tissue displayed a marked reduction in nuclear Maf1 expression. We further examined whether Maf1 expression might be deregulated in human prostate cancer where PTEN is frequently lost. Tissue specimens from four human prostate cancer cases that did not express PTEN were examined by immunostaining. A representative case is shown (Fig. 2B). Compared with the predominant nuclear staining of Maf1 in normal prostate epithelium, the adjacent cancerous tissue from the same individual exhibited a substantial decrease in Maf1 expression, correlated with loss of PTEN in the malignant tissue. Together, these results are the first to suggest the idea that Maf1 expression may be deregulated in human cancer. Given that a reduction in Maf1 expression is similarly observed in Pten-null mice prostate and liver (Fig. 1A), this suggests that the loss of PTEN drives the alterations in Maf1 expression. To further ascertain how alterations in Maf1 expression might affect the oncogenic state of cells, stable hepatoma Huh-7 cell lines were created to overexpress Maf1. Ectopic expression of the HA-tagged Maf1 did not alter endogenous Maf1 protein expression. As we could not detect the ectopically expressed higher molecular weight bands of the Maf1-HA protein with Maf1 antibodies, it likely represents a small fraction of the total cellular Maf1 protein. However, this enhanced Maf1 expression resulted in a decrease in Maf1 target gene expression (Fig. 2C). The doubling time of these cells was modestly increased compared with the vector control cells, whereas increased Maf1 expression resulted in a marked reduction in the ability of the cells to form colonies in soft agar (Fig. 2D and 2E). Tumorigenecity assays revealed that while all mice formed tumors, the onset of visible tumors as well as tumor growth was significantly delayed with increased Maf1 expression (Fig. 2F). Together these results reveal that Maf1 represses oncogenic transformation. A major function of PTEN is to repress the activation of PI3K signaling. We therefore assessed the potential role of PI3K in regulating Maf1 expression. MEFs and HepG2 human hepatoma cell lines were treated with the PI3K inhibitor, LY294002 (Fig. 3A). Increased Maf1 expression was observed with a corresponding decrease in the activation of AKT. As AKT2 is the predominant form of AKT in liver, we further analyzed its role in regulating Maf1 expression. Maf1 protein expression was measured in lysates derived from wild type livers, and those conditionally deleted for Pten, those null for Akt, or both (Fig. 3B). Loss of Akt2 resulted in an increase in Maf1 expression compared with liver lysates derived from wild type mice. Compared with Pten-deficient livers, additional loss of Akt2 restored Maf1 amounts to that observed in wild type mice, supporting a role for AKT2 in negatively regulating cellular Maf1 concentrations. Consistent with these results, expression of a constitutively activated form of AKT2 resulted in a reduction in Maf1 expression in Huh 7 cells (Fig. 3B). Together these results demonstrate that the ability of PTEN to negatively regulate PI3K/AKT signaling is responsible for PTEN-mediated regulation of Maf1 expression. To determine the biological relevance of Maf1-mediated regulation by PI3K signaling in vivo, we examined how diet-induced activation of this pathway would affect Maf1 expression in mice livers. Mice were fed a high carbohydrate diet (HCD) for 2 days and liver lysates were analyzed by immunoblot analysis. This diet results in insulin-induced activation of de novo lipogenesis in the liver [16]. Consistent with these previous results, we observed enhanced phosphorylation of AKT and induction of FASN and ACC1, two limiting enzymes in fatty acid biosynthesis (Fig. 3C). HCD-induced activation of PI3K signaling resulted in a corresponding decrease in Maf1 expression. Together, these results support the idea that Maf1 expression is negatively regulated both in vitro and in vivo through activation of PI3K signaling. Because FoxO1 is a direct downstream target of AKT, we sought to determine its role in regulating Maf1. AKT directly phosphorylates and inactivates FoxO1 by causing it to be translocated from the nucleus to the cytoplasm. shRNA-mediated down regulation of FoxO1 resulted in a decrease in Maf1 expression (Fig. 3D, left). Consistent with these results, expression of a constitutively nuclear-persistent and activated form of FoxO1, mimicking AKT inactivation, resulted in enhanced Maf1 expression (Fig. 3E, left). We further assessed how FoxO1 would affect the activity of Maf1-targeted RNA polymerase III-dependent genes. Decreased FoxO1 expression resulted in increases in tRNA gene transcripts (Fig. 3D, right) whereas the expression of the constitutively activated FoxO1 mutant produced a decrease in these transcripts (Fig. 3E, right). Analysis of Maf1 mRNA, however, showed that altered FoxO1 expression or activation produced a relatively modest change in Maf1 mRNA compared with an approximate 2. 5 fold change in Maf1 protein. This suggests that FoxO1-induced changes in Maf1 protein are not mediated through the ability of FoxO1 to regulate Maf1 transcription. Together our results reveal that Maf1 expression is positively regulated by FoxO1 and negatively regulated through PI3K/AKT inactivation of FoxO1. In addition, these results identify a new FoxO1-dependent signaling pathway that controls RNA pol III-dependent transcription. Given the important role of FoxO1 in regulating lipid metabolism [17], and our results indicating that a reduction of Maf1 expression was correlated with an increase in lipogenic enzyme expression in mice fed a high carbohydrate diet, we examined whether changes in Maf1 expression could modulate the expression of lipogenic enzymes. Maf1 expression was decreased in a mouse hepatocyte cell line (Fig. 4A) and we measured the expression of SREBP1c and the lipogenic enzymes, FASN and ACC1. Reduction in Maf1 expression resulted in the enhanced expression of both FASN and ACC1 mRNAs. In contrast, expression of SREBP1c remained unchanged. Ectopic expression of Maf1 in HepG2 cells resulted in a decrease in both FASN and ACC1 expression, but not SREBP1c (Fig. 4B). To determine whether changes in Maf1 expression would alter FASN promoter activity, we used a FASN promoter-reporter construct containing 178 bp upstream of the transcription start site. We observed an induction of the FAS promoter in association with decreased Maf1 expression, whereas ectopic expression of Maf1 resulted in a decrease in FAS promoter activity (Fig. 4C). These results reveal that Maf1 represses the expression of enzymes required for de novo lipogenesis. Furthermore, the Maf1-responsive region is contained within the first 178 bp upstream of the transcription start site. We next determined whether Maf1 repression of FASN transcription was mediated by the direct recruitment of Maf1 to the FASN promoter. Consistent with Maf1-mediated changes in mRNAs above, we found that down regulation of Maf1 in Huh7 cells resulted in an increase in both FASN and ACC1 protein expression, whereas ectopic expression of Maf1 in HepG2 cells produced a decrease in these proteins (Fig. 5A). Using chromatin immunoprecipitation assays we asked whether Maf1 was enriched in the region that conferred Maf1-mediated repression in comparison with Maf1 occupancy at sequences upstream of this region. Maf1 occupancy was enriched in sequences encompassing the transcription start site and SREBP1c binding site of the FASN promoter in both Huh7 and HepG2 cells (Fig. 5B, top). We next manipulated Maf1 expression in these cells. Analysis of Maf1 occupancy revealed that down regulation of Maf1 resulted in diminished recruitment of Maf1 to the FASN promoter (Fig. 5B, top left), whereas induction of Maf1-HA expression resulted in the enhanced recruitment of the HA-tagged Maf1 to the FASN promoter (Fig. 5B, top right). These changes in Maf1 occupancy on the FASN promoter observed when cellular Maf1 levels are altered further validate the ChIP signals and were similar to those observed for the Maf1-targeted tRNALeu gene (Fig. 5C). As expression of SREBP1c protein was unchanged despite altered Maf1 expression (Fig. 5A), we further assessed whether changes in Maf1 expression and promoter occupancy would affect SREBP1c binding. Analysis revealed that changes in Maf1 occupancy did not alter SREBP1c binding to the FASN promoter (Fig. 5B, bottom). These results indicate that at least one mechanism by which Maf1 represses lipogenic gene expression is through the ability of Maf1 to directly target the promoters of these genes. However, given that our previous work revealed that Maf1 represses the expression of the TATA-binding protein, TBP [9], we further assessed whether Maf1-mediated changes in TBP could indirectly regulate lipogenic gene expression. Cellular TBP amounts were increased in Huh7 cells or down-regulated in HepG2 cells (Supplemental Data, Figure S1). In both cases, altered expression of TBP had no affect on the expression of either FASN or ACC1 mRNA. Together, these results support the idea that Maf1 directly targets and negatively regulates genes required for de novo lipogenesis and that this effect does not impair the binding of SREBP1c at the promoter. Since the amount of lipogenic enzyme activity controls de novo lipogenesis and intracellular lipid accumulation, and FASN expression is limiting for lipogenesis, we further examined the biological consequence of Maf1-mediated repression on lipid biogenesis. As decreased Maf1 expression in Huh-7 cells resulted in an increase in both FASN and ACC1 protein expression (Fig. 5A), we asked whether Maf1-mediated changes in these lipogenic enzymes would affect intracellular lipid accumulation. Compared with control cells expressing non-silencing RNA, cells expressing Maf1 shRNA displayed an increase in the number of visible lipid droplets (Fig. 6A). These results support the idea that Maf1 negatively regulates intracellular lipid accumulation, at least in part, through its ability to directly repress the transcription of lipogenic enzymes. To further determine whether Maf1 can regulate de novo lipogenesis in vivo, Maf1 was ectopically overexpressed in mouse livers through adenoviral-mediated gene delivery. Mice were injected into the tail vein with adenovirus expressing an expression vector for HA-tagged human Maf1 or LacZ. Two days following adenovirus delivery, the mice were placed on a high carbohydrate diet for either 2 or 4 days and the livers of the mice were analyzed for RNA and protein expression. Expression of either LacZ or Maf1-HA protein was detected (Fig. 6C). In mice fed a HCD, both Maf1 mRNA (Fig. 6B) and Maf1 protein (Fig. 6C) was significantly increased in the mice injected with the adeno-Maf1-HA compared with the control LacZ construct. The enhanced expression of Maf1 in the liver resulted in a reduction in lipogenic enzyme mRNAs (Fig. 6B) and FASN protein (Fig. 6C) expression. Consistent with these results, there was a significant decrease in liver triglycerides when Maf1 was overexpressed (Fig 6D). Together these results reveal that overexpression of Maf1 in mouse liver is able to repress the expression of the lipogenic enzymes and reduce intracellular lipid accumulation. Previous studies demonstrated that PTEN negatively regulates RNA pol III-dependent gene transcription through alterations in the phosphorylation state of the transcription factor IIIB components [18]. Our current studies define an additional mechanism by which PTEN represses this class of genes through its ability to regulate Maf1 expression. Our studies identify Maf1 as a novel PTEN target and provide the first evidence that Maf1 functions as a tumor suppressor. Mammalian Maf1 has been shown to repress the expression of both RNA pol III-dependent genes and certain RNA pol II-dependent genes that drive oncogenic transformation [12], [13], [19]. Decreased expression of Maf1 could therefore result in the induction of these genes to promote cellular transformation. Consistent with this idea, enhanced expression of Maf1 significantly represses both anchorage-independent growth and tumor formation in mice. Thus, it is likely that the decrease in Maf1 expression observed in Pten-deficient mouse models and human cancer, and the concordant induction of Maf1 gene targets, contributes to the development or progression of the disease. Since deregulation of PTEN is one of the most common aberrations in many different human cancers, and our results show that PTEN-mediated regulation of Maf1 is not cell-type specific, a reduction in Maf1 expression is likely to be observed in many other types of cancers that possess alterations in PTEN function or mutations that activate PI3K signaling. However, it is conceivable that other pathways and mechanisms contribute to deregulation of Maf11 expression or function in human cancers. Collectively, these results support the idea that Maf1 is a key downstream target of PTEN that contributes to the ability of PTEN to function as a tumor suppressor. Our current studies identify an important new role for Maf1 in regulating PTEN' s metabolic functions through its ability to control intracellular lipid accumulation. PTEN-mediated affects on PI3K signaling regulate Maf1 expression to control lipogenic gene expression. Manipulation of Maf1 expression alters intracellular lipids in vitro. In vivo, overexpression of Maf1 in mouse liver is capable of abrogating insulin-induced de novo lipogenesis. The function of PTEN in repressing lipid metabolism has been well established. In mouse liver, PTEN loss leads to an increase in intracellular lipid accumulation, predominantly through the induction of lipogenesis [14]. In non-alcoholic fatty liver disease (NAFLD), de novo lipogenesis plays a significant role in its pathogenesis [20]. Down-regulation of PTEN in NAFLD contributes to steatosis and its progression towards fibrosis and hepatocellular carcinoma [21]. In addition, PTEN expression is repressed in steatotic livers of obese human subjects [22]. Furthermore, the fact that Maf1 is capable of regulating intracellular lipid concentrations in cells and mouse liver that express functional PTEN supports the idea that Maf1 functions downstream of PTEN in this process. As the cellular levels of PTEN are crucial for maintaining homeostasis, aberrant regulation of PTEN, and the subsequent changes in Maf1, may contribute to a variety of disease processes. Consistent with the importance of this newly identified role for mammalian Maf1 in regulating intracellular lipids, C. elegans MAFR-1 plays a central physiological function in organismal lipid homeostasis (Khanna et al., accompanying manuscript). Consistent with our findings, the insulin signaling pathway and daf-18/PTEN are required for MAFR-1 to regulate de novo lipogenesis. Collectively, our results establish a new and conserved Maf1-dependent regulatory node in the maintenance of lipid homeostasis. Our studies have uncovered a novel mechanism by which downstream PTEN targets, AKT2 and FoxO1, control lipogenic gene expression through Maf1. Our results further identify FoxO1 as a novel critical downstream target of PI3K signaling that controls Maf1 expression to modulate RNA pol III-dependent gene expression, identifying a new signaling pathway that controls both Maf1 expression and the biosynthetic capacity of cells. While Foxo1 is a transcription factor, the very modest effect on Maf1 mRNA relative to the protein changes observed when Foxo1 is manipulated suggests that Foxo1 is not functioning to regulate Maf1 at the level of RNA. There are various ways that Foxo1 might indirectly regulate Maf1 protein expression. FoxO1 could regulate the expression of proteins (s) that associate with Maf1 and/or modify it to regulate its stability. FoxO1 could potentially regulate Maf1 protein synthesis via its ability to regulate the expression of components that enhance the translation of Maf1 mRNA. Additionally, translational regulation could occur through FoxO1 negatively controlling the synthesis of micro RNAs that inhibit Maf1 translation. These various possibilities are currently under investigation. The physiological role of FoxO1 in regulating hepatic lipid metabolism has been unclear. Deletion of FoxO1 in mouse liver results in steatosis while Akt2 deletion is protective for intracellular lipid accumulation that is induced by diet [23] or Pten loss [24]. However, concomitant deletion of both Akt2 and FoxO1 results in a phenotype similar to the Akt2 deletion alone [25]. Ectopic expression of constitutively active FoxO1 in Pten-deficient mouse liver led to repression of lipogenic gene expression [24]. In addition, activation of mTOR and SREBP1c, a transcription factor that coordinates the induction of the lipogenic enzymes, is not sufficient to drive diet-induced lipogenesis without AKT2 [26]. These results suggest that when insulin signals are not present, alternative pathways may regulate basal lipogenesis independent of forkhead transcriptional factors and SREBP1c. The identification of Maf1 as a novel negative regulator of de novo lipogenesis may represent such a signal. Furthermore, AKT2 may control Maf1 expression and function to regulate hepatic lipid metabolism via additional downstream targets. Previous work established that PI3K/AKT activation stimulates de novo lipogenesis by inducing both the expression and processing of SREBP1c. One downstream AKT target, mTORC1, induces SREBP1c activation [27], [28] whereas another direct target of AKT, FoxO1, represses SREBP1c expression [29], [30] Our results reveal that in addition to its ability to regulate SREBP1c, FoxO1 regulates Maf1 expression to regulate lipogenic gene expression (see schematic, Fig. 7). Previous studies showed that mTORC1 directly phosphorylates Maf1 to decrease its ability to repress RNA pol III-dependent promoters [31], [32]. Together, these results suggest that both mTORC1 and FoxO1 function to control lipogenesis by coordinately regulating both SREBP1c and Maf1. In normal hepatocytes, low nuclear SREBP1c expression and high Maf1 expression and function are likely important for maintaining repression of lipogenic gene expression. Upon AKT activation, decreased cellular expression and function of Maf1 and enhanced nuclear SREBP1 accumulation would allow de-repression and induction of lipogenic genes. Together, our studies support opposing roles for SREBP1c and Maf1 in regulating lipogenic gene expression and lipogenesis. In yeast, Maf1 selectively represses genes transcribed by RNA polymerase III through its ability to associate with RNA polymerase III itself [8]. Initial studies in mammalian cells revealed that Maf1 also functions to directly repress select RNA pol II-dependent genes [9]. Here, we identify a new category of genes, those encoding lipogenic enzymes, as Maf1 targets. Maf1 is recruited to the FASN promoter within 200 base pairs upstream of the transcription start site to repress gene activity. This region encompasses a binding site for the master lipogenic gene inducer, SREBP1c. Interestingly, changes in Maf1 occupancy do not affect SREPB1c binding. Even in the presence of SREBP1c at the FASN promoter, recruitment of Maf1 is able to override the ability of SREBP1c to induce gene expression. This is consistent with our results demonstrating that increased Maf1 expression in mouse liver is able to diminish FASN and ACC1 expression. Previous studies revealed that Maf1 is recruited to the TBP promoter also within a region close to the transcription start site [9]. Since the Maf1 responsive regions within the TBP and FASN promoters contain distinct transcription factor binding sites, this suggests that Maf1 may repress RNA pol II-dependent gene expression independent of the composition of activator elements present. Maf1 is not known to have a DNA binding domain; therefore, its recruitment to promoters must occur through its association with other transcription components. While Maf1 genome targets and the mechanism by which Maf1 represses gene expression need to be further examined, it is possible that Maf1-mediated repression of RNA pol II-dependent promoters may involve its ability to repress the function or binding of coactivators or components of the general transcription machinery. Our findings establish a key molecular connection between de novo lipid biogenesis and cellular transformation that occurs through the ability of PI3K/AKT signaling to regulate Maf1 expression, which coordinately regulates lipid and protein biosynthesis. Liver-specific Pten deletion in mice causes spontaneous development of fatty liver disease starting by 1 month and HCC by 12 months of age [14], [33], [34]. Furthermore, knock out of Pten in the liver of Akt null mice delays the onset of both fatty liver and tumor formation supporting a role for AKT2 in this process and the notion that the induction of lipogenesis is needed to drive tumor formation [24], [33]. It is well documented that various tumors and their precursor lesions are re-programmed to undergo a significant induction of endogenous fatty acid biosynthesis independent of the levels of circulating lipids [35]. Overexpression and hyperactivity of lipogenic enzymes, such as ACC1 and FASN, have been shown to be critical in the growth and survival of cancer cells [2], [3]. Given our current findings that Maf1 represses intracellular lipid accumulation and cellular transformation, maintaining cellular Maf1 amounts is likely critical for co-repressing gene expression pathways in order to restrain oncogenic transformation. Collectively, our results support the idea that Maf1 serves as a central node in mammalian cells to co-repress genes involved in proliferative, biosynthetic, and metabolic processes (see schematic, Fig. 7). Future studies will be directed towards understanding how aberrant expression or function of Maf1 contributes to risk for metabolic diseases and cancer. Huh7, HepG2, and MEF cell lines were obtained from ATCC. Pten deficient MEF [36], murine hepatocyte cell lines [37], and PTEN and PTEN C124S-inducible U87 cell lines [18] were previously generated. PTEN and PTEN C124S cells were treated with 1 µg/ml doxycycline to induce PTEN expression. HepG2 cells were transfected with F1 transfection reagent (Invitrogen). Phosphoinositide 3-kinase inhibitor LY294002 [38] was purchased from Sigma-Aldrich. Mouse Maf1 siRNA target sequence 5′-TGTGACATCTACAGCTATA-3′ was identified using Dharmacon' s siDESIGN Center to NCBI accession number NM_026859. Non-silencing mismatch RNA (mmRNA) sequence is described previously [9]. Maf1shRNA2 and Maf1-HA plasmid [9], FoxO1-AAA [24] and FASN luciferase reporter constructs [39] were described previously. Stable Huh7 cell lines were generated by transfection of a Maf1-HA expression construct followed by selection with 200 µg/ml G418. After 4 weeks, G418-resistant cells were pooled. Multiple independent stable cell lines were generated for each construct and experimentally tested and representative results are shown for each. Non-silencing GIPZ lentiviral shRNAmir control vector and GIPZ lentiviral shRNAmir expression clones to human Maf1 or human TBP were from Thermo Open Biosystems (Maf1: Clone IDs V2LHS_138266,3′ UTR target; V3LHS_380771, cDNA target; V3LHS_380770, cDNA target; TBP: V2LHS_153794 (TBP shRNA 1), 3′ UTR target, V2LHS_262152 (TBP shRNA2), 3′ UTR target). Non-silencing empty vector control and pLKO. 1-mouse FoxO1 shRNA (clone IDs TRCN0000054880 and TRCN0000054879) were from Thermo Open Biosystems. Maf1-HA from pcDNA 3. 1 (−) Maf1-HA [9] was PCR amplified to add BamHI and a Kozak consensus sequence 5′ and XbaI 3′ of the Maf1-HA cDNA. E2-TBP from pLTR-E2TBP [11] was PCR amplified to add BamHI 5′ and XbaI 3′ of double HA epitope tagged human TBP. Inducible lentiviral vector pFTREW [40] was restriction digested with BamHI and XbaI to remove GFP and subclone in Maf1-HA (pFTREW-Maf1-HA) or E2TBP (pFTREW-E2TBP). FTREW and FUIPW-rtTA (lentiviral tetracycline transactivator) vectors were a gift from Wange Lu (USC) [40]. Lentiviral particles were produced by transfection of HEK293T using calcium phosphate. Lentiviral vectors were transfected with pMD2. G (vesicular stomatitis virus envelope protein (VSV-G) expression vector) and psPAX2 (packaging vector). After 48 h, conditioned media was collected, sterile filtered and centrifuged at 100,000×g (90 min, 4°). Pelleted virus was resuspended in media and sterile filtered. Cell lines were transduced with concentrated virus from conditioned media for 6–16 h. After 3 days, the infected cells were selected for with puromycin or GFP expression determined by fluorescence microscopy. Adenovirus (type 5 dE1/E3) overexpressing human Maf1-HA or LacZ cDNAs under control of the CMV promoter were purchased from Vector Biosystems. Virus diluted in PBS (1×109 plaque-forming units (pfu) ) was injected into the tail vein of 6–8 weeks old C57/BL6 male mice. Animals on a standard chow or high carbohydrate diet were sacrificed 4 and 6 days post injection and the livers were perfused with PBS and isolated. Three independent experiments were conducted with a minimum of 5 mice per group. Accumulation rates of Huh7 cells stably transfected with Maf1-HA or vector control expression plasmid were determined by plating 50,000 cells per 60 mm dish. Cells were harvested at daily and viable cells were counted using a hemacytometer and trypan blue exclusion. Growth in soft agar assays and tumorigenicity assays with athymic nude (nu/nu) mice were conducted as previously described [11]. Briefly, for the tumorigenecity assays, stable Huh7-Maf1-HA or vector control cells were resuspended in 1∶1 media: matrigel and 2×106 cells/100 µl injected per animal. Tumor volume was measured twice weekly. The first day for visible tumor was determined based when two consecutive tumor volume measurements increased 50% or more. The rate of tumor growth (tumor volume/day) was determined by linear regression on the tumor growth curves in log phase growth to measure the slope (R2>0. 8). Statistics to determine significant differences (p<0. 05) were performed using the Student' s T-test. PtenloxP/loxP; Alb-Cre+, and PtenloxP/loxP; Alb-Cre+; Akt2−/− double mutant mice were previously generated [14], [24]. Livers were harvested, flash frozen in liquid nitrogen, and prepared for protein analysis as previously described [14]. PtenloxP/loxP; PB-Cre+ mice were generated and prostate tissue was processed for protein analysis as previously described [15]. C57BL/6 mice (16 wk old; Jackson Labs) were fed standard chow diet or high carbohydrate diet for 2 days as described previously [41] and livers were harvested for protein analysis. All experiments were performed according to guidelines of the University of Southern California Institutional Animal Care and Use Committee. Protein lysates were subjected to immunoblot analysis as previously described [9] using the following antibodies: Maf1 (Abcam), PTEN, p-AKT, AKT2, FoxO1, ACC1 (Cell Signaling), SREBP-1c (Santa Cruz), HA (Roche), FLAG (Sigma), FASN (BD), TBP (N-12, Santa Cruz), and β-actin (Sigma). Bound primary antibody was visualized using HRP-conjugated secondary antibodies (Pierce) or biotinylated secondary antibodies complexed with avidin/peroxidase (Vector Labs) and enhanced chemiluminescence reagents (Pierce). Densitometry was performed using UN-SCAN-IT software (Silk Scientific). Total RNA was isolated and reverse transcribed as previously described [9]. RT-qPCR was performed with Brilliant II SYBR Green qPCR Mastermix (Stratagene) on the MX3000P System (Stratagene). Maf1 primer sequences are: (F) 5′-GTG GAG ACT GGA GAT GCC CA-3′; (R) 5′- CTG GGT TAT AGC TGT AGA TGT CAC A-3′ Primer sets for FASN, ACC1, SREBP1c [24], pre-tRNAiMet [12], pre-tRNALeu and GAPDH [9] have been described previously. Relative amounts of transcripts were quantified by the comparative threshold cycle method (ΔΔCt) using GAPDH as the endogenous reference control. Fold change was calculated from the control or vector cell lines. Chromatin immunoprecipitation was performed as previously described [9]. DNA was amplified by quantitative PCR using Terra qPCR Direct SYBR Premix (Clontech) or Brilliant II SYBR Green qPCR Mastermix (Stratagene) on the MX3000P System (Stratagene). FASN primer sequences are: −2479 to −2279 upstream forward 5′-CTG GTC ACA CTC TGC CCA CAG C-3′, reverse 5′-AGC TGC AAA GGT CCC ACG A-3′, and transcription start site -104 to +132 forward 5′-CAG CCC CGA CGC TCA TTG G-3′, reverse 5′-GGC TGC TCG TAC CTG GTG AG -3′. Primers for the tRNALeu gene were described previously [9]. Ct values for antibody pulldowns were normalized to input using the antibody IP*10/input calculation and IgG. For intracellular lipid staining, Huh7 stable cell lines were plated on coverslips and grown overnight at 37°. Cells were fixed and stained with Oil Red O and hematoxylin. Human tissues were obtained from the USC/Norris Comprehensive Cancer Center Tissue Procurement Core. Hematoxylin and eosin staining was performed to identify normal and cancerous tissue. Human liver sections and prostate sections were stained with antibodies against Maf1 and PTEN and counterstained with hematoxylin. The Maf1 antibodies (Santa Cruz FL-256) were validated for immunohistochemistry staining by Santa Cruz.
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Title: Maf1 Is a Novel Target of PTEN and PI3K Signaling That Negatively Regulates Oncogenesis and Lipid Metabolism Summary: Obesity is a strong risk factor for human cancers, yet the biological basis for this is unclear. In addition to aberrant growth, abnormal lipid synthesis is a hallmark of cancer cells. Our results have identified a novel role for Maf1 in suppressing both lipid biogenesis and tumor formation. Maf1 elicits these biological responses through its ability to repress genes that that synthesize lipids and regulate biosynthetic capacity. Maf1 amounts are regulated through a critical cellular pathway involving PTEN/PI3K/Akt/FoxO1, which is deregulated in many human cancers. Our results support the idea that deregulation of this pathway in cancer cells results in decreases in cellular Maf1, resulting in both abnormal growth and lipid synthesis. Thus, Maf1 represents a novel link between lipid metabolism and oncogenic transformation providing a new molecular basis for the strong association between obesity and cancer.
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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of Provisional U.S. Patent Application Ser. No. 61/874,442 entitled, “Lacrosse Training Apparatus”, filed in the name of Matthew Fitzgerald, on Sep. 6, 2013, the disclosure of which is also hereby incorporated herein by reference. FIELD OF INVENTION [0002] The present invention relates to sports equipment. More particularly, the present invention relates to apparatus for training persons to manipulate a lacrosse stick for athletic competition purposes. BACKGROUND OF THE INVENTION [0003] It is an axiom that all participants in competitive sports must practice their skills in order to improve their performance during the actual competitions. In many sports, a participant or player must also use and practice with specified sports equipment, such as a ball, a bat, a glove, a stick, etc. A player in the sport of lacrosse, in particular, is required to use a modified stick to catch, hold (i.e., carry or cradle) and release (i.e., throw or shoot) a lacrosse ball. As seen in FIG. 1, a typical lacrosse stick 10 comprises an elongated shaft 12 that has a straight segment 14 with its free end covered by an end cap 16, which may be detachable. The straight segment 14 serves as a handle for the player and is known as the handle, bottom, or butt-end of the lacrosse stick 10. The remainder of the shaft 12 is a second straight segment 18 that terminates into a curved or looped free end 20 that holds a flexible string or mesh pocket 22 for catching, holding/carrying, or throwing a lacrosse ball (not shown). In most lacrosse sticks 10 now manufactured, the second straight segment 18 has a free end that engages a hard structure 20 (called the “head”) that can have mesh pocket 22 installed or strung therein. Regardless, this second straight segment 18, along with the head 20, is generally referred to as the top or the head-end of the lacrosse stick 10. It is understood that the shaft 12 is, in almost all cases, a continuous element and there is no interruption between the first and second straight segments 14, 18. It is also understood that there is no clear demarcation between the two straight segments 14, 18 which are merely identified herein as separate segments for ease of description. [0004] A lacrosse player may hold the stick 10 with one or two hands, depending on the particular circumstance during the play of the lacrosse game. When both hands are holding or gripping the stick 10, the dominant hand of the player tends to be positioned at the head-end 18 and the non-dominant hand tends to be positioned at the butt-end 14. The dominant hand represents the preference of one hand (compared to the other) to perform fine and gross motor tasks. This so-called “dominant hand on top” grip permits a player to more proficiently control the action of the head-end 18 and, more specifically, the mesh pocket 22 for catching, holding/carrying, or throwing a ball. Accordingly, a right hand dominant, i.e., right-handed, player will tend to have the right hand positioned at the head-end 18 and the left hand positioned at the butt-end 14, and a left hand dominant, i.e., left-handed, player will tend to have the left hand positioned at the head-end 18 and the right hand positioned at the butt-end 14. [0005] Regardless of hand dominance, in the course of the play of a lacrosse game, a player invariably will be forced to switch the positions of the hands in gripping the stick 10 with both hands. This happens for several reasons, for example, when an opposing player physically pressures a player to give up a carried ball, or to move in a different direction, or to throw the carried ball in a different direction. The player may have several possible responses, such as moving his stick 10 away from the opposing player. Often, the player will switch his hand positions to maintain a controlled grip on the stick 10, and thus protect the carried ball from the opposing player or continue his intended play. For many players, the hand-switching will result in the non-dominant hand being positioned at the head-end 18 of the stick 10 with an accompanying decrease in the control of the action of the mesh pocket 22 for catching, holding/carrying, or throwing a ball. A player therefore would have a competitive advantage if lacrosse activities and stick movements can be accomplished with equal or more equal skill regardless of the positioning of the dominant hand on the stick 10. Accordingly, players customarily practice lacrosse activities and stick movements using different hand gripping positions. It would be advantageous if a player could utilize an apparatus to aid in training to manipulate a lacrosse stick with equal or more equal skill regardless of the positioning of the hands on the stick. SUMMARY OF THE INVENTION [0006] The above problems are obviated by an embodiment of the present invention which provides an apparatus for training users to manipulate a lacrosse stick, comprising a band that is adapted to snugly fit around a user's wrist or forearm; a fastening component that is adapted to be fastened to a bottom of the lacrosse stick; and a tie that connects the band and the fastening component together so as to restrict movement of the user's respective wrist or forearm to the vicinity of the bottom of the lacrosse stick. The band may be adapted to form an opening and may comprise a deformable material that expands to permit a user's hand to pass through the opening and that, once the hand passes through the opening, contracts to snugly fit around the user's respective wrist or forearm. The band may also comprise a non-stretchable material forming an opening of a selected size that permits a user's hand of similar or smaller size to pass through the opening and the band to snugly fit around the user's respective wrist or forearm. In such case, the size of the opening may be close-fitting with the size of the user's hand such that the use of force, the folding of the hand, or the combination thereof is required for the user's hand to pass through the opening. Also, the band may be adapted to form an opening and may comprise a first section of a non-deformable material and a second section of deformable material that expands to permit a user's hand to pass through the opening and that, once the hand passes through the opening, contracts so both band sections snugly fit around the user's respective wrist or forearm. [0007] The band may also comprise a deformable material and may be configured with a first free end and a second free end, each being adapted to stretch around a user's wrist or forearm and meet the other free end and to fasten, in a detachable manner, to the other free end, said band being maintained in a taut condition around the user's respective wrist or forearm upon fastening of the free ends. In such case, the free ends may comprise complementary detachable-fastening mechanisms. Also, the band may comprise a first free end of a non-deformable material and a second free end of deformable material that is adapted to stretch around a user's wrist or forearm and meet the first free end, at least one of said free ends being adapted to fasten, in a detachable manner, to the other said free end, said band being maintained in a taut condition around the user's respective wrist or forearm upon fastening of the free ends. In such case, the free ends may comprise complementary detachable-fastening mechanisms. [0008] The fastening component and the bottom of the lacrosse stick may comprise complementary detachable-fastening mechanisms. The fastening component may also comprise a detachable end cap for the bottom of the lacrosse stick. The tie may be adapted to be fixedly attached to the band, the fastening component, or both the band and fastening component. The tie may also be integral to the band, the fastening component, or both the band and fastening component. The tie may comprise a stretchable material. The tie may be adapted to be adjustable in length by the user to accommodate the user's usage of the apparatus. [0009] Another embodiment of the present invention provides an apparatus for training users to manipulate a lacrosse stick, comprising a fastening arrangement that connects a user's wrist or forearm to the lacrosse stick, said fastening arrangement adapted to restrict movement of the user's wrist or forearm relative to the lacrosse stick. The user's wrist or forearm may be anatomically proximate the user's dominant hand. The fastening arrangement may be adapted to be adjustable in length, tension, or both by the user to accommodate the user's usage of the apparatus. [0010] Another embodiment of the present invention provides a lacrosse stick apparatus, comprising a first band that is adapted to closely fit a user's wrist or forearm; a second band that is adapted to closely fit a bottom portion of a shaft of the lacrosse stick; and a joining member that links the first band and the second band, the movement of the user's respective wrist or forearm being restricted proximate the bottom portion of the shaft of the lacrosse stick. [0011] Another embodiment of the present invention provides a lacrosse stick apparatus, comprising a band that is adapted to closely fit a user's wrist or forearm; an end covering that is adapted to wrap and lock onto a bottom portion of a shaft of the lacrosse stick; and a joining member that links the band and the end covering, the movement of the user's respective wrist or forearm being restricted proximate the bottom portion of the shaft of the lacrosse stick. DESCRIPTION OF THE DRAWINGS [0012] For a better understanding of the present invention, reference is made to the following description of exemplary embodiments thereof, and to the accompanying drawings, wherein: [0013] FIG. 1 is a schematic representation of a lacrosse stick; [0014] FIG. 2 a is a schematic representation of a training apparatus for a lacrosse player constructed in accordance with an embodiment of the present invention; [0015] FIG. 2 b is a schematic representation of a training apparatus for a lacrosse player constructed in accordance with an alternative embodiment of the present invention; [0016] FIG. 3 is a schematic representation of the training apparatus of either FIG. 2 a or FIG. 2 b attached to a lacrosse player and to a lacrosse stick; [0017] FIG. 4 a is a schematic representation of a training apparatus for a lacrosse player constructed in accordance with a second alternative embodiment of the present invention; [0018] FIG. 4 b is a schematic representation of a training apparatus for a lacrosse player constructed in accordance with a third alternative embodiment of the present invention; [0019] FIG. 5 a is a schematic representation of a training apparatus for a lacrosse player constructed in accordance with a fourth alternative embodiment of the present invention; and [0020] FIG. 5 b is a schematic representation of the training apparatus of FIG. 5 a attached to a lacrosse stick. DETAILED DESCRIPTION [0021] FIG. 2 a shows a training apparatus 50 for a lacrosse player constructed in accordance with an embodiment of the present invention. The apparatus 50 comprises a band or bracelet 60 that forms an opening 62 and that is adapted to snugly fit around a player's wrist or lower forearm. The bracelet 60 may be made of a stretchable or deformable material that expands to permit a player's hand to pass through the bracelet opening 62. Once the player's hand passes through the bracelet opening 62, the bracelet 60 material contracts (for example, to return to its initial shape) and the bracelet 60 then snugly fits around the player's wrist or forearm. The bracelet 60 may also be made of non-stretchable material forming a bracelet opening 62 of a selected size that permits a player's hand of similar or smaller size to pass through the opening 62 and the bracelet 60 to snugly fit around the player's wrist or forearm. The size of the bracelet opening 62 may be close-fitting with the size of the player's hand such that the use of some force, the folding of the hand, or the combination thereof is required for the player's hand to pass through the opening 62. The bracelet 60 may also have one portion made of stretchable or deformable material and another portion made of non-stretchable material so that the portion made of stretchable or deformable material expands to permit a player's hand to pass through the bracelet opening 62 and then contracts so both bracelet portions snugly fit around the player's wrist or forearm. [0022] Alternatively, the bracelet 60 may be made of a stretchable or deformable material that is configured with two free ends 63, 64. This is shown in FIG. 2 b. The length of the bracelet 60 material is adapted to wrap around a player's wrist or forearm so that one free end 63 can be stretched to meet or overlap the other free end 64 that may be held in place against a player's wrist or forearm. The two free ends 63, 64 are adapted to fasten, in a detachable manner, to one another or to another portion of the bracelet 60 material. In this way, the bracelet 60 material may be stretched and then maintained in a taut condition around the player's wrist or forearm. Again, the bracelet 60 may also have one free end made of stretchable or deformable material and the other free end made of non-stretchable material so that the free end made of stretchable or deformable material can be stretched to meet or overlap the other free end made of non-stretchable material that may be held in place against the player's arm or forearm. The bracelet 60 material may use any type of appropriate detachable-fastening mechanism, for example, complementary Velcro strips. [0023] The apparatus 50 also comprises a patch of material 70 that is adapted to be detachably fastened to the end cap 16 of the butt-end 14 or some other portion of the butt-end 14. The patch 70 may use any type of appropriate detachable fastening mechanism. The patch 70 and the portion of the butt-end 14 to be affixed to the patch 70 (e.g., the end cap 16 ) may be configured with portions of a single detachable fastening mechanism, for example, complementary Velcro strips; hook and eyelet; etc. Alternatively, the patch 70 may simply be configured as a detachable end cap for a lacrosse stick 10. The detachable end cap may replace the end cap 16 of a lacrosse stick 10 or it may snugly fit or snap-fit over the end cap 16. [0024] The apparatus 50 also comprises a cord or tie 75 having one end attached to the bracelet 60 and another end attached to the patch 70. The tie 75 may be fixedly attached to either the bracelet 60 or the patch 70, or both. The tie 75 may be attached to the bracelet 60 or the patch 70 by various means. Alternatively, the tie 75 may be made integral to either the bracelet 60 or the patch 70, or both. For most uses of the apparatus 50, the tie 75 may be a fixed length, for example, approximately six to eight inches. Regardless of length, the tie 75 may be made of a material that may be slightly stretchable or deformable in length. One example is paracord (i.e., parachute cord). Alternatively, the tie 75 may be adjustable in length to fully accommodate a player's actual usage of the apparatus 50. Also, the tie 75 may be manufactured and configured in various ways, for example, as a single strand, multiple strands, braided, sheathed, etc. Note that the tie 75 is shown in FIG. 2 b as being attached to a particular end 64 of the bracelet 60 although it is understood that the tie 75 may be attached to any portion of the bracelet 60. [0025] FIG. 3 shows the training apparatus 50 attached to a lacrosse player and to a lacrosse stick 10. In operation, the player attaches the bracelet 60 to the player's wrist or forearm anatomically proximate the player's dominant hand. The player also fastens the patch 70 to the butt-end 14 of the lacrosse stick 10. With this usage of the apparatus 50, the player's dominant hand will only be able to grip the stick 10 at the butt-end 14 of the stick 10 and, in fact, only move the dominant hand up the shaft 12 of the stick 10 by the length of the tie 75. By restricting the player's dominant hand to the butt-end 14, the player will be forced to use the non-dominant hand, which is not attached to the stick 10, to grip the head-end 18 in order to control the mesh pocket 22 and perform appropriate lacrosse activities and stick movements during game play (for example, cradling, passing, shooting, fielding groundballs, etc.). The apparatus 50 may also help prevent the player from re-positioning the hands during breaks in lacrosse activities, or during use in non-lacrosse related activities, such as watching TV, allowing the player to become further conditioned to keeping the non-dominant hand positioned at head end 18 and the dominant hand positioned at the butt-end 14. Through consistent use of the apparatus 50, the player has the opportunity to increase the proficiency of manipulating a lacrosse stick 10 using the non-dominant hand and, over time, narrow the difference between the skill of manipulating a lacrosse stick 10 using the dominant hand and the skill of manipulating a lacrosse stick 10 using the non-dominant hand. [0026] In another usage of the apparatus 50, the player attaches the bracelet 60 to the player's wrist or forearm anatomically proximate the player's non-dominant hand and fastens the patch 70 to the butt-end 14 of the lacrosse stick 10. With this usage, the player's non-dominant hand will only be able to grip the stick 10 at the butt-end 14 and only move the non-dominant hand up the shaft 12 by the length of the tie 75. By restricting the player's non-dominant hand to the butt-end 14, the player will be forced to use the dominant hand, which is not attached to the stick 10, to grip the head-end 18 in order to control the mesh pocket 22 and perform appropriate lacrosse activities and stick movements during game play (for example, cradling, passing, shooting, fielding groundballs, etc.). This provides the player the opportunity, through consistent use of the apparatus 50, to increase the proficiency of manipulating a lacrosse stick 10 using the dominant hand. There are many reasons this may be needed. For example this may assist a player who needs to generally improve his stick movements. This may also assist a player who is rehabilitating an injured dominant hand. This may also assist a player who needs to overcome certain habits in positioning of the hands or in gripping a lacrosse stick 10. Additionally, the apparatus 50 can be used to help younger or less experienced players learn to keep two hands on the lacrosse stick 10, which is the preferred way to hold a lacrosse stick 10, especially when the player is just beginning to learn the game of lacrosse. When a player's non-dominant hand is attached to the butt-end 14, he/she will be more inclined to also keep the dominant hand at the head end 18, so as to achieve optimum control, when performing lacrosse activities such as scooping ground balls, cradling/carrying the ball, catching and throwing the ball, and playing defense, rather than just using the non-dominant hand to control the lacrosse stick 10. [0027] Other modifications are possible within the scope of the invention. The various components of the apparatus 50 may be conventional and known components. They may be configured and interconnected in various ways as necessary or as desired. For example, as noted above, the bracelet 60, the patch 70, and the tie 75 may be made as a single, unitary device. Also, as noted above, the components 60, 70, 75 may be adjusted in length, tension, or both by the user to accommodate the user's usage of the apparatus 50. [0028] Further, the apparatus 50 may use other components in combination with or in place of the components described. For example, as an alternative to the patch 70, the apparatus 50 may comprise a second band 80 that is similar to the band or bracelet 60. This is shown in FIGS. 4 a and 4 b. The second band 80 forms an opening 82 and is adapted to snugly fit around the butt-end 14 of a stick 10. The second band 80 may be made of a stretchable or deformable material that expands to permit the end cap 16 to pass through the second band opening 82. Once the end cap 16 passes through the second band opening 82, the second band 80 material contracts (for example, to return to its initial shape) and the second band 80 then snugly fits around the butt-end 14. The second band 80 may also be made of non-stretchable material, or have one portion made of stretchable or deformable material and another portion made of non-stretchable material, that permits the butt-end 14, in like fashion as described above with the band 60, to pass through the second band opening 82 so as to snugly fit the second band 80 around the butt-end 14. [0029] The second band 80 may also be made of a stretchable or deformable material that is configured with two free ends 83, 84. The length of the second band 80 material is adapted to wrap around a butt-end 14 so that one free end 83 can be stretched to meet or overlap the other free end 84 which may be held in place against the butt-end 14. The two free ends 83, 84 are adapted to be detachably fastened to one another or to another portion of the second band 80 material. In this way, the second band 80 material may be stretched and then maintained in a taut condition around the butt-end 14. Again, the second band 80 may also have one free end made of stretchable or deformable material and the other free end made of non-stretchable material so that the free end made of stretchable or deformable material can be stretched to meet or overlap the other free end made of non-stretchable material that may be held in place against the player's arm or forearm. The second band 80 material may use any type of appropriate detachable-fastening mechanism, for example, complementary Velcro strips. [0030] In another alternative to the patch 70, as noted above, the apparatus 50 may comprise an end cap or end covering 90. An embodiment is shown in FIGS. 5 a and 5 b. The end covering 90 may be a flexible container that has one end 90 a that opens into an interior space that is formed by a closed end 90 b and a side wall 90 c. The open end 90 a is adapted to receive the end cap 16 and butt-end 14 for insertion into the interior space and the covering 90 is adapted to envelop the end cap 16 and butt-end 14. The closed end 90 b is connected to the bracelet 60 via the tie 75. The closed end 90 b may be attached to the tie 75 by various means. Alternatively, as noted above, the tie 75 may be made integral to the closed end 90 b. Note that the figures show the closed end 90 b is attached to the tie 75 at or near the center of the closed end 90 b although the two elements 90 b, 75 may be connected to one another differently. The end covering 90 may be made of a flexible material and may be slightly stretchable or deformable. One example is textile that may be woven or otherwise manufactured. The end covering 90 may be configured to receive the end cap 16 and butt-end 14 in a close-fit manner and, in such case, the end covering 90 may be made with a material that deforms to accommodate the size and shape of an inserted end cap 16 and butt-end 14 of a stick 10. [0031] The end covering 90 also comprises a detachable-fastening mechanism 92, 94, for example, a single Velcro strip with complementary ends, complementary Velcro strips, buckle and strap, pair of laces, etc., formed on or attached to the exterior of the side wall 90 c proximate the open end 90 a. The mechanism 92, 94 may be fashioned on the side wall 90 c in a variety of ways depending upon the specific mechanism, for example, sewn, bonded, glued, laced, etc. The mechanism 92, 94 is adapted to permit the open end 90 a, and a portion of the side wall 90 c, to be pulled in opposing directions, stretched and/or gathered, and securely held (in a taut and/or gathered condition) so as to wrap and lock the open end 90 a around an inserted end cap 16 and butt-end 14. [0032] As shown in FIG. 5 b, the covering 90 is configured to locate the mechanism 92, 94 on the side wall 90 c so as to wrap the open end 90 a particularly around the shaft of the butt-end 14, avoiding wrapping around the end cap 16. The mechanism 92, 94, however, may be located so as to wrap around both the end cap 16 and butt-end 14. The shown arrangement is particularly useful though in assisting to secure the open end 90 a around the shaft of the butt-end 14. By interposing the raised edge of the end cap 16 between the wrapped open end 90 a and the closed end 90 b, the open end 90 a will not only be wrapped onto the shaft but also will be caught by the raised edge, preventing or hindering the open end 90 a from moving down the shaft and off the stick 10 as a player's motions of the arm or wrist attached to the bracelet 60 pulls on the closed end 90 b. Further, by having the tie 75 attached to the closed end 90 b rather than another part of the end covering 90 (and, further, at or near the center of the closed end 90 b ), any pulling motion has lesser effect in moving the open end 90 a down the shaft and off the stick 10.
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Summary: An apparatus ( 50 ) for training users to manipulate a lacrosse stick ( 10 ) having a fastening arrangement ( 60, 70, 75 ) that connects a user's wrist or forearm to the lacrosse stick ( 10 ) and restricts movement of the user's wrist or forearm relative to the lacrosse stick ( 10 ).
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Write a title and summarize: Severe dengue with severe plasma leakage (SD-SPL) is the most frequent of dengue severe form. Plasma biomarkers for early predictive diagnosis of SD-SPL are required in the primary clinics for the prevention of dengue death. Among 63 confirmed dengue pediatric patients recruited, hospital based longitudinal study detected six SD-SPL and ten dengue with warning sign (DWS). To identify the specific proteins increased or decreased in the SD-SPL plasma obtained 6–48 hours before the shock compared with the DWS, the isobaric tags for relative and absolute quantification (iTRAQ) technology was performed using four patients each group. Validation was undertaken in 6 SD-SPL and 10 DWS patients. Nineteen plasma proteins exhibited significantly different relative concentrations (p<0. 05), with five over-expressed and fourteen under-expressed in SD-SPL compared with DWS. The individual protein was classified to either blood coagulation, vascular regulation, cellular transport-related processes or immune response. The immunoblot quantification showed angiotensinogen and antithrombin III significantly increased in SD-SPL whole plasma of early stage compared with DWS subjects. Even using this small number of samples, antithrombin III predicted SD-SPL before shock occurrence with accuracy. Proteins identified here may serve as candidate predictive markers to diagnose SD-SPL for timely clinical management. Since the number of subjects are small, so further studies are needed to confirm all these biomarkers. The last five decades saw a dramatic geographic expansion to 119 countries with overall 30 times more incidence of dengue infection, a mosquito-borne viral disease [1,2]. The World Health Organization (WHO) estimates a mortality rate of dengue infection between 1 and 5%, with approximately 22,000 deaths, mainly among children with shock [2–4]. Most dengue infection are asymptomatic or self-limiting symptomatic while some patients, between days 4 and 7 of illness [5], may progress to severe disease, manifested with shock, serosal effusions and bleeding due to an increase of systematic vascular leak [1,5]. The 2009 WHO guidelines, classifying dengue as dengue without warning sign (D), dengue with warning sign (DWS) and severe dengue [6], facilitate the clinical application and provide useful research endpoints [7]. SD contains any of severe plasma leakage leading to shock and/or respiratory distress (SD-SPL); severe bleeding, and severe organ impairment [1]. Prompt replacement of the circulating plasma losses can reduced the mortality rate to less than 1% among severe cases [1]. Early detection of the individuals who will develop shock among DWS patients is thus critical for timely clinical management. However, there is no objective biomarker in the early stage that reflects the probability of developing SD-SPL. Previously, albumin and transferrin levels were found to be significantly reduced in plasma at presentation day of shock compared with convalescent values, and urinary heparan sulfate and creatinine excretion was shown significantly greater in shock children compared with that in healthy control subjects [8]. In our recent study [9], circulating plasma DNA was found significantly higher in shock patients compared to non-shock on day 3 and 4 after the onset. In addition, serum hyaluronan level was also reported to be significantly higher in patients with shock from day 3 to day 7, compared to healthy subjects [10] while decreased plasma level of inter-α inhibitor proteins was shown to be correlated with shock at the time of hospitalization, but specific day of infection course during sampling was not notified [11]. However, these markers were investigated at the time of shock occurrence or a combination of times during the febrile phase and defervescence of the disease. It is, thus, difficult for clinicians to make an accurate prediction of SD-SPL before shock development. “Warning signs” was mainly originated in the multicenter prospective study of more than 2000 dengue patients in seven countries in Asia and Latin American. However, not all DWS subjects progressed to severe dengue, just only 5% developed severe disease [12]. In an attempt to address helpful biomarkers for determining DWS patients at risk of SD-SPL, we conducted an observational study, using proteomic approach, from the hospital cohort called DENIM in Vietnam. Dengue patients were prospectively recruited and followed-up from febrile onset to defervescence as well as convalescent phase. The timing of early plasma sampling was carefully recorded and daily blood collection was carried out. In this study, we report for the first time the differential characterization of plasma proteome profiles and the identification of biomarkers for SD-SPL prediction among DWS children in the febrile phase, obtained from the DENIM cohort. The study was approved by Institutional Review Board of Institute of Tropical Medicine (NEKKEN), Nagasaki University (No. 11063072) and the Pasteur Institute in Ho Chi Minh City (PIHCM) (No. 602/QD-Pas 27/12/10). DENIM study is a longitudinal study of dengue fever, taking place in Nguyen Dinh Chieu Hospital in Ben Tre province, Vietnam. Children with age between 5 and 16 years hospitalized with clinically suspected dengue fever were eligible for enrollment. Written informed consent was given by a parent or guardian. As described details in the study protocol (S1 Fig), briefly, children presenting acute onset fever (≥38°C) for less than 72 hours were enrolled. No patients had severe symptoms before hospitalization because of concerns about the confounding of previous treatment. At study entry, demographic data, history, and examination findings were recorded, and venous blood were obtained for initial dengue diagnosis. After hospitalization, the patients were diagnosed using standardized diagnostic techniques, as described below. Venous blood samples were daily collected at the time of admission, one day, two days and three days after hospitalization, at the time of shock, in the convalescent phase prior to discharge and two weeks after discharge from the hospital. Plasma was separated by centrifugation at 3000 rpm for 10 min and then divided into two tubes: one used for dengue verification and the remaining one frozen at −80°C for storage. Routine laboratory tests includes complete blood counts, liver and renal function tests, ionograms were performed at the hospital laboratory. Hematocrit was measured every 8 hours in the day 1 and 2 from onset fever and every 4–6 hours in the day 3-day 6 from onset fever. During the time of severe dengue occurrence, hematocrit was measured every 0. 5–1 hour (as shown in S1 Fig). On the day of defervescent, a right lateral decubitus chest X-ray was obtained once and could be repeated thereafter if the patients had symptoms of dyspnea or signs of pleural effusion in clinical examination as dullness to percussion, decreased tactile fremitus and asymmetrical chest expansion, reduced or inaudible breath sounds, egophony, or a friction rub. Echography was performed once or twice, especially when patients had abdominal pain or abnormal physical findings as above. Spectra acquired from the iTRAQ experiment were submitted to the ProteinPilot Software (version 4. 0, AB Sciex), using Paragon protein database search algorithm [17], for generation of peak list, protein identification and quantification. Protein ontology classification was performed using PANTHER classification system (http: //pantherdb. org/, CA). Angiotensinogen and antithrombin III were selected for validation in sixteen subjects individually (ten DWS and six SD-SPL). Western blotting was performed after SDS-PAGE with primary antibodies: anti-AGT (Angiotensinogen, Abcam) and anti-AT III (Antithrombin-III, GeneTex Inc). For specific details, see S1 Appendix. All statistical analyses were performed using SPSS version 17. 0 and MedCalc version 14. 12. Statistical significance was determined by non-parametric Mann Whitney U-test for iTRAQ data comparison and validation data analysis. Experimental data of WB analysis were presented as median ± interquartile range. χ2 was used for categorical analysis, Fisher’s exact test was used when the expected counts were less than 5. A comparison of different methods for the selected feature was determined by the area under the curve (AUC) of receiver operating characteristics (ROC). An AUC value >0. 75 was used as a threshold for good diagnostic test [18]. The DeLong test was performed to compare AUCs. A multivariate logistic regression model was used to observe the independent predictive value of the biomarker on disease outcome. The difference was considered significant at p<0. 05. sp|P01019|ANGT_HUMAN, sp|Q9UFD9|RIM3A_HUMAN, sp|Q9Y4P8|WIPI2_HUMAN, sp|P01008|ANT3_HUMAN, sp|Q8WZ42|TITIN_HUMAN, sp|P00450|CERU_HUMAN, sp|P02765|FETUA_HUMAN, sp|P02787|TRFE_HUMAN, sp|Q68DK2|ZFY26_HUMAN, sp|P00734|THRB_HUMAN, sp|Q8WXH0|SYNE2_HUMAN, sp|O95255|MRP6_HUMAN, sp|Q9NUV7|SPTC3_HUMAN, sp|Q8TEM1|PO210_HUMAN, sp|Q8NGK0|O51G2_HUMAN, sp|Q7RTS5|OTOP3_HUMAN, sp|Q15582|BGH3_HUMAN, sp|Q8WWN8|ARAP3_HUMAN, sp|Q5THJ4|VP13D_HUMAN A flow chart of recruitment in the prospective DENIM study is presented in Fig 1. From January 2011 through June 2012,113 children were enrolled and 63 had confirmed dengue infection. After classification, 44 patients with diagnosis of dengue fever with warning sign without shock presentation (DWS) and seven patients with diagnosis of severe dengue with severe plasma leakage (SD-SPL) underwent study selection. One of SD-SPL was excluded due to the shock occurrence at the time of hospitalization. All of six cases of SD-SPL had clinical shock presentation with narrow pulse pressure, quick pulse rates (as in S2 Table). No patients showed fluid accumulation without or with respiratory distress and no abnormality in echography and chest X-ray were recorded at the time of shock occurrence. One case had fluid overload with pleural effusion after intravenous fluid therapy and needed treatment of small doses of diuretic (S2 Table). In this study, for addressing factors that can provide the early prediction of shock development in preceding 6–48 hours, a sub-set of 16 of the pediatric dengue patients (ten DWS and six SD-SPL) with pre-defervescence or pre-shock plasma were included. Patients selected in DWS and SD-SPL groups showed similar age and gender distribution (Table 2). No significant differences of maximal hematocrit (Hct) and minimal platelet count during the first three day from fever onset were found between the two groups. Hct and platelet count obviously changed from day 4 or 5 of disease course in all subjects, only one case of SD-SPL had maximal Hct on day 3. There was no difference of maximal hematocrit during disease course between two groups and the median day of maximal Hct was day 4 from onset fever for both of two groups. Hematocrit features and fluid management of sixteen patients were shown in S1 Table. The percentage of Hct rising of individual subjects was also recorded (S1 Table). Dengue serologic titration indicated that secondary infection was most common in both DWS (70%) and SD-SPL groups (100%). No difference was recorded in timing of sampling between DWS and SD-SPL subjects. The median duration of sampling time was 28. 5 hours prior to shock presentation for SD-SPL group and 28 hours before defervescence for DWS controls (Table 2). For discovery of potential markers of SD-SPL, we used eight plasma samples in the early stage of dengue infection including four SD-SPL and four DWS samples as controls, randomly selected from two groups of sixteen subjects. The plasma level of high abundant proteins was determined by BCA assays before high-abundance protein depletion (as shown in S3 Fig). No significant difference of the concentration of plasma protein between two patient groups was observed. As shown in S3 Table, the relative ratios for remaining amounts of abundant proteins (including albumin, immunoglobulin, α1-antitrypsin, transferrin, haptoglobin, α2-macroglobulin, fibrinogen, α1-acid Glycoprotein (orosomucoid), apolipoproteins A-I) between two groups of patients showed no difference, except for transferrin. Seventy proteins were identified and quantified with a confidence level of 95% from iTRAQ experiment (S3 Table). Fig 2 shows representative MS and peptide MS/MS spectrum of the corresponding amino acid sequences used in the identification and quantitation of angiotensinogen (AGT) and antithrombin III (AT-III) proteins in this study. Peaks of unique peptide sequence -ALQDQLVLVAAK- and -DPTFIPAPIQAK- corresponding to AGT protein, and -RVWELSK- and -ATEDEGSEQKIPEATNR-, corresponding to AT III proteins (Fig 2), revealed the increased signal intensities in the SD-SPL plasma. The relative intensities of iTRAQ reporter ions used to quantify the relative expression of these proteins were also shown in Fig 2. A statistical comparison revealed that 19 human proteins in both SD-SPL and DWS groups were significantly differentially expressed (p<0. 05), with 5 over-expressed and 14 under-expressed relative to DWS patients (Table 3). The fold-change of each plasma protein level between DWS and SD-SPL patients was also determined (Table 3) with the maximal and minimum ratios, 1. 2 and 2. 4, respectively. The proteins were found to participate in various biological processes including blood coagulation, vascular regulation, cellular and transport-related processes and immune response with expected function of protease, protease inhibitor, transaminase, membrane traffic/transfer/carrier proteins, cell adhesion molecule and signaling molecule as shown in Table 4. Based on the functional properties of these AGT and AT III (Table 3) we selected those for further study. Verification of those two proteins detected by iTRAQ method was performed by Western blotting in 16 individual plasma samples (10 DWS and 6 SD-SPL). Relative quantitation of AGT (Fig 3A and 3B) and AT III (Fig 3C and 3D) blots indicated that the levels of these proteins were significantly higher in early stage plasma of SD-SPL patients (n = 6) compared to those of the control group (n = 10) (p = 0. 031 for AGT and p = 0. 017 for AT III). The univariate logistic regression analysis revealed that level of the relative expression of AT III as well as AGT in plasma significantly correlated with the risk of SD-SPL (odds ratio (OR) 5. 93,95% confidence interval (CI) 1. 23–28. 68, p = 0. 02; and OR 20. 00,95% CI 1. 42–284. 45, p = 0. 03, respectively). ROC curve analysis indicated that SD-SPL prediction by AT III candidate performed more accurately with area under the curve (AUC) of 0. 85±0. 11 (p = 0. 0009) compared to AGT marker (AUC = 0. 83±0. 10, p = 0. 0013) although no statistically significant difference between the two curves was observed (Fig 4). Combination of AGT and AT III achieved an AUC of 0. 87±0. 10 (p = 0. 0004), however, there was no significant difference between this curve and AGT and AT III curves on their own (Fig 4). AT III has the potential to be a more precise marker for shock anticipation. Thus, the relative plasma level of AT III together with other variables (age, maximal hematocrit and minimal platelet count) recorded during the first three day from the onset fever were included in multivariate logistic regression analysis. The analysis demonstrated that the relative concentration of AT III in plasma was independently (p = 0. 03) associated with a greater risk of SD-SPL (Table 5). The severe dengue with severe plasma leakage (SD-SPL) is known as the most common of serious manifestation of severe dengue. Using iTRAQ and mass-spectrometry analysis, we have identified five proteins over-expressed and 14 proteins under-expressed in the early stage plasma of SD-SPL patients compared to that of dengue with warning signs (DWS) as shown in Table 3. Moreover, functionally selected two proteins from those, AGT and ATIII were confirmed by Western blotting analysis using more number of cases (Fig 3). While there was not much difference in the fold-change between iTRAQ experiment and Western blotting analysis of AGT, AT-III had higher change in immunoblot analysis than iTRAQ approach. These differences could be attributed to the use of depleted or whole plasma, the small sample size, or inherent differences in experimental techniques. Although iTRAQs approach was demonstrated as a robust and reproducible mean for simultaneous identification and quantitation of all peptides, and its advantage over other labelings (i. e., ICAT, SILAC, DIGE, mTRAQ) have been reported previously [19,20], underestimation of the change in iTRAQ analysis between samples has been recently proposed to depend on the MS-platform (QTOF, TOF, QTRAP) [21,22]. In concordance with our result, orthogonal analysis, known as biochemical assays such as antibody-based Western blots or enzyme-linked immunoassays, have previously shown a fold-change that is higher than the corresponding ratios derived from iTRAQ approach [22]. Angiotensinogen (AGT) detected increased here has never been reported as a predictive marker of shock in Dengue infection before. It is known as the renin substrate, a precursor molecule of angiotensin I & II, and plays an important role in the renin-angiotensin system [23]. Changes in mRNA expression levels of endothelial-like cells following infection with dengue virus type 2 showed the up regulation of angiotensinogen [24] and the high plasma levels of angiotensin II impair endothelial cell (EC) function [25], inhibit EC motility [26] and induce apoptosis of human ECs [27–29]. Although both of AGT and AT III showed correlation with SD-SPL predictive diagnosis, AT III candidate was the more precisely performing marker at discriminating circulatory collapse from DWS patients (AUC 0. 85). Through multivariate analysis, our study demonstrated that for prediction of SD-SPL in early time of disease course, AT III was independent of other features as hematocrit and platelet count, which have been usually used to monitor clinical progress. In the current study, they reveal obvious changes from day 4 of disease or around the time of shock occurrence, concordant with previous paper [5]. Besides, elevation of AT III accompanied with the low level of prothrombin in pre-shock plasma of SD-SPL patients may indicate the reduction of thrombin formation and that the clotting time for plasma is prolonged. That was consistent with the higher tendency of mucosal bleeding in SD-SPL group (20% of DWS versus 33. 3% of SD-SPL) during disease course. Among our patients with a hemorrhagic tendency, mucosal hemorrhage was mostly observed from day 4 of the disease with minor bleeding. However, children who will subsequently develop SD-SPL did have significantly abnormal levels of circulating level of AT III and prothrombin during the first few days. AT III has previously been found to have lower levels in the plasma of SD-SPL patients resuscitated from shock with colloid fluids compared with SD-SPL patients treated with electrolyte fluids [30]. Un-estimated dilution effect due to intravenously fluid perfusion before the sampling time may potentially explain the decrease of this coagulation inhibitor and the accuracy of that estimation is difficult to assess. In our study, plasma was collected before the patients had received fluids so the level of AT III in plasma of SD-SPL patients should reflect disease pathogenesis. Interestingly, AT III has also been shown to prevent the shedding of the endothelial glycocalyx which can causes a substantial increase in vascular permeability [31] and platelet adhesion [32]. In the pre-shock period the increase of AT III may be a natural human response to minimize the relative damage of glycocalyx layer. We have identified markers that could have a role in predicting severe dengue with severe plasma leakage among dengue patients with warning signs. Such biomarkers may help the institution of timely management and help guide dengue treatment. The study is limited by the small number of patients studied and this may a principle to apply the technique in further study with lager population for exploring the threshold of these markers in predictive diagnosis of severe dengue.
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Title: A Proteomic Approach Identifies Candidate Early Biomarkers to Predict Severe Dengue in Children Summary: Death outcome of dengue infection were mainly involved in pediatric patients with severe plasma leakage, or shock. No biomarker was addressed in the early stage of disease course for timely clinical management. Nineteen differentially expressed proteins were significantly detected in severe dengue patients with severe plasma leakage prior to the time of circulatory collapse occurrence. Angiotensinogen and antithrombin III level were verified to be significantly increased in early stage plasma of shock patients.
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Summarize: By. Ruth Styles. She was a promising film-maker on the verge of a stellar career. But rather than pursuing her Hollywood dream, Donna Hanssen chose to turn her back on the big time for a life on conservation's front line. Outraged by figures that revealed Namibia's carnivore population had halved during the 1980s, she and her siblings launched AfriCat - a pioneering organisation that has saved more than 1,000 lions, leopards and cheetahs since 1993. But, says the veteran conservationist, while numbers are picking up, the animals are still under threat - both from poachers and from farmers who shoot the animals as they move across their land for fear of what might happen to their cows if they don't. Scroll down for video. Hard at work: Donna Hanssen turned her back on a promising film career to dedicate her life to protecting Namibia's endangered lions, leopards and cheetahs. Beautiful: One of the rescued leopards living at the Okonjima Wildlife Reserve tucks into his lunch while perching precariously on a branch. Shocking: A leopard lies beaten and bloody after being killed by angry villagers (left) and right, a jubilant group of villagers show off a lion they have killed. 'What I love about big cats is their survival instinct, their independence, their patience and their beauty,' smiles Donna.'They're so essential for keeping the balance and they mostly take out the weak and the old, so that the strong are left to breed.' As in other African countries, Namibia's big cats are coming under increasing pressure from human farmers and, although not to the same extent as rhinos and elephants, are also threatened by poaching. Currently, the country is home to an estimated 6,000 cheetahs, 4,000 leopards and around 600 lions, while the number of African Wild Dogs - a species particularly loathed because of their vicious habit of ripping chunks of flesh out of living animals - is unknown but thought to be low. Most of those rescued by the AfriCat Foundation are the victims of traps and snares; some of which leave the animal so injured, it can never be released back into the wild. For the lucky ones, rescue means being given a new life in Etosha National Park, a stretch of wilderness in northern Namibia that covers 8,600sqm chunk of savannah and salt pan. New life: For the luckier animals rescued by Donna and her team, a new life at the gorgeous Etosha National Park in northern Namibia beckons. Healthy: Big cats, such as those relocated to Etosha by Donna and her team, help keep the springbok population in check and ensure only the strong breed. Safe: Etosha, one of the world's largest reserves, has been a haven for wildlife since 1907, when it was established by the German colonial authorities. The Africat Foundation is partially funded by guests staying at Okonjima Lodge. Exotic Travel and Safaris, which offers a 12-day tour that includes a night at the lodge and a chance to meet the animals at the reserve. Packages include accommodation, meals and transfers, with prices starting at £1,874 per person. For more information, see exotictravelsafaris.com. South African Airways offers return flights to Windhoek from London Heathrow via Johannesburg from £1,010. See flysaa.com for more. For further information on Namibia, please visit namibiatourism.com.na. For more on Africat, see africat.org. Those who can't be released go instead to the Okonjima Nature Reserve, a small stretch of land nestled among the Omboroko Mountains, which along with a small lodge, houses the AfriCat Foundation itself. From Okonjima, Donna and her colleagues run a programme that encompasses the whole of Namibia and combines rescuing trapped wildlife with educating farmers and schoolchildren - much of it paid for using the proceeds of the on-site safari lodge. 'The secret to conservation is habitat,' explains Donna. 'If you've got the space, animals make it, they survive. But we're also. dealing with farmers that have to survive. 'They have to farm with. leopards, cheetahs, elephants, spotted hyenas and brown hyenas and wild. dogs whereas a farmer in Europe might at the most have to deal with a wolf of a bear. Or if they're in the UK, a fox. 'What we do is say to the farmer, "Take a week to come in Okonjima and we will show you how to keep the cattle safe without using traps or shooting the wildlife". We're trying to change their minds and methods - that's conservation.' But while many have embraced the reforms championed by Donna and her team, others continue to take a harsher stance on dealing with wildlife - much to Donna's distress. 'The ugliest thing is the cruelty and the. greed,' she says, sadly. 'To destroy nature as much as you can just for the benefit of your own pocket -. just so you can make as much money as you can.' But that's not all. 'Cruelty is often a combination of. ignorance and fear,' she continues. 'So people do horrible things to animals because they. fear them, or because they don't realise, or ignore, the fact that animals get stressed. 'I've seen farmers keeping cheetahs in. tiny cages in the baking, 50°C summer heat just to punish it for killing. his calf. And then there's a new type of poaching, where people kill. mothers just to take the babies and raise them like they saved them. Then, when they grow up, they sell them to hunting farms to be shot for. money.' Under threat: African Wild Dogs are increasingly endangered thanks to farmers who loathe them for their cruel method of killing their prey. Tracking: A team from Okonjima hunt for one of the cheetahs living onto the property. All are given electronic tags before being released. Relaxed: One of the tagged cheetahs at Okonjima relaxes in a patch of long grass after tucking into a springbok supper. Getting angry, she adds: 'It. should be classified alongside human trafficking and illegal arms trafficking. Poaching needs to be attacked by a government level. We. can't fight that battle. Farmers earn so much money passing on trapped animals to the. poachers, it's impossible for us to fight.' And it's not just the big cats that suffer appalling cruelty. Whipping out a sheaf of photos, Donna runs through some of the other horrors she and her team have come across. One shows a. baby pangolin being turned into soup ('a cultural problem' says Donna),. a truly ghastly image of a pair of horses being worked into exhaustion. by two children and kicked when they fall over, an elephant lying. bloated and tusk-less after being mutilated by poachers and a snap of. two adult lions dead and bloody in a trap. Clearly,. there is much for Donna and her team to do. For all that, she remains. hopeful about the future and says she hopes that one day, people will. give Africa's wildlife the respect and protection it deserves. 'In. all the years that we've been doing this, we've managed to talk to. around five per cent of farmers. If we have managed to change the minds. of just that five per cent, then we're already happy. 'But. hopefully, with the rise of the internet, more people will find out. about what's going on and fewer animals will be shot. Hopefully the next. generation of farmers will realise that killing isn't fun and that it. certainly isn't a solution to their problems.' Education: One part of Donna's job involves teaching local children and farmers to appreciate the big cats with whom they share their lives. Long road: Although Donna and AfriCat have enjoyed some spectacular successes, she says there is still a very long way to go. Tiny: Jackals are also helped by AfriCat but suffer less at the hands of farmers, who don't see them as a huge threat to their livestock. Wonderful: One of the leopards living on AfriCat's Okonjima property blinks sleepily in the sunshine. On the prowl: A rescued cheetah goes about its business at the Okonjima Wildlife Reserve in Namibia. Lights out: A dramatic red and black sunset over AfriCat's Okonjima reserve. The property is a three-hour drive from Windhoek
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Summary: Donna Hanssen was on the verge of a Hollywood career but decided to become a conservationist instead. One of the founders of the AfriCat Foundation, which began life in 1993 and has saved more than 1,000 big cats. Says the animals are threatened by poachers who kill the mothers and sell the cubs to hunting farms to be shot. More are at risk from farmers who fear for the lives of their animals if leopards, lions and cheetahs are around.
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Summarize: Funding and Organizational Structure Funds for NBACC programs are provided through the DHS Science and Technology Directorate. Activities funded include both intramural and extramural efforts. The programs within NBACC, as well as the construction of the NBACC facility, have been reported as part of the Science and Technology Directorate's Biological Countermeasures portfolio. The NBACC program currently conducts research through partnerships and agreements with federal and private institutes. To provide a unique home for research overseen by the NBACC program, DHS is constructing an NBACC laboratory at Fort Detrick, MD as part of the National Interagency Biodefense Campus. Funds for the laboratory, estimated to cost $128 million, have been appropriated over FY2003-2005. Construction commenced on the NBACC facility in June 2006. The NBACC facility is operated as a federally funded research and development center (FFRDC). The U.S. Army Medical Research Acquisition Activity, acting as the contracting authority for DHS in this instance, released a Request for Proposals (RFP) for the operation of the NBACC facility. On December 20, 2006, DHS announced that Battelle National Biodefense Institute had been awarded the NBACC management contract. Similar to the Department of Energy National Laboratories, the NBACC facility is a government-owned, contractor-operated facility where the contract operator manages the facility and provides the technical expertise and program execution to support DHS's needs. The identity of the NBACC facility component centers has evolved since the facility's conception. Different information has been presented to Congress through various DHS testimony and documentation over the course of this evolution. In 2003, the FY2004 DHS budget justification and testimony by DHS Assistant Under Secretary for Science and Technology Albright stated that four centers would comprise NBACC: the Biodefense Knowledge Center (BKC), the Bioforensics Analysis Center, the Biothreat Assessment Support Center, and the Bio-Countermeasures Testing and Evaluation Center. In 2004, the FY2005 DHS budget justification and testimony by Assistant Under Secretary Albright stated that NBACC consisted of three organizational units: the Biodefense Knowledge Center, the National Bioforensic Analysis Center (NBFAC), and the Biological Threat Characterization Center (BTCC). In 2005, the FY2006 DHS budget justification and testimony by DHS Under Secretary for Science and Technology McQueary refer to the NBACC facility as having two component parts: the National Bioforensic Analysis Center and the Biological Threat Characterization Center. This two center configuration is reportedly the final configuration for the NBACC facility. The two NBACC component centers identified in the FY2006 DHS budget, the Biological Threat Characterization Center and the National Bioforensic Analysis Center, are operating in interim facilities pending construction of the NBACC laboratory building. The BTCC has ongoing activities in a number of government and private laboratories. The NBFAC is housed at the United States Army Medical Research Institute for Infectious Disease (USAMRIID), located in Fort Detrick, MD, and operates as a joint federal effort, including representatives of DHS, the Federal Bureau of Investigation, and the Army. The NBFAC is currently receiving, handling, and performing forensic analysis on biological samples. Apparently conceived as part of the NBACC facility, the Biodefense Knowledge Center was dedicated on September 10, 2004, and is located at the Department of Energy's Lawrence Livermore National Laboratory. This center operates as an independent center though its work is closely coordinated with that of the NBACC facility centers. While funding for the BKC originates from within the Biological Countermeasures portfolio, the BKC is funded independently, not as a component of the NBACC program. The BKC draws on the expertise of scientists at Lawrence Livermore National Laboratory and three additional national laboratories: the Pacific Northwest National Laboratory, Sandia National Laboratories, and Oak Ridge National Laboratory. Three Department of Homeland Security University Centers of Excellence, located at the University of Minnesota, the University of Southern California, and Texas A&M University, also collaborate with the Biodefense Knowledge Center. The NBACC facility is to be part of the federal biodefense research and development network. As such, its activities are to be coordinated with those of other network members, including the Plum Island Animal Disease Center, the National Institutes of Health (NIH), and USAMRIID. Missions of Component Centers The mission of the NBACC program is to understand current and future biological threats; assess vulnerabilities and determine potential consequences; and provide a national capability for conducting forensic analysis of evidence from bio-crimes and terrorism. Each of the NBACC facility component centers executes a piece of this overall program mission. Also, the Biodefense Knowledge Center appears to collaborate with these centers to assist them in meeting mission goals. The Biological Threat Characterization Center is to conduct studies and laboratory experiments designed to find and address critical gaps in understanding current and future biological threats, assess vulnerabilities, conduct risk assessments, and determine potential impacts. An apparent goal of this program is to provide a science-based assessment of possible biological threats, focusing on those pathogens deemed by the Centers for Disease Control and Prevention to have the potential for high consequence. Types of research to be performed in characterizing biological threats include, but are not limited to, investigating potential biothreat pathogens, studying pathogen stability and viability, and assessing lethality through dose/response studies. An earlier presentation on NBACC program activities also included developing strategies for defeating genetically engineered pathogens, and expanding current capabilities in testing non-human primates exposed to biological aerosols. The National Bioforensic Analysis Center was designated in Homeland Security Presidential Directive 10 (HSPD-10), Biodefense for the 21 st Century, as the lead federal facility to conduct and facilitate the technical forensic analysis and interpretation of materials recovered following a biological attack. The NBFAC conducts analysis of evidence from a bio-crime or terrorist attack to obtain a "biological fingerprint" in order to identify perpetrators and determine the origin and method of attack. Consequently, when housed in the NBACC facility, the NBFAC would provide the federal government with a centrally coordinated, validated bioforensic analysis facility. To meet this mission, NBFAC is developing forensic tools, methods, and strain repositories for pathogens of concern. The Biodefense Knowledge Center supports NBACC facility component centers and has its own functions and missions. One is to provide scientific assessments and information to the Homeland Security Operations Center regarding potential bioterrorism events. Another is to be a repository of biodefense information, including genomic sequences for pathogens of concern, the existence and location of vaccines, bioforensics information, and information about individuals, groups, or organizations that might be developing these pathogens. Finally, the BKC aids in assessing potential bioterrorism agents as "material threats" for the purpose of the Project Bioshield countermeasure procurement process. The BTCC and the BKC jointly make these assessments. Policy Issues NBACC Facility Construction and Operation The total construction cost for the NBACC facility has been determined by DHS to be $141 million, a $13 million increase from the initially requested $128 million. Original funds for the construction were appropriated in FY2003âFY2005. The additional $13 million were reprogrammed from other portfolios. Construction began in June 2006 and is projected to be finished in FY2008. Community response to the NBACC facility construction has been mixed. The construction of the NBACC facility, along with new laboratory space for other federal agencies, at the Fort Detrick site has been identified as beneficially spurring investment and development in the surrounding area. Some local advocates and citizen groups have protested the construction of the NBACC facility though. They cite concerns regarding security, safety, and secrecy surrounding the facility. The DHS operates the NBACC facility as an FFRDC, overseen in a manner akin to the Department of Energy National Laboratories. The NBACC FFRDC contractor has the responsibility for enacting the projects and program developed by the NBACC program office. This includes bringing the existing interim centers into the new NBACC facility, when constructed, and continuing those activities currently ongoing, such as the biological risk assessment process. Oversight of NBACC Research The degree to which the research programs of the NBACC program and component centers are transparent and actively overseen may become an area of Congressional interest. Two factors have contributed to an increased focus by biosecurity advocates on NBACC research activities: the degree to which classified research may be performed by the NBACC program and the extent and quality of review for compliance with the Biological and Toxin Weapons Convention (BWC). Because of the potential for classified research to be performed at the NBACC facility, some biodefense experts have identified the lack of transparency as problematic for international relations and treaty compliance. Other experts assert that such issues of transparency can be dealt with so long as a process for review and compliance with applicable treaties is developed and maintained. These issues and the Department's efforts to address these factors are described below. Classified Research Some research activities performed by the BTCC and the NBFAC, either in interim facilities or at the to-be-constructed NBACC facility, may be classified in nature. The NBACC facility is being constructed in such a manner that the entire building can be certified as a Sensitive Compartmented Information Facility (SCIF). The FFRDC contractor operating the NBACC facility must be capable of providing employees cleared at the Top Secret/Sensitive Compartmented Information (TS/SCI) level. The balance of classified and unclassified activities will change depending on agency need and future planning. Thus, the extent to which the capability to perform classified research will be utilized is undetermined. Initially, only a portion of the NBACC facility may operate under classified circumstances, with this amount increasing or decreasing depending on evolving research priorities. The NBACC FFRDC RFP provides some expectations for the FFRDC contractor's future capabilities though, stating: The Government has estimated final operations work force at 120 people, with virtually all of them (>95%) requiring TS/SCI clearance. The offerors are expected to propose a strategy and commensurate workforce for initial operations and transition. The Government estimates that 20-25% of the Contractor's workforce during initial operations will require TS/SCI clearances. The Government estimates that >40-60% of the Contractor's workforce during transition will require TS/SCI clearances. Requiring that virtually all of the NBACC FFRDC workforce be eligible for, and eventually possess, clearance for classified information may provide advantages over requiring only a sub-section of the workforce to obtain requisite clearance. One advantage may be increased flexibility in workforce management given changing workload. Another may be an increased ability to generate synergy between research skills and knowledge due to a larger pool of qualified researchers able to converse about a particular problem. Conversely, some experts are concerned about the potential proliferation of dual-use research results and biological techniques specific to such sensitive research topics. They argue that as more scientists are trained and brought into biological threat assessment studies, the risk of diversion of material, information, or scientific technique to others may increase. While acknowledging that the use of security background checks can reduce this risk, they assert that this risk can not be eliminated. Compliance and Oversight Some arms control experts and other stakeholders have raised concerns about the research to be performed by NBACC at the Fort Detrick facility. They assert that the research being undertaken might violate or might be interpreted as violating the Biological and Toxin Weapons Convention. While research activities may uphold both the letter and the spirit of the BWC, international observers may lack confidence in, or harbor suspicions about, those research activities being performed. Strong internal oversight and review of these research activities may allay some of these concerns or suspicions. As put by Petro and Carus, Thus, any federal program that focuses on threat characterization research will likely require strict administrative guidelines and procedures to ensure that all activities are legally compliant. The DHS asserts such a strong review process exists, as an internal process to the Department. The DHS has developed and implemented a management directive regarding compliance with arms control agreements. Adhering to this directive, DHS has established both a compliance assurance program office and a Compliance Review Group to determine whether the NBACC research activities are in compliance with the BWC, among other duties., The Compliance Review Group is composed of senior DHS officials, including those with oversight of pertinent research areas. The compliance assurance program office has been established separately from the research program offices, so that reseach management and compliance oversight activities do not become conflated. Currently, all NBACC research activities are reviewed, and compliance determinations are made, before the research activities begin. If questions persist about whether a research activity may pose a compliance concern, the compliance assurance program office and the Compliance Review Group are empowered to require additional, clarifying information be presented before a compliance determination is made. Should the Compliance Review Group not reach consensus regarding a particular research activity, the final judgement is reportedly made by the DHS Secretary, who is obligated to ensure DHS activities are in compliance. While such an internal compliance review process may be robust, some arms control experts have been critical of compliance processes that remain entirely internal to a single agency. Such critics assert that interagency review, or review performed or coordinated through the White House, for example through the National Security Council or the Homeland Security Council, would provide greater expert input and further divorce the compliance review from the programmatic and budgetary aspects of a research program. Other possible mechanisms for review of potentially contentious research exist outside of the Department. To assess federal research and development programs that may have potential dual-use capabilities, the Department of Health and Human Services has established the National Science Advisory Board for Biosecurity. This board's duties include providing expert advice on ways to minimize potential misuse of dual-use research. The NSABB is expected to generate guidance on assessing dual-use research through local oversight at research institutions. The NSABB was not given responsibility to view or assess classified research programs, so might be of limited utility in overseeing such research. Transparency Selective transparency in activities performed at the NBACC facility and funded by the NBACC program is considered both essential and difficult to enact. While the ability for outside observers to identify and understand the activities underway at the NBACC facility and funded through the NBACC program is deemed by some experts as key to maintaining international confidence in the US biodefense program, such openness must preserve the protection granted to information deleterious to national security. It is difficult to determine what an appropriate balance is when weighing the potential release of information relating to national vulnerabilities against assuring others of the munificent focus of biodefense research. How to best achieve needed transparency while preserving necessary information restriction is a matter of contention. Some arms control experts claim that openness in the US biodefense program should be held as the model for other countries. Other biosecurity experts assert that special care must be taken to assure that information that would disclose a potential vulnerability is not inadvertently released. Once possible mechanism is the inclusion of local community members into the oversight process for NBACC research. In other areas of contentious biological research, local review boards, such as institutional biosecurity boards, have been used to oversee research activities. Typically, the primary purpose of an IBC is to ensure that recombinant DNA research follows the NIH Guidelines for Research Involving Recombinant DNA Molecules. IBCs are required by NIH guidelines to seat community members on the committee, in addition to scientists and safety officials from the institution. IBCs have also been highlighted as possible mechanisms for implementation of NSABB recommendations. The DHS has stated that an IBC will be established at the NBACC facility, but it is unclear what role the IBC would have in assessing research programs. Inclusion of such persons may be problematic in light of the potential for classified or law-enforcement sensitive nature of some activities. The establishment of an IBC at NBACC may provide a potential public oversight mechanism, reassuring the local community and others with respect to the research being performed at NBACC. Some advocates have assailed the utility of the IBCs though, asserting that the IBCs often do not provide effective oversight of research facilities where they are established. Another possible mechanism might be developing independent, external oversight of research activities, using scientific experts, possibly using members of the National Academies, to assess research programs. The DHS has established an advisory committee through the National Academies to provide input into the NBACC research process. The advisory committee's suggestion and activities have not, as yet, been widely discussed or publicized. Formalizing the committee input mechanism or more widely disseminating the results of the advisory committee's activities may be considered by critics as sufficient to allay transparency concerns. The Department of Homeland Security states that research performed by the Department is solely for defensive purposes, will be in accord with treaty obligations, and will be published, to the maximum extent possible, in the open scientific literature. As such, they are committed to assessing and reducing biodefense vulnerabilities, adhering to scientific standards and practices, and exercising sufficient and appropriate levels of openness. The Department points to its robust internal compliance review process, the establishment of a standing advisory committee, and its plan to conduct much of its research in an unclassified manner as indicators that critics concerns are overstated. Prioritization of NBACC Research Activities Another area of concern amongst the arms control community and increasingly within Congress is the lack of a clear research plan for NBACC programs or for the NBACC facility. The research performed through the NBACC program is designed to fill in knowledge gaps regarding pathogens, test the effectiveness of biological countermeasures, and to assess the risk posed by new and future activities in biological science. Such research will likely span both classified and unclassified areas. Arms control experts have expressed concern that the research being considered at the NBACC facility may not be properly based on risk, but instead might be based on other characteristics, such as potential consequence or the ability of a nation state to develop such a weapon. These experts suggest that a more proper prioritization would focus on the capabilities of terrorists, rather than rogue nations. They also suggest that improper prioritization of research activities may lead to an arms race in the biodefense community, as scientists engaged in biodefense attempt to develop countermeasures to more dangerous pathogens developed in their own laboratories. Since the NBACC program deals with matters of homeland security, public oversight of its research activities might compromise homeland security. If that is the case, some advocates argue that a robust, prioritization and planning mechanism should be developed, so that the mechanism itself can be reviewed. They argue that such an activity will bolster confidence that proper prioritization and planning activities will occur, even if the results of those activities are not available. Others might argue that such structures already exist and are in place, citing the array of advisory boards and committees available to DHS in developing strategic planning. Since the efficacy of planning and prioritization is open to interpretation, developing appropriate metrics to assess this process may pose a challenge. Biosafety Level 4 Construction The NBACC facility is to include laboratory space at the highest level of biosafety containment, Biosafety Level 4 (BSL-4). Such laboratories are required for performing experiments using the most dangerous pathogens, like viral hemorrhagic fevers such as Ebola virus. The volume of BSL-4 laboratory space has historically been small, with federal facilities available at the Centers for Disease Control and Prevention in Atlanta, GA and at USAMRIID in Fort Detrick, MD. Federal efforts are increasing the available BSL-4 laboratory space. The National Institute of Allergy and Infectious Diseases (NIAID) has funded the construction of two new BSL-4 facilities, one at the University of Texas Medical Branch at Galveston and one at the Boston University Medical Center. Additional BSL-4 laboratory space is proposed for the National Bio- and Agro-defense Facility. The increase in BSL-4 laboratory space is likely to result in a corresponding increase in the number of scientists trained in the techniques required to handle contagious, deadly pathogens. Some posit that such an increase will lead to further dissemination of information regarding biothreat agents, possibly to scientists who oppose the United States. Others argue that the increase in BSL-4 laboratory facilities and trained scientists will lead to a more robust biodefense capability, providing more rapid breakthroughs in pathogen identification and countermeasure development. The construction of a DHS BSL-4 facility dedicated to threat characterization has raised community fears with regard to potential pathogen leakage, theft, or loss, and possible indirect health impacts. Similar concerns have been raised regarding the construction of the NIAID BSL-4 facilities. The DHS and DOD assert that such a release is unlikely, given the high safety requirements of a BSL-4 facility. Coordination with Other Federal Agencies How the NBACC program and NBACC facility coordinate their efforts with other federal agencies may attract Congressional interest. When the Department of Homeland Security was formed, most programs addressing medical countermeasures to biological threats remained under the authority of the Department of Health and Human Services (HHS). Most civilian programs addressing nonmedical countermeasures, such as those funded by the Department of Energy, were transferred to the Department of Homeland Security. With the establishment of the NBACC facility, research and development activities in some areas being pursued by the BTCC would be closely related to those supported by HHS. Results of such DHS research and development activities could also help inform and shape policy and research agenda in other departments. For example, the risk assessment activities undertaken by DHS could potentially aid in informing HHS strategic deliberations. The DHS Secretary is charged with coordinating homeland security research and development activities across the federal government. If coordination is well-managed, the effectiveness of research and development activities would be optimized. Results from DHS testing and evaluation of biological countermeasures, for example, might inform new research areas for HHS to support. On the other hand, if coordination is ineffective, significant overlap and duplication of effort may occur between agencies. Additionally, effective coordination between the NBFAC and the Federal Bureau of Investigation (FBI) would be necessary for a prompt forensics response following a bioterrorism incident. The NBFAC has, in its interim space, completed processing of thousands of forensic samples in support of bioterror/biocrime cases for the FBI, acting as the lead federal facility for bioforensic analysis. The FBI has entered into contracts with Department of Energy Laboratories to perform forensics and attribution activities for nuclear materials. Lessons learned from these activities may lower barriers to effective interagency actions following a biological attack. Some coordinative activities designed to leverage the NBACC facility capabilities are already underway. The DHS, acting through the Science and Technology Directorate, has entered into the Interagency Biomedical Research Confederation at Fort Detrick. This group consists of agencies and institutes engaged in medical or biotechnology research at Fort Detrick and also includes representatives from the National Institutes of Health, the Agricultural Research Service, the Centers for Disease Control and Prevention, and the Army Surgeon General. Through committees and subcommittees established under this interagency group, these participants attempt to coordinate work in scientific areas of mutual interest, so as to encourage efficient management, foster scientific interchange, and maximize research and development productivity. Interaction of the Biodefense Knowledge Center and NBACC Facility The feasibility study performed for NBACC identified several potential routes for the construction of NBACC. A phased approach, in which the BKC was initially formed outside of the Fort Detrick facility and then incorporated into the facility at a later date, was one route identified. Another was the construction of the NBACC facility with the BKC integrated within it. The BKC was, instead, established separately at Lawrence Livermore National Laboratory, and now appears to be a center independent of the NBACC facility and NBACC program. The BKC, in its data collection, analysis, and dissemination capabilities, appears to play a similar role to the NBACC program and facility. The degree to which information needs and gaps identified by one of the centers may be filled by the other center may rest heavily on internal communications and interactions. Efficient information sharing and planning may play a key role in maximizing the effectiveness of both centers.
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Summary: The mission of the National Biodefense Analysis and Countermeasures Center (NBACC) is to understand current and future biological threats; assess vulnerabilities and determine potential consequences; and provide a national capability for conducting forensic analysis of evidence from bio-crimes and bio-terrorism. The NBACC is operational, with a program office and several component centers occupying interim facilities. A laboratory facility dedicated to executing the NBACC mission and to contain two NBACC component centers is being built at Fort Detrick, Maryland, as part of the National Interagency Biodefense Campus. The laboratory facility, with an estimated construction cost of $141 million, will be the first Department of Homeland Security laboratory specifically focused on biodefense. Its programmatic contents and component organization appear to be evolving, as conflicting information has been provided during previous budget cycles. Congressional oversight of programs, especially those performed in federal facilities for homeland security purposes, is considered key to maintaining transparency in biodefense. Policy issues that may interest Congress include the operation of the NBACC facility as a federally funded research and development center, transparency and oversight of research activities performed through the center, and the potential for duplication and coordination of research effort between the Department of Homeland Security and other federal agencies. This report will be updated as circumstances warrant.
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Summarize: Shortly after being sworn in, Donald Trump went inside the Capitol and signed official documents making him president..@POTUS signing 3 things: Mattis waiver bill into law, formal nominations 2 Senate & proclamation for nat'l day of patriotism #Inauguration — Sean Spicer (@PressSec) January 20, 2017 He grabbed different pens and then handed them out as mementos of him becoming president of the United States in what’s known as the president’s room in the Capitol. He was surrounded by Republican leaders and Nancy Pelosi, the minority leader of the House. Trump’s children and grandchildren were also around at the official transfer of power. After the ceremony, Trump will go to lunch and then attend the parade. Trump and his staffers have said the weekend will be mostly social, and that he does not plan to start his official work until Monday. I wish people would read or listen to my words on the Border Wall. This was in no way a concession. It was taking care of millions of people who were getting badly hurt by the Shutdown with the understanding that in 21 days, if no deal is done, it’s off to the races! Less than hour after being sworn in as president at the U.S. Capitol — and roughly a half-hour after addressing the nation for the first time as president — Trump returned to his favorite platform, blasting a series of tweets to his 20 million-plus followers from his personal @realdonaldtrump account. Getty President Trump fires off tweets from his personal account President Donald Trump’s first tweets as commander in chief Friday captured key elements of the 16-minute inaugural address he just delivered. Less than hour after being sworn in as president at the U.S. Capitol — and roughly a half-hour after addressing the nation for the first time as president — Trump returned to his favorite platform, blasting a series of tweets to his 20 million-plus followers from his personal @realDonaldTrump account. Story Continued Below “Today we are not merely transferring power from one Administration to another, or from one party to another – but we are transferring power from Washington, D.C. and giving it back to you, the American People,” he wrote in the first pair of tweets. Trump inherited the White House’s @POTUS account after he was sworn in Friday afternoon. The handle, which has not yet sent any tweets, has more than 4.5 million followers as of 1:30 p.m., while his personal account boasts nearly 21 million followers. It’s unclear, however, if Trump will use his new handle at all. In the past week, Trump has said he doesn’t like tweeting, blaming the media for his proclivity to fire off messages in 140 characters or less. But he also told the Times of London last weekend that he would prefer to build up his personal account and “keep it at @realDonaldTrump.” In seven additional tweets, Trump highlighted other excerpts from his speech, including his proclamation that “January 20th 2017, will be remembered as the day the people became the rulers of this nation again” and that the forgotten men and women of America will no longer be forgotten because Trump’s administration will put “#AmericaFirst.” He also pledged to bring back jobs, as well as protect the nation’s borders, wealth and even Americans’ dreams. “It is time to remember that old wisdom our soldiers will never forget: that whether we are black or brown or white, we all bleed the same red blood of patriots!” Trump wrote in the last of two tweets, both linking to Facebook posts. His last message concluded with a message to every American. “[I]n every city near and far, small and large, from mountain to mountain, and from ocean to ocean, hear these words: You will never be ignored again!” His first tweets as president were each sent from an iPhone. In the past, tweets sent from an Android device usually indicated that the messages were coming from Trump himself, but The New York Times has reported that the president has swapped his Android with a new secure, encrypted, Secret Service-approved device.
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Summary: Donald Trump made his first official moves as president immediately after being sworn in Friday. The Hill reports he signed a number of acts into law during a signing ceremony following the inauguration. The first approved a law passed by Congress that lets Gen. James Mattis serve as secretary of Defense. The others created a national day of patriotism and made his cabinet nominees official. This will likely be the only official presidential business done by Trump until Monday. According to the Washington Post, the new president has said he wants to wait until after the weekend to get to work. Trump's other order of business post-inauguration? Tweeting, though not from the official @POTUS account. Politico reports Trump sent his first tweets as president-a series of quotes from his inauguration address-from his personal @realDonaldTrump account. Trump later tweeted from the @POTUS account, sending out a link to his Facebook page. It's so far the only tweet on the official account. Trump has said in the past he would prefer to continue using his personal Twitter account.
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Write a title and summarize: Genetic diversity across different human populations can enhance understanding of the genetic basis of disease. We calculated the genetic risk of 102 diseases in 1,043 unrelated individuals across 51 populations of the Human Genome Diversity Panel. We found that genetic risk for type 2 diabetes and pancreatic cancer decreased as humans migrated toward East Asia. In addition, biliary liver cirrhosis, alopecia areata, bladder cancer, inflammatory bowel disease, membranous nephropathy, systemic lupus erythematosus, systemic sclerosis, ulcerative colitis, and vitiligo have undergone genetic risk differentiation. This analysis represents a large-scale attempt to characterize genetic risk differentiation in the context of migration. We anticipate that our findings will enable detailed analysis pertaining to the driving forces behind genetic risk differentiation. Analyzing the impact of human migration on genetic disease susceptibility is critical to the understanding of complex disease. Moving to new environments causes adaptation, which can also affect disease susceptibility. The availability of methods to measure genomic differences between worldwide populations has increased our understanding of human migration. For example, a worldwide human relationships phylogenetic tree was constructed after genotyping over 50 worldwide populations [1]. This process has enabled researchers to characterize worldwide genetic variation and has provided information regarding migrations that founded entire populations [2], [3]. At the same time, Genome-Wide Association Studies (GWAS) have increased discoveries of disease-associated genetic loci. These developments have paved the way for studies investigating the effects of migration on the genetic basis of disease [4]–[6]. The human genome has been subjected to many selective pressures in recent history. They include changes brought about by the domestication of crops and animals, and the rise of urbanization [7]. These changes may increase the frequency of mutations that are beneficial in the new environment. They may also lead to disruptions of biological processes. When mutations confer a net increase in fitness, they are expected to increase in frequency in affected populations [8], [9]. A mutation increasing disease risk can accompany a beneficial mutation through linkage disequilibrium (LD). Studies have shown that unlinked single nucleotide polymorphisms (SNPs) associated with a single phenotype may be affected as a group if the phenotype undergoes differentiation [10], [11]. While a large set of loci may increase susceptibility to complex disease, individual loci generally make modest contributions, and their effect sizes indicate that they would not be expected to decrease reproductive success [12]. This situation allows differences in the genetic basis of disease to build naturally via genetic drift. However, deviations from genetic drift are expected when environmental changes occur due to migration [13]. Such changes provide an opportunity to learn about factors elevating disease risk in multiple populations. Adaptation to new environments may have caused genetic risk differences across many human populations. Despite the recent explosion of knowledge regarding disease-associated loci and the genetic structure of different world populations [14]–[19], few studies have examined population-based differences in the genetic risk factors for disease. Additionally, they have included only a modest number of diseases, populations, or genetic samples. For example, Myles et al. [6] genotyped 25 disease-associated SNPs in ∼1,000 individuals from 53 populations in the HGDP-CEPH Human Genome Diversity Cell Line panel [15]. The study measured allele frequency differences in the SNPs and concluded that while risk allele differentiation was unusually high in some cases, overall, disease SNPs were not more differentiated between populations than random SNPs. However, 25 SNPs may not be sufficient to determine whether disease-associated SNPs as a whole have undergone risk allele differentiation in worldwide populations. These 25 SNPs are a small subset of variants that influence a small number of specific diseases. Other studies have examined interactions between migration, selection, and disease. One examined the impact of selection on hypertension variants during the migration out of Africa [20]. Others reported increased allele frequency differentiation of type 2 diabetes variants [21]–[23]. It is also plausible that allele differentiation may have occurred in unexamined combinations of diseases and populations. One study probed 2,186 disease-associated SNPs in the HapMap CEU cohort and concluded that they were enriched for low-frequency alleles [24]. This study used a large number of SNPs, but was conducted on a single population. A study incorporating a comprehensive catalog of SNPs and a large genetically diverse cohort would implicitly place genetic risk in the context of migration and reveal worldwide genetic risk variation with respect to many diseases. In a previous study, we surveyed 8,377 SNPs representing 437 diseases in 11 subpopulations. We found differences in risk distribution and protective alleles in different diseases across many subpopulations [25]. The current study examines how the differences in these distributions could have arisen. We analyzed correlations between genetic risk and migration trajectories. Our goal was to identify populations with different genetic dispositions to disease, and to pinpoint migrations preceding these differences. Our analysis highlights the role evolution has played in changing disease susceptibility across populations. Importantly, our approach controls for genetic substructure within and among diverse populations [26]. To detect differences in the genetic basis of disease across migratory events, we integrated a large curated database of geographically-annotated, disease-associated SNPs with human variation measurements in 51 populations from the Human Genome Diversity Panel (HGDP). We controlled for established genetic similarity in subpopulations in order to detect differences in genetic risk exceeding those expected under genetic drift. Figure 1A summarizes our findings for type 2 diabetes on a world map. Consistent with our previous work [25], we found that African populations have the highest genetic risk for type 2 diabetes, followed by people from the Middle East, Europe, and Asia. The Mandinka population had the greatest risk, at a mean 0. 95 log likelihood ratio (LLR), while the Surui population had the lowest risk (LLR: −0. 87). This high-level view revealed stark differences in genetic risk between African and Asian populations. European and Middle-Eastern populations had an intermediate genetic risk for this disease. These patterns identified worldwide trends of increases or decreases in genetic risk and show a high-level view of variation in genetic risk for different diseases. An interactive version of Figure 1A is available for over 100 diseases at geneworld. stanford. edu. Placing genetic risk on a map does not reveal close relationships between populations. Figure 2 is a worldwide phylogenetic tree displaying the relative genetic risk for each population in the context of population relationships. Relationships were inferred in a previous study [1] by analyzing 650,000 SNPs using a maximum-likelihood approach. Results from that analysis have been incorporated here. Each branch in the figure represents the ancestral population common to all populations below it. Figure 1A and Figure 2A show that genetic risk for type 2 diabetes has undergone independent differentiation multiple times as humans migrated out of Africa to East Asia, and finally to the Americas. “Independent differentiation event” was defined as genetic risk differentiation occurring de novo in a population rather than via inheritance from an ancestral population. “Dependent risk differentiation” is a genetic risk inherited from an ancestor. Table 1 shows individual populations with genetic risk differences for type 2 diabetes that are larger than expected under genetic drift; however, it does not distinguish between dependent and independent genetic risk differentiation. We used a maximum likelihood method to identify branches in the phylogenetic tree representing independent genetic risk differentiation events in Figure 2A. The log-likelihood of having only one event for type 2 diabetes was l1 = −108. 848. The log-likelihoods for 2,3, 4,5, and 6 events were l2 = −66. 6083, l3 = −40. 7165, l4 = −33. 7324, l5 = −26. 8371, l6 = −23. 3415. We used the likelihood ratio test to determine the number of branches undergoing genetic risk differentiation independently that exceeded what would be expected under genetic drift. This test allows for the calculation of a p-value for n independent branches by converting two log-likelihood scores to a χ2 variable as follows: 2 (ln−ln−1) ∼χ21. The p-value for 2 branches versus 1 branch undergoing independent genetic risk differentiation for type 2 diabetes was less than 1. 00×10−16, meaning there was evidence for more than one independent genetic risk differentiation event. The p-values for 3,4, 5, and 6 branches were 6. 30×10−13,1. 86×10−4,8. 19×10−3, and 5. 64×10−2, respectively. There was evidence for 5 distinct genetic risk differentiation events (highlighted in green in Figure 2A). Figure 3 shows the genetic risk of all 1043 individuals. Each type 2 diabetes-associated genotype in each individual is displayed. The x-axis shows individual genetic risk; the y-axis corresponds to West (bottom) to East (top) migration. The figure shows that genetic risk for type 2 diabetes decreases as populations move into East Asia. The figure shows that genetic risk decreases steadily, as opposed to being caused by a single genetic risk differentiation event. Analysis of individuals showed that a person in the Mozabite population (HGDP1255) had the highest genetic risk (LLR: 2. 81), and an individual in the Han population (HGDP1291) had the lowest risk (LLR: −2. 21). The effect size of each individual variant is not necessarily the same in each population [27]. While the genetic risk of disease is currently computed using all available GWASs, there is a well-known European bias, as most GWASs are based on European-derived populations [28]. Table S1 displays the populations in which SNPs associated with type 2 diabetes in this study have been replicated. In order to ensure that effect sizes unique to European populations were not solely responsible for observed levels of genetic risk differentiation, the genetic risk for type 2 diabetes was recomputed using Asian-specific effect sizes for all variants. This was accomplished by using GWASs exclusively based on Asian populations. Table S2 compares the likelihood ratio computed using GWASs in which European populations are overrepresented with the Asian-specific likelihood ratio computed using Asian based GWASs. Table S3 compares the genetic risk estimates using all available GWASs and Asian-specific GWASs. Despite major differences in overall risk estimates, significant risk differentiation was still observed in Asian populations when risks were computed with Asian-specific GWASs. Figure 1B shows the genetic risk of biliary liver cirrhosis (BLC) across worldwide populations. In contrast to type 2 diabetes, no worldwide trend is apparent. Figure 2B shows risk differences that were larger than expected under genetic drift (q<0. 05). Branches were colored green if the deviation of a population below them shifted toward decreased risk compared to all other populations and red if the shift was toward increased risk. The maximum likelihood model was not applied to Figure 2B, as each population showing signs of genetic risk differentiation did not have descendant populations in the phylogenetic tree. The maximum likelihood test only distinguishes between inherited and independent genetic risk differentiation and is therefore only suitable for diseases displaying obvious worldwide deviation trends. Figure 2B shows that the Druze and Japanese populations have genetic risk differentiation exceeding what is expected under genetic drift. The Druze population shows significantly less risk, with only 28 out of 100,000 random draws showing less risk (q<0. 05) than all other populations combined. The genetic risk in the Japanese population was higher, with only 18 out of 100,000 random draws showing a higher risk. Genetic risk differentiation in BLC was localized in one European and an Asian population. We examined genetic risk in individuals to expose potential outliers (Figure 4) and to visualize variation in genetic risk estimates across all individuals. The person with the lowest risk (HGDP1201) was from the Mandinka population and had a risk score (combined LLR) of −4. 372. The person with the highest risk (HGDP1279) was from the Mozabite population and had combined LLR of 9. 521. This person appears to be an outlier. The individual with the second highest genetic risk (HGDP998) was from the Karitiana population (combined LLR: 5. 54). We observed genetic risk trends associated with migration in other diseases, including prostate cancer, alopecia areata, melanoma, asthma, neuroblastoma, polycystic ovary syndrome, and pancreatic cancer. One of the most extreme examples of genetic risk differentiation was observed in ulcerative colitis. Figure 5 displays the distribution of the expected amount of genetic risk difference for this disease between the Sinhdi population and all others. The red vertical line represents the actual observed genetic risk difference between the two populations. Only 15 out of 100,000 randomly generated genetic risk values had a larger ulcerative colitis genetic risk difference. We found genetic risk differentiation in multiple worldwide populations for pancreatic cancer and other diseases (Table 1). Detailed information relating to genetic risk differences for many other diseases are available on (geneworld. stanford. edu). Individual SNPs may have a large impact on overall levels of genetic risk differentiation (Text S1 and Figure S1). One notable case is rs13151961, associated with inflammatory bowel disease. Figure S1 shows examples of diseases in which a few SNPs have a disproportionate impact as well as examples in which all SNPs have a uniform impact on genetic risk differentiation. Our method can detect genetic-risk differentiation that is not captured by Fst calculations. Figure S2 shows that rank normalized Fst values, after binning with SNPs having matching allele frequencies, failed to detect population structure differentiation among SNPs associated with biliary liver cirrhosis. After combining p-values for each individual SNP, the combined p-value was 0. 91. Our method detects genetic-risk differentiation in the Japanese and Druze populations (Figure 2B). Figure S3 shows the rank normalized Fst values for SNPs associated with type 2 diabetes. The combined p-value for the observed type 2 diabetes Fst scores was not significant (p = 0. 30). Our method detects genetic-risk differentiation for multiple populations in type 2 diabetes, as can be observed in Figure 2A. Text S1 has additional details on the Fst analysis. Individuals may be outliers with respect to genetic risk, leading to false detection of genetic risk differentiation in a population. We defined outliers as individuals deviating >1. 5 times the interquartile genetic risk range from the median genetic risk and removed them from each population-disease sets showing genetic risk differentiation (Table S4). Each disease showed strong signs of genetic risk differentiation after removing outlying individuals. Disease-associated SNPs are likely to be biased towards genomic regions with higher LD levels where they are tagged more efficiently. We addressed this bias by resampling from SNPs reported to be associated with any phenotype (disease or not) as opposed to resampling from all SNPs. We discarded 614,563 and retained 46,192 of the genotyped SNPs in the HGDP cohort. Genetic risk differentiation was still detected after resampling exclusively from SNPs associated with a phenotype (Table S5). While the HGDP is an important sampling of the worldwide distribution of genotypes, it is possible that some populations are admixed. Detecting genetic risk differentiation in admixed populations may be more difficult, as the levels of genetic risk would most likely be less extreme. The genetic risk for any two populations in which admixed individuals were erroneously sampled is expected to be somewhere between the population with the least and most genetic risk. This explains why controlling for admixture is unlikely to decrease the significance of our findings. Multiple independent genetic risk differentiation events have occurred in various worldwide populations. Table 1 shows individual populations reported to have undergone or inherited an increase/decrease in genetic risk. We applied a maximum likelihood method to identify branches in a phylogenetic tree representing independent genetic risk differentiation events for type 2 diabetes (Figure 2A). Individuals migrate toward decreased genetic risk of type 2 diabetes as populations migrated East. Figure 3 shows that while there was great variability in risk within populations, there was still a steady decrease in susceptibility to type 2 diabetes. There were 44 SNPs associated with biliary liver cirrhosis used in this study. We found genetic risk differentiation in the Japanese and Druze populations (Figure 2B). Risk was increased in the Japanese population. The genetic risk score (combined LLR) was 1. 691, compared to 0. 026 for all other populations combined. The q-value for such a large risk difference was 0. 0112 (Table 1). Consistent with results in this study, our previous work shows significantly higher biliary liver cirrhosis risk in the Japanese population (p: 0. 013) in the Hap Map III cohort [29]. Like all the diseases discussed in this study, ulcerative colitis has genetic and environmental components [30]. Our results suggest that genetic risk differentiation for this condition is increased in South Asian Sindhis (Figure 5). Previous studies have reported that prevalence rates are not necessarily correlated to significant increases in genetic risk [31], [32]. However, our results imply that some event affected pathophysiology of this disease in a particular population. The environment, as well as currently unidentified loci, may also affect the absolute risk. In addition to the Sindhi population, genetic risk differentiation was detected in the Palestinian and Balochi populations (Table 1). Specific environmental differences inducing genetic risk differentiation in type 2 diabetes and other diseases have not been found. However, there is evidence that climate, diet, and living conditions have led to them [33]. For example, exposure to viruses may have increased risk for type 1 diabetes [34]. Autoimmune diseases show disproportionate positive selection in a trajectory toward increased versus decreased risk [35]. This finding has given rise to speculation that viral epidemics are likely to have increased the risk of these diseases by selecting for an overactive immune system. It is also established that modern cultural changes can cause drastic differences in disease prevalence in related populations [36]. However, little is known about how these changes modify risk profiles and disease prevalence over time. This study provides critical clues for the foundation for future analyses. The concepts discussed here could be used to link diseases that may share pathophysiology and environmental triggers. It is possible that modulation of environmental features in different global regions changed the genetic risk of certain diseases. If an environmental feature affects multiple diseases, risk estimates for the diseases should correlate across the same populations, even if no common genetic basis is apparent. If the environment increases risk for one disease and decreases it for another, a negative correlation is expected. Finding diseases with genetic risk estimates correlated across worldwide populations would represent a novel and potentially highly informative approach to uncover shared pathophysiologies. This type of analysis would benefit from the largest possible catalog of genetic variants. For example, in the HGDP, East Asia is biased to detect more genetic risk differentiation than the Americas, due to the increased sensitivity that comes from 17 East Asian versus 5 American populations. In addition, full genome sequence analysis enables the inclusion of copy number polymorphisms and rare variants that may be found to contribute to complex disease susceptibility. As sequencing costs decrease, analyses expanding the scope of this study will occur. We investigated >650,000 SNPs from the HGDP-CEPH, which has DNA from 1043 individuals in 51 populations on 8 continents [15]. We also used the HapMap Phase 3 cohort to analyze 1. 6 million SNPs from 11 populations [29]. Finally, we used VARIMED, a database of disease-associated SNPs [37]. VARIMED was built by curating 5,478 published studies with 4,573 disease associations. At the time of this study, it contained 67,678 unique phenotype-associated SNPs, of which 51,404 were associated with a disease. Of these, after filtering by p-value and other methods, 723 unique SNPs were on the Illumina genotyping array we used to represent disease phenotypes in the HGDP [15]. For this study, we used only GWAS SNPs that had been detected across ≥2 populations with p-values<10−6. SNPs were excluded if information about them was insufficient to compute likelihood ratios for the genotypes of associated SNPs. VARIMED was used to compute genetic risk estimates of the resulting diseases across all HGDP populations. The HapMap Phase 3 cohort includes 11 populations with 1. 6 million SNPs genotyped per person [29]. We used this cohort, in combination with results from previous work [25], to check our results in individual populations with elevated levels of genetic risk differentiation. All the methods with the exception of multiple hypothesis testing are applicable to this cohort. In this paper, the sample genetic risk in a population is referred to as genetic risk. Any computation of a population' s genetic risk is inaccurate, due to the inability to genotype all individuals in a population and the existence of many undiscovered disease-associated variants. The likelihood ratio (LR) represents the effect size of a particular genotype on genetic disease risk. SNPs in linkage disequilibrium (R2≥0. 2) in a population were excluded. For a given bi-allelic SNP, there are three possible genotypes: homozygous for the major allele, homozygous for the minor allele, or heterozygous. The function L (g) maps the genotype g to the estimated likelihood ratio. The LR used in our calculations represents the weighted mean LR reported across all studies [37]. Each LR was weighted by the square root of the sample size in each association study. The following is our method for combining multiple LRs from multiple GWASs. A is the vector of all sample sizes in each study. P (g | disease is present) i is the estimated probability that a genotype g is in the disease population of the ith GWAS. P (g | disease is not present) i is the estimated probability that a genotype g is present in the non-disease population of the ith GWAS. Once computed, the combined LR was used to compute the genetic disease risk for each individual, as follows. G is the vector of all genotypes in disease-associated SNPs in person m. The predicted genetic risk r for person m is the log of the combined likelihood ratios for all disease-associated variants present in that person. The genetic risk for a population was the mean risk of all its members. Derived populations created by combining multiple populations in the HGDP were treated as single populations, and the following computations applied equally. S is the vector of a population. The predicted genetic risk R for an entire population P is the mean predicted risk for everyone in the population: In order to compare genetic risk (D) between two populations A and B, we subtracted the genetic risk of one population from the other' s: Population A' s risk for a disease is higher than B' s if DAB is positive and lower if DAB is negative. We constructed a distribution of the expected difference of genetic risk between two populations in order to see if the observed difference was larger than expected by random chance:. The vector H represents all SNPs associated with a particular disease. Our goal was to assess the significance of a difference in risk DAB across two populations A and B. Each element in H was replaced by a SNP randomly drawn from the entire set of SNPs in the two populations. The global major allele frequency of the randomly drawn SNP was drawn to match original SNP' s global major allele frequency. In every case, the risk allele' s major or minor allele status in the randomly drawn SNP matched that of the SNP it replaced. In addition, each SNP was placed in one of eight functional categories (frameshift, nonsense, missense, untranslated, near-gene, intron, coding-synonymous, or unknown). Each randomly drawn SNP also matched the functional category of the SNP it replaced in vector H. Once all elements of H had been replaced, the genetic risk of all populations was recomputed, effectively assigning a randomly generated genetic risk score to each population. Since each population was assigned a genetic risk score from the same randomly drawn set of SNPs, the expected amount of correlation between genetic risk values among all populations was preserved. We created phylogenetic trees of our results with each branch representing a migration event. We computed the genetic risk difference of each migration event by subtracting the genetic risk of all descendant populations from the risk of all ancestral populations (those above the branch). Branches on the human phylogenetic tree created from the HGDP populations were tested for genetic risk differences. We computed the difference in risk between all ancestral and descendant populations. A phylogenetic tree of all the HGDP populations was used as described previously [1]. Each branch in the tree partitions an ancestral and descendant population. The ancestral population is made up of populations above a branch; the descendant population is below it. The expected difference in genetic risk between all possible ancestral and descendant comparisons was computed by randomly replacing all disease-associated SNPs by performing a random draw of H 100,000 times. We computed a matrix representing 100,000 randomly generated phylogenetic trees and compared it with the observed phylogenetic tree in the context of genetic risk. Let ri, k represent the genetic risk difference between ancestral and descendant populations in branch i computed from the kth randomly generated H. This matrix of 100,000 randomly generated phylogenetic trees was then compared to the observed tree. Oi is the observed genetic risk difference between ancestral and descendant populations in the ith branch of the phylogenetic tree. The probability of observing a genetic risk difference between ancestral and descendant populations in the ith branch is as follows: This equation gave a probability for a specific genetic risk difference observed on branch i. However, we tested multiple strongly correlated populations simultaneously. In addition, the genetic risk differences of nearby branches in the phylogenetic tree were strongly correlated due to genetic similarity between populations. One row in matrix R represented the genetic risk difference of all branches in the tree between ancestral and descendant populations in a single random draw. The genetic risk difference was calculated with the same set of SNPs in every branch in the phylogenetic tree in each row. This preserved the correlation of genetic risk among closely related populations and enabled us to detect independent genetic risk deviations. To assess the significance of a p-value α in the context of the entire tree, we first computed the number of branches having equal or greater significance for genetic risk differentiation. Significant genetic risk differentiation events were calculated follows. N (α) is the observed number of branches with a p-value for genetic risk differentiation at or below α. We next computed the expected number of branches with genetic risk differentiation at significance α from the matrix representing 100,000 randomly generated phylogenetic trees. Q (α) (the q-value) for a branch with p-value α for genetic risk differentiation was: While Q () can inform on the significance for a particular p-value, it does not estimate the total number of distinct genetic risk differentiation events in a phylogenetic tree. We estimated the most likely number of migration events independently contributing to observed genetic risk deviation in a phylogenetic tree using a maximum likelihood approach. P is the vector containing all probabilities of the observed genetic risk difference between ancestral and descendant populations of each branch of tree t. We defined a function that returns true if branch k is not a descendant of branch z: Branch z represents the branch in which an independent genetic risk differentiation event occurred. Applying the principle of maximum likelihood, the branch z that maximizes the likelihood function in tree t is shown below. The number of events refers to the number of independent genetic risk differentiation events. The log likelihood function produces the branch most likely to have undergone genetic risk differentiation, given that a single genetic risk differentiation event occurred. The ‘’ symbol represents a logical disjunction that returns true if one or more of the two operands is true. The branch that maximized likelihood was the one most likely to have caused the risk differentiation. However, there may have been multiple genetic risk differentiation events. This maximum likelihood method can be generalized to detect an arbitrary number of branches that underwent genetic risk differentiation. Find the indices z1, z2, …, zy for branches that maximize the following likelihood function: Due to the constraints this equation imposed, a very large number of random draws was required to produce an accurate estimate. All indicator functions in the numerator must return a non-zero value for the count to be incremented by one; this was computationally infeasible. In order to make the computation tractable, we made a simplifying assumption that only a single ancestral branch which has undergone de novo genetic risk differentiation (as opposed to an arbitrary combination of branches that have undergone de novo genetic risk differentiation) can pass a significantly modified genetic risk for a disease to a descendant branch. This led us to the method used for this study. The following calculation was used to find all branches with sufficient evidence of having undergone independent genetic risk differentiation. Find the indices z1, z2, …, zy that maximize the following likelihood function:
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Title: Analysis of the Genetic Basis of Disease in the Context of Worldwide Human Relationships and Migration Summary: The environment humans inhabit has changed many times in the last 100,000 years. Migration and dynamic local environments can lead to genetic adaptations favoring beneficial traits. Many genes responsible for these adaptations can alter disease susceptibility. Genes can also affect disease susceptibility by varying randomly across different populations. We have studied genetic variants that are known to modify disease susceptibility in the context of worldwide migration. We found that variants associated with 11 diseases have been affected to an extent that is not explained by random variation. We also found that the genetic risk of type 2 diabetes has steadily decreased along the worldwide human migration trajectory from Africa to America.
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Summarize: The schoolmaster was leaving the village, and everybody seemed sorry. The miller at Cresscombe lent him the small white tilted cart and horse to carry his goods to the city of his destination, about twenty miles off, such a vehicle proving of quite sufficient size for the departing teacher's effects. For the schoolhouse had been partly furnished by the managers, and the only cumbersome article possessed by the master, in addition to the packing-case of books, was a cottage piano that he had bought at an auction during the year in which he thought of learning instrumental music. But the enthusiasm having waned he had never acquired any skill in playing, and the purchased article had been a perpetual trouble to him ever since in moving house. The rector had gone away for the day, being a man who disliked the sight of changes. He did not mean to return till the evening, when the new school-teacher would have arrived and settled in, and everything would be smooth again. The blacksmith, the farm bailiff, and the schoolmaster himself were standing in perplexed attitudes in the parlour before the instrument. The master had remarked that even if he got it into the cart he should not know what to do with it on his arrival at Christminster, the city he was bound for, since he was only going into temporary lodgings just at first. A little boy of eleven, who had been thoughtfully assisting in the packing, joined the group of men, and as they rubbed their chins he spoke up, blushing at the sound of his own voice: "Aunt have got a great fuel-house, and it could be put there, perhaps, till you've found a place to settle in, sir." "A proper good notion," said the blacksmith. It was decided that a deputation should wait on the boy's aunt--an old maiden resident--and ask her if she would house the piano till Mr. Phillotson should send for it. The smith and the bailiff started to see about the practicability of the suggested shelter, and the boy and the schoolmaster were left standing alone. "Sorry I am going, Jude?" asked the latter kindly. Tears rose into the boy's eyes, for he was not among the regular day scholars, who came unromantically close to the schoolmaster's life, but one who had attended the night school only during the present teacher's term of office. The regular scholars, if the truth must be told, stood at the present moment afar off, like certain historic disciples, indisposed to any enthusiastic volunteering of aid. The boy awkwardly opened the book he held in his hand, which Mr. Phillotson had bestowed on him as a parting gift, and admitted that he was sorry. "So am I," said Mr. Phillotson. "Why do you go, sir?" asked the boy. "Ah--that would be a long story. You wouldn't understand my reasons, Jude. You will, perhaps, when you are older." "I think I should now, sir." "Well--don't speak of this everywhere. You know what a university is, and a university degree? It is the necessary hallmark of a man who wants to do anything in teaching. My scheme, or dream, is to be a university graduate, and then to be ordained. By going to live at Christminster, or near it, I shall be at headquarters, so to speak, and if my scheme is practicable at all, I consider that being on the spot will afford me a better chance of carrying it out than I should have elsewhere." The smith and his companion returned. Old Miss Fawley's fuel-house was dry, and eminently practicable; and she seemed willing to give the instrument standing-room there. It was accordingly left in the school till the evening, when more hands would be available for removing it; and the schoolmaster gave a final glance round. The boy Jude assisted in loading some small articles, and at nine o'clock Mr. Phillotson mounted beside his box of books and other _impedimenta_, and bade his friends good-bye. "I shan't forget you, Jude," he said, smiling, as the cart moved off. "Be a good boy, remember; and be kind to animals and birds, and read all you can. And if ever you come to Christminster remember you hunt me out for old acquaintance' sake." The cart creaked across the green, and disappeared round the corner by the rectory-house. The boy returned to the draw-well at the edge of the greensward, where he had left his buckets when he went to help his patron and teacher in the loading. There was a quiver in his lip now and after opening the well-cover to begin lowering the bucket he paused and leant with his forehead and arms against the framework, his face wearing the fixity of a thoughtful child's who has felt the pricks of life somewhat before his time. The well into which he was looking was as ancient as the village itself, and from his present position appeared as a long circular perspective ending in a shining disk of quivering water at a distance of a hundred feet down. There was a lining of green moss near the top, and nearer still the hart's-tongue fern. He said to himself, in the melodramatic tones of a whimsical boy, that the schoolmaster had drawn at that well scores of times on a morning like this, and would never draw there any more. "I've seen him look down into it, when he was tired with his drawing, just as I do now, and when he rested a bit before carrying the buckets home! But he was too clever to bide here any longer--a small sleepy place like this!" A tear rolled from his eye into the depths of the well. The morning was a little foggy, and the boy's breathing unfurled itself as a thicker fog upon the still and heavy air. His thoughts were interrupted by a sudden outcry: "Bring on that water, will ye, you idle young harlican!" It came from an old woman who had emerged from her door towards the garden gate of a green-thatched cottage not far off. The boy quickly waved a signal of assent, drew the water with what was a great effort for one of his stature, landed and emptied the big bucket into his own pair of smaller ones, and pausing a moment for breath, started with them across the patch of clammy greensward whereon the well stood--nearly in the centre of the little village, or rather hamlet of Marygreen. It was as old-fashioned as it was small, and it rested in the lap of an undulating upland adjoining the North Wessex downs. Old as it was, however, the well-shaft was probably the only relic of the local history that remained absolutely unchanged. Many of the thatched and dormered dwelling-houses had been pulled down of late years, and many trees felled on the green. Above all, the original church, hump-backed, wood-turreted, and quaintly hipped, had been taken down, and either cracked up into heaps of road-metal in the lane, or utilized as pig-sty walls, garden seats, guard-stones to fences, and rockeries in the flower-beds of the neighbourhood. In place of it a tall new building of modern Gothic design, unfamiliar to English eyes, had been erected on a new piece of ground by a certain obliterator of historic records who had run down from London and back in a day. The site whereon so long had stood the ancient temple to the Christian divinities was not even recorded on the green and level grass-plot that had immemorially been the churchyard, the obliterated graves being commemorated by eighteen-penny cast-iron crosses warranted to last five years. Slender as was Jude Fawley's frame he bore the two brimming house-buckets of water to the cottage without resting. Over the door was a little rectangular piece of blue board, on which was painted in yellow letters, "Drusilla Fawley, Baker." Within the little lead panes of the window--this being one of the few old houses left--were five bottles of sweets, and three buns on a plate of the willow pattern. While emptying the buckets at the back of the house he could hear an animated conversation in progress within-doors between his great-aunt, the Drusilla of the sign-board, and some other villagers. Having seen the school-master depart, they were summing up particulars of the event, and indulging in predictions of his future. "And who's he?" asked one, comparatively a stranger, when the boy entered. "Well ye med ask it, Mrs. Williams. He's my great-nephew--come since you was last this way." The old inhabitant who answered was a tall, gaunt woman, who spoke tragically on the most trivial subject, and gave a phrase of her conversation to each auditor in turn. "He come from Mellstock, down in South Wessex, about a year ago--worse luck for 'n, Belinda" (turning to the right) "where his father was living, and was took wi' the shakings for death, and died in two days, as you know, Caroline" (turning to the left). "It would ha' been a blessing if Goddy-mighty had took thee too, wi' thy mother and father, poor useless boy! But I've got him here to stay with me till I can see what's to be done with un, though I am obliged to let him earn any penny he can. Just now he's a-scaring of birds for Farmer Troutham. It keeps him out of mischty. Why do ye turn away, Jude?" she continued, as the boy, feeling the impact of their glances like slaps upon his face, moved aside. The local washerwoman replied that it was perhaps a very good plan of Miss or Mrs. Fawley's (as they called her indifferently) to have him with her--"to kip 'ee company in your loneliness, fetch water, shet the winder-shetters o' nights, and help in the bit o' baking." Miss Fawley doubted it.... "Why didn't ye get the schoolmaster to take 'ee to Christminster wi' un, and make a scholar of 'ee," she continued, in frowning pleasantry. "I'm sure he couldn't ha' took a better one. The boy is crazy for books, that he is. It runs in our family rather. His cousin Sue is just the same--so I've heard; but I have not seen the child for years, though she was born in this place, within these four walls, as it happened. My niece and her husband, after they were married, didn' get a house of their own for some year or more; and then they only had one till--Well, I won't go into that. Jude, my child, don't you ever marry. 'Tisn't for the Fawleys to take that step any more. She, their only one, was like a child o' my own, Belinda, till the split come! Ah, that a little maid should know such changes!" Jude, finding the general attention again centering on himself, went out to the bakehouse, where he ate the cake provided for his breakfast. The end of his spare time had now arrived, and emerging from the garden by getting over the hedge at the back he pursued a path northward, till he came to a wide and lonely depression in the general level of the upland, which was sown as a corn-field. This vast concave was the scene of his labours for Mr Troutham the farmer, and he descended into the midst of it. The brown surface of the field went right up towards the sky all round, where it was lost by degrees in the mist that shut out the actual verge and accentuated the solitude. The only marks on the uniformity of the scene were a rick of last year's produce standing in the midst of the arable, the rooks that rose at his approach, and the path athwart the fallow by which he had come, trodden now by he hardly knew whom, though once by many of his own dead family. "How ugly it is here!" he murmured. The fresh harrow-lines seemed to stretch like the channellings in a piece of new corduroy, lending a meanly utilitarian air to the expanse, taking away its gradations, and depriving it of all history beyond that of the few recent months, though to every clod and stone there really attached associations enough and to spare--echoes of songs from ancient harvest-days, of spoken words, and of sturdy deeds. Every inch of ground had been the site, first or last, of energy, gaiety, horse-play, bickerings, weariness. Groups of gleaners had squatted in the sun on every square yard. Love-matches that had populated the adjoining hamlet had been made up there between reaping and carrying. Under the hedge which divided the field from a distant plantation girls had given themselves to lovers who would not turn their heads to look at them by the next harvest; and in that ancient cornfield many a man had made love-promises to a woman at whose voice he had trembled by the next seed-time after fulfilling them in the church adjoining. But this neither Jude nor the rooks around him considered. For them it was a lonely place, possessing, in the one view, only the quality of a work-ground, and in the other that of a granary good to feed in. The boy stood under the rick before mentioned, and every few seconds used his clacker or rattle briskly. At each clack the rooks left off pecking, and rose and went away on their leisurely wings, burnished like tassets of mail, afterwards wheeling back and regarding him warily, and descending to feed at a more respectful distance. He sounded the clacker till his arm ached, and at length his heart grew sympathetic with the birds' thwarted desires. They seemed, like himself, to be living in a world which did not want them. Why should he frighten them away? They took upon more and more the aspect of gentle friends and pensioners--the only friends he could claim as being in the least degree interested in him, for his aunt had often told him that she was not. He ceased his rattling, and they alighted anew. "Poor little dears!" said Jude, aloud. "You SHALL have some dinner-- you shall. There is enough for us all. Farmer Troutham can afford to let you have some. Eat, then my dear little birdies, and make a good meal!" They stayed and ate, inky spots on the nut-brown soil, and Jude enjoyed their appetite. A magic thread of fellow-feeling united his own life with theirs. Puny and sorry as those lives were, they much resembled his own. His clacker he had by this time thrown away from him, as being a mean and sordid instrument, offensive both to the birds and to himself as their friend. All at once he became conscious of a smart blow upon his buttocks, followed by a loud clack, which announced to his surprised senses that the clacker had been the instrument of offence used. The birds and Jude started up simultaneously, and the dazed eyes of the latter beheld the farmer in person, the great Troutham himself, his red face glaring down upon Jude's cowering frame, the clacker swinging in his hand. "So it's 'Eat my dear birdies,' is it, young man? 'Eat, dear birdies,' indeed! I'll tickle your breeches, and see if you say, 'Eat, dear birdies,' again in a hurry! And you've been idling at the schoolmaster's too, instead of coming here, ha'n't ye, hey? That's how you earn your sixpence a day for keeping the rooks off my corn!" Whilst saluting Jude's ears with this impassioned rhetoric, Troutham had seized his left hand with his own left, and swinging his slim frame round him at arm's-length, again struck Jude on the hind parts with the flat side of Jude's own rattle, till the field echoed with the blows, which were delivered once or twice at each revolution. "Don't 'ee, sir--please don't 'ee!" cried the whirling child, as helpless under the centrifugal tendency of his person as a hooked fish swinging to land, and beholding the hill, the rick, the plantation, the path, and the rooks going round and round him in an amazing circular race. "I--I sir--only meant that--there was a good crop in the ground--I saw 'em sow it--and the rooks could have a little bit for dinner--and you wouldn't miss it, sir--and Mr. Phillotson said I was to be kind to 'em--oh, oh, oh!" This truthful explanation seemed to exasperate the farmer even more than if Jude had stoutly denied saying anything at all, and he still smacked the whirling urchin, the clacks of the instrument continuing to resound all across the field and as far as the ears of distant workers--who gathered thereupon that Jude was pursuing his business of clacking with great assiduity--and echoing from the brand-new church tower just behind the mist, towards the building of which structure the farmer had largely subscribed, to testify his love for God and man. Presently Troutham grew tired of his punitive task, and depositing the quivering boy on his legs, took a sixpence from his pocket and gave it him in payment for his day's work, telling him to go home and never let him see him in one of those fields again. Jude leaped out of arm's reach, and walked along the trackway weeping--not from the pain, though that was keen enough; not from the perception of the flaw in the terrestrial scheme, by which what was good for God's birds was bad for God's gardener; but with the awful sense that he had wholly disgraced himself before he had been a year in the parish, and hence might be a burden to his great-aunt for life. With this shadow on his mind he did not care to show himself in the village, and went homeward by a roundabout track behind a high hedge and across a pasture. Here he beheld scores of coupled earthworms lying half their length on the surface of the damp ground, as they always did in such weather at that time of the year. It was impossible to advance in regular steps without crushing some of them at each tread. Though Farmer Troutham had just hurt him, he was a boy who could not himself bear to hurt anything. He had never brought home a nest of young birds without lying awake in misery half the night after, and often reinstating them and the nest in their original place the next morning. He could scarcely bear to see trees cut down or lopped, from a fancy that it hurt them; and late pruning, when the sap was up and the tree bled profusely, had been a positive grief to him in his infancy. This weakness of character, as it may be called, suggested that he was the sort of man who was born to ache a good deal before the fall of the curtain upon his unnecessary life should signify that all was well with him again. He carefully picked his way on tiptoe among the earthworms, without killing a single one. On entering the cottage he found his aunt selling a penny loaf to a little girl, and when the customer was gone she said, "Well, how do you come to be back here in the middle of the morning like this?" "I'm turned away." "What?" "Mr. Troutham have turned me away because I let the rooks have a few peckings of corn. And there's my wages--the last I shall ever hae!" He threw the sixpence tragically on the table. "Ah!" said his aunt, suspending her breath. And she opened upon him a lecture on how she would now have him all the spring upon her hands doing nothing. "If you can't skeer birds, what can ye do? There! don't ye look so deedy! Farmer Troutham is not so much better than myself, come to that. But 'tis as Job said, 'Now they that are younger than I have me in derision, whose fathers I would have disdained to have set with the dogs of my flock.' His father was my father's journeyman, anyhow, and I must have been a fool to let 'ee go to work for 'n, which I shouldn't ha' done but to keep 'ee out of mischty." More angry with Jude for demeaning her by coming there than for dereliction of duty, she rated him primarily from that point of view, and only secondarily from a moral one. "Not that you should have let the birds eat what Farmer Troutham planted. Of course you was wrong in that. Jude, Jude, why didstn't go off with that schoolmaster of thine to Christminster or somewhere? But, oh no--poor or'nary child--there never was any sprawl on thy side of the family, and never will be!" "Where is this beautiful city, Aunt--this place where Mr. Phillotson is gone to?" asked the boy, after meditating in silence. "Lord! you ought to know where the city of Christminster is. Near a score of miles from here. It is a place much too good for you ever to have much to do with, poor boy, I'm a-thinking." "And will Mr. Phillotson always be there?" "How can I tell?" "Could I go to see him?" "Lord, no! You didn't grow up hereabout, or you wouldn't ask such as that. We've never had anything to do with folk in Christminster, nor folk in Christminster with we." Jude went out, and, feeling more than ever his existence to be an undemanded one, he lay down upon his back on a heap of litter near the pig-sty. The fog had by this time become more translucent, and the position of the sun could be seen through it. He pulled his straw hat over his face, and peered through the interstices of the plaiting at the white brightness, vaguely reflecting. Growing up brought responsibilities, he found. Events did not rhyme quite as he had thought. Nature's logic was too horrid for him to care for. That mercy towards one set of creatures was cruelty towards another sickened his sense of harmony. As you got older, and felt yourself to be at the centre of your time, and not at a point in its circumference, as you had felt when you were little, you were seized with a sort of shuddering, he perceived. All around you there seemed to be something glaring, garish, rattling, and the noises and glares hit upon the little cell called your life, and shook it, and warped it. If he could only prevent himself growing up! He did not want to be a man. Then, like the natural boy, he forgot his despondency, and sprang up. During the remainder of the morning he helped his aunt, and in the afternoon, when there was nothing more to be done, he went into the village. Here he asked a man whereabouts Christminster lay. "Christminster? Oh, well, out by there yonder; though I've never bin there--not I. I've never had any business at such a place." The man pointed north-eastward, in the very direction where lay that field in which Jude had so disgraced himself. There was something unpleasant about the coincidence for the moment, but the fearsomeness of this fact rather increased his curiosity about the city. The farmer had said he was never to be seen in that field again; yet Christminster lay across it, and the path was a public one. So, stealing out of the hamlet, he descended into the same hollow which had witnessed his punishment in the morning, never swerving an inch from the path, and climbing up the long and tedious ascent on the other side till the track joined the highway by a little clump of trees. Here the ploughed land ended, and all before him was bleak open down.
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Summary: The schoolmaster, Mr. Phillotson, is preparing to leave the village of Marygreen in Wessex. He is bound for Christminster, where he intends to take a university degree and then be ordained. He is helped in his preparation by Jude Fawley, an eleven-year-old boy who has been his student and who admires him. Phillotson has given the boy a book as a farewell gift, and the schoolmaster tells Jude to look him up if he ever comes to Christminster. After Phillotson leaves, Jude stands thinking of the schoolmaster at the old well, to which he has come originally to draw water for his great-aunt and which is one of few old parts of the village still remaining, the rest having been replaced by more modern structures, most notably the church. Returning to the house with water from the well, Jude hears his great-aunt, Drusilla Fawley, who runs a bakery in the house, in conversation with some friends. When he enters the room, his aunt explains to her friends the circumstances of Jude's life that brought him into her care a year before. She describes him as bookish, like his cousin Sue, and tells him that he should never marry, since the Fawleys are unlucky in matrimony. Jude goes off to his job in Farmer Troutham's cornfields, where he is supposed to scare off the rooks with a noisemaker. Depressed by the ugliness of the fields and sympathetic with the birds' hunger, he soon gives up his noisemaking and happily watches the birds eat. He is caught by Troutham, reprimanded, and punished for deserting his duties, and dismissed from his job. His aunt is annoyed by his now having nothing to occupy him and wonders aloud why he didn't go off with the schoolteacher to Christminster. Jude asks her about this city but is told he'll never be able to have anything to do with it. Jude leaves, reflecting on the difficulties of growing up and the incomprehensibility of life. He decides to see Christminster and starts off, his direction necessarily taking him back through Farmer Troutham's fields.
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Summarize: Federal law requires us to obtain and report your occupation and the name of your employer. If you are not employed, please enter None. By clicking "Contribute," I agree that the following statements are true and accurate: Processing, please wait. Contributions to Romney Victory, Inc. (“RV”) are not tax deductible for federal income tax purposes. RV is a joint fundraising committee composed of Romney for President, Inc. (“RFP”), the Republican National Committee (“RNC”), the official Republican Parties in four states (the Idaho Republican Party, the Massachusetts Republican Party, the Oklahoma Leadership Council, and the Vermont Republican Federal Election Committee), the National Republican Senatorial Committee, and the National Republican Congressional Committee. Proceeds shall be allocated by the following formula: Individuals and non-multicandidate PACs - The first $2,500 will go to RFP’s primary account. The next $2,500 will go to RFP’s general account. The next $30,800 will go to the RNC. The remaining amount will be split evenly among the federal accounts of the Republican Parties of Idaho, Massachusetts, Oklahoma, and Vermont, up to a maximum of $10,000 per committee. Multicandidate PACs - The first $5,000 will go to RFP’s primary account. The next $5,000 will go to RFP’s general account. The next $15,000 will go to the RNC. The remaining amount will be split evenly among the federal accounts of the Republican Parties of Idaho, Massachusetts, Oklahoma, and Vermont, up to a maximum of $5,000 per committee. Notwithstanding this allocation formula, a contributor may designate a contribution for a specific participant or participants. In addition, the allocation formula may change if any contribution would exceed applicable contribution limits. Contributions from corporations, labor unions, federal contractors, and foreign nationals are prohibited. Federal law requires us to collect and report the name, mailing address, occupation, and name of employer of each contributor whose contributions aggregate in excess of $200 in a calendar year (RNC and state parties) or election cycle (RFP). These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites.
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Summary: Yet another sign this is definitely a 21st-century campaign: Mitt Romney and Barack Obama have been snatching up dozens of web addresses to use as potential microsites that take a swing at some aspect of their opponent, each spending about $30,000 on domain-name purchases. And while these catchy, often issue- or gaffe-specific domain names are, in many cases, still sitting idle, USA Today's collection of ones purchased thus far is pretty entertaining. It recounts Romney's acquisitions in much greater detail. As far an Obama goes, USA Today notes that he takes a more defensive approach (attackwatch.com) but also claims some that definitely take aim at his rival (Mittbot.com). Romney owns: agingrockstar2012.com theapologistinchief.com o-the-amateur.com fortyfore.com (this one is up and running, and tweaks Obama's love for golf) Mom-Ney.com (purchased after the accusation that Ann Romney has "never worked a day in her life" IwilltransmityourflexibilitytoVladimir.com.
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Summarize: This application claims the benefit of Provisional Application No. 60/374,240, filed Apr. 19, 2002. FIELD OF THE INVENTION The present invention generally relates to storage and display items. More specifically, the present invention relates to shelving and even more specifically to adjustable shelving adapted to conform to a desired dimension. BACKGROUND OF THE INVENTION As our lifestyles become more and more complex, we desire devices that enable organization. This helps to bring our lives some sort of normality. Shelving products have evolved since the days of the uncovered “cup boards” in the late 18 th century to the modern cabinets with decorator doors and movable shelving as seen today. Though the shelving is typically “movable” it is not easy to move. The shelves must be unloaded, some sort of stop or mounting on two or more corners must be removed, and repositioned, the shelf reinstalled and then the shelf can again be loaded. This is a time consuming and labor-intensive project. As such, it is usually only done when the shelving is installed and never moved again. Many people are unaware that their shelving is movable. As far a width adjustment, there are very few choices. For the most part this is restricted to buying or building the shelf or bookcase at the desired width. That will never be changed. There are some “cup rack” type supports that offer a width adjustment. These usually include a base with an extension on one side. One of the problems is since there is an extension on a side the upper surface now has two levels, one for the base and one for the extension. This uneven surface is not only unattractive, but does not lend itself well to stacking items. Also, these devices do not provide for vertical or height adjustment. SUMMARY OF THE INVENTION In one aspect, the invention features a shelf with an adjustable height first leg and an adjustable height second leg positioned adjacent to the first leg. A leg extension is mounted to the first leg and the second leg, and positioned substantially orthogonal thereto. Also, a shelf portion is used that is capable of being received by the leg extensions, the shelf portion being movably mounted thereon, whereby width adjustment is enabled by varying placement of the shelf relative to the leg extensions. The shelf portion and the leg extensions may be movably mounted one to the other by a tongue in groove. In the preferred embodiment the leg extension includes the groove and the shelf portion includes the tongue. The system may also include the adjustable height first leg and adjustable height second leg as each being comprised of a first portion and a second portion, which are movably mounted one to the other. The two-part construction of the legs may also include a lock releasably securing the first portion to the second portion. The lock may be a device such as a pawl, a screw or a pin. The pawl can be pivotally mounted to the first portion, and may include a bias, such as a spring or more specifically a spring coil. The second portion would include a rack and the bias applies a force to enable engagement of the pawl and the rack. The device may also include a comprising an end cover, which is capable of receiving the leg extension and end cover may be modifiable in length. This can be accomplished by providing a plurality of undercuts on the end cover. The first leg and the second leg may include a foot positioned on a distal end thereof and opposite to the shelf portion, as a support on which the shelf may stand. The feet can be mounted to the legs by providing the first leg and the second leg each with a mounting tab positioned on a distal end thereof and opposite to the shelf portion. A mounting tab receiver is then positioned adjacent to the shelf portion on the leg opposite to the mounting tab. This also enables stacking of one shelf on the other by removing the feet and inserting the mounting tabs of one shelf in the mounting tab receivers of another shelf, thus releasably securing one to the other. In another aspect, the invention includes a method of providing an adjustable shelf as described, placing the device in said specific area and adjusting the height of the first leg and the second leg to provide a preferred vertical position of the shelf portion. The horizontal adjustment is then provided by adjusting the placement of the first leg and the second leg, thereby allowing the shelf portion to move relative to, and yet be supported by, the leg extensions. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, described: FIG. 1 is an isometric front, upper view of an adjustable shelf produced in accordance with the present invention. FIG. 2 is an isometric rear, lower view of an adjustable shelf produced in accordance with the present invention. FIG. 3 is an isometric front, upper view of an adjustable shelf with the end covers shortened to allow access to the tab receivers thus enabling stacking of multiple shelves, the shelves made in accordance with the present invention. FIG. 4 is an exploded isometric front, upper view of an adjustable shelf produced in accordance with the present invention. FIG. 5 is an exploded isometric upper view of the upper portion of a leg, a leg extension and a section of a shelf portion, showing the assembly, the shelf produced in accordance with the present invention. FIG. 6 is an exploded rear isometric view of a leg and foot assembly produced in accordance with the present invention. FIG. 7 is an exploded isometric view of a pawl and rack height adjustment lock produced in accordance with the present invention. FIG. 8 is an isometric view of a leg and foot assembly with a pin lock, the device produced in accordance with the present invention. FIG. 9 is an isometric view of a leg and foot assembly with a screw knob lock, the device produced in accordance with the present invention. FIGS. 10 a and 10 b are side views of an adjustable shelf in retracted and vertically extended positions respectively, the device produced in accordance with the present invention. FIGS. 11 a and 11 b are rear views of an adjustable shelf in a vertically retracted position showing both compact and extended horizontal positions respectively, the device produced in accordance with the present invention. For the most part, and as will be apparent when referring to the figures, when an item is used unchanged in more than one figure, it is identified by the same alphanumeric reference indicator in all figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention is an adjustable shelf that enables both vertical and horizontal adjustment. The fully assembled shelf 12 is shown in FIG. 1. This is a front, side, upper view that shows the preferred legs 14 which are comprised of a first portion 16 and a second portion 18. This two-part telescoping leg assembly allows for compact storage and an elegant appearance. A lock 20, shown here as a pawl 22, provides a set leg length that can be determined by the user. On the bottom of the second portion 18 of the leg 14, is positioned a foot 24. The foot 24 is optimally releasably mounted to the end of the second portion 18, but can be permanently fixed thereto. The advantage of the releasable mounting will be discussed later. The upper end of the first portion 16 supports a planar surface including a shelf portion 26 and may include one or more end covers 28. The end covers 28 and the shelf portion 26 comprise the supportive surface on which items can be stored. In FIG. 2 the “under side” of the shelf 12 is shown from the back. Here in the preferred embodiment the shelf portion 26 is shown to be open from the bottom. Leg extensions 30 are supported to the upper portions of the legs 14 and extend toward the other leg. This provides a “track” that enables the shelf portion 26 to move with respect to, while being supported thereon. The inside edges of the leg extensions 30 can be moved closer to or further apart from each other thus enabling a variation in shelf width. The height adjustment of the legs 14 is more clearly seen in this figure. In this, the preferred embodiment, the second portion 18 of the leg 14 includes a rack 32. This rack 32 can be molded into the second portion 18, as shown here or it can be a separate part that is mounted thereto. In either case, the rack includes teeth that provide a graduated vertical set of “steps” for attachment of the pawl to conditionally secure the first and second portions of the legs. This provides an easy and efficient method of adjustment of the vertical aspect of the shelf. In FIG. 3, at the top end of the first portion 16 of the leg 14 is a leg base 34. The leg base 34 has a primary function of providing a cap for the upper edge of the first portion 16 of the leg 14 as well as providing an attachment for the leg extension (item 30 shown in FIG. 2 ). The leg base 34 can be exposed by either shortening the end covers 28, or by extending the legs 14 further away from one another. The leg base 34 may include one or more mounting tab receivers 36. These receivers 36 can take a variety of shapes and configurations, but are intended to mate with mounting tabs (not shown here) on the bottom end of the second portion 18 of the leg 14. The foot 24 is releasably fastened to the second portion 18 by the foot also including tab receivers, similar to those in the leg base 34. This combination allows the user to stack a second shelf on a first shelf by remove the foot 24 from the second shelf and placing the mounting tabs exposed by removing the foot 24, and placing the tabs into the receivers 36 on the leg base 34 of the first shelf. This allows secure stacking of one shelf on the other. An exploded view of the invention 12 is shown in FIG. 4 to better shown the relationship between the parts. The leg extension 30 is shown here to be releasably secured to the leg base 34. This is done to provide for a more efficient “knock down” of the product to reduce shipping costs. A locking tab 38 is used to articulate with a hole in the bottom of the leg extension 30 and they are supported together by the leg protrusions 40 that mate with the protrusion cavities 42 on the leg extension 30. This is only one method of assembly of these parts and is not intended to limit the scope of the invention. The invention can also be manufactured such that the leg extensions and the leg base 34 are one part. The shape of the leg extension 30 is shown here to mate with and allow movement of the shelf portion 26 relative thereto. The ability of the shelf portion 26 to slide against the leg extension 30 and yet be supported by the leg extensions 30, allows for horizontal adjustment of the leg positions. The vertical adjustment is provided by the second portion 18, which is received by the first portion 16 of the leg 14. The lock in the form of a pawl 22 is shown to also be exploded from the first portion 16. The details of this and other locks will be discussed later. At the lower distal end of the second portion 18 are more clearly shown the mounting tabs 44. These tabs 44 are shown here to be substantially in the shape of a cylindrical pin, but this general shape and specific details are not critical to the novelty of the invention. The tab receivers 46 in the feet 24 are made to fit the tabs 44, thereby releasably locking them together. In a similar manner each leg base 34 also includes a receiver 36 to allow for stacking of the shelves 12, as previously noted. Further detail of the function and assembly of the leg 14 via the leg base 34 to the leg extension 30 is shown in FIG. 5. The leg protrusions 40 are received by the protrusion cavities 42 of the leg extension 30. The locking tab 38 includes a pin 48 which extends downward from the underneath side of the tab 38. This pin 48 also extends through a cavity 42 to be received by a hole in the bottom of the extension 30. With the protrusions 40 positioned within the cavities 42 (as shown by the arrow 50 ) and the pin 48 securing them in place, the structure of the leg 14 with the leg extension 30 via the leg base 34 is functionally one rigid unit. The shelf portion 26 is received by the leg extension 30 as shown by the second arrow 52. Though the shelf portion 26 can take a variety of forms, what is shown is considered by the applicants to be the preferred embodiment. The bottom side of the shelf portion 26 is open except for the “C” shaped edges 54. These “rails” run the length of the shelf portion 26 to provide for structural rigidity of the shelf portion 26 without excess material to cause potential part interference, added weight or cost. The shelf portion also includes a pair of tongues 56. The tongue 56 has a multi-fold purpose. First the material placement adds to the section modulus of the shelf portion about the axis that would see flexion when the shelf is loaded. This adds to the strength of the shelf portion especially when the shelf is at an extended position where a minimal amount of contact is made between the shelf portion 26 and the leg extensions 30. The second purpose to the tongue 56 is as a tracking guide along the grooves 58 located within the leg extensions 30. This tracking assistance reduces the likelihood for the shelf portion 26 to bind when moving along the leg extension 30 when the shelf width is being changed. The third advantage to the tongue 56 and groove 58 combination is during the loading of the shelf. When items are placed on the shelf portion 26, during its intended purpose of item storage, the weight of these items will cause the leg extensions 30 to flex slightly along an axis parallel to the long axis of the grooves 58. This is due to the reduced section at the grooves due to the presence of the grooves 58. The upper portion is open. When this happens the outside upper edges of the groove 58 will pinch together slightly, grabbing the tongue positioned there between. The friction due to the contact of the tongue and grooves acts as a “lock” to further stabilize the shelf and prevent it from moving from side to side when it is loaded. This eliminates the need for further locking of the shelf portion 26 to the leg extensions 30 when the shelf is in place. Under more extreme conditions, an additional lock mechanism may be used. As such, it is understood that any form of locking mechanism known in the art can be added between the shelf portion 26 and the leg extensions 30. A single leg 14 is shown in FIG. 6. Here the first portion 16 is shown with a pair of ears 60, which hold the pawl 22 as it is pinned through the pawl hole 62 and the ear holes 64. The pawl includes a pawl handle 66 and a pawl tip 68. The handle acts to enable the user to manipulate the pawl tip 68 to disengage it from the rack teeth 32 in the second portion 18 of the leg. The mounting tabs 44 are received by the tab receivers 46 in the foot 24. Also as previously noted, the mounting tabs 44 of another leg can be received by the receivers 36 in the leg base 34. A more detailed view of the locking mechanism is shown in FIG. 7. The pawl 22 is shown as removed from the first portion 16 showing the rack window 70. This window 70 allows access of the pawl tip 68 to the rack teeth 32. The pawl 22 can be manufactured from an number of materials but is preferably made from a plastic. This is inexpensive and allows for good elastic properties. The elastic properties are relevant in that in the preferred embodiment the pawl also includes a spring coil 72. This spring can be a separate item that is attached to the pawl 22 or as in this case a molded portion of the pawl 22. The free end of the spring coil 72 is positioned on the ridge 74 located above the window 70 and on the first portion 16. The spring then pushes the handle 66 of the pawl 22 out away from the rack 32, about the pivot of the pawl hole 62 and the ear holes 64, thus engaging the pawl tip 68 into the rack teeth 32. To disengage the pawl tip 68 from the rack 32, the handle 66 is pressed in toward the first portion 16, pulling the tip away form the rack teeth 32. This flexes the spring 72 so when the handle 66 is released by the user, the bias from the spring 72 reengages the tip 68 and the rack 32, locking one to the other. Another form of locking of the first portion 16 and the second portion 18 is accomplished by a pin 76 as shown in FIG. 8. Here the pin 76 is shown as it would be assembled into a first hole 78 located in the first portion 16 and also one of a plurality of second holes 80 located in the second portion 18. The first portion 16 is still able to move along the long axis of the second portion 18, as previously noted, only the locking mechanism is comprised of the pin 76 positioned through a pair of properly aligned holes ( 78 and 80 ). To achieve an infinite variety of height adjustments, a screw knob can be used for height adjustment. This is illustrated in FIG. 9. Here a knob 82 with a pressure pin 84 mounted to the end also includes a threaded portion 86. The threaded portion 86 is received by the mating threads 88 on a receiver 90, which is mounted to the first portion 16. A hole (not shown) is located within the receiver 90 to allow the pressure pin 84 access through the first portion 16 to contact the second portion 18. The friction between the pin 84 and the second portion 18 is provided and varied by the amount of tightening of the knob 82, thus pushing the pin 84 more firmly against the second portion 18. The height adjustment of the device is shown in a side view in FIGS. 10 a and 10 b. The minimum height of “Y 1 ” is shown in FIG. 10 a where the pawl 22 is at the bottom of the rack 32. The highest position “Y 2 ” is shown in FIG. 10 b. The difference being the relative positioning of the first portion 16 to the second portion 18 and as held in place by the pawl 22 and rack 32. The rack 32 design is also shown here to be of a saw-tooth design. This provides a vertical slant upward followed by a substantially horizontal “ledge”. The pawl tip 68 includes a shape that nearly matches this “V”, thus providing a locking of the pawl 22 to prevent downward movement of the first portion 16. In this application, this design is beneficial in that little resistance is offered to restrict vertical movement of the first portion relative 16 to the second portion 18. This allows the user to lift the shelf portion 26, and the weight of the second portion 18 and the feet 24 will allow the second portion to “fall” away from the first portion 16, thus “ratcheting” out to fit the height needs of the user. When the user desires a lower height, the user needs only to press the handle 66 of the pawl 22 in, releasing the pawl tip and allowing the first portion 16 to freely move relative to the second portion 18. FIGS. 11 a and 11 b show the horizontal width adjustment provided by the device. The narrow position is shown by the dimension “X 1 ” in FIG. 11 a and the widest dimension is depicted by “X 2 ” in FIG. 11 b. As can be seen here, the shelf portion 26 remains at a constant place in both positions. The relative position of the legs 14 and the gap between the leg extensions 30 increases from “Xa” to “Xb”. This provides the increased width. It is understood that both the height adjustment, as shown in FIGS. 10 a and 10 b, as well as the width adjustment, as shown here, can both be performed together in any combination allowed by the specific design of the elements of the device. Thereby providing both width and height adjustment in a single shelf to fit any of an infinite number of space requirements.
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Summary: A shelf stores items and provides for organized and attractive display with ease of item access. Conforming to a defined physical environment or variable storage space optimally requires the shelf to provide for both vertical and horizontal adjustment. Here this is accomplished by a pair of legs that are telescopic (to reduce size) and can be locked at a height (vertical adjustment) by use of a pawl, pin, screw or other fastener known in the art. The shelf includes a planar surface that is comprised of a shelf portion, which is supported on each distal end by a leg extension. The leg extensions are mounted to the upper area of the legs. The leg extensions are received by the shelf portion, being movably mounted thereto. This allows for horizontal (width) adjustment. An end cover can be used to provide a flat surface that is consistent with the upper surface of the shelf portion. Feet are added to provide a more solid footing, the feet being removable and providing a mounting tab at the end of the legs and a mounting tab receiver at the opposite end of the legs. This allows for multiple shelves to be securely mounted one on the other.
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Summarize: A few years ago, we were warned that the world’s helium reserves were running out. Today, researchers announced there may be several potential new sources of the precious gas hidden throughout the world. Helium, which is used widely in nuclear, medical and party industries, has become worryingly scarce. Despite it being the second most abundant element in the universe, here on Earth it’s rare - it is so light that it leaks away into space – and our major resources are running low. Practically all of the world’s helium reserves have been derived as a by-product from the extraction of natural gas from underground gas traps. “No-one could really pinpont where the next deposit would appear,” said Diveena Danabalan, a PhD student at the University of Durham. This has made it difficult to locate new sources of helium. To make the quest a little easier, Danabalan and her colleagues have identified how helium makes its way into gas traps. They analysed natural gas samples from 22 wells throughout the US and Canada and discovered that wherever helium was present, there was also a second chemical that is only ever associated with ground-water. Danabalan said: “This, we realised, meant that helium has to have been dissolved in groundwater and then transported throughout the US and Canada until it met a geological structure that drew it back out of the water and into an underground trap.” Danabalan presented the findings today at the Goldschmidt conference in Prague. Since known helium traps appear in regions such as the Rocky Mountains that were formed from ancient tectonic movements, the team reckon it may have been these climactic events occurring 135 million years ago that released helium into the groundwater in the first place. Since these tectonic events happened all over the planet, it means there is potentially several traps full of commercial quantities of helium throughout the world, said Danabalan’s colleague Christopher Ballentine of the University of Oxford. The problem is finding them. “We need to study maps that have been made for that period that infer groundwater direction, which will hopefully allow us pinpoint geological regions that might contain helium traps,” said Danabalan. The research looks interesting and is well done, said Jacob Lowenstern, a geologist at the US Geological Survey. “It shows that the accumulation of helium may occur in more geological settings than previously recognised.” Image of a helium filled discharge tube shaped like the element's atomic symbol. Image: Pslawinski/ Wikipedia. Helium gas - essential for MRI scanners, semiconductor manufacture (and according to some, party balloons) - may not be on the verge of running out after all. Previous studies had raised concerns that we were getting close to a world shortage of helium, but a new study shows that in many areas of North America, there is the potential for undiscovered quantities of helium to be associated with natural gas fields. This work is being presented in an award-winning poster at the Goldschmidt conference in Prague. A group of researchers from Durham and Oxford Universities, led by Diveena Danabalan, analysed natural gas samples from 22 wells in the USA and Canada. Using mass spectroscopy, they measured the full suite of noble gases, with an emphasis on helium, neon and argon. Diveena Danabalan said:"We identified neon isotope tracers which show a strong association between helium and groundwater. This means that in certain geological regions, groundwater transports large volumes of helium into natural gas fields, where trapping potential is greatest. This suggests that we have probably underestimated the volumes of helium which are actually available to explore". "On a continental scale, and we are talking about a line running right down the Rocky Mountains, we are seeing processes which are releasing the existing helium which has been built up deep underground over hundreds of millions of years. In some places, like in Yellowstone Park in Wyoming, the deep helium is released directly into the atmosphere. In others, we are seeing that the deep helium which was released when the Rocky Mountains formed has percolated via the groundwater into the same underground reservoirs where we find natural gas. This means that there are almost certainly reservoirs of helium which we had not anticipated. More importantly, understanding how and why helium arrives in these reservoirs means that we now know where to look for new helium resources" She continued:"Helium is the second lightest element in nature, it is so light that it leaks away into space. This is why it is ultimately a non-renewable, finite resource". Helium is the essential for use in the MRI body scanners and superconductor production. It is also used in super-hi-tech installations such as the Large Hadron Collider at CERN, which address questions on the origins of the universe Commenting for the Goldschmidt conference, Professor Barbara Sherwood Lollar, (University of Toronto) President of the Geochemical Society said: "This work shows that the amount of helium which is available is greater than we had thought. Helium is essential for many things, with the most important use being in body scanners and in microchip manufacture. So in many ways it is essential for modern medicine, and perhaps even for modern life. The lightness of helium means that it can escape off into space, to be lost forever, so a steady supply is needed. This work shows that it is trapped in natural processes, so the lightness of helium may not be as unbearable as we had thought". Explore further: Helium anomaly preceded Mount Ontake eruption More information: Noble gas evidence for the mechanisms creating commercial helium reservoirs, poster at the Goldschmidt conference 2015. ABSTRACT Since its first economic discovery in Kansas in 1903, helium has become an integral resource for today's society. With known reserves declining, discoveries of helium are still only serendipitously found with some petroleum discoveries; there are no viable exploration strategies in place for helium. Radiogenic 4He accumulates in the crust during quiescent periods and is subsequently released during periods of active tectonism [1]. In the Hugoton-Panhandle He-rich gas field (Texas/Oklahoma/Kansas), 4He correlates with water-derived 20Ne. This relationship is used to suggest that helium released from basement rock is dissolved in overlying groundwater and then quantitatively degassed following migration and contact with a pre-existing hydrocarbon gas phase [2]. To test whether this mechanism is common to other helium-rich natural gas fields, we have collected 22 natural gas samples to analyse for noble gas abundance and isotope determination. Preliminary data from the Kansas Basin, Central Kansas Uplift, the Harley Dome field in Utah, and two exploration wells in Montana and Saskatchewan, Canada, have helium contents that vary between 0.009 cc STP and 0.080 cc STP. The helium isotope ratio, 3He/4He, ranges from 0.08 RA to 0.66 RA (RA = 3He/4Heair) showing a predominantly crustal He source with a small mantle input for most samples. Argon isotope (40Ar/36Ar) ratios range from 589-1435 in the Kansas samples and from 4586-8963 in the Harley Dome, Montana and Saskatchewan samples. 4He/20Ne in the Hugoton- Panhandle averages 3.4 × 104 [2]. Kansas samples range from 4He/20Ne = 3.5-7.8 × 104, showing the same degree of water involvement as the Hugoton-Panhandle for the lowest values. Harley Dome, Montana and Saskatchewan samples have 4He/20Ne = 1.0-2.3 × 105 showing a 3-7 times lower involvement of the groundwater system, consistent with the elevated 40Ar/36Ar ratios.
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Summary: Don't mourn the loss of your humorously high-pitched voices quite yet. Despite years of warnings from scientists that Earth's supply of helium is quickly running out, the results of a study announced Wednesday in Prague show there could still be large deposits of the element hidden underground, the Guardian reports. Helium, as important to the nuclear industry as the birthday party industry, is essential to everything from MRIs to the Large Hadron Collider, according to Phys.org. But as PhD student Diveena Danabalan explains, "Helium is the second lightest element in nature, it is so light that it leaks away into space." That makes it a finite resource, and most of the helium we've used so far has come as a byproduct of natural gas extraction. But "no one could really pinpont where the next deposit would appear," says Danabalan. That problem that led researchers to look at 22 natural gas wells in the United States and Canada. According to Phys.org, whenever helium was found in those wells, so were neon isotope tracers, a chemical almost always associated with groundwater. "This, we realized, meant that helium has to have been dissolved in groundwater and then transported throughout the US and Canada until it met a geological structure that drew it back out of the water," Danabalan tells the Guardian. Knowing how and why helium got into natural gas reservoirs means scientists have a starting point in terms of where to look to find new helium deposits. Now, they just have to actually find them.
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Summarize: This application claims benefit under 35 U.S.C. 371 National Stage Application based on PCT/US98/19868, filed Sep. 23, 1998 which also claims benefit under 35 U.S.C. 119(e) of No. 60/059,791 filed Sep. 23, 1997 which claims benefit of No. 60/059,645, filed Sep. 24, 1997 which claims benefit of No. 60/061,136 filed Oct. 6, 1997. BACKGROUND OF THE INVENTION The commercial baking industry has long sought additives which improve softness and prevent staling of baked products. The ordinary consumer equates softness with freshness and any change in a baked product during storage which develops firmer texture is viewed as undesirable. Such change is, however, inevitable as time dependent effects which are largely associated with the starch component of flour begin to occur immediately after baking. This staling phenomenon ultimately restricts the lifetime of a baked product prior to use and has major impact on the economics associated with sales and distribution. While much study has been devoted to staling of baked products, and much progress has been made in the understanding of the process, there are still many unanswered questions regarding the staling mechanism. Flour contains predominantly two complex macromolecular structures—gluten and starch fraction. Each contributes both functional and nutritional aspects to baked products. Gluten is believed to be more dominant with respect to formation and stabilization of the developing gas cell structure, while starch is believed to be important in water absorption and stabilization of the skeletal cellular network that remains after the baking cycle. Mechanically developed gluten (produced during dough mixing) consists of a microscopically stranded network of thread like structures surrounding a discontinuous phase of dispersed starch granules. During the fermentation stage microscopic gas pockets nucleate and are stabilized by the continuous phase comprised of the gluten starch network. Ultimately a dispersed foam structure is formed. On baking the gluten is denatured and the starch is gelatinized about a thermally expanding gas cell to form a stable film compartmentalizing the dispersed gas cells. After baking the film begins to harden as the two principal starch components, amylose and amylopectin, begin to pass through their respective glass transition temperature ranges. The final baked product is best described as a microscopically networked sponge. Over time, profound physical changes begin to occur largely due to moisture migration from the central interior toward the periphery of the baked dough piece and oligomeric restructuring of the amylopectin and amylose starch components. Some ordering of the now partially denatured gluten structure may also begin to occur. There are three recognized ways to enhance softness and abate staling. Each has some limitation. Firstly, it has been found that alpha monoglycerides of C-16 and C-18 fatty acids (particularly glycerol monosterate, GMS) are very effective in the prevention of retrogradation (crystal formation) of the amylose fraction. Amylose forms strong helical clathrate structures about GMS which prevents or interrupts substantial crystalline domains from forming with this high molecular weight alpha 1-4 linear glucan. This is largely a one time fix as the effect is immediate after baking when there is still some mobility for the amylose chains to configurationally encase the GMS. There is a limit to the effectiveness of GMS, as the available sites for clathrate formation are rapidly saturated as retrogradation to form GMS inaccessible crystalline domains is kinetically competitive. The second recognized means of abating staling is the use of amylolytic enzymes. There are at least three important mechanisms of enzyme action which can occur during or after the baking cycle, depending on the heat stability of the enzyme type in question. Alpha amylases are complex enzyme mixtures usually containing several alpha 1-4 glucan hydrolase activities. They basically catalyze the random hydrolysis of long chain starch segments, breaking the chain endolytically to effect depolymerization. They are particularly effective on the linear amylose chains and extended linear segments of the branched amylopectin fraction. In both cases the ability to retrograde and form intermolecular crystalline regions is abated by depolymerization to shorter chain length and generation of a less interactive species designated dextrins. Alpha amylases are broadly classified by their microbial source and characterized by their thermal stability. Fungal alpha amylases are typically thermally labile and are inactivated by the temperatures achieved during the baking cycle. Bacterial alpha amylases are more thermally stable and can largely survive the baking cycle. This can be a problem if their concentration is too high as the residual activity after baking can lead to “gummy” baked products due to continued and excessive starch hydrolysis. In general there is a limit to the use of amylolytic enzymes as too much activity leads to sticky doughs or later a gummy baked product. A second class of amylolytic enzyme is designated glucoamylase. This enzyme operates on starch and dextrins in an exolytic fashion to hydrolytically cleave the pendent glucose moiety sequentially along the alpha 1-4 glucan chain starting at the reducing sugar end. This activity may be present in commercial alpha amylase products. It is not a major contributor to amylase function but can control dextrin accumulation which contributes to sticky doughs. It is thermally labile and inactivated by the baking process. A third class of amylotic enzymes is designated beta amylase or maltogenic amylase. These enzymes, like glucoamylase, are exoactive cleaving the dissacharide maltose sequentially from the reducing end of the polysaccharide chain. Amylose is ultimately converted to maltose, a readily fermented carbohydrate by yeast. Amylopectin is converted to a species denoted as a beta limit dextrin. Beta amylases cannot proceed beyond the 1-6 branch point found in amylopectin, hence the potentially intermolecular interactive alpha amylose chains which extend from the highly branched amylopectin structure are selectively degraded to maltose. Beta amylases are usually of fungal or vegetable origin and are inactivated during the baking cycle. The third means of softening bread and hence delaying staling is simply to add more water to the dough. While much of the water absorption is intimately associated with the starch granules present in the starch component of wheat flour, excess water which serves as a mediator of moisture depletion in the baked dough piece is largely controlled by the gluten fraction. Again there is a limit to water incorporation as the dough ultimately becomes sticky and cannot be processed by automated equipment. Typically white bread doughs range from 55 to 65˜ absorption on flour; however, variety breads and specialty diet bread doughs can have less or more absorption, respectively. The use of cellulose in bread and other baked products is not entirely new. Powdered and ground cellulosic substances have been used for the production of low or reduced calorie products for over thirty years. These forms of cellulose, however, constitute nutritionally unavailable carbohydrate that functions as an inert bulking agent replacing flour. The levels of cellulose incorporated, based on flour, are relatively high, ranging from 15 to 25%. At these high levels of flour dilution, additional gluten is required for dough performance. Moisture content in diet breads is somewhat elevated due to higher water absorption by the cellulose particles versus flour. However the additional moisture is intimately associated with hydration of the cellulose particle domains and not available to the surrounding continuous starch/gluten matrix. The incorporation of large amounts of ordinary refined cellulose into a reduced calorie bread does not impart any significant anti-staling or extraordinary softening effect. Insofar as is known, it has not previously been proposed to add cellulose to baked products as a means to improve softness or prevent staling. SUMMARY OF THE INVENTION It has now been found that structurally expanded celluloses can interact with developed doughs to incorporate significant increments of water above that which would be limiting in the absence of addition of the structurally expanded cellulosic matrix. The result is a softer product and increased yield. Another benefit is that these products display a tighter and more uniform grain which is desirable for sliced products. It is believed that the microfibrillated structure of the cellulose interacts with the microstructure of the gluten fibril network to enhance water immobilization within the developed dough. This allows increased absorption in the developed dough without stickiness and leads to higher water content in the baked dough piece. This additional water results in elevated moisture levels throughout the finished baked product. These effects abate migration and depletion of moisture which is believed to be a major factor in the staling process. There may also be physical participation of the structurally expanded cellulose in the abatement of the gelatinized starch retrogradation process, particularly at the level of the cellulose microfibril. BRIEF DESCRIPTION OF THE DRAWING FIGURE 1, the sole drawing FIGURE shows the results of softness test evaluations for four test breads up to 8 days after baking. DETAILED DESCRIPTION OF THE INVENTION The use of structurally expanded cellulose in baked products if novel. The use level, based on flour, is on the order of 500 to 1000 ppm (0.05 to 0.1% w/w) or in the range of 2 orders of magnitude less than that of powdered celluloses in reduced calorie baked products. A typical absorption increase for a bread dough is 2-4%, based on flour, or 40 to 400 fold on structurally expanded cellulose basis. Because structurally expanded cellulose, as used in the practice of this invention, displays vastly different properties from other forms of cellulose, differentiation of structurally expanded cellulose from ordinary powdered, refined cellulose is elaborated below. In chemical terms cellulose specifically designates a class of plant derived linear glucose homopolysaccharides with beta 1-4 glycosyl linkage. It is the dominant structural polysaccharide found in plants and hence the most abundant polymer known. The function of cellulose is to provide the structural basis for the supramolecular ensemble forming the primary wall of the plant cell. Differentiation and aggregation at the cellular level are highly correlated with cellulose biosynthesis and assembly. In combination with associated proteins, lignin and heteropolysaccharides such as pectin and hemicelluloses, the cellulosic containing primary cell wall defines the shape and spatial dimensions of the plant cell. Therefore cellulose is intimately involved in tissue and organelle specialization associated with plant derived matter. Over time the term “cellulose substance” or simply “cellulose” has evolved as a common commercial describer for numerous non-vegetative plant derived substances whose only commonality is that they contain large amounts of beta 1-4 linked glucan. Commercially, combinations of mechanical, hydrothermal and chemical processing have been employed to enrich or refine the beta 1-4 glucan content to various degrees for specific purposes. However, only highly refined celluloses are useful substrates for structural expansion. Examples of highly refined celluloses are those employed as chemical grade pulps derived from wood or cotton linters. Other refined celluloses are paper grade pulps and products used in food. The latter are typically derived from non-woody plant tissues such as stems, stalks and seed hulls. Refined cellulose can be considered a supramolecular structure. At the primary level of structure is the beta 1-4 glucan chain. All cellulose is similar at this level. Manipulation at this level would by necessity involve chemical modification such as hydrolysis or substitution on the glycosyl moiety. However, as discussed below this level of structure does not exist as an isolated state in other than special solvent systems which are able to compete with extremely favorable intermolecular association energies formed between beta 1-4 glucan chains. In contrast to primary structure, a stable secondary level of structure is formed from the nascent beta 1-4 glucan chains which spontaneously assemble into rodlike arrays or threads, which are designated as microfibrils. The number of chains involved are believed to vary from 20 to 100. The dimension of the microfibril is under the control of genetic expression and hence cellulose differentiation begins at this level. Pure mechanical manipulation is not normally practiced at this level of organization. However, reversible chemical modification is the basis for commercial production of reconstituted forms of cellulose fibers such as rayon. Chemical substitution by alkylation of the glycosyl moiety yields stable ether substituted beta 1-4 glycans which no longer self assemble. This reaction forms the basis for the production of commercial forms of cellulose ethers such as carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC) and methyl or ethyl cellulose (MC & EC). One further modification at the secondary structural level involves intensive acid hydrolysis followed by application of high shear to produce colloidal forms of microcrystalline cellulose (MCC). This modification is best deferred to the next level of structure as most forms of MCC are partially degraded microfibril aggregates. The third level of cellulose structure is that produced by the assemblage of microfibrils into arrays and ribbon-like structures to form the primary cell wall. As in the case of secondary structure, tertiary structure is under genetic control but additionally reflects cellular differentiation. It is at this level that other structural polymeric and oligomeric entities such as lignin and proteins are incorporated into the evolving structure. Selective hydrolytic depolymerization and removal of the noncellulose components combined with application of sufficient shear results in individually dispersed cellular shells consisting of the cellulosic skeletal matrix. With the removal of strong chemically and physically associated polymeric moieties which strengthen the cellulose motif, structural expansion by mechanical translation and translocation of substructural elements of cellulose can begin to occur. The process by which structural expansion occurs is that of rapid anisotropic application of mechanical shear to a dispersed phase. Particles of refined cellulose, consisting of cellular fragments, individual cells or aggregates of a few cells, are dispersed in a liquid. The continuous liquid phase serves as the energy transduction medium and excess enthalpy reservoir. While the individual forces maintaining secondary and tertiary structure of the refined cellulose particles are largely noncovalent and hence of relatively low energy, the domains of collective ensembles of such interactions possess extraordinary configurational stability due to the large number of interactions. Only by application of intense hydraulic gradients across a few microns and on a time scale that precludes or minimizes relaxation to mere translational capture, can sufficient energy be focused on segments of the refined cell wall to achieve disassembly of tertiary and secondary structure. In practice a small fraction of the applied energy is captured by structural expansion of the dispersed phase. The vast majority of useful energy is lost into enthalpy of the continuous phase and can complicate processing due to high temperature excursions. As disassembly progresses and the structures become smaller and selectively more internally ordered, disassembly rates diminish rapidly and the process becomes self limiting. Three general processes are known in the art of cellulose manipulation to provide structurally expanded celluloses useful for practicing this invention. The simplest is structural modification from intense shear resulting from high velocity rotating surfaces such as a disk refiner or specialized colloid mill, as described in U.S. Pat. No. 5,385,640. A second process is that associated with high impact discharge such as that which occurs in high pressure homogenization devices, such as the Gaulin homogenizer described in U.S. Pat. No. 4,374,702. The third process is that of high speed, wet micromilling whereby intense shear is generated at the collision interface between translationally accelerated particles as described in U.S. Pat. No. 4,761,203. It would be expected that anyone skilled in the art could apply one or combinations of the last-mentioned three processes to achieve structurally expanded forms of cellulose useful in the practice of this invention. The disclosures of U.S. Pat. Nos. 5,385,640, 4,374,702 and 4,761,203 are incorporated by reference in the present specification, as if set forth herein in full. Two other commercial modifications are commonly employed at this structural level and are mentioned to distinguish the resulting product from SECs. The first is a widely practiced approach which involves indiscriminate fragmentation by various dry grinding methods to produce powdered celluloses. Such processes typically result in production of multimicron dimensional particles as intraparticle fragmentation and interparticle fusion rates become competitive in the low micron powder particle size region. Typical powdered celluloses contain particle size distributions ranging from about 5 to 500 microns in major dimension and may be highly asymmetric in shape. These products are employed as anticaking or flow improvement additives for ground and comminuted forms of cheese. The second process involves strong acid hydrolysis followed by moderate dispersive shear producing colloidal microcrystalline cellulose (MCC). It is believed that certain less ordered regions comprising tertiary structure are more susceptible to hydrolytic depolymerization than highly ordered domains resulting in shear susceptible fracture planes. Dispersed forms of MCC are needlelike structures roughly three orders of magnitude smaller than powdered celluloses and range from about 5 nanometers in width to about 500 nanometers in longitudinal dimension. On spray drying MCC aggregates to form hard irregular clusters of microcrystals whose particle dimensions range from 1 to 100 microns. The resulting MCC clusters can serve as a precursor for a unique SEC best described as a microscopic “puff ball” reported in U.S. Pat. No. 5,011,701 and is reported to be a fat mimetic. MCC also finds application as a rheology control agent in processed cheese products. In contrast to mechanical disassembly of preformed secondary and tertiary structure of refined cellulose from plants, bacterial derived extracellular beta 1,4 glucans self assemble into microfibriller ensemblies and never reach a tertiary structural level. These materials can be isolated from fermentation and perform similarly to mechanically disassembled cellulose upon hydration and dispersion. For purposes of this invention, bacterially derived cellulose is incorporated by reference into the general category of structurally expanded cellulose. Finally, the quaternary or final structural level of cellulose is that of the cellular aggregate and is mentioned only for completeness. These substances may be highly lignified such as woody tissue or relatively nonlignified such as those derived from the structural stalks and seed hulls of cereal grain plants. Commercial types of these materials are basically dried forms of nonvegetative plant tissue. These moderately elastic substances respond to mechanical processing by deformation and ultimate fracture along the principal deformation vector. Consequently, these materials readily undergo macroscopic and microscopic size reduction and are reduced to flowable powders by conventional cutting, grinding or debridement equipment. Because of the cohensive strength of the molecular ensemble comprising quaternary structure, these materials are not candidates for systematic structural expansion at the submicron level without chemical intervention. Structural expansion as defined herein is a process practiced on refined celluloses involving mechanical manipulation to disassemble secondary and tertiary cellulose structure. The ultimate level of expansion would be to unravel the cell wall into individual microfibrils. Although plant specific, a typical microfibril is best described as a parallel array of 25 to 100 beta 1,4 glucan chains with diameter in the 50 nanometer range and variable length ranging from submicron to micron multiples. In practice, generation of a dispersed microfibril population is not a realistic objective and only of academic interest. What is usually achieved because of the relatively indiscriminate application of mechanical energy is a highly heterogeneous population of miniature fibrils, ribbon like and slab like structures. These structures display irregular distention of individual microfibrils and aggregates of microfibrils from their surfaces and at internal and external discontinuities. The ensuing collage consists of an entangled and entwined network of cell wall detritus to form a particle gel. Some of the larger structural features with dimensions in the micron range are discernable with the light microscope, however, higher resolution techniques such as scanning transmission electron microscopy are necessary for detailed observation of submicron features. This particle gel network exhibits a vast increase in surface area associated with the volumetric expansion and projection of cell wall structure into the continuous phase medium. Lastly, structurally expanded celluloses useful for purposes of this invention may be further characterized by possessing a water retention value greater than 350 and a settled volume of at least 50% for a 0.5% percent by weight dispersion of the structurally expanded cellulose in aqueous media. The following examples are illustrative of practice of the invention but are not intended to limit the scope of the invention. EXAMPLE 1 Four white, pan bread test doughs with the formulas set forth in Table 1 were produced and baked on a commercial scale bread line. The amounts of ingredients used were in pounds, unless otherwise indicated. The make up was a standard two stage sponge and dough. The sponge was mixed one minute at low speed and four minutes at high speed (1+4) and developed at 74° F. The doughside ingredients were then combined with the sponge (except salt) and mixed at low and high speeds (1+41/2). The salt was added and the dough again subjected to low and high speed mixing (1+41/2). The final temperature of the dough was 78° F. and it was given a floor time of 10 minutes prior to dividing and sheeting. The panned dough pieces were proofed to optimum and baked. The baked breads were bagged without slicing for Instron evaluation. TABLE 1 Test 1 Test 2 Test 3 Test 4 Sponge White flour 800.0 800.0 800.0 800.0 Water 445.0 445.0 445.0 445.0 Yeast 38.0 38.0 38.0 38.0 Conditioner 5.5 5.5 5.5 5.5 Softener 5.5 5.5 5.5 5.5 Dough White flour 300.0 300.0 300.0 300.0 Water 180.0 180.0 205.0 180.0 Liquid sugar 90.0 90.0 90.0 90.0 Shortening 28.0 28.0 28.0 28.0 Salt 20.0 20.0 20.0 20.0 †NFDM 17.0 17.0 17.0 17.0 solids Conditioner 5.5 5.5 5.5 5.5 *Oven Spring ™ 3 pkg 3 pkg 3 pkg 3 pkg *Ultracel ™ — — 0.7 — 3200 HV *Stay Soft 8 pkg — 8 pkg — 250 *Stay Soft 8 pkg — 8 pkg — 260 †Non-fat dried milk *Denotes proprietary products manufactured by Watson Foods Company, Inc., West Haven, Connecticut (WFC). Oven Spring ™ is an oxidizer (bromate replacer) system sold in 1 oz. water soluble packets (Solupak ™). Ultracel™ 3200 HV is a structurally expanded form of cellulose manufactured by WFC. Stay Soft 250 is a proprietary enzyme complex, largely maltogenic amylase activities, produced in a 5 gram Solupak™ by WFC. Stay Soft 2600 is a proprietary enzyme complex, largely alpha amylase activities, produced in a 5 gram Solupak™ by WFC. The test series was designed to isolate the contribution of enzyme addition and additional water (absorption) over the control bread (Test 4) containing 0.5% distilled mono- and diglycerides based on bakers percentage (flour 100%). The standard absorption was nominally 60%, based on flour accounting for water from yeast and liquid sugar in the control and Tests 1 and 2 and had been carefully optimized. Addition of 1 ounce/hundred weight (cwt) flour in Test 3 allowed incorporation of an additional 25 lbs of water into the dough, or 2.3% additional absorption based on flour. Addition of this incremental level of water to the control dough gave a “sticky” dough that was difficult to remove from the mixer and could not be processed by the high speed divider/rounder or sheeter. All four test doughs behaved similarly during mixing giving well developed, extensible doughs that handled well throughout the mechanical manipulations of the modern, automated high speed bread line. Bread firmness was measured by the Universal Testing Machine (Instron) Method as described in the AACC method 74-09. The results are shown in FIGURE 1. Two loaves each of Tests 1 through 4 were tested on days 1, 4, 6 and 8 after baking. For testing the ends of the loaf (2 inches) were discarded and a minimum of six, 1 inch slices distributed evenly throughout the loaf center were tested using a 25% (0.25 inch) compression. In addition, the center slice height was recorded and a subjective evaluation of eating quality was made on each of the test dates. Residual crumb moisture was determined in each of the samples by daying a preweighed portion of the crumb to a constant weight at 185° F. An analysis of variances (ANOVA) was used to determine whether each of the test results was statistically significant. Over 8 days of storage, the average compression values for Test 3 were significantly lower (bread was softer) than those for the other formulations tested. Average compression values for Test “4”, over the same storage period were significantly higher (bread was firmer) than those for the other formulations tested. Test 1 showed lower average compression values than Test 2 over 8 days of storage. Overall slicing was clean without piling for all formulations tested. The chewdown (time to masticate the bread, an indication of eating quality) was moderately fast (indicating that the crumb was not gummy) for all formulations tested. Average percent standard deviation for compression values of 12 slices per 2 loaves was 4% for Tests 1 and 2, 6% for Test 3, and 5% for Test 4, indicating even crumb properties both within and between loaves. Crumb moisture contents of the loaves of Day 1 was 42.9% for Test 1, 42.4% for Test 2, 43.8% for Test 3, and 42.3% for Test 4. Average center slice heights for all Tests were the same at 4.25 inches. In summary, incorporation of Ultracel, a structurally expanded form of cellulose, at a level of only 1 oz/cut of flour (620 ppm) allowed incorporation of 2.3% more absorption or an increase in yield of over 25 lb for the 1100 lb flour weight batch. The incorporation of the additional moisture is preserved throughout the baking process as indicated by the 1.5% moisture increase in the finished bread product containing Ultracel versus the control. The impact of the increased moisture is reflected in additional softness of the baked product and a reduction in the staling rate. The combined effect of increased yield and abatement of the staling phenomenon have important economic consequences to the baking industry.
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Summary: A method is disclosed to enhance the softness of baked products and retard staling. The method is based on the use of structurally expanded cellulose which is incorporated into the dough during mixing to increase the water absorption of the dough without imparting stickiness. The ability to supersaturate the dough without detrimental impact on processability results in softer character and better grain of the baked product. These properties are maintained over time resulting in reduced staling and superior quality of sliced product.
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Summarize: By. Sarah Griffiths for MailOnline. Amazon's streaming box, Fire TV, will launch in the UK in October. The device, which allows users to access on-demand TV, apps and smartphone content as well as games, debuted in the US in April. It will compete with the Apple TV box and Google’s Chromebox, but the tech giant says that none of the current TV boxes give consumers what they really want. Setting the world alight? Amazon's streaming box, Fire TV (pictured) will launch in the UK on October 23. The device allows users to access on-demand TV, apps and smartphone content as well as games. Speaking on the first day of the IFA technology show in Berlin, senior vice president of devices David Limp said: ‘We think that the products that are out there - and we sell a lot of them on Amazon - just haven't offered customers the value that we think you can, and this is an existence proof that you can put a lot of horsepower and performance into a box, at a great value.’ The Fire TV box will be shipped in Europe on October 23. It can be pre-ordered for £79 and Amazon Prime members can pre-order it for £49 for five days. The device will also be available at Argos, Dixons, Sainsbury’s and Tesco. Talking it up: Amazon said voice search (illustrated) will allow users to say the name of a film, TV show or actor, for example into their remote to find a film. This means no more scrolling through an alphabetical grid. Users will be able to buy a separate controller and download games to play through Fire TV (shown). ‘It doesn't replace an Xbox or a Playstation. There's a group of customers who live and die by those things, but there is a segment of customers that we think is out there that don't make the leap to those consoles,’ Mr Limp said. Size: 1.8cm thick. Processor: Quad core with three times the processing power of Apple TV. Graphics: Adreno 320 graphics engine for fast and fluid pictures. Memory: 2GB – four times that of Apple TV or Chromecast. Operating system: Fire OS, based on Android. Streaming: High definition up to 1080p with Support for Dolby Digital Plus Surround Sound. Set-up: Box comes pre-registered so there’s no set-up. Previously purchased films and TV shows from Amazon will automatically be places in a Watchlist. Remote control: Remote has the usual buttons and uses Bluetooth. Voice activated: Users can say the name of a film, TV show, actor, director, genre, app or game into the remote and the results will appear in seconds. Games controller: It has dual analogue sticks and the usual controls, connecting to the box via Bluetooth. Powered by two AA batteries for 55 hours, they costs £34.99 each and multiple controllers can be used for multiplayer games. Cost: £79. Amazon said that the box is designed to offer users access to films and bring photos, music and games to the living room in one place. As well as Netflix and Prime Instant Video, owners of the new box will be able to access Sky News, YouTube as well as music services - Amazon Music, Spotify and TuneIn. Voice search will allow users to say the name of a film, TV show or actor, for example into their remote to find a film. This means no more scrolling through an alphabetical grid. A function called ASAP (Advanced Streaming and Prediction) will suggest films and TV shows that people will want to watch and buffers them for playback before a user hits play. For film buffs, there’s a feature called X-Ray where they can see information about the cast and crew as well as movie trivia. Jeff Bezos, Amazon Founder and CEO said: ‘Tiny box, huge specs, tons of content, incredible price- customers love Fire TV and we’re thrilled to introduce it in the UK. ‘Voice search that actually works means no more typing on an alphabet grid. Our exclusive new ASAP feature predicts the shows you’ll want to watch and gets them ready to stream instantly. And our open approach gives you not just Amazon Instant Video and Prime Instant Video, but also Netflix, Sky News, YouTube, Spotify, and more.’ Users will be able to buy a separate controller for £34.99 and download games to play through Fire TV. Game on: The controller needed to play video games will be sold separately to the Fire TV box, and will cost £34.99 each. Hundreds games, such as Minecraft, Asphalt 8, NBA 2K14, and The Walking Dead will be available when the box launches next month. No need to flap: With the addition of a controller, Fire TV owners will be able to play games on their TV. A new multiplayer version of the popular Flappy Bird app (pictured) will be exclusively available to Fire TV. ‘It doesn't replace an Xbox or a Playstation. There's a group of customers who live and die by those things, but there is a segment of customers that we think is out there that don't make the leap to those consoles,’ Mr Limp said. A new multiplayer version of the popular Flappy Bird app will be exclusively available to Fire TV. Hundreds of high-quality, low-cost games, such as Minecraft, Asphalt 8, NBA 2K14, and The Walking Dead will be available at launch too, with the average game costing £1.07. The device has a quad-core processor with three times the processing power of Apple TV and four times the memory of Apple and Google’s offerings, so it’s faster and more fluid, according to Amazon. The little box offers 1080p HD video and supports Dolby Digital Plus surround sound. It measures just 1.8cm thick and comes with an even tinier remote. Content: The device offers users access to films and TV from Sky News, Netflix, Prime Instant Video, Curzon Home Cinema and more. Music: Users can either rent or purchase films from Amazon Instant Video, or choose a subscription service. Photos: Users will be able to view slideshows of photos on their TV set. Games: Games such as Minecraft, Asphalt 8, NBA 2K14, The Walking Dead and a multiplayer version of Flappy Bird will be available from launch with more added. The average cost of downloading a game is £1.07. Users could choose to play them on a remote with dual analogue sticks. ASAP: The ASAP (Advanced Streaming and Prediction) feature predicts which movies and TV episodes a user will want to watch and prepares them for playback before they hit play. Suggestions change according to a user’s viewing habits. Second Screen: Owners of a Kindle Fire HDX tablet can ‘Fling’ Amazon TV shows to their Fire TV using Second Screen, and use their tablet as a remote control. X-Ray: The feature gives film buffs relevant trivia on their tablet while a film is playing. They will be able to see information about cast and crew, the names of songs as they play, as well as a list of all music in the movie or TV show, trivia items in context with the action on the screen and character backstories. Whispersync: Amazon’s technology saves and synchronises video and music libraries so users can access them on multiple devices, including Kindle Fire tablets and iPhones. Archive: Digital content is backed up in the cloud automatically. Freebies: The box comes with a free month of Amazon Prime
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Summary: Streaming box will go on sale on October 23 and will cost £79. Device was launched in the US in April and sells for $99. Box will let users access on-demand channels and film services such as Twitch and Netflix on their TV as well as put phone content on the TV. With a separate controller, users will be able to play games via Fire TV. A new multiplayer version of the popular Flappy Bird app will be available.
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Write a title and summarize: The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H2O2 and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause diseases in several hosts [1]. P. aeruginosa acute infections are one of the major problems in immunodeficient subjects, burn victims and mechanical ventilation patients. Pulmonary infections caused by P. aeruginosa, including ventilator-associated pneumonia and chronic pulmonary infection in cystic fibrosis patients, are associated with high mortality rates, and chronic pulmonary infection impairs life quality and life expectancy of the infected individuals [2]–[4]. The high intrinsic resistance of P. aeruginosa to antibiotics adds to the difficulties of treating infections caused by this versatile opportunist [5]. Macrophages are the first line of defense in pulmonary infections and play a major role in the host response to P. aeruginosa infections. Pathogens are recognized by the immune system, which detects pathogen-associated molecular patterns (PAMPs) by the corresponding pattern recognition receptor. The activation of signaling cascades by the binding of PAMPs to Toll-like receptors (TLRs), except for TLR-3, depends on MyD88 and results in the activation of the NF-κB and MAPK pathways [6]; their activation leads to the production of cytokines, including TNF-α, IL-6 and IL-1. The role of TLR-4 and TLR-5 in triggering a protective immunity against P. aeruginosa has been shown in vivo; as mice lacking TLR-4/5 have increased susceptibility to pulmonary infections [7], [8]. After the recognition of bacteria by the TLRs in macrophages, the signaling cascade leads to the generation of reactive oxygen species (ROS) in a process known as the oxidative burst, which depends on NADPH oxidase [9]. The oxidative burst is bactericidal and can cause lipid, protein and DNA lesions, resulting in pathogen clearing. However, to overcome or prevent these lesions, pathogens have developed a complex detoxification system that includes superoxide dismutase, and catalase/peroxidases that have been extensively studied in several pathogens, including P. aeruginosa [10], [11]. Among hydroperoxide-reducing enzymes, the peroxiredoxins (Prxs) are considered cellular sensors due to their abundance and reactivity [12]. Prxs catalyze the reaction ROOH+2e−→ROH+H2O and reduce hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxide compounds [13]–[15]. Prxs are found in organisms belonging to all Domains of life, indicating their crucial physiological function, but their role in P. aeruginosa virulence remains uncharacterized. Prxs are a large family of proteins that can be divided into six sub-groups with distinct amino acid sequences, but all contain the thioredoxin fold and the PXXT (S) XXC motif [16]. Among these six-subgroups, Prx enzymes can display 2-Cys Prx or 1-Cys-Prx mechanisms, depending on the number of cysteine residues involved in catalysis [16]. AhpC, a 2-Cys Prx, is involved in the virulence of Helicobacter cinaedi and Staphylococcus aureus [17], [18], but it does not seem to be a virulence determinant for other bacteria that have been analyzed [19]–[21]. The genome of the highly virulent P. aeruginosa strain PA14 contains at least eight genes that encode Prxs, including AhpC and Tpx. Ohr is another Cys-based peroxidase from P. aeruginosa that has been structurally and enzymatically characterized [22], but it is not required for virulence [23]. Among the six Prx sub-groups, Prx6 is the least well studied. The precise physiological roles of the Prx6 sub-group remain unknown, with few reports addressing their kinetics and structural functions; most of these reports were from studies of eukaryotes [24]–[29]. Thus far, all of the Prx6 proteins characterized display the 1-Cys Prx mechanism. Remarkably, although the bacterial Domain contains hundreds of Prx6 representatives [16], no characterization of their roles has been reported. In the P. aeruginosa genome, only one gene coding for a putative Prx6 is present (PA14_19490 in PA14; PA3450 in PA01). It was named lsfA because its expression is up-regulated, together with a gene cluster coding for an ABC-transport system involved in organic sulfur uptake, in cells grown in low-sulfate medium [30]. However, there is no experimental evidence for the mechanism underlying the function of LsfA in this process. It has been suggested that LsfA up-regulation and AhpC expression in low sulfate conditions may be a response to the oxidative stress caused by the excess levels of reduced flavin nucleotides due to sulfonate utilization [31]. Several transcriptomic analyses have revealed that lsfA expression is up-regulated in other stressful conditions, including in the presence of sodium hypochlorite [32], a product of the macrophage oxidative burst. Proteomic analyses identified LsfA as differentially expressed during other stressful conditions. Three LsfA isoforms are induced by the superoxide-generating drug paraquat [33] and in P. aeruginosa biofilms [34]. In iron starvation conditions, Pseudomonas putida also showed increased levels of the LsfA ortholog protein [35]. Interestingly, indole treatment, which may mimic conditions of iron abundance, decreased lsfA expression as well as virulence-related traits [36]. A more recent report found that the oxidation responsive OxyR activator protein binds to the lsfA promoter region [37]; this observation supports the role of LsfA in the bacterial response to H2O2. Here, we show that the antioxidant function of the bacterial 1-Cys Prx LsfA is important for P. aeruginosa virulence, both in a macrophage model in vitro and in an acute pneumonia model in vivo. This work reveals the role of this protein as a novel virulence factor that contributes to the P. aeruginosa arsenal against host defenses and allows it address other stresses in various environmental conditions. A previous sequence alignment revealed that LsfA belongs to the Prx6 subfamily [16], with the Cys45 of LsfA as the putative peroxidasic cysteine (Fig. S1). To determine whether P. aeruginosa LsfA is indeed endowed with thiol-dependent peroxidase activity, the recombinant wild-type (His-LsfA) protein and a mutant protein without the putative catalytic cysteine (His-C45A) were expressed in Escherichia coli and purified by affinity chromatography. As predicted, H2O2 was reduced in the presence of wild-type His-LsfA but not when His-C45A was employed (Fig. 1A), showing that Cys45 is essential for catalysis and confirming that LsfA is an active 1-Cys Prx. Because 2-Cys Prxs play an important role in bacterial protection against H2O2, tert-butyl hydroperoxide and paraquat [13], [14], we assessed whether LsfA was also important for P. aeruginosa resistance to such oxidants. To test this hypothesis, an in-frame deletion mutant strain (ΔlsfA) and a strain with a point mutation in the catalytic cysteine (C45A) were constructed. Both mutant strains grow like wild-type in minimal medium and in biofilms, excluding any growth defects (Fig. S2). The wild-type, ΔlsfA and C45A strains were tested using disk diffusion halo assays in the presence of oxidants. Larger inhibition haloes due to H2O2 were observed for both mutant strains (12. 8±0. 7 mm for ΔlsfA, 13. 0±0. 6 mm for C45A) compared with PA14 (7. 4±0. 8 mm for PA14) (Fig. 1B), indicating that LsfA is important for oxidative stress resistance in P. aeruginosa and confirming that the C45 catalytic cysteine is essential for LsfA activity. Complementation of the mutant strains with a copy of the lsfA gene in a plasmid restored the wild-type phenotype (Fig. S3), confirming that the larger haloes were due to a lack of LsfA activity. The lsfA mutants did not demonstrate increased sensitivity to paraquat or tert-butyl hydroperoxide (data not shown), which may reflect a compensatory effect of other Prxs and/or Ohr. Because macrophages and neutrophils produce ROS and reactive nitrogen species in response to pathogens, bacterial antioxidant systems are important mechanisms that allow bacteria to overcome the deleterious effects of oxidative lesions and to survive during infection. To assess whether the P. aeruginosa LsfA is related to virulence, an in vitro model of infection in J774 macrophages was used. The macrophages were infected with PA14 and the lsfA mutants, and incubated for 1 hour prior to the gentamicin addition. At regular time points, the number of bacterial cells remaining in the culture supernatants was assessed (Fig. 2A), the macrophages were washed and lysed, and the released bacteria were counted (Fig. 2B). The phagocytosed bacteria counts were similar for all strains and showed a slight increase over time (Fig. 2B), suggesting that all of the strains were internalized by macrophages at the same extent. However, for the lsfA mutants, a 2-fold reduction in extracellular colony-forming units (CFUs) compared with the wild-type PA14 was observed (Fig. 2A). Thus, the peroxidase activity of LsfA contributes to bacterial viability in the presence of macrophages. The number of remaining macrophages at the end of the assay, assessed by a LDH release assay, was similar for all bacterial strains tested, showing that LsfA does not alter cytotoxicity (Fig. S4). The increased survival of PA14 compared with the lsfA mutants may reflect a change in the oxidative status of the macrophages in addition to an improved bacterial response to the oxidative burst. To address this hypothesis, macrophages were incubated with wild-type or mutant strains, and at different times, the cells were washed and incubated with 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA), a probe that can sense shifts in the cellular redox state. After 3 hours of treatment, an increase in the intracellular oxidative status in macrophages incubated with the lsfA mutant strains was observed, in comparison with macrophages infected with PA14 (Fig. 3A). This result suggests that LsfA participates in the response of P. aeruginosa to the oxidative insult caused by the macrophages, most likely due to its peroxidase activity. Because LsfA affects the macrophage redox status, it can also impact virulence by subverting the host signaling pathways [38]. The mutant for gacA, which encodes a protein that plays a role in P. aeruginosa pathogenicity, has been extensively studied [39] and caused a similar increase in the macrophage oxidative state (Fig. 3A), suggesting that LsfA may indirectly inhibit the oxidative burst. To evaluate the role of ROS generated by NADPH oxidase in the clearance of lsfA mutant strains, a gentamicin exclusion assay was performed again with macrophages, now pre-treated with DPI, a NADPH oxidase inhibitor. The CFU number corresponding to the bacteria that survive inside the DPI-treated macrophages is higher than in untreated ones (Fig. 2B and 3B), suggesting that they are no longer able to kill the bacteria. Moreover, the number of PA14 or the mutant cells under DPI treatment were similar both intra and extracellular, suggesting that LsfA counteracted NADPH oxidase activity during PA14 evasion from the macrophages (Fig. 3B and C). To understand whether P. aeruginosa LsfA modulates macrophage activation, J774 cells were infected with PA14 or the lsfA mutant strains. The supernatants were recovered after 3 hours, and cytokine levels were measured. Macrophages infected with the lsfA mutants secreted more TNF-α than macrophages infected with PA14 (Fig. 3D). This result suggests that the LsfA function decreases macrophage activation and, thus, its oxidative state because the oxidative burst is less pronounced when macrophages are infected with the wild-type strain. C45A-infected macrophages treated with the thiol-reductant N-acetylcysteine (NAC) showed a reduced oxidative state (Fig. S5) and lower TNF-α production, similar to that of PA14-infected macrophages either in the presence or absence of NAC (Fig. 3D). Both NAC and LsfA may prevent macrophage activation by decreasing the oxidative state of phagocyte and thereby subverting signaling pathways involved in the immunological response. Indeed, ROS can act as signaling molecules that lead to NF-κB and MAPK activation and increased cytokine production [40]. To determine which signaling pathways are up-regulated when the macrophages are infected with lsfA mutant strains, specific inhibitors of NF-κB or the MAPKs ERK1/2, JNK or p38 were used. In the presence of these inhibitors, no differences in TNF-α production were observed when macrophages were infected with either PA14 or C45A strains (Fig. 3D). These data suggest that LsfA peroxidase activity in PA14 decreases the activation of the NF-κB and MAPK pathways, and this decreased activation is reflected in the lower oxidative state of macrophages. Because LsfA is required for virulence in macrophages in vitro, the next step was to ascertain whether this 1-Cys Prx belonging to Prx6 group was also relevant in an acute pneumonia model in mice. Although all mice infected intratracheally (i. t.) with PA14 were dead 48 hours after infection, mice infected with lsfA strains had higher survival rates, with 37. 5% of animals still alive after 13 days (Fig. 4A). After 60 days, surviving mice that had previously been infected with ΔlsfA (37. 5%) and C45A (25%) seemed healthy, indicating that the infection had resolved or became chronic. In conclusion, the requirement of LsfA activity for P. aeruginosa virulence was confirmed. P. aeruginosa introduced i. t. into mice disseminates quickly and affects other organs, leading to death [8]. Bacterial loads were assessed in the primary site of infection as well as in secondary organs (liver and spleen) as an indication of sepsis. Lungs, spleen and liver were recovered 24 hours p. i., and bacterial counts were evaluated. Animals infected with the C45A mutant strain showed a reduced bacterial burden in all organs analyzed compared with PA14-infected animals (Fig. 4B). This result indicates an improved bacterial resolution in mice infected with the C45A strain and demonstrates the relevance of the 1-Cys Prxs to virulence. To ascertain whether LsfA would impact cytokine production in vivo, thus potentially decreasing the immune system activation and neutrophil recruitment to the infection site, the TNF-α and IFN-γ levels in the lungs of infected animals were determined by ELISA at 24 hours p. i. The wild-type strain induced local immunosuppression, with TNF-α and IFN-γ release similar to that observed in the control mice (Fig. 4C). However, mice infected with C45A released higher levels of these cytokines (Fig. 4C). No differences were observed in the anti-inflammatory cytokine IL-10 in mice infected with either PA14 or the lsfA mutant strain compared to the control (Fig. S6). In addition, C45A-infected mice showed an increased recruitment of neutrophils (Ly6G/Ly6C+; F4/80−) and macrophages (F4/80+; CD11b+) to the lungs compared with the controls (Fig. 4D). To determine if the recruited neutrophils were activated, myeloperoxidase (MPO) activity was present in all treatments, but it was higher when mice were infected with the C45A mutant strain (Fig. 4E), suggesting that thiol peroxidase activity is also involved in neutrophil activation. This set of data confirms that a better immune response that is reflected in the higher survival of the lsfA mutant-infected animals compared to those infected with PA14. In this study, we show for the first time the relevance of the 1-Cys Prx LsfA in bacterial virulence. LsfA, a Prx from P. aeruginosa, is the first protein in the Prx6 sub-group to be connected to pathogenicity. This protein, LsfA, has orthologues in other pathogens, including Burkholderia and Bordetella, suggesting that this host-pathogen interaction may be present in other bacteria. Biochemical analysis demonstrated that LsfA has peroxidase activity that depends on the catalytic cysteine (C45). Inactivating this peroxidase activity makes lsfA mutant cells more sensitive than wild-type cells to hydrogen peroxide but not to organic peroxides, including tert-butyl-hydroperoxide. In addition, LsfA may also be endowed with other enzymatic activity because LsfA displays a conserved lipase motif (GDSWG) that is also present in the Prx6 from humans and mice. Curiously, the lipase motif is not conserved in all 1-Cys Prxs, and a deeper analysis of the evolution and function of 1-Cys Prxs is in progress. However, the lipase motif is not required for virulence in the mice macrophage and lung models we used here, as a point mutation in the catalytic cysteine was sufficient to abrogate the virulence to the same extent as when the entire lsfA coding region was deleted from the P. aeruginosa chromosome. Prx proteins have been implied in several diseases in humans. The 1-Cys Prdx6 has been characterized as a tumor inhibitor because it protects mice and human skin cells against lipid peroxidation [41], and the levels of this protein are lower in papillary thyroid carcinomas than in normal thyroid tissue [42]. Prx6 may also be involved in degenerative neuronal disorders, including Alzheimer' s disease and prion diseases [43], [44]. In mice infected i. t. with P. aeruginosa, LsfA directly or indirectly downregulates the host innate immune response, enabling the pathogen to spread and colonize other organs, leading to an acute infection that results in death. In the macrophage infection model, LsfA is required for P. aeruginosa resistance to clearance, with lower TNF-α production in macrophages infected with the PA14 strain compared with macrophages infected with lsfA mutants, suggesting that LsfA plays a role in the PA14 immunomodulatory effect. This immunomodulation seems to be related to the macrophages' oxidative state, which is higher when the macrophages are infected with lsfA mutant strains than when they are infected with the wild-type PA14. We also found that NADPH oxidase activity is required for the clearance of the lsfA mutant, indicating that LsfA is important for PA14 resistance to ROS generated by macrophages. Other P. aeruginosa virulence factors also promote immunomodulation. ExoU, a secreted phospholipase, inhibits caspase-1 activation, which is related to pro-IL-1β maturation [45]. The quinolones HHQ and PQS can also negatively regulate the immune system, reducing NF-κB activation, bacterial clearance and TNF-α and IL-6 production [46]. Nevertheless, this is the first evidence of a Prx exerting an immunomodulatory function, protecting the pathogen against phagocytes, reducing phagocyte activation and leading to increased bacterial virulence. We also found that the oxidation of H2DCFDA was higher in macrophages infected with lsfA mutant strains than in macrophages infected with PA14. In support to our findings, LPS stimulated macrophages, carrying a knockout of the 2-Cys PrxII gene, released more pro-inflammatory cytokines, including TNF-α and IL-6, than wild-type macrophages. This increase in cytokine release is correlated with higher ROS production by the PrxII knockout macrophages, leading to activation of the MAPK and NF-κB pathways [47]. In this study, we show that the lack of a bacterial Prx seems to have the same effect as the lack of PrxII in macrophages, and we speculate that a balance between ROS production and turnover might be necessary to allow the outcome of a host-pathogen interaction to swing from an efficient immune response to virulence, favoring either the host or the bacteria. In addition to bactericidal activity, ROS can also act as signaling molecules, but the mechanisms involved are poorly understood. Further studies are required to investigate how oxidative stress leads to NF-κB and MAPK activation. However, it appears that bacterial antioxidants play a role in protecting microorganisms from oxidative insult [38] and subvert signaling pathways such as those involved in the immunological response [48]. With the increasing resistance of pathogens to antibiotics, it is crucial to explore new paradigms to develop novel anti-infective drugs, taking advantage of a deeper understanding of bacterial virulence and host defense mechanisms. One aspect that may be explored to achieve this goal is the ROS sensing and production that is employed by both pathogens and hosts. Understanding the role of LsfA in the P. aeruginosa immunomodulatory effect may lead to novel therapeutics to overcome the effects of infection by targeting LsfA itself or by improving the host immune response. All of the strains and plasmids used in this study are listed in Supporting Table S1. The P. aeruginosa strains were grown at 37°C in LB broth. The E. coli strains were grown in LB supplemented with 100 µg/mL of ampicillin, 50 µg/mL of kanamycin, or 10 µg/mL of gentamicin, when required. The P. aeruginosa strains were grown in 250 µg/mL of kanamycin, 20 µg/mL nalidixic acid or 30 µg/mL of gentamicin, when required. To construct the unmarked in-frame deletion of lsfA, primers flanking the upstream and downstream regions of lsfA were designed. Amplicons were cloned into pNPTS138 at the HindIII and EcoRI sites to generate pNPTS138ΔlsfA. The resulting construct was used to introduce the lsfA deletion into the wild-type PA14 genome by homologous recombination [49], resulting in the ΔlsfA mutant, that contains only the first eight N-terminal aminoacids and 31 aminoacids at the LsfA C-terminus. No polar effect is expected, because lsfA coding region is distant 249 bp from the next open reading frame in the PA14 genome and the frame of translation was maintained. The oligonucleotide-directed mutagenesis of the LsfA Cys45 to Ala was performed using the primer pairs listed in Supporting Table S2, and a two-step procedure was performed as previously described [50]. The resulting amplicon was cloned into pNPTS138 and introduced into the PA14 genome by homologous recombination. Mutant clones were screened by PCR followed by digestion or direct sequencing. Again, no polar effects are anticipated, because only one codon was changed. To construct the lsfA complementation strains, the lsfA coding region was amplified by PCR using the primer pairs listed in the Supporting Table S2. The resulting amplicon was cloned into pJN105 at the EcoRI and SpeI sites to generate pLsfA. pLsfA and pJN105 plasmids were introduced into the ΔlsfA and the C45A mutants, generating the ΔlsfA/pJN105, ΔlsfA/pLsfA, C45A/pJN105 and C45A/pLsfA strains. The lsfA or lsfAC45A coding regions were cloned into pProEX-Hta to overexpress His-LsfA or His-LsfAC45A in E. coli BL21. Briefly, E. coli cultures were grown in 250 mL of LB at 37°C until the culture reached an OD600 nm of 0. 5. IPTG was added to a final concentration of 0. 6 mM, and the cultures were grown at 30°C for 6 h. Cells were harvested by centrifugation and resuspended in 25 mL lysis buffer (20 mM sodium phosphate pH 7. 4,500 mM NaCl, 20 mM imidazole, 1 mM PMSF). Cell suspensions were lysed by ten 15 second sonication cycles in an ice bath. The lysate was centrifuged at 16000 g for 20 min at 4°C. The recombinant proteins His-LsfA and His-LsfAC45A were purified using an Ni-NTA column (Invitrogen) equilibrated with lysis buffer and eluted with an imidazole gradient (20–1000 mM). The eluted fractions were analyzed by SDS-PAGE and pooled, and the buffer was exchanged using a PD-10 column (GE) equilibrated with incomplete reaction buffer (20 mM sodium phosphate pH 7. 4,500 mM NaCl). The proteins were concentrated with Centriprep-Ultracel YM-10. 000 MWCO (Millipore). The protein concentration was determined by UV spectroscopy using an extinction coefficient of 33920 M−1 cm−1, calculated as described by Gill and von Hippel [51], and confirmed using the Bradford reagent (Sigma-Aldrich) according to the manufacturer' s protocol. The in vitro peroxidase activity was determined using the ferric-thiocyanate assay [15]. Briefly, 10 µM of purified recombinant protein was incubated at 37°C in 100 µL of reaction buffer (20 mM sodium phosphate pH 7. 4,500 mM NaCl, 1 mM DTT, 10 µM DTPA, 10 µM sodium azide) in the presence or absence of 200 µM H2O2. At the times indicated, the reaction was stopped by the addition of 20 µL of 2 M HCl and incubated for 10 min at 37°C. Then, 100 µL of 2. 5 M KSCN, 100 µL of 20 mM FeSO4 and 680 µL of H2O were added to the reaction. The absorbance was read at 480 nm. As controls, we used the same conditions in the absence of DTT or without recombinant protein. After the reaction times, the H2O2 concentration was determined by comparison with a standard curve with different H2O2 concentrations (15. 625–1000 µM). Halo inhibition assays were performed as described before with modifications [52]. Cultures of P. aeruginosa PA14 and the ΔlsfA and C45A mutant and complemented strains were grown overnight in LB broth. Cultures were diluted into fresh LB broth to an OD600 nm of 0. 1 and grown to an OD600 nm of 1. 0. Plate assays were performed by adding 200 µL of a cell culture to 3 mL of 0. 7% LB soft agar. The agar suspensions were spread on LB plates. Sterile paper disks (6 mm in diameter) were saturated with 10 µL of 2. 5% hydrogen peroxide and placed on the plates, which were incubated for 16 hours at 37°C. For the complementation assays, 30 ug/mL gentamicin (to maintain the plasmids) and 0. 2% arabinose (to induce expression from the ara promoter) were added into the LB. The macrophage cell line J774 was maintained in R-10 (RPMI 1640 supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS) and 40 µg/mL gentamicin) at 37°C in 5% CO2. Macrophages were counted using a Neubauer chamber, and dead cells were excluded by the trypan blue exclusion assay. Macrophages were seeded at 1×105 cells/well (96-well plates) or 2×106 (6-well plates) in R-10 without antibiotic and primed overnight with 10 ng/mL IFN-γ at 37°C in 5% CO2. PA14 and the ΔlsfA and C45A mutant strains were grown in LB broth to an OD600 nm of 2. 0. The bacteria were diluted in R-10 without antibiotics. Macrophages that had been previously seeded in 96-well plates were infected at a multiplicity of infection (MOI) of 10. After 1 hour of infection, the supernatant was removed and 200 µg/mL of gentamicin was added to cell cultures to kill extracellular bacteria. After 30 minutes the cells were washed once with PBS and fresh media were added. This point was defined as time 0 (t = 0 h). To determine the numbers of extracellular bacteria, the supernatant was collected, serially diluted, the cells were plated and the CFU were enumerated. To access intracellular bacteria, macrophages were lysed with PBS+0. 1% Triton X-100, the lysates were serially diluted, and the CFU were determined. Cytotoxicity was measured for samples taken at the indicated times by quantifying the release of lactate dehydrogenase (LDH) using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) following the manufacturer' s instructions. For NADPH oxidase inhibition, macrophages were treated with 10 µM DPI (diphenylene iodonium) 4 hours before infection and the same steps described above were performed. To quantify TNF-α, macrophages were seeded in six-well plates and infected at an MOI of 10. For inhibition experiments, where indicated, cells were pretreated for 4 hours before infection with an NF-κB inhibitor (10 µM BAY11-7085) or MAPK inhibitors of ERK1/2 (1 µM U0126), p38 (1 µM SB203580), or JNK (1 µM SP600125); alternatively, the cells were treated with 2 mM NAC. At 3 hours post-infection, the supernatants were removed, centrifuged and stored at −20°C. Cytokine quantification was performed by ELISA (R&D systems) following the manufacturer' s instructions. The macrophage oxidative state was determined as described previously [53]. Briefly, macrophages were pre-treated with or without 2 mM NAC for 4 hours before infection. Macrophages were infected with PA14 or with the ΔlsfA, C45A or gacA: : tn mutant strains at an MOI of 10. After the indicated time in post-infection culture medium, the cells were washed with PBS and then incubated with H2DCFDA (Invitrogen) at a 2. 5 µM final concentration for 30 min at 37°C. Cells were washed with warmed PBS (37°C), resuspended in cold PBS containing 1% FBS and analyzed by fluorescence-activated cell sorting (FACS). Unstained controls were treated similarly. For the baseline fluorescence control, macrophages were uninfected but stained according to the above procedure. The mean fluorescence intensity values were calculated by dividing the values of the infected macrophages by those of the uninfected control. The animal experiments were performed in agreement with the Ethical Principles in Animal Research adopted by the Conselho Nacional de Controle da Experimentação Animal (CONCEA) and in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Internal Animal Care and Use Committee of the Instituto de Química, Universidade de São Paulo (N°08/2012). Female BALB/c mice (8–12 weeks old) were obtained from the in-house animal facility (Biotério de Produção e Experimentação da Faculdade de Ciências Farmacêuticas e do Instituto de Química da Universidade de São Paulo). Mice were kept on a 12/12-h light/dark cycle with free access to food and water and were maintained under specific pathogen-free conditions. All mice were euthanized in CO2 chamber, and every effort was made to minimize suffering. PA14 and the ΔlsfA and C45A mutant strains were used for i. t. inoculation as described before [54], with few modifications. Bacteria were grown as described above, harvested by centrifugation at 12000 g for 2 min, washed twice in sterile PBS and resuspended in PBS at a concentration of 2×106 bacteria. The CFU/mL were validated by plating serial dilutions of the suspensions. Each mouse received 60 µL of a bacterial suspension. A ketamine/xylazine mixture was injected i. p. to anesthetize the mice before surgery. A midventral incision was made, and the trachea was exposed. The bacterial suspension was inoculated i. t. Controls were inoculated i. t. with 60 µL sterile PBS. At 24 hours after infection, the lungs, spleen and liver were harvested for CFU and cytokine measurements. The tissues were homogenized in 1 mL PBS for the lung and spleen and in 2 mL PBS for the liver. The supernatants were collected, and the CFUs were assayed by serial dilution and plating on LB plates. For cytokine measurements, the lung tissues were homogenized, and the supernatants were centrifuged at 12000 g for 10 min at 4°C. The cytokines TNF-α, IFN-γ and IL-10 were quantified by ELISA (R&D systems), following the manufacturer' s instructions. At 24 hours after infection, the lungs were harvested, minced and digested with collagenase for 30 min at 37°C. The RBCs were lysed by adding NH4Cl lysing buffer. The cells were resuspended in PBS with 3% FBS and stained with different combinations of conjugated antibodies, including F4/80-PECy5 (BM8), CD11c-FITC (HL3), CD11b-PE (M1/70) and Ly6G/Ly6C-APC (RB6-8C5), followed by incubation for 20 min on ice. Finally, the cells were washed and resuspended for flow cytometry analysis. FlowJo software (Tree Star) was used to analyze the data. The myeloperoxidase activity assay was performed as previously described with a few modifications [55], [56]. The animals were infected as described above, and the lungs were harvested, lysed mechanically in the presence of 50 mM sodium phosphate pH 5. 4,5 mM EDTA and 0. 5% cetyltrimethylammonium bromide, ultrasonicated and centrifuged. The supernatant (50 µL) was mixed with an equal volume of 3 mM 3,3′, 5,5′-tetramethylbenzidine dihydrochloride (TMB) for 2 minutes. The reaction was stopped by the addition of 25 µL of 2M H2SO4. The optical density (OD) was measured at 450 nm. After i. t. infection with wild-type PA14 or the ΔlsfA or C45A strains, the treated (N = 8) and control (N = 3) groups were observed for survival. All deaths reported were from moribund/euthanized mice. Mice with labored or rapid breathing, decreased motility, ruffled or abnormal-looking fur or other obvious signs of distress were considered to be moribund as described before [57]. Prism 5 (GraphPad Inc.) was used for all statistical analyses. Kaplan-Meier survival curves were plotted, and significance was calculated using the log-rank test. The data were compared using the one-way or two-way analysis of variance (ANOVA) followed by Bonferroni' s multiple comparison test.
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Title: Involvement of a 1-Cys Peroxiredoxin in Bacterial Virulence Summary: Pseudomonas aeruginosa is an important human pathogen that employs a vast arsenal of virulence factors and infects immunocompromised hosts, such as patients in intensive care units, causing pneumonia and other illnesses. Macrophages are cells in the first line of defense against pathogens in the lungs. After pathogen recognition, macrophages release pro-inflammatory cytokines to recruit other immune cells and employ a process known as oxidative burst to kill invading microbes. P. aeruginosa can counteract oxidative stress using antioxidant proteins, such as peroxiredoxins. We show here that LsfA, which belongs to the poorly characterized Prx6 subfamily of peroxiredoxins, is indeed endowed with a thiol-dependent activity that is required for full virulence. In vitro and in vivo infection models confirmed that LsfA peroxidase activity is required for the immunomodulation caused by P. aeruginosa and that its absence allows the host to overcome the infection. This study demonstrates for the first time the involvement of a bacterial Prx6 in virulence.
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Summarize: Sentenza definitiva Il nostro sistema processuale è ispirato in linea generale al principio secondo cui il giudice che delibera nel merito deve definire il giudizio, pronunciando su tutte le domande e le eccezioni proposte dalle parti (art. 277, comma 1, c.p.c.). Il nostro ordinamento prevede alcune eccezioni a questo principio con due (sotto)tipi di sentenza: la sentenza definitiva generica e la sentenza (definitiva) parziale. Il danneggiato può chiedere che sia accertata la responsabilità del danneggiante per il verificarsi dell'evento dannoso (condanna generica) e riservarsi di chiedere in un successivo giudizio la quantificazione del danno. La sentenza generica è una sentenza definitiva, che chiude il procedimento, ma il suo oggetto riguarda solo al generica responsabilità del danneggiante. Come si è detto il giudice che delibera nel merito deve definire il giudizio, pronunciando su tutte le domande e le eccezioni proposte dalle parti. Alla predetta regola può tuttavia derogarsi nei casi previsti dagli art. 277, comma 2, c.p.c. e art. 279 c.p.c., comma 2, n. 4, che contemplano la possibilità delle sentenze non definitive, vale a dire di quelle pronunce che non esauriscono il thema decidendum in quanto risolvono soltanto alcune delle questioni dibattute, disponendo per le altre la prosecuzione del giudizio In tale successiva fase del processo, il giudice che abbia emesso una sentenza parziale, rimane da questa vincolato, nel senso che non può rimetterne in discussione il decisum a meno che la stessa non sia stata riformata a seguito di impugnazione immediata (C. Cass. 1998/04821, 1999/05860, 2000/10101 e 2001/02332).
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Summary: Cassazione 18.6.2019 n 16289 Passata in giudicato la sentenza penale di condanna generica dell'imputato al risarcimento del danno da liquidarsi in sede civile a favore della persona offesa la successiva azione volta alla determinazione del quantum debeatur, per il disposto dell'art 2953 cc, non è soggetta alla prescrizione breve ex art 2947 cc, ma a quella decennale, decorrente dalla data in cui la sentenza penale è divenuta irrevocabile.
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Summarize: Progress on the KMCC Has Been Slow, Quality Problems Persist, and Project Costs Are Unknown Approximately 1 year after our initial testimony and over 2 years after the KMCC’s originally scheduled construction completion date, the project continues to experience significant cost and schedule uncertainty along with construction quality problems and ongoing criminal investigations. Since our testimony in June of 2007, limited progress has been made on KMCC construction, and estimates of how much the total project will cost or when it will be completed are uncertain. Total project costs are uncertain because the Air Force does not track all costs associated with the project. Specifically, tens of millions of dollars related to design, foreign currency fluctuation, rework, personnel, and furniture and equipment costs are not included in the Air Force’s cost to date or estimate for construction completion of the project. Contingencies to fund items such as potential hindrance claims are also not included in the estimated costs for construction completion. When including all estimated costs, the total cost of the project will likely exceed $200 million. Limited Progress Has Been Made Over the past year, limited progress has been made on constructing the KMCC, and the project’s construction completion date is uncertain. Figures 1 and 2 show comparisons of the project when we visited in 2007 and during our last visit in May 2008. The comparison of the KMCC food court area shows that other than some wall finishes and floor tiles being installed, the area remains essentially the same and still requires finishing work and the installation of the food court restaurants. Overall, relatively few contractor employees were on-site from the time of our initial site visit in May 2007 through early 2008, as LBB-Kaiserslautern experienced repeated delays in reaching agreements with major contractors to return to work. While LBB-Kaiserslautern reached completion agreements with its major contractors in March 2008, and the number of workers on-site has increased since that time, it is not clear if the number and type of workers are sufficient to sustain production and meet LBB- Kaiserslautern’s new estimated construction completion date of January 2009. This projected date represents construction completion; however, the facility will not be open to the public for about 4 months following construction completion. Moreover, because LBB-Kaiserslautern has not met earlier construction schedules and the estimated construction completion date has continued to slip, we are concerned about LBB- Kaiserslautern’s ability to manage actions needed to achieve the January 2009 estimated construction completion date. The comparison of the KMCC name-brand restaurant shows that the restaurant still requires painting, wiring, and other finishing work similar to that needed in 2007. In addition, major construction deficiency problems, such as the leaking roof and improperly installed kitchen exhaust ducts, which we discussed in our 2007 testimony, are just now being repaired. For example, contracts were recently awarded for the first two of four phases of repair work on the leaking roof, and most of the deficient ductwork has been removed. However, additional problems, such as several long cracks in the concrete floor of the AAFES store, have been identified by the Air Force since our last testimony. LBB-Kaiserslautern has retained an expert consultant to investigate the floor cracks, and the consultant’s report recommends that joints be cut into the floor to control further cracking. The actual scope of the floor repair work and associated cost and schedule impacts are not known at this time. Figure 3 shows an example of these cracks. The pen in the photograph is provided for scale. Final Costs of the KMCC Project Are Still Unknown Because the Air Force does not track the total cost of the KMCC project, there are no accurate estimates of how much the total KMCC project will cost. Cost estimates provided by the Air Force primarily include core construction-related costs and do not account for millions of dollars in other costs related to the KMCC project. When all project-related costs are taken into account, the total cost for the project will likely exceed $200 million. This figure was derived by consolidating numerous Air Force cost estimates captured in different accounts, such as military construction funds (MILCON), non-appropriated funds, and operations and maintenance funds. According to Air Force officials, approximately $121 million has been spent on core construction-related costs for the KMCC as of March 31, 2008. In addition, Air Force officials estimate that an additional $41 million in construction-related costs are required to complete the project. However, this estimate only relates to those costs that the Air Force tracks for reporting purposes. Not included in the Air Force’s estimate are design costs, costs of rework to repair known construction deficiencies, foreign currency fluctuation costs, costs for furniture and equipment, some costs for secondary services, and costs for the numerous Air Force personnel reassigned from other projects in order to help manage the KMCC construction project. For example, the Air Force cost estimate does not fully reflect the effect of the weakening dollar compared to the euro because payments from MILCON funded project components to cover losses due to foreign currency fluctuation are drawn from a separate account that the Air Force does not track as part of this project. Table 1 shows the Air Force cost estimate for core construction-related expenses along with additional costs associated with the project. As the table shows, an accurate estimate on the total cost of the project is not possible because of a number of unknown expenses, such as the Air Force’s liability for contractor hindrance claims. Additional clarification on selected project costs not included in the Air Force’s construction cost estimate is provided below. Construction deficiency repairs. Deficient construction has been identified on major building components, including the roof and kitchen exhaust ducts. LBB-Kaiserslautern is taking steps to contract for repairs to correct these deficiencies. Roof damage. Significant sections of the roof require repair or replacement. The current plan is to complete repairs to the roof in four phases. The first phase of repairs is expected to cost $2.7 million and is included in the Air Force’s construction cost estimate, which according to the Air Force is being paid for primarily with funds withheld from the original roof contractor. Costs for repair phases two through four are estimated by the Air Force to cost about $10.8 million and are not included in its construction cost estimate. U.S. government funds remaining from the original roof contract are being used to fund the majority of phase one repairs; therefore, the phase one repair cost is reflected in the Air Force’s construction cost estimate. Funds for repair phases two through four are initially being funded by the German government, and the Air Force excludes these costs in its construction costs to complete the project. Kitchen exhaust ducts. The kitchen exhaust ducts installed in the KMCC do not comply with U.S. National Fire Protection Association standards specified by the Air Force. LBB-Kaiserslautern has completed the design for repairs to the exhaust ducts and is negotiating with its contractor a final price for their installation. The estimated installation cost is approximately $1.8 million. In early 2007, the Air Force agreed to pay for $600,000 of the rework and has included that amount in its construction cost estimate, but has not included the rest of the repair cost as part its construction cost estimate. Secondary services. As the project has progressed, the Air Force has retained several contractor personnel to provide construction inspection, engineering, consulting, and other secondary services. For example, since January 2005 the Air Force has contracted for construction inspectors who are responsible for, among other things, ensuring that installed work meets the terms of the contract and checking that invoices correctly reflect the work performed. The cost for most of these secondary services is not included in the Air Force’s construction cost estimate. Air Force officials indicated that some of the Air Force’s technical consultants were also supporting other projects, but that the majority of their work involved support on the KMCC. Since the majority of work performed under these secondary services contracts relates to the KMCC project, we have included the cost of these contracts in the Air Force’s estimate. Foreign currency fluctuation. Since the start of the project in 2003, the euro has appreciated 35 percent relative to the U.S. dollar. Because KMCC contractors are paid in euros but KMCC project funds are budgeted in dollars, the Air Force is susceptible to paying more when the dollar loses value. In addition, this risk has been magnified by the 2-year delay in completion of the project. Since the June 2007 testimony alone, the euro has appreciated by 15 percent, from $1.35 to $1.55. As a result of the devaluation of the dollar, nonappropriated fund payers (AAFES and AFSVA) have paid about $31.9 million in foreign currency fluctuation expenses, and appropriated MILCON funds have paid $8.6 million since the project’s inception. The Air Force includes nonappropriated fund foreign currency fluctuation costs in its construction cost estimates. However, because MILCON foreign currency fluctuation costs are paid from a separate account, the effect of foreign currency fluctuation is not reflected in the Air Force’s construction cost estimate. Therefore we have included in the estimate based on the Air Force’s reported numbers the actual foreign currency fluctuation costs paid to date associated with MILCON funds. In addition, the Air Force also did not include an allowance for future foreign currency fluctuation costs associated with MILCON funding. Based on the trend of the strengthening euro, these foreign currency fluctuation costs will only increase as MILCON expenses increase. Figure 4 shows the trend in the strengthening of the euro against the U.S. dollar over the past several years. In addition to the estimated expenses listed above, there are still a number of significant costs associated with project completion that have not been estimated. These items include potential hindrance claims, repair of cracking concrete, and Air Force personnel costs for staff needed to manage the KMCC’s construction. Depending on the resolution of these unknown costs, the total cost of the KMCC project could increase substantially before completion. Hindrance claims. As a result of delays to the project, the Air Force expects that contractors will submit claims for additional costs incurred from being hindered in their performance. As of June 1, 2008, LBB-Kaiserslautern had forwarded change order requests for contractor hindrances totaling $8.2 million (5.4 million euros) to the Air Force. However, the Air Force has denied the requests because it contends that they are not substantiated. In anticipation of contractors making formal hindrance claims—that must meet a prescribed legal standard and would be adjudicated through the German courts—the Air Force has retained a consultant to evaluate the history of the project and prepare the Air Force’s claims defense strategy. At this time, the Air Force’s potential exposure to claims is unknown. Concrete cracking. Long cracks have been identified by the Air Force at various locations on concrete floors of the KMCC subsequent to our 2007 site visit. A German court appointed an expert consultant to determine the severity of the cracks and identify their causes. The consultant’s report, issued in March 2008, indicates that joints will need to be cut into the floor to keep additional cracks from occurring. LBB- Kaiserslautern subsequently initiated design work to detail the scope of the repairs. The Air Force expects that LBB-Kaiserslautern will award a contract for the repairs in July 2008. At this time, the cost of repairs is unknown. (See fig. 3 for an example of these cracks). Air Force personnel costs for staff managing the KMCC. According to the Air Force, a team of 29 military, civilian, and contractor personnel manage the KMCC construction project. They are responsible for financial management, claims management, design and construction management, and stakeholder operations. This team comprises 6 military officers and enlisted personnel, 10 civil service employees, and 13 contracted employees. The cost of the salaies and benefits for military and civil service employees who have been reassigned from other projects to assist in managing the KMCC project since its inception in 2003 is a real cost of the project but is not included in the Air Force’s overall cost estimate. This cost is difficult to quantify because the military and civilian personnel occasionally work on other projects, and it is not clear how their time is apportioned among projects. Federal Republic of Germany funds. The extent to which project cost increases will be borne by the Air Force and its funding partners is unknown because of uncertainties regarding the $37.9 million (25 million euros) committed by the German government for the project. The German government allocated $15.2 million (10 million euros) to pay contractors’ invoices against change orders that had not yet been approved by the Air Force. According to the Air Force, only $8.6 million (5.7 million euros) were actually expended by LBB-Kaiserslautern prior to the funds expiring at the end of calendar year 2007. According to the German officials, this initial $8.6 million amounts to a loan, and the Air Force stated that it will reimburse this amount when it approves the change orders and makes payment against the associated invoices. The additional $22.7 million (15 million euros) are planned to be used by LBB-Kaiserslautern to fund contracts for rework items, such as roof repairs and kitchen exhaust duct replacement, and may also be used to settle contractor hindrance claims. In discussions with us, German officials characterized the $22.7 million (15 million euros) as a loan as well and said that they expected full repayment of funds. However, Air Force officials contended that they will only pay for valid work under the original KMCC contract and will not pay for rework. In light of these opposing views, for transparency purposes, all expenses paid from the German funds are incorporated into the total project cost regardless of which party is ultimately responsible for financing them. Delayed Completion Has Caused Opportunity Costs to Grow As a result of the delayed KMCC project completion, AFSVA and AAFES, two major funding sources for the project, have experienced negative financial effects. Both AFSVA and AAFES invested in the project with the expectation of returns being generated in early 2006, the project’s original construction completion date. However, the delayed completion has resulted in lost profits and in turn reduced the amount of funds AAFES and AFSVA can provide to military communities for morale, welfare, and recreation. For example, AAFES estimates that every month the KMCC’s opening is delayed, AAFES loses approximately $500,000 in net profits from stores such as the base exchange and food court restaurants. In total, AAFES estimates it will have lost more than $14 million in net profits by the time construction is estimated to be completed in January 2009. Also, the unavailability of the 350 hotel rooms to be completed in the KMCC Visitors Quarters results in the U.S. government paying more for lodging on the local economy. According to an Air Force estimate, on average each month that the opening of the hotel is delayed results in the U.S. government paying an additional $90,000 for off-base lodging of displaced personnel traveling at the government’s expense. Estimated increases in off-base housing costs will total $2.9 million dollars if construction is completed in January 2009. Criminal and Civil Investigations Continue to Surround the KMCC and Other Projects In our June 2007 testimony, we reported that several Air Force and LBB- Kaiserslautern personnel involved in management of the KMCC and other RMTP projects were under investigation by AFOSI and German police for a variety of issues, both criminal and civil. AFOSI officials stated that the cases have matured significantly since that time; however, formal indictments have not yet been made. Because the investigations are still active, AFOSI is limited as to the information it can disclose. However officials did state that investigations involve U.S. military and civilian employees, employees of LBB-Kaiserslautern, and KMCC trade contractors. These individuals are being investigated for offenses such as dereliction of duty and bribery. The Air Force Has Improved Controls over the KMCC Project The Air Force has made significant improvements in its oversight and internal controls over the KMCC project. As stated in the June 2007 testimony, the Air Force had failed to institute effective management oversight and internal controls in order to mitigate the high risk of the project. Specifically, the Air Force lacked standardized policies, did not have sufficient staffing to oversee the project, was not thoroughly reviewing invoices, and was approving change orders and payments on the project without proper documentation. In the last year, the Air Force created the RDO, which centralized management oversight of the KMCC project and brought together appropriate personnel specializing in financial management, claims management, design and construction management, and stakeholder operations. The Air Force has also standardized its invoice and change order review processes to minimize future risks of paying for unapproved work. The Air Force trained and appointed certifying officers and accountable officials for the KMCC project to ensure that personnel authorizing payments are aware of their fiduciary responsibilities. Finally, during 2007, senior officials within the Air Force engaged officials from the Department of State and the Federal Republic of Germany in an effort to encourage progress on the project. Establishment of the RDO and Improvements in Invoice and Change Order Reviews In August 2007, the Air Force established the RDO to centralize control over all aspects of the KMCC project. According to the Air Force, the RDO has 29 personnel, which is a significant increase from the 8 oversight personnel initially assigned to the KMCC project. These 29 personnel are 6 military officers and enlisted personnel, 10 civil service employees, and 13 contracted employees. The RDO is divided into four branches: financial controls, ABG-75 administration, engineering, and operations/quality assurance. The Air Force also standardized policies and procedures for the four RDO branches, which are documented in an official operating instruction manual for the KMCC project. The documented policies were a measurable improvement from the one-page flowchart used during our last visit in 2007. As part of the establishment of the RDO, the Air Force also appointed and trained 9 certifying officers and accountable officials. Certifying officers are responsible for certifying vouchers for payment. Accountable officials provide source information to a certifying officer to support a decision to certify a voucher for payment or to disburse funds. Appointment and training of these types of officials are key to ensuring that appropriate controls are in place over payments made by the Air Force. The RDO also includes 6 quality assurance engineers and technicians responsible for performing technical reviews of designs and conducting daily surveillance of the work site. Since creating the RDO, the Air Force has also improved its review process for invoices. During our initial visit last year, we reported how Air Force management instructed its staff to certify for payment invoices with items included in unapproved change orders and items that exceeded quantity limitations specified in contracts. The Air Force has since revised its policy and now rejects invoices for quantities in excess of contracted amounts and also rejects invoices with items included in unapproved change orders. For example, if the Air Force receives an invoice that contains charges for items contained in unapproved project change orders, the Air Force will reject the invoice and refuse payment until LBB- Kaiserslautern submits an invoice that only lists items that have been approved by the Air Force. In addition, the Air Force has gone back on prior invoices and recouped amounts previously paid in excess of contracted quantities. Involvement of Senior Air Force Officials Beyond improvements over internal controls and the establishment of the RDO, senior Air Force, Department of State, and German government officials have also increased oversight of the project. Beginning in August 2007, the U.S. Air Forces in Europe Vice Commander, the U.S. Ambassador to Germany, and the heads of the German Ministry of Construction and the German Ministry of Finance started meeting regularly to discuss problems facing the KMCC. These repeated meetings between U.S. and German officials prompted the German government to provide $37.9 million (25 million euros) to the project for backlogged change orders and repair work. According to the Air Force, German support is essential for a successful completion of the KMCC project. The Air Force’s expectation is that by maintaining strong relationships among senior officials, it will ensure that those running the KMCC project are able to better organize, prioritize, and execute the project. Unfortunately, this joint effort has not yet expedited KMCC construction to an acceptable pace according to Air Force officials. Other Recent U.S. Projects on Ramstein Air Base Have Experienced Similar Problems Other projects recently completed in the KMCC area managed by LBB- Kaiserslautern have experienced problems similar to those affecting the KMCC. Air Force officials report that several recent projects on Ramstein Air Base experienced cost and schedule growth, including projects that were delivered by LBB-Kaiserslautern months after the planned delivery dates. In addition, several projects were delivered with construction flaws that have caused flights to be diverted and have affected the processing of freight shipments. According to the Air Force, it has experienced fewer problems with less sophisticated projects, such as the construction of more than 1,000 new military family housing units in the KMCC area and the construction of an indoor pool on Ramstein Air Base, that were managed by LBB-Kaiserslautern. However, construction of these projects is not comparable to the scope and complexity of constructing the KMCC building, runways, and other operational facilities. Cost and Schedule Growth of Other Projects Numerous recent projects associated with the RMTP built on Ramstein Air Base by LBB-Kaiserslautern have experienced significant cost growth, according to the Air Force. Several of these projects have also not received final bills from LBB-Kaiserslautern despite being turned over to the United States more than a year ago. Therefore, the final costs of the projects are still unknown. In addition, many of the same group of projects managed by LBB-Kaiserslautern also experienced significant schedule growth according to the Air Force, including projects that were delivered months after their scheduled completion dates. Construction Deficiencies of Other Recently Completed Projects Construction deficiencies experienced on several of the RMTP projects built by LBB-Kaiserslautern add to the concerns associated with cost and schedule growth. Specifically, there have been several projects where faulty construction has resulted in safety concerns, building evacuations, and potentially reduced useful lives of facilities. Below are examples of such problems. South runway. This runway was recently built on Ramstein Air Base in order to upgrade the base’s capabilities in support of overseas missions, including those in Iraq and Afghanistan. However, the runway lights are adversely affected by a construction defect that allows groundwater to collect in the underground ducts and manholes that contain electrical components used to control the lights. The intrusion of water has damaged electrical components resulting in power outages to the lights. These lights are crucial for operations because they allow aircraft to land during times of extremely limited visibility. According to the Air Force, because of the lighting failures, aircraft could not land during periods of limited visibility from November through December 2007 and flights were diverted to other bases in Europe. Portable lights have been used to temporarily resolve the problem. Additionally, water is pumped from the manholes as an ongoing maintenance activity that according to Air Force officials, has cost about $65,000 to date. Finally, during rainy periods, several large ponds developed between the south runway and adjacent runways and taxiways because of poor water drainage. These ponds, which were several acres in total area during our visit in March 2008, have attracted large waterfowl, which pose a safety hazard to aircraft using the runway. Figure 5 shows one of these large ponds. Studies and court actions are currently under way to resolve these issues. Freight terminal. This large 100,000 square foot material handling facility was constructed to assist in processing freight shipments to and from overseas locations. However, deficiencies with the structural steel that frames the freight terminal required evacuation of the building until additional support columns could be installed. According to the Air Force, because of material fabrication issues, insufficient welds, and undersized connections, the steel structure that supports the freight terminal building was determined to be at risk of collapsing. As an interim safety measure, 41 additional support columns were installed to bolster the structural integrity of the facility. According to the Air Force, these additional supports effectively reduce the open area in the center of the facility and affect freight processing operations. For example, forklifts are restricted in maneuvering and cannot access certain areas. Figure 6 shows an example of the additional support columns installed within the freight terminal building. A permanent resolution will be determined by legal proceedings that are currently ongoing in the German courts. In addition, officials were concerned that a building attached to the freight terminal was also structurally deficient as a result of potentially insufficient welds on the structural steel. Because of these concerns, the building was evacuated from July through September 2007 while additional support columns were installed. An inspection completed in March 2008 confirmed that the structural steel was, in fact, installed correctly and that the building is not at risk of collapsing. The attached building is currently being used as intended. Hot cargo pad. A large concrete area intended to be used for loading live munitions onto aircraft destined for overseas locations such as Iraq and Afghanistan was recently built near the Ramstein Air Base runway. However, when building the concrete pad, contractors did not install dowels between adjacent concrete slabs as is typically done in this type of construction. Because the dowels were not installed, Air Force officials stated that differential settling of the concrete slabs could result in damage to the cargo pad and reduce its useful life. The Air Force is currently negotiating with LBB-Kaiserslautern to determine what, if any, actions will be taken to address this issue. While this does not represent a safety risk, the reduced life span of the cargo pad could result in the Air Force spending money for repair or replacement earlier than would normally be expected. Concluding Comments Although the Air Force has made measurable improvements in its oversight and control of the KMCC since our last testimony, the project remains at risk because of schedule and cost uncertainties. With few visible changes, no reliable construction completion date, rising repair costs, and continuing construction quality problems, the KMCC will continue to be a high-risk project. Even after the KMCC is completed and open, it will likely take years before all issues related to this project, including litigation and potential construction quality problems, are resolved. Mr. Chairman and Members of the Committee, this concludes our statement. We would be pleased to answer any questions that you or other members of the committee may have at this time. GAO Contacts For further information about this testimony, please contact Gregory Kutz at (202) 512-6722 or [email protected] or Terrell Dorn at (202) 512-6293 or [email protected]. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this testimony. Appendix I: Scope and Methodology To determine the current status of the Kaiserslautern Military Community Center (KMCC) construction project, including projected costs, cost completion analyses, projected construction completion dates, and status of ongoing investigations, we interviewed officials from the Air Force at Ramstein Air Base in Germany, the Army and Air Force Exchange Service (AAFES), the Air Force Services Agency (AFSVA), the U.S. Army Corps of Engineers (USACE), the Air Force Audit Agency, the Air Force Office of Special Investigations (AFOSI), and the Department of State. In addition, we interviewed officials from Landesbetrieb Liegenschafts-und Baubetreuung office in Kaiserslautern (LBB-Kaiserslautern), the German government construction agency; the Federal Republic of Germany’s construction division; and the German police. We physically inspected the KMCC facility with an Air Force project manager and observed construction deficiencies. We also reviewed financial records and statements in the form of contracts, change orders, and invoices to the extent that they were available. We also compiled Air Force and LBB- Kaiserslautern cost estimates for the various cost elements associated with the KMCC project. To determine whether oversight and internal control improvements have been made by the Air Force since our last testimony, we interviewed Air Force officials from the KMCC Resident Director’s Office. We also interviewed officials from LBB-Kaiserslautern, AAFES, AFSVA, AFOSI and the Air Force Audit Agency. We obtained and reviewed project management plans, standardized policies and procedures, cost estimates, training materials for certifying officers and accountable officials, and other relevant documents related to project management. To determine if other projects recently completed in the KMCC area have experienced problems similar to those affecting the KMCC, we interviewed Air Force officials regarding their construction projects in Germany. We also conducted interviews with other organizations, including USACE, AFOSI, LBB-Kaiserslautern, and the Air Force Center for Engineering and the Environment to understand the cost, schedule, and construction flaws experienced by other military construction projects in the KMCC area and how they may affect the viability of future construction projects. We also reviewed available technical expert reports documenting construction deficiencies associated with these projects. Because of ongoing investigations, we were not able to fully explore and discuss the details of fraud investigations with AFOSI and the German police, which limited the scope of our audit. We conducted this performance audit from November 2007 through June 2008 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. This is a work of the U.S. government and is not subject to copyright protection in the United States. It may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately.
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Summary: The Kaiserslautern Military Community Center (KMCC) is one of many projects initiated at Ramstein Air Base to upgrade capabilities of the base as a result of the consolidation of military bases in Europe. The KMCC is intended to provide lodging, dining, shopping, and entertainment for thousands of U.S. military and civilian personnel and their families in the area. Construction on the project, which began in late 2003, was originally scheduled to be completed in early 2006. On June 28, 2007, GAO testified that construction deficiencies and mismanagement had drawn into question when the project would be completed and at what cost. This testimony discusses updated findings related to the KMCC project. The testimony describes (1) the current status of the KMCC construction project, (2) whether oversight and internal control improvements have been made by the Air Force since GAO's last testimony, and (3) if other projects recently completed in the KMCC area have experienced problems similar to those affecting the KMCC. To address the objectives, GAO interviewed officials from the U.S. Air Force, Army and Air Force Exchange Service (AAFES), Air Force Services Agency, U.S. Army Corps of Engineers, Department of State, and German government. GAO also conducted site visits and reviewed project plans, cost estimates, completion analyses, and other relevant KMCC documents. Approximately 1year after GAO'sJune 2007 testimony and over 2years after the KMCC's originally scheduled construction completion date, the project continues to experience significant cost and schedule uncertainty along with construction quality problems and ongoing criminal investigations. Limited progress has been made on KMCC construction, and there are still no accurate estimates of how much the total project will cost or when it will be completed. Major construction deficiencies GAO reported in 2007 are just now beginning to be corrected. In addition, the Air Force does not track the total cost of the KMCC. Specifically, tens of millions of dollars related to design, foreign currency fluctuation, rework, personnel, and furniture and equipment costs are not included in the Air Force's cost estimates. Contingencies to fund items such as repairs to cracking concrete are also not included in the Air Force's estimates. After including all estimated costs, the total cost of the project will likely exceed $200 million. Project delays have also resulted in additional costs to the U.S. government and lost profit for project funding partners. For example, AAFES estimates that it is losing $500,000 of profit for each month that the exchange facility is not open. Although these problems exist, the Air Force has made significant improvements in its oversight and control over the project. For example, the Air Force established standardized policies and procedures for reviewing change orders and invoices. Improvements in controls over payments and change orders have minimized future risks of paying for unapproved work or fraudulent billings for work not performed. Cost, schedule, and construction deficiencies affected other projects built by German government construction agents in the KMCC area. For example, underground electrical ducts at Ramstein Air Base flood with water causing runway lights to malfunction. A freight terminal on the air base was also built with structural deficiencies that resulted in its temporary evacuation.
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Summarize: "Roc, you got me on a good day," Dr. James Fallon announced. The horses over at Saratoga racetrack had been running in his favor all morning. It was also the day that his best friend from college had died. Fallon received the news just four hours before he met me. "I'm still waiting for it to hit," he claimed. I smiled gamely and raised my eyebrows. He returned the smile and threw in a shrug. We both knew that it would never hit. "When I really dissect my behavior," the neuroscientist later confided to me, "everything in a strict sense is a lie. Everything. Even though I think I never lie, I'm lying all the time. I seem like a nice guy, but I want to be clear—I'm not as nice as I seem to be." In 2006, Fallon was studying the brain scans of psychopathic killers when he happened to compare them to a scan of his own brain. The characteristic deactivation of emotional regions was unmistakable. He discovered for himself what friends and family had been insisting for years: Fallon was a psychopath—albeit a "pro-social" one, as he likes to say. The 66-year-old father of three is happily married, highly successful in his field, and has no criminal record. The aggression, the narcissism, the callousness, the recklessness typical of psychopaths—he can turn all of them off, he claims. The charm, however, is always on. At the door of his home in Irvine, California, Fallon greeted me like an old friend. He was on the phone with his bookie in Jersey—a six-foot-eight, 300-pound former Mafioso by the name of Big Moe. "His actual name is Joey," Fallon chuckled. "He thinks it's still 1965." Within a minute of ringing his doorbell, he had me laughing and feeling at ease. We took our drinks to the backyard where we could watch monarch butterflies feast on a milkweed garden—one of the scientist's favorite distractions. He noted that the seasons elicit very different behaviors from the insects. "When they're down in Mexico and up in Monterey hanging in the trees, they're very sociable. But, in between, when they're competing for food and sex—they're killers. Two very different behaviors, one animal." "You can ask me anything, Roc," he added through his beard as he spread his arms wide. "I am restricted somewhat, though. My mother is still alive, and my wife is still alive." "So, we'll do the follow-up after they pass away?" I teased. "Sure," he laughed. "That'll be quite a different interview." VICE: There are many definitions of psychopathy. What are the critical qualifications? James Fallon: Core psychopathy is basically lack of empathy and extreme manipulation of anyone to get what you want. You don't have to be sadistic like a lot of people think. You don't have to be glib, but you often see glibness because the psychopath doesn't have to make the loop into the limbic system [the brain's emotional center], which slows you down. Do you ever place pauses in your speaking in order to seem more authentic? Sometimes—and, sometimes I'll make up things so it appears I was wrong. I'll insert a red herring, so that I can go back and say, "Gee, I was wrong about that." It makes me more approachable, more believable. You're very good at building rapport. But, I'm trying to slow down—trying to say, "Am I really being truthful here?" The game for me now is: Can I manipulate myself? The challenge is: Can I astonish somebody with purely the truth? But you've also talked in your book about lying to achieve a certain effect. Yeah, yeah, yeah. I've lied for some effect, in fact, in the opposite direction. So, for example, if I caught a 20-pound tuna, I'll say I caught a 15-pound tuna, and somebody will say, "No, it was much bigger than that." Those are manipulative techniques. I remember this really funny book called How to Cheat at Tennis from the 70s. A tennis court is supposed to be a perfect rectangle, but you can manipulate the court—repaint the lines to make it a parallelogram—screw with them that way. So subtly that they won't realize it. Exactly. And then, of course, you do the obvious things—early in the match, if they hit a ball out, you call it in. That way, you can do the reverse later when you need it. Start lying the other way to set the person up later. I do that. It's just a game, in a sense—a practical joke, but it's still fucking with people. I never thought of myself as doing it in any malignant way. It was always fun. Was it fun for the other guy? Not all the time. It's kind of intellectually bullying people, I guess—playing with their heads. There's a darker side to it. In the past two years, I've come to realize how much I do that. I've never taken advantage of underdogs though. You know what I mean? I'm enough of the sportsman to play a fair game. Is that a moral stance? It's hard to imagine morality without empathy. Where does your sense of morality come from? You know, growing up Catholic around priests, and nuns, and my parents—I just never did anything wrong. Lying, cheating, stealing, squeezing girls' asses, I did none of that. But, it was part of an obsessive compulsiveness. I thought all behavior had to be perfect and in line with the universe. I had to keep everything in symmetry. So, the code itself was arbitrary? I mean, if you had been raised under a different system, would that have become your ideal? You know, there's one psychiatrist I spent time with in India. She goes, "Jim, you're actually a natural Buddhist. The type of empathy you have is not for people, but for mankind. That's very Buddhist." I think if I had been brought up in the Buddhist system, it might've been even easier. What made Catholicism sub-optimal? I had no problem with that strict code. I just went way beyond it. That's the problem. You never lie, steal, cheat—all that stuff seemed obvious to me. People would say, "Geez, Jim. Relax." The idea of adding the concept of morality to it was nonsense to me. The obsession with being perfect was just innate. You know, the first memory I had—and it may be from when I was two or three—is from when I would be going to sleep, and as I would close my eyes, I would see this… You ever live in the north? Aurora borealis? Right. Now, if you go really far north, and it's a really good storm, the aurora borealis is right overhead, so dramatic, and you just feel like an ant on the stage. These silver curtains come down—whoosh! Well, every time when I closed my eyes, it was just like that. It wouldn't have any color or brightness until it started to get closer and closer. It would come in like a funnel, faster and faster. And it would condense into something that felt, every time, like the entire universe was hitting me right in the forehead. It would go: ping! And, it was light as a feather. It's the most exquisite feeling, because it's infinity and infinitesimal all at once. It would happen just that way every night. It may have set up some kind of leitmotif, very early on, that really focused me on the whole universe and perfection. When did you lose the morality fixation? I was 19 or 20 when it suddenly switched. I went from thinking that everything was a moral issue to thinking that nothing was a moral issue. So, even though I can bring back the sense of that light, it's detached from morality completely. What's your religious affiliation now? I'm an agnostic atheist. I will manipulate groups, but I try to do it for ethical reasons. I don't think of it so much as a moral issue, but there's a certain beauty to it. You sound almost like the God of the Old Testament—appearing in order to restore balance. I've never heard it put that way. That's good. I identify with that personality of God the Father intervening at the right time to make things right, and he does it dispassionately. It's really important that there's no emotion. I can imagine that being a benefit in many situations. It seems to be helping you process the death of your friend. Yeah, there is no emotional flinching. I remember we had a great time together. He was an interesting guy, but something about it just doesn't emotionally connect with me. You can look at it objectively—I should be upset, and I'm not. In a way, it sounds freeing. Would you want to have empathy? No. I'm quite happy with who I am. I really love my life. And, actually, everybody's got a little piece of what I have—not everybody, but a lot of people. They're not categorical psychopaths, but they have some really nasty psychopathic behaviors. They're on the spectrum? Definitely. If you took the raw sense of how people feel about the people they're interacting with, it would be brutal! You could actually say that these behaviors are not psychopathic because so many people have them. Do you think an individual's behavior is consistent, or can different environments bring out morally conflicting behaviors from the same person? For example, the commandant of Auschwitz would go home and kiss his wife and kids at the end of the day. Is there a consistency to that? Well, that's why it's really hard for any psychiatrist to say that Hitler was a real psychopath, as well as most of the Nazis. They were close to their families. They had real empathy. They all did. They're not psychopaths. Hannah Arendt's concept about everyone participating a little bit means that they had to believe in the ethos of what they were doing. I'm sure they convinced themselves that they were helping the world—like the Norwegian shooter Breivik. If you read his manifesto, he was quite sane. Not a psychopath? Not quite a psychopath. He had empathy, but he had a vision. The problem is, that's the equivalent of what Gandhi had, what Mother Teresa had, what Mandela had. They really believed that they were going to fix the world, even if they had to walk over people to do it. They knew that it comes with a price. To save the children of the world, Mother Teresa would walk over people. Gandhi walked over his family all the time, so did Mandela. To them, what they were doing was good, even though in a local way they were just doing brutal things—things that would be considered psychopathic. But it's just a different kind of empathy. How does that play out neurologically? In neuroscientific terms, all behaviors that are reinforced, whatever they are, go to a little spot in the brain called the dorsal medial notch of the nucleus accumbens. It's where everything comes together: all the dopamine, endorphins, acetylcholine, oxytocin, vasopressin. It's hedonism central. So any behavior that can be reinforced has to go through this tiny little spot. And, people will all pick a different thing. Some people have a shopping addiction, some have an eating addiction, some have drug addictions—it just depends on the wiring. So, essentially, you're a slave to it? Exactly. So, if you look at evil behaviors—if it's going through that little spot, it basically takes the evil out of it, because it's driven the same as everybody. I think a candidate for a truly evil act is unhooking from that. If you can do something for no other reason than for the pure rationale of what it is—only then could you start to consider something pure evil. But if you're not rewarded, why do it? Exactly. Why do it? Because, if it all goes through that little spot, it means there is no evil, and there is no free will. Instead of evil, it's just behavior we don't agree with. And, that's a real quandary, because I don't know how you conduct a civilized society and have any kind of common ground. So in order for society to function, we have to participate in the illusion of free will? I think so. Participating in the illusion is important. But, it's funny, you know—I started treating my wife better as a little experiment. She liked it, and I told her—because I can really be honest with her—I said, "I don't mean this. I'm just doing this as a game." She said, "I don't care. You treat me better. Why would I care why you're doing it?" That, I don't understand, because it should be all about intention. What are your relationships like with women in general? I have a lot of female friends. When I go out roaming, they like me, even though I look like hell. It doesn't matter, because I act like I don't care, and I really don't care, and they love it. Why do you think that is? We have to look at everything in terms of sexual fitness. If you don't need them, it means that you can get anybody, therefore they want your genes. You can't get me, therefore you want me. It's a little too glib and slick, but it's probably true. And, if I have a conversation like this with my female friends who are really very, very smart neuroscientists—they have to keep from getting mad, even though they know there's some truth to it. Their emotionality blinds them to it? I love how my female friends, who are so smart, are fighting their emotions all the time. They know it, and they actually come asking for it. They want me to torture them. They want to push up against something. Most guys are always on the make. They'll say anything to get laid, but to have a guy push against her, and say something to her like, "You're never getting laid tonight," they go crazy for it. How do you think the world would be if everyone were like you? Well, it would be bad for people like us. I think the biggest thought experiment would be if we got rid of all aggression-related genes, and got rid of some psychopathic traits—as a species, we'd be screwed. We'd be completely screwed if everybody becomes Jimmy Carter. Any aggressive person could run the world. People say that it would be great to have peace and love. Their heaven, to me, is the end of humanity. You've written about the possible increase of aggressive genes in places of conflict. Is the reverse true? Is aggression being bred out in stable Western societies? I think so. Right here in California—I don't want to call it the feminization—but you have the idea that everybody wins. It's about getting along so much that you have no competition. I see it as a very, very negative force, if you care about the species. But there's always this dynamic about what's good for the species and what's good for the individual. These are very much at odds with one another. In a sense, we need psychopathy. We don't need full-blown psychopath dangerous fuckers, but having a prevalence of psychopathic traits is associated with leadership. It's in presidents, and prime ministers, and in people who take risks. They do things to protect against aggressors. So you're saying that they're doing it for themselves, but it just so happens to protect society as well? That's why people like Jimmy Carter do not belong in those positions. Obama's stuck on this a little bit. He's too good? He's not psychopathic enough. Almost all the great leaders have high levels of psychopathic traits. If they took the Psychopathic Personality Inventory, they'd score pretty high. In the end, this is a broader discussion that I'm not really qualified to talk about, but you are. What do you mean? See, what I did there was manipulative. I really don't think you are, but I said that you are. I don't think you are, and I don't think I am either. I wanted to ask about your brain scans. You have zero activity in your emotional regions in the presence of emotional stimuli, but obviously those regions are being used for something, right? Do you know what that might be? It's probably being used to inhibit my bad behavior. I guess there's a way of testing that. You'd have to get people like me, and then give them an opportunity to do something bad and then have them stop themselves and see if that area turns on. That's a very good experiment. Well, I'm sure you wouldn't have too much trouble finding subjects. I know you hang out with some rough characters. That's true. I get a lot of contacts from really bad guys and girls—people who are on the edge. I can't give medical advice, but they see me as somebody who would understand. So, it's almost like a brotherhood of psychopaths. They're usually very earnest. The ones that aren't earnest, I can sort out. I'll say something to them, and they go fucking crazy. I can find people very quickly—I have my ways. If somebody's in hiding and they're trying to screw with me, I just have ways of knowing exactly where they are at any time, and showing them that I know where they are. They hightail it. What's your ultimate objective in life? I'm actually trying to catch myself from trying to manipulate people all the time. I'm trying to get rid of it all. Why? Because, then I can beat myself. I'm my own best opponent. If I can beat myself, I've won. But then the game is over, isn't it? In a way, it's over. It's checkmate, but I still make mistakes like I did with you earlier. That comment you called yourself out on? Right, because that wasn't honest. And, you didn't set that up as a red herring? No, I didn't. I'm trying to be truthful here. The game for me now is to manipulate myself. In terms of the checkmate, I think the real prize would be ego death. The universe tapping you on the forehead? I think so. What do you think you'll find there? Probably some existential realization that this is just this. But, that ego-desire is very strong, to the point of obnoxiousness. So, the game is to try to strip that part away. If I had my druthers, I would get rid of all the ego, and be able to do something truly good, just for the sake of goodness. But, the drive is really just to prove that I can do it—not for the action itself. It loops within itself and becomes instantly phony. Still, I like it as a target, as a goal. It's the best game I can think of. Follow Roc Morin's latest project collecting dreams from around the globe at World Dream Atlas. No matter how confident you are in your own skin or how experienced you are at what you do, both in work and in life, many of us have times when we feel like we're faking it. This week, Piya asks: How do you deal with feeling like an impostor? Here are the stories from this week's episode... 'If you think about who is feeling like an impostor, it's people not in the privileged class' Dena Simmons says "it is the easy way out" to think of impostor syndrome as something that just happens at the individual level. She wants us to ask how certain environments often make racialized people feel like impostors. Dena tells Piya about her experience as a black girl in a school of mostly white kids, and how she's channeling it to change schools from within. 'Confronting the truth about yourself is really hard': Rediscovering a complicated youth through adult eyes Piper Weiss didn't feel like she belonged at the elite private all-girls school she attended. And stakes were high to fit in because her family had worked hard to get her a spot. So she was thrilled when she met a tennis coach who she identified with and felt supported by. But that man turned out to be an impostor himself, when he was revealed as a child predator. When my second child was born, I started to feel like a fraud Out in the Open producer Lisa Bryn Rundle's definition of motherhood has always included all the different ways that people become mothers. But three years after she gave birth, her partner became pregnant. And Lisa began to feel like an impostor. In a personal essay, she talks about the internal struggle of being the mom of a child she didn't birth herself. How a psychiatry professor accidentally discovered he was a psychopath In 2006, neuroscientist and professor James Fallon accidentally discovered he's a psychopath. But not the serial killer kind. Instead, he's emotionally unavailable, reckless and manipulative. He speaks with Piya about how he gets by around non-psychopaths on charm and what he calls "cognitive empathy" – the ability to understand what others are feeling, without actually feeling it himself. Even Canada's top law students can feel they aren't good enough The University of Toronto's prestigious law school draws some of Canada's best and brightest students. So when Nick Papageorge applied, his hopes weren't high. To his surprise, he was accepted. But that didn't assuage an impostor syndrome that would follow him through his three years at the school. No one told James Fallon he was a psychopath. Or maybe they had. When he was young, he'd heard again and again from people in positions of authority – a priest, a professor, a friend's father – that there was something off about him. Something dark that they couldn't quite name. But Fallon brushed it off each time. Many years later, as a professor of psychiatry at the medical school of the University of California, Irvine, Fallon discovered his psychopathic mind for himself. "I'm a little bit of a snake, but I'm not really a bad guy," Fallon told Out in the Open host Piya Chattopadhyay. "But you don't want to be close to me." Fallon made the discovery by accident. In the late 1980s, the university got a PET scanner. Accused murderers were coming into the school to get brain scans done as part of their defences. "They'd come in tied up in manacles," Fallon said. "We'd have these SWAT teams all over the roofs of the medical school." Over the decades that followed, the school accumulated these brain scans. And as Fallon studied them, he was noticing patterns. Certain areas that light up in normal brains were dark. "So I said, my god, there's something here." He gave talks on his findings. Bizarre coincidences Meanwhile, two other events in his life were converging. "All this happened at the same time," he said. "It was very bizarre." Lizzie Borden was tried and acquitted for the murders of her father and stepmother in 1892, though she remains infamous. James Fallon discovered that he was distantly related to Borden. The first coincidence came when his mother told Fallon about a historical book on his father's family. "And there's all these nasty guys in there," he said. People who weren't so different from the murderers he'd been studying. Thomas Cornell Jr., an early colonial settler who was convicted and hanged for killing his mother, was a direct ancestor. And Lizzie Borden, who was famously tried for the axe murders of her father and stepmother in the late 19th century, was a distant cousin. The second coincidence came through Alzheimer's research Fallon was conducting. The team had completed brain scans of patients. But they needed a control group. So Fallon put his family members, including himself, under the machines. And as he was flipping through the pile of his family's scans, he saw one that looked identical to the killers he'd been studying. "I said, okay guys, really funny. Ha ha." He thought the lab technicians had played a joke on him, slipping a psychopath's scan into the pile with his family. They assured him that this was no joke. "I said, whoever this person is shouldn't be walking around in society." The psychopathic markers were all there. The parts of the brain that regulate conscience, emotional empathy and inhibition were turned off. "This is probably a very dangerous person." "Well, I peeled back the tape over the name, and there it was. It was my name." He laughed it off. He still didn't believe it. He'd never been a violent guy. He was married with kids. He had plenty of friends, and a successful career. But when he got home and told his wife about it, she said to him: "It doesn't surprise me." He came around to the idea gradually. "I just started asking everybody, what do you think of me? I started with my wife, my sister, my brothers, my parents. On and on. All the people close to me, including psychiatrists who I'd worked with for years who really knew me well. They all said – except for my mother, who said 'No, you're a nice boy' – everybody else said, we've been telling you for decades, for years, that you do psychopathic things." He'd been emotionally unavailable. Reckless. Manipulative. Getting by on charm and what he calls "cognitive empathy" – the ability to understand what others are feeling, without actually feeling it himself. Assessments by his colleagues were what really convinced him. His brain scans, genetic markers and behaviours all pointed toward borderline psychopathy. If a cold-blooded killer is formed through both nature and nurture, Fallon's nature suggested he was capable of terrible things. Perhaps a lack of childhood trauma had prevented him from acting on his violent instincts, he thought. 'What would a good guy do?' Fallon now describes himself as a "pro-social" psychopath. He's not out to prey on people. And his psychopathic tendencies are relatively benign. Driven by what he describes as ego, Fallon put a challenge to himself. To try pretending to be a nice, normal, emotionally connected guy. He'd start with his wife. "Every time something came up where I was interacting with her socially, I just asked myself, 'What would a good guy do?'" Whereas in the past he might have made up an excuse to, say, ditch her uncle's funeral and head down to the beach bar, he was now going to try doing right by her, despite his nature. When she caught on that a sudden kindness had come over him, he assured her, "Don't take it seriously. It's just an experiment." But nice is nice. She didn't seem to mind. "Strangers are very safe around me," he said. "It's when you get close to me that it's a little more dangerous, because I'm going to get you to do something you don't want to do. "So, I'm trying to control that. I figure if I tell everybody I have this, then I can't get away with anything any more." This story appears in the Out in the Open episode "Impostors".
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Summary: James Fallon is still trying to be a friendly psychopath-just ask his wife. "Every time something came up where I was interacting with her socially, I just asked myself, 'What would a good guy do?'" the psychiatry professor at University of California, Irvine, tells the CBC. But when she notices his good behavior, he tells her, "Don't take it seriously. It's just an experiment." Fallon famously made a discovery about his brain after getting a PET scanner in the late 1980s. Initially he used it to scan the brains of accused murderers before their defense trials ( "They'd come in tied up in manacles," says Fallon) and then noticed certain patterns: Areas that lit up in healthy brains were dark in the brains of accused killers. "So I said, my god, there's something here," he says. Then Fallon learned about "nasty" people in his own family, including a colonial settler convicted of killing his mother and distant cousin Lizzie Borden, who famously hacked up her stepmother and father in the late 1800s. And when Fallon scanned his own brain to help with a 2006 study, Vice reported, he saw clear psychopathic markers. "The parts of the brain that regulate conscience, emotional empathy and inhibition were turned off," Fallon says. "This is probably a very dangerous person." A non-violent, successful family man, Fallon laughed it off, but his wife and colleagues weren't surprised, describing him as emotionally distant, manipulative, and reckless-if often charming. Fallon says he's "still trying to control" the darker side, but "I figure if I tell everybody I have this, then I can't get away with anything more."
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Summarize: The fiancee of a man on trial for murdering a black teenager in cold blood has told how they went back to their hotel, ordered a pizza and drank rum and Cokes straight after the killing. Michael Dunn, 47, is accused of murdering Jordan Davis, a 17-year-old, after he refused to turn down loud rap music he and his friends were playing in his car parked outside a store in Jacksonville, Florida. Rhonda Rouer, who was inside the store when the shooting happened, said she heard shots and was immediately told to get in the car by Dunn, who drove them back to their hotel. Prosecutor John Guy said Dunn (left) fired at the SUV 'with malice in his heart and intent in his hand' while the defense said it happened because he felt in imminent danger. Right: Jordan Davis, a 17-year-old from Marietta, Georgia. The couple then ordered a meal, made drinks and went to bed. Dunn was arrested the following morning. The shooting - which nobody denies led to Davis's death - happened after a shouting match between Dunn and the group of teenagers in the car. Dunn is accused of firing 10 times into the SUV, including several times as the driver tried to get away. One of the shots killed Davis. No one else in the SUV was injured. Earlier in the trial, prosecutor John Guy said Dunn fired at the SUV 'with malice in his heart and intent in his hand' while the defense said it happened because he felt in imminent danger. Michael Dunn, 47, (left) is on trial for a second time, charged with first-degree murder in the November 2012 killing of Jordan Davis, a 17-year-old from Marietta, Georgia. He has already been convicted of three counts of attempted second-degree murder and is facing 60 years in prison. Dunn is facing a retrial on the first-degree murder count after the jury deadlocked at the previous trial in February. Giving her testimony, Rouer said that she was buying chips and wine in the convenience store when she 'heard a pop, pop, pop'. She said she didn't know what was going on until she went outside and her fiancee Michael Dunn urged her to get into the car, then drove off quickly, before she could fasten her seatbelt. State Attorney Angela Corey shows witness Steven Smith a hat and asked him if he ever saw it above the roof line of the car that Jordan Davis was in during the shooting. Rouer repeated testimony she gave in the first trial, saying the couple, who live together, went back to their hotel, ordered a pizza and drank rum and Cokes, then she fell asleep. The next morning when she woke up, the TV was on and she said she saw that Davis had died and she told Dunn she wanted to go home. They drove straight back to Satellite Beach, canceling plans to have lunch with Dunn's son. Dunn was arrested after they returned home that afternoon. The prosecution spent a third day laying out its case that Dunn acted in cold blood when he killed Davis, who was sitting in the rear passenger seat of an SUV next to Dunn's car. Parents: Prosecutor John Guy, right, shakes hands with Jordan Davis's father Ron before the start of the Michael Dunn retrial. Dunn, who had just left his son's wedding, asked Davis and the other teens in the car to turn down the music and a shouting match erupted between Dunn and Davis. The trial was halted briefly Saturday when a juror was dismissed. The dismissal followed a column posted online by Jacksonville alternative publication Folio Weekly that quoted a one-time prospective juror who was not picked after he said he had written for the publication. Richard David Smith III told the publication that the juror who was picked had questioned State Attorney Angela Corey's competence during jury selection and complained about her sense of humor. Judge Russell Healey, Corey and other attorneys involved in the case questioned the columnist and Smith. One of four alternate jurors will replace the dismissed juror
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Summary: Michael Dunn 47, accused of murdering Jordan Davis, 17, in cold blood. Fired his gun at a car Davis was riding in after an argument about rap music. Told Davis and his friends to turn it down outside store in Jacksonville, FL. His fiancee, Rhonda Rouer, told court she was inside the store at the time. Told how Dunn got her in the car, drove then drove off to their hotel. The couple then ordered pizza and drank rum and Coke then went to sleep.
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Summarize: Israeli soldiers conduct a search for suspects of a shooting attack yesterday in the West Bank City of Ramallah, Monday, Dec. 10, 2018. Israeli officials say seven people have been wounded, one critically,... (Associated Press) Israeli soldiers conduct a search for suspects of a shooting attack yesterday in the West Bank City of Ramallah, Monday, Dec. 10, 2018. Israeli officials say seven people have been wounded, one critically, in a shooting by a suspected Palestinian assailant outside a Jewish settlement of Ofra in the... (Associated Press) JERUSALEM (AP) — Israeli forces killed a Palestinian suspect wanted in a drive-by shooting at a West Bank bus stop, shooting him Wednesday just hours after an Israeli baby delivered prematurely as a result of the weekend attack died. The Shin Bet security agency said Salah Barghouti, 29, was killed while "attempting to harm" Israeli forces as he sought to escape from an arrest raid. It said an unspecified number of additional suspects were arrested. In Sunday night's attack, assailants in a Palestinian vehicle opened fire at a bus stop outside a West Bank settlement, wounding seven people before speeding away. Israeli forces have been on a manhunt for the attackers in the West Bank since then. Among the wounded was a pregnant woman who was critically injured. Her baby was delivered by cesarean section after the attack. On Wednesday, Jerusalem's Shaare Zedek Hospital said the boy had died. Speaking to foreign reporters Wednesday night, Israeli Prime Minister Benjamin Netanyahu condemned the attackers as "the most deviant criminals on Earth." "The security forces are pursuing them and I hope that there will be news soon on this matter," he said. Jerusalem (CNN) A baby born prematurely to an Israeli mother who was wounded in a drive-by shooting in the West Bank has died, a Jerusalem hospital said in a statement Wednesday evening. The four-day-old baby boy was delivered early at 30 weeks, just hours after the shooting at the entrance to the Ofra settlement that left seven people injured on Sunday. The baby's mother, Shira Ish-Ran, 21, was critically injured in the shooting but is slowly improving. "The murderers are abhorrent, the most deviant criminals on earth. The security forces are pursuing them and I hope that there will be news soon on this matter," said Prime Minister Benjamin Netanyahu at an event in Jerusalem on Wednesday evening. "We will not slacken until we find them and deal with them to the fullest extent of the law." According to the Israel Defense Forces (IDF), shots were fired from a passing car toward people standing at a bus stop at the entrance to the settlement. Soldiers nearby responded by firing toward the vehicle, which managed to get away, the IDF said. Israeli security forces have focused their efforts around the West Bank city of Ramallah as they look for the gunmen in the shooting.
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Summary: A drive-by shooting at a West Bank bus stop Sunday night led to the premature birth of a baby, who died Wednesday, hours before a Palestinian suspected in the attack was killed by Israeli forces. Salah Barghouti, 29, was allegedly among assailants in a vehicle that opened fire outside a West Bank settlement, wounding seven people including a woman who was 30 weeks pregnant and was critically injured, the AP reports. Doctors delivered her baby via cesarean section, but the boy died at four days old, CNN reports. His mother's condition is slowly improving. As for Barghouti, the Shin Bet security agency said he was killed while trying to escape from an arrest raid and "attempting to harm" Israeli forces in the process. An unspecified number of other suspects were arrested.
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Summarize: [0001] This invention relates to a folding table, portable, in particular a massage table. TECHNICAL OVERVIEW [0002] Folding tables are known in which the plate is made of two elements folding over each other and with retractable legs (U.S. 2006/260516). We also know of folding tables in which the plate is comprising one element with retractable legs folding in two parts but requiring time and many manipulations to set up, not allowing a construction sufficiently strong and light, for example, for a portable massage table. Finally we know of folding tables whose plate comprises more than two elements (WO 2007/065919), but their general structure does not allow to make a portable massage tables, light, robust and manageable. In the particular field of portable massage tables, so far, one knows of folding tables with two halves which, when folded, have a large volume, making them bulky and difficult to transport. None of these tables were until now sufficiently compact, robust, lightweight and easy to use. [0003] This invention proposes a folding table, which is at the same time easy to use, compact, lightweight and very robust. [0004] To this end, the folding table according to the invention is characterized in that it comprises an elongated plate consisting of at least four elements mounted on rigid frames articulated together perpendicularly to the long side of the plate, means for locking the table in the open position, said plate being connected with legs, each leg being formed of at least two parts articulated together, the first parts being mounted with articulations on a part fixed to the end elements, the length of each part being less than the length of said plate elements; braces, comprising two arms articulated together by a hinge, are articulated to the second part of each leg at each inner end of the end parts of the plate, locking means are used between the braces of each end element and/or the legs of each end element to prevent lateral movement of the table; there are tension cables, linking articulations between the arms of the braces to the point situated above the articulation of the legs on the parts attached to the frame of the end elements, and means for locking the articulation between the parts of the legs to hold the legs in the open position and to make the whole rigid, the whole being designed in such a way that the legs and the articulated arms of each half of the table, once folded, fold into the frames of the adjacent elements, and that all the elements when folded, are superimposed. [0005] Thus we obtain a table whose size when folded is reduced, for example for a massage table, whose installation is quick and easy, and also ensures high strength and stability, supporting high workloads with a very light construction. [0006] According to one embodiment, the table is characterized in that it comprises an elongated plate made of at least four elements mounted on rigid frames articulated together perpendicularly to the long sides of the plate means for locking the table in the open position, said plate having legs, each leg being made of at least two parts articulated together, the first parts being articulated on a part attached to the end elements, the length of each part being less than the length of said elements of the plate, bracing, comprising two arms articulated together are articulated to the second section of each leg and to each inner end of the end elements of the plate, locking means are used between the braces of each end element and/or the legs of each end element to prevent lateral movement of the table, and there are tension cables connecting the articulations between the arms of the braces to the point located above the articulations of the legs on the parts attached to the frame of the end elements and tension cables connecting the legs, at a point above the articulation between the leg to the arm, to the articulations of the bracing with the inner ends of the end parts of the plate to hold the legs in the open position and make the whole rigid, the whole being designed in such a way that the legs and the articulated braces of each half of the table, once folded, fold into the frames of the adjacent elements and that all the elements, once folded, are superimposed. [0007] According to one embodiment, the table is characterized in that in the folded position, the walls of the frames ( 2 a ) covers at least approximately completely the said parts of the legs and said folded braces. [0008] According to one embodiment, the table is characterized in that each leg comprises a third part sliding on the second part and there are means to fix the two parts together in a desired position for adjusting the height of the table. [0009] According to one embodiment, the table is characterized in that the two end elements of the plate are of a smaller width than the central elements of the plate in a way that in the folded state the lateral parts of the end elements of the plate are covered at least approximately by the parts of the frames of the central elements of the plate. [0010] According to one embodiment, the table is characterized in that supporting means of the central elements comprise surface elements on the adjacent parts of the frames between the end elements and the central elements such that the vertical pressure exerted on the central elements is transmitted to the end elements and there are similar supporting means between the central elements. [0011] According to one embodiment, the table is characterized in that the side parts of the frames of the central elements of the plate are articulated at a point and form, in the open state of the table, a rigid assembly maintaining the central elements of the plate aligned with each other when applying a force F 1 on the table. [0012] According to one embodiment, the table is characterized in that means for locking the table in the open position comprise tension cables fixed on one side to the articulations between the two articulated arms of the braces and on the other side to the articulation of the braces with the end elements of the opposite side of the table, or tension cables respectively fixed between the articulations of the braces of the end elements and between the articulations between the two articulated arms of the braces of the end elements, said cables being fixed together by a part. [0013] According to one embodiment, the table is characterized in that means for locking the table in the open position comprise tension cables attached between the articulations located between the two articulated arms of the braces and a point of the adjacent elements respectively. [0014] According to one embodiment, the table is characterized in that means for locking the table in the open position comprise tension cables fixed firstly to the articulations between the two articulated arms of the braces with the end elements of the opposite side of the table and fixed together to their intersection by a part, the part passing through an orifice of a central part fixed and articulated to a part attached to the adjacent parts of the central elements, or tension cables respectively fixed between the articulations of the braces with the end elements and between the articulations located between the two articulated arms of the braces of the end elements, said cables being fixed together by a part, the part passing through the orifice of the central part. [0015] According to one embodiment, the table is characterized in that means for locking the table in the open position comprise tension cables fixed on one side to the articulations between the two articulated arms of the braces and on the other side to the articulations of the braces with the end elements of the opposite side of the table, the cables passing through an orifice of a central part fixed and articulated to a part attached to the adjacent parts of the central elements, or tension cables fixed respectively between the articulations of the braces with the end elements and between the articulations between the two articulated arms of the braces of the end elements, said cables being secured together by a part, the part passing through the orifice of the central part and a cable fixed between the articulations of the braces with the end elements and passing through an orifice situated on the central part. [0016] According to one embodiment, the table is characterized in that the attachment point of the tension cables and to the articulations of the braces to the end elements is removed to the point located above the articulations of the legs on a part of the frames of the end elements said cables passing through a fixed point of the articulation of the braces with the end elements. DESCRIPTION [0017] The invention will now be described in detail with the following description that refer to the accompanying drawings in which: [0018] FIG. 1 is an elevation view of one embodiment of the table in the unfolded state. [0019] FIG. 2 shows a view of the table in an intermediate folding state. [0020] FIG. 3 is a bottom view of an end element of the plate with the legs folded inside. [0021] FIG. 4 shows the folding step in which the two end elements are folded over the median elements. [0022] FIG. 5 is an elevation view of the table when closed. [0023] FIG. 1 b is an elevation view of the table according to a first embodiment. [0024] FIG. 6 is an elevation view of the table in the unfolded position of a second embodiment. [0025] FIG. 7 is an elevation view of the table shown in the intermediate position of folding according to the second embodiment. [0026] FIG. 8 is an elevation view of the table in the unfolded position in a third embodiment. [0027] FIG. 9 is an elevation view of the table shown in the intermediate position of folding according to the third embodiment. [0028] FIG. 10 shows the stage of folding according to the second and third alternative embodiments in which the two end elements are folded over the median elements. [0029] FIG. 11 is an elevation view of the table closed according to the second and the third embodiments. [0030] FIG. 12 shows a detail of the table illustrating one way to secure the four elements of the plate between them. [0031] According to the first embodiment illustrated in FIGS. 1 to 5, the massage table consists of a rectangular plate 1 made of four elements approximately of the same length, 1 a, 1 b, 1 c, 1 d, the four elements being articulated to each other with articulations 14, 20 c, the axis of which is perpendicular to the long side of the plate 1. [0032] Each element consists of a rigid frame 2 a, covered by a plate 2 b, its self covered with foam 2 c and upholstered. The two central elements 1 b, 1 c can be substantially wider than the end elements 1 a, 1 d. The parts 20 a, 20 b of the frames of the elements 1 b, 1 c are wider than the other parts of the frame so that the elements 1 a, 1 d can fold inside the elements 1 b, respectively 1 c, without projecting beyond these frames at these places ( FIG. 4 ), they allow to maintain the central elements 1 b, 1 c aligned, these elements are substantially wider than the end elements 1 a, 1 d as shown in FIGS. 1, 2, 5 and 11 in order to fold the table. [0033] Adjacent parts of the frames 2 a of the central elements 1 b, 1 c can be wider in order to increase the resistance of the centre of the table. In this case the central elements 1 b, 1 c are slightly longer than the end elements 1 a, 1 d in order to fold the table. [0034] At the four corners of the plate 1 are mounted the legs 3 each consisting of three parts. The first part 3 a is articulated at the point b by an articulation on a part B fixed to the free end 2 a of the frames of the end elements 1 a, 1 d. This first part 3 a is extended by a second part 3 b. This second part, connected to the first by an articulation 6 articulated at 6 c has longitudinal ribs. The third lower part 3 c of the leg has ribs corresponding to the ribs of the second part 3 b and can slide thereon without lateral play, making it possible to adjust the height of the table. There are a series of holes 3 x evenly distributed over the third part 3 c which can coincide with a single opening of the second part 3 b, fixation between the two parts can be made, for example, with a screw 30 passing through the two holes and a wing-nut, or similar, tightening the whole. [0035] Alternatively, if one renounces to the height adjustment, legs can comprise only two parts articulated together. [0036] On each side of the table there are two articulated braces, each one comprising several articulated arms, in the example shown there are two arms 7, 8 articulated in 30, 31, 32 connecting the inner part 2 a in 32 of the end element 1 a, respectively 1 d to the second part 3 b of the corresponding leg 3. There is also a mean, such as a part 9 connecting the articulated arms 7 and a part 10 connecting the articulated arms 8 associated with the element 1 a, respectively 1 d, making the whole rigid and preventing lateral movement of the table in its open position. [0037] The stability of the table in the open position is ensured by a system of cables C 1, C 2, C 3, exerting reverse tensions to the load distributed on the table on the different parts of the structure. A cable C 1 is attached between the point b 1 located above the leg 3 on a part B fixed to the frame 2 a, and the articulation 31 between the articulated arms 7 and 8 of the braces. A cable C 3 connects this same articulation 31 to the articulation 32 of the opposite end element. Finally, to lock the articulation between the two parts 3 a, 3 b of the legs 3 a cable C 2 connects the leg 3 at a point situated above the point 30 of articulation of the leg 3 to the brace 7, 8, to the articulation 32 of the same end element 1 a, respectively 1 d. The cable C 2 can be replaced by a hinge or self-locking hinge or a system to maintain aligned the two parts 3 a, 3 b of the legs 3. To improve the locking in the open position of the articulation 6 the cable C 2 can be attached to a point 6 b of the first part 3 a of the leg 3 and slide on a part 6 a of the second part 6 b of the foot 3, and so exert a force F 2 on the articulation 6. The elements 6 a and 6 b being offset from the articulation point 6 c of the articulation 6 the force F 2 will have the effect of holding the elements 3 a and 3 b aligned. [0038] The same cable system is planned for the other legs. The lengths of these cables are adjusted so that they are tensioned in the open position of the table. [0039] The end elements 1 a, 1 d of the plate 1 are kept aligned to their adjacent elements 1 b, 1 c by the two sets of triangulation made by the cables C 1 and C 3 : b 1, 31, 32 on one hand and 32, 31, 32 on the other hand. The two central elements 1 b, 1 c of the plate 1 are kept aligned together by the parts 20 a and 20 b of their frames, articulated in 20 c. The same device is provided for the opposite sides of the frames ( FIG. 1 ). Thus the weight of the patient on the table will result to tension the cables C 3 and C 1 which will exert a force opposite to the weight on the arm 8, involving triangulation b 1, 31, 32, 32, 31, 32. [0040] In the open position of the table, to stabilize the position of the legs, it suffices to exert a pressure on the articulation 31 in the direction of the arrow F 3, the braces 7 and 8 forming between them an obtuse angle. This pressure in 31 will cause tension to the cables C 1 and C 2 by the triangular forces and thus maintain aligned the elements 3 a and 3 b of the legs 3 perpendicular to the plate to make the whole rigid. To close the table, a pressure is exerted on the articulation 31 in the opposite direction of the arrow F 3, which will release the cable tension C 1 and C 2. Once the cables are not under tension anymore, the articulated braces 7 and 8 can be folded in the frame of the element 1 a, respectively 1 d, as illustrated in FIGS. 2 and 3. Once the legs are retracted into the elements 1 a and 1 d, the elements 1 a, respectively 1 c can rotate a 180° on the articulations 14. Then the two pairs of elements rotate 90° on the articulation 20 c ( FIGS. 4 and 5 ). In this position the four elements 1 a, 1 b, 1 c and 1 d are superimposed on each other and the side walls of the frames 2 a cover at least approximately completely the legs and braces folded, forming a compact structure not too bulky. [0041] Since the end elements 1 a and 1 d are bearing the central elements 1 b and 1 c, in order to relieve the pressure on the articulation 14, there are additional blocking means Z, for example mortise or tenon, to secure in the open position the central elements 1 b, 1 c, with bearing end elements 1 a, 1 d, as shown in FIGS. 11 and 12. The same locking means may also be used between the central elements 1 b, 1 c for rigidifying the central part of the table and also to allow the second and third embodiments. [0042] As shown in FIG. 1, parts 2 a of the frames, at both ends of the table, may be wider on a portion of their length and have orifices 13 allowing for example the use of a face cradle. [0043] A part S with a handle P can be fixed between the adjacent parts of the frames 1 b and 1 c ( FIG. 5 ). [0044] According to a first alternative embodiment shown in FIG. 1 b a cable C 3 replacing the cable C 30 can be attached between the articulation 31 of the arms of the braces and a point 40 of the Central elements 1 b, 1 c. [0045] According to a second embodiment ( FIGS. 6 and 7 ) the cables C 4 and C 5 can replace the cables C 3, the four elements ( 1 a, 1 b, 1 c, 1 d ) are all of the same width or approximately the same width and the parts 20 a and 20 b of the central elements are eliminated. Elements 0 of adjacent parts of the central elements 1 b, 1 c make the frames broader in some places and are connected by hinges 14 in order to replace the articulation 20 c. Parts M are fixed in N and articulated in n 1 on the adjacent inner parts of the central frames 1 b, 1 c and fold into the table, in the open position of the table part M can pivot on its frame to reach the centre of the table at the junction of the cables C 4 and C 5 or remain upright, which implicates to slightly remove the junction point of the cables C 4 and C 5. The cable C 4 is attached to the articulation 32 of the end elements 1 a to 1 d, the cable C 5 is attached to the articulation 31 of the end elements 1 a to 1 d, these two cables pass through the orifice m 1 in parts M, they are fastened together by a part X 1, there are means for locking the part M so that it can not go beyond the point X 1 when it unfolds: for example, the part X 1 is equipped with a larger part that can not pass through the orifice m 1, or the orifice m 1 is smaller at one of its ends in order that the part X 1, used to fixe the cables C 5 and C 4, can not cross it entirely. So the central elements 1 b and 1 c of the frame are kept aligned by the cable C 4 exerting a force on the part M, vertical and opposite to the load located on the table and the end elements 1 a, 1 d are kept aligned by the cables C 5 and C 1 exerting a pressure on the brace 8 vertical and opposite to the charge located on the table. As illustrated in FIG. 7, the cables C 4 and C 5 can slide in m 1 to let the part M fold into the table. There are also locking means Z in order to distribute the vertical load on the central adjacent elements 1 a, 1 c. If the cables C 3 are not replaced by the cables C 4 and C 5, they are fixed at their intersection, or near their intersection, by a part X 1 passing through the orifice ml and will have the same function as the cables C 4 and C 5, C 4 or C 3 cables can be fixed to the top of the legs at points b 1 and pass through a loop or a similar item attached to the articulation points 32 of the elements 1 a and 1 d. [0046] In a third alternative embodiment ( FIG. 8 ) cables C 3 are kept, they pass through an orifice m 1 of the part M that can be longer and have a second orifice m 2. A cable C 6 attached to the articulation 32 of the elements 1 a to 1 d passes through the point m 1, or to relieve the cable, through the point m 2. C 6 cable can also be attached in b 1 and pass through the articulation 32 or a part attached to the articulation 32. Cable C 6 has the effect of maintaining the central elements 1 b and 1 c aligned. If the part M is fixed to the element 1 c, the cable C 3, attached to the articulation 31 of the bracing of the element 1 a, may be fixed in m 1, if the part M is fixed to the element 1 b the cable C 3 attached to the articulation 31 of the element 1 d will be fixed in m 1, this in order to unfold automatically the part M if it folds in the longitudinal direction of the table, and to fix it when unfolding the table, there is also the same locking means Z between the central elements 1 b, 1 c and also the same articulation system as in the second embodiment attached to the elements O of the adjacent parts of the central frames. In order to fold the part M in its frame, the cables C 3 remained free and C 6 slide through the openings m 1 and m 2 ( FIG. 9 ). The cables C 3 can be replaced by the cables C 4 and C 5 arranged in the same way as in the second embodiment, the cables C 4 can also be attached to the top of the legs at the points b 1 and go through a loop or similar part set at the articulations 32 of the elements 1 a and 1 d.
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Summary: This invention relates to a folding table, portable, in particular a massage table, comprising a plate ( 1 ) made up of at least four elements ( 1 a, 1 b, 1 c, 1 d ) mounted on rigid articulated frames ( 2 a ), means for locking the table in the open position, said plate having legs ( 3 ) comprising at least two parts ( 3 a, 3 b ) articulated together, the first parts ( 3 a ) being articulated to the end elements ( 1 a, 1 d ), braces, comprising two arms ( 7, 8 ) articulated together, are articulated to the end elements ( 1 a, 1 d ), locking means ( 9, 10 ) are used between the arms ( 7, 8 ) and/or the legs ( 3 ). There are cables (C 1 ) for connecting the articulations ( 31 ) of said arms ( 7, 8 ) above (b 1 ) the articulations (b) of the legs ( 3 ) to the end elements ( 1 a, 1 d ), and means for locking the articulation ( 6 ) between the parts ( 3 a ) and ( 3 b ) of the legs to keep the legs erect and to make the whole rigid, the whole being designed in such a way that the articulated legs and arms of each half of the table, once folded, fold into the frames ( 2 a ).
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Write a title and summarize: Cytoplasmic dynein is a giant ATP-driven molecular motor that proceeds to the minus end of the microtubule (MT). Dynein hydrolyzes ATP in a ring-like structure, containing 6 AAA+ (ATPases associated with diverse cellular activities) modules, which is ~15 nm away from the MT binding domain (MTBD). This architecture implies that long-distance allosteric couplings exist between the AAA+ ring and the MTBD in order for dynein to move on the MT, although little is known about the mechanisms involved. Here, we have performed comprehensive molecular simulations of the dynein motor domain based on pre- and post- power-stroke structural information and in doing so we address the allosteric conformational changes that occur during the power-stroke and recovery-stroke processes. In the power-stroke process, the N-terminal linker movement was the prerequisite to the nucleotide-dependent AAA1 transition, from which a transition cascade propagated, on average, in a circular manner on the AAA+ ring until it reached the AAA6/C-terminal module. The recovery-stroke process was initiated by the transition of the AAA6/C-terminal, from which the transition cascade split into the two directions of the AAA+ ring, occurring both clockwise and anti-clockwise. In both processes, the MTBD conformational change was regulated by the AAA4 module and the AAA5/Strut module. Dynein is an ATP-hydrolysis driven molecular motor that linearly proceeds along the microtubule (MT) towards its minus end [1–3]. Cytoplasmic dynein is involved in the transport of many different types of cargo such as mRNAs, proteins, and vesicles [4,5] and is also involved in mitosis [6,7]. Of the two forms, cytoplasmic dynein-1 is the major form, and is the focus of this work. Unless otherwise denoted, we simply denote cytoplasmic dynein-1 as “dynein”. Mutations in dynein are associated with various diseases such as amyotrophic lateral sclerosis (ALS) and cancer [8,9]. The motility of dynein is often compared with that of kinesin, a better-understood major MT-based molecular motor. While most kinesin moves towards the plus end of the MT, dynein moves towards its minus end. Most kinesin molecules do not move backwards, but it is known that yeast dynein, for example, moves stochastically with about 20% molecules moving backward [10–12]. The step size is always 8 nm, the structural unit of MT [13], for kinesin but varies in the range of 8–32 nm for dynein [10–12,14]. The homo-dimeric kinesin is known to move in a hand-over-hand manner [15]: Starting from the two-head bound form, each step involves detachment of the rear kinesin head from the MT, a forward move of the detached head, and its re-binding to the MT ahead of the other head. Steps are realized alternately by the two kinesin heads. Even though dynein also forms a homo-dimer, it does not exhibit this clear hand-over-hand movement[11]; of the two molecules, the same dynein molecule can move successively, which is not consistent with the hand-over-hand mechanism [10]. When one dynein subunit is mutated to inhibit the ATP hydrolysis reaction [10], even as dramatically as removing the ATPase module [16], the dimeric dynein still moves although with a modestly affected velocity. In contrast, kinesin motility is severely affected when one head is inactivated [17]. Although these facts exemplify the sharp differences in the motility of dynein from that of kinesin, the molecular mechanisms underlying dynein motility remain obscure. Cytoplasmic dynein is a giant protein complex containing two heavy chains, two intermediate chains, two light-intermediate chains, and six light chains. The C-terminal fragment of the heavy chain contains the motor domain comprising of an ATPase module and a MT-binding domain (MTBD). The overall architecture of the motor domain consists of a hexagonal AAA+ (ATPases associated with diverse cellular activities) ring that hydrolyzes ATP [18], the MTBD, and the Stalk which connects the AAA+ ring and the MTBD [19,20] (Fig 1A). Remarkably, the AAA+ ring is ~15 nm away from the MTBD. The affinity of the MTBD to MT is regulated via a nucleotide-dependent structural change in the AAA+ ring, suggesting long-distance allosteric couplings between the two elements. Understanding the mechanisms behind this long-range coupling was the focus in this study. Notably, this is a distinct structural feature, which differentiates dynein from kinesin where the core module hydrolyzes ATP and binds MT, directly coupling them in the same module. The landmark X-ray crystallographic structure of Dictyostelium discoideum dynein-1 revealed a high-resolution structure of the full-length motor domain [21] (Fig 1A, right). The motor domain can be divided into eight distinct modules; six AAA+ modules making the hexagonal AAA+ ring (termed, in order of amino acid sequence, AAA1, AAA2, AAA3, AAA4, AAA5, and AAA6), the N-terminal linker which is docked on the planar surface of the AAA+ ring, and the Stalk/MTBD which protrudes from the AAA+ ring. At the C-terminal side of the AAA6, there is a C-terminal end segment, which is grouped with the AAA6 in this study. We refer to this module as the AAA6/C-terminal module. It should also be noted that the MTBD and the Stalk are inserted within the sequence of the AAA4 module. For the purposes of this study, we treated the MTBD and the Stalk together as a single module. At the junction between the AAA+ ring and the Stalk, a small element called the Strut/Buttress seems to augment/regulate the connection (Fig 1A). The Strut/Buttress sequence is embedded in the AAA5 sequence and thus is included in the AAA5 module in this study. Notably, even though the crystallographic diffraction indicates high resolution, the electron density near the MTBD is vague and the structural model for the MTBD region has low resolution. Only a combination of electron microscopy and computational modeling has provided a high resolution structural model of the MTBD with a high affinity for MT [22]. For the dynein motor function, nucleotide-dependent conformational changes have been partially characterized. Comparison of the above mentioned structure by Kon et al [21] with a recent crystallographic structure for human cytoplasmic dynein-2 by Schimidt et al [23] (Fig 1, left) reveals many structural changes, one of which is termed the “power-stroke” motion. In the pre-power-stroke state solved by Schmidt et al, the linker assumes a sharply bent form with its N-terminal tip approaching the AAA2 (Fig 1A left). Whereas in the post-power-stroke state solved by Kon et al, when looked from the side of the ring, the linker looks nearly straight so that its N-terminal tip extends to the AAA4 and AAA5 modules (Fig 1A right). The ATP- and ADP + Pi bound states are considered to form the pre-power-stroke state, while the ADP-bound and apo (nucleotide-free) states correspond to the post-power-stroke state [21,24–27]. Thus, the Pi-release event corresponds to the pre-to-post-power-stroke motions, while ATP binding couples the post-to-pre recovery-stroke. Among the six AAA+ modules, the first four AAA+ modules possess ATP binding sites, in particular the AAA1 is known to play a major catalytic role in ATP-driven movement; a mutation that abolishes ATP hydrolysis in the AAA1 causes a complete loss of dynein motility [28,29]. The other ATP-binding modules appear to have regulatory roles [30–33]. The affinity of the MTBD for MT is regulated by a nucleotide-dependent conformational change. Generally, it is thought that the pre-power-stroke state corresponds to a weak-binding state of the MTBD, while the post-power-stroke state corresponds to a strong-binding state of the MTBD [34]. However, the mechanisms underlying this are unclear. For allosteric communication between the AAA+ ring and the MTBD, the Strut and the Stalk structures are considered to be important elements. The Strut move may be transmitted via the Stalk, which may then serve to change the structure of the MTBD [35]. In this study, we address allosteric couplings within the full-length motor domain of dynein-1, using a molecular simulation approach. Previously, molecular dynamics (MD) simulations have been used for dynein in structural modeling, fluctuation analysis, and a study of the interactions with the MT [22,36–39]. While highly desirable, the gold-standard atomistic MD simulation cannot be directly used at the moment for the current purpose due to the large size of the molecules and the high degree structural changes. To this end, we employed a coarse-grained MD approach where each amino acid is represented by one bead. For the last two decades, such coarse-grained MD approaches have been successfully employed to study a broad range of large-scale protein dynamics, such as folding, binding, and motor motions [40–45]. Notably, using the software CafeMol [46], we can utilize atomistic interaction information present in the reference X-ray structures even though we performed coarse-grained MD simulations. Using high efficiency computation, we conducted comprehensive simulations for both the pre-to-post-power-stroke transition and the post-to-pre recovery-stroke transition multiple-times (10–30 times per one setup) with multiple setups (35 setups in total). Assuming that the full-length motor domain is made of eight modules, we investigated the sequence of transitions in these eight modules, from which we reveal the allosteric couplings between these modules. To simulate large-scale conformational changes in dynein in a systematic manner, we employed a structure-based coarse-grained model where each amino acid is represented by a single bead located at the Cα atom. Starting with the atomistic models for the two reference structures, i. e. the pre- and post- power-stroke structures of dynein, we first estimated approximate pairwise residue interaction energies at atomic resolution. Feeding these energy values into the coarse-grained model, we set up an atomic interaction-based coarse-grained model (AICG2+ model) for both the pre- and post- power-stroke states [47,48]. Finally, integrating the AICG2+ model for the two-states, we constructed the multiple-basin model that empirically connects the two energy basins smoothly [49–51] (See also S1A Fig). Specifically, for dynein, in constructing the multiple-basin model, we assumed that conformational change can occur module by module. We assigned eight modules within the dynein full-length motor domain: (from N-terminal to C-terminal) the N-terminal linker, the AAA1, the AAA2, the AAA3, the AAA4, the MTBD together with the stalk region, the AAA5 including the Strut, and the AAA6-C-terminal module (Notably, since the MTBD is inserted within the AAA4 module, the AAA4 module contains two disconnected segments). For each module, together with its interactions to other modules, we set up one double-basin sub-system that connects the pre- and post- conformations (S1 Table and Fig 1B). Neighboring sub-systems interact with each other via module-interface interactions. Thus, in the simulation of the entire molecule, we observed sequential transitions for eight sub-systems, from which we analyzed conformational change pathways among, in principle, 8! conceivable orders of transitions. We noted that, since the multiple-basin model is inherently empirical, we needed to introduce two parameters, ΔV for modulating the relative stability of the two states (pre- and post-) and Δ for deciding the height of the energy barrier between the two states for each module (S1A Fig). Through many preliminary simulations, we empirically tuned these parameters (See Method for details). Briefly, Δ’s were set so that the corresponding conformational transition occurs, on average, in the middle of the possible simulation time window. In contrast, ΔV‘s were tuned separately for power-stroke and recovery-stroke transitions so that we could observe the conformational transitions from the initial state to the target state. Although the tuned parameters were far from unique, we found that the allowed parameter ranges were rather narrow. We defined one set of these parameters that satisfied all the requirements as the standard set and these are listed in S2 and S3 Tables. Since the parameter choice was not unique, we varied ΔV values systematically (35 setups in total) repeating numerous simulations (10–30 trajectories for each setup), from which we sought the robust responses. We did not systematically change Δ value, because, by decreasing Δ significantly, we see virtually no structural change and, by increasing Δ, we often see too-frequent transitions or even appearance of an artificial minimum between the pre- and post- structures. First, using the standard set of parameters, we performed molecular simulations of dynein from the pre-power-stroke state to the post-power-stroke state 30 times with different samples of stochastic forces. Fig 2A illustrates a typical trajectory of the reaction coordinates χ of all modules, which assume negative and positive values at the initial and final states, respectively (see METHODS for the definition of χ. See also S1 Movie). The trajectory in Fig 2A showed a representative trend in the order of transitions, although the order was to some extent stochastic in other trajectories. In Fig 2A, the AAA3 made the pre-to-post-transition first, which is followed by a change in the linker conformation. The linker conformational change triggers conformational changes in the AAA1 and subsequently in the AAA2. Following this, the transition cascade continues, in the order AAA4, MTBD, AAA5, and finally the AAA6/C-terminal module. Thus, with the exception of the first AAA3 transition, the order of change was sequential in sequence and occurred in a clockwise manner from a structural point of viewed from the side of the linker. To statistically account for the order of the transitions of the modules, we calculated the probabilities, P-order (b ← a), that the transition in module “a” precedes that in “b” in 30 trajectory samples and the results are shown in Fig 2B (we refer to this map as an “order-map”). It should be noted that, when we observe multiple transitions between the pre and post states in a single trajectory, we use the time of the first transition for this analysis. In the figure, the dark square indicates that the module in the horizontal axis precedes the module in the vertical axis with a high probability (the p-value for the null hypothesis of the indistinguishable order of each pair of transitions is given in S2 Fig). For example, in the “linker” column, all rows except for AAA3 are dark, indicating that the linker conformational change occurs after AAA3, on average, and is before those of the other modules. The p-value for this particular order was 0. 002 including the multiple testing correction so that we can clearly state that the AAA3 changes earlier than linker followed by the other transitions with statistical significance. Indeed, the order of transitions observed in Fig 2A was a representative case. Generally, when a transition in one module occurs before a transition in another module, there can be two underlying mechanisms. In one, the two transitions are independent, but the characteristic time in one is shorter than that in the other (this is termed an “independent pair”). In the other mechanism, the transition in the first one is obligatory for the second transition (this is termed a “dependent pair”). We distinguished the two cases by the following two analyses. First, to infer whether a pair of transitions is independent or dependent, we calculated the average pair rate constants k (b|a), for the transition in “b” after the transition of “a”, defined as k (b|a) = (1/n) ∑i=1n1/Δtab (i) where the summation is taken over the trajectories in which “b” transited later than “a”. Δtab (i) is the time interval from the transition in a to that in b in the i-th trajectory, and n is the number of the corresponding trajectories. When transition “b” did not occur, we set the time of the “b” transition as the final time of the simulation. Fig 2C shows the pairwise rates k (b|a) for the standard set, in which the value of k (b|a) is represented by the darkness (empty if n < 7). Thus, the dark squares in the plot suggest that these pairs are “dependent pairs”. Additionally, we also performed bootstrapping (S3 Fig). These dependent pairs are discussed in more detail in subsequent sub-sections. As an additional analysis of the dependence, we consider a hypothetical and perfectly-independent pair of systems, A and B, both of which make transitions with single-exponential time courses. The rate constants for A and B were set as those obtained from the above MD simulations. Then, for the perfectly-independent system, we calculated the analytical probability density function of the difference in the time of transitions of the two modules A and B (“null” hypothesis). This distribution can directly be compared with the time difference histogram obtained from the actual MD simulations (S4 Fig). The difference between the hypothetical probability distribution (“null” hypothesis) and the observed histogram was assessed by Kolmogorov-Smirnov test (S4 Table). By sorting the eight modules in descending order of Pi = Σj P-order (j ← i) we obtained an average order of transitions, as shown in Fig 2D. Notably, for many pairs of modules, the orders of transitions were fully deterministic (i. e. ; the probabilities were zero or one), whereas, for some pairs, the orders of transitions were stochastic. Moreover, some orders of transitions depended heavily on the parameters ΔV and Δ. Hereafter, we focused on “robust” orders of transitions that were found in the majority of trajectories and were not sensitive to changes in the parameters. In the standard setup, the linker transition, i. e., the linker power-stroke, occurred before the AAA1 transition with a probability of one (P-order (AAA1 ← linker) = 1. 0). In most trajectories, the AAA1 transition occurred immediately after the conformational change in the linker: The pairwise rate k (AAA1|linker) = 0. 093 (95% confidence interval [0. 065,0. 13], S3A Fig) (hereafter, the rate is given in the unit 1/104 MD steps) is large, as shown in the map in Fig 2C. Importantly, the transition in the AAA1 is the nucleotide-dependent process corresponding to the release of phosphate from the AAA1 module in the power-stroke process. The current result that the linker movement is a prerequisite for the AAA1 transition implies that the transition in the AAA1 module needs to be unlocked by the thermally-activated spontaneous linker movement. To test the mechanical dependence of the two transitions, we examined the following two cases. First, we examined the effect of retarding the linker conformational change (denoted as “linker ↓”). The retarding effect can easily be introduced by increasing the ΔV value for the corresponding module (S2 Table). By introducing this change, we did indeed observe a delay in the timing at which the linker made its conformational change. Importantly, we found that AAA1 did not make its transition before the linker conformational change and this was true in all trajectories examined (P-order (AAA1 ← linker) = 1. 0) (Refer to setup 2 in S5 Fig which shows all of order map data obtained from power-stroke simulations). Second, we examined the effect of accelerating the AAA1 conformational change (denoted as “AAA1 ↑" ). Under this condition, we did not observe a significant change in the order of transitions; the linker conformational change almost always preceded the AAA1 transition (P-order (AAA1 ← linker) = 0. 8, while P-order (linker ← AAA1) = 0. 1) (see Fig 3A for a representative trajectory). We also performed another statistical test supporting these dependences (S4A Fig, S4 Table). Taken together, these data suggest that the conformational transition in AAA1 is dependent on the linker being in the post-stroke conformation. Fig 3B depicts the major interaction interface between the linker (purple) and the AAA1 module (blue) in the pre-stroke state (left) and in an intermediate snapshot (after the transition of the linker) (right). The figure also include some important residue-residue interaction, selected based on its large change between the pre- and post- structures, primarily electrostatic interactions, and relatively high sequence conservation (a multiple sequence alignment given in S6A Fig). In the pre-stroke state, a major interaction occurs between E1849 in the linker H17 helix and Q2116 in the loop between the H5 and H6 helices of AAA1. This interaction is lost in the intermediate and the post-stroke states. Instead, a contact between D1861 in H17 in the linker and Q2116 is observed in the intermediate and the post-stroke states. The dissociation of E1849 from Q2116 may be responsible for unlocking the conformational transition in the AAA1. We did not observe any marked structural changes in the N-terminal loop of AAA1, which connects the AAA1 with the linker. Technically, since we included the interaction energies between the linker and AAA1 into the linker multiple-basin system (S1 Table), this could, in principle, have affected the order of transitions. To check this possibility, we performed a control set of simulations in which the interaction between the linker and AAA1 is included into the AAA1 multiple-basin system. The result shown in S1C Fig is essentially unchanged from the standard setup. Thus, the result is not affected by the particular simulation setting. In all the trajectories in the standard setup, the AAA4 module state transition occurred after the AAA3 module conformational change (Fig 2B, P-order (AAA4 ← AAA3) = 1. 0). To test the robustness of this order of events, we examined the effect of retarding the AAA3 conformational change (referred to as" AAA3 ↓" ). The results demonstrated that the AAA3 module still changed its conformation before the AAA4 module changed its (P-order (AAA4 ← AAA3) = 0. 9 and P-order (AAA3 ← AAA4) = 0. 0). Next, we examined the effect of accelerating the AAA4 conformational change (referred to as" AAA4 ↑" ). Even in this case, the AAA3 made the transition earlier than the AAA4 with the probability one. However, the pair rates were modest in both directions. In the setup, AAA3 ↓, k (AAA4|AAA3) = 0. 019 (see setup 6 in S7 Fig), while in the setup AAA4 ↑, k (AAA4|AAA3) = 0. 047 (in comparison the fast response k (AAA1|linker) = 0. 093 in the standard setup (95% confidence interval [0. 0060,0. 011], S3B Fig) ). Thus, the allosteric coupling between the two transitions appears to be modest. Taken together, these data suggest that the conformational transition in the AAA3 promotes the conformational change in the AAA4, although the coupling is not as strong as that between the linker and the AAA1 (S4B Fig, S4 Table). We note that the summation of P-order does not always equal to 1 due to the trajectory where one of the two modules did not make transition at all until the end. Transitions in the AAA2 were uncoupled from other transitions in the current simulations. By retarding or accelerating the AAA2 transition, we observed the corresponding changes in the order of AAA2 transition and noted that these change did not have a clear effect on other transitions. These data suggest that the AAA2 transition does not play a crucial role in the power-stroke motion, which is in agreement with the experimental result that inhibition of ATP/ADP binding to the AAA2 module does not affect the motility of dynein [31]. From both functional and structural perspectives, the most important allosteric coupling must be the conformational transition order between the MTBD and the AAA+ ring, i. e., which occurs earlier. In most trajectories, the MTBD stayed in the pre-stroke state before the AAA4 transition, and then showed stochastic and reversible transitions between the pre- and post- stroke states after the AAA4 module transition (see for example Fig 2A). When the AAA5 module transitioned to its post-stroke state, the MTBD conformation was fixed in its post-stroke state. To demonstrate this behavior systematically, we used a scaled time, (scaled-time = (MDstep − τAAA4) / (τAAA5 − τAAA4), where τAAA4 and τAAA5 are the MD time steps of the AAA4 and AAA5 module transitions in each trajectory, respectively). The χ value for the MTBD was plotted against scaled-time for three additional trajectories in Fig 3C. These data suggest that the MTBD (including the Stalk) transition depends on both the AAA4 and AAA5 states. Naturally, this might be expected since the Stalk is inserted in the AAA4, and the Strut in the AAA5 interacts with the Stalk, thereby moving the Stalk (referred to as the ‘open-zipper’ model, see also Fig 1A). We also plotted the χ value for the MTBD averaged over 30 trajectories against scaled time in Fig 3D, showing that χ started to increase upon the AAA4 transition and peaking after the AAA5 transition. These data imply that the AAA5 module has a somewhat larger effect on the MTBD conformation than the AAA4 module. In the majority of trajectories, the last part of the transition cascade consisted of the transition of the AAA5 followed by that of the AAA6/C-terminal module. This final pair of steps was very robust. Even when a retardation in the AAA5 transition was introduced (referred to as" AAA5 ↓" ) ), the order of transitions did not change (P-order (AAA6 ← AAA5) = 0. 5 and P-order (AAA5 ← AAA6) = 0. 0). Moreover, even when we introduced an acceleration of the AAA6/C-terminal module transition (referred to as “AAA6 ↑" ), the order of the two events was completely unaffected (P-order (AAA6 ← AAA5) = 1. 0, P-order (AAA5 ← AAA6) = 0. 0). Next, we investigated the pathways for the recovery-stroke from the post- to pre-power-stroke states using the standard parameter set for this process. Fig 4A depicts a representative trajectory, where the transition occurred in the order of the AAA6/C-terminal module, the AAA1, the AAA5, the MTBD, the AAA2, the linker region, the AAA3, and finally the AAA4 (S2 Movie). As the average over 30 trajectories, the order-map for this recovery processes (see Fig 4B for the original index order and Fig 4D for the optimally sorted module order) supports the same order of transitions, on average, as those in Fig 4A. Structurally, this order of transitions contains two parallel cascades, both initiated from the AAA6/C-terminal module. In one pathway, the transition in the AAA6/C-terminal module propagated to that in the AAA5 and then to the MTBD (anti-clockwise in the viewing angle of Fig 1). In the other pathway that proceeds in the opposite orientation, the transition in the AAA6/C-terminal module propagated to transitions in the AAA1, the AAA2, the linker, the AAA3, and finally the AAA4 (clockwise). Of note, the AAA2 module is markedly independent of the other modules, meaning that the AAA2 can make transitions largely independent of other structural changes. The rate map in Fig 4C points out a strong dependency; namely that the AAA6/C-terminal module transition is immediately followed by the AAA1 transition. In nearly all of the trajectories in the recovery-stroke simulations, we observed that the conformational change cascade is initiated by the transition in the AAA6/C-terminal module. When we retarded the transition in the AAA6/C-terminal module, (referred to as “AAA6 ↓" ) we found that the order of transition events did not change (P-order (AAA6 ← AAA5) = 0), and that all of the subsequent events were retarded (see setup 2 in S8 Fig). Fig 5A shows that other modules did not make their transitions until AAA6/C-terminal module transited. As a result, due to this delay in all transitions, some modules did not complete their transitions within the simulation time. This result may be correlated to experimental data showing that a C-terminal truncated mouse dynein shows markedly larger stall-force compared to wild-type dynein [52]. This may suggest that the truncated dynein lacking the C-terminus tends to stay in its post-stroke state maintaining a strong affinity for tubulin. In the case of standard parameter set, as shown in Fig 4, the AAA1 module made the transition immediately after the transition in the AAA6/C-terminal module. Even when we introduced an accelerating effect in the AAA1 transition (referred to as “AAA1 ↑) the order of the pair of events did not change. (P-order (AAA1←AAA6) = 1. 0 and P-order (AAA6←AAA1) = 0. 0). Thus, this order is strictly adhered to. This dependence of the AAA1 transition on the conformational change of the AAA6/C-terminal module suggests that the ATP hydrolysis cycle in the AAA1 module is tightly regulated by the AAA6/C-terminal module conformation at the time of the recovery-stroke. The result suggests that thermally-activated transition in the AAA6/C-terminal unlocks the nucleotide-dependent AAA1 transition. In the post-stroke structure, the interface between the AAA1 and the AAA6 contains the salt bridge interaction between R2084 in the sensor-I loop in AAA1 and E4297 in the AAA6 module (Fig 5B left). In the simulation, when the AAA6/C-terminal module made the transition to the pre-stroke conformation, this salt bridge is lost and the interface became loose (Fig 5B right), which allows the sensor-I loop to move significantly, triggering the conformational transition in the AAA1 module. In the pre-stroke structure, the sensor-I loop in the AAA1 module reaches to the interface with the AAA2 module enabling the sensor-I motif (N2078) to contribute to ATP binding (see S6B for the sequence conservation of this site). The AAA5 module transition occurred after those of the AAA6 and AAA1 modules. However, the AAA1 does not interact directly with the AAA5 and thus is unlikely to trigger the AAA5 transition itself. Instead, most likely, the AAA5 transition is triggered by the AAA6 transition via the interface. As above, when the AAA6/C-terminal module transition was retarded, the AAA5 did not make its transition. Conversely, when the conformational change in the AAA5 was accelerated (referred to as “AAA5 ↑" ), we found that, in some cases, the AAA5 conformational change preceded that of the AAA6/C-terminal module (P-order (AAA6←AAA5) = 0. 3). Yet, in these cases, the conformational change in the AAA6/C-terminal module almost immediately followed that of AAA5. Thus, these data may also support the idea that other modules do not readily change their conformation prior to the change in the AAA6/C-terminal module. To understand the allosteric propagation from the AAA6/C-terminal module to the AAA5 module, we examined the interface between the C-terminal module and AAA5 during simulations (refer to Fig 5C where AAA5 is shown in orange and the AAA6/C-terminal module in red). In the early stage of the trajectories from the post-power-stroke structure, a long α helix (H1 helix) in the C-terminal module moved away from the AAA5 module (red to pink, indicated by an arrow with 1 in a circle). This H1 helix contains K4422 and K4425, which make salt bridges with D3962 and D3963, respectively, in the post-power-stroke structure (these sites are well conserved as in S6C Fig). As a result of this movement of the H1 helix, these interactions are lost. Later on, the H9 helix in the AAA5 module containing D3962 and D3963 moved further (indicated by an arrow with 2 in a circle). One end of the H9 helix (not depicted in Fig 5C) interacts with the C-terminal end of Strut so that movement of this helix transmits the change into Strut. The Strut shift with respect to Stalk affects the MTBD conformation. The conformational change in the AAA2 was observed after the transition in the AAA1 (Fig 4). To test this dependence, we introduced a retardation in the transition of the AAA1, (referred to as “AAA1 ↓”), which, in many cases, resulted in a change in the order of transitions between the AAA1 and AAA2 modules (P-order (AAA2←AAA1) = 0. 6 and P-order (AAA1←AAA2) = 0. 4). Next, we accelerated the transition of the AAA2 (referred to as “AAA2 ↑”), which resulted in a change of transition order; in half of the trajectories, the AAA2 changed its conformation prior to that of the AAA1. In these trajectories, the AAA2 transition occurred even earlier than that of the AAA6/C-terminal module. These results suggest that the AAA2 transition does not depend on other transitions. In the standard setup, the AAA1 module transited rather quickly after the AAA6/C-terminal module transition, while the transition of the AAA2 is inherently slower. Next, we investigated the role of the AAA2 module transition on subsequent processes, by introducing a retardation in the AAA2 transition (referred to as “AAA2 ↓”). The data revealed a weak correlation between the AAA2 module transition with that of the linker (k (linker|AAA2) = 0. 020, P-order (linker←AAA2) = 0. 6, P-order (AAA2←linker) = 0. 2) (refer to setup 12 in S9 Fig). We also accelerated the transition of the linker, (referred to as “linker↑”), which in most cases did not result in a change in the order of transitions (P-order (linker←AAA2) = 0. 8, P-order (AAA2←linker) = 0. 2). Moreover, the pair transition rate increased from that seen in the standard setup (k (linker|AAA2) = 0. 013 (95% confidence interval [0. 0041,0. 023], S3C Fig) ). These data suggest a weak dependence of the linker recovery-stroke motion on the AAA2 transition. Another statistical test supported these observations (S4C Fig, S4 Table). Next, we investigated the correlation between the AAA2 and AAA3 module transitions. In the “AAA2 ↓” simulations, we found a change in the order of transitions in two trajectories out of ten, (P-order (AAA3←AAA2) = 0. 5, and P-order (AAA2←AAA3) = 0. 2). When we accelerated the AAA3 transition (referred to as “AAA3↑”), the order of transitions did not change at all. However, their pair rates were small, k (AAA3|AAA2) = 0. 023 in the AAA3↑ setup, and k (AAA3|AAA2) = 0. 011 (95% confidence interval [0. 0044,0. 018], S3D Fig) in the standard setup (Fig 4C). Thus, we conclude that the AAA3 transition is only weakly dependent on the AAA2 transition (see also S4D Fig and S4 Table). Finally, we addressed the structural change in the MTBD, directly related to the dynein affinity for the MT. Fig 5D depicts three additional trajectories of MTBD motions along scaled time (defined as scaled-time = (MDstep − τAAA5) / (τAAA4 − τAAA5) ) in a manner similar to that in the power-stroke case; the scaled time is zero at the time of the AAA5 transition, and is one at the time of the AAA4 transition. Fig 5D shows that the AAA5 transition immediately triggers a high degree of fluctuation in MTBD between the pre- and post- states. These repetitive transitions ceased when the AAA4 module made its transition to the pre-stroke state. Thus, as was the case for the power-stroke, the MTBD motions in the recovery-stroke are determined by its two neighboring modules namely, AAA4 and AAA5. To address which of the AAA4 and AAA5 modules play a dominant role in the MTBD transition, we plotted the averaged reaction coordinate, χMTBD, against the scaled time both for the power-stroke and the recovery-stroke motions in the standard setup (Fig 5E). Here, the average was taken over 30 trajectories for each of the cases. We noted that χ MTBD moved more rapidly for the recovery-stroke case than for the power-stroke case. Of note, the scaled times zero and one correspond to the time of the AAA5 transition and that of the AAA4 transition in the recovery-stroke, respectively. On the other hand, over the same time range in the power-stroke case, the scaled times zero and one coincide with the time of the AAA4 and AAA5 transitions, respectively (Fig 3D). This implies that, of the two, the AAA5 module seems to have a stronger effect on the motion of the MTBD. We identify the major allosteric transitions that occur in cytoplasmic dynein during the power-stroke and the recovery-stroke motions. Using the standard set up and a statistical analysis of the order-map, we sorted the modules in the rows and the columns so that the summation of the probabilities in each column is in descending order (Fig 2D for the power-stroke and Fig 4D for the recovery-stroke). The resulting sequences in the rows/columns represent the dominant pathways that occur during the power-stroke and the recovery-stroke motions. Including sub-dominant pathways, we constructed a network of allosteric transitions for the power-stroke and recovery-stroke transitions as shown in Fig 6. In the power-stroke motion, the AAA3 transition precedes the linker transition in the standard setup, although this order is not obligatory in other setups. From a structural perspective, we see some interactions between the tip of the linker (helices H6 and H7) and a loop (called “pre-Walker B”) in the AAA3 in the pre-power-stroke state (S10 Fig), which may serve as a hook. Once these contacts are lost in the intermediate snapshot (S10 Fig), the linker motions are activated. However, as noted above, the precedence of the AAA3 transition is not a robust property in simulations and thus is not to be emphasized (S6D Fig). Then, the critical conformational transition occurred in the linker, which is immediately followed by the AAA1 transition. This order was very robust, being observed in any of the setups we applied. The nucleotide-dependent AAA1 transition may be unlocked by the precedent linker movement. Subsequently, the AAA4 module transition occurred. The transition of the AAA2 was stochastic and only weakly related to the other transitions. On average, the AAA2 transition occurred either before or after the AAA4 transition (Fig 2D). The transition cascade of linker, AAA1, AAA2, AAA3, and AAA4 constitutes the first half of the transition in the power-stroke process. The MTBD, including the Stalk, started to exhibit large-scale fluctuations after the transition of the AAA4. However, it took some time for this module to adopt its post-power-stroke structure. After this time interval had passed, the second half of the transition started with the AAA5 transition. The AAA5 transition appears to be dependent on the preceding transitions of the AAA4 and the linker via direct contacts in the post-power-stroke structure (S5 Table) [21]. Once the AAA5 module assumed its post-power-stroke structure, the Strut loop in the AAA5 stabilized the Stalk and MTBD in their post-power-stroke structure. Finally, the AAA5 transition triggered the AAA6~C-terminal module transition, which robustly terminated the whole process. Overall, the transition cascade proceeded in a clockwise manner looking from the linker attached side. In the recovery-stroke motions, the first transition to occur was that of the AAA6/C-terminal module. This order was very robust. In fact, no other module could make a conformational change before the AAA6/C-terminal module transition. The AAA6/C-terminal module transition unlocked a cascade of transitions in two directions on the AAA+ ring. In one, the AAA6/C-terminal module transition enabled the AAA1 module to make the nucleotide-dependent transition, which was followed by the AAA2 module and the linker transition (i. e. the clockwise direction). In the other direction, the AAA6/C-terminal module triggered the AAA5 transition, which was followed by the AAA4 and MTBD transitions (i. e. the anti-clockwise direction). The timing of the AAA3 transition was stochastic occurring either after the linker recovery or after the AAA4 transition. This is reasonable since the AAA3 module has direct contacts with both the AAA4 module and the linker (when the linker takes the pre-power-stroke structure). Recent experiments have shown that ATP hydrolysis in the AAA3 module stabilizes the weak-binding state of the MTBD [53]. The current simulation suggested that the state of the MTBD is largely regulated by the state of the AAA5 module. Taken together these data imply that for the AAA3 transition to affect the transition of the MTBD, the AAA3 module transition has to precede the AAA5 module transition. However, in our simulation this was not realized in most cases. This is probably because we used a pair of crystallographic information where the nucleotide state is different only in the AAA1 module, and thus the effect of ATP hydrolysis in the AAA3 module could not be reproduced. The same experimental work [53] showed that hydrolysis in the AAA3 module is not well correlated with the hydrolysis cycle in the AAA1 module, which is in accord with the current work where the AAA3 module transition shows little correlation with the AAA1 module transition in the power-stroke motion. As a sub-dominant pathway of the recovery stroke process, we observed the case where the linker recovery stroke motion occurred in the last step (drawn as the lower branch path in Fig 6). This pathway well correlates with a scenario that the linker movement is induced by the steric contact of the N-terminal tip of the linker with PS-I insert of the AAA4 large sub-module [23]. We note, however, that this pathway was sub-dominant observed in 4 cases out of 17 complete recovery-stroke trajectories. The scenario is motivated by the structural insight and is supported by the experimental data that the mutation in AAA4 reduces the dynein motility [23]. Notably, in our dominant pathway (13 out of 17, drawn as the upper path in Fig 6), the structural stability of MTBD/Stalk well depends on AAA4 so that it is also compatible with the same experiment. It should be emphasized that the power-stroke and recovery-stroke allosteric pathways are not merely the reverse order of each transition. The power-stroke allosteric transitions in the AAA+ ring occurred in a clockwise manner starting from the linker/AAA1 modules and ending at the AAA6/C-terminal module transition. The recovery-stroke started with the AAA6/C-terminal module transition, which is indeed opposite to the power-stroke step. However, the recovery allosteric transitions then propagated in a bidirectional manner, both clockwise and anti-clockwise. This asymmetry might have important functional implications for the uni-directional movement of dynein. To understand dynein motility from a structural perspective, it is important to understand the status of the MTBD when the linker makes its conformational changes. If the MTBD is in a high-affinity state for the MT at the timing of the linker transition, dynein transmits the linker stroke directly to the MT. Otherwise the linker stroke does not provide a strong force to the MT. In the power-stroke process, our simulation clearly predicts that the MTBD is in the low-affinity state for MT (i. e., the pre-power-stroke state) when the linker makes its conformational change. This is apparently in sharp contrast to the conventional model for dynein motility, in which the MTBD is assumed to take a high-affinity state for MT when the linker makes power-stroke swing [54]. Here, we discuss two points. 1) For dynein, there is no direct evidence, to our knowledge, for this conventional assumption. Instead, this assumption is based on its analogy to other linear dimeric molecular motors, kinesin and myosin. It should be noted that the motility of dynein is markedly different from those in kinesin and myosin. Thus, the analogy is not necessarily true. There are some evidences that show marked differences in dynein motility from kinesin motility. For example, dynein still proceed along MT even if one motor domain is replaced with a simple stick-like crutch [16]. Thus, the assumption that MTBD has a high affinity to the MT at the timing of power stroke has no support in dynein. 2) From structural perspectives, the linker is next to the AAA1 module where ATP hydrolysis drives the conformational transitions. Conversely, the MTBD is far away from the AAA1. If the conformational changes propagate through physical contacts at their interfaces, it is unlikely that the MTBD makes the transition earlier than that of the linker. Our simulation results are reasonable in that the conformational change cascade went through physical contacts, which resulted in the preceding transition of the linker with the MTBD in its low-affinity state for MT. However, we note that experimental data unambiguously show that the linker power-stroke motion is necessary for the dynein motility [55]. We consider two possible explanations. First, the linker may contribute to the stability of the AAA+ ring, which contains marked gaps between AAA+ modules. Secondly, if the linker power-stroke occurs while the MTBD being in the low-affinity state, the AAA+ ring is displaced or rotated with respect to the other monomer, which could induce the MTBD sliding towards the minus end of MT. After the sliding, the MTBD may changes its conformation to the high-affinity state for MT. Together, the dynein may achieve a move towards minus end of MT. In this model too, the linker power-stroke is an indispensable step, making it compatible with the experimental data. For the recovery-stroke, the situation is subtle. Starting from the AAA6/C-terminal conformational change, the clockwise cascade of transitions induces the linker conformational change, while the MTBD/Stalk structure change is a part of the anti-clockwise propagation from AAA6. Thus, the linker and the MTBD structural changes are not directly linked mechanistically so that their timings are not clearly predicted in the simulations. In the standard setup, P-order (MTBD←linker) = 0. 167 and P-order (linker←MTBD) = 0. 8, indicating that the MTBD tends to make transitions earlier, on average (Fig 4B). When we introduced an acceleration in the linker transition (linker↑), the order of transitions was P-order (MTBD←linker) = 0. 6 and P-order (linker←MTBD) = 0. 4. These data suggest that the order of these two events is not robust and thus is not well controlled in the current simulations. We note that the conformational change cascades given in Fig 6 are regarded as theoretical prediction, which needs to be verified experimentally. In principle, these can be examined by multicolor FRET for example. With regard to Fig 5C, by introducing the deletion mutant of the C-terminal helix 1, we can test how much that Helix 1 contributes to structure determination of MTBD over Strut in AAA5. While the current study provided a detailed description of the allosteric conformational changes that occur in dynein, it has some limitations. Since we relied on structure-based MD simulations, the result is largely dependent on the given pair of reference structures. First, the pre-power-stroke structure was obtained for human dynein-2, which differs in sequence and function from Dictyostelium discoideum dynein-1 for which the post-power-stroke structure is available (Identity is 28%). Second, in both of the reference structures, the AAA2, AAA3, and AAA4 modules have ADP bound. Therefore with these structures alone, we cannot address the role of ATP hydrolysis in these three modules. Once a high-resolution structure is obtained for the different nucleotide-bound forms of these modules, we can use them to address this point. Third, related to the first point, because the sequence is less conserved in the C-terminal module, we could not accurately model the Dictyostelium discoideum dynein-1 structure from the human dynein-2 C-terminal module. This precluded us from treating the C-terminal module as a separate unit of transition. It was desirable, but not possible, to treat the C-terminal module independently from the AAA6/C-terminal module. Fourth, the multiple-basin model contained the parameters, Δ and ΔV, to smoothly connect the two reference structures. These parameters were treated in this work as empirical parameters tuned to realize conformational changes within the simulation time. We can, in principle, determine these parameters based on either experimental data, if available, or atomistic molecular simulations. Finally, to understand the motility of dynein on the MT, ultimately we need to simulate the dynein-MT complex. Moreover, since the method used in this work is very general, we can apply the same approach to study other multi-domain proteins. The simulated protein was Dictyostelium discoideum cytoplasmic dynein-1 (UniProtKB (P34036). For modeling/analysis, we divide the motor domain Q1522-I4730 into eight structural units; the linker (Q1522~Y1935), AAA1 (Y1936~Q2229), AAA2 (P2230~V2631), AAA3 (P2632~R2948), AAA4 (P2949~D3262, A3597~L3638), Stalk~MTBD (F3263~S3596), AAA5 (S3639~Q4114), and AAA6/C-terminal end (E4115~I4730) (Fig 1). Of note, the AAA4 module contains two disconnected segments, between which the Stalk~MTBD is inserted. For the reference structure of the post-power-stroke state, we primarily used the X-ray crystal structure of Dictyostelium discoideum cytoplasmic dynein in the ADP-bound form (pdb ID: 3VKH) [21]. This structural model has low resolution near the MTBD region. Thus, for the MTBD region, we used a model obtained by a combination of cryo-electron microscopy and molecular simulations for Mus musculus cytoplasmic dynein (pdb ID: 3J1T) [22]. Clustal ω was used to align the two reference sequences [56,57]. MODELLER was used to make a homology model and augment missing residues [58]. Using CafeMol 2. 1 [46], we first converted the Cartesian coordinates of the two reference structures into internal coordinates. Combining all but the MTBD coordinates from 3VKH and the MTBD (V3350~K3514 in Dictyostelium discoideum and K3264~K3427 in Mus musculus) coordinates from 3J1T, we obtained a hybrid structure in the internal coordinate representation. Using CafeMol 2. 1 with this hybrid coordinate as the reference, we performed a very short simulated annealing simulation (essentially, minimization) to reach a stable three dimensional structure with the coarse-grained representation. PD2 ca2main [59] and Scwrl4 [60] were used to reconstruct the atomic model for the backbone and the sidechain, respectively. This reconstituted atomic structure was used as the reference structure of the post-stroke state. To test the modeled structure, we checked the local stability of the model by performing short atomistic MD simulations with GROMACS 5. 1. 1 [61,62]. For the pre-power-stroke structure, the full-length motor domain structure is solved only for human dynein-2 in the ADP + Pi bound form (pdb ID: 4RH7) [23], in which Q1255~V4308 was utilized as the template. Using this as the template, we obtained a homology model for Dictyostelium discoideum cytoplasmic dynein-1 using MODELLER 9. 15. In the same manner as described above for the pre-power-stroke state, after a short simulated annealing run with CafeMol, we used PD2 ca2main, and subsequently Scwrl4, to reconstruct an atomistic structure. This modeled atomic structure was used as the reference structure of the pre-power-stroke state. To test this structure, it was subjected to MD simulations with GROMACS 5. 1. 1, confirming that the modeled structure is locally stable. In the MD simulation using GROMACS, we used the amber99sb-ildn force field for dynein [63] and TIP3P for solvent water [64]. We added sodium and chloride ions to neutralize the system and to make the salt concentration approximately equal to 0. 1 M, which resulted in 668 sodium ions and 627 chloride ions in 322,307 (283,925) water molecules in the pre-power-stroke (post-power-stroke) conformations. The energy minimization by the steepest descent minimization algorithm was followed by equilibration of solvents with NVT and subsequently NPT ensemble simulations for 100 ps at 300 K. In the production run, we used NPT ensemble with 1 atm and 300 K. Long range electrostatics were calculated by the Particle-Mesh-Ewald method. The production run was conducted with 1 fs step for 10 ns. It should be noted that the purpose of this atomistic MD simulation was to simply check the local quality of the model. The RMSD from the initial structure (S11 Fig) shows saturation around 0. 5 nm, which, considering the large size of AAA+ ring, we regard it reasonably stable. With the atomic structures of the pre- and post- power-stroke structures of dynein motor domain, we performed coarse-grained MD simulations of the entire motor domain to investigate conformational change pathways between the two structures. The coarse-grained model represents each amino acid as a single bead located at its Cα position. Dividing the entire motor domain into eight modules, we assumed that each module adopts two locally stable states corresponding to the pre- and post- power-stroke structures. Thus, we assigned a double-basin potential for each region/module. Formally, we can write the entire potential energy function as, Vtotal = ∑I = 1,8 VMB, I where VMB, I represents the multiple (two in this work) basin potential for the I-th modules partially including the interactions with their neighbors. (The explicit forms will be described below). Since each double basin potential possesses two basins, corresponding to the pre- and post- power-stroke states, the entire motor domain can have, in theory, 28 local minima. Among all the minima, the one where all regions were in the pre- (or post-) power-stroke state coincides with the pre- (or post-) power-stroke structure of the entire motor domain. In this modeling, the entire conformational change occurs in a modular manner, region by region. From the pre-power-stroke to the post-power-stroke structure (and also in the recovery-stroke process), there can be 8! possible orders of transitions for every module. Before describing the double-basin model VMB, I, we start with the explanation of the energy function AICG2+ for a single basin model [47,48]. The AICG2+ potential is explicitly biased towards the reference structure, while all the terms are tuned to represent chemical interactions at the reference structure. The potential energy function is written as VAICG2+ (R|R0) =∑iKb, i (bi−bi, 0) 2+Vlocflp+∑j=i+2εloc, ijexp (− (rij−rij0) 22Wij2) +∑j=i+3εloc, ijexp (− (ϕij−ϕij0) 22Wϕ, ij2) +∑i<j−3natcontactεgo, ij[5 (rij0rij) 12−6 (rij0rij) 10]+∑i<j−3non−nativeεev (drij) 12 Here, each term represents, in order, the elasticity of the virtual bond, the sequence-dependent angle- and dihedral-angle potential, the structure-based local potential between i-th and i+2-th residues, the structure-based local potential for dihedral angles, the Gō potential for non-local natively interacting pairs, and the generic repulsion for the rest of the non-local pairs. The vector R represents the 3naa-dimensional Cartesian coordinates of the target protein where naa is the number of amino acids in the protein. R0 is the corresponding coordinates in the reference structure (All the variables with the subscript 0 refer to parameters with their corresponding value from the reference structure). bi is the i-th virtual bond length between i-th and i+1-th amino acids. Vlocflp is the sequence-dependent local potential [65]. rij is the distance between the i-th and j-th residues. ϕij is the dihedral angle defined as i-th, i+1-th, i+2-th, and i+3-th residues. Wij2 and Wϕ, ij2 are the parameters representing the widths of the attractive interaction. The parameters Kb, ibd, εloc, ij, εgo, ij are determined based on atomic-interactions evaluated by AMBER force field via a multiscale algorithm. The parameters εev and d are determined from a structural survey. The CafeMol manual should be consulted for the meaning and the default values of these parameters (http: //www. cafemol. org). Given the two single-basin potential functions V (R|Rν) with different reference structures ν = 1,2, we define the multiple-basin potential for each module VMB, I as the smaller eigenvalue of the eigenvalue equation, (VI (R|R1) ΔΔVI (R|R2) +ΔV) (c1c2) =VMB, I (c1c2) Here, the single basin potentials VI (R|Rν) for I-th modules are constructed from the AICG2+ model for each module with small modifications to adapt it with the multiple-basin model, as described in Okazaki et al [49]. Notably, we put interaction energies between neighboring modules in the multiple-basin potential of one module out of two interacting modules. The eigenvalue can be obtained by solving the secular equation. We can explicitly write the smaller eigenvalue as VMB, I=VI (R|R1) +VI (R|R2) +ΔV2− (VI (R|R1) −VI (R|R2) −ΔV2) 2+Δ2 Here, each multiple-basin potential contains two parameters, the coupling constant Δ that is related to the potential barrier height and ΔV that modulates the relative stability of the two basins (S1A Fig). For dynein, these parameters cannot be easily determined from experiments or in a bottom-up manner. Thus, we treat them as empirical parameters, the fine tuning of which is described in detail in the next subsection. Using the eigenvector for the smaller eigenvalue, we can define the reaction coordinate as χ = ln (c2/c1) which takes the negative (positive) value when the system stays in the basin 1 (2). As mentioned above, we divided the dynein motor domain into eight modules. Thus, the entire motor domain is described by eight multiple-basin potentials. For each module, we included the intra-module interaction and some inter-module interactions associated with the module into the multiple-basin potential (see S1 Table). Each inter-module interaction term is included in only one of the relevant multiple-basin potential systems so that no double counting occurs. Notably, even though the inter-module interaction is included only in one of two multiple-basin potentials, the coarse-grained MD is conducted by using the summation of all the terms so that the inter-module interaction affects conformational changes in both modules. Transitions in the regions/modules are described by χ values. Based on the sign of the χ, we can assign if the module is either in the pre-power-stroke state or in the post-power-stroke state. By combination, the entire motor domain can have, in theory, 28 states. Although the values of parameters Δ and ΔV can, in principle, be determined from all atom molecular mechanics calculations or from experimental data, if available, we cannot currently use these approaches for the dynein motor domain. In this study, we tuned these parameters purely empirically via many preliminary simulations, as described below. The parameter Δ modulates the energy barrier height between two basins. When one uses a small value of Δ, there is no observable conformational transition over a feasible computer time frame (An example is shown in S1B Fig top right panel). Conversely, at an extremely large value of the parameter Δ, the two basins are completely coalesced to form a single basin between the positions of the two reference structures (S1B Fig top left). Neither of these situations is desirable. Thus, we sought a range for the value of the Δ parameter, in which we could observe conformational transitions within the possible simulation time. In general, the range of acceptable Δ values is relatively narrow and thus we can determine the value of Δ with little uncertainty. The Δ parameter values thus decided are listed in S6 Table. The parameter ΔV modulates the relative stability of the two basins. Here, we defined ΔV as the energy shift in the final structure, relative to the initial structure. For example, in the power-stroke process, the final structure is the post-power-stroke state and thus an increase in the value of ΔV destabilizes the post-power-stroke state relative to the pre-power-stroke state. When one uses a very small ΔV value (often a negative value), one normally observes the conformational transition immediately at the beginning of simulations (as exemplified in S1B Fig bottom left). When one adopts a very large ΔV value, the transition is never observed (S1B Fig bottom right). Neither of these situations is desirable. For each of the power-stroke and recovery-stroke processes, we tuned the value of ΔV so that we could observe the conformational transition in the middle of the simulation time window. As was the case for Δ, the range of acceptable ΔV values was relatively narrow and so we could also assign values with confidence. We found one set of these values that match all these criteria, and refer to it as the standard set. Since the choice of parameters is far from unique, we comprehensively investigated the sensitivity/robustness of the results when we alter the parameter values ΔV. Specifically, relative to the standard set, we introduced either a destabilization in the final state (retarding effect), or a stabilization in the final state (accelerating effect) into each region/module, by the ΔV change of ±10 kcal/mol (S2 Table for the power-stroke and S3 Table for the recovery-stroke processes). Conformational change simulations were performed 30 times with different stochastic forces for the standard parameter set and 10 times for the other sets that introduced a retarding or accelerating effect. We utilized the underdamped Langevin dynamics for constant temperature MD simulations. The friction coefficient was 0. 02 (in CafeMol units) and the temperature was set at 300 K. Each MD simulation consists of 107 MD steps. One MD step was mapped very roughly to ~1 ps [45]. Thus, each trajectory corresponds to approximately 10 μs although the time mapping was a little inaccurate, especially because the transition rates depend on the empirical parameter Δ. All of the coarse-grained MD simulations were performed using an in-house modified version of CafeMol 2. 1. To infer atomic interaction at the interface between modules, we modeled the all-atom structures of representative intermediate states. The reconstruction method is the same as that used to model the pre/post structural models. First, PD2 ca2main [59] were used to reconstruct the atomic model for the backbone and we also did energy minimization as an option. Then, Scwrl4 [60] were used to reconstruct the atomic model for the sidechain. Additionally, we checked the stability of these reconstructed structures by performing short all-atom MD simulations from these structures. The setup of simulation is the same as that for the pre/post structures’ case (S11 Fig).
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Title: Allosteric conformational change cascade in cytoplasmic dynein revealed by structure-based molecular simulations Summary: The linear molecular motor dynein is an intriguing allosteric model protein. ATP hydrolysis, catalyzed by modules in the AAA+ ring, regulates the binding to the rail molecule, microtubule, which is ~15 nm away from the AAA+ ring. The molecular mechanisms underpinning this long-distance communication are unclear. Based on recently solved pre- and post- power-stroke crystal structure information, we performed, for the first time to our knowledge, molecular simulations of complete conformational changes between the two structures. The simulation revealed that module-by-module allosteric conformational changes occur. Interestingly, the transition cascade from the pre- to the post-power-stroke states propagated in a circular manner around the AAA+ ring, while that of the recovery transitions propagated in a bi-directional manner around the ring.
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Summarize: FIELD OF THE INVENTION The invention relates to aqueous organ preservation solutions and methods useful in surgical operations on the heart and in transplantation of the heart and other mammalian organs. BACKGROUND OF THE INVENTION Transplantation of vital organs such as the heart, liver, kidney, pancreas, and lung has become increasingly successful and sophisticated in recent years. Because mammalian organs progressively lose their ability to function during storage, even at ice temperatures, transplant operations need to be performed expeditiously after organ procurement so as to minimize the period of time that the organ is without supportive blood flow. This is particularly true for the heart in which the permissible storage time with present methods of preservation is limited to a maximum of about 4 to 6 hours. In clinical practice, the two situations in which cardiac preservation is required are heart transplantation and cardioplegia for open heart surgery. In heart transplantation, the donor heart is exposed through a midline sternotomy. After opening the pericardium, the superior and inferior vena cavae and the ascending aorta are isolated. The venous inflow is then occluded, the aorta is cross clamped, and approximately 1 liter of cold cardioplegic solution is flushed into the aortic root under pressure through a needle. As a result, the heart is immediately arrested, and cooling is supplemented by surrounding it with iced saline. The cold arrested heart is then surgically excised, immersed in cold cardioplegic solution, surround by ice and rushed to the recipient center. The recipient's chest is opened through a midline sternotomy, and after placing the patient on cardiopulmonary bypass, the diseased heart is excised. The preserved donor heart is then removed from the preservation apparatus, trimmed appropriately and sewn to the stumps of the great vessels and the two atria in the chest. After completion of the vascular anastomoses, blood is allowed to return to the heart. It then will either resume beating spontaneously or will require chemical and electical treatment to restore normal rhythm. When the heart is ready to take over the circulation, the cardiopulmonary bypass is discontinued and the recipient's chest closed. Most non transplant surgical procedures on the heart, such as coronary artery bypass grafting, require that the heart's action be arrested for a period ranging from 1 to 4 hours. During this time, the heart is kept cool by external cooling as well as by periodic reflushing a cardioplegic solution through the coronary arteries. The composition of the latter solution is designed to rapidly arrest the heart and to keep it in good condition during the period of standstill so that it will resume normal function when the procedure is finished. In the cardioplegic procedure, the heart is exposed in the chest, and as a minimum the aortic root is isolated. A vascular clamp is applied across the aorta and approximately 1 liter of cold cardioplegic solution is flushed into the aortic root through a needle. Venting is provided through the left ventricle, pulmonary artery or the right atrium and the effluent which may contain high levels of potassium is sucked out of the chest. This, together with external cooling, produces rapid cessation of contractions. During the period of arrest, the patient's circulation is maintained artificially using cardiopulmonary bypass. After completion of the surgical procedure, blood flow is restored to the coronary circulation and beating either returns spontaneously or after chemical and electric treatment. The ease with which stable function is restored depends to a large extent on the effectiveness of preservation by the cardioplegic solution. Once the heart is beating satisfactorily, cardiopulmonary bypass is discontinued and the chest closed. It is generally understood that "living" organs, including the heart, continue the process of metabolism after removal from the donor so that cell constituents are continuously metabolized to waste products. The accumulation of these metabolic waste products, depletion of cell nutrients and consequent derangement of cell composition lead to progressive loss of function and ultimately to cell death if the storage technique is inadequate. That is, the organ will lose its ability to function adequately after transplantation into the recipient. Several procedures have been successfully explored to enable organs to be preserved ex vivo for useful time periods. In one method the organ to be transplanted is rapidly cooled by flushing cold solutions through the organ's vascular system and maintaining the organ at temperatures near 0° C. for the purpose of greatly slowing the metabolic rate. In the case of the mammalian heart, the flush solution composition is designed to cause the heart to rapidly stop beating as well as to preserve it. Another method for organ storage utilizes continuous perfusion at temperatures in the range of 7°-10° C. with an oxygenated solution designed to support oxidative metabolism and to remove waste products. A suitable perfusate is delivered through the circulatory system of the isolated organ--usually from the arterial side--and as the perfusate is conveyed through the vascular system waste products are carried away from the organ. Kidneys and livers can commonly be preserved in this manner for several days. However, only limited success has been achieved in preserving the heart, and therefore this method is not used in clinical heart transplantation. The heart must function well enough to sustain a good circulation in the recipient immediately after the transplant operation, whereas some impairment of function can be tolerated in transplanted livers and kidneys. Since hearts can only be preserved for 4-6 hours using cardioplegic solutions, heart transplantation tends to be ruled out in certain situations in which the proposed donor and recipient are far distant from one another. The viability of a preserved organ depends on a number of factors, among which may be listed (1) cell swelling which occurs at low temperatures as water is transferred across cell membranes in a stored organ, (2) the degree of intracellular acidosis which occurs during non perfused ice storage as a consequence of continued cell metabolism, (3) derangement of internal cell composition which results from impaired metabolism, particularly with respect to cations such as calcium, potassium, magnesium and sodium, and (4) injury caused by oxygen-derived free radicals during oxygenated perfusion or after restoration of the circulation. Perfusate solutions containing hydroxyethyl starch ("HES") have been reported by Belzer and Southard in Transplantation, 45:673 676, April, 1988, for use in preserving the kidney, liver and pancreas. See also PCT Publication No. WO 87/01940 (published Apr. 9, 1987). The compositions differ depending on whether they are to be used for continuous perfusion or a single flush ice storage of the organ. In both cases, however, a central feature of the solution is the content of the colloid HES. SUMMARY OF THE INVENTION We have found that mammalian organs, and particularly hearts, can be preserved for comparatively long periods of time without significant loss in viability through the use of aqueous preservation solutions containing polyethylene glycol having a molecular weight above about 15,000 daltons. The solution ingredients, including an impermeant composition, are pharmacologically acceptable, and the solution desirably is buffered to a pH in the range of from about 7.1 to about 7.5. The polyethylene glycol is free of material capable of being removed by filtration through a 10 micron filter. It further has been found that a solution of the type described can be used for heart preservation as a perfusate in a process in which the perfusate is flowed through the circulatory system of a heart muscle at a low flow rate not exceeding about 6 ml per gram of heart weight per hour. The solution may be used for cardioplegia applications, and 4 hours of cardioplegic heart preservation can be achieved without significant loss of viability DESCRIPTION OF THE PREFERRED EMBODIMENTS The polyethylene glycol ingredient of solutions of the instant invention has an average molecular weight that is at least about 15,000 daltons and preferably is in the range of from about 15,000 to about 20,000 daltons. The high molecular weight polyethylene glycol desirably may be made through a process in which hydroxy-functional lower molecular weight polyethylene glycols are linked together using, as linking moieties, such diepoxidies as the diglycidyl ether of Bisphenol A. When polyethylene glycol having an average molecular weight of about 8000 daltons is linked in this manner, the resulting material will include some polymer having an average molecular weight of about 8000 daltons, some polymer having an average molecular weight of about 16,000 daltons, and some higher molecular weight polymers generally thought to be multiples of the 8000 dalton material. The initial 8000 dalton material, of course, itself has a molecular weight distribution, and as a result the high molecular weight polyethylene glycol used in the invention has a broad molecular weight distribution. At least about 60 weight percent of the high molecular weight polyethylene glycol, however, is about 15000 daltons or greater in molecular weight. High molecular weight polyethylene glycol is available commercially, as from Union Carbide Corporation. Of importance, the polyethylene glycol ingredient must be free of particulate or other material that is retained on a 10 micron (preferably a 5 micron) filter. During development of the instant invention, it was found that polyethylene glycol of the type described may contain particulate or gel or other material which, when employed in a perfusion operation, tends to block capillary flow, leading to rapid loss of viability of the organ being treated. The polyethylene glycol ("PEG") is water soluble, and commonly is dissolved at a low concentration in water, following which the solution may be filtered through appropriate filters such as Millipore brand filters to remove the unwanted material. Various filtering methods can be employed. In a preferred embodiment, the polyethylene glycol solution is filtered through ten micron filters and preferably two or more such filters in series. The purpose of the filtration step is to remove particulate or gel or other material that is harmful to organ preservation. Filtration of the PEG solution through 0.2 micron cartridge filters has the additional advantage of rendering the solution bacteriologically sterile. Desirably, the concentration of the PEG in organ preservation solutions of the invention is maintained in the range of about 4% to about 10% by weight. The desirable effects which are provided by the PEG material are displayed for the most part at concentrations of about 3% by weight or greater. On the other hand, the PEG employed in the instant invention is of sufficiently high molecular weight as to unduly increase the viscosity of an aqueous perfusate solution when the PEG is employed in concentrations exceeding about 10% by weight. Approximately 5% PEG concentrations have yielded excellent results. The high molecular weight polyethylene glycol employed in the instant invention provides perfusion and cardioplegic flush solutions with unexpectedly good organ preservation capabilities. Although it is believed that in some applications the polyethylene glycol functions as a colloid to keep the perfusate in the vascular system, and that it may interact in some beneficial way with cell membranes, the precise technical explanation for the surprisingly beneficial properties of this material in organ preservation is not known. The perfusates of the invention may include a variety of further ingredients common to organ preservation, e.g., perfusate solutions in general. Typical ingredients include: ______________________________________Ingredient Amount Function______________________________________NaOH 30-40 mM Provide buffering of ingredients to desired pHLactobionic acid 100 mM Impermeant anion, chelation of calcium and ironKH.sub.2 PO.sub.4 25 mM Provides potassium and phosphate for bufferingKOH 100 mM Potassium to prevent loss of intracellular cation and for cardioplegia, OH neutralizes lactobionic acidImpermeant non- 30 mM Further impermeant to controlelectrolyte, cell swellingtypically raffinoseGlutathione 3 mM Control of redox potential and protection against free radical injury______________________________________ The solution preferably is devoid of nutrients that are metabolized by the organ to be preserved. EXAMPLE 1 This example describes the use of an organ preservation solution of the invention for cardioplegia and short term heart preservation in an experimental model designed to imitate the conditions of open heart surgery likely to be encountered clinically in operations on the arrested heart. The test was conducted at 15° C. to simulate realistic conditions. Candidate cardioplegic solutions were prepared as shown in the following Table 1 in which a solution of the invention was designated Solution "A". Solution "B" was the well known and widely used St. Thomas II solution commonly employed in human heart surgery and for cardiac transplantation. Solution "C" was a perfusate solution containing hydroxyethyl starch ("HES") commonly known as the "UW" solution and reported in Heart Transplantation 7:456, 1988. TABLE 1______________________________________Ingredient Solution A Solution B Solution C______________________________________ mM mM mMNa (as NaOH) 40 120 30K (as KOH) 125 16 125Ca -- 1.2 --Mg 5 16 5H.sub.2 PO.sub.4 25 -- 25SO.sub.4 5 -- 5Lactobionate 100 -- 100Raffinose 30 -- 30Cl -- 160 --HCO.sub.3 -- 10 --Polyethylene glycol.sup.(1) 50 Gm/L -- --HES -- -- 50 Gm/LAdenosine -- -- 5Glutathione 3 -- 3Allopurinol -- -- 1Insulin -- -- 100 UnitsPenicillin -- --.133 Gm/LDexamethasone -- --.008 Gm/L______________________________________.sup.(1) Polyethylene glycol compound "20M", Union Carbide Corporation. New Zealand white rabbits weighing 2-3 kgs were anesthetized using sodium pentobarbital, and supported on a ventilator via a trachesotomy. A median sternotomy was performed and the heart rapidly excised (5-10 sec) and immersed in iced saline. An occlusive cannula was immediately placed in the aorta, and 15-25 ml of one of the above cardioplegic solutions were infused (4° C.) at a pressure of 100 cm H 2 O. Thereafter, hearts were kept in a water bath at 15° C. for a total of 4 hours. During the first two hours, each heart was reflushed with the cardioplegic solution every 30 minutes in order to simulate clinical practice in which the arrested heart is reflushed approximately every 30 minutes. After the preservation period, the aorta and left atrium (LA) were cannulated and the hearts were functionally evaluated on an ex vivo perfusion circuit filled with oxygenated Krebs Henseleit solution at 37° C. The test apparatus was similar to that originally described by Neely, et al. (Neely J. R., Liebermeister H., Battersby E. J., et al. Am. J. Physiol. 1967; 212: 804). The hearts were allowed 15 minutes of recovery while on retrograde Langendorff perfusion at a pressure of 100 cm H 2 O. This was converted to a working heart mode for a 45-minute test period: The LA pressure was kept at 20 cm H 2 O and the aortic outflow was connected to a column in which the overflow was set at 100 cm H 2 O. Measurements of aortic pressure, coronary and aortic flow rates were made and recorded at 1 hour and cardiac output was calculated. Hearts preserved by each of the three cardioplegic solutions were tested for cardiac output as described above, and that output was compared to the cardiac output of hearts flushed with each of these solutions and tested immediately with no storage period. The observed results were as follows: TABLE 2______________________________________Candidate Cardioplegic Mean Cardiac Output, ml/gSolution (No. of Hearts Tested)______________________________________Control Solution A 38.30 ± 2.42 (6)(no Solution B 20.51 ± 8.26 (5)storage) Solution C 26.47 ± 2.27 (6)Test Solution A 37.00 ± 6.53 (5)(4 hour Solution B 17.40 ± 0.86 (4)storage) Solution C 27.80 ± 11.07 (5)______________________________________ Tetrazolium staining revealed diffuse macroscopic infarcts in all hearts treated with Solution B and in one of the 5 hearts treated with Solution C. No infarcts were observed in any of the control hearts, or in any of the test hearts treated with Solution A. The results indicate that the solution of the invention provided substantially improved protection of hearts from injury under realistic conditions in comparison with conventional cardioplegic solutions. EXAMPLE 2 This example describes the use of an organ preservation solution of the invention employed in a technique involving continuous hypothermic perfusion at very slow rates. New Zealand white rabbits weighing 2-3 kgs were each anesthetized using sodium pentobarbital, and supported on a ventilator via a tracheostomy. Median sternotomies were performed and the hearts excised and immersed in iced-saline. An occlusive cannula was immediately placed in the aorta of each heart, and 15-25 ml of flush solution infused (4° C.). Extraneous tissue was removed and each heart was rapidly weighed. The aorta of each heart was tied to a cannula fitted through the lid of a small plastic specimen container in order to stabilize it during the subsequent perfusion period. The container was provided with an outlet port to permit effluent to escape, assuring that the aortic root remained stable and the aortic valve shut so that perfusate passed through the coronary vessels rather than the aortic valve. Control groups consisted of (a) 6 unstored hearts which had been rapidly removed cooled and arrested with "UW" (a hydroxyethyl starch perfusate reported in Table 3 below) and then immediately functionally evaluated, (b) 6 unstored hearts cooled and arrested with a solution identical to the UW solution but containing polyethylene glycol of molecular weight approximately 15,000-20,000 daltons in place of hydroxyethyl starch, (c) 9 hearts treated as in group (a) and then ice stored for 24 hours, and (d) 11 hearts subjected to low pressure oxygenated perfusion with a cardioplegic solution designated "WP" (Table 3). Hearts in the experimental groups were perfused at a rate of 3-6 ml/Gm/24 hours with the perfusates reported in Table 3 below. This very low delivery rate was achieved by the use of electrically driven syringe pumps or IV infusion pumps. The solutions were maintained at 0° C. Ten hearts (Experimental group (1)) were perfused with UW solution prepared within 24 hours of use. Experimental group (2) (16 hearts) were perfused with UW solution which had been shelf stored in double plastic bags for 4-6 months. Experimental group (3) (5 hearts) were perfused with UW solution containing no hydroxyethyl starch and Experimental Group (4) (14 hearts) were perfused with the UW solution in which the hydroxyethyl starch had been replaced with 5% polyethylene glycol of molecular weight in the range of approximately 15,000 to 20,000 daltons. The high molecular weight polyethylene glycol reported in this example was that identified in Example 1. In each case, the penicillin, dexamethasone and insulin were added immediately before use. The high molecular weight polyethylene glycol ingredient had been dissolved in water and passed three times through 10 micron Millipore brand filters to remove small amounts of contaminants. Finally, 6 hearts (Experimental Group 5) were perfused with the solution of Experimental Group 4 from which the insulin, penicillin, dexamethasone, adenosine and allopurinol had been omitted. After the 24-hour perfusion period, the aorta and left atrium were cannulated and the hearts were functionally evaluated at 37° C. on an ex vivo perfusion circuit as described in Example 1. The individual results are summarized in Table 4. The significance of the experimental group data was compared with fresh controls, groups (a) and (b), and 24-hour UW perfused hearts, group (1). The group (a) hearts achieved a cardiac output ("CO") of 26.48±2.25 ml/Gm/min. This was significantly inferior to the CO of group (b) (38.30±2.42 ml/Gm/min) indicating that the high molecular weight polyethylene glycol solution was superior to the standard UW solution (group (a)) for short term cardioplegia. Those hearts undergoing oxygenated perfusion with WP solution, group (d), had a mean CO of 21.19±6.20. This was significantly inferior to the CO of hearts perfused slowly for 24 hours with UW solution and the two high molecular weight polyethylene glycol solutions (Experimental Groups (1), (4) and (5)), respectively. Hearts perfused slowly with solution containing HES as the colloid showed excellent function after storage (CO 28.72±7.69), indistinguishable from unstored control group (a) but significantly inferior to the high molecular weight polyethylene glycol control group (b). The high molecular weight polyethylene glycol perfusate led to a CO after 24-hour perfusion (31.52±4.49 group (4), and 31.67±3.43 group (5)) that was actually significantly higher than the HES-UW fresh controls (group a). However, all of experimental groups (1,4, and 5) showed a significantly lower CO after 24-hour perfusion than was found in the fresh polyethylene glycol solution controls (group (b)). This implies that there was a measurable amount of loss of function after 24-hour storage by comparison with the best control group (group (b)). Omitting the HES or polyethylene glycol colloid from the UW solution was very detrimental in this model. The mean CO in Exp. group (3) fell to 11.44±5.24 ml/Gm/min, significantly below both fresh controls and the freshly prepared solutions in groups 1,4, and 5. Similarly, comparatively poor function followed simple ice storage with UW for 24 hours without microperfusion, (Control Group (c)). The mean CO in this group was 12.84±10.03 ml/Gm/min (P<0.01 vs group (a) and group (1)). Experimental group (2) in which the UW solution had been shelf stored in plastic bags for 4-6 months, yielded a mean CO significantly lower than with HES-UW made up fresh before use (Experimental Group (1)). It would appear that this effect is due to oxidation of the glutathione during shelf storage. These data suggest that simple ice storage of the heart with the flush solution generally accepted heretofore to have the best composition (UW solution) yields inadequate function after 24-hour storage. This failure is probably due in part to the accumulation of waste products and the development of acidosis. Although we do not wish to be bound by the following explanation, it appears that low flow rate perfusion and the UW solution and the solution of Experimental Groups 4 and 5 (at rates ranging from 3 to about 6 ml/Gm tissue/24 hours) offers support of anaerobic metabolism by provision of substrate, control of acidosis, and removal of waste products. This process leads to significant improvement in cardiac performance to levels that are either equal to or only a little below fresh unstored controls. In this low flow perfusion method the presence of a colloid appears to be essential. Of the colloids tested, high molecular weight polyethylene glycol yields surprisingly good results and would seem to be the best. Higher rates of perfusion in the presence of oxygen utilizing a modified cardioplegic solution (Control Group (d)) resembling St. Thomas II solution yielded results that were clearly inferior to hypoxic, low flow perfusion. Flow rates of conventional continuous perfusion methods (on the order of 1 ml/Gm/min) are approximately 500 times greater than the flow rates desirably employed utilizing the perfusate of the invention. TABLE 3__________________________________________________________________________COMPOSITION OF PERFUSION SOLUTIONS Amount mM/L Control Group Exp. Group Exp. Group (3) Exp. Group (4) Exp. Group (5)Substance "WP" (1)&(2) UW UW. No HES High MWPEG High MWPEG__________________________________________________________________________Na 126 30 30 30 30K 8 125 125 125 125Ca 0.6 -- -- -- --Mg 14 5 5 5 5Cl 127 -- -- -- --HCO.sub.3 -- -- -- -- --H.sub.2 PO.sub.4 8 25 25 25 25SO.sub.4 14 5 5 5 5Lactobionate -- 100 100 100 100Raffinose -- 30 30 30 30Glucose 11 -- -- -- --Mannitol -- -- -- -- --Taurine 4 -- -- -- --Glycerol 136 -- -- -- --Sucrose 7 -- -- -- --Polyethylene glycol.sup.1 1 Gm/L -- -- -- --Chlorpromazine 0.010 -- -- -- --Procaine 1 -- -- -- --Phenoxybenzamine 0.007 -- -- -- --Adenosine -- 5 5 5 5Glutathione -- 3 3 3 3Insulin -- 100 U/L 100 U/L 100 U/L --Penicillin --.133 Gm/L.133 Gm/L.133 Gm/L --Dexamethasone -- 8 mg/L 8 mg/L 8 mg/L --Hydroxyethyl starch -- 50 Gm/L -- -- --Allopurinol -- 1 mg/L 1 mg/L 1 mg/L --Polyethylene glycol.sup.2 -- -- -- 50 Gm/L 50 Gm/L__________________________________________________________________________.sup.1 Mol. wgt. of approximately 8,000 daltons..sup.2 Mol. wgt. of approximately 15,000-20,000 daltons. TABLE 4__________________________________________________________________________ # Reaching Cardiac OutputGroup 100 cm H.sub.2 O (ml/Gm/min) Significance__________________________________________________________________________Controls(a) (UW) 6/6 26.48 ± 2.25(b) (High MWPEG) 6/6 38.30 ± 2.42 vs (a) p < 0.01(c) ((a) + ice stored) 5/9 12.64 ± 10.03 vs (a) p < 0.01(d) (Oxygenated perfusion) 11/11 21.19 ± 6.20 vs (a) NSExperimental Groups(1) UW Fresh 10/10 28.72 ± 7.69 vs (a) NS vs (b) p < 0.01(2) UW Shelf stored 8/16 17.29 ± 9.27 vs (1 & 2) p < 0.01(3) UW No HES 2/5 11.44 ± 6.24 vs (1 & 2) p < 0.01(4) UW with high 14/14 31.52 ± 4.48 vs (a) p < 0.05MW PEG (5%) vs (b) p < 0.01(5) UW with high 6/6 31.67 ± 3.43 vs (4) NSMW PEG (5%) vs (1) NS(less insulin andother ingredients)__________________________________________________________________________ EXAMPLE 3 The ingredients of the organ preservation solutions of the invention can be varied widely, and compositions of the invention (in distilled water and sterilized by filtration through a 0.2 micron filter) have given good results in a small number of dog kidney and heart experiments, and pig heart experiments. Because of technical difficulties associated with large animal heart preservation studies, the data is qualitative rather than quantitative and involves the ability of the heart to beat vigorously and in some cases sustain the circulation of the animal for a few hours with minimum doses of cardiotonic drugs. Two dog kidneys have successfully been preserved for 48 hours using low flow perfusion, as described below. In one case, the perfusate was UW containing hydroxyethyl starch (solution of Experimental Group (1) from Example 2) and in the second the solution was the following solution containing 5% high molecular weight polyethylene glycol ("20M", Union Carbide): ______________________________________Ingredient Concentration, mM______________________________________Potassium lactobionate 100KH.sub.2 PO.sub.4 25MgSO.sub.4 5Raffinose 30NaOH.sup.1 30-40Glutathione (reduced) 3PEG, 5mol. wgt. approx. 5% by weight(15,000-20,000 daltons)______________________________________.sup.1 Added to provide a final pH 24 hours after initial formulation of the solution, in the range of 7.1-7.45. For each solution, the left kidney was removed from an anesthetized mongrel dog, immediately flushed with 150 ml of the solution and immersed in ice saline. Thereafter, a cannula was tied into the renal artery and the perfusate solution a 0° C. was perfused through the artery for 48 hours using an electrically driven syringe pump at a rate of 3 ml/Gm/24 hours. At the end of this time, the dog was again anesthetized, the contralateral kidney removed and the preserved kidney transplanted into the right side of the pelvis using conventional vascular and ureter-to bladder anastomosis techniques. Blood samples were taken daily for the following week and serum creatinine levels measured. The maximum serum creatinine in the dog receiving the kidney perfused with UW was 3.4 mg % and in the other dog receiving the kidney preserved with the high molecular weight polyethylene glycol solution was 3.0 mg %. This preliminary data suggests that the latter solution may be equally as effective as the UW solution for kidney preservation and that the low flow perfusion technique may be beneficial in the kidney as it has been shown to be for the heart. While a preferred embodiment of the present invention has been described, it should be understood that various changes, adaptations and modifications may be made therein without departing from the spirit of the invention and the scope of the appended claims.
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Summary: An organ preservation solution, particularly valuable in the preservation of mammalian hearts intended for transplantation, in aqueous solution, at least about 3% by weight of polypropylene glycol having an average molecular weight of at least about 15,000 daltons and being free of material retained by 10 micron filtration, a buffer buffering the pH of the solution perfusate to a value in the range of about 7.1 to about 7.5, and an impermeant composition for retarding the passage of water across cell membranes of an organ treated with the solution.
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Write a title and summarize: SECTION 1. DEFINITIONS FROM NUCLEAR WASTE POLICY ACT OF 1982. In this Act, the terms ``Commission'', ``disposal'', ``high-level radioactive waste'', ``repository'', ``Secretary'', ``State'', ``spent nuclear fuel'', and ``Yucca Mountain site'' have the meanings given those terms in section 2 of the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10101). SEC. 2. APPLICATION PROCEDURES AND INFRASTRUCTURE ACTIVITIES. (a) Application.--Section 114(b) of the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10134(b)) is amended-- (1) by striking ``If the President'' and inserting the following: ``(1) In general.--If the President''; and (2) by adding at the end the following ``(2) Required information.--An application for construction authorization shall not be required to contain information relating to any surface facility other than surface facilities necessary for initial operation of the repository.''. (b) Application Procedures and Infrastructure Activities.--Section 114(d) of the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10134(d)) is amended-- (1) in the first sentence, by striking ``The Commission shall consider'' and inserting the following: ``(1) In general.--The Commission shall consider''; (2) by striking the last 2 sentences; and (3) by inserting after paragraph (1) (as designated by paragraph (1)) the following: ``(2) Amendments to application for construction authorization.-- ``(A) In general.--If the Commission approves an application for construction authorization and the Secretary submits an application to amend the authorization to obtain permission to receive and possess spent nuclear fuel and high-level radioactive waste, or to undertake any other action concerning the repository, the Commission shall consider the application using expedited, informal procedures, including discovery procedures that minimize the burden on the parties to produce documents that the Commission does not need to render a decision on an action under this section. ``(B) Final decision.--The Commission shall issue a final decision on whether to grant permission to receive and possess spent nuclear fuel and high-level radioactive waste, or on any other application, by the date that is 1 year after the date of submission of the application, except that the Commission may extend that deadline by not more than 180 days if, not less than 30 days before the deadline, the Commission complies with the reporting requirements under subsection (e)(2). ``(3) Infrastructure activities.-- ``(A) In general.--At any time before or after the Commission issues a final decision on an application from the Secretary for construction authorization under this subsection, the Secretary may undertake infrastructure activities that the Secretary determines to be necessary or appropriate to support construction or operation of a repository at the Yucca Mountain site or transportation to the Yucca Mountain site of spent nuclear fuel and high level radioactive waste, including infrastructure activities such as-- ``(i) safety upgrades; ``(ii) site preparation; ``(iii) the construction of a rail line to connect the Yucca Mountain site with the national rail network, including any facilities to facilitate rail operations; and ``(iv) construction, upgrade, acquisition, or operation of electrical grids or facilities, other utilities, communication facilities, access roads, rail lines, and non-nuclear support facilities. ``(B) Compliance.-- ``(i) In general.--The Secretary shall comply with all applicable requirements under the National Environmental Policy Act of 1969 (42 U.S.C. 4321 et seq.) with respect to an infrastructure activity undertaken under this paragraph. ``(ii) EIS.--If the Secretary determines that an environmental impact statement or similar analysis under the National Environmental Policy Act of 1969 is required in connection with an infrastructure activity undertaken under this paragraph, the Secretary shall not be required to consider the need for the action, alternative actions, or a no-action alternative. ``(iii) Other agencies.-- ``(I) In general.--To the extent that a Federal agency is required to consider the potential environmental impact of an infrastructure activity undertaken under this paragraph, the Federal agency shall adopt, to the maximum extent practicable, an environmental impact statement or similar analysis prepared under this paragraph without further action. ``(II) Effect of adoption of statement.--Adoption of an environmental impact statement or similar analysis described in subclause (I) shall be considered to satisfy the responsibilities of the adopting agency under the National Environmental Policy Act of 1969 (42 U.S.C. 4321 et seq.), and no further action for the activity covered by the statement or analysis shall be required by the agency. ``(C) Denials of authorization.--The Commission may not deny construction authorization, permission to receive and possess spent nuclear fuel or high-level radioactive waste, or any other action concerning the repository on the ground that the Secretary undertook an infrastructure activity under this paragraph.''. (c) Connected Actions.--Section 114(f)(6) of the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10134(f)(6)) is amended-- (1) by striking ``or''; and (2) by inserting before the period at the end the following: ``, or an action connected or otherwise relating to the repository, to the extent the action is undertaken outside the geologic repository operations area and does not require a license from the Commission''. (d) Expedited Authorizations.--Section 120 of the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10140) is amended-- (1) in subsection (a)(1)-- (A) in the first sentence, by inserting ``, or the conduct of an infrastructure activity,'' after ``repository''; (B) by inserting ``, State, local, or tribal'' after ``Federal'' each place it appears; and (C) in the second sentence, by striking ``repositories'' and inserting ``a repository or infrastructure activity''; (2) in subsection (b), by striking ``, and may include terms and conditions permitted by law''; and (3) by adding at the end the following: ``(c) Failure to Grant Authorization.--An agency or officer that fails to grant authorization by the date that is 1 year after the date of receipt of an application or request from the Secretary subject to subsection (a) shall submit to Congress a written report that explains the reason for not meeting that deadline or rejecting the application or request. ``(d) Treatment of Actions.--For the purpose of applying any Federal, State, local, or tribal law or requirement, the taking of an action relating to a repository or an infrastructure activity shall be considered to be-- ``(1) beneficial, and not detrimental, to the public interest and interstate commerce; and ``(2) consistent with the public convenience and necessity.''. SEC. 3. REGULATORY REQUIREMENTS. (a) Material Requirements.--Notwithstanding any other provision of law, no Federal, State, interstate, or local requirement, either substantive or procedural, that is referred to in section 6001(a) of the Solid Waste Disposal Act (42 U.S.C. 6961(a)), applies to-- (1) any material owned by the Secretary, if the material is transported or stored in a package, cask, or other container that the Commission has certified for transportation or storage of that type of material; or (2) any material located at the Yucca Mountain site for disposal, if the management and disposal of the material is subject to a license issued by the Commission. (b) Permits.-- (1) In general.--The Environmental Protection Agency shall be the permitting agency for purposes of issuing, administering, or enforcing any new or existing air quality permit or requirement applicable to a Federal facility that is subject to the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10101 et seq.). (2) State and local activity.--A State or unit of local government shall not issue, administer, or enforce a new or existing air quality permit or requirement affecting a Federal facility or activity that is subject to the Nuclear Waste Policy Act of 1982 (42 U.S.C. 10101 et seq.). SEC. 4. CONFIDENCE IN AVAILABILITY OF WASTE DISPOSAL. Notwithstanding any other provision of law, in deciding whether to permit the construction or operation of a nuclear reactor or any related facilities, the Commission shall deem, without further consideration, that sufficient capacity will be available in a timely manner to dispose of the spent nuclear fuel and high-level radioactive waste resulting from the operation of the reactor and related facilities.
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Title: A bill to enhance the management and disposal of spent nuclear fuel and high-level radioactive waste, and for other purposes Summary: Amends the Nuclear Waste Policy Act of 1982 regarding Yucca Mountain site application procedures to provide that an application for construction authorization shall not be required to contain information relating to any surface facility other than those necessary for initial operation of the repository. Revises requirements governing Nuclear Regulatory Commission (NRC) actions on construction applications and infrastructure activities. Includes infrastructure activities within the scope of expedited authorizations. Declares specified federal, state, or local regulatory requirements referred to in the Solid Waste Disposal Act inapplicable to: (1) material owned by the Secretary of Energy if it is transported or stored in an NRC-certified package, cask, or other container for transportation or storage of that type of material; or (2) material located at the Yucca Mountain site for disposal, if its management and disposal is subject to an NRC license. Prohibits a state or local governmental entity from issuing, administering, or enforcing a new or existing air quality permit or requirement affecting a federal facility or activity subject to the Nuclear Waste Policy Act of 1982. Instructs the NRC, in deciding whether to permit the construction or operation of a nuclear reactor or any related facilities, to deem, without further consideration, that sufficient capacity will be available in a timely manner to dispose of the spent nuclear fuel and high-level radioactive waste resulting from the operation of the reactor and related facilities.
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Summarize: Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more (CNN) A pharmaceutical company executive defended his company's recent 400% drug price increase, telling the Financial Times that his company had a "moral requirement to sell the product at the highest price." The head of the US Food and Drug Administration blasted the executive in a response on Twitter. Nirmal Mulye, founder and president of Nostrum Pharmaceuticals, commented in a story Tuesday about the decision to raise the price of an antibiotic mixture called nitrofurantoin from about $500 per bottle to more than $2,300. The drug is listed by the World Health Organization as an "essential" medicine for lower urinary tract infections "I think it is a moral requirement to make money when you can," Mulye told the Financial Times, "to sell the product for the highest price." The Financial Times said Mulye compared his decision to increase the price to that of an art dealer who sells "a painting for half a billion dollars" and said he was in "this business to make money." According to the Financial Times, the executive defended "Pharma Bro" Martin Shkreli, who was once dubbed the "most hated man in America" after his company raised the price of an AIDS drug by more than 5,000% in 2015. Shkreli was recently sentenced to seven years in prison for fraud due to mismanaging money at his hedge funds. "I agree with Martin Shkreli that when he raised the price of his drug he was within his rights because he had to reward his shareholders," Mulye was quoted as saying. FDA Commissioner Dr. Scott Gottlieb issued a sharp rebuke of the CEO on Twitter shortly after the story published, saying "there's no moral imperative to price gouge and take advantage of patients." 1/2 Regarding @FT story today @bydavidcrow; there's no moral imperative to price gouge and take advantage of patients. FDA will continue to promote competition so speculators and those with no regard to public health consequences can't take advantage of patients who need medicine — Scott Gottlieb, M.D. (@SGottliebFDA) September 11, 2018 In comments to CNN on Wednesday, Mulye said he was not quoted accurately. "The word morality was used in the conversation, but not in the context of price increases," he said. "I said I have to raise prices when I can -- how to get the best prices for my products, so that I can survive," Mulye told CNN. "It is about the survival of the business. It has nothing to do with morality." The Financial Times said Wednesday it sticks by its story. In his remarks to CNN, Mulye went on to say he "nearly went bankrupt" twice and that he lost money after buying a plant in Ohio, which he said employs just under 100 people. "Is it moral for me to lose money?" Mulye asked. "If I don't make money, then I can't create those jobs. And where does the money go when I make it? It goes back into research and hiring new people right here in the US." He said the real villain is the "incompetent and corrupt" FDA, which he said has placed regulatory burdens on the industry, leading to higher drug prices. He said he fired off an email to Gottlieb after his tweet: "Basically, I said in a nutshell that he does not have the necessary competence to comment on the morality of drug price increases, which is a complex subject." "Honestly, Gottlieb should stay away from tweet, and he needs to stay in his office and listen to people like me and reform the agency," Mulye said. Mulye noted his drug is the generic version and less expensive than the branded version, listed at $2,800. "I'm being vilified for no good reason," he said. "It shows that the highest office at the FDA and the media doesn't understand how the drug pricing works." Get CNN Health's weekly newsletter Sign up here to get The Results Are In with Dr. Sanjay Gupta every Tuesday from the CNN Health team. The Trump administration has pledged to tackle the soaring costs of drug prices, with the president unveiling a plan in May to increase competition, reduce regulations and change incentives for players in the pharmaceutical industry. When he announced his plan, President Donald Trump slammed drug makers, health insurers, pharmacy benefit managers and others for profiting off American patients. "The drug lobby is making an absolute fortune at the expense of American patients," Trump said.
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Summary: The founder and president of a pharmaceutical company that last month raised the price of an "essential" antibiotic from $474.75 per bottle to $2,392 per bottle tells the Financial Times the 400% increase was not only justifiable but morally imperative. "I think it is a moral requirement to make money when you can... to sell the product for the highest price," says Nirmal Mulye, CEO of Nostrum Laboratories, the small Missouri-based drugmaker that makes a liquid version of nitrofurantoin. The World Health Organization lists the antibiotic mixture as an "essential" drug for lower urinary tract infections, CNN reports. Mulye says his company decided to raise its price after Casper Pharma, which makes another version of the drug, increased its own price 182% between 2015 and 2018. A bottle of that version now sells for $2,800: "The point here is the only other choice is the brand at the higher price. It is still a saving regardless of whether it is a big one or not," Mulye says, adding that he's in the pharmaceutical business "to make money." As for another former pharma exec who came under fire for a similar move, Mulye also defended Martin Shkreli during the interview. "If he's the only one selling it then he can make as much money as he can," said Mulye. The FDA commissioner responded to the interview on Twitter: "There's no moral imperative to price gouge and take advantage of patients."
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