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Summarize: Metro Nashville Police pleaded for tips from neighbors to help solve the brutal murder of a 12-year-old girl in a Goodlettsville mobile home. Yhoana Arteaga died from brutal blunt-force trauma in her mobile home at Hillview Acres last week. Police did not give details about her injuries but described the scene as 'brutal' and horrific.' "I've never seen anything this brutal in my entire career," MNPD Sgt. David Kautzman said. According to MNPD Spokesperson Don Aaron, Yhoana's last communication was with her mom. She texted her mom Thursday evening saying someone was knocking on the door. Yhoana had been at home on crutches that day after she was injured in a skating accident. After the text, her family members arrived home around 6:45 p.m. to find her dead. "Without being overly specific, there was blunt force trauma to her body and her clothing was in disarray," Don Aaron said. Due to the location of the home and Yhoana's skating injury, police believe she knew her killer. There was no obvious forced entry into Yhoana's home. Police stayed at the mobile home for hours to process the crime scene with extreme detail and placed the investigation as high priority. Metro Officers said the 911 calls for Yhoana's death are being held as evidence and will not be released. Police spoke in English and Spanish to try to encourage anyone with information to come forward about Arteaga's death. View the full news conference in Spanish below. Anyone with information should call the Emergency Communications Center at 615-862-8600 or Crime Stoppers at 615-742-7463. The Go Fund Me "Friends of Yhoana" described her as "kind, polite, caring, fun, intelligent, and beautiful she will be remembered as a wonderful daughter and great friend to all who knew her." Her school, Liberty Collegiate Academy said the community is heartbroken over Yhoana's death. GOODLETTSVILLE, Tenn. (WKRN) – Less than a week after a 12-year-old girl was found brutally murdered inside her family’s mobile home, Metro police say the case is a priority for the department. Yhoana Arteaga was found dead around 6:45 p.m. inside the Old Dickerson Pike home by family members last Thursday after her mother returned home from work with two of her siblings. Police said Yhoana’s mother last saw the girl around 12:30 p.m. while the two had lunch before she left for work. Her mother said her daughter texted her several hours later, around 5:30 p.m., saying someone was knocking on the door. Police said there have been few leads in the case, only a handful, and the department needs the public’s help finding Yhoana’s killer. “She was brutally murdered, and if anyone has any information whatsoever that could help in this investigation, please contact us,” Metro police spokesman Don Aaron said during a news conference Tuesday afternoon. According to Aaron, there was blunt force trauma to the girl’s body and her clothing was in disarray. “The killer knows how she was found. The killer knows what he did,” Aaron said, saying only the police department and the killer know certain details, which is helpful when fielding tips and information from the public. There was no obvious indication of forced entry into the mobile home and police said it is possible Yhoana may have known her killer. “At first blush, this would seem to indicate she would know the person responsible. We haven’t confirmed that yet, but it’s a good possibility,” Aaron explained. Sgt. David Kautzman added, “This is a horrible, brutal thing. I’ve never seen anything this brutal in my entire career. The police department is coming together like no case I’ve ever seen in my career.” The home was processed for hours, and it remains secured by police to preserve the crime scene in case scientists at the crime laboratory need anything else for their investigation. “We continue to hold the scene. The crime scene tape is still up. An officer is still stationed here,” Aaron told the media. A detective said they do have some persons of interest in the case, but they did not elaborate on who it may be. Police will remain in the neighborhood talking to residents about any suspicious activity they may have seen last Thursday. “This department is going to do everything we can, above and beyond, to make sure we find out who did this,” Sgt. Kautzman said. Authorities are also trying to speak with Yhoana’s friends and classmates to determine if they have any information that could help further the investigation. Anyone with information is urged to call Crime Stoppers at 615-74-CRIME. Spanish speaking operators are also available and callers can remain anonymous. Yhoana was registered at Liberty Collegiate Academy charter school. She was a student there for two years, though she had not attended this week due to injuring her leg while roller skating. Click here to visit the GoFundMe for Yhoana’s family. Yhoana Arteaga death investigation View as list View as gallery Open Gallery (Photo: WKRN) (Photo: WKRN) (Photo: WKRN) (Photo: WKRN) (Photo: WKRN) (Photo: WKRN) (Photo: WKRN) Yhoana Arteaga (Courtesy: Liberty Collegiate Academy via Facebook) (Photo: WKRN) (Photo: WKRN) (Photo: WKRN) (Photo: WKRN)

Summary: Police are begging for information from the public regarding the horrific murder of a 12-year-old girl in her family's mobile home last week in Tennessee. Fox 17 reports Yhoana Arteaga was on crutches and stuck at home in Goodlettsville last Thursday, having injured her leg in a skating accident the day before. According to WKRN, Yhoana's mother says her daughter texted her around 5:30pm to let her know someone was knocking on the door. She says she returned home from work a little over an hour later to find her daughter dead. Police, who are keeping some details private, say Yhoana died from blunt force trauma and her clothing "was in disarray," the Tennessean reports. "This is a horrible, brutal thing," Sgt. David Kautzman says. "I've never seen anything this brutal in my entire career." So far, police have received fewer tips than is typical with this type of crime, and police have few leads. "We believe there are persons in this community who have knowledge of a person who may be responsible for this," police spokesperson Don Aaron says. "Please, for the family's sake, for the sake of this 12-year-old deceased girl, let us know." Police say they believe Yhoana knew her killer, and they do have a number of persons of interest-though they are not identifying them. The family's home remains an active crime scene, and police are continuing to investigate. "This department is going to do everything we can, above and beyond, to make sure we find out who did this," Kautzman says.

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Summarize: Comedian Tamale Rocks posted a video to YouTube showing off a two-way mirror at Berwyn's Cigars and Stripes, but the bar owner said there's no "hanky panky going on." NBC Chicago's Katie Kim reports. (Published Monday, April 27, 2015) A bar owner in suburban Chicago is defending the years-long use of a two-way mirror in the women's restroom with full view of the toilet. "This is a giant funhouse, ladies and gentlemen," Ronnie Lotz told reporters Monday evening. "I put a lot of heart and soul into this business, and I am sorry to tell you this, but there is no hanky panky going on in that bathroom." The controversy surfaced when a comedian by the stage name of Tamale Rocks was performing a set at a bar in Chicago’s west suburbs when she visited the women’s restroom and noticed something strange – a full-length mirror directly across from the toilet. Rocks opened the door behind the mirror and inside found a utility closet large enough to stand in. From the other side she noticed you were able to see through the mirror and into the stall at Berwyn’s Cigars and Stripes. WATCH: Woman Discovers See-Through Mirror in Chicago Restroom A female comic discovered a full-length mirror directly opposite a private stall in the women's restroom of a bar in Chicago's west suburbs. Behind the mirror, she discovered a utility closet and a cutout behind the glass — directly viewing the toilet. (Published Monday, April 27, 2015) Shocked, Rocks recorded her findings and posted the video on YouTube. In less than 24 hours the video had garnered more than 70,000 views. "Why, when I go into a public bathroom, when there's an expectation of privacy, am I having to do that due diligence? It's a bathroom!" Rocks told NBC Chicago. Many women questioned by NBC Chicago were uncomfortable with the idea of the mirror, and some reported that they were not aware of the two-way mirror at all. The owner of Cigars & Stripes, located at 6715 Ogden Ave., Ronnie Lotz, first told Gawker’s Jezebel that they have a two-way mirror in their restroom, and it’s been there for nearly 15 years, since since 2001. In further comment to the publication, the bar's owner reportedly said the bar used to hang a witch's head in the closet as a Halloween gag so that women would look into the closet after using the restroom and "be all weirded out." He invited people to “come see my mirror; eat my chicken wings,” adding that he enjoyed the attention from those outraged by the mirror because, afterall, he's "selling chicken wings.” The bar has since shared Jezebel's article on their Facebook page. The owner told NBC Chicago that he has no plans to remove the mirror. While many are outraged, others don't see the issue. "Ronnie is a great guy and he's not doing anything wrong," said Steven Cusek, a bar patron who said he goes once a week for the last 2 years. "I'm kinda mad there's not one in the men's restroom," Cusek joked. However, Rocks and others aren't laughing. While the bar owner maintains that no recordings were made, Rocks isn't convinced that a two-way mirror with a full view of the toilet is a good idea. "I didn't see anyone, thankfully, at that point, there wasn't anyone," Rocks told NBC Chicago, "but there's nothing to say that there hadn't been in the past." The Berwyn Police Department is investigating. Tonight a reader tipped us off to a video posted by comic Tamale Rocks, who uncovered a two-way mirror in the women’s bathroom at Chicago bar Cigars and Stripes. Here’s the video, which shows the stall that has a second door equipped with a two-way mirror (on the other side is a supply closet; once you’re in it, you can clearly see whoever was/is using the toilet while in the stall): After watching, I gave the bar a call to see if they wanted to comment—and boy, did they. This all happened somewhat rapidly, with no time to get a voice recorder—luckily, I type really fucking fast and was able to capture almost everything the owner, Ronnie Lotz, had to say. The conversation really speaks for itself; the few questions I was able to edge in are in bold and italicized—insane shit Lotz (who is apparently obsessed about chicken wings) retorted with is also in bold, emphasis ours. Oh, and, he gave us his email address. You can find that below, too. (Phone rings) (A woman answers.) “Bathroom mirror, how can I help you?” Oh, excuse me, is this Cigars and Stripes? Yes it is. I’m calling in regards to a video posted about a two-way mirror in the bathroom— Hold on. (Man picks up) Hello? Hi, I’m with Jezebel, and I’m calling about the two-way mirror in your ladies’ restroom. Advertisement What does Jezebel do? I’ve never heard of her. And yes, absolutely: we have a two-way mirror in there, yes, yes; we’ve had it since 2001. Okay. So, I’m just wondering why it’s in the ladies’ room? Young lady, I have in my beer garden—I have a 10 and a half-foot hole. Did you know that it’s haunted. Did you know that a man fell dead. Do you know that we do seven days of Halloween. Do you know that mirror’s been there for the longest time. That woman opened the door. I mean she opened the door, there’s nothing in there. Open the door and pee and do what you want. Quite frankly I’m enjoying people calling, because I’m selling chicken wings. Advertisement Look, this woman is looking for a viral video. There’s a big movement in reality TV to be outraged about feminism. I’ve been in professional wrestling. So yes: come see my mirror; eat my wings—I don’t know what to tell you, honey. And you’re going to keep the mirror in there? I will burn this fucking place to the ground before I get rid of that mirror. Do you know how much joy that mirror has brought to us? We’re synonymous with Halloween. We do a freaky family fun day, and all the kids look in the mirror. This is a fun house, honey, and if you don’t like the two-way mirror, go fuck yourself; and if you come on my stage, have something to say. Everyone needs an angle. My angle is: I do barbeque; I promote it, and I break my ass for the local comics in Chicago. I pay them. I try to make them into rock stars. They do nothing, except for this. One girl said she wanted to pull her pants down and show her clitoris to me, and they all came and ate the wings. Advertisement So you’re the owner? I’ve been the goddamned fucking owner for the past 16 miserable years, blood, sweat and tears. Do you know if Lenny Bruce was alive, he’d have something to fucking say about this. I go to bed for these comics. But hey—how do you know this isn’t a publicity stunt I haven’t manufactured? You don’t, do you. You’re taking the word of someone else. How do you know I didn’t set that up? Advertisement Do you mind if I get your name? My name is Ronnie Lottz, and by the way, you’re on speaker. Remind me again: you’re with Jezebel? Is that a little blog? [LOL] Yeah, sure. It’s part of Gawker Media. But wait, what are you guys, a small entertainment company? What is Gawker? This thing would go so viral if she was smart and called me on the fucking phone, but honey, you’d get 3 million hits on YouTube. 300 views [at the time of the call] ain’t going to do shit. Advertisement I’ll give you my email address and promote the shit out of your story—a big blast; Facebook campaign. Twitter isn’t going to get on it; she needs to be a little bit more aggressive—she’s really gotta go for it. What’s your email address? cigarsandstripes@gmail.com Got it. Thanks. Look, she’s on the right track, she’s just gotta work harder. I can’t explain it to you. Everyone is looking for a viral video; you’ve gotta get one. She should have put a phone in there, or said I’m recording. What we have here is a comic with a bad set who’s looking for a viral video. She’s doing nothing with her career; she’s stuck in Chicago. We sell fucking chicken wings. They want attention. I’d love to be on the reciprocating end of this; I’ll take this to the next level. I’ve already got T-shirts being printed. Advertisement It’s like if you put something [about the mirror] into that Yelp thing: no bitch, you didn’t, somebody else did about fucking five years ago. It’s old news; the door is open; it’s for Halloween. We used to hang a witch’s head in there so that the girls would pee, and they’d look into a dingy closet and be all weirded out. We specialize in making people feel very uncomfortable. It’s why we’re successful today. If you get it, you get it. If you don’t, stay home. Screengrab courtesy YouTube Contact the author at karyn@jezebel.com A comedian who posted a video of a two-way mirror in the women's bathroom of a Berwyn club prompted a police investigation Monday, but authorities say it appears no one's privacy rights were violated. The performer, who identified herself as Tamale Rocks, took the smartphone video in the Cigars and Stripes bar, documenting her discovery of the mirror that is mounted on the door of a closet of cleaning supplies. The video showed that anyone standing inside the closet would have an unobstructed view of the women's toilet. The video quickly generated a storm of attention online and sparked the interest of Berwyn police detectives, who showed up Monday to ask questions. Club employees told police that regulars are aware of the mirror, Chief James Ritz said. During the Halloween season, the club will place a skull or mask behind the mirror and rig it so it can be seen, he said. He also said the club reported that the door is always kept unlocked, so that anyone using the bathroom can open the door and inspect the closet. But the chief said he could understand why the mirror could be a cause of concern. "Oh my God, absolutely," Ritz said. In an email to the Tribune, Rocks said she was overwhelmed by the positive response to the video and disappointed by those who didn't see the mirror as inappropriate. "The fact that a two-sided mirror EXISTS in a bathroom is NOT OK. Cover it? So that it can be made operational afterward? No. Remove it," she wrote. (Graphic language) Ron Lottz discussed allegations that a bathroom at his club, Cigars and Stripes bar, contains a two-way mirror in the women's restroom. (Graphic language) Ron Lottz discussed allegations that a bathroom at his club, Cigars and Stripes bar, contains a two-way mirror in the women's restroom. SEE MORE VIDEOS "What if your daughter, or mom or sister was in there? So there isn't someone in there right now. What if there has been? What if there will be? Get rid of the two-way mirror. How hard is this to understand?" As a few patrons arrived Monday, many defended the owner and said the two-way mirror is a longtime joke among regulars and never used inappropriately to their knowledge. Teresa Lucas said it began as a Halloween prank years ago. After a customer entered the bathroom, lights flickered, she said. Then a doll — typically Bride of Frankenstein or a vampire — lit up from behind the mirror to frighten the patron. Lucas said the door is never locked and patrons can tell it's a two-way mirror from either side. "It wasn't something that was hidden," Lucas said. "It was used as a gimmick, not as a Peeping Tom." The bar's owner, who identified himself as Ron Lottz, allowed reporters and photographers inside his bar late Monday to check out the infamous mirror. A woman discovers a two-way mirror at a comedy club in Berwyn. A woman discovers a two-way mirror at a comedy club in Berwyn. SEE MORE VIDEOS Inside the tiny one-stall women's bathroom, the interior of the closet is clearly visible when a patron looks into the mirror. The small closet does not lock from the inside and has a light. It is the only women's bathroom in the facility. Lottz described his eclectic bar as a "giant fun house," complete with horror-inspired decor and tales of haunted sightings, where the house specialty is BBQ wings and rib tips. Lottz said police searched "every nook and cranny" of his business and found nothing inappropriate. He insisted the door — with the two-way mirror cutout — will stay and said no one has ever complained before. "There is no hanky-panky going on in that bathroom," he said. "It's part of the gag. It's just old-school fun house fun." A female bartender, Kelly McElroy, argued it was much ado about nothing. "I think it's a little unsettling to the business," said McElroy, 37, of Naperville. "Social media has taken it way too far and made it into something it's not. People have too much time on their hands." Ward is a freelance reporter. Gutowski is a Tribune reporter.

Summary: The two-way mirror in the women's bathroom of a bar in suburban Chicago is only there for harmless Halloween pranks and it's not going anywhere, vows the owner of Cigars & Stripes. Ronnie Lottz's bar started getting attention when comedian Tamale Rocks posted a video on YouTube of the mirror, which would give anyone concealed in the utility closet behind it a full view of the toilet, NBC Chicago reports. Lottz tells the Chicago Tribune that cops searched the bar after receiving a complaint and found nothing wrong. "There is no hanky-panky going on in that bathroom," he says. "It's part of the gag. It's just old-school fun house fun." A police spokesman tells the Tribune that according to employees, regulars are aware of the mirror, and at Halloween, a hidden doll or mask behind it is lit up to scare people. When contacted by Jezebel, Lottz said the attention is helping him sell chicken wings and he would burn the place down before he got rid of the mirror. "I have in my beer garden-I have a 10-and-a-half-foot hole. Did you know that it's haunted? Did you know that a man fell dead?" he asked. "Do you know that we do seven days of Halloween? Do you know that mirror's been there for the longest time?"

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Summarize: By. David Williams. and Nick Fagge In Gyor, Hungary. Straddling a powerful motorbike in his leathers, this is the first picture of the ex-Hungarian policeman named as a suspect in the brutal murder of a former British model and friend of John Lennon at the remote animal sanctuary she ran. The family of Eva Rhodes gave the name of officer Zoltan Peter Horvath to her London inquest – claiming he had threatened to have her ‘eliminated’ – and told how he had once attacked the animal lover before beating her daughter so badly she feared she would die. But on Sunday the shaven-headed 47-year-old former officer dismissed the dead Briton’s family and their allegations, declaring : ‘Eva’s sister is just a mean b****, so is her daughter.’ Suspect: Hungarian ex-policeman Zoltan Peter. Horvath, pictured, denies murdering Eva Rhodes at the remote animal. sanctuary she ran in Gyor, north-west Hungary, despite claims from her. family. In chilling evidence to Westminster Coroner Dr Fiona Wilcox, members of Rhodes’s family alleged they believed Horvath was linked to both the death of the 65-year-old and an extensive cover-up of the killing at the sanctuary in Gyor, north-west Hungary, where her body was dismembered and set ablaze in September 2008. Speaking for the first time yesterday, there was only contempt from Horvath for Mrs Rhodes and her family, mocking them by suggesting the sanctuary – partly funded by donations from the UK and money given by Lennon’s widow Yoko Ono – had been used to run drugs and guns hidden inside dogs. ‘Of course I didn’t kill her,’ he said dismissively. ‘I was representing the law. I cannot be linked to her death. Celebrity: Animal lover Eva Rhodes, left, was. murdered in Hungary in 2008, and found fame in the 1960s as a model and. socialite. An inquest into her death held in London recorded a verdict of unlawful killing. ‘The only reason this case hasn’t died off yet is because she was English.’ He pointed out that Csaba Augusztinyi, 33, who worked at the sanctuary, had confessed and been sentenced to 13 years behind bars for the killing. Recording a verdict of unlawful killing, Dr Wilcox had said that while she was satisfied Augusztinyi was ‘at least partly responsible’, inconsistencies in evidence supported the belief that ‘others might have been involved’. This belief is strongly held by Rhodes’s sister Judith Majlath – a Nobel peace prize winner ¬– and daughter Sophia Barta. Eva Rhodes pictured in Tuscany (left). The. 65-year-old disappeared in September 2008 from her home in Gyor, near. Budapest in Hungary. Caretaker Csaba Augusztinyi admitted killing the. former model and socialite. Inquest: Eva Rhodes' daughter Sophia Barta (left) and Eva's sister Judith Majlath (right) at Westminster Coroner's Court during the inquest. The family has called on Scotland Yard to be allowed to investigate the death after the inquest heard of a disturbing conflict in the evidence of investigators opening the way for Horvath to be potentially questioned by British officers. Horvath insisted: ‘I’m also sure that when the authorities want to hear my side of the story I will be properly notified. But until then I cannot take these accusations seriously.’ The inquest was told that Horvath had never been questioned about the killing and had even taken part in the police investigation. But he stressed: ‘I have been questioned about this Eva Rhodes case so many times and I have had enough of it. I just want to leave it behind me.’ He accused Mrs Rhodes of using her animal sanctuary as a cover to ‘smuggle drugs and guns’ into Hungary from Austria. Friendship: Eva Rhodes became friends John Lennon and Yoko Ono, whose cash eventually helped pay for her animal sanctuary. Beauty: Ms Rhodes was a successful model and actress before moving to Hungary to look after animals. He claimed: ‘I filed reports about Eva’s suspicious activities but my superiors did not let me investigate properly. ‘She was collecting large dogs from the west to rehabilitate them after they were neutered, she claimed, but there was no rehabilitation. ‘Instead she burned the dogs at dawn. ‘How mysterious, they always ended up dying. I was never allowed to investigate. ‘What was she hiding inside the dogs – drugs, guns?’ He said Mrs Rhodes had refused to let him search her car. The cats and dogs sanctuary was funded partly by donations from animal lovers in England and a gift from Yoko Ono. Horrific crime: Ms Rhodes was murdered at her animal sanctuary in September 2008 near Gyor, 65-miles from Budapest. Her body was dismembered and burnt. ‘I stopped Eva in the middle of the night on a back road. She had been driving without her headlights on. She was clearly drunk. There were empty champagne bottles in the car and some big dogs with her. ‘I wanted to see the dogs, whether they were alive, but instead of opening the car, she just drove off. I had to jump out of the way so I wasn’t run over. Again, I was not allowed to investigate. ‘I’m sure there is a whole lot more to this story than what has been made public. But I do not want to get involved with the case ever again.’ Horvath left the police when investigations into the death were on-going – the family claim he was ‘pensioned off’ – and he went to live in Bony, a dust-blown country village off the main truck road to Vienna, near the town of Gyor. Star: Eva Rhodes being chased by the press in London at the height of her fame in the 1960s. Following the break-up of his marriage, he moved to neighbouring Slovakia where he set up home with his girlfriend in one of the many Hungarian-speaking villages on the north side of the river Danube. The pair worked together in a hotel. While he was happy to taunt those closest to Mrs Rhodes, he said nothing about his attack on the grandmother and Ms Barta – or the court ruling against him and legal wrangling that was on-going at the time of the murder in connection with the assaults. Ms Barta, from Chelsea, west London, told the inquest how the policeman had been responsible for a ‘Rambo’ style attack on her and her mother at the sanctuary in 2002. Crime: Caretaker Csaba Augusztinyi admitted killing Mrs Rhodes and setting. fire to her body, but her daughter Judith Majlath believes that the Hungarian. authorities covered up the investigation to protect a local officer. A dispute over a rescue dog had grown, she said, so that at one point the officer had attacked her mother in the courtyard and then turned on Ms Barta when she tried to photograph the assault. She said her head was ‘smashed’ against the wall and Horvath then hit her repeatedly with his baton. 'I crumpled into a heap and he began raining blows on me with his baton - an American baton designed to break bones,’ she said. 'I turned my head and looked at his face and his eyes were that of somebody taking great pleasure in what he was doing. ‘I thought I was going to die.’ Eventually, Mrs Rhodes calmed him, she recalled, but not before he had ‘crushed like a pancake’ a grey kitten with his foot that had been beside her head.’ The remains of Eva Rhodes was found in woods near her home (pictured) It was this incident and his subsequent ‘humiliation’ in court, the family believes, that lead to an on-going feud which ended in death. Last night a furious Ms Majlath hit back at Horvath calling his comments ‘cruel and outrageous.’ ‘It shows the quality of this man that he would say such things about the dead, about a woman who was murdered, butchered and burned on his patch, the very patch he was meant to police. ‘Like others in authority in Hungary, he has questions to answer – he had damned himself, what more does he need to do to the memory of a dead woman, whose only crime was to love animals and be prepared to stand-up to him? ‘With the evidence of the inquest and now these words, surely Hungary and Prime Minister Viktor Orban, who was in London last week given a speech on European Traditional Values, can’t continue to refuse Scotland Yard and Foreign Secretary William Hague’s call for all documents relating to the case to be handed over to Britain.’ She added: ‘Scotland Yard must be called in. The longer they delay, the more people will ask what Hungary is hiding.’ Mrs Rhodes, attracted the attention and friendship of John Lennon and Yoko Ono in the 1960s when the famous couple were looking to cast an actress in a film project

Summary: Eva Rhodes found dead in 2008 at the animal sanctuary she ran. Caretaker Csaba Augusztinyi, 33, confessed and was jailed for 13 years. Family believe others were involved in the brutal killing in Gyor, Hungary. Inquest in London hears her daughter believes police had a hand in death. Westminster coroner Dr Fiona Wilcox returns a verdict of unlawful killing. But Dr Wilcox said there is evidence 'others might have been involved' Family name ex-policeman Zoltan Peter Horvath as the new prime suspect. Pressure mounts for Scotland Yard to be allowed to investigate the death.

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Write a title and summarize: SECTION 1. TECHNICAL CORRECTIONS. (a) Advice of Counsel.--Notwithstanding section 35 of the Leahy- Smith America Invents Act (35 U.S.C. 1 note), section 298 of title 35, United States Code, shall apply to any civil action commenced on or after the date of the enactment of this Act. (b) Transitional Program for Covered Business Method Patents.-- Section 18 of the Leahy-Smith America Invents Act (35 U.S.C. 321 note) is amended-- (1) in subsection (a)(1)(C)(i), by striking ``of such title'' the second place it appears; and (2) in subsection (d)(2), by striking ``subsection'' and inserting ``section''. (c) Joinder of Parties.--Section 299(a) of title 35, United States Code, is amended in the matter preceding paragraph (1) by striking ``or counterclaim defendants only if'' and inserting ``only if''. (d) Dead Zones.-- (1) Inter partes review.--Section 311(c) of title 35, United States Code, shall not apply to a petition to institute an inter partes review of a patent that is not a patent described in section 3(n)(1) of the Leahy-Smith America Invents Act (35 U.S.C. 100 note). (2) Reissue.--Section 311(c)(1) of title 35, United States Code, is amended by striking ``or issuance of a reissue of a patent''. (e) Correct Inventor.-- (1) In general.--Section 135(e) of title 35, United States Code, as amended by section 3(i) of the Leahy-Smith America Invents Act, is amended by striking ``correct inventors'' and inserting ``correct inventor''. (2) Effective date.--The amendment made by paragraph (1) shall be effective as if included in the amendment made by section 3(i) of the Leahy-Smith America Invents Act. (f) Inventor's Oath or Declaration.--Section 115 of title 35, United States Code, as amended by section 4 of the Leahy-Smith America Invents Act, is amended-- (1) by striking subsection (f) and inserting the following: ``(f) Time for Filing.--The applicant for patent shall provide each required oath or declaration under subsection (a), substitute statement under subsection (d), or recorded assignment meeting the requirements of subsection (e) no later than the date on which the issue fee for the patent is paid.''; and (2) in subsection (g)(1), by striking ``who claims'' and inserting ``that claims''. (g) Travel Expenses and Payment of Administrative Judges.-- Notwithstanding section 35 of the Leahy-Smith America Invents Act (35 U.S.C. 1 note), the amendments made by section 21 of the Leahy-Smith America Invents Act (Public Law 112-29; 125 Stat. 335) shall be effective as of September 16, 2011. (h) Patent Term Adjustments.--Section 154(b) of title 35, United States Code, is amended-- (1) in paragraph (1)-- (A) in subparagraph (A)(i)(II), by striking ``on which an international application fulfilled the requirements of section 371 of this title'' and inserting ``of commencement of the national stage under section 371 in an international application''; and (B) in subparagraph (B), in the matter preceding clause (i), by striking ``the application in the United States'' and inserting ``the application under section 111(a) in the United States or, in the case of an international application, the date of commencement of the national stage under section 371 in the international application''; (2) in paragraph (3)(B)(i), by striking ``with the written notice of allowance of the application under section 151'' and inserting ``no later than the date of issuance of the patent''; and (3) in paragraph (4)(A)-- (A) by striking ``a determination made by the Director under paragraph (3) shall have remedy'' and inserting ``the Director's decision on the applicant's request for reconsideration under paragraph (3)(B)(ii) shall have exclusive remedy''; and (B) by striking ``the grant of the patent'' and inserting ``the date of the Director's decision on the applicant's request for reconsideration''. (i) Improper Applicant.--Section 373 of title 35, United States Code, and the item relating to that section in the table of sections for chapter 37 of such title, are repealed. (j) Financial Management Clarifications.--Section 42(c)(3) of title 35, United States Code, is amended-- (1) in subparagraph (A)-- (A) by striking ``sections 41, 42, and 376,'' and inserting ``this title,''; and (B) by striking ``a share of the administrative costs of the Office relating to patents'' and inserting ``a proportionate share of the administrative costs of the Office''; and (2) in subparagraph (B), by striking ``a share of the administrative costs of the Office relating to trademarks'' and inserting ``a proportionate share of the administrative costs of the Office''. (k) Derivation Proceedings.-- (1) In general.--Section 135(a) of title 35, United States Code, as amended by section 3(i) of the Leahy-Smith America Invents Act, is amended to read as follows: ``(a) Institution of Proceeding.-- ``(1) In general.--An applicant for patent may file a petition with respect to an invention to institute a derivation proceeding in the Office. The petition shall set forth with particularity the basis for finding that an individual named in an earlier application as the inventor or a joint inventor derived such invention from an individual named in the petitioner's application as the inventor or a joint inventor and, without authorization, the earlier application claiming such invention was filed. Whenever the Director determines that a petition filed under this subsection demonstrates that the standards for instituting a derivation proceeding are met, the Director may institute a derivation proceeding. ``(2) Time for filing.--A petition under this section with respect to an invention that is the same or substantially the same invention as a claim contained in a patent issued on an earlier application, or contained in an earlier application when published or deemed published under section 122(b), may not be filed unless such petition is filed during the 1-year period following the date on which the patent containing such claim was granted or the earlier application containing such claim was published, whichever is earlier. ``(3) Earlier application.--For purposes of this section, an application shall not be deemed to be an earlier application with respect to an invention, relative to another application, unless a claim to the invention was or could have been made in such application having an effective filing date that is earlier than the effective filing date of any claim to the invention that was or could have been made in such other application. ``(4) No appeal.--A determination by the Director whether to institute a derivation proceeding under paragraph (1) shall be final and not appealable.''. (2) Effective date.--The amendment made by paragraph (1) shall be effective as if included in the amendment made by section 3(i) of the Leahy-Smith America Invents Act. (3) Review of interference decisions.--The provisions of sections 6 and 141 of title 35, United States Code, and section 1295(a)(4)(A) of title 28, United States Code, as in effect on September 15, 2012, shall apply to interference proceedings that are declared after September 15, 2012, under section 135 of title 35, United States Code, as in effect before the effective date under section 3(n) of the Leahy-Smith America Invents Act. The Patent Trial and Appeal Board may be deemed to be the Board of Patent Appeals and Interferences for purposes of such interference proceedings. (l) Patent and Trademark Public Advisory Committees.-- (1) In general.--Section 5(a) of title 35, United States Code, is amended-- (A) in paragraph (1), by striking ``Members of'' and all that follows through ``such appointments.'' and inserting the following: ``In each year, 3 members shall be appointed to each Advisory Committee for 3-year terms that shall begin on December 1 of that year. Any vacancy on an Advisory Committee shall be filled within 90 days after it occurs. A new member who is appointed to fill a vacancy shall be appointed to serve for the remainder of the predecessor's term.''; (B) by striking paragraph (2) and inserting the following: ``(2) Chair.--The Secretary of Commerce, in consultation with the Director, shall designate a Chair and Vice Chair of each Advisory Committee from among the members appointed under paragraph (1). If the Chair resigns before the completion of his or her term, or is otherwise unable to exercise the functions of the Chair, the Vice Chair shall exercise the functions of the Chair.''; and (C) by striking paragraph (3). (2) Transition.-- (A) In general.--The Secretary of Commerce shall, in the Secretary's discretion, determine the time and manner in which the amendments made by paragraph (1) shall take effect, except that, in each year following the year in which this Act is enacted, 3 members shall be appointed to each Advisory Committee (to which such amendments apply) for 3-year terms that begin on December 1 of that year, in accordance with section 5(a) of title 35, United States Code, as amended by paragraph (1) of this subsection. (B) Deemed termination of terms.--In order to implement the amendments made by paragraph (1), the Secretary of Commerce may determine that the term of an existing member of an Advisory Committee under section 5 of title 35, United States Code, shall be deemed to terminate on December 1 of a year beginning after the date of the enactment of this Act, regardless of whether December 1 is before or after the date on which such member's term would terminate if this Act had not been enacted. (m) Clerical Amendment.--Section 123(a) of title 35, United States Code, is amended in the matter preceding paragraph (1) by inserting ``of this title'' after ``For purposes''. (n) Effective Date.--Except as otherwise provided in this Act, the amendments made by this Act shall take effect on the date of enactment of this Act, and shall apply to proceedings commenced on or after such date of enactment. Speaker of the House of Representatives. Vice President of the United States and President of the Senate.

Title: To correct and improve certain provisions of the Leahy-Smith America Invents Act and title 35, United States Code Summary: (This measure has not been amended since it was passed by the Senate on December 28, 2012. The summary of that version is repeated here.) Amends the Leahy-Smith America Invents Act (AIA) to make technical changes regarding the transitional program for covered business method patents and joinder of parties. Applies, to any civil action commenced on or after enactment of this Act, the AIA's bar on using an accused infringer's failure to obtain the advice of counsel to prove that any infringement was willful or induced. (Currently, the bar would not take effect until one year after the AIA's enactment.) Prohibits a nine-month waiting period for inter partes review from applying to issuances of first-to-invent patents ineligible for post-grant review. (Currently, the remaining first-to-invent patents to be issued prior to the effective date of the new first-to-file patent system have no avenue for review during the first nine months because they must wait nine months for inter parties review and are ineligible for post-grant review.) Revises the filing deadline for inter partes review to be after the later of either: (1) nine months after the grant of a patent (currently, nine months after such grant or the issuance of a reissue patent), or (2) the termination date of any post-grant review. Extends the time period for an applicant to file an inventor's oath or declaration, substitute statement, or recorded assignment until the date on which the issue fee for the patent is paid. (Current law permits a notice of allowance of a patent application only after such a filing.) Makes provisions concerning travel expenses for employees of the U.S. Patent and Trademark Office (USPTO) and the payment of administrative judges effective as of September 16, 2011. Modifies requirements and time periods for activities relating to patent term adjustments. Revises the patent extension period for certain international applications. Specifies that a civil action filed in the U.S. District Court for the Eastern District of Virginia is the exclusive remedy for challenging a USPTO decision on a request for reconsideration of a patent term adjustment determination. Repeals a provision prohibiting the USPTO from accepting certain international applications designating the United States from anyone not qualified under specified application requirements. Revises USPTO funding requirements to make all federal patent law fees available for expenses relating to patent processing and to permit patent and trademark fees to be used interchangeably to cover proportionate shares of the USPTO's administrative costs. (Currently, patent fees are used to cover administrative costs relating to patents while trademark fees are used to cover administrative costs relating to trademarks.) Modifies requirements for applicants filing petitions to institute derivation proceedings and delineates the criteria applied to deem an application as an earlier application with respect to an invention relative to another application. Sets forth authority for the Patent Trial and Appeal Board to conduct, and for courts to review appeals from, interference proceedings declared after enactment but before the effective date of certain AIA amendments replacing interference proceedings with derivation proceedings. Modifies the original appointment terms for members of the Patent Public Advisory Committee and the Trademark Public Advisory Committee. Directs the Secretary of Commerce to designate, from among the appointed members, a Chair and Vice Chair of each committee.

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Summarize: Introduction Federal financial regulation encompasses vastly diverse markets, participants, and regulators. As a result, regulators' goals, powers, and methods differ between regulators and sometimes within each regulator's jurisdiction. This report provides background on the financial regulatory structure in order to help Congress evaluate specific policy proposals to change financial regulation. Historically, financial regulation in the United States has coevolved with a changing financial system, in which major changes are made in response to crises. For example, in response to the financial turmoil beginning in 2007, the Dodd-Frank Wall Street Reform and Consumer Protection Act in 2010 (Dodd-Frank Act; P.L. 111-203 ) was the last act to make significant changes to the financial regulatory structure (see Appendix A ). Congress continues to debate proposals to modify parts of the regulatory system established by the Dodd-Frank Act. In the 115 th Congress, proposals to change the regulatory structure include the Financial CHOICE Act of 2017 ( H.R. 10 ), which would modify and rename the Consumer Financial Protection Bureau (CFPB) and the Federal Insurance Office (FIO), change the relationship between the Financial Stability Oversight Council (FSOC) and the regulators, eliminate the Office of Financial Research (OFR, which supports FSOC), and make other changes to how financial regulators promulgate rules and are funded. This report attempts to set out the basic frameworks and principles underlying U.S. financial regulation and to give some historical context for the development of that system. The first section briefly discusses the various modes of financial regulation. The next section identifies the major federal regulators and the types of institutions they supervise (see Table 1 ). It then provides a brief overview of each federal financial regulatory agency. Finally, the report discusses other entities that play a role in financial regulation—interagency bodies, state regulators, and international standards. For information on how the regulators are structured and funded, see CRS Report R43391, Independence of Federal Financial Regulators: Structure, Funding, and Other Issues, by [author name scrubbed], [author name scrubbed], and [author name scrubbed]. The Financial System The financial system matches the available funds of savers and investors with borrowers and others seeking to raise funds in exchange for future payouts. Financial firms link individual savers and borrowers together. Financial firms can operate as intermediaries that issue obligations to savers an d use those funds to make loans or investments for the firm's profits. Financial firms can also operate as agents playing a custodial role, investing the funds of savers on their behalf in segregated accounts. The products, instruments, and markets used to facilitate this matching are myriad, and they are controlled and overseen by a complex system of regulators. To help understand how the financial regulators have been organized, financial activities can be separated into distinct markets: Banking —accepting deposits and making loans; Insurance —collecting premiums from and making payouts to policyholders triggered by a predetermined event; Securities —issuing contracts that pledge to make payments from the issuer to the holder, and trading those contracts on markets. Contracts take the form of debt (a borrower and creditor relationship) and equity (an ownership relationship). One special class of securities is derivatives, which are financial contracts whose value is based on an underlying commodity, financial indicator, or financial instrument; and Financial Market Infrastructure —the "plumbing" of the financial system, such as trade data dissemination, payment, clearing, and settlement systems, that underlies transactions. A distinction can be made between formal and functional definitions of these activities. Formal activities are those, as defined by regulation, exclusively permissible for entities based on their charter or license. By contrast, functional definitions acknowledge that from an economic perspective, activities performed by types of different entities can be quite similar. This tension between formal and functional activities is one reason why the Government Accountability Office (GAO), and others, has called the U.S. regulatory system fragmented, with gaps in authority, overlapping authority, and duplicative authority. For example, "shadow banking" refers to activities, such as lending and deposit taking, that are economically similar to those performed by formal banks, but occur in the securities markets. The activities described above are functional definitions. However, because this report focuses on regulators, it mostly concentrates on formal definitions. The Role of Financial Regulators Financial regulation has evolved over time, with new authority usually added in response to failures or breakdowns in financial markets and authority trimmed back during financial booms. Because of this piecemeal evolution, powers, goals, tools, and approaches vary from market to market. Nevertheless, there are some common overarching themes across markets and regulators, which are highlighted in this section. The following provides a brief overview of what financial regulators do, specifically answering four questions: 1. What powers do regulators have? 2. What policy goals are regulators trying to accomplish? 3. Through what means are those goals accomplished? 4. Who or what is being regulated? Regulatory Powers Regulators implement policy using their powers, which vary by agency. Powers can be grouped into a few broad categories: Licensing, Chartering, or Registration. A starting point for understanding the regulatory system is that most activities cannot be undertaken unless a firm, individual, or market has received the proper credentials from the appropriate state or federal regulator. Each type of charter, license, or registration granted by the respective regulator governs the sets of financial activities that the holder is permitted to engage in. For example, a firm cannot accept federally insured deposits unless it is chartered as a bank, thrift, or credit union by a depository institution regulator. Likewise, an individual generally cannot buy and sell securities to others unless licensed as a broker-dealer. To be granted a license, charter, or registration, the recipient must accept the terms and conditions that accompany it. Depending on the type, those conditions could include regulatory oversight, training requirements, and a requirement to act according to a set of standards or code of ethics. Failure to meet the terms and conditions could result in fines, penalties, remedial actions, license or charter revocation, or criminal charges. Rulemaking. Regulators issue rules (regulations) through the rulemaking process to implement statutory mandates. Typically, statutory mandates provide regulators with a policy goal in general terms, and regulations fill in the specifics. Rules lay out the guidelines for how market participants may or may not act to comply with the mandate. Oversight and Supervision. Regulators ensure that their rules are adhered to through oversight and supervision. This allows regulators to observe market participants' behavior and instruct them to modify or cease improper behavior. Supervision may entail active, ongoing monitoring (as for banks) or investigating complaints and allegations ex post (as is common in securities markets). In some cases, such as banking, supervision includes periodic examinations and inspections, whereas in other cases, regulators rely more heavily on self-reporting. Regulators explain supervisory priorities and points of emphasis by issuing supervisory letters and guidance. Enforcement. Regulators can compel firms to modify their behavior through enforcement powers. Enforcement powers include the ability to issue fines, penalties, and cease and desist orders; to undertake criminal or civil actions in court, or administrative proceedings or arbitrations; and to revoke licenses and charters. In some cases, regulators initiate legal action at their own bequest or in response to consumer or investor complaints. In other cases, regulators explicitly allow consumers and investors to sue for damages when firms do not comply with regulations, or provide legal protection to firms that do comply. Resolution. Some regulators have the power to resolve a failing firm by taking control of the firm and initiating conservatorship (i.e., the regulator runs the firm on an ongoing basis) or receivership (i.e., the regulator winds the firm down). In other markets, failing firms are resolved through bankruptcy, a judicial process. Goals of Regulation Financial regulation is primarily intended to achieve the following underlying policy outcomes: Market Efficiency and Integrity. Regulators are to ensure that markets operate efficiently and that market participants have confidence in the market's integrity. Liquidity, low costs, the presence of many buyers and sellers, the availability of information, and a lack of excessive volatility are examples of the characteristics of an efficient market. Regulators contribute to market integrity by ensuring that activities are transparent, contracts can be enforced, and the "rules of the game" they set are enforced. Integrity generally also leads to greater efficiency. Regulation can also address market failures, such as principal-agent problems, asymmetric information, and moral hazard, that would otherwise reduce market efficiency. Consumer and Investor Protection. Regulators are to ensure that consumers or investors do not suffer from fraud, discrimination, manipulation, and theft. Regulators try to prevent exploitative or abusive practices intended to take advantage of unwitting consumers or investors. In some cases, protection is limited to enabling consumers and investors to understand the inherent risks when they enter into a contract. In other cases, protection is based on the principle of suitability—efforts to ensure that more risky products or product features are only accessible to sophisticated or financially secure consumers and investors. Capital Formation and Access to Credit. Regulators are to ensure that firms and consumers are able to access credit and capital to meet their needs such that credit and economic activity can grow at a healthy rate. Regulators try to ensure that capital and credit are available to all worthy borrowers, regardless of personal characteristics, such as race, gender, and location. Illicit Activity Prevent ion. Regulators are to ensure that the financial system cannot be used to support criminal and terrorist activity. Examples are policies to prevent money laundering, tax evasion, terrorism financing, and the contravention of financial sanctions. Taxpayer Protection. Regulators are to ensure that losses or failures in financial markets do not result in federal government payouts or the assumption of liabilities that are ultimately borne by taxpayers. Only certain types of financial activity are explicitly backed by the federal government or by regulator-run insurance schemes that are backed by the federal government, such as the Deposit Insurance Fund (DIF) run by the Federal Deposit Insurance Corporation (FDIC). Such schemes are self-financed by the insured firms through premium payments, unless the losses exceed the insurance fund, and then taxpayer money is used temporarily or permanently to fill the gap. In the case of a financial crisis, the government may decide that the "least bad" option is to provide funds in ways not explicitly promised or previously contemplated to restore stability. "Bailouts" of large failing firms in 2008 are the most well-known examples. In this sense, there may be implicit taxpayer backing of parts or all of the financial system. Financial Stability. Financial regulation is to maintain financial stability through preventative and palliative measures that mitigate systemic risk. At times, financial markets stop functioning well—markets freeze, participants panic, credit becomes unavailable, and multiple firms fail. Financial instability can be localized (to a specific market or activity) or more general. Sometimes instability can be contained and quelled through market actions or policy intervention; at other times, instability metastasizes and does broader damage to the real economy. The most recent example of the latter was the financial crisis of 2007-2009. Traditionally, financial stability concerns have centered on banking, but the recent crisis illustrates the potential for systemic risk to arise in other parts of the financial system as well. These regulatory goals are sometimes complementary, but other times conflict with each other. For example, without an adequate level of consumer and investor protections, fewer individuals may be willing to participate in the markets, and efficiency and capital formation could suffer. But, at some point, too many consumer and investor safeguards and protections could make credit and capital prohibitively expensive, reducing market efficiency and capital formation. Regulation generally aims to seek a middle ground between these two extremes, where regulatory burden is as small as possible and regulatory benefits are as large as possible. As a result, when taking any action, regulators balance the tradeoffs between their various goals. Types of Regulation The types of regulation applied to market participants are diverse and vary by regulator, but can be clustered in a few categories for analytical ease: Prudential. The purpose of prudential regulation is to ensure an institution's safety and soundness. It focuses on risk management and risk mitigation. Examples are capital requirements for banks. Prudential regulation may be pursued to achieve the goals of taxpayer protection (e.g., to ensure that bank failures do not drain the DIF), consumer protection (e.g., to ensure that insurance firms are able to honor policyholders' claims), or financial stability (e.g., to ensure that firm failures do not lead to bank runs). Disclosure and R eporting. Disclosure and reporting requirements are meant to ensure that all relevant financial information is accurate and available to the public and regulators so that the former can make well-informed financial decisions and the latter can effectively monitor activities. For example, publicly held companies must file disclosure reports, such as 10-Ks. Disclosure is used to achieve the goals of consumer and investor protection, as well as market efficiency and integrity. S tandard S etting. Regulators prescribe standards for products, markets, and professional conduct. Regulators set permissible activities and behavior for market participants. Standard setting is used to achieve a number of policy goals. For example, (1) for market integrity, policies governing conflicts of interest, such as insider trading; (2) for taxpayer protection, limits on risky activities; (3) for consumer protection, fair lending requirements to prevent discrimination; and (4) for suitability, limits on the sale of certain sophisticated financial products to accredited investors and verification that borrowers have the ability to repay mortgages. Competition. Regulators ensure that firms do not exercise undue monopoly power, engage in collusion or price fixing, or corner specific markets (i.e., take a dominant position to manipulate prices). Examples include antitrust policy (which is not unique to finance), antimanipulation policies, concentration limits, and the approval of takeovers and mergers. Regulators promote competitive markets to support the goals of market efficiency and integrity and consumer and investor protections. Within this area, a special policy concern related to financial stability is ensuring that no firm is "too big to fail." Price and R ate R egulat ions. Regulators set maximum or minimum prices, fees, premiums, or interest rates. Although price and rate regulation is relatively rare in federal regulation, it is more common in state regulation. An example at the federal level is the Durbin Amendment, which caps debit interchange fees for large banks. State-level examples are state usury laws, which cap interest rates, and state insurance rate regulation. Policymakers justify price and rate regulations on grounds of consumer and investor protections. Prudential and disclosure and reporting regulations can be contrasted to highlight a basic philosophical difference in the regulation of securities versus banking. For example, prudential regulation is central to banking regulation, whereas securities regulation generally focuses on disclosure. This difference in approaches can be attributed to differences in the relative importance of regulatory goals. Financial stability and taxpayer protection are central to banking because of taxpayer exposure and the potential for contagion when firms fail, whereas they are not primary goals of securities markets because the federal government has made few explicit promises to make payouts in the event of losses and failures. Prudential regulation relies heavily on confidential information that supervisors gather and formulate through examinations. Federal securities regulation is not intended to prevent failures or losses; instead, it is meant to ensure that investors are properly informed about the risks that investments pose. Ensuring proper disclosure is the main way to ensure that all relevant information is available to any potential investor. Regulated Entities How financial regulation is applied varies, partly because of the different characteristics of various financial markets and partly because of the historical evolution of regulation. The scope of regulators' purview falls into several different categories: 1. Regulate Certain Types of Financial Institutions. Some firms become subject to federal regulation when they obtain a particular business charter, and several federal agencies regulate only a single class of institution. Depository institutions are a good example: a new banking firm chooses its regulator when it decides which charter to obtain—national bank, state bank, credit union, etc.—and the choice of which type of depository charter may not greatly affect the institution's business mix. The Federal Housing Finance Authority (FHFA) regulates only three government-sponsored enterprises (GSEs): Fannie Mae, Freddie Mac, and the Federal Home Loan Bank system. This type of regulation gives regulators a role in overseeing all aspects of the firm's behavior, including which activities it is allowed to engage in. As a result, "functionally similar activities are often regulated differently depending on what type of institution offers the service." 2. Regulate a Particular Market. In some markets, all activities that take place within that market (for example, on a securities exchange) are subject to regulation. Often, the market itself, with the regulator's approval, issues codes of conduct and other internal rules that bind its members. In some cases, regulators may then require that specific activities can be conducted only within a regulated market. In other cases, similar activities take place both on and off a regulated market (e.g., stocks can also be traded off-exchange in "dark pools"). In some cases, regulators have different authority or jurisdiction in primary markets (where financial products are initially issued) than secondary markets (where products are resold). 3. Regulate a Particular Financial Activity. If activities are conducted in multiple markets or across multiple types of firms, another approach is to regulate a particular type or set of transactions, regardless of where the business occurs or which entities are engaged in it. For example, on the view that consumer financial protections should apply uniformly to all transactions, the Dodd-Frank Act created the CFPB, with authority (subject to certain exemptions) over financial products offered to consumers by an array of firms. Because it is difficult to ensure that functionally similar financial activities are legally uniform, all three approaches are needed and sometimes overlap in any given area. Stated differently, risks can emanate from firms, markets, or products, and if regulation does not align, risks may not be effectively mitigated. Market innovation also creates financial instruments and markets that fall between industry divisions. Congress and the courts have often been asked to decide which agency has jurisdiction over a particular financial activity. The Federal Financial Regulators Table 1 sets out the current federal financial regulatory structure. Regulators can be categorized into the three main areas of finance—banking (depository), securities, and insurance (where state, rather than federal, regulators play a dominant role). There are also targeted regulators for specific financial activities (consumer protection) and markets (agricultural finance and housing finance). The table does not include interagency-coordinating bodies, standard-setting bodies, international organizations, or state regulators, which are described later in the report. Appendix A describes changes to this table since the 2008 financial crisis. Financial firms may be subject to more than one regulator because they may engage in multiple financial activities, as illustrated in Figure 1. For example, a firm may be overseen by an institution regulator and by an activity regulator when it engages in a regulated activity and a market regulator when it participates in a regulated market. The complexity of the figure illustrates the diverse roles and responsibilities assigned to various regulators. Furthermore, financial firms may form holding companies with separate legal subsidiaries that allow subsidiaries within the same holding company to engage in more activities than is permissible within any one subsidiary. Because of charter-based regulation, certain financial activities must be segregated in separate legal subsidiaries. However, as a result of the Gramm-Leach-Bliley Act in 1999 (GLBA; P.L. 106-102 ) and other regulatory and policy changes that preceded it, these different legal subsidiaries may be contained within the same financial holding companies (i.e., conglomerates that are permitted to engage in a broad array of financially related activities). For example, a banking subsidiary is limited to permissible activities related to the "business of banking," but is allowed to affiliate with another subsidiary that engages in activities that are "financial in nature." As a result, each subsidiary is assigned a primary regulator, and firms with multiple subsidiaries may have multiple institution regulators. If the holding company is a bank holding company or thrift holding company (with at least one banking or thrift subsidiary, respectively), then the holding company is regulated by the Fed. Figure 2 shows a stylized example of a special type of bank holding company, known as a financial holding company. This financial holding company would be regulated by the Fed at the holding company level. Its national bank would be regulated by the OCC, its securities subsidiary would be regulated by the SEC, and its loan company might be regulated by the CFPB. These are only the primary regulators for each box in Figure 2 ; as noted above, it might have other market or activity regulators as well, based on its lines of business. Depository Institution Regulators Regulation of depository institutions (i.e., banks and credit unions) in the United States has evolved over time into a system of multiple regulators with overlapping jurisdictions. There is a dual banking system, in which each depository institution is subject to regulation by its chartering authority: state or federal. Even if state chartered, virtually all depository institutions are federally insured, so both state and federal institutions are subject to at least one federal primary regulator (i.e., the federal authority responsible for examining the institution for safety and soundness and for ensuring its compliance with federal banking laws). Depository institutions have three broad types of charter—commercial banks, thrifts, and credit unions. For any given institution, the primary regulator depends on its type of charter. The primary federal regulator for national banks and thrifts (also known as savings and loans) is the OCC, their chartering authority; state-chartered banks that are members of the Federal Reserve System is the Federal Reserve; state-chartered thrifts and banks that are not members of the Federal Reserve System is the FDIC; foreign banks operating in the United States is the Fed or OCC, depending on the type; credit unions—if federally chartered or federally insured—is the National Credit Union Administration, which administers a deposit insurance fund separate from the FDIC's. National banks, state-chartered banks, and thrifts are also subject to the FDIC regulatory authority, because their deposits are covered by FDIC deposit insurance. Primary regulators' responsibilities are illustrated in Figure 3. Whereas bank and thrift regulators strive for consistency among themselves in how institutions are regulated and supervised, credit unions are regulated under a separate regulatory framework from banks. Because banks receive deposit insurance from the FDIC and have access to the Fed's discount window, they expose taxpayers to the risk of losses if they fail. Banks also play a central role in the payment system, the financial system, and the broader economy. As a result, banks are subject to safety and soundness (prudential) regulation that most other financial firms are not subject to at the federal level. Safety and soundness regulation uses a holistic approach, evaluating (1) each loan, (2) the balance sheet of each institution, and (3) the risks in the system as a whole. Each loan creates risk for the lender. The overall portfolio of loans extended or held by a bank, in relation to other assets and liabilities, affects that institution's stability. The relationship of banks to each other, and to wider financial markets, affects the financial system's stability. Banks are required to keep capital in reserve against the possibility of a drop in value of loan portfolios or other risky assets. Banks are also required to be liquid enough to meet unexpected funding outflows. Federal financial regulators take into account compensating assets, risk-based capital requirements, the quality of assets, internal controls, and other prudential standards when examining the balance sheets of covered lenders. When regulators determine that a bank is taking excessive risks, or engaging in unsafe and unsound practices, they have a number of powerful tools at their disposal to reduce risk to the institution (and ultimately to the federal DIF). Regulators can require banks to reduce specified lending or financing practices, dispose of certain assets, and order banks to take steps to restore sound balance sheets. Banks must comply because regulators have "life-or-death" options, such as withdrawing deposit insurance or seizing the bank outright. The federal banking agencies are briefly discussed below. Although these agencies are the banks' institution-based regulators, banks are also subject to CFPB regulation for consumer protection, and to the extent that banks are participants in securities or derivatives markets, those activities are also subject to regulation by the SEC and the Commodity Futures Trading Commission (the CFTC). Office of the Comptroller of the Currency The OCC was created in 1863 as part of the Department of the Treasury to supervise federally chartered banks ("national" banks; 13 Stat. 99). The OCC regulates a wide variety of financial functions, but only for federally chartered banks. The head of the OCC, the Comptroller of the Currency, is also a member of the board of the FDIC and a director of the Neighborhood Reinvestment Corporation. The OCC has examination powers to enforce its responsibilities for the safety and soundness of nationally chartered banks. The OCC has strong enforcement powers, including the ability to issue cease and desist orders and revoke federal bank charters. Pursuant to the Dodd-Frank Act, the OCC is the primary regulator for federally chartered thrift institutions. Federal Deposit Insurance Corporation Following bank panics during the Great Depression, the FDIC was created in 1933 to provide assurance to small depositors that they would not lose their savings if their bank failed (P.L. 74-305, 49 Stat. 684). The FDIC is an independent agency that insures deposits (up to $250,000 for individual accounts), examines and supervises financial institutions, and manages receiverships, assuming and disposing of the assets of failed banks. The FDIC is the primary federal regulator of state banks that are not members of the Federal Reserve System and state-chartered thrift institutions. In addition, the FDIC has broad jurisdiction over nearly all banks and thrifts, whether federally or state chartered, that carry FDIC insurance. Backing deposit insurance is the DIF, which is managed by the FDIC and funded by risk-based assessments levied on depository institutions. The fund is used primarily for resolving failed or failing institutions. To safeguard the DIF, the FDIC uses its power to examine individual institutions and to issue regulations for all insured depository institutions to monitor and enforce safety and soundness. Acting under the principles of "prompt corrective action" and "least cost resolution," the FDIC has powers to resolve troubled banks, rather than allowing failing banks to enter the bankruptcy process. The Dodd-Frank Act also expanded the FDIC's role in resolving other types of troubled financial institutions that pose a risk to financial stability through the Orderly Liquidation Authority. The Federal Reserve The Federal Reserve System was established in 1913 as the nation's central bank following the Panic of 1907 to provide stability in the banking sector (P.L. 63-43, 38 Stat. 251). The system consists of the Board of Governors in Washington, DC, and 12 regional reserve banks. In its regulatory role, the Fed has safety and soundness examination authority for a variety of lending institutions, including bank holding companies; U.S. branches of foreign banks; and state-chartered banks that are members of the Federal Reserve System. Membership in the system is mandatory for national banks and optional for state banks. Members must purchase stock in the Fed. The Fed regulates the largest, most complex financial firms operating in the United States. Under the GLBA, the Fed serves as the umbrella regulator for financial holding companies. The Dodd-Frank Act made the Fed the primary regulator of all nonbank financial firms that are designated as systemically significant by the Financial Stability Oversight Council (of which the Fed is a member). All bank holding companies with more than $50 billion in assets and designated nonbanks are subject to enhanced prudential regulation by the Fed. In addition, the Dodd-Frank Act made the Fed the principal regulator for savings and loan holding companies and securities holding companies, a new category of institution formerly defined in securities law as an investment bank holding company. The Fed's responsibilities are not limited to regulation. As the nation's central bank, it conducts monetary policy by targeting short-term interest rates. The Fed also acts as a "lender of last resort" by making short-term collateralized loans to banks through the discount window. It also operates some parts and regulates other parts of the payment system. Title VIII of the Dodd-Frank Act gave the Fed additional authority to set prudential standards for payment systems that have been designated as systemically important. National Credit Union Administration The NCUA, originally part of the Farm Credit Administration, became an independent agency in 1970 (P.L. 91-206, 84 Stat. 49). The NCUA is the safety and soundness regulator for all federal credit unions and those state credit unions that elect to be federally insured. It administers a Central Liquidity Facility, which is the credit union lender of last resort, and the National Credit Union Share Insurance Fund, which insures credit union deposits. The NCUA also resolves failed credit unions. Credit unions are member-owned financial cooperatives, and they must be not-for-profit institutions. Consumer Financial Protection Bureau Before the financial crisis, jurisdiction over consumer financial protection was divided among a number of agencies (see Figure A-1 ). Title X of the Dodd-Frank Act created the CFPB to enhance consumer protection and bring the consumer protection regulation of depository and nondepository financial institutions into closer alignment. The bureau is housed within—but independent from—the Federal Reserve. The director of the CFPB is also on the FDIC's board of directors. The Dodd-Frank Act granted new authority and transferred existing authority from a number of agencies to the CFPB over an array of consumer financial products and services (including deposit taking, mortgages, credit cards and other extensions of credit, loan servicing, check guaranteeing, consumer report data collection, debt collection, real estate settlement, money transmitting, and financial data processing). The CFPB administers rules that, in its view, protect consumers by setting disclosure standards, setting suitability standards, and banning abusive and discriminatory practices. The CFPB also serves as the primary federal consumer financial protection supervisor and enforcer of federal consumer protection laws over many of the institutions that offer these products and services. However, the bureau's regulatory authority varies based on institution size and type. Regulatory authority differs for (1) depository institutions with more than $10 billion in assets, (2) depository institutions with $10 billion or less in assets, and (3) nondepositories. The CFPB can issue rules that apply to depository institutions of all sizes, but can only supervise institutions with more than $10 billion in assets. For depositories with less than $10 billion in assets, the primary depository regulator continues to supervise for consumer compliance. The Dodd-Frank Act also explicitly exempts a number of different entities and consumer financial activities from the bureau's supervisory and enforcement authority. Among the exempt entities are merchants, retailers, or sellers of nonfinancial goods or services, to the extent that they extend credit directly to consumers exclusively for the purpose of enabling consumers to purchase such nonfinancial goods or services; automobile dealers; real estate brokers and agents; financial intermediaries registered with the SEC or the CFTC; and insurance companies. In some areas where the CFPB does not have jurisdiction, the Federal Trade Commission (FTC) retains consumer protection authority. State regulators also retain a role in consumer protection. Securities Regulation For regulatory purposes, securities markets can be divided into derivatives and other types of securities. Derivatives, depending on the type, are regulated by the CFTC or the SEC. Other types of securities fall under the SEC's jurisdiction. Standard-setting bodies and other self-regulatory organizations (SROs) play a special role in securities regulation, and they are overseen by the SEC or the CFTC. In addition, banking regulators oversee the securities activities of banks. Banks are major participants in some parts of securities markets. Securities and Exchange Commission The New York Stock Exchange dates from 1793. Following the stock market crash of 1929, the SEC was created as an independent agency in 1934 to enforce newly written federal securities laws (P.L. 73-291, 48 Stat. 881). Thus, when Congress created the SEC, stock and bond market institutions and mechanisms were already well-established, and federal regulation was grafted onto the existing structure. The SEC has jurisdiction over the following: Participants in securities markets issuers—firms that issue shares, asset managers—investment advisers and investment companies (e.g., mutual funds and private funds) that are custodial agents investing on behalf of clients, intermediaries (e.g., broker-dealers, securities underwriters, and securitizers) experts who are neither issuing nor buying securities (e.g., credit rating agencies and research analysts); Securities markets (e.g., stock exchanges) and market utilities (e.g., clearinghouses) where securities are traded, cleared, and settled; and Securities products (e.g., money market accounts and security-based swaps). The SEC is not primarily concerned with ensuring the safety and soundness of the firms it regulates, but rather with "protect[ing] investors, maintain[ing] fair, orderly, and efficient markets, and facilitat[ing] capital formation." This distinction largely arises from the absence of government guarantees for securities investors comparable to deposit insurance. The SEC generally does not have the authority to limit risks taken by securities firms, nor the ability to prop up a failing firm. Firms that sell securities—stocks and bonds—to the public are required to register with the SEC. Registration entails the publication of detailed information about the firm, its management, the intended uses for the funds raised through the sale of securities, and the risks to investors. The initial registration disclosures must be kept current through the filing of periodic financial statements: annual and quarterly reports (as well as special reports when there is a material change in the firm's financial condition or prospects). Beyond these disclosure requirements, and certain other rules that apply to corporate governance, the SEC does not have any direct regulatory control over publicly traded firms. Bank regulators are expected to identify unsafe and unsound banking practices in the institutions they supervise, and they have the power to intervene and prevent banks from taking excessive risks. The SEC has no comparable authority; the securities laws simply require that risks be disclosed to investors. Registration with the SEC, in other words, is in no sense a guarantee that a security is a good or safe investment. Besides publicly traded corporations, a number of securities market participants are also required to register with the SEC (or with one of the industry SROs that the SEC oversees). These include stock exchanges, securities brokerages (and numerous classes of their personnel), mutual funds, auditors, investment advisers, and others. To maintain their registered status, all these entities must comply with rules meant to protect public investors, prevent fraud, and promote fair and orderly markets. The degree of protection granted to investors depends on their level of sophistication. For example, only "accredited investors" that have, for example, high net worth, can invest in riskier hedge funds and private equity funds. Originally, de minimis exemptions to regulation of mutual funds and investment advisers created space for the development of a trillion-dollar hedge fund industry. Under the Dodd-Frank Act, private advisers, including hedge funds and private equity funds, with more than $150 million in assets under management must register with the SEC, but maintain other exemptions. Several provisions of law and regulation protect brokerage customers from losses arising from brokerage firm failure. The Securities Investor Protection Corporation (SIPC), a private nonprofit created by Congress in 1970 and overseen by the SEC, operates an insurance scheme funded by assessments on brokers-dealers (and with a backup line of credit with the U.S. Treasury). SIPC guarantees customer accounts up to $500,000 for losses arising from brokerage failure or fraud. Unlike the FDIC, however, SIPC does not protect accounts against market losses or examine broker-dealers, and it has no regulatory powers. Some securities markets are exempted from parts of securities regulation. Prominent examples include the following: Foreign exchange. Buying and selling currencies is essential to foreign trade, and the exchange rate determined by traders has major implications for a country's macroeconomic policy. The market is one of the largest in the world, with trillions of dollars of average daily turnover. Nevertheless, no U.S. agency has regulatory authority over the foreign exchange market. Trading in currencies takes place among large global banks, central banks, hedge funds and other currency speculators, commercial firms involved in imports and exports, fund managers, and retail brokers. There is no centralized marketplace, but rather a number of proprietary electronic platforms that have largely supplanted the traditional telephone broker market. Treasury securities. Treasury securities were exempted from SEC regulation by the original securities laws of the 1930s. In 1993, following a successful cornering of a Treasury bond auction by Salomon Brothers, Congress passed the Government Securities Act Amendments of 1993 ( P.L. 103-202, 107 Stat. 2344), which required brokers and dealers that were not already registered with the SEC to register as government securities dealers, extended antifraud and antimanipulation provisions of the federal securities laws to government securities brokers and dealers, and gave the U.S. Treasury authority to promulgate rules governing transactions. (Existing broker-dealer registrants were simply required to notify the SEC that they were in the government securities business.) Nevertheless, the government securities market remains much more lightly regulated than the corporate securities markets. Regulatory jurisdiction over various aspects of the Treasury market is fragmented across multiple regulators. The SEC has no jurisdiction over the primary market. Municipal securities. Municipal securities were initially exempted from SEC regulation. In 1975, the Municipal Securities Rulemaking Board was created and firms transacting in municipal securities were required to register with the SEC as broker-dealers. The Dodd-Frank Act required municipal advisors to register with the SEC. However, issuers of municipal securities remain exempt from SEC oversight, partly because of federalism issues. Private securities. The securities laws provide for the private sales of securities, which are not subject to the registration and extensive disclosure requirements that apply to public securities. However, private securities are subject to antifraud provisions of federal securities laws. Private placements of securities cannot be sold to the general public and may only be offered to limited numbers of "accredited investors" who meet certain asset tests. (Most purchasers are life insurers and other institutional investors.) There are also restrictions on the resale of private securities. The size of the private placement market is subject to considerable variation from year to year, but at times the value of securities sold privately exceeds what is sold into the public market. In recent decades, venture capitalists and private equity firms have come to play important roles in corporate finance. The former typically purchase interests in private firms, which may be sold later to the public, whereas the latter often purchase all the stock of publicly traded companies and take them private. Commodity Futures Trading Commission The CFTC was created in 1974 to regulate commodities futures and options markets, which at the time were poised to expand beyond their traditional base in agricultural commodities to encompass contracts based on financial variables, such as interest rates and stock indexes. The CFTC was given "exclusive jurisdiction" over all contracts that were "in the character of" options or futures contracts, and such instruments were to be traded only on CFTC-regulated exchanges. In practice, exclusive jurisdiction was impossible to enforce, as off-exchange derivatives contracts such as swaps proliferated. In the Commodity Futures Modernization Act of 2000 ( P.L. 106-554 ), Congress exempted swaps from CFTC regulation, but this exemption was repealed by the Dodd-Frank Act following problems with derivatives in the financial crisis, including large losses at the American International Group (AIG) that led to its federal rescue. The Dodd-Frank Act greatly expanded the CFTC's jurisdiction by eliminating exemptions for certain over-the-counter derivatives. As a result, swap dealers, major swap participants, swap clearing organizations, swap execution facilities, and swap data repositories are required to register with the CFTC. These entities are subject to reporting requirements and business conduct standards contained in statute or promulgated as CFTC rules. The Dodd-Frank Act required swaps to be cleared through a central clearinghouse and traded on exchanges, where possible. The CFTC's mission is to prevent excessive speculation, manipulation of commodity prices, and fraud. Like the SEC, the CFTC oversees SROs—the futures exchanges and the National Futures Association—and requires the registration of a range of industry firms and personnel, including futures commission merchants (brokers), floor traders, commodity pool operators, and commodity trading advisers. Like the SEC, the CFTC does not directly regulate the safety and soundness of individual firms, with the exception of a net capital rule for futures commission merchants and capital standards pursuant to the Dodd-Frank Act for swap dealers and major swap participants. Standard-Setting Bodies and Self-Regulatory Organizations National securities exchanges (e.g., the New York Stock Exchange) and clearing and settlement systems may register as SROs with the SEC or CFTC, making them subject to SEC or CFTC oversight. According to the SEC, "SROs must create rules that allow for disciplining members for improper conduct and for establishing measures to ensure market integrity and investor protection. SRO proposed rules are published for comment before final SEC review and approval." Title VIII of the Dodd-Frank Act gave the SEC and CFTC additional prudential regulatory authority over clearing and settlement systems that have been designated as systemically important (exchanges, contract markets, and other entities were exempted). One particularly important type of SRO in the regulatory system is the group of standard-setting bodies. Most securities or futures markets participants must register and meet professional conduct and qualification standards. The SEC and CFTC have delegated responsibility for many of these functions to official standard-setting bodies, such as the following: FINRA —The Financial Industry Regulatory Authority writes and enforces rules for brokers and dealers and examines them for compliance. MSRB —The Municipal Securities Rulemaking Board establishes rules for municipal advisors and dealers in the municipal securities market; conducts required exams and continuing education for municipal market professionals; and provides guidance to SEC and others for compliance with and enforcement of MSRB rules. NFA —The National Futures Association develops and enforces rules in futures markets, requires registration, and conducts fitness examinations for a range of derivatives markets participants, including futures commission merchants, commodity pool operators, and commodity trading advisors. FASB —The Financial Accounting Standards Board establishes financial accounting and reporting standards for companies and nonprofits. PCAOB —The Public Company Accounting Oversight Board oversees private auditors of publicly held companies and broker-dealers. These standard-setting bodies are typically set up as private nonprofits overseen by boards and funded through member fees or assessments. Although their authority may be enshrined in statute, they are not governmental entities. Regulators delegate rulemaking powers to them and retain the right to override their rulemakings. Regulation of Government-Sponsored Enterprises Congress created GSEs as privately owned institutions with limited missions and charters to support the mortgage and agricultural credit markets. It also created dedicated regulators—currently, the Federal Housing Finance Agency (FHFA) and the Farm Credit Administration (FCA)—exclusively to oversee the GSEs. Federal Housing Finance Agency The Housing and Economic Recovery Act of 2008 ( P.L. 110-289, 122 Stat. 2654 ) created the FHFA during the housing crisis in 2008 to consolidate and strengthen regulation of a group of housing finance-related GSEs: Fannie Mae, Freddie Mac, and the Federal Home Loan Banks. The FHFA succeeded the Office of Federal Housing Enterprise Oversight (OFHEO), which regulated Fannie and Freddie, and the Federal Housing Finance Board (FHFB), which regulated the Federal Home Loan Banks. The act also transferred authority to set affordable housing and other goals for the GSEs from the Department of Housing and Urban Development to the FHFA. Facing concerns about the GSEs' ongoing viability, the impetus to create the FHFA came from concerns about risks—including systemic risk—posed by Fannie and Freddie. These two GSEs were profit-seeking, shareholder-owned corporations that took advantage of their government-sponsored status to accumulate undiversified investment portfolios of more than $1.5 trillion, consisting almost exclusively of home mortgages (and securities and derivatives based on those mortgages). OFHEO was seen as lacking sufficient authority and independence to keep risk-taking at the GSEs in check. The FHFA was given enhanced safety and soundness powers resembling those of the federal bank regulators. These powers included the ability to set capital standards, to order the enterprises to cease any activity or divest any asset that posed a threat to financial soundness, and to replace management and assume control of the firms if they became seriously undercapitalized. One of the FHFA's first actions was to place both Fannie and Freddie in conservatorship to prevent their failure. Fannie and Freddie continue to operate, under agreements with FHFA and the U.S. Treasury, with more direct involvement of the FHFA in the enterprises' decisionmaking. The Treasury has provided capital to the GSEs (a combined $187 billion to date), by means of preferred stock purchases, to ensure that each remains solvent. In return, the government received warrants equivalent to a 79.9% equity ownership position in the firms and sweeps the firms' retained earnings above a certain level of net worth. Farm Credit Administration The FCA was created in 1933 during a farming crisis that was causing the widespread failure of institutions lending to farmers. Following another farming crisis, it was made an "arm's length" regulator with increased rulemaking, supervision, and enforcement powers by the Farm Credit Amendments Act of 1985 ( P.L. 99-205 ). The FCA oversees the Farm Credit System, a group of GSEs made up of the cooperatively owned Farm Credit Banks and Agricultural Credit Banks, the Funding Corporation, which is owned by the Credit Banks, and Farmer Mac, which is owned by shareholders. Unlike the housing GSEs, the Farm Credit System operates in both primary and secondary agricultural credit markets. For example, the FCA regulates these institutions for safety and soundness, through capital requirements. Regulatory Umbrella Groups The need for coordination and data sharing among regulators has led to the formation of innumerable interagency task forces to study particular market episodes and make recommendations to Congress. Three interagency organizations have permanent status: the Financial Stability Oversight Council, the Federal Financial Institution Examination Council, and the President's Working Group on Financial Markets. The latter is composed of the Treasury Secretary and the Fed, SEC, and CFTC Chairmen; because the working group has not been active in recent years, it is not discussed in detail. Financial Stability Oversight Council Few would argue that regulatory failure was solely to blame for the financial crisis, but it is widely considered to have played a part. In February 2009, then-Treasury Secretary Timothy Geithner summed up two key problem areas: Our financial system operated with large gaps in meaningful oversight, and without sufficient constraints to limit risk. Even institutions that were overseen by our complicated, overlapping system of multiple regulators put themselves in a position of extreme vulnerability. These failures helped lay the foundation for the worst economic crisis in generations. One definition of systemic risk is that it occurs when each firm manages risk rationally from its own perspective, but the sum total of those decisions produces systemic instability under certain conditions. Similarly, regulators charged with overseeing individual parts of the financial system may satisfy themselves that no threats to stability exist in their respective sectors, but fail to detect systemic risk generated by unsuspected correlations and interactions among the parts of the global system. The Federal Reserve was for many years a kind of default systemic regulator, expected to clean up after a crisis, but with limited authority to take ex ante preventive measures. Furthermore, the Fed's authority was mostly limited to the banking sector. The Dodd-Frank Act created the FSOC to assume a coordinating role, with the single mission of detecting systemic stress before a crisis can take hold (and identifying firms whose individual failure might trigger cascading losses with system-wide consequences). FSOC is chaired by the Secretary of the Treasury, and the other voting members are the heads of the Fed, FDIC, OCC, NCUA, SEC, CFTC, FHFA, and CFPB, and a member appointed by the President with insurance expertise. Five nonvoting members serve in an advisory capacity—the director of the Office of Financial Research (OFR, which was created by the Dodd-Frank Act to support the FSOC), the head of the Federal Insurance Office (FIO, created by the Dodd-Frank Act), a state banking supervisor, a state insurance commissioner, and a state securities commissioner. The FSOC is tasked with identifying risks to financial stability and responding to emerging systemic risks, while promoting market discipline by minimizing moral hazard arising from expectations that firms or their counterparties will be rescued from failure. The FSOC's duties include collecting information on financial firms from regulators and through the OFR; monitoring the financial system to identify potential systemic risks; proposing regulatory changes to Congress to promote stability, competitiveness, and efficiency; facilitating information sharing and coordination among financial regulators; making regulatory recommendations to financial regulators, including "new or heightened standards and safeguards"; identifying gaps in regulation that could pose systemic risk; reviewing and commenting on new or existing accounting standards issued by any standard-setting body; and providing a forum for the resolution of jurisdictional disputes among council members. The FSOC may not impose any resolution on disagreeing members, however. In addition, the council is required to provide an annual report and testimony to Congress. In contrast to some proposals to create a systemic risk regulator, the Dodd-Frank Act did not give the council authority (beyond the existing authority of its individual members) to respond to emerging threats or close regulatory gaps it identifies. In many cases, the council can only make regulatory recommendations to member agencies or Congress—it cannot impose change. Although the FSOC does not have direct supervisory authority over any financial institution, it plays an important role in regulation because it designates firms and financial market utilities as systemically important. Designated firms come under a consolidated supervisory safety and soundness regime administered by the Fed that may be more stringent than the standards that apply to nonsystemic firms. (FSOC is also tasked with making recommendations to the Fed on standards for that regime.) In a limited number of other cases, regulators must seek FSOC advice or approval before exercising new powers under the Dodd-Frank Act. Federal Financial Institution Examinations Council The Federal Financial Institutions Examination Council (FFIEC) was created in 1979 ( P.L. 95-630, 92 Stat. 3641) as a formal interagency body to coordinate federal regulation of lending institutions. Through the FFIEC, the federal banking regulators issue a single set of reporting forms for covered institutions. The FFIEC also attempts to harmonize auditing principles and supervisory decisions. The FFIEC is made up of the Fed, OCC, FDIC, CFPB, NCUA, and a representative of the State Liaison Committee, each of which employs examiners to enforce safety and soundness regulations for lending institutions. Federal financial institution examiners evaluate the risks of covered institutions. The specific safety and soundness concerns common to the FFIEC agencies can be found in the handbooks employed by examiners to monitor lenders. Each subject area of the handbook can be updated separately. Examples of safety and soundness subject areas include important indicators of risk, such as capital adequacy, asset quality, liquidity, and sensitivity to market risk. Nonfederal Financial Regulation Insurance42 Insurance companies, unlike banks and securities firms, have been chartered and regulated solely by the states for the past 150 years. There are no federal regulators of insurance akin to those for securities or banks, such as the SEC or the OCC, respectively. The limited federal role stems from both Supreme Court decisions and congressional action. In the 1868 case Paul v. Virginia, the Court found that insurance was not considered interstate commerce, and thus not subject to federal regulation. This decision was effectively reversed in the 1944 decision U.S. v. South-Eastern Underwriters Association. In 1945, Congress passed the McCarran-Ferguson Act (15 U.S.C. §1011 et seq.) specifically preserving the states' authority to regulate and tax insurance and also granting a federal antitrust exemption to the insurance industry for "the business of insurance." Each state government has a department or other entity charged with licensing and regulating insurance companies and those individuals and companies selling insurance products. States regulate the solvency of the companies and the content of insurance products as well as the market conduct of companies. Although each state sets its own laws and regulations for insurance, the National Association of Insurance Commissioners (NAIC) acts as a coordinating body that sets national standards through model laws and regulations. Models adopted by the NAIC, however, must be enacted by the states before having legal effect, which can be a lengthy and uncertain process. The states have also developed a coordinated system of guaranty funds, designed to protect policyholders in the event of insurer insolvency. Although the federal government's role in regulating insurance is relatively limited compared with its role in banking and securities, its role has increased over time. Various court cases interpreting McCarran-Ferguson's antitrust exemption have narrowed the definition of the business of insurance, whereas Congress has expanded the federal role in GLBA and the Dodd-Frank Act through federal oversight. For example, the Federal Reserve has regulatory authority over bank holding companies with insurance operations as well as insurers designated as systemically important by FSOC. In addition, the Dodd-Frank Act created the FIO to monitor the insurance industry and represent the United States in international fora relating to insurance. Various federal laws have also exempted aspects of state insurance regulation, such as state insurer licensing laws for a small category of insurers in the Liability Risk Retention Act of 1986 (15 U.S.C. §§3901 et seq.) and state insurance producer licensing laws in the National Association of Registered Agents and Brokers Reform Act of 2015 ( P.L. 114-1, Title II). Other State Regulation In addition to insurance, there are state regulators for banking and securities markets. State regulation also plays a role in nonbank consumer financial protection, although that role has diminished as the federal role has expanded. As noted above, banking is a dual system in which institutions choose to charter at the state or federal level. A majority of community banks are state chartered. There are more than four times as many state-chartered as there are nationally chartered commercial banks, but state-chartered commercial banks had less than one-half as many assets as nationally chartered banks at the end of 2016. For thrifts, nationally chartered thrifts make up less than half of all thrifts, but have 65% of thrift assets. Although state-chartered banks have state regulators as their primary regulators, federal regulators still play a regulatory role. Most depositories have FDIC deposit insurance; to qualify, they must meet federal safety and soundness standards, such as prompt corrective action ratios. In addition, many state banks (and a few state thrifts) are members of the Fed, which gives the Fed supervisory authority over them. State banks that are not members of the Federal Reserve System are supervised by the FDIC. However, even nonmember Fed banks must meet the Fed's reserve requirements. In securities markets, "The federal securities acts expressly allow for concurrent state regulation under blue sky laws. The state securities acts have traditionally been limited to disclosure and qualification with regard to securities distributions. Typically, the state securities acts have general antifraud provisions to further these ends." Blue sky laws are intended to protect investors against fraud. Registration is another area where states play a role. In general, investment advisers who are granted exemptions from SEC registration based on size are overseen by state regulators. Broker-dealers, by contrast, are typically subject to both state and federal regulation. The National Securities Markets Improvement Act of 1996 ( P.L. 104-290, 110 Stat. 3416) invalidated state securities law in a number of other areas (including capital, margin, bonding, and custody requirements) to reduce the overlap between state and federal securities regulation. States also play a role in enforcement by taking enforcement actions against financial institutions and market participants. This role is large in states with a large financial industry, such as New York. Federal regulation plays a central role in determining the scope of state regulation because of federal preemption laws (that apply federal statute to state-chartered institutions) and state "wild card" laws (that allow state-chartered institutions to automatically receive powers granted to federal-chartered institutions). For example, firms, products, and professionals that have registered with the SEC are not required to register at the state level. International Standards and Regulation Financial regulation must also take into account the global nature of financial markets. More specifically, U.S. regulators must account for foreign financial firms operating in the United States and foreign regulators must account for U.S. firms operating in their jurisdictions. Sometimes regulators grant each other equivalency (i.e., leaving regulation to the home country) when firms wish to operate abroad, and other times foreign firms are subjected to domestic regulation. If financial activity migrates to jurisdictions with laxer regulatory standards, it could undermine the effectiveness of regulation in jurisdictions with higher standards. To ensure consensus and consistency across jurisdictions, U.S. regulators and the Treasury Department participate in international fora with foreign regulators to set broad international regulatory standards or principles that members voluntarily pledge to follow. Given the size and significance of the U.S. financial system, U.S. policymakers are generally viewed as playing a substantial role in setting these standards. Although these standards are not legally binding, U.S. regulators generally implement rulemaking or Congress passes legislation to incorporate them. There are multiple international standard-setting bodies, including one corresponding to each of the three main areas of financial regulation: the Basel Committee on Banking Supervision for banking regulation, the International Organization of Securities Commissions (IOSCO) for securities and derivatives regulation, and the International Association of Insurance Supervisors (IAIS) for insurance regulation. In addition, the G-20, an international body consisting of the United States, the European Union, and 18 other economies, spearheaded international coordination of regulatory reform after the financial crisis. It created the Financial Stability Board (FSB), consisting of the finance ministers and financial regulators from the G-20 countries, four official international financial institutions, and six international standard-setting bodies (including those listed above), to formulate agreements to implement regulatory reform. There is significant overlap between the FSB's regulatory reform agenda and the Dodd-Frank Act. The relationship between these various entities is diagrammed in Figure 4. Appendix A. Changes to Regulatory Structure Since 2008 The Dodd-Frank Act of 2010 created four new federal entities related to financial regulation—the Financial Stability Oversight Council (FSOC), Office of Financial Research (OFR), Federal Insurance Office (FIO), and Consumer Financial Protection Bureau (CFPB). OFR and FIO are offices within the Treasury Department, and are not regulators. The Dodd-Frank Act granted new authority to the CFPB and transferred existing authority to it from other regulators, as shown in Figure A-1 (represented by the arrows). (With the exception of the OTS, the agencies shown in the figure retained all authority unrelated to consumer protection, as well as some authority related to consumer protection.) The section above entitled " Consumer Financial Protection Bureau " has more detail on the authority granted to the CFPB and areas where the CFPB was not granted authority. In addition, the Dodd-Frank Act eliminated the Office of Thrift Supervision (OTS) and transferred its authority to the banking regulators, as shown in Figure A-2. The Housing and Economic Recovery Act of 2008 (HERA) created the Federal Housing Finance Agency (FHFA) and eliminated the Office of Federal Housing Enterprise Oversight (OFHEO) and Federal Housing Finance Board (FHFB). As shown in Figure A-3, HERA granted the FHFA new authority, the existing authority of the two eliminated agencies (shown in orange), and transferred limited existing authority from the Department of Housing and Urban Development (HUD). Appendix B. Experts List

Summary: The financial regulatory system has been described as fragmented, with multiple overlapping regulators and a dual state-federal regulatory system. The system evolved piecemeal, punctuated by major changes in response to various historical financial crises. The most recent financial crisis also resulted in changes to the regulatory system through the Dodd-Frank Wall Street Reform and Consumer Protection Act in 2010 (Dodd-Frank Act; P.L. 111-203) and the Housing and Economic Recovery Act of 2008 (HERA; P.L. 110-289). To address the fragmented nature of the system, the Dodd-Frank Act created the Financial Stability Oversight Council (FSOC), a council of regulators and experts chaired by the Treasury Secretary. At the federal level, regulators can be clustered in the following areas: Depository regulators-Office of the Comptroller of the Currency (OCC), Federal Deposit Insurance Corporation (FDIC), and Federal Reserve for banks; and National Credit Union Administration (NCUA) for credit unions; Securities markets regulators-Securities and Exchange Commission (SEC) and Commodity Futures Trading Commission (CFTC); Government-sponsored enterprise (GSE) regulators-Federal Housing Finance Agency (FHFA), created by HERA, and Farm Credit Administration (FCA); and Consumer protection regulator-Consumer Financial Protection Bureau (CFPB), created by the Dodd-Frank Act. These regulators regulate financial institutions, markets, and products using licensing, registration, rulemaking, supervisory, enforcement, and resolution powers. Other entities that play a role in financial regulation are interagency bodies, state regulators, and international regulatory fora. Notably, federal regulators generally play a secondary role in insurance markets. Financial regulation aims to achieve diverse goals, which vary from regulator to regulator: market efficiency and integrity, consumer and investor protections, capital formation or access to credit, taxpayer protection, illicit activity prevention, and financial stability. Policy debate revolves around the tradeoffs between these various goals. Different types of regulation-prudential (safety and soundness), disclosure, standard setting, competition, and price and rate regulations-are used to achieve these goals. Many observers believe that the structure of the regulatory system influences regulatory outcomes. For that reason, there is ongoing congressional debate about the best way to structure the regulatory system. As background for that debate, this report provides an overview of the U.S. financial regulatory framework. It briefly describes each of the federal financial regulators and the types of institutions they supervise. It also discusses the other entities that play a role in financial regulation.

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Summarize: FIELD OF THE INVENTION The present invention relates in general to systems for performing photoplethysmographic measurements of various blood analytes and other hemodynamic parameters. BACKGROUND OF THE INVENTION In the science of photoplethysmography, light is used to illuminate or trans-illuminate tissue for the purpose of measuring blood analytes or other hemodynamic or tissue properties. In this monitoring modality, light is injected into living tissue and the light which is not absorbed by the tissues is detected a short distance from the entry point. The detected light is converted into an electronic signal, indicative of the received light signal from the tissue. This electronic signal is then used to calculate one or more physiologic parameters such as arterial blood oxygen saturation, heart rate, cardiac output, or tissue perfusion. Other blood analytes that may be measured by photoplethysmography include the percentages of oxyhemoglobin, carboxyhemoglobin, methemoglobin, and reduced hemoglobin in the arterial blood. The first commercial use of photoplethysmography in medicine was in the pulse oximeter, a device designed to measure arterial blood oxygen saturation. Since the inception of this device, this monitoring modality has been used to detect more and more different parameters. For example, a device has recently been disclosed which is capable of measuring the percentages of four different analytes in the arterial blood, including oxyhemoglobin, carboxyhemoglobin, methemoglobin and reduced hemoglobin. To make these measurements a number of different bands of light must be used, with each light band possessing a unique spectral content. Each spectral band is usually referred to by the center wavelength, or sometimes by the peak wavelength, for the given band. In the case of pulse oximetry for instance, two different light emitting diodes (LEDs) are typically used to generate the sensing light, the first with a center or peak wavelength around 660 nanometers (nm) and a second with a center or peak wavelength around 940 nm. As the number of different parameters measured by photoplethysmography increases, so too does the number of different bands of light required to make the measurements. Further, because a fairly high intensity of light, over a fairly narrow spectral range is needed for these measurements, it has been found that the most successful sources of light for these measurements have been discrete, narrow-band emitters such as LEDs or laser diodes. These types of light sources are typically used because broadband sources have too little energy over the desired narrow spectral ranges to provide sufficient signal amplitude for photoplethysmographic measurements. As the number of light sources, or emitters, used in a single device increases, the problems associated with how to deliver the light to the tissue-under-test also increases. These problems are created by several different factors. These include the ergonomic constraints placed on photoplethysmographic instrumentation by the medical community, cost constraints, reliability concerns, emitter operating parameters, and technical feasibility considerations. In the design of a multi-parameter photoplethysmographic device, it is desirable to use a selection of emitters with the best optical properties possible for the analyte measurements of interest and in an opto-mechanical configuration that best meets the technical, ergonomic, and cost constraints of the instrument. BRIEF SUMMARY OF THE INVENTION In the science of photoplethysmography, light is used to illuminate, or trans-illuminate, tissue for the purpose of measuring blood analytes or hemodynamic properties or parameters. In making these measurements it can become necessary to use light from a number of different sources including but not limited to LEDs, incandescent bulbs, or lasers. Each of these different sources has different properties that dictate how the light will be delivered to the tissue under test. For example, use of LEDs allows the light source to be placed in the sensor and directly proximate to the tissue-under-test. Additionally, it is difficult to couple LED light into a secondary light-delivering apparatus such as a fiber optic cable, making it technically difficult to position the LEDs at a distance from the sensor. Note that for the purposes of this document the ‘sensor’ is defined as that portion of the system that is placed directly on the tissue-under-test. By comparison, laser light sources (particularly currently-available conventional semiconductor lasers) require thermal stabilization to maintain a proper spectral output and, when mounted with their necessary thermal controllers and heat sinks, are too bulky to be placed directly on the sensor. Instead the laser or lasers are preferably placed in the instrument or in a housing at some intermediate point on the patient cable. The laser light can be transmitted to the sensor via some type of light pipe, typically a fiber optic cable of some type. Laser light sources are also typically more expensive and considerably more fragile than LEDs, which are additional reasons to place these types of components at a distance from the sensor and in a housing that can adequately protect them. Incandescent light sources are also difficult to position at the sensor. They tend to be large, hot, and require a certain amount of optics to make them useful for photoplethysmographic devices. Again, these properties make it desirable to place any incandescent sources some distance from the sensor. With the increasing complexity of photoplethysmographic devices detecting and measuring more and more parameters, it becomes necessary to design systems with large numbers of emitters. In an effort to maximize measurement accuracy, it can be beneficial to utilize a multitude of emitters of different types. For example in the specific instrument of this invention, one capable of monitoring four different species of hemoglobin and the heart rate, it is desirable to use three or more laser diodes and one or two LEDs. This allows for the measurement system to utilize the different optical properties of the different emitters in the same system. For the specific instrument described herein, the optical stability of the LEDs' output light is required for measurement of low perfusion patients, and the extremely narrow-band output of the laser diodes makes possible the measurement of optical absorption of a hemoglobin species on very steep portions of its extinction curve. A hybrid optical system, containing multiple emitters of more than one type, makes photoplethysmographic measurements possible that are otherwise not feasible or which could otherwise not be made to clinically acceptable levels of accuracy and precision. One aspect of this invention therefore is the use of multiple types of emitters in the same photoplethysmographic system. A second aspect of this invention is the optimal placement of the different emitters, with some type or types typically positioned at the sensor and other types positioned in an intermediate housing or inside the instrument housing, to create the most optimal configuration both mechanically and optically while maximizing reliability and minimizing product cost. This emitter layout creates a problem for the instrument designer in that the lasers are difficult to position at the sensor and the LEDs are difficult to position at a distance from the sensor. In past designs of photoplethysmographic devices, the emitters were all of one type and were all positioned either at the sensor with their output light directly incident on the tissue-under-test or the emitters were all positioned at a distance from the sensor and the light piped to the sensor by a light guide. This invention allows the photoplethysmographic instrument designer to utilize the beneficial optical properties of multiple and different types of emitters in the same design while permitting the positioning of these emitters at different locations in the photoplethysmographic system for optimal coupling of the output light into the tissue-under-test. A further aspect of this invention is the co-location of the output light from the various sources to cause the light from all emitters to be incident on the same small area of the tissue-under-test, thus forcing the output light from all the light sources to follow the same optical path. This is a necessary condition for accurate photoplethysmographic measurements in tissue. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of the photoplethysmographic instrument showing the control flow, front panel connector, cable connector, and sensor cable. FIG. 2 is a sketch of the sensor cable, sensor connector, and a durable finger sensor used in conjunction with the photoplethysmographic instrument. FIG. 3 is a detailed drawing of the sensor connector, and sensor. DETAILED DESCRIPTION OF THE INVENTION Prior to this device all photoplethysmographic devices have housed the emitters, or sources, for the system at the same physical location. They have also used only one type of emitter in any given system. Thus if we examine an early photoplethysmographic device, the Minolta/Marquest Oxygen Saturation Monitor, Model SM-32, we see that a single incandescent light source was used. The output light from this lamp was transmitted by fiber optics to the tissue-under-test. The light that was not absorbed or scattered by the tissues was then collected and carried back to the instrument via a second fiber bundle. Note that the light collected was limited to the light that exited the tissue at the contact point for the second fiber bundle. This light was then transmitted back to the instrument where it was filtered through two interference filters to create two separate bands of light each with a distinct spectral content. It was the analysis of these two different bands of light that allowed the photoplethysmographic measurements. Unfortunately, filtering broadband sources of light into narrowband sources, for use in the photoplethysmographic measurements eliminates the majority of the light intensity and barely leaves a usable quantity of light. As a result the Minolta/Marquest Oxygen Saturation Monitor had a great deal of difficulty functioning on a broad range of patients with a broad range of optical densities. An improvement over this design was the next generation of pulse oximeters that utilized LEDs to generate the different bands of light necessary for the photoplethysmographic measurements. The first of these devices, the BIOX II, was made by Bioximetry Technology, Inc. and used two LEDs installed in the sensor. The LEDs generated light centered at approximately 660 nm and 940 nm with much greater intensities then was possible with the earlier incandescent system. Since BIOX II all commercially available pulse oximeters have used LED sources, typically housed in the sensor itself. Some photoplethysmographic systems have been made that position the sources at a distance from the sensor and use fiber optics to pipe the light to the tissue-under-test. While these LED-based systems utilizing fiber optics to transmit and receive the light have been shown to work, they suffer from extremely low signal levels and are generally only used in devices designed to work in a magnetic resonance imaging (MRI) environment without interfering with the MRI readings. More recently in a patent by Pologe, J A (U.S. Pat. No. 5,891,022), a device was revealed that utilizes two or more emitters, typically laser diodes, where the output light is coupled into individual fibers and then coupled into a single fiber or fiber bundle to then be carried to the tissue-under-test. The emitters and their accompanying thermal control and heat sinking elements were housed at a distance from the sensor. In this device, once again, all light sources were housed in the same location. The present invention relates to a system for performing photoplethysmographic measurement utilizing multiple types of emitters positioned at multiple locations, both proximal to and at a distance from the tissue-under-test. This configuration allows for optimizing the optical properties of the selected emitters, maximizing the light intensities incident on the tissue-under-test, maximizing system reliability, and minimizing system noise. In one embodiment, as illustrated in FIGS. 1 through 3, both light emitting diodes (LEDs) and laser diodes are used in the same instrument. In FIG. 1 the Photoplethysmographic Instrument 1 is shown. The arrows in this figure show the control and signal flow in the instrument. The Digital Processing Section 10 controls all operations in the instrument, calculates the measured analyte levels and the other physiological parameters, and runs the user interface accepting input from the Keypad 12 and displaying the measured parameters on the instrument Display 11. In alternate embodiments, output might also be provided to other interfaces such as a digital interface for connection to an external computer system or in analog form for connection to such analog devices as an external strip chart recorder. The Digital Processing Section 10 also controls the Emitter Drive Electronics 13. Most photoplethysmographic systems are run in a time division multiplexed manner with the emitters turned on one at a time in a repetitive cycle. This allows a single Photodiode 27, shown in FIG. 3, to receive light from all the different emitters and for the instrument to be able to discern from which emitter the light is being received at any given point in time. Unique to this invention, the Emitter Drive Electronics 13 (FIG. 1) control the timing of two different sets of emitters, each set positioned in a physically different location. The first set of emitters is housed in the Photoplethysmographic Instrument 1 and a second set is housed in the Sensor 5. In the preferred embodiment, the emitters housed in the instrument are contained in the Laser Module 14. This module contains a set of laser diodes that are coupled into a fiber, a fiber bundle, or some other type of light guide 16, for transmission to the sensor and on to the tissue-under-test. A second set of emitters is housed in the Sensor 5 and the Emitter Drive Electronics 13 provides control signals for these emitters as well. It should be noted that this embodiment shows the first set of emitters housed within the Photoplethysmographic Instrument 1. It is also possible to house this set of emitters in a separate housing positioned between the Photoplethysmographic Instrument 1 and the Sensor 5. This would not change the intent of this invention, as the output light from this set of emitters would still need to be conducted or piped to the sensor through some sort of light guide. Finally, the light from the Laser Module 14 and the emitter drive signals, for the LEDs, from the Emitter Drive Electronics 13 are passed to the Front Panel Connector 15 to interface with the Patient Cable 3. The Patient Cable 3 connects to the Photoplethysmographic Instrument 1 by way of the Patient Cable Connector 2, which plugs directly into the Photoplethysmographic Instrument 1. Returning from the Sensor 5 is an electronic signal representative of the received light intensities picked up by the Photo-Detector 27 in the Sensor 5. This signal is passed on to the Analog Signal Processing Section 17. The Analog Signal Processing Section 17 along with the Digital Processing Section 10, process (processing refers to filtering, amplification, analog-to-digital conversion, and mathematical calculations performed on the signal.) the signal received from the Sensor 5 to measure and calculate the parameters of interest. In FIG. 2 a Sensor 5 is shown which is used to attach the photoplethysmographic system to the tissue-under-test. This figure shows a sketch typical of the type of durable Sensor 5 that might be used for connection to a patient's finger. The Sensor 5 is connected to the Patient Cable 3 by means of the Sensor Connector 4. In the current embodiment, the Patient Cable 3 and the Sensor Connector 4 are a fixed unit that is detachable from the Sensor 5. In this way, alternate sensor types may be attached to same patient cable. It is not uncommon for manufacturers of photoplethysmographic devices to have five or more different types of sensors for attachment in different ways and to different parts of the body as well as a variety of sensor sizes for use on patients ranging from neonates to adults. FIG. 3 shows, in detail, the current embodiment for the Patient Cable 3, Sensor Connector 4, and the Sensor 5. The signal controlling the second set of emitters, originating in the emitter drive electronics, enters the printed circuit board (PCB) 23 by way of the Drive Cable 22. These are the electronic signals that, in this embodiment, drive the LEDs 20 housed on the PCB 23. These LEDs make up the second set of emitters that are housed directly at the sensor. The light from these emitters can be directly incident on the Output Aperture 26, or, as in this embodiment, reflected off the Mirror 24 used to direct all light from all emitters toward the output aperture. The PCB 23, shown in cross section in FIG. 3. In the current embodiment, it is designed as a small circular PCB that positions the LEDs radially around the Light Guide 21 which protrudes through the center of the PCB. The goal of this particular design is to create a small and homogeneous set of light sources that, from the point of view of the output aperture, appear to be a single point source as required for photoplethysmographic measurements. In this invention two sets of emitters are used. One is located at the Sensor 5 and a second set is located in the Photoplethysmographic Instrument 1. A design of this type allows the use of different types of emitters in the same instrument. The Photoplethysmographic Instrument 1 houses the laser diodes, whereas the LEDs are housed at the Sensor 5. Laser diodes are expensive and have a considerably shorter lifetime then LEDs, therefore it is important to minimize the number of laser diodes utilized in a given design. It can also be beneficial to house the lasers inside the instrument housing to allow for additional physical protection of these components and to provide the space needed for heat sinks or cooling devices such as fans or thermoelectric coolers. Because lasers have a very narrow bandpass they are necessary for the measurement of certain analytes where the broader bandpass of other emitter types such as LEDs would not allow sufficient accuracy of measurement or may make the desired measurement impossible to attain. LEDs can be used when extremely short bandpass is not required for measurement accuracy. They also have the advantages of being extremely durable, very inexpensive, and extremely stable in their output light levels. Unfortunately LEDs have a very wide spatial irradiance pattern and are not physically small enough to behave like a point source when being coupled into optical fibers. Due to these properties it is difficult to obtain high efficiency coupling from LEDs into optical fibers. Since the intensity of the light incident on the tissue-under-test is directly proportional the signal-to-noise ratio it is optimal to allow the LEDs to shine directly onto the output aperture to avoid the losses in intensity associated with fiber optic coupling. Thus this invention combines the two modalities for supplying light to the tissue-under-test allowing multiple types of emitters to be used in the same photoplethysmographic system. This provides the benefits derived from each type of emitter enhancing the number of analytes that can be measured and the accuracy with which they can be measured. It is important in photoplethysmographic systems that the light incident on the tissue-under-test enters the tissue from the same aperture regardless of which emitter generated the light. The optical and geometric configuration of the Sensor 5 and the Sensor Connector 4 therefore must be designed such that this condition is met. In the embodiment shown in FIG. 3, the Mirror 24 reflects the light generated remotely, i.e. by the emitter set housed in the photoplethysmographic instrument and transmitted to the sensor via the Light Guide 21, and locally, i.e. by the local set of LED emitters, and reflects these signals towards the Output Aperture 26. The light guide in this embodiment consists of a set of optical fibers each coupled to one of the remote emitters. The type of light guide used can be individual optical fibers, either glass or plastic, a fiber bundle, or even a liquid light guide. It is only important that the type of light guide used has properties that make it functional in a medical instrument. In general the light guide should be as thin and flexible as possible and minimize the losses to the light it is transmitting. It is also possible to couple more than one emitter into any given light guide so that there is not necessarily a one to one correspondence between the number of remote emitters and the number of light guides used. The Mirror 24 also allows for the light output from the light guide and the LEDs to spread out, fully filling the output aperture, and therefore co-locating the light from all sources. With this design, the criterion that all light sources enter the tissue-under-test through the same aperture is met. This is necessary to ensure that the path the light takes through the tissue-under-test to the Detector Aperture 28 is essentially identical for all emitters. The light that exits the tissue via the Detector Aperture 28 is incident upon the Photo-detector 27, where it is converted to an electronic signal representative of the incident light. In the preferred embodiment of this device, the photo-detector of choice is a silicon-based photodiode. This device type is utilized because it is an inexpensive device that has a very low noise level, making it ideal for the low-level signals received by the photo-detector in typical photoplethysmographic devices. These low-level signals consist of two primary components of the light transmitted by the tissue-under-test, after attenuation by the targeted blood analytes, other light absorbers, and scattering elements in the tissue. These two signal components are a DC, or constant signal, component and an AC, or a time-varying signal, component. The time-varying component is that portion of the light signal that fluctuates up and down in amplitude as the blood volume in the tissue-under-test pulsates down and up, i.e. out of phase, with each heartbeat. It is this AC component that can be extremely small in amplitude, sometimes less than 0.1% of the DC component. Both the AC and DC components must be accurately measured many times a second to allow for accurate photoplethysmographic measurement of the targeted parameters. These components of the received light signal, or their electronic equivalents, can be measured independently or as single, combined amplitude. Thus it is this electronic signal, representative of the light incident on the Photo-detector 27, that is transmitted back to the Photoplethysmographic Instrument 1, via the Detector Cable 25, for processing. The previous discussion of the invention has been presented for the purposes of illustration and description. The description is not intended to limit the invention to the form disclosed herein. Variations and modifications commensurate with the above are considered to be within the scope of the present invention. The embodiment described herein is further intended to explain the best mode presently known of practicing the invention and to enable others skilled in the art to utilize the invention as such, or in other embodiments, and with the particular modifications required by their particular application or uses of the invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.

Summary: A hybrid optical system for photoplethysmographic measurements is disclosed. The system delivers light from a plurality of emitters to the tissue-under-test. A set of one or more of the emitters resides in close proximity to the sensor. A second set of one or more emitters resides at some distance from the sensor with the light from this second set of emitters being delivered to the sensor by a light pipe typically consisting of a fiber optic cable. This invention allows the photoplethysmographic instrument designer to use two or more different types of emitters in the same photoplethysmographic device while still generating a technically feasible and cost effective design.

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Summarize: CROSS-REFERENCE [0001] This application is a continuation of U.S. patent application Ser. No. 11/141,669, filed May 31, 2005, which claims benefit of U.S. Provisional Patent Application Ser. No. 60/575,913, filed Jun. 1, 2004, and of U.S. Provisional Patent Application Ser. No. 60/604,139, filed Aug. 23, 2004, which applications are incorporated herein by specific reference in their entirety. BACKGROUND [0002] Although chemical pesticides are valuable in the control of pests, their use poses many problems. They tend to harm non-target organisms such as humans, domestic animals, beneficial insects, and wildlife. In addition, their residues tend to remain on the crop and may accumulate in the soil, water, or air. Another concern is the development of resistance to pesticides by the targeted organisms. Due to the serious environmental problems associated with chemical pesticides, the demand for safer pesticides and alternate pest control strategies is increasing. [0003] It has become widely known in the art that certain live biological control agents (bacteria, phage, bacteriophages) have some use in pest control. The success of these biological control agents depends on understanding the biology of these agents and of the target pest. Several limitations are associated with the use of these biological agents according to conventional techniques. One of the limitations is that some of the biologic control agents are opportunistic pathogens against humans or animals. Another constraint is the competition between the native organisms (beneficial or pathogens) in the ecosystem and the control agents. In many cases, the control agent is not able to compete in the environment and the efficacy of the treatment is questionable. [0004] U.S. Pat. No. 5,767,090 describes the use of rhamnolipids against the transitional zoosporic stage of zoosporic fungi. Although rhamnolipids are very effective against this motile stage of the pathogenic fungi, successful treatment in commercial crops production depends greatly on application time and repetitive applications are sometimes necessary to achieve the desired control. During wet conditions foliar application may be impractical and repetitive applications significantly increases production cost. SUMMARY [0005] The present invention relates to methods and compositions, based upon natural microbial biosurfactants, for controlling pests. Examples of the pests treated with the biocontrol agents include insects such as ants, aphids, thrips, whiteflies, scales, lice, cockroaches, termites, houseflies, mosquitoes, etc; arachnids such as mites, spiders, ticks; nematodes; mollusks; amoeba; parasites; and algae. The agents for controlling pests according to the invention include natural pesticides (biopesticides) derived of microbial origin. Biopesticides are inherently less harmful than conventional pesticides. They often biodegrade quickly, therefore resulting in low exposures and largely avoiding the pollution problems caused by conventional pesticides. [0006] Embodiments of the invention extend to the use of naturally occurring microbial biosurfactant compounds to control pests. The invention also extends to methods of controlling pests using biosurfactant-producing microorganisms. The invention further encompasses the compositions themselves, as well as the methods for manufacturing or producing the compositions and their application to pest control as disclosed herein. Other objects of the invention may be apparent to one of skill in the art upon learning of the invention described herein. [0007] In another embodiment of the invention, formulations and treatments to control pests are achieved by the addition of carbon, or organic, substrates, such as oil or fatty acids, to target areas (before or during infestation) to support the growth of biosurfactant-producing organisms as well as to produce the desired biosurfactants on site to achieve the objective of the invention. Natural biosurfactant-producing organisms at the site of application (soil, aquatic system, plant parts, etc) will produce biosurfactants while utilizing the carbon substrate. During this process, the biosurfactant produced will destroy or paralyze pests in the targeted area. According to the invention, it is not necessary to pre-inoculate the targeted site or the carbon substrate mixture with biosurfactant-producing organisms. [0008] Microbial biosurfactants are compounds produced by variety of microorganisms such as bacteria, fungi, and yeasts. Biosurfactants form an important class of secondary metabolites that occur in many microorganisms such as Pseudomonas species ( P. aeruginosa, P. putida, P. florescens, P. fragi, P. syringae ); Flavobacterium spp.; Bacillus spp. ( B. subtilis, B. pumillus, B. cereus, B. licheniformis ); Candida species ( C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C. torulopsis ); Rhodococcus sp.; Arthrobacter spp.; campylobacter spp.; cornybacterium spp. and so on. [0009] Biosurfactants are biodegradable and can be easily and cheaply produced using selected organisms on renewable substrates. Most biosurfactant-producing organisms produce biosurfactants in response to the presence of hydrocarbon source (e.g. oils, sugar, glycerol, etc) in the growing media. Other media components can also affect biosurfactant production significantly. For example, the production of rhamnolipids by the bacteria Pseudomonas aeruginosa can be increased if nitrate is used as a source of nitrogen rather than ammonium. Also the concentration of iron, magnesium, sodium, and potassium; the carbon:phosphorus ratio, and agitation greatly affect rhamnolipid production. [0010] Biosurfactants include low-molecular-weight glycolipids (GLs), lipopeptides (LPs), flavolipids (FLs), phospholipids, and high-molecular-weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. Biosurfactants have a great deal of structural diversity. The common lipophilic moiety of a biosurfactant molecule is the hydrocarbon chain of a fatty acid, whereas the hydrophilic part is formed by ester or alcohol groups of neutral lipids, by the carboxylate group of fatty acids or amino acids (or peptides), organic acid in the case of flavolipids, or, in the case of glycolipids, by the carbohydrate. [0011] According to the embodiments of the invention, these compounds may also be synthesized by standard organic synthesis methods. [0012] In one embodiment, a single biosurfactant or a mixture of different biosurfactants may be used in a formulation to perform the functions and achieve the results disclosed herein. [0013] Co-pending U.S. Provisional Patent Application Ser. No. 60/604,139, filed Aug. 23, 2004, is incorporated herein by reference and describes biosurfactants (e.g. GLs, FLs, and LPs etc) having a powerful penetrating capabilities. It has been discovered that an important characteristic of these biosurfactants or biopesticides is that they are able to penetrate tissues or cells. This is a very important factor that influences effectiveness of pesticides. In general, the effectiveness of pesticides can be significantly enhanced if they are able to readily spread on the treated surface and to penetrate into the pest (e.g., into the insects' cuticle). According to embodiments of this invention, the biopesticide is able to penetrate through pests' tissues sufficiently and to be effective in lesser amounts without the use of adjuvants. It has been found that at concentrations above the critical micelle concentration, the biosurfactant are able to penetrate more effectively into treated objects. [0014] In this invention, we have unexpectedly discovered that some natural and non-toxic biosurfactants such as rhamnolipids have unique mode of action against some pests and parasites and this broad-spectrum activity differs from conventional pesticides mode of action. [0015] The development of the natural biopesticides of the invention, which can be produced in high amounts using selected microorganisms for pest control, represents a significant advancement in the art. As noted above, pests can be controlled using either the biosurfactant-producing organisms as a biocontrol agent or by the biosurfactants themselves. In addition, pests control can be achieved by the use of specific substrates to support the growth of biosurfactant-producing organisms as well as to produce biosurfactants pesticidal agents. Thus, according to the invention, the microbial biosurfactants' glycolipids, such as rhamnolipids, sophorlipids, trehalose lipids; flavolipids; lipopeptides; etc., as pesticidal agents have the potential to reduce the need for and use of synthetic pesticides. [0016] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE FIGURES [0017] The foregoing and following information as well as other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which: [0018] FIG. 1 is a flow diagram illustrating a method of applying microbial biosurfactants to control pests according to an embodiment of the invention; and [0019] FIG. 2 is a flow diagram illustrating a method of obtaining biosurfactants in a concentration that is sufficient to be used to control pests; [0020] arranged in accordance with at least one of the embodiments described herein, and which arrangement may be modified in accordance with the disclosure provided herein by one of ordinary skill in the art. DETAILED DESCRIPTION [0021] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. [0022] The present invention is directed to compositions and methods for controlling pests, and, more particularly, to pesticides derived from natural substances, such as microbial or fermentation metabolites. According to embodiments of the invention, the produced fermentation broth containing the microbial biosurfactant may be used without extraction or purification. If desired, extraction and purification of the biosurfactants can be easily achieved using standard extraction methods or techniques described in the literature. [0023] In one aspect of the invention, fermentation broth or the purified biosurfactants, e.g., GLs, FLs, LPs, etc., may be used to protect crop plants, homes, structures, soils, aquatic systems, ponds, fish aquariums, humans, or animals by controlling harmful pests. As used herein, the term “control” used in reference to the activity produced by the biosurfactants or biosurfactant-producing organisms extends to the act of killing, disabling or immobilizing pests or otherwise rendering the pests substantially incapable of causing harm. [0024] In another aspect of the invention, biosurfactant-producing organisms, e.g., Pseudomonas spp. may be added to the soil, plants' growing medium, plants, aquatic medium, or any area to be treated. The organisms can grow onsite and produce the biosurfactants to control the pests targeted as described in this invention. The cultures may be mixed with growth enhancement substances to aid in their growth and the production of the microbial biosurfactants. Substances such as oil, glycerol, sugar, or other nutrients may also be used. [0025] In another embodiment of the invention, carbon substrate to support the growth of biosurfactant producing organisms is added to the pest infested areas, soil, plants' growing medium, plant parts, aquatic medium, or any area to be treated. Biosurfactant producing organisms can grow on the substrate to produce biosurfactant in place and control the targeted pests as described in this invention. It is not necessary to add biosurfactant-producing organisms to the substrate. Natural biosurfactant producing organisms found at the site of application will be able to grow and produce the biosurfactant. Examples of carbon substrates that can be added to the targeted areas include, but not limited to, organic carbon sources such as natural or synthetic oil including used frying oil; fat; lipid; wax (natural or paraffin); fatty acids such as lauric; myristic, etc; fatty acid alcohol such as lauryl alcohol; amphiphilic esters of fatty acids with glycerol such as glyceryl monolaurate; glycol esters of fatty acid such as polyethylene monostearate; fatty acid amines such as lauryl amine; fatty acid amides; hexanes; glycerol; glucose; etc. It is preferable to use water insoluble carbon substrate to encourage excessive production of the biosurfactants. In addition to the carbon substrate, nutrients such as vitamins, inorganic minerals may also be added to the substrate to encourage biosurfactant-producing organisms growth. Although it is not necessary, it is preferable to spike or amend the carbon substrate with a sufficient amount of specific biosurfactant to initiate the emulsification process and to inhibit or reduce the growth of other competing organisms for the biosurfactant-producing organism and to control pests. An illustrative but not restrictive example would be the addition of 60-100 mg/l rhamnolipid biosurfactant in the final diluted oil substrate mixture. The diluted mixture is applied to the area to be treated. This method aids in growth establishment of Pseudomonas aeruginosa or florescens (rhamnolipid producing organisms) population and reduces the chance of growth of its competing or disease causing organisms, the Phytophthora, nematodes, Bacillus sp. If it is desired to produce Syringotoxin lipopeptide biosurfactants, a small amount of syringotoxins (less than few mg/l) is added to the oil-glycerol substrate. Syringotoxin will eliminate many competing organisms and maintain Pseudomonas syringae growth while producing the lipopeptide toxins. Pseudomonas syringae and Bacillus subtilis for instance produce series of lipopeptides biosurfactants referred to as porens. These lipopeptide porens include pseudomycin, syringomycin, tabtoxin, phaseolotoxin, and surfactin. Some lipopeptides are capable of creating holes in cell membranes, cells, and tissues. Due to their powerful activity on cells and tissues, these biosurfactants are very useful in controlling algae, nematodes, insects and other pests. Pseudomycin can be applied as a pre-plant treatment for nematode or insects larvae control in crop production. If it is desired to encourage the growth of Bacillus subtilis, a small amount of surfactin biosurfactant is added to the carbon substrate medium to aid in establishment of subtilis population and the production of more surfactin on-site. [0026] The use of carbon substrates to produce biosurfactants at the targeted sites especially in the presence of minute amount of biosurfactant as a biocontrol starting point, greatly enhances the efficacy of the treatment, broadens the biocontrol spectrum against many pathogens, and reduces the frequency and cost of application of the biosurfactant. As it will be described in the examples section, this is very essential for soil treatment applications. [0027] Synthetic surfactants such as alkyl betaines e.g. Lauryl betaine, alkyl sulfates as lauryl sulfate or its salt, alkyl ammonium bromide derivatives, alkyl phenol ethoxylates, alkyl ethylene (or polyethylene) ethoxylates may be used to lower the surface tension and facilitate the utilization of the carbon substrate by the natural biosurfactant-producing organisms but it is preferable to use natural biosurfactants that are able to inhibit the growth of competing organisms and enhance the growth of the specific biosurfactant producing organisms as described in the invention. [0028] Derivatives of these microbial biopesticides or compounds with similar structures or characteristics and able to control pests as also disclosed herein and are encompassed by embodiments of the invention. [0029] It has been observed that some of the mentioned synthetic surfactants above may have inhibitory effects against some pathogens and may also be used as active agents to control pests such as insects, algae, parasitic amoeba, nematodes, weeds or other pests as described in this invention. They may also be used in conjunction with the natural biosurfactants. [0030] According to this invention, biosurfactants (e.g. GLs, FLs, and LPs etc) have a powerful biopesticidal activity against many pests and diseases affecting plants and these biosurfactants also have similar biopesticide activity against pests and diseases affecting humans and animals. Pests controlled include insects, their larvae and eggs; mites; algae (seaweeds, pond algae, and the microscopic algae such as blue-green algae); microbial pests (nematodes, bacteria, fungi, parasites, amoeba, protozoa, viruses, etc); mollusks; worms; and plant weeds. In addition, these biosurfactants may be used to treat human diseases such as ova-parasites and cysts, hair dandruff, etc. In addition, rhamnolipid biosurfactant is an effective spennicide at a concentration of 250 ppm. Examples of animal diseases include, but not limited to, dog's heart worm; fish parasites and microbial infections such as whirling disease caused by the amoeba Myxobolus, fish fungal disease (water mold) or green algae; fish protozoa disease such as Chilodonella ; fish parasites as gill and skin flukes. Also cattle hoof diseases can also be controlled as described in this invention. Animals are treated by dipping or bathing in a biosurfactant solution alone or in the presence of other compounds such as copper or zinc. [0031] The natural biosurfactants' active components may be used according to the invention either alone or combined with other acceptable active or inactive (inert) components that may be used as adjuvants or may have pesticidal activity. It is preferable to use adjuvants or pesticidal components of natural source to complement the natural aspects of the biosurfactants. These components can be either an oil component such as cinnamon oil, clove oil, cottonseed oil, garlic oil, or rosemary oil; another natural surfactant such as Yucca or Quillaja saponins ; or the component may be an aldehyde such as cinnamic aldehyde. Other oils that may be used as a pesticidal component or adjuvants include: almond oil, camphor oil, castor oil, cedar oil, citronella oil, citrus oil, coconut oil, corn oil, eucalyptus oil, fish oil, geranium oil, lecithin, lemon grass oil, linseed oil, mineral oil, mint or peppermint oil, olive oil, pine oil, rapeseed oil, safflower oil, sage oils, sesame seed oil, sweet orange oil, thyme oil, vegetable oil, and wintergreen oil. Other suitable additives, which may be contained in the formulations according to the invention, are all substances, which are customarily used for such preparations. Example of such additives include adjuvants, surfactants, emulsifying agents, plant nutrients, fillers, plasticizers, lubricants, glidants, colorants, pigments, bittering agents, buffering agents, solubility controlling agents, pH adjusting agents, preservatives, stabilizers and ultra-violet light resistant agents. Stiffening or hardening agents may also be incorporated to strengthen the formulations and make them strong enough to resist pressure or force in certain applications such as soil, root flare or tree injection tablets. [0032] Examples of buffering agents include organic and amino acids or their salts. Suitable buffers include citrate, gluconate, tartarate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine and a mixture thereof. Phosphoric and phosphorous acids or their salts may also be used. Synthetic buffers are suitable to be used but it is preferable to use natural buffers such as organic and amino acids or their salts listed above. [0033] Examples of solubility control agents or excipients may be used in the formulations to control the release of the active substances may include wax, chitin, chitosan, C12-C20 fatty acids such as myristic acid, stearic acid, palmitic acid; C12-C20 alcohols such as lauryl alcohol, cetyl alcohol, myristyl alcohol, and stearyl alcohol; amphiphilic esters of fatty acids with glycerol, especially monoesters C12-C20 fatty acids such as glyceryl monolaurate, glyceryl monopalmitate; glycol esters of fatty acids such as polyethylene monostearate or polypropylenemonopalmitate glycols; C12-C20 amines such as lauryl amine, myristyl amine, stearyl amine, and amides C12-C20 fatty acids. [0034] Examples of pH adjusting agents include Potassium hydroxide, ammonium hydroxide, Potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid or a mixture. [0035] Additional components such as an aqueous preparation of a salt as polyprotic acid such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, sodium biphosphate, can be included in the formulation. [0036] According to embodiments of this invention, the microbial biopesticides can be produced and formulated in a variety of ways, including liquid, solids, granular, dust, or slow release products by means that will be understood by those of skill in the art upon learning of the invention disclosed herein. [0037] They may be applied by spraying, pouring, dipping, in the form of concentrated or diluted liquids, solutions, suspensions, powders, and the like, containing such concentrations of the active agent as is most suited for a particular purpose at hand. They may be applied as is or reconstituted prior to use. For example, they may be applied by direct injection into trees or root flares. [0038] Solid formulations of the invention may have different forms and shapes such as cylinders, rods, blocks, capsules, tablets, pills, pellets, strips, spikes, etc. Solid formulations may also be milled, granulated or powdered. The granulated or powdered material may be pressed into tablets or used to fill pre-manufactured gelatin capsules or shells. Semi solid formulations can be prepared in paste, wax, gel, or cream preparations. [0039] For human or animal applications, the formulations may be prepared in liquid, paste, ointment, suppository, capsule or tablet forms and used in a way similar to drugs used in the medicinal drugs industry. The formulations can be encapsulated using components known in the pharmaceutical industry. Encapsulation protects the components from undesirable reactions and helps the ingredients resist adverse conditions in the environment or the treated object or body e.g. stomach. [0040] The compositions according to the invention can be applied to the plants, pests, or soil using various methods of application. Each method of application may be preferred under certain circumstances. [0041] The compositions according to the invention may be used to introduce the active compounds into the soil. These preparations could be incorporated into the soil in the vicinity of the roots of the plants. This could be in the form of liquid, bait, powder, dusting, or granules, or they are inserted in the soil as tablets, spikes, rods, or other shaped moldings. [0042] The compositions according to the invention can be used for treating individual trees or plants. For example, the formulations can be molded in different shapes or forms (solid, paste or gel, or liquid) and introduced into the vascular tissue of the plants. Moldings forms can be as tablets, capsules, plugs, rods, spikes, films, strips, nails, or plates. The shaped moldings can be introduced into pre-drilled holes into the plants or root flares, or they can be pushed or punched into the cambium layer. [0043] Another method of application of the invention is the use of dispensing devices such as syringes, pumps or caulk guns, paste-tubes or plunger tubes for delivering semi-solid formulations (paste, gel, cream) into drilled holes in tree trunks or root flares. [0044] The compositions of the invention can be applied in the form of paste, gel, coatings, strips, or plasters onto the surface of the plant. In one method, a plaster or strip may have the semi-solid formulation, e.g., insecticide placed on the side that will contact the tree, bush, or rose during the treatment. The same strip may have glue or adhesive at one or both ends to wrap around or stick to the subject being treated. [0045] The compositions according to the invention can be sprayed or dusted on the leaves in the form of pellets, spray solution, granules, or dust. [0046] The solid or semi-solid compositions of the invention can be coated using film-coating compounds used in the pharmaceutical industry such as polyethylene glycol, gelatin, sorbitol, gum, sugar or polyvinyl alcohol. This is particularly essential for tablets or capsules used in pesticide formulations. Film coating can protect the handler from coming in direct contact with the active ingredient in the formulations. In addition, a bittering agent such as denatonium benzoate or quassin may also be incorporated in the pesticidal formulations, the coating or both. [0047] The compositions of the invention can also be prepared in powder formulations and filled into pre-manufactured gelatin capsules. [0048] The concentrations of the ingredients in the formulations and application rate of the compositions may be varied widely depending on the pest, plant or area treated, or method of application. As described in greater detail below, the compositions and methods of the invention can be used to control a variety of pests, including insects and other invertebrates, algae, microbial pests, and, in some situations, weeds or other plants. [0049] A purified mixture of rhamnolipids (supplied by Jeneil Biotech of Saukville, Wis.) and Pseudomonas spp. fermentation broth filtrate was tested for their activity on different pests such as thrips, aphids, houseflies, mosquitoes, box-elder bugs, nematodes, spider mites and algae. The cultured material was effective at a concentration as low as 0.005% on some pests. The following are examples to illustrate the procedures of practicing the invention. These examples are illustrative and should not be construed as limiting. EXAMPLES [0050] Fermentation broth containing rhamnolipids was tested for its effectiveness as insecticide to control houseflies. Ten houseflies were confined in petri dishes covered with screen through which the insecticide was sprayed. Another set of ten houseflies were sprayed with water and kept as control. Table 1 shows the results of the test. [0000] TABLE 1 Effect of rhamnolipid treatments on housefly survival. Rhamnolipid Treatments 0% 3% Time(min.) Houseflies Survival Rate 2 100 100 5 100 90 7.5 100 70 10 100 30 12 100 20 15 100 0 [0051] In another test, purified rhamnolipid material at 2.5% concentration was sprayed directly on spiders in naturally infested area. Treatment included six spiders sprayed with the pesticide as a test, while six spiders were sprayed with water as control. Full control of the spider mites was achieved in less than fifteen minutes after treatment. [0052] A naturally infested lemon tree with spider mites was sprayed with a 1.25% rhamnolipid solution. The mites were observed using magnifying glass for movement. Death was noted in less than 15 minutes. [0053] Naturally infested tomato plants with whiteflies were sprayed with 0.1% rhamnolipid mixture diluted in water. Control plants were sprayed with water only. Whiteflies sprayed with the rhamolipids stuck to the leaves and weren'table to move after the treatment. Full control was achieved in less than 6 minutes. [0054] Into each 1-liter water bottle a tablet containing 0, 0.075, or 0.2 grams rhamnolipid (put example) was added. Ten-mosquitoes larvae were transferred into each of the bottles. Total death of the larva was observed in about 2 hours and 40 minutes in the bottle containing 0.2 grams rhamnolipid. In the bottle containing 0.075 grams rhamnolipid, only one live mosquito larva was left after 24 hours of study initiation. No death was observed in the control treatment. This significant discovery is critical in the control and spread of the West Nile Virus Vector. In another study we found that rhamnolipid addition at concentration of 100 ppm prevented mosquitoes eggs from hatching. [0055] A Petri dish containing fifty ml of water infested with amoeba was treated with 250 ppm rhamnolipid. Examining the amoeba under the microscope before and after the treatment showed that within five minutes of rhamnolipid addition that the amoeba collapsed and disintegrated. [0056] An infested red ants mound was drenched with 0.5% rhamnolipid solution. The treatment was effective and the mound was free of ants for more than 2 weeks. [0057] Pesticidal Preparations: Example A [0058] A 5% rhamnolipid solution was prepared using 25% purified rhamnolipid concentrate supplied by Jeneil Biotech. Example B [0059] A concentrated solution was prepared by mixing 20 grams sesame oil, 30 grams canola oil, 10 grams glycerol, and 40 grams water. The mixture is diluted 10, 50 or 100 times with water prior to use. Example C [0060] A concentrated solution was prepared by mixing 5 grams rhamnolipids, 20 grams sesame oil, 30 grams canola oil, 10 grams glycerol, and 35 grams water. The mixture was diluted 10, 50, or 100 times with water prior to use. Example D [0061] Water used as a control treatment. Example E [0062] A concentrated Solution of 10% phosphite and 8% potassium was prepared by mixing 70% phosphorous acid, 45% potassium hydroxide, and water. The solution was buffered with citrate/gluconic acid to pH of 5.8. The mixture was diluted 100 times with water prior to use. Example F [0063] A concentrated Solution of 5% rhamnolipid, 10% phosphite and 8% potassium was prepared by mixing 70% phosphorous acid, 45% potassium hydroxide, and water. The solution was buffered with citrate/gluconic acid to pH of 5.8. The mixture was diluted 100 times with water prior to use. [0064] Grasshopper Study: [0065] For each treatment, six grasshoppers were sprayed with solutions prepared from Examples A, B, C, or D diluted 10 or 100 times with water. At ten times dilution, Example C treatment was the most effective and killed all the treated grasshoppers within ten minutes. Example A treatment at 10 times dilution instantly slowed down the movement of the grasshoppers, but half of the treated recovered within 20 minutes of application. Example B treatment at 10 times dilution had similar effect like that of rhamnolipid alone. At 100 times dilution, example A was not effective. Example B treatment was 33% effective and example C treatment was 84% effective. Water treatments had no effect on grasshoppers. [0066] Powdery Mildew Study: [0067] Diluted solutions of Examples A, B, C, or D were sprayed on squash and roses plants heavily infected with Powdery mildew. Results of rose treatments are presented in Table 2. It was interesting to note that upon spraying the roses with example C formulation at 100 times dilution, the infected area washed out completely from the leaves. The results of squash treatments were similar to rose treatments, but squash plants were more sensitive to the spray solutions. At 10 times dilution, squash leaves developed necrotic tissues within 24 hours of the spray application and the plants shut down and died within three days. Examples E and F were tested on roses only. An important finding in powdery mildew treatments is that neither rhamnolipid nor phosphite alone was very effective against the powdery mildew at the concentrations used; however, the rhamnolipid/phosphite combination was very effective in the treatment of powdery mildew disease. Although powdery mildew does not belong to the zoosporic fungi group, it is believed that the rhamnolipid enhances the activity and mode of action of phosphite through membrane disturbance or by penetrating the fungus protective layers. [0000] TABLE 2 Effect of different formulations on the control of powdery mildew on roses. DILU- DISEASE TION SEVER- PHYTO- TREATMENT FACTOR ITY* CONTROL TOXICITY** EXAMPLE A 10x 4 Limited 2 suppression 50x 5 No Effect 1 100x 5 No Effect 1 EXAMPLE B 10x 2 Effective 4 suppression Total leaf burn 50x 4 Limited 3 Suppression 100x 4 Limited 2 Suppression Localized spots EXAMPLE C 10x 2 Effective 4 suppression 50x 1 Effective 2 suppression 100x 1 Very effective 1 control EXAMPLE D Water 1 No Effect 1 CONTROL EXAMPLE E 100x 5 No Effect 1 EXAMPLE F 100x 2 Suppression for 1 more than 10 days *Treated plants were visually examined for disease symptoms on the leaves. Evaluation was documented on scale of 1-5, where 1 = No Powdery Mildew, 2 = 1-25% infection, 3 = 26-50% infection, 4 = 51-75% infection, 5 = 76-100% infection (all the leaves are infected). Phyto-toxicity was documented on scale of 1-4 where 1 = No necrosis, 2 = 25% of leaf is necrotic, 3 = 50% leaf damage, and 4 = total leaf damage. [0068] Herbicidal Activity: [0069] Due to the powerful micro-emulsifying and penetrating activity of the biosurfactants, especially in combination with oil, they can be used as nonselective herbicides to control weed pests. At concentrations of 0.5% rhamnolipid and higher, necrosis was observed on some plants. This effect is extremely magnified in the presence of oil especially sesame or cottonseed oil. At a concentration of 0.05% rhamnolipid and 2% oil, many treated weeds or plants were destroyed within few days of the treatment. [0070] Nematodes Treatment: [0071] Preliminary tests on nematodes were conducted according to the following procedure. The soil used in this test was isolated from a potato field naturally infested with nematodes. Seventy-five grams of soil (15% initial water content) were wrapped in double folded piece of cheesecloth and fitted in a strainer. The strainer containing the soil was gently suspended in a plastic funnel containing 450 ml water (control), or 0.75% Rhamnolipid mixture. The bottom surface of the strainer containing the soil was maintained in contact with the treatment solutions throughout the study. Twenty-five ml samples were collected at different times through the clamped tubing connected to the stem of the funnel. The supernatant solutions were directly transferred to a petri dish for examination using a microscope. The number of nematode pests surviving was recorded at 24 hrs intervals for a period of seven days. Mortality was concluded if individual nematodes are immobile and fail to respond to disturbance with an eyelash cemented to a needle. The test was done in three replicates. The results are presented in Table 3. [0000] TABLE 3 Effect of rhamnolipid mixture treatments on nematodes control. Rhamnolipid Treatment 0% 0.75% Time (hrs) Total Nematode Control Rate (%) 24 7.7 16.3 48 23.1 35.4 72 27.7 48.3 96 34.6 60.5 120 39.2 74.8 144 47.7 93.0 [0072] Different formulations and concentrations have been tested for activity against potato, tomato, and sugar beet nematodes and other pests. [0073] After the nematode experiment was terminated, a surprise finding was observed on the nematodes treatment solutions present in the plastic funnel. It was observed that the control (water only) solution supported the growth of algae after it was left in the sun for few weeks. On the contrary, rhamnolipid treatment maintained clear solution with no algae growth. To verify the results, another set of treatments at 0 (water as control), 0.005, 0.01, 0.1 and 1% rhamnolipid concentrations were conducted. The water used in the experiment was collected from an algae infested pond. All rhamnolipid treatments did not support the growth of algae. However, at the lowest concentration of rhamnolipid, algae growth was re-established after 6 weeks of the initiation of the study. The other treatments were clean of algae during the three months study. Control treatment (pond water) turned greenish in color and the algae flourished in the water. [0074] Root Knot Eggs Nematode Study: [0075] A set of nematode eggs taken from the roots of tomato plant infested with nematode galls were transferred into petri dishes containing either 25 ml water or 250 ppm rhamnolipid in 25 ml water. The eggs were periodically examined under the microscope. Rhamnolipid treated eggs' color changed to brownish color during the course of the study and the eggs collapsed and disintegrated after 7 days. No change in eggs' color or shape was observed in the water treatment. [0076] Gel preparation: A 5% rhamnolipid Gel formulation is prepared by impregnating 1.0% gum or carboxyvinyl carbopol polymer with purified rhamnolipid dissolved in water. The material is mixed using a vortex to yield a paste in less than 30 minutes. This treatment can be used to rub on animals for Ticks treatment. [0077] FIG. 1 is a flow diagram that illustrates an example of the methods of the invention for controlling pests. The method begins by obtaining a microbial biosurfactant ( 102 ). As described herein, the biosurfactant can be obtained by a manufacturing or cultivation process that occurs prior to applying the biosurfactant ( 104 ). Alternately, the biosurfactant can be obtained by applying a carbon substrate to the environment of the pests ( 106 ) and permitting naturally-occurring microbes to grow on the substrate ( 108 ) and to thereby produce the biosurfactant. In either case, the biosurfactant is applied to the pests or to the environment of the pests ( 110 ), such that the pests are substantially controlled. [0078] FIG. 2 is a flow diagram that illustrates an example of the methods of the invention for producing biosurfactants that can be used to control pests. The method begins by cultivating a biosurfactant-producing microbe, including producing a fermentation broth containing the biosurfactant ( 202 ). The biosurfactant is then obtained ( 204 ) from the fermentation broth in a concentration that can be applied to pests or to an environment in which the pests are located in an amount such that the pests are substantially controlled. Obtaining the biosurfactant from the fermentation broth can be performed in one of a variety of ways illustrated in FIG. 2. For instance, in certain embodiments, the fermentation broth includes the biosurfactants at a suitable concentration ( 206 ) without requiring purification or extraction. Alternately, the fermentation broth can be purified ( 208 ) or the biosurfactant can be extracted from the fermentation broth ( 210 ). Although these exemplary methods illustrated in FIG. 2 are suitable for obtaining biosurfactants, the methods of controlling pests disclosed herein can be performed regardless of the methods used to obtain the biosurfactants. [0079] The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. [0080] The processes, methods of use and examples of components listed in the invention are illustrative and not inclusive. The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The appended claims are presented to illustrate the embodiments of the invention disclosed herein. [0081] One skilled in the art will appreciate that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing order. Furthermore, the outlined steps and operations are only provided as examples, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments. [0082] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [0083] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. [0084] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an”, should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” [0085] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [0086] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. [0087] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. All references recited herein are incorporated herein by specific reference in their entirety.

Summary: Biosurfactants produced by microbes are used to control pests. The biosurfactants can be produced by cultivating a biosurfactant-producing microbe, producing a fermentation broth, and obtaining the biosurfactant from the fermentation broth. Alternately, the biosurfactants can be produced in situ in the environment of the pests by applying a carbon substrate to the pests' environment, which permits naturally-occurring biosurfactant-producing microbes to grow and to generate the biosurfactants. The biosurfactants have pesticidal qualities, and can be used to control a variety of pests, while being biodegradable and otherwise avoiding adverse environmental effects that have often been associated with conventional synthetic pesticides.

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Write a title and summarize: The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models. Amyloids are protein aggregates with a cross-β structure. Amyloid folds are gradually being recognized as important components in the structural landscape of peptides and proteins. The amyloid fold has been envisioned as a generic primordial fold from which globular folds had to emancipate in prebiotic times to attain structural and functional diversification into globular proteins [1], [2], [3], [4], [5]. Amyloid folds also fulfill a variety of biological functions in relation to their specific structural properties [2], [6], [7]. Importantly, amyloids represent the underlying cause of a number of age-related protein deposition diseases which impose a major burden to modern societies [8], [9]. Yet, the determinants that govern amyloid folding and stability are much less well understood than in the case of globular proteins in part because of the scarcity of available high resolution structures of amyloid proteins. Amyloids have the inherent property of being self-perpetuating and as such can represent the mechanistic basis for prion formation [10], [11], [12], [13]. Many prions are amyloids that are self-perpetuating in vivo. Amyloid prions cause fatal neurodegenerative diseases in mammals and can be encountered as epigenetic elements in fungi [14], [15]. The [Het-s] prion of the filamentous fungus Podospora anserina represents an example of such fungal prions [16], [17], [18]. Highly prevalent in nature, [Het-s] is involved in a non-self recognition process that takes place when cells of unlike genotype undergo fusion [16], [19]. This process termed heterokaryon incompatibility leads to the cell death of the mixed fusion cells. The het-s gene exists as two incompatible alleles termed het-s and het-S. When a het-s and a het-S strain are confronted, the incompatibility cell death response leads to the formation of a macroscopic demarcation line termed barrage. It is proposed that this cell death reaction might have a more general function in fungal defense [20], [21], [22]. Strains of the het-s genotype exist as two alternate epigenetic states: [Het-s*] (the non-prion-state) and [Het-s] (the prion state). Transition to the prion state can occur spontaneously at a low rate or can be induced systematically by contact with a prion-infected ([Het-s]) strain. The prion form then invades the fungal hyphal network at a rate an order of magnitude higher than the linear growth rate of the fungus [23]. Incompatibility is only triggered when HET-s is in the prion form ([Het-s]). Thus in this system, the prion state corresponds to the active state of the protein. The [Het-s] prion also displays a specific effect in the sexual cycle, where presence of the prion form leads to specific abortion of the het-S spores in a sexual cross, a process designated spore-killing [24]. The HET-s protein displays two distinct domains, an N-terminal globular domain termed HeLo and a C-terminal prion forming domain [25], [26]. The PFD encompassing residues 218 to 289 is natively unfolded in the soluble form of the proteins and adopts a specific β-solenoid fold in the amyloid prion form of the protein [25], [27], [28], [29]. Cell death is triggered when [Het-s] interacts with HET-S because the HET-s PFD templates the folding of the homologous region of HET-S into the β-solenoid fold which in turn induces a refolding of the globular domain and exposition of a N-terminal hydrophobic helix which targets the cell membrane [30], [31], [32]. A second mode of activation of HET-S, apparently involves the NWD2 STAND protein encoded by the gene adjacent to HET-S which contains an N-terminal region homologous to the PFD region [21], [33]. A high-resolution solid state NMR structure of the amyloid form of the HET-s PFD (HET-s (218–289) based on more than 2500 distance constraints has been reported [28], [29]. This domain adopts a β-solenoid fold in which β-strands delimit a triangular hydrophobic core. The domain is composed of two 21 amino acid long pseudo-repeats each of which forms one layer of β-strand in the β-solenoid structure, the two repeats are connected by a 15 amino acid long flexible loop. At the C-terminus of the domain, a C-terminal loop folds back onto the core region and forms a semi-hydrophobic pocket which can be considered as an extension of the hydrophobic core [28], (Figure 1A). The β-solenoid fold contains two aspargine ladders at the beginning of the first and last β-strand of each repeat and three salt bridges per monomer. The hydrophobic core contains essentially aliphatic residues and serine and threonine residues residing in different β-strand layers and forming a hydrogen bond within the core. A 8. 5 Å cryo-EM model of HET-s (218–289) fibrils has also been reported and largely agrees with the ssNMR data. HET-s (218–289) are singlet fibrils with a left-handed twist and a helical pitch of 410 Å [34]. Sequence comparisons suggest an evolutionary conservation of the β-solenoid fold [35], [36]. Characterization of a HET-S homolog of a different fungal species, the plant pathogen Fusarium graminearum revealed a conservation of the β-solenoid fold and of the prion formation ability [37], [38]. Thus, the HET-s PFD sequence has been evolutionarily shaped to adopt this fold and the overall fold and the prion forming ability have been conserved over an extended evolutionary period. Perhaps, as a consequence of this evolutionary process, the HET-s PFD sequence does not to lead to the formation of amyloid polymorphs in physiological conditions as generally observed for disease causing amyloids or for yeast prions [13], [39]. HET-s (218–289) adopts the same structure in vitro and when forming inclusion bodies during heterologous expression in E. coli [40]. Only at highly acidic pH, when the native β-solenoid fold cannot be attained, HET-s (218–289) adopts an alternate non-infectious amyloid structure [34], [41], [42], [43], [44]. Alanine scanning approaches have been widely used to analyze folding and stability of globular proteins and such approaches have also been used in the context of amyloid fibril formation and stability [45], [46]. In the case of yeast prion proteins such as the well studied Sup35 model, the applicability of such approaches is complicated by the absence of a full structure model, the primary sequence independence in prion formation and the existence of multiple prion strains corresponding to different amyloid polymorphs [47], [48], [49]. Nevertheless, a recent study has revealed the importance of a glycine pair in [PSI+] prion formation as a structural determinant in the soluble form of the PFD [50]. The HET-s model constitutes a favorable system for such studies because of the lack of prion strain variants and the availability of a high resolution structure. A previous study has revealed the functional importance of the β-strand elements in the β-solenoid structure using a proline mutant approach [27]. Here, we relied on the more subtle alanine scanning approach to analyze the functional and structural role of specific residues and structural components of the HET-s PFD fold. Previous studies have analyzed the evolutionary conservation of the β-solenoid fold [21], [35], [36], [37], [38]. Due to the rapid increase in the number of fully sequenced fungal genomes, many additional HET-S homologs sequences have become recently available, we have thus conducted a database search for HET-s/HET-S homologs in current fungal genome sequences. We could identify a total of 51 het-s/het-S homologs (file S1). Based on residues found at 33 of the Helo domain which is known to define HET-s and HET-S allele specificities [51], [52], the homologs found in the other species are of the HET-S, rather than HET-s-type (Figure S1), as suggested previously [18], [21], [30], [37]. Some species like Nectria haematococca and some strains of the Fusarium oxysporum species complex contained up to 4 het-S paralogs. The majority of the sequences showed conservation of the 21 amino acid repeat regions (R1 and R2) but in a fraction of the sequences there was a two amino acid deletion in the first repeated motif (R1). Figure 1B shows an alignment of the 22 non redundant sequences for which both 21-amino acid repeats are conserved. The level of identity between the sequences is in the range of 30% which is about the level of identity between the P. anserina HET-s (218–289) and F. graminearum FgHET-s (218–289) sequences that were previously found to display closely related structures [38]. It can thus be reasonably inferred that the sequences presented in the alignment share similar folds, as previously suggested by homology modeling approaches [35], [36]. The positions showing strict conservation are two of glycines (G242/G278, position 17 of the 21 aa repeat) located in the arc between the third and fourth β-strand of each rung, (Figure 1A and B). The N226/N262 asparagine pair (forming the asparagine ladder at the start of the first β-strand, position 1) was also conserved although some exceptions occurred. The second asparagine ladder (N243/N279, position 18) is not as strongly conserved as the first ladder. There is also a strong conservation of the hydrophobic residues in the core, large hydrophobic residues are found at core positions 231/267 (position 6), 239/275 (position 14) and 241/277 (position 16). There is an interesting trend of mutual exclusion of large hydrophobic side chains at position 228 and 264 (position 3), suggesting that the presence of large residues at both positions might lead to sterical hindrance. Interestingly, two sequences (Fo_4287-2 and Cg_HET-S) constitute exceptions in that regard with an isoleucine found both at 228 and 264. But in these sequences, an Ala residue is found in 231 which might allow for accommodation of the larger residues in position 3 (i. e. residue 231 in Figure 1A). Although the residues forming the three salt bridges identified in HET-s (in positions 4,9 and 11) are not strictly conserved, there is an overall preference for favorable charge interactions at these positions. In the 22 listed sequences, favorable charge combination occurs in 13 and 15 sequences at position 4 and 9 respectively while repulsive interactions are never found. At position 11, favorable charge interactions are less conserved and occur in five sequences. Also of note, is the conservation of the glycine-rich C-terminal loop region encompassing the W287 and F286 aromatic residues. Conservation occurs not only on the aromatic positions but also the glycines and the charged residues (K284 and D288) are strongly conserved. This region is however entirely missing in a fraction of the sequences. Of note is the fact that the sequences missing this region are found in species that contain other HET-S paralogs that do display the loop region. The conservation is poor in the central loop region between R1 and R2. This region generally contains glycine residues and the length of the loop region varies from 12 to 18 residues. It was shown experimentally that three to five amino acid deletions in the loop region could be tolerated without affecting [Het-s] and HET-S function [26], [27]. In order to achieve an overall comparison of the two repeats, we devised a separate consensus sequence for repeats R1 and R2 using MEME [53] (Figure 1C). The consensus sequence reveals some marked differences. In the turn at position 10, there is a preference for a Glu in R1 and a Gly residue in R2. In position 12, there is a preference for an Ala and Ser in R1 and R2 respectively, which is reflected by the reciprocal distribution in position 8. Also, there is a strong conservation of the Gly at the end of R2 in the region leading into the C-terminal semi-hydrophobic pocket. The charge complementary mentioned above is also apparent in these consensus sequences; with a preference in R1 for positive charges in position 4 and negative charges in 9 and the opposite preference in R2. This differential conservation of specific residues in the first and the second repeat suggest that both repeats are not strictly functionally equivalent. Globally, many aspects of the sequence conservation and variation in HET-s homologs are well explained by the structural information available. Yet, the role of certain features such as for instance the conservation of the Arg residue at position 13 is not directly apparent. We set out to analyze the structure-function relation in the HET-s PFD by generating a series of 32 alanine mutants along the HET-s PFD and by testing their prion activity in vivo in Podospora anserina. Based on the sequence conservation described above the β-strand region and the C-terminal pocket were specifically targeted (Figure 1A). The mutation coverage amounts to one mutation every 1. 8 residue in these regions. Mutations were introduced in a plasmid expressing full length HET-s and the corresponding plasmid was introduced into a Δhet-s strain as previously reported for proline and deletion mutants of the PFD [27]. For each mutation, at least 24 individual transformants were assayed for [Het-s] incompatibility function and for their ability to infect a [Het-s*] strain. In this experimental setting, transformants express the transgene from ectopic integration sites and in multiple copies. This approach allows a rough categorization of the mutants. Results are presented in Figure 2. In most cases and as previously observed, the number of transformants able to infect a [Het-s*] strain is equivalent or higher than those producing a barrage reaction to [Het-S] [27]. This is likely due to the fact, that higher prion titers are required to produce an incompatibility reaction than to infect a [Het-s*] strain. We have categorized the mutants into three functional classes: mutations that lead to percentages of active transformants in the range of wild-type (>60% of active transformants), those significantly reducing the number of transformants with [Het-s] activity (>30%and <60%) and those strongly affecting the number of active transformants (<30%). The majority of the mutations (18/32) did not significantly affect [Het-s] activity in this assay. Nine mutants were slightly affected and five were strongly affected. The latter corresponded to two group of mutants, the mutants of the glycine residue at the sharp turn in position 17 (G242A/G278A) and the aromatic F286 and W287 residues in the C-terminal loop. The Ala replacement of the strongly conserved Tyr residue ending the second rung (Y281) led also to a marked decrease in the number of active transformants. The other mutations having a lesser effect on activity were the mutation of the Asn pair forming the first asparagines ladder (N226/N262, position 1). Mutation of the second Asn pair (N243/N279, position 18) had no effect in agreement with the lower conservation of this second N-pair. In general, mutation of the core hydrophobic residues did not reduce activity significantly with the exceptions of I231A and I277A. Also the Ala replacement at positions E234, K270 and R236 affected the prion function. These residues are involved in the second and third salt bridges in the HET-s fold. Mutations in the residues forming the first salt bridge (K229A/E265A, position 4) had no detectable effect on function in this assay. The variant Q240A showed a reduced activity. Q240 is located close to W287 in the structure [28] and it might be that Q240A also affects this pocket region. Indeed, solid state NMR studies of the variants at the C-terminus results in chemical shift changes of Q240 (as discussed below and Figure S3 and S4). The D288 residue is also located in that spatial neighborhood. The mutational approach points to an instrumental role of this region in prion propagation although this region is not part of the β-solenoid core. Based on this first series of mutants, we generated a second set of mutants to specifically test several hypotheses. First, because our results suggest that the salt bridges are not essential for activity, we generated a triple mutant that leads to inactivation of all three salt bridges (E234A, E236A, E265A) and found it to be active (Figure 2). In respect to this observation it is noteworthy that, there appears to be an overall conservation of the salt-bridges at position 4 and 9 of the repeat motif, but some HET-S orthologs such as Cg_HET-S lack these salt bridges completely (Figure 1B). Next, we have generated a double mutant with both N ladders replaced (N226A and N243A). This mutant lacked activity suggesting that at least one N-ladder is required for function and thus providing evidence for a functional role also for the second N-ladder. In both rungs there is a conservation of an Arg residue (R238/R274, position 13). Yet, single Ala replacements at these positions showed normal function. In a R238A/R274A double variant however the prion function is affected. This result could explain conservation at that position. Finally, we reasoned that if a reduced activity of I231A is caused by a reduction of the hydrophobicity of the core, one might be able to introduce a compensatory mutation increasing hydrophobicity elsewhere in the core. We thus changed the A228 residue of the core by a larger residue. We generated a I231A/A228V double variant and found that this double mutant recovered wild-type like activity. We concluded that in this multicopy assay, the function of HET-s appears to be very robust with very few mutations having a very strong effect on prion activity, namely G242A and G278A in the turns after the triangular core and F286A and W287A in the C-terminal pocket. Double mutants could also demonstrate the functional importance of other elements such as the N-ladders, the hydrophobic core (i. e. I231), and the Arg row at position 238/274. The multicopy assay allows an initial functional characterization of the mutants but does not allow for a detailed and sensitive analysis of phenotypic differences. We have thus selected a number of mutants for a more detailed functional characterization as single copy integrants. In order to have more subtle and sensitive assays for prion function, we have re-introduced the mutant allele at the resident het-s locus. We chose to further analyze a subset of seven single mutants N226A, Q240A, E272A, K284A, F286, W287, D288A and the R238A/R274A and T233A/S273A double mutants and the E234A/R236A/E265A triple mutant. Q240A, K284A, F286A, W287A and D288A were chosen to investigate the role of the C-terminal loop region. The triple E234A/R236A/E265A mutant was chosen to analyze in more detail the effect of the elimination of all three salt-brigdes. R238A/R274A and T233A/S273A double mutants were included to analyze respectively the role of the conserved Arg residues close to the loop and the role of the partner hydroxyl residues in the core. Strains with the corresponding gene replacements were generated and tested for [Het-s] function. The three mutants F286A, W287A and R238A/R274A led to a null phenotype as single copy integrants. This result confirms the functional importance of the aromatic residues of the C-terminal pocket and also stresses the functional importance of the conserved Arg residues at position 13 of each repeat. The remaining mutants were able to express the [Het-s] phenotype (ie. infect a [Het-s*] strain and produce a barrage reaction to het-S) and were tested for three other criteria of prion formation and propagation efficacy. First, we determined the spontaneous prion formation rate (Table 1). Secondly, we measured the rate of spreading of the prion infection in the mycelium, reflecting prion propagation efficiency (Figure 3). This propagation rate is measured in an experimental setting where the length of the barrage line corresponds to the distance of prion spreading in a given period of time [54] (Figure S2). Third, the rate of spore-killing in the sexual cycle (a measure of prion maintenance during the sexual cycle) was determined [24], (Table 2). These three criteria have been compared previously between wt and a ΔPaHsp104 mutant. All three were found to be diminished in this mutant background [55]. In these assays, all mutants were affected for at least one of the measured criteria. There was in general a good correlation between the effect of the mutations on propagation rate and spore killing activity (Figure 3 and Table 2). Quantitatively the effects of the mutations were however stronger on spore-killing rates than on propagation rates. For instance N226A and Q240A were affected both in propagation rate and spore-killing activity. While E272A, D288A and T233A/S273A showed close to wt propagation and killing rates. K284A was an exception as this mutation affected spore-killing rates although the propagation rate was close to wt. The E234A/R236A/E265A triple mutant lacking all three salt bridges was significantly affected in both assays, suggesting that the salt bridges participate in [Het-s] function although the effect of the mutation went unnoticed in the more basic multicopy assay (Figure 2). When spontaneous prion formation rates were measured, it appeared that N226A and K284A had significantly reduced spontaneous prion formation rate (Table 1). For K284A, 700 strains were tested but none had spontaneously acquired [Het-s]. Interestingly, we measured an increase in spontaneous prion formation rate in D288A and the T233A S273A double mutant. These mutants represent to our knowledge the first HET-s mutants with enhanced prion behavior (Table 1). In order to get insights into the structural consequences of the Ala replacements, 2D 13C-13C dipolar assisted rotational resonance (DARR) solid state NMR spectra of amyloid fibrils of the 15N, 13C-labeled HET-s (218-289) variants K229A, I231A, V239A, Q240A, L241A, N262A, V264A, V267A, E272A, S273A, R274A, I277A, G278A, N279A, F286A, D288A and the double variant F286A/W287A were recorded and compared with the corresponding spectrum of 15N, 13C-labeled wild-type HET-s (218-289) amyloid fibrils [27], [56]. Since the chemical shift is a highly sensitive probe of the electronic surrounding structure, the measurement of the 2D 13C-13C DARR spectrum can be regarded as a fingerprint of the 3D structure. Thus, a comparison of the DARR spectrum of wild-type and Ala-variant fibrils enables a straight forward analysis of the preservation or alteration of the β-solenoid fold (Figure 1A). This atomic-resolution analysis is of particular importance in the establishment of a structure-activity relationship of amyloids because many amyloidogenic systems are prone to polymorphism and thus a secondary structure analyses or structural comparisons conducted at the mesoscopic level may not be not sufficient to reveal structural preservation or alterations. As shown in Figure 4, the 2D DARR spectra of K229A, I231A, V239, Q240A, L241A, N262A, V264A, E265A, V267A, E272A, S273A, R274A, I277A, E279A, F286A, W288A, and D288A closely resemble the wild-type spectrum. This indicates that the overall structure of the listed variants is conserved. A detailed inspection of the individual spectra shows in general, small chemical shift differences of residues close in space to the replaced amino acid side chain (in addition to the lack of the signal of the replaced side chain), which is attributed to local structural changes. For example, in the 2D DARR spectrum of the N262A variant, cross peak changes of the spatially close residues I231, T261, S263, V264, and I277 are observed (Figure 4, Figure S3, Figure S4, Figure 1A). In the case of E265A located at the surface, no significant chemical shift changes are evident (Figure 4, Figure S3, Figure S4, Figure 1A). Similar findings are observed for all the other Ala variants (but G278A, and the double variant F286A/W287A, see below) as discussed in details in the figure caption of Figure S3. In the case of I231A, a substantial amount of small local chemical shift perturbations are observed for A228, K229, D230, T233, V239, L241, T266, V267, V268, V275 and I277 as well as for the more remote residues E235, V244, A247, A248, K270, R274, and L276, hinting that I231 is a structurally important hydrophobic core residue. In addition to these local chemical shift alterations, (slight) perturbations of chemical shifts of I277 are found for many Ala variants (i. e. I231A, Q240A, N262A, V264A, V267A, R274A, F286A, W287A) indicating that I277, which is the second Ile side chain in the center of the core of the β-solenoid structure (Figure 1A), responses sensitively to slight structural alterations. In striking contrast to all the variants discussed, the 2D DARR spectrum of the 15N, 13C-labeled variant G278A is reproducibly distinct from the corresponding spectrum of wild-type HET-s (218–289) amyloids (Figure 4). The spectrum appears to be less well dispersed and the cross peaks are broad when compared to wild-type HET-s (218–289) amyloids. The spectrum is actually reminiscent to the spectrum of the non-infectious HET-s (218–289) amyloid grown at pH 3 which was also found to be less dispersed and to have broad cross peaks [42]. Hence, it is concluded that the G278A variant does form a distinct amyloid structure, which may serve as an explanation of the lack of a prion phenotype (Figure 2; and see below). The nature of the misfolding of the G278A variant may be that the Gly residue is key for the arc between the two β-strands β4a and β4b. In addition, the F286A/W287A double variant shows a 2D DARR NMR spectrum quite different from the wild-type DARR spectrum (Figure 4). There are major chemical shift perturbations spread over the β-solenoid structure (i. e. I231, S273, E235, K229, T233, N262, R238, A247, V264, V244, I277, and Q240), making it difficult to assess whether the double variant forms the β-solenoid fold (Figure 4). In conclusion, the comparative analysis of the solid state NMR spectra suggests that all the single Ala variants with the exception of G278A form the HET-s prion β-solenoid fold. With the exception of G278A, all the single Ala variants can therefore be used to explore the detailed structure-infectivity relationship of the HET-s prion. One of the consequences of a side chain replacement with Ala may be a change in the stability of the protein fold, which may influence function [57], [58], [59], [60]. Applied to HET-s this would mean that a change of the stability of the β-solenoid may alter heterokaryon incompatibility, spontaneous prion formation, prion propagation and spore killing (Figure 2, Figure 3, Tables 1 and 2). In order to get insights into the individual contribution of the amino acid side chains to the stability of the HET-s prion, Ala variants K229A, I231A, V239A, Q240A, L241A, N262A, V264A, V267A, E272A, S273A, G278A, F286A, D288A and F286A/W287A were measured by a fibril denaturation assay using GuHCl (Figure 5 and Table 3) following the concept by Santoro and Bolen to study protein folding and unfolding [61]. However, here we studied only the fibril denaturation process. This was monitored by measuring the light scattering at 500 nm (OD500) [62] versus GuHCl concentration (Figure 5) detecting fibril disassembly. Since the β-solenoid amyloid structure is composed of many inter-molecular interactions it is assumed that disassembly and unfolding occur together and that this detection method measures thus both fibril disassembly and β-solenoid unfolding. Such a fibril denaturation reaction mechanism can be of complex nature but here a simplified two-state process was assumed for the quantitative analysis. HET-s fibril denaturation studies have been performed previously using Trp fluorescence as probe for fibril denaturation [41], because the unique Trp residue (W286) is located in the C-terminal loop rather than in the actual core, we favored the use of light scattering because it might constitute a better probe for the overall unfolding of the fibril since Trp fluorescence might be affected by local unfolding of the loop. The individual denaturation curves in triplicates show a sigmoidal behavior (Figure 5). [61]. For most of the variants (i. e. wild-type, K229A, I231A, V239A, Q240A, L241A, N262A, V264A, V267A, E272A, S273A, F286A, D288A) the measurements appear to be reproducible. Only for G278A and possibly the double variant F286A/W287A a lack of reproducibility is observed (Figure 5). The lack of reproducibility is attributed to the altered, possibly polymorphic structure as evidenced by the solid state NMR spectra (Figure 4). A further qualitative inspection of Figure 5 suggests that the variants K229A, V239A, L241A, V264A, V267A, E272A, S273A, and D288A (labeled group 1 in the following) show sigmoidal denaturation curves similar to wild-type, while the denaturation curves of the variants I231A, Q240A, N262A, G278A, F286A (group 2), and the double mutant F286A/W287A have a different form and properties and are sometimes less reproducible (as just stated) (see also Table 3, fifth column). The two-state analysis of the denaturation curves yields two parameters for the unfolding transition: the m-value and the Gibbs free energy ΔG. The m-value is the steepness of the transition and is generally thought of as a measure of the change in solvent accessible surface area upon unfolding and reflects how cooperative the unfolding transition is [63], [64], [65]. The change in Gibbs free energy (ΔΔG) is a measure of the contribution of the amino acid side chain to the protein unfolding energy (given that the structures of the folded and unfolded states as well as the unfolding pathway are otherwise unperturbed). The variants of group 1 can all be fitted with a two-state model, most of them with an unchanged m-value (i. e. the m-value of the wildtype HET-s (218–289) prion). All the group 1 variants had an unaltered ΔΔG value or were up to 2 kcal/mol more stable (Table 3). The positive ΔΔG value for K229A of 1. 5 kcal/mol indicates that the salt bridge between K229 and E265 does not play a favorable stability effect on the β-solenoid structure. Similarly, the replacement E272A (removing the E272-R236 salt bridge) results in a positive stability effect of approximately 2 kcal/mol. The amino acid replacements in the hydrophobic core at position V239, L241, V264 also had a 1 kcal/mol positive effect on stability V267 had no effect. This is in line with the in vivo data showing that none of these variants were affected for the prion phenotype (Figure 2). In combination with the solid state NMR spectra (Figure 4 and see above), these unfolding measurements indicate that the Ala variants of group 1 are composed of the same β-solenoid 3D structure and the amino acid side chain replacement had no negative effect on its unfolding transition. In contrast, the GuHCl denaturation curves of group 2 variants (Figure 5) are distinct from wild-type showing a flattened slope (lower m-value) (i. e. I231A, Q240A, and N262A) indicative of a decrease in unfolding cooperativity or a change of the surface. The latter potential explanation finds support in the surface location of the Q240. Some replacements comprise a complex unfolding transition exemplified by an immediate loss of light scattering at low GuHCl concentrations (i. e. I231A, Q240A, N262A, and F286A) or poor reproducibility (i. e. G278A and F286A/W287A), thus for these variants a Gibbs free energy calculation has not been considered (see Table 3). It is worth mentioning, that the less reproducible nature of the denaturation curves of G278A and F286A/W87A is reflected in the less well defined solid state NMR spectra (Figure 4). Together the data strengthen the notion that these two variants are not able to fold properly into the β-solenoid structure. The effect on the denaturation curve of N262A is attributed to the loss of the conserved Asn ladder. The I231A in the hydrophobic core replacement appears to influence the stability and unfolding pathway (Table 3). The NMR analyses also indicated an important structural role of this residue as the I231A replacement leads to a substantial amount of chemical shift perturbations in the β-solenoid fold (Figure 4, Figure S3 and S4). Most interestingly, with the exception of D288A there appears to be a perfect correlation between the decrease in prion phenotype formation in vivo determined by the heterokaryon incompatibility assay (Figure 2) and the change in the denaturation curve profile (Table 3). The F286A and W287A mutants affecting the C-terminal semi-hydrophobic pocket stand out in virtue for their drastic effect on [Het-s] activity although the corresponding residues reside outside the rigid β-solenoid core and do not alter the β-solenoid structure significantly (Figure 4). Only the double variant F286A/W287A may be unable to form the β-solenoid structure as suggested by the solid state NMR studies (Figure 4) and the GuHCl denaturation curves (Table 3, Figure 5). Moreover, two further mutants in the same region K284A and D288A also modulate prion behavior in a subtle way as the first mutation decreases prion formation while the other in fact increases the prion formation rate (Table 2). However, the D288A variant shows a wild-type-like solid state NMR spectrum and is ca. 1. 2 kcal/mol more stable than wild-type, having otherwise a wild-type-like GuHCl denaturation curve (Figure 4 and 5). We chose to further analyze the mutants in that region for their ability to form aggregates in vivo as GFP fusion proteins. We found that neither F286A nor W287A led to the formation of dot-like aggregates in vivo, however when the mutant allele as GFP fusion proteins were introduced in a strain expressing wild-type HET-s (Figure 6), the mutant alleles were found to form dot-like aggregates presumably by being integrated into wild-type aggregates, a situation already described for the K284P mutant residing in the same conserved C-terminal loop region [27]. We also tested infectivity of the variants F286A and W287A fibrils generated in vitro and found them to be infectious (Table 4). The level of infectivity of the recombinant fibrils was at least as elevated as for wild-type fibrils. Infectivity could be detected in the same level of dilutions as for wild-type fibrils. These results indicate that once the amyloid fold is acquired, these mutants display wild-type seeding activity in vivo. The determination of the HET-s β-solenoid structure exposed the existence of a number of structural elements defining this fold, such as the hydrophobic core, the asparagines ladders, the sharp turns connecting the β-strands termed arches [66], the salt bridges, the internal hydrogen bond between the Thr and Ser residing in different β-strand layers and the C-terminal loop folding back onto the core [28], [29]. The functional importance of the individual β-strands was documented before in a mutational approach involving proline substitutions [27]. Here, it is shown that to various degrees, the other described structural determinants also participate in prion function. Ala replacement of the strictly conserved G278, leads to a null prion phenotype and this variant is unable to form the β-solenoid structure as evidenced by the solid state NMR studies. This effect is very likely due to the importance of the glycine residue in the arc between β-strands β4a and β4b for the β-solenoid fold (Figure 1). Ala replacements in the asparagine ladder affected function in vivo and the N262A variant shows an altered denaturation curve. The hydrophobic core residues I231 and I277 appear to be important for prion infectivity (Figure 2) and in the case of I231A also for β-solenoid unfolding and structure (Table 3, Figure 4). Also, the substitution of the highly conserved Thr/Ser pair leads to a detectable alteration of prion function but only in the spore-killing assay, while the S273A variant is 1. 4 kcal/mol more stable. For the prion function in vivo, the solvent exposed salt bridges are important determinants, but their contribution to the β-solenoid stability is negative (i. e. E272A is 1. 9 kcal/mol more stable than wild-type). Sequence comparisons shows a preference for a positively charged residue in position 13 of each repeat (R238 and R274 in HET-s) (Figure 1). The functional significance of these conserved Arg residues is not directly evident. While the single Ala replacement did not change the prion activity, the R238A/R274A double mutant leads to a null phenotype thus providing a functional justification for the sequence conservation. A possible explanation of the role of these positively charged residues could be an interaction with the C-terminal loop and in particular the penultimate conserved Asp residue. In support of this hypothesis, is the fact that the two sequences that are exceptions to the conservation of positively charged residues at these positions also lack the C-terminal glycine-rich loop like Fo_CL52-2 and Nh_mpVI-1, (Figure 1B). In the presented Ala mutagenesis scan, two mutants were found for which spontaneous prion formation rate was increased rather than decreased, the D288A mutant and the T233A/S273A double mutant. This would indicate that the HET-s PFD does not have an optimal behavior in terms of prion formation rates. It might be that the increased prion formation rate of D288A is due to the higher stability of the variant (i. e. ΔΔG = +1. 2 kcal/mol, Table 3) or/and a modification of the properties of the C-terminal loop which is functionally important for a prion phenotype (see below). The T233A/S273A double mutation increases the overall hydrophobicity of the core. Possibly, this increase in hydrophobicity favors a hydrophobic collapse and subsequent prion nucleation and/or stabilizes the β-solenoid fold since already the amyloid of S273A alone is 1. 4 kcal/mol more stable (Table 3). Interestingly, the D288 and the S273 residues (and to a lesser extend T233) are conserved positions (Figure 1B). Thus, one may hypothesize that conservation at these positions reflects the need to reduce spontaneous β-solenoid folding because a too strong propensity for spontaneous folding might lead to uncontrolled activation of the HET-S pore-forming toxin. Similarly, the fact that several replacements increase rather than decrease stability also suggests that the HET-s PFD sequence is not optimized for highest possible stability. The theoretical model of prion propagation by the Weissman group [67] and others [68], [69] highlights that for the prion propagation, amyloid fibril fragmentation is necessary. Thus, highest possible amyloid stability may not be compatible with the function of the HET-s fold. One finding of this study is the detection of the functional importance of a 7 amino acid residues C-terminal loop starting at the end of β-strand β4b and ending at the C-terminus of the protein (GKGFWDN, residue 283 to 289). The only previous indication of the functional importance of this region came for the observation that the K284P mutation affects HET-s aggregate formation in vivo [27]. Structurally, this region forms a semi-hydrophobic pocket that folds back onto the space delimited by the third and fourth strand of each rung [28]. This part of the structure is less well defined and appears to be more dynamic than the core region. The individual mutations of the two aromatic residues F286A and W287A have a very strong effect on activity. Actually, these two mutations are the most severe ones in our set of 32 mutants together with the mutations of the Gly in the arc positions (i. e. G242/G278). In addition, the mutations of the conserved charged residues in that region K284A, D288A (and the structurally nearby Q240A) also affect function. As noted above, the D288A mutation produced an unexpected effect on prion function as this mutation increased spontaneous [Het-s] prion formation. This is the first mutation found to increase [Het-s] prion formation. Of note however is that other criteria such as the spore-killing activity are in fact slightly reduced in that mutant. A recent molecular dynamics simulation study of the HET-s (218–289) β-solenoid suggested that the F286A and W287A mutations reduce flexibility at the fibril ends and that this flexibility is an important determinant for prion fibril infectivity [70]. However, F286A and W287A are able to form fibrils in vitro that display the same levels of infectivity as wild-type fibrils suggesting that mutation of the aromatic residues does not critically affect templating activity and the 3D structure of the β-solenoid fold. Alternatively, the hydrophobic nature of F286 and W287 may be important for an initial non specific hydrophobic interaction of the incoming molecule with the prion template. This hypothesis is supported by the solid state NMR spectra of both variants indicating no significant structural changes of the β-solenoid fold. Rather it appears that this region might be required for the prion formation process without actually being part of the cross-β part of the fold. These interpretation illustrates that amyloid folding can be dramatically modulated in a functionally relevant way by a sequence stretch that is not actually part of the cross-β structure per se. Strikingly, this part of the HET-s PFD sequence turns out to be the region of the protein with the highest level of sequence conservation. While, the repeat region can accommodate a significant level of divergence and still retain the ability to adopt the β-solenoid fold, the ability for this short C-terminal region to act possibly as a folding inducer of the repeat region appears highly constrained at the sequence level. The β-solenoid forming region of HET-s/HET-S is thus located between the HeLo domain which can have a prion inhibitory function and the C-terminal pocket region which acts as a prion promoting region [26]. These results revealing the existence of a short amyloid folding modulator stretch that is not directly involved in the cross-β core could be of general importance in the identification of sequence determinants for relevant amyloid formation and in efforts to control amyloid aggregation in disease related systems. Since many proteins are able to aggregate into amyloid-like entities and since the inter-molecular β-sheet formation with its many hydrogen bonds is an essential determinant of the cross-β-sheet motif, it has been suggested that the formation of amyloid fibrils is mainly a generic property of the polypeptide backbone and that the side chains play a minor role [71]. On the contrary, amyloid aggregation is highly amino-acid sequence specific as demonstrated by the intermolecular side chain interactions observed in the crystal structures [72], [73], [74] and the essential involvement of side chain interactions in the aggregation process as evidenced for example by the experimentally-derived scale of amino-acid aggregation-propensities (ranging from the aggregation-prone hydrophobic residues to the aggregation-interfering charged side chain [75], [76], [77]). These observations accompanied with the predictive power of several algorithms for the cross-β aggregation propensity of polypeptide sequences [78], [79], [80] suggests that the cross-β state is less complex than most structures of soluble proteins, indicating that the complexity of the cross-β fold may fall somewhere between a secondary and a tertiary structure [2]. This notion of intermediate folding complexity of amyloids is now supported by the stability measurements of the HET-s Ala variants. Indeed, our study reveals both the absence of destabilizing effect for many replacements but also the critical importance of certain key residues. While the loss of side chain hydrogen bonds, salt bridges and hydrophobic interactions in soluble proteins yields in general a ΔΔG loss of ∼−2 kcal/mol [58], [59], [60], in the case of the HET-s prion, the ΔΔG for the group 1 variants with K229A and E272A deleting a salt bridge, S273A getting rid of a side chain hydrogen bond, and V239A, L241A, V264A, and V267A reducing the hydrophobicity of the core, is either insignificant or with a positive value of up to +2 kcal/mol (Table 3). In particular, hydrophobic core residue replacements have little effect (+0. 6 kcal/mol in average; Table 3) compared to the average ΔΔG of ∼−2 kcal/mol predicted from data of soluble proteins. Only a few side chains appear to be crucial for the formation of the β-solenoid structure (such as for instance I231 and N262). Side chain replacements of such residues lead to in vivo effects but also to noticeable changes of the GuHCl denaturation curve. Thus, it appears that alteration of the β-solenoid structure can be achieved either by Pro insertions [27] having a drastic consequences on the backbone structure or by replacing side chains of a limited number of key residues that appear critical for directing the peptide backbone into the β-solenoid fold. P. anserina strains used in this study were wild-type het-s, het-S, Δhet-s strains and the ΔPaHsp104 strain [55]. Growth medium for barrage assays and prion transmission assays was standard corn meal agar DO medium. The Δhet-s strain was constructed by inserting the nat1 cassette from the pAPI508 plasmid [81] in place of the het-s ORF. The nat1 cassette was amplified with oligonucleotides 5′ CTTCCCTTCCACTTCTTCACAC 3′ and 5′ ATCCTAGATGACTTAAGACGACAGG 3′. The sequences upstram and downstream of the het-s ORF were amplified respectiviely with oligonucleotide pairs 5′ AAGCTTTTCGAATTGGTCTCTCAG 3′ and 5′GGGCAGTTTGAGGGGAAAGCGAAG 3′ and 5′ GGGACTAGTACCCTCCAGCAAGGATAGC 3′ and 5′ GCGGCCGCCATGGGCACTGCATCTGGG 3′. The fragments were ligated to create the het-s: : nat-1 cassette cloned as a HindIII-NcoI fragment in a pSK plasmid. This cassette was used to transform a het-s ΔPaKu70 strain [81]. Nourseothricine resistant transformants compatible with a het-S tester were selected. Inactivation of het-s was verified by PCR using oligonucleotides 5′ CGACGATCACAGCTATAGCGTGGTG 3′ and 5′ ATCCGGCTTCCCTGGACCTGCTTC 3′. The strain was then backcrossed once to a het-s wild-type strains and Δhet-s (NourR and het-S compatible) Δhet-s ΔKu70 strains (NourR, PhleoR and het-S compatible) were selected in the progeny. Methods for determination of incompatibility phenotypes, prion formation and prion propagation were as previously described [54]. In brief, incompatibility phenotypes were determined by confronting strains of solid corn meal agar medium to [Het-s] and [Het-S] tester strains and visualizing the formation of barrages (abnormal contact lines forming upon confrontation of incompatible strains). [Het-s] prion propagation was assayed as the ability to transmit the [Het-s] prion from a [Het-s]-donor strain to a [Het-s*] prion-free tester strain after confrontation on solid medium. Prion formation rates of [Het-s] were determined by measuring the fraction of [Het-s*] or subcultures that spontaneously acquired the prion phenotype after 5 days of growth at 26°C. For fluorescence microscopy, synthetic medium containing 2% (wt/vol) agarose was poured as two 10 ml layers of medium. P. anserina hyphae were inoculated on this medium and cultivated for 16 to 24 h at 26°C. The top layer of the medium was then cut out and the mycelium was examined with a Leica DMRXA microscope equipped with a Micromax CCD (Princeton Instruments) controlled by the Metamorph 5. 06 software (Roper Scientific). The microscope was fitted with a Leica PL APO 100X immersion lens. HET-s (218–289), HET-s (218–289) Ala variants proteins were expressed in E. coli and purified as previously described [27]. Both proteins had a C-terminal 6 histidine tag and expressed as insoluble proteins and purified under denaturing conditions using Qiagen columns. Yields were in the range of 10 mg/L of culture. Proteins were eluted in 6 M GuHCl 50 mM Tris–HCl pH 8,150 mM NaCl, 200 mM imidazole. Elution buffer was replaced by 175 mM acetic acid by passage on a 5 ml Hitrap column (Amersham). After lyophilisation the samples were stored at −20°C. The protein variants were dissolved in diluted HCl (45 mM) and the concentration adjusted to 90 µM which was affirmed by UV absorption at 280 nm. The solution was filtered through a 0. 2 µM filter and Buffer E (Tris [3 M]; NaCl [1 M] HCl [45 mM] 1/19 V) was added such that the end concentration of NaCl and Tris were 150 and 50 mM respectively. If necessary pH was adjusted to 7. 4 using NaOH (4 M). Samples were rotated at 37°C on a rotator at constant speed for at least one week. Protein transfection experiments with amyloid fibrils of recombinant HET-s (218–289) variants were performed using a cell disruptor (Fast-prep FP120, Bio101, Qbiogen Inc.). For each test, ∼0. 5 cm3 of [Het-s*] mycelium grown on solid medium is sheared (run time 30 s, speed 6) in 500 µl of STC50 buffer (0. 8 M sorbitol, 50 mM CaCl2,100 mM Tris HCl pH 7. 5) and the sonicated HET-s (218–289) amyloids assembled at pH 7 (20 µl at 1 mM) in a 2 ml screw cap tube. The sheared mycelium is then diluted with 600 µl of STC50 buffer and then plated onto DO−0. 8 M sorbitol medium and incubated at 26°C until being confluent (7–8 days). Several implants (at least two per mycelium) are checked for the [Het-s] phenotype in barrage tests. The fibril containing samples of the various HET-s (218–289) variants were concentrated after centrifugation (20000 g; 10 min) to 500 µM. The fibrils were then diluted in the different GuHCl stock solutions (between 0–8 M in 0. 5 M steps) to an end concentration of 20 µM. It is important to note that the fibril samples were added to the GuHCl solution by an Eppendorf pipette with a single extrusion. The samples were incubated at room temperature without shaking for one day or if stated for one week, respectively. The extent of amyloid disassembly was monitored by the measurement of the optical density at 500 nm (OD500) on a JASCO V-650 spectrophotometer with quartz cuvettes and an adaptor from Eppendorf. The OD500 is believed to be an indirect measure of the amount of protein aggregation. Each experiment has been measured three times starting in part from different sample preparations. The analysis and extraction of the ΔG and m-value was done by the software package MATLAB following the mathematical formula and procedures of Santoro and Bolen [61]. As stated above the samples were incubated before the OD500 measurement at the various concentrations of GuHCl for one day in order to reach equilibrium conditions. Longer incubation periods such as one week did either not alter the measurements as demonstrated for the V267A variant in Figure S5, or a flattening of the denaturation curve was observed as shown for the V264A variant (Figure S5). The flattening of the curve is interpreted as a restructuring of the amyloid aggregate during the long incubation time of one week, which let us concentrate on the use of the denaturation measurements with one day incubation for the quantitative analysis. Although in the case of V264A the extracted ΔG value did not alter between the two type of measurements (Figure S5), the change of the denaturation curve indicates that in GuHCl only a pseudo-equilibrium is obtained. Another point worth mentioning is that highly reproducible denaturation curves were obtained by incubating the protein in GuHCl solution without shaking, while in presence of shaking the reproducibility was only moderate (data not shown). All the solid-state NMR experiments were conducted on a AVANCE II Bruker 600 MHz (14. 1 T) spectrometer using a Bruker 3. 2 mm probe. DARR spectra with 20 ms mixing time were obtained from 15N, 13C-labeled HET-s (218–289) variants [82]. All the spectra were processed using Topspin 2. 0 (Bruker Biospin), using a shifted cosine squared window function as indicated in the Figure captions and so called zero-filling was done to the next power of two in both dimensions. Automated baseline correction was applied in the direct dimension. For spectral analysis the software package CcpNMR Analysis [83] was used and the chemical shift list was taken from [56]. HET-S homologs were recovered from available genome databases at ncbi (http: //blast. ncbi. nlm. nih. gov/Blast. cgi) and jgi (http: //genome. jgi-psf. org/) using BLAST searches. Multiple alignments were performed with CLUSTALW2 at http: //www. ebi. ac. uk/Tools/msa/clustalw2/. The consensus sequence of the R1 and R2 repeats were generated using MEME at meme. nbcr. net/, [53]. The MEME output gives a graphical representation of the consensus sequence as a weighted consensus in which the size of the letter designating a given amino acid is proportional to the conservation of the residue in the different sequences used to generate the motif. The size of the character reflects the information content measured in bits.

Title: Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function, Amyloid Structure and Stability Summary: Prions are infectious protein particles causing fatal diseases in mammals. Prions correspond to self-perpetuating amyloid protein polymers. Prions also exist in fungi where they behave as cytoplasmic infectious elements. The [Het-s] prion of the fungus Podospora anserina constitutes a favorable model for the analysis of the structural basis of prion propagation because a high resolution structure of the prion form of [Het-s] is available, a situation so far unique to this prion model. We have analyzed the relation between [Het-s] structure and function using alanine scanning mutagenesis. We have generated 32 single amino acid variants of the prion forming domain and analyzed their prion function in vivo and structure by solid-state NMR. We find that the PFD structure is very robust and that only a few key mutations affect prion structure and function. In addition, we find that a C-terminal semi-flexible loop plays a critical role in prion propagation although it is not part of rigid amyloid core. This study offers insights on the structural basis of prion propagation and illustrates that accessory regions outside of the amyloid core can critically participate in prion function, an observation that could be relevant to other amyloid models.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``National Bone Marrow Donor Registry Reauthorization Act''. SEC. 2. NATIONAL BONE MARROW DONOR REGISTRY. (a) National Registry.--Section 379 of the Public Health Service Act (42 U.S.C. 274k) is amended-- (1) in subsection (a)-- (A) in paragraph (1), by striking ``except that'' and all that follows and inserting ``except that-- ``(A) such limitations shall not apply to the Chair of the board (or the Chair-elect) or to the member of the board who most recently served as the Chair; and ``(B) 1 additional consecutive 2-year term may be served by any member of the board who has no employment, governance, or financial affiliation with any donor center, recruitment group, transplant center, or cord blood bank.''; and (B) in paragraph (4)-- (i) by striking ``the Naval Medical Research and Development Command'' and inserting ``the Department of Defense Marrow Donor Recruitment and Research Program operated by the Department of the Navy''; and (ii) by striking ``Organ'' after ``Division of''; (2) in subsection (b)-- (A) in paragraph (4), by inserting ``at least'' before ``annually''; (B) in paragraph (7), by striking ``and comparisons of transplant centers regarding search and other costs that prior to transplantation are charged to patients by transplant centers; and''; (C) in paragraph (8), by inserting ``and outreach'' after ``and demonstration''; (D) at the end of paragraph (8), by striking the period and inserting a semicolon; (E) by redesignating paragraphs (3) through (8) as paragraphs (4) through (9); (F) by inserting after paragraph (2), the following: ``(3) maintain and expand medical emergency contingency response capabilities in concert with Federal programs for response to threats of use of terrorist or military weapons that can damage marrow, such as ionizing radiation or chemical agents containing mustard, so that the capability of supporting patients with marrow damage from disease can be used to support casualties with marrow damage;''; and (G) by adding at the end the following: ``(10) conduct and support research to improve the availability, efficiency, safety, and cost of transplants from unrelated donors and the effectiveness of Registry operations; ``(11) increase the number of umbilical cord blood units listed in the Registry and assist cord blood banks in the Registry program in accordance with subsection (c); and ``(12) establish bylaws and procedures-- ``(A) to prohibit any member of the board of directors of the Registry who has an employment, governance, or financial affiliation with a donor center, recruitment group, transplant center, or cord blood bank from participating in any decision that materially affects the center, recruitment group, transplant center, or cord blood bank; and ``(B) to limit the number of members of the board with any such affiliation.''; (3) in subsection (c)-- (A) in clause (ii) of paragraph (2)(A), by striking ``, including providing updates''; and (B) in paragraph (3), by striking ``the availability, as a potential treatment option, of receiving a transplant of bone marrow from an unrelated donor'' and inserting ``transplants from unrelated donors as a treatment option and resources for identifying and evaluating other therapeutic alternatives''; (4) in subsection (d)-- (A) in paragraph (2)(C), by inserting ``and assist with information regarding third party payor matters'' after ``ongoing search for a donor''; (B) in paragraph (2)(F)-- (i) by redesignating clause (v) as clause (vi); and (ii) by inserting after clause (iv) the following: ``(v) Information concerning issues that patients may face after a transplant regarding continuity of care and quality of life.''; and (C) in paragraph (3)(B), by striking ``Office may'' and inserting ``Office shall''; (5) in subsection (g), by striking ``the bone marrow donor program of the Department of the Navy'' and inserting ``the Department of Defense Marrow Donor Recruitment and Research Program operated by the Department of the Navy''; (6) in subsection (h)-- (A) by striking ``Application.--'' and inserting ``Contracts.--''; (B) by striking ``To be eligible'' and inserting the following: ``(1) Application.--To be eligible''; and (C) by adding at the end the following: ``(2) Considerations.--In awarding contracts under this section, the Secretary shall give substantial weight to the continued safety of donors and patients and other factors deemed appropriate by the Secretary.''; (7) in subsection (i), by striking ``include'' and inserting ``be''; and (8) by striking subsection (l). (b) Bone Marrow Scientific Registry.--Section 379A of the Public Health Service Act (42 U.S.C. 274l) is amended-- (1) in subsection (a), by adding at the end the following: ``The scientific registry shall participate in medical research that has the potential to improve transplant outcomes.''; (2) in subsection (c), by striking ``Each such report shall in addition include the data required in section 379(l) (relating to pretransplant costs).''; and (3) by adding after subsection (c) the following: ``(d) Publicly Available Data.--The scientific registry shall make relevant scientific information not containing individually identifiable information available to the public in the form of summaries and data sets to encourage medical research and to provide information to transplant programs, physicians, and patients.''. (c) Bone Marrow and Marrow Defined.--Part I of title III of the Public Health Service Act (42 U.S.C. 274k et seq.) is amended-- (1) by redesignating section 379B as section 379C; and (2) by inserting after section 379A the following: ``SEC. 379B. BONE MARROW AND MARROW DEFINED. ``For purposes of this part, the terms `bone marrow' and `marrow' include bone marrow and any other source of hematopoietic progenitor cells the acquisition or use of which is not inconsistent with Federal law.''. (d) Authorization of Appropriations.--Section 379C of the Public Health Service Act, as redesignated by subsection (c), is amended to read as follows: ``SEC. 379C. AUTHORIZATION OF APPROPRIATIONS. ``(a) In General.--For the purpose of carrying out this part, there are authorized to be appropriated $32,000,000 for fiscal year 2004, and such sums as may be necessary for each of the fiscal years 2005 through 2008. ``(b) Emergency Contingency Response Capabilities.--In addition to the amounts authorized to be appropriated under subsection (a), there are authorized to be appropriated such sums as may be necessary for the maintenance and expansion of emergency contingency response capabilities under section 379(b)(3).''. Passed the House of Representatives October 1, 2003. Attest: Clerk.

Title: To amend the Public Health Service Act to reauthorize the National Bone Marrow Donor Registery, and for other purposes Summary: National Bone Marrow Donor Registry Reauthorization Act - Amends the Public Health Service Act to authorize: (1) specified FY 2004 appropriations, and FY 2005 through 2008 appropriations, for the National Bone Marrow Donor Registry; and (2) appropriations for emergency contingency response capabilities. Revises Registry provisions respecting: (1) board of directors term of office; and (2) Registry functions, including emergency response to terrorist threats injurious to bone marrow, and umbilical cord blood units and blood banks. Defines "bone marrow" and "marrow" to include bone marrow and any other source of hematopoietic progenitor cells the acquisition or use of which is not inconsistent with Federal law.

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Write a title and summarize: Protocell multilevel selection models have been proposed to study the evolutionary dynamics of vesicles encapsulating a set of replicating, competing and mutating sequences. The frequency of the different sequence types determines protocell survival through a fitness function. One of the defining features of these models is the genetic load generated when the protocell divides and its sequences are assorted between the offspring vesicles. However, these stochastic assortment effects disappear when the redundancy of each sequence type is sufficiently high. The fitness dependence of the vesicle with its sequence content is usually defined without considering a realistic account on how the lower level dynamics would specify the protocell fitness. Here, we present a protocell model with a fitness function determined by the output flux of a simple metabolic network with the aim of understanding how the evolution of both kinetic and topological features of metabolism would have been constrained by the particularities of the protocell evolutionary dynamics. In our model, the sequences inside the vesicle are both the carriers of information and Michaelis-Menten catalysts exhibiting saturation. We found that the saturation of the catalysts controlling the metabolic fluxes, achievable by modifying the kinetic or stoichiometric parameters, provides a mechanism to ameliorate the assortment load by increasing the redundancy of the catalytic sequences required to achieve the maximum flux. Regarding the network architecture, we conclude that combinations of parallel network motifs and bimolecular catalysts are a robust way to increase the complexity of the metabolism enclosed by the protocell. The RNA world hypothesis [1–3] describes a stage in the early evolution of life where RNA constituted both the catalytic and the informational component of living systems. This scenario has a reasonable degree of plausibility accounted for by the evidence gathered from both prebiotic chemistry [4,5] and the analytic approach of evolutionary biology [6] to the study of the origins of life. Constrained by this assumption, elucidating the evolutionary dynamics of primordial replicators becomes a fundamental question, initially addressed by the quasi-species model [7,8]. This formalism assumed that error rates for early self-replicators must have been inherently high leading to a distinct evolutionary behavior, namely that a sequence, regardless of its fitness, never fixates in the population, but instead is always surrounded by its mutant neighborhood forming a quasispecies. Furthermore, given a sharply peaked fitness landscape, if the mutation rate is increased to a critical value, known as the error threshold, the extinction of the population known as the error catastrophe will occur. One possible solution would be to reduce the mutation rate, but that would almost certainly involve a more complex self-replicator, encoded by a sequence of considerable length, which is in turn limited by the error rate. This evolutionary cul-de-sac, known as Eigen’s paradox, required a reassessment of the model of protobiotic systems in light of their poor evolutionary potential. Trying to address this issue led to the consideration of distributing the information encoded by the sequences in a mutualistic community of replicators known as the hypercycle model [9] in order to relax the error rate. However, the large amplitude of the oscillations of the stationary state of the hypercycle model could lead to its extinction upon stochastic perturbation which, together with the susceptibility of the hypercycle model to the invasion of selfish molecules, make it difficult for the community of replicators to emerge without a form of population viscosity [10]. Viscosity could have been provided by limited spatial dispersal over a substrate like clay [11,12] or as a limiting case, the compartmentalization of the sequences in a protocell acquiring a multi-level selection [13,14] structure when a the vesicle survival is coupled to its sequence content. The hypothetical protocell organization has been extensively modelled in the past decades, from population genetics multilevel selection models to explicit biophysical accounts of chemical reactions that contribute to the formation of the vesicle membrane [15,16]. These two avenues have remained relatively separated in the literature and the population genetic models have not fully explored the consequences of deriving the fitness function from realistic models of catalysed reaction networks that could have occurred inside the vesicle. On the other hand, the biophysically inspired models are mainly concerned with the coupling of chemical reactions to the division of the membrane but do not incorporate population genetic considerations such as mutation, recombination or variations in heredity mechanisms. The population genetics multilevel selection protocell models have their origins in the package model [17,18] and the stochastic error corrector model [19,20]. These models describe the evolutionary dynamics of a set of replicating sequences encapsulated in a protocell and consider the processes that such a system would have had to endure at the dawn of life such as random assortment of the sequences upon fission, mutations to non-functional sequence types and conflict between selection at the sequence level and the vesicle level. All of these processes suppose that the protocell population exhibits an average fitness lower than the maximum achievable which is commonly referred as genetic load. In these models, the protocell level fitness is commonly specified by a simple function, like the geometric average, of the frequencies of the sequence types contained in the vesicle. The dynamics at the sequence level inside the protocell is described by a replication-mutation process. Deleterious mutation is typically introduced into the system in the form of irreversible mutation to a defective type that does not directly affect the protocell fitness except by reducing the effective total number of functional sequences. These protocell models account, in a non-systematic manner, for the study of additional complexities and processes to the protocell dynamics. For example, the incorporation of mutation schemes exhibiting trade-offs in catalytic activities [21,22], modifications in the mechanism of inheritance or the incorporation of protocell fusion [23] and recombination [24]. Few of them have directly considered a model of the structure of the underlying metabolic network and those that have, did so in a limited manner by studying a linear chain of unsaturated Michaelis-Menten enzymes [25]. Most of the vesicle models depict a scenario where the nature of the encapsulated sequences is RNA, corresponding to the idea of the ribocell as a solid candidate for the protocellular stage in the early evolution of life [26]. The coupling between metabolism and replication would have been particularly strong in the hypothesized ribocells as the RNA sequences are supposed to have operated as both the catalysts and the carriers of the genetic information. We conjecture that this must have limited the space of possible catalysed chemical reaction networks which could be encapsulated within the protocells. In this work, we incorporate a simple implementation of enzymatic metabolism to the protocell model to address the problem of determining the complexity and architectural characteristics of early metabolic networks. The approach of considering the organism structure has been taken in other studies of the evolution of biological networks [27,28] that highlight the relevance of accounting for some level of biological detail to study evolutionary dynamics. Metabolic networks are modelled with different degrees of dynamic and kinetic detail. With a high level of kinetic detail, metabolic networks can be modelled with systems of differential equations incorporating aspects of the reaction mechanisms and properties of their catalysts. These same reactions can be studied in the stochastic limit when the number of particles considered is low. A framework that connects both the stochastic and the deterministic limits of a reaction network is that of chemical reaction network theory [29]. With a lower level of kinetic detail, one can use modelling techniques such as flux balance analysis to understand the general behavior of reaction fluxes at stationary states [30]. As a compromise between realism and computability we modeled the protocellular metabolism as a mass action irreversible reaction network with a small number of catalysts. In our model, each of the metabolic reactions is catalyzed by a sequence of a given type with a Michaelis-Menten kinetic mechanism and thus exhibiting saturation behavior. The output of this metabolic network determines the fitness of the protocell. We are interested in the transition to the coexistence of all sequence types at the protocell population level and how this depends on the kinetics and architecture of the network. For simple protocell models, the transition to coexistence has been extensively studied analytically [31]. Sequence coexistence provides a way to increase the complexity of protocellular metabolism by harboring different types of catalysts. We find that saturated metabolic fluxes provide a way to increase the coexistence of sequence types in the protocell by allowing the necessary redundancy to alleviate the fitness load produced by assortment when the protocell divides. We also find that the combination of parallel fluxes with a terminal bimolecular catalyst provides a way of increasing the complexity of the protocellular metabolism. We model the protocell evolutionary dynamics as a Wright-Fisher process following the approach in [32]. We augment the package model with a fitness function given by the flux of a critical metabolite, ϕ (M), coming from a small metabolic network comprised of a set of chemical reactions, r, catalysed by the sequences inside the protocell. Each protocell contains a number of sequences from a finite set of sequence types, or genes, g, of cardinality |g| = |r| + 1, meaning that there is a sequence type catalysing each reaction of the metabolic network plus a mutational sink, the type ω, that does not contribute to the metabolic activity. The population dynamics is derived from the following protocell life-cycle. At the beginning of the life-cycle, N sequences are found in the vesicle distributed amongst the set of sequence types and each sequence is equally likely to replicate. The total number of sequences at the beginning of the protocell life-cycle will be herein referred as the protocell ploidy. When the sequences replicate they can mutate to the defective type, ω, with a probability traditionally unpacked in terms of the sequence length, L, and the mutation probability per base, μ, so that the accuracy of the replication process is given by q = (1 − μ) L. Hence, the sequences have a probability (1 − q) to mutate to the defective type. After reaching twice the ploidy, 2N, the vesicle splits in half and the sequences randomly assort between the offspring protocells regaining the starting total number of sequences N. At the population level, once all the protocells have divided, half of them are selected with a survival probability proportional to the critical metabolite flux. A simplified treatment of metabolic networks is used to define the protocell fitness function in our simulations. We evaluated the consequences on the protocell evolutionary dynamics of varying the kinetic parameters and the organization of the catalysts in different network topologies. Our model assumes the separation of the dynamics at three time scales. The quickest time scale is that of metabolic reactions catalysed by the sequences. The next time scale is that of the replication of sequences and division of the protocell. The third and longest time scale is that of the dynamics of the population of protocells. We make use of simplifying assumptions based on each of the time scales being much longer than the previous time scale. We illustrate the effects of the variations in the kinetic and stoichiometric parameters of the network with the example of a protocell containing two catalysts (Fig 1A). This network is the starting point for the construction of larger networks which allow us study the effects of the network architecture on the protocell evolutionary dynamics. The first catalyst, the keystone (denoted as α), takes an environmental input A that diffuses inside the protocell to produce the critical metabolite, M, that is required for the protocell survival. The keystone catalyst also produces certain amount, c w α, of a waste product, W that can be converted by a second catalyst, the recycler (denoted as β), into c M β molecules of critical metabolite. A protocell composed solely of keystone catalysts is viable and thus constitutes an absorbing boundary of the stochastic process. For all of the networks studied, we assume that the environmental input is present at sufficiently large quantities such that its concentration is not affected by the protocell population growth and the keystone catalysts that use it are in the saturation regime. The corresponding metabolic flux for this scenario according to our formalism is given by (see Methods, section 4. 1. 2): Φ = k cat α [ α ] + c M β min (c w α k cat α [ α ], k cat β [ β ]) (1) Here kcat denotes the catalytic constant of the catalysts. With the total ploidy constraint, the fitness function defined as the critical metabolite flux of this metabolic network (Fig 1B) will have a single maximum with a position on the space of protocell types given by the kinetic parameters and the protocell composition, more concretely when c w α k cat α [ α ] = k cat β [ β ]. The position of the maximum determines the behaviour of the observables of the population like the coexistence fraction; the fraction of the population with protocells containing the maximum number of functional sequence types. This dependence can be shown by choosing the kinetic and stoichiometric parameters so that the flux is closer or further away from the absorbing boundary of protocells with no recycler sequences. One simple way of achieving this is by increasing the catalytic constant, k cat β, of the recycler sequence to get closer to the boundary or increasing the waste output of the keystone sequence by changing the stoichiometry, c w α, to get further away from it. The remaining stoichiometric coefficients can be adjusted to achieve the same maximum flux for comparison as the fitness difference between the maximum in the interior and the fitness of the absorbing boundary of the stochastic process (that is when the protocell lacks any recycler sequences and just harbors the keystone) also affects the ploidy at which the coexistence transition occurs. The coexistence fraction of the stationary population for the flux with the maximum further away from the absorbing boundary is always higher than the one closer to it (Fig 1C). This is due to the effects of stochastic assortment when protocells divide that can bring a fraction of the population to fixate at the boundary. This probability is smaller as we get further away from the boundary and it vanishes upon increasing the ploidy of the protocell leading to a stationary population of coexisting types only. Hence, fluxes with a maximum further away from the absorbing boundary benefit coexistence as they are less affected by the loss introduced by the assortment. In terms of the average fitness of the stationary population, the flux further away from the boundary also presents a higher average fitness for all ploidy values and consequently if the two fluxes were to be competed with no interaction between the protocells the flux further from the boundary would dominate at the infinite population size limit (see S1 Fig). This is because the flux that is further away from the boundary is also flatter than the other flux and thus suffers from less assortment load as the population can spread over a larger region of viable coexisting types. When mutations leading to the defective type are taken into account, the decay of the coexistence fraction of the flux closer to the boundary is faster when increasing the mutation rate than that of the flux further away from it (Fig 1D). In terms of average fitness of the populations, both landscapes show similar values at low mutation rates but the flux further to the boundary becomes fitter as the mutation rate increases and the coexisting types are lost in the flux closer to the boundary. As noted by [32], the way mutation towards a defective sequence type is usually included in the protocell model has no other effect at the protocell level than reducing the total number of functional types in the vesicle. Therefore, the impact of mutation on the coexistence fraction of the stationary population will be equivalent to that of reducing the ploidy to the effective ploidy given the mutation rate and the starting ploidy of the protocell. For a given flux function and mutation rate, the effective ploidy will saturate at a constant value as the total starting ploidy is increased. At high mutation rates the effective ploidy becomes sensitive to the assortment in the flux closer to the boundary and thus the flux further away from the boundary is also more robust to the effects of introducing non-functional mutants. We now focus on analysing effects of network architecture by considering two different arrangements of three catalytic sequences (Fig 2A). We will add the third recycler catalyst in serial or parallel with respect to the second recycler sequence of the previous example. In both cases studied, we assume for simplicity that the keystone catalyst produces two different waste products to have non-competing reactions. In order to discern effects derived from the topology of the networks alone, the total fitness benefit a protocell containing all three catalysts and the marginal fitness benefit of the third sequence with respect to the other two given by the kinetic and stoichiometric parameters was set to be the same for each of the studied architectures. According to our simplifying treatment (see Supplement, section 7. 1), the output metabolite fluxes for these two architectures are: Φ S e r i a l = k cat α [ α ] + c M β min (k cat α [ α ], k cat β [ β ]) + c M γ min (k cat α [ α ], k cat β [ β ], k cat γ [ γ ]) (2) Φ P a r a l l e l = k cat α [ α ] + c M β min (k cat α [ α ], k cat β [ β ]) + c M γ min (k cat α [ α ], k cat γ [ γ ]) (3) The fluxes over the simplex of protocell types all have their maxima at the center for the parameters chosen removing the effect of the proximity to the absorbing boundaries. The transition to coexistence for the parallel addition of the third catalyst occurs at a lower ploidy values than that of the serial addition (Fig 2B). The coexistence fraction also grows faster with ploidy for the parallel architecture. Both observations can be explained in terms of the assortment load, as the parallel architecture has a larger region of viable protocell types surrounding the maximum making it more robust to assortment by allowing the population to spread over this region (Fig 2C). The serial architecture, on the other hand, has a smaller region of viable types surrounding the maximum and becomes more sensitive to the assortment. The mean fitness of the population reflects also this pattern given by the assortment load (S2 Fig). It can be concluded that for our model the population observables are mostly determined by the features of the fitness landscape given by the metabolic flux. The protocell population can be seen as a quasispecies with a cloud of vesicles around the fittest type spread by the assortment at a particular ploidy. Adding mutation to the system does not change the conclusion of the parallel addition higher robustness as mutations factor in the dynamics by effectively reducing the ploidy of the system. The process of increasing the complexity of the studied networks by sequentially connecting to them additional recycler catalysts given the considered motifs, raises the question about what is the best way to perform this addition in order to guarantee a transition to coexistence of the resulting networks at low ploidy values. Adding a new sequence type to the protocell always comes at the cost of increasing the effects of the assortment, which depends on ratio the protocell ploidy and the total number of sequence types. This means that any increase in the coding capacity of the protocell, in terms of the number of coexisting sequence types, requires in turn an increase in the vesicle ploidy. It has been shown that by making the ploidy sufficiently large, the numbers of types that can coexist in the protocell can be made to increase arbitrarily [32,33]. This is, however, just for the cases with low competition at the sequence level and no mutations. The requirement of increasing the ploidy becomes a burden once mutation is considered, as higher ploidy protocells accumulate mutants at a faster rate (S3 Fig). If certain amount of mutants are considered deleterious as in the early versions of the package model, the mutation process will set a limit to the ploidy increase. The mutation process also establishes a limit on the number of catalytically active sequences in the protocell by saturating to a constant effective ploidy regardless of the starting ploidy value. This can bring the population to the absorbing boundaries if the effective ploidy is low enough for the assortment to dominate. A way of keeping the ploidy moderate while adding new catalysts to the network is by increasing the marginal fitness benefit of each added sequence (changing the stoichiometric coefficient cM). This can substantially lower the critical ploidy for the coexistence transition and its effects will depend on both the fitness benefit and the topology of the previous architecture in place. For instance, if we were to add another catalyst with a high marginal fitness benefit to our two previous examples, the parallel architecture would become unbalanced leading to the fixation of one of the branches while the serial addition would still be viable. In order for the parallel architecture to accept the new sequence with a higher marginal fitness benefit without becoming unbalanced a bimolecular catalyst should be considered. A bimolecular catalyst can be added to two independent keystone catalysts and allow them to coexist. We consider the case of two keystone catalysts converting two independent environment metabolites into the critical metabolite and also producing two different waste products that are in turn required for the third recycler catalyst in a bimolecular reaction to produce more metabolite of interest (Supplement, section 7. 2. The parameters were chosen to have the same marginal fitness benefit and maximum fitness than the previously studied cases. According to our simplifying the flux would be: Φ B i m o l e c u l a r = c M α k cat α [ α ] + c M β k cat β [ β ] + c M γ min (k cat α [ α ], k cat β [ β ], k cat γ [ γ ]) (4) The bimolecular motif flux exhibits a large region of viable types around the maximum however, the protocell population is also able to spread to an absorbing boundary with a large neutral space as the two keystone catalyst have the same marginal fitness benefit (Fig 3A). These features of the fitness landscape contribute to a large assortment load and a substantial fraction of the population is close to the absorbing boundary even at relatively high ploidy values (Fig 3C). Therefore, the coexistence transition for this architecture occurs at larger ploidy values than the other two architectures in spite of a larger region of viable types surrounding the flux maximum (Fig 3B). On the other hand, the third bimolecular catalyst is able to provide a way for the coexistence of these two keystone catalysts. Moreover, the bimolecular reaction gives us a way of adding a catalyst with a large fitness benefit to the parallel architecture without unbalancing it. We now consider the fluxes derived from the addition of a fourth catalytic sequence to the previous networks as a unimolecular or bimolecular recycler (Supplement, section 7. 3). We again consider the production of an additional waste metabolite to have non-competing reactions. When the added recycler catalyst has the same marginal fitness benefit as the other two recyclers, the parallel architecture shows the transition to coexistence at the lowest ploidy value, as in the three catalyst networks previously analysed (Fig 4A). The parallel architecture is followed by the two other architectures with parallel motifs showing that for the case of equal marginal fitness benefit the parallel architectures are better at supporting the coexistence of catalyst and thus maintaining metabolic complexity. This is due to the larger region of viable types surrounding the flux maximum for the parallel architectures. When the marginal fitness benefit of the fourth recycler catalyst is greater than the other two, the parallel architectures are no longer the ones showing coexistence transitions at lower ploidy values. The bimolecular and the serial architectures will be the ones showing a markedly earlier transition to coexistence than the architectures with parallel motifs (Fig 4B). The addition of a recycler with larger marginal fitness benefit unbalances the parallel architectures unless it is added as a bimolecular motif. Increasing the complexity of metabolic networks in the protocell model requires the balance between the limitations given assortment load, the marginal fitness benefit and the risk of unbalancing the previously existing architectures. We find that combining bimolecular and parallel pathways one can achieve networks with transitions to coexistence at lower ploidy values that would avert the problems introduced by mutations at large ploidy values. Our finding is interesting in the light of the prevalence of bow-tie modules in extant metabolic networks [34,35] as the concatenation of the parallel and bimolecular motifs could produce such a network feature. Mathematical models of early life forms constitute a powerful tool to test the feasibility of proposed hypothetical scenarios in the first stages of life. They allow us to increase our understanding of the conjectured scenarios and provide intuition about how to overcome their limitations. Our metabolic model of the protocell serves to analyse the effects of considering details of the kinetic mechanism of the catalysts encapsulated and their network architecture. Our model provides an example of how the fitness load introduced by protocell fission assortment process and its effects on the coexistence of catalysts can be alleviated by modifications in their kinetic parameters and their network organization. The requirement to withstand the assortment load limits at low ploidy values the metabolic architectures that can be effectively encoded in the protocell. Although the networks analyzed here are simple and small, they already show interesting consequences of accounting for the saturation of the catalysts in different architectures. We demonstrate that variations in the kinetic parameters that keep the catalyst saturated provide a way to increase the catalyst redundancy and diminish the assortment effects. The saturation of the catalysts means that the reaction they control becomes limiting and additional catalytic sequences will consequently increase the output flux. The other major limitation for the evolution of a complex protocellular metabolism is the interplay of the effects of the assortment and the network architecture. We demonstrate that architectures with a larger region of viable types surrounding the maximum in the interior favour the coexistence transition as they allow for the quasispecies to spread. The architectures that are favoured also depend on how they are assembled, more specifically, the marginal fitness benefit of the new catalyst with respect to the previous network catalysts. This benefit can happen directly by a higher stoichiometric benefit or indirectly by increasing the catalytic efficiency of the enzyme which allows the limiting catalyst to increase their numbers in the vesicle virtue of the flux saturation. For architectures containing parallel motifs it will be detrimental to accept the new catalyst if it has a higher marginal fitness benefit unless it is involved in a bimolecular reaction. The bimolecular motif prevents the system from becoming unbalanced and becomes a fundamental building block for larger architectures. Nevertheless, the assortment load together with the coupling of genes and catalysts, limit the architectures of the metabolic networks that can be maintained in the vesicle by coexistence of its catalysts. This limit in the metabolic evolutionary potential of the protocell can be alleviated, as it has been previously pointed out [36], by the evolution of the decoupling between genetic information and catalytic activity, either with the advent of proteins or DNA in a ribozyme world or by the divergence of complementary RNA sequences [37]. The evolution of correlated inheritance like the chromosomal organization would be another mechanism that would allow the exploration of a larger space of network architectures and catalytic efficiency [38] as it would diminish the assortment load. An example of releasing this constraint can be found in [25] which makes use of the unsaturated linear metabolic chain formulation of [39] to address the effects of chromosomal organization in the evolution toward specialization of promiscuous catalysts. The model presented incorporates assumptions about the biochemistry of the hypothetical protocell and standard population dynamics that can be sophisticated or relaxed with interesting consequences. For example, the incorporation of trade-off between catalytic and replication activity has shown interesting dynamical features at the population level such as sustained oscillations [40]. The metabolism model could be further enriched in the direction of the whole cell model [41] by increasing the biochemical details considered. The only limitation is that the nature of early living things is purely conjectural and it would be preferable to understand general features of the evolution of metabolic networks rather than a concrete instantiation mirroring extant life. Together with this consideration, a better model for the development stage of the protocell and the corresponding population dynamics would allow us to decompose the summary statistic of the evolutionary dynamics, that is fitness, in terms of the whole interplay of the network flux and the replication of catalytic sequences throughout the developmental stage. A more detailed account on protocell development would require a full stochastic treatment that properly accounts for the separation of metabolic and replicative dynamic time scales. The effects of introducing other processes in the population dynamics like alternative division mechanisms in the metabolic protocell model as described by [42,43] or modification in the heredity mechanisms already studied in previous protocell models could also yield interesting results. Further incorporating considerations about environment would lead us in the direction of understanding the ecology of the protocells. If our intermediates that leak out accumulate significantly in the environment [44], interesting frequency dependent relations can be established in the protocell populations and one could add protocell fusion to study the assembly of networks in this augmented model. The protocell organization is considered a solid candidate for a transitional form of life and, as such, has been extensively explored computationally and is starting to be addressed in experimental settings [45]. The utility of the protocell theoretical models has been recently validated in helping the development of chemical implementations of protocell structures [46]. This also provides the experimental set-up to test the conclusions of the theoretical works in the protocell literature. Furthermore, construction of the first synthetic autonomous protocell is becoming a reality and we believe that theoretical models can be useful to further this endeavour. We will model the metabolic activity of our protocell as a well-stirred reactor in which ribozymes, environmental inputs, and metabolites interact. More specifically, the metabolic dynamics will be modelled as Michaelis-Menten enzyme dynamics where each flux is given by the expression v = k cat [ E ] [ S ] K m + [ S ] (5) Where, [E] and [S] denote the concentrations of enzyme and substrate respectively and kcat is the turnover number (catalytic constant) of the enzyme and Km its Michaelis constant. We will also study bimolecular reactions with mechanisms of the following form: (6) The velocity will have the functional form v r = k cat [ S 1 ] [ S 2 ] K m + a 1 c 1 + a 2 c 2 + c 1 c 2 (7) where the values of a1 and a2 can be determined from either the forward and the backward rates of the underlying reactions or from thermodynamic potentials. Special values of these constants correspond to the cases of random order, definite order, non-interacting. We will model the depletion reactions as exponential decay or diffusion v δ = δ i [ S ] (8) where δi is the decay rate or diffusion constant for S. We assume that even though the protocell development will be accompanied by an increase in the vesicle volume, this will be negligible and will not dilute significantly the concentrations of the catalysts and intermediates of the reaction network. Therefore we can identify the catalysts’ concentrations with the catalyst number in our model of metabolism. We will augment this hypothesis with three further assumptions. We assume that the supply of this input in the environment is high enough that we can disregard depletion and treat its concentration as constant (but note that the concentration of the same inputs within the protocell may vary due to the permeability of the membrane). We assume that the intermediates that leak out to the environment are degraded and do not accumulate. We assume that the characteristic timescale of the metabolic reactions is sufficiently faster than the timescale of sequence replication that we may ignore initial transients, approximate the metabolic reactions by their (dynamic) equilibrium solution, and describe the metabolic outputs by their averaging their rates of production over a metabolic timescale. Our aim is to enrich the evolutionary population genetic models with relevant biochemical features rather than go on the direction of a full-blown simulation of a cellular system. However, in order to introduce these features realistically, we will derive them from a simplified “whole protocell” model in which the metabolic reactions described above are augmented with reactions that describe the replication of the sequences: α + M → 2 α β + M → 2 β (25) We will model the replication as mass action kinetics. As described in the previous section, we will model the enzymatic reactions using Michaelis-Menten kinetics. The only difference is that now we will augment the equation for the critical metabolite with a term which accounts for the depletion of metabolite as the sequences replicate. d [ M ] d t = Φ - r [ M ] ([ α ] + [ β ]) For the two catalyst keystone recycler network, we have the system of equations: d [ α ] d t = r [ M ] [ α ] d [ β ] d t = r [ M ] [ β ] d [ M ] d t = k cat α [ α ] [ A ] + c M β k cat β [ β ] [ W ] K m β + [ W ] - r [ M ] ([ α ] + [ β ]) d [ W ] d t = k cat α [ α ] [ A ] - δ [ W ] - k cat β [ β ] [ W ] K m β + [ W ] For convenience, we introduce two rescaled variables—the ratio of types x and the scaled flux ϕ: x = [ α ] [ α ] + [ β ] ϕ = Φ [ α ] + [ β ] = k cat α x [ A ] + c M β k cat β (1 - x) [ W ] K m β + [ W ] Combining the first two equations, we conclude that d x d t = ([ β ] d [ α ] d t - [ α ] d [ β ] d t) ([ α ] + [ β ]) 2 = r [ M ] ([ β ] [ α ] - [ α ] [ β ]) ([ α ] + [ β ]) 2 = 0 so the ratio α to β remains constant during development. Since ϕ only depends upon α and β through their ratio, ϕ will also remain constant. As per the “slow-fast” minority control hypothesis of Kaneko and Yomo [48], we shall assume that the counts and reaction rates of the sequences are much smaller than the counts and reaction rates of the metabolites. In terms of the constants, this means that k cat α, k cat β > > r. As a consequence, the concentrations of M and W will approach an equilibrium values after an initial transient. Combining the equations, we conclude that d d t ([ α ] + [ β ]) = r [ M ] ([ α ] + [ β ]) = ϕ ([ α ] + [ β ]). The general protocell model for our study will be built following a similar approach to the package model [17,32]. We will add to it an explicit representation of the metabolism by considering a set of chemical reactions, R, catalyzed by the sequences contained in the protocell. Each protocell will contain a number of sequences from a finite set of sequence types, g, that, in principle will be of cardinality | g | = | R | + 1, indicating that there will be a sequence catalyzing each reaction, r ∈ R, of the metabolic network plus a mutational sink, the type ω, that will not contribute to the metabolic activity. We will model the protocell development process as 2N independent samplings with replacement from the initial contents of the vesicle to represent the replication of sequences. Let ji denote the number of number of sequences of type i present at the beginning of the cycle. The probability of sampling a sequence of type i equals ji/N. The probability that a replication will produce a sequence of non-error type i equals q j i N (26) and the probability of producing an error type ω equals 1 - q + q j ω N. (27) Hence the probability of an outcome in which there are ki offspring of the sequence type i is given as P dev (k i | j i) = (2 N k 1, k 2, … k ω) ∏ i = 1 g (q j i N) k i (1 - q + q j ω N) k ω (28) The probability that a protocell of this composition will then assort into an vesicle containing mi sequences of type i and an offspring with ki − mi sequences of type i equals to Passt (mi; ki−mi|ki) =∏i=1g (miki) (N2N) (29) The probability of a particular outcome at the end of the lifecycle equals the product of the development and assortment probabilities. Defining m i ′ = k i - m i and simplifying with the aid of binomial identities, we obtain the result P life (m i; m i ′ | j i) = P asst (m i; m i ′ | k i) P dev (m i + m i ′ | j i) = (N m 1, … m ω) ∏ i = 1 g (q j i N) m i (1 - q + q j ω N) m ω × (N m 1 ′, … m ω ′) ∏ i = 1 g (q j i N) m i ′ (1 - q + q j ω N) m ω ′ (30) The way this probability factors means that the type of one vesicle offspring is independent of the type of the other offspring given the initial protocell composition and that both follow a Wright-Fisher process. This does not mean that the two daughter protocells are independent after development. Our population is comprised of a number of non-interacting protocells each of which undergoes a lifecycle as described above. To describe this population, we introduce the summary statistic F (ji) which is the frequency in the population of protocells which began their development process with ji copies of the i-th sequence. Let W (ji) denote the survivability of a typical protocell which begins its development process with ji copies of the i-th sequence. Such a population may be modelled either as a branching process to describe a growing population or as a Moran process to describe a population which has reached its carrying capacity. In the limit of large population size, the dynamics of either of these models become deterministic and the equilibrium frequencies of both models are determined by the same eigenvalue equation ∑ j i | ∑ i j i = N K (m i | j i) F (j i) = W ¯ F (m i) (31) where K (m i | j i) = W (m i) (N m 1, … m ω) ∏ i = 1 g (q j i N) m i (1 - q + q j ω N) m ω (32) and W ¯ is the mean fitness. Since all the components of the matrix K (mi|ji) are non-negative, this equation may be solved numerically by using Perron-Frobenius iteration for small ploidy values (up to 100) and few sequence types (up to three). For higher ploidy values, both the Perron-Frobenius iteration or the Moran Process become cumbersome, so an agent based simulation which also approaches the same limit for large population size was used instead. As in [24], the agent based model algorithm starts with an homogenous populations of protocells with the same number of sequences for each catalytic type. The entire population undergoes development and assortment doubling the number of protocells. Half of the population is selected without replacement with a probability proportional to its fitness. We found that 5000 generations were sufficient to reach the stationary behavior of the model in all the cases studied. Comparing instances the Perron-Frobenius iteration and the agent-based model available shows that even for small population sizes of 1000, the results are already in reasonable agreement with the infinite population limit. Using these values of 5000 generations of 1000 individuals, the computation proceeds sufficiently rapidly that it becomes feasible to scan across ranges of parameter values and generate plots which show how observables vary across parameter space. The model was implemented in Julia version 0. 3. 11 [49] and the plots were generated in python 2. 7. The code used for the simulations can be found in GitHub as: https: //github. com/jopejor/protocell_metabolism.

Title: The evolutionary dynamics of metabolic protocells Summary: The protocell hypothesis conjectures the existence of a vesicle containing catalytic and replicating sequences as the primordial cellular organization during the early stages of the evolution of life. Mathematical models of protocells traditionally consider RNA sequences being encapsulated and having both an informational and a catalytic role in the same molecule. Because of this dual function, the protocell sequences are evolutionarily constrained. Mathematical models have been extensively used to study the evolutionary dynamics of protocells with a focus on the processes, like mutation or stochastic sequence assortment upon division, that affect the protocell information capacity in terms of the coexistence of different sequence types. Here we introduce a simple model of metabolic networks whose output determines the survival of the protocell with the aim of studying the effect of modifying the kinetic and architectural properties of the network on sequence coexistence. We find that stochastic assortment and mutation limit the architectures able to be encapsulated by the protocell with a given fraction of the population harbouring all possible sequence types.

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Summarize: Background Surveillance radars allow air traffic controllers to manage aircraft operating in the airspace around airports and to expedite the flow of air traffic into and out of airports by reducing the separation between aircraft. Currently, radar coverage for the Cherry Capital Airport is provided by a long-range surveillance radar in Empire, Michigan, 20 miles away from the airport. Although the radar is located near the Cherry Capital Airport, its signals are transmitted over 300 miles away to the Air Route Traffic Control Center in Minneapolis, where the controllers there are responsible for using instrument flight or radar rules to control the aircraft approaching and departing the airport outside a 5-mile radius of the airport. Controllers at the Cherry Capital Airport use visual flight rules or visual procedures to manage aircraft within the 5-mile radius during the normal tower operating hours from 7 a.m. to 10 p.m. However, aircraft are allowed to take off and land at the airport when the tower is closed. FAA conducted a study in 1994 to assess the benefits and costs of installing a surveillance radar at the airport. The results showed that the potential benefits of installing a radar exceeded the costs. Therefore, FAA concluded that the airport qualified for a radar. Because no radar was available and funds were unavailable to purchase a new radar, FAA added the airport to a waiting list of other qualifying airports. At the request of Members of Congress, FAA conducted another benefit-cost study in 1996 to determine whether the airport still qualified for a radar. The results of that study showed that the costs exceeded the benefits, thereby disqualifying the airport for a radar, and FAA removed the airport from its waiting list of qualifying airports. At our request, FAA conducted another benefit-cost study in 1997 to determine whether the airport qualified for a surveillance radar. That study’s results also showed that the costs exceeded the benefits and that the airport did not qualify for a radar. FAA’s Decision-Making Process for Installing Surveillance Radars at Airports FAA uses a multifaceted process to determine which airports should get surveillance radars. (See fig. 1.) First, FAA officials at the airport identify an operational need—such as the need to reduce delays to aircraft taking off and landing and the risks of midair and terrain collisions—that they believe a surveillance radar would satisfy. They then submit a written request to the appropriate FAA regional office. FAA airport officials identify need? Did regional FAA officials validate airports? Did airport meet the benefit-cost ratio of 1.0 or greater? Airport no longer considered for radar needs? available? Surveillance radar installed Second, FAA regional officials review the request to determine whether an operational need exists, assess the airport’s need relative to those of other airports in the region, and prioritize all airports within the region that have valid radar needs. If regional officials determine that a need exists, the request is forwarded to FAA headquarters. They also include an estimate of the equipment and annual operating costs in the region’s annual budget. If they determine that an operational need does not exist, the airport is no longer considered a potential candidate for a surveillance radar. Third, FAA headquarters officials use the agency’s Investment Criteria for Airport Surveillance Radar, dated May 1983, to determine whether an airport identified by the regional officials as a candidate for a radar meets FAA’s cost-effectiveness criteria. Specifically, the officials conduct a detailed study using site-specific air traffic data, along with estimated equipment and operating costs, to assess the potential benefits and costs for installing a radar at the airport. If the benefits exceed the costs, further consideration is given to the request. If the costs exceed the benefits—that is, if the benefit-cost ratio is less than 1.0—the airport is no longer considered a potential candidate for a surveillance radar. Fourth, FAA headquarters officials validate the operational needs by considering, among other things, the level of air traffic operations at the airport and the complexity of its airspace compared with those of other airports nationwide. If the officials conclude that a radar is needed, the request is approved. If FAA headquarters cannot validate the operational needs, the airport is no longer considered a potential candidate for a surveillance radar. Finally, if a radar is available from another airport where an upgraded radar has been installed, or if funds are available to purchase a new radar, the radar is acquired and installed at the airport. Otherwise, the airport is placed on a waiting list. Once radars or funds become available, however, FAA must determine whether the airports on the waiting list still meet its cost-effectiveness criteria by using the latest air traffic operations data. Airports that do not meet the criteria are no longer considered candidates for a surveillance radar. In addition to the radar requests initiated by FAA airport and regional officials, the Congress may mandate that a surveillance radar be installed at an airport. If the Congress designates funds with the mandate, the request does not have to follow FAA’s decision-making process. If the Congress does not designate funds, however, the request must follow the process, according to FAA headquarters officials. The Congress has mandated that FAA install surveillance radars at eight airports. These airports are included in appendix I. Although FAA’s decision-making process was in place in 1994, agency officials did not follow it before concluding that the Cherry Capital Airport qualified for a radar. For example, after conducting the 1994 benefit-cost study and determining that the airport met FAA’s cost-effectiveness criteria, agency officials prematurely concluded that Cherry Capital qualified for a radar. They did not assess the airport’s operational needs relative to the needs of other airports or consider the radar coverage already provided by the long-range surveillance radar nearby in Empire, Michigan. According to FAA officials, if these factors had been considered, the Cherry Capital Airport would not have qualified for a surveillance radar. The officials also told us that even if the airport had a benefit-cost ratio of 1.0 or greater, it still would not get a surveillance radar because other airports have greater operational needs and the airport already receives better radar coverage than many airports that have surveillance radars on site. They added that if a radar was installed at the airport, its signal would most likely be transmitted to another air traffic control facility where other controllers would be responsible for controlling aircraft approaching and departing the Cherry Capital Airport, an arrangement similar to the present one at the airport. Factors FAA Considered When Conducting Benefit-Cost Studies In accordance with its decision-making process, FAA used its investment criteria to identify the factors to consider when conducting the 1994, 1996, and 1997 benefit-cost studies for the Cherry Capital Airport. The officials calculated benefit-cost ratios of 1.66 in 1994, 0.68 in 1996, and 0.78 in 1997, which resulted in the airport meeting FAA’s cost-effectiveness criteria in 1994, but not in 1996 and 1997. We found that an overstatement of air traffic growth was the primary reason the airport met the investment criteria in 1994. FAA officials considered the potential efficiency and safety benefits, estimated the equipment and annual operating costs, and projected air traffic operations when conducting the benefit-cost studies. To calculate the efficiency and safety benefits of installing a surveillance radar, FAA considered travelers’ time saved because of the potential reductions in the delays to aircraft and the lives saved and injuries avoided because of the reductions in the risks of midair and terrain collisions. To compute the benefits represented by reduced delays to aircraft and collision risks, FAA used projections of air traffic operations at the airport, the average time required for aircraft takeoffs and landings, and the percentage of time that weather conditions at the airport would require controllers to use radar to manage the air traffic. To compute the equipment and annual operating costs, FAA estimated the costs for the acquisition and installation of the radar and the annual costs for controller and support staff salaries, training, utilities, and for maintenance. The benefits and the annual operations and maintenance costs were estimated over a 15-year period and discounted to the present time using the discount rate published by the Office of Management and Budget. FAA used both national and site-specific data to compute the benefits and costs. For example, the values for travelers’ time saved, lives saved, and injuries avoided were national data published annually by the Department of Transportation. The estimated costs for acquiring the radar were FAA’s purchase price for the surveillance radar plus other necessary equipment and personnel training costs. The projections of air traffic operations were specific to the Cherry Capital Airport. Although the results of benefit-cost studies depend on several factors, FAA officials told us that the projections of air traffic operations—particularly aircraft operations controlled by instrument flight or radar rules—were the most critical factors because they affect the level of benefits that would be achieved as a result of having a surveillance radar at the airport. They commented that there was a direct correlation between the projections of air traffic operations and the benefits—as air traffic increases, so do the potential for delays to aircraft and the risks of collision, and, thus, the benefits of installing a radar at the airport also increase. In particular, we found that FAA’s criteria give more weight to aircraft, such as air carriers and commuter aircraft, that carry the largest number of passengers because the higher the number of passengers, the greater the potential efficiency and safety benefits to be achieved from saving travelers’ time and avoiding collisions that could cause injuries and deaths. Therefore, according to FAA headquarters officials, the potential efficiency and safety benefits calculated for having a surveillance radar at the Cherry Capital Airport, which is mainly a general aviation airport, would be less than those calculated for airports that service a larger number of commercial air carriers and commuter aircraft. Equipment and Operating Costs Differed in the Benefit-Cost Studies FAA considered the installation of the same type of surveillance radar in all three of its studies on the Cherry Capital Airport. We found, however, that the estimated equipment costs in the 1997 study were over $8 million higher than the costs included in the other studies. Specifically, the equipment costs in the 1994 and 1996 studies totaled about $12.9 million and $13.5 million, respectively; whereas, the equipment costs totaled $22 million in the 1997 study. In contrast, the annual operating costs in the 1994 and 1996 studies totaled $611,000 and $677,000, respectively, compared with $167,000 in the 1997 study. FAA could not explain why such significant differences existed in the cost figures or provide documentation to support the costs included in the 1994 and 1996 studies. They did, however, provide support for the costs included in the 1997 study. FAA headquarters officials speculated that the costs differed because the 1994 and 1996 studies only included the costs for a surveillance radar and not the costs for the necessary auxiliary equipment. Air Traffic Projections Differed in the Benefit-Cost Studies To develop the air traffic projections in the 1996 and 1997 studies, FAA officials considered the historical air traffic growth at the Cherry Capital Airport and the mix of aircraft using the airport. As shown in table 1, they assumed that air traffic at the airport would grow, on average, about 1 percent annually. The FAA officials were uncertain about how the higher projections in the 1994 study were developed. They told us that the original projections were probably based on historical data, but were adjusted upward based on input from headquarters, regional, and district officials to reflect a 4.2-percent projected average annual growth rate, also shown in table 1. We could not determine the basis for the adjustments because FAA did not maintain supporting documentation. Nevertheless, FAA headquarters and regional officials, as well as the FAA officials and controllers at the Cherry Capital Airport, all agreed that the 1994 projections were overstated. For the 1996 and 1997 studies, FAA based its projections on actual air traffic growth at the airport over the 10-year periods preceding the 1996 (1986 through 1995) and 1997 (1987 through 1996) studies. As shown in table 2, the actual annual growth of air traffic from fiscal year 1986 through fiscal year 1996 ranged from an increase of 22.5 percent to a decrease of about 6.5 percent. According to FAA officials, the large increase in air traffic in fiscal year 1987 was due to the introduction of new air carrier service at the airport. Because the officials did not expect such a large increase in air traffic to reoccur in future years, they excluded the surge in air traffic in fiscal year 1987 from the air traffic projections in the 1996 and 1997 studies. Therefore, the resulting average annual growth rate used in the 1996 and 1997 studies was about 1 percent. Also, as illustrated in tables 1 and 2, the 128,704 projected air traffic operations included in the 1996 study more closely tracked the 128,419 actual operations that occurred in 1996 than the 148,000 operations projected in the 1994 study. Even so, the 123,957 actual air traffic operations reported for fiscal year 1997 were considerably less than the 152,000 projected in the 1994 study, the 130,078 projected in the 1996 study, and the 130,318 projected in the 1997 studies. Impact of Other Air Traffic Projections on the 1997 Benefit-Cost Study Since air traffic projections were the most critical factors influencing the results of the benefit-cost studies for the Cherry Capital Airport, we requested air traffic projections developed by the state of Michigan and Traverse City transportation planning officials to determine what impact their projections would have had on the results of FAA’s 1997 study. We found, however, that the state and local officials relied routinely on FAA’s air traffic projections and, therefore, that using their projections would not have had any impact on the 1997 study results. We did, however, identify another set of air traffic projections developed in 1996 (based on 1994 actual air traffic data), which had been used by two consulting firms. The firms used the projections in studies conducted for the Michigan Department of Transportation and the Northwestern Regional Airport Commission to identify facility improvements needed at the Cherry Capital Airport, such as expanding the terminal building and parking areas. The projections the firms used were based on a higher annual air traffic growth rate and a higher baseline of air traffic operations than FAA’s projections. Whereas FAA projected an average annual growth rate of 1 percent in its 1996 and 1997 studies, the firms projected a growth rate of about 1.5 percent. Also, FAA’s actual air traffic count of 124,000 for 1994 included only aircraft operations that were managed by the Cherry Capital and the Minneapolis controllers. The firms added 18,000 operations to FAA’s air traffic count by including an estimate of aircraft operations that were not managed by the controllers because they occurred at Cherry Capital when the tower was closed. While the firms’ count might have been appropriate for determining facility needs, FAA’s count was more appropriate for determining radar needs. Nonetheless, we asked FAA to conduct a benefit-cost study using the firms’ projections to determine the impact on the 1997 study. When the air traffic projections developed by the firms were used, they produced a benefit-cost ratio of 1.35, which exceeded the minimal threshold for meeting FAA’s cost-effectiveness criteria to qualify for a surveillance radar. However, as mentioned previously, FAA officials told us that even if the airport were to achieve a benefit-cost ratio of 1.0 or greater, it still would not get a surveillance radar because other airports have greater operational needs and the airport already receives better radar coverage than many other airports that have surveillance radars. Actions FAA Has Taken to Address Safety Concerns at the Cherry Capital Airport In response to the safety concerns raised by Members of Congress and controllers at the Cherry Capital Airport, such as the greater risk of aircraft collisions that results from increased air traffic, FAA installed a Terminal Automated Radar Display and Information System (TARDIS) in 1997 to help the controllers locate and identify aircraft approaching or departing the airspace around the airport. The TARDIS is a commercial, off-the-shelf system that consists of a computer, monitor, and software costing about $23,000. Although the system displays data, such as aircraft speed and altitude, received directly from the surveillance radar in Empire, Michigan, the Cherry Capital controllers can only use it as a visual aid and cannot use it to control or separate aircraft. According to FAA regulations, the Cherry Capital Airport controllers can only use visual procedures or visual flight rules to track aircraft. Controllers at the Cherry Capital Airport told us that the TARDIS has helped them manage air traffic better, but that they have had difficulty using it. They said that, on occasion, the information the TARDIS has displayed on aircraft identification and altitude, for example, has overlapped and has sometimes been unreadable. FAA headquarters and regional officials agreed that the data display problem exists occasionally but said that it is not unique to the TARDIS at the Cherry Capital Airport. They commented that the problem does not compromise safety at the airport because the additional equipment is only intended to be used as a visual aid and not to control air traffic. Moreover, the Minneapolis controllers use the radar in Empire to track aircraft flying under instrument flight rules until control of the aircraft is switched, via radio contact, to the Cherry Capital controllers. The switch usually occurs within a 5- or 10-mile radius of the airport. Also, FAA’s regulations require that pilots contact the Cherry Capital controllers prior to entering the airport’s airspace. According to the officials, the TARDIS provides two benefits to the Cherry Capital controllers—enhanced traffic monitoring capabilities and data directly from the radar in Empire. Even if the automated system at the Minneapolis facility fails, the TARDIS would still receive data from the Empire radar. FAA Plans to Replace Surveillance Radars at Airports With Fewer Total Air Traffic Operations Than the Cherry Capital Airport Beginning in 1999 and continuing through 2004, FAA plans to retire all of the older airport surveillance radars (ASR), specifically ASR-7 and ASR-8, which were installed in the 1960s and 1970s. These radars, currently located at 101 airports, will be replaced as part of FAA’s efforts to modernize its air traffic control system with new, technologically advanced ASR-11 radars, which cost over $5 million each. During our review, we found that 75 of the 101 airports scheduled to have their radars upgraded had fewer total air traffic operations than the Cherry Capital Airport in 1996 and that FAA will spend well over $375 million to purchase replacement radars for these airports. This cost does not include the additional expenditures for auxiliary equipment and for the modifications to airport infrastructure required for the effective operation of the radars. We noted that FAA officials routinely conduct benefit-cost studies using air traffic operations as one of the critical factors in deciding whether it would be cost-effective to install surveillance radars at airports without radars. Yet FAA officials did not conduct similar studies to determine whether it would be cost-beneficial to replace all of the existing ASR-7 and ASR-8 radars, to prioritize replacement of the radars, or to assess whether the circumstances that initially warranted installation of the radars at the airports had changed over the years. The officials agreed that the results of benefit-cost studies would be a relevant factor in deciding whether to install the replacement radars. But they said they have no plans to conduct such studies because they believe that it would be very difficult to discontinue radar operations at an airport found not to qualify because the public’s perception would be that safety was being reduced, even if safety was not compromised and other circumstances warranted the discontinuance of radar operations. FAA’s past practice has been that once an airport gets a radar, it qualifies for a replacement radar regardless of changes in the air traffic or the other circumstances that initially warranted the radar. Although FAA has criteria for discontinuing radar operations, the agency has never done so. FAA officials also explained that there may be other important reasons, besides cost-effectiveness, for replacing or installing a radar at an airport. These reasons include an airport’s location; the complexity of the airspace surrounding an airport; the capacity of an airport to serve multiple satellite airports; the capacity of an airport to provide relief capacity to hub or major airports on an as needed basis; and national security. We asked FAA for documentation of the operational needs that showed why the radars were installed initially at the 75 airports with fewer total air traffic operations than the Cherry Capital Airport that are scheduled to have their radars replaced. In response, FAA headquarters officials contacted the airports to obtain information on the rationale for installing the radars. Among the reasons FAA provided were that some of the airports provide radar services to the Air National Guard, military bases, and multiple satellite airports or serve as alternates for major airports or that the radars are the only sources for radar coverage in mountainous areas. FAA also cited congressional interest as a reason for installing surveillance radars at some airports. We were unable to verify the validity of FAA’s rationales because FAA did not have records dating back to the 1960s and 1970s to document why the radars were installed. FAA’s information, however, shows that at some of the airports, the circumstances that originally justified the installation of radars no longer exist. See appendix II for a list of the 75 airports and more details about FAA’s justifications for the initial installation of the radars in 1960s and 1970s. Although installing and retaining radars at some of the airports with fewer total air traffic operations than the Cherry Capital Airport might be justified, conducting benefit-cost studies and revalidating the operational needs would ensure that (1) radars are installed or replaced first at the airports that have the greatest needs and (2) FAA is not spending millions of dollars to replace radars when continued operation of the existing radars might not be justified. Since FAA already has a process in place for conducting benefit-cost studies, we believe that the time and costs associated with conducting similar studies to determine the effectiveness of replacing existing radars would be minimal. Conclusions An overstatement of projected air traffic growth was the primary reason the Cherry Capital Airport met FAA’s cost-effectiveness criteria in 1994, and agency officials prematurely concluded that the airport qualified for a surveillance radar. FAA officials expected a higher rate of growth for air traffic at the airport in future years, and as a result, the potential benefits of installing a radar were greater than the costs. If FAA had included less optimistic air traffic projections in its 1994 study, the Cherry Capital Airport would not have met the agency’s cost-effectiveness criteria. Furthermore, if FAA had followed its decision-making process by assessing the airport’s needs relative to other airports’ needs and considered the existing radar coverage, the airport would not have been considered for a surveillance radar. Even if the benefits exceeded the costs, there was no guarantee that the airport would get a radar because of the competing needs of other airports within the region and the quality of service that the radar in Empire, Michigan, already provides to the Cherry Capital Airport. Safety and confidence in the national airspace system are very important, and several factors must be considered when making decisions regarding the installation and replacement of surveillance radars. However, FAA’s current plans to install replacement radars without conducting benefit-cost studies and revalidating operational needs may result in the agency spending millions of dollars to replace radars at airports with fewer air traffic operations than the Cherry Capital Airport, which does not meet FAA’s cost-effectiveness criteria for having a radar. FAA’s perceived difficulties in discontinuing radar operations at an airport only elevate the need for conducting benefit-cost studies and assessing the operational needs. We believe that conducting benefit-cost studies and assessing operational needs before replacing the radars would allow FAA to obtain the convincing data needed to ensure that the equipment is installed at the airports that have the greatest needs and that FAA could use the data to prioritize the installation of the radars at qualifying airports. In addition, conducting these analyses would give FAA the opportunity to reassess the benefits and costs of replacing the equipment and ensure that funds are not spent to modernize radars at airports where continued radar operations might not be justified. Recommendation Because of current budget constraints and the future expenditures associated with installing radars as part of the effort to modernize the nation’s air traffic control system, we recommend that the Secretary of Transportation direct the Administrator of the Federal Aviation Administration to conduct benefit-cost studies to validate the cost-effectiveness and revalidate the need for the radars at airports scheduled to receive replacement radars and to use the results of the studies in prioritizing the replacement of the radars at qualifying airports. Furthermore, the Federal Aviation Administration should advise the Congress on the results of these studies for its consideration during deliberations on the Department of Transportation’s budget request. Agency Comments and Our Evaluation We provided copies of a draft of this report to the Department of Transportation and the Federal Aviation Administration for review and comment. We met with Federal Aviation Administration officials, including the Project Leader, Integrated Product Team/Terminal Surveillance Program, Communications, Navigation, Surveillance, and Infrastructure Directorate, Air Traffic Services; and Business Manager, Integrated Product Team/Terminal Surveillance Program, Office of Communication, Navigation, and Surveillance Systems, Research and Acquisitions. We also met with Department of Transportation officials from the Offices of the Assistant Secretaries for Administration and for Budget and Program Performance. The agencies generally agreed with the findings, conclusions, and recommendation presented, but commented that we should include information in the report on instrument flight rule operations and ASR-9 radars located at airports that had fewer total air traffic operations than the Cherry Capital Airport in 1996. Specifically, the agencies noted that instrument flight rule operations may be a better indicator of the need for a radar at airports than total air traffic operations and, thus, could have an impact on the results of benefit-cost studies. In addition, they commented that some airports that currently have ASR-9 surveillance radars, which were installed in the 1980s, also had fewer total air traffic operations than the Cherry Capital Airport did in 1996. Although the Federal Aviation Administration currently has no plans to replace these radars, the agencies noted that the equipment will need to be replaced over the next 10 years. The Federal Aviation Administration reiterated that the results of benefit-cost studies also could be used to revalidate the operational needs for the radars before they are replaced. However, the agency has no plans to conduct such studies for these airports. In response to the agencies’ comments, we included more detailed information about the airports that currently have ASR-9 radars in appendix I and information about airports’ instrument flight rule operations in appendix II. The agencies also suggested several changes to improve the accuracy and clarity of the report that we incorporated where appropriate. Scope and Methodology We performed audit work at FAA’s headquarters in Washington, D.C.; the Great Lakes Regional Office in Chicago; the Air Route Traffic Control Center in Minneapolis; and the Cherry Capital Airport in Traverse City, Michigan. To determine what process FAA currently has in place for determining which airports that do not have radars may be eligible for surveillance radars, we interviewed officials at FAA’s headquarters, regional, and airport offices; and reviewed and analyzed pertinent FAA criteria, regulations, procedural, and other guidance documents. To identify the factors FAA considered when conducting the 1994, 1996, and 1997 benefit-cost studies, we analyzed the studies and supporting documents, FAA’s Investment Criteria for Airport Surveillance Radar, dated May 1983, and other guidance documents for conducting such studies. We interviewed FAA headquarters officials currently responsible for conducting benefit-cost studies. We also obtained information on the factors FAA considered when developing air traffic projections, analyzed the projections, and compared actual and projected air traffic operations. In addition, we interviewed representatives of local planning and public interest groups located in the Traverse City area that were familiar with the Cherry Capital Airport’s air traffic operations to obtain information on past and anticipated air traffic growth, the need for a surveillance radar, and the safety concerns at the airport. To determine the impact other air traffic projections would have had on the results of FAA’s 1997 benefit-cost study, we interviewed FAA officials and controllers working at the Cherry Capital Airport, officials of the Michigan Department of Transportation and the Traverse City Planning Commission, and representatives of two aviation consulting firms. We obtained air traffic projections from the consulting firms and had FAA headquarters officials conduct sensitivity analyses using the projections. Although we evaluated what impact the projections would have had on the results of the 1997 study, we did not evaluate the methodologies used by the consulting firms to develop their projections because this was not part of the scope of our review. To determine what actions FAA has taken to address the safety concerns raised by Members of Congress, air traffic controllers, and local citizens, we obtained information on the operational capabilities of the TARDIS and on how the equipment is intended to be used through interviews with FAA headquarters and regional officials, the Cherry Capital controllers, and airport officials. In addition, we collected data from FAA that identified the airports with fewer total air traffic operations than the Cherry Capital Airport in 1996 that are scheduled to receive replacement surveillance radars. We discussed with FAA headquarters officials the rationales for initially installing surveillance radars at the airports and when the existing radars are scheduled to be replaced. However, we did not contact representatives at the airports to verify the information provided by FAA headquarters officials. We also obtained data on airports that currently have ASR-9 radars and fewer total air traffic operations than the Cherry Capital Airport. We performed our review from October 1997 through May 1998 in accordance with generally accepted government auditing standards. We are providing copies of this report to interested congressional committees; the Secretary of Transportation; the Administrator, FAA; and the Members of Congress representing the Traverse City area. We will also make copies available to others on request. If you or your staff have any questions or need additional information about this report, please call me at (202) 512-2834. Major contributors to this report are listed in appendix III. Airports With ASR-9 Radars That Had Fewer Total Air Traffic Operations in 1996 Than the Cherry Capital Airport Nantucket Memorial (Nantucket, MA) Theodore Francis Green State (Providence, RI) Portland International (Portland, ME) Spokane International (Spokane, WA) Atlantic City International (Atlantic City, NJ) Fort Wayne International (Fort Wayne, IN) Roswell Industrial Air Center (Roswell, NM) Charlottesville-Albermarle (Charlottesville, VA) Cedar Rapids Municipal (Cedar Rapids, IA) Harrisburg International (Harrisburg, PA) — Provides coverage and services in challenging terrain environment (continued) Walker Field (Grand Junction, CO) Huntsville International-Carl T. Jones Field (Huntsville, AL) Rogue Valley International (Medford, OR) Rio Grande Valley International (Brownsville, TX) Lynchburg Regional-Preston Glenn Field (Lynchburg, VA) Fayetteville Regional/Grannis (Fayetteville, NC) Missoula International (Missoula, MT) Eastern West Virginia Regional-Shepard Field (Martinsburg, WV) — Congressional mandate in 1991 — Radar signal will be remoted to the terminal radar approach control facility at Dulles International Airport (Table notes on next page) Not applicable. Not available. Airports With Fewer Total Air Traffic Operations in 1996 Than the Cherry Capital Airport That Have ASR-7 and ASR-8 Radars Scheduled for Replacement Scheduled date for installing radarreliever airport for Detroit Metropolitan Airport — Provides services to corporate travelers, including General Motors and services for a large naval flight training center interest— Radar was installed after an accident and services for a military base — Former Air Force base and services for a military base — Former Air Force base provided approach control services for the Department of Defense’s Strategic Air Command Base and services in mountainous terrain — Flight school at airport — Provides services to numerous satellite airports — Low-visibility airport during winter months (continued) Scheduled date for installing radarand services to the Air National Guard and military bases and services to the Air National Guard and services for military bases 95,129 — Air route traffic control center does not have adequate coverage of the airspace — Formerly a hub for Ozark Airlines and services to Air National Guard base — Airport has capability to provide air route traffic control services interest — Provides services to corporate travelers, including Goodyear Corporation and Timkin Roller Bearing 123,894 — Air National Guard (continued) Scheduled date for installing radarand services for the Camp Grant military base — Provided services for military training flights because Chicago O’Hare could not accommodate these aircraft -A hub for United Parcel Service 93,875 — Provides coverage in mountainous terrain — Air route traffic control center does not have adequate coverage of the airspace 80,435 — Provides coverage in mountainous terrain — No long-range surveillance radar coverage available and services for military training interest — Provides coverage and services for the Air Force Reserves interest — Provides coverage in mountainous terrain (continued) Scheduled date for installing radarand services to the largest B1 bomber base — Provides services to five satellite airports and services for military practice approaches — Provides services to satellite airports — Provides services to the largest civil fleet of helicopters 27,441 — Alternate airport for Honolulu — Island with highest terrain; heavy rainfall area, limited visibility — Stopover for flights to and from Australia and New Zealand 97,804 — Stopover airport for flights from Europe — Alternate airport for Boston Logan International 61,011 — Air National Guard fighter wing — Minihub for air cargo operations — Largest city in South Dakota interest — Provides services to satellite airports 144,338 — FAA takeover of a when new airport was built in 1963 to cover growth in general aviation, military, and air carrier traffic (continued) Scheduled date for installing radarand services to the military — Previously provided approach control services to the Department of Defense’s Strategic Air Command base to 12 satellite airports — Provides coverage and services to 100 scheduled air carriers daily 66,418 — Only airport with primary radar within 120 miles — Provides services to the Air National Guard for a combined air traffic control center and terminal radar approach control facility in San Juan, PR to 18 satellite airports — Alternate airport for Chicago O’Hare — Provides overflight services to and from Chicago O’Hare congressional mandate; however, no documentation available — Provides support for surrounding restricted military area activities (continued) Scheduled date for installing radarinterest — Provides coverage in mountainous terrain — Moody Aviation trains bush pilots for missionary work to 10 satellite airports — Air route traffic control center does not have adequate coverage of the airspace for Longview and Tyler, TX, airports — Provides services for oil industry business jet air traffic 102,407 — Provides service to corporate travelers, including John Deere Corporation for air taxi and military approaches 103,273 — Provides coverage in mountainous terrain 221,673 — Provides coverage and services to an Air National Guard base — Provides approach control services for northwest Arkansas, including Fayetteville, AR to four satellite airports and to military air traffic (continued) Scheduled date for installing radar69,160 — Provides coverage in mountainous terrain — Provides services to satellite airports — Previously provided approach control services to the Seneca Army Air Depot to nine satellite airports — Third-largest approach control facility and fourth-busiest airport in the state and services to the Air National Guard — Provides coverage and services to overflow and diverted traffic from Minneapolis — Previously provided services to two Air Force base squadrons and services to the Air National Guard and four satellite airports (continued) Scheduled date for installing radarfor the Mayo Clinic, including lifeguard flights — Alternate airport for Minneapolis airport — Airport has one of the few Global Positioning System Heliport instrument approaches — Provides services for large cargo operations to the Air National Guard and satellite airports — Provides tower air route traffic control services and services to Fort Benning Military Base — Provides services to 19 satellite airports — Sequences turboprops and props into Atlanta Hartsfield and services to the Air National Guard — Provides coverage for detecting and interdicting aircraft involved in illegal drug activities 82,573 — FAA takeover of a Department of Defense site — Provides services for military practice approaches (continued) Dallas-Fort Worth Metroplex — Provides air traffic relief services to an air route traffic control center and services to the Air National Guard — Indiana State University student pilot training — Midnight freight operations coverage gap for terminal radar approach control in Houston interest — Provides services for oil industry related air traffic — Provides coverage in mountainous terrain 63,842 — Provides coverage in mountainous terrain (continued) Not applicable. Major Contributors to This Report Resources, Community, and Economic Development Division Janet Barbee Sharon Dyer Wanda Hawkins Mehrzad Nadji John Thomson 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. Each day, GAO issues a list of newly available reports and testimony. 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Summary: Pursuant to a legislative requirement, GAO reviewed the benefit-cost studies that the Federal Aviation Administration (FAA) conducted for the Cherry Capital Airport in 1994, 1996, and 1997, focusing on: (1) FAA's decisionmaking process for installing surveillance radars at airports; (2) the factors, including costs, benefits, and air traffic projections, that FAA considered when conducting the 1994, 1996, and 1997 studies; (3) the impact, if any, that air traffic projections developed by other sources would have had on the results of the 1997 study; (4) actions FAA has taken to address safety concerns at the airport; and (5) FAA's plans to replace surveillance radars at airports with fewer total air traffic operations than the Cherry Capital Airport. GAO noted that: (1) FAA uses a multifaceted process to determine which airports should get surveillance radars; (2) this process includes completing a benefit-cost study, assessing an airport's need for a surveillance radar compared with the needs of other airports, and determining the availability of radar equipment or funds to purchase needed radar equipment; (3) in its 1994 benefit-cost study for the Cherry Capital Airport, FAA officials overstated the projected air traffic growth; (4) this overstated growth was the primary reason FAA concluded that the airport met its cost-effectiveness criteria; (5) moreover, in 1994, FAA officials did not follow the agency's decisionmaking process and prematurely concluded that the Cherry Capital Airport qualified for a surveillance radar; (6) when conducting the 1994, 1996, and 1997 benefit-cost studies, FAA considered the potential efficiency and safety benefits; (7) with the higher growth rate used in the 1994 study, the benefits exceeded the costs of installing a surveillance radar, so the Cherry Capital Airport met FAA's cost-effectiveness criteria; but with the lower growth rate used in the 1996 and 1997 studies, it did not qualify; (8) the air traffic projections were the most critical factors influencing the results of FAA's benefit-cost studies; (9) to address the safety concerns, FAA installed an automated display and information system at the Cherry Capital Airport in 1997; (10) while the controllers told GAO that the equipment can help them better manage air traffic and improve safety, they have difficulty using it because information on aircraft identification and altitude is sometimes unreadable on the display monitor; (11) beginning in 1999, FAA plans to replace the existing surveillance radars installed in the 1960s and 1970s at 101 airports as part of its efforts to modernize its air traffic control system; (12) seventy-five of the 101 airports had fewer total air traffic operations in 1996 than the Cherry Capital Airport did; (13) although FAA conducts benefit-cost studies and uses air traffic operations as a basis for determining the cost-effectiveness of installing surveillance radars at airports, agency officials did not conduct similar studies to determine whether it would be cost-effective to replace existing radars at the 101 airports or to prioritize the replacement of the radars; and (14) FAA has no plans to undertake such efforts because agency officials believe that it would be very difficult to discontinue radar operations at an airport because of the public's perception that safety would be reduced.

gov_report

en

Summarize: There is a speech which George Osborne hoped to deliver today which will have to remain in the Treasury drawer for a few years yet. It is the one where the Chancellor declares that the deficit has been eliminated and the government is raising more in tax than it spends. In 2010 Mr Osborne confidently declared that he would balance the books by 2014-15, but today he was forced admit that borrowing remains stubbornly high and balancing the books is still a long way off. In June 2010, the Office for Budget for Responsibility predicted that in 2014-15 borrowing would fall to just £37billion, but it has remained high at around £100billion and is not expected to return a surplus until 2018-19. In 2010 George Osborne remained confident that borrowing would fall sharply, but in 2011 and 2012 a deterioration in the economic picture meant the prospect of balancing the books slipped further into the future. In 2010, the Office for Budget Responsibility predicted that borrowing would be just £37billion in 2014-15. Instead it will be £91.3billion, which is still £5billion higher than the OBR predicted in March this year. While there are record numbers of people in work, many are in low paid jobs. These workers have benefitted most from the coalition policy of not paying income on the first £10,000 of earnings. But it means the Treasury is raising less than expected from taxes to offset spending. Four years ago in June 2010 Mr Osborne boasted that he would have 'eliminated' the deficit by 2014-15. 'We have set the course for a balanced budget and falling national debt by the end of this Parliament,' he predicted in the Coalition's emergency budget. Instead borrowing has remained stubbornly high, and barely changed in the last 12 months. Revealing that the deficit has been halved over this Parliament, Mr Osborne said the OBR now forecasts the deficit will fall slightly from £97.5 billion last year to £91.3 billion this year - rather than the £86.4billion previously expected. Mr Osborne will boast that the economy is now growing strongly, and faster than most other countries. But its has failed to keep pace with the predictions made in 2010. National debt as a proportion of the economy (GDP) was supposed to start falling in 2013-14, but has instead been rising and is not expected to peak until next year. Borrowing for 2015-16 is expected to fall to £75.9 billion, worse than the £68.3 billion previously expected. But the outlook for 2016-17 of a £40.9 billion shortfall is better than the £41.5billion previously set out. For 2017-18 expected borrowing is likely to be £14.5billion, an improvement on the previously pencilled-in £15.8 billion. Mr Osborne told MPs that by 2019-20 the UK will be spending £23billion less than it raises in tax. 'Out of the red and into the black for the first time in a generation - a country that inspires confidence around the world because it seeks to live within its means,' he added. It still means that the country will not be in a position where it is spending less than it raises in tax, until 2018-19 - a decade after the financial crash. The OBR said: 'Despite stronger growth, we have revised up our estimate of the budget deficit this year. 'In headline terms, the UK economy has outperformed our March forecast. 'But wage and productivity growth have once again disappointed, while national income and spending have outperformed most in those areas that yield least tax revenue.' Several times Mr Osborne claimed that he had brought the British economy back from the brink over the last four years. The Chancellor used his Autumn Statement to plead for time to 'finish the job', warning a change of course now would'squander' prosperity. Four years ago the OBR hoped for strong growth in pay, but low wages have persisted and have only just started to outstrip inflation. He revealed growth in this year is now expected to hit 3 per cent, up from 2.7 per cent forecast in March this year. And he told MPs that under the coalition, Britain has created 1,000 jobs every day since 2010. But in future years, growth is forecast to be lower than previously thought. The OBR now it forecasts growth of 2.4 per cent in 2015, 2.2 per cent in 2016, then 2.4 per cent, 2.3 per cent and 2.3 per cent in subsequent years. Unemployment has also fallen sharply, dropping to just 6 per cent for the first time since autumn 2008. Pay is also rising faster than the cost of living for the first time in five years. Average earnings are 1.3 per cent higher than a year ago, compared to inflation running at 1.2 per cent, with workers in the private sector enjoying even higher pay rises. The OBR predicts wages will rise by 1.8 per cent this year, 2 per cent next year and 3.1 per cent in 2016. Employment has surged above expectations, breaking through 30million a year earlier than expected. Latest figures show there are 30.8million in work, a figure which was not expected to be reached until 2016. With total debt still rising to the vicinity of £1.4 trillion, Mr Osborne is set to have borrowed more in five years at the Treasury than Labour chancellors did in 13. Time and again the Chancellor has seen the economic forecasts shift, with hopes of balancing the books drifting further into the future. Britain will still not be back in the black, spending less than it raises in tax, until 2018-19, a decade after the financial crash. Labour's shadow chancellor Ed Balls said: 'It's all here in black and white, hard evidence from the Budget for Responsibility that the Chancellor's borrowing targets are all in tatters. 'We all know he's changed the way he styles his hair but he can't brush away the facts. People are worse off and he's failed to balance the books in this Parliament. 'I've got to say, for all his strutting, for all his preening, for all his claims to have fixed the economy, he promised to make people better off, working people are worse off. 'He promised we were all in this together and he cut taxes for millionaires. He promised to balance the books in this Parliament and that commitment is now in tatters. Every target missed, every test failed, every promise broken.'

Summary: In 2010, Osborne boasted he would have 'eliminated' the deficit by 2014-15. But new figures show Britain will borrow around £91billion this year. Treasury is raising less in income tax than thought earlier this year. Economic growth has been strong but failed to match 2010 forecasts. Pay rises have also been well below expectations four years ago. But employment has soared above 30million much sooner than expected.

cnn_dailymail

en

Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application is the national phase of PCT/EP2011/005200, filed Oct. 17, 2011 which claims the benefit of German Patent Application No. 10 2010 054 009.9, filed Dec. 9, 2010. FIELD OF THE INVENTION [0002] The invention relates to a cleaning device comprising a carrier with at least one cleaning surface. Raised cleaning islands are disposed on the cleaning surface in spaced relation to each other. The cleaning islands are surrounded on all sides by storage spaces for holding soil particles. BACKGROUND OF THE INVENTION [0003] A cleaning device as described above is made, for example, as a cleaning towel on the cleaning surface of which congruently shaped cleaning islands are disposed in a uniformly distributed pattern. Congruently shaped storage spaces for holding soil particles are disposed around the cleaning islands. [0004] However, the soil particles that are removed from the surface being cleaned have different sizes, and such soil particles of different sizes cannot be held very effectively by the congruently shaped cleaning islands and the correspondingly shaped storage spaces. Larger soil particles, for example, long hairs, cannot be held in small storage spaces, while fine dust is not retained in large storage spaces. In each of these cases the cleaning result is not very satisfactory. SUMMARY OF THE INVENTION [0005] A general object of the invention is providing a cleaning device of the kind described above that is configured so that soil particles of different sizes can be effectively removed from the surface being cleaned and held and retained in the storage spaces. [0006] To this end, the storage spaces are made steadily smaller starting from at least one edge of the cleaning surface in a wiping direction at least up to the middle of the cleaning surface. It is advantageous if during wiping, at first larger soil particles, for example, long hairs, can be held in the large storage spaces that lie at the front in the wiping direction. Smaller soil particles, for example, dust, pass by the larger storage spaces largely unhindered and then reach the smaller storage spaces lying behind them in the wiping direction. The smaller soil particles then accumulate in these smaller storage spaces. The smallest soil particles, for example, fine dust, pass by even these storage spaces and accumulate either in the smallest storage spaces or in the cleaning islands themselves. A single cleaning device is thus suitable for removing and holding soil particles of different sizes from the surface that is to be cleaned and thereby achieving an especially good cleaning result. [0007] The storage spaces that are adjacent to each other in a direction transverse to the wiping direction can have a uniform size. Because of this configuration, the production of the cleaning device can be manufactured simply and affordably. [0008] To produce a steady decrease in the size of the storage spaces in the wiping direction at least up to the middle of the cleaning surface, the cleaning islands can be configured to become steadily larger in the wiping direction at least up to the middle of the cleaning surface, starting from at least one edge of the cleaning surface. It is advantageous if large cleaning islands are provided that bind very small soil particles (e.g., fine dust particles) in the islands themselves, in addition to being captured in the small storage spaces that surround the large cleaning islands. [0009] In order to be able to wipe large, evenly soiled surfaces in the wiping direction and produce a good, uniform cleaning result, only the cleaning islands that are adjacent to each other in the direction transverse to the wiping direction can be configured with a uniform size. With such an arrangement, substantially the same effect as described above is achieved as with the arrangement in which the storage spaces adjacent to each other in the direction transverse to the wiping direction have a uniform size. [0010] The cleaning surface can be configured with one substantially centrally disposed central cleaning island, which is concentrically surrounded by other cleaning islands. The cleaning islands may be configured and arranged such that the radial distances between the adjacent cleaning islands become steadily larger proceeding in a radial direction towards each edge, starting from the central cleaning island. With such an arrangement, the wiping direction extends in the radial direction with respect to the central cleaning island. Practically independent of the wiping direction, this results in the storage spaces lying around the central cleaning island being smaller than the storage spaces disposed radially further in the direction towards the edge. [0011] Each of the cleaning islands and/or the central cleaning island can have a substantially round configuration. With such a configuration, the soil particles can easily pass by the cleaning islands without getting caught on them and thus blocking the flow in the direction of the smaller storage spaces. [0012] The cleaning islands disposed next to each other in a direction transverse to the wiping direction can form an island row. The cleaning islands of the island row can be staggered in the gaps between the cleaning islands of the next island row in the wiping direction. Arranging the cleaning islands that are next in the wiping direction in the gaps between the previous row of cleaning islands can ensure that no un-wiped strips will remain on the surface being cleaned if the sizes of the cleaning islands are appropriate matched. [0013] The distances and/or the radial distances between the adjacent cleaning islands can be 0.1 mm to 50 mm. Such distances are advantageous for forming storage spaces that are well suited for holding ordinary household soil. [0014] The cleaning islands can cover 1% to 80% of the cleaning surface. More preferably, the cleaning islands can cover 5% to 50% of the cleaning surface. Coverage of up to 50% is sufficient for most uses. Because of the material of which the cleaning islands consist is often quite expensive, such a design is advantageous. Thus, with this configuration, the cleaning device can be made cheaply. [0015] The cleaning islands can include flock fibers. The use of flock fibers helps achieve a good cleaning performance. However, hand flocking of carrier materials with flock fibers is expensive compared to the carrier material itself. A large portion of the costs associated with flocking comes from the materials, namely the flock fibers and the adhesive that is necessary to affix the flock fibers to the carrier material. Due to the arrangement of the storage spaces and the cleaning islands, only a comparably small amount of flock fibers is necessary to achieve a good cleaning performance, so the cleaning device can be made cheaply. [0016] The flock fibers of each cleaning island can have different lengths and can be oriented substantially perpendicular to the cleaning surface. For example, a cleaning island can have flock fibers with two different lengths. For example, the lengths can be 1 mm and 3 mm. This type of arrangement provides a further way to produce a three-dimensional structure for soil removal and soil holding. Each cleaning island thus has a more highly structured surface. It was found that when flock fibers of 1 mm and 3 mm are used standard household soil is held on average 23% better than with a cleaning device that has cleaning islands with flock fibers of the same length. [0017] The flock fibers of different lengths can be substantially homogeneously distributed throughout the cleaning island. [0018] The flock fibers of a cleaning island can be formed by viscose fibers and/or polyamide fibers. It is advantageous for the cleaning islands to maintain their three-dimensional flocking structure even in wet cleaning A flock fiber mixture of viscose and polyamide has good water absorbency properties. The viscose fibers are very water absorbent and would, without the additional use of fibers with a support effect (for example, polyamide fibers), lie against the carrier material in a wet state resulting in a loss of the pronounced three-dimensional structure of the flocking Flock fibers of polyamide, and also polypropylene or polyethylene, absorb only small amounts of water and therefore remain in their upright position projecting from the surface of the carrier even in a wet state. Such fibers therefore perform a supporting function for the viscose fibers. In order to achieve this supporting function, a fraction of support fibers ≧20% with respect to the viscose fibers is necessary. [0019] In each case depending on how the cleaning cloth is used, particularly depending on how the surface to be cleaned is made and its material of construction and depending on the kind of soil that is supposed to be removed and held by the cleaning device, it was found to be advantageous if a mixture ratio of 80:20 to 20:80 of viscose fibers to polyamide fibers per cleaning island is used. [0020] The carrier also can be flocked with flock fibers. Preferably, the carrier is a nonwoven material and is made as a towel or sponge. The flocking of the carrier with flock fibers can take place, for example, electrostatically, with the flock fibers affixed generally perpendicularly to the carrier material with one end of the fibers in an adhesive layer. With such a configuration, the carrier with the flocked fibers has a three-dimensional structure with an increased surface area. Because of this increase in surface area, soil can be removed from the surface to be cleaned and held in the storage spaces especially well both in dry cleaning and wet cleaning. An electrostatic flocking has the advantage that it achieves a high flocking density. In order to achieve a sufficient three-dimensionality of the surface structure and to keep the production costs of the mop as low as possible, the coverage of the carrier with the cleaning islands preferably does not exceed 80%. BRIEF DESCRIPTION OF THE DRAWINGS [0021] Two exemplary embodiments of a cleaning device in accordance with the invention are described in more detail below with reference to FIGS. 1 and 2. [0022] FIG. 1 includes a schematic top view of a first exemplary embodiment of a cleaning device in which the storage spaces disposed next to each other in the direction transverse to the wiping direction have the same size. FIG. 1 also includes an enlarged view of a portion of the cleaning cloth. [0023] FIG. 2 includes a schematic top view of a second exemplary embodiment of a cleaning device according to the invention, in which the cleaning surface has only one substantially centrally disposed central cleaning island, which is surrounded by other cleaning islands. FIG. 2 further includes an enlarged view of a portion of the cleaning cloth. DETAILED DESCRIPTION OF THE INVENTION [0024] Each of FIGS. 1 and 2 shows a top view of a cleaning towel. A carrier 1 of the cleaning towel includes a nonwoven material with raised cleaning islands 3 disposed on the cleaning surface 2 of the carrier 1. The cleaning islands 3 are surrounded on all sides by storage spaces 5, where both the cleaning islands 3 themselves as well as the storage spaces 5 can hold soil particles. [0025] In the illustrated embodiment, the distances 4, A, B, C, D, E, 9 between the adjacent cleaning islands 3 are between 0.1 mm and 50 mm, with the cleaning islands 3 covering about 50% of the cleaning surface 2. [0026] The cleaning islands 3 include flock fibers. The flock fibers of each cleaning island 3 have different lengths, in order to be able to make available more surface for holding soil. In the illustrated embodiment, the flock fibers of each cleaning island 3 include viscose fibers and polyamide fibers, where the mixture ratio of viscose fibers to polyamide fibers for each cleaning island 3 is about 50:50. [0027] In the illustrated embodiment, the storage spaces 5 become steadily smaller in the wiping direction 7 up to the middle of the cleaning surface 2, starting from at least one edge 6 of the cleaning surface 2. Also, the cleaning islands 3 become steadily larger in the wiping direction 7 up to the middle of the cleaning surface 2, starting from at least one edge 6 of the cleaning surface 2. [0028] A first exemplary embodiment of a cleaning towel is shown in FIG. 1. The storage spaces 5, which surround the cleaning islands 3 on all sides, become steadily smaller in the wiping direction 7 starting from edge 6. In addition, the cleaning islands 3 become steadily larger in the wiping direction 7 starting from the edge 6. [0029] When the cleaning towel is moved in the wiping direction 7, coarse soil particles like hairs or crumbs first arrive at the large storage spaces 5, while finer soil particles like dust or pollen pass by the larger storage spaces 5 at the small cleaning islands 3 and are held in the smaller storage spaces 5. Fine dust particles are held not only in the storage spaces 5, but also in the storage islands 3 themselves. [0030] Sets of the cleaning islands are disposed next to each other in a direction transverse to the wiping direction 7 so as to form an island row. A plurality of adjacent island rows 10 extending transverse to the wiping direction 7 are provided on the cleaning cloth. The cleaning islands 3 of each island row 10 are staggered in the gaps of the cleaning islands 3 of the next island row 10 in the wiping direction 7. With this arrangement, when wiping in the wiping direction 7, no un-wiped strips remain on the surface being cleaned. [0031] The cleaning towel of FIG. 1 has two wiping directions 7. 1 and 7. 2. The wiping direction 7. 1 extends from edge 6. 1 up to the middle of the wiping towel, and the wiping direction 7. 2 extends from edge 6. 2, likewise to the middle of the cleaning towel. The function of the cleaning towel is accordingly connected with the wiping direction. [0032] The reverse side of the cleaning towel, not shown here, can also be flocked with flock fibers like the illustrated cleaning surface 2. Alternatively, the reverse side of the cleaning towel can be flocked in a manner different from the illustrated cleaning surface 2. [0033] The cleaning surface 2 of FIG. 2 is designed differently from the cleaning surface 2 of FIG. 1. The entire cleaning surface 2 consists of one pattern of cleaning islands 3 in which the cleaning surface 2 has only one centrally disposed central cleaning island 8, which is concentrically surrounded by other cleaning islands 3. [0034] In contrast to the cleaning towel shown in FIG. 1, the cleaning towel of FIG. 2 has not just two wiping directions 7, but rather a plurality of wiping directions 7. 1, 7. 2, 7. 3, 7. 4, and 7. 5, each of which extends in the radial direction with respect to the central cleaning island 8. The radial distances 9 between the cleaning islands 3 that are next to each other in the radial direction become steadily larger in the direction of each edge 6. 1, 6. 2, 6. 3, and 6. 4 of the cleaning surface 2, starting from the central cleaning island 8. The flocking used in the cleaning islands 3 can be the same as used in the embodiment of FIG. 1. Additionally, the coverage with which the cleaning islands 3 cover the cleaning surface 2 can be the same as the embodiment of FIG. 1.

Summary: Cleaning device comprising a support ( 1 ) with at least one cleaning surface ( 2 ) on which raised cleaning islands ( 3 ) are arranged at certain distances ( 4, A, B, C, D, E, 9 ) from each other such that the cleaning islands ( 3 ) are surrounded on all sides by storage spaces ( 5 ) for holding dirt. The size of the storage spaces ( 5 ) decreases steadily from at least one edge ( 6 ) of the cleaning surface at least to the middle of the cleaning surface ( 2 ) in the cleaning direction ( 7 ).

big_patent

en

Summarize: It’s a story that gets more tragic by the hour. The Sewol ferry, carrying 476 passengers, mostly youth from one high school in Ansan, South Korea, capsized last Wednesday while en route to Jeju Island. Shortly after the sinking, 174 passengers were rescued. More than a week later, at the time of this writing, 175 are confirmed dead; 127 are missing. It’s also a story that’s downright mystifying. A captain that jumped ship. Untouched lifeboats. Orders to stay put as the vessel rolled. As bodies continue to be pulled from the sea, and as families desperately seek answers, the Sewol disaster has created a cultural flashpoint, forcing many to ask, “Who is to blame?” It’s a question that is haunting a nation, baffling pundits, and challenging a highly militarized government hailed for its organization and efficiency. And while Sewol’s captain remains Public Enemy No. 1, media outlets are factoring in a more amorphous villain: South Korean culture. “Media coverage has portrayed the ferry disaster either as a terrible tragedy that any person can sympathize with,” says Ju Hui Judy Han, a professor of cultural geography of travel at the University of Toronto, “or as a bizarre accident that could have only happened somewhere else.” According to several English-language media reports, the Asian country’s “culture of obedience” and youth deference to authority is the culprit. The Los Angeles Times reports that the disaster has “cast a harsh light on a Confucian culture in which young people are taught to respect the older generation,” while Reuters cites, “Many of the children did not question their elders, as is customary in hierarchical Korean society. They paid for their obedience with their lives.” The Brief Newsletter Sign up to receive the top stories you need to know right now. View Sample Sign Up Now Others echo the sentiment: If that was a boatload of American students, you know they would have been finding any and every way to get off that ferry. But in Asian cultures…compliance is de rigueur. — Dallas Morning News While children from any culture might well obey orders in such an unfamiliar and terrifying situation, one expert says Korean teenagers are particularly conditioned to do so: “Korean teenagers are very accustomed to being told what to do and what to think.” — South China Morning Post “What this culture prizes in its children, in its students, is obedience. And so when they were told to stay put by an adult, of course they would stay put.” — CNN It’s called “culture blaming,” and the media embraces it – especially the mainstream. “There’s two issues at play,” says Elizabeth Spayd, editor and publisher of Columbia Journalism Review. “First, there are many correspondents who parachute in to write about disasters who aren’t that familiar with the culture they drop into. (And it’s also true, in most American newsrooms, that the staff is not as diversified as the world.) But another reality is that, after a big disaster, when everyone’s looking for answers, people grab for whatever they can find.” We saw culture blaming, too, when South Korean norms were incriminated for the Asiana Airlines crash in San Francisco last year, while Korean male anger and aggression served as an “explanation” for Seung-Hui Cho’s shooting spree on the Virginia Tech campus in 2007. Call it a journalistic hit: Sewol coverage transforms into a spectacle of groundless theories on how a culture is partially guilty for the wiping out of an entire sophomore class. On the surface, it appears like an innocuous and thoughtful attempt to offer insight, but at the root is the suggestion that these cultures are inferior, broken, or backwards. It paints a portrait of Asian youth without humanity and individual agency. Should aspects of Korean society be scrutinized? Of course. Poor communication, disorganization, and complacency – compounded with fumbling bureaucracies and the lack of protocol and proper training – resulted in a botched rescue mission that has South Korea reeling. But to theorize that the high death toll is linked to a perceived cultural flaw or deficiency is a lazy journalistic shortcut. It fits a stereotype. Meanwhile, what’s missed in all this culture blaming is the real cause. “Culture is not to blame for the Sewol disaster,” says Jaehwan Cho, a South Korean reporter covering the story in Seoul and Ansan. “It represents the problem of government structure.” The worst offense: It places blame on the victims and their families as a nation grieves. If this happened to American teens, would we point to American culture as the source of the accident, or criticize our youth for not knowing what to do? Would we have blamed them for not surviving? We wouldn’t. Contact us at editors@time.com. ANSAN, South Korea — For South Korea, a country that pulled itself out of abject poverty to become the world's 15th-largest economy, the most stinging accusation about last week's ferry sinking is that it looks like a Third-World disaster. While the captain escaped and the crew dithered and bickered with emergency officials, hundreds of passengers, most of them high school students, obediently remained in their cabins as the ferry rolled and slipped beneath the surface of the cold, gray sea. Mistake piled atop mistake turned a near-shore mishap into the nation's worst maritime disaster in decades. The calamity has shamed many South Koreans and left them with serious doubt about their political leaders at a moment when they were preparing for a high-profile visit from President Obama, who is slated to arrive Thursday night. "We are supposed to be a prosperous middle power, but the fundamentals are still weak," said Choi Kang, vice president of the Asan Institute for Policy Studies in Seoul. "There was no control tower, nobody in charge." The botched rescue also has cast a harsh light on a Confucian culture in which young people are taught to respect the older generation. "I feel embarrassed as a Korean. We failed our children," said Kim Seun-tae, a 50-year-old minister whose son attends Danwon High School, which had 350 students and faculty members among the 476 people aboard the ship. Kim's son was not on the ferry. The minister said he was struck by video from survivors' cellphones that showed the mostly 16- and 17-year-old students sitting dutifully in their seats. "They were good, well-behaved kids. They followed instructions," Kim said. "Everybody is in a state of shock and depression. We can't look each other in the eye or speak." Some events initially planned for the presidential visit have been toned down to avoid any appearance of festivities at a time when divers will probably still be plucking bodies from the sunken vessel, according to people involved in the planning. More than 160 passengers remained missing as of Tuesday, with 139 confirmed dead. South Korean President Park Geun-hye, who made the protection of youths one of the themes of her administration, lashed out Monday at the crew members who abandoned the ship, saying the "behavior of the captain and some crew members is beyond understanding and no better than homicide." But Park faces accusations that her newly restructured Ministry of Security and Public Administration failed at its first disaster response. "They were civil servants with no experience or expertise. When it came to a time of crisis, people were paralyzed," said Jeong Chan-gwon of the Seoul-based Korea Institute for Crisis Management Analysis. "When she called the captain a'murderer,' it showed clearly how much political pressure she is under," said Scott Snyder, a Korea specialist with the Council on Foreign Relations, who was visiting Seoul last week. A panicked and inexperienced crew failed to call the coast guard, instead notifying the vessel traffic service at their destination on Jeju Island, about 50 miles away. It took 53 minutes for the Central Disaster and Safety Countermeasures Headquarters to mobilize. Meanwhile, the 69-year-old captain had already left the ship. Most of the crew members survived, while more than two-thirds of the passengers appear to have been lost. Even before the official call for distress, students used their cellphones to call their parents and the South Korean emergency number. "Help us. The boat is sinking," one boy reportedly told emergency dispatchers, according to a report Wednesday on Joongang Tongyang Broadcasting. "The kids had never been on a ferry like that before. They didn't know what to do. They could have been saved," said Kim Seong-kyu, 19, a school student who was among the mourners leaving white chrysanthemums and handwritten messages at a makeshift shrine outside the school. Among those being mourned was one of the surviving teachers from the doomed expedition who hanged himself from a tree last week in Jindo, the port city where families are staying. Seven crew members have been arrested, including the captain, a semi-retired and part-time pilot who had been called at the last minute to command the 6,825-ton ship. He was in his cabin when the boat ran into trouble, with a 25-year-old third mate at the helm. Investigators believe that a sharp right turn by the novice might have caused the ferry's cargo to shift, putting it in an irreversible list. I’ve never been a big believer in the, “Because I’m the Daddy,” school of parenting. My view has always been that, if I, a thinking adult, could not impose a rule or condition on my child through logic and persuasion, perhaps the rule or condition wasn’t such a good idea. On the flip side, if my kids can convince me – again, through logic and persuasion – of a particular cause or request, I’d consider acquiescing to it. After all, my goal as a parent isn’t to always be right. It’s to get my kids to think. I’m taken back to those core values as I read about the tragic sinking of the Sewol, the South Korean ferry in which 86 people are confirmed dead and another 220 are still missing — and increasingly presumed dead. Most of the victims are from a single high school. The tragic tale was made even more heartbreaking amid reports that the high number of victims may have been linked to the captain instructing passengers to stay in their rooms. It’s believed that the youngsters complied, and may have been entombed in those rooms by the cold, rising water. Here in the United States, it’s hard to imagine that, first of all, a bunch of high school kids would even listen, much less totally and faithfully comply with an adult’s instructions – even as a ship sinks. That’s because we Americans – and certainly Texans – value the individual over the group. If that was a boatload of American students, you know they would have been finding any and every way to get off that ferry. But in Asian cultures, which place the needs of the group over the needs of the individual, compliance is de rigueur. So, you end up with this horrendous death by obedience. But there’s a second part to the group-first formula. If compliance and obedience are expected, then much responsibility is required of the people giving orders. Which is why South Korean President Park Geun-hye said Monday that the actions of the captain and some crew amounted to “unforgivable, murderous behavior.” Yep. Some adults make bad decisions, shouldn’t be trusted. And that’s why I prefer to teach my kids to think rather than simply obey. Police officers are seen as a rescue and salvage team helicopter (top) flies over a port where family members of missing passengers from the capsized passenger ship Sewol gathered to wait for news from rescue and salvage teams, in Jindo April 22, 2014. SEOUL (Reuters) - The first distress call from a sinking South Korean ferry was made by a boy with a shaking voice, three minutes after the vessel made its fateful last turn. He called the emergency 119 number which put him through to the fire service, which in turn forwarded him to the coastguard two minutes later. That was followed by about 20 other calls from children on board the ship to the emergency number, a fire service officer told Reuters. The Sewol ferry sank last Wednesday on a routine trip south from the port of Incheon to the traditional honeymoon island of Jeju. Of the 476 passengers and crew on board, 339 were children and teachers on a high school outing. Only 174 people have been rescued and the remainder are all presumed to have drowned. The boy who made the first call, with the family name of Choi, is among the missing. His voice was shaking and sounded urgent, a fire officer told MBC TV. It took a while to identify the ship as the Sewol. "Save us! We're on a ship and I think it's sinking," Yonhap news agency quoted him as saying. The fire service official asked him to switch the phone to the captain, and the boy replied: "Do you mean teacher?" The pronunciation of the words for "captain" and "teacher" is similar in Korean. The captain of the ship, Lee Joon-seok, 69, and other crew members have been arrested on negligence charges. Lee was also charged with undertaking an "excessive change of course without slowing down". Authorities are also investigating the Yoo family, which controls the company that owns the ferry, Chonghaejin Marine Co Ltd, for possible financial wrongdoing amid growing public scrutiny. An official at the Financial Supervisory Service (FSS) told Reuters it was investigating whether Chonghaejin or the Yoo family engaged in any illegal foreign exchange transactions. The official did not elaborate. Another person familiar with the matter told Reuters that prosecutors were looking into suspected tax evasion by the firm, its affiliates or the Yoo family with assistance from the National Tax Service. A spokesman at the tax agency declined to comment on the matter. "There are lots of reports in the media, so as the regulator we need to check if they are true," another FSS official said. Neither the Yoo family nor the company was immediately available for comment. ONLY OBEYING ORDERS Several crew members, including the captain, left the ferry as it was sinking, witnesses have said, after passengers were told to stay in their cabins. President Park Geun-hye said on Monday that instruction was tantamount to an "act of murder". Many of the children did not question their elders, as is customary in hierarchical Korean society. They paid for their obedience with their lives. Four crew members appeared in court on Tuesday and were briefly questioned by reporters before being taken back into custody. One unidentified second mate said they had tried to reach the lifeboats, but were unable to because of the tilt. Only two of the vessel's 46 lifeboats were deployed. Two first mates, one second mate and the chief engineer stood with their heads lowered and it was impossible to tell who was speaking. One said there had been a mistake as the boat made a turn. Another said there was an eventual order to abandon ship. He said the crew gathered on the bridge and tried to restore balance, but could not. "Maybe the steering gear was broken," one said. Media said the ship lost power for 36 seconds, which could have been a factor. Public broadcaster KBS, quoting transcripts of the conversation between the crew and sea traffic control, the Jindo Vessel Traffic Services Centre, said the passengers were told repeatedly to stay put. For half an hour, the crew on the third deck kept asking the bridge by walkie-talkie whether or not they should make the order to abandon ship, KBS said. No one answered. "We kept trying to find out but... since there was no instruction coming from the bridge, the crew on the third floor followed the instructions on the manual and kept making'stay where you are' announcements," KBS quoted a crew member as saying. "At least three times." Lee was not on the bridge when the ship turned. Navigation was in the hands of a 26-year old third mate who was in charge for the first time on that part of the journey, according to crew members. In a confused exchange between the sinking Sewol and maritime traffic control released by the government, the crew said the ship was listing to port. "Make passengers wear life jackets and get ready in case you need to abandon ship," traffic control said. The Sewol answered: "It's difficult for the passengers to move now." (Additional reporting by Jungmin Jang, Se Young Lee, Joyce Lee and Miyoung Kim; Writing by Nick Macfie; Editing by Robert Birsel)

Summary: The Sewol ferry disaster has left the world looking for answers, wondering who to blame. And an uncomfortable number of English-language media outlets have hit on an answer, Kai Ma at Time observes: South Korea's "culture of obedience." For example... LA Times: The disaster and failed rescue have "cast a harsh light on a Confucian culture in which young people are taught to respect the older generation." Reuters: "Many of the children did not question their elders, as is customary in hierarchical Korean society. They paid for their obedience with their lives." Dallas Morning News: "If that was a boatload of American students, you know they would have been finding any and every way to get off that ferry. But in Asian cultures... compliance is de rigueur." "It's called 'culture blaming,' and the media embraces it-especially the mainstream," Ma complains. "On the surface, it appears like an innocuous and thoughtful attempt to offer insight, but at the root is the suggestion that these cultures are inferior, broken, or backwards." Certainly aspects of Korean society failed here-like its "fumbling bureaucracies and the lack of protocol." But blaming it on some cultural flaw is lazy journalism, and, more heinously, "It places blame on the victims and their families as a nation grieves." Click for the full column.

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Write a title and summarize: Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e. g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1. 5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i. e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. Salmonella enterica subspecies 1 serovar Typhimurium (referred to as S. Typhimurium in this study) is a common food-borne pathogen. Central to the pathogenesis of S. Typhimurium is its ability to invade intestinal cells, namely M-cells, epithelial cells and possibly dendritic cells [1], [2], [3]. Normally, only a small fraction of the mucosal cells are being invaded [4], [5], [6], [7]. The mechanisms focusing S. Typhimurium invasion to particular sites are not completely understood. Host-cell invasion by S. Typhimurium is the result of a multistep process. This includes: i) 3-dimensional movement in the gut lumen (motility, chemotaxis, diffusion); ii) transient interactions with the mucosal surface and particulate matter within the gut lumen; iii) reversible binding via adhesins like type 1 fimbriae (fimH, [8], [9]); iv) irreversible “docking” mediated via type III secretion system 1 (TTSS-1; [8], [9]). This step commits wild type S. Typhimurium to invasion. v) secretion of bacterial virulence factors, so called effectors, via TTSS-1 into the host cytosol; key effectors include SopE, SopE2, SopB and SipA. ; vi) manipulation of the host cell by S. Typhimurium effectors leading to the emergence of prominent membrane ruffles in epithelial- and M-cells [10]; vii) host cell invasion, which often features the simultaneous entry of several bacteria through the same ruffle. While many steps have been studied in detail before, those steps targeting the pathogen to a particular site (i. e. steps ii and iii) have remained enigmatic. Thus, it is still unclear whether S. Typhimurium actively “selects” target sites and which mechanism would enable such a preference. We speculated that motility might affect target site selection. Like many other pathogens, S. Typhimurium employs flagella to orient and move in 3D space [11], [12]. This has multiple well documented effects on the pathogen-host interaction. The coupling to chemo-sensing systems allows swimming towards nutrient sources (“chemotaxis”; [13], [14]). Motility is important for the invasion of tissue-culture cells and in the induction of gut inflammation by Salmonella spp. (in vitro: [15], [16], [17], [18], [19]; animal model: [20], [21], [22], [23], [24]). Furthermore, the flagella might mediate adhesion [25] or elicit host-cellular signaling responses. It remained unclear whether flagella may also serve additional tasks, i. e. in target site selection. Flagellar rotation propels the bacterium with a velocity of at least 25–55 µm/s (“motility” [12], [26]). If encountering a host cell, S. Typhimurium is generally assumed to either be “deflected” back into the medium or to initiate a productive infection. However, so far, this step of the infection process has not been studied in detail. In contrast, the interaction of motile E. coli strains and solid surfaces has been extensively studied. On solid surfaces, E. coli slides in large circles, remaining in contact with the surface for extended time periods, a phenomenon called near surface swimming (NSS). Two mechanisms explaining this ability of E. coli to swim along solid surfaces have been proposed (Fig. 1A, inserts I and II). According to the hydrodynamic entrapment theory [27], [28], the bacteria experience extensive drag stress at the part of their body close to the surface, causing a “forward” rotation. This rotates the rod-shaped bacterium into an “upright” position. The upright position in turn increases the drag resistance against the fluid, resulting in an opposing rotational force. Ultimately, these two forces are in equilibrium, keeping the bacterial rod at a constant angle towards the surface, thus entrapping the organism in a tilted swimming position. The alternative DLVO model (Derjaguin, Landau, Verwey, and Overbeek; for a review, see [29]) explains NSS via electrostatic and van der Waals forces. Nevertheless, both models predict that motile bacteria encountering a solid surface would be “trapped” at the surface and perform a NSS motion. It remains unclear whether NSS may also occur on cellular surfaces and whether this might affect target site selection. Here, we studied target site selection by S. Typhimurium. The initial stages of S. Typhimurium-interaction with cellular or artificial surfaces were analyzed in real time. In this “pre-docking” phase of the infection, bacterial motility was of key importance. It led to characteristic near surface swimming patterns on host-cell surfaces and targeting to sites with a prominent surface topology. Our data suggest a model, in which physical forces emanating from the flagella-driven motility facilitate near-surface swimming and explain the pathogen' s target preference during infection. We are discussing possible implications for the disease and for infections by other flagellated pathogens. To study the initial interactions of S. Typhimurium with cellular surfaces, we employed time-lapse microscopy (supplementary Videos S1 and S2). HeLa cells, a commonly used tissue-culture model for studying S. Typhimurium invasion, were infected with S. TmΔ4 (SL1344 sopEE2B sipA; Table 1). S. TmΔ4behaves like S. Tmwt in all aspects of the early host-cell interaction (steps i to v), but cannot trigger membrane ruffling or invasion as it lacks the key effector proteins SopE, SopE2, SopB and SipA [8], [9], [30]. Therefore, S. TmΔ4 allowed us to focus on the initial surface interactions and docking (Fig. 1A). First, the initial surface interactions of S. TmΔ4were analyzed using time-lapse differential-interference contrast (DIC) microscopy (Fig. 1B). Strikingly, S. TmΔ4was swimming along the cellular surface for extended time periods (supplementary Videos S1 and S2). Our subsequent frame-by-frame analysis of the time-lapse videos identified several “stages” of this interaction: In most cases, bacteria went through all stages of interaction before taking off again. Please note that some bacteria stopped until the end of the time-lapse movie. In this experiment, we could not distinguish whether these bacteria were “docking” (i. e. bacteria that bound irreversibly, e. g. via the TTSS-1 apparatus [8], [9], [30]), or “stopping” (bacteria transiently stopping but continuing NSS or taking off after the end of the movie). Stopping did not happen randomly but occurred frequently at “obstacles” encountered during NSS. In particular, S. Typhimurium stopped and docked at cells with a round morphology (i. e. a mitotic cell; see supplementary Video S1). This provided a first indication that a transient stop may “preselect” specific sites for subsequent docking of S. Typhimurium. This would be in line with the preferential docking of S. Typhimurium onto mitotic cells observed in earlier studies ([31], [32], see below). For a quantification of these initial bacteria surface interactions we used S. TmΔ4harboring a plasmid conferring constitutive gfp expression (pGFP; Table 1) and time lapse fluorescence microscopy. This allowed precise quantification of all stages of the bacteria surface interaction including landing and take-off, since the fluorescent bacteria moving out of the focus layer appear as “rings” in the movie (see T0s in Fig. 1C). Hence, the “landing” stage was defined as the time between the first detection of a “ring” (followed by a continuous downward movement) and the change of the direction and speed typically observed when NSS started (T0–0. 4 s in Fig. 1C). In analogy, the “take off”-stage describes the time between the end of stopping or NSS and the disappearance of the “ring” (T41. 9–42. 3 s in Fig. 1C). Additionally, we tracked the time spent stopped or engaged in NSS. Take-off and landing occurred within <3. 1 and <4. 2 s, respectively (median 0. 4 s for both; Fig. 1D), while the time engaged in NSS was significantly longer (median 1. 5 s; range 0. 3–41. 5 s; Fig. 1D). Overall, the bacteria covered significant distances swimming along the host cellular surface (6. 7–325 µm; see also below). Furthermore, we observed a variety of “behaviors” with respect to stopping. 33% of all imaged S. TmΔ4 (pGFP) bacteria did never stop, while 67% made one or more stops (“NSS and stop”; Fig. 1E). Some bacteria stopped up to 5 times on the cell surface and some S. TmΔ4remained stopped (or docked) until the end of the imaging experiment, with the longest observed stop lasting 280 s (Fig. 1F). Overall, the bacterial density at the cellular surface was significantly increased compared to the overlaying media (Fig. 1G). Accumulation of bacteria at the cellular surfaces was not attributable to gravity, as indicated by a comparison between motile (affected by gravity and NSS) and non-motile bacteria (affected only by gravity; suppl. Fig. S1). Much rather, flagella-driven NSS and ensuing stops accounted for the increased local S. Typhimurium density at the host cell surface. This suggested that the prolonged contact time might contribute to the target site selection by S. Typhimurium. In order to determine the role of bacterial adhesins in the initial phases of the surface interaction, we analyzed two S. Typhimurium mutants, S. Tm-T1 (SL1344 invG) and S. Tm-T1-Fi (SL1344 invG fimD; Tab. 1). These mutants lack one or two surface structures, namely type 1 fimbriae and the TTSS-1 apparatus, which are known to mediate reversible binding and irreversible docking of S. Typhimurium to HeLa and other host cells [8], [9], [30]. However, a role of these adhesins for near surface swimming or stopping had not been addressed. HeLa cells were infected with S. Tm-T1 (pGFP) and S. Tm-T1-Fi (pGFP) and transient interactions were monitored by time lapse fluorescence microscopy as in Fig. 1. All analyzed parameters, including the number of stops, were indistinguishable from those of S. TmΔ4 (Fig. 2A–D; compare to Fig. 1). These results indicated that none of the initial surface interactions were affected by TTSS-1or by fimD (Fig. 2A, C). Strikingly, this also pertained to the transient stops and clearly distinguishes the initial surface interactions from later stages of the infection, i. e. reversible binding and docking. Stopping thus seems to be attributable to a different mechanism. Nevertheless, as indicated by earlier data [8], [9], [30], some of the stops must result in reversible binding and docking. To estimate the relative frequency of stopping and of the docking events, the total number of stops observed on the cell layer (= the number of potential docking events), during a 5 minute period was calculated: the total number of S. TmΔ4bacteria landing on the cell surface during a 5 min period was multiplied by the number of stops using the values determined in as Fig. 1E. After the end of the5-minute real time imaging experiment, the cells were washed, fixed and stained for DNA (DAPI), actin (TRITC-phalloidin) and S. Typhimurium (anti-LPS antibody). This protocol removed all “stopped” bacteria, while “reversibly bound” and “docked” bacteria remained on the cells and were enumerated by fluorescence microscopy [8], [9], [30]. Comparing the results from both types of analysis revealed that no more than 1–2% of the total stops (as detected by time lapse microscopy) resulted in a docking event. Hence, transient stops are approx. 50- to 100-fold more frequent than docking events, at least in the 5-minuteinfection experiments that we have performed, here. In conclusion, these data established that NSS requires neither TTSS-1nor type I fimbriae. The initial pathogen host cell interaction is thus clearly distinct from subsequent stages, i. e. reversible binding and docking. Bacterial flagellar movement is guided by chemotaxis. It had remained unclear, if NSS was dependent on chemotaxis or whether non-directed motility would suffice. To address this issue, HeLa cells were infected with S. TmΔ4 cheY (pGFP) (SL1344 ΔsipAsopBEE2 cheY; Table 1). This isogenic mutant is a “straight-swimmer”, expresses wild type numbers of functional flagella, but cannot swim along chemical gradients. The initial surface interactions were monitored by time lapse microscopy as described in Figs. 1 and 2A–D. All analyzed parameters were indistinguishable from those ofS. TmΔ4, S. Tm-T1 or S. Tm-T1-Fi (compare Fig. 2E, F to Fig. 1D, E and Fig. 2A–D). Therefore, in our tissue culture assays non-directed motility was sufficient for facilitating the initial surface interactions. Our data suggested that basic physical principles may explain the initial interactions of S. Typhimurium with host cellular surfaces. In this case, the movement patters on cellular and acellular surfaces should be quite similar. Therefore, we inoculated glass-bottom tissue culture dishes seeded with HeLa cells (or not) with S. TmΔ4 (pGFP). The bacterial surface interactions were analyzed by time-lapse fluorescence microscopy as described in Figs. 1 and 2. S. TmΔ4 (pGFP) performed NSS with equivalent speed on glass and on cellular surfaces (approx. 30 µm/s; Fig. 3A). However, the median duration of an episode of NSS (6. 3 s on glass vs. 2. 95 s on cells; Fig. 3B) and the median distance travelled during this time (221 µm on glass vs. 95 µm on cells; Fig. 3C) were slightly larger on glass than on the cellular surface. These observations were in line with our hypothesis that the initial surface interactions may be governed by equivalent physical principles. Strikingly, the bacteria followed “right-handed” curved tracks on glass and, to a lesser extent, on cellular surfaces (Fig. 3D, E). It is thought that this curvature of the NSS tracks is attributable to the shear force between the flagella-mediated rotation of the bacterial body and the respective surface [28], [33]. So far, our data were in line with the hypothesis that general physical principles are responsible for NSS on cellular and on glass surfaces. In this case, other types of host cells or motile bacteria should yield similar results. Therefore, we have extended our analysis to MDCK cells, a commonly used polarized epithelial cell line, and E. coliNissle which was transformed with the GFP expression plasmid (E. coliNissle (pGFP); Suppl. Fig. S2). Both, E. coliNissle (pGFP) and S. TmΔ4 (pGFP) engaged in NSS and displayed similar movement patterns on MDCK, on HeLa cells and on glass surfaces (Fig. 3D, E and suppl. Fig. S2A–D). Nevertheless, the shape of the NSS-tracks may differ slightly. This might be attributable to different morphological features displayed by the different types of surface and represents an interesting topic for future research. In conclusion, these data lend further support to the notion that initial surface interactions are governed by general physical principles and suggest that near surface swimming might be a general strategy for target selection employed by different flagellated bacteria. Analysis of our movies so far suggested that during NSS, rounded cells (e. g. dividing cells) represent preferential sites for stopping. We hypothesized that bacterial stopping can be explained by the prominent topological features of these rounded cells. If so, bacteria should also stop at artificial topological obstacles and bacteria should accumulate at such sites. This was tested in two different ways, i. e. in a simplified experimental setup and by a computer simulation (see below). In order to experimentally test our hypothesis, we have analyzed the initial surface interactions of bacteria with small glass beads which were placed as artificial obstacles onto a glass surface. Glass-bottom tissue-culture dishes harboring glass beads (Ø = 500 µm) were therefore inoculated with S. Tmwt (mCherry) and bacterial movement patterns were analyzed by time-lapse fluorescence microscopy, as described in Figs. 1,2 and 3. Again, the bacteria were moving for long distances along the glass surface, but were stopping in the immediate vicinity of the glass beads (Fig. 4A, B; suppl. Video S3). 45–70% of these bacteria continued to swim or took off again before the end of the movie, indicating that these were truly stopping (not binding/docking). Thus, topological obstacles can facilitate site-specific stopping of bacteria engaged in NSS. In addition, these data allowed a rough estimation of the “altitude” at which S. Typhimurium swims above the glass surface. Based on the geometry of the glass bead, the center of the bacterial cell was 0. 43+/−0. 07 µm away from the surface. Similar results were obtained for beads with smaller diameters (150 and 30 µm, respectively). The rod-shaped S. Typhimurium cell has a radius of approx. 0. 5 µm. Therefore, most of the “distance” is attributable to the bacterial cell and the NSS “altitude” is most likely <150 nm above the glass surface. These observations were in line with earlier work [27], [28], [29], [34] describing bacterial NSS. The data presented above indicated that NSS affects target cell selection in two ways: by increasing the local pathogen density on surfaces and by enhancing the probability of surface contacts (stopping) at topological obstacles projecting from this surface. A computer simulation was used to verify whether these two phenomena are sufficient for explaining target site selection. We modeled the interaction of S. Typhimurium with a three dimensional landscape consisting of a flat surface and one spherical obstacle, partially submerged into the surface (Materials and Methods). The particles (motile, but non-chemotactic “bacteria”) were introduced and moved linearly within the 3D virtual space above the surface. Upon contact with the sphere, the particles were either “stopping” (10% likelihood) or reflected (90% likelihood). Three different scenarios were analyzed with respect to the particles encountering the flat surface. (1) the “random” scenario: particles were reflected back into 3D space and randomly assigned a new direction of movement. (2) the “billiard” scenario: particles were reflected with an angle of reflection identical to the angle of impact. (3) the “NSS” scenario: particles encountering the surface did not leave but followed the surface via NSS. If the sphere was encountered during NSS, the particles stopped at this site with a likelihood of 10%. In the NSS scenario (scenario 3) but not the random or the billiard scenario, particle accumulation occurred right at the topological obstacle (Fig. 5). Therefore, NSS and stopping at physical obstacles are sufficient for explaining the target site selection observed in simplified model systems (Fig. 4A, B) and tissue culture infection experiments (Fig. 1, [31], [32]). So far, our analyses of the target site preference of S. Typhimurium had focused on the initial surface interactions, i. e. landing, take-off, NSS and stopping. Next, it was important to establish how these initial interactions may affect the subsequent steps of the infection process, i. e. docking. If stopping increases the probability of binding and irreversible docking at the respective site, stopping (as observed by time lapse microscopy; Fig. 1,2) and docking should display an equivalent target site preference. To assess the target site preference of binding/docking on host cell layers quantitatively, we have employed a well-established “standard” infection protocol [9], [32]. HeLa cells were infected for 6 min with S. TmΔ4 at the indicated m. o. i., washed gently, fixed and stained (see Materials and Methods). As binding/docking occurred approximately 50-fold less frequently than stopping (see above), we employed higher multiplicities of infection than in the time lapse microscopy experiments. Visual inspection indicated that S. Typhimurium has a pronounced targeting preference for rounded cells (Fig. 6A, top panel; “mitotic” nuclei with condensed DNA highlighted in yellow). In particular, the bacteria were found to dock to the base of rounded cells (suppl. Fig. S3A). In order to quantify this phenotype, we employed automated fluorescence microscopy, and an automated image-analysis routine (Materials and Methods). Rounded cells were targeted (docked) with significantly higher efficiency than non-dividing cells (Fig. 6C, left panel). This targeting preference for rounded cells was also observed by manual quantification (S. TmΔ4; suppl. Fig. S3B) and in infection experiments with S. Tmwt (data not shown). These findings were quite similar to our observations during the initial surface interactions (Figs. 1 and 2). Rounded mitotic cells seem to represent topological obstacles within the cellular landscape. In fact, the rounded mitotic cells were significantly “higher” than the interphase cells (12±2 µm vs. 5. 4±1. 1 µm; n = 20 each; p<0. 0001, Mann-Whitney-U test). Taken together, NSS-driven stopping and binding/docking displayed equivalent preferences for topological obstacles like rounded cells. Next, we have addressed the role of flagellar-driven motility. It is well established that flagella are required for cellular invasion of S. Typhimurium [15], [16], [17], [18], [19]. The data presented above suggested that flagella-driven NSS might determine the target site preference. In order to study the role of bacterial motility in the targeting preference of binding/docking, we analyzed the host-cell interaction patterns of the non-motile mutant S. TmΔ4 fliGHI (SL1344 fliGHI) which does not express flagella. This mutant and other non-motile S. Typhimurium mutants did not dock efficiently at all (Fig. S4). Importantly, the few HeLa cells that were infected harbored just one bacterium, even at high m. o. i. (Fig. 6A, bottom panel; Fig. 6C, right panel). In line with our hypothesis, S. TmΔ4 fliGHI did not display a targeting preference for rounded cells (Fig. 6A, C). In order to increase the chances of a host cell encounter by S. TmΔ4 fliGHI, equivalent infection experiments were performed applying mild centrifugal force (500 g for 10 min) to increase the collision rate between non-motile S. TmΔ4 fliGHI and the host cells (Materials and Methods). This strategy is commonly used for “rescuing” invasion defects of non-motile S. Typhimurium mutants, (e. g. [16], [35]). As expected, centrifugation increased the number of binding/dockingS. TmΔ4 fliGHI in our assay (Fig. 6B, data not shown). Nevertheless, S. TmΔ4 fliGHI did not display any target preference for the rounded (mitotic) cells, even at the highest m. o. i., tested (Fig. 6D; right panel). This was in line with our hypothesis that flagella-driven NSS determines not only the targeting preference of “stopping”, but also that of binding/docking. Finally, we analyzed the role of chemotaxis, i. e. directed motility along chemotactic gradients. S. TmΔ4 cheY is an isogenic and motile mutant incapable of chemotaxis (compare Fig. 2E, F). This mutant yielded equivalent results as S. TmΔ4including a highbinding/docking efficiency and a pronounced targeting preference for rounded cells (data not shown). Taken together, these results suggested that preferential infection of rounded cells is attributable to simple non-directed motility and that chemotaxis was not required, at least in this simple tissue culture model. During NSS, stopping and docking occurred preferentially at rounded cells. If targeting was indeed dictated by topological obstacles, manipulation of the host cell morphology should affect both, the initial surface interactions and docking by S. Typhimurium. To test this hypothesis, we manipulated the host cellular morphology with cytochalasin D (Cyt. D). This drug depolymerizes actin filaments causing the cells to round up. First we analyzed the effect of cytochalasin D treatment on binding/docking. HeLa cells, pretreated with the indicated concentration of cytochalasin D for one hour, were infected for 12 minutes with S. TmΔ4 (pGFP), S. Tm-T1 (pGFP) or S. Tm-T1-Fi (pGFP) at the indicated m. o. i.. Afterwards, cells were fixed and stained and we analyzed the actin-based cytoskeleton and bacterial docking (Materials and Methods). Since the automated evaluation now focused on the percentage of infected cells (not the number of individual bacteria per infected cell), overlapping but somewhat higher m. o. i. s were used than in Fig. 6. As shown in Fig. 7A, HeLa cells were partially rounded at 2 µM and fully rounded at 10 µM cytochalasin D. All three S. Tm strains bound/docked to the rounded cells with an increased efficiency (Fig. 7B). Equivalent results were obtained with another actin-disrupting drug (latrunculin B; suppl. Fig. S5). This verified that the host cell topology has a profound effect on the target site preference of binding/docking by S. Typhimurium. Next, we analyzed the effects of the cytochalasin D treatment on the initial surface interactions. HeLa cells pretreated with 10 µm cytochalasin D were infected with S. TmΔ4 (pGFP), S. Tm-T1 (pGFP) or S. Tm-T1-Fi (pGFP) and landing, NSS, stopping and take-off were analyzed by time-lapse microscopy as described in Fig. 1 and Fig. 2. Strikingly, on the cytochalasin D-treated cells, nearly all bacteria engaging in NSS made at least one stop. Only very few bacteria displayed uninterrupted NSS (“NSS only”; Fig. 8A, D, G). This was quite different from our data on untreated HeLa cells where 33% of all bacteria displayed uninterrupted NSS (see Fig. 1E). Furthermore, on cytochalasin D-treated cells, the time spent at each stop was significantly longer and some of these bacteria “stopped” for the entire course of the 5-min experiment (Fig. 8B, E, H). In a similar analysis, the fraction of time thatS. TmΔ4 (pGFP), S. Tm-T1 (pGFP) or S. Tm-T1-Fi (pGFP) spent stopping at the host-cell surface was significantly higher than in untreated HeLa cells. In fact, on the cytochalasin D-treated cells, a majority of the bacteria spent most of their time “stopping” (“only stoppers”; Fig. 8C, F, I). Again, no differences were observed between S. TmΔ4 (pGFP), S. Tm-T1 (pGFP) and S. Tm-T1-Fi (pGFP), confirming that “classical” adhesins do not significantly affect these transient initial bacteria-host cell interactions. In conclusion, the shape of host cells has a profound effect on initial surface interactions and binding/docking. This provides further evidence that NSS leads to a target site preference for physical obstacles. Membrane ruffles triggered by the TTSS-1 virulence system represent a well-known topological obstacle encountered on infected cell layers [36], [37], [38], [39]. We hypothesized that membrane ruffles might enhance local stopping, binding and docking at pre-existing ruffles, thus leading to cooperative invasion. To study the targeting of ruffles, we have focused on binding and docking. These two steps of the infection process can be analyzed using “standard” fluorescence microscopy assays [9], [32]. First, we employed a co-infection strategy using a “helper strain” and a “reporter strain”. S. TmSopE (without plasmid) was chosen as the helper strain, since its effector SopE is able to trigger pronounced membrane ruffling [5], [32]. S. TmSopE carries deletions of the effectors sipA, sopB and sopE2, thus eliminating confounding pleiotropic actions of these effectors on the host cell. SopE-induced ruffles appeared within 5 minutes, are known to have a prominent shape, and represent a large physical obstacle on an otherwise much flatter cellular surface (Fig. 9A; [40]). S. TmΔ4 (pGFP), which does not trigger ruffles itself, was used as a “reporter” to assess docking to the ruffles. In a time lapse microscopy experiment employing DIC and fluorescence imaging both strains engaged in NSS and stopped frequently at ruffles (Fig. 9B; supplementary VideoS4; data not shown). To analyze a preference of S. Typhimurium for cellular ruffles in a quantitative manner, HeLa cells were infected with a 1∶1 mixture of S. TmSopE andS. TmΔ4 (pGFP) at a high or a lower m. o. i for 6 min. In control experiments (no ruffles), HeLa cells were infected with a mixture of S. TmΔ4 and S. TmΔ4 (pGFP). Subsequently, cells were fixed followed by staining of extracellular bacteria (using an anti-Salmonella antibody; Materials and Methods). After permeabilization of the cell membrane, the actin cytoskeleton was stained. The data evaluation strategy is depicted in Fig. 9C. In the experiments using S. TmSopE as the helper strain (ruffling occurs), we determined the number ofS. TmΔ4 (pGFP) (Fig. 9D, red bars) and S. TmSopE (Fig. 9E, red bars) residing on the respective ruffle. In the negative controls (no ruffling; co-infection with S. TmΔ4 and S. TmΔ4 (pGFP) ) we quantified all bacteria located on the respective cell (Fig. 9D, D grey bars). Comparing targeting to a ruffle as opposed to targeting to a whole cell (control w/o ruffle) is a conservative strategy for detecting ruffle-specific target site preferences, since the whole cell has a much larger area than an individual ruffle. Furthermore, it should be noted that the anti-Salmonella antibody was applied before permeabilization (Materials and Methods). This allowed us to discern internalized and external reporter bacteria. In all experiments, S. TmSopE and S. TmΔ4 (pGFP) docked more efficiently to ruffles than to non-ruffling cells (Fig. 9D, E compare red and grey bars). This was true for the reporter strain (Fig. 9D; S. TmΔ4 (pGFP) ) as well as for the helper strain (Fig. 9E; i. e. S. TmSopE). A similar effect was also observed in 12-min infection experiments (data not shown). Therefore, ruffling stimulated docking of bacteria to cellular ruffles. Moreover, ruffling facilitated internalization of S. TmΔ4 (pGFP). Internalized S. TmΔ4 (pGFP) was detected in all co-infections with S. TmSopE (1. 4 and 1. 6 bacteria per ruffle on average for the experiment in Fig. 9D for m. o. i. s of 62 and 250, respectively). No internalizedS. TmΔ4 (pGFP) was detected in the negative controls (no ruffles; S. TmΔ4 as helper strain). Equivalent data were obtained in an analogous experiment using automated image acquisition and analysis methods (suppl. Fig. S6). In another control we tested immotile bacteria (S. TmΔ4 fliGHI (pGFP) ). As expected, these bacteria did not attach to ruffles or normal cells (Fig. S7). Again, cellular attachment was rescued upon centrifugation; however, this did not lead to any targeting preference for ruffles vs. non-ruffling cells (Fig. S7). Finally, we explored the targeting to ruffles by E. coli Nissle, a non-invasive motile bacterium. In co-infection experiments with S. Tmwt, E. coli Nissle displayed a targeting preference for host cellular ruffles (Fig. S8). Taken together, the effector protein SopE is required for triggering ruffles. Once the ruffles are formed, they seem to represent prominent physical obstacles which facilitate stopping, binding, docking and internalization of motile pathogens. However, please note that we cannot exclude that other factors, besides ruffle topology (e. g. altered membrane structure or composition in the ruffle) might also contribute to S. Typhimurium targeting to these sites. Finally, we reasoned that S. Typhimurium recruitment onto ruffles might lead to cooperative invasion. If multiple bacteria dock to the same ruffle, more effector proteins are delivered, thus increasing the ruffle size and enhancing the chance for stopping, binding and docking of additional bacteria. To test this hypothesis, HeLa cells were infected for 9 min with S. TmSopE (pGFP) at increasing multiplicities of infection, fixed and stained. We first quantified the fraction of ruffling cells (Fig. 10A, B, left panel). Next we determined (at each m. o. i.) the number of intracellular and extracellular bacteria residing in an individual ruffle (Fig. 10A, B, middle panel). Finally, we determined the number of “invaded S. TmSopE” per cell (Fig. 10 A, B, right panel). This was achieved by multiplying the percentage of ruffling cells with the number of intracellular bacteria per ruffle. All these parameters increased as a function of the m. o. i. Next we wanted to determine the invasion efficiency mediated by a single bacterium without “support” from other bacteria. We therefore focused at low m. o. is., where typically one bacterium was observed per ruffle. The few ruffles with two associated bacteria were excluded from the subsequent analysis. The single bacterium was either located outside or within the host cell (Fig. 10B, m. o. i. 4 and 8). To estimate invasion efficiency by this single bacterium alone without possible support by other bacteria, this invasion efficiency was extrapolated to higher m. o. i., assuming a linear increase of invasion efficiency with increasing m. o. i. (Fig. 10B, right panel, black line). Strikingly, this extrapolated invasion efficiency was much lower than the observed invasion efficiency at higher m. o. i. (Fig. 10B, right panel, red line). This indicated that S. Typhimurium invasion occurred in a cooperative fashion, most likely by provoking stopping, binding and docking of additional bacteria engaged in NSS at sites of membrane ruffling. The mechanism of target site selection by S. Typhimurium had remained enigmatic. We have analyzed the initial surface interactions between the pathogen and the host by time-lapse microscopy, by comparative analysis of pathogen movements on cells and glass surfaces as well as by standard infection experiments. Upon encounter with a host-cell layer, we identified a distinct early phase of the infection characterized by landing, near surface swimming and stopping, which preceded later events such as fim- or TTSS-1-mediated binding and docking. In this initial phase, flagellar motility has at least two functions in establishing the contact with the host cell, i. e. propelling the pathogen towards the host cell layer and facilitating NSS. NSS is likely attributable to physical forces emanating from the pathogen movement along the surface. Here we found that NSS increased the local pathogen concentration on the host-cell surface, and lead to stopping at topological obstacles. NSS thereby mediates preferential docking at the base of rounded cells and on pre-existing membrane ruffles. Thus, the target-cell selection for dividing cells and cooperative infection at membrane ruffles can be explained simply by physical forces between the host cellular surface and the motile pathogen, which govern the initial phase of the pathogen-host cell interaction. Different models have been proposed to explain NSS [27], [28], [41], [42], [43], [44]. Recent approaches have combined hydrodynamic entrapment and DLVO interactions with Brownian motion, which leads to random changes of the bacterial NSS-altitude [37]. Random variations in surface distance also lead to predictable changes of the angle of bacteria towards the surface and the radius of the curved track. Therefore, Brownian motion can enable probing of the surface following different curvatures at different heights when the bacteria are swimming along. This variability may help to further expand the surface probing capacity of a motile pathogen [37]. However, it should be noted that the Brownian motion-forces are much weaker than the thrust provided by the flagella. This may explain why stopping and docking on open glass surface areas is much less frequent than at the base of elevated obstacles located on the surface. Only at these obstacles, the force provided by the flagella is fully employed to counteract repulsive forces thus driving the bacterium as close to the surface as possible. This may increase the chances for the formation of stable contacts as required for binding and docking. Are “stopping” and “docking” related? In both cases, S. Typhimurium stays at one particular spot on the host cell surface for at least some time. However, the retention mechanisms seem to differ. Docking and binding, i. e. long-term association with host cells are mediated by adhesins [45], [46]. In the case of HeLa cells, docking is mediated mainly viaTTSS-1 and type-1 fimbriae which is why the mutant strainS. Tm-T1-Fi has a reduced docking efficiency [9]. In contrast, stopping was not significantly affected in the case ofS. Tm-T1-Fi, implying that neither TTSS-1 nor type-1 fimbriae mediate stopping (Fig. 2). Furthermore, washing (as performed in docking experiments; see Materials and Methods), removed ≥98% of all “stopped” bacteria from the host cell surface. Presumably, the remaining ∼2% were docked, suggesting that stopping is mediated by a weaker force and that it is approximately 50-fold more frequent than docking, at least during the first minutes of infection. In spite of these differences, both stopping and docking required motility (but not chemotaxis) and occurred with high probability at rounded cells and membrane ruffles. Based on these considerations, we propose that stopping at topological obstacles may simply extend the residence time at a given location (close to the surface), thereby increasing the probability of adhesin-mediated binding and docking. It is tempting to speculate that Brownian motion might randomly drive stopping bacteria into even closer proximity of the host cell surface, thus increasing the chances for a successful engagement of TTSS-1 or type-1 fimbriae [34]. In this way, prolonged stopping and Brownian motion would foster the preferential infection of rounded cells and cell ruffles. Thus, landing, NSS and stopping may allow prolonged probing ata very limited area of the 2D surface. If docking is unsuccessful, S. Typhimurium continues NSS or takes off into and may engage in initial surface interactions at another site. NSS recruits S. Typhimurium into membrane ruffles, thus promoting cooperative invasion. Invasion of more than one bacterium at a single ruffle has been described before [47], [48], [49] but no mechanistic explanation and no evidence of cooperativity has been provided. Our data show that ruffles represent topological obstacles favoring stopping and productive invasion. Therefore, ruffles might be regarded as a site of “communication” between individual bacteria. Firstly, this implicates the exploitation of invasive strains by non-invasive strains, which can subsequently invade. This type of “rescue” has been described previously [37], [50]. Secondly, it implicates cooperation between invasive strains, in two respects. When S. Typhimurium makes a favorable docking interaction and induces membrane ruffling in the host cell, this increases the chance that additional bacteria can “find” this invasion-permissive site. Furthermore, in some hosts or cell types, higher dosages of effector proteins might be required for triggering successful invasion. In this case, recruitment into ruffles might allow larger amplitudes of stimulation. Certainly, this would be of importance for the mechanistic interpretation of results from tissue-culture cell-infection experiments. In animal experiments, flagella and motility are also required for efficient gut infection [21], [22], [24], [51]. Here, the flagella serve additional functions not observed in tissue culture. In the inflamed gut, flagella facilitate chemotactic movement thus mediating access to the nutrient-rich molecules secreted by the gut wall [24]). Thereby, chemotactic motility propels the pathogen towards the gut surface. For this reason, both, chemotaxis and motility are required in the gut [24]. This additional function of flagella, which does not affect the tissue-culture infection, has precluded straightforward animal-infection experiments addressing the “within host” importance of the NSS-based, chemotaxis-independent targeting mechanism described here. Does NSSor stopping also occur at the gut surface? In this study we demonstrated NSS in vitro at the surface of HeLa cells and polarized epithelial cells. However, in previous studies, S. Typhimurium was found to accumulate at the surface of the gut epithelium during the first phase of the infection [24]. Furthermore, S. Typhimurium swimming along the epithelial surface has also been visualized by in vivo live microscopy in the cecum of infected mice [6]. At 4 h post infection, the pathogen was found to swim along the surface of infected crypts at a speed of 5–50 µm/sec. Subsequently bacteria stopped at the epithelium and entered into enterocytes. While these experiments did not elaborate on the individual phases of the initial surface interactions, they clearly demonstrate that NSS occurs within the host' s intestine. Later on, upon triggering mucosal inflammation, S. Typhimurium seems to invade into neutrophils which are transmigrating in large numbers into the gut lumen [52]. Intriguingly, neutrophil infection was found to require motility. It is tempting to speculate that neutrophils are infected by S. Typhimurium during transmigration. While crossing the epithelial barrier, the luminal part of the neutrophil may form a physical obstacle stopping S. Typhimurium swimming along the epithelial surface. These observations suggest that NSS-mediated targeting of host cells may occur in vivo. A detailed analysis of these processes during the course of a real infection will be an interesting topic for future research. The NSS-mediated targeting mechanism identified in our work is based on general physical forces which act on any particle moving on a 2D surface. Therefore, it should pertain to many other motile bacteria, including pathogens (e. g. enteropathogenic E. coli, Yersinia spp.), commensals, as well as environmental bacteria. Accordingly, we found that the motile strain E. coli Nissle can engage in NSS and target Salmonella-induced membrane ruffles. Our results imply that the NSS-mediated targeting should result in preferential binding to physical obstacles present on the respective surface, including abiotic obstacles as well as prominent topological features of surface-exposed cells of a given host. Thus, the initiation of biofilm formation and the infection of animals at particular sites might be governed by the same basic principles. Deciphering these principles will be of great interest for basic microbiology and might allow the development of countermeasures prohibiting the initial steps of bacterial colonization. Published bacterial strains are listed in Table 1. The construction of additional strains is described, below. Cells were seeded into glass-bottomed culture dishes (Mat Tek) in DMEM (Invitrogen), 10% FCS (Omnilab) containing streptomycin (50 µg/ml), 24 hours prior to the experiment at 300 000 cells (HeLa Kyoto, if not mentioned otherwise) per 35 mm well; experiments were performed in HBSS (Invitrogen), 10% FCS, 20 mM Hepes (Invitrogen), pH 7. 2–7. 5 (Invitrogen). After exchange of media to HBSS, cells were incubated at 37°C, 5% CO2 and infected with the indicated S. Typhimurium strain (derivative of SL1344, [53]) carrying plasmid pCJLA-GFP at an estimated m. o. i. of 1. 5. If not stated otherwise, movies were acquired on a Leica DMI-6000B microscope, either using the differential interference contrast mode or the fluorescence mode. DIC-movies were acquired using a 63× oil objective (HCX PLAN Apochromat from Zeiss, NA 1. 4); for fluorescence imaging, a 20×-objective (HC PLAN Apochromat from Zeiss, NA 0. 75) with a 2-fold optovar was used. If not indicated otherwise, movies were acquired at 10 frames per second for 5 minutes. Movies were analyzed using the program Volocity (Improvision, UK) and the manual tracking mode. For Fig. 4, we used a Zeiss Axio Observer equipped with a spinning disc confocal head and a 100× objective (oil NA1. 4,). For suppl. Fig. S1, we used a Zeiss Axiovert 200 m inverted microscope equipped with an Ultraview confocal head (Perkin Elmer) and a krypton-argon laser (643-RYP-A01 Melles Griot, The Netherlands) and a 20× objective, 0. 75NA (Optovar 1. 6). In these experiments estimating the influence of gravity, glass-bottom culture dishes containing HBSS but no cells were inoculated with bacteria as described for the other time lapse experiments; For testing of bacterial near surface swimming on MDCK cells, cells were seeded in glass-bottom culture dishes in DMEM, supplemented with 10% FCS, grown to confluence and let polarize for five days. Infection with S. TmΔ4 was done exactly as described for HeLa cells. Movies were acquired on a Leica DMI-6000B microscope using a 20×-objective with a 1. 6 optovar. The movie was acquired for 5 min at 10 frames per second and an overlay of the movie is shown. S. Typhimurium strains were grown before infection as described [32]. E. coli Nissle was grown identically except that normal Luria-Bertani media was used. For quantification of the contact of S. Typhimurium with the cells, the interaction was divided into 4 phases: 1) landing: the time of appearance of a S. Typhimurium until interruption of the continuous downward movement, indicated by changes in focus, direction and speed. 2) stopping: episodes without changes in any direction for at least 3 frames. 3) take off: the continuous upward movement until disappearance. 4) NSS: a continuous movement in the xy-direction other than 1) or 3). S. Typhimurium that left the field of view during quantification, as well as a few cases with ambiguities were excluded. For quantification of the number of S. TmΔ4 (pGFP) within the focus depth, either at the cell surface or >100 um above the surface (“in solution”), 40 time points from 2 independent experiments were analyzed. The same time points were used for each movie analyzed and they were spaced throughout the duration of the movie. For each bacterium within focus depth at the specific time point, the entire contact time spent on the surface was analyzed. Each bacterium was thus either counted as engaged in “NSS” or “NSS and stopping”. For the quantification of bacteria swimming in solution, any non-motile bacteria were excluded. For quantification of the distance, time and speed of S. TmΔ4 (pGFP) on the glass and cell surface, 20 bacteria from 2 independent experiments were manually tracked from their first contact with the cell to their last contact with the cell (i. e. NSS quantification excluding the landing and take-off stages) or until they left the field of view. S. TmΔ4 were selected at random from those arriving in the center of the field of view. The time indicated is NSS-time only and does not include any intermittent time spent stopping at the surface. For high-resolution images, HeLa cells were seeded on glass cover slips for 24 hours and infected with S. Typhimurium carrying plasmid pM965 for constitutive gfp expression at the indicated m. o. i.. After fixation, S. Typhimurium were stained by indirect immunofluorescence using an anti-LPS antibody (Difco) and goat anti-rabbit-Cy5 (Jackson) as a secondary antibody; the actin cytoskeleton was stained by tetra methyl rhodamine isothiocyanate (TRITC) -phalloidin after 5′ permeabilization with 0. 1% Triton Tx100. Images were taken on a Zeiss Axiovert 200 m inverted microscope equipped with an Ultraview confocal head (PerkinElmer) and a krypton argon laser (643-RYP-A01, Melles Griot) using a 100× oil immersion objective (PLAN-Apochromat Zeiss with an NA of 1. 4). Stacks of 0. 2 µm were acquired; deconvolution was performed in the actin channel with the program Volocity and a calculated point spread function. 3D-reconstruction and reconstruction of zx-layers was done using Volocity. Docking experiments were done as described [9]. In brief: cells were seeded in 96-well micro-clear plates, (half size, Greiner), at 6000 cells per well 24 hours prior to the experiment and infected with the indicated S. Typhimurium strain followed by fixation, staining of nuclei and bacteria using DAPI and an anti-Salmonella antibody (Difco), respectively. Images were acquired on a MD-Image Xpress microscope (Molecular Devices) using a 4×-objective in the DAPI- and Salmonella-channel. Images were analyzed using the open source program CellProfiler [58] and customized Matlab-scripts, available upon request. For automated analysis of individual docked bacteria (Fig. 1) the analysis consisted of three major steps. First, cells and nuclei were detected using the program CellProfiler [58] and cellular properties, such as morphology, texture and intensity were extracted. Second, single bacteria were identified using the “a trous” wavelet transform [59] and every bacterium was assigned to the corresponding host cell. In a third step, mitotic cells were distinguished from interphase cells using supervised machine learning technique. For this purpose, the “Advanced Cell Classifier program” was applied and the classification was performed using the extracted cellular features with an artificial neural network classifier [60]. Finally the average amount of bacteria on mitotic and interphase cells was calculated. Docking onto mitotic cells: HeLa cells were seeded in 96-well Microclear plates (full size, Greiner) at 6000 cells per well 24 hours prior to infection. After infection and fixation, extracellular S. Typhimurium was stained using an anti-Salmonella LPS antibody and a Cy5-labelledsecondary antibody (Fig. 9). Due to the bright staining of extracellular bacteria, the unique bacterial shape and the high resolution used for quantification bacterial staining was clearly distinguishable from actin staining. For illustrative purposes (Fig. 9C), in one experiment bacteria were stained using a Cy3-labelled secondary antibody. Subsequently, nuclei and actin were stained using DAPI and TRITC-phalloidin, respectively. Finally, bound S. Typhimurium bacteria were manually quantified using a 40×-objective (EC Plan-Neofluar objective with a NA of 1. 3). Docking onto ruffles: The gfp-labeled S. Typhimurium strain and the helper strain were mixed at equal dilutions prior to infection. Staining and fixation was done as described for mitotic cells. Testing of cooperativity: S. TmSopE-carrying plasmid pM965 was incubated at the indicated concentration with HeLa cells; fixation and staining was done as described. To calculate invasion efficiency of S. Typhimurium without cooperativity, invasion was calculated at the lowest m. o. i., excluding ruffles with more than one associated S. Typhimurium. At higher m. o. i., this number was assumed to increase proportionally with the number of added S. Typhimurium. In our calculations we modeled a three-dimensional environment within a cubic space. A round sphere was placed into this space; the radius of the sphere was chosen to be 1/10 of the length of the surrounding cube. The sphere was partially submerged into the bottom surface of the cube and 75% of the height of the sphere remained within the cube. In addition, 100 single particles were modeled into the cubic space. For simplicity the size of the particles was assumed to be zero in all dimensions. The whole volume of the cube was assumed to be accessible to the particles except the interior of the sphere. All particles were assigned the same constant speed but a randomly chosen vector of movement. The positions of the particles where integrated after travelling a fraction of 1. 2*10−5 of the length of the square. Upon hitting the bottom surface, particles followed the rules according to the chosen scenario: 1) Reflection at a randomly chosen angle in the “random” scenario, where the new vector of movement was randomly chosen (“impossible” movements, for instance into the surface were excluded). 2) Reflection with an angle identical to the angle of infliction in the “billiard” scenario, the vector of “take off” thus mirroring the vector of “landing” or 3) particles started swimming along the surface in the “NSS” scenario. In this scenario the z-vector of movement was set to zero (particles thus following the surface); the x and y-direction of movement remaining unchanged. When encountering the remaining 5 limiting surfaces of the cube, particles were simply reflected. Upon hitting the limits of the sphere, particles had a 10% chance of being attached (simulating stopping/docking); otherwise particles were simply reflected. Particles hitting the sphere during NSS also stopped/docked to the sphere with a likelihood of 10%. Upon each change of direction the movement vectors were adjusted to achieve a constant overall velocity. After an identical number of calculated increments of particle movements the simulation was interrupted and screenshots were acquired. Scatter3D is implemented in C++ and has previously been used to simulate light scattering [61]. Calculations were performed on a desktop computer. Further details are available upon request. Strains S. TmΔ4 fliGHI, S. Tm-T1 fliGHI, S. Tm-T1 -Fi fliGHI were constructed by P22 transduction [62] of the tetracycline allele of SB245 (sipABCDsptP: : aphT, fliG/H: : Tn10, K. Kaniga and J. E. Galan, unpublished data) into strains S. TmΔ4, S. Tm-T1 and S. Tm-T1 -Fi, respectively. flgK was deleted in SL1344 (SB300) as described in [35]. Strains S. TmΔ4 flgK, S. Tm-T1 flgK, S. Tm-T1 -Fi flgK were constructed by P22 transduction of the chloramphenicol resistance containing the flgK deletion into the respective host strain. MotA/B were deleted in SL1344 (SB300) using the method of Datsenko and Wanner [63] by insertion of a chloramphenicol resistance cassette that was amplified using the forward primer: ATGCTTATCTTATTAGGTTACCTGGTGGTTATCGGTACAGTGTAGGCTGGAGCTGCTTC and the reverse primer: TCACCTCGGTTCCGCTTTTGGCGATGTGGGTACGCTTGCATGGGAATTAGCCATGGTCC. S. TmΔ4 motAB, S. Tm-T1 -motAB and S. Tm-T1 -Fi motAB were obtained by P22 transduction of the chloramphenicol resistance into the respective host strain. Lack of motility of the respective strains was tested on motility agar. pM2120 (expressing mCherry constitutively) was constructed by PCR amplification of the mCherry gene using forward primer: CGCGGATCCCCCGGGCTGCAGGAATTCAGGAAACAGTATTCATGGTGAGCAAGGGCGAGGAG (BamHI) and reverse primer: GGGAAGCTTGATATATCGGAATTCTTACTTGTACAGCTCGTCCATG (HindIII). Subsequently the PCR product as well as plasmid pM975 [22] was digested using BamHI/HindIII and ligated. fliG (11765205), fliH (11765206), fliI (11767468), flgK (11767368), motA (11765169), motB (11765168), cheY (11765165), sopE (11765807), sipA (11765948), sopE2 (11768039), sopB (1252609), invG (11765959), fimD (11764167).

Title: Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion Summary: The animal body is protected by physical, chemical and immunological barriers. Identification of "promising" target sites is therefore of importance for any pathogen. This crucial step of the infection is still poorly understood. Here, we have studied target site selection by the flagellated Gram-negative gut pathogen Salmonella Typhimurium. Using a well-established tissue culture model system, we found that flagella-driven motility forces the bacterium into a "near surface swimming" mode which facilitates "scanning" of the host cell surface. The near surface swimming was found to target the pathogen towards sites with particular topological features, i. e., rounded cells and membrane ruffles. This explains how S. Typhimurium "identifies" particular target sites and infects membrane ruffles in a cooperative manner. Interestingly, the near surface swimming is attributable to generic physical principles acting on moving particles. Therefore, our findings might be of general importance for the infection by motile pathogens.

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Summarize: REFERENCE TO CO-PENDING APPLICATION [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 12/480,111 entitled “Insect Protection Garment” that was filed Jun. 8, 2009 by Gary Hunt, the entire contents of which are herein incorporated by reference. OVERVIEW [0002] This invention is for protection from bothersome and biting insects normally occurring in outdoor areas. It is a one piece, self-supporting total body covering. It will be made mostly of see-through netting material impervious to insects. Most importantly it will create a large enough space, inside the netting, to allow the wearer to eat, drink and relax while doing normal activities, without having to remove the garment, thus reducing exposure to annoying and harmful insects. The netting will be well away from the face making it comfortable to wear for long periods of time. It will be easy to deploy, wear, remove and transport. FIELD OF DISCLOSURE [0003] This invention is an improvement on all other types of insect protection garments for anyone in an outdoor area where bothersome insects are present. It is designed to be easy to use and comfortable to wear. BACKGROUND OF THE INVENTION [0004] Previously users of the outdoors have had to use either a head net or a head net in combination with a full body suit to protect themselves from insects. Head nets are uncomfortable, especially if you have to wear one for hours, or all day, or day after day as is the case when working or camping outdoors. The netting is only inches from the face, which gets annoying very quickly and makes activities such as eating very difficult. To eat, the head net must either must be removed thus exposing the person to the insects, or the user can try to pull the neck elastic out far enough to get a fork full of food into the mouth, which is difficult, and also creates an opening for insects to enter, thus undermining the protective aspect of the head net. Some try to solve this problem by eating in a tent, this is not recommended because animals are attracted to food smells and bears, skunks etc may come to the tent looking for a meal. [0005] Suit type body protection must be worn over full length clothing as the netting cannot be held away from the skin. This makes suit types very uncomfortable on hot days when insects are present. They also must be used in conjunction with a head net which fails to address the problem of how to eat and perform other necessary tasks without creating an opening for insects to enter or removing the head net. [0006] Some users of the outdoors rely on chemical repellents on their skin or clothing. Recent reports on these chemicals safety on the body make many reluctant to place it on the skin or inhale the vapors given off by these harsh chemicals even when placed on clothing. Some have label warnings of chemical stains if placed on clothing, and have to be re-applied frequently to be effective. [0007] The Alquist et al, U.S. Pat. No. 5,717,990 is a hood arrangement, and fails to address the need for a larger inside area to eat, read etc. The Alquist hood also hangs very close to the face which is annoying. It also fails to address the ability to be folded into a small and more pack able size. Also the crown piece is made of mesh material that provides little shade and no rain protection. It can be raining hard enough to get a person wet, yet not deter the insects. It also appears that the reinforcing cross strips on top lay directly on the head potentially making it uncomfortable after many hours of use. The Alquist et al hood has no openings for convenience, necessitating it to be opened or removed frequently to perform simple tasks. [0008] The Merrill, U.S. Pat. No. 5,091,993 and Myers U.S. Pat. No. 4,395,781 are both suit type of garments that would be extremely hot on even moderately warm days. The face netting can be opened for eating etc, but there is nothing to prevent insects from flying inside the head piece while opened, thus undermining the protective aspect of the garment. [0009] So what is desired is a garment that is one piece, light weight, covers the whole body and has a large enough inside space so you can eat, read etc and has openings for convenience so the garment will need to be removed as little as possible. It has a top that not only blocks insects from entering, but also provides shade and rain protection and allows the user to wear shorts and tee shirt on hot days. SUMMARY [0010] This invention is intended to be worn to provide protection from insects and to be easy to deploy, wear, remove and transport. It is to be used by people who wish to engage in outdoor activities where enjoyment of such activities may be compromised by bothersome or biting insects. [0011] It can also be used by military personnel deployed in areas where biting insects are a big problem and they are faced with long exposure times and where the possibility of contracting malaria is a factor. This invention could also help the populations in poorer countries or any place where there is a danger of malaria. There are bug nets for the heads and beds but none for the body, this invention fills that need. [0012] A key of this invention is to have insert protection that can be worn over regular clothing, including light weight shirts and shorts that are normally worn outdoors when the weather is hot. Biting insects thrive in hot weather and having to wear a long sleeve shirt and long pants on 90 degree plus days is dreadful and possibly dangerous as such clothing could facilitate heat stroke. [0013] It is the object of this invention to provide a large enough inside space as to not be annoyed by netting close to the face. Also to allow a large enough inside space for activities such as eating, drinking, smoking, sewing, repairing, cleaning gear or reading a map or book or writing in a journal, without fear of biting bugs. [0014] A key of this invention is the spring steel, or plastic ring of the top to be the only support necessary for this garment. Also, the ring of the top will hold the netting well away from the face of the wearer creating a space inside impervious to bugs. Another key to this invention is that the ring on top will be sewn into the outer edge of the top material so as to allow it to be twisted and folded to approximately one third of its unfolded size to make it much easier to pack and transport. [0015] Another important aspect to this invention is the opening in the front, at approximately waist level, will have a fabric hook and loop fastener or similar closure that is large enough to pass a plate of food, a book, etc inside. The opening for men will be located where a zipper on a regular pair of men&#39;s pants is located to facilitate the use of a restroom as needed. This will also have a fabric hook and loop fastener or similar closure. [0016] The seating area, which is in back, at waist level, extending down to just above the back of the knee, will be made of a durable material such as nylon so as to reduce the possibility of wear and tear when sitting on abrasive surfaces such as rocks or logs. The opening in the seating area will also be large enough to allow the use of a restroom without removing the garment and will have a fabric hook and loop fastener or similar closure. [0017] There will also be a round piece of cotton or other soft material sewn onto the inside of the top to deny insects the ability to bite the wearer on the head. There will also be a chin strap made of elastic strap material or cloth string with a cord lock, permanently attached to the round piece of cloth material attached to the top. It will be long enough to comfortably reach under the chin of the wearer to hold the garment in place. [0018] There will be a strip of the durable nylon material at the bottom to prevent wear if dragged on the ground. There will be elastic straps sewn to the bottom strip that can be looped under the shoes to hold the garment down far enough to prevent insects from entering from the bottom. [0019] The garment can be rolled up to waist level or to shoulder or neck level depending on how much of the body the wearer wants to protect. [0020] The entire bottom of the garment will be open to allow entry into the garment and make removal easy. Also the garment can be closed at the bottom around the shoes to keep insects from getting inside. This also will allow the wearer to walk around the campsite, etc. Also, if the wearer prefers to bring the feet inside, the entire bottom of the garment can be sealed with the fabric hook and loop fastener. BRIEF DESCRIPTION OF THE DRAWINGS [0021] The above-mentioned aspects of the present invention and the manner of obtaining them will become more apparent and the invention itself will be better understood by reference to the following description of the embodiments of the invention, taken in conjunction with the accompanying drawings, wherein: [0022] FIG. 1 is the front elevation of the garment. [0023] FIG. 2 is the right elevation of the garment. [0024] FIG. 3 is the rear elevation of the garment. [0025] FIG. 4 demonstrates how a nylon strip of material should be attached to the top and the bottom of the netting material. [0026] FIG. 5 demonstrates how the spring steel or plastic hoop should be sewn into the material on the top piece of the garment. [0027] FIG. 6 illustrates the lower details of the insect protection garment. [0028] FIG. 7 illustrates a bottom view of the insect protection garment. [0029] FIG. 8 illustrates a lower perspective view of the insect protection garment. [0030] FIG. 9 illustrates a side perspective view of the insect protection garment with a portion of the mesh netting removed. [0031] FIG. 10 illustrates a front view of the insect protection garment with cross-sections at various elevations. DETAILED DESCRIPTION [0032] The embodiments of the present invention described below are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art may appreciate and understand the principles and practices of the present invention. [0033] In FIG. 1 the front elevation of the garment for the protection from insects is illustrated, it is a one piece total body covering. It can be made as a large size for adults or smaller sizes for smaller adults or children. It will be constructed mostly out of see-through insect barrier netting and nylon. The top piece 1 is made of nylon or other similar material that will provide a barrier from insects and shade and rain protection for the wearer. Sewn into the outer edge of top piece 1 is preferably a narrow spring steel hoop, but maybe a strong plastic hoop 2 that will provide support for the garment and should have the ability to be twisted and folded for packing convenience. [0034] The top piece 1 will be made of a circular piece of nylon or similar material that is commonly used to make outdoor clothing or tents. The edge will be sewn in such a way as to prevent fraying. [0035] The spring steel or plastic hoop 2 will be sewn into the outer edge of top piece 1. Top piece 1 will be folded over at the edge so the hoop 2 will be between the layers of material. Double stitching for strength is recommended. [0036] The netting 11, that extends down below the top piece 1 will be sewn to the outer edge of the top piece 1. Before sewing the netting 11 to the top piece 1, a strip of nylon 12 should be sewn to the top of the netting 11 where it will be attached to top piece 1. The strip of nylon 12 should be folded over so the top of the netting 11 will be between the layers of the folded nylon 12 and then double stitched for strength. Then the strip of nylon 12 with the netting 11 attached can be sewn to top piece 1 and double stitched for strength. The top piece 1 is tightly stretched across hoop 2 (or resilient loop) to transfer the weight of the netting 11 and hoop 2 to the head piece 8 with minimal sagging of the top piece 1. [0037] The arm openings 3 and 7 may have a permanently attached sleeve (not shown) and allows the wearer to extend the hand and arm outside of the netting to cook, operate a camera, set up a tent, etc. If a sleeve is attached to openings 3 and 7, a strip of nylon should be sewn on the end of the sleeve that will be attached to the garment and then the nylon sewn to the garment. The sleeve should be made of netting or nylon material. With or without the sleeve the opening can be closed, preferably with a fabric hook and loop fastener, or with a zipper or other types of fabric closing systems. The upper front opening 4 should be large enough to pass a plate of food or an article of clothing, etc, inside the garment or passing such items outside of the garment. This opening will be closable with a fabric hook and loop fastener or similar system. The lower front opening 5 will be for the males convenience when using the restroom and can be closed with A fabric hook and loop fastener or similar system. FIG. 3 illustrates an example of a fabric hook and fastener 17 being secured adjacent to one of convenience openings. [0038] The bottom opening 6 can be opened to a size large enough to allow the wearer to put the garment on and off. It can also be closed at the bottom with a fabric hook and loop fastener or similar system. It will have a strip of nylon sewn around the entire bottom to prevent wear. It should be sewn to netting 11 the same way as the nylon strip 12 is sewn to netting 11 in FIG. 4. Fabric hook and loop fastener can then be attached to the bottom strip of nylon so the entire bottom of the garment can be opened or closed. If the wearer wishes to pull the feet inside the garment the entire bottom of the garment can be closed with a fabric hook and loop fastener. If the wearer wishes to walk with the garment on, the bottom opening can be closed around the tops of the shoes and remain closed between the feet to allow mobility while still denying insects entry. The bottom strip may have two loops of elastic (not shown) that can be looped under the shoes to hold the garment down close to the shoes. [0039] Before a fabric hook and loop fastener is attached to the garment it should be sewn to a piece of nylon first rather than sewing a fabric hook and loop fastener directly to the garment. Then the nylon with the fabric hook and loop fastener attached can be sewn to the garment. Double stitching for strength is recommended everywhere anything is attached. [0040] The circular head piece 8 is sewn to the top piece 1 to keep insects from reaching the wearers head and as an attachment for chin strap 9. The chin strap 9 keeps the garment from sliding around and keeps the garment in place if wind is present. It can be made of an elastic material or cotton, like a shoe string with an adjustable stop so the wearer can adjust for comfort under the chin. [0041] FIG. 2 shows the garment from the right elevation, the left elevation is a mirror image of the right elevation. The circular head piece 8 is shown and should be sewn to the inside center of top piece 1 and should be large enough and thick enough to prevent insects from reaching the head when trying to bite through from the top of top piece 1. [0042] Chin strap 9 is made of either of thin elastic material or cotton material such as a shoe string that has adjustable cord lock, like the type found on stuff sacks, so the chin strap can be adjusted for comfort. It will be sewn to head piece 8 just above the ears. It will prevent the garment from sliding around and keeps the garment in place if wind is present. [0043] The arm opening 7 is shown for the right arm to extend out as needed and close with a fabric hook and loop fastener. [0044] FIG. 3 shows the rear elevation. The rear convenience flap 10 is shown. It will be a flap type or trap door type opening similar to the rear opening seen in one piece long underwear. It will have a fabric hook and loop fastener or similar closer on the left, right and bottom that can be opened for restroom use. The fabric hook and loop fastener should be sewn to the nylon flap along the left, right and bottom edges, and fabric hook and loop fastener sewn to nylon strips and then sewn to the left, right and bottom edges of the opening in the garment. The top of flap 10 will be sewn to the garment. [0045] FIG. 4 shows the way the nylon strip of material 12 is folded over netting material 11 so that the netting material 11 is sandwiched between the folded piece of nylon strip 12. Then with double stitching die folded piece of nylon strip 12 with the netting material 11 between the fold of nylon strip 12 is all sewn together. Then nylon strip 12 can be attached along the outer edge of top piece 1, double stitching is recommended. [0046] FIG. 5 demonstrates how spring steel hoop 2 is sandwiched between the folded over edge of top piece 1. The folded over edge of top piece 1 with spring steel hoop 2 between the fold of the edge of top piece 1 is all sewn together. Double stitching is recommended on both sides of spring steel hoop 2. FIG. 6 highlights the lower portions of the garment. [0047] FIG. 7 illustrates a bottom view of the garment with the head piece 8 fully circumscribed by the outer perimeter 15 of the top piece 1. Substantially all of the outer perimeter 15 of the top piece 1 is secured to the hoop 2 (also referred to as a loop). The head piece 8 is substantially separated from the outer perimeter 15 of the top piece and the hoop 2 in that there is a significant minimum separation of the head piece 8 from both the outer perimeter 15 and the hoop 2. Substantially separated is herein defined to mean having a significant minimum separation between objects. In the illustrated example, the head piece 8 is separated from the hoop by a minimum hoop distance 20 of twelve inches. In an exemplary embodiment, the diameter 22 of the top piece is at least two feet. The head piece 8 is separated from the outer perimeter 15 by a minimum perimeter distance 25 of twelve and a quarter inches. It should be understood that these distance are examples, and other distances, such as 6 inches, 9 inches, 18 inches, and 24 inches may also be used as the minimum separation distances between the head piece 8 and the outer perimeter 15 and hoop 2. [0048] FIG. 8 illustrates a lower perspective view of the garment with the netting 11 in the form of a tube 30 fully circumscribing the head piece 8. FIG. 9 illustrates a lower perspective view of an insect protection garment with a portion of the netting cut away. The netting 11 is in the shape of a cylindrical tube 30 that has a height 35 of at least three feet; however other embodiments will have a height 35 of between 5 and 7 feet. The outer perimeter 15 of the top piece 1 in the illustrated example has a length of at least six feet and the tube 30 has a similar circumference. [0049] FIG. 10 illustrates an example of a mesh tube 30 having a bottom opening 6 near the feet of a user and a top opening 40 at the top piece 1. Adjacent to the top opening the tube 30 has a first horizontal perimeter 45 that has a first length. Midway between the top opening 40 and the bottom opening 6, the tube 30 of netting 11 has a second horizontal perimeter 50 that is the same length as the first horizontal perimeter 45. Adjacent to the bottom opening 6, the tube 30 has a third horizontal perimeter 55 that has the same length as the other two horizontal perimeters. In the illustrated example, the first and second horizontal perimeters ( 45 and 50 ) are substantially circular while the third horizontal perimeter has an erratic shape due to the netting being gathered around the feet of the user. In the illustrated example, the first horizontal perimeter 45 and the second horizontal perimeter 50 are separated by a distance 60 of at least two feet. In an exemplary embodiment, the first and second horizontal perimeters are separated by at least three feet with the total height of the mesh tube being approximately six feet. In an exemplary embodiment, the top piece is constructed of a canvas material that provides shade to the user while the mesh tube is see-through and only minimally obstructs the user&#39;s vision. [0050] It should be understood that the descriptions of the invention were made as specific as possible and that changes in materials, openings, support systems and closer systems, fall within the true scope of the invention and are covered by the stated claims. [0051] While exemplary embodiments incorporating the principles of the present invention have been disclosed herein, the present invention is not limited to the disclosed embodiments. Instead, this application is intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.

Summary: A one piece, self-supporting total body covering for protection against insects to be used by campers, hikers, fisherman, hunters or by professionals who work in the outdoors such as scientists and photographers. The garment will be mostly made of a see-through material impervious to insects. What is new and different about this garment is that it will create and area inside the netting large enough to eat, read, etc, and keep the user protected from insects and comfortable for long periods of time. It will be easy to deploy, wear, remove and transport.

big_patent

en

Summarize: FIELD OF THE INVENTION This invention relates to an improved method for preventing and treating idiopathic inflammatory bowel disease (IIBD), including Crohn&#39;s Disease and Ulcerative Colitis in human patients by sterilizing the intestinal tract by the use of multiple antibiotics, and the use of select, well-characterized intestinal bacteria taken from normal humans to replace the patient&#39;s intestinal bacteria. DESCRIPTION OF THE PRIOR ART Idiopathic Inflammatory Bowel Disease (IIBD) is a disease of unknown etiology, manifested by destruction of the wall of the intestine, and an abnormal and excessive immune response to a person&#39;s own intestinal cell molecules. Until now, all therapies of this disease have been directed toward suppression of the general immune response, often with poor success, and always with little or no selectivity, and with unwanted and sometimes dangerous consequential side effects. The original defect is thought to be genetic, but penetrance of the abnormal gene or genes is believed to be caused by phenomena occurring inside the lumen of the intestine. Every person normally has numerous genera and many more species as well as particular strains of bacteria in his/her intestinal tract. These strains are called the indigenous microflora. Their purpose is not well understood. One theory of the etiology of IIBD is that the immune system may be unusually stimulated by one or another strain of one&#39;s own intestinal indigenous bacterial microflora, and that the configuration of those antigenic bacterial molecules may be similar to the configuration of that person&#39;s own cells&#39; molecules (molecular mimicry). As a result, there would be a &#34;cross-reactivity&#34;, with the immune system attacking and destroying the person&#39;s own similarly-shaped cellular protein and/or glycoprotein molecules. A second theory is that toxic metabolic products are produced by one&#39;s indigenous microflora which alter or destroy one&#39;s intestinal cells. A third theory is that some of the natural intestinal digestive secretions coming from the liver and pancreas are not properly neutralized by one&#39;s indigenous microflora, so the intestinal epithelium continues to be exposed to their erosive and digestive process. We believe that by suppressing or killing all, or the vast majority, of the indigenous microflora of the intestinal tract, and replacing them (a &#34;re-florestation&#34;) with a selection of known, specific, non-pathogenic bacteria, there will be subtle, but significant changes in the composition of the indigenous microflora. Changes in the metabolic pathways, processes, products and functions of fats, proteins, carbohydrates, and of intestinal secretions will then follow. In addition, the new bacteria will also offer different molecular configurations to the immune receptors. With the absence or change of immune stimulation, the destructive process will stop, and there will no longer be pathological inflammation of the intestinal wall. SUMMARY OF THE INVENTION The invention is directed to a method of preventing and treating IIBD in humans, comprising replacing the former (presumed pathologically antigenic) bacterial flora with selected, new, non-pathogenic human intestinal bacteria. There should be a normalization of the immune responses, and a cessation of inflammation. Compared with other methods to reduce or stop inflammation, this method is non-toxic to the human or animal, and offers the hope of completely stopping inflammation at the molecular level. DETAILED DESCRIPTION OF THE INVENTION In order to attempt to sterilize the intestinal wall and intestinal lumenal contents, concurrent administration of several antibiotics is necessary. Several combinations of antibiotics may be used. Vancomycin, metronidazole, an aminoglycoside (such as gentamycin), a monobactam (such as aztreonam), and an anti-fungal agent (such as nystatin); or a third generation cephalosporin (such as cefaperzone), nystatin metronidazole, ampicillin-sulbactam (or ampicillin-clavulanate), must be used for 72 to 120 hours (days 2 through days 4, 5, or 6) depending on the severity of the inflammation and infection, given orally or parenterally. All antibiotics are given in standard doses according to weight, age, or surface area of the patient. Prior to antibiotic administration, the patient&#39;s room (not in a hospital, to avoid possible contamination with antibiotic-resistant bacteria) is disinfected by using any standard bactericidal agent. Floors, walls, window sills, table, chairs, etc. are swabbed with this agent each 24 hours (from day 1 through day 10). Bedsheets and blankets are washed just prior to use. Air entering the room is filtered with a HEPA-type filter system, although some fresh air is permitted. Vents should be closed if their contained air comes from another part of the building thought to harbor potential pathogenic bacteria. The intestine is cleaned, using any standard cathartic or laxative (during day 1). Magnesium citrate or &#34;Go-LYTELY&#34; are two examples. Enemas are not given. Oral rinses of a germicidal solution, such as chlorhexidine gluconate are given every 2 hours (staring after the cathartics, on day 1 through days 11, 12, or 13). Teeth are brushed 4 times a day, using brushes sterilized by microwave heating before each usage, (without tooth paste, followed by chlorhexidine rinses). Facial cleansing is done 4 times a day with ordinary soap and water. On day 1, after the bowel has been emptied from the laxatives, a germicidal soap shower is taken, including hair wash. Clean masks, gowns, and caps are worn by all personnel in contact with the patient or supplies, from day 1 through days 11, 12 or 13. Clear, sterile liquids (such as soda pop, water, and elemental preparations, such as Vivonex) are given, starting from day 1 (after bowel evacuation) through days 4, 5 or 6. Diet is increased to sterile, full liquids on days 4, 5 or 6 (beginning 6 hours after the last antibiotic dosage), for 24 hours. Thereafter, diet consists of solid, sterile, normal food. Sterilization of food is achieved by microwaving, or boiling, or frying, or broiling, etc., at temperatures greater than 212 degrees F. for a least 5 minutes. Some easily sterilized foods are: hamburgers, potatoes, cooked vegetables, rice, spaghetti, and soups. Sterile solid diet is continued until day 15, 16, or 17. Bacteria are obtained and maintained in freeze-dried (lyophilized) form, or frozen in liquid nitrogen, or in suitable culture media. The anaerobic bacteria require expert handling to avoid contamination from opportunistic bacteria to avoid damage from oxygen. Suitable sterile, commercially available oxygen-free solid or liquid culture media tubes or bottles are used to maintain the mixture of anaerobes. Aerobes are maintained with the same precision, in suitable sterile, solid or liquid, culture media tubes or bottles (for example, tryptocase soy broth) containing normal air. Materials: Specific strains of the following genera and species of bacteria will be used to accomplish the &#34;re-florestation&#34;: Bacteroides vulgatus, distasonis, stercoris, ovatus, caccae, and uniformis; Bifidobactertum adolescentis, longum, and bifidum; Eubacterium aerofaciens, rectale; Fusobacterium prausnitzii; Ruminococcus obeum, bromii, gnavus; Lactobacillus acidophilus, leichmannii; Streptococcus oralis; Escherichia Coli (Pingel, Nissl 1917, and other strains). After ingestion of 1/2 to 1 teaspoon of sodium bicarbonate (heat treated/new box carefully opened) to neutralize stomach acid, at least approximately 10 to the 8th power of each bacteria in the mixture, in isotonic saline, is swallowed (separately or together) 3 times per day for 1 week (days 4, 5, or 6 through 11, 12, or 13). Rectal infusions of the same bacteria are given twice per day for 5 days. Sterile gloves are used to manipulate a sterile catheter and/or syringe for the rectal insertions. The anus is first swabbed with a germicidal preparation (such as povidone-iodine, 10%). The patient is placed upside down, over a chair, and tilted side to side for 30 minutes, so that gravity will disperse the infusion as proximally as possible. Approximately 100 cc of sterile isotonic saline is used to suspend the infusion mixture of bacteria. The patient&#39;s general medical condition, blood pressure, pulse, temperature, is monitored throughout the procedure, by appropriately trained and motivated staff. Four days after stoppage of the antibiotics, the patient is allowed to leave his or her room. All medicine taken previously for this IIBD procedure are allowed to be gradually withdrawn over a period of time, being determined by the clinical picture. Maintenance doses of the same bacteria should be taken once per week by mouth for an unlimited length of time

Summary: A new method for the prevention and treatment of Idiopathic inflammatory Bowel Disease (IIBD), including Crohn&#39;s Disease and Ulcerative Colitis, in human patients is provided. The key steps include sterilizing the intestinal tract with multiple antibiotics to kill the pre-existing bacterial flora, and replacing the latter with different, select, well-characterized bacteria taken from normal humans. The new microflora serve to generate more normal metabolic and immune responses, remitting thereby the IIBD.

big_patent

en

Summarize: In the fight against obesity, counting up the calories in every chip, biscuit or stick of celery can be a faff too far. However, a US company claims to have developed a smart wristband that does the adding up for you - by measuring changes in the glucose levels and liquid in the skin cells. The HealBe GoBe wristband is the latest twist in the wearable technology revolution, most of which is built on a theme of improving health. Scroll down for video. The HealBe GoBe wristband (pictured) was designed by a team of Russian developers and product managers led by Artem Shipitsyn and George Mikaberydze. The wristband uses an algorithm that analyses data collected by three sensors, designed to monitor changes in the body through the skin. It was designed by a team of Russian-based developers and product managers led by Artem Shipitsyn and George Mikaberydze. The wristband uses an algorithm that analyses data collected by three sensors. These sensors monitor changes in the body through the skin as people go through their day, walking, eating, sitting and sleeping. The wristband uses an algorithm that analyses data collected by three sensors. These sensors monitor changes in the body through the skin as people go through their day, walking, eating, sitting and sleeping. There is a pulse sensor which measures blood flow and heart rate, plus an accelerometer which measures body movement and activity, such as the number of steps taken. The third is called a bioimpedance sensor, which the company claims can give a good measure calorie intake. The theory is that when people eat, carbohydrates are turned into glucose or sugar in their blood. Cells absorb the glucose and use it for energy, displacing liquid in the process. The bioimpedance sensor measures this change using patented software to translate this into the number of calories consumed, which are shown on the band’s screen. There is a pulse sensor which measures blood flow and heart rate, plus an accelerometer which measures body movement and activity, such as the number of steps taken. The third is called a bioimpedance sensor, which the company claims can measure calorie intake. The theory is that when people eat, carbohydrates are turned into glucose or sugar in their blood. Cells absorb the glucose and use it for energy, displacing liquid in the process. The bioimpedance sensor measures this change using patented software to translate this into the number of calories consumed, which are shown on the band’s screen. As well as coming up with a figure for calorie intake, the other information collected by the sensors gives a total for calories burned, which allows the wearer to see whether they have done enough to lose weight during the day. The information on the pulse is used to deliver a stress level measure, while the device can also measure whether you have slept soundly or been tossing and turning through the night. While the theory sounds remarkable and useful, there are questions about whether there is sound science to back up the calorie count claims. The device initially launched on Indiegogo, and raised more than $1 million dollars - 10 times its target. Following the launch of the campaign, doctors and nutritionists disputed the claims made by Mr Shipitsyn, saying that even if the sensors could accurately measure the energy changes, it wouldn't be a reliable guide to calorie intake. At this week's Consumer Electronics Show, BBC reporter Leo Kelion was given the opportunity to test the company's claims. The built-in pulse sensor measures blood flow and heart rate, plus an accelerometer measures activity. The bioimpedance sensor measures calories. The theory is that when people eat, carbohydrates turn into glucose. Cells absorb glucose and use it for energy, displacing liquid in the process. The bioimpedance sensor measures changes in glucose using patented software to translate the levels into the number of calories consumed. This is then shown on the band’s screen and app (pictured). The other information collected by the sensors gives a total for calories burned, letting users compare the figures. During the test, Mr Kelion ate half a cheese and turkey sandwich (125 calories), a small Snickers chocolate bar (220 calories), and drank a bottle of apple juice (210 calories), giving a total of 555. The wristband started adding up the calories after 15 minutes and following an experiment lasting two and half hours, it came up with a figure of 515 calories. The US manufacturer, HealBe, said the science behind the wristband has been verified by studies. An independent report, published in September, gave an accuracy rating of 84 per cent to 93 per cent - but only involved five volunteers. If that error margin is correct, the wristband, which will go on sale through the company’s website later this year with a $299.99 (£196 plus international postage), may be of limited use for calorie counting. The device initially launched on Indiegogo, and raised more than $1 million dollars, but was soon criticised by doctors and nutritionists. While the theory sounds remarkable and useful, there are questions about whether there is sound science to back up the calorie count claims. The device initially launched on Indiegogo, and raised more than $1 million dollars - 10 times its target. Following the launch of the campaign, doctors and nutritionists disputed the claims made by Mr Shipitsyn and his team, saying that even if the sensors could accurately measure the energy changes, it wouldn't be a reliable guide to calorie intake. The US manufacturer, HealBe, said the science behind the wristband has been verified by studies. An independent report, published in September, gave an accuracy rating of 84 per cent to 93 per cent - but only involved five volunteers. At this week's Consumer Electronics Show, BBC reporter Leo Kelion was given the opportunity to test the company's claims. During the test, Mr Kelion ate half a cheese and turkey sandwich (125 calories), a small Snickers chocolate bar (220 calories), and drank a bottle of apple juice (210 calories), giving a total of 555. The wristband started adding up the calories after 15 minutes and following an experiment lasting two and half hours, it came up with a figure of 515 calories

Summary: HealBe GoBe wristband was designed by a team of Russian developers. Wristband uses an algorithm that analyses data collected by three sensors. The theory is that when we eat, carbohydrates are turned into glucose. Cells absorb glucose and use it for energy, displacing liquid in the process. GoBe's sensors track these changes through the skin, and translate it into calorie intake and output. But doctors have disputed the claims, saying the technology isn't reliable.

cnn_dailymail

en

Summarize: CROSS-REFERENCE TO RELATED DOCUMENTS This application is a divisional of U.S. patent application Ser. No. 11/442,371 filed May 26, 2006 entitled Intravascular Cuff, which is related to and claims priority benefit of U.S. Provisional Patent Application Ser. No. 60/685,433, filed May 27, 2005, entitled Intravascular Cuff, both of which are hereby incorporated by reference herein in their entireties. This application also incorporates by reference U.S. patent application Ser. No. 11/443,814 filed May 30, 2006 entitled Stentless Support Structure; U.S. Provisional Patent Application Ser. No. 60/685,349, filed May 27, 2005, entitled Stentless Support Structure; and U.S. Provisional Patent Application Ser. No. 60/709,595, filed Aug. 18, 2005, entitled Stentless Support Structure. BACKGROUND OF THE INVENTION There has been a significant movement toward developing and performing cardiac and other surgeries using a percutaneous approach. Through the use of one or more catheters that are introduced through, for example, the femoral artery, tools and devices can be delivered to a desired area in the cardiovascular system to perform any number of complicated procedures that normally otherwise require an invasive surgical procedure. Such approaches greatly reduce the trauma endured by the patient and can significantly reduce recovery periods. The percutaneous approach is particularly attractive as an alternative to performing open-heart surgery. Valve replacement surgery provides one example of an area where percutaneous solutions are being developed. A number of diseases result in a thickening, and subsequent immobility or reduced mobility, of valve leaflets. Valve immobility leads to a narrowing, or stenosis, of the passageway through the valve. The increased resistance to blood flow that a stenosed valve presents eventually leads to heart failure and death. Treating severe valve stenosis or regurgitation has heretofore involved complete removal of the existing native valve followed by the implantation of a prosthetic valve. Naturally, this is a heavily invasive procedure and inflicts great trauma on the body leading usually to great discomfort and considerable recovery time. It is also a sophisticated procedure that requires great expertise and talent to perform. Historically, such valve replacement surgery has been performed using traditional open-heart surgery where the chest is opened, the heart stopped, the patient placed on cardiopulmonary bypass, the native valve excised and the replacement valve attached. A proposed percutaneous valve replacement alternative method is disclosed in U.S. Pat. No. 6,168,614 (the entire contents of which are hereby incorporated by reference) issued to Andersen et al. In this patent, the prosthetic valve is collapsed to a size that fits within a catheter. The catheter is then inserted into the patient&#39;s vasculature and moved so as to position the collapsed valve at the location of the native valve. A deployment mechanism is activated that expands the replacement valve against the walls of the body lumen. The expansion force pushes the leaflets of the existing native valve against the lumen wall thus essentially “excising” the native valve for all intents and purposes. The expanded structure, which includes a stent configured to have a valve shape with valve leaflet supports, is then released from the catheter and begins to take on the function of the native valve. As a result, a full valve replacement has been achieved but at a significantly reduced physical impact to the patient. However, this approach has decided shortcomings. One particular drawback with the percutaneous approach disclosed in the Andersen &#39;614 patent is the difficulty in preventing leakage around the perimeter of the new valve after implantation. As the tissue of the native valve remains within the lumen, there is a strong likelihood that the commissural junctions and fusion points of the valve tissue (as pushed against the lumen wall) will make sealing of the prosthetic valve around the interface between the lumen and the prosthetic valve difficult. Other drawbacks of the Andersen &#39;614 approach pertain to its reliance on stents as support scaffolding for the prosthetic valve. First, stents can create emboli when they expand. Second, stents are typically not effective at trapping the emboli they dislodge, either during or after deployment. Third, stents do not typically conform to the features of the native lumen in which they are placed, making a prosthetic valve housed within a stent subject to paravalvular leakage. Fourth, stents can be hard to center within a lumen. As to the first drawback, stents usually fall into one of two categories: self-expanding stents and expandable stents. Self-expanding stents are compressed when loaded into a catheter and expand to their original, non-compressed size when released from the catheter. Balloon expandable stents are loaded into a catheter in a compressed but relaxed state. A balloon is placed within the stent. Upon deployment, the catheter is retracted and the balloon inflated, thereby expanding the stent to a desired size. Both of these stent types exhibit significant force upon expansion. The force is usually strong enough to crack or pop thrombosis, thereby causing pieces of atherosclerotic plaque to dislodge and become emboli. If the stent is being implanted to treat a stenosed vessel, a certain degree of such expansion is desirable. However, if the stent is merely being implanted to displace native valves, less force may be desirable to reduce the chance of creating emboli. As to the second drawback, if emboli are created, expanded stents usually have members that are too spaced apart to be effective to trap any dislodged material. Often, secondary precautions must be taken including the use of nets and irrigation ports. The third drawback is due to the relative inflexibility of stents. Stents rely on the elastic nature of the native vessel to conform around the stent. Stents used to open a restricted vessel do not require a seal between the vessel and the stent. However, when using a stent to displace native valves and house a prosthetic valve, a seal between the stent and the vessel is necessary to prevent paravalvular leakage. Due to the non-conforming nature of stents, this seal is hard to achieve, especially when displacing stenosed valve leaflets. The fourth drawback is that stents can be hard to center within a lumen. Stenosed valves can have very irregular shapes. When placing a stent within an irregularly shaped, calcified valve, the delivery catheter can become misaligned causing the stent to be delivered to an off-center location, such as between two calcified valve leaflets. Expanding the stent in such a location can result in poor seating against the lumen walls and significant paravalvular leakage or a non-functioning prosthetic valve. BRIEF SUMMARY OF THE INVENTION The present invention addresses the aforementioned drawbacks by providing a tubular or toroidal cuff that surrounds a native valve and creates an ideal implantation site for a stent. The cuff is constructed of at least one fine braided strand of a material having super-elastic or shape memory characteristics, such as Nitinol. The cuff is tubular when in an extended configuration within a delivery catheter. When released from the delivery catheter, the ends of the cuff curl back on themselves, trapping the native valve leaflets between the curled ends. The center of the cuff does not expand as much as the ends, thereby leaving a reduced diameter lumen that is ideal for receiving an intravascular device. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a side view of a preferred device of the present invention; FIG. 2 is an end view of the device of FIG. 1 ; FIGS. 3-7 are cutaway views of a device of the present invention being deployed in a native vessel; FIG. 8 is a side view of a preferred device of the present invention; FIG. 9 is an end view of the device of FIG. 1 in an expanded state; and, FIG. 10 is a side view of the device of FIG. 1 in an expanded state. DETAILED DESCRIPTION OF THE INVENTION Referring now to the Figures and first to FIGS. 1 and 2, there is shown an intravascular cuff 10 of the present invention. The cuff 10 is shown in its relaxed, expanded configuration and comprises a generally tubular structure having two flared ends 12 and 13 and a narrow tubular body 14. The elongated tube that is used to construct the cuff 10 is formed from at least one braided strand capable of exhibiting super-elasticity or shape memory. In one embodiment, the elongated tube is folded in half upon itself such that the first end 12 becomes a folded end and the second end 13 includes a plurality of unbraided strands. The tubular body is thus two-ply. The strand or strands may be fibrous, non-fibrous, multifilament, or monofilament. Nitinol is an example of a preferable material for the strand(s). The strand(s) are braided to allow the device to be expanded longitudinally into a very long, thin tube capable of being placed in a very small delivery catheter. Preferably, the cuff 10 can be inserted into a delivery catheter that is sized 16 Fr or smaller. The braids are tight enough to catch emboli that may be dislodged from a lumen wall, while still allowing the thin, elongated configuration. The cuff 10 includes a central lumen 16, which extends through the entire cuff 10. The central lumen 16 is sized to receive a delivery catheter for a prosthetic device such as a stent. Preferably, the lumen 16 has ends that flare or mushroom gently, thereby creating a funnel for guiding a delivery catheter into the center of the lumen 16. FIGS. 1 and 2 show that, even when the cuff 10 is in a radially expanded, relaxed configuration, the lumen 16 is small and very well defined. Thus, when deployed, the mushroom-like ends 12 and 13 expand and conform to the shape of the target vessel lumen while the cuff lumen 16 remains well defined and relatively centered within the cuff 10. Thus, the cuff 10 presents an ideal target and guide for a physician placing a prosthetic valve or stent within the cuff. Preferably, the cuff 10 is radiopaque, making the target it presents even more accessible. The deployment of the cuff 10 is illustrated in FIGS. 3-7. Beginning with FIG. 3, a cuff 10 is percutaneously delivered to a targeted stenosed valve 18 via a delivery catheter 20. The catheter 20 is advanced until a distal end 22 of the catheter is past the targeted valve 18. As seen in FIG. 4, the delivery catheter 20 is then retracted relative to the cuff 10. Doing so releases the distal end 12, which immediately flares outwardly. With the end 12 in contact with the vessel walls, the physician may pull gently on the catheter 20 and the cuff 10 to abut the end 12 of the cuff 10 against the stenosed valve 18, thereby ensuring proper placement of the cuff 10. Next, as shown in FIG. 5, the catheter 20 is retracted fully, allowing the proximal end 13 of the cuff 10 to expand against the vessel walls on a proximal side of the stenosed valve 18. The stenosed valve 18 is now completely encased in the braided mesh of the cuff 10, and the cuff is ready to receive a prosthetic device such as a stented prosthetic valve 24 ( FIGS. 6 and 7 ). Notably, despite the irregular shape of the stenosed valve 18, the central lumen 16 of the cuff presents a path through the targeted site and provides an ideal receiving seat for the prosthetic valve 24. In FIG. 6, the stented prosthetic valve 26 is percutaneously delivered to the cuff 10 via a catheter 28. The catheter 28 is inserted directly into the cuff lumen 16, using the funneled end 13 as a guide. In FIG. 7, the prosthetic valve 26 is expanded and the catheter 28 removed. Expanding the stented prosthetic 26 necessarily expands the central lumen 16. Doing so causes the cuff 10 to shorten and the flared ends 12 and 13 to fold back further, placing a more secure grip on the stenosed valve 18. Furthermore, any plaque or other material dislodged during the expansion of the prosthetic 26 is trapped by the ends 12 and 13. The cuff 10 provides an optimal seat for the prosthetic 26 and prevents any blood from leaking around the prosthetic valve 26. Over time, the braided strand(s) promote ingrowth, further improving the seal provided by the cuff 10. One embodiment of the present invention uses a non-woven fabric to further enhance the seal created between the cuff 10 and the vessel walls. FIG. 8 shows a cuff 10 having a two-ply body with a material 32 trapped between the two layers. The non-woven fabric expands easily such that the expansion characteristics of the cuff 10 are not affected. Additionally, the material 32 may be impregnated with a therapeutic compound. The material 32 can consist of a non-woven material, a woven fabric, a polymer or other material. Referring now to FIGS. 9 and 10, the cuff 10 originally depicted in FIGS. 1 and 2 is shown with a plug 30 expanding the central lumen 16 of the cuff 10. This demonstrates how the cuff 10 shortens and the ends 12 and 13 fold back when the lumen 16 is expanded. Expanding the central lumen 16 thus causes the ends 12 and 13 to create a strong grip on native tissues, such as valve leaflets, lodged between the ends 12 and 13. In another embodiment, on deployment from a catheter, the ends of the elongate tube roll outwardly toward the middle of the device. Alternatively, the end can roll inwardly toward the middle of the device. This action would be facilitated by use of a super-elastic or shape memory material such as Nitinol. Although the invention has been described in terms of particular embodiments and applications, one of ordinary skill in the art, in light of this teaching, can generate additional embodiments and modifications without departing from the spirit of or exceeding the scope of the claimed invention. Accordingly, it is to be understood that the drawings and descriptions herein are proffered by way of example to facilitate comprehension of the invention and should not be construed to limit the scope thereof.

Summary: An intravascular cuff acts as a lining between a native vessel and an intravascular prosthetic device. During deployment, the ends of the cuff curl back upon themselves and are capable of trapping native tissue, such as valve leaflet tissue, between the ends. The cuff creates a seal between the vessel and the prosthetic, thereby preventing leakage around the prosthetic. The cuff also traps any embolic material dislodged from the vessel during expansion of the prosthetic.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Holocaust Victims Insurance Act''. SEC. 2. STATUS OF POLICIES. (a) Report.--Any person engaged in the business of insurance in the United States directly or through a related company during the period 1920 to 1945 shall, within 90 days of the enactment of this Act, report to the Secretary of the Commerce on-- (1) the number of insurance policies issued during such period; (2) the holder, beneficiary, and current status of those policies; (3) the attempts made by such person to locate the beneficiaries of such policies for which no claim of benefits has been made; (4) a comparison of the names of the holders and beneficiaries of such policies and the names of the victims of the Holocaust; (5) the reason such person is unable, after reasonable and good faith efforts, to provide information described in paragraphs (1) through (4); and (6) any money contributed to a fund established to compensate victims of the Holocaust as discussed in section 3(b) of this Act. (b) Penalty.--Any person who does not file a report as required by subsection (a) shall be subject to a civil penalty of $1,000 for each day such a report is not filed as required. (c) Transmission of Report.--The Secretary of Commerce shall immediately upon receipt transmit the report required by subsection (a) to the Committees on Commerce of the House of Representatives and the Senate. SEC. 3. PAYMENT OF PROCEEDS OF INSURANCE. (a) Payments.--If such a person engaged in the business of insurance determines that it issued insurance policies to individuals who were victims of the Holocaust, such person shall pay to the beneficiaries or descendants of the victims the proceeds of such policies. (b) Fund.--Any such person who does not have records of the individuals to whom it issued life insurance policies during the period 1920 to 1945 is strongly encouraged to establish a substantial monetary fund to compensate victims of the Holocaust. Such fund should be derived from sources including the policies for which there is no one entitled to its proceeds because the holder and all beneficiaries perished in the Holocaust. SEC. 4. REPORT. The Secretary of Commerce, in consultation with State insurance commissioners, shall, within 180 days of the enactment of this Act, report to the Committees on Commerce of the House of Representatives and the Senate, to the extent possible, on-- (1) the number of individuals who were victims of the Holocaust who held life insurance policies during the period 1920 to 1945; (2) the number of such individuals for whom payment was made under such policies and the amount of such payments; (3) the number of such policies for which no payment has been made; (4) the number of such policies for which payment was made to persons other than the policy holder or their beneficiaries; (5) the names of the companies which issued such policies; and (6) the relationship (if any) between such companies and the governments of Germany, Italy, Austria, Croatia, Vichy France, Hungary, Romania, Switzerland, and other provisional governments in Nazi occupied countries. SEC. 5. REGISTRY. The United States Holocaust Memorial Museum shall develop a registry of those who died in the Holocaust which shall be made available to the general public, including insurance companies preparing a report under section 2(a). Such sums as may be necessary are authorized to be appropriated for this purpose. SEC. 6. STATUTE OF LIMITATIONS. Any action brought by Holocaust victim or heir or beneficiary of a Holocaust victim, seeking proceeds of such policies issued or in effect between 1920 and 1945 shall not be dismissed for failure to comply with the applicable statute of limitations or laches provided the action is commenced on or before December 31, 2010. SEC. 7. DEFINITIONS. For the purpose of this Act: (1) Related company.--The term ``related company'' means any parent, subsidiaries, or affiliated companies at least 50 percent of whose stock is in common ownership with an insurance carrier doing business in the United States. (2) Victim of the holocaust.--The term ``victim of the Holocaust'' means any person who lost his or her life or property as a result of discriminatory laws, policies, or actions targeted against discrete groups of persons between April 1933 and May 1945 in Nazi Germany, areas occupied by Nazi Germany, and or countries allied with Nazi Germany. (3) Insurance policies.--The term `insurance policies'' means, but is not limited to, life insurance, property insurance, dowry, or education policies. (4) Proceeds of such policies.--The term ``proceeds of such policies'' means the face or other payout value of policies and annuities plus reasonable interest to date of payment without diminution for wartime or immediate postwar currency devaluation.

Title: Holocaust Victims Insurance Act Summary: Holocaust Victims Insurance Act - Requires any person engaged in the insurance business in the United States directly or through a related company during the period 1920 to 1945 to report to the Secretary of Commerce within 90 days of the enactment of this Act on the current status of the insurance policies issued during such period. Imposes a civil penalty upon any person who does not file a report. Directs the Secretary to immediately upon receipt transmit such reports to the House and Senate Committees on Commerce. Requires a person engaged in the insurance business that determines that it issued insurance policies to individuals who were Holocaust victims to pay to the beneficiaries or descendants of the victims the proceeds of such policies. Encourages strongly any such person who does not have records of the individuals to whom it issued life insurance policies during the period 1920 to 1945 to establish a substantial monetary fund to compensate Holocaust victims. Requires the Secretary to report to the House and Senate Committees on Commerce on the number of individuals who were Holocaust victims who held life insurance policies during the period 1920 to 1945. Directs the U.S. Holocaust Memorial Museum to develop a registry of those who died in the Holocaust which shall be made available to the public, including insurance companies preparing a report under this Act. Authorizes appropriations. Prohibits any action brought by a Holocaust victim or heir or beneficiary of a Holocaust victim seeking proceeds of such policies issued or in effect between 1920 and 1945 from being dismissed for failure to comply with the applicable statute of limitations or laches provided the action is commenced on or before December 31, 2010.

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Summarize: By. James Rush. Police investigating the death of a father-of-two found dead in a chest freezer at home are examining the possibility he may have fallen in and frozen to death. Architect Colin Machin, who was 50, was found dead at his home in Alva, Clackmannanshire, last week, but police do not believe there is a criminal reason for his death. Instead they are hoping that a post-mortem - expected to be carried out this week - will shed some light on whether Mr Machin froze to death or died of other causes. A spokesman for Police Scotland said: 'We have got to keep an open mind about everything now.' Tributes have been paid to father-of-two Colin Machin, right, who was found dead in a freezer at the home he shared with his wife Maria, to his left. Also in the picture are their daughters Sinead, left, and Erin, right. Mr Machin was found at his home in Alva, Clackmannanshire, last week. Dundee University Graduate Mr Machin, who ran architecture firms Machin, Dunn and Macfarlane and Machin Associates, and ran Boll Holiday Cottages with his wife, Maria, 51, was a popular figure locally. Friends and family have now paid tribute to the architect, who leaves daughters Sinead, 23, and Erin, 21, online. Mr Machin's younger daughter Erin changed her profile picture on Facebook to a photo of her and her father, and posted lyrics from the song Caledonia, saying: 'Let me tell you that I love you. And I think about you all the time.' Friend Kevin Hogg wrote: 'Such a great guy with a huge heart. Such a privilege having being a friend, so sad to have have lost a great friend.' Margaret Paterson added: 'There are not enough words to say how sad Jock and I are to hear about Colin. 'A wonderful person and loyal friend whom I will miss very much. My love to you all.' Police are treating the death as 'unexplained'. It is understood officers are working on a theory that no crime was involved in his death. Another friend wrote: 'I did not want to believe what I was hearing, just heartbroken for you all. 'Colin will be so missed by everyone who knew him.' Pauline Fisher told Erin: 'So so sorry to hear about your dad. He was a great friend and so much fun to be around. We will miss him. All our love to you.'

Summary: Colin Machin was found in the freezer at his home in Alva, Clackmannanshire. Police treating the father-of-two's death as 'unexplained', but not criminal. They hope a post-mortem will shed more light on how the tragedy occurred. Daughter Erin, 21, changed Facebook picture to photo of her with her father. Friend Kevin Hogg described Mr Machin as 'a great guy with a huge heart'

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Summarize: Background Medicare FFS consists of Part A, hospital insurance, which covers inpatient stays, care in skilled nursing facilities, hospice care, and some home health care; and Part B, which covers certain physician visits, outpatient hospital treatments, and laboratory services, among other services. Most persons aged 65 and older, certain individuals with disabilities, and most individuals with end-stage renal disease are eligible to receive coverage for Part A services at no premium. Individuals eligible for Part A can also enroll in Part B, although they are charged a Part B premium. MA plans are required to provide benefits that are covered under the Medicare FFS program. Most Medicare beneficiaries who are eligible for Medicare FFS can choose to enroll in the MA program, operated through Medicare Part C, instead of Medicare FFS. All Medicare beneficiaries, regardless of their source of coverage, can choose to receive outpatient prescription drug coverage through Medicare Part D. Beneficiaries in both Medicare FFS and MA face cost-sharing requirements for medical services. In Medicare FFS, cost sharing includes a Part A and a Part B deductible, the amount beneficiaries must pay for services before Medicare FFS begins to pay. Medicare FFS cost sharing also includes coinsurance—a percentage payment for a given service that a beneficiary must pay, and copayments—a standard amount a beneficiary must pay for a medical service. Medicare allows MA plans to have cost-sharing requirements that are different from Medicare FFS’s cost-sharing requirements, although an MA plan cannot require overall projected average cost sharing that exceeds what beneficiaries would be expected to pay under Medicare FFS. MA plans are permitted to establish dollar limits on the amount a beneficiary spends on cost sharing in a year of coverage, although Medicare FFS has no total cost-sharing limit. MA plans can use both out-of-pocket maximums, limits that can apply to all services but can exclude certain service categories, and service-specific maximums, which are limits that apply to a single service category. These limits help provide financial protection to beneficiaries who might otherwise have high cost- sharing expenses. MA Plans Projected They Would Allocate Most of the Rebates to Beneficiaries in the Form of Reduced Cost Sharing and Reduced Premiums MA plans projected that, on average, they would allocate most of the rebates to beneficiaries as reduced cost sharing and reduced premiums for Part B services, Part D services, or both. In 2007, almost all MA plans in our study (1,874 of the 2,055 plans, or 91 percent) received a rebate payment from Medicare that averaged $87 PMPM. MA plans projected they would allocate 69 percent of the rebate ($61 PMPM) to reduced cost sharing and 20 percent ($17 PMPM) to reduced premiums. MA plans projected they would allocate relatively little of the rebates (11 percent or $10 PMPM) to additional benefits that are not covered under Medicare FFS. (See fig. 1.) On average, for plans that provided detailed cost estimates, the projected dollar amounts of the common additional benefits ranged from a low of $0.11 PMPM for international outpatient emergency services to $4 PMPM for dental services. Additional benefits commonly offered included dental services, health education services, and hearing services. About 41 percent of beneficiaries, or 2.3 million people, were enrolled in an MA plan that also charged additional premiums to pay for additional benefits, reduced cost sharing, or a combination of the two. The average additional premium charged was $58 PMPM. Based on plans’ projections, we estimated that about 77 percent of the additional benefits and reduction in beneficiary cost sharing was funded by rebates, with the remainder being funded by additional beneficiary premiums. MA Plans Projected that MA Beneficiaries, on Average, Would Have Lower Cost Sharing than if They Were in Medicare FFS, but Some MA Beneficiaries Could Pay More For 2007, MA plans projected that MA beneficiary cost sharing, funded by both rebates and additional premiums, would be 42 percent of estimated cost sharing in Medicare FFS. Plans projected that their beneficiaries, on average, would pay $49 PMPM in cost sharing, and they estimated that the Medicare FFS equivalent cost sharing for their beneficiaries was $116 PMPM. Although plans projected that beneficiaries’ overall cost sharing was lower, on average, than Medicare FFS cost-sharing estimates, some MA plans projected that cost sharing for certain categories of services was higher than Medicare FFS cost-sharing estimates. This is because overall cost sharing in MA plans is required to be actuarially equivalent or lower compared to overall cost sharing in Medicare FFS, but may be higher or lower for specific categories of services. For example, 19 percent of MA beneficiaries were enrolled in plans that projected higher cost sharing for home health services, on average, than in Medicare FFS, which does not require any cost sharing for home health services. Similarly, 16 percent of MA beneficiaries were in plans with higher projected cost sharing for inpatient services relative to Medicare FFS. (See table 1.) Some MA beneficiaries who frequently used these services with higher cost sharing than Medicare FFS could have had overall cost sharing that was higher than what they would pay under Medicare FFS. Cost sharing for particular categories of services varied substantially among MA plans. For example, with regards to inpatient cost sharing, more than half a million beneficiaries were in MA plans that had no cost sharing at all. In contrast, a similar number of beneficiaries were in MA plans that required cost sharing that could result in $2,000 or more for a 10-day hospital stay and $3,000 or more for three average-length hospital stays. In Medicare FFS in 2007, beneficiaries paid a $992 deductible for the first hospital stay in a benefit period, no deductible for subsequent hospital stays in the same benefit period, and a 20 percent coinsurance for physician services that averaged $73 per day for the first 4 days of a hospital stay and $58 per day for subsequent days in the stay. Figure 2 provides an illustrative example of an MA plan that could have exposed a beneficiary to higher inpatient costs than under Medicare FFS. While the plan in this illustrative example had lower cost sharing than Medicare FFS for initial hospital stays of 4 days or less as well as initial hospital stays of 30 days or more, for stays of other lengths the MA plan could have cost beneficiaries more than $1,000 above out-of-pocket costs under Medicare FFS. The disparity between out-of-pocket costs under the MA plan and costs under Medicare FFS was largest when comparing additional hospital visits in the same benefit period, since Medicare FFS does not charge a deductible if an admission occurs within 60 days of a previous admission. Some MA plans had out-of-pocket maximums, which help protect beneficiaries against high spending on cost sharing. As of August 2007, about 48 percent of beneficiaries were enrolled in plans that had an out-of- pocket maximum. However, some plans excluded certain services from the out-of-pocket maximum. Services that were typically excluded were Part B drugs obtained from a pharmacy, outpatient substance abuse and mental health services, home health services, and durable medical equipment. MA Plans Projected Approximately 87 Percent of Total Revenue Would be Spent on Medical Expenses For 2007, MA plans projected that of their total revenues ($783 PMPM), they would spend approximately 87 percent ($683 PMPM) on medical expenses. Plans further projected they would spend approximately 9 percent of total revenue ($71 PMPM) on nonmedical expenses, such as administration expenses and marketing expenses, and approximately 4 percent ($30 PMPM) on the plans’ profits, on average. There was variation among individual plans in the percent of revenues projected to be spent on medical expenses. For example, about 30 percent of beneficiaries—1.7 million—were enrolled in plans that projected spending less than 85 percent on medical expenses. While there is no definitive standard for the percentage of revenues that should be spent on medical expenses, Congress adopted the 85 percent threshold to require minimum thresholds for MA plans in the Children’s Health and Medicare Protection Act of 2007. MA plans projected expenses separately for certain categories of nonmedical expenses, including marketing and sales. One type of MA plan—Private Fee-for-Service (PFFS)—allocated a larger percentage of revenue to marketing and sales than other plan types. On average, as a percentage of total revenue, marketing and sales expenses were 3.6 percent for PFFS plans compared to 2.4 percent for all MA plans. Concluding Observations Medicare spends more per beneficiary in MA than it does for beneficiaries in Medicare FFS, at an estimated additional cost to Medicare of $54 billion from 2009 through 2012. In 2007, the average MA plan receives a Medicare rebate equal to approximately $87 PMPM, on average. MA plans projected they would allocate the vast majority of their rebates—approximately 89 percent—to beneficiaries to reduce premiums and to lower their cost- sharing for Medicare-covered services. Plans projected they would use a relatively small portion of their rebates—approximately 11 percent—to provide additional benefits that are not covered under Medicare FFS. Although the rebates generally have helped to make health care more affordable for many beneficiaries enrolled in MA plans, some beneficiaries may face higher expenses than they would in Medicare FFS. Further, because premiums paid by beneficiaries in Medicare FFS are tied to both Medicare FFS and MA costs, beneficiaries covered under Medicare FFS are subsidizing the additional benefits and lower costs that MA beneficiaries receive. Whether the value that MA beneficiaries receive in the form of reduced cost sharing, lower premiums, and extra benefits is worth the increased cost borne by beneficiaries in Medicare FFS is a decision for policymakers. However, if the policy objective is to subsidize health-care costs of low-income Medicare beneficiaries, it may be more efficient to directly target subsidies to a defined low-income population than to subsidize premiums and cost sharing for all MA beneficiaries, including those who are well off. As Congress considers the design and cost of the MA program, it will be important for policymakers to balance the needs of beneficiaries—including those in MA plans and those in Medicare FFS—with the necessity of addressing Medicare’s long-term financial health. Mr. Chairman, this completes my prepared remarks. I would be happy to respond to any questions you or other Members of the Subcommittee may have at this time. For further information about this testimony, please contact James Cosgrove at (202) 512-7114 or cosgrovej@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this statement. Christine Brudevold, Assistant Director; Jennie Apter, Alexander Dworkowitz, Gregory Giusto, Drew Long, and Christina C. Serna made key contributions to this statement. 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.

Summary: Although private health plans were originally envisioned in the 1980s as a potential source of Medicare savings, such plans have generally increased program spending. In 2006, Medicare paid $59 billion to Medicare Advantage (MA) plans--an estimated $7.1 billion more than Medicare would have spent if MA beneficiaries had received care in Medicare fee-for-service (FFS). MA plans receive a per member per month (PMPM) payment to provide services covered under Medicare FFS. Almost all MA plans receive an additional Medicare payment, known as a rebate. Plans use rebates and sometimes additional beneficiary premiums to fund benefits not covered under Medicare fee-for-service; reduce premiums; or reduce beneficiary cost sharing. In 2007, MA plans received about $8.3 billion in rebate payments. This testimony is based on GAO's report, Medicare Advantage: Increased Spending Relative to Medicare Fee-for-Service May Not Always Reduce Beneficiary Out-of-Pocket Costs ( GAO-08-359, February 2008). For this testimony, GAO examined MA plans' (1) projected allocation of rebates, (2) projected cost sharing, and (3) projected revenues and expenses. GAO used 2007 data on MA plans' projected revenues and covered benefits, accounting for 71 percent of beneficiaries in MA plans. GAO found that MA plans projected they would use their rebates primarily to reduce cost sharing, with relatively little of their rebates projected to be spent on additional benefits. Nearly all plans--91 percent of the 2,055 plans in the study--received a rebate. Of the average rebate payment of $87 PMPM, plans projected they would allocate about $78 PMPM (89 percent) to reduced cost sharing and reduced premiums and $10 PMPM (11 percent) to additional benefits. The average projected PMPM costs of specific additional benefits across all MA plans ranged from $0.11 PMPM for international outpatient emergency services to $4 PMPM for dental care. While MA plans projected that, on average, beneficiaries in their plans would have cost sharing that was 42 percent of Medicare FFS cost-sharing estimates, some beneficiaries could have higher cost sharing for certain service categories. For example, some plans projected that their beneficiaries would have higher cost sharing, on average, for home health services and inpatient stays, than in Medicare FFS. If beneficiaries frequently used these services that required higher cost sharing than Medicare FFS, it was possible that their overall cost sharing was higher than what they would have paid under Medicare FFS. Out of total revenues of $783 PMPM, on average, MA plans projected that they would allocate about 87 percent ($683 PMPM) to medical expenses. MA plans projected they would allocate, on average, about 9 percent of total revenue ($71 PMPM) to nonmedical expenses, including administration and marketing expenses; and about 4 percent ($30 PMPM) to the plans' profits. About 30 percent of beneficiaries were enrolled in plans that projected they would allocate less than 85 percent of their revenues to medical expenses. As GAO concluded in its report, whether the value that MA beneficiaries receive in the form of reduced cost sharing, lower premiums, and additional benefits is worth the additional cost to Medicare is a decision for policymakers. However, if the policy objective is to subsidize health care costs of low-income Medicare beneficiaries, it may be more efficient to directly target subsidies to a defined low-income population than to subsidize premiums and cost sharing for all MA beneficiaries, including those who are well off. As Congress considers the design and cost of MA, it will be important for policymakers to balance the needs of beneficiaries and the necessity of addressing Medicare's long-term financial health.

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Summarize: [0001] This application is a continuation of U.S. application Ser. No. 11/492,328, filed Jul. 24, 2006, which in turn claims priority benefit under 35 USC § 119(e) to U.S. Provisional Application No. 60/702,239, filed Jul. 25, 2005, entitled STEERABLE ENDOLUMINAL PUNCH, the entire contents of which are hereby incorporated herein by reference. FIELD OF THE INVENTION [0002] The invention relates to devices and methods for performing endovascular access to the cardiovascular system or other body vessels or body lumens, especially procedures performed in the fields of cardiology, radiology, electrophysiology, and surgery. BACKGROUND OF THE INVENTION [0003] During certain interventional procedures that are directed at cardiac access, the patient is catheterized through an access point in a vein or artery. A catheter is routed to the heart or other region of the cardiovascular system through the access point, which may be created by a cutdown or a percutaneous access procedure. The catheter may be routed to a target location within the heart, cerebrovasculature, or other region of the cardiovascular system. In certain cases, it becomes necessary to create a hole in a cardiovascular structure so that catheters or devices can be routed through a wall so as to provide for placement on the other side of the wall. One such case is the need to punch a hole in the septum that divides the right atrium of the heart from the left atrium. Such atrial septal punctures are increasingly used to gain access to the left atrium by way of the central venous system and the right atrium. Access to the left atrium of the heart is often useful in therapeutic and diagnostic procedures such as, but not limited to, valve replacement, valve repair, electrophysiology mapping, cardiac ablation, atrial appendage plug placement, and the like. [0004] The currently accepted procedure for left atrial access involves routing a needle called a Brockenbrough TM needle into the right atrium with the Brockenbrough needle pre-placed within a guiding catheter. The guiding catheter specifically preferred for use with a Brockenbrough needle is called a Mullins TM catheter. The Brockenbrough needle is a long punch formed from a stainless steel wire stylet that is surrounded by a stainless steel tube. The distal end of the stainless steel tube forms a relatively sharp circular punch capable of penetrating certain vascular structures such as the inter-atrial septum. Brockenbrough needle stylets are typically 0.013 to 0.014 inches in diameter while the stainless steel tube is generally between 0.045 and 0.050 inches in outside diameter. The Brockenbrough needle outside diameter is configured to slidingly fit within the central lumen of the Mullins catheter. The stainless steel tube is substantially straight along most of its length but is pre-bent into a curved or “J” shape at its distal end. A loop at the proximal end of the Brockenbrough stylet facilitates grasping of the stylet and performing manual advance or retraction. The current art considers the access to the right atrium from the femoral vein to be relatively straight so the current devices are straight except for the distal curve, which is shaped for the approach to the atrial septal wall. [0005] The Brockenbrough needle, a relatively rigid structure, is operated by advancing the device, with its stylet wire advanced to blunt the sharp tip, within its guiding catheter through the inferior vena cava and into the superior vena cava. Under fluoroscopic guidance, the Brockenbrough needle, retracted inside the distal tip of the Mullins catheter, is withdrawn caudally into the right atrium until it falls or translates medially into the Fossa Ovalis. The force of the Brockenbrough needle/Mullins catheter assembly pushing against the relatively weak atrial septal wall causes the Fossa Ovalis to become tented toward the left atrium. The Brockenbrough needle, protected by the blunt distal tip of the Mullins catheter, is firmly held against the Fossa Ovalis of the atrial septum. Pressure monitoring and dye injection are carried out through the central lumen of the punch following removal of the stylet wire. The circular or hollow punch is next advanced distally to puncture a hole through the atrial septum. Erroneous placement of the punch can lead to penetration of adjacent structures such as the aorta, damage to which would cause potentially severe hemorrhage and potentially compromise the health of the patient. Thus, extreme care is exercised to verify location prior to the actual punching step. The Brockenbrough needle is next advanced through the atrial septum. The guide catheter, which includes a removable, tapered, distal dilator, having a central lumen for the Brockenbrough needle, is advanced over the Brockenbrough needle system and into the left atrium. The Brockenbrough needle is next removed from the Mullins guide catheter along with the central dilator or obturator. [0006] A main disadvantage of this system is that the Brockenbrough needle system is pre-curved at its distal end and is relatively rigid. This pre-curving, rigidity, and necessary distal sharpness causes the Brockenbrough needle system to carve material from the interior wall of the otherwise straight guiding catheter when the Brockenbrough needle assembly is inserted therethrough. The material carved from the guide catheter could potentially be released into the cardiovascular system and generate emboli with any number of serious clinical sequelae. Should this embolic catheter material enter the left atrium it could flow into and block important arterial vasculature such as the coronary arteries or cerebrovasculature. Furthermore, advancing a pre-curved, rigid punch through the cardiovascular system is difficult and could potentially damage the vessel wall or any number of significant cardiovascular structures, during the advancement. [0007] It would be desirable to have a Brockenbrough needle system that was initially straight and then became curved after being inserted into the guiding catheter. Such a straight Brockenbrough configuration would be advantageous during ex-vivo insertion as well as insertion after the guide catheter has already been placed into the cardiovascular system. During ex-vivo insertion, the debris can be flushed from the lumen of the guide catheter but complete removal is not assured and emboli can still be generated by the device. However, if the guide catheter was already inserted into the cardiovascular system, the debris could not be flushed out ahead of time and could easily flow toward or be released into the cardiovascular system with potentially catastrophic or fatal results. Furthermore, the needle or punch could be more easily advanced into the body lumen if it were not pre-curved. SUMMARY OF THE INVENTIONS [0008] In an embodiment, the invention is a transvascularly or endovascularly placed tissue punch, with internal deflectability or the ability to articulate, at its distal end, in a direction away from its longitudinal axis. The punch can also be termed a catheter, needle, or cannula. The punch is generally fabricated from stainless steel and comprises an outer tube, an intermediate tube, a central stylet wire, and a distal articulating region. The deflecting or articulating mechanism is integral to the punch. The punch, needle, or catheter is sufficiently rigid, in an embodiment, that it can be used as an internal guidewire or internal guide catheter. The punch is useful for animals, including mammals and human patients and is routed through body lumens or other body structures to reach its target destination. [0009] In an embodiment, the punch comprises an inner core wire or stylet, an intermediate tube and an outer tube. In an embodiment, the stylet can be removable or non-removable. The punch further comprises a hub at its proximal end which permits grasping of the punch and also includes a stopcock or valve to serve as a lock for the stylet, or inner core wire, as well as a valve for control of fluid passage into and out from the innermost lumen within which the stylet or inner core wire resides. The proximal end further comprises one or more control handles to manipulate the amount of articulation at the distal end of the catheter. The proximal end further is terminated with a female luer or luer lock port, which is suitable for attachment of pressure monitoring lines, dye injection lines, vacuum lines, a combination thereof, or the like. [0010] The punch is fabricated so that it is substantially straight from its proximal end to its distal end. Manipulation of a control mechanism at the proximal end of the punch causes a distal region of the punch to bend or curve away from its longitudinal axis. The bending, steering, or articulating region is located near the distal end of the punch and can be a flexible region or structure placed under tension or compression by pull wires or control rods routed from the control handle at the proximal end of the punch to a point distal to the flexible region. In another embodiment, the bending or articulating mechanism can also be created by pre-bending the outer tube in one direction and bending the intermediate tube in another direction. The two tubes can be rotated relative to each other, about their longitudinal axis, by turning knobs or grips at the proximal end of the punch. When the curvatures of both tubes are aligned, the tubes will generally cooperate and not oppose each other, thus, maximum curvature or deflection is generated. When the tubes are rotated so their natural curvatures are aligned 180 degrees from each other, the curves will oppose each other or cancel out. Thus, the nested tubes will be substantially straight when the curvatures of the two concentric tubes oppose each other. Alignment marks or graduations at the proximal end can be used to assist with proper rotational alignment of the two tubes. The central core wire or stylet is generally straight and flexible and does not contribute to the curvature. In another embodiment, however, the stylet can be imparted with a curvature to assist with steering or articulation. Rotation of the two concentric tubes at relative angles between 180 degrees and 0 degrees will result in intermediate amounts of deflection so the amount of deflection can be increased or decreased in an analog, continuously variable, digital, or stepwise fashion. The stepwise or digital response can be generated using detents or interlocks that weakly engage at specific pre-determined locations. A locking mechanism can be further utilized to hold the two tubes in rotational alignment once the desired amount of curvature has been achieved. [0011] In another embodiment, steerability can be obtained using actuators on the surface or within the interior of the cannula to force bending of the cannula. These actuators can be typically electrically powered. In an embodiment, an actuator can comprise electrical leads, a power source, a compressible substrate, and shape memory materials such as nitinol. Such actuators may be distributed along the length of the cannula. The actuators may be placed so as to oppose each other. Opposing actuators are activated one at a time and not simultaneously and can generate a steering effect or back and forth motion. [0012] Other embodiments of the inventions comprise methods of use. One method of use involves inserting the central core wire so that it protrude out the distal end of the punch. A percutaneous or cutdown procedure is performed to gain access to the vasculature, either a vein or an artery. An introducer and guidewire are placed within the vasculature and the guidewire is routed proximate to the target treatment site. The introducer can be removed at this time. A guiding catheter, preferably with a central obturator or dilator is routed over the guidewire to the target site. In an embodiment, the target site can be the atrial septum. The guidewire can be removed at this time. The punch is adjusted so that it assumes a substantially straight configuration. The punch can be advanced through the central lumen of the already placed catheter. By making the punch as straight as possible, there is no curvature to force the sharpened distal edges of the punch to scrape the inside of the catheter lumen as the punch is advanced distally inside the guide catheter and potentially dislodge or scythe away debris or material which could cause embolic effects to the patient. Carefully ensuring that the punch does not protrude beyond the distal end of the catheter or its obturator, the punch is next deflected so that it forms a curve. The curve is oriented so that it is medially directed toward the atrial septum. Alignment with any curvature of the catheter can be completed at this time. The punch and guide catheter/obturator are withdrawn caudally, as a unit, into the right atrium. The punch and guide catheter are positioned using fluoroscopy or other imaging system against the Fossa Ovalis. The Fossa Ovalis is a relatively thin structure and the force of the punch will tent the Fossa Ovalis toward the left atrium. In one embodiment, the central core wire or stylet, initially advanced, can next be withdrawn to expose the sharp distal edge of the punch. When correctly positioned under fluoroscopy, ultrasound, or other imaging system, dye can be injected into the central lumen of the punch at its proximal end and be expelled out of the distal end of the punch and obturator to paint or mark the Fossa Ovalis. A generally “V-shaped” mark can be observed under fluoroscopy, which denotes the location of the Fossa Ovalis. [0013] The curvature of the punch can be increased or decreased by articulation to gain optimal alignment with the Fossa Ovalis. This steering function can be very beneficial in device placement. [0014] Maintaining the position of the guiding catheter against the Fossa Ovalis, the punch is advanced distally against and through the atrial septum, in the region of the Fossa Ovalis, so that it penetrates and protrudes into the left atrium. In order to stabilize the atrial septal tissue prior to coring, a distally protruding corkscrew tipped wire or a vacuum head operably connected to the proximal end of the punch, can be used to grasp and retract the septal tissue. Once the initial penetration is completed, the guide catheter is next advanced, with its tapered obturator leading the way, across the atrial septum until it resides within the left atrium. The tapered obturator or dilator along with the punch can be removed at this time to allow for catheter placement through the guiding catheter. In another embodiment, a calibrated spacer can be used between the guide catheter hub and the punch hub to ensure that the punch does not protrude beyond the distal end of the guide catheter tip until the desired time for punching the hole. At this point, the spacer is unlocked and removed from the punch or catheter. [0015] In another embodiment, the core wire or stylet is sharpened and serves as a tissue punch. In this embodiment, the distal end of the hollow tubes of the punch are blunted and made relatively atraumatic. Once the core wire punch has completed tissue penetration, the outer tubes are advanced over the central punch wire through the penetration and into the left atrium. In another embodiment, a pressure monitoring device such as a catheter tip pressure transducer, or a pressure line terminated by a pressure transducer, can be affixed to a quick connect, generally a luer fitting, at the proximal end of the punch hub. By monitoring pressure, it is possible to determine when the distal end of the punch has passed from, for example, the right atrium into the left atrium, because the pressure versus time curves in these two chambers are measurably, or visually, different. The proximal end of the hub further has provision for attachment to a dye injection line for use in injecting radiographic contrast media through the central lumen of the punch. Typically a manifold can be attached to the luer fitting on the proximal end of the hub, the manifold allowing for pressure monitoring, for example on a straight through port, and for radiopaque dye injection, for example through a side port. A stopcock, or other valve, can be used to control which port is operably connected to the central lumen of the punch. [0016] For purposes of summarizing the invention, certain aspects, advantages and novel features of the invention are described herein. It is to be understood that not necessarily all such advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves one advantage or group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein. These and other objects and advantages of the present invention will be more apparent from the following description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0017] A general architecture that implements the various features of the invention will now be described with reference to the drawings. The drawings and the associated descriptions are provided to illustrate embodiments of the invention and not to limit the scope of the invention. Throughout the drawings, reference numbers are re-used to indicate correspondence between referenced elements. [0018] FIG. 1 illustrates a side view of a trans-septal punch assembled so that the intermediate tube is bent in a direction 180 degrees opposite that of the outer tube, resulting in a substantially straight punch configuration; [0019] FIG. 2 illustrates a side view of the disassembled trans-septal punch showing the central core wire or stylet, the intermediate tube bent in one direction and the outer tube bent in another direction; [0020] FIG. 3 illustrates a side view of the trans-septal punch assembled so that the intermediate tube bend is aligned in the same direction as the outer tube bend, resulting in a curved distal end on the punch assembly; [0021] FIG. 4 illustrates a side view of a trans-septal punch comprising a flexible region proximal to the distal end and pull-wires disposed between the distal end and the proximal end of the punch; [0022] FIG. 5 illustrates a side view of the trans-septal punch of FIG. 4 wherein one of the pull-wires is placed in tension causing the distal flexible region of the punch to deflect into an arc away from the longitudinal axis of the punch; [0023] FIG. 6 illustrates an adjustable, spacer, which sets and maintains the distance between the distal end of the punch hub and the proximal end of a guide catheter hub. DETAILED DESCRIPTION OF THE INVENTION [0024] In accordance with current terminology pertaining to medical devices, the proximal direction will be that direction on the device that is furthest from the patient and closest to the user, while the distal direction is that direction closest to the patient and furthest from the user. These directions are applied along the longitudinal axis of the device, which is generally an axially elongate structure having one or more lumens or channels extending through the proximal end to the distal end and running substantially the entire length of the device. [0025] FIG. 1 illustrates a side view of a punch, needle, or catheter assembly 100, with an integral articulating or bending mechanism. The punch assembly 100 comprises a stylet or obturator wire 102, an intermediate tube 104, an outer tube 106, an obturator grasping tab 108, a stopcock 110, an intermediate tube pointer 112, an outer tube pointer 114, an intermediate tube hub 116, and an outer tube hub 118. [0026] Referring to FIG. 1, the obturator wire 102 is affixed to the obturator grasping tab 108. The stylet or obturator wire 102 is inserted through the central lumen of the intermediate tube 104 and is slidably disposed therein. The stopcock 110 is affixed to the intermediate tube hub 116 and the through lumen of the stopcock 110 is operably connected to the central lumen of the intermediate tube 104. The intermediate tube pointer 112 is affixed to the intermediate tube hub so that it is visible to the user. The outer tube pointer 114 is affixed to the outer tube hub 118 so that it is visible to the user. The intermediate tube hub 116 and the intermediate tube 104 are able to rotate about the longitudinal axis within the outer tube hub 118 and the outer tube 106. In an embodiment, the intermediate tube 104 is restrained from longitudinal motion relative to the outer tube 106. In another embodiment, the intermediate tube 104 can be advanced distally relative to the outer tube 106. In this latter embodiment, advancement of the inner tube 104 can be used to facilitate punching. The distal end of the intermediate tube 104 can be sharpened and serve as a punch. The distal end of the intermediate tube 104 is sheathed inside the outer tube 106 to protect the tissue from the sharp distal edge of the intermediate tube 104 until the intermediate tube 104 is advanced distally outside the distal end of the outer tube 106. A releaseable lock can be used to maintain the axial or longitudinal position of the intermediate tube 104 relative to the outer tube 106 until punching is required. A releaseable lock can further be used to maintain the rotational position of the intermediate tube hub 116 and thus the intermediate tube 104 relative to the outer tube hub 118 and the outer tube 106. [0027] All components of the punch assembly 100 can be fabricated from metals such as, but not limited to, stainless steel, Elgiloy™, cobalt nickel alloy, titanium, nitinol, or the like. The nitinol can be shape-memory or it can be superelastic. The metals used in the obturator wire 102, the intermediate tube 104 and the outer tube 106 are advantageously cold rolled, heat treated, or otherwise processed to provide a full spring hardness. The intermediate tube 104, the outer tube 106, or both, are relatively rigid, resilient structures. Polymeric materials, such as, but not limited to, polycarbonate, ABS, PVC, polysulfone, PET, polyamide, polyimide, and the like, can also be used to fabricate the stopcock 110, the intermediate tube hub 116, the outer tube hub 118, the intermediate tube pointer 112, and the outer tube pointer 114. The materials are beneficially radiopaque to maximize visibility under fluoroscopy during the procedure. Additional radiopaque markers fabricated from tantalum, platinum, iridium, barium sulfate, and the like can be added to improve visibility if needed. The intermediate tube 104 is curved or bent near its distal end into a gentle curve, preferably with a radius of between 1 to 5 inches and so that the distal tip is deflected through an angle of approximately 10 to 90 degrees from the longitudinal axis of the intermediate tube 104. The outer tube 106 is curved or bent near its distal end into a gentle curve, preferably with a radius of between 1 to 5 inches and so that the distal tip is deflected through an angle of approximately 10 to 90 degrees from the longitudinal axis of the outer tube 106. The intermediate tube hub 116 is welded, silver soldered, bonded, crimped, or otherwise fastened to the proximal end of the intermediate tube 104 so that the intermediate tube pointer 112 points in the direction of the bend in the intermediate tube 104. The outer tube hub 118 is welded, silver soldered, bonded, crimped, or otherwise fastened to the proximal end of the outer tube 106 so that the outer tube pointer 114 points in the direction of the bend in the outer tube 106. When the intermediate tube pointer 112 is oriented 180 degrees away from the direction of the outer tube pointer 114, the bend in the intermediate tube 104 substantially counteracts or opposes the bend of the outer tube 106 and the coaxial assembly 100 is substantially straight, as shown in FIG. 1. The stopcock 110 can also be a ring seal, Tuohy-Borst valve, membrane valve, hemostasis valve, gate valve, or other valve, generally, but not necessarily manually operated. The stiffness of the intermediate tube 104 and the outer tube 106 are sufficient that the punch can be used as a guide for other catheters through which the punch 100 is passed. [0028] FIG. 2 illustrates a side view of a stylet or obturator 140 further comprising the obturator wire 102 and the obturator-grasping tab 108. The obturator wire 102 is blunted at its distal end to render it as atraumatic as possible. In another embodiment, the obturator wire 102 can be tapered in diameter to render it very flexible and therefore atraumatic at its distal end. The obturator wire 102, in another embodiment, can be sharpened and serve as a needle or primary punching mechanism. FIG. 2 also illustrates an intermediate punch assembly 120 further comprising the intermediate tube 104, the stopcock 110, the intermediate tube pointer 112, the intermediate tube hub 116, an intermediate tube seal 124, an intermediate tube pointer ball 126, a through lumen port 128, a beveled distal tip 132, and a pre-set curve 136. FIG. 2 further illustrates an outer tube assembly 122 further comprising the outer tube 106, the outer tube hub 118, the outer tube pointer 114, an outer tube distal curve 130, and an outer tube pointer ball 134. [0029] Referring to FIG. 2, the obturator-grasping tab 108 is affixed, either integral to, silver soldered, welded, crimped, adhered, pinned, or otherwise attached, to the proximal end of the obturator wire 102. The intermediate tube 104 is affixed to the intermediate tube hub 116 by silver soldering, welding, potting, crimping, setscrew, pin, or other fixation method, such that the hub 116 rotates 1 to 1 with the intermediate tube 104. An optional intermediate tube pointer ball 126 is affixed to the intermediate tube pointer 112 and provides additional visual and tactile rotational positioning sense for the intermediate punch or needle assembly 120. A curve or bend 136 is heat set, or cold worked into the intermediate tube 104 at or near its distal end. The distal end of the intermediate tube 104 comprises a bevel 132 which helps serve as a punch or cutting edge for the intermediate tube 104. The angle of the bevel 132 can range between 20 and 70 degrees from the direction perpendicular to the longitudinal axis of the intermediate tube 104. In another embodiment, the bevel is removed and the distal tip of the intermediate tube 104 is a gentle inward taper or fairing moving distally that serves as a dilator should the obturator wire 102 be used as the punching device rather than the blunt distal tip obturator of the intermediate tube 104. The intermediate tube hub 116 further comprises a circumferential groove with an “O” ring 124 affixed thereto. The “O” ring 124 serves to form a fluid (e.g. air, blood, water) tight seal with the inner diameter of the outer sheath hub 118 central lumen and allows for circumferential rotation of the intermediate tube hub 116 within the outer tube hub 118. The “O” ring 124 is fabricated from rubber, silicone elastomer, thermoplastic elastomer, polyurethane, or the like and may be lubricated with silicone oil or similar materials. The stopcock 110 can be a single way or a three-way stopcock without or with a sideport, respectively. [0030] The outer punch assembly 122 comprises the bend 130, which is heat set or cold worked into the outer tube 106 in the same longitudinal location as the bend 136 of the intermediate tube. The wall thicknesses of the intermediate tubing 104 and the outer tubing 106 are chosen to provide bending forces that cancel out when the curves 136 and 130 are oriented in opposite directions and the intermediate tubing 104 is inserted fully into the outer tubing 106. The wall thickness of the outer tube 106 and the intermediate tube 104 can range between 0.003 inches and 0.20 inches, preferably ranging between 0.004 and 0.010 inches. The outer diameter of the outer tube 106 can range between 0.014 and 0.060 inches and preferably between 0.025 and 0.050 inches. The outer diameter of the obturator wire 102 can range between 0.005 and 0.030 inches and preferably range between 0.010 and 0.020 inches. [0031] FIG. 3 illustrates a side view of the punch assembly 100 fully assembled and aligned so that both the intermediate tube distal curve 136 (Refer to FIG. 2 ) and the outer tube distal curve 130 are aligned in the same direction resulting in a natural bend out of the axis of the punch 100. The punch assembly 100 comprises the obturator wire 102, the intermediate tube 104, the outer tube 106, the obturator grasping tab 108, the stopcock 110, the intermediate tube pointer 112, the outer tube pointer 114, the intermediate tube hub 116, the intermediate tube pointer ball 126, and the outer tube pointer ball 134. [0032] Referring to FIG. 3, the outer tube pointer 114 and intermediate tube pointer 112 are aligned together and in this configuration, the tubing assembly possesses its maximum curvature, which is oriented in the same directions as the pointers 112 and 114. The pointer balls 126 and 134 are aligned together to provide additional tactile and visual indices of curvature direction. In an embodiment, the curvature of the tube assembly 104 and 106 is unbiased with no net force exerted therebetween and an angle of approximately 45 degrees is subtended by the device in the illustrated configuration. Further curvature can also occur out of the plane of the page so that the curvature takes on a 3-dimensional shape, somewhat similar to a corkscrew. In another embodiment, the curvature of the aligned inner tube 104 and the outer tube 106 subtends an angle of 90-degrees or greater. Again, the intermediate tube 104 and the outer tube 106 have stiffness sufficient that the assembly is capable of guiding any catheter through which the punch 100 is passed. In another embodiment, the intermediate tube 104 and the outer tube 106 have different degrees of curvature so that when they are aligned, a net force still is generated between the two tubes, although a maximum curvature configuration is still generated. This embodiment can be advantageous in permitting articulation in a direction away from the direction of primary curvature. The radius of curvature of the punch 100 can range from substantially infinity, when straight, to as little as 0.5-cm, with a preferred range of infinity to as little as 2-cm radius when fully curved or articulated. One embodiment permits a substantially infinite to a 3-cm radius of curvature. The overall working length of the punch, that length from the proximal end of the outer tube hub to the distal most end of the punch, can range from 10 to 150-cm and preferably between 60 and 100-cm, with a most preferred range of between 70 and 90-cm. A preferred curve has a radius of about 3-cm and is bent into an arc of about 45 to 90 degrees. [0033] FIG. 4 illustrates a side view of another embodiment of a needle or punch assembly 400 comprising an obturator wire 102, an obturator wire grasping tab 108, a stopcock 110, an intermediate tube 404, an outer tube 406, a plurality of deflecting wires 412, an outer tube hub 414, a deflecting lever 416, a weld 420, an axis cylinder 424, a plurality of deflecting wire channels 426, and a flexible region 430. The distal end of the region just proximal to the flexible region 430 is shown in breakaway view. Furthermore, the distal end of the region just proximal to the flexible region 430 as well as the flexible region 430 has been expanded in scale so that certain details are more clearly visible. [0034] Referring to FIG. 4, the flexible region 430 is affixed to the outer tube 406 by a weld 420. The flexible region 430 can also be fixed to the outer tube 406 by a crimp, pin, setscrew, adhesive bond, interference fit, mechanical interlock, thread, or the like. The attachment between the flexible region 430 and the outer tube 406 is made at the proximal end of the flexible region 430 and a second attachment or weld 420 can be made at the distal end of the flexible region 430 so as to attach to a length of distal outer tube 406. The flexible region 430 can comprise a length of coiled wire such as that used in guidewires, it can be a tube that comprises cutouts to provide a backbone configuration to impart flexibility, it can be a length of polymeric tube with elastomeric characteristics, or it can be another type of structure that is known in the art as providing flexibility. These preferred structures also advantageously provide column strength and kink resistance to the flexible region 430. The center of the flexible region 430 is hollow and comprises a lumen, which is operably connected to the central lumen of the outer tube 406 at both the proximal and distal end of the flexible region 430. The stopcock 110 is affixed, at its distal end, to the outer tube hub 414. The outer tube hub 414 further comprises a deflecting lever 416 that is affixed to the axis cylinder 424, which serves as an axle or rotational pin, and can be moved proximally or distally by manual action on the part of the operator or by a motor or other electromechanical actuator (not shown). The deflecting lever 416 is operably connected to the proximal ends of the deflecting wires 412. In an exemplary embodiment, one of the deflecting wires 412 is affixed to the top of the axis cylinder 424 and the other deflecting wire is affixed to the bottom of the axis cylinder 424. When the deflecting lever is pulled proximally, for example, the top wire 412 is placed under tension and the tension on the bottom wire is relieved causing tension to be exerted on the distal end of the punch 400. The deflecting wires 412 are slidably routed through the deflecting wire channels 420 within the outer tube 406. The deflecting wires 412 also run through the deflecting wire channels 420 within the flexible region 430. The deflecting wires 412 can also be routed through the internal lumen of the outer tube 406 and the flexible region 430. [0035] Referring to FIG. 4, the outer tube hub 414 is affixed to the proximal end of the outer tube 406 by a crimp, pin, setscrew, adhesive bond, interference fit, mechanical interlock, thread, or the like. The intermediate tube 404 is affixed to the distal end of the outer tube 406 by a crimp, pin, setscrew, adhesive bond, interference fit, mechanical interlock, thread, or the like. In another embodiment, the intermediate tube 404 is routed throughout the length of the outer tube 406. In this embodiment, the intermediate tube can comprise grooves (not shown) that serve as deflecting wire channels 420 when the intermediate tube 404 is inserted inside the outer tube 406. Such grooves can also be disposed on the interior surface of the outer tube 406, rather than on the exterior surface of the inner tube 404. The obturator wire 102 and the attached grasping loop 108 are slidably disposed within the inner lumen of the outer tube 406, or the intermediate tube 404. The intermediate tube 404 is gently tapered up to the outer tube 406 at the distal end of the outer tube 406 in a transition region so that a dilator effect can be created during distal advancement of the punch 400. The distal end of the intermediate tube 404 can comprise a bevel 132 ( FIG. 2 ) or other sharp point for punching through biological tissue. The distal end of the intermediate tube 404 preferably forms a non-coring needle or punch that does not excise a tissue sample. The non-coring punch feature is achieved by keeping the central lumen closed or very small. The non-coring punch 400 embodiment can comprise filling the lumen of the intermediate tube 404 with the obturator or stylet wire 102 to prevent the sharp edge of the intermediate tube from functioning as a trephine. [0036] FIG. 5 illustrates a side view of the punch assembly 400 wherein the deflecting lever 416 has been withdrawn proximally causing increased tension in one of the deflecting wires 412, causing the flexible region 430 to bend 422 out of the longitudinal axis. The punch assembly 400 further comprises the obturator wire 102, the obturator wire grasping tab 108, the stopcock 110, the deflecting lever 416, an axis cylinder 424, the hub 414, the outer tubing 406, the intermediate tubing 404, and the bend 422. [0037] Referring to FIG. 5, the deflecting lever 416 has been moved proximally and the axis cylinder 424 causing the top deflecting wire 412 to be placed in tension while the bottom deflecting wire 412 is relaxed. The deflecting wires 412 are affixed at their distal end to the outer tubing 406 or the intermediate tubing 404 at a point substantially at or beyond the distal end of the flexible region 420. The distal fixation point (not shown) of the deflecting wires 412 is off-center from the axis of the outer tubing 406 or intermediate tubing 404. When uneven tension is created in the opposing deflecting wires 412, the uneven tension on the distal end of the punch 400 causes the bendable region 430 to undergo deflection into a curve or bend 422. Similarly, forward movement of the deflecting lever 416 will place the bottom deflecting wire 412 in tension while the upper deflecting wire 412 will be relaxed, causing the punch 400 to undergo a bend in the opposite direction (downward). The deflecting lever 416 can further comprise a ratchet and lock, a friction lock, a spring-loaded return, or other features to hold position or cause the lever and the bendable region 430 to return to a neutral deflection configuration (substantially straight). The spring nature of the outer tube 406 and the bendable region 430 can advantageously be used to cause a return to neutral once the deflection force is removed from the deflecting lever 416. The stylet or obturator wire 102 can be withdrawn or extended to expose or protect (respectively) the distal end of the intermediate tube 404 which can be sharpened or blunted. The obturator wire 102 can further be used as the primary punch, especially if the distal tip of the obturator wire 102 is sharpened. If the obturator wire 102 is used as the primary punch, the proximal end of the intermediate tube hub is fitted with a Tuohy-Borst or other hemostatic valve to permit the obturator wire 102 to remain in place. In this embodiment, sidearms affixed proximal to the proximal end of the punch, and operably connected to the central lumen, serve to permit pressure monitoring and dye contrast injection without compromising hemostasis or air entry into the punch assembly 400. [0038] FIG. 6 illustrates a side view of an adjustable spacer 600 interconnecting a guide catheter 620 and a punch assembly 100. The spacer 600 further comprises a proximal connector 602, a rotating nut 604, an inner telescoping tube 608, a threaded region 606, a distal locking connector 610, and an outer telescoping tube 614. The guide catheter further comprises a tube 622, a hub 624, and a proximal connector 626. The punch assembly 100 further comprises the stopcock 110, the distal rotating locking connector 612, the intermediate tube pointer 112, the outer tube pointer 114, and the intermediate tube hub 116. The spacer 600 can comprise an optional slot 630. [0039] Referring to FIG. 6, the punch assembly 100 is inserted through the central lumen of the adjustable spacer 600. The distal end of the punch assembly 100 is then inserted through the central lumen of the guide catheter 620. The hub 624 of the guide catheter 620 is affixed to the proximal end of the guide catheter tube 622. The distal end of the hub 624 comprises a female luer lock connection, which is bonded to, or integrally affixed to the hub 624. The hub 624 can further comprise a seal or hemostasis valve such as a Tuohy-Borst fitting. The punch 100 hub 116 is terminated at its distal end by a swivel male luer lock connector 612. The adjustable spacer 600 comprises an outer telescoping tube 614, shown in partial cutaway view that is terminated at its proximal end with a female luer lock 602. The proximal end of the outer telescoping tube 614 has a flange that permits rotational attachment of the rotating nut 604, shown in partial cutaway view, so that the rotating nut is constrained in position, longitudinally, relative to the outer telescoping tube 614 but is free to rotate. The inner telescoping tube 608 is affixed at its distal end with a swivel male luer connector 610, or equivalent. The proximal end of the inner telescoping tube 608 is affixed to, or comprises, the integral threaded region 606. The threaded region 606 mates with the internal threads on the rotating nut 604. As the rotating nut 604 is rotated, either manually or by an electromechanical device, it moves forward or backward on the inner telescoping tube 608 and threaded region 606 thus changing the space between the hub 116 of the punch 100 and the proximal end of the hub 624 of the guide catheter 620. The system is preferably set for spacing that pre-sets the amount of needle or stylet travel. In an embodiment, the rotating nut 604 comprises a quick release that allows disengagement of the inner telescoping tube 608 from the outer telescoping tube 614 so that collapse is permitted facilitating the tissue punching procedure of advancing the punch 100 distally relative to the hub 624. The system further comprises hemostatic valves at some, or all, external connections to prevent air leaks into the punch 100. The telescoping tube 608 can be set to disengage from the outer telescoping tube 614 to allow for longitudinal collapse so that the punch 100 can be advanced distally to provide its tissue punching function. In another embodiment, the spacer 600 comprises the slot 630 that permits the spacer to be removed sideways off the punch 100. The slot 630 is wide enough to allow the outer tube 106 to fit through the slot 630 so the spacer 600 can be pulled off, or removed from, the punch 100 prior to the punching operation. Thus, the slot 630 can be about 0.048 to 0.060 inches wide and extend the full length of the spacer 600. With the slot 630, the spacer 600 comprises a generally “C-shaped” lateral cross-section. The spacer 600 can further comprise a slot closure device (not shown) to prevent inadvertent removal of the punch 100. [0040] In another embodiment, the threaded region 606 and the rotating nut 604 are replaced by a friction lock on telescoping tubes, a ratchet lock, or other suitable distance locking mechanism. In yet another embodiment, a scale or series of markings (not shown) is incorporated into the adjustable spacer 600 to display the exact distance between the proximal end and the distal end of the spacer 600. In another embodiment, the proximal end and the distal end of the spacer 600 do not comprise one or both of the female luer lock 602 or the rotating male luer lock 610. In this embodiment, the spacer 600 provides positional spacing but does not affix the punch 100 to the guide catheter 620 so that the two devices move longitudinally as a unit. In another embodiment, the pull wires 412 of FIG. 4, which are strong in tension but cannot support compression are replaced by one or more control rods, which are flexible but which have column strength. Thus, deflection can be generated by imparting either tension on the control rod or compression and such tension and compression is capable of deflecting the distal tip of the punch 400 without the need of a separate control rod to impart tension in the other direction. The intermediate tube hub 116 is terminated at its proximal end by a female luer, luer lock, threaded adapter, bayonet mount, or other quick release connector. The quick connect or female luer can be releasably affixed to a hemostasis valve, other stopcock, pressure transducer system, “Y” or “T” connector for pressure and radiopaque contrast media infusion, or the like. [0041] In another embodiment, a vacuum line can be connected to a port affixed to the proximal end of the punch. The port can be operably connected to a bell, cone, or other structure at he distal end of the punch by way of a lumen, such as the central lumen of the intermediate tube or an annulus between the intermediate and outer tube, within the punch. By application of a vacuum at the proximal end of the punch, the distal structure can be releasably secured to the atrial septum prior to punching through. In another embodiment, a corkscrew structure projects out the distal end of the punch and is operably connected to a knob or control at the proximal end of the punch by way of a control rod slideably or rotationally free to move within a lumen of the punch. The corkscrew structure can be screwed into tissue to releasably secure the distal end of the punch to the tissue, for example, to enhance stability of the punch prior to, during, or after the punching operation. [0042] Referring to FIG. 1, in another embodiment, the intermediate tube 104, the outer tube 106, or both, are fabricated from shape memory nitinol. In this embodiment, electrical energy can be applied to the pre-bent regions of the inner tube 104, the outer tube 106, or both. Upon application of electrical energy, Ohmic or resistive heating occurs causing temperature of the tubes to increase. The nitinol changes its state from martensitic to austenitic, with the increase in temperature, and can assume a pre-determined configuration or stress state, which is in this case curved. The austenite finish temperature for such a configuration is approximately 40 degrees centigrade or just above body temperature. In yet another embodiment, the austenitic finish temperature can be adjusted to be approximately 28 to 32 degrees centigrade. The punch 100 can be maintained at room temperature where it is substantially martensitic and non-rigid. Upon exposure to body temperatures when it is inserted into the core lumen of the guide catheter, it will assume its austenitic shape since body temperature is around 37 degrees centigrade. This can cause the punch 100 to curve from substantially straight to substantially curved. In this configuration, only a single tube, either the intermediate tube 104 or the outer tube 106 is necessary, but both tubes, while potentially beneficial, are not required. [0043] The punch can be used to create holes in various structures in the body. It is primarily configured to serve as an articulating or variable deflection Brockenbrough needle. [0044] However, the steerable punch can be used for applications such as transluminal vessel anastomosis, biopsy retrieval, or creation of holes in hollow organs or lumen walls. The punch can be used in the cardiovascular system, the pulmonary system, the gastrointestinal system, or any other system comprising tubular lumens, where minimally invasive access is beneficial. The punch can be configured to be coring or non-coring in operation, depending on the shape of the distal end and whether an obturator or the circular hollow end of the punch is used to perform the punching operation. In the coring configuration, a plug of tissue is removed, while in the non-coring configuration, substantially no tissue is removed from the patient. The punch facilitates completion of transseptal procedures, simplifies routing of the catheters, minimizes the chance of embolic debris being dislodged into the patient, and improves the ability of the cardiologist to orient the punch for completion of the procedure. The punch of the present invention is integral and steerable. It is configured to be used with other catheters that may or may not be steerable, but the punch disclosed herein does not require external steerable catheters or catheters with steerability to be steerable as it is steerable or articulating on its own. The punch is capable of bending and unbending a practically unlimited number of times. The punch is especially useful with catheters that are not steerable since the punch comprises its own steering system. [0045] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. For example, the deflecting wires 412 can be replaced by an electromechanical actuator and external control unit. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Summary: This invention is a transvascularly placed punch, further including internal steerability and the ability to articulate in a direction at right angles to its longitudinal axis at or near its distal end. The punch is generally fabricated from stainless steel and includes an outer tube, an intermediate tube, a central stylet, hub structures, and a distal articulating region. The punch can be advanced through a body lumen in its straight configuration and then be selectively articulated or curved to permit negotiation of tortuous curvature or to permit optimal approach or access to a puncture site. The punch is able to create holes in the atrial septum of the heart or other structures and is easier to use than punches that are pre-curved near their distal tip since it is easier to advance through accessory catheters.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Armed Forces Behavioral Health Awareness Act''. SEC. 2. ELIGIBILITY OF MEMBERS OF THE ARMED FORCES WHO SERVE IN OPERATION IRAQI FREEDOM OR OPERATION ENDURING FREEDOM FOR READJUSTMENT COUNSELING AND SERVICES THROUGH VET CENTERS. (a) In General.--A member of the Armed Forces, including the Reserve components, who is deployed in support of Operation Enduring Freedom or Operation Iraqi Freedom shall be eligible for readjustment counseling and related mental health services under section 1712A of title 38, United States Code, through the centers for readjustment counseling and related mental health services (commonly known as ``Vet Centers'') operated by the Secretary of Veterans Affairs under that section. (b) Eligibility.-- (1) Regulations.--Except as provided in paragraph (2), the eligibility of a member of the Armed Forces, including the Reserve components, for counseling and services under subsection (a) shall be subject to such regulations as the Secretary of Defense and the Secretary of Veterans Affairs shall jointly prescribe for purposes of this section. (2) Duty status.--The duty status of a member may not have an effect on the eligibility of the member to receive counseling and services under subsection (a). SEC. 3. GRANTS FOR NONPROFIT ORGANIZATIONS FOR THE PROVISION OF EMOTIONAL SUPPORT SERVICES TO FAMILY MEMBERS OF MEMBERS OF THE ARMED FORCES. (a) In General.--The Secretary of Defense shall carry out a program to award grants to nonprofit organizations that provide emotional support services for family members of members of the Armed Forces, including the Reserve components. (b) Award of Grants.-- (1) Eligibility.--To be eligible for a grant under the program under this section, a nonprofit organization shall meet such criteria as the Secretary shall establish for purposes of the program. (2) Application.--A nonprofit organization seeking a grant under the program shall submit to the Secretary an application for the grant in such form and manner as the Secretary shall specify for purposes of the program. (c) Grants.-- (1) Amount.--The amount of each grant awarded to a nonprofit organization under the program under this section shall be such amount as the Secretary determines appropriate for purposes of the program. (2) Duration.--The duration of each grant awarded to a nonprofit organization shall be such period as the Secretary determines appropriate for purposes of the program. (d) Use of Grant Funds.--Each nonprofit organization awarded a grant under the program under this section shall use amounts under the grant to provide emotional support services for family members of members of the Armed Forces, including the Reserve components, through certain programs as the Secretary shall specify in the grant. (e) Funding.--Amounts for grants under the program under this section shall be derived from amounts authorized to be appropriated to the Department of Defense for military personnel. SEC. 4. PILOT PROGRAM TO ENHANCE AWARENESS OF POST-TRAUMATIC STRESS DISORDER. (a) Pilot Program Required.--The Secretary of the Army shall carry out a pilot program to enhance awareness of post-traumatic stress disorder among members of the Army. The Secretary shall carry out the pilot program in the following locations: (1) Fort Huachuca, Arizona. (2) Fort Carson, Colorado. (3) Fort Leonard Wood, Missouri. (b) Activities.-- (1) In general.--In carrying out the pilot program, the Secretary shall implement activities that-- (A) for a member of the Army who will be deployed in support of a contingency operation, increase the understanding of-- (i) the neurophysiological effects of stress and trauma associated with combat, including post-traumatic stress disorder; and (ii) the means of eliminating or mitigating such effects after returning from combat; (B) for a member of the Army deployed in support of a contingency operation, reinforce the information provided under subparagraph (A); (C) for a member of the Army who returns from being deployed in support of a contingency operation, assist the member in reintegrating into noncombat life; and (D) for the family of a member of the Army covered under this subsection, include training and assistance (including Internet-based training and assistance) at each stage of deployment in order to assist the family and member in recognizing and addressing post-traumatic stress disorder. (2) Development of activities.--In developing activities under this subsection, the Secretary shall consider methods to address stress and trauma used by other appropriate populations, including special operations forces and elite athlete communities. (c) Duration.--The Secretary shall carry out the pilot program for a period of three years. (d) Report.--Not later than two years after the date of the enactment of this Act, the Secretary shall submit to Congress a report assessing the pilot program, including the effectiveness of the activities under subsection (b).

Title: To improve the mental health care benefits available to members of the Armed Forces, to enhance counseling available to family members of members of the Armed Forces, and for other purposes Summary: Armed Forces Behavioral Health Awareness Act - Makes any member of the Armed Forces who is deployed in support of Operations Iraqi Freedom or Enduring Freedom eligible for readjustment counseling and related mental health services through Vet Centers (centers for such counseling and services for veterans), regardless of the member's duty status. Directs the Secretary of Defense to award grants to nonprofit organizations that provide emotional support services for family members of members of the Armed Forces, including members of the reserves. Requires the Secretary of the Army to carry out a pilot program to enhance awareness of post-traumatic-stress-disorder (PTSD) among members of the Army.

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Summarize: Stone Movie Review By Prairie Miller A murky Bible belt noir in which typically nobody is what they seem, even when inadvertently, Stone mixes evangelism, midlife crisis lust, and serious parole violations by, well, somebody on the parole board. Along with Edward Norton in a weirdly over the top performance in what is usually referred to in less than polite circles as a 'wigger.' Directed by John Curran (The Painted Veil) and steeped in what seems like mystical heartland evangelical voodoo more suited for sci-fi, the movie sets up a convoluted cat and mouse prison caper among predators and prey alike, in a continuously alternating switching up of sides and hidden agendas. Edward Norton is Stone, a Michigan prison inmate obsessed with manipulating his parole officer Jack (Robert De Niro) into recommending his release, related to a long prison term he's been serving as an accomplice in the murder of his own grandparents and the heartless torching of their home. Decked out in dreads and spouting a borderline clownish ghetto-speak, Stone moves on to Plan B when he gets hip to Jack not being in the least impressed with his intimidating jailhouse freedom rap. Enter Milla Jovovich as Lucetta, Stone's flirty trailer park scantily clad slutty wench back home, in an equally caricature-driven performance, who is sent to seduce Jack into a more receptive attitude towards her arsonist spouse's pending parole hearing. Jack, who is up for parole himself so to speak and about to retire shortly, is not without his own dark secrets, including a damaged relationship with an alcoholic wife Madylyn (Frances Conroy) who sits at home all day drinking and reading scriptures. That is, ever since her control freak hubby dangled their infant out the window and threatened to drop him, if Madylyn ever made good on her determination to leave him. At some point Jack seems to turn into Travis Bickle once again minus his taxi, and Lucetta makes toy birds' nests between make out sessions with the grumpy lawman. While Stone, a con in evidently more ways than one, finds Jesus, loses his dreads, and becomes a self-described tuning fork for God. And the audience may be left to wonder if all these prominent stars should be indicted and convicted as well, for making such bad choices in movies. Overture Films Rated R 2 [out of 4] stars Prairie Miller is a multimedia journalist online, in print and on radio. Contact her through NewsBlaze. As 1995 slouches into 1996, winter holds Montreal in an unrelenting grip. Adding to the chill are a series of unsolved rapes and murders occurring in the west-end neighbourhood of Notre-Dame-de-Grâce, all of which, it seems, have been perpetrated by the same man. That’s the set-up for Jacob Tierney’s third feature, using a self-penned screenplay adapted from Chrystine Brouillet’s novel Chère Voisine. The action revolves around three tenants of a run-down apartment: a cat-obsessed waitress (Emily Hampshire) working in a Chinese restaurant that never seems to have any customers, a wheelchair-bound widower (Scott Speedman) whose suite is filled with aquariums, and an emotionally needy, newly arrived elementary-school teacher (Jay Baruchel). Tierney, who scored big at TIFF last year with The Trotsky, garners great (and not a little creepy) turns from each of his leads to concoct a twisty tale that’s part-mystery, part-psychological thriller, part-comedy. His assured mise-en-scène echoes Polanski’s The Tenant and Boyle’s Shallow Grave. J.A. Sept. 18, 12:45 p.m., AMC 6 Trigger Bruce McDonald (Canada) 3 STARS Sometimes an on-screen pairing just clicks. Though filled with recognizable faces – Sarah Polley, Don McKellar, Callum Keith Rennie, Caroline Gillis – Trigger leans almost entirely on its leading ladies, Molly Parker and the late Tracy Wright. They play reunited rocker chicks who have taken widely divergent paths in life and are wrangling demons born of a youth spent with sex, drugs and rock ’n’ roll. Looking gaunt, Wright is splendid as Vic, the sharp and sarcastic half of the duo wasted by a taste for heroin. Parker is a charming and convincing foil as the cheerier Kat, now a well-paid TV executive still battling demons of her own. Trigger was rushed into production after Wright was diagnosed with cancer, but it doesn’t feel that way. Shot simply but compellingly, it feels like a polished and practised portrait of rekindled bonds. J.B. Sept. 18, 12:45 p.m., Scotiabank 2 Dhobi Ghat (Mumbai Diaries) Kiran Rao (India) 3 STARS Move over Bollywood, because India is going indie. More precisely, it's showing some early signs of returning to the auteur-driven sensibilities of a director like Satyajit Ray. Here, making her feature debut, Kiran Rao takes us to teeming Mumbai in the monsoon season, then glides fluidly through the urban strata embodied in her three principal characters – an affluent Indian-American woman with photographic ambitions; a dour artist with a troubled marital past; and a hunky laundry boy (a dhobi) keen to climb out of the slums and into the movies. Perched on their different rungs, each is looking up or down the social ladder and doing the same thing but from different motives: peering, either curiously or enviously, into the lives of others. As they do, the film means to be the cinematic answer to Suketu Mehta's book, Maximum City – that is, it wants to draw a vibrant portrait of Mumbai in all its cruel beauty. Wants to, and almost succeeds. R.G. Sept. 18, 12 p.m., Bell Lightbox 1 Stone John Curran (USA) 3 STARS Does sin come naturally to human beings? That, and how to look to be forgiven for it, is at the heart of Stone, especially where crimes that never reach a courtroom are concerned. Edward Norton steals the show as Gerald (Stone) Creeson, a convict of eight years looking for freedom through his parole officer, Jack Mabry (Robert De Niro). Norton is thoughtful and clever, but reptilian and rough. As Mabry, De Niro grapples ably with his own demons, entangled with the efforts of Lucetta Creeson (Milla Jovovich), a seductive manipulator trying to spring her husband. This film isn’t without flaws: For example, its religious overtones, framed by the devout rituals of Jack’s wife Madylyn and squawked out over Christian radio, don’t quite strike the right chord. But it’s worth the price of admission to see Norton and De Niro sparring across a prison-house desk. J.B. Enlarge By Ron Batzdorff, Overture Films You talkin' to me? Robert De Niro plays a corrections officer, and Edward Norton is an imprisoned arsonist. ABOUT THE MOVIE ABOUT THE MOVIE Stone * * (out of four) Stars: Robert De Niro, Edward Norton, Milla Jovovich, Frances Conroy Director: John Curran Distributor: Overture Films Rating: R for strong sexuality and violence and pervasive language Running time: 1 hour, 45 minutes Opens Friday in select cities Nothing is etched in anything remotely resembling a hard surface in Stone. TRAILER: Watch a preview of 'Stone' Moral ambiguity and ethical compromise are at the heart of this meandering prison drama, but at a certain point we simply don't care anymore who is base and baser. The thin line between lawman and criminal has been explored a fair amount in films. Stone probes the blurring of the two roles and bleeding of black and white into gray. It tries to raise the specter of spiritual emptiness, but the characters come off like dramatic devices rather than real people in this ponderous cat-and-mouse game directed by John Curran (The Painted Veil) and written by Angus Maclachan (Junebug). Robert De Niro plays corrections officer Jack Mabry, a taciturn and inarticulate guy who, like most law enforcement types of a certain age in movies, is about to retire. But before he's officially free, he must review one last case. But this is no open-and-shut matter. Mabry is slyly manipulated by wily, cornrowed arsonist Gerald "Stone" Creeson, ably played by Edward Norton. Given the caliber of these actors, their back-and-forth should be fascinating. But somehow it never becomes the profound character drama it aims to be. Further complicating things is Stone's scheming wife, Lucetta (Milla Jovovich), who is working to spring Stone. Out of the loop but in the background is Mabry's reclusive and religious wife, Madylyn (Frances Conroy in a thankless part). Though outwardly religious, a jarring flashback of Mabry's not-so-random act of pure viciousness reveals his menacing nature and emotional baggage. We understand why his wife is hollow-eyed. It's no wonder that he's chosen a job in a prison. He needs to contain his worst tendencies. Somehow, the fast-talking Stone unlocks Mabry's darkest impulses. Or maybe they were just there below the surface, ready to reveal themselves at the slightest provocation. During their rambling discussions, Stone insists he has had a religious epiphany and struggles to convey honest emotion in his stream-of-consciousness way. He may be getting to Mabry. Or not. Clarity is not the point here. Lucetta gets to Mabry, in several ways, triggering some gratuitous nudity. But despite the verbal fireworks and steamy scenes, the film is mired in tedium. De Niro's restrained Method acting is a natural for the role and Norton always is an arresting actor, but what they're given to say and ponder never quite catches fire. Both men are spiritually bereft and seeking some kind of salvation, but the incessant background soundtrack of Christian talk radio seems a heavy-handed way to get this notion across. Stone muddles along, like an idea in search of a plot. Guidelines: You share in the USA TODAY community, so please keep your comments smart and civil. Don't attack other readers personally, and keep your language decent. Use the "Report Abuse" button to make a difference. You share in the USA TODAY community, so please keep your comments smart and civil. Don't attack other readers personally, and keep your language decent. Use the "Report Abuse" button to make a difference. Read more There are crimes and there are sins, and while society demands atonement for the former, many terrible sins are perfectly legal and atonement is up to the sinner. Writer-director John Curran explores these questions of official and personal morality in Stone, an intelligent but brutally portentous drama in the style of his 2004 marital drama We Don’t Live Here Anymore. He also gets a real, committed performance out of Robert De Niro, who’s spent much of the last decade lazily mugging his way through Meet The Parents comedies and genre stinkers like Hide And Seek and Righteous Kill—all of which suggest either good roles for him are limited or he simply doesn’t care like the De Niro of old. Whatever the case, De Niro plays a deeply flawed and hypocritical corrections officer with just the right mix of self-righteousness and vulnerability, embodying a man whose soul has withered from decades of corrosion. De Niro goes toe-to-toe with Edward Norton, who stars as a corn-rowed (though non-racist) iteration of the wily, hyper-intelligent thug he immortalized in American History X. Having served eight years in prison for arson, Norton is up for parole, but his path to freedom goes through De Niro, whose recommendation will ultimately determine his fate. Mere weeks before his retirement, De Niro has been on the job long enough to see through Norton’s abrasive, confrontational pleas, so the inmate turns to a more drastic form of coercion, enlisting his sultry wife, played by Milla Jovovich, to coax out a favorable report. It happens that De Niro is a uniquely vulnerable mark, and as he gets drawn into their web, his past sins begin to resurface. The title Stone is double-meaning, referring both to Norton’s nickname and the end of the Biblical tenet that begins, “Let him who is without sin…” At its best, the film works as a morally freighted film noir, with Jovovich particularly good as a breathy femme fatale who seduces De Niro with a mere change in inflection. But Curran resists Stone’s genre elements to his peril, suffocating the drama with a tone of high seriousness and unnecessary elements, like an omnipresent AM religious talk show that spells out themes that are already apparent. He doesn’t trust the story or his actors to bring the film across—and worst of all, he doesn’t trust the audience.

Summary: Prison drama Stone isn't light viewing, critics say, but top-notch performances from stars Robert De Niro and Edward Norton save it from collapsing under the weight of its themes of morality and spirituality. De Niro, who plays wily inmate Norton's parole officer, "seems to turn back into Travis Bickle minus his taxi" in this "murky Bible Belt noir steeped in evangelical voodoo," writes Prairie Miller at News Blaze. Stone works well as a "morally freighted noir" but director John Curran sometimes suffocates the drama "with a tone of high seriousness and unnecessary elements," writes Scott Tobias at the AV Club, praising Milla Jovovich's performance as "a breathy femme fatale who seduces De Niro with a mere change in inflection." "Moral ambiguity and ethical compromise are at the heart of this meandering prison drama" but it never quite catches fire, writes Claudia Puig at USA Today, complaining that the "characters come off like dramatic devices rather than real people." The movie has its flaws, including religious overtones that don't hit the right note, but "it's worth the price of admission to see Norton and De Niro sparring across a prison-house desk," decides James Bradshaw at the Globe and Mail.

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Summarize: One Texas couple is finally expecting not just one, but two babies, after struggling with infertility for two years. Lauren Walker shared her story, with a moving photo featuring two onesies and 452 needles used for her In-Vitro Fertilization treatments in a photo that has since gone viral on Facebook. "We prayed for 953 days...452 Needles, 1000's of tears, 1 corrective surgery, 4 clomid/letrozole attempts, 2 IVF rounds, 3 failed transfers and & 1 Amazing GOD," the yoga instructor wrote as a caption before explaining her inspirational journey. Walker, 28, had been trying to have a child with her high school sweetheart, Garyt, since 2014. "When we started, we knew off the bat that I was having issues," Walker told ABC News, "which I guess is a blessing." Courtesy of Lauren and Garyt Walker So Walker decided to undergo IVF treatments at Houston Fertility Institute "and we expected it to work." Still, she miscarried two embryos on Sept. 10, 2014. After another round of treatment, Walker miscarried two more embryos three months later. "It's every mother's job to be able to protect their children and keep them safe," Walker said through tears. "And every time they kept putting them inside me I couldn't do it." The couple had one embryo left and decided to "give it one more shot," Walker said. But two days before Christmas in 2014, they discovered they still weren't pregnant. Courtesy of Lauren and Garyt Walker Walker said she made her husband take the call from her fertility nurse because she was too afraid to hear any more bad news. "He went into the bedroom to take the call. He came out and just looked at me and he started to tear up [and said,] 'I'm so sorry, sweetie,'" Walker recalled. "We just held each other and I let out this blood curdling scream. I was completely broken." It didn't help that, by then, they had spent approximately $30,000 on treatments. Thankfully, their marriage was still in tact. "We have heard stories of how going through infertility can really cause wear and tear in a marriage," Walker said. "[We decided] we come first. We need to make sure we are always taking care of each other first and foremost." Courtesy of Lauren and Garyt Walker The couple credits the strength of their marriage and their faith in God for giving them the courage to try to have a baby again. They moved to The Woodlands, Texas, from Houston, in May 2016. After taking out a $14,000 loan, they began treatments again last October. This time, they decided not to tell family and friends they were trying again to have a baby. Instead, they surprised their family with the news that Walker was indeed pregnant -- with twins -- just a week before Christmas by handing them the pregnancy test wrapped in a bow. Walker said that despite her long journey, she wouldn't want it any other way. Courtesy of Lauren and Garyt Walker "Life happens the way that it's supposed to happen," she said. "Had this all happened the way I wanted to back in 2014, we would have different children and we would have a different life, and I know that these babies right now are meant to be here." "The reason why we were waiting so long is that we were waiting for them," she gushed. Walker is due in August and she said she's looking forward to introducing her twins, that she's named Duke and Diana Walker, to her 6-year-old goldendoodle, Fenway -- and of course they rest of their family. "They're the first grandchildren," Walker said. "Everyone's just so excited." Walker reflected on some of the hardest moments in her journey to conceiving the twins. “My darkest hour was going through an entire cycle and losing embryo after embryo; five,” she recalled. “Coming so far only to miscarry. Getting to where all the hope, the money, all of what could have been your children were gone.” Though the experience left her feeling physically, emotionally and spiritually drained, Walker said she ultimately made it through with the help of her Christian faith and her husband’s unwavering support. Describing Garyt’s role in this multiyear struggle, she wrote:

Summary: After more than two years of failed attempts, a woman in Texas is finally pregnant, and her heartfelt Facebook post has gone viral with thousands of likes and hundreds of comments. It took, as Lauren Walker writes, more than 953 days, 452 needles, two IVF rounds, multiple miscarriages, and thousands of tears, but she and her husband are expecting twins, Duke and Diana, in August. "The reason why we were waiting so long is that we were waiting for them," she says. It's been a rocky road. ABC News reports that the couple had spent around $30,000 on treatments in Houston by Christmas of 2014. They gave it "one more shot" with their final embryo, but when her husband took the call from the fertility nurse two days before Christmas that year, his face said it all. "I let out this bloodcurdling scream," Walker says. "I was completely broken." But they strived to keep their marriage intact, and after moving to the Woodlands in Texas in May 2016, they took out a $14,000 loan and began treatments again. They announced the pregnancy to family on Christmas, with the test wrapped in a bow. And Walker's Facebook post is a smash. "I wanted to show people not just how much we had been through, but to hopefully instill a sense of hope and comfort for anyone who is or has struggled to get pregnant," she tells the Huffington Post.

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Summarize: BACKGROUND OF THE INVENTION The present invention relates to a surgical instrument commonly referred to as a trocar, or an obturator and cannula, often used in laparoscopic or arthroscopic surgery. More particularly, the invention relates to new and improved designs for shields, seals, and coatings, and to the use of disposable seal and stopcock assemblies on an otherwise reusable instrument. Many surgical procedures are now being performed with the use of trocars and cannulas. Originally these devices were used for making a puncture and leaving a tube to drain fluids. As technology and surgical techniques have advanced, it is now possible to insert surgical instruments through the cannulas and perform invasive procedures through openings less than half an inch in diameter. Previously these procedures required incisions of many inches. By minimizing the incision, the stress and loss of blood suffered by patients is reduced and the patients&#39; recovery times are dramatically reduced. Surgical trocars are most commonly used in laparoscopic surgery. Prior to use of the trocar, the surgeon will usually introduce a Veress needle into the patient&#39;s abdominal cavity. The Veress needle has a stylet which permits the introduction of gas into the abdominal cavity. After the Veress needle is properly inserted, it is connected to a gas source and the abdominal cavity is insufflated to an approximate abdominal pressure of 15 mm Hg. By insufflating the abdominal cavity, pneumoperitoneum is created separating the wall of the body cavity from the internal organs. A trocar is then used to puncture the body cavity. The piercing tip or obturator of the trocar is inserted through the cannula or sheath and the cannula partially enters the body cavity through the incision made by the trocar. The obturator can then be removed from the cannula and an elongated endoscope or camera may be inserted through the cannula to view the body cavity, or surgical instruments may be inserted to perform ligations or other procedures. A great deal of force is often required to cause the obturator to pierce the wall of the body cavity. When the piercing tip breaks through the cavity wall, resistance to penetration ceases and the tip may reach internal organs or blood vessels, with resultant lacerations and potentially serious injury. The creation of the pneumoperitoneum provides some free space within which the surgeon may stop the penetration of the trocar. To provide further protection, trocars have more recently been developed with spring loaded shields surrounding the piercing tip of the obturator. Once the piercing tip of the obturator has completely pierced the body cavity wall, the resistance of the tissue to the spring loaded shield is reduced and the shield springs forward into the body cavity and covers the piercing tip. The shield thereby protects internal body organs and blood vessels from incidental contact with the piercing tip and resultant injury. Trocars including various safety shield designs are described in Yoon, U.S. Pat. Nos. 4,535,773; Moll, 4,654,030; and Moll, 4,601,710. An improved piercing tip comprised of a pointed blade with an improved safety shield is also the subject of commonly owned pending U.S. Ser. No. 08/038,904. Once the cannula has been introduced into the opening in the body cavity wall, the pneumoperitoneum may be maintained by introducing gas into the abdominal cavity through the cannula. Various seals and valves have been utilized to allow abdominal pressure to be maintained in this fashion. Maintaining abdominal pressure is important both to allow working room in the body cavity for instruments introduced through the cannula, and to provide free space for the puncturing of the body cavity wall by one or more additional trocars as may be required for some procedures. While the existing trocars and cannulas have proven useful, several disadvantages remain. The force required to cause the piercing tip to penetrate a body cavity wall is often so great that some physicians must strain to use the trocar with resulting loss of control over the depth of penetration of the instrument. In addition, the existing shield mechanisms do not spring forward to cover the obturator tip as quickly as possible. The delay in covering the piercing tip exposes the patient to unnecessary risk of internal laceration. Also, with the current emphasis on cost controls in health care, it is desirable to utilize reusable medical instruments whenever possible. The difficulties of cleansing, disinfecting and otherwise decontaminating used trocars has made this a time consuming or impossible task, especially for shielded trocars. Therefore, a need exists for an improved apparatus for performing laparoscopic and similar surgical procedures. SUMMARY OF THE INVENTION Accordingly, it is the primary object of the invention to provide a safer trocar for piercing body cavity walls. It is also an object of the invention to provide an improved shield design which will provide faster coverage of the obturator&#39;s piercing tip. It is a further object of the invention to provide improved coatings for shields and cannulas which will permit easier penetration of body cavity walls and faster coverage of the obturator&#39;s piercing tip. It is another object of the invention to provide a cannula which may be substantially reused without the need for excessive labor in cleaning and decontamination. It is still another object of the invention to provide a seal or valve which permits easy insertion of surgical and exploratory instruments through the cannula yet still operates effectively to maintain the pneumoperitoneum in the body cavity. Accordingly, the present invention provides a cannula with a detachable cap and disposable seal assembly and stopcock. To achieve faster shield protection, the forward or distal end of the shield is bottleshaped and is biased to move forward to cover the obturator&#39;s piercing tip when the piercing tip penetrates the body cavity wall. The shield may also be fabricated from or treated with hydrophilic materials so that when it is dipped in sterile water or other wetting solution before use, there is less friction between the shield and the body cavity wall allowing faster coverage of the piercing tip. To achieve easier penetration of body cavity walls by the cannula, the cannula may also be treated with a hydrophilic coating. A novel and inexpensive flexible valve is also provided which facilitates insertion of surgical instruments. BRIEF DESCRIPTIONS OF THE DRAWINGS FIG. 1A is a cross sectional side view of an improved cannula according to the invention with a disposable gland retainer and end cap. FIG. 1B is a side view of a conventional obturator which is adapted for use with the cannula of FIG. 1A. FIG. 2 is an exploded perspective view of the improved cannula of FIG. 1. FIG. 3A is a cross sectional side view of the end cap of the improved cannula of FIG. 1 in isolation. FIG. 3B is an end view of the end cap of the improved cannula of FIG. 1. FIG. 4A is a cross sectional side view of the flexible flapper valve of the improved cannula of FIG. 1 in isolation. FIG. 4B is an enlarged side plan view of the button which is mounted in the center of the preferred embodiment of the flexible flapper valve. FIG. 4C is an end view of the flexible flapper valve of the improved cannula of FIG. 1. FIG. 5 is a cross sectional side view of the gland retainer of the improved cannula of FIG. 1 shown in isolation. FIG. 6A is a side view in partial cross section of an obturator with an improved shield design and mechanism according to the present invention. FIG. 6B is a cross sectional side view of a cannula with a flexible flapper valve for use with the obturator of FIG. 6A. FIG. 7A illustrates a side view of the improved shield design in its fully extended position covering the pointed blade of the obturator. FIG. 7B illustrates a top view of the shield of FIG. 7A. FIG. 8A illustrates a side view of the improved shield design in a partially retracted position exposing only portions of the sharpened edges of the pointed blade of the obturator, but covering the sharp tip of the blade. FIG. 8B illustrates a top view of the shield of FIG. 8A. FIG. 9A illustrates a side view of the improved shield design in a fully retracted position exposing the tip and sharpened edges of the pointed blade of the obturator. FIG. 9B illustrates a top view of the shield of FIG. 9A. FIGS. 10A through 10D show, in sequential diagrammatic sectional views, an embodiment of the present invention as used to puncture a body cavity wall of a patient. DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention commonly known as a trocar is comprised of two major components. These are a cannula 13 such as those illustrated in FIGS. 1A and 6B, and an obturator such as the traditional obturator 11 in FIG. 1B or the shielded obturator 12 illustrated in FIG. 6A. The obturator 11 of FIG. 1B and cannula 13 of FIG. 1A are interfitting and as explained below are used together to penetrate a body cavity wall. Once the body cavity wall is penetrated, however, the obturator 11 may be removed and other medical instruments may be introduced into a lumen 19 of the cannula 13. The cannula 13 of FIG. 1A consists of three components and two seal assemblies. The components are the cannula tube 14, the gland retainer 42 and the cap 44. The outer surface of the cannula tube 14 of FIG. 1A is shown with a helically wound thread 49 preferably beginning at a reduced height a short distance from the distal end 68 of the cannula tube 14 and gradually increasing to its full height as it proceeds toward the proximate end 69. At the proximate end 69 of the cannula tube 14, there is a raised flange 36 for convenience in handling the cannula 13. The proximal end of the cannula above the raised flange 36 forms a handle. Above the flange 36 is a gas port 16 which can be connected to a gas supply, not shown, to supply gas through the lumen 19 of the cannula tube 14 into a body cavity to create or maintain pneumoperitoneum. Above the gas port 16 are male threads 65 which allow the cannula tube 14 to be securely coupled with cap 44 which has corresponding female threads 66. The cap 44 also has an aperture 55 to permit insertion of an obturator 11, and gripping protrusions 67 to facilitate fastening and unfastening the cap 44. Mounted concentrically mostly within the proximate end 69 of the cannula tube 14 and held in place by the cap 44 is the gland retainer 42. The cap 44 is shown in isolation in FIGS. 3A and 3B. The gland retainer 42 shown in isolation in FIG. 5, holds two seals, 43 and 41, in place. Flexible flapper seal 43 shown in FIG. 4A has an annular outer casing 56 which engages the gland retainer 42. A raised edge 45 at the distal end of the gland retainer 42 fits within a channel 47 shown in FIG. 4A formed by the lips of the C shaped edge formed by a lip of the outer casing 56 of flexible flapper seal 43, shown in FIGS. 1A and 2. A second raised edge 46 at the proximate end of the gland retainer 42 fits with a channel formed by a similar C shaped edge of the outer casing of wiper seal 41. Both the membrane seal 41 and flexible flapper seal 43 are fabricated from materials having sufficient elasticity that the outer casings of the seals 41 and 43 can be stretched over ridges 46 and 45 respectively. The wiper seal 41 is of conventional design, however, the flexible flapper valve 43 is of novel construction. As shown in FIGS. 4A and 4C, a hinge 57 extends inward from the outer casing 56 of the flexible flapper valve 43 and mounted on the hinge is the flapper 58. In the preferred embodiment a hard plastic or metal button 59 is mounted on or through the flapper. When the piercing tip 80, shown in FIG. 1A, of an obturator 11 is inserted through the opening 55 in the cap 44, and through the wiper seal 41, the piercing tip contacts the hard button 59 of the flexible flapper valve 43 and begins to deflect the flapper 58 from its normal closed position flush against the distal end of the gland retainer 42. Because the flapper 58 is preferably molded or stamped in one piece with the hinge 57 and outer casing 56 of an elastomeric material, the piercing tip of the obturator might otherwise penetrate or cut into the flapper 58 which could cause resistance to entry of the obturator or damage the flapper 58 so that it would no longer seal the cannula 13 or gland retainer 42 effectively against loss of air through the opening 55 in the cap 44. The hard button 59 alleviates these possible problems. When the obturator 12 or other endoscopic instrument is removed from the cannula 13, the resiliency of the hinge 57 causes the flapper 58 to move to a partially closed position. The flapper 58 is then firmly closed and sealed by action of the air pressure from the inflated body cavity, such air pressure pushing the flapper 58 against the edges of the distal end of the gland retainer 42 and thereby forming an effective seal against further loss of gas. In its preferred embodiment, the cannula tube 14 portion is manufactured of a durable material such as stainless steel or titanium alloys, capable of withstanding repeated high temperature cleaning and sterilization, while the gland retainer 42 is made of an inexpensive plastic. The cap 44 may be made of either type of material. The gland retainer 42, containing the flapper valve and possibly also containing a wiper seal, is difficult to clean and sterilize. However, the gland retainer 42 and seals 41 and 43 are relatively inexpensive to manufacture and can be discarded after each use. The cannula tube 14, and optionally the cap 44, are relatively easy to clean and sterilize and need not be discarded. By reusing the cannula tube 14, and the cap 44 if manufactured of an appropriate material, cost and waste can be minimized. If desired the cap 44 can also be manufactured of inexpensive plastic and discarded with the gland retainer 42 after use. A shielded obturator 12 is shown in greater detail in FIG. 6A. The shielded obturator 12 in FIG. 6A includes an obturator housing 17 which forms a grip or handle. The shielded obturator 12 also includes a hollow obturator sheath 18, the proximal end of which is mounted to the obturator housing 17. The distal end of the obturator sheath 18 is fitted with a piercing tip 80. Housed within the obturator sheath 18 is the shield 15, shown in its extended position with the distal end 51 covering the piercing tip 80. The proximal end 50 of the shield 15 is received some distance within the hollow obturator sheath 18 where it is outwardly biased by a biasing means such as the coiled spring 21 interposed between the proximal end 50 of the shield 15 and the inner wall 20 of the obturator housing 17. Still referring to FIG. 6A, a double action latch 34 for locking the shield 15 in both the extended position and a partially retracted position is illustrated. The latch 34 comprises a biasing means such as the spring 27, a blocking member such as the illustrated pivoting chock 25, an actuator such as the trigger 48, shown in FIG. 6B, and a linkage between the actuator and the blocking member such as the linking member 24. The illustrated latch operates to keep the shield 15 locked in the extended position except when armed or actuated. To lock the shield 15, a solid portion 30 of the pivoting chock 25 engages with a first lug 32 on the proximal end 50 of the shield 15 to prevent the shield 15 from retracting further toward the inner wall 20 of the obturator housing 17 and thereby exposing the piercing tip 80. In order to permit the shield 15 to be retracted, the latch 34 must first be armed or actuated. To accomplish this, the shielded obturator 12 and cannula 13 must first be interfitted. In FIGS. 6A and 6B, the distal end 22 of the linking member 24 from the shielded obturator 12 is received through opening 62 and engaged in the trigger 48 mounted in the cannula 13. Also a guiding tab 60 from the shielded obturator 12 is received in an opening 61 in the cannula 13. By depressing push buttons 63 and 64, the guiding tab 60 and linking member 24 can be disengaged, allowing for easy separation of the shielded obturator 12 from the cannula 13. To arm the latch 34 in the embodiment illustrated in FIG. 6A, the distal end 22 of the linking member 24 engages with a trigger 48, shown in FIG. 6B. When the trigger 48 is depressed and pushed forward toward the piercing tip 80, the linking member 24 is likewise pulled forward. A hook 29 on the proximate end of the linking member 24 is engaged in a cradle 28 on the pivoting chock 25. As the linking member 24 is pulled forward, it causes the pivoting chock 25 to turn on its pivot 26 and thereby rotates the solid portion 30 away from the lug 32 on the proximal end 50 of the shield 15. This arms or actuates the obturator 12. As the trigger 48 continues forward, the linking member 24 also travels forward until a stop block 23 halts further movement. When the stop block 23 halts further movement, the linking member 24 is in measured close proximity to a cam 31 protruding from the proximal end 50 of the shield 15. As soon as the surgeon applies pressure to the trocar 10, the proximal end 50 of shield 15 is pushed back into the housing 17. After the shield 15 is pushed back sufficiently that a second lug 33 on the proximal end 50 of the shield 15 has reached the solid portion 30, the cam 31 on the proximal end 50 of the shield 15 then dislodges the hook 29 of the linking member 24 from the cradle 28 of the pivoting chock 25. A biasing means such as the spring 27 acting on the pivoting chock 25 now causes the solid portion 30 of the pivoting chock 25 to try to return to its original locking position. When the shield 15 is fully retracted, the pivoting chock 25 cannot return to a locking position. Yet if the shield 15 should extend even partially, so that it is no longer fully retracted, the solid portion 30 of the pivoting chock 25 can engage the second lug 33, and become locked against the shield again becoming fully retracted. This is referred to as a partially locked or semi-protective position. In this fashion the second lug 33 acts as a detent allowing the shield to rachet forward to an extended position. If the distal end 51 of the shield 15 and piercing tip 80 are properly designed, the partially locked position can provide substantial protection to the patient. With the pointed blade designs discussed in FIGS. 7-9, it is possible to have the shield cover the sharp tip 87 of the blade and allow only the sharpened edges 91 of the blade to remain exposed as shown in FIG. 8. This allows the sharpened edges 91 to continue cutting the body cavity wall but without risk of exposing the sharp tip 87 thus reducing the chance of inadvertent injury during the incision. When the entire piercing tip 80 of the obturator 12 has passed through the body cavity wall and there is no resistance to the spring loaded shield the shield 15 will spring completely forward to cover the piercing tip 80, as shown in FIGS. 6A and 7, and the pivoting chock 25 will pivot into its original locking position, where the solid portion 30 of the pivoting chock 25 acts to block rearward movement of the first lug 32 on the proximate end 50 of the shield 15. In this fashion, once the body cavity wall has been pierced by the sharp tip 87 and the shield 15 has partially locked, the shield 15 cannot again retract to expose the sharp tip 87 of the piercing tip 80 unless the operating physician uses the trigger 48 to rearm the trocar 10. Once the piercing tip 80 has completely penetrated the body cavity wall and the shield 15 has fully locked, the shield 15 cannot again retract to oppose any portion of the piercing tip 80. Referring again to FIG. 6B, the cannula 13 includes an outer housing 40, an inner housing 39, and a cannula tube 84 which is secured to and extends from the inner housing 39. In addition, the inner housing 39 is mounted within the outer housing 40. The inner housing 39 contains a gas sealing means such as the illustrated flexible flapper valve 43. The flexible flapper valve 43 will allow the introduction of the obturator sheath 18 of a shielded obturator 12, or other endoscopic instruments (not illustrated) without substantial loss of gas from the body cavity. As shown in FIG. 6B, when no instrument is inserted, the flapper 43 closes against the end plate 37 to maintain the gaseous pressure in the body cavity. FIGS. 7-9 illustrate the presently preferred embodiment of the piercing tip 80, which is comprised of a pointed blade 81. The illustrated pointed blade 81 is substantially planar and has a central sharp tip or pointed distal end 87 and straight sharpened edges 91 proceeding from both sides of said distal end 87 to the shoulders 88. In alternative constructions, the blade 81 may have slight convex or concave curve to the sharpened edges 91. The point of the blade may be acute as illustrated or rounded. Other variations of a pointed blade or blades may also be utilized. As shown in FIGS. 7A and 7B, the distal end 51 of the shield 15 used with the illustrated pointed blade is &#34;bottle shaped&#34; or &#34;dolphin nose shaped&#34; in that it has a blunt end 92 at its most distal point which widens slowly at first and then more rapidly so that there is a slight concavity 94 as the distal end 51 of the shield 15 proceeds from the blunt end 92 to a shoulder 93. As shown in FIGS. 8A and 8B, when the shield end 51 is of this shape, it is possible for the blunt end 92 to cover the pointed distal end 87 of the blade 81 so that no additional penetration is possible, while still leaving portions of the sharpened edges 91 exposed. In this fashion, when the pointed distal end 87 of the blade 81 has penetrated the body cavity wall, but before the blade 81 is entirely through the wall, the blunt end 92 of the shield can move forward and be partially locked while still allowing the sharpened edges 91 of the pointed blade 81 to expand the incision in the body cavity wall so that the obturator and cannula may be introduced to communiate with the body cavity. FIGS. 10A through 10D illustrate the piercing tip 80 and shield end 51 of the trocar 10 formed from the obturator 12 and cannula 13 of FIGS. 6A and 6B in use. In FIG. 10A, a small incision 70 is made in the surface of the skin or epidermis &#34;a&#34; of the body cavity wall 71 of a patient. The piercing tip 80, in this case the pointed blade 81, of the trocar 10 with extended shield 15 may then be placed against the incision 70 as shown. When the trocar 10 is in this position, it may be armed or actuated by depressing the trigger 48 (shown in FIGS. 6A and 6B) and sliding the trigger 48 forward as explained above. When manual force is applied to the obturator housing 17 which forms the trocar handle (shown in FIG. 6A) pushing the trocar to the left, the resistance of the body cavity wall 71 holds back the distal end 51 of the shield 15 and the pointed blade 81 is exposed as shown in FIG. 10B. The pointed blade 81 then slices through the dermis &#34;a&#34; and subcutaneous tissue including fat cells, muscle, and facia, depicted as &#34;b,&#34; &#34;c,&#34; &#34;d&#34; and &#34;e&#34; in FIGS. 10A-10D. The various layers of the body cavity wall 71 vary depending upon the cavity being pierced, but in general, the layers of facia are particularly resistant to penetration. In FIG. 10C, immediately after the sharpened tip 87 of the pointed blade 81 has penetrated the innermost layer &#34;e&#34; of the body cavity wall 71, the blunt end 92 of the distal end 51 of the shield 15 is forced forward through the incision 70 by the bias of the coil spring 21. When the shield 15 reaches this partially retracted position and the preferred double action latch 34 of the obturator 12 illustrated in FIG. 6A is used, the shield 15 will lock so that it cannot be retracted to again expose the sharp tip 87 of the pointed blade 81, yet the shield 15 permits the sharp edges 91 of the blade 81 to continue cutting. This is a semi-protected shield position. In this partially locked or semi-protected position the interior of the body cavity 72 and organs, blood vessels and other anatomical structures are protected against puncture by the sharp end 87 of the blade 81. FIG. 10D shows that the shield end 51, the obturator sheath 18, and the distal end 68 of the cannula tube 84 all proceed through the incision 70 in the body cavity wall 71 as manual force continues to be applied to the trocar 10. Just as the shoulder 93 of the distal end 51 of the shield 15 passes through the inner layer &#34;e&#34; of the body cavity wall 71, the resistance of the body cavity wall 71 is significantly reduced and the shield 15 is pushed to its fully extended position with the distal end 51 covering the pointed blade 81. This action completely protects the interior of the body cavity 72 immediately after the full penetration of the pointed blade 81 through the body cavity wall 71 and protects organs, blood vessels and other anatomical structures from incidental contact even with the sharpened edges 91 of the blade 81. Simultaneously with the full extension of the shield 15 into its protective position covering the pointed blade 81, the pivoting chock 25 engages the first lug 32 on the proximate end 50 of the shield 15, which prevents the shield 15 from being retracted to expose the pointed blade 81 until the trocar 10 is again actuated by depressing and moving the trigger 48 forward. In order to improve the performance of the shield 15 in moving forward through the body cavity wall, it is possible to either mold the shield 15 or shield end 51 from a hydrophilic plastic or to coat the shield with a hydrophilic coating. By providing the shield end 51 with appropriate hydrophilic properties, the shield end 51 can be dipped in a wetting solution, usually of water or an appropriate alcohol solution, and the shield end 51 will then have a lower coefficient of friction and will more easily slide through the incision 70 in the body cavity wall 71 to cover the piercing tip 80. Appropriate coatings or compounds must be substantially nonreactive with respect to living tissue and non-thrombogenic when in contact with blood. Appropriate hydrophilic coatings would include polyvinylpyrrolidone-polyurethane or polyvinybutyrol interpolymers as described in U.S. Pat. Nos. 4,100,309 and 4,119,094. Appropriate molding compounds, which could alternatively also be applied as coatings, include hydrophilic polymer blends with thermoplastic polyurethane or polyvinylbutyrol and hydrophilic polyvinylpyrrolidone or other poly(N-vinyl lactans) as described in U.S. Pat. Nos. 4,642,267 and 4,847,324. An appropriate hydrophillic coating will reduce the coefficient of friction for stainless steel by over 60% and can reduce the coefficients of friction for plastics by over 90%. This permits hydrophillic plastics to be used for the shield 15 rather than steel. The same hydrophilic coatings or compounds may also be used in the cannula 13 which will facilitate the entry of the distal end 68 of the cannula 13 into the incision made by the obturator 12. Although the sharpened blade 81 does not provide substantial resistance, some minor improvement might also be achieved by applying a hydrophillic coating to this blade 81. Numerous alterations of the structures herein described will suggest themselves to those skilled in the art. It will be understood that the details and arrangements of the parts that have been described and illustrated in order to explain the nature of the invention are not to be construed as any limitation of the invention. All such alterations which do not depart from the spirit of the invention are intended to be included within the scope of the appended claims.

Summary: A trocar is formed from a cannula and an interfitting obturator for penetrating body cavity walls in laparoscopic and endoscopic surgery. The obturator is provided with an improved piercing tip having a pointed blade and an improved double locking blunt nosed shield design. Hydrophilic coatings or components are also employed on the shield and cannula to facilitate quicker shield action covering the pointed blade and ease of entry of the cannula tube through the body cavity wall. The cannula also has an improved inexpensive flexible flapper valve and can be manufactured with a reusable cannula tube but disposable flapper valve assembly to minimize cost.

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Summarize: A Brazilian man arrested for stabbing a woman to death calmly confessed to murdering another 41 people - putting him among history's most prolific serial killers, it was reported today. Sailson Jose das Gracas, 26, told police in Rio de Janeiro he would go out 'on the hunt' for white women after studying each victims for up to a month. In an unreported reign of terror spanning nine years, Gracas claims to have murdered 37 women, two men and one two-year-old child in the outlying area of Rio de Janeiro known as Baixada Fluminense. Scroll down for video. Sailson Jose das Gracas, 26, told police in Rio de Janeiro he would go out 'on the hunt' for white women after studying each victims for up to a month. He told police: 'I would watch the victim, study her. I would wait a month, sometimes a week, depending on the place. I would get to know where she lived, how her family were, if she passed by on the street, I would watch, have a look at her house, studying her. 'In the early hours of the morning, I'd get through a gap in her house, really easily, and take advantage.' In an interview in front of TV cameras at the police station in Nova Iguacu, Garcas said he found pleasure in seeing the life drain out of the women he murdered. He said: 'I would stop in the bakery, or in a square, reading a newspaper. I would look at a woman and say to myself, "it's that one". 'I would follow her to her house, but I never raped anyone. Women for me has to be white, not black, because of my colour.'I got pleasure from them fighting, screaming and scratching me. I thought that I was crazy, sometimes normal.' Chillingly, Garcas admitted he had become 'addicted' to murder. In an unreported reign of terror spanning nine years, Gracas claims to have murdered 37 women, two men and one two-year-old child in the outlying area of Rio de Janeiro (pictured) known as Baixada Fluminense. 'The first woman I killed I strangled with my hands. I felt pleasure, I liked it. I felt a very strong desire. I've already killed 38 women, I'm addicted,' he said. He also told camera he was eleven when he lost his father, who was electrocuted during an accident at work. He said: 'It marked me a lot. But that wasn't the reason why I committed these crimes.' Marcelo Machado, the Rio police chief investigating the supposed serial killer, said Sailson found pleasure in stragulating his victims, and masturbated after his murders. Amelia Dyer - known as 'The Baby Butcher' Killed 200 children in the 1800s in Bristol, UK. Luis Garavito - known as 'The Beast' Confessed to 140 child murders in Colombia in the 1990s, but may have killed as many as 400. Pedro Lopez - known as 'The Monster of the Andes' Between 1969 and 1980 in Colombia, Ecuador and Peru he killed 110 young girls. He was released from jail in 1998. No one knows where he is. Daniel Camargo. He confessed to killing 72 people in Colombia and Ecuador between 1974 and 1986. Pedro Rodrigues Filho. Between 1967 and 2003 this Brazilian murdered 71 people and was sentenced to 128 years. Forty seven of his victims were inmates. Kampatimar Shankariya. He murdered at least 70 with a hammer in India between 1977 and 1978. Yang Xinhai. Chinaman Xinhai used axes, meat cleavers and hammers to butcher 67 people between 2000 and 2003. Abul Djabar. He used turbans to strangle 65 men and boys up in 1970 and the years leading up to it in Afghanistan. Andrei Chikatilo. Known as the Butcher of Rostov, he murdered 53 between 1978 and 1990. Anatoly Onoprienko. Known as the Beast of Ukraine, between 1989 and 1996 he murdered 52. Source: Wikipedia. He said: 'On occasions, he killed his victims and masturbated on seeing them with their eyes open.' Police chief Pedro Henrique Medina, from the Baixada Fluminense Homicide Division, said Gracas voluntarily confessed to the murders have being caught in the act of murdering his latest victim. He said he believed his testimony because only the author of the crimes could recall them in so much detail. He said: 'His desire to kill was for women, and he didn't kill black women, just white. He followed the victim, studied her step by step until he managed to commit the crime.' Gracas was arrested after stabbing to death a 26-year-old woman identified as Fatima Miranda, in her home in the town of Nova Iguaçu, Greater Rio de Janeiro. During the investigation into her death, police officers arrested Gracas and two other people, Cleusa Blabina and Jose Messias, who were with Ms Miranda on the night of her death. The three were arrested after giving contradictory versions of what happened when questioned by police. After they were taken to the police station, Gracas admitted that he had been paid by the other two accomplices to murder Ms Miranda. He also admitted killing two men for money, and then detailed the dozens of other women he had murdered for his own pleasure in his serial killing spree. Gracas claimed commiting murder would help him 'calm down'. He said: 'When I didn't do it, I'd become nervous, I'd pace up and down inside the house. And when I'd done it I'd become calmer. I'd do a victim, and I could stay two months without doing it, or three. I'd stay fine, I'd just be thinking of that one that I'd killed. Then I'd go out on the hunt. 'I didn't kill worrying about going to jail. I did everything well, it's because I really like it doing it. I was most concerned with fingerprints, or if the place had a camera. I wouldn't take any documents with me so I wouldn't leave any clues for the police.' During his testimony Gracas also confessed to killing a two-year-old child because he was afraid her cries might wake up neighbours after he had killed her mother. He said his taste for murder began as a teenager. He said: 'I started to steal small things, do small robberies. And as I grew up I started having other thoughts. My thinking started to change, understand? From robbing, I started to think about killing. Aged 15, I stole handbags. After that, aged 17, I killed the first person. 'It gave me that adreneline rush, the first woman. And I thought about jail. Will I get caught? The things flowed well. So I started thinking about doing it more and more. And I liked it and started to get used to it.' Chillingly, Gracas claimed he did not regret any of the murders. Tiago Henrique Gomes da Rocha, another Brazilian, confessed to murdering at least 39 people in just three years. He said: 'I have no regrets, no. For me what I did is done. I wouldn't change anything, I don't regret anything. If I leave prison in 10, 15 or 20 years, I'll start doing the same thing again. It's my will, there's no other way. I leave, choose my 'women', the women I like to choose, and if I think that it has to be, it will.' According to police records, Sailson was arrested twice in 2007 for robbery and theft, carrying illegal firearms in February 2010 and theft again in February this year. But his murders remained undiscovered until today. Police have now launched an investigation into Gracas' claims and are calling on families of murder victims over the last nine years to get in touch. Rocha began killing in 2011, at first targeting homeless people, gays and prostitutes. Gracas spent last night in a police cell and will be transferred to a maximum security jail in Rio de Janeiro today. His arrest comes after Tiago Henrique Gomes da Rocha, another Brazilian, confessed to murdering at least 39 people in just three years. Rocha, 26, was arrested in October after being pulled over in the city of Goiania, when local police found he was facing trial for stealing a motorcycle's license plate in January. He was stopped while riding with fake plates on the motorcycle he often used during the murders, when he would drive by and attack random victims before speeding away. A day later, he confessed to killing 39 people — 15 young women as well as prostitutes and random people he thought to be gay or homeless. In a sick twist, it has been revealed that Rocha was in a romantic relationship with a pretty church girl with long hair, her appearance similar to that of his victims, the newspaper Folha de S. Paulo reported. Rocha began killing in 2011, at first targeting homeless people, gays and prostitutes. His first victim was a man, the first of 17 males he would eventually kill. This year, he turned his attention to young women. The list of the 15 most prolific modern serial killers is topped by Colombian Luis Garavito, a child murderer and rapist known as 'La Bestia' or 'The Beast.' He is thought to have killed more than 400 people, mostly street children. His proven victim count is 138. Pedro Rodrigues Filho, Brazil's most prolific killer, claimed to have killed more than 100 victims, 47 of them inmates. He was convicted of killing 71 people. He killed his first two victims at the age of 14 and murdered his father and ate a piece of his heart

Summary: Sailson Jose das Gracas, 26, was arrested for stabbing a woman to death. He then told police in Rio de Janeiro that he had murdered another 41. Gracas claims to have murdered 37 women, two men and one two-year-old. He said he got pleasure from them 'fighting, screaming and scratching me' Rio police chief said Sailson found pleasure in strangulating his victims. 'Killing gave me that adrenaline rush,' Gracas said in his testimony. It comes after another Brazilian confessed to at least 39 murders.

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Write a title and summarize: L'OTAN devait dévoiler, lundi 17 mai, à Bruxelles, les conclusions d'un groupe de douze experts mandatés, en 2009, par le secrétaire général Anders Fogh Rasmussen, pour redéfinir le concept stratégique de l'Alliance atlantique. Confrontée à la guerre de Géorgie, en 2008, aux doutes quant à l'issue de la mission du corps expéditionnaire en Afghanistan et, plus généralement, à la question de ses missions futures dans un monde de menaces globalisées, l'OTAN doit adopter officiellement à Lisbonne, en novembre, son nouveau plan d'action. D'ici là, M. Rasmussen aura rédigé son propre texte et consulté les 28 Etats membres, pour dégager un consensus au niveau intergouvernemental. Le groupe de réflexion, qui a procédé à diverses consultations - y compris à Moscou - était présidé par l'ancienne secrétaire d'Etat américaine Madeleine Albright. Bruno Racine, président de la Bibliothèque nationale de France et président du conseil de la Fondation pour la recherche stratégique (FRS), en faisait partie, au titre des spécialistes choisis par M. Rasmussen. "Le dernier concept stratégique, défini en 1999, était marqué par l'optimisme de l'après-guerre froide. En onze ans, l'environnement a changé, explique-t-il : les relations avec la Russie n'ont pas pris la direction souhaitée, de nouvelles menaces sont apparues, comme le terrorisme, les cyberattaques ou le développement de capacités balistiques." Et puis, surtout, l'intervention en Afghanistan s'est avérée "beaucoup plus lourde et plus risquée que celle en Bosnie". Le document dévoilé lundi maintient les grands principes, dont la permanence du lien Europe-Amérique du Nord, vu comme un facteur de la sécurité internationale, et l'article 5 du traité qui affirme la solidarité entre tous les membres en cas d'attaque contre l'un ou plusieurs d'entre eux. "On a senti chez les nouveaux membres un besoin de réassurance, de réaffirmation que cet engagement restait bien la pierre angulaire", souligne M. Racine. Parallèlement, les experts soulignent toutefois le consensus sur le dialogue avec Moscou et "une volonté de faire du Conseil OTAN-Russie un véritable outil, là où nos intérêts convergent : la lutte contre le terrorisme, contre les trafics, la piraterie, etc." Quant aux nouvelles perspectives d'élargissement de l'Alliance à l'Est, qui irritent tellement les dirigeants russes, le document "réaffirme la politique de la porte ouverte, y compris pour la Russie, si un jour elle le souhaite", indique M. Racine.

Title: "L'OTAN veut s'adapter aux" "menaces diffuses" "d'un monde globalisé Summary: Le groupe de réflexion présidé par l'ancienne secrétaire d'Etat américaine Madeleine Albright suggère qu'à l'avenir, les pays européens seront exposés à des menaces balistiques et plaide en faveur d'un bouclier antimissile. Diverses questions, dont le financement et le contrôle du dispositif, demeurent toutefois en suspens.

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fr

Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to crop harvesters, and more particularly to such a harvester which is adapted to travel along a row of crops, and to engage said crops and bring them more closely together to enhance the harvesting operation of the crops. 2. Background Art There are in the prior art harvesting machines which are adapted to harvest such crops as berries, grapes, fruit, other produce from standing plants. Normally such machines comprise a chassis having two side portions which define therebetween a crop engaging area. As the machine travels down a crop row, the crops pass between the two side portions of the machine, and suitable harvesting implements, such as beater rods or the like engage the crop to shake the crop and thus cause removal of the berries or other food product. It has been found that this harvesting operation can be enhanced if the lower portions of the crops or plants can be engaged so as to squeeze the lower portions of the crops toward the row centerline. Accordingly, one prior art device for accomplishing this is to have two crop engaging belts positioned on opposite sides of the crop, with these belts being urged toward a center location so as to engage the crops with the appropriate pressure. These two belts are then driven so that the crop engaging portions thereof travel rearwardly at substantially the same speed as the forward travel of the vehicle, so that the crop engaging belt portions are &#34;stationary&#34; relative to the crop. Thus, there arises a need to coordinate the rearward speed of the belt engaging portions with the forward travel of the machine. Such an aggregating device is disclosed on pages 33 and 34 of a book entitled &#34;Mechanical Harvesting of Raspberries: Development of a System for Scottish Conditions&#34;, authored by A. M. Ramsay, published by the Scottish Institute of Agricultural Engineering, Technical Report No. 7. Also, illustrations of this same system are shown in photographs appearing in different parts of this publication. U.S. Pat. No. 4,204,389 (Delfosse) shows an endless conveyor in a harvester, the conveyor being timed to be stationary relative to the crop. One means of monitoring the ground speed of the harvesting machine so that the speed of a crop engaging member can be controlled is by actually measuring the forward speed of the machine and then providing control means responsive to the forward speed of the machine. One method of accomplishing this is by means of a ground engaging wheel which engages the ground and thus rotates at a speed proportional to the forward velocity. One such system is shown in U.S. Pat. No. 4,176,511 (Scudder et al), entitled &#34;Conveyor System for a Harvester&#34;, where there are shown two conveyors in the form of endless belts which are positioned on opposite sides of the crop row and which travel rearwardly relative to the machine so that these are &#34;stationary&#34; relative to the crop row. With reference to FIGS. 8, 16 and 17, there is a hydraulic motor 30a which rotates the two belt conveyors, with this motor 30a being driven by a motor 49 which powers a pump 50 that in turn delivers hydraulic fluid through a throttle control valve 53 that in turn delivers the hydraulic fluid to the motor 30a. To control the speed of the motor 30a, the hydraulic fluid that passes from the motor 30a is directed through a feedback system to control operation of the throttle valve 53. More specifically, there is an unloaded ground engaging wheel 52, the speed of rotation of which is a measure of the speed of forward travel of the machine. This wheel 52 connects through a shaft 59 to a drive transmission comprising gears 61, 62 and 63. The hydraulic motor 55 which is a positive drive motor driven from the pump 30a rotates a gear 64 which in turn rotates a gear 58 which in turn rotates the case 60 of the differential transmission. When the speed of the hydraulic motor 30a matches the ground speed as indicated by the wheel 52, the rotational speed of the differential casing 60 matches the rotational speed of the shaft 59 so that there is no output from the differential transmission, and the shaft 54 leading to the throttle valve 53 remains stationary. However, when a difference in the two speeds is sensed, the shaft 54 will be cause to rotate to change the setting of the throttle valve 53 so that the ground speed does match the speed of the hydraulic motor 30a that in turn drives the belt conveyors. One of the problems of this type of system is that not only must the operating components be closely matched, but there is some margin of error in that the ground wheel (such as a ground wheel 52) may not be a truly reliable indication of ground speed. For example, if the ground surface has some irregularities where the ground wheel must travel upwardly and downwardly over the ground surface, the rate of rotation will increase relative to the forward speed of the vehicle. Further, even though these ground wheels can be arranged with devices to increase friction (high friction treads or even pins or the like) which would engage the ground, there can under certain circumstances be slippage of such wheels. Various other systems and method time the speed of a conveyor or the like to the rotation of a ground wheel, by using a governor responsive to rotation of the ground wheel to control a throttle valve in a hydraulic conveyor drive system, U.S. Pat. No. 3,414,200 (Savory), monitors for monitoring the speed of the conveyor belt and of a vehicle which use electro-optical aperture discs, U.S. Pat. No. 3,550,866 (Swenson), magnetic sensors 46 for sensing and equalizing the speeds of a driven axle and a non-driven axle, U.S. Pat. No. 4,441,848 (Bailey), chains and sprockets, U.S. Pat. Nos. 4,195,570 (Rodriquez) and 3,901,005 (Rohrbach et al) and an intermediate wheel 101 that meshes with a ground wheel and a conveyor to drive the conveyor, U.S. Pat. No. 4,081,094 (Pereira et al). Additionally, U.S. Pat. No. 4,212,428 (Walker) shows a ground wheel that drives a pump 52. The pump 52 provides fluid pressure to operate a motor 36 that drives a conveyor belt. SUMMARY OF THE INVENTION The present invention relates to a system for positioning crops in a row which is being harvested by a machine. A system for positioning crops in a row which is being harvested by a machine in a manner to minimize damage to the crops, comprises crop aggregating means, crop aggregating drive means, and control means. The crop aggregating means, connected to the harvester, aggregates the crops by bringing first and second crop engaging means into engagement with the crops as the crops pass rearwardly with respect to the machine in a pathway defined between the first and second crop engagement means. The crop aggregating drive means drives the crop engaging means rearwardly along the crop engaging path with respect to the machine as the machine travels forwardly relative to the crops. The control means responds to a force imparted by the engaging means by engagement with the crops along the crop engaging path, thereby matching the speed of the crop engaging means with the speed of the machine. This protects the crops from the effects of friction between the crop engaging means and the crops. The drive means comprising a hydraulic motor, receives hydraulic fluid the pressure of which is controlled by the control means. The control means comprises pressure relief valve means operably connected to the hydraulic motor, thereby controlling fluid pressure directed to the hydraulic motor. The hydraulic pump means is operably connected to the hydraulic motor means so as supply hydraulic fluid under pressure to the hydraulic motor means. At a given volumetric flow rate of hydraulic fluid, the hydraulic pump means drives the machine at a given linear speed which is less than the linear speed at which the crop aggregating drive means drives linearly the crop engaging means, at the given rate of volumetric flow. Accordingly, with the speed of travel of the machine being equal to the linear speed of the crop engaging means, the pressure relief valve means remains in an open position to bypass a portion of hydraulic flow around the aggregating drive means. The pressure relief valve means is connected in parallel with the hydraulic motor means in a manner that fluid flow above a preset pressure level is bypassed from around the hydraulic motor. The pressure relief valve means comprises pressure differential valve means which opens in response to a predetermined difference in pressure at locations upstream and downstream of the hydraulic motor. Alternatively, the crop aggregating drive means comprises first and second hydraulic motors connected to, respectively, the first and second crop aggregating means, the control means comprising pressure relief valve means positioned in parallel with the first and second hydraulic motors. The pressure relief valve means comprises pressure differential valve means which senses pressure at locations upstream and downstream of the first and second hydraulic motors. Also, there is selectively operable valve means connected between the hydraulic pump means and the crop aggregating drive means to selectively drive hydraulic fluid from the hydraulic pump means to the hydraulic motor means, bypassing the crop aggregating drive means, or on a path through the crop aggregating drive means to the hydraulic motor means. In a second embodiment, the crop aggregating drive means comprises a hydraulic motor means which delivers power to the crop aggregating means and hydraulic pump means which delivers hydraulic fluid to the hydraulic motor, the system further comprising ground speed monitoring means to measure speed of the machine and to control output of the hydraulic motor means in accordance with the speed of the machine. The control means comprises pressure relief valve means operably connected to the hydraulic motor means thereby controlling fluid pressure directed to the hydraulic motor means, wherein at a given volumetric flow rate of hydraulic fluid, the hydraulic pump means drives the machine at a given linear speed which is less than a linear speed at which the crop aggregating drive means drives linearly the crop engaging means, at the given rate of volumetric flow. Accordingly, the speed of travel of the machine is equal to the linear speed of the crop engaging means, with the pressure relief valve means remaining in an open position to bypass a portion of hydraulic flow around the aggregating drive means. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a top plan view of a harvesting machine employing teachings of the present invention with the top removed and with portions of the components removed for purposes of illustration, also including a schematic representation of a drive system for the crop aggregating belts; FIG. 1a is a detail of a positioning member which is used to support the crop aggregating belt; FIG. 2 is a front end view of the harvester of FIG. 1; FIG. 3 is a view similar to the view of FIG. 1 showing a second embodiment of the invention; FIG. 4 is a schematic view of the pump and drive components in a hydraulic system in the present invention; FIG. 5 is a view similar to FIG. 4 showing a second embodiment of the invention. DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 is a top plan view showing generally a harvesting machine 10 suitable for incorporating the system of the present invention. As is common in the prior art, the operator&#39;s section 14 provides a seat 18 for the operator and a manually operated steering wheel 20. In addition, the operator&#39;s section 14 could provide a location for collecting the food product that is being harvested and possibly serve other functions. The harvesting section 16 comprises right and left frame portions 22 and 24, respectively, which define therebetween a middle crop harvesting area or region 26. For purposes of description, the harvesting section 16 will be considered as having a center longitudinal axis 28, and this axis 28 will generally coincide rather closely with a centerline of a row of the crops which are being harvested. In describing the present invention, the terms &#34;inward&#34; or &#34;inwardly&#34; will denote a direction toward, or proximity to, the longitudinal center axis 28, while the terms &#34;outward&#34; or &#34;outwardly&#34; will denote the opposite. In general, such harvesting machine 10 will comprise a turning means, such as at least one forward steerable wheel (not shown for ease of illustration) located at a forward corner of the machine, and being steerable to properly align the machine 10, and a pair of rear wheels, one of which is shown at 30, with at least one of these rear wheels being a power driven wheel to move the machine. Also, as is common in the prior art, there are provided suitable harvesting elements such as beater rods 32 which reciprocate to come into engagement with the crop so as to shake the food product from the bushes or plants. A suitable automatic steering means can be provided. Specifically, as shown herein, there are two crop engaging members 34 positioned at a forward location on the machine 10 on opposite sides of the longitudinal axis 28. These crop engaging members have converging end portions 36 that define a converging throat to receive the crop, and lateral displacement of these crop engaging members 34 operate a suitable control mechanism which automatically steers the front steerable wheel to maintain the machine 10 in close alignment with the crop row. Such a steering mechanism is disclosed in a U.S. Patent Application, entitled &#34;METHOD AND SYSTEM FOR AUTOMATICALLY STEERING ALONG ROW CROPS&#34; with the inventors being the same as the inventors herein, this application being filed on May 19, 1989, the contents of which are hereby incorporated by reference. As indicated previously, the present invention is particularly directed toward a means of aggregating the crop (i.e. squeezing the crop inwardly) to enhance the harvesting operation. As shown herein, there is a crop aggregating assembly 38 which comprises a pair of endless belts 40 which are mounted at a lower location of the chassis 12 and are positioned on opposite sides of the longitudinal axis 28. Each belt 40 has a forward belt portion 42, which engages a related hydraulic drive motor 44 and extends rearwardly and moderately inwardly to a crop engaging belt portion 46 that is positioned more closely to the longitudinal center axis 28. Rearwardly of the crop engaging belt portion 46, the belt extends at 48 around a pair of rear idler wheels 50, with the belt having a return run 52 that leads to the aforementioned forward belt section 42 that engages the drive motor 44. To urge each of the crop engaging belt sections 46 into proper engagement with the crop, there are provided along the length of the belt section 46 a plurality of positioning arms 54 as best shown in FIG. 1A. Each positioning arm 54 is mounted at 56 and coiled to form a coiled spring portion 57 leading to an arm portion 58 which extends from the mounting 56 rearwardly and inwardly to a respective roller 60 mounted at the rear swing end of the arm portion 58, with this roller 60 engaging its related crop engaging belt portion 46, the coil portion 57 bringing the arm portion 58 into proper yielding engagement with the belt section 46. Thus, it is imperative as shown in FIG. 1 that as the crops enter into the harvesting area of the chassis 12, the two belt engaging portions 46 engage the crop (a couple of plants of this crop having the lower portions thereof indicated at 62 in FIG. 2) with the appropriate pressure as determined by the strength of the respective coiled spring portions 57) to hold the lower stems of the crop plant 62 together. As a preliminary comment, is to be understood that the main components of the crop aggregating assembly 38 as described above are already known in the prior art, and actually are shown in the publication noted under Background Art, entitled &#34;Mechanical Harvesting of Raspberries: Development of a System for Scottish Conditions&#34;. One of the important considerations in incorporating such a crop aggregating assembly 38 is that the linear speed of each of the crop aggregating belts 40 closely match the forward speed of travel of the machine 10 so that there is no scruffing action which might damage the somewhat sensitive stems of the plants. Further, it is desirable that the force exerted by the crop engaging portions 46 be substantially a lateral inward force, and not a longitudinally directed force which would have the tendency of possibly damaging the stems. With the foregoing in mind, there will now be a description of the drive and control system of the present invention which, in combination with the elements described above, provide unique advantages over the prior art. To describe a first embodiment of the present invention, reference is made to the schematic drawing of FIG. 4. There is a fluid reservoir 64 which provides hydraulic fluid for makeup oil for a main hydraulic pump 66 which in turn delivers fluid under pressure through a selectively operable control valve 68. This control valve 68 has a first position (shown in FIG. 4) where the fluid is moved directly through a line 70 to a main hydraulic motor 72 that in turn rotates the one or more of the drive wheels 30. The valve 68 can be moved to a second position where no fluid is delivered through the line 70, the fluid is directed through a second line 74 that leads to the two aforementioned hydraulic motors 44 which are connected in series with one another. The hydraulic line 76 from the second of these drive motors 44 then leads to the main hydraulic motor 72. There is provided a differential pressure relief valve 78 that is connected through a first line 80, and a second line 82 which extends from the valve 78 to the line 76 that leads from the second motor 44 to the main hydraulic motor 72. This relief valve 78 is arranged so that it responds to a pressure differential in the lines 80 and 82. This function is indicated somewhat schematically, by the broken line 84 extending from the first line 80 into one side of a valve element 86, and a second valve line 88 extending from the line 82 to an opposite side of the valve element 86. A spring member 90 urges the valve element 86 toward its closed position, as shown in FIG. 4. Thus, when the pressure differential in the two lines 80 and 82 is at a lower level, the force of the spring 90 will be sufficient to maintain the valve element 86 in its closed position. However, when the pressure in the line 80 exceeds the pressure of the line 82 by a predetermined margin, the hydraulic pressure will be sufficient to move the valve element 86 (downwardly as shown in FIG. 4) to its open position to cause hydraulic fluid to bypass the two motors 44. Also, the spring member 90 is adjustable so that the level of the pressure differential at which the bypass valve 78 will open can be accurately adjusted. With regard to the setting of the control valve 78, it is to be understood that the pressure drop at locations upstream and downstream of the drive motors 44 will be proportional to the resistance (primarily internal) against the movement of the belts 40 of the crop aggregating assemblies 38. As indicated previously, it is desirable that the engagement of the belt portions 46 against the crops 62 be such that there is essentially an inwardly directed force (i.e. a &#34;squeezing&#34; force toward the longitudinal center locating plane 28), and very little, if any, longitudinal force which would tend to cause the belt portions 46 to possibly scruff away bark or possibly damage the plants in some other way. Thus, the setting of the pressure relief valve 78 should be such that it will react to a pressure differential level which is just at the level at which the motors 44 are able to generate sufficient force to overcome the internal resistance or power losses due to the operation of the assemblies 48 themselves. In actual practice, a fairly close estimate of the setting of the valve 78 can be achieved simply by operating the belt aggregating assembly 38 without engaging any of the crop 62, and then manually grasping one of the belts 40 to determine if the belt can be stopped simply by a moderate force exerted by the person&#39;s hand. At such time as the person is manually able to stop the belt without exerting an excessive amount of force on the belt, the setting of the valve 78 would be at the level where the motors 44 are just able to overcome the internal operating resistance of the assembly 38 and possibly deliver just slightly greater power to compensate for any additional losses which might be contributed, for example, to greater internal frictional resistance of the belt 40 against the positioning rollers 58. To describe the overall operation of the present invention, let it be assumed that the machine 10 is traveling toward a crop, but is not engaged in a harvesting operation. In this instance, the main control valve 68 is set to the position shown in FIG. 4 so that there is no operation of the crop aggregating assemblies 38. At such time as the machine 10 begins moving into engagement with a crop row, the valve 68 is moved to its second position (i.e. moved upwardly in the showing of FIG. 4) so that hydraulic power is delivered first through the belt drive motors 44 and thence through the main drive motor 72 which powers the drive wheel or wheels 30 and possibly performs other functions of the machine 10. It is to be understood that since the fluid flow path is in series between the motors 44 and the motor 72, the relative speeds of these motors 44 and 72 are fixed. Desirably, these relative speeds are such that the linear speed of the belts 40 is just greater than the speed of travel of the machine 10. Thus, with the relief valve 78 closed, there is a tendency for the belts 40 to travel faster than the forward motion of the machine 10. For this reason, in normal operation, the valve 78 will generally remain open just to the extent to provide the proper balance of pressure on the upstream and the downstream sides of the motors 44. Under a situation where the pressure relief valve 78 is set at a level just below the desired pressure level at which it should open, there could be a situation where the power delivered by the hydraulic fluid to the motors 44 would not be sufficient to overcome the internal resistance of the operation of the aggregating assembly 38. Under these conditions, the belt engaging portions 46 would engage the crop 62, but there would not be quite enough power to move the belt portions 44 to match the forward speed of the machine 10. Under these circumstances, there would be a force exerted by the plants 62 engaging the belts 40 to help move the belt portions 46 rearwardly so as to supplement the power supplied by the motors 44. Thus, it is to be understood that while the setting of the pressure relief valve 78 is desirably set at the precise operating level, and even if it is at a level just below the desired operating level, the scruffing action against the plants is substantially alleviated. Let us now examine a situation where the machine 10 requires that greater power be delivered by the main hydraulic motor 72 to maintain an adequate ground speed, with such a situation arising when the machine 10 is traveling up an incline. Under these circumstances, the hydraulic pump 66 must necessarily deliver hydraulic fluid at higher pressure, and this would in turn increase the pressure on the upstream side of the motors 44. However, since the control valve 78 is responsive to differential pressure on the upstream and downstream side of the motors 44, the power delivered to the motors 44 would be substantially the same as if the machine 10 were operating over a level surface where the drive power requirements are less and the fluid pressure in the system upstream of the main drive motor 72 would be less. A second embodiment of the present invention is illustrated in FIGS. 3 and 5, FIG. 3 being a top plan view of the machine and FIG. 5 being a schematic drawing of the control system of the second embodiment. Components of this second embodiment which are similar to those of the first embodiment will be given like numerical designations, with an &#34;a&#34; suffix distinguishing those of the second embodiment. This second embodiment is distinguished from the first embodiment primarily in that the drive system for the belts second embodiment is separated from the main drive system of the machine. Thus, there is provided a relatively small hydraulic pump 92 which draws fluid from a reservoir 94. This pump 92 is connected directly to a speed monitoring ground wheel 96 so that the volume output of the pump 92 corresponds to the rotational speed of the ground wheel 96. As shown herein, there is a direct drive connection between the ground wheel 96 and the pump 92 so that the power for the pump 92 is derived from the ground wheel 96. However, power for the pump could be derived from another source. The pump 92 leads through an on/off valve 98 to the motors 44a which are, as in the first embodiment, connected in series with one another and which are operatively connected to the belts 40a, and the flow from the second motor 44a leads directly back to the pump 92. There is a pressure relief control valve 78a connected in parallel with the two motors 44a, but this pressure relief valve 78a leads directly to the pump 92. As shown herein, this valve 78a is again a pressure relief differential valve. On the assumption (which is not shown) that the reservoir 94 is connected in series between the motors 44a and the main pump 92, and that the fluid in the reservoir 94 is maintained at substantial ambient pressure this valve 78a could be a pressure relief valve that opens simply at a predetermined hydraulic pressure upstream of the valve 78. It is believed that the operation of this second embodiment is evident from the description of the operation if the first embodiment, so it will be described briefly herein. The ground engaging wheel 96 rotates at a speed corresponding to the ground speed of the vehicle 10, causing the pump 92 to operate. The speed of this pump 92, relative to the speed of the ground wheel 96, and of the motors 44a are selected so that with the by-pass valve 78a being closed, the pump 44 will drive the belts 38a at a linear speed greater than the speed of travel of the machine 10a over the ground surfaces. However, as the fluid pressure upstream of the motors 44a increases, this valve 78a opens to an extent just sufficient to drop the pressure in the line upstream of the motors 44a so that there is just enough power delivered to cause linear travel of the belts 40a, but not so much power to enable the belts 40a to scruff against the stems of the plants. It is understood that various modification could be made to the present invention without departing from the basic teachings thereof.

Summary: A system carried in a harvesting machine and adapted to align crops in a row so as to enhance harvesting, comprises a crop aggregating means which aggregates the crops by bringing first and second crop engaging means into engagement with right and left sides of the crops as the crops pass rearwardly. The system further comprises a locomotion means, which provides the main locomotion for the harvesting machine, an aggregating drive means to drive the crop engaging means rearwardly and a control means, connected together in a fluid circuit. The control means comprises a pressure differential valve, and first and second sensors. The pressure sensors are adapted to sense first and second fluid pressures, respectively, at first and second sides of the aggregating drive means and to actuate the pressure differential valve. The pressure differential valve relieves pressure in the fluid circuit when there is a pre-determined pressure difference between the first and second fluid pressures. In a second embodiment the locomotion means is separated from the fluid circuit that provides motive force to the aggregating means. The fluid circuit comprises the pump, the aggregating drive means and pressure control means. The pump operates at a rate in accordance with a rotation of a ground engaging wheel.

big_patent

en

Summarize: XV. HESTER AND PEARL. So Roger Chillingworth--a deformed old figure, with a face that haunted men's memories longer than they liked--took leave of Hester Prynne, and went stooping away along the earth. He gathered here and there an herb, or grubbed up a root, and put it into the basket on his arm. His gray beard almost touched the ground, as he crept onward. Hester gazed after him a little while, looking with a half-fantastic curiosity to see whether the tender grass of early spring would not be blighted beneath him, and show the wavering track of his footsteps, sere and brown, across its cheerful verdure. She wondered what sort of herbs they were, which the old man was so sedulous to gather. Would not the earth, quickened to an evil purpose by the sympathy of his eye, greet him with poisonous shrubs, of species hitherto unknown, that would start up under his fingers? Or might it suffice him, that every wholesome growth should be converted into something deleterious and malignant at his touch? Did the sun, which shone so brightly everywhere else, really fall upon him? Or was there, as it rather seemed, a circle of ominous shadow moving along with his deformity, whichever way he turned himself? And whither was he now going? Would he not suddenly sink into the earth, leaving a barren and blasted spot, where, in due course of time, would be seen deadly nightshade, dogwood, henbane, and whatever else of vegetable wickedness the climate could produce, all flourishing with hideous luxuriance? Or would he spread bat's wings and flee away, looking so much the uglier, the higher he rose towards heaven? [Illustration: "He gathered herbs here and there"] "Be it sin or no," said Hester Prynne, bitterly, as she still gazed after him, "I hate the man!" She upbraided herself for the sentiment, but could not overcome or lessen it. Attempting to do so, she thought of those long-past days, in a distant land, when he used to emerge at eventide from the seclusion of his study, and sit down in the firelight of their home, and in the light of her nuptial smile. He needed to bask himself in that smile, he said, in order that the chill of so many lonely hours among his books might be taken off the scholar's heart. Such scenes had once appeared not otherwise than happy, but now, as viewed through the dismal medium of her subsequent life, they classed themselves among her ugliest remembrances. She marvelled how such scenes could have been! She marvelled how she could ever have been wrought upon to marry him! She deemed it her crime most to be repented of, that she had ever endured, and reciprocated, the lukewarm grasp of his hand, and had suffered the smile of her lips and eyes to mingle and melt into his own. And it seemed a fouler offence committed by Roger Chillingworth, than any which had since been done him, that, in the time when her heart knew no better, he had persuaded her to fancy herself happy by his side. "Yes, I hate him!" repeated Hester, more bitterly than before. "He betrayed me! He has done me worse wrong than I did him!" Let men tremble to win the hand of woman, unless they win along with it the utmost passion of her heart! Else it may be their miserable fortune, as it was Roger Chillingworth's, when some mightier touch than their own may have awakened all her sensibilities, to be reproached even for the calm content, the marble image of happiness, which they will have imposed upon her as the warm reality. But Hester ought long ago to have done with this injustice. What did it betoken? Had seven long years, under the torture of the scarlet letter, inflicted so much of misery, and wrought out no repentance? The emotions of that brief space, while she stood gazing after the crooked figure of old Roger Chillingworth, threw a dark light on Hester's state of mind, revealing much that she might not otherwise have acknowledged to herself. He being gone, she summoned back her child. "Pearl! Little Pearl! Where are you?" [Illustration: Pearl on the Sea-Shore] Pearl, whose activity of spirit never flagged, had been at no loss for amusement while her mother talked with the old gatherer of herbs. At first, as already told, she had flirted fancifully with her own image in a pool of water, beckoning the phantom forth, and--as it declined to venture--seeking a passage for herself into its sphere of impalpable earth and unattainable sky. Soon finding, however, that either she or the image was unreal, she turned elsewhere for better pastime. She made little boats out of birch-bark, and freighted them with snail-shells, and sent out more ventures on the mighty deep than any merchant in New England; but the larger part of them foundered near the shore. She seized a live horseshoe by the tail, and made prize of several five-fingers, and laid out a jelly-fish to melt in the warm sun. Then she took up the white foam, that streaked the line of the advancing tide, and threw it upon the breeze, scampering after it, with winged footsteps, to catch the great snow-flakes ere they fell. Perceiving a flock of beach-birds, that fed and fluttered along the shore, the naughty child picked up her apron full of pebbles, and, creeping from rock to rock after these small sea-fowl, displayed remarkable dexterity in pelting them. One little gray bird, with a white breast, Pearl was almost sure, had been hit by a pebble, and fluttered away with a broken wing. But then the elf-child sighed, and gave up her sport; because it grieved her to have done harm to a little being that was as wild as the sea-breeze, or as wild as Pearl herself. Her final employment was to gather sea-weed, of various kinds, and make herself a scarf, or mantle, and a head-dress, and thus assume the aspect of a little mermaid. She inherited her mother's gift for devising drapery and costume. As the last touch to her mermaid's garb, Pearl took some eel-grass, and imitated, as best she could, on her own bosom, the decoration with which she was so familiar on her mother's. A letter,--the letter A,--but freshly green, instead of scarlet! The child bent her chin upon her breast, and contemplated this device with strange interest; even as if the one only thing for which she had been sent into the world was to make out its hidden import. "I wonder if mother will ask me what it means?" thought Pearl. Just then, she heard her mother's voice, and flitting along as lightly as one of the little sea-birds, appeared before Hester Prynne, dancing, laughing, and pointing her finger to the ornament upon her bosom. "My little Pearl," said Hester, after a moment's silence, "the green letter, and on thy childish bosom, has no purport. But dost thou know, my child, what this letter means which thy mother is doomed to wear?" "Yes, mother," said the child. "It is the great letter A. Thou hast taught me in the horn-book." Hester looked steadily into her little face; but, though there was that singular expression which she had so often remarked in her black eyes, she could not satisfy herself whether Pearl really attached any meaning to the symbol. She felt a morbid desire to ascertain the point. "Dost thou know, child, wherefore thy mother wears this letter?" "Truly do I!" answered Pearl, looking brightly into her mother's face. "It is for the same reason that the minister keeps his hand over his heart!" "And what reason is that?" asked Hester, half smiling at the absurd incongruity of the child's observation; but, on second thoughts, turning pale. "What has the letter to do with any heart, save mine?" "Nay, mother, I have told all I know," said Pearl, more seriously than she was wont to speak. "Ask yonder old man whom thou hast been talking with! It may be he can tell. But in good earnest now, mother dear, what does this scarlet letter mean?--and why dost thou wear it on thy bosom?--and why does the minister keep his hand over his heart?" She took her mother's hand in both her own, and gazed into her eyes with an earnestness that was seldom seen in her wild and capricious character. The thought occurred to Hester, that the child might really be seeking to approach her with childlike confidence, and doing what she could, and as intelligently as she knew how, to establish a meeting-point of sympathy. It showed Pearl in an unwonted aspect. Heretofore, the mother, while loving her child with the intensity of a sole affection, had schooled herself to hope for little other return than the waywardness of an April breeze; which spends its time in airy sport, and has its gusts of inexplicable passion, and is petulant in its best of moods, and chills oftener than caresses you, when you take it to your bosom; in requital of which misdemeanors, it will sometimes, of its own vague purpose, kiss your cheek with a kind of doubtful tenderness, and play gently with your hair, and then be gone about its other idle business, leaving a dreamy pleasure at your heart. And this, moreover, was a mother's estimate of the child's disposition. Any other observer might have seen few but unamiable traits, and have given them a far darker coloring. But now the idea came strongly into Hester's mind, that Pearl, with her remarkable precocity and acuteness, might already have approached the age when she could be made a friend, and intrusted with as much of her mother's sorrows as could be imparted, without irreverence either to the parent or the child. In the little chaos of Pearl's character there might be seen emerging--and could have been, from the very first--the steadfast principles of an unflinching courage,--an uncontrollable will,--a sturdy pride, which might be disciplined into self-respect,--and a bitter scorn of many things, which, when examined, might be found to have the taint of falsehood in them. She possessed affections, too, though hitherto acrid and disagreeable, as are the richest flavors of unripe fruit. With all these sterling attributes, thought Hester, the evil which she inherited from her mother must be great indeed, if a noble woman do not grow out of this elfish child. Pearl's inevitable tendency to hover about the enigma of the scarlet letter seemed an innate quality of her being. From the earliest epoch of her conscious life, she had entered upon this as her appointed mission. Hester had often fancied that Providence had a design of justice and retribution, in endowing the child with this marked propensity; but never, until now, had she bethought herself to ask, whether, linked with that design, there might not likewise be a purpose of mercy and beneficence. If little Pearl were entertained with faith and trust, as a spirit messenger no less than an earthly child, might it not be her errand to soothe away the sorrow that lay cold in her mother's heart, and converted it into a tomb?--and to help her to overcome the passion, once so wild, and even yet neither dead nor asleep, but only imprisoned within the same tomb-like heart? Such were some of the thoughts that now stirred in Hester's mind, with as much vivacity of impression as if they had actually been whispered into her ear. And there was little Pearl, all this while, holding her mother's hand in both her own, and turning her face upward, while she put these searching questions, once, and again, and still a third time. "What does the letter mean, mother?--and why dost thou wear it?--and why does the minister keep his hand over his heart?" "What shall I say?" thought Hester to herself. "No! If this be the price of the child's sympathy, I cannot pay it." Then she spoke aloud. "Silly Pearl," said she, "what questions are these? There are many things in this world that a child must not ask about. What know I of the minister's heart? And as for the scarlet letter, I wear it for the sake of its gold-thread." In all the seven bygone years, Hester Prynne had never before been false to the symbol on her bosom. It may be that it was the talisman of a stern and severe, but yet a guardian spirit, who now forsook her; as recognizing that, in spite of his strict watch over her heart, some new evil had crept into it, or some old one had never been expelled. As for little Pearl, the earnestness soon passed out of her face. But the child did not see fit to let the matter drop. Two or three times, as her mother and she went homeward, and as often at supper-time, and while Hester was putting her to bed, and once after she seemed to be fairly asleep, Pearl looked up, with mischief gleaming in her black eyes. "Mother," said she, "what does the scarlet letter mean?" And the next morning, the first indication the child gave of being awake was by popping up her head from the pillow, and making that other inquiry, which she had so unaccountably connected with her investigations about the scarlet letter:-- "Mother!--Mother!--Why does the minister keep his hand over his heart?" "Hold thy tongue, naughty child!" answered her mother, with an asperity that she had never permitted to herself before. "Do not tease me; else I shall shut thee into the dark closet!" [Illustration] [Illustration]

Summary: Hester and Pearl As Chillingworth walks away, Hester goes to find Pearl. She realizes that, although it is a sin to do so, she hates her husband. If she once thought she was happy with him, it was only self-delusion. Pearl has been playing in the tide pools down on the beach. Pretending to be a mermaid, she puts eelgrass on her chest in the shape of an "A," one that is "freshly green, instead of scarlet. Pearl hopes that her mother will ask her about the letter, and Hester does inquire whether Pearl understands the meaning of the symbol on her mother's chest. They proceed to discuss the meaning of the scarlet letter. Pearl connects the letter to Dimmesdale's frequent habit of clutching his hand over his heart, and Hester is unnerved by her daughter's perceptiveness. She realizes the child is too young to know the truth and decides not to explain the significance of the letter to her. Pearl is persistent, though, and for the next several days she harangues her mother about the letter and about the minister's habit of reaching for his heart.

booksum

en

Summarize: A Manchester man accused of robbing a convenience store while armed with a sharp stone was arrested after returning to the store to apologize. Click to watch News 9's coverage. Manchester police said Carlos Reyes, 46, went to the Shop-N-Go store twice Tuesday. The first time, he took money from the store, and the second time, he came back to return it, they said. Police said the store on Massabessic Street was robbed just after noon Tuesday. "Just after noontime yesterday, an individual walked into the store, allegedly armed with a sharp stone," Lt. Brian O'Keefe said. "He threatened the clerk and took roughly $200 worth of cash and fled." Police released a surveillance photo of the suspect holding the weapon in front of the man behind the counter. Police said they recovered the sharpened rock inside the store. Police were called back to the store later that night for a report of a suspicious person who turned out to be Reyes. "About seven hours later, he decided that he was feeling bad about his actions, and he allegedly went back there to apologize, at which time he was placed into custody by a Manchester police officer who arrived on scene," O'Keefe said. Court records said Reyes told police that he robbed the store earlier in the day, because he was hungry and that he was trying to return the money to the clerk. Police said Reyes only had a small portion of the money that was originally taken, but what happened is unusual. "In 20 years, I haven't seen that," O'Keefe said. "So no, it's not a very frequent occurrence in the Queen City." Bail was set at $5,000. MANCHESTER, N.H. (AP) — A man is accused of robbing a New Hampshire convenience store while armed with a sharp rock, then returning hours later to give back the money. Police say 46-year-old Carlos Reyes, of Manchester, was charged with robbery and was scheduled to be arraigned Wednesday. It wasn't immediately known if he had lawyer, and a phone number couldn't be found for him. Officers responded to a Shop-N-Go just after noon Tuesday. Witness Saoul Haney tells WMUR-TV (http://bit.ly/1spGUG7 ) that the robber apologized for the theft as he fled the scene and said he needed the money. The clerk called police hours later and said the man had arrived to "turn himself in" and began crying. Reyes was arrested after police reviewed surveillance photos.

Summary: He may have robbed a Stop-N-Go, but it looks like a New Hampshire man decided to stop and turn back instead-and return the money he was said to have stolen, WMUR reports. Carlos Reyes, 46, allegedly entered the Manchester convenience store just after noon on Tuesday, armed with a sharp rock, per the AP. A witness says the suspect said he was sorry while demanding the loot, explaining he needed the cash. Reyes then reportedly took off, but a few hours later, according to a Stop-N-Go clerk, Reyes trudged back into the store weeping and gave the money back. The cops were called once again, and after they reviewed the surveillance tape, Reyes was arrested on charges of armed robbery. He's due in court Wednesday.

multi_news

en

Summarize: By. Lizzie Parry. PUBLISHED:. 04:34 EST, 30 January 2014. |. UPDATED:. 05:32 EST, 30 January 2014. Fatimah Lim is accused of stealing diamonds worth £12million from the ex-wife of the Sultan of Brunei. A female bodyguard stole rare diamonds worth £12million from the ex-wife of one of the world's richest men, replacing them for near-worthless replicas, a court was told. Fatimah Lim, 35, is accused of stealing two large diamonds from the safe at the London home of Mariam Aziz, a former wife of the Sultan of Brunei. She is accused of replacing the pear-shaped 12.71 carat blue diamond - valued at £7.6million - and a rectangular 27.1 carat yellow diamond - worth £600,000 - with replicas worth £150 each, to clear a gambling debt. The Singapore national is said to have stole the gems from her employer’s £1.7 million home in Kensington, West London, after duping her daughter into letting her borrow them for a few hours. She is also accused of stealing a £3.3 million diamond bracelet after Mariam Aziz gave it to her for safe-keeping while out gambling. When Miss Aziz wanted it back, Lim denied ever being given it and suspicion fell on a housemaid. Later she stole and sold the diamonds given to Miss Aziz by the Sultan of Brunei during their marriage, with the blue diamond being bought from the Hilton Park Lane jewellers in 1986, the court heard. The Sultan, one of the world’s richest men, and Miss Aziz, a former airline stewardess who is of Bruneian, Japanese and Scottish ancestry had four children during their 21 years of marriage. Gareth Patterson, prosecuting, said: 'The prosecution case is that this defendant breached the trust that was placed in her by her employer, Miss Aziz. 'These three items were a diamond bracelet, a large yellow coloured diamond and a large blue coloured diamond. 'It appears that the defendant may have decided to sell these diamonds, because she - the defendant - had been gambling and needed to clear her debts.' Isleworth Crown Court heard that after stealing the gems, Lim took them to a jeweller in Hatton Garden to sell, claiming they been a gift from Miss Aziz to her mother, who she claimed had been a PA to the sultan’s former wife. To cover her tracks she had fakes worth just £300 made and put these back into the safe at Miss Aziz’s home, it is claimed. The alleged theft was only discovered in December 2009 when Miss Aziz asked her adopted daughter, Afifa Abduallah, to take the diamonds, which had now been set in rings, back to the Hilton for resizing. Jurors heard Lim started working for Miss Aziz in 2003 firstly as a badminton coach, but that her role eventually evolved to that of a personal assistant and bodyguard to the mother-of-four. In her capacity she travelling between her boss’ houses in Singapore, or Brunei, or in London. Mr. Patterson added: 'The background, sadly, to this case is that Miss Aziz. came to consider the defendant to be one of her most trusted employees. and considered her essentially as a friend.' The. pair would visit casinos across the world, including the Clermont Club. in Berkeley Square and Les Ambassadeurs, near Park Lane and such was the. trust placed in her Lim 'was allowed to become a signatory on Miss. Aziz’s accounts'. If Lim reached her credit limit on any of her own accounts, she would be able to use Miss Aziz's. Lim, a former bodyguard to Mariam Aziz, right, the ex-wife of the Sultan of Brunei, left, is said to have duped the couple's daughter into letting her 'borrow' the diamonds, before selling them and replacing them with near-worthless replicas. It was at the Les Ambassadeurs casino in May 2008 that Miss Aziz gave her an eight-diamond bracelet worth around $5.5million dollars bought from Graff jewellers in Bond Street for safe keeping. Two or three weeks later the sultan’s former wife wanted to wear the bracelet, but that it could not be found, the court heard. Miss Aziz’s staff were questioned and Lim denied having ever been given the item. Instead suspicions were raised against another housemaid, jurors heard. However, Mr Patterson said that this housemaid claims that Lim had later come to her and said she knew someone who could make replica jewellery. Lim eventually took the bracelet, as well as another one, to a jeweller in London’s famous Hatton Garden. Giving her name as Sue, but later identifying herself, she claimed she had authorisation from her mother to sell the items. But Mr Patterson told the court that Lim’s mother had never worked for Miss Aziz and that this was just a story she had made up to 'cover her tracks'. It is alleged that she then played on her close relationship with Miss Aziz’s adopted daughter, Afifa Abduallah, who saw the two as 'best friends'. Twice she asked Miss Abdullah to borrow the diamonds as collateral on a property deal on the promise to return them in a short time. Mr Patterson said: 'In order to do that, what she did is she abused her friendship with the young and naove adopted daughter.' The second time when she asked for the same favour toward the end of the year she switched the diamonds with £300 replicas made out of cubic zirconia. She later sent Miss Abdullah Facebook messages asking her to keep it secret from her mother. When the thefts were discovered Miss Abdullah eventually told her mother what had happened and Lim was confronted by Miss Aziz’s nephew Idris Ja’afar in Brunei, the court heard. She initially denied being in London in December 2009, but 'came clean' when Miss Abdullah arrived at the meeting and asked 'if Miss Aziz might forgive her and pardon her'. However, Lim, of no fixed address, denies three counts of theft. The trial continues

Summary: Fatimah Lim'stole two rare gems and bracelet to pay off a gambling debt' Former bodyguard 'tricked Mariam Aziz's daughter into letting her borrow the gems before selling them' A pear-shaped 12.71 carat blue. diamond, worth £7.6m, and a rectangular 27.1 carat yellow diamond, worth. £600,000, were replaced with £150 replicas. Singapore national Lim denies three counts of theft.

cnn_dailymail

en

Summarize: CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of co-pending U.S. provisional patent application Ser. No. 61/672,955 filed Jul. 18, 2012. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates generally to an animal collar accessory and, more specifically, to a programmable alarm device which can be removably attached to a dog collar. [0004] 2. Description of the Prior Art [0005] Properly caring for a pet requires more than simply providing food, water and bathroom accommodations. On the most basic level, pet care requires maintaining custody and/or keeping track of the location of one&#39;s pet. Moreover, pet care, particularly as it relates to dogs, will also typically require periodic visitation to veterinary offices, adherence to a vaccination schedule, and administration of pest and parasite prevention and treatment formulas. Though pet ownership in itself is sufficiently rewarding so as to not cause such reoccurring obligations to discourage many from owning pets, it is unmistakable that when the obligations incident to pet ownership are added to the already busy schedules of pet owners, many find it these obligations difficult to track and remember. [0006] It is well known that dogs kept as pets typically wear collars all or most of the time. This collar is usually the only thing the dog will wears with such frequency. Therefore, if one were seeking to track and be alerted to certain reoccurring obligations as it related to the dog, it would be desirable to utilize the collar to attach the reminder(s) to the dog. U.S. Pat. No. 6,283,065 issued Sep. 4, 2001 entitled “Animal Collar and Stud Assembly that Promotes Animal Safety and Well-Being,” discloses a collar stud which attaches to the collars, wherein the studs can function as tags, collar locks, and/or electronic identification devices. A constraint on existing attachments for dog collars which provide information relating to the dog is that they typically lack an accompanying user interface, lack a display, and are not programmable to provide visual and/or audible alerts. [0007] The Applicant&#39;s invention described herein an electronic collar attachment attachable to a dog&#39;s collar allows a user to directly enter, store and view pet related information as well as to directly input reminders (such as vaccination dates, medications etc.) into the device and be subsequently notified of such reminders. The electronic collar attachment includes a rigid and waterproof casing which houses and protects its key components of a display interface, input interface, a speaker, and solid state memory. The electronic collar attachment can be programmed using its input interface with information such as the date and time, the dog&#39;s name, the contact information for the dog&#39;s owner, the dog&#39;s veterinarian, the dog&#39;s medications, the dog&#39;s vaccines, the dog&#39;s microchip number, and upcoming or past appointments. The electronic collar attachment can also display this information on its display interface. SUMMARY OF THE INVENTION [0008] An electronic collar attachment for attaching to a dog&#39;s collar which allows the direct entry, storing, and viewing of pet related information and reminders. The electronic collar attachment comprises a rigid outer casing on which its interface components are located and in which its electronic components are disposed. The interface components include a display interface, input interface, and a speaker. The electronic components include a controller, non-volatile memory, and a power source. Data is entered into the electronic collar attachment through its input interface or through a computer via a USB connection. Information concerning the same can be then conveyed using the electronic collar attachment&#39;s display interface or speakers. [0009] The collar attachment utilizes the electronic components to control its internal operations and the operation of its interface components. Several applications are stored in the memory and provide the instructions to the controller to operate the electronic and interface components of the electronic collar attachment. For example, a system application provides instructions for the controller&#39;s handling of the interaction between the electronic components and the interface components as well as between interface components. A display application provides instructions to the controller for converting the information (being entered or previously stored) sought to be displayed on the display interface to a format which can be viewed intelligibly over the collar attachment&#39;s specific five screen display interface. Additionally, a menu application provides instructions to the controller for organizing the data received or stored and other applications on the device so as to provide intuitive navigation and programming for a user and proper interfacing between the applications and the data. The menu application thus provides the general navigation logic and a search routine. Components of the menu application include a me application and a calendar application. [0010] It is an object of this invention to provide a device that is attachable to a dog&#39;s collar which can be used to directly enter certain information that can be stored and retrieved at a later point in time. [0011] It is another object of this invention to provide such an attachable device which can directly display stored information on its face. [0012] It is yet another object of this invention to provide such an attachable device which can provide visual or audible alerts to a user at a preset time or on a preset day. [0013] And yet another object of this invention is to provide such an attachable device which is waterproof. [0014] These and other objects will be apparent to one of skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIG. 1 is a top elevational view of the front side of an electronic collar attachment built in accordance with the preferred embodiment of the present invention. [0016] FIG. 2 is a top elevational view of the back side of an electronic collar attachment built in accordance with the preferred embodiment of the present invention. [0017] FIG. 3 is a top elevational view of the back side of an electronic collar attachment built in accordance with the preferred embodiment of the present invention with its USB interface cover removed. DETAILED DESCRIPTION OF THE INVENTION [0018] Referring now to the drawings and in particular FIGS. 1 and 2, the outer casing of an electronic collar attachment 10 for attaching to a dog&#39;s collar is shown from the front side and the back side. The casing of the electronic collar attachment 10 includes a ring attachment 11 structural component for physically attaching the electronic collar attachment 10 to a dog&#39;s collar, as well as interface components such as a display interface and an input interface. In this manner, the casing of the electronic collar attachment 10 provides a housing means for attaching to the collar or harness of a dog. The display interface, which provides a display means disposed on said housing means for displaying information and alerts, includes five discrete display screens 27 (shown as 27 a, 27 b, 27 c, 27 d, and 27 e ), which allow messages and other information to be viewable by a user. In addition, the display interface includes a plurality of light bulbs 31 a located on the face of the collar attachment to provide for additional visual stimuli in the preferred embodiment. The input interface, in providing an input means disposed on said housing means for entering information and commands, includes the following components: [0019] a menu button 12, as a navigational button, for navigating the collar attachment&#39;s display interface to its main menu; [0020] an enter (or set) button 13, as a command button, for indicating completion of the a current display interface frame so that the next frame can be navigated to; [0021] two vertical scrolling (or up/down) buttons 14 (shown as 14 a and 14 b ), as navigational buttons, which allow a user to scroll up and down through different screen choice options and keys on the display interface; [0022] two horizontal scrolling (or reverse/forward) buttons 15 (shown as 15 a and 15 b ), as navigational buttons, which allow a use to move between the discrete display screens 27 which comprise the display interface; [0023] a back button 16, as a navigational button, which allows a user to go to the frame or five characters displayed which immediately preceded the present frame or five characters; [0024] an alarm button 17, as a navigational button, for navigating the display interface to the alarm application (or alarm setting routine); [0025] a display toggle switch 18, as a configuration button, for controlling whether the display screens 27 operate with a normal backlight or a neon backlight; [0026] a sound toggle switch 19, as a configuration button, for selecting between an audible tone alarm, a vibration alarm, and a silent alarm; [0027] an alarm signal button 28, as a configuration button, for selecting the type and volume of any tone alarm which is operational; and [0028] an AM/PM toggle switch (or flag) 29, as a configuration button, for switching between military and civilian timekeeping and for allowing am/pm to be set when civilian timekeeping is used. [0029] In addition, the collar attachment 10 includes two speakers 20, an area for a vendor or other entity logo 21, and area for a unique unit number 22, a battery compartment 23, a power toggle switch 24, a light switch 25, and a USB interface cover 26. The speakers 20 provide an audio output means for disposed on said housing means for indicating conditions. It is contemplated that the USB interface cover 26 and the cover the battery compartment 23, when both are engaged with the collar attachment 10, form a sufficiently bond to allow the collar attachment 10 to be water resistant Referring now to FIG. 3, when the USB interface cover 26 is removed, a USB port 30 is accessible to allow the collar attachment to be connected to a computer or other USB ready device. The USB port provides an interface means for connecting said collar attachment 10 to an external electronic device. [0030] Inside the collar attachment 10, electronic components including a controller, non-volatile memory, and a power source operate to control the device&#39;s operations. Together, the controller, non-volatile memory, and power source provide an electronic control means disposed in said housing means for operating the display means and the input means. In the preferred embodiment, the power source utilized is a battery. A system application provides instructions for the controller&#39;s handling of the interaction between the electronic components and the interface components as well as between interface components. [0031] Information is entered into the device using the vertical scrolling buttons, horizontal scrolling buttons, and the enter button. The function of these buttons is controlled by the system application so as to coordinate the interaction between the processor, memory, power, input interface, and the display interface. One feature available due to these components is that users can program custom messages to be alerted and/or displayed at a later point. For example, one owner or caregiver who may have already fed the dog but is leaving the location where the dog is kept can program and cause to be displayed a custom message indicating such (i.e. “Good morning, I already ate breakfast and was walked”). [0032] In the preferred embodiment, five LED screens are utilized. In an alternate, low power embodiment, BCD screens may be used for one or more of the five screens. A display application provides instructions to the controller for taking information being entered or previously stored and formatting it so that it is understandable over the collar attachment&#39;s specific five screen display interface. In this manner, the display application applies specific rules concerning the handling of such information. For example, information is provided on the screen with each screen working in concert and displaying no more than one character at any given time. The five screens together, collectively referred to as a frame, can operate sequentially to display messages which require more than five characters. The display of information can be either a display of up to five characters at a time for three seconds or the scrolling display of characters across each screen sequentially (again, a frame being the display of all five screens at a given point of time). The collar attachment automatically separates words or phrases which require more than five characters, including spaces between words, and displays them sequentially over multiple frames. For example, in the 3 second display mode, the phrase “dog to groomer” would be spread over several frames (separated by semicolons) as follows: - D - O - G -_-_-; - T - O -_-_-_-; - G - R - O - O - M -; - E - R -_-_-_-. In this case, each frame would display for three seconds before the next frame was shown and such would repeat until enter is pressed. When messages concerning events are auto scrolled in a calendar function of the collar attachment, a blank frame in inserted between each messages. [0033] As with operating systems generally, many of the system application&#39;s operations have significant intersection with the operation of the other applications. For example, as the display application controls scrolling displays, the enter button, who operation is controlled by the system application, is used to finish with a current frame and advance to the next. In situations where enter is pressed while on a frame that has no frames to advance to, however, it causes the display application to move the display interface back to the main display. Similarly, while such scrolling is ongoing, pressing either vertical scrolling button causes the character scrolling to pause and freeze at whatever frame was active when the button was pressed. Subsequent presses of vertical scrolling button causes the characters to scroll at the user&#39;s discretion. [0034] A menu application provides the general navigation logic and a search routine. The menu application&#39;s instructions allow the controller to organize the data received or stored and other applications on the device so as to provide intuitive navigation and programming for a user and proper interfacing between the applications and the data. Components of the menu application include a me application and a calendar application. [0035] In operation, when an alarm is triggered, the collar attachment utilizes is audible notification function in the form of a tone over its speakers or its vibration notification function along with causing its light bulbs to flash to alert a user visually. The user can then use the vertical scrolling buttons, horizontal scrolling buttons, and the enter button to select a sleep mode or general display mode from the alarm application. [0036] The menu display of the collar attachment allows the user to navigate to time, date, me, calendar, and application settings. As with the previous navigation, the vertical scrolling buttons, horizontal scrolling buttons, and the enter button are used to navigate to and through these screens and/or frames. Notably, the me application allows a user to save information concerning the pet&#39;s name, owner (including contact information), veterinarian, vaccine status, chip number and unit number. Similarly, the calendar application provides for the display of a present date and upcoming appointments as well as the entry of appointments, setting of reminders, and the searching of appointments already entered. [0037] It is contemplated that the USB port 30 allows for the direct entry of information and data from an external electronic device, such as a computer or other USB compatible device. Typically, the collar attachment would be plugged into USB and allow the user to enter scheduling and other information on the collar attachment (through XML or file uploads or through text entry) and display all the information from the collar attachment, including alerts. Such information and data includes medical records and digital diagnostics and it is desirable for such to be entered (and subsequently accessible) at a treating veterinarian office. In an alternate embodiment, the collar attachment will also be able to receive and store electrical power from an active USB connection. [0038] In an alternate embodiment, a proximity sensor is included within the collar attachment. The proximity sensor can work in conjunction with a separate proximity base to cause the alarm application in the collar attachment to activate anytime the collar moved further than a predetermined distance from the proximity base. In the alternative (or in addition), the proximity sensor may also include a transponder which can utilize existing GPS systems to allow for the collar&#39;s location to be tracked. [0039] In another embodiment, the information stored on the collar attachment can be uploaded to a website and stored in a cloud setting by the respective collar attachment&#39;s unique unit number. [0040] In yet another embodiment, the collar attachment is configured to be able to connect to a computer or other computing device (i.e. smart phone) wirelessly. In such an embodiment, an app would be availed to users which would allow the user to pair and connect the collar attachment to the computer or other computing device wirelessly and allow the user to enter scheduling and other information on the collar attachment and display all the information from the collar attachment, including alerts, on any medium, including the collar attachment, computer, smart phone, or a web site. [0041] In still another embodiment, the collar attachment is configured to be attachable anywhere on the dog, such as other dog wear (i.e. harnesses, backpacks). [0042] The instant invention has been shown and described herein in what is considered to be the most practical and preferred embodiments. It is recognized, however, that variations and departures may be made therefrom within the scope of the inventions and that obvious modifications will occur to a person of ordinary skill in the art.

Summary: An electronic collar attachment attachable to a dog&#39;s collar which allows a user to directly enter, store and view pertinent information relating to, among other things, the dog&#39;s identity, medical history, and upcoming appointments and medications, as well as scheduled meal times, medications, and walks. The collar attachment also selectively provides audible, visual, and/or vibration notifications of such upcoming events. The electronic collar attachment includes a rigid and waterproof casing which houses its key components of a display interface, input interface, a speaker, and solid state memory. Data is entered into the electronic collar attachment through its input interface or through a computer via a USB connection. This data is displayed using the electronic collar attachment&#39;s display interface.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Chesapeake Bay Science, Education, and Ecosystem Enhancement Act of 2009''. SEC. 2. REAUTHORIZATION OF NOAA CHESAPEAKE BAY OFFICE. Section 307 of the National Oceanic and Atmospheric Administration Authorization Act of 1992 (15 U.S.C. 1511d) is amended-- (1) in subsection (a)-- (A) in paragraph (1) by striking ``(in this section'' and all that follows and inserting a period; (B) by amending paragraph (2) to read as follows: ``(2) The Office shall be headed by a Director, who-- ``(A) shall have knowledge and experience in research or resource management efforts in the Chesapeake Bay; and ``(B) shall be responsible for the administration and operation of the office and the implementation of this Act.''; and (C) by striking paragraph (3); (2) in subsection (b)-- (A) by striking so much as precedes paragraph (1) and inserting the following: ``(b) Purpose.--The purpose of this section is to focus the relevant science, research, and resource management capabilities of the National Oceanic and Atmospheric Administration as they apply to the Chesapeake Bay and to utilize the Office to--''; (B) in paragraph (2), by striking ``Secretary of Commerce'' and inserting ``Administrator''; (C) in paragraph (3)-- (i) by striking the matter preceding subparagraph (A) and inserting the following: ``(3) coordinate the programs and activities of the various organizations within the National Oceanic and Atmospheric Administration in furtherance of such administration's coastal resource stewardship mission, including--''; (ii) in subparagraph (A), by striking ``and'' after the semicolon at the end of clause (vi), and by inserting after clause (vii) the following: ``(viii) coastal hazards and climate change; and''; and (iii) in subparagraph (B), by striking ``and'' after the semicolon at the end of clause (iii), by inserting ``and'' after the semicolon at the end of clause (iv), and by adding at the end the following: ``(v) integrated ecosystem assessments;''; (D) in paragraph (4)-- (i) by striking ``Environmental Protection Agency'' and inserting ``Chesapeake Executive Council''; and (ii) by inserting before the semicolon at the end the following: ``as appropriate to further purposes of this section''; (E) by striking paragraphs (5) and (7); (F) by redesignating paragraph (6) as paragraph (5); and (G) by adding at the end the following: ``(6) perform any functions necessary to support the programs referred to in paragraph (3).''; and (3) by striking subsection (c) and all that follows through the end of the section and inserting the following: ``(c) Program Activities.-- ``(1) In general.--The Administrator, through the Director, shall implement the program activities authorized by this subsection to support the activity of the Chesapeake Executive Council and to further the purposes of this section. ``(2) Ensuring scientific and technical merit.--The Director shall-- ``(A) establish and utilize an effective and transparent mechanism to ensure that projects funded under this section have undergone appropriate peer review; and ``(B) provide other appropriate means to determine that such projects have acceptable scientific and technical merit for the purpose of achieving maximum utilization of available funds and resources to benefit the Chesapeake Bay area. ``(3) Consultation with chesapeake executive council.--The Director shall, in the implementation of the program activities authorized under this section, consult with the Chesapeake Executive Council, to ensure that the activities of the Office are consistent with the purposes and priorities of the Chesapeake Bay Agreement and plans developed pursuant to the Agreement. ``(4) Integrated coastal observations.-- ``(A) In general.--The Administrator, through the Director, may collaborate with scientific and academic institutions, State and Federal agencies, non- governmental organizations, and other constituents in the Chesapeake Bay watershed, to support an integrated observations system for the Chesapeake Bay consistent with the purposes of subtitle C of title XII of Public Law 111-11 (33 U.S.C. 3601 et seq.). ``(B) Specific requirements.--To support the system referred to in subparagraph (A) and provide a complete set of environmental information for the Chesapeake Bay, the Director shall-- ``(i) coordinate existing monitoring and observing activities in the Chesapeake Bay; ``(ii) identify new data collection needs and deploy new technologies, as appropriate; ``(iii) collect and analyze the scientific information necessary for the management of living marine resources and the marine habitat associated with such resources; ``(iv) manage and interpret the information described in clause (iii); and ``(v) organize the information described in clause (iii) into products that are useful to policy makers, resource managers, scientists, and the public. ``(C) Chesapeake bay interpretive buoy system.--To further the development and implementation of the Chesapeake Bay Interpretive Buoy System, the Director may-- ``(i) support the establishment and implementation of the Captain John Smith Chesapeake National Historic Trail; ``(ii) delineate key waypoints along the trail and provide appropriate real-time data and information for trail users; ``(iii) interpret data and information for use by educators and students to inspire stewardship of Chesapeake Bay; and ``(iv) incorporate the Chesapeake Bay Interpretive Buoy System into the Integrated Ocean Observing System regional network of observatories. ``(5) Chesapeake bay watershed education and training program.-- ``(A) In general.--The Administrator, through the Director, may establish a Chesapeake Bay watershed education and training program. The program shall-- ``(i) continue and expand the Chesapeake Bay watershed education programs offered by the Office immediately before the enactment of the Chesapeake Bay Science, Education, and Ecosystem Enhancement Act of 2009; ``(ii) improve the understanding of elementary and secondary school students and teachers of the living resources of the ecosystem of the Chesapeake Bay; ``(iii) provide community education to improve watershed protection; and ``(iv) meet the educational goals of the Chesapeake 2000 Agreement. ``(B) Grant program.--The Director may award grants for the purposes of this paragraph. Grants awarded under this subparagraph may be used to support education and training projects that enhance understanding and assessment of a specific environmental problem in the Chesapeake Bay watershed or a goal of the Chesapeake Bay Program, or protect or restore living resources of the Chesapeake Bay watershed, including projects that-- ``(i) provide classroom education, including the development and use of distance learning and other innovative technologies, related to the Chesapeake Bay watershed; ``(ii) provide watershed educational experiences in the Chesapeake Bay watershed; ``(iii) provide professional development for teachers related to the Chesapeake Bay watershed and the dissemination of pertinent education materials oriented to varying grade levels; ``(iv) demonstrate or disseminate environmental educational tools and materials related to the Chesapeake Bay watershed; ``(v) demonstrate field methods, practices, and techniques including assessment of environmental and ecological conditions and analysis of environmental problems; ``(vi) build the capacity of organizations to deliver high quality environmental education programs; and ``(vii) educate local land use officials and decision makers on the relationship of land use to natural resource and watershed protection. ``(C) Collaboration.--The Director shall implement the education and training program in collaboration with the heads of other relevant Federal agencies. ``(6) Coastal and living resources management and habitat program.-- ``(A) In general.--The Administrator, through the Director, may establish a Chesapeake Bay coastal living resources management and habitat program to support coordinated management, protection, characterization, and restoration of priority Chesapeake Bay habitats and living resources, including oysters, blue crabs, and submerged aquatic vegetation. ``(B) Activities.--Under the program, the Director may, subject to the availability of appropriations, carry out or enter into grants, contracts, and cooperative agreements and provide technical assistance to support-- ``(i) native oyster restoration; ``(ii) fish and shellfish aquaculture that is carried out in accordance with a valid Federal or State permit; ``(iii) establishment of submerged aquatic vegetation propagation programs; ``(iv) the development of programs that protect and restore critical coastal habitats; ``(v) habitat mapping, characterization, and assessment techniques necessary to identify, assess, and monitor restoration actions; ``(vi) application and transfer of applied scientific research and ecosystem management tools to fisheries and habitat managers; ``(vii) collection, synthesis, and sharing of information to inform and influence coastal and living resource management issues; and ``(viii) other activities that the Director determines are appropriate to carry out the purposes of such program. ``(d) Reports.-- ``(1) In general.--The Administrator, through the Director, shall submit a biennial report to the Congress and the Secretary of Commerce on the activities of the Office and on progress made in protecting and restoring the living resources and habitat of the Chesapeake Bay. ``(2) Action plan.--Each such report shall include an action plan for the 2-year period following submission of the report, consisting of-- ``(A) a list of recommended research, monitoring, and data collection activities necessary to continue implementation of the strategy under subsection (b)(2); and ``(B) recommendations to integrate National Oceanic and Atmospheric Administration activities with the activities of the partners in the Chesapeake Bay Program to meet the commitments of the Chesapeake 2000 agreement and subsequent agreements. ``(e) Agreements.-- ``(1) In general.--The Administrator, through the Director, may, subject to the availability of appropriations, enter into and perform such contracts, leases, grants, or cooperative agreements as may be necessary to carry out the purposes of this Act. ``(2) Use of other resources.--For purposes related to the understanding, protection, and restoration of Chesapeake Bay, the Director may use, with their consent and with or without reimbursement, the land, services, equipment, personnel, and facilities of any Department, agency, or instrumentality of the United States, or of any State, local government, Indian tribal government, or of any political subdivision thereof. ``(3) Donations.--The Director may accept donations of funds, other property, and services for use in understanding, protecting, and restoring the Chesapeake Bay. Donations accepted under this section shall be considered as a gift or bequest to or for the use of the United States. ``(f) Definitions.--In this section: ``(1) Administrator.--The term `Administrator' means the Administrator of the National Oceanic and Atmospheric Administration. ``(2) Chesapeake bay agreement.--The term `Chesapeake Bay Agreement' means the formal, voluntary agreements executed to achieve the goal of restoring and protecting the Chesapeake Bay ecosystem and the living resources of the Chesapeake Bay ecosystem and are signed by the Chesapeake Executive Council. ``(3) Chesapeake executive council.--The term `Chesapeake Executive Council' means the representatives from the Commonwealth of Virginia, the State of Maryland, the Commonwealth of Pennsylvania, the Environmental Protection Agency, the District of Columbia, and the Chesapeake Bay Commission, who are signatories to the Chesapeake Bay Agreement, and any future signatories to that agreement. ``(4) Director.--The term `Director' means the Director of the Office. ``(5) Office.--The term `Office' means the Chesapeake Bay Office established under this section. ``(g) Authorization of Appropriations.--There is authorized to be appropriated to the Secretary to carry out this section-- ``(1) $17,000,000 for fiscal year 2011; ``(2) $18,700,000 for fiscal year 2012; ``(3) $20,570,000 for fiscal year 2013; and ``(4) $22,627,000 for fiscal year 2014.''. Passed the House of Representatives September 30, 2009. Attest: LORRAINE C. MILLER, Clerk.

Title: To reauthorize the Chesapeake Bay Office of the National Oceanic and Atmospheric Administration, and for other purposes Summary: Chesapeake Bay Science, Education, and Ecosystem Enhancement Act of 2009 - Amends the National Oceanic and Atmospheric Administration Authorization Act of 1992 to revise research, management, and program provisions of the Chesapeake Bay Office of the National Oceanic and Atmospheric Administration (NOAA). States that the Director of the Office shall be responsible for the administration and operation of the Office and implementation of such Act. Requires the Director to: (1) implement the program activities of the Chesapeake Executive Council; (2) ensure that projects have scientific and technical merit and have undergone appropriate peer review; (3) consult with the Council; and (4) report biennially to Congress and to the Secretary of Commerce regarding activities to protect the Chesapeake Bay. Authorizes the Director to: (1) collaborate with scientific and academic institutions, state and federal agencies, nongovernmental organizations, and other constituents to support an integrated observations system for the Chesapeake Bay; (2) support the establishment and implementation of the Captain John Smith Chesapeake National Historic Trail; (3) incorporate the Chesapeake Bay Interpretive Buoy System into the Integrated Ocean Observing System regional network of observatories; (4) establish a Chesapeake Bay watershed education and training program (authorizes related grants); (5) establish a Chesapeake Bay coastal living resources management and habitat program to support management of priority Chesapeake Bay habitats and living resources, including oysters, blue crabs, and submerged aquatic vegetation; and (6) accept donations of funds, property, and services. Authorizes FY2011- FY2014 appropriations for the Office and related activities.

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Write a title and summarize: Pattern classification of human brain activity provides unique insight into the neural underpinnings of diverse mental states. These multivariate tools have recently been used within the field of affective neuroscience to classify distributed patterns of brain activation evoked during emotion induction procedures. Here we assess whether neural models developed to discriminate among distinct emotion categories exhibit predictive validity in the absence of exteroceptive emotional stimulation. In two experiments, we show that spontaneous fluctuations in human resting-state brain activity can be decoded into categories of experience delineating unique emotional states that exhibit spatiotemporal coherence, covary with individual differences in mood and personality traits, and predict on-line, self-reported feelings. These findings validate objective, brain-based models of emotion and show how emotional states dynamically emerge from the activity of separable neural systems. Functional neuroimaging offers unique insight into how mental representations are encoded in brain activity [1,2]. Seminal cognitive neuroscience studies demonstrated that distributed patterns of cortical activity measured with functional magnetic resonance imaging (fMRI) contain information capable of differentiating among visual percepts, including object categories [3] and basic visual features [4]. Extending findings from these studies, subsequent work demonstrated that machine learning models trained on stimulus-evoked brain activity, termed “decoding” or “mind-reading” [5], can be used to predict the contents of working memory [6–8] and mental imagery [9,10], even during sleep [11]. Thus, pattern recognition approaches can identify defining features of mental processes, even when driven solely on the basis of endogenous brain activity. The approach was further shown to accurately discriminate among multiple cognitive processes (e. g., decision-making, working memory, response inhibition, among others) in independent subjects [12], establishing the efficacy of assessing diverse mental states with fMRI across individuals. Paralleling cognitive studies decoding task-evoked brain activity, multivariate decoding approaches have recently been used to map patterns of neural activity evoked by emotion elicitors onto discrete feeling states [13,14]. However, a key piece of missing evidence is whether categorically distinct emotional brain states occur intrinsically [15,16] in the absence of external eliciting stimuli. If so, then it should be possible to classify the emotional status of a human being based on analysis of spontaneous fluctuations of brain activity during rest. Successful classification would validate multivariate decoding of unconstrained brain activity and provides insight into the nature of emotional brain activity during the resting state. Adapting the logic of other cognitive imaging studies [16,17], we postulate that the presence of spontaneous emotional brain states should be detectable using multivariate models derived from prior investigations of emotion elicitation. We previously developed decoding algorithms to classify stimulus-evoked responses to emotionally evocative cinematic films and instrumental music [13]. These neural models (Fig 1) accurately classify patterns of neural activation associated with six different emotions (contentment, amusement, surprise, fear, anger, and sadness) and a neutral control state in independent subjects, generalizing across induction modality. Importantly, these neural biomarkers track the subjective experience of discrete emotions independent of differences in the more general dimensions of valence and arousal [18]. By indexing the extent to which a pattern of neural activation to extrinsic stimuli reflects a specific emotion, these models can be used to test whether intrinsic spatiotemporal patterns of brain activity correspond to stimulus-evoked emotional states. Here, we evaluate whether these neural models of discrete emotions generalize to spontaneous brain activation measured via fMRI in two experiments. The first experiment assesses if model predictions are convergent with individual differences in self-reported mood and emotional traits. Because individual differences are linked to mental health and subjective well-being [19–21], this evaluation provides insight into the potential clinical utility of quantifying spontaneous emotional states, as they may be associated with risk factors for mental illness. The second experiment employs an experience sampling procedure to evaluate whether model predictions based on brain activity during periods of rest are congruent with on-line measures of emotional experience. Together, these studies probe how brain-based models of specific emotion categories quantify changes in extemporaneous affect both between and within individuals. We applied the multivariate models of emotional experience to brain activation acquired from young adults during resting-state fMRI (n = 499; Fig 2A). Two consecutive runs of resting-state scans were acquired, spanning a total duration of 8. 53 min. Following preprocessing of data, we computed the scalar product of the resting-state signal and emotion category-specific model weights at every time point of data acquisition. This procedure yielded scores that reflect the relative evidence for each of seven emotional states across the full scanning period. A confirmatory analysis revealed that voxels distributed across the whole brain informed this prediction, as opposed to activity in a small number of brain regions (S1 Fig). If emotional brain states occur spontaneously, the frequency of classifications from our decoding models should be more varied than the uniform distribution that would be expected by chance. To test this hypothesis, we sought to identify whether the total time (or absolute frequency) in each state differed across emotion categories. Such an analysis informs the degree to which discrete emotional brain states may spontaneously occur and, by extension, could contribute to the identification of individual differences that map onto the likelihood of experiencing specific spontaneous states. To perform this comparison, we identified the single model with the maximum score at each time point (one-versus-all classification) and summed the number of time points assigned to each category. The frequency of emotional states clearly differed across categories (Fig 2B, χ2 = 1491. 52, P <. 0001, Friedman test), in contrast to the uniform distribution that would be expected if emotional brain-states did not occur in spontaneous activity (see S2 Fig). Follow-up comparisons revealed that neutral states occurred more frequently than chance rates (20. 1 ± 3. 59% [s. d. ], z = 20. 50, Punc = 2. 03E-93), followed by states of surprise (18. 37 ± 3. 87% [s. d. ], z = 16. 38, Punc = 2. 47E-60) and amusement (14. 71 ± 3. 78% [s. d. ], z = 1. 25, Punc = 0. 21). States of sadness (13. 49 ± 3. 76% [s. d. ], z = -3. 31, Punc = 9. 24E-4), fear (13. 26 ± 3. 42% [s. d. ], z = -5. 28, Punc = 1. 28E-7), and anger (11. 31 ± 3. 62% [s. d. ], z = -13. 07, Punc = 4. 78E-39) occurred with lower frequency, while states of contentment occurred the least often (8. 74% ± 3. 42% [s. d. ], z = -19. 61, Punc = 1. 33E-85; see Table 1). Although patterns of neural activation were most often classified as neutral as a whole, it is possible that consistent fluctuations in the time course of emotional states occur against this background. Research on MRI scanner-related anxiety has shown that self-report [22,23] and peripheral physiological [24] measures of anxiety peak at the beginning of scanning, when subjects first enter the scanner bore. This literature predicts that brain states indicative of fear should be most prevalent at the beginning of resting-state runs, and that neutral states should emerge over time, given their overall high prevalence (Fig 2B). To assess gradual changes in the emotional states over time, we performed Friedman tests separately for each emotion category, all of which revealed significant effects of time (see S1 Table). Next, we quantified the direction of these effects using general linear models to predict classifier scores using scan time as an input. We found the scores for fear decreased over time (β^=−0. 001, t498 = -4. 92, Punc = 1. 20E-006, Fig 3 gray lines), whereas neutral states exhibited an increasing trend throughout the scanning period (β^=0. 0017, t498 = 7. 36, Punc = 7. 66E-013), consistent with predictions (additional effects were observed for scores for contentment [β^=0. 0017, t498 = 7. 37, Punc = 7. 05E-13], surprise [β^=0. 0010, t498 = 4. 07, Punc = 5. 51E-05], anger [β^=−0. 0007, t498 = -3. 36, Punc = 0. 00085], and sadness [β^=−0. 0034, t498 = -15. 59, Punc < 2. 52E-038]). To determine whether emotional states exhibited consistent dynamics over the course of the scanning period, we fit smoothing spline models [25] for each subject and assessed the correlation between each subject and the average time course of other subjects in a cross-validation procedure. This analysis showed that there is substantial moment-to-moment variability in the time course of emotional states across subjects (which cannot simply be explained by scaling differences in the emotion models or resting-state data; see S3 Fig). Consistent with the linear models using time as a predictor, evidence for neutral brain states was most prevalent in the second scanning session, especially during a peak at the beginning of the run, whereas the time course for fear peaked at the beginning of the first run and decreased throughout the scanning session. The model for surprise exhibited a similar time course as neutral states but peaked at the end of the second run. Additionally, this analysis showed that evidence for sad classifications peaked in the middle of the first run and decreased over time. Overall, these time series revealed a gradual change in evidence from negative emotions (fear and sadness in run 1) to non-valenced or bi-valenced emotions (neutral and surprise in run 2). To ensure that our emotion-specific brain states are not proxies for more general resting-state networks thought to subserve other functions, we examined the spatial overlap between our models and those commonly derived by connectivity-based analysis of resting-state fMRI data [26]. On average, we observed little overlap (Jaccard index = 13. 1 ± 1. 97% [s. d. ]; range 10. 8%–16. 7%) with the seven most prominent networks found in resting-state data, implicating a substantial degree of independence. To further establish the construct validity of the spontaneous emotional brain states, we reasoned that their incidence should vary with individual differences in self-reported mood and personality traits associated with specific emotions. We assayed depressive mood with the Center for Epidemiologic Studies Depression Scale (CESD) [27] and state anxiety using the State-Trait Anxiety Inventory State Version (STAI-S) [28], instructing participants to indicate how they felt during the resting-state scan itself. Binomial regression models revealed that higher depression scores were associated with increases in the frequency of sadness (β^=0. 0025, t497 = 2. 673, Punc =. 0075, Fig 4A, see S4 Fig for scatter plots of predictions) and no other emotional state (all Punc >. 24). State anxiety was associated with increasing classifications of fear (β^=0. 0033, t497 = 2. 608, Punc =. 0091) and decreasing frequency of contentment (β^=−0. 0031, t497 = -2. 015, Punc =. 0439). Viewing these beta estimates as odds ratios (computed as eβ^) reveals how a one-unit increase in self-reported mood is associated with differences in the occurrence of spontaneous emotional states. Applying this approach to CESD scores reveals that individuals with a score of 16 (the cutoff for identifying individuals at risk for depression) have 5. 92% increased odds of being in a sad state compared to those with a score of 0. In more practical terms, this corresponds to approximately seven extra minutes a day of exhibiting a brain state that would be classified as sadness. Drawing from the Revised NEO Personality Inventory (NEO-PI-R) [29], we focused personality trait assessment on the specific Neuroticism subfacets of Anxiety, Angry Hostility, and Depression, due to their discriminant validity [30], heritability [31], universality [32], and close theoretical ties to the experience of fear, anger, and sadness. We found that increasing Anxiety scores were associated with more frequent classification of fear (β^=0. 003, t497 = 1. 978, Punc = 0. 0479, Fig 4B) and fewer classifications of anger (β^=−0. 004, t497 = -2. 407, Punc = 0. 0161). Angry Hostility scores were positively associated with the number of anger classifications (β^=0. 0042, t497 = 2. 400, Punc = 0. 0164). Depression scores were positively associated with the frequency of fear (β^=0. 003, t497 = 2. 058, Punc = 0. 0396) and sadness (β^=0. 0037, t497 = 2. 546, Punc = 0. 0109). These results provide converging evidence across both state and trait markers that individual differences uniquely and differentially bias the spontaneous occurrence of brain states indicative of fear, anger, and sadness. Finally, we examined whether the predictions of our decoding models were consistent with self-report of emotional experience during periods of unconstrained rest. We conducted a separate fMRI experiment in which an independent sample of young adult participants (n = 21) performed an experience sampling task in the absence of external stimulation (Fig 5A). Participants were instructed to rest and let their mind wander freely with their eyes open during scanning. Following intervals of rest of at least 30 s, a rating screen appeared during which participants moved a cursor to the location on the screen that best indicated how they currently felt. If spontaneous emotional states are accessible to conscious awareness, then scores should be greater for emotion models congruent with self-report relative to scores for models incongruent with self-report. Contrasting emotion models in this manner is advantageous from a signal detection standpoint because it minimizes noise by averaging across emotions, as some were reported infrequently or not at all in some subjects (see [33] for an analogous approach to predict the contents of memory retrieval during similarly unconstrained free-recall). To test our hypothesis, we extracted resting-state fMRI data from the 10-s interval preceding each self-report query and applied multivariate models to determine the extent to which evidence for the emotional brain states in this window predicted the participants’ conscious emotional experience. Consistent with our hypothesis, we found that scores for models congruent with self-report were positive (0. 016 ± 0. 0093 [s. e. m. ], z = 2. 068, Punc = 0. 0386; Wilcoxon signed rank test), whereas scores for incongruent models were negative (-0. 0048 ± 0. 0017 [s. e. m. ], z = -3. 041, Punc = 0. 0024). Classification of individual trials into the seven emotion categories exhibited an overall accuracy of 27. 9 ± 2. 1% (s. e. m.) of trials, where chance agreement is 21. 47% (Punc = 0. 001; binomial test). Not only do these results demonstrate that classification models are sensitive to changes in emotional state reported by participants, but also that there is selectivity in their predictions, as negative scores indicate evidence against emotion labels that are incongruent with self-report. Establishing both sensitivity and selectivity is important for the potential use of these brain-based models as diagnostic biomarkers of emotional states. As an additional validation of our decoding models, we examined the correspondence between the prevalence of individual emotional brain states as detected via pattern classification and participant self-report. Classifications based on self-report and multivariate decoding yielded similar frequency distributions (Fig 5C), in which neutral and amusement were the most frequent. We found a positive correlation between the frequency of classifications based on participant ratings and multivariate decoding (r =. 3876 ± 0. 102 [s. e. m. ], t20 = 2. 537, Punc =. 0196; one sample t test), further demonstrating a link between patterning of brain states and subjective ratings of emotional experience in the absence of external stimuli or contextual cues. Converging findings from our experiments provide evidence that brain states associated with distinct emotional experiences emerge during unconstrained rest. Whereas prior work has decoded stimulus-evoked responses to emotional events, our study demonstrates that spontaneous neural activity dynamically fluctuates among multiple emotional states in a reliable manner over time. Observing such coherent, emotion-specific patterns in spontaneous fMRI activation provides evidence to support theories that posit emotions are represented categorically in the coordinated activity of separable neural substrates [34,35]. Validating the neural biomarkers in the absence of external stimulation suggests that they track information of functional significance, and do not merely reflect properties of the stimuli used in their development. It is possible that these classifiers detect the endogenous activity of distributed neural circuits, consistent with recent views that emotions are not represented in modular functional units [36,37]. However, the extent to which such activity is the result of innate emotion-dedicated circuitry, a series of cognitive appraisals, or constructive processes shaped by social and environmental factors remains to be determined (for a review of these viewpoints, see [38]). Regardless of the relative influence of such factors, the present findings suggest that the emotion-specific biomarkers track the expression of functionally distinct brain systems, as opposed to idiosyncrasies of the particular machine-learning problem. Our findings complement recent studies demonstrating that a variety of emotion manipulations have lasting effects on resting brain activity [39–41]. For instance, one study revealed elevated striatal activity following gratifying outcomes in a decision-making task—an effect that was diminished in individuals with higher depressive tendencies [39]. Because these effects immediately followed emotional stimulation, they could plausibly reflect regulatory processes or lingering effects of mood. The present results, on the other hand, show that resting brain activity transiently fluctuates among multiple emotional states and that these fluctuations vary depending on the emotional status of an individual. Thus, emotional processes unfolding at both long and short time scales likely contribute to spontaneous brain activity. Findings from our resting state experiment stand in contrast to recent work investigating emotion-specific functional connectivity [42]. In this study, whole-brain resting-state functional connectivity was assessed using seeds identified from a meta-analytic summary of emotion research [43]. This latter approach failed to reveal unique patterns of resting-state connectivity for individual emotions but showed that seed regions were commonly correlated with domain-general resting-state networks, such as the salience network [44]. In light of the present results, it is important to consider methodological differences between studies. Seed-based correlation highlights connectivity between brain regions whose time course of activation is maximally similar to the activity of a small number of voxels (which are averaged together to create a single time series), whereas pattern classification identifies combinations of voxels that maximally discriminate among mental states. Because individual voxels sample diverse neural populations [45], it is plausible that seed-based correlation is biased towards identifying networks that have large amplitudes in seeded regions as opposed to exhibiting specificity (e. g., see [46]). Thus, our approach may have greater sensitivity to detect discriminable categorical patterns. Results of the experience sampling study provide external validation of our emotion-specific biomarkers [13]. Consistent with the resting-state study, the overall distribution of emotional states was clearly non-uniform, and classifications of neutral states occurred with high frequency. Beyond these commonalities, the inclusion of behavioral self-report led to differences in emotion-related brain activity. States of contentment and amusement were more frequently predicted during experience sampling compared to resting-state (46. 31% versus 23. 45%), a finding that was corroborated by higher ratings for these emotions in the self-report data. It is possible that this difference in the frequency of positive brain states is the result of a self-presentation bias [47], wherein participants may have employed emotion regulation in order to project a more positive image. Alternatively, it is possible that the self-reporting task requirement elicited more introspection between trials, which contributed to the pattern of altered emotional states [48]. Future work will be necessary to fully characterize how such cognitive-emotional interactions shape the landscape of emotional brain states [36,49]. We found that individual differences in mood states and personality traits are associated with the relative incidence of brain states associated with fear, anger, and sadness. These findings further establish the construct validity of our brain-based models of emotion and link subfacets of Neuroticism to the expression of emotion-specific brain systems. Given their sensitivity to individual differences linked to the symptomology of anxiety and depression, spontaneous emotional brain states may serve as a novel diagnostic tool to determine susceptibility to affective illness or as an outcome measure for clinical interventions aimed at reducing the spontaneous elicitation of specific emotions. This tool may be particularly useful to objectively assess the emotional status of individuals who do not have good insight into their emotions, as in alexithymia, or for those who cannot report on their own feelings, including patients in a vegetative or minimally conscious state. All participants provided written informed consent in accordance with the National Institutes of Health guidelines as approved by the Duke University IRB. The resting state experiment was approved as part of the Duke Neurogenetics Study (Pro00019095) with an associated database (Pro00014717). The experience sampling project was approved separately (Pro00027404). Classification of emotional states was performed using neural biomarkers that were developed based on blood oxygen level dependent (BOLD) responses to cinematic films and instrumental music [13]. This induction procedure was selected because it reliably elicits emotional responses over a 1 to 2 min period, as opposed to longer-lasting moods. These models were developed to identify neural patterning specific to states of contentment, amusement, surprise, fear, anger, and sadness (in addition to a neutral control state). These particular emotions were modeled to broadly sample both valence and arousal, as selecting common sets of basic emotions (e. g., fear, anger, sadness, disgust, and happiness) undersamples positive emotions. In selecting these particular emotions, we verified that the accuracy of these models tracked the experience of specific emotion categories (average R2 across emotions =. 57) independent of subjective valence and arousal. Thus, the models offer unique insight into the emotional state of individuals and characterize the likelihood they would endorse each of the seven emotion labels, independent of general factors such as valence or arousal. A total of 499 subjects (age = 19. 65 ± 1. 22 years [mean ± s. d. ], 274 women) were included as part of the Duke Neurogenetics Study (DNS), which assesses a wide range of behavioral and biological traits among healthy, young adult university students. For access to this data, see information provided in S1 Text. This sample was independent of that used to develop the classification models. This sample size is sufficient to reliably detect (β =. 01) a moderate effect (r =. 2) with a type-I error rate of. 05, which is particularly important when studying individual differences in neural activity. All participants provided informed consent in accordance with Duke University guidelines and were in good general health. The participants were free of the following study exclusions: (1) medical diagnoses of cancer, stroke, head injury with loss of consciousness, untreated migraine headaches, diabetes requiring insulin treatment, chronic kidney or liver disease, or lifetime history of psychotic symptoms; (2) use of psychotropic, glucocorticoid, or hypolipidemic medication; and (3) conditions affecting cerebral blood flow and metabolism (e. g., hypertension). Diagnosis of any current DSM-IV Axis I disorder or select Axis II disorders (antisocial personality disorder and borderline personality disorder), assessed with the electronic Mini International Neuropsychiatric Interview [50] and Structured Clinical Interview for the DSM-IV subtests [51], were not an exclusion, as the DNS seeks to establish broad variability in multiple behavioral phenotypes related to psychopathology. No participants met criteria for a personality disorder, and 72 (14. 4%) participants from our final sample met criteria for at least one Axis I disorder (10 Agoraphobia, 33 Alcohol Abuse, 3 Substance Abuse, 25 Past Major Depressive Episode, 5 Social Phobia). However, as noted above, none of the participants were using psychotropic medication during the course of the DNS. Participants were scanned on one of two identical 3 Tesla General Electric MR 750 system with 50-mT/m gradients and an eight channel head coil for parallel imaging (General Electric, Waukesha, Wisconsin, USA). High-resolution 3-dimensional structural images were acquired coplanar with the functional scans (repetition time [TR] = 7. 7 s; echo time [TE] = 3. 0 ms; flip angle [α] = 12°; voxel size = 0. 9 × 0. 9 × 4 mm; field of view [FOV] = 240 mm; 34 contiguous slices). For the two 4 min, 16 s resting-state scans, a series of interleaved axial functional slices aligned with the anterior commissure—posterior commissure plane were acquired for whole-brain coverage using an inverse-spiral pulse sequence to reduce susceptibility artifact (TR = 2000 ms; TE = 30 ms; α = 60°; FOV = 240 mm; voxel size = 3. 75 × 3. 75 × 4 mm; 34 contiguous slices). Four initial radiofrequency excitations were performed (and discarded) to achieve steady-state equilibrium. Participants were shown a blank gray screen and instructed to lie still with their eyes open, think about nothing in particular, and remain awake. Preprocessing of all resting-state fMRI data was conducted using SPM8 (Wellcome Department of Imaging Neuroscience). Images for each subject were slice-time-corrected, realigned to the first volume in the time series to correct for head motion, spatially normalized into a standard stereotactic space (Montreal Neurological Institute template) using a 12-parameter affine model (final resolution of functional images = 2 mm isotropic voxels), and smoothed with a 6 mm FWHM Gaussian filter. Low-frequency noise was attenuated by high-pass filtering with a 0. 0078 Hz cutoff. A total of 22 subjects (age = 26. 04 ± 5. 16 years [mean ± s. d. ], 11 women) provided informed consent and participated in the study. Data from one participant was excluded from analyses because of excessive head movement (in excess of 1 cm) during scanning. While no statistical test was performed to determine sample size a priori, this sample size is similar to those demonstrating a correspondence between self-report of affect and neural activity [13,52,53]. Participants engaged in an experience sampling task in which they rated their current feelings during unconstrained rest. Participants were instructed to keep their eyes open and let their mind wander freely and that a rating screen [54] would occasionally appear, which they should use to indicate the intensity of the emotion that best describes how they currently feel. This validated assay of emotional self-report consists of 16 emotion words organized radially about the center of the screen. Four circles emanate from the center of the screen to each word (similar to a spoke of a wheel), which were used to indicate the intensity of each emotion by moving the cursor about the screen. During four runs of scanning, participants completed 40 trials (10 per run) with an inter-stimulus interval (ISI) of 30 s plus pseudo-random jitter (Poisson distribution, λ = 4 s). Self-report data were transformed from two-dimensional cursor locations to categorical labels. Polygonal masks were created by hand corresponding to each emotion term on the response screen. A circular mask in the center of the screen was created for neutral responses. Because terms in the standard response screen did not perfectly match those in the neural models, the item “relief” was scored as “content, ” whereas “joy” and “satisfaction” were scored as “amusement. ” The items “surprise, ” “fear, ” “anger, ” “sadness, ” and “neutral” were scored as normal. Scanning was performed on a 3 Tesla General Electric MR 750 system with 50-mT/m gradients and an eight channel head coil for parallel imaging (General Electric, Waukesha, Wisconsin, USA). High-resolution images were acquired using a 3D fast SPGR BRAVO pulse sequence (TR = 7. 58 ms; TE = 2. 936 ms; image matrix = 2562; α = 12°; voxel size = 1 × 1 × 1 mm; 206 contiguous slices) for coregistration with the functional data. These structural images were aligned in the near-axial plane defined by the anterior and posterior commissures. Whole-brain functional images were acquired using a spiral-in pulse sequence with sensitivity encoding along the axial plane (TR = 2000 ms; TE = 30 ms; image matrix = 64 × 64; α = 70°; voxel size = 3. 8 × 3. 8 × 3. 8 mm; 34 contiguous slices). Four initial radiofrequency excitations were performed (and discarded) to achieve steady-state equilibrium. Processing of MR data was performed using SPM8 (Wellcome Department of Imaging Neuroscience). Functional images were slice-time-corrected, spatially realigned to correct for motion artifacts, coregistered to high resolution anatomical scans, and normalized to Montreal Neurologic Institute (MNI) space using high-dimensional warping implemented in the VBM8 toolbox (http: //dbm. neuro. uni-jena. de/vbm. html). Low-frequency noise was attenuated by high-pass filtering with a 0. 0078 Hz cutoff. To rescale data for classification, preprocessed time series were standardized by subtracting their mean and dividing by their standard deviation. Maps of partial least squares (PLS) regression coefficients from stimulus-evoked decoding models [13] were resliced to match the voxel size of functional data. These coefficients are conceptually similar to those in multiple linear regression, only they are computed by identifying a small number of factors (reducing the dimensionality of the problem) that maximize the covariance between patterns of neural activation and emotion labels (for specifics on their computation, see [55]). Classifier scores were computed by taking the scalar product of functional data at each time point and PLS regression coefficients from content, amusement, surprise, fear, anger, sad, and neutral models. Individual time points were assigned categorical labels by identifying the model with the maximal score. In order to determine if relatively focal or diffuse patterns of resting-state activity informed classification, we computed importance maps for each subject (S1 Fig). This was accomplished by calculating the voxel-wise product between PLS regression coefficients for each emotion model and the average activity of acquisition time points labeled as the corresponding emotion. We made inference on these maps by conducting a mass-univariate one-sample t test for each of the seven models, thresholding at FDR q =. 05. To address the potential overlap of the emotion classification models and canonical resting-state networks of the brain, we computed the maximal Jaccard index for each emotion model and the seven most prominent resting-state networks identified in Yeo et al [26]. This index is computed as the intersection of voxels in the two maps (voxels above threshold in both maps) relative to their union (the number of voxels above threshold in either map). Thresholds for classification models were adaptively matched to equate the proportion of voxels assigned to each resting state network. When conducting inferential tests on classification frequency (count data), non-parametric tests were conducted. To test whether classifications were uniformly distributed across the emotion categories, a Friedman test was performed (n = 499 subjects, k = 7 emotions). Wilcoxon signed-rank tests were performed to test for differences in frequency relative to chance rates (14. 3%) in addition to pairwise comparisons between emotion models, and corrected for multiple comparisons based on the false-discovery rate. Because the models have different levels of accuracy when used for seven-way classification [13], we additionally conducted wavelet resampling of classifier scores in the time domain [33,56] over 100 iterations to ensure that differences in the sensitivity of models did not bias results. This procedure involved scrambling the wavelet coefficients (identified using the discrete wavelet transform) of classifier scores (time series in Fig 3) to generate random time series with similar autocorrelation as the original data. Classifications were then made on these surrogate time series, and Friedman tests were performed to test for differences in frequencies across categories. This procedure yielded a null distribution for the chi-square statistic against which the observed statistic on unscrambled data was compared. To test whether classifier scores changed over time, Friedman tests were conducted on the outputs of the emotion models separately (concatenating the time series across runs), as classifier scores were found to violate assumptions of normality. Follow-up tests on the direction of these changes (either as increases or decreases) were conducted using general linear models with one constant regressor and another for linearly increasing time for each subject. Inference on the parameter estimate for changes over time was made using a one-sample t test (498 degrees of freedom). In addition to testing gradual changes over time, smoothing spline models [25] were used to characterize more complex dynamics of emotional states. Because spline models are flexible and may include a different number of parameters for each subject, cross-validation was conducted to assess the coherence of spline fits across subjects. In this procedure, a smoothing spline model was fit for each subject, and its Pearson correlation with the mean fit for all other subjects was computed. The average of resulting correlations accordingly reflects the coherence of nonlinear changes in emotional states across all subjects. The influence of individual differences in mood and personality was assessed using generalized linear models with a binomial distribution and a logit link function. Multiple models were constructed, each using a single measure from either the CESD, STAI, or facets from the NEO-PI-R to predict the frequency of classifications for the seven emotion categories (seven models per self-report measure). Inference on parameter estimates (characterizing relationships between individual difference measures and classification frequency) was made using a t distribution with 497 degrees of freedom. To control for multiple comparisons, FDR correction (q =. 05) [57,58] was applied for targeted predictions. For individual differences in mood, this procedure included correction for positive associations between the frequency of sad classifications and CESD scores and between fear classification and STAI values (Pthresh =. 0091). For differences in emotional traits, correction was applied to models predicting the frequency of fear classification on the basis of Anxiety scores, anger classification using Angry Hostility scores, and sad classifications on the basis of Depression scores (Pthresh =. 0479). Scatterplots and predicted outcomes for these regression analyses are displayed in S4 Fig. To assess concordance in the experience sampling study, classifier scores were averaged for trials congruent and incongruent with self-report for each subject. For instance, all trials in which a participant self-reported “fear, ” the classifier outputs from the neural model predicting fear were considered congruent, whereas the remaining six models were averaged as incongruent. Because the frequency of self-report varied across emotions (e. g., endorsement of fear and sadness were very infrequent), scores were averaged across all trials to reduce noise. In a supplemental analysis, scores were extracted separately for all trials and classified by identifying the model with the highest score. Accuracy was assessed on data from all subjects, using self-reports of emotion as ground truth. Because the frequency of self-reported emotions was non-uniform, chance agreement between self-report and neural models was calculated based on the product of marginal frequencies, under the assumption of independent observer classifications [59]. Inference on the observed classification accuracy was tested against this value using the binomial distribution B (480,0. 2147). Due to infrequent self-reports of surprise, fear, and anger, accuracy on individual models was not computed. Scores were initially assessed by averaging the 10 s preceding each rating. Subsequent analyses increasing the window length up to 20 s did not alter results. Because the scores for congruent (p = 0. 0186, Lilliefors test against normal distribution) and incongruent (p = 0. 0453) trials exhibited non-normal distributions, Wilcoxon signed rank tests were used to test each sample against zero mean rank. The correspondence between the frequencies of classification labels from self-report and neural decoding was assessed by computing the Pearson correlation for each subject. The correlation coefficients were Fisher transformed and tested against zero using a one-sample t test. To ensure that population differences (i. e., inclusion of individuals with psychopathology) did not contribute to differences in the prevalence of emotions in the resting-state and experience sampling studies, we re-calculated the frequency of classifications using repeated random subsampling of healthy participants in the resting-state sample (1,000 iterations, sampling 21 participants without replacement). The average correlation between the healthy subsamples and the full sample was very high (ravg =. 981, s. d. =. 013), making it unlikely that clinical status accounts for differences in the frequency of classifications across studies.

Title: Decoding Spontaneous Emotional States in the Human Brain Summary: Functional brain imaging techniques provide a window into neural activity underpinning diverse cognitive processes, including visual perception, decision-making, and memory, among many others. By treating functional imaging data as a pattern-recognition problem, similar to face- or character-recognition, researchers have successfully identified patterns of brain activity that predict specific mental states; for example, the kind of an object being viewed. Moreover, these methods are capable of predicting mental states in the absence of external stimulation. For example, pattern-classifiers trained on brain responses to visual stimuli can successfully predict the contents of imagery during sleep. This research shows that internally mediated brain activity can be used to infer subjective mental states; however, it is not known whether more complex emotional mental states can be decoded from neuroimaging data in the absence of experimental manipulations. Here we show that brain-based models of specific emotions can detect individual differences in mood and emotional traits and are consistent with self-reports of emotional experience during intermittent periods of wakeful rest. These findings show that the brain dynamically fluctuates among multiple distinct emotional states at rest. More practically, the results suggest that brain-based models of emotion may help assess emotional status in clinical settings, particularly in individuals incapable of providing self-report of their own emotional experience.

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Summarize: by Fox News September 26, 2011 If Mitt Romney was in New York City Monday to kiss the ring of business mogul Donald Trump, he didn't seem to want the press to capture his reaction afterwards. According to Fox News producer Nicole Busch, Romney's director of operations stood with the media the entire time they waited to catch a glimpse of the former Massachusetts Governor...only to tell reporters and cameras after an hour that Romney had already come and went. "Chicken Mitt!!" DNC spokesperson Brad Woodhouse wrote in a rapid fire email to reporters upon learning the Romney aide had misled the press while the candidate departed another exit. "Bwok-Bwok!" he continued. But in contrast to the DNC's caricature of Romney's actions, Donald Trump told Fox News Channel anchor Neil Cavuto afterwards that he and Romney had a "really had a great hour together" and that he was "impressed by certain things" Romney said during their meeting. "I thought he was very good at the debate, I think he did a good job, I did tell him that," Trump said, although he has yet to endorse a candidate. Mitt Romney requested the one-on-one meeting with Donald Trump, their first since Romney declared a run for the Oval Office; fellow Republican hopefuls Michele Bachmann and Rick Perry have already met with the reality television host. Herman Cain has a meeting scheduled next week. In the interview with Cavuto, Trump walked back from less than flattering comments he has made about Romney in the past. "Mitt Romney is a basically small business guy, if you really think about it," Trump said of his potential rival in April as he pondered the idea of a presidential run. "He was a hedge fund. He was a fund guy. He walked away with some money from a very good company that he didn't create. He worked there. He didn't create it." The hither-to lack of fuzzy feelings between the two men did not stop the DNC from trying to lump the two men together in a web video released Monday morning, entitled "Trump, Romney: You're Fired!" You can watch the DNC web video here. Fresh off a victory in the Michigan straw poll, Mitt Romney will be reaching out to another sizable part of the Republican base Monday -- Donald Trump Romney will be meeting with His Trumpness in New York on Monday afternoon, becoming the latest GOP presidential aspirant to court the developer/TV host/former potential candidate/birth certificate investigator. Rick Perry dined with Trump earlier this month at pricey, exclusive Jean-Georges in Manhattan. (You might recall that in June, Trump and Sarah Palin shared some slices of pizza in Times Square. Read into that what you will.) Michele Bachmann has also met with the Donald and plans to do so again.Why court the blessings of Trump? That’s a good question. Well, for one thing, there are still those out there who believe Trump should be president of the United States. There’s a whole website and everything. (It includes items such as “Trump Answers Boy’s Prayer for New Bike.”) For another, say what you will about the man, but he has a platform. He can pretty much show up on Piers Morgan ’s CNN show or Fox News just about whenever he likes. (And don’t forget “Celebrity Apprentice” on NBC. Don’t worry. He’s not going to let you forget it.) Third, he has money. And he knows other people with money. Presidential candidates like money.But Democrats are just as eager to associate Trump with the GOP, as the candidates seem to be to meet with him. Part of it is that it makes Perry, Romney and the like look a little less, well, serious. The other is that Trump is sort of the epitome of the fatcatness that Democrats argue the GOP protects. (This is what GOP calls "class warfare.")The Democratic National Committee, in fact, has released a video in advance of the Romney-Trump summit, imaginatively titled “You’re Fired” that paints both men as uber-capitalists who care little about the middle-class, education, Medicare or Social Security.Romney is on a small roll. He was widely viewed as handily winning the Fox News- Google debate Thursday night while his top rival, Perry, turned in a shaky performance. Then Perry lost a Florida straw poll he had been expected to win to Herman Cain. On Sunday, Romney trounced Perry and all others in a straw poll in his home state of Michigan.But can he win the coveted Trump endorsement? If there’s one thing to be sure of, it’s that Trump will keep America in suspense as long as possible before answering that question.

Summary: If you're looking for the Republican presidential nomination, you'd better get the Donald's blessing. Both Rick Perry and Michele Bachmann (as well as Sarah Palin, hmmm) have paid visits to Trump, and Mitt Romney met Trump this afternoon, the Los Angeles Times reports. But Romney ducked out rather than face reporters lingering outside: Hanging out with the journos, Romney's operations director suddenly told them the candidate had left an hour before, Fox News reports. "Chicken Mitt!!" wrote DNC spokesperson Brad Woodhouse in a quick email. "Bwok-Bwok!" Democrats are even using the opportunity to ridicule both men-check out the DNC ad in the gallery. But Trump took a calmer tone in a Fox News chat after the duck-out: "I thought he was very good at the debate... I did tell him that," Trump said. Better than his characterization of Romney back in April as "a basically small business guy... [who] walked away with some money from a very good company that he didn't create."

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Write a title and summarize: Several rodent-associated Bartonella species are human pathogens but little is known about their epidemiology. We trapped rodents and shrews around human habitations at two sites in Kenya (rural Asembo and urban Kibera) to determine the prevalence of Bartonella infection. Bartonella were detected by culture in five of seven host species. In Kibera, 60% of Rattus rattus were positive, as compared to 13% in Asembo. Bartonella were also detected in C. olivieri (7%), Lemniscomys striatus (50%), Mastomys natalensis (43%) and R. norvegicus (50%). Partial sequencing of the citrate synthase (gltA) gene of isolates showed that Kibera strains were similar to reference isolates from Rattus trapped in Asia, America, and Europe, but that most strains from Asembo were less similar. Host species and trapping location were associated with differences in infection status but there was no evidence of associations between host age or sex and infection status. Acute febrile illness occurs at high incidence in both Asembo and Kibera but the etiology of many of these illnesses is unknown. Bartonella similar to known human pathogens were detected in small mammals at both sites and investigation of the ecological determinants of host infection status and of the public health significance of Bartonella infections at these locations is warranted. Bartonella species are Gram-negative haemotrophic bacteria that infect mammalian erythrocytes and are transmitted between hosts by blood-sucking arthropods. Over 30 species of Bartonella have been described and members of this genus infect a broad range of mammalian hosts including rodents, bats, carnivores and ruminants [1]. Arthropod vectors including fleas, sandflies, lice, ticks, bat flies and ked flies are implicated in the transmission of these pathogens [2–4]. The genus Bartonella has a global distribution. The Bartonella elizabethae complex includes several Bartonella genotypes and strains (including B. elizabethae, B. tribocorum, B. rattimassiliensis and B. queenslandensis) that have been isolated from Rattus and Bandicota species around the world [1]. Recent analyses indicate that this complex has south-east Asian origins and has been globally dispersed by Rattus species [5]. Several Bartonella species are recognized as human pathogens that cause diverse clinical presentations [6]. Among rodent-associated Bartonella species, B. elizabethae is a known cause of human endocarditis [7]. Other rodent-associated species including B. tribocorum, B. vinsonii subsp. arupensis, B. washoensis and B. alsatica have been associated with a range of symptoms in humans including fatigue, muscle and joint pain, and serious complications, such as endocarditis and neurological signs, particularly in immunocompromised patients [8,9]. Bartonella species have been identified as important causes of febrile illness in some settings. In two studies conducted in Thailand, 15% of febrile patients were diagnosed with confirmed Bartonella infection based on a four-fold rise in antibody titres, and six different Bartonella species were identified by culture from blood clots collected from febrile patients [10,11]. Non-specific clinical signs and difficulties in culturing the organism present substantial challenges to the diagnosis of bartonellosis. Consequently, Bartonella species may well be under-recognized as a cause of human disease [12]. This is particularly true for Africa, where very few data on the etiology of febrile illness are currently available [13]. In the Democratic Republic of Congo (DRC), a seroprevalence study identified IgG antibodies against Bartonella (B. henselae, B. quintana or B. clarridgeiae) in 4. 5% of febrile patients [14]. Bartonella bacteraemia was detected by PCR in 10% of HIV-positive patients in South Africa [15]. Apart from these studies however, there is little information on the impact of Bartonella on human health on the African continent. A variety of Bartonella species have been detected in animal and ectoparasite populations in Africa. Considering rodents and small mammals specifically, B. elizabethae and two other Bartonella lineages were detected in Namaqua rock mice sampled in South Africa, where 44% of the 100 individuals sampled were positive by PCR for Bartonella species [16]. B. elizabethae, B. tribocorum and a Bartonella species with intermediate species classification based on sequence data were detected in 28% of rodents and hedgehogs (n = 75) sampled in Algeria [17]. B. elizabethae, B. tribocorum and novel Bartonella species were also detected in rodents sampled in the Democratic Republic of Congo (DRC) and Tanzania [18]. Small mammals trapped in Ethiopia, had an overall Bartonella infection prevalence of 34% (n = 529) and were infected with multiple genotypes including genotypes very closely related to B. elizabethae [19]. B. elizabethae has also been detected in invasive and indigenous rodents sampled in Uganda [20] and genotypes related to B. rochalimae, B. grahamii and B. elizabethae have been detected in Mearn’s pouched mice studied in Kenya [21]. Bartonella have also been detected in fleas collected in Egypt, Morocco, DRC and Uganda [20,22–24]. The first objective of this study was to determine the presence and prevalence of Bartonella infections in small mammals trapped at rural and urban locations in Kenya. We also aimed to characterize the Bartonella isolates obtained using partial sequences of the citrate synthase (gltA) gene and to compare the Bartonella genotypes detected in these distinct Kenyan populations with each other and with Bartonella detected in small mammals in other parts of the world. Cross-sectional rodent trapping surveys were conducted within two locations: Asembo, a rural area on the northern shore of Lake Victoria in Nyanza Province western Kenya (Latitude-0. 1443, longitude 34. 3468) and Kibera, an urban informal settlement in Nairobi City (Latitude-1. 3156, longitude 36. 7820, Fig. 1). These locations are the study sites for ongoing population-based human health surveillance [25]. In Asembo, subsistence farming is the primary occupation for 65% of household heads, 13% work in the informal economy and 5% are salaried [25]. Households are clustered into compounds of closely related family units. Livestock ownership is common: 44% of Asembo households own cattle and 43% own at least one sheep or goat. In contrast, in urban Kibera, 53% of heads of household are salaried and 43% work in the informal sector [25]. Ownership of large livestock species in Kibera is very rare and prohibited by City Council law. In Asembo, trapping was conducted over the period of July—August 2009. Traps were placed at 50 compounds that were a randomly selected subset of livestock-owning compounds enrolled in a larger study of zoonoses epidemiology [26]. Within each selected compound, five or six medium-sized foldable Sherman traps (H. B. Sherman Traps Inc., Tallahassee, FL) were placed for three or four nights. Traps were placed in three categories of habitat: within occupied dwellings; within outbuildings, which included unoccupied dwellings, stores, latrines or kitchens separate from the main dwelling; and outside, in areas within the compound yard. In Kibera, trapping was conducted over the period of September—November 2008. The overall study site was divided for this study into five trapping zones of similar area and within each zone a 50m x 50m trapping area was defined (Fig. 2). Within each of the five trapping zones, medium-sized foldable Sherman traps were placed for a minimum of two consecutive nights and a maximum of six nights with the aim of trapping approximately 50 rodents per zone. In Kibera, all traps were placed indoors at 270 occupied dwellings. All trapped animals from both locations were euthanized by overdose of the inhalant anesthetic halothane and whole blood was collected by cardiocentesis using aseptic technique. Blood samples were processed to remove serum and the remaining blood clots frozen at -80°C prior to testing. Blood clots were shipped on dry ice to the Bartonella laboratory at the Centers for Disease Control and Prevention, Fort Collins, Colorado for laboratory testing. Morphometric data were collected from each trapped animal for species identification at the National Museums of Kenya. The Asembo small mammals were submitted for archiving under accession numbers NMK 171860—NMK 171922. The Kibera rodent population included in this study is as described previously [27]. Culture was performed using previously described techniques [28]. Briefly, blood clots were re-suspended 1: 4 in brain heart infusion broth supplemented with 5% amphotericin B, then plated onto agar supplemented with 5% sterile rabbit blood and incubated at 35°C in an aerobic atmosphere of 5% carbon dioxide for up to 30 days. Bacterial colonies were presumptively identified as Bartonella based on their morphology. Subcultures of Bartonella colonies from the original agar plate were streaked onto secondary agar plates and incubated at the same conditions until sufficient growth was observed, usually between 5 and 7 days. Pure cultures were harvested and stored in 10% ethanol. The identity of presumptive Bartonella isolates was confirmed by PCR amplification and sequencing of a specific fragment of the Bartonella citrate synthase (gltA) gene. Crude DNA extracts were obtained from bacterial cultures by heating a heavy suspension of the microorganisms. Two oligonucleotides (BhCS. 781. p and BhCS. 1137. n) were used as PCR primers to generate a 379-bp amplicon of the Bartonella gltA gene [29]. PCR products were separated by 1. 5% agarose gel electrophoresis and visualized by ethidium bromide staining. Sequencing reactions were carried out in a PTC 200 Peltier Thermal cycler (Applied Biosystems; Foster City, California) using the same primers as the initial PCR assay. Sequences were analysed using Lasergene 12 Core Suite (DNASTAR, Madison, WI) to determine sequence consensus for the gltA amplicons. Unique gltA sequences generated through this study were submitted to GenBank (accession numbers KM233484—KM233492). The Clustal V program within the MegAlign module of Lasergene was used to compare homologous Bartonella gltA sequences generated in this study with others available from the GenBank database. Phylogenetic trees were constructed using the neighbor-joining method with the Kimura’s 2-parameter distance model and bootstrap calculations were carried out with 1000 replicates. B. tamiae was used as the outgroup. A criterion of >96% homology was used to define similarity of study sequences to known Bartonella species [30]. Generalized linear models were used to examine associations between individual Bartonella infection status (culture positive or negative for Bartonella) and host and environmental variables in R (Version 3. 0. 3) [31]. Binomial family models with a logit link function were used and p values ≤ 0. 05 were considered statistically significant. Variables examined included host species, sex, mass and trapping location. Data from the Asembo (S1 Table) and Kibera (S2 Table) sites were analysed separately. Written informed consent for trapping was obtained from representatives of the study households. The protocols and consent forms were reviewed and approved by the Animal Care and Use and Ethical Review Boards of the Kenya Medical Research Institute (#1191). The study protocols were also approved by the Institutional Animal Care and Use Committee and Institutional Review Board of the U. S. Centers for Disease Control and Prevention (#5410) and complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. A total of 49 small mammals trapped at 29 compounds in Asembo and 220 rodents trapped at 143 households in Kibera were included in this study. The small mammals trapped in Asembo included Crocidura olivieri (n = 16, African giant shrew), and rodents of the species Lemniscomys striatus (n = 2, striped grass mouse), Mastomys natalensis (n = 14, Natal multimammate mouse), Mus minutoides (n = 1, pygmy mouse), and Rattus rattus (n = 16, black rat). All of the rodents trapped in Kibera were Mus musculus (n = 178, house mouse), Rattus norvegicus (n = 10, brown rat) or Rattus rattus (n = 32) (Table 1). Ten of the 49 (21%) animals trapped in Asembo were culture-positive for Bartonella, including: Crocidura olivieri (n = 1,7%); Lemniscomys striatus (n = 1,50%); Mastomys natalensis (n = 6,43%); and Rattus rattus (n = 2,13%). Overall, 24 of the 220 (11%) animals trapped in Kibera were culture positive: including Rattus norvegicus (n = 5,50%) and R. rattus (n = 19,60%). None of the 178 samples collected from Mus musculus in Kibera were positive (Table 2). Culture-positive Rattus species were trapped in four of the five trapping grids established at the Kibera site (Fig. 2). Information on gltA sequences was obtained from all 34 culture-positive animals. The phylogenetic relationships between the isolates obtained in this study and previously described Bartonella species are shown in Figs 3 and 4. Bartonella detected in one M. natalensis and two R. rattus trapped in Asembo belong to the B. elizabethae species complex based on the similarity of the gltA sequences (Fig. 3 & Table 2). Five additional sequences detected in M. natalensis and one detected in L. striatus were most closely related to B. tribocorum. None of the strains obtained from M. natalensis or L. striatus were identical (≥ 96% sequence identity) to reference strains of the Bartonella species described previously in Rattus species trapped elsewhere. The strain of Bartonella cultured from a C. olivieri was not very similar to any previously described Bartonella reference species but has 93. 8% similarity B. birtlesii. Three pathogenic Bartonella species (B. elizabethae, B. tribocorum and B. queenslandensis) were detected in the two Rattus species sampled at the Kibera site. The gltA sequences for all Bartonella strains from Kibera rodents were identical (≥ 96% sequence identity) to reference isolates that are typical of Bartonella detected in Rattus populations globally (Table 2 & Fig. 4). At the Asembo location, there was a weak association between individual infection status and host species (likelihood ratio test p = 0. 053) where infection probability was higher in M. natalensis individuals than in the reference species C. olivieri (OR = 11. 25,95% CI = 1. 15–110. 47, p = 0. 038). Approximately half (24/49) of the small mammals trapped in Asembo were trapped outside (Table 1). None of the Bartonella positive animals trapped in Asembo were trapped within occupied dwellings. Two positive R. rattus were trapped in outbuildings but all other positive animals (one C. olivieri, one Lemniscomys striatus and six M. natalensis) were trapped outside. There were no statistically significant associations between the probability of Bartonella infection and small mammal sex or mass within the Asembo population. At the Kibera location, there was a clear influence of genus upon infection probability. None of the 178 Mus trapped were Bartonella positive but 24/42 Rattus were positive indicating much higher infection probability in Rattus (OR = Infinite). Considering the data for Rattus individuals only, there were no statistically significant associations between the probability of Bartonella infection and rodent species, sex, or mass. The proportion of infected Rattus and proportion of infected rodents overall varied by trapping zone in Kibera (Fig. 2). There was no statistically significant difference in the probability of Bartonella infection in Rattus from different trapping zones. However the sample size for this analysis was small and the existence of zones where no positive individuals were trapped complicate this analyses and its interpretation. Descriptively, the trapping data from Kibera fall into two groups. In zones A, B and D few Rattus were trapped (Fig. 2), the rodent populations in these zones were dominated by Mus musculus and only four Bartonella isolates were identified in the combined rodent populations from these three zones (Fig. 2). In contrast, in trapping zones C and E, Rattus made up larger proportions of the total trapped population (51% in zone C and 40% in zone E) and more Bartonella isolates of several species were identified in these populations. This study reports isolation of Bartonella strains from rodent and shrew species in Asembo and Kibera, Kenya. Bartonella strains were found in several small mammal species with variation observed in the infection prevalence and in the strains of Bartonella detected between host species and study sites. The majority of Bartonella isolates obtained from these Kenyan mammals are genetically similar to reference strains of known human pathogens. Several recent studies indicate that the prevalence of Bartonella infection in Rattus in Africa may be low in contrast to the frequently high prevalences observed in Asian Rattus populations [18–20,32]. It has been argued that this pattern of lower prevalence in African Rattus populations could be attributed to host escape during colonization [19,20], a phenomenon where relatively small founding populations of invading species can leave their parasites behind when colonizing new areas [33]. Consistent with this, a relatively low prevalence was seen in R. rattus from Asembo (13%). However, the high infection prevalence observed in Rattus trapped at the Kibera site (e. g. 50% R. norvegicus and 60% R. rattus) is more similar to prevalence values observed in studies of Asian Rattus populations than to other African populations [19]. There are multiple possible explanations for the difference in the prevalence observed in Rattus at these two sites. Phylogenetic analyses indicate that B. elizabethae complex strains originated in Southeast Asia and have been disseminated throughout Asia, Europe, Africa, Australia and the Americas through multiple dispersal events of commensal Rattus species [5]. The Kibera study site is near the centre of Nairobi, the Kenyan capital, and is likely to have greater international connectivity (in terms of international rodent movement through trade etc.) than the Asembo site, which is more rural. The higher prevalence observed at the Kibera site could therefore be explained by repeated introduction of Rattus and their associated Bartonella species to this site [19,33]. Further analyses would however be needed to elucidate the colonization history of Rattus and their associated Bartonella at these sites specifically. The number of species trapped in Kibera was smaller than the number trapped in Asembo, indicating a simpler species composition at this site and these data could also suggest a possible dilution effect of the increased community complexity in Asembo on Bartonella prevalence [34]. Finally, temporal dynamics in host and ectoparasite population structure are known to affect Bartonella infection prevalence [21,35]. This study involved cross-sectional trapping surveys conducted at different times of year in the two study locations. There are few data on the seasonal variation in the abundance or diversity of the rodent populations at these sites but it is likely that there are seasonal influences upon rodent abundance and diversity with differences between the urban Kibera site and the more rural Asembo site in the seasonal population dynamics observed [36]. It is therefore possible that differences in the sampling time may have contributed to the differences in infection prevalence seen between these two Rattus populations. Notably, no bartonellae were detected in Mus musculus trapped in Kibera despite a large number of tested animals and high infection prevalence observed in Rattus trapped in the same locations. Low-level Bartonella infection has been reported from Mus trapped in Ethiopia but the absence of Bartonella in Mus was also reported in a small-scale study from Nigeria [19,37]. All of the Kibera rodents were trapped within residents’ homes. In contrast, although nearly half of the animals trapped in Asembo were trapped indoors or in outbuildings, none of the positive animals at this site were trapped indoors, and only two positive animals were trapped in outbuildings. Approximately one third of the animals trapped outside in Asembo however were culture positive for Bartonella. The two culture positive animals trapped in outbuildings were R. rattus and they were carrying Bartonella similar to the B. elizabethae reference strain (Fig. 2). This species was most commonly found indoors or in outbuildings (14/15 records) and therefore may pose a risk due to closer human contact, even though only 2/16 were positive. Many of the Bartonella detected at in this study (except the birtlesii-like isolates from Crocidura) belong to the Bartonella elizabethae complex and many of the strains identified in invasive Rattus hosts particularly are closely related to known human pathogens. All of the Bartonella strains isolated from Kibera rodents have ≥ 96% sequence identity with strains that are common in Rattus species sampled in Asia and on several other continents [5]. In contrast, several strains isolated from Asembo rodents and shrews were less similar to the international reference strains from Rattus and were more similar to isolates gathered previously from Ugandan rodents, suggesting a longer history of circulation of these strains within these species at the Asembo site. The identification of similar B. tribocorum sequences in Mastomys and Lemniscomys individuals trapped in Asembo suggests an absence of strong host-pathogen associations in these populations. There is a high incidence of acute febrile illness in people in both Asembo and Kibera [25]. A variety of pathogens are known to account for a proportion of febrile illness in Asembo and Kibera but considerable proportions remain unexplained [38–41]. Bartonella species have been identified as important causes of human febrile illness in several global settings but there has been little investigation of the impact of bartonellosis upon human health in Africa particularly and it is conceivable that Bartonella may be an important cause of febrile illness in these study populations. The data presented from the Asembo and Kibera sites indicate clear differences in: the prevalence of Bartonella infection in the same host (Rattus species) at the two sites; the prevalence of infection in different hosts trapped at the same sites; the abundance of different infected hosts between the two locations and also between trapping zones in Kibera; the strains of Bartonella detected and finally in the locations within communities where rodents overall and Bartonella infected rodents were trapped. The impact of this variation in rodent host community composition, infection prevalence, ectoparasite vector preferences, and other ecological factors need to be understood to evaluate human Bartonella infection risks at these sites. The data presented here suggest that investigations of the multi-host infection dynamics of Bartonella and the public health significance of Bartonella infections at these Kenyan locations and others where there are close associations between people and small mammals are warranted.

Title: Prevalence and Diversity of Small Mammal-Associated Bartonella Species in Rural and Urban Kenya Summary: Bartonella are bacteria that infect many different mammal species and can cause illness in people. Several Bartonella species carried by rodents cause disease in humans but little is known about their distribution or the importance of bartonellosis as a cause of human illness. Data from Africa are particularly scarce. This study involved trapping of rodents and other small mammals at two sites in Kenya: Asembo, a rural area in Western Kenya, and Kibera, an informal urban settlement in Nairobi. Blood samples were collected from trapped animals to detect and characterize the types of Bartonella carried. At the Kibera site over half of the trapped rats were infected with Bartonella very similar to human pathogenic strains isolated from rats from other global regions. In Asembo, Bartonella were detected in four of the five animal species trapped and these Bartonella were less similar to previously identified isolates. All of the small mammals included in this study were trapped in or around human habitations. The data from this study show that Bartonella that can cause human illness are carried by the small mammals at these two sites and indicate that the public health impacts of human bartonellosis should be investigated.

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Summarize: Background In October 2004, Congress included a provision in the Ronald W. Reagan National Defense Authorization Act for Fiscal Year 2005 that required the Secretary of Defense to develop a comprehensive policy for DOD on the prevention of and response to sexual assaults involving members of the armed forces. In part, the legislation required DOD to develop a uniform definition of “sexual assault” for all the armed forces and submit an annual report to Congress on reported sexual assault incidents involving members of the armed forces. The statute also required the Secretaries of the military departments to prescribe procedures for confidentially reporting sexual assault incidents. DOD issued its first annual report to Congress in May 2005, and in August 2008 we conducted a review that, among other things, evaluated the extent to which DOD had visibility and exercised oversight over reports of sexual assault involving servicemembers. We found that DOD’s annual reports to Congress may not effectively characterize incidents of sexual assault in the military services because the department had not clearly articulated a consistent methodology for reporting incidents and because the means of presentation for some of the data did not facilitate their comparison. Further, we found that while DOD’s annual reports to Congress included data on the total number of restricted and unrestricted reported incidents of sexual assault, meaningful comparisons of the data could not be made because the offices providing the data to DOD measured incidents of sexual assault differently. As a result, we recommended that DOD improve the usefulness of its annual report as an oversight tool by establishing baseline data to permit analysis of data over time. DOD concurred with this recommendation and has taken steps to develop baseline data through the development of DSAID. Also, in 2008, Congress mandated that DOD implement a centralized, case-level database for the collection and maintenance of information regarding sexual assault involving a member of the armed forces. Additional mandates have since required the DOD-wide collection of additional data, such as case disposition and military protective orders for annual reporting purposes. We conducted a review of DOD’s efforts to implement a centralized sexual assault database, and in 2010 we reported that while DOD had taken steps to begin acquiring a centralized sexual assault database it did not meet the statutory requirement to establish the database by January 2010. Moreover, we found that DOD’s acquisition and implementation of DSAID did not fully incorporate key information technology practices related to the following: economic justification, requirements development and management, risk management, and test management. DOD concurred with all of our findings and recommendations and has taken some actions to address them. Economic justification: We found that DOD’s cost estimate for DSAID ($12.6 million) did not include all costs over the system’s life cycle and had not been adjusted to account for program risks. In 2012, DOD reassessed DSAID’s costs to include additional expenses not included in the original estimate; however, as of November 2016, DOD has not been able to provide DSAID life cycle documentation that would demonstrate that DOD had taken steps to ensure that all costs and program risks were accounted for. Requirements development and management: We found that DOD had taken initial steps to engage some users in the development of DSAID requirements and in 2009 had developed its initial requirements management plan. DOD’s initial requirements management plan established processes and guidelines for requirements management activities. This plan has been updated three times with the latest update in January 2016. DOD has some systematic methods in place for tracking user feedback, which is a key step in identifying system requirements. In addition, DOD has elicited feedback on users’ experience with DSAID since the database’s implementation. For example, in 2012, 2013, and 2015 DOD collected non generalizable feedback from DSAID users, including SARCs, SAPR program managers, and the military services legal officers. Risk management: We found that during development of the system, DOD had begun to identify key risks such as staffing shortages and competing priorities among the military services. In 2011, DOD developed a risk management plan that identified risks associated with DSAID. In the risk management plan, DOD assigned probability and impact ratings to some of the identified risks. In addition, DOD reported discussing program risks and technical risks to the database at its management meetings and has an issues tracker to track, among other things, risks to the database. However, as of August 2016, DOD had not demonstrated that it had established and implemented defined processes for mitigating risks identified in its risk management plan. Test management: At the time of our review in 2010, DOD officials told us that they were planning, but had not started to work with a development contractor to establish an effective test management structure, develop test plans, and capture and resolve problems found during testing. As of October 2016, DOD had developed several test management plans. DOD Has Implemented DSAID across the Military Services and Taken Steps to Standardize Its Use and Monitor Data Quality DOD Has Implemented DSAID across the Military Services As of October 2013, DOD had implemented DSAID across the military services, and the military services were using it to track and collect data on sexual assault cases. DSAID has since been used to generate data included in DOD’s Annual Reports on Sexual Assault in the Military for Fiscal Years 2014 and 2015, DOD’s Fiscal Year 2014 Report to the President of the United States on Sexual Assault Prevention and Response, and DOD’s Annual Report on Sexual Harassment and Violence at the Military Service Academies for Academic Program Year 2014-15. DSAID captures DOD-wide data on reports of sexual assault that allow victims to receive treatment and services. Reports can be “restricted” (i.e., confidential reporting of alleged sexual assault without initiating an investigation) or “unrestricted” (i.e., nonconfidential reporting that may initiate an investigation). Reports of sexual assault included are those in which either the victim of the assault or the subject of the investigation are members of the armed forces, or in some cases, when a victim is a servicemember’s spouse or adult family member, or is a DOD civilian or contractor. Data are input into DSAID through both manual and automated data entry processes, and include, as applicable, victim and referral support information; investigative and incident information; and case outcome data. DSAID cases are originated by SARCs, based on a report of sexual assault made by a victim to a SARC, a military service Sexual Assault Prevention and Response (SAPR) victim advocate, or military criminal investigative organization (MCIO) investigator. Generally, victim data are manually input into DSAID by SARCs and investigative data are collected by each military service’s MCIO and transferred into DSAID through an automated interface process. For details on DOD’s process for inputting data elements into DSAID, see figure 1. DSAID can be accessed only by authorized users, who are assigned different access rights depending on their roles and responsibilities pertaining to the collection of sexual assault data. SARCs with DSAID access are required to have a valid DOD Sexual Assault Advocate Certification, and all DSAID users must meet background check and Privacy Act/Personally Identifiable Information training requirements as well as complete user-role specific system training. According to DOD officials, as of July 19, 2016, DSAID had 1,009 users, including 938 SARCs; 34 program managers; 11 SAPRO analysts; 25 military service legal officers; and 1 SAPRO super user See table 1 for a description of the roles and access rights for each of these user groups. DOD Has Taken Steps to Standardize the Use of DSAID across the Military Services Development of Policies, Processes, and Procedures Since 2012, DOD has taken several steps to standardize the use of DSAID throughout the department, including the development of (1) policies, processes, and procedures for use of the system; (2) training for system users; and (3) processes for monitoring the completeness of data. Since its implementation, DOD has developed multiple policies, processes, and procedures to guide the use of DSAID. Specifically, DOD’s sexual assault prevention and response instruction requires that information about sexual assaults reported to DOD involving persons covered by the instruction be entered into DSAID, and also established rules for DSAID access and procedures for entering data. Similarly, three of the services have added and officials from one of the services told us that they are in the process of adding language to their military service- specific sexual assault guidance requiring the use of DSAID. To assist users, DOD also developed a DSAID user manual that is revised with each new system update. Further, DOD’s instruction on the investigation of adult sexual assault requires MCIOs to ensure that data obtained through unrestricted reports of sexual assault, such as the investigative case number, are available for incorporation into DSAID. According to DOD officials, this instruction is currently being reviewed to provide more specific instructions to investigators. Further, DOD has instituted formal processes to facilitate changes to DSAID. In 2011—prior to DSAID becoming fully operational—DOD established its DSAID Change Control Board, which provides a framework to formally manage updates or modifications to the system, and includes representation from each of the military services. The board has a formal charter, is to use established processes and procedures, and members are to meet monthly to discuss proposed changes to the system. In order for a change to be approved, a majority of members must agree to the modification unless there is a legislative or DOD mandate for modification. Through its change control processes, the CCB approves, prioritizes, and implements change requests. As of October 2016, there have been 135 change requests submitted since the system became operational in 2013—56 of which have been implemented through the change control process. Development of Training for DSAID Users DOD has developed and conducted several training courses for DSAID users. Initially, DSAID users were required to attend in person training on DSAID prior to being granted access to the system. However, as of April 2013, DOD converted this required training from in person to a web- based self-guided training that consists of simulations demonstrating DSAID’s capabilities. Further, a DOD official told us that SAPRO conducts in-person training for program managers as well as virtual training for military services’ legal officers. In addition, since June 2013, DOD has hosted a regular webinar series to inform and train users on a range of DSAID topics, including policy, new releases, or updates to DSAID. According to DOD officials, as of April 2016, DOD had implemented required annual refresher training for program managers and military service legal officers and, according to DOD officials, they are considering conducting required refresher training for SARCs. Development of Processes for Monitoring the Completeness of Data DOD and each of the military services have developed processes for monitoring the completeness of data input into DSAID. The primary tool used to monitor DSAID data is DOD’s DSAID quality assurance tool. This tool allows users to run point-in-time reports that identify missing data in DSAID; validate the accuracy of selected data fields; and perform cross- checks of selected data fields to identify potential conflicts of information. Officials from each of the services’ headquarters-level SAPR offices said that they distribute quality assurance reports monthly to their installations and request that SARCs correct any issues identified before the next monthly report is generated. According to DOD and SAPR officials for two of the military services, these reports allow them to identify trends in data quality issues. SAPR officials for two of the military services also told us that they use quality assurance reports to perform more targeted training to address installation-specific needs. According to DOD officials, data errors identified by the quality assurance tool provides DOD and the military services the opportunity to fix or improve the data entry quality or processes. For example, DOD recently used the quality assurance tool to identify some cases without any subject record. As a result, DOD officials have made plans to meet and develop solutions. Additionally, DOD officials conduct regular manual and automated data validation checks of DSAID to help ensure that sensitive information is protected as well as to help ensure the general integrity of the data. SAPR officials for three of the military services’ SAPR offices also told us that they conduct military service-specific reviews of DSAID data on an ongoing basis to help ensure their completeness and accuracy, and to identify any systemic issues. Users Have Identified Technical Challenges with DSAID and DOD Plans to Implement Modifications to Alleviate Challenges DSAID users have identified a variety of technical challenges with the system and DOD officials told us they have plans to spend approximately $8.5 million to address most of these issues in fiscal years 2017 and 2018. Some of the key technical challenges users have reported experiencing with the system are related to DSAID’s system speed and ease of use; interfaces with MCIO databases; utility as a case management tool; and users’ ability to query data and generate reports. DOD has plans in place to implement modifications to DSAID that are expected to alleviate these challenges; however, officials stated that they will not get approval to fund these modifications until after having conducted an analysis of alternatives in line with DOD’s acquisition policy framework. This framework, and the GAO Cost Estimating and Assessment Guide outline key elements that should be included in this analysis, such as relative lifecycle costs and benefits; the methods and rationale for quantifying the lifecycle costs and benefits; the effect and value of cost, schedule, and performance tradeoffs; the sensitivity to changes in assumptions; and risk factors for any proposed modifications. DOD plans to complete the first draft of this analysis by the end of November 2016. DSAID Users Have Experienced Technical Challenges That Hinder the Use of the System Based on our review of the nearly 600 DSAID help desk tickets that were generated from January 2015 through April 2016; DSAID change requests; user feedback reports; interviews with SARCs, program managers, and DOD officials; and our first-hand observations made during visits to selected installations, we identified technical challenges that users reported with DSAID that hinder its use across the military services. These challenges are related to, DSAID’s system speed and ease of use; interfaces with MCIO databases; DSAID’s utility as a case management tool; and users’ ability to query data and generate reports. System speed: According to our review of DSAID help desk tickets and interviews with service SAPR officials and SARCs, DSAID’s slow system speed presented a challenge in efficient use of the database. Specifically, users report that slow system speed caused them to spend an inordinate amount of time on data input, and limited their ability to save data and run reports because the system is programmed to time out after a certain period. In our review of the DSAID developer’s monthly system performance reports from December 2014 through January 2016, we learned that DSAID is rebooted on an almost daily basis to prevent or minimize system slow down. Further, in our review of nearly 600 help desk tickets, we found that DSAID’s slow system speed was one of the challenges cited by users. Users reported that, due to issues with system speed, it was cumbersome to perform their required DSAID functions along with other job responsibilities. For example, SARCs we interviewed representing 7 of the 13 installations said that in their estimation, DSAID’s slow system speed regularly resulted in data input taking up to two to three times longer than it should have. Additionally, according to interviews with military service officials and SARCs representing 8 of the 13 installations, computers would frequently time out during the lengthy period of time it took to input and save data in DSAID and, if all required fields to save were not complete, the time- out would result in the need to reenter the data. In addition, officials from the Department of the Army told us they were unable to run all- Army reports during the last half of 2015 because DSAID would time- out before a full report could be generated. Therefore, according to DOD SAPRO officials, they ran the Army’s reports for them on a regular basis, and in February 2016, they resolved this immediate issue by implementing a report scheduler capability in DSAID. According to DOD SAPRO officials, this capability allowed the Army and the other military services to run the full report without timing out. DOD SAPRO officials acknowledge the latency issue overall and are addressing it with software and server upgrades that are designed to reduce page load time and ease the burden of data entry on SARCs. DOD SAPRO officials stated that the software upgrades are scheduled for completion in December 2016 and server upgrades in early 2017, but DOD officials emphasized that when DSAID users experience DSAID slow system speeds it can also be an issue with the user’s local network, and not with DSAID. Ease of use: DSAID users we interviewed and DOD documents that we reviewed cited the inability to easily navigate DSAID as a challenge. According to a DSAID user feedback report, in 2015 the biggest issue SARCs reported to their military service headquarters officials was that the DSAID user interface and navigation could be improved. This was supported by SARCs we met with from 7 of the 13 installations who said that it is easy to miss or skip data fields and pages because the logic flow from one page to the next in DSAID is not intuitive, often leaving those SARCs unsure of how much progress they have made in completing a case record. For example, during our site visit to one installation, we observed instances in which the selection of certain data elements would trigger other data fields that needed to be completed, but the system did not prompt the user that additional data were required. Additionally, Army headquarters officials raised concerns with DSAID’s ease of use, stating that improvements to the system’s flow would increase data accuracy by ensuring that users enter relevant information when a case was initiated and also decrease the frequency that “relevant data missing” is noted in DOD’s annual report. However, DOD officials stated that they are limited in their ability to make some changes to DSAID’s workflow because DSAID is a commercial-off-the-shelf system, which does not allow for such customization. Automated interfaces with military service investigative databases: Based on DSAID quality assurance tool reports generated by the military services for the first three quarters of fiscal year 2016 and discussions we had with DSAID users, we found that DSAID data that are populated through interfaces with MCIO databases vary in completeness. According to military service officials and SARCs, this is because MCIO data systems are not required to capture the same information that is required for DSAID. For example, an official from one MCIO said that investigators are not required to complete the data field in their database for whether alcohol was used by the subject or victim; however, this same data field in DSAID is designed to be populated automatically with data from the MCIO databases. Further, in September 2014, DSAID was modified to address technical issues with the interface by allowing SARCs or program managers to manually enter these data in instances where MCIO data are regularly omitted. However, according to DOD officials, any additional data received from the MCIOs during the weekly interface will overwrite what the SARC or program manager has entered as the MCIO is the authoritative source for such data. Utility as a case management tool: According to DOD documents, DSAID is intended to be a case management tool, and according to DOD’s Fiscal Year 2015 annual report, DSAID enhances a SARC’s ability to provide comprehensive and standardized victim case management; however, users of DSAID told us that the system is of limited usefulness for case management. According to the DSAID user manual, the system allows for case management in that it enables a victim’s incident and referral data to seamlessly transition between locations or SARCs. While DSAID is DOD’s system of record and the only system in which SARCs are permitted to maintain case data, according to headquarter-level officials from the Army, the Navy, and the Air Force, and according to Marine Corps SARCs, DSAID is of limited usefulness to the personnel working with victims. Specifically, officials stated that DSAID does not provide the requisite functionality, such as the ability to input case notes to manage individual cases. Officials from one service’s headquarters-level SAPR office said that this functionality would be helpful in ensuring continuity of victim case management. However, according to DOD SAPRO officials, as of December 2016, a change request to the change control board to add the functionality has not been submitted. SARCs we met with from 9 of the 13 installations similarly stated that DSAID is missing basic elements of standard case management systems, such as the ability to document victim outreach or record unique incident details that may inform referrals for care or other support services. Further, SARCs we interviewed from 8 of the 13 installations indicated they would, at a minimum, like the ability to document how and when they met with victims to track the level of service victims were provided. DOD officials told us that the decision to limit narrative information in DSAID was by design to protect the victim. According to DOD officials, there is concern that their phrasing of narrative information could inadvertently harm the victim were DSAID data subpoenaed in the course of legal proceedings. Additionally, DOD officials noted that there have not been any change requests made to the change control board for the addition of case management elements to DSAID. Data query and reporting: According to DOD officials, DSAID has been used to provide data for congressionally mandated reports, produce ad-hoc queries, facilitate trend analysis, and support program planning analysis and management. However, officials from three services’ headquarters-level SAPR offices told us that DSAID’s reporting capabilities are limited. As a result, many users told us they have developed their own tools to track sexual assault outside of DSAID. For example, the Army’s and the Marine Corps’ SAPR offices have each developed “dashboard” systems that use raw data from DSAID to identify specific trends that are useful to their leadership and accessible by SARCs for their military service. Further, SARCs we met with from 11 of the 13 installations told us that they keep informal “databases” or hard copy documents on their cases in order to brief their leadership, because they cannot run ad-hoc queries and reports of cases in DSAID for which they are responsible. According to the SARCs, these trackers duplicate key data points input into DSAID (e.g., victim demographics, type of assault, involvement of alcohol, etc.), but do not include any personally identifiable information. DOD Officials Have Plans to Modify DSAID in Fiscal Years 2017 and 2018 to Alleviate Most of the System’s Technical Challenges, and Plan to Conduct an Analysis of Alternatives of System Changes According to DOD officials, they have planned to spend $8.5 million in funding for fiscal years 2017 and 2018 for DSAID modifications, which officials stated will allow them to implement specific modifications that should alleviate most of the technical challenges identified by users. See table 2 for a complete list of DOD’s initiatives planned for fiscal years 2017 and 2018 and the purpose of these initiatives. DOD plans to spend $3.59 million in fiscal year 2017 and $4.916 in fiscal year 2018 for specific modifications to DSAID to support these initiatives, and officials are in the process of beginning to conduct a formal analysis to identify the costs and benefits of alternative options for implementing each modification. (See app. I for a list of modifications to DSAID that DOD plans to implement to support the initiatives in fiscal year 2017.) For example, a DOD official stated that DOD plans to implement an encrypted file storage mechanism for DSAID, but they have not yet determined how they plan to do this. Rather, this DOD official stated that the analysis of alternatives will establish options for this mechanism and weigh the costs and benefits. This DOD official also stated that the Defense Human Resources Activity—which is responsible for funding DSAID—will not approve DOD to spend resources on the individual modifications until an analysis of alternatives addressing each modification is conducted. According to this DOD official, the initial draft of this analysis will be completed by the end of November 2016, and all planned modifications can be implemented within the planned available budgetary resources. DOD officials based these budgets on rough-order-of-magnitude cost estimates that were derived from costs they have experienced in recent years. For example, costs for adding a module in DSAID to document reports involving retaliation were based on DOD’s costs for building DSAID’s legal officer module, which was purchased in 2013.The GAO Cost Estimating and Assessment Guide states that rough-order-of- magnitude estimates are useful to support “what-if” analyses and can be developed for a particular phase or portion of an estimate, but unlike an analysis of alternatives, they do not rise to the level of analysis recommended by best practices to support an investment decision and are not considered budget-quality estimates. In addition to DOD acquisition requirements, an analysis of alternatives is supported by the GAO Cost Estimating and Assessment Guide. These documents identify key elements that should be included in this analysis. For example, an organization should identify relative lifecycle costs and benefits; methods and the rationale for quantifying the lifecycle costs and benefits; the effect and value of cost, schedule, and performance tradeoffs; sensitivity to changes in assumptions; and risk factors. Further, according to GAO guidance, a comparative analysis of alternatives is essential for validating decisions to sustain or enhance a program. Because these elements are part of DOD’s acquisition requirements, if DOD’s analysis of alternatives complies with these requirements, it should incorporate these key elements. Conducting a comparative analysis of alternatives, including identifying and quantifying lifecycle costs and benefits and weighing the cost, schedule, and performance tradeoffs, is key to ensuring that DOD appropriately manages its modifications to DSAID. In 2010, we found that DOD had failed to demonstrate adherence to these key elements in the initial development and implementation of DSAID. By the end of fiscal year 2018, DOD spending on DSAID will exceed initial cost estimates by over $13 million. In 2010, we reported that DOD estimated development and implementation of DSAID to cost $12.6 million, but DOD’s estimate did not include costs for program management or sustainment and for lifecycle costs such as operations and maintenance. In December 2012, DOD documentation shows that DOD had adjusted its estimate to $17.9 million to reflect research and development costs for fiscal years 2011 and 2012 and operations and maintenance costs for fiscal years 2013 through 2018. DOD projected it will have spent a total of approximately $31.5 million as of November 2016 on implementing and maintaining DSAID through fiscal year 2018. This is approximately $13 million more than the revised 2012 estimate. If DOD conducts an accurate and complete analysis of alternatives, it should result in more precise cost estimates for planned enhancements. DOD’s plan to conduct an analysis of alternatives that adheres to the department’s acquisition framework and adequately considers key elements identified in the GAO Cost Estimating and Assessment Guide, as DOD officials have stated that their analysis will do, should position DOD to more accurately assess whether planned modifications to DSAID can be implemented within budget and with the desired outcome. DOD’s Process for Managing Changes to DSAID Substantially Aligns with Key Elements of Industry Standards DOD manages modifications to DSAID through its change management process, which we found, based on our review of DOD documentation, substantially align with the elements described in the project management and information technology industry standards that we reviewed. “Change management” is the process of controlling changes requested to work products to help ensure that project baselines are maintained. According to the PMBOK® Guide, the activity of change control allows for documented changes within the project to be considered in an integrated fashion while reducing project risk, which often arises from changes made without consideration to the overall project objectives or plans. Configuration management activities can be included as part of an organization’s change control process. While change control is focused on managing project change such as identifying, documenting, and approving or rejecting changes to the project documents, deliverables, or baselines, configuration management is typically focused on managing changes to a configuration item or system. Industry standards include descriptions of the following elements of change and configuration management that are applicable to DOD’s efforts to manage DSAID: (1) managing change requests; (2) configuration status accounting, tracking and communicating to stakeholders the changes made to the database; (3) interface control, managing the database interfaces; and (4) release management, managing the publication and communication of updates to users. (1) Managing change requests: According to the PMBOK® Guide, changes may be requested by any stakeholder involved with the project. Although changes may be initiated verbally, they should be recorded in written form and entered into the change management and/or configuration management systems. Every documented change request—which may include corrective actions, preventive actions, and defect repairs—needs to be either approved or rejected by a responsible individual who is identified in the project management plan or by organizational procedures according to the PMBOK® Guide. When required, the change control process includes a change control board, which is a formally chartered group responsible for meeting and reviewing the change requests and approving, rejecting, or otherwise disposing of those changes and for recording and communicating such decisions. According to the PMBOK® Guide, the roles and responsibilities of this board are to be clearly defined and agreed upon by appropriate stakeholders and documented in the change management plan. Further, the disposition of all change requests, approved or not, are to be updated in the change log that is used to document changes that occur during a project. DOD has established a change request process in the DSAID change control management plan and DOD has documented and formally chartered its Change Control Board. The board’s roles and responsibilities are defined in the DSAID Change Control Board charter and board members include representation from SAPRO and each military service’s SAPR office. Change requests can be submitted only by board members or their designees. The board meets monthly to evaluate and vote on change requests. The DSAID Change Control Board charter outlines the requirement that change requests will be captured through a change request form, which will then be uploaded to the board’s website and made available to the DSAID community. Both the DSAID Change Control Board charter and the DSAID change control management plan outline DOD’s procedures for evaluating these change requests. During its evaluation of each proposed change request, DOD conducts an impact analysis that includes an assessment of the change’s potential impact on requirements, development, training, communications, policy, and testing. This impact analysis also assesses the expected level of effort to implement the request. Documentation from the Change Control Board meetings shows that DOD considers approximate costs and time to implement in change request discussions. DOD also documents and tracks testing and implementation of approved changes in a requirements log. The log includes the approval status, prioritization, and tracking notes for each change request as each moves through the approval process. Once a change request is implemented, DOD updates the requirements log to note which baseline requirement was affected and which system release was included the change. In the requirements log, DOD also documents baseline requirement changes associated with the change requests that have been disapproved and closed. (2) Configuration status accounting: According to the IEEE standard on configuration management, the purpose of configuration status accounting is to track the status of configuration items. In this process, organizations track baseline requirements and total changes requested and implemented. This information should provide objective insights into a system’s performance overtime and the status of the system as changes are implemented. DOD has documented and established baseline requirements for DSAID and, through the change request tracker, DOD tracks total changes requested, implemented, disapproved, deferred, and pending. As previously discussed, DOD conducts an impact analysis of each proposed change request as part of the evaluation process. DOD tracks DSAID’s requirements and change requests until release and monitors and documents identified defects at each stage until they are resolved, which allows DOD to monitor the system’s status as changes are implemented. (3) Interface control: According to the IEEE standard on configuration management, organizations use interface controls to manage the interfacing effects that hardware, system software, and other projects and deliverables have on the project. Interface control activities include identifying the product’s key interfaces and controlling the interface specifications. DOD is currently managing interfaces between DSAID and the MCIO databases to collect sexual assault case information, and DOD plans to incorporate additional interfaces with other DOD systems to collect more case information. DOD documentation shows that the department has identified DSAID’s key interfaces and specifications. Specifically, DOD SAPRO has established a memorandum of understanding with each service investigative agency that describe roles and responsibilities and data mapping parameters, which includes a technical description of the fields and types of data that will be interfaced between DSAID and each service investigative agency’s system. Through these mechanisms, DOD manages the parameters of these interfaces that provide key information to DSAID. While DOD has met industry standards for identifying key interfaces and controlling interface specifications, as discussed earlier in this report, some DOD users reported some technical challenges with data from the MCIO database interfaces overwriting manually input DSAID data. According to DOD documentation, DOD is taking steps to mitigate them in enhancement efforts, which include improving how investigative data transferred into DSAID, and adding additional database interfaces. (4) Release management: According to the IEEE standard on configuration management, release management allows an organization to ensure that the proper deliverables such as changes and fixes to a system are delivered to the designated receiving party, in the designated form, and to the designated location. Release management activities include delivering approved releases and defining the following: a release policy, release planning, release contents, release format and distribution, and release tracking. In line with defining release policy, DOD’s change control board charter defines board members as the authority for establishing DSAID release schedules and prioritize and assign changes to a release. With respect to release planning, we found that DOD has defined the types of releases it delivers and the activities conducted during DSAID’s formal release process. DOD has also defined the content, format, and distribution materials to be included in each release. Communication of the release follows a defined process starting with limited distribution to select users and then distribution to the full user community. DOD uses its master project schedule for DSAID to track and monitor release activities. Agency Comments We are not making recommendations in this report. We provided a draft of this report to DOD for review and comment. DOD provided technical comments, which we incorporated as appropriate. We are sending copies of this report to the appropriate congressional committees and to the Secretary of Defense; the Under Secretary of Defense for Personnel and Readiness; the Secretaries of the Army, the Navy, and the Air Force; and the Commandant of the Marine Corps. 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) 512-3604 or farrellb@gao.gov. 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: Change Requests Prioritized for Implementation in Fiscal Year 2017 Table 3 describes change requests to the Defense Sexual Assault Incident Database (DSAID) that the Department of Defense (DOD) has prioritized for implementation in fiscal year 2017. These change requests were approved through DOD’s change control process and determined to be priority modifications by the DSAID Change Control Board. Appendix II: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the staff named above, key contributors to this report include Kim Mayo (Assistant Director); Michael Holland; Jim Houtz; Mae Jones; Anh Le; Amie Lesser; Oscar Mardis; Shahrzad Nikoo; Tida Reveley; Monica Savoy; Jasmine Senior; Maria Staunton; and Randall B. Williamson. Related GAO Products Sexual Assault: Actions Needed to Improve DOD’s Prevention Strategy and to Help Ensure It Is Effectively Implemented. GAO-16-61. Washington, D.C.: November 4, 2015. Military Personnel: Actions Needed to Address Sexual Assaults of Male Servicemembers. GAO-15-284. Washington, D.C.: March 19, 2015. Military Personnel: DOD Needs to Take Further Actions to Prevent Sexual Assault during Initial Military Training. GAO-14-806. Washington, D.C.: September 9, 2014. Military Personnel: DOD Has Taken Steps to Meet the Health Needs of Deployed Servicewomen, but Actions Are Needed to Enhance Care for Sexual Assault Victims. GAO-13-182. Washington, D.C.: January 29, 2013. Military Personnel: Prior GAO Work on DOD’s Actions to Prevent and Respond to Sexual Assault in the Military. GAO-12-571R. Washington, D.C.: March 30, 2012. Preventing Sexual Harassment: DOD Needs Greater Leadership Commitment and an Oversight Framework. GAO-11-809. Washington, D.C.: September 21, 2011. Military Justice: Oversight and Better Collaboration Needed for Sexual Assault Investigations and Adjudications. GAO-11-579. Washington, D.C.: June 22, 2011. Military Personnel: DOD’s and the Coast Guard’s Sexual Assault Prevention and Response Programs Need to Be Further Strengthened. GAO-10-405T. Washington, D.C.: February 24, 2010. Military Personnel: Additional Actions Are Needed to Strengthen DOD’s and the Coast Guard’s Sexual Assault Prevention and Response Programs. GAO-10-215. Washington, D.C.: February 3, 2010. Military Personnel: Actions Needed to Strengthen Implementation and Oversight of DOD’s and the Coast Guard’s Sexual Assault Prevention and Response Programs. GAO-08-1146T. Washington, D.C.: September 10, 2008. Military Personnel: DOD’s and the Coast Guard’s Sexual Assault Prevention and Response Programs Face Implementation and Oversight Challenges. GAO-08-924. Washington, D.C.: August 29, 2008. Military Personnel: Preliminary Observations on DOD’s and the Coast Guard’s Sexual Assault Prevention and Response Programs. GAO-08-1013T. Washington, D.C.: July 31, 2008. Military Personnel: The DOD and Coast Guard Academies Have Taken Steps to Address Incidents of Sexual Harassment and Assault, but Greater Federal Oversight Is Needed. GAO-08-296. Washington, D.C.: January 17, 2008.

Summary: GAO has reported that DOD has not collected uniform data on sexual assaults involving members of the armed forces. In 2008, Congress required DOD to implement a centralized, case-level database for the collection and maintenance of these data. In 2012, DSAID reached initial operational capability to capture sexual assault data. House Report 112-479 included a provision for GAO to review DSAID no sooner than 1 year after it was certified compliant with DOD standards by the Secretary of Defense. This report (1) describes the current status of DOD's implementation of DSAID and steps DOD has taken to help standardize DSAID's use, (2) assesses any technical challenges DSAID's users have identified and any DOD plans to address those challenges, and (3) assesses the extent to which DOD's change management process for modifying DSAID aligns with information technology and project management industry standards. GAO reviewed DOD documents, and interviewed DOD program officials as well as DSAID users. Specifically, GAO conducted site visits to 9 military installations and met with 42 DSAID users. Views obtained are nongeneralizable. Installations were selected based on their use of DSAID, number of users, geographic diversity, and other factors. GAO is not making recommendations in this report. DOD provided technical comments, which GAO incorporated as appropriate. As of October 2013, the Department of Defense's (DOD) Defense Sexual Assault Incident Database (DSAID) was fully implemented and in use across the military services, and DOD had taken several steps to standardize DSAID's use throughout the department. Sexual assault incident data are input into DSAID through both manual and automated data entry processes and include, as applicable, victim and referral support information, investigative and incident information, and case outcome data for certain incidents of sexual assault that involve a servicemember. Additionally, in some instances DSAID includes sexual assault cases involving a servicemember spouse, an adult family member, and DOD civilians and contractors. Further, DOD has taken several steps to standardize DSAID's use through the development of (1) policies, processes, and procedures for using the system; (2) training for system users; and (3) processes for monitoring the completeness of data. DSAID users have identified technical challenges with the system and DOD officials stated that they have plans to spend approximately $8.5 million to implement modifications to DSAID that address most of these challenges in fiscal years 2017 and 2018. Some of the key technical challenges users have identified experiencing with the system relate to DSAID's system speed and ease of use; interfaces with other external DOD databases; and users' ability to query data and generate reports. DOD has plans in place to implement modifications to DSAID that are expected to alleviate these challenges; however, officials stated that they will not be approved to fund these modifications until they have conducted an analysis of alternatives that is in line with DOD's acquisition policy framework. This framework, as well as the GAO Cost Estimating and Assessment Guide, outline key elements of this analysis, such as relative lifecycle costs and benefits and the effect and value of cost and schedule, among others. Conducting an analysis of alternatives including these elements is key to ensuring that DOD appropriately manages its modifications to DSAID. In 2010, GAO found that DOD had failed to demonstrate adherence to these key elements in the initial development and implementation of DSAID, and, DOD projects it will have spent a total of approximately $31.5 million on implementing and maintaining DSAID through fiscal year 2018. This is approximately $13 million more than the 2012 estimate. DOD's plan to conduct an analysis of alternatives that adequately considers key elements should position DOD to more accurately assess whether planned modifications to DSAID can be implemented within budget and result in the desired outcome. DOD manages modifications to DSAID through its change management process, which GAO found substantially aligns with key applicable elements established in the industry standards that GAO reviewed. Specifically, DOD has established processes for managing change requests, such as developing a process to evaluate requested changes to the database and establishing a board that approves, tracks, and controls changes to the database. DOD has also established processes for configuration management, including a process to track, communicate, and deliver changes to the database.

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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. — Authorities have arrested a man in the bizarre killing of his stepfather, who was found with his own underwear stretched around his head and neck. The stepson called it an “atomic wedgie.” Brad Davis, 33, of McLoud, was arrested on a murder complaint Tuesday evening in the death of Denver Lee St. Clair, 58. St. Clair was the husband of Tressia St. Clair, Davis' mother. McLoud police and Pottawatomie County deputies were called about 10:10 p.m. Dec. 21 to 4 Shadow Lake. Davis said he and St. Clair fought, and he thought St. Clair was dead, according to a probable cause affidavit. When authorities questioned Davis, he said St. Clair asked him to come over for drinks. The men began arguing and Davis told officers that St. Clair “came at him,” and they began exchanging blows. Davis told investigators that he hit St. Clair's head, causing him to lose consciousness. Then, Davis said, he grabbed his stepfather's underwear and gave him an “atomic wedgie” by pulling the underwear over his stepfather's head. “I'd never seen this before, but when we first looked at our victim seeing the waistband of his underwear was around his neck,” Pottawatomie County Sheriff Mike Booth said. The cause of death has been determined to be from blunt force trauma to the head and asphyxiation. The death has been ruled a homicide, said Amy Elliott, Oklahoma state medical examiner's spokeswoman. While he was at St. Clair's home, Davis sent a friend a text message saying he was planning to harm Denver, according to the affidavit. According to court documents, Tressia St. Clair, who was in the hospital during the confrontation, told investigators that Davis lived in a trailer house at 3 Shadow Lake Drive owned by Tressia and Denver St. Clair. She told investigators that Denver St. Clair thought Davis owed him $7,000 for living in the trailer home. Brad Davis' brother told investigators that on Dec. 20, Denver St. Clair said “he was going to pull Brad out of that trailer by his throat.” St. Clair was under a permanent protective order filed in 2008 by his wife, Tressia Ann St. Clair. She lived in the home where Denver St. Clair was killed. In the 2008 protective order, Tressia St. Clair claimed she found her husband embraced in a “sexual way,” with a drinking buddy and that he later grabbed her by the neck and threw her through a bathroom door. A domestic assault and battery misdemeanor case against Denver St. Clair filed in Pottawatomie County District Court that year was dismissed, according to the Oklahoma Supreme Court Network website. Denver St. Clair filed for divorce on Sept. 30, 2013; this was the third time the couple filed for divorce. The first two divorce filings were dismissed and were filed in 2004 and 2010 by Tressia St. Clair. Contributing: Staff Writer LeighAnne Manwarren

Summary: A completely nuts crime report out of Oklahoma: Police say a man died after being given an "atomic wedgie" by his stepson. "I'd never seen this before, but when we first looked at our victim... the waistband of his underwear was around his neck," says Pottawatomie County Sheriff Mike Booth. The victim, Denver Lee St. Clair, was already dead when police found him at his home on Dec. 21. His stepson, Brad Davis told investigators an altercation occurred after he went to the house. Davis, 33, says St. Clair, 58, "jumped" him when he arrived. It appears there was a fight at that point: St. Clair had a head wound and his underwear had been pulled up his back, over his head, and around his neck. His cause of death was found to be blunt force trauma to the head and asphyxiation. In other words, as the Oklahoman puts it, he was "suffocated with his own underwear." Davis faces a murder charge. It's not clear what sparked the argument, but WOWK-TV says the men had been drinking and St. Clair may have insulted his estranged wife, Davis' mother.

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Summarize: A unanimous Supreme Court ruling has invalidated an Arkansas state prison rule that barred inmates from growing beards measuring more than a quarter of an inch long. The rule had been challenged by inmate Gregory Holt, a Muslim man who had asked for permission to grow a half-inch-long beard as a compromise from the full beard he believes is required by his faith. In a ruling announced Tuesday, the Supreme Court said the policy violated Holt's religious beliefs. Arkansas was virtually alone in having such a restrictive rule on beards. Holt — who is also known as Abdul Maalik Muhammad — had argued in a handwritten plea to the court that the state's refusal to grant exceptions was oppressive and forced inmates "to either obey their religious beliefs and face disciplinary action on the one hand, or violate those beliefs in order to acquiesce" to the grooming policy. The Supreme Court had earlier granted Holt's request to maintain a half-inch long beard while his case worked its way through the appeals process. Holt, who did time for making a threat against President Bush's daughters before being convicted in 2010 of knifing his girlfriend, was sentenced to life in prison in 2010. The opinion, written by Justice Samuel Alito, said the state has a legitimate need to prevent the concealment of contraband, which was its stated reason for the policy. But, Alito said, "the argument that this interest would be seriously compromised by allowing an inmate to grow a 1⁄2-inch beard is hard to take seriously." "An item of contraband would have to be very small indeed to be concealed by a 1⁄2-inch beard, and a prisoner seeking to hide an item in such a short beard would have to find a way to prevent the item from falling out. Since the Department does not demand that inmates have shaved heads or short crew cuts, it is hard to see why an inmate would seek to hide contraband in a 1⁄2-inch beard rather than in the longer hair on his head," Alito said. Gregory Houston Holt, an Arkansas prisoner who says he should be allowed to keep a beard on religious grounds. Arkansas department of Corrections Facebook Twitter Google Plus Embed 0:42 autoplay autoplay Copy this code to your website or blog IN-DEPTH — Pete Williams and Jon Schuppe 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.

Summary: The Supreme Court was unanimous today in a decision that will allow an Arkansas inmate to grow a half-inch beard. The state's rule specified that beard length must be kept to a quarter inch; Gregory Holt, a Muslim man also known as Abdul Maalik Muhammad, had sought permission to grow the slightly longer beard in a compromise: He believes his faith demands a full beard, but was willing to accept the half-inch length in keeping with what California prison policy afford Muslim inmates there. In what NBC News terms a "handwritten plea," he noted that Arkansas' restriction put inmates in a catch-22: grow the full beard he sees as required by his religion and be disciplined, or give in and violate his faith. Arkansas argued that contraband smuggling would become a concern with the longer beard. Wrote Justice Samuel Alito in the opinion, that "argument... is hard to take seriously. An item of contraband would have to be very small indeed to be concealed by" such a beard, and the inmate would have to devise a way to keep the item in there to boot. Further, "Since the Department does not demand that inmates have shaved heads or short crew cuts, it is hard to see why an inmate would seek to hide contraband in a half-inch beard rather than in the longer hair on his head." The AP notes that more than 40 states allow inmates to keep beards, and NBC frames Arkansas as "virtually alone in having such a restrictive rule." Holt has previously served time for threatening President Bush's daughters; he's now serving a life sentence for a brutal assault on his girlfriend.

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Summarize: The lone survivor of an ill-fated sunset flight over the Manhattan skyline made a frantic cry for help as his helicopter plunged into the frigid East River with five passengers on board. “Mayday, mayday, mayday,” said the pilot, identified by police sources as Rick Vance, shouting to air traffic controllers Sunday night. “East River, engine failure.” A helicopter crashed into the East River, near E. 86th St. and Roosevelt Island, late Sunday. (JJ Magers / Twitter) A LaGuardia Airport dispatcher heard that plea, according to air traffic audio of the tragic crash, and asked him to repeat what had happened — twice — after his signal apparently broke up. The dispatcher asked if Vance needed “any assistance.” Five dead after helicopter crashes into East River Another frustrated voice joined the chaotic mix to clue in the dispatcher. “It was a mayday call, LaGuardia,” the man said. Deadly Helicopter crash in New York City's East River The radio traffic revealed that the dispatchers were among several who called the NYPD with news of the fatal impact, hoping that first responders could reach survivors by boat. Video footage of the crash showed the Eurocopter AS350 gliding into the water and tipping forward as the rotors continued to spin. Then, the helicopter flipped. Passenger bag may have caused deadly East River helicopter crash Rescue boats work at the scene where the helicopter came down in the East River. (Susan Watts/New York Daily News) Two of the five passengers strapped inside for a photoshoot of the city’s towering skyscrapers were dead as rescuers reached them. Three more died later after being pulled from the water, officials said Monday. Nearby pilots, including one with another photography tour that took off with the ill-fated flight, swarmed the river almost immediately to find the crashed chopper. From above, they found the harrowing sight near E. 86th St. and Roosevelt Island. “It appears that the aircraft is inverted in the water,” said another pilot hovering above the crash site, as a boat approached the helicopter. “The aircraft is upside down.” Another man with eyes on the wreckage said he could see the surviving pilot on top of his partially submerged aircraft. Video captures take-off of helicopter that crashed in East River Medics move a victim through the East 34th St. ferry dock following the crash. (Gardiner Anderson/for New York Daily News) Police sources said investigators have spoken to Vance since the crash and that he’s been released from a nearby hospital. The National Transportation Safety Board will be investigating what went wrong. Sign up for BREAKING NEWS Emails privacy policy Thanks for subscribing! New York (CNN) The New York helicopter crash that killed everyone on board except the pilot may have been caused by a passenger's piece of luggage, the pilot told investigators. The pilot said one of the passenger's bags may have inadvertently hit the emergency fuel shutoff button, leading to the crash that killed five passengers, a senior law enforcement official said. A police source identified the pilot as 33-year-old Richard Vance. The passengers were on a Liberty Helicopters chopper that had been chartered for a private photo shoot, authorities said. All the victims were between 26 and 34 years old, according to the New York Police Department. @cnn @FoxNews just witnessed a helicopter crash into the East River.. hope everyone's ok. Caught it all on tape! pic.twitter.com/saHOMTLR69 The helicopter was lifted from the water on Monday afternoon and is being examined by experts from the National Transportation Safety Board, NTSB member Bella Dinh-Zarr said. Investigators will inspect the helicopter, its flotation devices, the weather and other factors in their attempt to determine the cause of the crash, she said. The crash was the third for Liberty Helicopters in the past 11 years. US Sen. Chuck Schumer of New York called on the FAA to suspend Liberty Helicopters' FAA operating certificate until the company's safety record and this crash are fully assessed. "Three (crashes) is too many," Schumer said. "There are too many allegations. No one knows what's happened. I don't think Liberty should be flying until we get to the bottom of this." Chopper was upside down and submerged The pilot was the sole survivor of the helicopter crash Sunday evening. The Federal Aviation Administration said the helicopter, a Eurocopter AS350, went down in the East River near Roosevelt Island at 7 p.m. In an audio recording of a mayday call to LaGuardia Airport, the pilot said the helicopter was experiencing engine failure. When emergency workers responded, the helicopter was upside down and submerged, authorities said. Police called for a barge with a crane to pull the chopper out of the water near 23rd Street. "One of the most difficult parts of the rescue were that five people were tightly harnessed," Fire Department of New York Commissioner Daniel Nigro said. "People had to be cut out." The pilot was able to free himself and was rescued, Nigro said. Passengers are identified Richard Vance, the pilot in Sunday's crash, is a licensed helicopter pilot from Danbury, Connecticut, FAA records show. His current commercial pilot license was issued in September 2011, according to FAA records. Anthony Vance, the pilot's brother, told CNN in a phone interview that "he did his job and got out alive." "He's a true f---ing pilot, so just let him be," he said. The NYPD identified the victims as Carla Vallejos Blanco, 29; Daniel Thompson, 34; Tristian Hill, 29; Trevor Cadigan, 26; and Brian McDaniel, 26. Carla Vallejos Blanco was killed in the helicopter crash on Sunday. Vallejos Blanco, a tourist from Argentina, was on a vacation taking a photographic tour of the city when she was killed, according to deputy consul general Eduardo Almirantearena. The consulate said her family is working with the New York City medical examiner to bring her body back to Argentina. Cuenca del Plata University in Argentina said she studied art, design and communications. Cadigan was an intern at media organization Business Insider until a few weeks ago, according to a company spokesman. "He was a smart, talented, and ambitious young journalist and producer who was well-liked and made a big contribution. Our hearts go out to his family and friends," the company said. Cadigan was the son of Jerry Cadigan, the production manager for WFAA in Dallas, and had interned at WFAA previously. "The entire WFAA family is heartbroken by the sudden and tragic loss of Trevor Cadigan," Brad Ramsey, WFAA president and general manager, said in a statement. "We would like to thank the many friends and former employees of WFAA who have reached out to offer your condolences and support. Our thoughts and prayers are with all of Trevor's family and friends, and with the families of all of the victims of yesterday's tragic accident." Officer Brian McDaniel of Dallas-Fire Rescue died in a helicopter crash on March 11, 2018 in New York. McDaniel was a fire rescue officer for Dallas Fire-Rescue who had been hired in May 2016, the department said in a statement. "Despite his short tenure, hearts are heavy with grief as we not only try to come to grips with his loss departmentally; but to also be there in every way that we can for his family." Company had 3 crashes in 11 years A helicopter is hoisted by crane from the East River onto a barge, Monday, March 12, 2018, in New York. Liberty Helicopters describes itself as "the largest and most experienced helicopter sightseeing and charter service in New York City." The company has "a fleet of 10 state-of-the-art Airbus helicopters (formerly American Eurocopter)," according to the website. "We have been in business and flying safely for over 30 years," the website says. This is the company's third crash since 2007. In August 2009, nine people were killed after a helicopter and a small, private plane crashed into each other over the Hudson River. The FAA said the helicopter and small plane were communicating on different radio frequencies "and were not aware of each other's positions." Two years before that, in July 2007, a Liberty sightseeing chopper carrying eight people suffered a rotor blade separation midflight and crashed into the Hudson River. All passengers were wearing inflatable life vests, and the pilot said she deployed pop-out floats, the National Transportation Safety Board said. Neither the pilot nor the seven passengers were seriously injured. In all, the FAA has documented 16 accidents or incidents involving Liberty Helicopters since 1995. The 2009 crash was the only previous incident with fatalities. There are no accident or incident histories or closed enforcement actions for the Eurocopter AS350 helicopter, the N350LH, or for pilot Richard Vance, according to FAA spokesman Jim Peters. Crash investigation First responders carry a person to an ambulance after the helicopter crashed into the East River. The NTSB will likely look at three things: the pilot's training, experience and immediate response during the crash; what, if anything, on the helicopter caused the crash; and what environmental factors may have contributed to the crash, said Gary C. Robb, an aviation attorney based in Missouri. Robb said the NTSB would then release a preliminary report, and a probable-cause accident report would follow detailing what happened during the crash. He said that any helicopter operating around water should have floats so it can land on water and stay upright. Based on the video of the crash, Robb speculated that one of the floats on the helicopter did not activate, which may have caused it to turn sideways in the water. Liberty Helicopters posted a statement on its website, saying it is "focused on supporting the families affected by this tragic accident and on fully cooperating with the FAA and NTSB investigations." It said it was referring all press inquiries to federal agencies.

Summary: It will take some time before investigators can say for sure what caused a helicopter to crash into the East River Sunday evening, but CNN reports on one possible explanation. The pilot told investigators that a passenger's bag may have hit the emergency fuel shutoff button, according to a law enforcement source cited by the network. The pilot, identified as 33-year-old Richard Vance, survived the crash, but all five passengers on the aircraft operated by Liberty Helicopters were killed. In air traffic audio just before the crash, the pilot can be heard saying, "Mayday, mayday, mayday," reports the Daily News. "East River, engine failure," he says. A LaGuardia Airport dispatcher, having trouble understanding him, asked him to repeat what he said and asked whether he needed any assistance. At that point, another voice chimes in, "It was a mayday call, LaGuardia."

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Summarize: CLOSE Hundreds of activists came to Bedminster to protest President Donald Trump during the national Families Belong Together rally. Editorial Intern Alexander Lewis contributed to this video. Nick Muscavage, @nmuscavage Activists gather to protest the Trump administration's approach to illegal border crossings and separation of children from immigrant parents in Washington. (Photo: Alex Brandon, AP) BEDMINSTER, NJ – President Donald Trump will have to watch the news or surf the Internet to see the tens of thousands of people gathering outside the White House protesting his "zero tolerance" immigration policies. He's 220 miles from the official residence, ensconced in Trump National Golf Club, his private resort in Bedminster, New Jersey. The president has no public events this weekend and is not expected to make comments in the press. So some protesters decided to come to him. More than 100 people lined up along the side of Route 206 a few miles away from the president's club in Bedminster, waving anti-Trump signs and chanting, “Where are the children!” on Saturday during the "Families Belong Together" protest near his club. Weekly protests have been going on here since the president’s 2016 election but this is one of the largest turnouts here. Out of sight, Trump turned to his favorite medium – Twitter – to push back on some Democrats' calls for the abolition of the U.S. Immigration and Customs Enforcement which has been carrying out the president's directive. "The Democrats are making a strong push to abolish ICE, one of the smartest, toughest and most spirited law enforcement groups of men and women that I have ever seen," he tweeted. "The radical left Dems want you out. Next it will be all police. Zero chance, It will never happen!" The Democrats are making a strong push to abolish ICE, one of the smartest, toughest and most spirited law enforcement groups of men and women that I have ever seen. I have watched ICE liberate towns from the grasp of MS-13 & clean out the toughest of situations. They are great! — Donald J. Trump (@realDonaldTrump) June 30, 2018 To the great and brave men and women of ICE, do not worry or lose your spirit. You are doing a fantastic job of keeping us safe by eradicating the worst criminal elements. So brave! The radical left Dems want you out. Next it will be all police. Zero chance, It will never happen! — Donald J. Trump (@realDonaldTrump) June 30, 2018 After receiving backlash from immigrants, Democrats and Republicans, Trump signed an executive order on June 20 to end the practice of separating parents from their children after being arrested. But the order did not end the administration's policy of referring all migrants caught illegally crossing the border for criminal prosecution. The order says the administration will "rigorously enforce" immigration laws and "initiate proceedings" accordingly. And Trump said after signing the order that officials will have "zero tolerance" for people crossing the border illegally. What has changed is that immigration agents have temporarily stopped charging border crossers if they arrive with children. Trump has gone back and forth on whether he wants Congress to pass an immigration bill. On Saturday, he tweeted that he "never pushed" lawmakers to pass a hardline immigration bill or a more moderate version sponsored by House Judiciary Committee Chairman Bob Goodlatte, R-Va., "because it could never have gotten enough Democrats as long as there is the 60 vote threshold." That's a reference to the filibuster-proof margin needed to pass bills in the Senate. I never pushed the Republicans in the House to vote for the Immigration Bill, either GOODLATTE 1 or 2, because it could never have gotten enough Democrats as long as there is the 60 vote threshold. I released many prior to the vote knowing we need more Republicans to win in Nov. — Donald J. Trump (@realDonaldTrump) June 30, 2018 Both of Goodtlatte's bills to fund President Trump's border wall and dramatically decrease legal immigration died in the GOP-controlled House after conservative and hard-line Republicans split their support. Democrats voted against both of them. On Wednesday, several hours prior to the defeat of the second bill, Trump tweeted that the House "SHOULD PASS THE STRONG BUT FAIR IMMIGRATION BILL, KNOWN AS GOODLATTE II, EVEN THOUGH DEMS WON'T LET IT PASS IN THE SENATE. PASSAGE WILL SHOW THAT WE WANT STRONG BORDERS & SECURITY..." Contributing: Alan Gomez, USA TODAY; Nick Muscavage, Bridgewater (NJ) Courier-News More: As thousands prepare to rally, here's where things stand on immigration More: Timeline: Immigrant children separated from families at the border More: In 50 states and extreme heat, thousands march for 'Families Belong Together' rallies Read or Share this story: https://usat.ly/2KDRJ4p Breaking News Emails Get breaking news alerts and special reports. The news and stories that matter, delivered weekday mornings. Dec. 29, 2017, 5:00 PM GMT / Updated Nov. 19, 2018, 1:31 PM GMT By Liz Johnstone President Donald Trump frequently uses his luxury properties for government business and leisure, prompting ethics concerns over a president appearing to promote his private enterprise at public cost. The NBC News digital politics team and the White House unit are tracking Trump's visits to his properties and golf courses since his inauguration. Where does Trump go? Trump's trips have so far been concentrated in Palm Beach, Florida, where he mainly visits his Mar-a-Lago club and golf course, Trump International Golf Club. He's spent "working vacations" at his Bedminster, New Jersey, golf club, and as well as a handful of days at Trump Tower in Manhattan. He also frequents his golf course in Sterling, Virginia, near Washington, and his luxury hotel, Trump International, just blocks from the White House — and sometimes will visit multiple properties on the same day. On a July 2018 trip to the U.K., the president visited his Trump Turnberry golf property in Scotland. How many times has Trump gone golfing as president? Trump visited his golf clubs more than 150 times in his first official year in office. However, the precise number of times Trump has actually played golf is difficult to track. His administration has tried to hide Trump's activity, keeping his traveling press pool away and often refusing to confirm whether he has played golf. Instead, social media has become a source of crowd-sourced reporting into the president's whereabouts. Related: How Donald Trump's Old Tweets Haunt Him Today During the campaign, Trump argued that Americans should vote for him because he would rarely leave Washington. He promised that he wouldn't go golfing or take vacations because there was too much work to do. And prior to his run for president, Trump spent years attacking President Barack Obama for golfing and taking vacations while in office. When does Trump visit his properties? With the exception of extended stays at his golf resort in Bedminster, Trump usually frequents his properties on weekends. Trump has referred to his Mar-a-Lago club in Palm Beach, Florida, as the "Southern White House" — and that's where he's hosted foreign leaders like President Xi Jinping of China and directed the launch of a strike against a Syrian airbase. How much does Trump's travel cost? The president's trips can cost taxpayers millions of dollars because of the use of Air Force One and the expenses of the Secret Service, the Air Force, local sheriff's departments, the Coast Guard and other agencies. However, a precise accounting can't be made because the expenses aren't required to be disclosed. In response to a recent report in USA Today that Secret Service's funding problems were partly caused by the president's large family and their multiple, oft-visited Trump properties both in the U.S. and overseas, agency director Randolph "Tex" Alles said it was not an issue "that can be attributed to the current Administration’s protection requirements alone." Presidents other than Trump incurred similar questions about travel costs. Much of what is known about Obama's travel costs was obtained by the conservative watchdog group Judicial Watch, which aggressively sought receipts through Freedom of Information Act requests. In October 2016, the Government Accountability Office (GAO) issued its own report breaking out the costs of one specific trip taken by Obama, finding that a trip to Chicago and Florida cost $3.6 million. This tracker automatically counts Trump's days in office. When Trump arrives at one of his properties, the tracker updates accordingly.

Summary: As thousands gathered near the White House to protest the Trump administration's "zero tolerance" immigration policy on Saturday, the president was 220 miles away, at the Trump National Golf Club in Bedminster, NJ. But he couldn't entirely escape the demonstrators, reports USA Today. About 100 protesters lined up along Route 206, near president's private resort, holding up signs and calling out, "Where are the children!?" Protesters are not an unusual sight in Bedminster; they've been gathering weekly near the club for the last 18 months, but Saturday's crowd was the largest so far. Although the president's visit to Bedminster was his first this year, he spent 40 days there in 2017, according to NBC. In all, he has spent 31% of his time in office at his properties.

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Summarize: An anonymous caller warned authorities Monday that his roommate was on his way to Yale University to shoot people, leading officials to lock the Ivy League campus down as police searched for a gunman. At least two witnesses reported seeing someone on or near campus with a long gun. An emergency call was received around 9:30 a.m. local time from a man at a pay phone about a mile (two kilometers) from the campus, said Officer David Hartman, a New Haven Police spokesman. "All he really said was that his roommate was on his way to the university, to Yale University, to shoot people," he said. Police later received reports from witnesses who reported seeing someone with a gun, Hartman said. He said that there was nothing specific about the threat and that the call lasted just several seconds. There were no reports of shots fired or anyone injured. Police blocked off several streets near the Ivy League university, located in the heart of New Haven. Several local schools also were placed in lockdown. Yale advised students and staff to shelter in place. The school also issued an advisory asking people off campus to stay away from the area. The school is on November break, with undergraduate classes to resume Dec. 2. Many students and staff had left campus for the American holiday of Thanksgiving. Pedestrian traffic in the normally bustling area also was sparse, with cold and windy weather keeping many people inside. Police in tactical gear entered several buildings, and a helicopter hovered over the area. Yale has been the target of violence in the past. In May 2003, a bomb damaged an empty classroom and adjacent reading room at the law school. A Yale professor, David J. Gelernter, was seriously injured in 1993, when a mail bomb mailed by Theodore Kaczynski, the man known as the Unabomber, exploded in his campus office. Friday's search came about two weeks after a scare on another Connecticut campus. Central Connecticut State University was in lockdown for several hours Nov. 4 after reports by witnesses of a masked man carrying a gun or sword. Police arrested a student, David Kyem, who said he had been wearing a ninja-like Halloween costume and meant no harm. He faces charges including breach of peace. A day-long lockdown of the Yale University campus was lifted this evening after authorities determined that a call warning of a gunman intent on shooting people was likely a hoax. Yale's New Haven, Conn., campus was put on lockdown after an anonymous male called 911 this morning to say his roommate was on his way to the campus with the intention of shooting people, according to officials. New Haven Police Chief Dean Esserman said there was "malicious intent" behind the call. However, after a search of the campus did not reveal any threats, he said authorities are "leaning toward this being a hoax." Esserman said once police identify the caller, that person "will face every charge possibly imaginable." The anonymous call was placed from a phone booth and the university sent out an alert at 10:17 a.m. ET. At a news conference earlier today, New Haven Police Officer David Hartman said authorities interviewed several witnesses who said they saw someone with a "long gun." After speaking with some of the witnesses, Hartman said, "Some of those witnesses in all probability saw police officers with long guns instead of a suspect." Yale Police Chief Ronell Higgins said this incident showed that the school's emergency preparedness plan worked well. "You have to exchange business cards with local law enforcement and schools before incidents like this one," he said. "We are always in constant communication." An email was also sent by Yale's dean to notify parents of undergraduate students of the threat. The school is on its November break, meaning there are fewer people on campus than usual. An alert send by the university this morning instructed people to shelter in place. "Confirmed report of person with a gun on/near Old Campus. SHELTER IN PLACE. This is NOT a test," the alert said. A search of the campus dorms was hampered because some students refused to open their doors to police, fearing that the person knocking was not really a police officer. The university has over 11,000 students.

Summary: Yale University has lifted its lockdown after police said an anonymous call about a gunman headed to campus was probably a hoax, reports the AP. "New Haven is safe. The Yale campus is safe," said New Haven's police chief. "Though it is starting to tilt in the direction of an innocent mistake, it started with a purposeful and malicious call." A New Haven police officer said earlier that the caller claimed "that his roommate was on his way to the university, to Yale University, to shoot people." Several witnesses reported seeing someone with a "long gun," but there were no reports of shots fired or injuries, ABC News reports. During the lockdown, police blocked off several streets near the school and advised students and faculty to either stay off campus, or stay where they were on campus.

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Summarize: By. Harriet Arkell. PUBLISHED:. 05:21 EST, 14 January 2014. |. UPDATED:. 06:02 EST, 14 January 2014. A lawyer defending a woman who was caught drink-driving while her small child was at home alone has accused Madeleine McCann's parents of child neglect. In what has been described as an 'outrageous' slur on the missing girl's parents, defence lawyer Gareth Morgan compared them unfavourably to his client, who left her five-year-old son at home by himself before being arrested after she crashed her car at traffic lights. He told magistrates at Merthyr Tydfil, south Wales: 'The parents of Madeleine McCann were guilty of child neglect for leaving their little girl much longer than my client. They were never prosecuted.' Kate and Gerry McCann, pictured, were accused of child neglect by a lawyer defending a drink-driving mother. The lawyer said that his client, whose son has Asperger's, was ashamed of what she had done. The 44-year-old defendant, who cannot be named, hit the back of another car at traffic lights. When police followed her home, she told them they couldn't arrest her because her son was by himself in the house, and they found the child asleep under a blanket on the sofa. The court heard that the woman was more than two-and-a-half times over the limit when she drove to a cashpoint machine just before midnight. On her way home, she ran into a car driven by Bronnie-May Dunn, 21, which was stopped at traffic lights. Miss Dunn suffered head injuries in the crash but magistrates heard that the driver shouted at her, saying it was her fault. The woman admitted child neglect, drink-driving and leaving an accident and was given a 12-month supervision order, 120 hours of unpaid work, a 22-month driving ban and told to attend a course. Today a friend of the McCanns, whose daughter went missing in Portugal in May 2007 while they ate in a nearby restaurant with friends, hit out at Mr Morgan's comments. Resort: Madeleine McCann went missing from her apartment in The Ocean Club in Praia Da Luz in 2007. British police are hoping to go to Portugal to interview three new 'prime suspects' in the hunt for Madeleine, pictured. The friend told the Mirror: 'If Kate and Gerry were guilty of child neglect, and they were absolutely not, they would have been dealt with. 'As it is, there are two police inquiries in two different countries trying to find out what happened to Madeleine, and comments made by a lawyer in an unrelated case is offensive, outrageous and inaccurate.' It was revealed today that Portuguese police are under mounting pressure to give British detectives access to the three 'prime suspects' wanted for questioning over the abduction of Madeleine McCann. The Crown Prosecution Service has sent them a second 'International Letter of Request' after identifying three burglars who were operating in the Algarve resort where the McCanns were staying. Analysis of mobile phone records at the time show that they made a high number of calls at around the time the small girl was taken. It is now known that between January and May 2007 there had been a four-fold increase in the number of burglaries in the area around the Praia da Luz resort where the family from Rothley, Leicestershire, was staying. Police said in October that in the 17 days before she disappeared there was one burglary and one attempted burglary in the block where the McCann family were staying

Summary: Defence lawyer Gareth Morgan compared them to his drink-driving client. The woman was over limit when she crashed on way back from cashpoint. He told Merthyr Tydfil magistrates McCanns left Maddie for much longer. His client, 44, who can't be named, admitted drink-driving and child neglect. She also admitted leaving an accident and was given 22-month driving ban. Friend of Kate and Gerry McCann said slur was 'offensive and inaccurate'

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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation application of U.S. patent application Ser. No. 15/179,289 filed Jun. 10, 2016, which is a continuation of U.S. patent application Ser. No. 14/293,621 filed Jun. 2, 2014, now U.S. Pat. No. 9,387,348, which is a continuation application of Ser. No. 13/322,327 filed Nov. 23, 2011, now U.S. Pat. No. 8,835,877, which is a U.S. National Application of International Patent Application No. PCT/US2010/50850 filed Sep. 30, 2010, which claims the benefit of U.S. Provisional Patent Application No. 61/277,792 filed Sep. 30, 2009, U.S. Provisional Patent Application No. 61/365,175 filed Jul. 16, 2010, U.S. Provisional Patent Application No. 61/365,449 filed Jul. 19, 2010, which are incorporated by reference herein in their entirety. STATEMENT CONCERNING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under NSF CBET-0755054. The government has certain rights in the invention. FIELD OF THE INVENTION [0003] The present invention relates to photon-based radiosurgery and more specifically to dynamic photon painting. The present invention provides for using a dynamically changing radiation beam (such as speed, direction, and/or dose) to irradiate a target thereby significantly increasing a radiation dose falloff rate. BACKGROUND OF THE INVENTION [0004] Radiosurgery is a non-invasive medical procedure for various kinds of tumors and one of the most effective means for treating local and regional targets such as brain tumors. Instead of a surgical incision, radiosurgery delivers a high dose of high energy photons in radiated beams to destroy the tumor. Radiosurgery is a very efficient method for treating cancers and avoids loss in quality of life compared to other more invasive methods such as surgery or chemotherapy. Since radiated high energy photons can also damage normal cells that are irradiated as the beam passes through a patient to irradiate a tumor, the key of a good radiosurgery plan is to maintain a sharp radiation dose falloff from the high radiation dose regions (high dose regions) inside the tumor to the low radiation dose regions (low dose regions) of nearby healthy structures. The steep radiation falloff rate of dose distribution—known as the “dose falloff rate”—guarantees that normal, healthy tissue and other body parts or structures near the target receive a low dose of radiation while the center of the target or tumor receives a high dose of radiation. Sharper radiation dose falloff will results in better tumor control and less damage to the normal tissue and other body parts surrounding the tumor that are irradiated by the radiation beams. [0000] Focused Beam Geometry: Currently, most radiosurgeries are performed in a “step-and-shoot” manner and use a number of precisely focused external beams of radiation that are aimed at the target from different directions to increase the dose falloff rate (see FIG. 1 ). In this technique, as the number of radiation beams increases, the dose falloff rate improves. Therefore, a large number of radiation beams focus on a target to create a high dose region around the target at the point of intersection of the beams. Intuitively, if the number of beams is increased, the contribution of each beam inevitably decreases, resulting in a lower dose to the tissues and structures some distance away from the target. This is because more beams pass through different parts of the body at lower radiation doses but collectively provide the same radiation dose to the target. [0005] However, in these conventional radiation treatments the number of radiation beams is constrained to several hundred beams due to various spatial and physical constraints. For example, in Gamma Knife® radiosurgery, the number of radiation beams is limited to about two hundred beams. Physically, it is not possible to drill a large number of apertures in a fixed size metal screen without eventually causing interference among the beams escaping from the apertures. [0006] For intensity-modulated radiation therapy (IMRT), it is usually not practical to deliver more than a dozen beams due to prolonged treatment time. Even with rotational techniques, such as Tomotherapy, intensity-modulated arc therapy (IMAT), volumetric modulated arc therapy (VMAT), and arc-modulated radiation therapy (AMRT), the maximum number of radiation beams is still limited to a few hundred. Fundamental Physics Underlying Photon Radiosurgery: [0007] The fundamental physics underlying photon-based radiosurgeries includes high energy photon production and photon interactions with matter. [0008] Generally, high energy photons used in current radiosurgeries are produced either by radioactive decay from Cobalt-60 sources or bremsstrahlung interactions in a linear accelerator. In the linear accelerator, electrons are accelerated in an electric field to a high energy and then collide with a metal target. This generates radiation particles or photons in a bremsstrahlung process. The photons produced from Cobalt-60 are called “γ-ray” or gamma rays whereas the photons produced from a linear accelerator are called “X-ray” or X-rays. [0009] Typically photons produced by different sources are heterogeneous in energy. For example, the energies of γ-rays emitted by Cobalt-60 are 1.17 and 1.33 MeV. The energy spectrum of X-rays from a linear accelerator shows a continuous distribution of energies for the bremsstrahlung photons superimposed by characteristic radiation of discrete energies. The energies of photon beams created by a 6MV accelerator are continuous from 0 to 6 MeV with a large number of photons having energy around 2 MeV. For examples, Gamma Knife® (see FIG. 3 ) uses γ-rays emitted from radioactive Cobalt-60 sources to irradiate tumors, while Cyberknife® (see FIG. 4 ), which is essentially a linear accelerator carried by a robotic arm, uses X-rays to irradiate tumors. [0010] When photons pass through matter, they interact in one of three ways: Photoelectric effect, Compton effect and Pair production. For radiosurgery, the predominant interaction is the Compton effect, where the incident photons collide elastically with orbit electrons. During this elastic collision, energy is imparted from the incident photons to orbiting electrons and sets off a chain of reactions. These electrons know as secondary electrons, as they travel through matter, produce ionization and excitation along their path. On a cellular level, these ionizations damage DNA and cause cell death in the body. Important Beam Characteristics for Treatment Planning: [0011] A percent depth dose curve relates the absorbed dose deposited by a radiation beam into a medium. FIG. 2A shows the percent depth dose curve of Cobalt-60 with an 80 cm Source Surface Distance (SSD). Two parameters of a radiation beam are its Tissue Maximum Ratio (TMR) and Off Center Ratio (OCR). TMR is defined as the ratio of the dose at a given point in phantom to the dose at the same point at the reference depth of maximum dose. OCR is the ratio of the absorbed dose at a given off-axis point relative to the dose at the central axis at the same depth. FIG. 2B shows the TMR of Cobalt-60 and a 6MV accelerator. FIG. 2C shows the OCR of a 6MV accelerator. SUMMARY OF THE INVENTION [0012] The present invention improves the quality of radiosurgery by increasing the dose fall-off rate. The dose fall-off rate is determined from high dose regions inside a target such as a tumor to low dose regions of nearby healthy tissues and body parts or structure. [0013] In order to further improve the focusing power of radiosurgery, dynamic strategies are implemented in the present invention. A beam source is directed around a focused point in a three dimensional (3D) trajectory and may provide a constant change of dose rate, speed, and beam directions to create kernels. The dynamic motion is equivalent to focusing tens of thousands of beams at a focus point and therefore creates kernels with a much sharper dose falloff. [0014] The present invention uses a new optimization paradigm called “kernelling and de-convolution”. The paradigm uses two key steps: (1) kernelling, in which a subset of beams is “grouped” together by convolution to form “dose kernels”, and then optimized based on the kernels. (2) de-convolution, in which, once kernel level optimization is done, the kernels are de-convolved into individual beams to form a final dynamic plan. Instead of relying on numerical optimization, the present invention uses a hybrid geometric technique that involves geometric routing in both steps, and thus avoids the daunting task of optimizing hundreds of thousands of beams numerically. [0015] Specifically, a radiation beam is moved along a helical type trajectory to dynamically irradiate a target and thereby further improve the dose falloff rate. This approach according to the present invention is termed herein as “dynamic photon painting” (DPP). The dose distribution from this convergence of tens of thousands of beams on a small volume is used as the DPP kernel. [0016] As mentioned previously, the key to radiosurgery is the dose falloff rate. According to the present invention, DPP moves a beam source around an isocenter in a 3D trajectory, which is equivalent to focusing thousands of beams on a single point, to increase the dose falloff rate. FIG. 5 illustrates the trajectories of the radiation beam in DPP. The beam source rotates around the center of the target from latitude angle φ 1 to φ 2, and 360° around in longitude angle. [0017] The present invention also overcomes computational problems using the DPP approach. The least square problem often occurs as a key sub-problem of some larger computational problem, such as radiosurgery treatment planning. The least square problem is defined as min ∥Ax−b∥ 2. Intuitively in this model, each column of A represents a radiation beam, the column vector b represents the ideal dose distribution and the goal of the optimization is to find the optimal “beam on time” for each column (i.e. X) to create a distribution as close to b as possible. Since in reality, “beam on time” must be non-negative, it is required x≧0, which gives the Non-Negative Least Square (NNLS). The following is a brief discussion of the solution of a least square problem and NNLS problem. If the total treatment time must stay under a given threshold T, we end up with the constrained least square problem with the constraint [0000] ∑ x ∈ X   x ≤ T. [0000] There are many algorithmic solutions to the least square problems as is known to those skilled in the art. [0018] The present invention and its attributes and advantages further understood, are further appreciated with reference to the detailed description below of some presently contemplated embodiments, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0019] The preferred embodiments of the invention are described in conjunction with the appended drawings provided to illustrate and not to the limit the invention, where like designations denoted like elements, and in which: [0020] FIG. 1 illustrates the cross-firing technique used in radiosurgery; [0021] FIG. 2A illustrates the Percent Depth Dose of Cobalt-60 and 6MV accelerator sources of radiosurgery; [0022] FIG. 2B illustrates the Tissue Maximum Ratio of Cobalt-60 and 6MV accelerator sources of radiosurgery; [0023] FIG. 2C illustrates the Off Center Ratio curves of Cobalt-60 and 6MV accelerator sources of radiosurgery; [0024] FIG. 3 illustrates a conventional Gamma Knife® machine used in radiosurgery; [0025] FIG. 4 illustrates a conventional CyberKnife® machine used in radiosurgery; [0026] FIG. 5 illustrates the trajectories of the radiation beam in dynamic photon painting according to the present invention; [0027] FIG. 6A illustrates curve fitting results according to the present invention; [0028] FIG. 6B illustrates curve fitting results according to the present invention; [0029] FIG. 7A illustrates radiation dose profile comparisons between dynamic photon painting (DPP) kernels and Gamma Knife® perfexion 4 mm kernels according to the present invention; [0030] FIG. 7B illustrates radiation dose profile comparisons between dynamic photon painting (DPP) kernels and Gamma Knife® perfexion 4 mm kernels according to the present invention [0031] FIG. 8A illustrates isodose comparisons between DPP kernels and Gamma Knife® perfexion 4 mm kernels according to the present invention; [0032] FIG. 8B illustrates isodose comparisons between DPP kernels and Gamma Knife® perfexion 4 mm kernels according to the present invention; [0033] FIG. 8C illustrates isodose comparisons between DPP kernels and Gamma Knife® perfexion 4 mm kernels according to the present invention; [0034] FIG. 8D illustrates isodose comparisons between DPP kernels and Gamma Knife® perfexion 4 mm kernels according to the present invention; [0035] FIG. 9A illustrates dose profile comparisons between kernels created by Cobalt-60 source and CyberKnife® cone beam according to the present invention; [0036] FIG. 9B illustrates dose profile comparisons between kernels created by Cobalt-60 source and CyberKnife® cone beam according to the present invention; [0037] FIG. 10A illustrates comparisons between the DPP kernel and 116 MeV proton according to the present invention; [0038] FIG. 10B illustrates comparisons between the DPP kernel and 116 MeV proton according to the present invention; [0039] FIG. 10C illustrates comparisons between the DPP kernel and 116 MeV proton according to the present invention; [0040] FIG. 11A illustrates the impact of lateral angular range on the dose gradient of DPP kernels according to the present invention; [0041] FIG. 11B illustrates the impact of lateral angular range on the dose gradient of DPP kernels according to the present invention; [0042] FIG. 12A illustrates isodose distributions of DPP kernels of different latitude angular ranges according to the present invention; [0043] FIG. 12B illustrates isodose distributions of DPP kernels of different latitude angular ranges according to the present invention; [0044] FIG. 12C illustrates isodose distributions of DPP kernels of different latitude angular ranges according to the present invention; [0045] FIG. 12D illustrates isodose distributions of DPP kernels of different latitude angular ranges according to the present invention; [0046] FIG. 13A illustrates the impact of complementary error function (ERFC) parameter on the dose gradient of DPP kernels according to the present invention; [0047] FIG. 13B illustrates the impact of complementary error function (ERFC) parameter on the dose gradient of DPP kernels according to the present invention; [0048] FIG. 14A illustrates the isodose distributions of DPP kernels of different ERFC parameters according to the present invention; [0049] FIG. 14B illustrates the isodose distributions of DPP kernels of different ERFC parameters according to the present invention; [0050] FIG. 14C illustrates the isodose distributions of DPP kernels of different ERFC parameters according to the present invention; [0051] FIG. 14D illustrates the isodose distributions of DPP kernels of different ERFC parameters according to the present invention; [0052] FIG. 15A illustrates treatment planning of painting a three-dimensional (3D) tumor volume with a spherical “paintbrush” according to the present invention; [0053] FIG. 15B illustrates treatment planning of painting a three-dimensional (3D) tumor volume with a spherical “paintbrush” according to the present invention; [0054] FIG. 15C illustrates treatment planning of painting a three-dimensional (3D) tumor volume with a spherical “paintbrush” according to the present invention; [0055] FIG. 16 illustrates comparisons of dose-volume histograms (DVH) according to the present invention; [0056] FIG. 17A illustrates dose profile comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0057] FIG. 17B illustrates dose profile comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0058] FIG. 18A illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0059] FIG. 18B illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0060] FIG. 18C illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0061] FIG. 18D illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0062] FIG. 19A illustrates a plot of C-shaped tumor phantom according to the present invention; [0063] FIG. 19B illustrates a plot of C-shaped tumor phantom according to the present invention; [0064] FIG. 20 illustrates comparisons of DVH according to the present invention; [0065] FIG. 21A illustrates dose profile comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0066] FIG. 21B illustrates dose profile comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0067] FIG. 21C illustrates dose profile comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0068] FIG. 22A illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0069] FIG. 22B illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0070] FIG. 22C illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0071] FIG. 22D illustrates isodose comparisons between a DPP plan and a Gamma Knife® plan according to the present invention; [0072] FIG. 23 illustrates comparisons of DVH according to the present invention; [0073] FIG. 24A illustrates dose profile comparisons between various DPP plans according to the present invention; [0074] FIG. 24B illustrates dose profile comparisons between various DPP plans according to the present invention; [0075] FIG. 24C illustrates dose profile comparisons between various DPP plans according to the present invention; [0076] FIG. 25A illustrates isodose comparisons between various DPP plans according to the present invention; [0077] FIG. 25B illustrates isodose comparisons between various DPP plans according to the present invention; [0078] FIG. 25C illustrates isodose comparisons between various DPP plans according to the present invention; [0079] FIG. 25D illustrates isodose comparisons between various DPP plans according to the present invention; and [0080] FIG. 26 illustrates an exemplary computer system, or network architecture, that may be used to implement the methods according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0081] The present invention is now described in detail with reference to preferred embodiments as illustrated in the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It is apparent, however, to one skilled in the art, that the present invention may be practiced without some or all of these specific details. In other instances, well known process steps and/or structures are not described in detail in order to not unnecessarily obscure the present invention. [0082] The present invention uses a new optimization paradigm, in which “kernelling and de-convolution” occurs in the following steps: Step 1—(Kernelling) Dose kernels approximating the radiation dose distributions of about 10,000 focused beams are created by convolving thousands of equivalent beams via preset 3D trajectories; Step 2—(Dose painting) The dose kernel is viewed as a 3D “paintbrush” and an optimal route of the paintbrush is calculated to dynamically “paint” the targeted tumor volume; and Step 3—(De-convolution) The kernel is de-convolved along the route from Step 2 into a single or a few merged trajectories extending into a 4π solid angle of varying source-to-focal distances, which are connected into a dynamic treatment plan using geometric routing algorithms. [0083] One advantage of the present invention kernelling and de-convolution paradigm is routing convolved kernels rather than numerically optimizing individual beams. By doing this process, the daunting task of optimizing hundreds of thousands of beams simultaneously is avoided, which even if implemented may prove to be too computational intensive to be practical. [0084] Kernels are created by convolving 2,000 to 10,000 individual beams along preset 3D trajectories. This step requires a determination of the kind of beam profiles, cross-section shapes, and 3D trajectories that are mostly suited for dynamic radiosurgery in terms of creating the most sharp dose fall-offs in the kernels. [0085] As an integral part of the planning system, a library of kernels is created using different beam shapes, profiles, and trajectories. The characteristics of each kernel in the library can be investigated for producing useful dose focusing powers. [0086] By convolving individual beams into kernels and optimizing kernels rather than individual beams, directly optimizing a large number of beams is avoided, and treatment planning is shifted to routing the kernels to dynamically cover the target. To solve this routing problem, techniques from computational geometry are utilized. [0087] The route calculated in the dose painting step will create a high quality plan, however to deliver it using robotic radiosurgery, this route of the kernels must be converted to a feasible dynamic route of a single beam. To accomplish this process, the kernels to individual beams along the route are de-convolved, which results in a set of beams with different orientation and locations. These individual beams are then connected into a tour, which will be the final dynamic radiosurgery plan. Specifically, the following problem is solved: Given a planar region with the presence of polygonal obstacles (e.g., the robotic arm in a CyberKnife® unit is not allowed in certain region for fear of collision with patient or patient table) and a set of sites, find a tour to visit all the sites. [0088] Turning now to FIG. 5, the above process as dynamic photon painting (DPP) may be performed by using a CyberKnife® cone radiation beam that is revolved in a hemispherical trajectory around a target. As shown in FIG. 5 and described above, the beam source rotates around the center of a target from latitude angle φ 1 to φ 2, and 360° around in a longitude angle. The CyberKnife® beam model is obtained from curve fitting of measured Tissue Phantom Ratio (TPR) and Off Center Ratio (OCR) tables. [0089] FIG. 6A illustrates the curve fitting results for TPR and FIG. 6B illustrates the curve fitting results for OCR. The functions used for curve fitting are: [0000] TPR  ( d ) = { ∑ i = 1 5   a i  d i - 1 for   d &lt; d max  - a 6 · ( d - a 7 ) for   d &gt; d max   and   OCR  ( SAD, r ) = 0.5 · ( erfc ( a · ( r · 800 SAD - b ) ) + erfc ( a · ( r · 800 SAD + b ) ) ), [0000] where d is the depth and r is the off-center radius of the calculation point, Source to Axis Distance (SAD)=Source Surface Distance (SSD)+d, and [0000] erfc  ( x ) = 2 π  ∫ x ∞   - t 2    t [0000] is the error function. For a 10 mm cone, the curve fitting parameters for TPR are a 1 =0.8185, a 2 =0.0203, a 3 =0.004, a 4 =−0.0006, a 5 =0.00002, a 6 =0.0061, a 7 =15, and for OCR a=0.4317 and b=4.9375. (Note that the parameter b here is essentially the radius of the field at 800 mm standard SAD). [0090] The motion trajectory of the beam source (see FIG. 5 ) is described using the following parameters: (1) latitude angular range [φ 1,φ 2 ], (2) longitude angular range [θ 1, θ 2 ], and (3) source to axis distance. [0091] By rotating the radiation beam in a dynamic manner, DPP kernels are created. Comparisons were carried out with Gamma Knife® kernels and proton Bragg Peaks. The DPP kernels were compared with Gamma Knife® Perfexion 4 mm kernels. The Gamma Knife® kernel is a 41×41×41 matrix with 0.5 mm steps. [0092] FIGS. 7A, 7B show the dose profile comparisons between DPP kernels and Gamma Knife® kernels. As shown, the DPP kernels were created using a 10 mm cone of the CyberKnife® beam model, a SAD of 320 mm, and a latitude angular range of 1 to 50°. The SAD was chosen so that the diameter of the DPP kernel at the isocenter is 4 mm. FIG. 7A illustrates the dose profiles in the XY plane (along lateral directions) and FIG. 7B illustrates the dose profiles in the XZ plane (along longitudinal directions). [0093] FIGS. 8A-8D show the isodose comparisons of the two kernels, specifically between the DPP kernel and Gamma Knife® Perfexion 4 mm kernels. In these plots, the planes are defined as in FIG. 5. FIG. 8A illustrates the DPP kernel in the XY plane. FIG. 8B illustrates the Gamma Knife® kernel in the XY plane. FIG. 8C illustrates the DPP kernel in the XZ plane. FIG. 8D illustrates the Gamma Knife® kernel in the XZ plane. The plot shown contains isodose lines from 10% to 100% with 10% steps. The DPP kernel of the present invention has a sharper lateral fall off than the conventional Gamma Knife® kernel. [0094] In order to understand whether the DPP strategy or a specific beam source makes the kernel better, the same DPP trajectory was evaluated using a Cobalt-60 Gamma Knife® beam source as the beam source to create kernels and compared to DPP kernels created with the CyberKnife® cone beam. FIGS. 9A-9B show the dose profile comparisons between kernels created by the Cobalt-60 source and the CyberKnife® cone beam. FIG. 9A illustrates the dose profiles in the XY plane (along the lateral direction). FIG. 9B illustrates the dose profiles in the XZ plane (along the longitudinal direction). The dose profiles are almost identical, which means the impact of beam source is not significant and the DPP strategy causes kernels to have better dose falloff rates. [0095] The same DPP kernels were compared with a pristine 116 MeV proton beam. The proton beam was generated in a water phantom with 10 6 primary protons. The proton beam had a circular Gaussian profile with σ=2 mm. The kernel had a 40 mm radius and bins with 0.5 mm sides and was calculated using the Fluka simulation program. FIG. 10A shows the dose profile comparison in the longitudinal direction. FIG. 10B shows the dose profile comparison in the lateral direction. FIG. 10C shows the VDH comparison. As can be seen, the DPP kernel deposits most of its energy in a small region. [0096] The impact of latitude angular ranges [φ 1,φ 2 ] on the dose gradient of the DPP kernels is also considered. By varying φ 1 and φ 2, a set of kernels is obtained and their dose profiles and isodose distributions are compared as discussed below. [0097] FIGS. 11A-11B show the comparisons of dose profiles with latitude angular ranges of 1 to 40°, 1 to 45°, 1° to 50°, 1° to 55°, 1 to 60°, and 1 to 65°. As Δφ=φ 1 −φ 2 increases, the dose gradient increases in the XY plane (i.e., along the latitude direction) and decreases in the XZ plane (i.e., along the longitudinal direction). The optimal angular range is a tradeoff between the sharpness of dose in the XY plane to that in the XZ plane. In addition to the above comparisons, the impact of φ 1 is considered, the starting latitude angle when Δφ is fixed. The comparisons of the XZ isodose distributions of DPP kernels of different latitude angular ranges are shown in FIGS. 12A-12D. [0098] FIG. 12A illustrates a latitude angular range of 1° to 50°. FIG. 12B illustrates a latitude angular range of 5° to 55°. FIG. 12C illustrates a latitude angular range of 10° to 60°. FIG. 12D illustrates the Gamma Knife® 4 mm kernel. The plots shown contain isodose lines from 5% to 100% with 5% steps. As φ 1 increases, the isodose distributions in the XZ plane become more and more irregular at low dose levels in comparison to that of the Gamma Knife® kernels. The impact of the error function (ERFC) sharpness parameter on DPP kernels is also considered as discussed below. [0099] The Off Center Ratio (OCR) curve is fitted using function ƒ=0.5*(erfc(a(x−b))+erfc(a(x+b))), where erfc(x) is defined as: [0000] erfc  ( x ) = 2 π  ∫ x ∞   - t 2    t. [0000] Mathematically, the parameter “a” reflects the sharpness, while “b” represents the width or radius of the field. FIGS. 13A-13B show the comparison of dose profiles with a=1 and a=10. Specifically, FIG. 13A illustrates the profile comparison in the XY plane. FIG. 13B illustrates the profile comparisons in the XZ plane. FIGS. 14A-14D show the isodose comparison of DPP kernels with different ERFC parameters. Specifically, FIG. 14A illustrates the isodose distributions of the DPP kernel with a=10 in the XY plane. FIG. 14B illustrates the isodose distributions of the DPP kernel with a=1 in the XY plane. FIG. 14C illustrates the isodose distributions of the DPP kernel with a=10 in the XZ plane. FIG. 14D illustrates the isodose distributions of the DPP kernel with a=1 in the XZ plane. The plots shown contain isodose lines from 10% to 100% with 10% steps. As can be seen from these figures, the dose falloff rate increases as “a” increases. [0100] To demonstrate the advantage of DPP approach, the DPP kernels are replaced with the Gamma Knife® kernels and the resulting radiation dose distributions are compared. Gamma Knife® has long been considered the “gold standard” of various radiosurgery modalities. Since the DPP approach can outperform Gamma Knife®, the DPP approach is advancing the state of the art. [0101] Two examples comparing the treatment planning result when using DPP kernels versus Gamma Knife® kernels are now discussed. In the first embodiment, a 3D spherical phantom is used with a 80 mm radius and a spherical tumor with a 7.5 mm radius at the center. Both optimizations ran with identical parameters. To ensure that the best possible Gamma Knife® plan is obtained, only 4 mm shots were used in the planning phase. The current Gamma Knife® system can produce kernels ranging from 4 mm to 16 mm, with the 4 mm kernel being the sharpest kernel. FIG. 16 shows the DVH comparisons. FIGS. 17A-17B and FIGS. 18A-18D show the comparisons between dose profiles and isodose distributions. [0102] FIG. 17A illustrates the dose profiles in the XY plane with the DPP plan shown by line 10 and the Gamma Knife® plan shown by line 12. FIG. 17B illustrates the dose profiles in the XZ plane, again, with the DPP plan shown by line 10 and the Gamma Knife® plan shown by line 12. FIG. 18A illustrates the isodose distributions of the DPP plan in the XY plane. FIG. 18B illustrates the isodose distributions of the Gamma Knife® plan in the XY plane. FIG. 18C illustrates the isodose distributions of the DPP plan in the XZ plane. FIG. 18D illustrates the isodose distributions of the Gamma Knife® plan in the XZ plane. The plot shown contains isodose lines from 10% to 100% with 10% steps. As can be seen from these plots, the DPP plan and the Gamma Knife® plan are very similar with the DPP plans being slightly better and more uniform. [0103] However, the precision of these comparisons is limited by the resolution of the Gamma Knife® kernels obtained from Zlekta at 5 mm. With such a sharp dose gradient, the numerical limit is approached. If these comparisons could be conducted at a much higher resolution, the sharper dose gradient of DPP plans of the present invention would be more pronounced. [0104] The DPP kernels and Gamma Knife® kernels are also considered for a more challenging phantom, which contains a C-shaped tumor surrounding a spherical critical structure as shown in FIGS. 19A-19B with the line 14 defining the outer perimeter of the tumor, surrounding a spherical critical structure having an outer perimeter defined by line 16. Specifically, FIG. 19A illustrates the phantom in the XY plane and FIG. 19B illustrates the phantom in the XZ plane. [0105] The goal is to have the tumor receive a 2100 cGy radiation dose. FIG. 20 shows the DVH comparison. FIGS. 21A-21C and FIGS. 22A-22D show the comparisons between dose profiles and between isodose distributions. FIGS. 21A-21C illustrate the dose profiles with the DPP plan shown by line 18 and the Gamma Knife® plan shown by line 20. Specifically, FIG. 21A illustrates the dose provides along the X direction; FIG. 21B illustrates the dose profiles along the Y direction; and FIG. 21C illustrates the does profiles along the Z direction. FIG. 22 A illustrates the isodose distribution of the DPP plan in the XY plane; FIG. 22B illustrates the isodose distributions of the Gamma Knife® plan in the XY plane; FIG. 22C illustrates the isodose distributions of the DPP plan in the XZ plane; FIG. 22D illustrates the isodose distributions of the Gamma Knife® plan in the XZ plane. The plots shown contains isodose lines from 10% to 100% with 10% steps. The DPP plan is better than the Gamma Knife® plan. This is because, in the DPP plan, the target receives a higher dose and critical structures receive a lower dose than with the Gamma Knife® plan. [0106] Since the DPP approach uses a single cone beam to dynamically treat a target, it is possible to modify the beam profiles of the cone beam (e.g., beam sharpness) to further improve the dose gradient. To demonstrate this, two sets of DPP kernels are created with two different ERFC sharpness parameters a=1 and a=10. These kernels are used in the Dynamic Gamma Knife® Radiosurgery Treatment Planning System. The goal is to let the tumor receive a 2100 cGy dose. FIG. 23 shows the DVH comparison. FIGS. 24A-24C illustrates the dose profiles with the DPP plan shown by line 22 and the Gamma Knife® plan shown by line 22. FIGS. 25A-25D show the comparisons between dose profiles and isodose distributions. FIG. 24A illustrates dose profiles along the X direction; FIG. 24B illustrates dose profiles along the Y direction; and FIG. 24C illustrates dose profiles along the Z direction. FIG. 25A illustrates the isodose distributions of the DPP plan with a=10 in the XY plane; FIG. 25B illustrates the isodose distributions of the DPP plan with a=1 in the XY plane; FIG. 25C illustrates the isodose distribution of the DPP plan with a=10 in the XZ plane; and FIG. 25D illustrates the isodose distribution of the DPP plan with a=1 in the XZ plane. The plots shown contain isodose lines from 10% to 100% with 10% steps. As the ERFC sharpness parameter increases, the target receives a higher dose and critical structures receive a lower dose, which results in an improved treatment plan using the present invention as compared to conventional treatment plans. In reviewing the profile comparisons shown in FIGS. 24A-24C, it can be seen that the DPP plan with a=10 has a lower dose at a low dose region than a DPP plan with a=1. This means the critical structure receives a lower dose as the ERFC parameter increases. [0107] CyberKnife® robotic radiosurgery may be used to implement dynamic photon painting according to the present invention. In one embodiment, it is contemplated that the computational challenge of optimizing thousands of beams can be solved using one or more of cloud computing, GPU technologies, vector instructions, and multithreading. [0108] Dynamic photon painting for radiation therapy and radiosurgery may be used in place of proton therapy and Gamma Knife® radiosurgeries. [0109] In addition to CyberKnife® robotic radiosurgery, FIG. 26 illustrates an exemplary computer system 100, or network architecture, that may be used to implement certain methods according to the present invention. One or more computer systems 100 may carry out the methods presented herein as computer code. One or more processors, such as processor 104, which may be a special purpose or a general-purpose digital signal processor, is connected to a communications infrastructure 106 such as a bus or network. Computer system 100 may further include a display interface 102, also connected to communications infrastructure 106, which forwards information such as graphics, text, and data, from the communication infrastructure 106 or from a frame buffer (not shown) to display unit 130. Computer system 100 also includes a main memory 105, for example random access memory (RAM), read-only memory (ROM), mass storage device, or any combination thereof. Computer system 100 may also include a secondary memory 110 such as a hard disk drive 112, a removable storage drive 114, an interface 120, or any combination thereof. Computer system 100 may also include a communications interface 124, for example, a modem, a network interface (such as an Ethernet card), a communications port, a PCMCIA slot and card, wired or wireless systems, etc. [0110] It is contemplated that the main memory 105, secondary memory 110, communications interface 124, or a combination thereof function as a computer usable storage medium, otherwise referred to as a computer readable storage medium, to store and/or access computer software and/or instructions. [0111] Removable storage drive 114 reads from and/or writes to a removable storage unit 115. Removable storage drive 114 and removable storage unit 115 may indicate, respectively, a floppy disk drive, magnetic tape drive, optical disk drive, and a floppy disk, magnetic tape, optical disk, to name a few. [0112] In alternative embodiments, secondary memory 110 may include other similar means for allowing computer programs or other instructions to be loaded into the computer system 100, for example, an interface 120 and a removable storage unit 122. Removable storage units 122 and interfaces 120 allow software and instructions to be transferred from the removable storage unit 122 to the computer system 100 such as a program cartridge and cartridge interface (such as that found in video game devices), a removable memory chip (such as an EPROM, or PROM) and associated socket, etc. [0113] Communications interface 124 allows software and instructions to be transferred between the computer system 100 and external devices. Software and instructions transferred by the communications interface 124 are typically in the form of signals 125 which may be electronic, electromagnetic, optical or other signals capable of being received by the communications interface 124. Signals 125 are provided to communications interface 124 via a communications path 126. Communications path 126 carries signals 125 and may be implemented using wire or cable, fiber optics, a phone line, a cellular phone link, a Radio Frequency (“RF”) link or other communications channels. [0114] Computer programs, also known as computer control logic, are stored in main memory 105 and/or secondary memory 110. Computer programs may also be received via communications interface 124. Computer programs, when executed, enable the computer system 100, particularly the processor 104, to implement the methods according to the present invention. The methods according to the present invention may be implemented using software stored in a computer program product and loaded into the computer system 100 using removable storage drive 114, hard drive 112 or communications interface 124. The software and/or computer system 100 described herein may perform any one of, or any combination of, the steps of any of the methods presented herein. It is also contemplated that the methods according to the present invention may be performed automatically, or may be invoked by some form of manual intervention. [0115] The invention is also directed to computer products, otherwise referred to as computer program products, to provide software to the computer system 100. Computer products store software on any computer useable medium. Such software, when executed, implements the methods according to the present invention. Embodiments of the invention employ any computer useable medium, known now or in the future. Examples of computer useable mediums include, but are not limited to, primary storage devices (e.g., any type of random access memory), secondary storage devices (e.g., hard drives, floppy disks, CD ROMS, ZIP disks, tapes, magnetic storage devices, optical storage devices, Micro-Electro-Mechanical Systems (“MEMS”), nanotechnological storage device, etc.), and communication mediums (e.g., wired and wireless communications networks, local area networks, wide area networks, intranets, etc.). It is to be appreciated that the embodiments described herein can be implemented using software, hardware, firmware, or combinations thereof. [0116] The computer system 100, or network architecture, of FIG. 26 is provided only for purposes of illustration, such that the present invention is not limited to this specific embodiment. It is appreciated that a person skilled in the relevant art knows how to program and implement the invention using any computer system or network architecture. [0117] The invention is also directed to computer products (also called computer program products) comprising software stored on any computer useable medium. Such software, when executed, at least in part, in one or more data processing devices, causes the data processing device(s) to operate as described herein. Embodiments of the invention employ any computer useable or readable medium, known now or in the future. Examples of computer useable mediums include, but are not limited to, primary storage devices (e.g., any type of random access memory), secondary storage devices (e.g., hard drives, floppy disks, CD ROMS, ZIP disks, tapes, magnetic storage devices, optical storage devices, MEMS, nanotechnological storage device, etc.), and communication mediums (e.g., wired and wireless communications networks, local area networks, wide area networks, intranets, etc.). It is to be appreciated that the embodiments described herein can be implemented using software, hardware, firmware, or combinations thereof. [0118] While the disclosure is susceptible to various modifications and alternative forms, specific exemplary embodiments thereof have been shown by way of example in the drawings and have herein been described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure as defined by the appended claims.

Summary: Photon-based radiosurgery is widely used for treating local and regional tumors. The key to improving the quality of radiosurgery is to increase the dose falloff rate from high dose regions inside the tumor to low dose regions of nearby healthy tissues and structures. Dynamic photon painting (DPP) further increases dose falloff rate by treating a target by moving a beam source along a dynamic trajectory, where the speed, direction and even dose rate of the beam source change constantly during irradiation. DPP creates dose gradient that rivals proton Bragg Peak and outperforms Gamma Knife® radiosurgery.

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Summarize: The Family Council It was at eleven o'clock the next morning that the aunts and uncles came to hold their consultation. The fire was lighted in the large parlor, and poor Mrs. Tulliver, with a confused impression that it was a great occasion, like a funeral, unbagged the bell-rope tassels, and unpinned the curtains, adjusting them in proper folds, looking round and shaking her head sadly at the polished tops and legs of the tables, which sister Pullet herself could not accuse of insufficient brightness. Mr. Deane was not coming, he was away on business; but Mrs. Deane appeared punctually in that handsome new gig with the head to it, and the livery-servant driving it, which had thrown so clear a light on several traits in her character to some of her female friends in St. Ogg's. Mr. Deane had been advancing in the world as rapidly as Mr. Tulliver had been going down in it; and in Mrs. Deane's house the Dodson linen and plate were beginning to hold quite a subordinate position, as a mere supplement to the handsomer articles of the same kind, purchased in recent years,--a change which had caused an occasional coolness in the sisterly intercourse between her and Mrs. Glegg, who felt that Susan was getting "like the rest," and there would soon be little of the true Dodson spirit surviving except in herself, and, it might be hoped, in those nephews who supported the Dodson name on the family land, far away in the Wolds. People who live at a distance are naturally less faulty than those immediately under our own eyes; and it seems superfluous, when we consider the remote geographical position of the Ethiopians, and how very little the Greeks had to do with them, to inquire further why Homer calls them "blameless." Mrs. Deane was the first to arrive; and when she had taken her seat in the large parlor, Mrs. Tulliver came down to her with her comely face a little distorted, nearly as it would have been if she had been crying. She was not a woman who could shed abundant tears, except in moments when the prospect of losing her furniture became unusually vivid, but she felt how unfitting it was to be quite calm under present circumstances. "Oh, sister, what a world this is!" she exclaimed as she entered; "what trouble, oh dear!" Mrs. Deane was a thin-lipped woman, who made small well-considered speeches on peculiar occasions, repeating them afterward to her husband, and asking him if she had not spoken very properly. "Yes, sister," she said deliberately, "this is a changing world, and we don't know to-day what may happen tomorrow. But it's right to be prepared for all things, and if trouble's sent, to remember as it isn't sent without a cause. I'm very sorry for you as a sister, and if the doctor orders jelly for Mr. Tulliver, I hope you'll let me know. I'll send it willingly; for it is but right he should have proper attendance while he's ill." "Thank you, Susan," said Mrs. Tulliver, rather faintly, withdrawing her fat hand from her sister's thin one. "But there's been no talk o' jelly yet." Then after a moment's pause she added, "There's a dozen o' cut jelly-glasses upstairs--I shall never put jelly into 'em no more." Her voice was rather agitated as she uttered the last words, but the sound of wheels diverted her thoughts. Mr. and Mrs. Glegg were come, and were almost immediately followed by Mr. and Mrs. Pullet. Mrs. Pullet entered crying, as a compendious mode, at all times, of expressing what were her views of life in general, and what, in brief, were the opinions she held concerning the particular case before her. Mrs. Glegg had on her fuzziest front, and garments which appeared to have had a recent resurrection from rather a creasy form of burial; a costume selected with the high moral purpose of instilling perfect humility into Bessy and her children. "Mrs. G., won't you come nearer the fire?" said her husband, unwilling to take the more comfortable seat without offering it to her. "You see I've seated myself here, Mr. Glegg," returned this superior woman; "_you_ can roast yourself, if you like." "Well," said Mr. Glegg, seating himself good-humoredly, "and how's the poor man upstairs?" "Dr. Turnbull thought him a deal better this morning," said Mrs. Tulliver; "he took more notice, and spoke to me; but he's never known Tom yet,--looks at the poor lad as if he was a stranger, though he said something once about Tom and the pony. The doctor says his memory's gone a long way back, and he doesn't know Tom because he's thinking of him when he was little. Eh dear, eh dear!" "I doubt it's the water got on his brain," said aunt Pullet, turning round from adjusting her cap in a melancholy way at the pier-glass. "It's much if he ever gets up again; and if he does, he'll most like be childish, as Mr. Carr was, poor man! They fed him with a spoon as if he'd been a babby for three year. He'd quite lost the use of his limbs; but then he'd got a Bath chair, and somebody to draw him; and that's what you won't have, I doubt, Bessy." "Sister Pullet," said Mrs. Glegg, severely, "if I understand right, we've come together this morning to advise and consult about what's to be done in this disgrace as has fallen upon the family, and not to talk o' people as don't belong to us. Mr. Carr was none of our blood, nor noways connected with us, as I've ever heared." "Sister Glegg," said Mrs. Pullet, in a pleading tone, drawing on her gloves again, and stroking the fingers in an agitated manner, "if you've got anything disrespectful to say o' Mr. Carr, I do beg of you as you won't say it to me. _I_ know what he was," she added, with a sigh; "his breath was short to that degree as you could hear him two rooms off." "Sophy!" said Mrs. Glegg, with indignant disgust, "you _do_ talk o' people's complaints till it's quite undecent. But I say again, as I said before, I didn't come away from home to talk about acquaintances, whether they'd short breath or long. If we aren't come together for one to hear what the other 'ull do to save a sister and her children from the parish, _I_ shall go back. _One_ can't act without the other, I suppose; it isn't to be expected as _I_ should do everything." "Well, Jane," said Mrs. Pullet, "I don't see as you've been so very forrard at doing. So far as I know, this is the first time as here you've been, since it's been known as the bailiff's in the house; and I was here yesterday, and looked at all Bessy's linen and things, and I told her I'd buy in the spotted tablecloths. I couldn't speak fairer; for as for the teapot as she doesn't want to go out o' the family, it stands to sense I can't do with two silver teapots, not if it _hadn't_ a straight spout, but the spotted damask I was allays fond on." "I wish it could be managed so as my teapot and chany and the best castors needn't be put up for sale," said poor Mrs. Tulliver, beseechingly, "and the sugar-tongs the first things ever I bought." "But that can't be helped, you know," said Mr. Glegg. "If one o' the family chooses to buy 'em in, they can, but one thing must be bid for as well as another." "And it isn't to be looked for," said uncle Pullet, with unwonted independence of idea, "as your own family should pay more for things nor they'll fetch. They may go for an old song by auction." "Oh dear, oh dear," said Mrs. Tulliver, "to think o' my chany being sold i' that way, and I bought it when I was married, just as you did yours, Jane and Sophy; and I know you didn't like mine, because o' the sprig, but I was fond of it; and there's never been a bit broke, for I've washed it myself; and there's the tulips on the cups, and the roses, as anybody might go and look at 'em for pleasure. You wouldn't like _your_ chany to go for an old song and be broke to pieces, though yours has got no color in it, Jane,--it's all white and fluted, and didn't cost so much as mine. And there's the castors, sister Deane, I can't think but you'd like to have the castors, for I've heard you say they're pretty." "Well, I've no objection to buy some of the best things," said Mrs. Deane, rather loftily; "we can do with extra things in our house." "Best things!" exclaimed Mrs. Glegg, with severity, which had gathered intensity from her long silence. "It drives me past patience to hear you all talking o' best things, and buying in this, that, and the other, such as silver and chany. You must bring your mind to your circumstances, Bessy, and not be thinking o' silver and chany; but whether you shall get so much as a flock-bed to lie on, and a blanket to cover you, and a stool to sit on. You must remember, if you get 'em, it'll be because your friends have bought 'em for you, for you're dependent upon _them_ for everything; for your husband lies there helpless, and hasn't got a penny i' the world to call his own. And it's for your own good I say this, for it's right you should feel what your state is, and what disgrace your husband's brought on your own family, as you've got to look to for everything, and be humble in your mind." Mrs. Glegg paused, for speaking with much energy for the good of others is naturally exhausting. Mrs. Tulliver, always borne down by the family predominance of sister Jane, who had made her wear the yoke of a younger sister in very tender years, said pleadingly: "I'm sure, sister, I've never asked anybody to do anything, only buy things as it 'ud be a pleasure to 'em to have, so as they mightn't go and be spoiled i' strange houses. I never asked anybody to buy the things in for me and my children; though there's the linen I spun, and I thought when Tom was born,--I thought one o' the first things when he was lying i' the cradle, as all the things I'd bought wi' my own money, and been so careful of, 'ud go to him. But I've said nothing as I wanted my sisters to pay their money for me. What my husband has done for _his_ sister's unknown, and we should ha' been better off this day if it hadn't been as he's lent money and never asked for it again." "Come, come," said Mr. Glegg, kindly, "don't let us make things too dark. What's done can't be undone. We shall make a shift among us to buy what's sufficient for you; though, as Mrs. G. says, they must be useful, plain things. We mustn't be thinking o' what's unnecessary. A table, and a chair or two, and kitchen things, and a good bed, and such-like. Why, I've seen the day when I shouldn't ha' known myself if I'd lain on sacking i'stead o' the floor. We get a deal o' useless things about us, only because we've got the money to spend." "Mr. Glegg," said Mrs. G., "if you'll be kind enough to let me speak, i'stead o' taking the words out o' my mouth,--I was going to say, Bessy, as it's fine talking for you to say as you've never asked us to buy anything for you; let me tell you, you _ought_ to have asked us. Pray, how are you to be purvided for, if your own family don't help you? You must go to the parish, if they didn't. And you ought to know that, and keep it in mind, and ask us humble to do what we can for you, i'stead o' saying, and making a boast, as you've never asked us for anything." "You talked o' the Mosses, and what Mr. Tulliver's done for 'em," said uncle Pullet, who became unusually suggestive where advances of money were concerned. "Haven't _they_ been anear you? They ought to do something as well as other folks; and if he's lent 'em money, they ought to be made to pay it back." "Yes, to be sure," said Mrs. Deane; "I've been thinking so. How is it Mr. and Mrs. Moss aren't here to meet us? It is but right they should do their share." "Oh, dear!" said Mrs. Tulliver, "I never sent 'em word about Mr. Tulliver, and they live so back'ard among the lanes at Basset, they niver hear anything only when Mr. Moss comes to market. But I niver gave 'em a thought. I wonder Maggie didn't, though, for she was allays so fond of her aunt Moss." "Why don't your children come in, Bessy?" said Mrs. Pullet, at the mention of Maggie. "They should hear what their aunts and uncles have got to say; and Maggie,--when it's me as have paid for half her schooling, she ought to think more of her aunt Pullet than of aunt Moss. I may go off sudden when I get home to-day; there's no telling." "If I'd had _my_ way," said Mrs. Glegg, "the children 'ud ha' been in the room from the first. It's time they knew who they've to look to, and it's right as _somebody_ should talk to 'em, and let 'em know their condition i' life, and what they're come down to, and make 'em feel as they've got to suffer for their father's faults." "Well, I'll go and fetch 'em, sister," said Mrs. Tulliver, resignedly. She was quite crushed now, and thought of the treasures in the storeroom with no other feeling than blank despair. She went upstairs to fetch Tom and Maggie, who were both in their father's room, and was on her way down again, when the sight of the storeroom door suggested a new thought to her. She went toward it, and left the children to go down by themselves. The aunts and uncles appeared to have been in warm discussion when the brother and sister entered,--both with shrinking reluctance; for though Tom, with a practical sagacity which had been roused into activity by the strong stimulus of the new emotions he had undergone since yesterday, had been turning over in his mind a plan which he meant to propose to one of his aunts or uncles, he felt by no means amicably toward them, and dreaded meeting them all at once as he would have dreaded a large dose of concentrated physic, which was but just endurable in small draughts. As for Maggie, she was peculiarly depressed this morning; she had been called up, after brief rest, at three o'clock, and had that strange dreamy weariness which comes from watching in a sick-room through the chill hours of early twilight and breaking day,--in which the outside day-light life seems to have no importance, and to be a mere margin to the hours in the darkened chamber. Their entrance interrupted the conversation. The shaking of hands was a melancholy and silent ceremony, till uncle Pullet observed, as Tom approached him: "Well, young sir, we've been talking as we should want your pen and ink; you can write rarely now, after all your schooling, I should think." "Ay, ay," said uncle Glegg, with admonition which he meant to be kind, "we must look to see the good of all this schooling, as your father's sunk so much money in, now,-- 'When land is gone and money's spent, Then learning is most excellent.' Now's the time, Tom, to let us see the good o' your learning. Let us see whether you can do better than I can, as have made my fortin without it. But I began wi' doing with little, you see; I could live on a basin o' porridge and a crust o' bread-and-cheese. But I doubt high living and high learning 'ull make it harder for you, young man, nor it was for me." "But he must do it," interposed aunt Glegg, energetically, "whether it's hard or no. He hasn't got to consider what's hard; he must consider as he isn't to trusten to his friends to keep him in idleness and luxury; he's got to bear the fruits of his father's misconduct, and bring his mind to fare hard and to work hard. And he must be humble and grateful to his aunts and uncles for what they're doing for his mother and father, as must be turned out into the streets and go to the workhouse if they didn't help 'em. And his sister, too," continued Mrs. Glegg, looking severely at Maggie, who had sat down on the sofa by her aunt Deane, drawn to her by the sense that she was Lucy's mother, "she must make up her mind to be humble and work; for there'll be no servants to wait on her any more,--she must remember that. She must do the work o' the house, and she must respect and love her aunts as have done so much for her, and saved their money to leave to their nepheys and nieces." Tom was still standing before the table in the centre of the group. There was a heightened color in his face, and he was very far from looking humbled, but he was preparing to say, in a respectful tone, something he had previously meditated, when the door opened and his mother re-entered. Poor Mrs. Tulliver had in her hands a small tray, on which she had placed her silver teapot, a specimen teacup and saucer, the castors, and sugar-tongs. "See here, sister," she said, looking at Mrs. Deane, as she set the tray on the table, "I thought, perhaps, if you looked at the teapot again,--it's a good while since you saw it,--you might like the pattern better; it makes beautiful tea, and there's a stand and everything; you might use it for every day, or else lay it by for Lucy when she goes to housekeeping. I should be so loath for 'em to buy it at the Golden Lion," said the poor woman, her heart swelling, and the tears coming,--"my teapot as I bought when I was married, and to think of its being scratched, and set before the travellers and folks, and my letters on it,--see here, E. D.,--and everybody to see 'em." "Ah, dear, dear!" said aunt Pullet, shaking her head with deep sadness, "it's very bad,--to think o' the family initials going about everywhere--it niver was so before; you're a very unlucky sister, Bessy. But what's the use o' buying the teapot, when there's the linen and spoons and everything to go, and some of 'em with your full name,--and when it's got that straight spout, too." "As to disgrace o' the family," said Mrs. Glegg, "that can't be helped wi' buying teapots. The disgrace is, for one o' the family to ha' married a man as has brought her to beggary. The disgrace is, as they're to be sold up. We can't hinder the country from knowing that." Maggie had started up from the sofa at the allusion to her father, but Tom saw her action and flushed face in time to prevent her from speaking. "Be quiet, Maggie," he said authoritatively, pushing her aside. It was a remarkable manifestation of self-command and practical judgment in a lad of fifteen, that when his aunt Glegg ceased, he began to speak in a quiet and respectful manner, though with a good deal of trembling in his voice; for his mother's words had cut him to the quick. "Then, aunt," he said, looking straight at Mrs. Glegg, "if you think it's a disgrace to the family that we should be sold up, wouldn't it be better to prevent it altogether? And if you and aunt Pullet," he continued, looking at the latter, "think of leaving any money to me and Maggie, wouldn't it be better to give it now, and pay the debt we're going to be sold up for, and save my mother from parting with her furniture?" There was silence for a few moments, for every one, including Maggie, was astonished at Tom's sudden manliness of tone. Uncle Glegg was the first to speak. "Ay, ay, young man, come now! You show some notion o' things. But there's the interest, you must remember; your aunts get five per cent on their money, and they'd lose that if they advanced it; you haven't thought o' that." "I could work and pay that every year," said Tom, promptly. "I'd do anything to save my mother from parting with her things." "Well done!" said uncle Glegg, admiringly. He had been drawing Tom out, rather than reflecting on the practicability of his proposal. But he had produced the unfortunate result of irritating his wife. "Yes, Mr. Glegg!" said that lady, with angry sarcasm. "It's pleasant work for you to be giving my money away, as you've pretended to leave at my own disposal. And my money, as was my own father's gift, and not yours, Mr. Glegg; and I've saved it, and added to it myself, and had more to put out almost every year, and it's to go and be sunk in other folks' furniture, and encourage 'em in luxury and extravagance as they've no means of supporting; and I'm to alter my will, or have a codicil made, and leave two or three hundred less behind me when I die,--me as have allays done right and been careful, and the eldest o' the family; and my money's to go and be squandered on them as have had the same chance as me, only they've been wicked and wasteful. Sister Pullet, _you_ may do as you like, and you may let your husband rob you back again o' the money he's given you, but that isn't _my_ sperrit." "La, Jane, how fiery you are!" said Mrs. Pullet. "I'm sure you'll have the blood in your head, and have to be cupped. I'm sorry for Bessy and her children,--I'm sure I think of 'em o' nights dreadful, for I sleep very bad wi' this new medicine,--but it's no use for me to think o' doing anything, if you won't meet me half-way." "Why, there's this to be considered," said Mr. Glegg. "It's no use to pay off this debt and save the furniture, when there's all the law debts behind, as 'ud take every shilling, and more than could be made out o' land and stock, for I've made that out from Lawyer Gore. We'd need save our money to keep the poor man with, instead o' spending it on furniture as he can neither eat nor drink. You _will_ be so hasty, Jane, as if I didn't know what was reasonable." "Then speak accordingly, Mr. Glegg!" said his wife, with slow, loud emphasis, bending her head toward him significantly. Tom's countenance had fallen during this conversation, and his lip quivered; but he was determined not to give way. He would behave like a man. Maggie, on the contrary, after her momentary delight in Tom's speech, had relapsed into her state of trembling indignation. Her mother had been standing close by Tom's side, and had been clinging to his arm ever since he had last spoken; Maggie suddenly started up and stood in front of them, her eyes flashing like the eyes of a young lioness. "Why do you come, then," she burst out, "talking and interfering with us and scolding us, if you don't mean to do anything to help my poor mother--your own sister,--if you've no feeling for her when she's in trouble, and won't part with anything, though you would never miss it, to save her from pain? Keep away from us then, and don't come to find fault with my father,--he was better than any of you; he was kind,--he would have helped _you_, if you had been in trouble. Tom and I don't ever want to have any of your money, if you won't help my mother. We'd rather not have it! We'll do without you." Maggie, having hurled her defiance at aunts and uncles in this way, stood still, with her large dark eyes glaring at them, as if she were ready to await all consequences. Mrs. Tulliver was frightened; there was something portentous in this mad outbreak; she did not see how life could go on after it. Tom was vexed; it was no _use_ to talk so. The aunts were silent with surprise for some moments. At length, in a case of aberration such as this, comment presented itself as more expedient than any answer. "You haven't seen the end o' your trouble wi' that child, Bessy," said Mrs. Pullet; "she's beyond everything for boldness and unthankfulness. It's dreadful. I might ha' let alone paying for her schooling, for she's worse nor ever." "It's no more than what I've allays said," followed Mrs. Glegg. "Other folks may be surprised, but I'm not. I've said over and over again,--years ago I've said,--'Mark my words; that child 'ull come to no good; there isn't a bit of our family in her.' And as for her having so much schooling, I never thought well o' that. I'd my reasons when I said _I_ wouldn't pay anything toward it." "Come, come," said Mr. Glegg, "let's waste no more time in talking,--let's go to business. Tom, now, get the pen and ink----" While Mr. Glegg was speaking, a tall dark figure was seen hurrying past the window. "Why, there's Mrs. Moss," said Mrs. Tulliver. "The bad news must ha' reached her, then"; and she went out to open the door, Maggie eagerly following her. "That's fortunate," said Mrs. Glegg. "She can agree to the list o' things to be bought in. It's but right she should do her share when it's her own brother." Mrs. Moss was in too much agitation to resist Mrs. Tulliver's movement, as she drew her into the parlor automatically, without reflecting that it was hardly kind to take her among so many persons in the first painful moment of arrival. The tall, worn, dark-haired woman was a strong contrast to the Dodson sisters as she entered in her shabby dress, with her shawl and bonnet looking as if they had been hastily huddled on, and with that entire absence of self-consciousness which belongs to keenly felt trouble. Maggie was clinging to her arm; and Mrs. Moss seemed to notice no one else except Tom, whom she went straight up to and took by the hand. "Oh, my dear children," she burst out, "you've no call to think well o' me; I'm a poor aunt to you, for I'm one o' them as take all and give nothing. How's my poor brother?" "Mr. Turnbull thinks he'll get better," said Maggie. "Sit down, aunt Gritty. Don't fret." "Oh, my sweet child, I feel torn i' two," said Mrs. Moss, allowing Maggie to lead her to the sofa, but still not seeming to notice the presence of the rest. "We've three hundred pounds o' my brother's money, and now he wants it, and you all want it, poor things!--and yet we must be sold up to pay it, and there's my poor children,--eight of 'em, and the little un of all can't speak plain. And I feel as if I was a robber. But I'm sure I'd no thought as my brother----" The poor woman was interrupted by a rising sob. "Three hundred pounds! oh dear, dear," said Mrs. Tulliver, who, when she had said that her husband had done "unknown" things for his sister, had not had any particular sum in her mind, and felt a wife's irritation at having been kept in the dark. "What madness, to be sure!" said Mrs. Glegg. "A man with a family! He'd no right to lend his money i' that way; and without security, I'll be bound, if the truth was known." Mrs. Glegg's voice had arrested Mrs. Moss's attention, and looking up, she said: "Yes, there _was_ security; my husband gave a note for it. We're not that sort o' people, neither of us, as 'ud rob my brother's children; and we looked to paying back the money, when the times got a bit better." "Well, but now," said Mr. Glegg, gently, "hasn't your husband no way o' raising this money? Because it 'ud be a little fortin, like, for these folks, if we can do without Tulliver's being made a bankrupt. Your husband's got stock; it is but right he should raise the money, as it seems to me,--not but what I'm sorry for you, Mrs. Moss." "Oh, sir, you don't know what bad luck my husband's had with his stock. The farm's suffering so as never was for want o' stock; and we've sold all the wheat, and we're behind with our rent,--not but what we'd like to do what's right, and I'd sit up and work half the night, if it 'ud be any good; but there's them poor children,--four of 'em such little uns----" "Don't cry so, aunt; don't fret," whispered Maggie, who had kept hold of Mrs. Moss's hand. "Did Mr. Tulliver let you have the money all at once?" said Mrs. Tulliver, still lost in the conception of things which had been "going on" without her knowledge. "No; at twice," said Mrs. Moss, rubbing her eyes and making an effort to restrain her tears. "The last was after my bad illness four years ago, as everything went wrong, and there was a new note made then. What with illness and bad luck, I've been nothing but cumber all my life." "Yes, Mrs. Moss," said Mrs. Glegg, with decision, "yours is a very unlucky family; the more's the pity for _my_ sister." "I set off in the cart as soon as ever I heard o' what had happened," said Mrs. Moss, looking at Mrs. Tulliver. "I should never ha' stayed away all this while, if you'd thought well to let me know. And it isn't as I'm thinking all about ourselves, and nothing about my brother, only the money was so on my mind, I couldn't help speaking about it. And my husband and me desire to do the right thing, sir," she added, looking at Mr. Glegg, "and we'll make shift and pay the money, come what will, if that's all my brother's got to trust to. We've been used to trouble, and don't look for much else. It's only the thought o' my poor children pulls me i' two." "Why, there's this to be thought on, Mrs. Moss," said Mr. Glegg, "and it's right to warn you,--if Tulliver's made a bankrupt, and he's got a note-of-hand of your husband's for three hundred pounds, you'll be obliged to pay it; th' assignees 'ull come on you for it." "Oh dear, oh dear!" said Mrs. Tulliver, thinking of the bankruptcy, and not of Mrs. Moss's concern in it. Poor Mrs. Moss herself listened in trembling submission, while Maggie looked with bewildered distress at Tom to see if _he_ showed any signs of understanding this trouble, and caring about poor aunt Moss. Tom was only looking thoughtful, with his eyes on the tablecloth. "And if he isn't made bankrupt," continued Mr. Glegg, "as I said before, three hundred pounds 'ud be a little fortin for him, poor man. We don't know but what he may be partly helpless, if he ever gets up again. I'm very sorry if it goes hard with you, Mrs. Moss, but my opinion is, looking at it one way, it'll be right for you to raise the money; and looking at it th' other way, you'll be obliged to pay it. You won't think ill o' me for speaking the truth." "Uncle," said Tom, looking up suddenly from his meditative view of the tablecloth, "I don't think it would be right for my aunt Moss to pay the money if it would be against my father's will for her to pay it; would it?" Mr. Glegg looked surprised for a moment or two before he said: "Why, no, perhaps not, Tom; but then he'd ha' destroyed the note, you know. We must look for the note. What makes you think it 'ud be against his will?" "Why," said Tom, coloring, but trying to speak firmly, in spite of a boyish tremor, "I remember quite well, before I went to school to Mr. Stelling, my father said to me one night, when we were sitting by the fire together, and no one else was in the room----" Tom hesitated a little, and then went on. "He said something to me about Maggie, and then he said: 'I've always been good to my sister, though she married against my will, and I've lent Moss money; but I shall never think of distressing him to pay it; I'd rather lose it. My children must not mind being the poorer for that.' And now my father's ill, and not able to speak for himself, I shouldn't like anything to be done contrary to what he said to me." "Well, but then, my boy," said Uncle Glegg, whose good feeling led him to enter into Tom's wish, but who could not at once shake off his habitual abhorrence of such recklessness as destroying securities, or alienating anything important enough to make an appreciable difference in a man's property, "we should have to make away wi' the note, you know, if we're to guard against what may happen, supposing your father's made bankrupt----" "Mr. Glegg," interrupted his wife, severely, "mind what you're saying. You're putting yourself very forrard in other folks's business. If you speak rash, don't say it was my fault." "That's such a thing as I never heared of before," said uncle Pullet, who had been making haste with his lozenge in order to express his amazement,--"making away with a note! I should think anybody could set the constable on you for it." "Well, but," said Mrs. Tulliver, "if the note's worth all that money, why can't we pay it away, and save my things from going away? We've no call to meddle with your uncle and aunt Moss, Tom, if you think your father 'ud be angry when he gets well." Mrs. Tulliver had not studied the question of exchange, and was straining her mind after original ideas on the subject. "Pooh, pooh, pooh! you women don't understand these things," said uncle Glegg. "There's no way o' making it safe for Mr. and Mrs. Moss but destroying the note." "Then I hope you'll help me do it, uncle," said Tom, earnestly. "If my father shouldn't get well, I should be very unhappy to think anything had been done against his will that I could hinder. And I'm sure he meant me to remember what he said that evening. I ought to obey my father's wish about his property." Even Mrs. Glegg could not withhold her approval from Tom's words; she felt that the Dodson blood was certainly speaking in him, though, if his father had been a Dodson, there would never have been this wicked alienation of money. Maggie would hardly have restrained herself from leaping on Tom's neck, if her aunt Moss had not prevented her by herself rising and taking Tom's hand, while she said, with rather a choked voice: "You'll never be the poorer for this, my dear boy, if there's a God above; and if the money's wanted for your father, Moss and me 'ull pay it, the same as if there was ever such security. We'll do as we'd be done by; for if my children have got no other luck, they've got an honest father and mother." "Well," said Mr. Glegg, who had been meditating after Tom's words, "we shouldn't be doing any wrong by the creditors, supposing your father _was_ bankrupt. I've been thinking o' that, for I've been a creditor myself, and seen no end o' cheating. If he meant to give your aunt the money before ever he got into this sad work o' lawing, it's the same as if he'd made away with the note himself; for he'd made up his mind to be that much poorer. But there's a deal o' things to be considered, young man," Mr. Glegg added, looking admonishingly at Tom, "when you come to money business, and you may be taking one man's dinner away to make another man's breakfast. You don't understand that, I doubt?" "Yes, I do," said Tom, decidedly. "I know if I owe money to one man, I've no right to give it to another. But if my father had made up his mind to give my aunt the money before he was in debt, he had a right to do it." "Well done, young man! I didn't think you'd been so sharp," said uncle Glegg, with much candor. "But perhaps your father _did_ make away with the note. Let us go and see if we can find it in the chest." "It's in my father's room. Let us go too, aunt Gritty," whispered Maggie.

Summary: The next day the aunts and uncles gather for consultation. Mrs. Tulliver, "with a confused impression that it was a great occasion, like a funeral," makes the house look its best. Mrs. Deane arrives first. Her husband, who is rising in the world, is away on business. She offers to send jelly for Mr. Tulliver if the doctor orders it. This reminds Mrs. Tulliver of her cut jelly glasses which must be sold. The Gleggs and Pullets come soon after. Mrs. Pullet is much interested in Tulliver's illness, but sister Glegg recalls her to the subject of the meeting -- "for one to hear what the other 'ull do to save a sister and her children...." There is no sympathy for Mrs. Tulliver's desire to keep her "best things." It is felt that she should be content with the bare necessities. As none of the family personally covets Mrs. Tulliver's things, there is no need to try to keep them in the family. As for Mrs. Tulliver's plea that she has never asked them to do anything for her, Mrs. Glegg replies that she should have, for "how are you to be provided for, if your own family don't help you?" Mr. Pullet ventures that Mr. and Mrs. Moss should help, and notes that they are absent. This reminds Mrs. Glegg that Tom and Maggie are not there, and that someone should tell them "what they're come down to." Mr. Glegg observes to Tom that now some good will have to come of his schooling. He repeats his ditty: When land is gone and money spent,Then learning is most excellent. Mrs. Glegg tells Tom that he must work hard and be "humble and grateful to his aunts and uncles," and she includes Maggie in the remark. Tom says that since it is a disgrace to the family for them to be sold up, it should be prevented. He proposes that any legacies to be left to him and Maggie be given now. Mr. Glegg admires the proposal, but Mrs. Glegg objects that she would have to alter her will and leave less behind when she dies. Then Mr. Glegg agrees that it is useless to save the furniture when the law debts remain. At last Maggie angrily demands why they interfere if they don't mean to help. Her mother is frightened by the outburst, and Tom is "vexed; it was no use to talk so." Mrs. Moss comes in and goes at once to the children. She discloses that she has three hundred pounds of theirs and is incapable of repaying it without being "sold up" herself. This was unknown to the family, and they inquire for details. Mr. Glegg warns that if there is a note, the creditors will force payment. Tom objects that it would not be right for them to pay, for his father didn't wish it. He says his father has told him that the loan was not to be paid back. Uncle Glegg then says that the note must be made away with. Mrs. Tulliver would like to sell the note and save her things, but Tom asks his uncle to help him destroy it. They go into Mr. Tulliver's room to search for the note. Mrs. Moss vows that the debt will be paid as soon as it is possible.

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Summarize: By. Katie Davies. PUBLISHED:. 18:07 EST, 2 March 2013. |. UPDATED:. 15:50 EST, 4 March 2013. A top Sixties model is suing makers of the hit TV show Mad Men for using her image without consent in the opening credits, it emerged today. The stunning image of Gita Hall May, well-known for her eye-catching red hair, is seen projected on a skyscraper in the Emmy-winning show's iconic opening credits. During the show's title music a silhouette of an 'ad man' is seen falling among the Manhattan skyline with famous adverts shown all around him. May's image is included and comes from an advert she did for Revlon hairspray in the 1950s. Scroll down for video. Controversy: Gita Hall May is suing Lionsgate for this image in the opening credits of Mad Men. The sequence shows an image of the model, pictured, she claims was used without her consent. Top model: Gita Hall May, pictured, was a popular 1950s and 1960s model. She claims Mad Men used her image to give the show credibility and she is entitled to damages as a result. In a complaint filed to Los Angeles Superior Court on Friday, showbiz website Deadline reports, Hall May. 79, claimed Mad Men studio Lionsgate failed to request her permission to use the image shot by famous fashion photographer Richard Avedon. 'I wished they had had the courtesy to get in touch with me,” she told ABC News on Monday. 'I was surprised because nobody had checked with me about it– they must have thought I was dead.' She says the now well-known opening sequence put together by design firm Imaginary Forces helped the drama, starring January Jones and John Hamm, become a success. 'At no time did she agree to allow, forty years later, her image to be cropped from the photo, in secret, and inserted as a key element in the title sequence of a cable television series, without her consent and for commercial purposes,' her complaint says. 'The Main Titles were integral to the success of Mad Men'. Upset: Hall said on a morning show that no one tried to get in touch with her and she assumes they thought she must be dead. Award-winner: Mad Men which first appeared on screens in 2007 has been both critically acclaimed and popular among the public. It follows central character Don Draper, pictured. Redhead: Character Joan is seen as the epitome of fifties glamor in the hit series. Gita Hall May was a model well-known for her striking red hair. It goes on to point out the titles themselves won an Emmy and the show's producers have made in excess of $1billion as a result of the series' success. May says she only became aware of the use of her image last May despite it appearing in the credits since the show's pilot in 2007. She claims the show used her image to give the series credibility. 'Defendants sought to include the likeness of a famous celebrity from the 1950s in the credits in order to promote and burnish the image of the show in the eyes of the public and to create the 'feel' of the program that was critical to its commercial success... Defendants sought Plaintiff's image because she was among the top models of her day,' the lawsuit says. Lionsgate were unavailable to comment on the legal action which seeks unspecified damages. Fifties beauty: Gita Hall May, pictured, is claiming unspecified damages in the lawsuit filed today. Lawsuit: Hall May filed the lawsuit against the hit TV show in an LA court on Friday. The highly anticipated sixth season of the popular show which follows the lives and affairs of Don Draper and his ad world colleagues is due to return on April 7. It is said to be the second to last season of the popular AMC drama. Gita Hall May, 79, was a former Miss Sweden who became an actress and model. She was famously married to the actor Barry Sullivan and appeared in numerous adverts and fashion magazines throughout the fifties and sixties shot by photographers such as Avedon and Irving Penn

Summary: Gita Hall May, 79, is suing Lionsgate for 'failing to seek permission' to use an advert she appeared in in the 1950s. The beauty queen turned model filed a lawsuit seeking damages for the use of her image on the Emmy winning show's famous opening sequence. The striking redhead poses in a Revlon advert as the silhouette of an ad man is seen falling in front of her from a Manhattan skyscraper.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Make it in America: Create Clean Energy Manufacturing Jobs in America Act''. SEC. 2. REQUIREMENTS FOR PURCHASE OF GREEN TECHNOLOGIES WITH 85 PERCENT DOMESTIC CONTENT FOR USE BY FEDERAL GOVERNMENT AND STATES. (a) Requirement for Purchases by Federal Government.-- Notwithstanding chapter 83 of title 41, United States Code (popularly referred to as the Buy American Act), and subject to subsection (c), only green technologies that are 85 percent manufactured in the United States, from articles, materials, or supplies 85 percent of which are grown, produced, or manufactured in the United States, may be acquired for use by the Federal Government. (b) Requirement for Purchases by States Using Federal Funds.-- Subject to subsection (c), Federal funds may not be provided to a State for the purchase of green technologies unless the State agrees that the funds shall be used to purchase only green technologies that are 85 percent manufactured in the United States, from articles, materials, or supplies 85 percent of which are grown, produced, or manufactured in the United States. (c) Phase-In of Requirement.--During the first three fiscal years occurring after the date of the enactment of this Act, subsections (a) and (b) shall be applied-- (1) during the first fiscal year beginning after such date of enactment, by substituting ``30 percent'' for ``85 percent''; (2) during the second fiscal year beginning after such date of enactment, by substituting ``50 percent'' for ``85 percent''; and (3) during the third fiscal year beginning after such date of enactment, by substituting ``80 percent'' for ``85 percent''. (d) Green Technologies Defined.--In this Act, the term ``green technologies'' means renewable energy and energy efficiency products and services that-- (1) reduce dependence on unreliable sources of energy by encouraging the use of sustainable biomass, wind, small-scale hydroelectric, solar, geothermal, and other renewable energy and energy efficiency products and services; and (2) use hybrid fossil-renewable energy systems. (e) Effective Date.--This section shall apply to purchases of green technologies on and after October 1 of the first fiscal year beginning after the date of the enactment of this Act. SEC. 3. RENEWABLE ENERGY PRODUCTION AND INVESTMENT TAX CREDITS LIMITED TO DOMESTICALLY PRODUCED PROPERTY. (a) Credit for Electricity Produced From Certain Renewable Resources.--Subsection (d) of section 45 of the Internal Revenue Code of 1986 is amended by adding at the end the following new paragraph: ``(12) Domestic content requirement.-- ``(A) In general.--In the case of any facility originally placed in service after the date of the enactment of the Make it in America: Create Clean Energy Manufacturing Jobs in America Act, such facility shall not be treated as a qualified facility for purposes of this section unless such facility is 85 percent manufactured in the United States, from articles, materials, or supplies 85 percent of which are grown, produced, or manufactured in the United States. ``(B) Transitional rule.--In the case of any facility originally placed in service before January 1, 2015, subparagraph (A) shall be applied-- ``(i) in the case a facility originally placed in service during 2012, by substituting `30 percent' for `85 percent' both places it appears, ``(ii) in the case a facility originally placed in service during 2013, by substituting `50 percent' for `85 percent' both places it appears, and ``(iii) in the case a facility originally placed in service during 2014, by substituting `80 percent' for `85 percent' both places it appears.''. (b) Investment Energy Credit.--Section 48 of such Code is amended by adding at the end the following new subsection: ``(e) Domestic Content Requirement.-- ``(1) In general.--In the case of any property for any period after the date of the enactment of the Make it in America: Create Clean Energy Manufacturing Jobs in America Act, such property shall not be treated as energy property for purposes of this section unless such property is 85 percent manufactured in the United States, from articles, materials, or supplies 85 percent of which are grown, produced, or manufactured in the United States. ``(2) Transitional rule.--In the case of any property for any period before January 1, 2015, paragraph (1) shall be applied-- ``(A) in the case of any period during 2012, by substituting `30 percent' for `85 percent' both places it appears, ``(B) in the case of any period during 2013, by substituting `50 percent' for `85 percent' both places it appears, and ``(C) in the case of any period during 2014, by substituting `80 percent' for `85 percent' both places it appears.''. (c) Effective Dates.-- (1) Production credit.--The amendments made by subsection (a) shall apply to facilities originally placed in service after the date of the enactment of this Act. (2) Investment credit.--The amendments made by subsection (b) 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).

Title: To require 85 percent domestic content in green technologies purchased by Federal agencies or by States with Federal funds and in property eligible for the renewable energy production or investment tax credits Summary: Make it in America: Create Clean Energy Manufacturing Jobs in America Act - Authorizes federal acquisition of, or the provision of federal funds to states for purchase of, only green technologies that are 85% manufactured in the United States from articles, materials, or supplies that are 85% grown, produced, or manufactured in the United States beginning in the fourth fiscal year after enactment of this Act. Provides that such percentage shall be 30% in the first fiscal year after enactment, 50% in the second fiscal year, and 80% in the third fiscal year. Defines "green technologies" to mean renewable energy and energy efficiency products and services that: (1) reduce dependence on unreliable sources of energy by encouraging the use of sustainable biomass, wind, small-scale hydroelectric, solar, geothermal, and other renewable energy and energy efficiency products and services; and (2) use hybrid fossil-renewable energy systems. Amends the Internal Revenue Code to prohibit treating any facility originally placed in service after the enactment of this Act as a qualified facility for purposes of the renewable energy production and investment tax credits unless such facility is 85% manufactured in the United States from articles, materials, or supplies that are 85% grown, produced, or manufactured in the United States. Provides that such percentage shall be 30% for a facility placed in service during 2012, 50% for a facility placed in service during 2013, and 80% for a facility placed in service during 2014.

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Summarize: If you had $20,000 to spare, what would you spend it on? A car? Your child's education? Investments? Donations to charities? Maaaybe, but you might also use it to pay to have your yard filled up with fake snow. Yes, that is a real thing! They've been doing it at ski hills for ages, but you can also have a snow machine come to your very own home—and apparently business is booming, especially in the South. David Bryant, the president of a Florida company called The Ice Man, has been in the fake snow business for 27 years and says demand is higher than ever: It's just absolutely exploded this year. We don't know why.... We booked about 140 tons of snow in December 2010 and we've booked over 230 tons of snow this year in December. Maybe because our society is going down the tubes, and the road to hell is paved with fake snow (and Kardashians and a few other things)? Just a guess, but I digress. The privilege of having your yard covered in white powder can cost anywhere from $1,700 to $20,000, depending on how much snow you require. If you're paying that much, you can expect a real winter wonderland filled with light, fluffy snow, right? Actually, not quite, according to Bryant, It's not the white powdery stuff but for the kids down here, it's the closest thing they'll see to snow. It's flaked ice. It's not chunks of ice. It's real, fine ground up ice. That doesn't sound that dreamy, though Bryant's specialty, a 70-foot snow slide that you can toboggan down, does sound pretty cool. Still, he says an icy yard is enough to satisfy the snow-starved citizens of Florida. And one of his customers, Mark Pierce agrees. Every year he throws a "Christmas Eve Eve Snow Blow" in his yard, and more than 200 guests show up to play in the show. The kids, he says, love it: They're just excited. They don't know what to do. It's like watching a baby zebra learn to walk and they just don't know what to do. They'll just have the operator blow it in the air for the kids to dance around in, just giving them a winter experience they're not used to having. Advertisement Ahh, yes, we all know what it's like to watch a baby zebra learn to walk...? Anyway, it is nice that these kids get to have a novel experience. Still, it seems a bit extreme to spend so much money to have snow in your very own yard when you've purposefully chosen to live in a place without wintery weather—especially in these tough economic times. Besides, if you have that kind of money to shred into shaved ice, you know what else you could spend it on? A vacation to a place that is already filled with snow, like a ski resort in the Rockies! That way your children could see what snow looks like in its natural habitat, instead of having it blasted at them from a big truck. Fake Snow Business Booming as Customers Pay Cool Bucks for Flakes [ABC News] Image via Martin Novak/Shutterstock. While many people in the Northeast would likely never dream of paying for snow, the snow business is actually booming, largely in the South, and many of people are willing to pay big bucks for the white stuff. "It's just absolutely exploded this year. We don't know why," said David Bryant, the president of the Jacksonville, Fla.-based company, The Ice Man. "In past years, we've always been busy in December, but we were crazy busy this year. We have January and February bookings." Bryant has been in the ice business for 27 years and has never seen the demand as high. "We booked about 140 tons of snow in December 2010 and we've booked over 230 tons of snow this year in December." Bryant said the company has never before been double or triple booked for Friday and Saturday nights, but they are this year, almost every weekend. The service ranges in cost from $1,700-$20,000, depending on the amount of snow. Bryant's company uses shredded ice for its events. A bag of ice goes into a macerator that softens it. Then it is mashed down and turned into flakes. Finally, it goes into a blower that blows it 40-45 feet in the air. "It's not the white powdery stuff but for the kids down here, it's the closest thing they'll see to snow," Bryant said. "It's flaked ice. It's not chunks of ice. It's real, fine ground up ice." The company gets the snow to stick even in warm temperatures by putting a lot of it down very quickly. "Ice is the greatest insulator in the world," Bryant said. "When you get a layer down, the bottom layer protects the next layer, which protects the layer above and so on." An average snow blow runs between five and seven tons, which covers an area of 25 square feet in eight inches of the stuff. Bryant's specialty is a 70-foot snow slide that people can toboggan down. "So many people in Florida have never seen snow, especially children. Kids will play and play and play," Bryant said. "They will absolutely go all night long if you let them." "It's a blast. The best part of the snow blow is to watch the children," he said. "Their eyes light up and they're just in awe." One of Bryant's customers is Jacksonville chiropractor Mark Pierce. He has an annual "Christmas Eve Eve Snow Blow" on Dec. 23 in his front yard for friends and neighbors. Pierce has more than 200 guests at the party, many of whom are children. "They're just excited. They don't know what to do. It's like watching a baby zebra learn to walk and they just don't know what to do," Pierce said. "They'll just have the operator blow it in the air for the kids to dance around in, just giving them a winter experience they're not used to having." In addition to the slide, he said the children have snowball fights, build snowmen for the first time and make snow angels. "Christmas in the south is a pretty festive occasion," Pierce said. "To bring the snow in is such a unique novelty that the kids go nuts. They start knocking on the door around Thanksgiving wanting to know if the ice man is coming." Pierce would not say how much he spends on the snow blow, but said, "I'm blessed and I'm fortunate. And just to see these children, to watch the excitement they have…it's my personal gift to the neighborhood." Another popular Florida company is Zigmont Magic F/X, a full-fledged special effects company that works with the Wizarding World of Harry Potter and Sea World, in addition to malls and private parties. "It seems like every year it gets a little busier," said Lisa Zigmont who owns the company with her husband Steve Zigmont. "We have over 100 machines and they're all out on rentals." Zigmont rents out the machines to people and companies all over the country. She said that the most frequent customers are churches and shopping malls. The machines range from $275-$650 a day, depending on the type and size. The snow from the machines does not land on the ground or accumulate, it evaporates in 30-90 seconds, but it does produce the effect of a snowfall. The effect was featured on the Christmas episode of ABC's "Modern Family," where the family danced around their California yard as the fake snow fell. "It's a magnificent effect," said Chris Konios, the owner of CK Design Build, which rents the machines for private parties. "If you looked at their snow, you can't tell the difference and you're talking to a guy from New York. I've seen snow. It looks exactly the. It really looks like a snowstorm, but it does not accumulate. For people that don't see it every year, it makes it a little more Christmassy." Last year, Konios threw a party that included a horse-drawn carriage through the snow. "It's magical to see snow, people dreaming of a white Christmas," Zigmont said. "Everybody wants snow falling in the air, seeing the twinkling, it shines like diamonds. It's really beautiful."

Summary: Weird trend story of the holidays: ABC News says people in the South are apparently very happy to shell out big money to have their yards covered in fake snow. One company says it has "booked" more than 230 tons of the stuff this month, up from 140 tons last December. "It's just absolutely exploded this year," says the president of the Ice Man. "We don't know why." It's even harder to fathom when you consider that the cost of the service runs from $1,700 to $20,000, the latter presumably for those with massive yards. Blogger Cassie Murdoch at Jezebel is a wee bit skeptical that this is such a great idea. "If you have that kind of money to shred into shaved ice, you know what else you could spend it on? A vacation to a place that is already filled with snow." At least then, kids will see it come out of the sky instead of a truck, she adds.

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Summarize: A convicted murderer sentenced to more than a half-century behind bars is suing the state of Connecticut, saying its ban on pornography in prison violates his constitutional rights. Dwight Pink Jr., 44, says in the lawsuit filed in U.S. District Court in July that guards have used the policy to deny him an art book, The Atlas of Foreshortening, which uses nude models to help show how to draw the human form. Pink says the ban is a violation of his free speech rights and serves no meaningful objective in prison. The state filed its response Monday, saying Pink has not been harmed by the ban and none of his rights were violated. My right to porn: Dwight Pink, Jr., 40, said he is suing the state of Connecticut because the ban on pornography in prison violates his constitutional rights. 'Any injury or harm, if any, was caused solely by plaintiff's own acts, omissions, or conduct and was not due to any wrongful conduct by the defendants,' Assistant Attorney General Steven Strom wrote. The state Department of Correction put out the administrative directive in 2011. It bans all material that contains 'pictorial depictions of sexual activity or nudity' from the prisons. But it also says the ban should not apply to'materials which, taken as a whole, are literary, artistic, educational or scientific in nature'. A prison spokesman at the time said the ban was intended to improve the work environment for prison staffers, especially female staffers, who might be inadvertently exposed to pornography. It led to a letter-writing campaign and threats of lawsuits from inmates upset with the policy. Jaclyn Falkowski, a spokeswoman for the attorney general's office, said about a half dozen lawsuits have been filed since then challenging the ban in both federal and state courts. She said those cases are still being litigated. But a judge in 2012 refused to issue a preliminary injunction allowing inmate Akove Ortiz to possess magazines like Playboy. 'Although prisoners do not forfeit all of their constitutional rights upon incarceration, the fact of incarceration and the needs of the prison system impose limitations on prisoners' constitutional rights, even those derived from the First Amendment,' the judge wrote in his ruling. Prison: A convicted murderer at Cheshire Correctional Institution in Connecticut thinks he should be able to view sexually explicit materials. William Dunlap, a law professor at Quinnipiac University, said courts have generally sided with prison officials in such cases if they can prove the ban has a legitimate goal other than to simply suppress material that some people might find objectionable — such as maintaining safety in the prisons or keeping the material out of the hands of sex offenders. But he said the lawsuit has a chance of succeeding if Pink can show it was improperly used in his case to suppress art or literature. 'I think that's a much stronger argument than saying the statute itself is facially unconstitutional,' he said. Pink is serving a 56-year sentence for his part in the 1998 slaying of a 35-year-old father of two in Old Saybrook. Scott Rufin was shot up to five times in the head with two guns and stabbed in the heart seven times with a sword, authorities said. Pink led authorities to the skeletal remains two years after the killing, which he said was committed by another man

Summary: Dwight Pink Jr., 44, was sentenced to 56 years in prison for the 1998 execution-style murder of a father-of-two. He is serving the sentence at Cheshire Correctional Institution in Connecticut. In 2012, the state Department of Corrections banned printed material depicting sexual activity or nudity. Pink filed a lawsuit in July saying the porn ban violates his constitutional rights. He claims he wants an art book called The Atlas Of Foreshortening. The state says Pink has not been harmed by the ban.

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Summarize: The wife of Clarke Carlisle has praised her campaigning husband as the ex-footballer lies in a hospital bed following a road accident. After tweets about his work raising awareness of mental illness, Gemma Carlisle wrote: 'He's been helping others for too long he forgot to help himself.' Her comments came as it emerged the 35-year-old ex-player is facing court action over an alleged drink driving offence for which he was charged just 36 hours before he was hit by a lorry. The former Premier League star was seriously injured following the collision on the A64, near York, on December 22 and remains in a Leeds hospital. Scroll down for video. Ex-footballer Clarke Carlisle and his wife Gemma, who has thanked fans for messages of support after her husband was seriously injured in a road accident hours after he was charged with driving offence. Praising her husband's charity work, she told fans he been caring for other rather than thinking about himself. The Metropolitan Police have confirmed that he was charged with failing to provide a sample on December 20 and is due to appear in court next month. Mother-of-two Mrs Carlisle has used her Twitter account to thank fans for their support as her husband continues to receive treatment. Shortly after the accident, she wrote: 'Don't normally use this account anymore. But have been overwhelmed with your thoughts and prayers. Thank you. 'Clarke's very poorly but he's alive and stable. Best Christmas gift we as a family could wish for. Thanks again.' She added yesterday: 'Thank you for continuing messages of support and for media restraint. Clarke still very poorly but we're hopeful he'll make a full recovery.' She also tweeted other messages highlighting her husband's work around depression and with stroke victims. After news of Carlisle's charges emerged this morning, former England international Kenny Samson told The Sun it was vital for Carlisle to get support and understanding from others in the game. A picture of the A64 road in North Yorkshire, near to where Carlisle was hit by a lorry last Monday. Samson said: 'I know what Clarke is going through at the moment and just how he is feeling. People shouldn't kick him while he's down.' He added: 'It's not for others to judge him because they don't understand the battles he is facing. At the moment he needs everyone close to him to rally round and support him through this.' A statement from the Metropolitan Police said: 'Clarke Carlisle, 35, was charged on Saturday, 20 December with failing to provide a specimen and driving otherwise than in accordance with a licence. 'He is due to appear at Highbury Corner Magistrates' Court on 20 January 2015.' North Yorkshire Police launched an investigation into the circumstances of the incident and appealed for witnesses. Since the collision, a host of footballers, former clubs, organisations and charities have used social media to send messages hoping former Professional Footballers' Association chairman Carlisle makes a quick recovery. Over a 16-year career, Carlisle, who is also a former chairman of the Professional Footballers' Association, made more than 500 appearances for nine clubs. QPR fans show their appreciation for their former defender with a round of applause at Loftus Road. Carlisle has been inundated with message from fans. Pictured, a show of support from a Burnley fan. His most notable stint as a Premier League player came during his five years with Burnley, with him featuring for them in 2009-10, their first season in the division. As a sign of the Clarets' support for their former player, fans at Turf Moor clapped in the fifth minute of their game against Liverpool on Boxing Day to match the squad number Carlisle wore at the club. As well as Burnley, there have also been messages of support for Carlisle from the other clubs he played for, which include Blackpool, QPR, Leeds, Watford and Northampton. The Football Association, Football League and anti-racism organisation Kick It Out, for which Carlisle is an ambassador, all tweeted their support as well, while representatives from the PFA visited him in hospital. During his time in football and since retiring last year, Clarke Carlisle has campaigned to shed a light on what he has called the 'dark side of the beautiful game'. He was once named 'Britain's cleverest footballer' and has appeared on the BBC's flagship political programme, Question Time, and quiz show Countdown. In a TV documentary called Football's Suicide Secret he spoke to those working in the game about problems of depression and stress. He said: 'Everybody's perception of being a footballer is that you've made it. You're a superstar. Football provides some magical highs, but it also provides some really, really dark lows. 'As a footballer, I've experienced all the ups and downs of the game, but there's one taboo no-one wants to talk about, mental illness.' As part of his work highlighting racism and mental illness in football, Carlisle appeared on Question Time. In the documentary, he spoke about his own attempt at suicide after he was injured early in his career. He said: 'I thought "I'm going to take all these pills and kill myself because I'm of no use to anyone, because I know without football, they are going to see me for what I really am", and that was nothing.' Carlisle, who is on the Management Committee of the Professional Footballers' Association, used the programme to call on footballing authorities to do more to support players. Carlisle has also campaigned against racism in the game and is an ambassador for the Kick It Out scheme. As part of his work, he visits schools to speak to students and he has criticised Fifa president Sepp Blatter for claiming racism is no longer an issue in the game. In February 2012, he was part of a PFA delegation who spoke to David Cameron to discuss issues of racism and homophobia in football

Summary: Wife has been thanking fans for their support as he lies in hospital. She praises his campaigning work drawing attention to mental illness. Carlisle hit by lorry while walking a dual carriageway in the dark last week. The 35-year-old is in hospital in Yorkshire recovering from the accident. He was pulled over by police while driving on Saturday, December 20. Charged with failing to provide specimen and driving without correct licence. For confidential support call the Samaritans in the UK on 08457 90 90 90, visit a local Samaritans branch or click here.

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Summarize: SCENE 5. The Tower of London Enter MORTIMER, brought in a chair, and GAOLERS MORTIMER. Kind keepers of my weak decaying age, Let dying Mortimer here rest himself. Even like a man new haled from the rack, So fare my limbs with long imprisonment; And these grey locks, the pursuivants of death, Nestor-like aged in an age of care, Argue the end of Edmund Mortimer. These eyes, like lamps whose wasting oil is spent, Wax dim, as drawing to their exigent; Weak shoulders, overborne with burdening grief, And pithless arms, like to a withered vine That droops his sapless branches to the ground. Yet are these feet, whose strengthless stay is numb, Unable to support this lump of clay, Swift-winged with desire to get a grave, As witting I no other comfort have. But tell me, keeper, will my nephew come? FIRST KEEPER. Richard Plantagenet, my lord, will come. We sent unto the Temple, unto his chamber; And answer was return'd that he will come. MORTIMER. Enough; my soul shall then be satisfied. Poor gentleman! his wrong doth equal mine. Since Henry Monmouth first began to reign, Before whose glory I was great in arms, This loathsome sequestration have I had; And even since then hath Richard been obscur'd, Depriv'd of honour and inheritance. But now the arbitrator of despairs, Just Death, kind umpire of men's miseries, With sweet enlargement doth dismiss me hence. I would his troubles likewise were expir'd, That so he might recover what was lost. Enter RICHARD PLANTAGENET FIRST KEEPER. My lord, your loving nephew now is come. MORTIMER. Richard Plantagenet, my friend, is he come? PLANTAGENET. Ay, noble uncle, thus ignobly us'd, Your nephew, late despised Richard, comes. MORTIMER. Direct mine arms I may embrace his neck And in his bosom spend my latter gasp. O, tell me when my lips do touch his cheeks, That I may kindly give one fainting kiss. And now declare, sweet stem from York's great stock, Why didst thou say of late thou wert despis'd? PLANTAGENET. First, lean thine aged back against mine arm; And, in that ease, I'll tell thee my disease. This day, in argument upon a case, Some words there grew 'twixt Somerset and me; Among which terms he us'd his lavish tongue And did upbraid me with my father's death; Which obloquy set bars before my tongue, Else with the like I had requited him. Therefore, good uncle, for my father's sake, In honour of a true Plantagenet, And for alliance sake, declare the cause My father, Earl of Cambridge, lost his head. MORTIMER. That cause, fair nephew, that imprison'd me And hath detain'd me all my flow'ring youth Within a loathsome dungeon, there to pine, Was cursed instrument of his decease. PLANTAGENET. Discover more at large what cause that was, For I am ignorant and cannot guess. MORTIMER. I will, if that my fading breath permit And death approach not ere my tale be done. Henry the Fourth, grandfather to this king, Depos'd his nephew Richard, Edward's son, The first-begotten and the lawful heir Of Edward king, the third of that descent; During whose reign the Percies of the north, Finding his usurpation most unjust, Endeavour'd my advancement to the throne. The reason mov'd these warlike lords to this Was, for that-young Richard thus remov'd, Leaving no heir begotten of his body-- I was the next by birth and parentage; For by my mother I derived am From Lionel Duke of Clarence, third son To King Edward the Third; whereas he From John of Gaunt doth bring his pedigree, Being but fourth of that heroic line. But mark: as in this haughty great attempt They laboured to plant the rightful heir, I lost my liberty, and they their lives. Long after this, when Henry the Fifth, Succeeding his father Bolingbroke, did reign, Thy father, Earl of Cambridge, then deriv'd From famous Edmund Langley, Duke of York, Marrying my sister, that thy mother was, Again, in pity of my hard distress, Levied an army, weening to redeem And have install'd me in the diadem; But, as the rest, so fell that noble earl, And was beheaded. Thus the Mortimers, In whom the title rested, were suppress'd. PLANTAGENET. Of Which, my lord, your honour is the last. MORTIMER. True; and thou seest that I no issue have, And that my fainting words do warrant death. Thou art my heir; the rest I wish thee gather; But yet be wary in thy studious care. PLANTAGENET. Thy grave admonishments prevail with me. But yet methinks my father's execution Was nothing less than bloody tyranny. MORTIMER. With silence, nephew, be thou politic; Strong fixed is the house of Lancaster And like a mountain not to be remov'd. But now thy uncle is removing hence, As princes do their courts when they are cloy'd With long continuance in a settled place. PLANTAGENET. O uncle, would some part of my young years Might but redeem the passage of your age! MORTIMER. Thou dost then wrong me, as that slaughterer doth Which giveth many wounds when one will kill. Mourn not, except thou sorrow for my good; Only give order for my funeral. And so, farewell; and fair be all thy hopes, And prosperous be thy life in peace and war! [Dies] PLANTAGENET. And peace, no war, befall thy parting soul! In prison hast thou spent a pilgrimage, And like a hermit overpass'd thy days. Well, I will lock his counsel in my breast; And what I do imagine, let that rest. Keepers, convey him hence; and I myself Will see his burial better than his life. Exeunt GAOLERS, hearing out the body of MORTIMER Here dies the dusky torch of Mortimer, Chok'd with ambition of the meaner sort; And for those wrongs, those bitter injuries, Which Somerset hath offer'd to my house, I doubt not but with honour to redress; And therefore haste I to the Parliament, Either to be restored to my blood, Or make my ill th' advantage of my good. Exit

Summary: The action shifts to the Tower of London. Mortimer is brought in by his jailors in a chair. Now an old man, he has been a prisoner since his youth and is dying. He asks a jailor if his nephew, Richard Plantagenet, will come to visit him. The jailor replies that he will. At that moment, Richard Plantagenet enters. Plantagenet tells his uncle that he has quarrelled with Somerset, who attacked him with his father's execution for alleged treason. Plantagenet asks Mortimer to explain what the Earl of Cambridge was supposed to have done. Mortimer replies that it is this same deed that led to his own imprisonment. Mortimer says that he was next in line to the throne after Richard II. But Richard II was deposed by Henry IV, so Henry's line has occupied the throne instead. Mortimer tried to press his claim to the throne but was imprisoned. During the reign of Henry V, Plantagenet's father, the Earl of Cambridge, raised an army to put Mortimer on the throne. He was captured and executed, and the Mortimers were suppressed. As Mortimer has no children, Richard Plantagenet is his heir. In deliberately cryptic wording, Mortimer implies that he wants Richard to restore their birthright. But he begs him to be wary, as the House of Lancaster is firmly in control of the throne. Mortimer dies. Richard is determined to redress the wrongs that have been done to his family, and goes off to Parliament to make his case..

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Summarize: By. Ryan Gorman. and Associated Press Reporter. PUBLISHED:. 11:24 EST, 10 March 2014. |. UPDATED:. 12:47 EST, 10 March 2014. An 11-year-old girl abducted by her father last week after her he'murdered her mother' has been returned home. Caitlyn Marie Virts was found with father Timothy Virts, 38, in a seedy South Carolina motel hundreds of miles from home. She has now been reunited with twin sister Cayla, police said, adding that she was unharmed,. The young girl and her sister are currently in state care, grandmother Anna Trainor-Goodwin told the Baltimore Sun. Caitlyn and her father went missing Thursday last week after mother Bobbie Jo Cortez was found dead in her home. Found: Timothy Virts faces a first-degree-murder charge after skipping town with daughter Caitlyn just after the stabbing death of her mother. A family torn apart: Mother Bobbie Jo Cortez. (pictured with twin daughters Cayla and Caitlyn) is dead, their father. awaits a murder charge and their stepfather is in jail on a child sex assault. charge. 'I'm hoping and praying that they let me have them,' Trainor-Goodwin said. 'They said they want to be with me. They miss me.' Virts. and his daughter were located at the Colonial Inn, according to local. police in Florence. They first rented a room on Thursday night, said. owner Carol Gause. Timothy Virts remains in custody in South Carolina awaiting extradition to Maryland to face a first-degree murder charge in the woman's stabbing death, according to the Sun. Stepfather: Registered sex offender Daniel Cortez was hit last week with 11 new counts related to child sex abuse - authorities would not say if the allegations were related to Timothy Virts' actions. Timothy Virst is listed in court documents as homeless, according to ABC 2, but his most recent profession was as a butcher. A court hearing to determine his fate is expected Monday. It has also been revealed that stepfather Daniel Cortez was arraigned Wednesday on 11 counts including attempted rape, perverted practices, assault and sex abuse of a minor, court records showed. Cortez is already a registered sex offender following a 2009 conviction on a charge of sexual conduct against a middle-school-aged child, according to the Maryland Sex Offender Registry. Authorities will not confirm or deny if either of the Virts twins were abused by the pedophile. Timothy Virts also has a history of child custody battles, and even had an order of protection issued against him by father Peter Virts in 2012. Peter Virts stated in court documents that he feared for the lives of his children as well as his own, according to court documents cited by ABC 2. 'Every time [Timothy] gets drunk or on drugs, he believes that I cause his problems,' he said. 'He's threatening to break my jaw and my life itself.' Timothy Virts also threatened his sons, saying he made them and they belonged to him, the father said. The accused killer's custody agreement with Bobbie Jo Cortez allowed him to see the twins every other weekend and on holidays, but she went to great lengths to avoid seeing him. 'I can get someone to bring them to him,' she said in court, according to ABC 2. 'I really don't want any contact between him and I.' Seedy: The father and daughter were found at this roadside motel 400 miles away in South Carolina. These startling revelations come as Timothy Virts now faces a first-degree murder charge for Bobbie Jo Cortez's death and details of the Caitlyn's rescue have started coming out. 'They. looked fine. She just walked right up with her father. I didn't notice. anything,' a hotel worker said by phone from the hotel on Saturday. The employee checking her Facebook page from the front desk around 7 p.m. Friday. when she saw the Amber Alert posted by a friend and immediately. recognized Virts and his daughter. She called police and officers. quickly arrived. Virts was arrested without problems and his. daughter appeared to be OK, the employee recalled. 'I. was startled, but I wasn't worried or frightened. You don't expect to. ever be involved in something like this,' said the employee. 'But I called. police and they took over.' The. search for Timothy Virts and his daughter, Caitlyn, become a nationwide. operation, with police believing his daughter could be in danger. Police. spokeswoman Elise Armacost said probable cause for the murder charge. was based on physical evidence and interviews with people in the house. at the time Ms Cortez was killed. Caitlyn's twin sister and Ms Cortez's young son, as well as two other adults, were also living at the home. Virts had also been staying there for the past several weeks, Armacost added.. Victim: Bobbie Jo Cortez was found in her home, covered in multiple cuts, on Thursday. The agency put up digital billboards across the country as it searched for Caitlyn Virts. An Amber Alert issued in Maryland was expanded late Thursday to include West Virginia. A police spokesman told WUSA9 they believed Virts had connections to West Virginia and were 'doing our due diligence' to follow tips. Authorities. said anyone who saw Caitlyn, Virts or the vehicle Virts was believed to. be driving should call 911. 'Virts may be armed and is dangerous,' the alert. warned. He was thought to be driving a black 1999 Dodge Durango registered to Ms Cortez's husband, Daniel Williams Cortez. Mr Cortez, 38, is currently being held at the Baltimore County Detention Center on sexual assault charges. Police said they do not believe he has anything to do with the murder of his wife

Summary: Caitlyn Virts, 11, has been reunited with her twin sister after being found with father Timothy Virts in a South Carolina motel. Timothy Virts remains in South Carolina custody, but an extradition hearing is expected to be held Monday. He faces a first-degree murder charge after Bobbie Jo Cortez, the girl's mother, was found Thursday stabbed to death. The accused killer's most recent job was as a butcher. Daniel Cortez, the girls' registered sex offender stepfather, was arraigned last week on 11 new charges of child sex abuse.

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Summarize: By. Lizzie Parry. Sexually transmitted infections in the over-60s has soared in the last year, as experts claim the 'baby boom' generation rarely use a condom. New figures published by Public Health England for 2013 reveal diagnoses of chlamydia, gonorrhoea, herpes and genital warts, in those aged over-65, increased 8.2 per cent on the previous year - a total of 1,125 cases. And in the 45 to 64-year-old group there was an overall increase in diagnoses of 7 per cent from 2012 to 2013 - a total of 16,411 cases. In total there were 446,253 STIs diagnosed in England last year, a slight decrease from the 448,775 cases diagnosed in 2012. New figures released by Public Health England reveal the overall number of cases of STIs fell last year from 448,775 cases diagnosed in 2012 to 446,253 cases in 2013. Experts warned of an 'undeniable growth' in cases among the over-60 age group. Chlamydia, pictured under the microscope, was the most common STI, making up 47 per cent of all diagnoses - a total of 208,755 cases in 2013. Chlamydia was the most common STI, making up 47 per cent of all diagnoses - a total of 208,755 cases. Meanwhile there was a large rise in cases of gonorrhoea, up 15 per cent from 2012, with a total of 29,291 cases recorded in 2013. Public Health England said young people aged 15 to 24 years still experience the highest rates of STIs, accounting for 63 per cent of chlamydia cases, 54 per cent of genital warts, 42 per cent of genital herpes and 56 per cent of gonorrhoea cases. However experts at the Family Planning Association warned of the 'undeniable growth' of STIs in older people. FPA’s Policy and Parliamentary. Manager, Harry Walker, said while many of the 'baby boom' generation, growing up in the 1960s when the contraceptive pill was introduced may have never, or rarely used a condom. With a rise in the number of older people using dating websites, and enjoying sex later in life, he urged older people to be aware of the dangers of unprotected sex. He said: 'While increases in STI diagnoses can in. part be attributed to greater awareness and better testing programmes,. there has been an undeniable growth of STIs among older age groups. 'Some. of these people will be from the ‘baby boom’ generation who grew up in. the 1960s when the contraceptive pill was introduced and may have never. or rarely used condoms. 'And. with sexual health messaging often targeted at young people, men and. women who come out of long-term relationships might find themselves with. very little information and knowledge about STIs. 'We. know that many are enjoying a new lease of life through internet. dating, and enjoying sex is by no means the preserve of the young. 'While. they might not need to worry about the risks of becoming pregnant. anymore, they might not think about the other type of protection which. is offered by condoms. The highest number of cases of all STIs was among the 15-24 age group. 'What. is particularly worrying is the fact that people over 45 now will have. been sexually active around the time of the high-profile and effective. condom-use messages of the 1980s and 90s, but perhaps these have been. forgotten.' Dr Catherine Lowndes, consultant scientist in PHE’s STI surveillance team, said: 'Sustained efforts to encourage people to regularly get checked for STIs means we are now finding and treating more infections – which is good news. 'Nevertheless these data show too many people are still getting STIs each year, especially young adults and gay men. 'Investment in promoting good sexual health awareness, contraception and condom use, and STI testing is vital, as is ongoing investment in easy to access sexual health services that meet the needs of local populations. 'Not only will this help bring down STI rates but abortion rates and under 18 conceptions as well.' National guidance recommends local services routinely offer chlamydia screening to young adults but only 15 per cent of young men and 35 per cent of young women were tested in 2013. Wide variations were seen across the country in the rates of chlamydia testing and diagnoses – with only around a third of local authorities reaching the recommended chlamydia screening levels (2,300 diagnoses per 100,000 young adults per year). Clamydia in males was most prevalent in the 20 to 24 age group, while in women the highest number of cases was among those aged 15 to 19. Dr Lowndes, added: 'Chlamydia can have serious consequences, including infertility, if it’s not treated. 'These data show we need to do more to encourage young adults to ask for testing every year when they attend health services. 'Local areas can look at embedding screening into a variety of settings to make it as easy as possible for this group to get tested. 'Offering a young adult a chlamydia test opens the door to conversations about other important aspects of good sexual health, such as contraception and condom use.' People can significantly reduce their risk of catching or passing on an STI by consistently and correctly using condoms until all partners have had a sexual health screen, by reducing the number of sexual partners, and by avoiding overlapping sexual relationships

Summary: Total number of STIs fell from 448,775 in 2012 to 446,253 cases in 2013. 7% increase in cases among 45-64-year-old age group. 8.2% increase in STI diagnoses among the over-65 age group. The 15-24 age group still account for the highest number of cases. FPA warned of 'undeniable growth' of cases in older 'baby boom' generation. Claims many people in the 1960s may have never or rarely used condoms.

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Write a title and summarize: Linkage analysis is useful in investigating disease transmission dynamics and the effect of interventions on them, but estimates of probabilities of linkage between infected people from observed data can be biased downward when missingness is informative. We investigate variation in the rates at which subjects' viral genotypes link across groups defined by viral load (low/high) and antiretroviral treatment (ART) status using blood samples from household surveys in the Northeast sector of Mochudi, Botswana. The probability of obtaining a sequence from a sample varies with viral load; samples with low viral load are harder to amplify. Pairwise genetic distances were estimated from aligned nucleotide sequences of HIV-1C env gp120. It is first shown that the probability that randomly selected sequences are linked can be estimated consistently from observed data. This is then used to develop estimates of the probability that a sequence from one group links to at least one sequence from another group under the assumption of independence across pairs. Furthermore, a resampling approach is developed that accounts for the presence of correlation across pairs, with diagnostics for assessing the reliability of the method. Sequences were obtained for 65% of subjects with high viral load (HVL, n = 117), 54% of subjects with low viral load but not on ART (LVL, n = 180), and 45% of subjects on ART (ART, n = 126). The probability of linkage between two individuals is highest if both have HVL, and lowest if one has LVL and the other has LVL or is on ART. Linkage across groups is high for HVL and lower for LVL and ART. Adjustment for missing data increases the group-wise linkage rates by 40–100%, and changes the relative rates between groups. Bias in inferences regarding HIV viral linkage that arise from differential ability to genotype samples can be reduced by appropriate methods for accommodating missing data. Interest has been growing in the use of viral linkage analysis to investigate disease transmission dynamics and the effect of interventions on them [1]–[8]. To optimize interventions intended to control the HIV epidemic, it will be useful to identify host characteristics (e. g. disease status and demographics) that are associated with high rates of clustered or genetically-linked infections. Many studies attempt to make inferences about linkage patterns in a larger population than that represented by the set of observed viral genetic sequences without considering the effect of sampling or missing data (see, e. g. [5], [9]). However, estimates of probabilities of linkage that ignore the impact of missing data (henceforth referred to as unadjusted estimators) can be biased downward. In order to estimate the amount of linkage in communities or compare rates of linkage across groups we must properly account for the presence of missing data. The work presented here arose from a desire to compare linkage rates between demographic groups found via a household survey from the Mochudi study, an HIV prevention program for Mochudi, Botswana (R01 AI083036; PI: M. Essex; www. aids. harvard. edu/news/spotlight/archives/v6i3_mochudi_project. html). Young males were found to be severely underrepresented, making inferences about linkage involving this group unreliable. As information regarding the size of this subpopulation is available, it is possible to leverage it to improve inferences. This household survey is part of a pilot project leading to a large community-randomized trial, also in Botswana, of a combination HIV prevention intervention, the Botswana Combination Prevention Project (BCPP; U01 GH000447; PIs: M. Essex & V. De Gruttola) [10], [11]. One of the goals of the BCPP study is to leverage viral linkage to understand the patterns of mixing across communities and the relative contributions of within-community and outside-community sources to new infections. This paper develops estimators for linkage probabilities under the assumption that unobserved sequences are missing at random conditional on observed information. We consider analyses in which linkage is defined by a threshold on the pairwise distance between viral sequences. The choice of the threshold is an important scientific question in the analysis of viral genetic data, but the methods developed here apply regardless of the particular value of the threshold chosen, or can be applied to range of thresholds of interest. We first show that the probability that randomly selected sequences are linked can be estimated unbiasedly from observed data. We then derive an estimate of conditional probabilities of linkage between groups given the existence of a link, and consider estimation of group-level probabilities of linkage. We first develop estimators under the assumption that indicators of linkage are independent across pairs of individuals who may be linked – an assumption that could be appropriate in situations with either a very sparse graph or sparse sampling in the population. We then develop a bootstrap resampling approach that is approximately correct under general assumptions about the structure of correlations of linkage indicators across pairs. Finally, we propose a diagnostic approach for assessing the reliability of the method. We apply the methods developed to analyses of viral sequences from the northeast sector of the village of Mochudi in Botswana, the site of a pilot study intended to determine the feasibility of testing for HIV infection in a household setting and linking infected subjects to care. Our investigation focuses on assessment of whether rates at which subjects' HIV genotypes link with others depends on ART treatment status and viral load levels (low/high) among the untreated. Such clustering reflects underlying HIV transmission dynamics; a tendency for subjects with high viral load to link more frequently with others might suggest an increased role of subjects with elevated levels of viral replication in HIV transmission. This is also consistent with high viremia in early infection; the contribution of those with elevated viral load to onward spread is difficult to assess in samples of prevalent cases due to the fact that a subject' s category varies over time. With high prevalence, however, it is unlikely that a high proportion of subjects in the sample are newly infected; nonetheless, this approach will be particularly useful in the analysis of data from the BCPP, which will identify incident cases and permit comparison of their linkage rates with the groups discussed here. Our first goal is to estimate the probability of linkage between viral sequences from two individuals selected at random from their respective groups,. Under the assumption of missingness at random (MAR) conditional on group membership, the hosts for whom viral sequences are available represent a random sample of the total population of their group, and by extension the observed linkage indicators are a random sample of the linkage indicators for the full population. Thus, the Law of Large Numbers tells us that the sample average,, converges to the population mean,. As a result, under the assumption of MAR conditional on group, it is possible to obtain an unbiased estimate of the probability that a pair of sequences are linked without adjustment for missing data. One quantity of interest in the analysis of a community randomized trial such as the BCPP is the relative probability that a new infection arises from contact with an infected person from within a community versus from outside the community. Therefore we may wish to estimate the conditional probability,, that a pair of sequences are from groups, given that are linked. If missingness is completely at random (unconditional on group), then we can use the observed proportions of links in each group pair,, with, to estimate the conditional probabilities. If missingness is MAR conditional on group, as we assume, this estimate requires adjustment for the differing missingness rates between groups. In a population of size, there are total possible pairs. The probability of linkage for a randomly selected pair is given byThe probability that a randomly selected pair is from groups and is linked isThus, the conditional probability we desire isWe substitute into these formulas to obtain a plug-in estimator of. Note that this derivation does not require an assumption of independence, so we can consistently estimate the conditional probability of linkage regardless of the underlying correlations of linkage indicators across pairs. Based on the results above, we now focus on estimation of the probability that a randomly selected sequence from group links with at least one sequence from group (excluding itself if),. In this case the unadjusted estimate of the probability of linkage between groups will be an underestimate of the true rate: any sequence that does not link with any other in the observed data may in fact link with sequence (s) from the community that were not observed. Thus, the proportion of observed sequences in group that do not link with any sequence in group will be higher than the proportion in the population. For the purposes of exposition, we begin with an assumption of independence among linkage estimators, but we extend to a case with individual-by-group random effects driving the correlations among indicators. This flexible model accounts for correlations due to individual factors – biology, behavior, network position – as well as differential interactions of individuals with different groups. As a first step in validating the performance of the approach, we perform a simulation study applying our methods to data simulated from an evolutionary model. To implement the simulation, we used SeqGen v1. 3. 2 [17]. We obtained the tree required as input to the program by fitting a maximum likelihood tree to the 423 observed sequences from Mochudi, and parameterized the evolutionary model by fitting the general time-reversible model with gamma distributed rate heterogeneity to those sequences and using the estimated parameters (both using MEGA version 5 [18]). Each node maintained the group assignment it had in the Mochudi data. The simulation proceeds as follows: The threshold ranged from 0. 17 to 0. 24, which corresponds roughly to the 0. 04th to 0. 41st percentiles of the distance distribution. The expected number of links per sequence ranged from 0. 04 to 0. 79. We simulated 100 sets of sequences, and for each set, we simulated 100 different observed data sets for each threshold, for a total of 10,000 simulations per threshold. Figure 1 plots the mean relative bias (|estimate–truth|/truth) of the unadjusted and adjusted estimators across the range of thresholds. The unadjusted estimator has uniformly higher bias than the adjusted, and the differences in the degree of bias is often large; averaged across subpopulations (weighting by their size) and thresholds, the relative bias of 25. 7% in the unadjusted analyses is reduced to 6. 5% in adjusted analyses. For higher thresholds, the adjustment reduces the bias to under 5% in the majority of cases and to under 10% in all. For the lower thresholds (where linkage rates are lower), the bias in unadjusted analyses is generally greater than for higher thresholds-exceeding 35% in some cases. By contrast the bias in the adjusted analyses is below 10% in the majority of cases and below 20% in all but one. The worst performance for the adjusted analyses (low thresholds for LVL to LVL) still shows a considerable reduction in bias. In the analysis of the Mochudi household survey data, we consider three groups: HVL, LVL and ART. We observe sequences for out of individuals in each group, yielding. We use p-distance as our distance measure: the proportion of compared sites at which two sequences differ. Viral linkage in this analysis is defined by a p-distance below a specified value. We present the results in two ways: first, using a range of thresholds from 0. 085 to 0. 12 (corresponding to the 0. 03rd to 0. 54th percentiles), and second, focusing on a threshold of 0. 1 for more detailed consideration. This latter threshold yields an overall rate of linkage of 18% within the observed sample. Using the results for the probability of linkage between individuals, we find the given in Figure 2 and Table 1. As one would expect, the overall probability of linkage increases with the more generous thresholds, but the pattern of relative probabilities appears to be maintained. In the table, we can see more clearly that linkage is most likely with the HVL group for all groups, while the LVL group demonstrates less linkage overall. We now move to estimation of the conditional probability that a linked pair are from groups,. First, we examine the performance of the estimator via simulation from real data. Treating the 423 observed sequences in the Mochudi data as a full population, we sample with probability (0. 7,0. 6,0. 8) from the (HVL, LVL, ART) groups. We can then record the true conditional probabilities from the full data and the unadjusted and adjusted estimates from the sampled data. Figure 3 gives the distribution of estimates of the conditional probabilities, compared against the probabilities observed in the full sample. The MLE is quite accurate, as we would expect given the generality of the results in Methods for conditional probabilities of linkage. The adjusted estimates of the conditional probabilities for the full sample are given in Figure 4 and Table 2. The relative probabilities vary more with the threshold in this case than in the individual-to-individual case, likely because the probabilities of linkage are extremely small (particularly when involving the ART group) and thus minor differences in the distribution of distances by group pair could lead to widely varying conditional probability estimates. It does appear to be most likely that a given link occurs between HVL and ART or LVL, and it is least likely to be between two LVL individuals. Before we proceed to estimate group-wise linkage rates for the Mochudi data, it is useful to examine the estimated correlation under the exchangeable model, which we will consider in development of a diagnostic tool for assessing the reliability of our methods. For the Mochudi data, we obtain a population-wide estimate of; group-specific estimates are given in Table 3. Most are close to the population-level estimate, but there is some variability. We can also see how the realized values of change with the sampling fraction. Figure 5 show boxplots of these realized values for subsamples of 5 to 95% of the Mochudi data. Each boxplot represents 500 samples. The estimates become increasingly variable as the sample gets smaller, but remain centered about the value of from the full data (red line) until the sample size falls below 40%, at which point the estimates decline sharply. This is likely due to an increased probability of obtaining a sample with very few observed links between the two groups. In the extreme case when no links are observed, this yields and, and we can expect the estimated correlation to be extremely small in cases with only a handful of links as well. This decline in the estimated correlation,, for very small samples has implications for bootstrap bias correction. We propose as a diagnostic creation of a plot similar to Figure 5 from the observed data by group. If the estimated median of appears to remain fairly constant over a range of sampling fractions including the size of the appropriate subsample, the estimated is likely to be similar to the true value, and the bootstrap bias correction should work well, since the estimated adjustment ratio described in the Methods depends upon being similar across the population, observed sample, and bootstrap subsample. We first assess the performance of the bootstrap estimator via simulation. Figures 6 and 7 show the resulting estimates for 70% and 30% samples of the observed data, respectively, including one modification: if the adjustment reduces the unadjusted estimate, we take the unadjusted value rather than the bias-adjusted value. This rule follows from knowing that the unadjusted estimate is an underestimate, implying that any reductions are likely due to very small bootstrap sample sizes or disparate correlations in the observed data and the subsample. This restriction has no effect on the 70% sample, but does impact the 30% sample substantially; we see that the bias-corrected results are not very different from the unadjusted estimates. We can now compare the estimates of for each pair of groups using the unadjusted estimator based on observed data and using the bootstrap adjustment method. The estimated probabilities of inclusion are 65,54, and 45% for HVL, LVL and ART, respectively. Based on the decline of in Figure 8, we would expect that the observed correlation in the subsamples is likely to be different from the sample correlation for the LVL group, despite the theoretical possibility of obtaining the 8% subsample needed for interval subsampling. Therefore, we use interval subsampling for the HVL group, but use proportionate subsampling for the LVL and ART groups. We calculate a confidence interval for the bootstrap adjusted estimate using a bootstrap quantile interval. Because the adjustment is made by taking the inverse of the bootstrap samples, the upper (lower) bound of the interval will be given by taking the () quantile of the bootstrap distribution of the ratio of the unadjusted estimate to the bootstrapped value (raised to the power of if using proportionate subsampling) and calculating with this quantile. Simulation results show that the coverage of this interval is likely to be good as long as the sampling percentage is at least 65% and may be anti-conservative if the percentage is lower. Intervals for the unadjusted estimator are found using a traditional binomial interval. We first present the results of applying the adjustment across a range of possible thresholds. As can be seen in Figure 9, the adjusted estimates are consistently higher than the unadjusted, regardless of threshold. In many cases, particularly for higher thresholds, the confidence interval for the unadjusted estimator excludes the point estimate from the bootstrap adjustment. The bootstrap quantile interval is consistently narrower than the unadjusted interval in the cases where interval subsampling was used (column 1). To see the effects of adjustment in more detail, we focus on a single cutoff of 0. 1 in Table 4, where we see both estimates for the probability that a member of group A (rows) is linked with at least one member of group B (columns). The adjusted estimates range from 40 to 100% larger than those of the unadjusted estimator. The relative values of the probabilities change as a result - for example, using the unadjusted estimator, it appears that someone with high viral load is nearly twice as likely to cluster within the high viral load group as to cluster with anyone on ART. After adjustment, an HVL individual is only half again as likely to cluster within group as with ART. In this case, qualitative comparisons - specifically, the ranking of the prevalence of various combinations - remain unchanged, although it is possible in other applications that this would not be the case. Unlike the sequence-to-sequence or conditional probabilities of linkage, the probability of linkage from group to group is not equal to the probability of linkage from group to group. This is a function both of the sizes of the groups (if group is much bigger, there are more chances for someone in group to have a link with than the other way around) and of the distribution of links. Consider, for example HVL/LVL linkage; it is much more likely for someone with high viral load to link with the low viral load group than for someone with LVL to link with the HVL group. This could arise simply due to the size of the LVL group, but it is also possible that several individuals in the LVL group link with multiple individuals in the HVL group. For each such configuration, only one person in LVL is counted as having link (s) to HVL, but multiple people in HVL are counted as having link (s) to LVL. The results for group-wise linkage rates suggest that individuals with high viral load have more links; by extension, this suggests they are involved in more transmissions or more recent transmissions. This group should include both individuals in the chronic phase of infection with poor viral suppression and recent infections [21]. Of 75 HVL with a CD4 count available, 44% had CD4 count below 250 cells/mm3, while only 19% of the 122 LVL individuals with a CD4 count measurement were below 250. Individuals on ART are most likely to link within their own group, suggesting links from older transmissions in which both individuals have progressed to the point of needing treatment. Individuals in the low viral load group link relatively little. Without information on the relative timing of infections (such as will be available from the BCPP), we cannot make inference about transmission contributions. However, taken together, these results are at least consistent with the hypothesis that those with low viral load, either from natural suppression or treatment, are not transmitting as efficiently as those with high viral load. Data on prevalence and incident cases collected over time will permit more formal testing of this hypothesis. Many different methods are available for calculating distances between genetic sequences [22]. As the definition of linkage is a simple threshold on the distance between sequences, distance models that give different results could result in rate estimates that vary widely. We compared four different distance calculation methods on the set of 423 sequences from the first year household survey in Mochudi, Botswana. The methods compared were: Codon positions included were 1st+2nd+3rd+Noncoding. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 1050 positions in the final dataset. Analyses of uncorrected pairwise distances and corrected by different evolutionary models were conducted in MEGA version 5 [18]. The scale or mean of the distance distribution might be expected to vary over the methods used to calculate the distances. To reduce this source of variability, we treat the threshold for linkage as a quantile of the distribution (i. e., the bottom 10% of distances cluster), thereby ensuring that measures that maintain the same ranking of distances provide equivalent results. Table 5 gives the Spearman rank correlation matrix of the five methods listed above. As the Spearman correlation considers only ranks, any measures that are equivalent up to a monotonic increasing transformation will have a correlation of 1. All four methods used have nearly perfect correlation, indicating that applying the analysis methods described here with a quantile-based cutoff will result in nearly identical results regardless of the distance model used. Genetic linkage analyses have been useful in making inferences about important HIV epidemic drivers, including the impact of acutely or recently infected subjects [1], [2], [4]. Application of these methods to community randomized trials of HIV prevention interventions such as the cluster randomized trial of HIV prevention in Botswana [10], [23] may be useful not only for this purpose but also to provide information regarding the subpopulations in which these interventions are succeeding or failing. For example if newly infected subjects in communities randomized to the intervention cluster only with viruses that infect people living outside the community, this knowledge would imply that the intervention is succeeding in stopping transmission within communities. The implications for the success of the intervention are very different from the setting in which newly infected cases are in fact being infected with viruses circulating within communities. For the latter, it is important to know what subgroups contribute most to onward transmission of virus, whether these subgroups be defined by plasma HIV levels, ART treatment status, or demographic or behavioral factors. All such analyses, however, are very much impacted by potentially informatively missing data. This paper proposes methods to adjust for such biases. Our methods adjust for the presence of missing viral sequences in estimates of viral linkage rates under the assumption that sequences are missing at random conditional on group membership. We show that we can consistently estimate the probability that two sequences are linked without adjustment for missing data, and can consistently estimate conditional probabilities of linkage between two sequences from a pair of groups given the existence of a link via a minor adjustment using the (known) sizes of the groups in the population. In settings where it is reasonable to assume that the linkage status of pairs of sequences are all independent conditional on group, the estimator presented for estimation of group-wise linkage probabilities under independence is in fact the MLE and provides an exact solution. This assumption might be reasonable in investigations of airborne pathogens, or in settings with sparse sampling. For settings in which the assumption of independence is not reasonable, we propose a bootstrap resampling approach to adjust for the bias in the unadjusted estimator. If linkage indicators are exchangeably correlated or if their correlations can reasonably be modeled as functions only of individual effects (a random-effects type model) and we can use interval subsampling, then the resampling method can adequately adjust for bias. When using proportionate subsampling under the random-effects model the bootstrap may under-correct, but the resulting estimates are still preferable to those provided by unadjusted estimators. We note that departures from the assumption of a random effects structure in the correlation would arise if the probability of linkage depended not only on the individual characteristics of sequences and the people infected with them, but also, in unspecified ways, on the interactions between these characteristics. In such cases, unbiased adjustment for missing data is not possible, because such departures would imply that unobserved linkages followed a different process from those that are observed. Even in this case, however, it would be useful to employ our methods, because they at least provide estimates that are valid under much broader assumptions than in the case for unadjusted analyses and they demonstrate the effect of the broadening of assumptions on results. Large changes in estimates provide caution against overinterpretation of results. Furthermore, our simulation results using the Mochudi data suggest that the adjustment may be adequate in some realistic settings where the assumption of the random effects structure may not hold perfectly. To provide guidance on appropriate usage of the method, we propose a diagnostic tool that provides assessment of the likely reliability of the bootstrap resampling approach to adjust estimates of clustering rates. The choice of the threshold defining linkage will vary broadly with the goal of analysis and methods of data collection. This choice is critical to any linkage analysis, and sensitivity to the choice of threshold should be examined. The methods developed here can be applied to any threshold or range of thresholds in order to obtain linkage rate estimates that are adjusted for the presence of missing data. Considering adjusted results for a range of thresholds will permit more reliable comparisons between groups and between thresholds. Although the groups of interest for linkage and those of relevance for the missingness model were the same in our example, this condition is not required. A more general missingness model could be formed by creating a partition into subgroups such that pairs of observations are missing at random given subgroup membership. Our method would then proceed by first estimating linkage rates for each of these subgroups, and then aggregating across them to obtain the estimates for the groups of interest (as suggested in Methods). As an example, to address our fundamental goal of estimating the relative contributions of within-community and outside-community partners to new infections, we would include community as one of the variables that defines our groups. We might, for example, define groups as community by sex by age category, for example. Given age- and sex-specific prevalence estimates for each community, we can adjust for missing data within these categories, and then aggregate to the level of community, yielding estimates of the proportion of individuals in community 1 who cluster with community 2 and vice versa, as well as the proportion who cluster within their own communities. Such an analysis will provide an indication of the relative force of infection from within versus outside the community, especially if we have separate groups for incident infections. The methods can also be extended to allow the model for missingness to depend on continuous-valued variables. The approach discussed here is not restricted to linkage indicators defined by a pairwise distance cutoff. The rate of occurrence of any feature of interest that can be coded as an indicator variable for each pair of sequences can also be estimated with adjustment for missing data. Beyond the change in the definition of a link, the application of the method is identical. The bootstrap method described here is similar in spirit to inverse-probability weighting in that adjustment for bias makes use of information on the probability of observation to estimate a scaling factor. In our setting, however, it is not possible to express the weight in closed form because of the complex correlation structure induced by the vagaries of HIV evolution and of patterns of viral transmission.

Title: Linkage of Viral Sequences among HIV-Infected Village Residents in Botswana: Estimation of Linkage Rates in the Presence of Missing Data Summary: The analysis of viral genomes has great potential for investigating transmission of disease, including the identification of risk factors and transmission clusters, and can thereby aid in targeting interventions. To make use of genetic data in this way, it is necessary to make inferences about population-level patterns of viral linkage. As with any rigorous statistical inference from sampled data to a population, it is important to consider the effect of the sampling strategy and the occurrence of missing data on the final inferences made. In this paper we highlight the effects of missing data on the resulting estimates of population level linkage rates and develop methods for adjusting for the presence of missing data. As an example, we consider comparing the rates of linkage of HIV sequences from subjects with high viral load, low viral load, or on antiretroviral treatment, and show that comparative inferences are compromised when adjustment is not made for missing sequences and bias in inferences can be reduced with proper adjustment.

lay_plos

en

Summarize: FIELD OF THE INVENTION [0001] The invention lies in the field of rehabilitation and exercise devices. BACKGROUND [0002] Orthopedic physical therapy and athletic training may seek to reduce the amount of joint and muscle strain by countering the effects of gravity upon the body. This can be accomplished by structural supports that the user can rest against. Some suspension frames aim to lift the user, shifting the user&#39;s weight to the suspension frame and off of the ground. Suspension frames are beneficial in the case of lower body rehabilitation because of the reduction of strain on the muscles and joints of the legs. [0003] Ambulation therapy may be desirable in the case of lower body injuries or neurological damage that compromises the patient&#39;s balance. In addition, ambulation therapy may be useful for athletic training to lower the risk of said lower body injuries. Ambulation therapy devices typically use moving treadmills in combination with a means of suspension. In order to support the full weight of a human body and include a mechanical treadmill for ambulation, these devices are typically extremely heavy and have a large footprint. As a result, they are often too large and too expensive for ordinary consumer purchase. Similarly, modest training facilities, gyms and physical therapy centers may be unable to afford existing therapy device. [0004] Currently known ambulation therapy devices use air or harnesses for buoyancy. For instance, U.S. Pat. No. 7,341,543 to Dandy discloses an apparatus for shifting the body weight of a runner from the runner&#39;s legs to a wheeled frame using a pelvic harness that is secured to the frame with an elastic material. Another example, the AlterG® Anti-Gravity Treadmill®, suspends the user on an inflatable cushion, which the user must enter into from the top of the machine. Securing the user in these devices can be dangerous, burdensome and inefficient, and moreover, involve straps and fabric that press into parts of the body uncomfortably as the user is suspended above the ground. SUMMARY [0005] An exercise and physical therapy device is described comprising a suspension frame and garment combination that is easy to enter and comfortable for the user when lifted by the frame. The garment is structured so that it distributes the user&#39;s weight to the frame effectively while minimizing uncomfortable pressure from the garment as the user&#39;s body strains against the garment due to gravity. The garment comprises an elastic fabric such that the user can ambulate freely, and several integrated support panels comprised of a less elastic material, such that due to their resistance to deformation the support panels integrate with the frame and lift the user. The garment comprises thigh support panels that fit the thigh snugly and connect to the suspension frame at waist level, causing the suspension frame to lift the user from the thigh support panels. This alleviates pressure as the user&#39;s body strains against the garment due to gravity, concentrating any squeezing effect at the thigh. Furthermore, the garment comprises a posterior support panel, which connects to the suspension frame at waist level, lifting the user from under the buttocks and counterbalancing the thigh panels that lift the user from the front. [0006] The device also comprises a wheeled or stationary suspension frame comprising a waist-level ring to which the garment connects, and which is vertically movable. The ring surrounds the user at waist level, attaches to the garment, and can elevate a user wearing the garment slightly off of the ground, thus enabling the user to ambulate while minimizing impact on the user&#39;s lower body. Because the ring is lowerable, the user can safety and comfortably enter the ring and/or step into the garment. [0007] The frame may be wheeled, or it may be fixed in place such that the user is held in place by the frame. The wheeled embodiment comprises hinges to allow vertical adjustment of the suspension ring. The fixed embodiment may be comprised of a hydraulic suspension ring, or any other means for vertical movement of the suspension ring. In the preferred stationary embodiment, the frame is collapsible, comprising on each side: rotatably connected legs that form an X-frame that expands and collapses due to operation of a mechanical actuator. [0008] Where the stationary frame is used, the device can also include a low-friction plate that is positionable under the user&#39;s feet, enabling the user to ambulate in place, sliding against the low-friction plate as though it were the ground. The low-friction surface eliminates the need for a costly and oversized treadmill. Furthermore, through the addition of slidable layers of resistance, the friction of the surface may be adjustable. Adjustable friction controls resistance against the user&#39;s feet and therefore can serve as an incremental therapy or training device. BRIEF DESCRIPTION OF THE DRAWINGS [0009] FIG. 1A shows a front view of an example of the garment. [0010] FIG. 1B shows a side view of an example of the garment. [0011] FIG. 1C shows a rear view of an example of the garment. [0012] FIG. 2 is an example exercise apparatus depicted in use. [0013] FIG. 3A is an example of a collapsible suspension frame in an expanded position. [0014] FIG. 3B is an example of a collapsible suspension frame in a collapsed position. [0015] FIG. 4 is an example of a friction-adjustable plate. DETAILED DESCRIPTION [0016] An example of an exercise apparatus is depicted in FIG. 2. Support ring 21 surrounds a user 11 at waist level and is vertically movable by hinges 21 and 23. With reference to FIGS. 1A through 1 C, garment 1 wearable by user comprises a posterior band 4, a left leg anterior band 8, and a right leg anterior band 7. Garment 1 is attachable to support ring 21 such that lifting the support ring suspends the user above the ground. The user is lifted and fixed in place by the support ring, but elasticity of the garment allows ambulatory movement. Because the combination of the garment and frame bear the user&#39;s weight, the user moves without strain on the lower body. [0017] FIG. 2 shows a friction-adjustable plate 30 positioned under the user&#39;s feet. Because the friction of the pad 30 is adjustable, resistance can be added or subtracted to change the way the user&#39;s feet slide over the pad. The friction-adjustable plate comprises a low-friction surface, such as a surface that has been coated with dry film lubricant, wherein friction can be increased by the introduction of a high-friction material such as rubber or felt. A cloth foot garment may further decrease the coefficient of friction between the user&#39;s feet and the friction-adjustable plate. An example of a friction-adjustable plate is shown in FIG. 4, comprising a first layer 33 with openings 31 and a high-friction section 33, and a second layer 36 with high-friction sections 35 and low-friction section 36. The second layer 36 is slidable under the first layer 33 such that the high-friction section can be exposed through openings 31. Increasing the level of exposure of the low-friction section 36 through openings 31 results in increasing the resistance against the user&#39;s feet, while exposing only the high-friction section 35 results in decreasing resistance. An alternative to the two-layer friction-adjustable plate is a single-layer friction-adjustable plate comprising a low-friction material and vertical strips of high-friction material, such that rotating the plate results in increasing the amount of friction against the user&#39;s feet. [0018] An example of the attachable garment 1 is shown in three views in FIGS. 1A through 1C. The garment 1 comprises a posterior band 4, a left leg anterior band 8, and a right leg anterior band 7. The posterior band 4 lifts the user&#39;s posterior from the front side of the user&#39;s waist, extending from under the buttocks to the user&#39;s front, where the posterior band 4 can be attached to the support ring 21. The anterior bands 7 and 8 extend from the user&#39;s right and left front thigh to the back side of the user&#39;s waist, where the anterior bands 7 and 8 are attachable to the support ring 21. The posterior and anterior bands counterbalance each other, due to the fact that they extend from the front of the frame to the back of the user, and the back of the frame to the front of the user, criss-crossing at the user&#39;s side. The user&#39;s center of gravity is maintained above the ground and the user does not tip forward or backwards when elevated off the ground by the frame. Furthermore, because the bands are less elastic than the rest of the garment, their attachment to the frame creates a structure that lifts the user from the garment itself, distributing the user&#39;s weight to the frame evenly and comfortably. [0019] Right and left anterior bands 7 and 8 are securable around the leg via right and left braces 2 and 3, respectively. Braces 2 and 3 are coupled to anterior bands 7 and 8, respectively, such that when the garment is attached to the frame, the user is lifted by the anterior bands pulling the user up from the braces. By hoisting the user by the thighs and the user&#39;s weight being distributed to the frame via the bands, pressure is alleviated from the crotch, which would typically receive the force of the garment as the garment is hoisted by the frame. Moreover, the right and left braces 2 and 3 may be adjustable to accurately fit the user and to allow the user to adjust for comfort. The braces may be adjustable according to many forms currently known in the art, such as by Velcro as depicted in FIGS. 1A through 1C. [0020] In the preferred embodiment, the garment 1 comprises a cloth or cloth-like fabric that is elastic to allow the user full range of movement. The posterior 4 and/or anterior bands 7 and 8 comprise a less elastic material than the rest of the garment 1, to facilitate weight distribution. For instance, one or more of the posterior and anterior bands may be comprised of materials such as limited-stretch polyester or nylon. Posterior and/or anterior bands may be comprised of a denser weave than the rest of the garment 1, or the bands may be reinforced with a less elastic material, or the bands may be sewn into the rest of the garment. [0021] The garment may be attachable to the frame by a variety of known means suitable for hoisting human weight, such as hooks, carabiners, and cam buckles. In the example depicted in FIGS. 1 through 3, posterior band 4 is attachable at the front section of ring 21 at attachment points 25 and 27, anterior band 7 is attachable at the rear section of ring 21 at attachment point 26, and anterior band 8 is attachable at the rear section of ring 21 at attachment point 24. Ring 21 surrounds the user at waist level, and is vertically movable such that the user 11 is lifted by the garment attached to ring 21. [0022] The frame is vertically movable to suspend the user off of the ground, and lowerable to allow the user to step into the ring 21 with minimal effort. For instance, hinges 22 and 23 can be used to raise and lower the front and rear legs of the frame. In the preferred embodiment, the frame is collapsible as depicted in FIGS. 3A and 3B. A right ring support 53 is rotatably connected by pin connection 47 to the right leg 51 of frame 20, while left ring support 54 is rotatably connected by pin connection 48 to the left leg 52 of frame 20, such that frame 20 is vertically movable by rotation of the supports and legs in their pin connections. By forcing frame 20 away from the right and left ring supports 53 and 54, actuator rods 45 and 46 rotate the supports and legs in their pin connections to vertically move support ring 21. The actuator rods 45 and 46 can be operated manually using rotatable knobs 28 and 29, as depicted, or they can automatically operated. Electronic control unit 39 may be used to facilitate automatic operation of the frame. [0023] The frame is elevatable to fully suspend the user off of the ground such that the user can walk or run over the ground with less joint and muscle strain. As such, the frame only needs to be capable of suspending the user up to 5 inches off of the ground. The low-friction surface 30 allows the user to ambulate in place within a stationary frame, but the frame 20 may also comprise a front and rear wheel such that the user can push against the ground and move with the wheeled frame with reduced body weight on the user&#39;s legs.

Summary: A garment attachable to an exercise or therapeutic apparatus that is capable of elevating the user slightly above the ground, reducing potential for injury and strain by shifting the user&#39;s weight to the frame of the apparatus. The garment is incorporated into the apparatus by comprising panels under the buttocks and around the thighs, providing balance and lifting the user comfortably in conjunction with the frame. A low-friction plate is positioned under the user&#39;s feet so that the user can ambulate freely while held in place by the frame.

big_patent

en

Summarize: BACKGROUND OF THE INVENTION This invention relates to herbicidal compositions and methods of use. In particular, this invention relates to herbicidal compositions comprising an herbicidally active thiolcarbamate in combination with certain substituted carbamates, the latter serving to prolong the effectiveness of a single application of the thiolcarbamate in controlling undesired plant growth. Thiolcarbamates are well known in the agricultural art as herbicides useful for weed control in crops such as corn, potatoes, beans, beets, spinach, tobacco, tomatoes, alfalfa, rice, and others. Thiolcarbamates are primarily used in pre-emergence application, and are particularly effective when incorporated into the soil prior to the planting of the crop. The concentration of the thiolcarbamate in the soil is greatest immediately after application of the compound. How long thereafter the initial concentration is retained depends in large part on the particular soil used. The rate at which the thiolcarbamate concentration declines following its application varies from one type of soil to the next. This is evident both in the observable extent of weed control and in the detectable presence of undegraded thiolcarbamate remaining in the soil after considerable time has elapsed. It is therefore an object of this invention to increase the soil persistence of thiolcarbamate herbicides and thus improve their herbicidal effectiveness. BRIEF DESCRIPTION OF THE INVENTION It has now been discovered that the soil persistency of certain herbicidally active thiolcarbamates is significantly extended by the further addition to the soil of certain extender compounds in the form of substituted carbamates, which have little or no herbicidal activity of their own and have no adverse effect on the spectrum of herbicidal activity of the thiolcarbamate. This improvement in the soil persistence of thiolcarbamates manifests itself in a variety of ways. It can be shown, for example, by soil analyses taken at regular intervals, that the rate of decrease of the thiolcarbamate content of the soil is substantially reduced. Improved soil persistence can also be shown by improvements in herbicidal efficacy, as evidenced by a higher degree of weed injury, brought about when the extender compound increases the soil persistence of the thiolcarbamate, prolonging its effective life. In particular, this invention relates to a novel herbicidal composition comprising (a) an herbicidally effective amount of a thiolcarbamate having the formula ##STR2## in which R 1, R 2, and R 3 are independently C 2 -C 4 alkyl; and (b) an amount of a carbamate having the formula ##STR3## in which R 4 and R 5 are each methyl, R 4 and R 5 conjointly form C 1 -C 4 alkylenedioxy, R 4 is --CF 3 and R 5 is hydrogen, or R 4 is --CH 2 Br and R 5 is hydrogen, the amount of said carbamate being sufficient to extend the soil life of said thiolcarbamate. Within the scope of the present invention, certain embodiments are preferred, namely: In formula I, R 1 is preferably ethyl, and R 2 and R 3 are each preferably propyl. In formula II, it is preferred that: R 4 and R 5 are each methyl, R 4 and R 5 conjointly form C 2 -C 4 alkylenedioxy, or R 4 is --CF 3 and R 5 is hydrogen. It is more preferred that: R 4 and R 5 are each methyl, or R 4 is --CF 3 and R 5 is hydrogen. It is most preferred that R 4 and R 5 are each methyl, and located on the 3- and 5-positions, respectively, of the phenyl ring. This invention further relates to a method of controlling undesirable vegetation comprising applying the above composition to the locus where control is desired. The term &#34;alkyl&#34; is intended to include both straight-chain and branched-chain groups, such as ethyl, n-propyl, isopropyl, butyl, etc. The term &#34;alkylenedioxy&#34; is used herein to denote multiples of the --CH 2 group with an oxygen atom at each end, the --CH 2 groups optionally substituted with alkyl groups. Examples include --O--CH 2 --O--(methylenedioxy), --O--CH 2 CH 2 O--(ethylenedioxy), --O--CH 2 CH 2 CH 2 --O--(trimethylenedioxy), --O--CH 2 CH 2 CH 2 CH 2 --O--(tetramethylenedioxy), --O--CH(CH 3 )CH 2 --O--(α-methylethylenedioxy), --O--CH(C 2 H 5 )CH 2 --O--(α-ethylethylenedioxy), etc. All carbon atom ranges recited in this specification and the appended claims are intended to be inclusive of their upper and lower limits. The term &#34;herbicide&#34;, as used herein, means a compound or composition which controls or modifies the growth of plants. By the term &#34;herbicidally effective amount&#34; is meant any amount of such compound or composition which causes a modifying effect upon the growth of plants. By &#34;plants&#34; is meant germinant seeds, emerging seedlings and established vegetation, including roots and above-ground portions. Such controlling or modifying effects include all deviations from natural development, such as killing, retardation, defoliation, desiccation, regulation, stunting, tillering, stimulation, leaf burn, dwarfing and the like. The phrase &#34;to extend the soil life of said thiocarbamate&#34; as used herein means to retard the rate at which molecules of thiolcarbamate are broken down into decomposition products when in contact with soil, and/or to prolong the period of time following application in which herbicidal effects can be observed. This applies both to field sites where repeated applications of thiolcarbamates have resulted in decreasing herbicidal effectiveness, and to field sites where a decline in activity is detected over time regardless of the prior history of herbicidal applications. An extended soil life can be manifest in a slower rate of decline of weed-killing activity, or an increased half-life of thiolcarbamate concentrate in the soil. Other techniques of determining soil life are readily apparent to one skilled in the art. DETAILED DESCRIPTION OF THE INVENTION The objects of the present invention are achieved by applying the carbamate extender compound to the soil at an agricultural field site in conjunction with the thiolcarbamate herbicide. The two compounds can be applied simultaneously in a single mixture or in separate formulations, or they may be applied in succession, with either one following the other. In successive application, it is preferable to add the compounds as close in time as possible. The herbicide extending effect is operable over a wide range of ratios of the two compounds. It is most convenient, however, to apply the compounds at a ratio of about 1:1 to about 20:1 (herbicide:extender) on a weight basis, preferably about 1:1 to about 10:1, and most preferably about 1:1 to about 5:1. Thiolcarbamate herbicides useful in the present invention include S-ethyl di-n-propylthiolcarbamate, S-ethyl diisobutylthiolcarbamate, S-n-propyl di-n-propylthiolcarbamate, and S-n-propyl ethyl-n-butylthiocarbamate. Such thiolcarbamates can be prepared by the process described in U.S. Pat. No. 2,913,327 (Tilles et al., Nov. 17, 1959). Carbamates useful as extenders in the present composition include 5&#39;-[N-methylcarbamoyloxy] benzodioxane, 5&#39;-[N-methylcarbamoyloxy] 2-ethyl benzodioxane, N-methyl-(3,4-methylenedioxyphenyl) carbamate, N-methyl-(4-α-bromomethylphenyl) carbamate, N-methyl-(3-α-bromoethylphenyl) carbamate, N-methyl-(2,4-dimethylphenyl) carbamate, N-methyl-(3,5-dimethylphenyl) carbamate, N-methyl-(2,5-dimethylphenyl) carbamate, N-methyl-(3-trifluoromethylphenyl) carbamate, N-methyl-(4-trifluoromethylphenyl) carbamate, and N-methyl-(5-trifluoromethylphenyl) carbamate. Such carbamates can be prepared by the process described in U.S. Pat. No. 3,304,225 (Szabo et al., Feb. 14, 1967). The variety of crops on which the present composition is useful can be significantly broadened by the use of an antidote to protect the crop from injury and render the composition more selective against weeds. For antidote descriptions and methods of use, reference is made to U.S. Pat. No. 3,959,304, issued to E. G. Teach on May 25, 1976; U.S. Pat. No. 3,989,503, issued to F. M. Pallos et al. on Nov. 2, 1976; U.S. Pat. No. 4,021,224, issued to F. M. Pallos et al. on May 3, 1977; U.S. Pat. No. 3,131,509, issued to O. L. Hoffman on May 5, 1964; and U.S. Pat. No. 3,564,768, issued to O. L. Hoffman on Feb. 3, 1971. Examples of useful antidotes include chloroacetamides such as N,N-diallyl-2,2-dichloroacetamide and N,N-diallyl-2-chloroacetamide, oxazolidines such as 2,2,5-trimethyl-N-dichloroacetyl oxazolidine and 2,2-spirocyclohexyl-N-dichloroacetyl oxazolidine, and 1,8-naphthalic anhydride. For maximum effect, the antidote is present in the composition in a non-phytotoxic, antidotally effective amount. By &#34;non-phytotoxic&#34; is meant an amount which causes at most minor injury to the crop. By &#34;antidotally effective&#34; is meant an amount which substantially decreases the extent of injury caused by the pesticide to the crop. The preferred weight ratio of pesticide to antidote is about 0.1:1 to about 30:1. The most preferred range for this ratio is about 3:1 to about 20:1. The following examples are offered to illustrate the utility of the present invention, and are intended neither to limit nor define the invention in any manner. EXAMPLE 1 Soil Persistence Tests This example shows, by soil analysis, the effectiveness of the compounds of the present invention in extending the soil life of the thiolcarbamate herbicides. The herbicide used in these tests was S-ethyl di-n-propylthiolcarbamate, known by the common name EPTC. The soil was a sandy loam soil obtained from Sunol, Calif., containing approximately (on a weight basis) 64% sand, 29% silt, and 7% clay, determined by mechanical means. The total organic content of the soil was approximately 4% by weight and the pH was 6.8, both determined by chemical analysis. The test procedure involved an initial pretreatment of the soil to simulate field conditions where the soil had been previously treated with EPTC, followed by a soil persistence test, as described below. A. Soil Pre-Treatment An emulsion was prepared by diluting an emulsifiable liquid concentrate containing 6 lb/gal (0.72 kg/l) of the thiolcarbamate in 100 ml of water, such that the concentration of thiolcarbamate in the resulting emulsion was 4000 mg/l. Five ml of this emulsion was then added to 10 lb (4.54 kg) of soil and the mixture was mixed in a rotary mixer for 10-20 seconds. Round plastic containers, 9 inches (22.9 cm) in diameter by 9 inches (22.9 cm) deep, were then filled with the sandy loam soil, which was tamped and leveled with a row marker to impress three rows across the width of each container. Two rows were seeded with DeKalb XL-45-A corn Zea mays (L.), and one row was seeded with barnyardgrass Echinochloa crusgalli (L.). Sufficient seeds were planted to produce several seedlings per row. The containers were then placed in a greenhouse maintained at 20° C. to 30° C. and watered daily by sprinkler. Five weeks after treatment, the soil was allowed to dry out and the plant foliage was removed. The soil was then passed through a 0.25 inch (0.64 cm) screen, followed by a 2 millimeter (mm) screen, to remove plant roots and clods. B. Soil Persistence Test A 100-gram quantity (air-dry basis) of the pretreated soil was placed in an 8-ounce (0.24-liter) wide-mouth glass bottle. The same emulsifiable concentrate described in Part A above was appropriately diluted in water such that a 5-ml portion added to the soil would produce a herbicide concentration of 6 ppm (weight) in the soil. This is equivalent to an application rate of 6 pounds per acre (6.7 kilograms per hectare) in a field where the herbicide is incorporated into the soil through a depth of about 2 inches (5.08 cm) soon after application. A selected extender compound in technical (nonformulated) form was then diluted in an acetone-water mixture such that a one-ml portion added to the soil would produce a concentration of 4 ppm by weight, equivalent to 4 pounds per acre (4.5 kilograms per hectare). On these bases, the herbicide and extender were added to the bottle containing the soil. The bottle was then sealed with a lid and shaken manually for approximately 15 minutes. Following such treatment, the soil was moistened with 20 ml deionized water. The bottle was then covered with a watch glass to maintain aerobic conditions and to prevent rapid soil drying, and placed in a controlled environmental chamber in darkness, where the temperature was maintained constant at 25° C. Four days later, the bottle was removed from the environmental chamber and 50 ml of water and 100 ml of toluene were added. The bottle was then tightly sealed with a lid containing a cellophane liner, and vigorously shaken on a variable speed, reciprocating shaker (Eberbach Corp. Model No. 6000) set at approximately 200 excursions per minute for one hour. After shaking, the bottle contents were allowed to settle, and a 10 ml aliquot of toluene was transferred by pipette into a glass vial and sealed with a polyseal cap. The toluene extract was analyzed for herbicide content by gas chromatography. The chromatogram data was then converted to equivalent soil concentrations in parts per million (ppm) by weight of the herbicide. The results are shown in the table below, where six compounds were tested in three separately treated batches of soil. A control run without an extender compound was conducted for each soil batch, to show how the drop in herbicide concentration was affected by the extender compound. In each case, the quantity of herbicide remaining in the soil after four days was dramatically increased when the extender compound was added. TABLE I______________________________________4-DAY SOIL PERSISTENCE DATAHerbicide: S-ethyl di-n-propylthiolcarbamate (EPTAM) at 6 lb/A (6 ppm in soil) ##STR4## EPTAM ResidueExtender After 4 Days (ppm)Compound With WithoutNo. R.sup.1 R.sup.3 Extender Extender______________________________________Soil Batch A:1 3-CH.sub.3 5-CH.sub.3 2.27 0.052 2,3- ##STR5## 2.14 0.05Soil Batch B:3 3-CF.sub.3 H 2.50 0.034 2,3- ##STR6## 2.06 0.03Soil Batch C:5 4-CH.sub.2 Br H 1.29 0.046 3-CH.sub.2 Br H 1.03 0.04______________________________________ EXAMPLE 2 Herbicidal Activity Improvement Tests This example offers herbicidal activity test data to show the effectiveness of the carbamate additives in improving the herbicidal activity of thiolcarbamates. The effect is observed by comparing the extent of weed control in test flats treated with a thiolcarbamate against that occurring in similar flats treated with both the thiolcarbamate and carbamate. The antidote N,N-diallyl dichloroacetamide was also included to improve the selectivity of the thiolcarbamate in controlling weeds. As in Example 1, the thiolcarbamate used in this test was S-ethyl di-n-propylthiolcarbamate applied in the form of an emulsifiable liquid concentrate containing 6 lb/gal (0.72 kg/l) active ingredient. The concentrate also containing 0.5 lb/A (0.06 kg/l) of the above-mentioned antidote. The carbamates were used in technical form. Stock solutions were prepared by adding appropriate amounts of the formulated test chemicals to 100 cc mixtures containing equal parts of water and acetone. Five cc of a stock solution was then added to three pounds of soil containing approximately 5% moisture in a 5-gallon (18.9 liter) rotary mixer where the soil and stock solution were mixed for 10 to 20 seconds. Sandy loam soil from Sunol, Calf., was used, containing approximately 49% sand, 44% silt, and 7% clay (on a weight basis), with an organic content of 1.8% by weight and a pH of 7.6. The treated soil was then placed in aluminum flats which were 2.5 inches deep, 3.5 inches wide, and 7.5 inches long (6.4×8.9×19.0 cm). The soil was tamped and leveled with a row marker to impress six rows across the width of the flat. The test weeds were as follows: ______________________________________COMMON NAME SCIENTIFIC NAME______________________________________yellow foxtail Setaria lutescens (Weigel) Hubb.barnyardgrass Echinochloa crusgalli (L.) Beauv.milo Sorghum bicolor (L.) Moenchbarley Hordeum vulgare (L.)giant foxtail Setaria faberi Herrmwild cane Sorghum bicolor (L.) Moench (variant of milo)______________________________________ DeKalb XL-45A corn of species Zea mays (L.) was also planted. Sufficient seeds were planted to produce several seedlings per inch in each row. The flats were then placed in a greenhouse maintained at 70° to 85° F. and watered daily by sprinkler. Two weeks after treatment, the degree of weed control and corn injury were estimated and recorded as percentage control compared to the growth of the same species in an untreated check flat of the same age. The rating scale ranges from 0 to 100%, where 0 equals no effect with plant growth equal to the untreated control, and 100 equals complete kill. Table II lists the results of these tests. Control experiments are included for comparison. Substantial improvements in average percent weed control over the control experiments are evident at each application rate of both the herbicide and the extender. It is clear from the table that the herbicidal efficacy of the thiolcarbamate two weeks after application was much improved by the use of the extender. TABLE II______________________________________ Herbicidal Activity Data______________________________________Herbicide S-ethyl di-n-propylthiolcarbamate as 6 lb/gal emulsifiable liquid, further containing 0.5 lb/gal N,N-diallyl 2,2- dichloroacetamideExtender N-Methyl 3,5-dimethylphenylcarbamate in technical formEvaluation 2 weeks after treatmentTime______________________________________Application Rates (lb/A) Plant InjuryHerbicide Extender Weed Average* Corn______________________________________Control data:3.0 0 4 0Test data:3.0 0.25 13 03.0 0.5 37 03.0 1.0 74 03.0 2.0 93 0Control data:6.0 0 19 0Test data:6.0 0.25 32 06.0 0.5 49 06.0 1.0 69 06.0 2.0 91 0______________________________________ *Average over 6 weeds (yellow foxtail, barnyardgrass, milo, barley, giant foxtail, and wild cane), 3 replications each. METHODS OF APPLICATION The herbicidal compositions of the present invention are useful in controlling the growth of undesirable vegetation by pre-emergence or post-emergence application to the locus where control is desired, including pre-plant and post-plant soil incorporation as well as surface application. The compositions are generally embodied in formulations suitable for convenient application. Typical formulations contain additional ingredients or diluent carriers which are either inert or active. Examples of such ingredients or carriers are water, organic solvents, dust carriers, granular carriers, surface active agents, oil and water, water-oil emulsions, wetting agents, dispersing agents, and emulsifying agents. The herbicidal formulations generally take the form of dusts, emulsifiable concentrates, granules and pellets, or microcapsules. A. DUSTS Dusts are dense powder compositions which are intended for application in dry form. Dusts are characterized by their free-flowing and rapid settling properties so that they are not readily windborne to areas where their presence is not desired. They contain primarily an active material and a dense, free-flowing, solid carrier. Their performance is sometimes aided by the inclusion of a wetting agent, and convenience in manufacture frequently demands the inclusion of an inert, absorptive grinding aid. For the dust compositions of this invention, the inert carrier may be either of vegetable or mineral origin, the wetting agent is preferably anionic or nonionic, and suitable absorptive grinding aids are of mineral origin. Suitable classes of inert solid carriers for use in the dust compositions are those organic or inorganic powders which possess high bulk density and are very freeflowing. They are also characterized by low surface area and poor liquid absorptivity. Suitable grinding aids are natural clays, diatomaceous earths, and synthetic mineral fillers derived from silica or silicate. Among ionic and nonionic wetting agents, the most suitable are the members of the group known to the art as wetting agents and emulsifiers. Although solid agents are preferred because of ease in incorporation some liquid nonionic agents are also suitable in the dust formulations. Preferred dust carriers are micaceous talcs, pyrophyllite, dense kaolin clays, tobacco dust and ground calcium phosphate rock. Preferred grinding aids are attapulgite clay, diatomaceous silica, synthetic fine silica and synthetic calcium and magnesium silicates. Most preferred wetting agents are alkylbenzene and alkylnaphthalene sulfonates, sulfated fatty alcohols, amines or acid amides, long chain acid esters of sodium isothionate, esters of sodium sulfosuccinate, sulfated or sulfonated fatty acid esters, petroleum sulfonates, sulfonated vegetable oils, and ditertiary acetylenic glycols. Preferred dispersants are methyl cellulose, polyvinyl alcohol, lignin sulfonates, polymeric alkylnaphthalene sulfonates, sodium naphthalenesulfonate, polymethylene bisnaphthalenesulfonate, and sodium-N-methyl-N-(long chain acid) taurates. The inert solid carriers in the dusts of this invention are usually present in concentrations of from about 30 to 90 weight percent of the total composition. The grinding aid will usually constitute 5 to 50 weight percent of the compositions, and the wetting agent will constitute from about 0 to 1.0 weight percent of the composition. Dust compositions can also contain other surfactants such as dispersing agents in concentrations of up to about 0.5 weight percent, and minor amounts of anticaking and antistatic agents. The particle size of the carrier is usually in the range of 30 to 50 microns. B. EMULSIFIABLE CONCENTRATES Emulsifiable concentrates are usually solutions of the active materials in nonwater-miscible solvents together with an emulsifying agent. Prior to use, the concentrate is diluted with water to form a suspended emulsion of solvent droplets. Typical solvents for use in emulsifiable concentrates include weed oils, chlorinated hydrocarbons, and nonwater-miscible ethers, esters, and ketones. Typical emulsifying agents are anionic or nonionic surfactants, or mixtures of the two. Examples include long-chain alkyl or mercaptan polyethoxy alcohols, alkylaryl polyethoxy alcohols, sorbitan fatty acid esters, polyoxyethylene ethers with sorbitan fatty acid esters, polyoxyethylene glycol esters with fatty or rosin acids, fatty alkylol amide condensates, calcium and amine salts of fatty alcohol sulfates, oil soluble petroleum sulfonates or, preferably, mixtures of these emulsifying agents. Such emulsifying agents will comprise from about 1 to 10 weight percent of the total composition. Thus, emulsifiable concentrates of the present invention will consist of from about 15 to about 50 weight percent active material, about 40 to 82 weight percent solvent, and about 1 to 10 weight percent emulsifier. Other additives such as spreading agents and stickers can also be included. C. GRANULES AND PELLETS Granules and pellets are physically stable, particulate compositions containing the active ingredients adhering to or distributed through a basic matrix of a coherent, inert carrier with macroscopic dimensions. A typical particle is about 1 to 2 millimeters in diameter. Surfactants are often present to aid in leaching of the active ingredients from the granule or pellet. The carrier is preferably of mineral origin, and generally falls within one of two types. The first are porous, absorptive, performed granules, such as preformed and screened granular attapulgite or heat expanded, granular, screened vermiculite. On either of these, a solution of the active agent can be sprayed and will be absorbed at concentrations up to 25 weight percent of the total weight. The second, which are also suitable for pellets, are initially powdered kaolin clays, hydrated attapulgite, or bentonite clays in the form of sodium, calcium or magnesium bentonites. Water-soluble salts, such as sodium salts, may also be present to aid in the disintegration of granules or pellets in the presence of moisture. These ingredients are blended with the active components to give mixtures that are granulated or pelleted, followed by drying, to yield formulations with the active component distributed uniformly throughout the mass. Such granules and pellets can also be made with 25 to 30 weight percent active component, but more frequently a concentration of about 10 weight percent is desired for optimum distribution. The granular compositions of this invention are most useful in a size range of 15-30 mesh. The surfactant is generally a common wetting agent of anionic or nonionic character. The most suitable wetting agents depend upon the type of granule used. When preformed granules are sprayed with active material in liquid form the most suitable wetting agents are nonionic, liquid wetters miscible with the solvent. These are compounds more generally known to the art as emulsifiers, and comprise alkylaryl polyether alcohols, alkyl polyether alcohols, polyoxyethylene sorbitan fatty acid esters, polyethylene glycol esters with fatty or rosin acids, fatty alkylol amide condensates, oil solution petroleum or vegetable oil sulfonates, or mixtures of these. Such agents will usually comprise up to about 5 weight percent of the total composition. When the active ingredient is first mixed with a powdered carrier and subsequently granulated, or pelleted, liquid nonionic wetters can still be used, but it is usually preferable to incorporate at the mixing stage one of the solid, powdered anionic wetting agents such as those previously listed for the wettable powders. Such agents will comprise from about 0 to 2 weight percent of the total composition. Thus, the preferred granular or pelleted formulations of this invention comprise about 5 to 30 percent by weight active material, about 0 to 5 weight percent wetting agent, and about 65 to 95 weight percent inert material carrier, as these terms are used herein. D. MICROCAPSULES Microcapsules consist of fully enclosed droplets or granules containing the active materials, in which the enclosing material is an inert porous membrane, arranged to allow escape of the enclosed materials to the surrounding medium at controlled rates over a specified period. Encapsulated droplets are typically about 1 to 50 microns in diameter. The enclosed liquid typically constitutes about 50 to 95% of the weight of the entire capsule, and may contain a small amount of solvent in addition to the active materials. Encapsulated granules are characterized by porous membranes sealing the openings of the granule carrier pores, trapping the liquid containing the active components inside for controlled release. A typical granule size ranges from 1 millimeter to 1 centimeter in diameter. In agricultural usage, the granule size is generally about 1 to 2 millimeters in diameter. Granules formed by extrusion, agglomeration, or prilling are useful in the present invention as well as materials in their naturally occurring form. Examples of such carriers are vermiculite, sintered clay granules, kaolin, attapulgite clay, sawdust, and granular carbon. Useful encapsulating materials include natural and synthetic rubbers, cellulosic materials, styrene-butadiene copolymers, polyacrylonitriles, polyacrylates, polyesters, polyamides, polyurethanes, and starch xanthates. E. IN GENERAL Each of the above formulations can be prepared as a package containing both the herbicide and the extender together with the other ingredients of the formulation (diluents, emulsifiers, surfactants, etc.). The formulations can also be prepared by a tank mix method, in which the ingredients are obtained separately and combined at the grower site. The herbicide and extender may both be used in the same type of formulation or a different formulation may be used for each, e.g., the herbicide may be in microcapsule form while the extender may be an emulsifiable concentrate, or vice versa. As a further alternative, the herbicide and extender can be applied sequentially, with either being applied first. This is a less preferred method, however, since more effective results are obtained with simultaneous application. In general, any conventional method of application can be used. The locus of application can be soil, seeds, seedlings, or the actual plants, as well as flooded fields. Soil application is preferred. Dusts and liquid compositions can be applied by the use of powder dusters, boom and hand sprayers, and spray dusters. The compositions can also be applied from airplanes as dusts and sprays because they are effective in very low dosages. In order to modify or control the growth of germinating seeds or emerging seedlings, as a typical example, the dust and liquid compositions are applied to the soil according to conventional methods and are distributed in the soil to a depth of at least one-half inch below the soil surface. It is not necessary that the phytotoxic compositions be admixed with the soil particles. Instead, these compositions can be applied merely by spraying or sprinkling the surface of the soil. The phytotoxic compositions of this invention can also be applied by addition to irrigation water supplied to the field to be treated. This method of application permits the penetration of the compositions into the soil as the water is absorbed therein. Dust compositions, granular compositions or liquid formulations applied to the surface of the soil can be distributed below the surface of the soil by conventional means such as discing, dragging or mixing operations. The herbicide/extender compositions can also be applied to the soil through irrigation systems. According to this technique, the compositions are added directly to irrigation water immediately prior to irrigation of the field. This technique is applicable in all geographical areas regardless of rainfall, since it permits supplementation of the natural rainfall at critical stages of plant growth. In a typical application, the concentration of the herbicide/extender composition in the irrigation water will range from about 10 to 150 parts per million by weight. The irrigation water can be applied by the use of sprinkler systems, surface furrows, or flooding. Such application is most effectively done before the weeds germinate, either early in the spring prior to germination or within two days after cultivation of the field. The compositions of this invention can also contain further pesticides, such as fungicides, insecticides, other herbicides, etc. Suitable pesticides include 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and the salts, esters and amides thereof; triazine derivatives, such as 2,4-bis-(3-methoxypropylamino)-6-methyl-thio-s-triazine; 2-chloro-4-ethylamino-6-isopropylamino-s-triazine; 2-ethylamino-4-isopropylamino-6-methylmercapto-s-triazine; urea derivatives such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea; acetamides such as N,N-diallyl-α-chloroacetamide, N-(α-chloroacetyl) hexamethyleneimine, and N,N-diethyl-α-bromoacetamide; and benzoic acids such as 3-amino-2,5-dichlorobenzoic acid. Fertilizers such as ammonium nitrate, urea, and superphosphate can also be included. The amount of composition of the present invention which constitutes a herbicidally effective amount depends upon the nature of the seeds or plants to be controlled. The rate of application of active ingredient varies from about 0.01 to about 50 pounds per acre, preferably about 0.1 to about 25 pounds per acre with the actual amount used depending on the overall cost and the desired results. It will be readily apparent to one skilled in the art that compositions exhibiting lower herbicidal activity will require a higher dosage than more active compounds for the same degree of control.

Summary: Herbicidally active thiolcarbamates are employed in combination with certain substituted carbamates having the formula ##STR1## in which R 4 and R 5 are each methyl, R 4 and R 5 conjointly form C 1 --C 4 alkylenedioxy, R 4 is --CF 3 and R 5 is hydrogen, or R 4 is --CH 2 Br and R 5 is hydrogen. In a typical application, the carbamate is included in sufficient quantity to lessen the rate of soil degradation of the thiolcarbamate. As a result, the herbicidal effectiveness of the thiolcarbamate is enhanced and prolonged, rendering a single application of the herbicide effective over a longer period of time.

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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from and is a continuation-in-part of U.S. Non-provisional patent application Ser. No. 10/366,387, filed Feb. 14, 2003 now U.S. Pat. No. 6,659,838, entitled RIGID HELIUM BALLOONS, the contents of which are incorporated by reference herein in their entirety. BACKGROUND 1. Field of the Invention The present invention relates to lighter-than-air balloons, and more particularly, to lighter-than-air balloons having a rigid skeleton. 2. Description of the Related Art Generally, it has been difficult to fabricate balloons with continuously curved shapes, and well-defined corners, or edges. Most balloons are formed in spherical shapes in order to allow the greatest volume for the least surface area. Also, the thin material of the balloon naturally becomes spherical as pressure is increased. To achieve the desired non-spherical shape, then, it is necessary to provide a supporting frame to maintain the thin material of the balloon. However, in the past, the weight of such frames, even when the most efficient materials for such purposes were selected, typically required a displaced volume of such size that fabrication for home use or the like would have been impractical. Consequently, helium balloons are typically formed in spherical shapes with some type of tethering device attached for maintaining control of the balloon&#39;s elevation. U.S. Pat. No. 4,032,086, issued Jun. 28, 1977 to W. Cooke, discloses an aerostat or aquastat in which a sealed envelope of flexible material is mounted on a flexible frame which can be caused to expand the envelope after it has been evacuated of internal gas, thereby setting up a vacuum or partial vacuum condition in the envelope. By controlling the frame to adjust the volume of the envelope, the lift or buoyancy of the device can be controlled in flight or precisely determined before ascent. U.S. Pat. No. 4,038,777, issued Aug. 2, 1977 to S. Schwartz, discloses a gas filled, balloon-like object capable of defining a non-spherical shape. A high modulus graphite impregnated epoxy material is used to prevent distortion of the inflated object. Strings or weights are required to prevent upward ascent of the balloon. U.S. Pat. No. 4,113,206, issued Sep. 12, 1978 to D. Wheeler, discloses a lighter-than-air apparatus, including a thin, pliable air-tight cuter envelope disposed in overlying relationship over a light-weight, coarse-opening inner frame of a spherelike shape. Other devices relating to balloons and lighter-than-air apparatuses include U.S. Pat. No. 2001/0003505 A1 issued Jun. 14, 2001 to T. Bertrand, which discloses a lighting apparatus secured to a balloon by string under tension; U.S. Pat. No. 4,925,426 issued May 15, 1990 to C. Lovik, which discloses an open skeletal frame of rigid rod-like formers made of thin strands of plastic, wire, or the like and which permits the insertion of an uninflated balloon of conventional shape and size into the interior thereof so that upon inflation of the balloon, the latex sidewall material of the balloon projects outwardly through the openings of the formers to produce bulbous projections; U.S. Pat. No. 5,115,997, issued May 26, 1992 to J. Peterson, which discloses a tethered surveillance balloon having a relatively low lift-to-weight ratio; U.S. Pat. No. 5,115,998, issued May 26, 1992 to L. Olive, which discloses a double-walled, annular balloon which requires less gas to inflate than its volume would indicate; U.S. Pat. No. 5,334,072, issued Aug. 2, 1994 to M. Epstein, which discloses an inflatable body, such as a balloon, and holder assembly therefore; U.S. Pat. No. 5,882,240, issued Mar. 16, 1999 to B. Larsen, which discloses a toy blimp; U.S. Pat. No. 6,276,984, issued Aug. 21, 2001 to K. Komaba, which discloses a balloon having adhering members disposed upon its surface; Japanese Patent No. 1238890, published Sep. 25, 1989, which discloses plastic film balloons in animal and other complex shapes. BRIEF DESCRIPTION OF THE DRAWINGS The invention is illustrated by way of example and not by way of limitation in the figures of the accompanying drawings in which like references indicate similar elements. It should be noted that references to “an” or “one” embodiment in this disclosure are not necessarily to the same embodiment, and such references mean at least one. FIG. 1 is an environmental, perspective view of a rigid helium balloon according to the present inventor. FIG. 2 is a section view along lines 2 - 2 of FIG. 1. FIG. 3 is a perspective view of a rigid helium balloon according to the present invention. FIG. 4 is a diagram of an alternative embodiment of the rigid balloon. FIG. 5 is a diagram of an alternative embodiment of the rigid balloon. FIG. 6 is a diagram of a sleeve to retain a structural member in one embodiment of the rigid balloon. Similar reference characters denote corresponding features consistently throughout the attached drawings. DETAILED DESCRIPTION OF THE INVENTION FIG. 1 is an environmental, perspective view of a rigid helium balloon according to the present inventor. As shown in FIG. 1, one embodiment of the balloon, generally designated as 10, is relatively small and can be easily adapted as a toy for indoor use. As depicted in FIG. 2, the balloon 10, is made from skin portions 12 and 14, e.g., a top half and a bottom half of the balloon 10. The skin portions 12 and 14 may be formed in any shape desired for the balloon 10. In the embodiment depicted in FIGS. 1-2, the skin portions 12 and 14 are shaped so that when the top half 12 and bottom half 14 are joined, the resulting balloon 10 is a lenticular-shaped balloon which resembles a flying saucer. Skin portions 12 and 14 can be made from any suitable heat sealable material which has low gas permeability. In one embodiment, skin portions 12 and 14 are made from polyethylene terephthalate (sold under the trademark Mylar®, a trademark of E.I. duPont de Nemours &amp; Co. of Wilmington, Del.). FIG. 2 is a section view along lines 2 - 2 of FIG. 1. As can be more clearly seen in FIG. 2 in this embodiment, the skin portions 12 and 14 are sealed together in a double seam about their periphery, including a first peripheral seam 16 and a parallel or concentric second seam 18. First seam portion 16 and second seam portions 18 are disposed near the peripheral edges of the first and second skins 12 and 14, and are spaced from one another. First seam portion 16 and second seam portion 18 are formed by heat sealing or any other suitable means. A channel portion 20 is defined between seam 16 and seam 18 and extends about the periphery of the balloon 10. Skin portions 12 and 14, when joined, define a chamber 22 therebetween which may be filled with a lighter than air gas such as helium. The chamber 22 includes a valve 24 through which the balloon 10 may be filled with the lighter than air gas. The valve 24 may be one which is commonly used in Mylar balloons, although any suitable valve may be used. FIG. 3 is a perspective view of a rigid helium balloon according to the present invention. As can be seen in FIG. 3, at least one structural member 26 is inserted into the channel portion 20 through apertures 28. While the structural member 26 can be formed from any acceptable material, in one embodiment it is made from fiberglass. In another embodiment, the structural member 26 is molded or extruded from a thermoplastic or other polymer. Once the structural member 26 has been inserted through the channel portion 20, opposing ends 30 of the structural member 26 can be joined together by a connector 32 to secure the structural member 26 in place. Any suitable connector 32 may be used to join the ends 30 of the structural member 26. In one embodiment, a brass fitting having a diameter slightly larger than the diameter of the structural member 26 is used. Alternatively, the structural member may be manufactured in a desired shape such as a ring. The ring may be placed adjacent to first seam 16 around the chamber before second seam 18 is formed. Second seam 18 may then be formed to retain the structure member 26. In such an embodiment, no connector is required. Once the structural member 26 is secured in the channel portion 20, the structural member 26 provides a substantially rigid skeleton for the balloon 10 so that the balloon 10 may maintain its desired shape once it has been inflated with gas. The rod member 26 has a weight which is calculated to counterbalance the buoyant effect of the gas so that the balloon 10 is prevented from floating upwards when filled, the balloon 10 simply floats at the height at which it is released. Stated differently, in one embodiment, the weight of the rod (and any connector) is selected to cause the balloon to be neutrally buoyant under ambient conditions when the chamber is inflated to a known pressure with a lighter than air gas. Although only one structural member 26 is depicted in the drawings, for some shapes, it may be necessary to use a plurality of structural members 26 of varying sizes (not shown). For such shapes, for example those with a plurality of curves or angles, a plurality of apertures may be provided at various points on the balloon 10 so that the structural members 26 may be easily inserted into the channel portion 20. The structural members 26 can then be connected to one another using the connector 32, as previously described. FIG. 4 is a diagram of an alternative embodiment of the rigid balloon. As shown in FIG. 4, instead of creating (or in addition to) a channel for the structural member at the junction between the two skins, a plurality of strips 130 may be attached to the external surface of the flexible material covering the chamber and by either threading the structural member 126 through the loops formed by attaching the strips 130 around the structural member, the structural member 126 is retained and provides a skeleton for the balloon 110. FIG. 5 is a diagram of an alternative embodiment of the rigid balloon. In this embodiment, the chamber is again constructed of one or more pieces of flexible low permeability material. The flexible material may be assembled to form the chamber by heat welding; adhesive or any other manner that results in a low gas permeability ultimate chamber. In one embodiment, one or more sleeves may be coupled to the external surface of the material defining the chamber to provide receptacles for one or more structural members 226. Again, this coupling may be accomplished with adhesive, heat welding or any manner that does not substantially degrade the structural integrity of the chamber. Alternatively, pockets may be formed in a manner analogous to that described above. FIG. 6 is a diagram of a sleeve to retain a structural member in one embodiment of the rigid balloon. The sleeve 232 may have one end sealed such as by heat welding. An aperture 336 is defined distal to the sealed end 338, but short of the opposing end 340. The structural member 226 having some elasticity may then be inserted into the sleeve 232 to the sealed end 338. The structural member may then be flexed so the other end of the member can be inserted past the aperture. The natural elasticity of the structural member will then hold it in place against the opposing ends of the sleeve 232. In one embodiment, additional heat welds are used within the sleeve to provide a well-defined seat 334 for the ends of the structural member 226 to reduce movement of the structural member 226 in the sleeve 232. In one embodiment, the sleeve is open at both ends and defines a channel for the structural member. A throughway connector may be used to hold the structural member 226 together. For example, the sleeve may run circumferentially around the lenticular shaped balloon described with reference to FIGS. 1-4. In one embodiment, the sleeve may be completely sealed at the time of manufacture with the structural member enveloped within. In one embodiment, a structural member may be a rod having substantially any shaped cross section. While rod with circular cross section is suitable for use in embodiments of the invention, square, triangular, dogbone and substantially any other cross sections are contemplated. Structural members having a thickness much less than their length or width are also contemplated. In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however be evident that various modifications and changes can be made thereto without departing from the broader spirit and scope of the invention as set forth in the appended claims. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Summary: The rigid balloon has a chamber to hold lighter-than-air gas portions to retain a structural member or members and a separate light gas is filled into the chamber through a valve in the balloon. Structural members are inserted into the portion to help retain the desired shape of the balloon. The structural members also provide a counterbalancing weight which prevents the balloon from floating upward. Thus, the balloon, once released into the air, will retain its shape and remain floating at the height from which it was released unless repositioned.

big_patent

en

Summarize: By. Ellie Zolfagharifard. PUBLISHED:. 04:48 EST, 2 October 2013. |. UPDATED:. 05:12 EST, 2 October 2013. Satellite images of the mysterious island that rose up from the sea last week reveal that it is round, with cracks and remarkably flat, similar to a'mud pie'. The island, named Zalzala Koh, emerged after a devastating 7.8 magnitude earthquake struck 145 miles southeast of Dalbandin in. Pakistan's quake-prone province of Baluchistan. It has become an attraction for tourists and locals who have visited the area despite toxic, flammable gas being emitted from its cracks. The surface is covered in sea creatures such as dead fish and is a mixture of mud, sand and rock. Scroll down for videos... An aerial photo of the island suggests the grey-coloured mound is about 15 to 20 meters tall. The surface is covered in sea creatures such as dead fish and is a mixture of mud, sand and rock. It is solid enough for people to walk on. The latest images were taken by Nasa's Earth Observing-1 and Landsat 8 satellites just days after the island emerged. The lighter shades of green and tan in the water reveal shallow seafloor or suspended sediment. The water depth around the new island is roughly 15 to 20 meters, according to marine geologist Asif Inam of Pakistan’s National Institute of Oceanography. 'The floor in that area is generally flat, but the gradient in this area changes quite abruptly,' said Mr Inam. The aerial photograph above provides a close-up of the landform,. estimated to stretch 75 to 90 meters across and standing 15 to 20 meters. (60 to 70 feet) above the water line. The below image is also clear enough to show the parallel ripples of waves marching toward the shore. The island rose from the seafloor near Gwadar on September 24, shortly after the earthquake struck about 380 kilometers (230 miles) inland. This image was captured by Nasa's Advanced Land Imager on the Earth Observing-1 satellite. Scientists. believe the 7.8 magnitude earthquake, which struck 145 miles southeast. of Dalbandin in Baluchistan, triggered what is known as'mud volcano'. They occur where there is a reservoir of. loosely compacted sediments buried beneath harder, denser rock and a. path is made to the surface. The. seismic waves caused a movement of gases locked in the earth under the. sea, pushing mud and earth up to the surface along with gas. These sudden islands are usually only. spotted after strong earthquakes, at least 7- or 8-magnitude events. 'The island is really just a big pile of mud from the seafloor that got pushed up,' said Bill Barnhart, a geologist at the U.S. Geological Survey who studies earthquakes in Pakistan and Iran. 'This area of the world seems to see so many of these features because the geology is correct for their formation. 'You need a shallow, buried layer of pressurised gas—methane, carbon dioxide, or something else—and fluids. 'When that layer becomes disturbed by seismic waves (like an earthquake), the gases and fluids become buoyant and rush to the surface, bringing the rock and mud with them.' Dead fish have been spotted floating on the surface of the waters surrounding the island and visitors have heard hissing noises from the escaping toxic gas. Dead fish have been spotted floating on the surface of the waters surrounding the island and visitors have heard hissing noises from the escaping toxic gas. An aerial view of the island taken soon after it formed. The life of this island is likely to be short, according to Nasa. The underground pocket of gas will cool, compress, or escape over time, allowing the crust to collapse and settle back down. Richard Luckett a seismologist at the. British Geological Survey said that these mud volcanoes are relatively. common in this area on both land and at sea. ‘They. occur where there is a reservoir of loosely compacted sediments buried. beneath harder, denser rock and a path is made to the surface,’ he told. MailOnline. ‘There is a. subducting plate boundary in this region where the Arabian plate is. converging on the Eurasian plate at about 2cm a year and being pushed. beneath it. ‘This is the same plate boundary responsible for the magnitude 7.7 earthquake.’ Scientists. believe gases associated with the melting at the plate boundary. contribute to heating the mud in the reservoirs and making it more. fluid. ‘Certainly mud volcanoes are most common near this type of plate boundary, such as in Japan,’ said Dr Luckett. Such masses are sometimes created by the movement of gases locked in the earth under the sea, pushing mud and earth up to the surface in something akin to a mud volcano. Waves, storms, and tidal action from the Arabian Sea will wash away the loose sand, soft clay, and mud on the island. ‘It is known that mud volcanoes can be. triggered by large nearby earthquakes – in fact the same thing happened. in this area in 2001. ‘The. exact mechanism for this triggering is poorly understood but the fact. that mud volcanoes often occur without a triggering earthquake suggests. that little extra impetus is required.’ The life of this island is likely to be short, according to Nasa. The underground pocket of gas will cool, compress, or escape over time, allowing the crust to collapse and settle back down. Waves, storms, and tidal action from the Arabian Sea will also wash away the loose sand, soft clay, and mud. The 7.8 magnitude quake struck 145 miles southeast of Dalbandin in Pakistan's quake-prone province of Baluchistan, which borders Iran, on Tuesday. Dozens of people had already visited the island despite concerns over toxic gas. Such land masses have appeared before off Pakistan's Makran coast, said Muhammed Arshad, a hydrographer with the navy. After quakes in 1999 and 2010, new land masses rose up along a different part of the coast about 282 kilometers (175 miles) east of Gwadar, he said. He said each of those disappeared back into the sea within a year during the monsoon season, a period of heavy rain and wind that sweeps Pakistan every summer. He said that in the area where the island was created on Tuesday, the sea is only about six to seven meters (23 feet) deep. Older residents of the coastal town recalled an earthquake in 1968 produced an island that stayed for one year and then vanished

Summary: Images show island to be remarkably flat and round, with huge fissures. The mountain-like island appeared 600m off Pakistan's Gwadar coast. The earthquake triggered a movement of gases in the earth under the sea. This pushed earth up to the surface in something akin to a'mud volcano' Scientists believe the island won't last long due to erosion and a cooling of the gas which will cause its crust to collapse.

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Write a title and summarize: Tumors are defined by their intense proliferation, but sometimes cancer cells turn senescent and stop replicating. In the stochastic cancer model in which all cells are tumorigenic, senescence is seen as the result of random mutations, suggesting that it could represent a barrier to tumor growth. In the hierarchical cancer model a subset of the cells, the cancer stem cells, divide indefinitely while other cells eventually turn senescent. Here we formulate cancer growth in mathematical terms and obtain predictions for the evolution of senescence. We perform experiments in human melanoma cells which are compatible with the hierarchical model and show that senescence is a reversible process controlled by survivin. We conclude that enhancing senescence is unlikely to provide a useful therapeutic strategy to fight cancer, unless the cancer stem cells are specifically targeted. Cancer cells are characterized by their persistent proliferation, but just as for normal cells [1] tumor cells can go senescent, halting their growth [2], [3]. The molecular basis for the induction of senescence appears to be a combination of several mechanisms such as telomerase shortening, DNA-damage and oxidative stress [3]. It has been suggested that senescence should be present only in pre-neoplastic cells [3] but there is evidence that senescence markers increase during tumor progression [4]. This is puzzling since it is usually assumed that tumors can only grow if senescence is avoided. Recent experiments are challenging this conventional view of cancer, showing that only a small fraction of cancer cells, the cancer stem cells (CSCs) [5], actively drive tumor growth. The implications of this finding for tumor cell senescence have hitherto not been explored. According to the CSC hypothesis, tumors behave in analogy with normal tissues, whose growth is controlled by a small population of slowly replicating stem cells with the dual capacity of either self-renewal or differentiation into the more mature cells required by the tissue. The crucial difference between tissue stem cells and CSCs lies in their proliferation properties. Stem cells in tissues tend to keep their number constant either deterministically by enforcing asymmetric division, or stochastically by balancing proliferation and depletion probabilities [6], [7]. This restriction does not hold for CSCs. The role of CSCs has important implications for therapeutic approaches. According to the conventional view, the success of a treatment is measured by the number of tumor cells killed; in contrast, according to the CSC hypothesis, only the CSC subpopulation matters in the end for complete eradication. First evidences for the existence of CSCs came from hematological tumors [5] and later from solid tumors such as breast cancer [8] and melanoma [9]–[16]. The presence of CSCs in melanoma is currently debated. It was argued that to obtain reliable estimates of the number of tumor initiating cells one should use highly immunocompromised mice for tumor xenografts [17]. Different groups, however, reported conflicting results with slight differences in assay conditions and mouse models [14], [17]. Furthermore, several putative CSC markers appears to be reversibly expressed in melanoma [18] and in breast cancer [19]. In this paper, we analyze cell senescence during the growth of melanoma cells both in vitro and in tumor xenografts. We find that the fraction of senescent cells increases considerably after a few months of cultivation, slowing down the growth of the cell population. This process is, however, only transient: after some time senescence almost disappears and growth resumes at the initial rate. We also show that senescence is a reversible process controlled by survivin: by overexpressing survivin in senescent cells, we are able to decrease senescent markers and increase cell proliferation. These results can be interpeted in terms of the hierarchical cancer model where only CSCs replicate indefinitely and senescence reflects the loss of proliferative capacity of other cancer cells. To prove this point we sort cancer cells according to a putative CSC marker, ABCG2, and show that senescence is more prevalent for negative cells, suggesting that CSC are able to rejuvenate an otherwise senescent cell population. To understand quantitatively the experimental results, we propose a mathematical model for cancer growth that is compatible with the existence of CSCs in melanoma, provides an unambiguous interpretation of experimental data, and explains quantitatively the occurrence of senescence in tumors. While in the stochastic cancer model, senescence is due to spontaneous mutations of tumor cells, in the hierarchical model only CSCs replicate indefinitely and senescence reflects the loss of proliferative capacity of other CCs. These observations can be recast in a mathematical framework using the theory of branching processes, a generic model for a growing population [20], [21]. Branching processes were first proposed at the end of the XIX century and found wide application in physics and biology with examples ranging from nuclear reactions [20] to evolution theory [21], tissue growth [6], [22]–[24] and cancer progression [25], [26]. The main limitation of branching processes is that they do not account for interactions between cells which could be relevant for the growth of a tumor, especially in vivo. Despite this shortcoming, our model allows for a good description of the experiments, providing an indirect confirmation of the CSC hypotesis for melanoma with important implications for therapeutic strategies based on the induction of senescence in cancer. Our theory has biologically motivated ingredients characterizing the probabilities for cells to duplicate, become senescent or die: According to the CSC hypothesis, cells are organized hierarchically, with CSCs at the top of the structure. CSCs can divide symmetrically with rate giving rise to two new CSCs with probability, two CCs with probability, or asymmetrically with probability giving rise to a CSC and a CC. While CSCs can duplicate for an indefinite amount of time, CCs become senescent after a finite number of generations and then eventually die rate (Fig. 1). The model kinetics depends on the combination, the average relative increase in the number of CSCs after one duplication, rather than on, and separately. In normal tissues, the stem cell population should remain constant which implies that [6], [27], while in tumors we expect. The kinetics of the cell populations for this model can be solved exactly. To this end, we first derive recursion relations linking the average cell populations at each generation. Denoting by the average number of CSCs after generations, by the average number of CCs, where indicates the “age” of the CCs (i. e. the number of generations separating it from the CSC from which it originated), and by the average number of senescent cells, we obtain (1) These relations are derived considering that CSCs can only originate from other CSCs either by a symmetric division – two CSCs are generated with probability – or by an asymmetric one, in which case a single CSC is generated with probability. Hence each CSC generates an average of new CSCs. CSCs also generate CCs () by asymmetric CSC divisions, with probability or by symmetric CSC division with probability, yielding an average of CCs. Two CCs are generated by duplication of other normal cells with unit probability (). Senescent cells accumulate at each generation when normal cells (with) lose the ability to duplicate and die with probability. Eqs. 2 can be solved explicitly. We consider first an initial condition with CSC only (i. e.) and obtain (2) Eqs. 2 implies that the average CSC population grows exponentially fast, and that after a large number of generations (i. e. for), all cells populations, and hence the size of the tumor, are proportional to the CSC population. This result confirms that the population of CSCs is the driving force behind tumor growth. Note that the population of senescent cells is also driven by the growth of CSCs and therefore cancer growth and senescence are inextricably linked. To interpret experimental data, it is important to consider the case in which the initial condition is composed of a mixed population of CSCs and CCs. The solution in this case can be obtained as a linear superposition of the solution of Eqs. 2 and the solution with initial conditions, and. For simplicity, we consider the case of a uniform distribution for the ages of CSCs:, and obtain (3) The complete solution for the total number of cells, corresponding to an initial condition and, is given by (4) and is plotted in Fig. 2A, while in Fig. 2B we report the fraction of senescent cells. Eqs. 2 represent only a transient contribution to the cell population and does not influence the asymptotic fractions (i. e. for) of senescent cells and CSCs which are readily obtained dividing the results in Eqs. 2 by the total number of cells at each generation: (5) (6) The asymptotic solutions are plotted in Fig. S1 as a function of for different values of and. It is instructive to use the solution to compare how cells become senescent in normal tissues and in cancers. In the long time limit, the asymptotic fraction of senescent cells is equal to and is independent of the number of duplications needed to induce senescence (see Fig. S1). For normal stem cells, and the percentage of senescent cells is expected to be large, reaching 100% in the limit of, when senescent cells never die. For cancer cells, and the fraction of senescent cells is smaller but still non-vanishing. Similarly, the CSC fraction is given by which is smallest for normal stem cells and increases as a function of in tumors (see Fig. S1). Aggressive tumors such as melanoma should be characterized by high values of and therefore by relatively high values of the CSC fraction. Beside asymptotic fractions, the model allows one also to characterize the evolution of the populations, i. e. the time-dependent evolution of the senescent cell fractions (see Fig. 2). The results show that the level of senescence in tumors strongly depends on the fraction of CSCs and on time. To quantitatively confirm that the model is able to predict the growth properties of stem cells, we first compare its results with experimental data for normal stem cells, and then turn to cancers. It has been shown that long-term in vitro culture of mesenchymal stem cells (MSC) induces replicative senescence with important implication for the therapeutic application of MSC preparations [28], [29]. In particular, the authors show a rapidly increasing fraction of cells that are positive to, a senescence marker; after roughly 50 days, the fraction of senescent cells reaches 80% of the population. By looking at the growth curves for cells extracted from donors of different ages, the authors could not establish a correlation between senescence and aging [29]. To validate our model, we have used it to fit the growth curves reported in Ref. [28]. In order to compare the model with the experiments, we should first express the evolution equations as a function of time rather than generation number. This is easily done introducing the rate of cell division per day and replacing in Eqs. 2–3 the generation index by, where is time expressed in days. In this way, Eq. 4 can directly be compared with the growth curves reported in Refs. [28], [29]. In particular, we use Eq. 4 for, and. Under this conditions the expression reduces to a much simpler form (7) We then compute the CPD based on Eq. 14 and perform a two parameters fit in terms of and (see Figs. 3 and S2). We have also analyzed similar data reported in Ref. [29] for the growth of MSC isolated from human bone marrow for the iliac crest (BM) and from the femoral head (HIP) (see Fig. 2). Although significant fluctuations between donors are revealed, the results show in general a decrease of the parameter with the age of the donor (see Fig. 4), older people have fewer generations of reproduction before senescence. The plot also shows for donors of similar age that is smaller for BM-MSC than for HIP-MSC, suggesting that the former are more prone to become senescent. On the other hand, the cell division rate fluctuates around the value of divisions/day, as reported in Ref. [27], without any apparent correlation with age. In recent publications, one of us identified aggressive subpopulations expressing two stem cell markers, ABCG2 [9] and CXCR6 [16], in human melanoma cell lines, making this tumor a suitable candidate to test our theory. Here, we analyze the level of expression of the senescence marker in three human melanoma cell lines, WM115, IGR39 and IGR37 [9], [16]. IGR39 and IGR37 cells have been sorted into two subpopulations according to the expression of the ABCG2 marker [16]. In Fig. 5A and Table S1, we report the level of the senescent marker as a function of time for ABCG2 positive (ABCG2+) and negative cells (ABCG2−). In both cases, we observe an increase in the percentage of positive cells after 80–90 days of growth followed by a decrease to the initial level after 120 days (see Table S1). While the data follow a similar trend for the two subpopulations, the peak for IGR39 ABCG2− cells is much higher, reaching 90% of the cells. In order to confirm that the difference in the expression of the senescence marker is related to a different proliferative behavior for the two subpopulations, in Fig. 5B we report the long-term growth curves corresponding to IGR39. The results show that ABCG2+ cells grow substantially more than ABCG2− cells (i. e. 23 times more). Furthermore, the peak percentage of coincides in time with a marked slow down in the growth (Fig. 5). When the level of expression falls back to the initial value, growth is also restored (Fig. 5B). These data can be described by our theoretical model, if we assume that ABCG2+ and ABCG2− data are only distinguished by a different initial fractions of CSCs. Fitting simultaneously the growth curves and the gal concentrations with the model (see Fig. 5 and Materials and Methods for details), we obtain for ABCG2− and for ABCG2+ cells. Hence, our model suggests that expression of ABCG2 is strongly correlated with CSCs (almost a factor of thirty), but only a relatively small fraction of ABCG2 expressing cells are CSCs. Notice that the final CSC fraction is different from the initial one: using the fit parameters we estimate. As shown in Fig. 5A the fraction of senescent cells predicted by the model is in good agreement with the experimental data, predicting a large senescence peak for ABCG2− cells. Notice that we could easily find parameters producing a clear peak in the concentration also for ABCG2+ cells. Our multicurve fit, however, is dominated by the (higher precision) growth curves and the resulting best fit parameters yield no peak in the plot. For ABCG2− cells, the peak in the theory curves arises after generations. The experimental peak in senescence and the associated dip in doubling rate after generations directly implies a long-term memory of the initial preparation, embodied by our senescence generation, a parameter that we expect may be smaller for other growth conditions and tumor types. The peak in cellular senescence is quantitatively described by the hierarchical model and it would be hard to reconcile this result with the stochastic cancer model, where cancer cells are not organized hierarchically. To illustrate this point, we reformulate the stochastic model in mathematical terms assuming that senescence can only occur as a result of random mutation. In this model, we distinguish between two cell populations, cancer cells (CC) and senescent cells. CC duplicate with probability, die with probability and become senescent with probability, while senescent cells do not duplicate but can die with probability. The average numbers of CC and senescent cells after generations follow recursion relations (8) (9) We can solve the recursion relations explicitly obtaining: (10) (11) where we have assumed for simplicity that. The solution implies that the number of CC grows as long as and shrinks otherwise, yielding a condition for tumor progression. Dividing and by the total population size, we obtain the relative fractions and of CC and senescent cells, respectively. In the asymptotic limit (i. e. large), we obtain steady-state solutions (12) (13) The approach to the steady-state is reported in Fig. 6A and the steady-state solutions are plotted in Fig. 6B in the case. These results show that the fraction of senescent cells should evolve rapidly towards a steady state in way that is incompatible with the experiments, providing additional evidence supporting the existence of CSCs in melanoma. The present stochastic model is clearly oversimplified and one could think of more elaborate models involving heterongeneous cell populations without a hierarchy. We do not see, however, how we can explain a peak in senescence after 90 days without assuming the presence of at least two distinct subpopulations one of which undergoes senescence after this long interval. In Fig. 7, we report the evolution of the fraction of senescent cells in human melanoma WM115 cells according to the marker. Fig. 7 shows an increase in the fraction of cells expressing this marker, reaching more than 40% after 80 days of cultivation. We find a similar level of also in tumor xenografts after 60 days (Fig. 7D). This confirms that our finding of senescent melanoma cells is not restricted to growth in vitro. In order to confirm that the observed cell senescence in xenografts (Fig. 7) is not due to hypoxia, we grow WM115 cells under normal or hypoxic conditions (see Materials and methods). Only a slight inhibition on cell growth was observed 48 hrs after incubation under hypoxic condition. This behavior is explained by the induction of the hypoxia inducible factor () after 18 hours of hypoxic conditions (see Fig. 3). is a pleiotropic transcription factor typically activated in response to low oxygen tension as well as other stress factors in normoxic conditions. Upon activation mediates the transcriptional activation of target genes involved in a variety of processes comprising stress adaptation, metabolism, growth and invasion, but also apoptotic cell death. While we cannot exclude that some form of environmental stress factor affect our results, we conclude that hypoxia is not a likely cause for the senescence we observe in xenografts; our in vitro cells do not experience hypoxia. Since it has been recently shown that survivin allows cells to escape senescence [30], we transfect IGR37 ABCG2− cells at the peak of (corresponding to the 98th day for IGR37 cells, see Table S1) with a survivin-GFP or GFP alone [31]. We then quantify the resulting senescence with activity and visualize cell proliferation with crystal violet assays (see supplement for more details). In Fig. 8A, we show that the overexpression of survivin induces a dramatic decrease of activity. Importantly, under the same conditions survivin enhances cell proliferation as shown by the colony size distributions (Fig. 8B), the number of colonies and the fraction of area covered by colonies (see Table S2). The effect of survivin is absent for unsorted IGR37 cells when the level of is low (see Fig. 8B). Moreover, when the percentege of positive cells is below the peak the effect is very small (see Fig. 5). These data provide a further confirmation of the presence of senescent cells in growing tumors. It is illuminating to use the CSC model to simulate the effect of a treatment on the progression of a tumor. We consider two possible strategies: (i) try to stop tumor growth by stimulating cell senescence, and (ii) eradicate the tumor by inducing cell death. Case (i) can be described by decreasing the parameter, representing the number of generation needed for a CC to turn senescent. The responses to treatment is summarized in Fig. 9A showing that the tumor size stops growing for a short while, but eventually the growth resumes at the previous rate. This is due to the action of CSC, whose fractional population increases dramatically in response to the treatment and sustains cancer growth. Our model quantifies the common-sense statement that if the cancer stem cells are the only parties that double forever, then a treatment that does not remove them will be fruitless in the long term. Case (ii) can be described by introducing a parameter, as the probability that a CC cell dies (before senescence). If we assume that CSCs are drug resistant and therefore do not die, we predict that the tumor size would initially shrink, but the growth would starts again after some time (Fig. 9C), due to the persistent growth of the CSC subpopulation (Fig. 9D) Again, the model predicts that the only possible strategy to stop cancer growth is to target CSCs. The present results have powerful implications for our picture of cellular senescence in tumors. The stochastic cancer model suggests that since cancer cells proliferate indefinitely, they must have evaded senescence by some yet unknown biological mechanism. Our results show instead that cancer cells in general become senescent, but this fact is ultimately irrelevant for tumor progression since long-term proliferation eventually resumes. This finding can be explained by the hierarchical cancer model where a subpopulation of CSCs drives tumor growth and the other cancer cells turn senescent after a fixed number of duplications. The peak in cell senescence should correspond precisely to the time at which the initial population of cancer cells turn senescent slowing tumor growth. At this point, CSCs are able to restart the growth process by symmetric duplication leading eventually to the decrease of the fraction of senescent cells as observed in the experiments. It would be hard to reconcile our experimental data with the conventional cancer model hypothizing that a random mutation would lead to massive senescence roughly at the same time for all the different cancer cell populations considered. The fact that CSCs do not become senescent is probably a signature that they originate from normal stem cells and are therefore already immortal. This observation may have implications for therapeutic strategies trying to stop tumor growth by inducing senescence: such an approach is bound to fail unless senescence is induced in CSCs. The data on the ABCG2 sorted population can be explained by the hierarchical cancer model if we assume that ABCG2+ and ABCG2− data are distinguished by a different initial fractions of CSCs. A larger fraction of CSCs in ABCG2+ cells should reduce the occurrence of senescence and increases proliferation with respect to ABCG2− cells. Furthermore, the observation that both cell populations eventually resume their growth at the same rate suggests that some CSCs are also present in ABCG2− cells, whose growth would otherwise stop. This implies that while ABCG2 is able to select a CSC rich population, by itself it is not a clean sorting criterion. This is in agreement with the results of Ref. [16] showing that ABCG2− cells yield a tumor xenograft in immunodeficient mice that is smaller than the one produced by ABCG2+ cells. The fact that ABCG2 is not an absolute CSC marker implies that its expression is reversible after sorting. We have checked this by sorting ABCG2+ and ABCG2− cells after 139 days finding that both express ABCG2 (with percentage 0. 92% and 0. 45%, respectively). The same problems are likely to affect putative CSC markers shown in the literature to be reversibly expressed and hitherto considered incompatible with the CSC model [18]. Similar observations in breast cancer cells have been recently interpreted as a signature of stochastic phenotypic switching, assuming that CC have a small probability to revert to the CSC state [19]. The same data could possibly be interpreted in our framework by assuming that the marker was not perfect. It would be interesting to develop quantitative tools to distinguish between phenotypic switching and imperfect markers. The presence of CSC in melanoma is debated because conflicting data are reported in the literature [14], [17], [18] showing that slightly different assay conditions lead to different CSC fractions, that can sometimes be relatively large [17]. There is in fact no reason to believe that the CSC population must be small. This idea comes from the analogy with tissue stem cells that replicate homeostatically, keeping their population constant either by asymmetric division or stochastically [6], [7], leading to a vanishing concentration of stem cells in the total cell population. CSCs do not replicate homeostatically and therefore their population grows exponentially. Changes in assay conditions can change the duplication rate of CSC, leading for extreme conditions (i. e. for example the use of matrigel, mice permissive conditions etc.) to a relatively large concentration of CSCs, perhaps resolving previous controversies [14], [17], [18]. Human IGR37 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and cultured as previously described [16]. IGR39 was derived from a primary amelanotic cutaneous tumor and IGR37 was derived from an inguinal lymph node metastasis in the same patient. Human primary melanoma WM115 (ATCC CRL 1675) was cultured as in previous work [9]. Cells were analyzed for fluoroscein isothiocyanate (FITC) mouse anti-human ABCG2 (R&D Systems, Minneapolis, MN) expression. All samples were analyzed using one- or two-color flow cytometry with non-specific mouse IgG used (Invitrogen, Carlsbad, CA) as isotype controls. For each flow cytometry evaluation, a minimum of cells were stained and at least 50000 events were collected and analyzed (cells were stained for sorting). Flow cytometry sorting and analysis was performed using a FACSAria flow cytometer (Becton, Dickinson and Company, BD, Mountain View, CA). Data were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA). Expression of pH-dependent senescence associated activity was analysed simultaneously in different passage of cells using the Senescence galactosidase Staining Kit (Sigma) according to the manufacturers protocol. cells were injected subcutaneously into five-week-old NOD-SCID mice (Charles River Laboratories, Boston, MA). Animals were maintained on standard laboratory food ad libitum fed and free access to water. Tumor mass was excised after two months and digested with 0. 2% collagenase type I (Gibco), 0. 2% bovine serum albumin (BSA; SIGMA, St. Louis, MO) for 30 minutes at on an orbital shaker. Cells are maintained in culture for no more than two passages. WM115 cells were grown in standard medium under normal or hypoxic conditions (99. 8% /0. 2%) for up to 48 hours. Total RNA was extracted from WM115 cells or prostate cancer cell lines (PC-3 used as control) [32] under normal and hypoxic conditions. Reverse transcription (RT) -PCR reactions were done as previously described [16]. The oligonucleotide primer sequences and gene-specific PCR amplification programs used are following: sense -CCTATGTAGTTGTGGAAGTTTATGC; antisense -ACTAGGCAATTTTGCTAAGAATG for 40 cycles at for 30 s, for 15 s, and for 30 s. The integrity of each RNA and oligodeoxythymidylic acid-synthesized cDNA sample was confirmed by the amplification of the -actin housekeeping gene. Ten microliter of each RT-PCR sample was run on a 2% agarose gel and visualized by ethidium bromide staining. 200000 cells were plated on 6 multiwell and the day after transfected by lipofectamin with cytoplasmic survivin-GFP plasmid or GFP (both plasmids were a gift of Dr Wheatley, [31]). 24 hours later the GFP-positive cells were counted by flow cytometry. More than 95% of cells were GFP-positive. These cells were immediately used for crystal violet or actosidase assays. Cells were plated on 6 multiwell and stained after 8 days. Briefly, they were fixed with 3. 7% paraformaldeide (PFA) for 5 minutes and then stained for 30 min with 0. 05% crystal violet solution. After two washings with tap water, the plates were drained by inversion for a couple of minutes and then photographed. To quantify cell proliferation, we threshold the images and apply the Hoshen-Kopelman algorithm to identify individual clusters (see Fig. 4 for a visual explanation of the method). We then compute the number of colonies, the area fraction covered by colonies (see Table S2) and the colony size distribution (see Figs. 8B and S5). The last method is the most accurate and complete, avoiding possible experimental artifacts due to local cell detachments. Long-term growth curves are obtained by splitting the cells at each passage and rescaling the result of the counting by an appropriate factor. Cells are counted using a burken chamber in the presence of trypan blue. It is convenient to express the growth curves in terms of cumulative population doublings (CPD), expressing the number of times the populations has doubled. CPD is directly related to the number of cells at time by the expression: (14) where is initial number of cells. In order to fit the data with model, the total number of cells can be expressed as the sums of two terms derived from Eq. 2 and Eq. 3, respectively. In particular, we have (15) and (16) Furthermore, we can also compare the model with the concentration, assuming that it is equal to the fraction of senescent cells. The experimental comparison involves the fit of four curves in terms of the parameters,,, and. Since ABCG2+ and ABCG2− cells only differ by their relative fraction of CSC, we use the same values of,,, for the two subpopulations and vary only the parameter. We thus perform a multiple curve fitting in which the two growth curves and the two curves are fitted simultaneously in terms of six parameters (,,,, and), where is the value of for ABCG2 cells. To this end, we use the pyFitting software (https: //github. com/gdurin/pyFitting) developed by G. Durin. The result is (17) (18) (19) (20) (21) (22) with a reduced of. In Table S3, we report the covariance matrix and the t-statistic. We can express the parameters and in terms of the initial fractions of CSC as for positive and negative cells, where the arbitrary scale is set equal to one in the fits. We obtain (23) (24) Hence the ABCG2+ cells have more CSCs than ABCG2− cells, as expected. We have also checked how much the choice of the initial condition affect the results. For simplicity, we have imposed a uniform distribution of (for) to obtain a closed form solution (Eqs. 2–3). To assess the robustness of our results, we have compared the growth curves obtained under this assumption with those obtained using the same fit parameters and imposed as the steady-state solution of the model with the same parameters but smaller. The resulting curves are very close to each other indicating that at least for this case there is a weak dependence on the initial conditions.

Title: Senescent Cells in Growing Tumors: Population Dynamics and Cancer Stem Cells Summary: It is commonly believed that cell senescence - the loss of replicative capacity of cells - acts as a barrier for tumor growth. Here we follow the evolution of senescence markers in melanoma cells and find that while most cancer cells eventually turn senescent, this is at root irrelevant for the long-term growth rate of a tumor. To demonstrate this, we construct a mathematical population dynamics model incorporating cancer stem cells which is able to reproduce quantitatively the experimental data. Our results support the existence of cancer stem cells in melanoma and explain why it is difficult to fight cancer by inducing senescence in cancer cells. Only a fraction of the cells are susceptible to senescence, but those cells are irrelevant for tumor growth. A successful therapeutic strategy should instead target cancer stem cells, which are, however, likely to be strongly resistant to drug induced senescence.

lay_plos

en

Summarize: BACKGROUND OF THE INVENTION RELATED APPLICATIONS This application is a continuation-in-part of copending patent application Ser. No. 825,672, filed Aug. 18, 1977, now abandoned. FIELD OF THE INVENTION This invention relates generally to the field of packages for matches, and more particularly concerns safety packages adapted to prevent children from inadvertently striking cardboard matches thereon. DESCRIPTION OF THE PRIOR ART The potential disasters engendered when young childern, particularly in the age group of one to four years old, play with matches are well known and feared by parents. Heretofore, however, there has not been developed a package for cardboard matches which is designed to defeat a young child&#39;s efforts to produce a flame by striking a match from a package which he has come across in his interminable searching and wandering. The only pertinent prior art of which applicant is aware is the well-known matchbook comprising a deck of cardboard matches attached to a base along a perforated line, and a cardboard cover is folded so as to provide a back portion, an upturned lip, and front flap. The base of the match deck is interposed between the lip and back portion and secured there with a staple extending through the lip, base and back. The front flap portion is openable and closable by tucking its free end between the lip and the match deck base. In most such matchbooks, a striking strip is provided on the front, presented surface of the lip portion of the cover. The striking strip is comprised of a layer of an appropriately abrasive material affixed to the cardboard cover material, which generally has a non-abrasive, decorative finish. The striking strip is usually in a contrasting color to the remainder of the cover--usually the strip is a very dark brown--and is completely accessable. The only &#34;safety features&#34; provided with such matchbooks are the fact that the cardboard matches contained therein may only be struck on the provided striking strip, and the familiar legend printed on the match cover, &#34;Please close cover before striking&#34;. The accessability and noticeability of the match striking strip, together with the ease with which the cover may be opened and the matches obtained, make such matchbooks hazardous in the hands of inquisitive children. After having seen an adult open the cover, pull out a match and draw the colorful head of the match across the dark, abrasive strip to produce a flame, a curious child will find it relatively easy to emulate those actions when given the opportunity to pick up such a book of matches carelessly left lying about. The potential danger goes without saying. It is an object of the invention herein disclosed to provide means for striking matches which are not easily used by young children, and which require more than casual observance of an adult utilizing the striking means in order for a child to successfully strike a match himself. It is a further object of the invention to combine the striking means with a standard matchbook to produce an improved, safe matchbook, which is also an inexpensive-to-produce article of manufacture. SUMMARY OF THE INVENTION In accordance with the present invention, the abrasive, match-striking strip is provided with a cover flap extending longitudinally thereof. By this arrangement, a two-fold safety factor is accomplished. First, the dark colored, abrasive strip is hidden from the view of a child either observing an adult&#39;s use of the invention, or playing with it himself: he cannot see what it was that the adult struck his match on, and consequently, will have difficulty striking a match himself. Second, the striking strip, being covered, is not easily accessable to a child&#39;s random poking and prodding with an unspent match, and, therefore, accidental striking of a match is much less likely than with the common matchbook presently in use. The only way a match can be struck with the present article of manufacture is by placing the unspent match end in the recess formed between the safety flap and the striking strip--a deliberate action which is likely to be beyond the dexterity of a large number of small children. By constructing the inventive article so that the material used and the transverse dimensions of the match-covering cavity bounded by the walls of the protective &#34;tube&#34; are such that insufficient friction can be generated by simply placing a match head in contact with the striking strip and moving it back and forth, another safety feature is achieved. To strike a match placed in such a cavity, the match head must be pinched between the striking strip and the inside surface of the safety flap with one hand as it is drawn out of the cavity with the other. This pinching action is carried out rapidly and is virtually unnoticeable to a child observing it, thus, not only will a child have trouble emulating the action, the limits of his dexterity and strength will also deter him from efficiently copying the requisite motion. Finally, the &#34;tubular&#34; striking means can be easily and cheaply manufactured simply by extending the upturned lip portion of the presently known matchbooks, and making several easy folds in the extension material to define a safety flap. The matchbook is completed by a staple extending through the lip, match deck base, and back portion of the cover. The vastly improved safety cover herein disclosed can be manufactured with little or no change in the presently used method of manufacture. It will be appreciated that an effective safety package for matches has been disclosed which hinders a child&#39;s observation of the method of striking matches, prevents accidental striking of a match randomly contacted with the package, and which requires that several deliberate steps be taken before a match may be struck, which steps are beyond the ability of most young children to perform due to limits on their strength and dexterity. These and other features of the invention will be discussed in more detail in the following sections. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view in perspective, indicating the proper use of a matchbook constructed in accordance with the present invention; FIG. 2 is a view in perspective of the article of manufacture of the present invention, with the safety flap folded back so as to reveal the striking strip; FIG. 3 is a view in end elevation of a matchbook constructed in accordance with the present invention as it appears with safety flap flattened against striking strip; FIG. 4 is a view similar to that shown in FIG. 3 of the match cover with safety flap spaced apart from striking strip. FIG. 5 is a view in perspective of another embodiment of the invention, showing the safety flap in its unfolded state. FIG. 6 is a view similar to that shown in FIG. 5 with the safety flap partially folded. FIG. 7 is a view similar to that shown in FIGS. 5 and 6 with the safety flap folded and in its normal, flattened position. FIG. 8 is a view in perspective showing the operation of the invention. FIG. 9 is an enlarged sectional view of the invention as seen from the line 9--9 of FIG. 7 showing its normal position in solid lines, and indicating its second, use position in dotted lines. DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to FIG. 2, there is shown an improved, safe matchbook 10, constructed in accordance with the present invention. As in conventionally known matchbooks, matchbook 10 comprises a cardboard cover 12, enclosing a plurality of cardboard matches 14 (shown in FIGS. 3 and 4) which are perforatedly attached to one or two cardboard base members 16. Cover 12 includes a back portion 18, a front flap 20, and an upturned lip portion 22. Affixed or coded, on the front presented surface of lip portion 22 is a match-striking strip 24. Striking strip 24 may be of sandpaper or emery paper, or some other suitable abrasive material which is well-known in the art. As opposed to the rough texture of strip 24, the remaining surfaces of cover 12 are generally smooth, and often polished and printed with decorative material, so that a match may only be struck on strip 24. On a conventional matchbook, lip 22 terminates at the edge indicated by fold line 26, and front cover flap 20 is tucked between lip 22 and the base or bases 16 to secure the flap in a closed position. In the present invention, a safety flap 28 extends from lip 22, as shown in FIG. 2. Safety flap 28 is folded down along fold line 26 and secured to lip portion 22 by means of staple 30 which extends through flap 28, lip 22, base or bases 16 and back 18. By folding flap 28 over and securing it to lip portion 22, a generally tubular structure is achieved, whereby striking strip 24 is situated within a longitudinally extending, match-receiving cavity 32, which is open at both ends to facilitate insertion of a match 34 therein, as indicated in FIG. 1. It will be appreciated that flap 28 could be secured to lip 22 by means other than a staple--glue, for instance, would be appropriate--or that a continuous tubular wall-something on the order of that provided by a soda straw--having a striking portion on the interior wall would be within the scope of my invention. However, the preferred form, especially for the purpose of ease of manufacture, is the flap and staple arrangement shown in the drawings. Cavity 32 extends, in longitudinal dimension, over the length of striking strip 24. In its transverse dimensions, cavity 32 must be such that, when a match is inserted therein, with its head resting against striking strip 24, there will be no appreciable force exerted by the interior wall of flap 28 tending to press match head 36 against strip 24. In general, this means that cavity 32 is slightly larger in transverse dimension than the corresponding dimensions of the matchhead portion 36 of match 34. This dimensioning allows match 34 to be easily inserted into cavity 32; it also ensures that there is not such a tight fit of the match between the interior wall of flap 28 and striking strip 24 that sufficient striking friction can be generated by simply pulling the match out of the match receiving space. Since cardboard matches are not very rigid in their construction, there is also no way to &#34;lever&#34; matchhead 36 against strip 24 by applying a moment around the shaft portion of match 34 while it is inserted in space 32--such a moment would only buckle the shaft of match 34. Consequently, the only way in which sufficient striking friction can be generated is to insert match 34 into cavity 32, and pinch matchhead 36 between flap 28 and strip 24 as it is drawn out of cavity 32. This operation is illustrated in FIG. 1. As there suggested, the requisite striking action requires use of both hands, and a level of dexterity, coordination, and timing, which, although simple for adults to master, is beyond the capabilities of most children in the age group which is the target of the invention. To accomplish the pinching of the matchhead, safety flap 28 is flattenable against lip 22, and strip 24 and illustrated in FIGS. 3 and 4. This is primarily due to the generally non-rigid characteristic of the cardboard used in the construction of cover 12. This &#34;flattenability&#34; could be accomplished by other means with other materials, however--by scoring, hinging, spring-biasing, etc. Referring to FIGS. 5-9 a second embodiment of the present invention is shown. The matchbook 10&#39; of this embodiment includes a cardboard cover 12&#39; enclosing a comb of matches 14&#39;, having a base member 16&#39;. Cover 12&#39; comprises a back cover portion 18&#39; attached to a front cover portion 20&#39; by means of a hinge portion 21&#39;, which not only allows front cover 20&#39; to be opened, but also provides for a sliding motion of front cover 20&#39; between the first and second position shown in FIG. 9. An upturned lip 22&#39; curls around base member 16&#39;, and a staple 30&#39; extends through lip 22&#39;, base 16&#39; and back cover 18&#39; to secure the match comb 14&#39; within cover 12&#39;. Cover 12&#39; further comprises a safety flap portion 40&#39; extending from lip 22&#39; above the staple point. Safety flap 40&#39; is folded along fold lines 42&#39;, 44&#39; and 46&#39; as shown in FIGS. 5, 6 and 7 in a &#34;reverse-S&#34; manner to define panels 48&#39; and 50&#39;, and second upturned lip 52&#39;. Striking strip 24&#39; may be mounted on the interior surface of panel 48&#39;, as shown in FIG. 5, or on the interior surface of panel 50&#39; (not shown). Upturned lip 52&#39; and panel 50&#39; cooperate to define a receiving groove for front cover 20&#39;, as is best shown in FIGS. 7-9. Safety flap 40&#39;, when folded, is normally in the flat position shown in FIG. 7, whereby there is no access to striking strip 24&#39;. A sliding movement of front cover 20&#39;, as shown in FIG. 8 to a second position, bulges panel 50&#39; outwardly to define a generally tubular member, thus giving access to striking strip 24&#39;. This sliding action is best shown in FIGS. 8 and 9. With panel 48&#39; bulged outwardly, a match 34&#39; may be inserted in the longitudinal, flattenable cavity defined by panels 48&#39; and 50&#39;, and struck as previously described. It will be appreciated that the addition of a safety flap over the striking strip of a conventional matchbook--an inexpensive operation requiring no additional parts and very few modifications in the present method of manufacture-- provides several extremely beneficial safety factors. First, the striking surface of the match cover and the entire striking operation are effectively hidden from the casual observance of a young child watching an adult strike a match. Second, the striking strip is covered so that a child who picks up the book cannot accidentally strike a match on an easily accessable, exposed surface of the book. Third, the presence of the open ended tube formed by the safety flap in the lip portion requires that a match be held between thumb and forefinger and inserted longitudinally therein in order for the match head to be brought into contact with the striking strip; this step requires manual dexterity and hand-eye coordination that very young children may not have developed, thus precluding their being able to strike a match on the safety package. Finally, the dimensions of the tube formed by the safety flap--particularly the transversely measured distance between the interior surface of the safety flap and the striking surface are such that a match head once inserted into the tube, must be pinched between the flap and striking strip by exerting a force on the outside surface of the flap with the fingers of one hand, while drawing the match rapidly across the strip and from the tube with the other hand--an easily disguised motion which a child may not learn by casual observance, and a difficult-to-perform step for a young child with limited strength and dexterity. All of their safety features previously described are present in this embodiment of the invention. In addition, a further step of bulging the safety flap by sliding the cover is required, so as to further hamper a child&#39;s attemp to strike a match, and, also, if safety flap 40&#39; should for some reason be torn from the matchbook 10&#39;, the striking strip 24&#39; will also be removed, thus rendering the remaining assembly strike proof.

Summary: An improved package for matches is disclosed, in which the striking surface of the package is protected by a flattenable, generally tubular enclosure. The enclosure hides the striking surface from view, and prevents a match from being struck unless it is pinched between the striking surface and the interior wall of the enclosure as it is drawn across the striking surface. The tubular enclosure may include a front cover receiving recess, and normally be in a flattened position, whereby a sliding motion of the front cover towards the tubular enclosure while in engagement with the receiving recess expands the tubular enclosure allowing access to the striking surface.

big_patent

en

Summarize: M. Spencer Green, AP Sponsored Links WASHINGTON -- The Hispanic population topped 50 million for the first time, new census data shows, as Latinos became the second-largest population group in the United States.The Census Bureau today released its final numbers for the 2010 census, which show how rapid growth of the Asian and Hispanic population dramatically transformed the U.S. into a more ethnically diverse country than it was 10 years ago.The swelling Hispanic population made up more than half the 27.3 million increase in population in the U.S. since 2000. While the nation's population grew by 9.7 percent in the past decade to 308.7 million people, the Hispanic population grew by 43 percent to 50.5 million.In at least nine states, the Hispanic population more than doubled. Large concentrations of Latinos live along the Atlantic seaboard, across the South, from Texas to California, and along the Pacific coast, said Nicholas Jones, a Census Bureau demographer.A Fox News reporter asked what part of the soaring Hispanic numbers can be attributed to Hispanics in the country illegally, but Jones declined to speculate. He said the Census Bureau is still analyzing the figures.The Asian population grew faster than any other race group. About 14.7 million people, or 5 percent, identified themselves as Asian alone.In contrast, the non-Hispanic population grew by about 5 percent, and the white population all but stagnated, with an anemic 1 percent growth, the data shows.The black population now numbers 38.9 million, or 13 percent.Census Bureau Director Robert Groves announced the population center of the United States is still in Missouri -- where it has been for the past four decades. But it has moved 23.4 miles south and west from Edgar Springs, the population center in 2000, reflecting the general southwesterly migration of the population at large. The center is now in Texas County, 2.7 miles northeast of the town of Plato, population 109, Grave said.In the last of the state counts to be released, the data showed New York state grew slightly to 19.4 million since 2000, while New York City's population inched up to 8,175,133.Mayor Michael Bloomberg disputed the count as too low.Groves offered sympathy but said a more intensive dissection of data for New York and other cities where officials think the count is too low will occur later this year."This is the time that mayors receive counts that disappoint," Groves said, adding that Census Bureau demographers were seeing the numbers themselves for the first time today. "We haven't drilled down into them," he said.Washington, D.C., grew by 5.2 percent in the past decade -- the first growth the city has experienced since 1950, when it experienced a 21 percent increase as a result of the boom in government jobs during World War II. The population reached 802,178 that year, and then began a 50-year decline, dropping to 572,059 in 2000. The American landscape shifted dramatically during the past 10 years and the population center of the country moved about 40 miles west from Edgar Springs, Mo., in 2000, to Plato, Mo., population 109. The Census Bureau announced the mean center of population as 2.7 miles northeast of Plato -- an hour's drive south and west of the population center 10 years go. In 2000, the population center of the country rested in Phelps County, Mo., and in 1790, the year Washington, D.C. was named the nation's capital, the mean center of population sat in Kent County, Md. The mean center represents the middle of the nation's population distribution. Over the past decade, the country's population bent further west and became more Hispanic, multi-racial and mobile, with people moving toward the Sun Belt and away from cities to suburbs. The final local data from the 2010 Census released today reveals a significant change in the racial composition of the country. "The minority population is going to make up an even larger share of the nation's growth than it did in the 1990s," said William Frey, a demographer at The Brookings Institution. "People are going to appreciate more why the minority growth is important in the U.S." The expansion in the Hispanic or Latino population crossed the 50 million mark for the first time in 2010, making people of Hispanic origin the second largest group in the country. The total Hispanic population in 2010 was 50,477,594 or 16.3 percent of the total population, a 43 percent increase from a decade ago. In Alabama, Kentucky, Maryland and six other states, the Hispanic population more than doubled, and in the booming Southwest, Hispanics accounted for the greatest spike in growth. In the state of New Mexico, the Hispanic population eclipsed the white population for the first time, rising to make up more than 46 percent of the state's population compared with the 40 percent for whites. Hispanic populations in California and Texas inched closer to becoming their states' majorities. Some states would have lost a significant amount of their population without the boost in minority growth. Massachusetts grew by just a bit more than 198,000 people while its white population fell by 194,000. The 46 percent growth in both the Hispanic and Asian populations boosted its overall population and prevented the state from experiencing a negative growth rate. A large portion of the minority growth is attributed to the rise of Hispanics in the youth population. In Nevada, 61 percent of children are minorities compared with 41 percent of adults. "As a nation, our youth is looking a lot different than the middle age population," Frey said. "The younger part of the population is becoming more diverse than the older part." In addition to the general uptick in minorities across the country, the locations where many Americans choose to live changed, specifically within the black population as more people identifying themselves as black or African American headed south. "The black population is moving to the South in bigger numbers than it did in earlier decades," Frey said. "The South has become a much more prosperous and open place for African Americans." Overall, the Sun Belt experienced some of the greatest population growth in the country. Nevada outpaced the country, expanding by more than 35 percent while Arizona, Utah and Texas followed with growth more than 20 percent. Along with the shift of population to the South has come a shift from cities to suburbs. The suburbs of southern cities such as Atlanta, Dallas and Houston saw record gains in overall populations. More than half of the cities with large black populations experienced declines in their populations because of an exodus to the suburbs. As some cities recorded a loss in population to the suburbs, many cities experienced a racial rebalancing with steadily growing numbers of Hispanics and shifting numbers of whites and blacks. In data set to be released today, demographers expect Washington, D.C. and New York City to increase in the number of whites in their cities for the first time since the 1950s. While many large cities transformed in their racial makeup and size, two stood out with dramatic losses in population. Due in large part to damage inflicted by Hurricane Katrina in 2005, New Orleans' population shrunk by 30 percent over the course of 10 years. In Michigan, Detroit shed nearly 65 people a day for a decade, causing its population to plummet 25 percent because of economic strife and a shift to the suburbs. And then there were the oddballs from the 2010 Census. Sarah Palin's hometown of Wasilla, Alaska, experienced the largest growth in the state, expanding its population size by 43 percent while Maricopa, Arizona, the new hometown of her daughter Bristol and grandson Tripp, increased by a whopping 4,000 percent. The trends of this changing American landscape will come into play during redistricting decisions state legislatures will make in the coming months. Analysts expect new congressional districts to reflect the overall trends revealed by the local Census data, beginning with the rise in Hispanic population. "There's little doubt that the growth in the Latino population is going to eventually result in a more Hispanic representation in legislatures and Congress, as well," said Tim Storey, a redistricting analyst with the National Council of State Legislatures. "But it's going to take time. The maps are being drawn now." Storey argued shifts from cities to suburbs and slow growth in rural areas will also spur new redistricting patterns. "From a national perspective, there will be fewer rural legislators, period, because those areas of the country continue to not grow or lose population," Story said. "There will be a shift of political power to suburban areas, period." Ten states -- Ohio, New York, Illinois, Iowa, Louisiana, Massachusetts, Michigan, Missouri, New Jersey and Pennsylvania -- lost a total of 12 congressional seats to eight states -- Texas, Florida, Arizona, Georgia, Nevada, South Carolina, Utah and Washington -- in the reapportionment process. And with GOP gains from the 2010 election, redistricting might favor Republicans in this Census cycle. "Republicans are in their best position politically for redistricting than they've ever been in, period," Storey said. "Republicans are positioned very well to not be on the short end of the redistricting process."

Summary: The latest Census Bureau numbers mark a milestone for the Hispanic population in the US-the figure exceeds 50 million for the first time. The total (50,477,594 to be exact) is a 43% increase from a decade ago, reports CNN. Related stats: Hispanics, now the nation's second largest population group, make up 16% of the population. Latinos accounted for more than half of the nation's overall growth of 27.3 million. (The new total is 308.7 million.) About 1 in 4 kids under 18 are Hispanic. Whites grew by 2.2 million to 196.8 million, though their share of the overall population shrank from 69% to 64%. Asians grew 43%, from 10.2 million to 14.7 million. That's 5% of the population. African-Americans grew by 4.3 million to 40 million, or about 12.6% of the population. The Hispanic population more than doubled in nine states, including Alabama, Kentucky, and Maryland, notes ABC. In New Mexico, the Hispanic population (46%) exceeds the white population (40%) for the first time. Click here for more on the Hispanic surge or here to see the nation's new population center.

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Summarize: RELATED APPLICATIONS [0001] The application claims the benefit of U.S. Provisional Application No. 60/539,661, filed on Jan. 27, 2004, which is incorporated herein in its entirety. FIELD [0002] The invention relates to tissue generation. More particularly, the invention relates to three-dimensional tissue generation by ex vivo three-dimensional cell culture methods using porous, three-dimensional tissue scaffold-implants. BACKGROUND [0003] The current trend of tissue engineering technology is toward the development of biomaterials for repairing tissue defects or to enhance fixation of implants to the host tissue. Basic requirements include a scaffold-implant conductive to cell attachment and maintenance of cell function, together with a rich source of progenitor cells. Biomaterials in combination with cells from ex-vivo cultures will not only accelerate the tissue healing, but also increase the biocompatibility of scaffold-implants to shorten the hospitalization, and improved long-term function of the devices. In particular, patients with large defects, impaired bone healing and cancer disease in the region of repair shall benefit from this new technology. One regenerative tissue engineering approach involves a process known as “tissue induction”, whereby a two or three-dimensional polymer or mineral scaffold-implant without cells is implanted into a patient. With tissue induction, tissue generation occurs through ingrowth of surrounding tissue into the scaffold-implant. [0004] Another approach to tissue generation, known as “cell transplantation”, involves seeding a scaffold-implant with cells, cytokines, and other growth-related molecules, then culturing and implanting the scaffold-implant into the subject to induce the growth of new tissue. Cultured cells are infused in a biodegradable or non-biodegradable scaffold-implant, which may be placed in a bioreactor in-vitro to allow the cells to proliferate before the cells containing scaffold-implant is implanted in the patient. Alternatively, the cell-seeded scaffold-implant may be directly implanted, in which case the patient&#39;s body acts as an in-vivo bioreactor. Once implanted, in-vivo cellular proliferation and, in the case of absorbable scaffold-implants, concomitant bio-absorption of the scaffold-implant, proceeds. [0005] In both types of tissue engineering, i.e., tissue induction and cell transplantation, the scaffold-implant, whether or not bio-absorbable, must be biocompatible, such that it does not invoke an adverse immune response from, or result in toxicity to, the patient. [0006] Several types of materials have been investigated for use as seeding scaffold-implants, including metals, ceramics, polymers, and polymer-coated metals and ceramics. Existing scaffold-implants may be manufactured by solvent casting, shaping sections with machining, 3D printing, or molded collagen/cell constructs. While the aforementioned scaffold-implant materials are primarily for industrial applications, the fabrication of hydroxyapatite scaffold-implants using selective laser sintering and polymer-coated calcium phosphate powder, have been investigated. Additional post-processing, such as high temperature heating which burns out the binder, and then higher temperature sintering which fuses the powder together, is required to strengthen the scaffold-implant. [0007] Whichever type of scaffold-implant is selected, a purpose of the scaffold-implant is to support cells, which, after being seeded into the device, cling to the interstices of the scaffold-implant, replicate, produce their own extra-cellular matrices, and organize into the target tissue. For example, in the case of bone regeneration, the optimal pore size for maximum tissue growth ranges from 200-400 mircons (μm). Therefore, the material or materials used for fabricating the scaffold-implant should have this pore size (200-400 μm) and have sufficient rigidity and biomechanical properties to support loads that are used for generating bone tissue. [0008] Scaffold-implants fabricated from a material such as hydroxyapatite, which is useful for supporting bone cells, are too brittle and non-pliable to act as scaffolding for muscle or tendons. Many of the polymers, and the polymer-coated metals and ceramics present a challenge to seeding cells in three-dimensional scaffolds. None of the known scaffold-implant materials allow growth of cells to a depth of greater than about 250 μm, which is a generally accepted practical limit on the depth to which cells and nutrients can diffuse into scaffold-implants having the desired porosities. Even if cells could be made to diffuse to greater depths, it is generally believed that to support cell growth and avoid or at least curtail apoptosis at these depths, the scaffold-implant must also support some form of vasculature to promote angiogenesis; none of the earlier mentioned scaffold-implant fabrication methods, however, allow for incorporation of blood vessels. [0009] Porous, three-dimensional metallic structures have recently been developed for potential application in reconstructive orthopaedics and other surgical disciplines. Such structures are described in U.S. Pat. No. 5,282,861 entitled “Open Cell Tantalum Structures For Cancellous Bone Implants And Cell And Tissue Receptors” issued to Kaplan; U.S. Pat. No. 5,443,515 entitled “Vertebral Body Prosthetic Implant With Slidably Positionable Stabilizing Member” issued to Cohen et al.; U.S. Pat. No. 5,755,809 entitled “Femoral Head Core Channel Filling Prothesis” issued to Cohen et al.; U.S. Pat. No. 6,063,442 entitled “Bonding Of Porous Materials To Other Materials Utilizing Chemical Vapor Deposition” issued to Cohen et al.; and U.S. Pat. No. 6,087,553 entitled “Implantable Metallic Open-Celled Lattice/Polyethylene Composite Material And Devices” issued to Cohen et al., the disclosures of which are incorporated herein by reference. The porous, three-dimensional metallic structure is a bio-compatible material having a three-dimensional network of continuously interconnected channels or pores which define a three-dimensional porosity, i.e., volume porosity, ranging from 50 to 90% (higher than all other known implant materials). This high bulk volume porosity readily facilitates nutrient diffusion and media circulation. [0010] The porous, three-dimensional metallic structures may be fabricated using a vapor deposition/infiltration process wherein tantalum, which has a long history of medical uses, or other bio-compatible metal or material is vaporized at high temperature and precipitated as a thin layer onto a carbon lattice. The coating of tantalum or other metal enhances or improves the strength or mechanical characteristics of the carbon lattice. [0011] As a scaffold for “bone induction,” preliminary animal studies with transcortical (bone conduction) porous, three-dimensional metallic structures have been shown to support rapid and extensive bone ingrowth. For example, tissue response to porous tantalum acetabular cups indicates that the porous tantalum material is effective for biologic fixation. The biomechanical property of the porous tantalum biomaterial is sufficient to withstand physiological load for specific applications, such as an acetabular cup, a spinal fusion, and a vertebral body replacement in fractures or in metastatic cancer disease. As a scaffold for “cell transplantation,” porous, three-dimensional metallic structures can extend culturing of multipotent hematopoietic progenitors without cytokine augmentation and enhance maintenance and retroviral transduction of primitive hematopoietic progenitor cells. [0012] Hyaluronan possesses biochemical and physical properties suitable to perform an important role in the early events of osteogenesis as well as in many other tissues. A low-molecular weight hyaluronan fully expresses the in-vitro steogenic potential of mesenchymal cells through the subsequent proliferation and differentiation of osteoprogenitor cells using proper conditions. Locally applied high-molecular hyaluronan of MW 1900 kDa also has been shown to be capable of accelerating new bone formation through mesenchymal cell differentiation in femur wounds. Hyaluronan at a low concentration (0.5 mg/mL) has been shown to increase the development of porcine embryos in culture. SUMMARY [0013] One aspect is a three-dimensional tissue scaffold-implant for supporting tissue (e.g., bone) on-growth. The tissue scaffold-implant comprises a lattice having a matrix of interconnected pores which form surfaces in three dimensions; and an inert, biocompatible material covering the surfaces. The surfaces of the scaffold-implant define a high surface area relative to a volume of the scaffold-implant. [0014] Another aspect is a method of forming tissue (e.g., bone). The method comprises providing a three-dimensional tissue scaffold comprising a lattice having a matrix of interconnected pores which form surfaces in three dimensions, and an inert, biocompatible material covering the surfaces; covering the biocompatible material covered surfaces of the scaffold with cells; and culturing the scaffold to grow tissue on and in the scaffold. [0015] Yet another aspect is a method of making an implant for supporting tissue (e.g., bone) on-growth. This method comprises providing a three-dimensional tissue scaffold comprising a lattice having a matrix of interconnected pores which form surfaces in three dimensions, and an inert, biocompatible material covering the surfaces; and covering the biocompatible material covered surfaces of the scaffold with cells. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 is a flow chart depicting an embodiment of the method of the present invention. [0017] FIG. 2 is a bar graph quantifying stem cells binding to a coralline hydroxyapatite disc and uncoated and coated porous tantalum discs, after a 24-hour incubation at 37° C. and normalized to the uncoated TA disc. [0018] FIGS. 3A and 3B are Hoechst stained fluorescent micrographs at 50× original magnification and 20× original magnification respectively, showing the growing stem cells in the pores of porous tantalum after 7 days of incubation. [0019] FIGS. 4A and 4B are histological micrographs at 20× original magnification and 6.25× original magnification respectively, after 8 weeks of implantation in pigs. [0020] FIG. 5 is a scanning electronic micrograph of the three-dimensional tissue scaffold-implant. [0021] FIG. 6 is a histological micrograph of the three-dimensional tissue scaffold-implant after 12 weeks of implantation in a pig. DETAILED DESCRIPTION [0022] The tissue formation method of the present invention utilizes an ex vivo cell culture system and a porous, three-dimensional metallic structure or tissue scaffold of a desired shape and size, which will be implanted into the body of an animal or human being (hereinafter scaffold-implant). The cell culture system induces early stage cell proliferation and differentiation on and in the tissue scaffold-implant, resulting in tissue generation. The tissue formation method of the present invention is especially useful for generating bone tissue. The method of the invention may also be used to generate connective tissue and hematopoietic tissue. [0023] The flow chart of FIG. 1 depicts an embodiment of the tissue formation method of the present invention. In step 10 of the method, a porous, three-dimensional tissue scaffold-implant is fabricated in a desired shape and size, e.g., hip implant, spinal implant, knee implant, etc. For example, the scaffold-implant may be shaped and sized as a prosthetic acetabular cup such as the one disclosed in U.S. Pat. No. 5,443,519 entitled “Prosthetic Ellipsoidal Acetabular Cup,” issued to Averill et al. In another example, the scaffold-implant may be shaped and sized as a prosthetic femoral component such as the one disclosed in U.S. Pat. No. 5,702,487 entitled “Prosethetic Device” issued to Averill et al. [0024] In one embodiment, the scaffold-implant may be fabricated as a single unitary member. In an alternative embodiment, the scaffold-implant may be fabricated as a single, integral member formed by two or more separately fabricated sections which are mechanically assembled together in a conventional manner. In still another embodiment, the scaffold-implant may be fabricated as an assembly of two or more cooperating, unitary and/or integral members (e.g., acetabular cup and femoral stem/ball assembly). [0025] The porous, three-dimensional tissue scaffold-implant may comprise a carbon lattice having a strut or ligament skeleton which forms a three-dimensional network of continuously interconnected channels or pores each roughly approximating a dodecahedron, which create a series of continuous microniches and form surfaces of the lattice in three dimensions; and a thin film of an inert, bio-compatible metal or other bio-compatible material, which covers the surfaces. [0026] The carbon lattice may be formed as a single, unitary member of a desired shape and size, or in sections of desired shapes and sizes to be mechanically assembled. The carbon lattice is substantially rigid, therefore, it may be machined into a bone regeneration tool of a desired shape and size using conventional machining methods. [0027] The inert, bio-compatible metal or other bio-compatible material may be applied to the surfaces of the carbon lattice using conventional vapor depositing and infiltrating methods. In a preferred embodiment, the inert, biocompatible metal comprises tantalum. In other embodiments, the inert, biocompatible metal may comprise niobium or alloys of tantalum and niobium. [0028] The completed porous, three-dimensional tissue scaffold-implant forms a three-dimensional network of continuously interconnected, channels or pores which define a three-dimensional porosity (volume porosity). In one embodiment, the tissue scaffold implant may comprise channels or pores having an average diameter of 400 to 500 μm and a volume porosity ranging from about 50 to about 90%. The geometry of the interconnected pores and surface texturing arising from the metal vapor deposition process produce high surface area-to-volume ratio. The large pores and surfaces allow attachment of proteins, peptides and differentiated and undifferentiated cells. After fabrication, the scaffold-implant may be coated with substrate molecules such as fibronectin and collagens which aid in the attachment of the proteins, peptides and differentiated and undifferentiated cells. [0029] In step 20, a hyaluronan (also referred to as hyaluronic acid or sodium hyaluronate) or a hyaluronan, dexamethasone, one or more growth factors and/or osteogenic genes is (are) applied to the surfaces of the tissue scaffold-implant to stimulate early cell proliferation and differentiation, therefore accelerating tissue generation. Sodium hyaluronate is a natural high-viscosity anionic mucopolysaccharide with alternating beta (1-3) glucuronide and beta (1-4) glucosaminidic bonds. It is commonly found in the umbilical cord, in vitreous humor, in synovial fluid, in pathologic joints, in group A and C hemolytic streptococci, and in Wharton&#39;s jelly. Dexamethasone is a synthetic steroid compound. In one embodiment, the tissue scaffold-implant may be treated with a low concentration (4 mg/mL) of sodium hyaluronate to induce in-vitro, early stage stem cell proliferation and differentiation on and in the tissue scaffold-implant (after performing steps 30 and 40 to be described further on). In another embodiment, the tissue scaffold-implant may be treated with a high concentration (10-20 mg/mL) of sodium hyaluronate which forms a hydro gel with the stem cells in the tissue scaffold-implant intraoperatively. [0030] In step 30 of the method, a cell transplantation process is performed on the porous, three-dimensional tissue scaffold-implant. In an embodiment of the cell transplantation process, the tissue scaffold-implant is seeded with living cells, which may comprise differentiated, undifferentiated or gene transfected cells. Examples of differentiated or undifferentiated cells include without limitation bone marrow cells, osteoblasts, mesenchymal stem cells, embryonic stem cells, endothelial cells. In another embodiment of the cell transplantation process, the tissue scaffold-implant is seeded with living cells and proteins, peptides, transcript factors, osteogenic genes, cytokines, therapeutic agents, and growth factors. [0031] The living cells and other factors can be entrapped and delivered in the tissue scaffold-implant by means of a versatile self-assembly method. In this self-assembly method cellular matrix fibrils are formed with methylated collagen (type I) and hyaluronic acid, or chitosan, which entrap and deliver living cells and other factors. The cellular matrix fibrils are then combined with an outer-layer membrane comprising a polymer such as alginate, hydroxylethyl methacrylate (HEMA), or a terpolymer of hydroxylethyl methacrylate (HEMA), methy methacrylate (MMA) and methylacric acid (MAA) by complex sandwich conjugation achieved, for example, using a complex coacervation process, to protect transplanted allogeneic cells from immune attacks and to sustain release of the stimulating factors and therapeutic agents. In one embodiment, the membrane may be several micrometers to about 100 micrometers thick. The thickness of the membrane may be adjusted by controlling the concentrations and contact time of polyelectrolytes in the complex sandwich conjugation process. [0032] The surface features (the texture on the surface of the metal resulting from the CVD of the metal) and the open, highly interconnected pores of the tissue scaffold-implant readily facilitate nutrient diffusion and media circulation and thus will operate as conduits for cell infusion, adhesion, mass transfer, or to stimulate angiogenesis for blood flow. [0033] In an alternate embodiment, the application of the hyaluronan or the hyaluronan, dexamethasone, one or more growth factors and/or osteogenic genes, to the surfaces of the tissue scaffold-implant (step 20 ) may be performed during the cell transplantation process of step 30. [0034] In step 40, after seeding, the scaffold-implant is cultured in a bioreactor to generate the desired tissue. In one embodiment, the culturing step is an ex-vivo process. Ex-vivo culturing may be performed in a broth medium, e.g., Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) available from HyClone, plus 10% fetal calf serum, which is placed in an incubator e.g., perfusion or spiner flask bioreactor or a rotating bioreactor. The broth medium and incubator operate as an in-vitro bioreactor. In one embodiment, the incubator may provide a humidified atmosphere of 95% air and 5% CO 2 at 37° C. In addition, the incubator may be of the type which provides static, dynamic medium flow, pulsatile air flow, microgravity and multidirectional gravity culturing conditions. The scaffold-implant may then be implanted (in-vivo) into an animal or patient&#39;s body. [0035] In another embodiment, the culturing step is an in-vivo process. In-vivo culturing may be performed in an animal or a patient by directly implanting the scaffold-implant in the animal or the patient. In this embodiment, the animal or the patient&#39; body operates as an in-vivo bioreactor. [0036] In still an alternate embodiment, the culturing step can be performed intraoperatively. In this embodiment, cells are taken from the animal or patient and applied to the scaffold-implant. The scaffold-implant is then implanted in the animal or patient. [0037] FIG. 2 is a bar graph quantifying stem cells binding to 1) a coralline hydroxyapatite (HA) disc; 2) an uncoated porous, tantalum-based, three-dimensional tissue scaffold-implant (TA) configured as a disc; 3) a TA disc coated with gelatin; 4) a TA disc coated with type I collagen; and 5) a TA disc coated with fibronectin, n=9 (repeated test), after a 24-hour incubation at 37° C. and normalized to the uncoated TA disc. In the graph, the stem cells are 3 H-thymidine labeling cells. [0038] FIGS. 3A and 3B are fluorescent micrographs showing the growing cells in the pores of a porous, tantalum-based, three-dimensional tissue scaffold-implant configured as a disc after 7 days of incubation (Hoechst staining). As can be seen in FIG. 3A, at day 7, porcine bone marrow stem cells depicted funicular proliferations of spindle cells on the pore surface and within the pores. As shown in FIG. 3B, growing stem cells in the pores mainly distributed on the surface areas of disc (superior) where the cells were loaded on. Only a few stem cells had grown into the central pores and down to other surface areas of the disc (inferior) where the disc was seated on a well. [0039] FIGS. 4A and 4B are histological micrographs which show, after 8 weeks of implantation in pigs, ectopic bone formation after autologous bone marrow stem cells cultured with a tantalum-based, three-dimensional tissue scaffold-implant for 7 days of incubation. Basic fuchsin and light green staining revealed the bone is green G and fibrous tissue is red R. The black structure B is porous tantalum strut. Specifically, FIG. 4A shows bone forming in the pore surface and the pores and FIG. 4B shows a layer of de novo bone formation at the surface area of the scaffold-implant. [0040] FIG. 5 is a scanning electronic micrograph of the three-dimensional tissue scaffold-implant. As can be seen, the scaffold-implant has a volume porosity of about 50% to about 90% with interconnecting pores, allowing approximately 2-3 times greater bone ingrowth compared to conventional porous coatings. [0041] FIG. 6 is a histological micrograph of the three-dimensional tissue scaffold-implant after 12 weeks of implantation in a pig. As can be observed, there is bone formation from intraoperative conjugation of autologous bone marrow stem cells and hyaluronic acid gel in the tissue scaffold-implant. Basic fuchsin and light green staining revealed the bone is green G and fibrous tissue is red R. The black structure B is porous tantalum strut. [0042] While the foregoing invention has been described with reference to the above, various modifications and changes can be made without departing from the spirit of the invention. Accordingly, all such modifications and changes are considered to be within the scope of the appended claims.

Summary: Adult autologous stem cells cultured on a porous, three-dimensional tissue scaffold-implant for bone regeneration by the use of a hyaluronan and/or dexamethasone to accelerate bone healing alone or in combination with recombinant growth factors or transfected osteogenic genes. The scaffold-implant may be machined into a custom-shaped three-dimensional cell culture system for support of cell growth, reservoir for peptides, recombinant growth factors, cytokines and antineoplastic drugs in the presence of a hyaluronan and/or dexamethasone alone or in combination with growth factors or transfected osteogenic genes, to be assembled ex vivo in a tissue incubator for implantation into bone tissue.

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Write a title and summarize: There is a growing interest in predicting the social and ecological contexts that favor the evolution of maternal effects. Most predictions focus, however, on maternal effects that affect only a single character, whereas the evolution of maternal effects is poorly understood in the presence of suites of interacting traits. To overcome this, we simulate the evolution of multivariate maternal effects (captured by the matrix M) in a fluctuating environment. We find that the rate of environmental fluctuations has a substantial effect on the properties of M: in slowly changing environments, offspring are selected to have a multivariate phenotype roughly similar to the maternal phenotype, so that M is characterized by positive dominant eigenvalues; by contrast, rapidly changing environments favor Ms with dominant eigenvalues that are negative, as offspring favor a phenotype which substantially differs from the maternal phenotype. Moreover, when fluctuating selection on one maternal character is temporally delayed relative to selection on other traits, we find a striking pattern of cross-trait maternal effects in which maternal characters influence not only the same character in offspring, but also other offspring characters. Additionally, when selection on one character contains more stochastic noise relative to selection on other traits, large cross-trait maternal effects evolve from those maternal traits that experience the smallest amounts of noise. The presence of these cross-trait maternal effects shows that individual maternal effects cannot be studied in isolation, and that their study in a multivariate context may provide important insights about the nature of past selection. Our results call for more studies that measure multivariate maternal effects in wild populations. Since selection often varies both over space and time [1]–[3], evolutionary mechanisms that increase adaptation to changing environments are considered to be highly advantageous [4], [5]. Conventional studies focus on mechanisms such as bet-hedging [6]–[8] and, in particular, within-generational phenotypic plasticity [5], [9]–[11] as major adaptations to changing environments. However, a growing number of recent studies suggest that nongenetic effects provide an additional way of adaptation to changing environments [12]–[14]. Here, nongenetic effects refer to any effect on the offspring phenotype that is brought about by the transmission of factors (other than sequences of DNA) from parents or more remote ancestors to the offspring [13], [15]. Nongenetic effects can be realized through a variety of mechanisms, such as social learning [16], the transmission of DNA methylation variants [17] or the transmission of maternal factors such as antibodies or hormones [18], [19]. Importantly, when nongenetic effects are present in a population, an individual' s phenotype becomes a function of the phenotypes (or the environment) of its parents or previous ancestors, giving rise to a form of transgenerational plasticity that is suggested to allow for increased flexibility when coping with environmental change [20]–[22]. Theoretical studies indeed predict that nongenetic effects are selectively favored in fluctuating environments [23]–[25], particularly when the parental phenotype provides information about selective conditions encountered by future generations [23], [24]. The role of parental information has been particularly well studied in the context of maternal effects, whereby the maternal phenotype or environment affects the offspring' s phenotype through the provisioning of resources, antibodies or hormones [26]–[29]. However, most studies on maternal effects focus mainly on univariate scenarios, in which a single maternal factor influences a single offspring character. By contrast, studies in plants and animals suggest that maternal effects typically have a multivariate nature, involving suites of interacting parental and offspring characters (e. g., [30]–[33]). Indeed, this multivariate nature has long been appreciated by those models that assess the consequences of (non-evolving) nongenetic effects to phenotypic evolution [34]–[38]. As yet, however, no theoretical predictions exist about the evolution of these multivariate maternal effects themselves. We believe that taking a multivariate view on the evolution of maternal effects is insightful for at least two different reasons. First, as stated before, the main prediction of univariate models is that maternal effects evolve when the parental phenotype correlates with selective conditions encountered by offspring [23], [24], [29], [39]. It is currently unclear how these predictions play out when offspring are not influenced by a single, but multiple components of a parental phenotype, raising the question how offspring should weigh information that results from the presence of multiple maternal effects (e. g., the presence of multiple maternal hormones and immonoglobulins in avian eggs [33], [40], [41]). A second reason for considering the evolution of nongenetic effects in a multivariate context is the finding [42] that the multivariate configuration of all maternal effects (here assumed to be captured in the matrix M, [34], [36], [43]) plays an analogous role in determining the course of evolution as the genetic variance-covariance matrix G that describes the scope for correlated selection between traits [44]–[46]. The role of the G-matrix in multivariate evolution has been the focus of a vast body of research for the last 40 years, and a substantial set of predictions exists on the ecological and social contexts that give rise to specific configurations of G (e. g., [47]–[49]) and ensuing evolutionary constraints [46], [50]–[54]. By contrast, there are yet no studies which investigate the selective conditions that lead to different configurations of M, which may similarly constrain phenotypic evolution [25], [34], [42]. As a first step towards a more inclusive theory that aims to incorporate both nongenetic and genetic constraints on evolution, the current study therefore aims to investigate which structures of M are likely to evolve across different ecological contexts. To this end, we develop a formal model to make predictions about the evolution of multiple maternal effects in a periodically fluctuating environment. In the current model, a maternal effect reflects any causal influences of the maternal phenotype on the offspring' s phenotype [18], [55]. Prominent examples of maternal effects are the modulation of offspring phenotypes by the maternal adjustment of a variety of egg hormones [41], [56] or the transmission of maternal antibodies to the embryo [57], [58]. In the current study, we focus on the evolution of multivariate maternal effects in the context of fluctuating selection. Specifically, we are interested in scenarios where the nature of selective fluctuations diverges between different maternal characters. For example, different maternal traits endure selection at different timepoints, because some maternal traits endure selection at different seasons than others, or because some selection on some characters may be more predictable (i. e., less stochastic noise) than selection on other characters. The current study thereby investigates whether such contexts lead to the interaction among different maternal characters, which cannot be inferred from studying the evolution of single traits in isolation. We study the evolution of multivariate maternal effects in a fluctuating environment using individual-based evolutionary simulations. Although analytical approaches would be preferred to provide a general evolutionary model of multivariate phenotypes, an analytical assessment of multivariate evolution quickly becomes prohibitively difficult, even when only the evolution of the genetic variance-covariance matrix G is considered (while maternal effects are absent). It is thus no surprise that individual-based simulations have been the method of choice when considering more complicated scenarios of multivariate evolution (e. g., [49], [59]–[61]). Moreover, maternal effects involve the additional complexity that phenotypic evolution depends on past generations, so that an analytical description requires a number of strong equilibrium assumptions (such as a constant covariance between genotypes and phenotypes, [34], [43]). Here, however, we use the flexibility of individual-based, evolutionary simulations to study the evolution of maternal effects in a dynamical fashion without these limiting assumptions. To assess the evolution of multiple maternal effects, we study the simplest possible case, in which an individual that breeds in generation t expresses two phenotypic traits, (where T denotes transposition). Each individual bears six unlinked, diploid loci: two of which correspond to the breeding values of both phenotypic characters, that determine the baseline elevation of both phenotypic traits. The remaining four loci code for the entries of the 2× maternal effect matrix, of which the entry reflects the maternal influence from maternal phenotype j to offspring phenotype i (see eq. [1] below). For all six loci, the two alleles at each locus interact additively (i. e., dominance effects are absent) and inheritance is biparental. During each generation, each allele mutates with probability, upon which a value drawn from a normal distribution with mean 0 and variance is added to the current allelic value (i. e., a continuum of alleles model [62]). Each individual simulation run started from the initial values and continued for generations. Throughout, we found that values of and attained stable values after approximately a tenth of this total timespan (e. g., see Figure S4). Extending seminal quantitative genetics models that focus on non-evolving multivariate maternal effects [34], [35], [43], [63]–[65], the phenotype of an individual in generation t is then given by the following recursion: (1) Here, is the breeding value of trait, while ε (t) reflects the contribution of environmental noise, which is normally distributed with mean 0 and variance as in classical quantitative genetics models [44], [66]. The starred value denotes the value of the maternal phenotype after selection. As noted before, is the evolving maternal effect coefficient, describing how maternal character influences offspring phenotype i. Note that we assume that the maternal effect loci are controlled by the offspring, representing a scenario in which entry of M (t) reflects the offspring' s sensitivity to maternal trait zj [67], [68]. For example, the vector z (t) may reflect titers of different offspring hormones, and the entry specifies how the maternal titer of hormone j (e. g., measured during pregnancy or added to the egg) affects the titer of offspring hormone i (e. g., mediated by the number of hormone binding sites present in the offspring endocrine cells). Indeed, studies indicate that maternal hormone and protein titers affect offspring hormone concentrations, often in a multivariate fashion (e. g., [33], [56], [69]). While multivariate maternal effects M (t) are assumed to be genetically expressed by the offspring, they still give rise to nongenetic effects on the multivariate phenotype z (t). This is because the offspring' s phenotype is not merely influenced by M (t) itself, but the product of maternal effects and the parental phenotype. Consequently, the involvement of the maternal phenotype gives rise to the well-known ‘cascading nature’ of maternal effects [34], [35], [70], whereby the maternal phenotype is itself a function of the multivariate grandmaternal phenotype, which in turn is a function of the phenotypes of previous ancestors. As the offspring' s phenotype z (t) is thus not only a function of DNA sequences it received from its ancestors (i. e., genes coding for a (t) and M (t) ), but also of the phenotypes of its ancestors, M (t) gives rise to nongenetic effects on the offspring phenotype (see also [38]). After birth and phenotype determination (as in eq. 1), each newborn enters a survival stage during which it endures survival selection. The survival probability ws decreases nonlinearly with a displacement of the character away from the selective optimum, according to the Gaussian function (2) where all individuals are assumed to have a baseline survival probability of c, while the strength of stabilizing selection on both phenotypes is proportional to the remainder. α2 measures the width of the selection function and is therefore inversely proportional to the strength of selection. Throughout, we assume that. After survival selection, the surviving individuals reproduce: each surviving individual produces a number of n = 10 ova, which are all fertilized by a randomly chosen other survivor that acts as a sperm donor. From the zygotes, new individuals are sampled with replacement to maintain a population of constant size N. Subsequently, the cycle starts anew with mutation and phenotype determination, as depicted in Figure 1A. So far, we have assumed that fluctuations in both selective optima and are identical, whereas different forms of fluctuating selection may act on each maternal trait, dependent on the ecological context that each trait experiences. Figure 4A shows, for example, how delays between the selective optima of both phenotypes impact on the evolution of multivariate maternal effects matrix M. First, the presence of a delay of φ timesteps between the two selective optima leads to a collapse from the two alternatively stable outcomes observed in Figure 3 to a single evolutionary outcome, unless both optima fluctuate in an exactly opposing fashion (depicted by the large error bars when). Second, Figure 4A shows that both cross-trait maternal effects (m12 or m21, which reflect maternal influences from one maternal trait to a different offspring character) evolve to opposing positive and negative values, dependent on the delay φ between both optima. The maternal effect m21 is positive (and m12 negative) when fluctuating selection on trait z1 is advanced relative to selection on z2 (grey region in Figure 4A), whereas m21 is negative (and m12 positive) when fluctuating selection on trait z2 is advanced relative to selection on z1. Insights on the adaptive significance of cross-trait maternal effects can be derived from the cross-correlations of selective conditions between different traits in parents and offspring, which reflect to what extent one maternal phenotype is able to predict future selection on a different offspring phenotype (Figure 4B). Focusing on the grey region in Figure 4B where the selective optimum is advanced relative to, we find that. In other words, selection on maternal phenotype is overall positively associated to selection on offspring phenotype (or when it attains negative values these are always smaller in magnitude than the other cross-correlation). Since the evolving maternal effect m21 reflects the influence of maternal trait to offspring trait, it evolves to positive values so that matches its future environment. By contrast, selection on the maternal phenotype is overall negatively associated to selection on offspring phenotype (or when it attains positive values these are always smaller in magnitude than the other cross-correlation). Hence, the maternal effect m12 evolves to negative values instead. The reverse scenario applies for the white region in Figure 3B, where selective optimum is advanced relative to. Indeed, we find that m12 then evolves to positive values, while m21 evolves to negative values. In other words, those maternal traits enduring fluctuating selection that is advanced relative to selection on other maternal traits are more likely to develop positive cross-trait maternal effects, whereas delayed fluctuating selection on maternal traits is more likely to lead to negative cross-trait maternal effects. Lastly, we investigated whether such cross-trait maternal effects also evolve in multivariate stochastic environments. As cross-correlations are easily measurable in time series analyses [76], we chose to vary the cross-correlation directly in Figure S3 (although results are similar when adjusting the time-lag between both optima). In a stochastic environment without any cross-correlation between both selective optima, we find that cross-trait maternal effects mij generally do not evolve, while the within-trait maternal effects mii track the autocorrelation of its associated selective optimum θi (Figure S3D, E). By contrast, when selective cross-correlations are nonzero, cross-trait maternal effects evolve to substantial values (Figure S5). For example, when varying the cross-correlation from −1 to +1, we find that the associated cross-trait maternal effect m12 evolves from strongly negative to strongly positive (Figure S5A). Instead, when varying the cross-correlation, we find a similar pattern for the other cross-trait maternal effect m21. Similar to the periodic environment in Figure 4, we find that cross-trait maternal effects evolve in concordance to the prevailing cross-correlations between both fluctuating selective optima. Next, we assess the combined influence of environmental stochasticity and periodicity, by considering a scenario in which selection on trait z1 fluctuates only periodically whereas selection on trait z2 fluctuates both stochastically and periodically (e. g. see Figure 1). Such a scenario reflects, for example, a case in which some traits (e. g., z1) are associated with more predictable selective conditions than other traits (e. g., z2). In Figure 5A we vary the degree of stochastic noise d in selection acting on character z2. Increasing levels of noise lead to the evolution of substantial cross-trait maternal effects m21 from maternal character to offspring character. By contrast, the value of the other cross trait maternal effect m12 (from maternal character to offspring character) evolves towards zero. Both the same-trait maternal effects m11 and m22 evolve towards positive and negative values respectively, corresponding to a positive dominant eigenvalue that is expected when the frequency of noise-free periodic fluctuations is relatively low (e. g., in Figure 5). In order to explain the marked evolution of the cross-trait maternal effect m21, note that selection on maternal trait is characterized by white noise, while noise is absent on maternal trait. Consequently, maternal trait is a less reliable source of information about future selective conditions experienced by offspring. As a result, offspring are selected to obtain their information about periodic fluctuations from the other maternal trait, thereby evolving large, positive values of the cross trait maternal effect m21 while its counterpart m12 evolves towards a value of 0. Hence, asymmetries in information content between both maternal characters favor the evolution of cross-trait maternal effects from the most reliable maternal character, phenotype. When the degree of noise in θ2 increases to ever larger levels, we find that m21 attains either positive or negative values of a large absolute magnitude, (Figure 5A). In line with analytical models that show that values of are associated with very large phenotypic variances [34], we find indeed that results in a large phenotypic variance of trait z2,, while the variance of the other trait is generally small (Figure 5B). Large phenotypic variances are selectively favored in unpredictable environments, since a large diversity of phenotypes among siblings warrants the survival of at least a subset of them in the event of environmental change (bet-hedging: [6], [8], [77], [78]). Given that maternal effects may thus drive the evolution of large phenotypic variances, large values of relative to can only be realized by evolving a large cross-trait maternal effect m21. By contrast, evolving a large within-trait maternal effect m22 would also generate large values of, but also gives rise to highly detrimental carry-over effects in which extreme values of the maternal phenotype will lead to values of an even larger magnitude in the next generation. Such detrimental carry-over effects are absent for the cross-trait maternal effect m21, since it has the more constant maternal phenotype as its input. Hence, cross-trait maternal effects may facilitate the evolution of large phenotypic variances without giving rise to deleterious carry-over effects that are associated with large absolute values of within-trait maternal effects mii. Maternal effects give rise to an interaction between parental and offspring phenotypes that is considered to be an important adaptation to changing environments [19], [22], [25], [79]. Yet, few formal models predict how selection shapes the strength and sign of maternal effects in different ecological contexts (see [80] for similar remarks in the context of social interactions). The current study is therefore the first to show that different forms of environmental fluctuations can have a profound impact on the evolution of multivariate maternal effects. When fluctuating selection acts in an identical fashion on all maternal traits, we find that the shape of the maternal effects matrix M (measured by its eigenvalues) evolves to be closely aligned with the degree of autocorrelation between parental and offspring phenotypes. Hence, we expect that slow fluctuations (leading to a positive autocorrelation) select for positive real parts of, while rapid fluctuations select for negative real parts of. This finding corroborates a number of univariate models which showed that the rate of environmental fluctuations corresponds to the degree with which offspring should copy or diverge from their parent' s phenotype in the case of discrete phenotypic variation [23], [39], [81]. Interestingly, our multivariate model shows that the same shape of M can sometimes be achieved in multiple ways, thereby leading to alternatively stable states in which entries of M can differ substantially in sign and magnitude between different subpopulations. Consequently, hybrid crosses between subpopulations are likely to lead to suboptimal values of maternal effects, potentially leading to postzygotic reproductive isolation. Nonetheless, as alternatively stable states collapse to a single outcome when fluctuating selection diverges between both traits, it remains to be seen whether maternal effects can indeed contribute to reproductive isolation across a broad range of contexts. The most important result of the current study is that cross-correlations between selective fluctuations acting on different traits can select for striking configurations of cross-trait maternal effects. One way in which such cross-correlations can arise is due to time lags between selection acting on one trait and selection acting on another trait. Essentially, lag-times between fluctuating optima create asymmetries in the information content between both maternal characters, so that characters which endure selective fluctuations which are ‘advanced’ (relative to selective fluctuations on other traits) are more informative about future selective conditions than other characters. As a consequence, cross-trait maternal effects from such maternal traits should evolve to positive values, whereas cross-trait maternal effects based on traits enduring delayed selection should evolve to negative values. Hence, our study suggests that pairs of positive and negative maternal effects are indicative of lag-times (or cross-correlations in the action of fluctuating selection between different traits. Another context in which asymmetries in information content occur between both maternal traits is when fluctuating selection on one character contains more stochastic noise relative to selection on other characters. In this case, we expect that offspring characters benefit from information contained in those maternal traits that endure the most predictable form of fluctuating selection, whereas offspring are selectively favored to ignore information from maternal characters with more selective noise when alternative maternal traits are available. Henceforth, we may expect cross-trait maternal effects to evolve that rely on those maternal traits which endure the most predictable forms of fluctuating selection. In addition, when stochastic noise in selective conditions acting on one particular trait is substantial, cross-trait maternal effects of large absolute magnitude may give rise to increased phenotypic variance in the trait that experiences noisy selection (see Figure 5B). Such increased phenotypic variation is selectively advantageous in fluctuating environments, as it gives rise to bet-hedging [6], [8], [82]. Following the seminal model of Kirkpatrick and Lande [34] our study indeed shows that large phenotypic variances are associated with relatively large magnitudes of maternal effects coefficients, i. e.,. Henceforth, such ‘large’ maternal effects may provide an efficient means to increase phenotypic variation among offspring, which is in line with a number of studies that have identified maternal effects as a mechanism that gives rise to bet-hedging [23], [83], [84]. On the other hand, large maternal effects will also imply that any fluctuations in current trait values resonate to future generations, generating ever larger mismatches to future environmental conditions. To use maternal effects to increase phenotypic variance, individuals are selectively favored to use other maternal traits, which are less affected by stochastic fluctuations in selective conditions. Consequently, cross-trait maternal effects used in the context of bet-hedging are expected to rely on maternal characters that are stably inherited across generations. While numerous empirical studies have measured nongenetic effects in animal and plant populations (reviewed in [15], [18], [79]), almost all of these studies have taken a univariate perspective and measured only single maternal effects coefficients [31], [85], [86], or alternatively, measured different maternal traits lumped into a single ‘maternal performance’ character ([30], [87]). Consequently, it is currently premature to assess whether our predictions correspond to any empirical measurements of maternal effects matrix M. The current study indicates, however, that overlooked components of M, such as cross-trait maternal effects, could potentially be an important adaptation to fluctuating selection, and may provide a signature of past selective differences between traits. We therefore hope that the current model provides an incentive for future studies to assess maternal effects in a multivariate context. Such measurements of M are facilitated by the recent advent of multivariate methods developed within the context of indirect genetic effects (IGEs, [87]–[90]) that allow for the estimation of M using variance components [91]. To our knowledge, only a single study so far has used these multivariate methods to measure M: Galloway and coworkers ([32], see also [70]) measured maternal effects across four different life history traits in the plant Campanulastrum americanum, finding that some cross-trait maternal effects have magnitudes similar to or sometimes even larger than any within-trait maternal effects on similar characters. Moreover, cross-trait maternal effects often differ considerably in magnitude and even in sign, suggesting that observed variability among components in M is present and might be matched to past selective conditions to test our hypotheses. Moreover, a follow-up study in the same population [70] highlights another distinctive feature of maternal effects, namely that evolutionary change in phenotypes in a given generation is the result of both current and past selection gradients. We therefore hope that more studies follow the example by Galloway, McGlothlin and coworkers [32] and measure maternal effects in multivariate contexts. The current model has made a number of limiting assumptions that suggest possible directions for future work. The current study considers the simplest possible genetic architecture, where the breeding values and maternal effects are represented by single, diploid loci. It is well established that the relative number of loci coding for each trait may affect the magnitudes of the additive genetic (co) variances (e. g., [92], [93]), which in turn may either enhance or constrain adaptation [46]. Apart from the potential effects of additive genetic (co) variances, however, we note that previous comparisons of multilocus and single-locus approaches (e. g., [94]–[97]) have shown that evolutionary endpoints are often remarkably similar regardless of the approach taken, so we expect the evolution of M to be robust to more complicated genetic architectures. Future studies should assess whether this prediction indeed bears out. In addition, we exclusively focus on the evolution of cross-trait maternal effects due to temporal cross-correlations, thereby generating interactions between two different characters in a transgenerational context. By contrast, conventional studies on multivariate evolution focus exclusively on genetic correlations as the main (within-generational) signature of interactions between traits [98]–[102]. It would therefore be interesting to investigate whether those selective contexts that give rise to genetic correlations, such as trade-offs [103], [104], phenotypic plasticity [105], [106], developmental interactions [99], [100], [102] or sexual selection [97], [101], [107] also affect the evolution of cross-trait maternal effects. In turn, the potential role of cross-trait maternal effects as constraints on phenotypic evolution is currently virtually unexplored [35], as conventional studies have focused almost exclusively on genetic correlations that constrain the multivariate response to selection [45], [46], [54]. When cross-trait maternal effects are present, however, past selection on a particular character in the previous generation may lead to a correlated selective response on other characters even when genetic correlations themselves are absent [34], [35]. In addition, a recent study on univariate maternal effects shows that maternal effects expose populations to stronger transient perturbations in response to sudden environmental shifts than populations without maternal effects [25], [73]. More work is therefore needed to single out those ecological conditions in which significant cross-trait maternal effects are expected to evolve, as well assessing their consequences to phenotypic adaptation.

Title: The Evolution of Multivariate Maternal Effects Summary: In numerous organisms, mothers influence the phenotype of their offspring by transmitting hormones, antibodies and nutrients to the embryo. Evolutionary studies that make predictions about the evolution of these maternal effects typically focus, however, on single maternal characters only, in isolation of other traits. This contrasts with insights from quantitative genetics where reliable predictions about evolutionary change can only be made when measuring multiple traits simultaneously. The current study is therefore the first to make formal predictions about the evolutionary properties of multiple maternal effects. We show that maternal phenotypic characters generally give rise to developmental interactions in which one maternal character affects multiple offspring characters. In turn, such interactions can give rise to correlations between different traits in parent and offspring, which constrain evolutionary responses to sudden change. In addition, we find that the rate of environmental change directly affects some of the measurable properties of maternal effects: in rapidly changing environments, multivariate maternal effects are negative, so that offspring attain phenotypes that are different from their mothers, whereas positive maternal effects where offspring are more similar to their mothers occur in slowly changing environments. Hence, multivariate maternal effects provide a clear signature of the past selective environment experienced by organisms.

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Write a title and summarize: Visually induced neuronal activity in V1 displays a marked gamma-band component which is modulated by stimulus properties. It has been argued that synchronized oscillations contribute to these gamma-band activity. However, analysis of Local Field Potentials (LFPs) across different experiments reveals considerable diversity in the degree of oscillatory behavior of this induced activity. Contrast-dependent power enhancements can indeed occur over a broad band in the gamma frequency range and spectral peaks may not arise at all. Furthermore, even when oscillations are observed, they undergo temporal decorrelation over very few cycles. This is not easily accounted for in previous network modeling of gamma oscillations. We argue here that interactions between cortical layers can be responsible for this fast decorrelation. We study a model of a V1 hypercolumn, embedding a simplified description of the multi-layered structure of the cortex. When the stimulus contrast is low, the induced activity is only weakly synchronous and the network resonates transiently without developing collective oscillations. When the contrast is high, on the other hand, the induced activity undergoes synchronous oscillations with an irregular spatiotemporal structure expressing a synchronous chaotic state. As a consequence the population activity undergoes fast temporal decorrelation, with concomitant rapid damping of the oscillations in LFPs autocorrelograms and peak broadening in LFPs power spectra. We show that the strength of the inter-layer coupling crucially affects this spatiotemporal structure. We predict that layer VI inactivation should induce global changes in the spectral properties of induced LFPs, reflecting their slower temporal decorrelation in the absence of inter-layer feedback. Finally, we argue that the mechanism underlying the emergence of synchronous chaos in our model is in fact very general. It stems from the fact that gamma oscillations induced by local delayed inhibition tend to develop chaos when coupled by sufficiently strong excitation. An increase of activity in the gamma band (30–100 Hz) is observed in Local Field Potential (LFP) and Multi-Unit Activity (MUA) recordings [1]–[14], as well as in EEG and Electrocorticogram studies [15], [16] in primary visual cortex (V1) upon visual stimulation. Gamma activity is modulated by properties of the presented stimulus, such as orientation [2], [14], [17], contrast [7], [9], [18], velocity [3], [4] or size [12], much more strongly than the change in power in other frequency bands [19], [20]. Local GABA-ergic interneuronal networks are thought to play a key role in the production of neuronal activity in the gamma range ([21], see [22] for a review), as upheld as well by recent results obtained through optogenetic techniques in-vivo [23], [24]. Modeling works have provided a theoretical basis to account for the way in which networks of inhibitory interneurons can generate synchronous oscillatory activity in the gamma range [25]–[30]. In brief, in one possible scenario, the dynamics of the inhibitory post-synaptic currents is non-instantaneous (due to axonal delays, but also simply to finite synaptic time-constants). This contributes to create narrow time-windows in which excitatory and inhibitory neurons can fire closely in-phase, before being prevented to do so by a delayed inhibitory feedback. Therefore delayed inhibition, without need of an active involvement of excitatory populations, is capable inducing collective synchronous oscillations in neuronal activity. The frequency of these oscillations falls in the gamma band if the synaptic time constant of the inhibition is in an appropriate range. If a network operates in such a synchronous regime the neurons are engaged into approximately periodic collective oscillations involving a macroscopically large number of neurons. Therefore these oscillations are weakly affected by local noise and they maintain coherence over arbitrarily long time intervals. Power spectra of population observables of the network activity (e. g. LFP or MUA) exhibit narrow harmonic-like peaks and the damping of the corresponding autocorrelograms is slow. Peaks in the gamma-band have been identified in the LFP or MUA spectra of induced activity in-vivo in V1 [1]–[4], [12]. However, in general these peaks are very broad and in many cases they are virtually indistinguishable as the stimulus-modulated gamma power of the signals spreads across a broad-band frequency interval [7], [9], [10], [31]–[33]. Characterization of the spatio-temporal structure of the gamma induced activity by means of auto-correlations (AC) and cross-correlations (CC) of single-unit, multi-unit and LFP signals has also revealed that the neuronal activity has a tendency to oscillate, which can be stronger or weaker, depending on the considered experiment. In some cases the oscillatory components of ACs and CCs of the induced activity display many cycles before getting damped [1], [2], [12], [14]. In other cases, however, the oscillations are completely damped after one or two cycles [3], [8], [13], [17]. The existence of different dynamical regimes might underlie this observed diversity. For the mathematical abstraction of infinitely large networks, sharp boundaries between asynchronous and synchronous dynamical states exist [34], but for networks of a finite size such transitions are fuzzier [25], [34], [35]. Consequently, if the network does not operate too far from the instability to collective oscillations, in a regime which is formally defined as asynchronous –see [34], [35] and below for the definition–, the dominant normal modes of the network, which describe its response to small perturbations, can display damped oscillations at gamma frequencies. Local noise can excite these modes, inducing short-lived episodes of synchronous oscillatory activity. However, since these episodes are transient, the subsequent increase in power at gamma frequencies is broad-band. Induced broad band gamma power increases in V1 can therefore be accounted for if one assumes that the V1 network operates in such an asynchronous regime at the edge of developing synchrony [36]–[38]. In this regime, correlations in the spikes as well as in the membrane potentials of pairs of neurons are in general weak unless the neurons are connected via strong and direct synapses. However, in order to get a significant, although damped, oscillatory component in the macroscopic activity, the network must be “at the edge of synchronization”. In models, parameters have to be tuned in such a way to be close to an instability toward fully-developed synchronous oscillations, and this tuning have to be tighter, the larger the size of the recruited network [25], [29]. It is not clear how the required fine tuning would be satisfied given the range of experimental conditions in which gamma oscillations have been observed. In the present study we explore another scenario which reconciles collective synchronous activity with broad-band spectral modulations and robust fast decoherence. It is based on a mechanism proposed recently for the emergence of synchronous chaos in recurrent neural networks [39]–[41]. In this mechanism, clusters of neuron undergo a synchronous gamma oscillation due to local mutual inhibition. These collective gamma oscillations become chaotic when the neuronal clusters are allowed to interact through longer-range excitation. The resulting overall patterns of activity are characterized by synchrony at the population level, but at a same time display a characteristic lack of temporal regularity due to chaos. As a consequence, the power of this activity spreads over a broad interval of frequencies and the oscillatory components of the autocorrelograms of neuronal activity and LFP signals are rapidly damped within a few tenths of a millisecond. In this alternative regime, correlations in the spikes of pairs of neurons are still weak and go together with the sparseness of the firing, but correlations in their membrane potentials can be strong. We present here a model of a hypercolumn in V1, endowed with a simplified multi-layer architecture. In order to explain broad-band contrast-dependent spectral modulations in terms of synchronous chaos, we need to identify distinct interacting oscillators within the local cortical circuit. We hypothesize that neuronal populations within different thalamo-recipient cortical layers are set into oscillation by increased driving and that the mutual interaction between these populations, mediated by inter-layer synaptic connections, supports the development of synchronous chaos. This hypothesis is backed up by anatomical evidence. Thalamo-cortical synapses, providing direct sensory-induced driving, indeed target cortical layer IV but also, to a lesser extent, layer VI [42]–[46]. Extensive networks of recurrent inhibitory connections are present within each thalamo-recipient layer [45], [47], [48], supporting local generation of oscillations at multiple depths in the cortical tissue. Finally, stereotyped circuit motifs provide a bidirectional poly-synaptic connection loop between thalamo-recipient layers [44]–[46], [49]–[51]. Relying on extensive numerical simulations, we show that our model displays broad-band gamma modulations of the spectra of LFPs upon stimulation of the network at low as well as at high contrast. Whereas this induced activity is asynchronous at low contrast, it develops synchrony on a macroscopic scale when the contrast increases. Therefore we argue that the broad band gamma power observed in recorded LFP spectra in V1 is compatible with the existence of visually induced synchronous oscillatory neuronal dynamics. We model a functional hypercolumn in primary visual cortex as a large recurrent network of spiking integrate-and-fire-type neurons. To account in a simplified way for the layered structure of the visual cortex –a cartoon of which is shown in Figure 1A– the model network consists of two sub-networks, schematically representing layers I to IV and layers V to VI. We denote these two sub-networks as the upper and lower layer respectively (Figure 1B). Each of these layers comprises excitatory and inhibitory neurons, for a total number of neurons in the network. Most of the simulations in this study are performed taking excitatory and inhibitory neurons per layer, leading a total of neurons in the model hypercolumn. This number is one order of magnitude smaller than estimates of the number of neurons in a real V1 hypercolumn based on neuronal densities recently measured by [52]. However, it leads to dynamical behaviors similar to larger network sizes (see following scaling analyses) and constitutes a compromise for efficient and fast simulations. Each layer is described by a network with the geometry of a ring as depicted in Figure 1C, with neurons labeled by angular coordinates,, ranging from −90 to +90 degrees [53], [54]. The connections between neurons within each layer are random, with connection probabilities that depend on the angular distance between pre- and post-synaptic neurons. Spatial averages and spatial modulations of connection probabilities are set independently for the various kinds of connections (e. g. excitatory-to-excitatory, excitatory-to-inhibitory, inhibitory-to-excitatory or inhibitory-to-inhibitory), thus making it possible to vary the spatial profiles of net synaptic interactions (see Figure 1D, E, F). Excitatory and inhibitory inter-layer connections are also random and spatially modulated. All the external inputs to the network are modeled as stochastic processes (see Methods section). The neurons receive an external non-selective noisy current representing background inputs to V1 from other brain areas and a weakly tuned noisy current which represents visually induced inputs to V1 from converging Lateral Geniculate Nucleus (LGN) synapses [55]. Note that the two main thalamo-recipient layers, i. e. layers VI and IV, are embedded within two distinct model layers. Our two-layer circuit embeds in a simplified manner several known features of the stereotypical interlaminar anatomy of the columnar microcircuit, in particular, the existence of a layers IV to VI to IV feedback loop [44], [46], [50]. Furthermore, a different degree of spatial modulation for inter-layer excitation and inhibition mimic the on-center off-surround arrangement of layers VI to IV projections [56]. In the simulations described below we assume that the LGN input to the lower layer is weaker (by a factor of 2) than the input to upper layer to account for the fact that thalamo-cortical synapses reaching layer VI are smaller in number than those reaching layer IV [45]. We also assume that latencies for inter-layer connections are longer than for intra-layer connections, thus accounting for the multisynaptic nature of this coupling. Our assumptions on the connectivity, external inputs and latencies are further commented upon in the Discussion section. In order to analyze the role of the interlayer interactions in shaping the spatiotemporal dynamics of our model hypercolumn, we introduce a parameter which homogeneously rescales the strength of excitatory and inhibitory connections between layers. For the interactions between the layers assume their maximum strength. For the layers are completely independent. In the following, we consider first the dynamics of the network at full coupling strength,. In absence of “visual” stimuli (contrast level), the model hypercolumn is driven only by the non-selective background input. The resulting spontaneous activity is heterogeneous across the neurons with average firing rates of and for excitatory and inhibitory neurons, respectively. Differences in the spontaneous firing rate distributions for upper and lower layers are not statistically significant at the 5% confidence level. The spontaneous firing of the neurons is highly irregular due to the stochasticity of the inputs. For instance, the average coefficient of variation (CV) of the interspike histogram of excitatory and inhibitory neurons in the upper layer is. More details about rate and CV distributions can be found in Figure S1. The profile of the activity induced by an oriented stimulus in both layers, is localized and centered at an angular coordinate corresponding to the stimulus orientation. Hence, the neuronal responses are selective to the stimulus orientation. The tuning curves of individual neurons display some heterogeneity in their broadness, as exhibited by distributions of peak response rates, circular variance and skewness of the tuning curves (reported in Figure S2). Figure 2A displays the population average tuning curve for various contrast levels for excitatory neurons in the upper layer. Comparison between tuning curves at different contrasts reveals that tuning width is approximately contrast invariant and that the larger deviations are observed for small contrast levels (tuning curves normalized to the peak are plotted in Figure S3). This invariance is achieved as an effect of noise in synaptic inputs [57], [58]. The preferred responses of the excitatory neurons vary non-linearly with the contrast as depicted in Figure 2B, where the population average Contrast Response Functions (CRFs) are plotted for excitatory neurons in the upper layer. It can be fitted by an hyperbolic ratio function (see Methods section), with mid-range contrast % and an exponent of (upper layer neurons). This nonlinear dependence stems from the fact that increased sensory-driving yields larger inhibitory neurons activity which in turn is responsible for the saturation of the excitatory population response [59]. The CRFs of inhibitory neurons show a much weaker tendency to saturation at large stimulus contrasts which is due to the logarithmic dependency on the contrast of their external input. The CRFs of single neurons are heterogeneous, in qualitative agreement with experimental reports [60] (see Figure S4). The contrast response functions of the lower layer are homologous, but the induced responses are approximately twofold smaller, due to the weaker LGN driving. For zero contrast, the synchrony level in the spontaneous neuronal activity is small, as denoted by a small value of the synchrony factor. This factor, defined in the Methods section, quantifies global synchrony over a network and is bounded between 0 and 1. For a network of size, the synchrony factor for spontaneous activity assumes the value. Furthermore, it vanishes consistently as for larger network sizes, allowing us to classify formally the state of the network as “asynchronous” (see later discussion, Methods section and [34], [35]). The single neuron and population responses of the network induced by visual stimulation are illustrated in Figure 3, for a low contrast stimulus (), and in Figure 4, for a large contrast stimulus (). We focus first on the low contrast case. The raster plot of the spike activity of all the excitatory neurons in the upper layer is plotted in Figure 3A. It suggests that the firing is highly irregular (the mean CV of the upper layer excitatory neurons is, see Figure S1) and that the network activity of the network is only weakly synchronized. This is confirmed in Figure 3B where the spike trains of six upper layer cells stimulated within from their preferred orientation are plotted. The neurons fire without any noticeable synchrony. Figure 3C displays the voltage traces of two of these neurons. The comparison between the sub-threshold fluctuations in the two traces does not reveal any significant correlation. To further quantify the correlations in the supra and subthreshold activity of the neurons we compute the zero delay pairwise correlation coefficients (CCos) of the spikes and the membrane potential traces for a large number of pairs formed by highly active neurons with preferred orientation within from the presented stimulus (see Methods section and Figure S5 for details). The resulting histograms are shown in Figure 3D (spikes: left, cyan color; voltage: right, blue color). They are peaked around zero with a mean statistically indistinguishable from zero (for spikes and voltage). Almost all the CCos are weak for the spikes as well as for the voltage traces (CCos larger than 0. 25 occur only for 2% of the pairs when considering spike CCos, and for 0. 1% of the pairs when considering voltage trace CCos). These results are consistent with a very weak synchronization in the network activity. This is in line with the small value of the synchronization factor, which is only. Auto- and crosscorrelograms of spike trains and membrane potential traces of three representative neurons are also shown in Figure 5A, B. The pairwise crosscorrelograms of both spikes and voltages do not display any persistent oscillatory component, even when two cells share a same orientation preference. The dynamical state of the network is qualitatively different for a high contrast stimulus. For the neurons are engaged into a collective pattern of synchronous oscillations in contrast to what happens for. This is clear from the raster plot in Figure 4A. Figure 4C plots the membrane potential traces of two neurons. Comparison of these traces suggests that now the subthreshold membrane fluctuations of the neurons are strongly correlated across the network. As a matter of fact, the synchrony factor,, which characterizes the degree of synchrony in the subthreshold activity at the network level, is. However action potentials are much less synchronized, as suggested by the comparison of the spike trains of the six neurons plotted in Figure 4B: although multi-neuron coincidences in firing (denoted by vertical grey bars) can be detected, the overall synchrony is weak. This substantial difference in the strength of the pair correlations in supra and subthreshold activities is clear in Figure 4D. All the CCos of the subthreshold membrane potentials (red histogram) are large and sharply distributed around 0. 75 (standard deviation of) whereas the distribution of the spike trains CCos (magenta histogram) has a mean which is only. Remarkably, the firing activity continues to be highly irregular, despite the high degree of synchrony (mean CV of upper layer excitatory neurons is, see Figure S1). Auto- and crosscorrelograms of spike trains and membrane potential traces of three representative neurons are shown in Figure 5C, D. The pairwise crosscorrelograms of voltages display now a clear oscillatory structure, which is however completely damped after only two or three cycles. Note that oscillatory correlations are evident even when the difference of preferred orientation is large (). Note however that pairwise crosscorrelograms of spike trains do not display any marked oscillation even when the two considered cells have similar preferred orientations. We stress that the small mean value CCos and the lack of a clear oscillatory structure in the crosscorrelograms for spike trains, in both the low and the strong contrast case, is associated to the irregularity and the sparseness of single neuron firing. These results indicate that synchrony in the population activity increases with the contrast. As a matter of fact, the synchrony measure varies abruptly around a contrast value of, as shown in Figure 6A. This is even sharper with larger network sizes (compare in Figure 6A, the solid line which is for with the dashed line which is for). Moreover, a systematic analysis of the dependency of on the size reveals that for, (low contrast regime) vanishes consistently with,, while for (large contrast regime) it converges toward a constant non zero value (Figure 6B). Hence, the network operates in qualitatively different regimes at low and high contrast. Whereas the network state can be classified as asynchronous in the low contrast regime (and in the spontaneous activity regime), it is synchronous in the high contrast regime. This sharp variation of synchrony is indicative of a phase-transition occurring for increasing contrast, due to an increased drive to the network (see Discussion). To characterize further how the population dynamics depend on the contrast we compute the autocorrelation, of the LFP signals induced by stimuli oriented at the preferred orientation of the recording site (see Methods for the way we define the “LFP” signals in the framework of our model and Figure S6 for examples of LFP traces). The result for low contrast,, is plotted in Figure 7A, B. The amplitude of the (non-normalized) AC at zero delay,, is small and decreases with the network size as. Similarly, the small oscillatory component of the AC disappears gradually for increasing network sizes (Figure 7B). This is because the network state is asynchronous and in a larger network more cells contribute to the LFP signal (see also Methods section). The fact that at high contrast,, the network is engaged in collective synchronous activity is manifest in Figure 7C, D: is now large, it does not vanish in the large limit and is almost independent of for. However, and remarkably, the induced dynamics exhibit a spatio-temporal structure which is more complex than a periodic regular oscillation of the population activity: the time interval between consecutive episodes of synchronous activity displays cycle-to-cycle fluctuations as can be observed in the raster plotted in Figure 4A). As a result, the LFP autocorrelogram is rapidly damped. Although it displays some secondary peaks their amplitudes are very small as shown in Figure 7C. The damping of the AC oscillations is even faster for larger network sizes (Figure 7D). Note that autocorrelations for intermediate contrast values are also rapidly damped (see Figure S6). A moderate tendency to period doubling, manifested by a second autocorrelogram peak slightly larger than the first autocorrelogram peak, is observed in our model. To our knowledge this has not been observed in experimental studies. However, this feature disappears for larger network sizes or stronger inter-layer coupling. LFPs induced by non-preferred stimulus directions display as well oscillatory components, for both low and high contrasts. Induced LFPs are correlated over the entire ring network as revealed by crosscorrelation analysis, confirming that sub-threshold coherence can exist independently from correlations in spiking activity (see Figure S6). Finally, we consider the spectral properties of induced LFPs, and their relation with MUA observed at a same location. The dependency on the contrast of the power spectra of the LFPs induced by preferred-orientation stimuli is shown in Figure 8A. The low-frequency part of the power spectra is weakly dependent on the stimulus contrast. Rather, it is shaped by the properties of cortical background activity, modeled as a stochastic Ornstein-Uhlenbeck noise with a frequency cutoff (see Methods section and [61]). This should be compared to the boosting of the power as the contrast increases for frequencies Hz. Although the network activity becomes much more synchronous at large contrast as explained above, power spectrum modulations are not limited to narrow peaks, but, even at the highest contrast, the whole frequency range comprised between 30 and 100 Hz is boosted. In this same broad frequency range in which contrast-dependent power modulations occur, the LFP displays phase-synchronization with the MUA at a same location, as measured by a MUA-LFP coherence increasing with contrast (see Figure 8B). Interestingly, the MUA-LFP coherence, even at full contrast, rises only at an average peak level of approximately 0. 3, compatible with physiologic ranges of synchronization [9], [62]. This can be explained by the random-like variability of single neuron firing –inherited by the MUA signal, which reflects the spiking activity of only a limited number of single units (see Methods section) –, but also by the lack of phase autocoherence in the LFP signal itself (see [63]). The spatio-temporal structures of the induced activity in the lower and in the upper layers are similar. In our simulations, the lower layer average firing rate is approximately half of that in the upper layer, reflecting weaker driving from LGN. Cross-correlation analysis of the LFPs in the two layers shows that the lower layer oscillations lag behind those in the upper layers (see Figure S7). Note that larger response latencies in deep layers have been experimentally observed in specific conditions [64], [65]. However, the multi-layer structure in our model is too schematic to capture quantitatively such inter-layer relations. In particular, the difference in response rate and the exact locking pattern between layers depend in our model on the parameters of LGN input and inter-layer coupling. On the contrary, the synchronization and the fast decorrelation of induced oscillations are robust against changes in these parameters (see later Discussion). In order to explore the role played by the inter-layer interactions, we investigate in the following how the dynamics in the high contrast regime is affected by a change of this coupling. More specifically, we rescale the peak conductances of all the synapses between cells in different layers by a same factor (and correspond respectively to fully coupled and fully decoupled layers). Upon layer-decoupling the mean firing rate of the excitatory and inhibitory cells increases in the upper layer (Figure 9A). However response rate changes are highly heterogeneous across cells and, in some cases, the peak rate is even slightly reduced. An analogous heterogeneity is observed in the changes in the preferred orientation, skewness and tuning width. However, even though changes after complete layer decoupling can be significant for specific cells, the distribution of tuning curve parameters over the entire upper layer excitatory neurons population is only weakly altered. Details are shown in Figure S8. Another effect of layer decoupling, albeit moderate, is that the degree of synchrony in induced activity decreases monotonically with (Figure 9B). For instance, the synchrony factor is for, but decreases to when, and drops further to for fully decoupled layers. The most striking consequence of the reduction in inter-layer coupling is the progressive qualitative change in the shape of the LFP autocorrelograms and power spectra as decreases. This is depicted in Figure 9. For 80% coupling strength (), the autocorrelogram of LFP and the corresponding power spectrum are similar to what is found in the fully-coupled case (fast temporal decorrelation and broad plateau-like peak in the gamma spectral band, see Figure 9C, D). However, for a 60% coupling strength (), the LFP temporal decorrelation becomes considerably slower and the envelope of the autocorrelogram displays amplitude modulations indicating that the LFP signal is quasi-periodic. In parallel, the gamma-band spectral plateau is replaced by a system of narrow peaks at incommensurate frequencies. The raster plot of activity (not shown) continues to display a temporally irregular oscillation; however spatial fluctuations in the width of consecutive bumps of spiking activity are reduced with respect to the fully-coupled case. For further reduction of the interlayer coupling to, the LFP autocorrelogram starts revealing periodicity of the signal over long time scales. The multiple narrow spectral resonances merge into a single prominent resonance in the gamma-band and secondary harmonic peaks also appear. Finally, for (Figure 9E, F), the LFPs are still substantially autocorrelated after several hundredths of ms. Spectra in the synchronous regime are harmonic at any contrast level. More details about the high contrast regime for completely uncoupled layers are presented in Figure S9. Interestingly, qualitative modifications of the population dynamics when is varied do not occur in the low contrast regime, in which collective oscillations do not develop. As a matter of fact, independently of the coupling strength, induced activity is asynchronous. Spiking and LFP responses to a low contrast stimulus between completely uncoupled or fully coupled layers are practically indistinguishable (not shown). Up to now we have focused on the response of the network to a time independent stimulus. Here we show that the inter-layer coupling also strongly affects the response of the model hypercolumn induced by an external input which varies periodically, representing visual stimuli to V1 in the form of flashed or drifting gratings. In this situation, we characterize the neuronal responses by means of peristimulus time histograms (PSTHs) which express the probability of observing the firing of a spike at a given time relative to the onset of each stimulus presentation (see Methods section). In the following, we focus on high contrast stimuli. The PSTH for is shown in Figure 10A. At the onset of the stimulus the probability of firing increases sharply, followed by a transient phase of reduced firing. This feature is not evident in experimental PSTHs. It is due to the strongly synchronous recruitment of recurrent inhibition which follows the initial burst of activity, triggered by the rise of external inputs (instantaneous in our model). Notwithstanding, after a few tenths of a ms the firing probability rises again and remains then almost constant. This reflects the fact that the population responses are highly variable across trials as is clear in Figure 10B. In each trial the response of the network consists of a sequence of episodes in which the neurons tend to fire together. However, there are substantial trial-to-trial fluctuations in the timing of these episodes and their amplitude (i. e. the numbers of recruited cells). Consequently, although the presentations of the stimulus do give rise to synchronous activity, the PSTH histogram averaged over many trials is almost flat after a peri-stimulus time on the order of the short temporal decorrelation time of the induced oscillation. In contrast, for fully decoupled layers (), the PSTH averaged over many trials exhibits a long-lasting, although damped population oscillation, as plotted in Figure 10C. This is because when the layers are decoupled the oscillations generated inside the layers are close to being periodic and they maintain coherence over several hundred milliseconds. Hence the timing of the oscillations does not fluctuate much across trials (Figure 10D). Population oscillations are thus masked by averaging across multiple stimulus repetitions only after many cycles. The large trial-to-trial variability displayed by the network for (Figure 10C) indicates a strong sensitivity to initial conditions (i. e. the network configuration at the onset of the stimulus). To further illustrate this sensitivity, we perturb the dynamics of the system by omitting artificially a single spike in a single neuron (out of) at the center of the bump of induced activity and we compare then the perturbed and the unperturbed dynamics. The results of this numerical simulation are illustrated by Figure 11. As visible from the raster plot (Figure 11A) and the population rate histogram (Figure 11B) of the upper layer induced activity (at full contrast), the perturbed and the unperturbed collective oscillations can be distinguished already after one oscillation cycle. After a few cycles, they have completely diverged. Such extreme sensitivity to perturbations or initial conditions is strongly indicative of dynamical chaos [66]. The sequence of states observed in our model for decreasing (from irregular to quasi-periodic to periodic, see Figure 9C, D) also suggests that chaos might emerge for strong inter-layer coupling and that its onset might occur according to a quasi-periodic scenario [66], [67]. This is indeed one of the possible scenarios for the transition to chaos occurring in a related rate model [41]. As we discuss in detail in the Text S1 and in the Figure S10, the chaotic nature of the dynamics of the network for and high contrast stimuli can be assessed by an estimation of its largest Lyapunov exponent [66]. A positive value of this Lyapunov exponent is the manifestation of deterministic chaos, denoting exponentially fast separation of trajectories. Using techniques of non-linear time-series analysis [68] applied to very long stationary time-series of LFP from our model (see Methods section, Text S1), we obtain the estimate, which is indeed positive. Interestingly, the dynamics of the network with uncoupled layers () fails to display a positive Lyapunov exponent (see Figure S10), and it is therefore non chaotic, confirming the role of inter-layer coupling in inducing (see also the Discussion section). Most of the simulations described above were performed in networks with a significantly smaller number of neurons (excitatory neurons and inhibitory neurons per layer) than in a real hypercolumn in V1. However, we checked that our results are robust against increases in network size. In particular, this is the case for the existence of two dynamical regimes induced respectively by low and high contrast stimulations and for the two distinct mechanisms underlying the fast temporal decorrelation and broad-band spectral modulations in these two regimes. In the low contrast regime, the dynamics are asynchronous. However, the network tends to resonate at a specific frequency, producing an increase of power in the gamma frequency band, without developing stable oscillations. Weakly coherent oscillatory modes are excited only transiently by local noise and then quickly damped. On the other hand, in the high contrast regime the network activity is synchronous. However the collective rhythm undergoes random variations in the time interval between consecutive activity episodes in the network. This temporal irregularity is not due to local noise (note that, in our model, recurrent inputs dominate over feed-forward inputs at low as well as at full contrast). It is produced intrinsically by the dynamics by virtue of the interaction between distinct oscillating populations localized in the two subnetworks representing different depths in the cortical section. This results in rapid temporal decorrelation of the induced activity. The contrast at which the transition between these two regimes takes place depends on the strength of fluctuations in the background noise. For our choice of parameters, the transition occurs for. However, as discussed in detail in Figures S14, S15 and S16, if the variance in the LGN input current is increased consistently without changing its mean value (Figure S14, parameters in Table S1), the transition can occur for an external drive, which is so large that it cannot be reached even for stimuli at full contrast (Figure S15). In such a condition, the induced activity is still asynchronous at high contrast and only transient oscillations can be detected (Figure S16), as in the recent modeling study by Mazzoni et al. [37]. It has been observed experimentally that the gamma-band synchronization of membrane potential fluctuations of nearby cells in V1 is larger in visually-induced activity than in spontaneous activity. Furthermore it is sustained over long stimulation durations, independently from stimulus properties or from the simultaneous observation of synchronized spiking activity. This leads to voltage crosscorrelograms with a manifest oscillatory component at gamma-range frequencies, damped quickly within only two or three oscillation cycles [80]. These observations are compatible with the occurrence of a transition between an asynchronous low contrast regime and a synchronous high contrast regime. Indeed, pairwise CCos between membrane potentials are small in the low contrast regime (Figures 3D and 5B), but large in the high contrast regime (Figure 4D and 5D), even if spike CCos are always small, in agreement with many experimental reports [5], [13], [81]–[86]. We remark that if the dynamics at high contrast would be asynchronous as the dynamics in absence of stimuli or for low contrast stimuli, then the pairwise crosscorrelations of both spikes and voltages should be weak. Therefore, the coexistence of weak correlations between spikes with stronger correlations between membrane potentials (displaying furthermore a damping oscillatory component) is suggestive of the existence of a synchronous, rather than of an asynchronous, regime. The dynamics at high contrast of our model, characterized by irregular spiking (leading to weak spike crosscorrelations) and by temporally irregular collective oscillations (leading to quickly damped oscillatory voltage crossocorrelograms) is therefore compatible qualitatively with the experimental regime observed in [80]. Conversely, this compatibility could not be claimed for the other two types of induced dynamics that our model can generate at full contrast, i. e. asynchronous, in the case of a large variance noise, or synchronous but approximately periodic (and therefore too slowly decorrelating), in the case of suppressed inter-layer interactions (). The rapid loss of temporal coherence of the synchronous induced activity at high contrast is a remarkable property of our model. Features of the model such as inter-layer inhibition, asymmetric interaction latencies in the lower-to-upper or in the upper-to-lower direction or different LGN driving levels to the different layers are not required for this decoherence to occur. In contrast, the strong local inhibition responsible for the local generation of the rhythm within each layer and the net excitatory interactions between neurons in close vertical alignment are crucial for this to occur. In fact, if the inter-layer excitation profile is altered by suppressing its modulation with orientation distance while keeping its average strength constant, the decorrelation does not take place (see Figure S17). A similar mechanism underlies the temporal decorrelation of synchronous oscillations in the network models studied by [39]–[41]. These papers showed that collective oscillations induced in two populations of neurons by local delayed inhibitory feedback can lose coherence when the two populations interact in an excitatory manner. In [41], we studied a rate model consisting of two networks, each composed of one excitatory and one inhibitory populations. Each of the networks was able to sustain synchronous oscillatory activity by virtue of the local inhibition. We computed the maximum Lyapunov exponent of the system (see e. g. [66]) to show that it undergoes a transition to a chaotic dynamical state when the two networks are coupled by sufficiently strong excitatory connections. In this state the network displays synchronous activity, but instead of being periodic, the temporal variations of the network activity are chaotic and thus the oscillations that the network tends to develop lose temporal coherence within a few cycles. A network operating in such a regime is said to be in a synchronous chaotic state. In [39], [40] a single ring network with strong local inhibition was considered. The decoherence of the oscillations occurred as the network underwent a spontaneous clustering into groups of oscillating neurons effectively interacting in an excitatory manner. In agreement with the positivity of its largest Lyapunov exponent, also the dynamics of our hypercolumn model in the high contrast regime displays typical features of chaos: exponentially fast damping of the local oscillations autocorrelograms (Figure 7C, D), spreading of the oscillation-related power over an extended continuous interval (Figure 8), and extreme sensitivity to initial conditions (Figure 10B and Figure 11). Therefore the decoherence of the population activity which occurs at high contrast stems in the present model from the fact that the network operates in a synchronous chaotic regime. We cannot exclude, obviously, that other mechanisms are contributing to the decorrelation of synchronous cortical oscillations. However, such a global decorrelation, characterized by the coexistence of elevated instantaneous synchrony and fast loss of collective phase autocoherence, could not be induced by local external noisy inputs, unless they are spatially correlated over a range matching the size of the local circuit which generates the ongoing soscillation. We also conjecture that the underlying mechanism of synchronous chaos is very general as it occurs in models in which neurons are described in term of rate, integrate-and-fire or conductance-based dynamics, with a simplified as well as more complex multi-layer network architecture. We also conjecture that a similar mechanism should act in even more realistic models, incorporating for instance a two-dimensional spatial structure, similarly to the one used in [87], [88], provided that local inhibition is strong enough to induce local oscillations and that excitation couples these local oscillators at a longer range. Chaotic dynamics as well as stable chaotic-like dynamics can occur in asynchronous states of activity [89]–[97]. In this cases, the network dynamics explores a high-dimensional manifold in the phase-space, while, in our model, the irregular sparse firing of many neurons give rise to collective synchronous chaos (SC) with a lower dimensionality [98]–[100] (the fractal dimension of the chaotic attractor is likely to be smaller than five, as discussed in Figure S10). SC has also been found in previous models of local circuits in V1 which consisted of only one single network with a ring architecture. The model studied by Hansel and Sompolinsky in [101] considered one neuronal population coupled with excitatory instantaneous synapses. It displayed a SC state in some appropriate range of parameters. However, in this model, SC was sensitive to the incorporation of synaptic time constants since it was destroyed with the introduction of synaptic time constants as small as 0. 5 ms. The model by the same authors considered in [34] considered two populations of neurons, one excitatory and one inhibitory, coupled via synapses with realistic synaptic time constants. The dynamics of the neurons were based on a Hodgkin-Huxley type model with several cellular and synaptic conductances. The pattern of connectivity had a “Mexican hat” with local excitation and broad range inhibition. Numerical simulations of the model showed that in an appropriate parameter range, the network settled in a SC state, characterized by strong temporal variability of the neural activity which was correlated across the hypercolumn. In both of these models, the SC state was characterized by strong neuronal pairwise spike correlations and wide variability in the firing of individual neurons which was induced by the chaotic nature of the population activity. This is essentially different from what happens in our two layers hypercolumn, in which, in the SC state at high contrast, the spike pairwise correlations are only slightly larger than in the low contrast asynchronous state, whereas the degree of irregularity in the spike trains are similarly large in both states (). As a matter of fact, in the present model, the spike train irregularities are mostly due to the local noise generated by the external inputs and to a lesser extent by the internal dynamics. Voltage CCos are large due to macroscopically correlated chaotic sub-threshold fluctuations, but spike CCos are still small. Another essential difference is that in [34] the excitation was local and inhibition was broad, whereas the opposite is required in the present model, as well as in the single ring model in [40]. Last but not least, it is not clear to what extent the chaotic dynamics found in [34], [101] were specific to the model adopted there for the single neuron dynamics. The increase in synchrony of the activity with the contrast displayed by our model is in agreement with experimental results reported recently in monkey V1 [9], [18]. More generally we should expect that varying a feature of a stimulus in a way that increases the external drive on V1 network should have a similar effect. This is consistent with other recent results showing that varying the size of a visual stimulus [12] or attention [6], [11] strengthens the coherence in the activity of V1 neurons. In the low and large contrast regimes identified in our model the increased gamma power in the LFP spectra is broadband. At low contrast, the loss of coherence of the oscillations in the LFP in a few tenths of a milliseconds is due to noise. At large contrast, it is a consequence of the chaoticity of the LFP time-series. The behavior of our model in both these regimes is compatible with recent results by Burns et al [63], because of its lack of sustained auto-coherence of induced oscillations. Our simulations predict that infra-granular layer inactivation should globally affect the experimentally observed spectral properties of induced LFPs by enhancing its periodicity. Single-layer inactivation experiments based on pharmacological or local cooling techniques [79], [102], [103] or with optogenetic techniques [23], [24] might be used to test this prediction. Furthermore, manipulations in which the firing of a single additional spike is induced (or suppressed, analogously to the simulation of Figure 11) can be performed. Extreme sensitivity to single-spike perturbations was experimentally proved using such a manipulation in the case of asynchronous spontaneous cortical dynamics [104]. It would be interesting to repeat similar experiments in a stimulus-induced regime of oscillatory activity, in order to study the impact of the addition of a single spike on the time-course of ongoing LFPs. In the present study we focused on the role of the interactions between cortical layers in promoting temporal decoherence of gamma oscillations via the generation of synchronous chaos in a network with the size of a typical classical receptive field in V1. It would be interesting to investigate whether horizontal interactions which extend at distances beyond the classical receptive field also contribute to the loss of temporal coherence via a similar mechanism when the visual stimuli are extended. The basic two-ring network developed in this paper can be replicated into a bi-dimensional architecture including long-range excitatory interactions in order to investigate this potential contribution. This framework can be also applied to assess how the phase relationship between activity at different locations in V1 (e. g. between center and surround of an extended stimulus) depend on the polarity of long range interactions. Furthermore, an additional source of decorrelation might be inter-areal interactions occurring at an even longer range. Finally, we have here considered temporal decorrelation induced by excitatory interactions between populations oscillating due to delayed mutual inhibition between interneurons. It would be interesting to investigate whether a similar decorrelation phenomenon can arise when the mechanism for the local generation of oscillations is different, and is based for instance on circuit loops with active involvement of pyramidal cells [27], [30], [75]–[77], [105]–[107]. We use single-compartment Exponential Integrate-and-Fire model neurons (EIF; [108]). In this model the membrane potential is given by the equation: (1) where is the membrane capacitance, the membrane time-constant, the leak potential, the total synaptic input current to the neuron. The function is: (2) For a constant input above a threshold current (nA for the parameters adopted here) the solution of (1) diverges to infinity in finite time. This divergence is identified with the firing of a spike. The parameters and characterize how sharp the initiation of the spike is and the voltage at which it occurs. The spike downswing is not explicitly modeled. After each spike event, the voltage needs to be reset. A refractory period must then follow. We model this refractoriness in a different way for excitatory and inhibitory neurons. In the case of excitatory neurons, following the emission of a spike at time, the parameters, and are updated according to the equations [109], (3) (4) (5) The membrane potential is reset to a value which is sub-threshold. Furthermore is strongly depolarized after a spike. Therefore the event that two spikes are closely emitted in time by a same neuron is extremely unlikely and, in practice, never occurs. For inhibitory interneurons, we use a “hard” refractory period instead, suspending the numerical integration for a time after voltage reset [108]. Therefore,, and. Parameters for excitatory neurons are chosen to coincide with fits of pyramidal neurons traces, following [109]. We use analogous parameters for inhibitory neurons, apart from halved membrane capacitance and time constant, consistent with experimental evidence [110] and fits of interneuronal traces presented in [111]. All single neuron parameters are given in Tables 1 and 2. We use three kinds of synaptic currents, modeling inhibitory (GABA-type), fast excitatory (AMPA-type) and slow excitatory (NMDA-type) synaptic inputs. No voltage dependence is introduced for the parameters of the slow excitatory synaptic current. A spike in an inhibitory pre-synaptic neuron evokes a GABA-type post-synaptic potential (PSP) in all the post-synaptic neurons; a spike in an excitatory presynaptic neurons evokes composite AMPA- and NMDA-type PSPs. The synaptic current produced by a single incoming spike is described as, where is the peak synaptic conductance, the reversal potential of the synapse (mV,). Denoting as the time of pre-synaptic firing and with the synaptic latency, the function is: (6) where the constant is such that it normalizes to unity the peak of. All the synaptic conductances in the network are calibrated to give unitary PSPs at resting potential in a range compatible with experimental observations [72]. The values of the synaptic times and synaptic peak conductances are given in Table 3, for a network including excitatory neurons and inhibitory neurons per layer. Synaptic peak conductances are rescaled for larger networks, according to Eqs. (8) and (9). Synaptic latencies are given in Table 4. Each of the two layers of the hypercolumn is modeled by a ring-network [34], [53], [54], [112]. Unless specified otherwise, the simulations described in this paper were performed for a network comprising excitatory cells and inhibitory cells per ring, for a total of neurons in the hypercolumn. Note that a very similar network architecture was used in [113], [114] but with a completely different interpretation. Intra-layer and inter-layer excitatory and inhibitory connections are random. The probability of connection between two neurons is spatially modulated and depends on the angular coordinates and of the pre- and post-synaptic neurons. It also depends on the nature (excitatory or inhibitory) of pre- and post-synaptic cells and on their absolute (lower or upper layer) and relative (intra-layer or inter-layer) depth. All the profiles of connection probability are parameterized as: (7) Here, denotes rectification; i. e. if, else. The probabilities of connection for intra-layer excitatory and inhibitory connection are identical for each of the two layers. In order to study the scaling properties of the dynamics it is important to guarantee that the spatial mean and spatial fluctuations of the time averaged recurrent synaptic currents received by each neuron are preserved when considering networks of different sizes. This requires a suitable modification of the probabilities of connection and of the peak synaptic conductances when passing from a network of size to a network of size [35]. For an arbitrary peak recurrent synaptic conductance, the probabilities of connection (and, correlatively the average number of pre-synaptic cells of each type) are scaled as: (8) and peak conductances as: (9) Here the index stands for different kinds of synaptic connections, each one potentially characterized by different mean probabilities of connections and connection strengths (i. e. originating from upper or lower layer excitatory or inhibitory neurons and directed toward upper or lower layer excitatory and inhibitory neurons). Sizes between –for a total network size of neurons– and –for a total network size of neurons– are compared in scaling analysis of synchrony properties. The parameters for and are given in Table 5. Probabilities of connection are compatible with the ranges reported by [73], [74]. We assume that the firing rate of a single LGN neuron, is related to the stimulus contrast,, () by the equation [112]: (10) where is the spontaneous activity of the neuron in dark conditions. Subsequently, we model the LGN input to a cortical cell as an AMPA-type synaptic connection with peak conductance, driven by homogeneous Poisson spike trains with rate, (11) with: (12) Here the parameter controls the broadness of tuning of the LGN input. It is set to 1 in all our simulations. Note that is maximum when. The contrast and, correspondingly, the term can also be time-dependent (see later section on peristimulus time histograms). The LGN input targets both layers. There is anatomical evidence that thalamo-cortical synapses target mainly layer IV and to a lesser extent layer VI [42], [45]. Accordingly, in all the simulations presented in this paper, in the lower layer is smaller by a factor of two than in the upper layer. Parameters describing LGN input properties are given in Table 6. For the adopted parameters, feed-forward inputs from LGN never dominate over recurrent inputs from the two layers of the network, consistently with the massively larger number of cortico-cortical synapses than thalamo-cortical synapses in the primary visual cortex [45]. The relative weight of feed-forward inputs with respect to recurrent inputs depends on contrast, doubling in our model from no more than 20% at low contrast to no more than 40% at high contrast stimulation (not shown). An alternative parameter choice for the tuned component of the LGN input, leading to noisy input current with a larger variance, is reported in Table S1. For the noisy inputs used in this paper as well as for the ones of Table S1, the resulting sub-threshold voltage fluctuations are on the order of at full contrast, compatible with experimentally observed fluctuation ranges [115], [116]. Voltage fluctuations are comparable in the two regimes, because the increase in amplitude of external input current fluctuations is paralleled by a decrease in amplitude of net input conductance fluctuations, due to reduced synchrony among the recurrent inputs (see Figure S14). More details about the mapping from stimulus contrast to input rates can be found in Text S2 and Tables S2 and S3. In addition to the LGN input, excitatory and inhibitory cells are driven by an untuned noisy input, representing the background firing of other cortical areas. This input is modeled by a single AMPA-type synapse per cell, with peak conductance activated by Ornstein-Uhlenbeck processes [61]. Input spikes are generated independently for each cell; however all the cells share the same instantaneous input rate obeying the stochastic differential equation: (13) where stands for Gaussian white noise and is the mean, the volatility and the filtering time-constant of the stochastic process. Parameters are given in Table 7. The dynamical equations are integrated using a fourth-order non-adaptive step Runge-Kutta scheme. Integration step was 0. 2 ms. Because of the exponentially fast divergence of the membrane in relation with firing, particular care is needed to ensure the stability of the numerical integration of equation (1). Following [108], the numerical integration of the membrane potential of a given neuron is stopped as soon as reaches a finite cutoff voltage. In our simulations, we use. This choice ensures that the non-linear term is the dominant contribution to the neuronal currents for. Under this condition, the leakage and the synaptic currents can be neglected, making it possible to compute analytically the time left before the actual divergence of the potential. Assuming that the integration is stopped at when, the time of the next spike is given by. In addition, for our choice of, is large compared to the integration-step, thus avoiding numerical errors in spike-time estimation due to the exponentially fast growth of in proximity of the divergence. The membrane potential is then reset to a value immediately after a spike. The Ornstein-Uhlenbeck process giving is computed using the properties of the exact solution to equation (13). This means that is normally distributed with mean and standard deviation [117]. In order to study the tuning properties of the neuronal responses we present stimuli at 12 different orientations in an interval between −90 degrees and +90 degrees, at least five different contrast values per each orientation. Tuning curves are derived for each neuron by measuring their average firing rate for each of the tested orientations and contrasts and are characterized by computing their skewness and their circular variance [118]. The peak rate is defined as the maximum response rate generated by each single neuron over the stimulus set. Denoting by the response firing rate for a stimulus of orientation, we define the complex vector: (14) The broadness of a tuning curve is quantified by circular variance (Mardia 1972; Ringach et al. 2003): (15) where is the modulus of. It is therefore a quantity bounded between 0 and 1. Deviation from symmetric tuning is quantified by a circular skewness coefficient (Kenney and Keeping 1962): (16) where is the angle (in degrees) of the complex vector and the preferred stimulus orientation. A skewness of 0 means a symmetric tuning curve and larger (or smaller) values denote tuning curves skewed toward the right (or the left). Single neuron tuning curves and the corresponding parameters for the upper layer excitatory population are shown in Figure S2. Population average tuning curves are computed after rotating single neuron tuning curves so that their maximum is at (see Figure 2A). The contrast-response functions, CRF, are computed for each neuron by measuring its peak firing rate (i. e. its firing response to a preferred orientation stimulus) at each given level of contrasts. Each individual CRF is fitted to a hyperbolic ratio function [60]: (17) Single neuron CRFs and the corresponding parameters for the upper layer excitatory population are shown in Figure S4 and population average CRFs in Figure 2B. To measure the degree of macroscopic synchrony in the steady state of a network comprising an arbitrary number of neurons, we follow the method used in [34], [35]. It is grounded on analysis of the temporal fluctuations of macroscopic observables of the networks such as the instantaneous activity or the instantaneous membrane potential averaged over a population of neurons of size. For instance, for the latter, one evaluates at a given time,, the quantity: (18) The variance of the time fluctuations of is (19) where denotes time-averaging over a large time,. After normalization of to the average over the population of the single cell membrane potentials: (20) one defines a synchrony measure, by: (21) This measure takes values between 0 and 1. In the limit it behaves as: (22) where is some constant, between 0 and 1. In particular,, if the system is fully synchronized (i. e., for all), and if the state of the system is asynchronous. Asynchronous and synchronous states are unambiguously characterized in the thermodynamic limit (i. e., when the number of neurons is infinite). In the asynchronous state,. By contrast, in synchronous states,. To characterize the degree of synchrony in the membrane potentials of neurons and, we compute the cross-correlation function: (23) The value of the normalized cross-correlogram for zero time-lag gives the pairwise crosscorrelation coefficient (CCo): (24) To estimate the degree of synchrony in the spiking activity of these neurons, discrete spike trains are first convolved with a square window of width, thus generating a continuous spike-count signal. Equations (23) and (24), with replaced by such smoothed spike trains, is used to compute crosscorrelograms and CCos for spiking activities [119]. We use a smoothing window size of. CCos and crosscorrelograms are estimated over simulated recordings lasting 100 s of real time. For CCos between membrane potentials only pairs of neurons within a 18 region centered on an angular coordinate matching the orientation of the presented stimulus are considered. In the case of spike trains, neurons in this region whose spike train contained fewer than 100 spikes are further excluded. Various stimulus orientations are pooled together to improve the estimation of CCo distributions. LFPs are believed to be an aggregate measure of the synaptic activity of several hundreds of neurons in the vicinity of the recording electrode [120], [121]. To evaluate the LFP in a given site, we thus arbitrarily average input synaptic currents in a small angular sector of centered on the considered angular position. LFPs are estimated over neurons of the upper layer only, reflecting the fact that superficial neurons should supply the largest contribution to the signal recorded by an applied recording tip. For the normally used size of excitatory neurons and inhibitory neurons per layer, this corresponds to averaging over 200 excitatory and 50 inhibitory upper layer neurons for each considered LFP recording site. Autocorrelograms of the LFPs are computed as: (25) the zero-lag value measures the variance of the temporal fluctuations of the LFP and has known size-scaling properties, which are different in synchronous and asynchronous regimes (see previous discussions and [35]). Power spectra are computed using conventional FFT techniques, as the square modulus of the Fourier Transform of signal autocorrelation. Windowing is applied to LFP-like signal time-series to reduce unwanted frequency leakage, following the Welch method [122]). An additional moving average smoothing is applied for visualization purposes. We measure power in arbitrary logarithmic units. Since we are interested in qualitative analysis of the overall shape of the spectra rather than in absolute power estimations, for each considered regime we assign a unit value at the power at 0 Hz frequency for 0% of contrast. Autocorrelation and spectral analysis of LFP-like signals are based on time-series lasting 100 s of real time, with a sampling rate of 5 kHz. The MUA signal reflects the spiking activity of few neurons in the immediate vicinity of the recording electrode [123]. Typically, the recorded MUA is separated in only a small number of contributing single units [124]. To evaluate MUA at a given site, following [114], we sum together the spike trains of three randomly selected cells within a small angular sector of centered on the considered angular position (the same used for the evaluation of the LFP). This discrete signal is then transformed into a continuous signal by convolving it with a gaussian window (1 ms of variance). We compute then the coherence [125] between the LFP and the MUA at a same site by taking the modulus of the normalized product of their complex Fourier Transform, using the Welch method [122], as in the case of the LFP power spectrum estimation: (26) where and denote the (complex) Fourier transform of the autocorrelograms of the LFP and of the MUA signals, respectively. Such MUA-LFP coherence is a real quantity in the unit interval, and provides an absolute (linear) measure of the phase synchronization between the two signals in different frequency bands. We average then the result over twenty different randomly chosen triplets of cells, in agreement with the experimental habit to average together different MUA recordings with only approximately similar selectivity properties [114]. Layer decoupling experiments are performed by multiplying the peak conductances of all the AMPA-type, NMDA-type and GABA-type synapses from the lower layer to the upper layer by a factor varying between 1 and 0. A value of 1 corresponds to the case of fully-interacting layers, and a value of 0 corresponds to fully uncoupled layers. For each excitatory neuron in the upper layer, at high contrast, the peak response after layer decoupling is compared with the peak response of the same neuron in the fully coupled network case. In comparing peak responses, we take into account the fact that the tuning curves of many neurons change their preferred orientation after full or partial lower layer inactivation. To simulate the flashing of a grating, for a given network realization we perform numerical simulations in which the tuned LGN input rate is not constant. More specifically, this tuned component is modeled according to equations (11) and (12), with a contrast modulated in time: (27) Phases lasting 0. 5 s in which are therefore alternated with phases lasting 1 s in which, leading to a square wave time-course of the input LGN rate. We consider only cells whose preferred orientation falls within a sector wide centered on the orientation of the presented stimulus and we use four different stimulus orientations. For each of the orientations, the stimulus is flashed 1000 times. An overall sample of 800 cells (200 per orientation) is thus considered. Spikes across stimulus repetitions and cells are binned into 2 ms bins according to their timing relative to stimulus onset. The bars in the peristimulus time histograms (PSTHs) are then evaluated as (number of spikes in a time-from-stimulus bin) / (number of stimuli repetitions) / (number of sampled cells). To study the sensitivity of induced dynamics to a small perturbation, we perform a simulation in which just a single spiking even is omitted and we compare it with the unperturbed simulation. We select a putative spiking time of a neuron whose preferred orientation matches the one of the applied 95% of contrast stimulus. No spike is then sent to the post-synaptic targets, we only reset the potential and the other time-dependent parameters of the failing presynaptic neuron to their just-after-spike values. Precisely the same realizations for all the stochastic noisy input processes are taken for the unperturbed and the perturbed dynamics. We measure the largest Lyapunov exponent of the induced dynamics of the system at high contrast through a non-linear analysis of a long time-series (600 minutes of real time) of the associated LFP signal. For a thorough introduction and a rationale to the used methodologies the reader is invited to refer to textbooks like [68]. The first step is the construction of proper “embeddings” of this time-series. Given a discretely sampled time-series, a reconstruction delay and an embedding dimension, we construct a new -dimensional sequence: (28) It can be proven [126], [127] that the latter time-delay embedding provide in general a one-to-one image of the original phase-space attractor of the dynamics generating the measured time-series, provided that the used embedding dimension is large enough. The general idea of the method is then to identify by systematic search pairs of points and which lie at a (euclidean) distance in the delay-embedding space smaller than a specified very small. Such points are said to be neighbors. It is therefore possible to consider the distance as a “small perturbation”, which should grow exponentially in time if the dynamics is chaotic. The eventual divergence of the trajectories originating by neighbor points can be monitored by the quantity. If there is a time range for which then coincides with the maximum Lyapunov exponent [128], [129]. We select a reconstruction delay of, much larger than the decorrelation time of the induced LFP oscillation. The minimum embedding dimension for a consistent estimation of the largest Lyapunov exponent can be estimated by monitoring the fraction of “false neighbors” pairs, i. e. pairs of points that are neighbors in a -dimensional embedding (due to a projection of the attractor to a space with a too small dimensionality) but that there are no more such in an embedding with a larger dimension [130]. Such analysis, summarized in Figure S10, indicates a critical embedding dimension lower than five and probably larger than three (even if a precise estimation is difficult due to the presence of noise). Practically, we estimate the largest Lyapunov exponent by evaluating the quantity: (29) for various, and, where is the set of points at a distance from and denotes average over time. If displays a linear increase in a reasonable range of with matching slopes for different sufficiently large and for few decades of, then the average slope of the linear sections of provides a robust estimation of. More details on our estimation of for the high contrast induced LFPs for and are given in the Text S1.

Title: Synchronous Chaos and Broad Band Gamma Rhythm in a Minimal Multi-Layer Model of Primary Visual Cortex Summary: Visual stimulation elicits neuronal responses in visual cortex. When the contrast of the used stimuli increases, the power of this induced activity is boosted over a broad frequency range (30-100 Hz), called the "gamma band." It would be tempting to hypothesize that this phenomenon is due to the emergence of oscillations in which many neurons fire collectively in a rhythmic way. However, previous models trying to explain contrast-related power enhancements using synchronous oscillations failed to reproduce the observed spectra because they originated unrealistically sharp spectral peaks. The aim of our study is to reconcile synchronous oscillations with broad-band power spectra. We argue here that, thanks to the interaction between neuronal populations at different depths in the cortical tissue, the induced oscillatory responses are synchronous, but, at the same time, chaotic. The chaotic nature of the dynamics makes it possible to have broad-band power spectra together with synchrony. Our modeling study allows us formulating qualitative experimental predictions that provide a potential test for our theory. We predict that if the interactions between cortical layers are suppressed, for instance by inactivating neurons in deep layers, the induced responses might become more regular and narrow isolated peaks might develop in their power spectra.

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Summarize: Introduction The Education for Homeless Children and Youth Program (EHCY) is intended to ensure that all homeless children and youth have equal access to the same free and appropriate public education, including public preschool education that is provided to other children and youth. It is the only federal education program exclusively focused on homeless children and youth. Federal education funding for homeless children and youth is provided by EHCY and through required set-asides for homeless children and youth from Elementary and Secondary Education Act (ESEA) Title I-A funds received by local educational agencies (LEAs). Reauthorization of EHCY may be considered by the 113 th Congress as part of the reauthorization of ESEA. This report provides an overview of the purposes and program structure of EHCY; the history of the program's funding; issues that have arisen regarding the implementation of ESEA Title I-A set-asides for homeless students; data on the number of LEAs receiving EHCY grants and on the characteristics of homeless students; and a discussion of proposed changes to EHCY included in bills introduced in the 112 th Congress to reauthorize the ESEA. The report also includes two appendices: the first provides information on the number and percentage of homeless students by state; the second discusses legislation adopted in the 110 th -111 th Congresses that impacts EHCY. Education for Homeless Children and Youth Program: McKinney-Vento Homeless Assistance Act, Title VII, Part B Program Overview EHCY provides grants to state educational agencies (SEAs) to help ensure that all homeless children and youth have equal access to the same free and appropriate public education, including public preschool education that is provided to other children and youth. The text box above provides the definition of "homeless" that is used in the program. SEAs competitively subgrant funds to local educational agencies. Not all LEAs receive EHCY grants. In school year (SY) 2010-2011, 3,651 LEAs, out of a total of 16,290, received awards. In order to receive EHCY funds, each state must submit a plan indicating how homeless children and youth will be identified; how the state will ensure that homeless children will participate in federal, state, and local food programs, if eligible; and how issues such as transportation, immunization, residency requirements, and the lack of birth certificates or school records will be addressed. EHCY grants are allocated to SEAs in proportion to grants made under Title I-A of the ESEA, except that no state can receive less than the greater of $150,000, 0.25% of the total annual appropriation, or the amount it received in FY2001. The Department of Education (ED) reserves 0.1% of the total appropriation to provide grants to outlying areas (Virgin Islands, Guam, American Samoa, and the Commonwealth of the Northern Mariana Islands), and 1.0% for the Department of the Interior for services to homeless children and youth provided by the Bureau of Indian Education. The law also authorizes the reservation of funds for national activities but does not specify a percentage. Authorized national activities include program evaluation, technical assistance, and information dissemination. States may reserve up to 25% of their EHCY funding for state activities. "Minimally funded" states are permitted to reserve up to 50% of their funding for state activities. After state reservations, remaining state funds are subgranted to LEAs competitively, based on the LEA's need and the quality of its application. LEAs may receive subgrant funds for a period of up to three years, and they may reapply for a new subgrant when their current subgrant expires. LEAs are authorized to use the funds for the following activities: The excess costs of transportation so that a student can attend his/her school of origin; Supplemental educational services, including tutoring; Provision of expedited evaluations on the strengths and needs of homeless students; Professional development to assist school personnel in understanding the needs and rights of homeless students; Referral services for medical, dental, mental, and other health services; Provision of early childhood education not otherwise available; Services and assistance to retain homeless students, including unaccompanied youth, in school; Mentoring and out-of-school time programs; Payment of costs for tracing, obtaining, and transferring needed records; Education and training of parents of homeless students regarding their student's rights; Coordination between schools and service agencies; Provision of, or referral for, services such as violence prevention counseling; Activities to address domestic violence; Adapting school space and purchasing supplies needed to provide services; Provision of school supplies; and Other emergency assistance needed to enable homeless students to attend school. Functions of the State EHCY Coordinator and Local EHCY Liaisons Each state is required to designate an EHCY coordinator, and each LEA is required to appoint a local EHCY liaison (whether they have an EHCY grant or not). The law is silent on the issue of whether a state coordinator must work full time on EHCY duties—most coordinators are responsible for additional programs. The law explicitly states that the local liaison may be a coordinator of other federal programs in addition to EHCY. The state EHCY coordinator is responsible for implementing the state's plan for the education of homeless children and youth; gathering reliable, comprehensive data on the difficulties homeless students have enrolling in public school, including public preschools; identifying any progress made by the state in ensuring homeless students are enrolled in, attending, and succeeding in school; submitting reports that the Secretary of Education (Secretary) determines are necessary; providing technical assistance to LEAs in the state; and facilitating coordination between the SEA and other entities and providers including state social service agencies, educators, preschool providers, and other community organizations and agencies that provide services to homeless children and youth and their families. Local EHCY liaisons are responsible for identifying and enrolling homeless students and ensuring that these students have a full and equal opportunity to succeed in school; ensuring that these students are receiving all the education services for which they are eligible; providing referrals to health care, dental, mental health, and other appropriate services; informing parents of the educational and other opportunities available to their children and providing meaningful opportunities for them to participate in their children's education; providing families and youth with information on their right to transportation, including transportation to the school of origin; widely disseminating public notice of the rights of homeless students and youth; and ensuring that enrollment disputes are mediated. Program Funding Regular Appropriations Appropriations for EHCY increased following the program's most recent reauthorization by ESEA ( P.L. 107-110 )—from $35.0 million in FY2001 to $50.0 million in FY2002. This increase was, however, substantially lower than the amount authorized for EHCY in FY2002 by P.L. 107-110. It authorized "$70,000,000 for EHCY for FY2002 and such sums as may be necessary for each of fiscal years 2003 through 2007." Funding continued to increase in FY2003-FY2005, then decreased to $61.9 million in FY2006 and FY2007. Regular appropriations have remained at approximately $65 million since FY2009. (See Table 1.) American Recovery and Reinvestment Act (P.L. 111-5) Funding In addition to regular appropriations, the EHCY program received $70 million in American Recovery and Reinvestment Act (ARRA, P.L. 111-5 ) funds in FY2009. Funds were awarded to SEAs on April 10, 2009; SEAs were required to award ARRA funds to LEAs within 120 days (by August 8, 2009). LEAs were required to obligate funds by September 30, 2011. States and LEAs were required to keep a separate record of how ARRA funds were used and states were required to report this information to ED quarterly. ARRA funds were to be used for the same local activities as those authorized by the regular EHCY program. After reservations (0.1% for the outlying areas and 1% for the Bureau of Indian Education), ARRA funding was distributed by formula to states. The formula used to distribute ARRA funds to states was different from the formula used to distribute regular appropriations. ARRA funds were distributed to states in proportion to their reported share of total enrolled homeless students in SY2007-2008. Unlike regular EHCY funding that is awarded by SEAs to LEAs competitively, states could choose to award subgrants to LEAs competitively or by formula (based on numbers of homeless students), or they could use a combination of both methods. States were permitted to calculate their FY2009 reservation for state activities based on the total of their ARRA allocation and their regular FY2009 program allocation. However, any funding reserved for state activities had to be drawn from regular EHCY program allocations, not from ARRA funding. First Focus and the National Association for the Education of Homeless Children and Youth (NAEHCY) jointly issued a report in 2010 that examined the increasing number of homeless students reported as enrolled by LEAs, and how ARRA funds were being used by LEAs. The report was based on a survey of 2,200 LEAs from 47 states and 45 Homeless Education state coordinators. Survey respondents indicated that ARRA funds were being used for a variety of purposes, including academic support, transportation, outreach and identification, increasing support for specific subpopulations of homeless students, and enhancing collaboration with other community organizations. According to the report: Prior to the receipt of ARRA funds, many school districts had never received funding to support homeless children and youth. For the first time, these districts have been able to dedicate resources to procedures and programming to ensure that homeless children and youth are identified, enrolled, and attending school. Respondents frequently commented that loss of funding would result in loss of educational access, stability, and success. Due in large part to ARRA funding, there was a 76% percent increase in the number of LEAs receiving EHCY grants between SY2008-2009 and SY2009-2010 (from 1,729 to 3,046). Elementary and Secondary Education Act (ESEA), Title I-A Provisions on Homeless Education Children and youth who are homeless are automatically considered eligible for ESEA Title I-A services, whether or not they meet the performance requirements normally required for Title I-A eligibility. In addition, LEAs are required to set aside a portion of their Title I-A funds for homeless students who do not attend schools receiving Title I-A funds: A local educational agency shall reserve such funds as are necessary under this part to provide services comparable to those provided to children in schools funded under this part to serve—(A) homeless children who do not attend participating schools including providing educationally related support services to children in shelters and other locations where children may live. However, ED non-regulatory guidance has indicated that the Title I-A funding set aside for homeless students not attending Title I-A schools may also be used to provide services for homeless students attending Title I-A schools. ED non-regulatory guidance also states that comparable services provided to homeless students may include services that are specifically tailored to the needs of homeless students: An LEA has the discretion to use reserved funds to provide a homeless student with services that are not ordinarily provided to other Title I students and that are not available from other sources. For example, where appropriate, a LEA at its discretion may provide a student with an item of clothing to meet a school's dress or uniform requirement so that students may effectively take advantage of educational opportunities. The guidance also indicates that Title I-A funds may not be used to provide transportation to the school of origin for homeless students, since this would violate the supplement-not-supplant provisions applicable to Title I-A funds (because EHCY requires that transportation to the school of origin be provided for homeless students, if requested). However, non-regulatory guidance states that "it may be appropriate in certain circumstances for an LEA to use title I, Part A funds to transport formerly homeless students to or from their school of origin for the remainder of the school year in which they become permanently housed." National data on the amount of ESEA Title I-A funding being set aside by LEAs for homeless students are not available. Some states require LEAs to report this information. ED does not require that the amount set aside be reported. Program and Related Data LEA and Participant Data All states are required to report data to ED on the number of homeless students enrolled in school each year, regardless of whether or not they receive an EHCY grant. In SY2009-2010, there were 16,290 LEAs; 3,651 (22%) of these LEAs received EHCY subgrants in that year. Although only 22% of LEAs received EHCY grants in SY20010-2011, they accounted for 71% of all enrolled homeless students in that year. It is important to note, however, that the number of homeless children and youth discussed here only includes those who are enrolled in school. As a consequence, because these data do not include the number of homeless students not enrolled in school, they do not represent the total number of homeless children and youth. In SY2008-2009, 956,914 homeless students were reported enrolled; in SY2009-2010, the number of enrolled homeless students reported was 939,903; and in SY2010-2011, 1,056,794 homeless students were reported enrolled (see Figure 1 ). The total number of homeless students enrolled declined by 2% between SY2008-SY2009 and SY2009-2010, but it increased by 13% between SY2009-2010 and SY2010-2011. The SY2008-2010 decline was due in part to changes in data collection procedures in California. If California were excluded from the total, there would have been an increase of 11% in enrolled homeless students between SY2008-2009 and SY2009-2010. According to the First Focus and NAEHCY report discussed above, between SY2006-2007 and SY2008-2009, the number of homeless students increased from 679,724 to 956,914. Sixty-two percent of respondents attributed the increase in homeless students to the economic downturn; some of the additional factors cited included greater awareness of homelessness (40%), the foreclosure crisis (38%), improved outreach and identification (33%), and an increase in factors affecting mental or physical health, such as incidents of domestic violence or substance abuse (29%). Four states accounted for 42% of the total number of students enrolled in both LEAs with EHCY subgrants and those without in SY2010-2011. Those states, and their percentages of total homeless student enrollment, were California (21%), New York (9%), Texas (8%), and Florida (5%). Forty-four states reported an increase in the number of homeless students enrolled between SY2009-2010 and SY2010-2011; nine states reported a decrease in the number of homeless students enrolled during this period. (See Appendix A for SY2008-2011 data on homeless student enrollment by state.) Homeless Preschool Students States are required to report on the number of homeless children (ages 3-5, not in kindergarten) enrolled in public preschool programs. For SY2008-2009, 33,433 preschoolers were reported enrolled in public preschool programs; this compares to 30,955 enrolled in SY2009-2010, and 36,308 in SY2010-2011. It is important to note, however, that obtaining accurate counts of homeless preschoolers is even more difficult than it is for homeless students in grades K-12. (See the discussion below on reauthorization issues.) Primary Nighttime Residence States must also report data on the primary nighttime residence of each homeless student who is counted as enrolled in school. The list of reporting options for primary nighttime residence includes shelters (this category includes both transitional housing and awaiting foster care); doubled-up (e.g., living with another family); unsheltered (e.g., cars, parks, campgrounds, temporary trailers, or abandoned buildings); and hotels/motels. In SY2010-2011, all LEAs reporting indicated that doubled-up accounted for the largest share of homeless students (72%). Doubled-up has accounted for the largest share of reported primary nighttime residences during the entire three-year period from SY2008-2009 through SY2010-2011. Additional primary residences reported for SY2010-2011, by share of total homeless students, were shelters (18%); hotels/motels (5%); and unsheltered (5%). Subpopulations of Homeless Students Each LEA that receives an EHCY grant is required to provide additional data on homeless students not required of LEAs without an EHCY grant. The additional information required includes data on subpopulations of homeless students served, and information on academic achievement. Data are reported on the following subpopulations of homeless students served: English language learners, children with disabilities, unaccompanied youth, and migratory children and youth. During the three-year period between SY2008-2009 and SY2010-2011, the percentage increase in homeless students served was 55% for migratory children and youth, 51% for English language learners, 51% for children with disabilities, and 4% for unaccompanied youth. Academic Progress LEAs with EHCY subgrants report on both the number of homeless students tested each year and on the proficiency of these students in reading and mathematics. Homeless students must be tested annually in grades 3-8 and once in high school. Data on homeless students' proficiency based on state tests are reported in each state's annual Consolidated State Performance Report (CSPR) submitted to the Department of Education. However, there are no data on the academic performance of homeless students that rely on the same measure of proficiency across states. As a consequence, these reported proficiency rates are not comparable across states. As of SY2010-2011, academic performance data was required to be reported for homeless children enrolled in all LEAs. Previously, data were reported only for students served by LEAs with EHCY grants. As a consequence, data from earlier years are not comparable with data for SY2010-2011. Data for SY2010-2011 are reported in Table 2. For FY2010-2011, out of all enrolled homeless students, 71% of students in grades 3-8 and 15% of high school students took the reading and mathematics assessment tests. In reading, 52% of students in grades 3-8 and 49% of high school students met or exceeded state proficiency standards. In mathematics, 51% of students in grades 3-8 and 44% of high school students met or exceeded state proficiency standards. Reauthorization Issues The final section of this report discusses changes to EHCY proposed by legislation introduced in the 112 th Congress. Reauthorization of EHCY may be considered by the 113 th Congress as part of ESEA reauthorization. Where relevant, background information on the context for proposed changes is also included. On October 20, 2011, the Senate Committee on Health, Education, Labor, and Pensions (HELP) ordered reported a bill to reauthorize the ESEA, titled the Elementary and Secondary Education Reauthorization Act of 2011 ( S. 3578, hereinafter referred to as the Senate HELP Committee bill). Reauthorization of EHCY was included as part of this legislation. On February 28 th, 2012, the House Committee on Education and the Workforce (Education and Workforce) ordered reported two bills to reauthorize portions of the ESEA. One of these bills H.R. 3990, the Encouraging Innovation and Effective Teachers Act (hereinafter referred to as the House Education and Workforce Committee bill ( H.R. 3990 )), would have reauthorized the EHCY program. The other bill H.R. 3989, the Student Success Act (hereinafter referred to as the House Education and Workforce Committee bill ( H.R. 3989 )), included amendments to ESEA Title I-A that would have impacted EHCY. In addition, H.R. 32, The Homeless Children and Youth Act of 2011, was ordered reported by the Subcommittee on Insurance, Housing, and Community Opportunity of the House Committee on Financial Services on February 6, 2012. This bill is discussed under the Definition heading in this section of the report. The EHCY issues discussed in this section include: funding; transportation of homeless students to their school of origin; ESEA Title I-A set asides; separate schools for homeless students; clarifying the "best interest" school selection process; enhancing the ability of LEA homeless liaisons and state coordinators to meet the needs of homeless students; improving identification and services for preschool students and unaccompanied youth; increasing access to education and related services for homeless students; the definition of homeless children and youth in EHCY and other federal programs; and additional issues. Funding There has been some concern that funding for the EHCY program may not be sufficient to meet the educational needs of the increasing number of homeless children and youth. Although the number of homeless students reported enrolled by LEAs increased 11% between SY2008-2009 through SY2010-2011, EHCY regular appropriations have been stagnant in recent years (see Table 1 ). Nevertheless, in the current context of limited federal resources, funding increases in the near future for many federal programs, including EHCY, may be unlikely. The Senate HELP Committee bill would have authorized "such sums as may be necessary for fiscal year 2012 and each of the 6 succeeding fiscal years." The House Education and Workforce Committee bill ( H.R. 3990 ) would have authorized "$65,173,000 for fiscal year 2013... " and stated that this amount "shall be increased for each of fiscal years 2014 through 2018 by a percentage equal to the percentage of inflation according to the Consumer Price Index, for the calendar year ending prior to the beginning of that fiscal year." Transportation of Homeless Students to Their School of Origin As discussed earlier in this report, transportation is cited as the most serious barrier to the enrollment of homeless students. Many school districts have limited funds, and transportation is an expensive service to provide. Current law prohibits the use of ESEA Title I-A funds to provide EHCY transportation, except under limited circumstances (see earlier discussion). National level estimates of the costs of transporting homeless students are not available. Therefore, data from a survey of eight districts in Washington state are included to provide an illustration of the potential costs of transporting homeless students. These data indicate that: The majority of homeless student trips (79 percent) cost the school districts from just under $3 per one-way trip to over $40 per one-way trip, as opposed to an average of just $.67 for the general student population. Transportation of homeless students by school bus accounted for the largest percentage of student rides provided overall (38 percent), with costs ranging from $4.24 to $53.79 per one-way trip. In addition to the costs involved in transportation, there are often disputes among LEAs regarding how costs of transportation are to be allocated if a student is temporarily housed in one district, but is transported to the school of origin in another district. These disputes can potentially delay the enrollment of homeless students in their school of origin. Some have suggested that this situation could be improved if the following issues were addressed as part of ESEA reauthorization: more specific requirements were included on the rules for cost sharing by LEAs; a separate funding stream was provided for transportation; and LEAs were permitted to use their ESEA Title I-A set aside for transportation costs. The Senate HELP Committee bill included new language that would have required states and LEAs to ensure that transportation to the school of origin was provided for as long as the student had the right to attend that school. Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3989 ) included new language that would have permitted ESEA Title I-A funds to be used to provide transportation for homeless students to their school of origin. ESEA Title I-A Set-Aside for Homeless Students LEAs are responsible for developing their own procedures for determining the amount of ESEA Title I-A funds that are set aside for homeless students; as a consequence, the amount of Title I-A funding set aside for this purpose varies considerably across LEAs. Homeless advocates argue that the lack of clarity regarding Title I-A set-asides has led to inadequate set-asides. Although no national data on set-asides are available, a survey of local homeless liaisons by the National Association for the Education of Homeless Children and Youth (NAEHCY) in January of 2010 included a question asking liaisons whether their district (LEA) set aside Title I-A funds for homeless students, and if so, how much was set aside. Out of the 2,234 liaisons who answered the question, 67% indicated that their district did set aside Title I-A funds; 23% indicated that their district did not set aside funds; and 11% did not know. The Senate HELP Committee bill included new provisions under ESEA Title I-A that would have require d Title I-A set-asides to be based on a needs assessment that reflected the number of homeless children and youth identified in the previous year and included the collaboration and input from other agencies. It also would have expanded the allowable uses of these funds to include, among other things, funding for local liaisons and the provision of transportation to the school of origin. The House Education and Workforce Committee bill ( H.R. 3989 ) included new provisions under ESEA Title I-A stating that Title I-A EHCY set-asides may be based on: a needs assessment that reflected the number of homeless children and youth identified in the previous year; and included collaboration and input from other agencies. It also would have expanded the allowable uses of these funds to include, among other things, the provision of transportation to the school of origin. Separate Schools for Homeless Students The most recent reauthorization of the ESEA ( P.L. 107-110 ) amended EHCY to explicitly prohibit states that receive program funding from segregating homeless students from non-homeless students, except for short periods of time for health and safety emergencies or to provide temporary, special, supplementary services. An exception was made for four counties that operated separate schools for homeless students in FY2000 (San Joaquin, Orange, and San Diego counties in California, and Maricopa County in Arizona), as long as (1) those separate schools offer services that are comparable to local schools; and (2) homeless children are not required to attend them. Four schools operate under this exception. Some argue that segregation is harmful to homeless students and no exceptions should be permitted. They believe that homeless students are better served, and perform better academically, if they attend the same schools as other students. Others argue that homeless students benefit from the wider array of services that are provided in separate schools, and that the greater sense of community and belonging provided by these schools is critical for homeless students. Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) would have continued the overall prohibition on the segregation on homeless students; they would have also retained the limited exception for those schools in exempt counties that were in operation in FY2000, as under present law. The House Education and Workforce Committee bill ( H.R. 3990 ) also included language that would have increased the reporting frequency (to once a year) for the Secretary's required report on separate schools. Clarifying the "Best Interest" Determination Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) would have required that the LEA presume that keeping a homeless student in the school of origin would be in the student's best interest unless the parent or guardian disagrees. In addition, both bills would have required that the "best interest" determination by an LEA consider student-centered factors "including factors related to the impact of mobility on achievement, education, health, and safety of homeless children and youth... " Both bills included new language stating that if an LEA determined it to be in the best interest of the homeless student to attend a different school, it must provide the parent, guardian, or unaccompanied youth with its decision in writing and must include information on appeal rights in a form that is understandable to parents and guardians of homeless children and youth. The House Education and Workforce Committee bill ( H.R. 3990 ) also included new language stating that "the term'school of origin' should include the designated receiving school at the next grade level for all feeder schools." This would have permitted, for example, homeless students graduating from elementary school to be eligible to attend the same middle school serving their elementary school as non-homeless students. The Senate HELP Committee bill stated that if parents and guardians have exhausted all available dispute procedures, they could appeal to the SEA. It also would have required that until there is a final resolution of the dispute, the LEA must enroll the student and provide transportation to the school of origin. In addition, it would have expanded the issues subject to dispute by explicitly adding: eligibility, services in a public school or public preschool, or any other issues relating to services. Homeless Liaisons and State Coordinators The EHCY program requires that each LEA, whether it receives an EHCY subgrant or not, designate a homeless liaison for the LEA. Many state coordinators and homeless liaisons are responsible for the coordination of several programs, and some argue that they have insufficient time to perform their duties under the EHCY. Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) would have included new language requiring that training be provided for local liaisons and other school personnel providing services under EHCY. In addition, the Senate HELP Committee bill stated that the Office of Coordinator for EHCY must have "sufficient knowledge, authority, and time to carry out the duties described in this title," and that local liaisons must have "sufficient training and time" to carry out their duties. The House Education and Workforce Committee bill ( H.R. 3990 ) stated that the Office of Coordinator for EHCY must be able to "sufficiently carry out the duties described in this subtitle." It would have also required each state coordinator to annually post data on the number of homeless children and youth identified in the state. Improving Identification and Services for Preschool Students and Unaccompanied Youth Homeless Preschoolers Homeless preschoolers are particularly difficult to identify, in part because they are not yet old enough to be covered by state compulsory attendance laws. For this reason, although preschool students are guaranteed equal access to public preschool and Pre-K programs under EHCY, they do not have the same guarantee of being able to attend public school as K-12 homeless students do. Although homeless preschoolers are eligible to attend public preschool programs, placement of homeless preschoolers in public programs is limited by the fact that there is not a sufficient supply of these programs, and programs that do exist often do not have sufficient vacancies to fill the need. In addition, preschoolers, unlike K-12 children and youth, are only guaranteed transportation if it is provided to non-homeless preschoolers at the school. This creates a significant barrier to school attendance for homeless preschoolers, because many homeless families are often unable to provide the needed transportation for their preschoolers. The Senate HELP Committee bill included an increased emphasis on school readiness for homeless preschoolers. New provisions would have required each state plan to ensure that preschools that are funded, administered, or overseen by an SEA or LEA prioritize the enrollment of homeless preschoolers, and coordinate the review of educational and related needs of homeless children and their families with the local liaison in the relevant service area. However, these preschools would not be required to immediately enroll homeless preschoolers if they are at full capacity. Unaccompanied Youth The term 'unaccompanied youth' includes a youth not in the physical custody of a parent or guardian. These youth are covered by the same protections that are provided to other homeless youth under EHCY. However, outreach to these youth is difficult because they usually do not stay in shelters, and in some cases they are fearful of being identified, even though they may wish to complete their education. Unaccompanied homeless youth have often run away from a difficult home situation, or been forced to leave by a parent or guardian. These students often struggle to find a place to sleep and meet their basic needs. In addition, many of these youth worry that attending school would be unsafe because the school might involve child welfare agencies or law enforcement. According to advocates, providing programs that make schools safe and welcoming to subgroups of unaccompanied youth who are overrepresented can help. Because of their situation, these youth benefit from receiving more flexibility in their school schedule than other students, and from diversified learning opportunities such as vocational education. Promising approaches to serving these students include providing a mentor; coordinating with other agencies to help meet the basic needs of these youth; as well as policies that facilitate the awarding of partial credit or recovery of previous credits. Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) included new language that would have required that homeless liaisons ensure that unaccompanied youth are enrolled and have the same opportunities to meet state academic standards as other students. In addition, both bills would have required that unaccompanied youth be told about their rights to apply for financial aid as an independent student. Both bills also included language that would have protected LEAs from liability for enrolling an unaccompanied youth without the consent of a parent or guardian. Increased Access to Education and Related Services Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) would have required that homeless students be able to participate in all the educational programs for which they meet the eligibility requirements. Both bills included language that stated that homeless children are to be immediately enrolled even if they have missed application or enrollment deadlines. In addition, the Senate HELP Committee bill would have required states to ensure that homeless students receive credits for previous coursework and that they not be hindered from enrolling due to overdue fines or fees. It also would have required that previous school records be released, even if a student owed fines or fees, or had not met the previous school's withdrawal requirements. Both bills would have required LEAs to coordinate services for homeless children and youth with disabilities with the Individuals with Disabilities Act and Section 504 of the Rehabilitation Act. Definition of Homeless Children and Youth under EHCY and other Federal Programs On February 7, 2012, H.R. 32, The Homeless Children and Youth Act of 2011, was reported out by the Subcommittee on Insurance, Housing, and Community Opportunity of the House Committee on Financial Services. This bill would have amended HUD's definition of homelessness to include children and families that have been verified as homeless through the following federal programs: the EHCY program, the IDEA Part C program, Early Head Start program, and the Runaway and Homeless Youth program. This change would have made these newly identified individuals and their families eligible for HUD housing assistance services, without requiring that they meet the detailed criteria or be subject to the funding cap included in the HEARTH Act ( P.L. 111-22 ). However, it would not have guaranteed that they would receive services. Receipt of services would have been based on local providers' needs assessments. An amendment (identified as Franken Title I-Amendment 3) introduced by Senator Franken and subsequently adopted as part of the final Senate HELP Committee bill, would have included the provision of services for foster children and youth. Among other things, the Franken amendment would have changed the definition of homeless in the EHCY by striking children who are "awaiting foster care placement" from the definition of homeless. This would have meant that children "awaiting foster care placement," as defined by each state, would no longer have been eligible for services under EHCY. The amendment would also have created a new program under ESEA Title I-A to ensure that foster children and youth would have improved access to education and related services. Only the proposed changes to the EHCY definition of homeless are discussed in the text of this report. However, a brief summary of the entire amendment is available in the separate text box below. Additional Changes Both the HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) would have incorporated changes to EHCY to increase cooperation across agencies and within ED to better serve homeless children. They both would have established privacy requirements to protect information on homeless children and youth's living situation from being released to anyone not authorized to have such information. Both the Senate HELP Committee bill and the House Education and Workforce Committee bill ( H.R. 3990 ) would have increased the amount of time that states have to submit required information for an EHCY grant to ED to 120 days. The Senate HELP Committee bill would have reduced the state set-aside to the greater of 20% of the state allocation or $85,000. It also would have expanded the time available for ED (from to 60 days to 90 days after enactment) to issue and publish school enrollment guidelines for states in the Federal Register. It also stated that the Secretary's required report on homeless children and youth must be published at least every two years (no timeline is specified in current law.) In addition, the Senate HELP Committee bill included provisions that would have allowed the Secretary to reserve EHCY funds for emergency assistance to homeless children, youth and their families, if appropriations for the program exceeded $70 million. The House Education and Workforce Committee bill ( H.R. 3990 ) would have removed the clause in current law requiring that an LEA provide an assurance in its application that it would maintain its fiscal effort at a percentage specified in law. Appendix A. Number and Percentage of Enrolled Homeless Students by State for Selected Years Appendix B. Legislation Adopted in the 110 th -111 th Congresses that affects the Education for Homeless Children and Youth Program Several bills were passed in the 110 th -111 th Congresses that will potentially increase the programs and/or services available to homeless students. This appendix summarizes the portions of these bills with relevance for homeless students. The Homeless Emergency and Rapid Transition to Housing Act (HEARTH Act) was passed as part of the Helping Families Save Their Home Act ( P.L. 111-22 ). This legislation amended and reauthorized the Housing and Urban Development (HUD) homeless programs under the McKinney-Vento Act, among other things. The HEARTH Act expanded the definition of homeless used by HUD and several other agencies. However, the definition of homeless used by ED in administering the EHCY program remains more expansive than the definition included in the HEARTH Act. The amended definition of homeless included in the HEARTH Act does allow children and youth that meet EHCY eligibility criteria (or eligibility under certain other federal programs) to be considered eligible under the HEARTH Act in limited circumstances. It requires that youth and families who are defined as homeless under another federal program meet each of the following criteria: They have experienced a long-term period without living independently in permanent housing. In its final regulation, HUD defined "long-term period" to mean at least 60 days. They have experienced instability as evidenced by frequent moves during this long-term period, defined by HUD to mean at least two moves during the 60 days prior to applying for assistance. The youth or families with children can be expected to continue in unstable housing due to factors such as chronic disabilities, chronic physical health or mental health conditions, substance addiction, histories of domestic violence or childhood abuse, the presence of a child or youth with a disability, or multiple barriers to employment. Under the final regulation, barriers to employment may include the lack of a high school degree, illiteracy, lack of English proficiency, a history of incarceration, or a history of unstable employment. The HEARTH Act includes additional provisions relevant for EHCY. The most important are the new requirements that in order to receive funds: Continuum of care (CoC) applicants must show that they have collaborated with LEAs in identifying homeless families, and in informing families and homeless youth of eligibility for EHCY services. CoC applicants must also show that they have considered the educational needs of homeless students when determining shelter placements, including placing families with children in shelters as close to the school of origin as possible. CoCs must obtain certification from each project applicant that provides housing or services that they have designated a staff person to ensure that children are enrolled in school and are connected to appropriate community services, and that these services are consistent with the provisions of Title VII of McKinney-Vento, as well as other relevant laws. The reauthorization of the HEAD Start Act through the Improving Head Start for School Readiness Act of 2007 ( P.L. 110-134 ) strengthened requirements regarding homeless preschoolers. Two of the most important changes are the requirement that homeless preschoolers be automatically deemed eligible for Head Start and Early Head Start; and a requirement that homeless children be identified and prioritized for enrollment in these programs. The Higher Education Opportunity Act, ( P.L. 110-315 ) included amendments prioritizing the provision of services for homeless students in the TRIO programs (several programs that provide support so that at-risk junior high and high school students graduate from high school and attend and complete college); and in the Gaining Early Awareness and Readiness for Undergraduate Programs (GEAR-UP) program. It also permits these programs to offer programs specifically designed for at risk groups, including homeless youth. The College Cost Reduction and Access Act ( P.L. 110-84 ) included provisions that allow youth who are verified as homeless and unaccompanied to be considered independent, thereby allowing them to apply for financial aid without the requirement that they obtain a parent's signature or provide information on family income. These students often face considerable obstacles to attending college, including the cost of attendance. This law is intended to ensure that these students will be able to apply for financial aid. It specifies that verification of independent student status must be made by a LEA homeless liaison, the director (or designee) of the Runaway Homeless Youth Act program, the director (or designee) of a Housing and Urban Development homeless shelter program, or a college financial aid administrator.

Summary: The Education for Homeless Children and Youth program (EHCY) provides formula grants to state educational agencies (SEAs) to help ensure that all homeless children and youth have equal access to the same free and appropriate public education, including public preschool education that is provided to other children and youth. It is the only federal education program exclusively focused on homeless children and youth. SEAs competitively subgrant funds to local educational agencies (LEAs). Not all LEAs receive EHCY grants. In school year (SY) 2010-2011, 3,651 LEAs, out of a total of 16,290, received awards. Although only 22% of LEAs received EHCY grants in SY2010-2011, they enrolled 71% of all homeless students in that year. Education and related services for homeless children and youth are also funded through required set-asides from Title I-A of the Elementary and Secondary Education Act. National data on the amount of funding set aside are not available. The EHCY program was most recently reauthorized as part of the Elementary and Secondary Education Act (ESEA, P.L. 107-110). Reauthorization of EHCY may be considered by the 113th Congress as part of the reauthorization of ESEA. EHCY received $65.2 million in funding for FY2012. EHCY is currently funded through March 27, 2013, by a government-wide Continuing Resolution (P.L. 112-175) at the FY2012 level plus 0.612%. All LEAs are required to report data to the Department of Education on the number of homeless students enrolled in school each year, regardless of whether or not they receive an EHCY grant. In SY2008-2009, 956,914 homeless students were reported enrolled in school; in SY2009-2010, 939,903 homeless students were reported enrolled; and in SY2010-2011, the number of enrolled homeless students reported was 1,065,794. The total number of homeless students enrolled decreased by 2% between SY2008-2009 and SY2009-2010; it increased 13% between SY2009-2010 and SY2010-2011. During the three-year period between SY2008-2009 and SY2010-2011, it increased by 11%. Four states accounted for 42% of the total number of students enrolled in both LEAs with EHCY subgrants and those without in SY2010-2011. Those states, and their percentages of total homeless student enrollment were, California (21%), New York (9%), Texas (8%), and Florida (5%). Legislation to reauthorize EHCY as part of the reauthorization of ESEA was reported by both House and Senate committees in the 112th Congress, and may be considered in the 113th Congress. Some of the issues that are under consideration include EHCY program funding; costs of transporting homeless students to their school of origin; implementation of the ESEA Title I-A set-aside for EHCY; whether to permit separate schools for homeless students; clarification of the "best interest" school selection process; how to enhance the ability of LEA homeless liaisons and state coordinators to meet the needs of homeless students; how to improve the identification of, and services provided to, preschool students and unaccompanied youth; how to increase access to education and related services for homeless students; and the impact of potential changes to the definition of homeless in EHCY and other legislation.

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Summarize: Credit: Paul Brennan/public domain A team of forensic linguistics experts believe they have solved a longstanding mystery, using a method similar to the one which discovered that Robert Galbraith – the author of detective story The Cuckoo's Calling—was in fact an alias for Harry Potter writer JK Rowling. In November 1864, a woman named Lydia Bixby received a letter supposedly written by President Abraham Lincoln telling her that her sons had been killed fighting in the Civil War. However, the letter—which subsequently became famous as one of the best letters written in the history of the English language – also became contentious, as historians have wondered whether it was, in fact, written by John Hay, Lincoln's secretary. With both men long dead and the original missing for more than 150 years, there seemed to be little hope of settling the question with any certainty. In a paper submitted to the journal Digital Scholarship in the Humanities, the researchers—including the University of Manchester's Dr Andrea Nini—say that a technique they developed leads them to believe that the letter was 'almost certainly' written by Hay. N-gram tracing, which involves using a computer to look for sequences of linguistic forms, is able to accurately analyse even very short pieces of writing in order to distinguish between different authors. The method was used by the researchers at Aston University's Centre for Forensic Linguistics to test 500 texts by Hay and 500 by Lincoln, before being used to analyse the Bixby letter. Nearly 90 percent of the time, the method identified Hay as the author of the letter, with the analysis being inconclusive in the rest of the cases. The researchers believe that the decades of uncertainty can now come to an end, leaving historians free to appreciate the letter's beauty, as well as the uncontested writing skills of both men, without the distraction of this lingering question. "Often, historical cases of disputed authorship involve very long texts, and there are several well-tested techniques that can be employed to solve these problems," said Dr Andrea Nini. "Because of its shortness the Bixby letter presented many challenges, and we had to devise a completely new method to analyse it." "We believe that this new method can now be also successfully applied to other cases—especially present-day forensic cases involving short threatening or malicious texts." Dr Nini is now hoping to solve another mystery—this time, to analyse letters supposedly written by Jack the Ripper. Explore further: New findings on debated authorship In November of 1864, a woman named Lydia Bixby received a letter from President Abraham Lincoln. He had been told that she had lost five sons to the then-ongoing Civil War. A Massachusetts state official had, learning of her plight, passed along her story. His request eventually made it to the White House. Though Bixby’s original copy of the letter was quickly destroyed or lost, the state official had also shared the text with the Boston Evening Telegraph, which published it. “I pray that our Heavenly Father may assuage the anguish of your bereavement,” the letter noted, “and leave you only the cherished memory of the loved and lost, and the solemn pride that must be yours to have laid so costly a sacrifice upon the altar of freedom.” A copy of the Bixby Letter. Library of Congress, Rare Book and Special Collections Division, Alfred Whital Stern Collection of Lincolniana. The brief, but eloquent missive struck a chord for many in the war-torn nation, and it has since become famous as one of the best letters written in the history of the English language. But it’s also one of the most “controversial” documents in Lincoln’s large body of writings. Though the letter has other complications to its history — such as, for example, the fact that it wasn’t true that Bixby had lost five sons, and despite her Boston address, her family said she was a Confederate sympathizer — the main point of contention has been whether or not Lincoln actually wrote it. Many historians have wondered whether perhaps it was written instead by his secretary, John Hay. Now, a team of forensic linguistics researchers think they have arrived at an answer once and for all. In a paper submitted to the journal Digital Scholarship in the Humanities, which will be presented at a linguistics conference that begins next week, they explain why they believe that the numbers show that the letter was written by Hay. Get your history fix in one place: sign up for the weekly TIME History newsletter “We’d never heard of Hay but we’d heard of Lincoln, obviously, and there’s loads of data,” says Jack Grieve, who worked on the project with colleagues Emily Carmody, Isobelle Clarke, Hannah Gideon, Annina Heini, Andrea Nini and Emily Waibel, as part of a working group at the Center for Forensic Linguistics at Aston University. Such teams have worked on everything from trying to use forensic linguistics to identify the creator of Bitcoin to using the same techniques to trace cybersecurity breaches by identifying hackers’ native languages. Using a technique they developed, called n-gram tracing, they arrived at the conclusion that the Bixby letter was was “almost certainly” written by Hay. But why was there ever any doubt? As Michael Burlingame has recounted in the Journal of the Abraham Lincoln Association, the doubt wasn’t just a matter of the lack of an original. Hay, who lived for decades after Lincoln’s assassination, apparently told several people privately during his lifetime that he had written it. The rumor spread, and by about a century ago, it had made its way to history books. On the other hand, Lincoln’s famed eloquence and the fact that the rumor only spread after both men’s deaths have made others reluctant to give anyone else credit for the missive, preferring instead to believe that if Hay said he had written it, he merely meant he had transcribed his boss’ dictation or copied it from a draft. With both parties long dead and the original missing for more than a century and a half, there seemed to be little hope of settling the question with any certainty. Enter forensic linguistics. This field — perhaps most famous in recent years for helping to out J.K. Rowling as Robert Galbraith — relies on the theory that, just as people from different regions may speak different dialects of the same language, each individual speaks and writes an even more subtle personal version of their language, known as an idiolect. “We pick up these idiolects over our lifetimes, not just because of where we grew up, but where we went to school, what kind of job we do, our personal history,” Grieve explains. Though the naked ear can’t often pick them up, computers can find and compare them by picking apart details such as the frequency of use of words as common as “the” or “and.” But while efforts have been made to analyze the Bixby letter to see whether it matches Lincoln’s or Hay’s writing style more, those attempts have never been conclusive, in particular because — though both Lincoln and Hay left countless examples of their writing styles — the letter itself is so short, containing only 139 words. “To make an analogy, if you take 10 people walking down the street in some American city and you look at their demographics, how many men you saw and how many women, different age groups and ethnicities, you wouldn’t get a very good estimate of the U.S. population,” Grieve says. “It’s so little data that you’re not getting a good measure of the real features of the population.” A solution to this problem, Grieve and his colleagues posit, can be found by breaking the language down into components that are inherently more numerous: n-grams. An n-gram is a “sequence of one or more linguistic forms,” as they phrase it in the paper. How to understand that? Well, on the word level, that last sentence contains one 4-gram (how-to-understand-that), two 3-grams (how-to-understand and to-understand-that), three 2-grams (how-to, to-understand, understand-that) and four 1-grams (each word). The same principle can also be applied to the individual characters in words. In n-gram tracing, the computer looks to see whether any individual n-gram that shows up in the sample text shows up at all in the comparison text. In this case, the Bixby letter is the disputed sample. The researchers tested 500 texts by Hay and a commensurate random sample from texts from the much larger body of work known to be by Lincoln. Because the question is whether something shows up or not — rather than asking how frequently it shows up — a short text can still be broken down by this algorithm and yield a good result. The authors of the paper claim their method is able to distinguish between the two writers “with a very high degree of accuracy,” even for very short texts. And their result? Nearly 90% of the time, the n-gram tracing method identified Hay as the author of the Bixby letter. The other roughly 10% of the time, the analysis was inconclusive. (Those times were when the researchers used groupings of just 1 or 2 letters at a time, rather than whole words, and those combinations proved extremely common overall.) That means, they believe, that the century of wondering about the Bixby letter can come to an end — leaving history buffs free to appreciate the letter’s beauty, as well as the uncontested writing skills of both men, without the distraction of this lingering question. It also leaves Grieve and his colleagues free to apply their techniques to other problems. One of his colleagues wants to tackle letters supposedly written by Jack the Ripper, for example. And what’s Grieve’s dream project in his field? “This,” he says, “was kind of it.”

Summary: Abraham Lincoln may have been a world-class orator, but one of his famous letters-known as one of the best English-language letters ever written-was "almost certainly" not authored by the former president. According to a press release, Lincoln sent a letter to a woman named Lydia Bixby in November 1864 after Bixby lost her sons in the Civil War. "I feel how weak and fruitless must be any words of mine which should attempt to beguile you from the grief of a loss so overwhelming," Newsweek quotes the letter as saying. "But I cannot refrain from tendering to you the consolation that may be found in the thanks of the Republic they died to save." As the letter gained notoriety for its prose, historians started to debate if it was actually written by Lincoln's secretary, John Hay. A team of forensic linguistic experts may have finally solved the mystery. With the original letter missing for more than 150 years and both Hay and Lincoln long dead, the researchers turned to a process called n-gram tracing, in which a computer searches for recurring strings of words, or linguistic forms. Time has an in-depth explainer of how n-gram tracing works, and researchers have published a presentation on their work here. A similar method was used in 2013 when sussing out that author Robert Galbraith was actually JK Rowling. Researchers used n-gram testing on 500 texts by both Hay and Lincoln, then tested the Bixby letter. In 90% of tests, Hay was identified as the author of the letter. The other 10% were inconclusive, with Lincoln never once being identified as the letter's writer.

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Summarize: Laura Perspicace, sceneggiatrice catanese di 37 anni, oltre alla scrittura, coltiva da 10 anni un'altra passione: quella di aiutare i figli adottivi a ritrovare i propri genitori biologici. Lo fa gratuitamente attraverso il suo sito web e una pagina social, da dove chiunque sia alla ricerca di una mamma naturale può lanciare il suo appello. “Ho creato un gruppo Facebook per mia madre, che è stata adottata – racconta Perspicace a Fanpage.it – volevo potesse conoscere le sue origini. Successivamente molte persone mi hanno cercata per pubblicare il loro appello e nel tempo, si è creata una solidarietà virtuale che oggi consente ad un adottato su tre di ritrovare la sua famiglia”. Le richieste arrivano da tutta Italia, e anche dall'estero. In tutto questo tempo, sono stati migliaia gli appelli pubblicati da Laura. C’è chi vuole avere notizie dei genitori naturali, ma ci sono anche tante mamme che cercano i figli dati in adozione. E non mancano fratelli o sorelle, divisi da adozioni diverse durante la loro infanzia. “In dieci anni – assicura – sono riuscita a risolvere oltre 800 casi”. A differenza dei bambini riconosciuti alla nascita, e in seguito adottati, ai figli nati in un parto in anonimato l'attuale ordinamento italiano non consente di accedere all'identità dei propri genitori biologici. Viene considerato prevalente il diritto alla segretezza della donna, rispetto all'interesse del figlio di conoscerne l'identità. In questi anni, tuttavia, i tribunali dei minorenni sono intervenuti e hanno stabilito che il figlio, al compimento del 25˚anno di età (per gravi e comprovati motivi attinenti alla salute psico-fisica dell’adottato bastano 18 anni), possa far pervenire alla madre una richiesta per sapere se vuole continuare a rimanere sconosciuta. Ma in mancanza di una legge che disciplini la materia, senza un protocollo univoco da seguire, i tempi e le modalità variano da città a città. Ecco perché sempre di più internet e i social sono diventati il mezzo per riuscire a trovare risposte alle tante domande sulla propria origine. “Le persone si rivolgono al web – sottolinea Perspicace – poiché oggi è diventato uno strumento veloce ed efficace con il quale si possono rintracciare i propri cari dati in adozione oppure la famiglia biologica in tutto il mondo. A volte, nella procedura di interpello, le mamme non si presentano in tribunale – aggiunge – perché non vogliono rendere pubblico il loro passato fatto di ricordi dolorosi però poi accettano di incontrare il figlio o la mamma biologica attraverso internet o la mia pagina Facebook”. “Di solito – spiega la scrittrice – vengo contattata attraverso un messaggio privato sulla pagina o tramite e-mail al mio sito. Le persone, a cui garantisco la privacy più assoluta, mi raccontano la loro storia. Una volta in mano i dati essenziali, scrivo l’appello e lo pubblico prima sul web e poi lo condivido in tutti miei i social network. Questo vale anche nel caso si tratti di stranieri. Ad esempio, se sono cittadini russi lo condivido sulle pagine che ho aperto in quel Paese”. “Dopo la pubblicazione dell’appello – continua Perspicace – iniziano le varie segnalazioni che verifico con la massima attenzione. Se realmente sono attendibili le comunico alla persona che ha chiesto il mio aiuto. Il tutto per arrivare ad un primo contatto tra i due che sarò io a gestire affinché avvenga con molta delicatezza e serenità da entrambe le parti e vada a buon fine”. Le storie che Laura Perspicace sente ogni giorno sono tutte commoventi e, allo stesso tempo, dolorose. “Ricordo ancora dei fratelli che cercavano disperatamente le due sorelle adottate in famiglie diverse. Questi ragazzi furono sottratti alla famiglia di origine e divisi in due orfanotrofi diversi: le ragazze furono adottate ma i 2 maschi no, un dramma nel dramma. Una volta cresciuti hanno cercato le sorelle ma invano, fino a quando hanno trovato me che in pochi giorni sono riuscita a riunirli di nuovo. Dopo 40 anni si sono ritrovati grazie alla mia pagina, una storia piena di ingiustizie, di dolori, ragazzi segnati da eventi che non dimenticheranno mai”.

Summary: Laura Perspicace è un "angelo" per molte persone che cercano i propri familiari. Da 10 anni, questa scrittrice catanese si dedica gratuitamente, attraverso la sua pagina web, ad aiutare figli adottivi a rintracciare la propria madre naturale. In tutti questi anni ha risolto più di 800 casi e a Fanpage.it ha spiegato come ci riesce. "Un adottato su tre ritrova la sua famiglia grazie alla mia pagina Facebook".

fanpage

it

Summarize: The parents of badly injured F1 driver Jules Bianchi have arrived by his bedside at the Japanese hospital where he is fighting for his life in intensive care. Philippe and Christine Bianchi arrived at the Mie General Hospital in Yokkaichi last night, a day after their son was badly hurt in a smash during the rain-soaked Japanese Grand Prix. 'When we have an update with Saillant, we will speak to you,' Philippe Bianchi told waiting reporters, referring to French brain injury specialist Professor Gerard Saillant, who has flown to Japan to assist in Bianchi's care. Scroll down for video. Worry: Philippe and Christine Bianchi leave the Mie General Medical Centre in Yokkaichi this evening. Stress: Mr and Mrs Bianchi arrived in Japan last night, but these are the first pictures of them at the hospital. Fears: They speak with Nicolas Todt (3rd L), Bianchi's manager, and assistant manager Alessandro Alunni Bravi (2nd L) at the entrance of the Mie General Medical Centre. Mie General Medical Centre: Hospital sources said Bianchi was breathing but only with the help of a ventilator. Prof Saillant, president of the FIA medical commission, arrived in Yokkaichi early this morning and went straight to the hospital where Bianchi is said to be in a 'critical' condition. On Sunday, the Marussia driver suffered a severe head injury when his car smashed into a tractor rescuing the crashed Sauber of Adrian Sutil, who had spun off on the previous lap of the Japanese Grand Prix. Prof Saillant arrived at Mie General Hospital with Nicolas Todt, Bianchi's agent, and went straight in via the emergency entrance to avoid crowds of waiting reporters, Le Parisien reports. Top doctor: French brain injury specialist Professor Gerard Saillant, president of the FIA medical commission, has flown in to assist in Bianchi's care. Bianchi, who is just 25, remains in intensive care today after emergency surgery on a severe head injury following his high speed crash on the rain-soaked Suzuka circuit. Sources at the hospital said Bianchi was breathing but only with the help of a ventilator, not on his own as was originally reported in the French media on Sunday night. Famous in the world of sport and one of France's leading neurologists, Prof Saillant is a brain and spinal injury expert with a particular specialism in motorsports. It was he who helped to treat Michael Schumacher after the retired F1 ace's devastating head injury while skiiing in December last year. He also oversaw Schumacher's medical care when he broke his leg in the 1999 British Grand Prix. A spokesman for the FIA, Formula One's governing body, said last night Bianchi's condition was ‘critical but stable’ and it is understood his family will finally see him on Tuesday. They had been delayed in getting to him due to Typhoon Phanfone. The first video of the horrific moment Bianchi crashed into the digger has now been distributed online. The clip was released as Jules Bianchi was revealed to be in a ‘critical but stable’ condition following the crash. An ambulance was sent to the scene straight after Bianchi's crash - which knocked him unconscious - and the 25-year-old Frenchman was taken to the Mie General Medical Center in Yokkaichi. Last night Camille Marchetti, 24, the French girlfriend of Bianchi, sent up a prayer through Twitter for her 'champion' as he fought for his life in hospital. Bianchi's father, Philippe, told French TV that his son's condition was 'critical' and he had undergone surgery for serious head injuries. Miss Marchetti, who has dated Bianchi for a number of years, said: 'You are my champion! You are the strongest’. She ended the tweet with emoticons of a heart and two hands locked in prayer. Crash: The clip was taken yesterday in the aftermath of another accident at the Japanese Grand Prix. Clip: The driver was severely injured when his car ploughed into the back of a tractor rescuing another vehicle. Girlfriend: Camilee Marchetti (pictured with Bianchi at an event) is an osteopathy student at the Collège Ostéopathique de Provence in France and originally hails from the Mediterranean island of Corsica. 'My champion'! Bianchi's long-term partner, Camille Marchetti, led prayers for the race ace's recovery as the world waited for news on his condition. Yesterday, Marussia issued a statement giving thanks for the support Bianchi had received. They also asked for ‘patience and understanding’ with regard to further medical updates. This was after an initial release was made by the FIA on Sunday confirming the driver suffered ‘a severe head injury’ and was to undergo surgery before being moved to intensive care. Marussia's statement read: ‘Following the accident involving Jules Bianchi during yesterday's 2014 Formula One Japanese Grand Prix at Suzuka Circuit, the Marussia F1 Team would like to acknowledge the huge outpouring of support and affection for Jules and the team at this very difficult time. ‘With regard to the communication of information concerning Jules' medical condition, we will respect, and be guided by, the wishes of the Bianchi family. Together with Jules' care, they will remain our highest priority. ‘Therefore, we would ask for patience and understanding with regard to further medical updates, which will be communicated in conjunction with the Mie General Medical Center in Yokkaichi, where Jules is being treated, when they feel it is appropriate. ‘Representatives of the Marussia F1 Team and Scuderia Ferrari will remain at the hospital to support Jules and the Bianchi family.’ Unconscious but breathing for himself, Bianchi was taken to Mie University hospital, ten miles from the track in Suzuka, and the race was stopped nine laps early. Bianchi's father has since told French television that his son's condition is 'critical' and he has undergone surgery for serious head injuries. The F1 season moves on to Sochi in Russia for next weekend's race and it remains to be seen whether or not Marussia will take part. Bianchi's crash saw Sunday's event halted at lap 44, with victory being awarded to Lewis Hamilton, who has opened up a ten-point lead in the championship over his second-placed Mercedes team-mate Nico Rosberg with four races remaining. The race had been under threat of cancellation in the build-up as Typhoon Phanfone headed towards Suzuka and the wet weather led to it being red-flagged after just two laps, despite starting under safety car conditions. Once enough standing water had been cleared to allow the race to begin in earnest, Briton Hamilton caught and passed Germany's Rosberg on lap 29 before disappearing into the distance. The rain then came down again, driving conditions deteriorated and Bianchi crashed out, prompting the red flags to return and the race to be ended with nine laps to go. Williams driver Felipe Massa has said he had been ‘screaming’ over his radio during the race about the amount of water on the track, but former FIA president Max Mosley believes ‘everything was done as it should have been’ in relation to safety. Supportive: Miss Marchetti (far left) watches Bianchi prepare to take part in the Amber Lounge Fashion Show ahead of the Monaco Formula One Grand Prix at Circuit de Monaco last May. Asked if F1 is doing everything right to secure the safety of its drivers, Mosley told Sky Sports News HQ on Monday: ‘I think it is. I think what happened in Suzuka was very unfortunate, a freak accident. ‘But I can't really fault any of the people involved - the marshals or the race director, any of those people. I think everything was done as it should have been.’ Massa said he was trying his best to get the race called off in the run up to Sutil and Bianchi's accidents. ‘I was already screaming on the radio five laps before the safety car that there was too much water on the track, but they took a bit too long and it was dangerous,’ the Brazilian said on Sunday. A subdued Hamilton said: ‘Our first thoughts go to Jules. It overshadows everything else when one of our colleague is injured and we are praying for him. ‘Next to this, the race result doesn't seem significant at all. Nico and I had a tough battle, we were pushing really hard... But like I said, that's not what matters - our thoughts are with Jules.’ Bianchi's long term partner, Miss Marchetti, is an osteopathy student at the Collège Ostéopathique de Provence in France who originally hails from the Mediterranean island of Corsica. She is well-known in the Formula 1 community and is often seen cheering Bianchi on from the pits as he races around the world. Known also for her glamorous attire and interest in fashion, she regularly accompanies him to F1 events and parties. Bianchi is a graduate of the Ferrari academy and former Ferrari driver Felipe Massa and team principal Marco Mattiacci went with him to hospital. It is the most serious accident to hit a race weekend since Felipe Massa fractured his skull and spent a week in a coma while qualifying for the Hungarian Grand Prix, in 2009

Summary: Philippe and Christine Bianchi arrived at Mie General Hospital last night. Their son is 'critical but stable' after Japanese Grand Prix crash on Sunday. Marussia driver was injured when his car ploughed into the back of a tractor. One of F1's top brain doctors has also flown to Japan to help treat Bianchi. Professor Gerard Saillant advised after Michael Schumacher's ski crash. A top neurologist in France, he is president of the FIA medical commission. He arrived in Yokkaichi early this morning and went straight to the hospital. Bianchi's girlfriend Camille Marchetti, 24, sends up prayer for her 'champion'

cnn_dailymail

en

Summarize: FIELD OF THE INVENTION The present invention relates to a dialysis machine for the removal of toxic substances from the blood with agents for the decalcification of a dialysis liquid circulation. The invention further relates to processes for determination of the degree of calcification of a dialysis machine. BACKGROUND OF THE INVENTION In bicarbonate dialysis for the removal of toxic substances from the blood, the problem arises that bicarbonate and calcium form a poorly soluble precipitate. The components in bicarbonate dialysis are therefore kept separate as sodium bicarbonate in the basic component and as calcium chloride, in the acid component, and then mixed only in the machine itself or shortly before use. In spite of this measure, seed crystals form in the dialysis machine, which in time leads to extensive deposits of calcium carbonate. For this reason, it becomes necessary to decalcify a dialysis machine from time to time. The usual decalcifying agents are, for example, acetic acid or citric acid. U.S. Pat. No. 5,326,476 teaches how to introduce a manually operable or time-controlled decalcification cycle. These decalcification methods prove to be disadvantageous to the extent that the manual selection of a decalcifying program must be made by service personnel, which as a rule turns out to be very time-consuming. Time-controlled decalcification, moreover, has the disadvantage that decalcification is effected even when the degree of calcification would not yet require such decalcification. This leads to unnecessary demands being placed on the decalcification device. Furthermore, the dialysis machine is not available for actual treatments during this period. OBJECTS AND SUMMARY OF THE INVENTION The object of the invention is therefore to create a dialysis machine with a decalcifying device which will obviate the above disadvantages. The invention has the further goal of providing indicating processes that can be advantageously used in determining the degree of calcification of a dialysis machine. Pursuant to the invention, this object is achieved by a dialysis machine as described and claimed herein. With the dialysis machine according to the invention, decalcification is automatically initiated only as a function of the state of calcification actually measured. This prevents placing demands on the operating personnel as well as unnecessary use of the decalcifying agents. Preferred embodiments are the subject of subclaims. According to one advantageous further development, the degree of calcification of the dialysis machine can be determined through the clouding of the window of a blood leak detector. The fact exploited here is that, with increasing calcification of a dialysis liquid circulation, lime deposits will also form on the window of the blood leak detector of the dialysis machine, which results in the weakening of a light ray passing through such a window. According to a further advantageous embodiment, the degree of calcification of the dialysis machine can be determined by determining the clouding of the dialysis fluid used; the clouding of the dialysis fluid is associated with an increasing degree of calcification of the dialysis liquid circulation and/or increasing lime deposition. This represents a simple and reliable method for determining the degree of calcification of a dialysis machine, which can be carried out, for example, continuously or discontinuously, i.e., at specified intervals. It is of particular advantage to use a blood leak detector of a dialysis machine which has a signal output that reacts to clouding of the dialysis liquid and/or clouding of the window of the blood leak detector. This represents an especially simple process in which an available blood leak detector need only be equipped with another signal output. According to a further advantageous embodiment, the degree of calcification of the dialysis machine can be determined by means of characteristic parameters of a pump, especially a gear pump, which serves to convey the dialysis liquid through the dialysis machine. This determination process can also be carried out in a very simple and reliable way, since certain characteristic parameters of a gear pump are changed in specific ways by increasing calcification. It is found to be particularly advantageous to determine the degree of calcification on the basis of a change in the dialysis liquid flow discharged by the pump. It is also possible to determine the degree of calcification on the basis of a change in the pump voltage and/or the pump delivery. According to a further advantageous development, the dialysis machine has means which, when a predetermined degree of calcification is detected, store this information and do not initiate automatic decalcification until a dialysis is terminated. This ensures that the dialysis operation is in no way hindered. According to a further design of the invention, provision is made to use the acid component of the bicarbonate dialysis fluid as the decalcifying agent. This provides an especially simple means for the decalcification of a dialysis machine. According to one process of the invention for determining the degree of calcification of a dialysis machine, the clouding of a window, such as an observation window, of a blood leak detector of a dialysis machine is determined, and the data thus obtained is correlated with a degree of calcification of the dialysis machine. This is shown to be an advantage because the degree of calcification and, hence, the clouding of an observation window of a blood leak detector, is dependent on the degree of calcification of the dialysis liquid circulation. In further accordance with a process of the invention for determining the degree of calcification of a dialysis machine, the degree of clouding of a dialysis liquid used is determined, and the data thus obtained is correlated with a degree of calcification of a dialysis machine. This makes use of the fact that there is a definite relation between the degree of clouding of the dialysis liquid used and the actual degree of calcification of a dialysis machine. Of particular advantage in this invention, is to determine the degree of clouding by means of a blood leak detector signal output reacting to clouding. To this end it is only necessary to provide a conventional blood leak detector with a signal output or signal channel which reacts to largely color-unspecified clouding, as may be caused, for example, by the clouding of a window and/or of the dialysis liquid. By arranging a suitable sensor of the blood leak detector inside the dialysis liquid circulation, such clouding is produced by CaCO 3 deposits on the sensor itself. However, it is also possible to arrange such a sensor in some other position in relation to the dialysis liquid circulation. The invention further provides a process in which at least one characteristic parameter of a pump, especially a gear pump, that serves to convey dialysis liquid through the dialysis machine, is determined, and the parameter value thus determined is correlated with a degree of calcification. It has been determined, in fact, that the operating or performance parameters of gear pumps change in some way as a result of calcification. It has proven particularly advantageous to use as a characteristic parameter the dialysis liquid flow delivered by the gear pump, or the delivery or voltage of the gear pump. These parameters can be easily and reliably measured and can be correlated unequivocally with the actual degree of calcification of the dialysis machine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the invention are described in greater detail with reference to the appended drawing. FIG. 1 is a schematic view of the design of a blood leak detector that, in accordance with a preferred embodiment of the invention, can be used to determine the degree of calcification of a dialysis machine. A blood leak detector has, for example, a bicolor LED 1, which alternately radiates an orange-red and a green light (light ray 10). Approximately 50% of this light is decoupled via a prism 3 and led to the reference receiver 5. This reference receiver 5 forms the input of a reference channel. The reference channel regulates the light intensity of the two colors in such a way that they are in a fixed ratio to one another. The remaining light radiates through a cell 7 and the medium in it (e.g., dialysis liquid). Afterwards this light hits a measuring and cell receiver 9. If there is blood in the dialysis liquid, the green light undergoes a loss of intensity when it radiates through the cell. The thereby changed intensity ratio is evaluated in a measuring channel following the measuring receiver 9. According to a preferred embodiment of the present invention, a dialysis machine has a blood leak detector equipped with an additional signal output or signal channel which reacts to clouding (clouding receiver 9a or clouding channel). An electronic circuit or its balance is hereby preferably so designed that when no clouding is present the output voltage is 5 volt. With increasing calcification during bicarbonate dialysis this output voltage decreases because of increased clouding. If this voltage drops below a specific threshold value, e.g., 3.5 volts, a decalcification cycle is automatically started. For this, a microprocessor-controlled valve, 24 for example, can be activated in such a way that a suitable decalcifying agent is introduced into the dialysis liquid circulation 26. In particular, it is thereby possible, for example by computer 22 control of the system, to set or generate a flag or trigger signal for the decalcification cycle that initiates the actual decalcification only after the dialysis procedure. The presence of blood in the dialysis liquid is therefore detected via a measuring channel, as is the onset of clouding of the dialysis liquid via a clouding channel. Signal processing of the measuring channel or the blood leak channel occurs when a photoelectric cell converts the light signals received from the two-color LED to a logarithmic voltage with reference to the light intensity received, so that square-wave amplitude produced by the alternating red-green lighting is proportional to the logarithm of the red-green quotient and that color-unspecific clouding, which attenuate the two colors equally, do not affect the signal. However, if blood is found in the dialysis liquid, the green light undergoes a loss of intensity, as described above, so that the changed ratio can be evaluated by the measuring channel. The clouding channel is so designed that its output voltage continues to decrease with increased clouding. According to a further preferred form of embodiment, the degree of calcification of a dialysis machine is determined by evaluation of specific parameters of a gear pump 32 used to deliver the dialysis liquid. Dialysis machines are known to insert such gear pumps for the delivery of dialysis fluid. Because of the increasing calcification in bicarbonate dialysis, the characteristic parameters of this gear pump change. If such calcification occurs, for example, with the use of dialysis machines with constant-voltage operation of the gear pump or feed pump, the calcification is expressed, for example, by an increase of the dialysis liquid flow. When a certain level of flow is exceeded, for example by 20%, it is then possible to start a decalcification cycle automatically. Here, too, it is possible to generate an alarm signal and to set or generate a flag signal or trigger signal for a decalcification cycle that would initiate the actual decalcification only after the dialysis ends. It is likewise possible to use other pump 32 parameters to determine the degree of calcification, for example the increase or decrease of the delivery and/or the voltage of the pump 32, depending on the pump 32 control used. Common to the invention embodiments is that they provide agents for starting automatic decalcification when a predetermined degree of calcification of the dialysis machine is detected. As examples, decalcifying agents such as acetic acid, peracetic acid, or citric acid may be used. According to a preferred form of embodiment, acid components used in bicarbonate dialysis can also be used for the decalcification. This is possible because these components for the most part contain acetate. It is thus possible to largely avoid complete disinfection cycles with other lime-dissolving disinfecting agents. Substantial advantages here are that neither a supplementary reservoir for the decalcifying agent nor a supplementary device for delivering the decalcifying agent is needed. Moreover, no connection or change in the connection of the dialysis machine need be made prior to the start of the decalcification cycle since the acid component is already connected to the dialysis machine, whether by being supplied from a canister or via a central supply attachment. When the above-mentioned criteria for decalcification are present, for example after recognition of the state where the &#34;dialysis has ended,&#34; the dialysis machine can then initiate the decalcification cycle fully automatically. According to the invention, therefore, the decalcification of a dialysis machine is simplified in such a way that a simple, user-friendly, and reliable regeneration of the dialysis machine is assured. The success of the decalcification itself as well as the safety of the patient is thereby assured. To further enhance safety, it is possible to provide means 34 which are capable, after the end of the decalcification cycle, to detect remnants of the decalcifying agent used, so as to be able to effectively avoid that decalcifying agent residues remain in the dialysis machine.

Summary: A dialysis machine for the removal of toxic substances from biological fluids with agents for the decalcification of a dialysis liquid circulation is disclosed. The dialysis machine allows measurement of the degree of calcification of the dialysis liquid through the use of a blood leak detector or by measuring the dialysis machine pump flow or pump voltage. The invention also includes a process for automatically decalcifying the dialysis liquid circulation when a predetermined degree of calcification of the dialysis machine is measured.

big_patent

en

Summarize: By. Anthony Bond and Sara Nathan. PUBLISHED:. 23:52 EST, 1 January 2013. |. UPDATED:. 21:02 EST, 2 January 2013. Justin Bieber has called for new laws for paparazzi following the 'tragic' death of a photographer who was run over and killed while taking pictures of the star's sportscar. A 29-year-old freelance photographer, named by TMZ as Chris Guerra, was struck by a vehicle on Tuesday night after taking pictures of the singer's white $200,000 Ferrari in Los Angeles. The star's friend, 19-year-old rapper Lil Twist, was behind the wheel of the 458 Italia Ferrari when it was pulled over by police - prompting Guerra to get out of his own car as he believed Bieber was in the vehicle. The 18-year-old today released a statement in which he said his 'thoughts and prayers' were with Guerra's friends and family. SCROLL DOWN FOR VIDEO. Tragic: Freelance photographer Chris Guerra died after being knocked over in LA while tailing Justin Bieber. Horrific: The photographer was struck by another car as he was running back to his own vehicle after taking pictures of the star's car. This picture shows the man's hat and shoe lying in the road where he was hit. Shattered: The car that struck and killed photographer Chris Guerra in Los Angeles on Tuesday night. Death: A photographer who was following Justin Bieber's white Ferrari in Los Angeles was killed Tuesday evening as he was trying to snap photos of the singer's vehicle. This picture shows the crash scene. In his statement, Bieber said: 'While I was not present nor directly. involved with this tragic accident, my thoughts and prayers are with the. family of the victim. 'Hopefully this tragedy will finally. inspire meaningful legislation and whatever other necessary steps to. protect the lives and safety of celebrities, police officers, innocent. public bystanders, and the photographers themselves.' Chris Guerra was tailing Bieber’s white Ferrari when it was pulled over by California Highway Patrol officers on an I-405 off ramp in L.A. He got out of his car to take pictures but was struck by a Toyota Highlander SUV as he was running back to his own vehicle after being told to return to his vehicle repeatedly by police. On the job: Photographer Chris Guerra pictured with his camera in Aspen, Colorado, in December. Memorials: Flowers and a hand-made cross were left by the roadside on Wednesday following Chris Guerra's death. Photographs taken from the scene show Guerra's hat and shoe lying in the road where he was hit. He was wearing the same baseball cap on one of the last time he was photographed last month in Aspen, Colorado. It is believed the female driver of the car which struck the photographer stopped other cars from hitting the man's body after she had called 911. Guerra was a freelance photographer who submitted pictures to AKI images/GSI Media for syndication, the company confirmed today. Los Angeles police officer Sergeant Rudy Lopez said the highway patrol men had noticed the photographer taking pictures close to the scene. He said one of the officers asked him to return to his vehicle. A paparazzo was struck and killed after he got out of his car to shoot pictures of Justin Bieber's white Ferrari (file photo) Bieber's Ferrari costs upwards of $200,000, according to reports (file photo) Sgt Lopez said: 'He verbally ordered. him to return to the vehicle, he had to order him twice and he seemed. reluctant to go back, but eventually he did. 'When. the photographer returned to the vehicle it did not appear that he was. looking and he was struck by a vehicle traveling southbound.' Guerra died at a hospital shortly after the crash. The. sports car - which contained two of Bieber's friends, with rapper Lil Twist believed to be behind the wheel - was parked on the side of Sepulveda Boulevard near Getty. Center Drive after the traffic stop. According to sources, Lil Twist - real name Christopher Moore - was with Bieber at the Four Seasons in Beverly Hills on Tuesday afternoon when he left in the white Ferrari without Justin. Twist, who is a Lil Wayne protege, and Bieber have been hanging out and partying together for the past few weeks. Justin Bieber performed last night on Dick Clark's New Year's Rockin' Eve at CBS studios in Los Angeles. Close friends: Rapper Lil Twist was behind the wheel of Justin Bieber's Ferrari when it was pulled over in Los Angeles. The pair are seen here on December 12 at Power 96.1's Jingle Ball 2012 in Atlanta, Georgia. Best buds: Justin Bieber and Lil Twist share drinks on New Year's Eve in Mexico. Prank: Lil Twist sneaks a picture alongside a sleeping Justin Bieber onboard a private jet. They even celebrated New Year's Eve together by popping champagne bottles in Mexico, where they're both of legal drinking age. No charges are likely to be filed against the motorist - believed to be female - who hit the man. The LAPD is continuing to investigate the man’s death. A representative for the department did not immediately return MailOnline's request for comment. According to TMZ, Guerra claimed to friends earlier in the day that he had spotted Bieber smoking what he believed to be marijuana from a pipe while driving the Ferrari. A source said the photographer told him he was following the singer back to the Four. Seasons and was continuing to pursue what he believed was a big story. The. source says after 20 minutes, Guerra hung up as he said the Ferrari was. pulling out of the hotel parking lot and he needed to hang up and follow. it. Apparently he had no idea Bieber in the car. The longtime friend, who spoke with Chris at 5:16 PM - less than an hour before he was killed - was one of his closest friends told TMZ he belived Bieber is partly to blame for Guerra's death because of the singer's reckless driving history and alleged pot smoking which made it a big story to follow. Bieber had already been pulled over by police in his Ferrari while driving through West Hollywood in November. He was initially pulled over for making an unsafe left turn. During the stop, the officer discovered Bieber's registration was expired and he was given a ticket. Bieber had helped ring in the new year, performing on 'Dick Clark's Rockin' New Year's Eve' on Monday night. Fury: the singer and actress didn't hold back as she made her opinions clear. Anti-paparazzi: Miley Cyrus vented her anger over the tragic accident in Los Angeles. Reacting to the accident, former child star Miley Cyrus took to Twitter to call the paparazzi 'fools'. The Disney star, now 20, tweeted: 'Hope this paparazzi/JB accident brings on some changes in '13 Paparazzi are dangerous! Wasn't Princess Di enough of a wake up call?! 'It is unfair for anyone to put this on to Justin's conscious as well! This was bound to happen! Your mom teaches u when your a child not to play in the street! 'The chaos that comes with the paparazzi acting like fools makes it impossible for anyone to make safe choices.' Bieber has had a gruelling couple of months.Only last month, a gruesome plot to murder the Canadian crooner was revealed. Trouble: Justin Bieber was pulled over in his Ferarri by cops in West Hollywood in November and given a ticket. Mark Staake and his nephew, Tanner Ruane, were allegedly plotting to kidnap, torture, and kill the singer while he was in New York to perform at Madison Square Garden. Martin had ordered for ‘the murder and castration’ of the 18-year-old singer. And in November, the ‘Baby’ singer had a run-in with a paparazzo who claimed Bieber was driving too fast. ‘What do your parents think about what you do? Do you tell them, “I stalk people for a living”?’ the singer asked. The incident held up traffic in Hollywood. Before speeding off, Bieber said, ‘You guys love this, don’t you?’

Summary: 29-year-old Chris Guerra was hit. by a vehicle after taking pictures of singer's white $200,000. Ferrari. The star's car was being driven by rapper Lil Twist and had been pulled over by police officers in Los Angeles. The 18-year-old has now called for new laws for paparazzi photography. Female driver of vehicle which struck the man stopped other cars from hitting his body. Miley Cyrus wades in to dub paparazzi 'fools' and says 'Wasn't Princess Di enough of a wake up call?!' Photographer tailed Bieber after he thought he saw him'smoking marijuana while driving'

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Summarize: A health and safety expert was too ill too drive when he blacked out at the wheel and smashed into a bright A-Level student's car, a High Court judge has ruled. Peter Haynes will have to pay substantial damages to 19-year-old Simon Green, who was left with a permanent brain injury after the horror crash on the A11 in Norfolk. Mr Haynes, 56, told London's High Court he was not to blame because he was in a state of 'automatism', similar to sleepwalking, after abdominal pain caused him to pass out. Horror crash: Health and safety consultant Peter Haynes (left) will have to pay substantial damages to Simon Green (right), who was left with life-changing brain damage after Mr Haynes passed out at the wheel on the A11. But today Mr Justice Supperstone ruled Mr Haynes was liable for the crash, because he should not have driven once he realised he was ill. The businessman, of Metfield, Suffolk, was on his way home from a conference in Norwich in March 2012 when he passed out on a slip road to the dual carriageway near the village of Cringleford. The self-employed health and safety consultant's Toyota Land Cruiser hit the rear of student Mr Green's Volkswagen Polo, shunting it under the Land Rover in front. Mr Green, a high-achieving A-level student, suffered life-changing brain damage in the crash and it was'remarkable' that he survived at all, the case heard. He launched a civil case to reclaim damages from the health and safety expert, insisting he was responsible for causing the pile-up. Mr Green sued Mr Haynes at the High Court (pictured), where he relied on the rare defence of automatism. People claiming 'automatism' - where they had no control over their actions through no fault of their own - has made headlines after defendants accused of sex offences claimed they acted in their sleep. It has its origins in 19th Century definitions of 'insanity' but now stands as a separate defence for people with no mental health issues. The defence is wide-ranging and open-ended - it can include sleep disorders or medical conditions such as an epileptic fit or a hypoglycaemic attack. In one criminal case in August, a driver was acquitted after a jury heard he caused a crash which killed a married couple because he was choking on a sweet. Other examples could include a swarm of bees flying into a car. The defence is almost completely unheard of in civil cases, however. One person involved in Mr Haynes' case said it the first example he had seen in more than 20 years. There are no statistics on how often the defence is pleaded or succeeds, and last year Parliament's Law Commission said it should be scrapped in criminal cases altogether. The five-strong panel led by Lord Justice Lloyd Jones said the wording should be changed to 'not criminally responsible by reason of recognised medical condition', making the law less open to interpretation. In his defence, Mr Haynes said the crash occurred after he had a blackout triggered by abdominal pain and he was in a state of 'automatism', meaning he was carrying out actions over which he had no control. Lawyers for Mr Green accepted that Mr Haynes passed out seconds before the collision - but said he was negligent in choosing to drive when he did, and and in the manner of his driving. The judge said Mr Haynes had felt very unwell with an upset stomach when he was still at the conference and rang his wife, asking her to make him a doctor's appointment. But he still decided to drive home, and 'in all likelihood' drove close to 70mph in the outside lane which would have made it more difficult to pull over. Had he been driving slowly and in the inside lane, ready to pull over, the accident would have been avoided. The judge said: 'In my judgment, having regard to the true state of the defendant's health when he left the conference centre, he should have appreciated the risk of his condition getting worse and that, if it did, his judgment and ability to drive was likely to be impaired. 'On a balance of probabilities, I find that the defendant should not have driven on leaving the conference centre after he felt so unwell, and that he was in breach of his duty to other road users in so doing.' The amount of damages to be awarded to Mr Green, now 22, of Swaffham, Norfolk, will be assessed at a later date. There was no mention in court of whether Mr Haynes ever faced a criminal investigation

Summary: Peter Haynes blacked out in his Toyota Land Cruiser on A11 in Norfolk. He hit 19-year-old Simon Green's car, shunting it under a Land Rover. Brain-damaged Mr Green sued for damages in civil case at High Court. Mr Haynes said he wasn't responsible because he was'sleep-driving' But he will have to pay out after judge ruled he was too ill to get on road.

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Summarize: Drew Peterson Admitted to Killing His Missing Wife Stacy Peterson, Inmate Testifies Alleged Attempt to Kill Prosecutor For nearly a decade, Drew Peterson has steadfastly maintained his innocence in the disappearance of his fourth wife, Stacy. In public statements and court records, he has alleged that the then-25-year-old woman left him for another man.He insisted that Stacy Peterson was still alive, even as he faced trial for killing his third wife, Kathleen Savio. He was eventually sentenced to 38 years in prison for Savio's murder, but has never faced trial for Peterson's disappearance.But now an inmate has come forward alleging that Peterson admitted that Stacy Peterson is a "dead woman," and that he had killed her.The allegations came during the first day of Peterson's solicitation-for-murder trial. The 62-year-old former cop is charged with attempting to hire a prisoner to kill James Glasgow, the attorney who successfully prosecuted Peterson in Savio's death.In court on Monday, prisoner Antonio Smith testified that Peterson spoke about Rev. Neil Schori, a spiritual advisor to Stacy Peterson.According to Smith, Peterson "made a comment stating that the clergyman didn't know he was talking to a dead woman.""He admitted to me that he killed Stacy Peterson," Smith testified.Stacy Peterson has never been found and nobody has been charged in her disappearance.Smith testified he was playing basketball in the prison yard in 2013 when Peterson allegedly approached him and made a proposition."He said he needed me to have someone kill James Glasgow," Smith told the court, adding that Peterson "despised" the lawyer.Smith testified that he led Peterson on for weeks, and even recorded some of the conversations at the direction of the authorities.On the recordings, Peterson appeared to blame Glasgow for his incarceration and the loss of his police pension. Peterson also claimed that his son lost his job as a cop due to Glasgow's actions.Peterson's attorneys dispute that their client wanted to kill Glasgow, and accuse the prosecution of "sensationalizing" the claims. On Monday, the judge ruled that the defense would be able to highlight Smith's criminal history during cross examination on Tuesday.If convicted, Peterson faces an additional 62 years in prison. The trial is expected to last all week. As Drew Peterson watched from across the prison yard, fellow inmate Antonio Smith picked up a phone and pretended to call his uncle. That fake call, Smith testified, led Peterson to believe the plan had been set in motion to kill prosecutor James Glasgow. The subsequent conversation between the inmates was recorded by Smith, with the help of authorities, and played Tuesday in Randolph County court. "I told him what you said, that it's the green light on, that basically go ahead and kill him," Smith says on the Nov. 15, 2014, recording. "That's what you wanted, right?... It ain't no turning back." "OK, all right. I'm in," Peterson responds. "From the first time we talked about it, there was no turning back. … If I get some booze in here, we'll celebrate that night." "You know this (expletive) gonna be all over the news," Smith says. "This is about to be huge." "But the first thing they will identify him as the guy that got me," Peterson says. "That's what he's known for, the guy that prosecuted Drew Peterson." Peterson is on trial for allegedly trying to arrange for a hit man to kill Glasgow, the Will County state's attorney who helped put the former Bolingbrook police officer behind bars for the murder of Peterson's third wife, Kathleen Savio. The recordings, made Nov. 13-15, 2014, include no direct mentions by Peterson of killing Glasgow, although there are references to Glasgow being "gone." The defense has said the recordings are nonspecific and typical prison talk. Drew Peterson listened to his own words when the first audio tape of his recorded conversations with a fellow prison inmate was played Tuesday at Peterson's murder-for-hire trial. May 24, 2016. (WGN-TV) Drew Peterson listened to his own words when the first audio tape of his recorded conversations with a fellow prison inmate was played Tuesday at Peterson's murder-for-hire trial. May 24, 2016. (WGN-TV) SEE MORE VIDEOS "I can't wait to get, if I can get out of prison," Peterson says in a conversation recorded Nov. 13, 2014. "And you said … you said with Glasgow gone, right?" Smith asks. "Yes, it'll get me out," Peterson says. Smith testified that he told Peterson the plan was to have Smith's uncle kill Glasgow. "Before you give the green light to your uncle," Peterson tells Smith on the Nov. 14 recording, "talk to me." The next day, Peterson asks, "So, how long before you think your uncle can take care of business?" Smith: "It'll be done by Christmas, if, if you say, if you say." Peterson: "It'd be a nice Christmas present." Smith: "If you say it's a green light." Peterson: "Go." That's when Smith went to make the pretend phone call to his uncle, he testified. The conversations between the inmates depict Peterson as angry at Glasgow and confident in his murder appeal. On one recording, Peterson laments that Glasgow was cozying up to judges, making it harder for Peterson to win his appeal. "All the laws are on my side. I mean, it's clear-cut," he says in a Nov. 14 conversation. While Peterson appeared to believe that his appeal chances were good, he feared that even if he were freed from prison Glasgow would charge him in the disappearance of his fourth wife, Stacy. Peterson: "But the thing is, he'll never leave me alone. If I get out, he's gonna charge me with Stacy." Smith: "If you get out, he's gonna charge you with what?" Peterson: "Stacy." Stacy Peterson has been missing since 2007, and Drew Peterson told Smith on the recordings that "Stacy's still alive running around out there." In that same conversation, Drew Peterson talked of flying to Mexico if he's freed from prison. Peterson: "I have a guy that's got a … a … his family's got property on the other side of Mexico. We can land right on his property with a plane." Smith: "You know how to do that?" Peterson: "Oh yeah, I got a pilot's license." Smith: "I'm saying, but you won't crash?" Peterson, laughing: "No." Smith: "What are you talking about? What you talking about? A cartel?" Peterson: "Yeah, there's that guy that's got that cartel connections. They can get us into dope." At one point, Smith asks: "If Glasgow is dead by Christmas, when will that put you out? Approximately, like, what do you think? Are you worried about his assistant district attorneys or anything?" "No, they're idiots," Peterson says in response. He then goes on to talk about the details of his appeal and never specifically addresses Glasgow being dead by Christmas. Smith testified that he never had any intention of helping arrange a hit on Glasgow, and he continued to talk to Peterson to gain his trust before going to authorities. Smith testified Monday that Peterson admitted to killing his wife, Stacy, but there was no such admission in the recordings played in court. The conversations between the inmates included prison gossip and Peterson's thoughts on his murder trial. Peterson refers to his former defense attorney Joel Brodsky as an "idiot" and recalls joking with his defense team about Glasgow, calling him Glass Cow. Peterson also claims Glasgow won re-election to his state's attorney post on a campaign of "I got Drew Peterson." "So it's like (President Barack) Obama getting (Osama) bin Laden," Smith responds, and the inmates laugh. Peterson boasted on the recordings about insulting the Illinois State Police and Will County jail guards, how he was in line to be head of security at a well-known Las Vegas brothel and how a Russian billionaire wanted him to be his head of security and personal helicopter pilot. And there was talk about Peterson's sexual exploits. Peterson, 62, was convicted of Savio's murder in 2012 and was sentenced to 38 years in prison. He faces up to 60 years if convicted of solicitation of murder for hire along with solicitation of murder. After Stacy Peterson disappeared, Drew Peterson's bizarre behavior and media interviews made international headlines. The case prompted a TV movie and led authorities to reopen the investigation into Savio's 2004 death. Her body was found in her bathtub, and her death was originally ruled an accident. Smith, known as "Beast" because of his size, is serving 40 years for attempted murder, home invasion and robbery. His testimony is expected to continue Wednesday. mwalberg@tribpub.com Twitter @mattwalberg1

Summary: Drew Peterson is on trial again and his chances of leaving prison before his 100th birthday appear to be growing slimmer by the day. Fellow inmate Antonio Smith testified on Tuesday that he secretly recorded the Illinois ex-cop after convincing him that a plan to kill Will County State's Attorney James Glasgow was underway, the Chicago Tribune reports. In November 2014 recordings played in court, Peterson tells Smith about his plans for after Glasgow is "gone" and he is out of prison. "I have a guy that's got a... his family has property on the other side of Mexico. We land right there with a plane," says Peterson, who went on to tell Smith that the friend has cartel connections who "can get us into dope." Peterson, 62, is serving a 38-year sentence for the murder of his third wife, Kathleen Savio. He is charged with soliciting the murder of Glasgow, who was the prosecutor in that case. On the recordings, he complains that even if he wins an appeal, Glasgow will charge him with the murder of Stacy Peterson, his fourth wife, who has been missing since 2007. In the conversations played in court Tuesday, Peterson says, "Stacy's still alive running around out there," though Smith, his former cellmate, testified on Monday that Peterson told him in another conversation that he had killed her, People reports. If Peterson is convicted of solicitation of murder for hire, he could get another 60 years added to his sentence.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Coal Ash Reclamation, Environment, and Safety Act of 2009''. SEC. 2. REQUIREMENTS FOR SURFACE STORAGE AND DISPOSAL OF COVERED WASTES IN IMPOUNDMENTS. (a) Regulations.--The Secretary of the Interior shall, within 180 days after the date of enactment of this Act, promulgate regulations that establish design, engineering, and performance standards that provide for safe storage and disposal of covered wastes in impoundments. (b) Impoundment Requirements.--The regulations under subsection (a) shall require that an impoundment for the storage or disposal of covered wastes shall be designed, constructed, and maintained in accordance with requirements that are substantially similar to the requirements that apply to impoundments under paragraphs (8), (11), and (13) of section 515(b) and section 515(f) of the Surface Mining Control and Reclamation Act of 1977 (30 U.S.C. 1265(b), 1265(f)). (c) Prohibition.--No person shall construct or operate any impoundment for the storage or disposal of covered wastes on any land in any State except in accordance with regulations promulgated under subsection (a). (d) Inspections, Penalties, and Enforcement.--For purposes of sections 517, 518, and 521 of the Surface Mining Control and Reclamation Act of 1977 (30 U.S.C. 1267, 1268, 1271)-- (1) this section and permitting, design, performance, and other requirements and prohibitions established by the regulations and orders under this section shall be treated as requirements and prohibitions under that Act; and (2) an impoundment for the deposit and maintenance of covered wastes in violation of this section, the regulations this section, or any order under subsection (e) shall be treated as a surface coal mining operation. (e) Pre-Existing Impoundments.-- (1) Limitation on application.--Except as provided in an order under this subsection, the regulations under subsection (a) and subsections (c) and (d) shall not apply to an impoundment for the deposit and maintenance of covered wastes that is in existence on such date of enactment. (2) Inventory.--The Secretary shall, within 12 months after the date of enactment of this Act, complete an inventory of all impoundments for the deposit and maintenance of covered wastes in existence on such date of enactment. The inventory shall include-- (A) an assessment of the design, stability, and engineering of embankments and basin characterization and design of each such impoundment; (B) an assessment of risks to surface and groundwater posed by each such impoundment; and (C) a determination on the degree of risk each such impoundment poses to human and environmental health. (3) Orders.--Based on the assessments and determination of degree of risk under paragraph (2), the Secretary may issue any order necessary to ensure that any such impoundment complies with requirements established by the regulations under this section. (4) Report to congress.--Not later than one year after the date of completion of the inventory under paragraph (2), the Secretary shall report to Congress on the findings and determinations of the inventory. (f) State Programs.--The Secretary shall implement the requirements of this Act pursuant to the regulations promulgated pursuant to subsection (a), except that any State with an approved State program under section 503 of the Surface Mining Control and Reclamation Act of 1977 (30 U.S.C. 1253) may submit to the Secretary a revision to such State program to incorporate the regulations under subsection (a) subject to the terms and conditions of section 503 of the Surface Mining Control and Reclamation Act of 1977 (30 U.S.C. 1253). (g) Relationship to Other Law.-- (1) Federal law.--Nothing in this section shall affect any authority under any other Act of Congress to prohibit the construction or operation of any impoundment for the storage or disposal of covered wastes. (2) State law.--Any reclamation, land use, environmental, or public health protection standard or requirement in State statute or regulation with respect to the regulation of impoundments or of the storage or disposal of covered wastes that meets or exceeds the requirements and prohibitions of this section and the regulations issued under this section shall not be construed to be inconsistent with this section or any regulation under this section. (h) In General.--In this section: (1) Covered wastes.--The term ``covered wastes''-- (A) means material referred to as ``other wastes'' in section 515(b)(11) of the Surface Mining Control and Reclamation Act of 1977 (30 U.S.C. 1265(b)(11)); and (B) includes coal ash, slag, and flue gas desulfurization materials stored or disposed of in liquid, semi-liquid, or solid form. (2) Impoundment.--The term ``impoundment'' means any dam or embankment used to retain covered wastes. (3) Secretary.--The term ``Secretary'' means the Secretary of the Interior.

Title: To direct the Secretary of the Interior to promulgate regulations concerning the storage and disposal of matter referred to as "other wastes" in the Surface Mining Control and Reclamation Act of 1977, and for other purposes Summary: Coal Ash Reclamation, Environment, and Safety Act of 2009 - Directs the Secretary of the Interior to promulgate regulations that: (1) establish design, engineering, and performance standards that provide for safe storage and disposal of covered wastes in impoundments; and (2) contain requirements that are substantially similar to the requirements that apply to impoundments under provisions in the Surface Mining Control and Reclamation Act of 1977 concerning environmental protection performance standards for surface coal mining and reclamation operations and standards and criteria for coal mine waste piles. Prohibits persons from constructing or operating impoundments for the storage or disposal of covered wastes on any land in any state except as in accordance with such regulations. Provides for the monitoring and enforcement of such requirements and prohibitions. Requires the Secretary to complete an inventory of existing impoundments for the deposit and maintenance of covered wastes. Authorizes the Secretary to issue orders necessary to ensure that impoundments comply with requirements. Defines the term "covered wastes" to mean wastes in areas other than the mine working or excavations, including coal ash, slag, and flue gas desulfurization materials stored or disposed of in liquid, semi-liquid, or solid form.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Nuclear Black-Market Elimination Act''. TITLE I--SANCTIONS FOR NUCLEAR ENRICHMENT AND REPROCESSING TRANSFERS BY FOREIGN PERSONS SEC. 101. AUTHORITY TO IMPOSE SANCTIONS. Notwithstanding any other provision of law, the President is authorized to prohibit, for a period of not less than three years, any transaction or dealing by a United States person or within the United States with any foreign person or entity that the President determines sells, transfers, brokers, finances, assists, delivers or otherwise provides or receives, on or after the date of the enactment of this Act-- (1) nuclear enrichment or reprocessing equipment, materials, or technology to-- (A) any country for which an additional protocol with the International Atomic Energy Agency for the application of safeguards (as derived from IAEA document INFCIRC/540 and related corrections and additions) is not in force; or (B) to any country that the President determines is developing, constructing, manufacturing, or acquiring a nuclear explosive device; or (2) designs, equipment, or specific information to assist in the development, construction, manufacture, or acquisition of a nuclear explosive device by a non-nuclear weapon state. SEC. 102. PRESIDENTIAL DETERMINATION. (a) Determination.--If the President receives credible information or evidence regarding any activity described in section 101(a) by a foreign person or entity, the President shall promptly make a determination as to whether, in his judgment, such activity occurred. (b) Report.--If the President makes an affirmative determination under subsection (a), the President shall, within 5 days after making the determination, report the fact and substance of the determination to the appropriate congressional committees. (c) Publication of Determination.--If the President makes an affirmative determination under subsection (a), the President shall publish in the Federal Register, not later than 15 days after reporting such determination to the Committees under subsection (b), the identity of each foreign person or entity that is subject to that determination and on whom sanctions have been imposed under section 101(a), the reasons for the sanctions, and period during which the sanctions will be in effect. SEC. 103. ADDITIONAL REPORTS. (a) Possible Activity.--The President shall submit to the appropriate congressional committees, not later than January 30th of each year, a report containing all credible information regarding the activities described in section 101(a), regardless of whether the President determines that such activities did in his judgment occur. (b) Transactions by Foreign Persons.--The President shall submit to the appropriate congressional committees, not later than June 30th of each year, a report that identifies any foreign person or entity that engages in transactions or dealings with foreign persons or entities on whom sanctions are in effect under section 101(a) that-- (1) would be prohibited transactions or dealings subject to sanctions under section 101(a) if those transactions or dealings had been conducted by United States persons or within the United States; and (2) could make material contributions to a nuclear enrichment, reprocessing, or nuclear weapon development program. The report under this subsection shall be unclassified to the maximum extent feasible, but may also include a classified annex. TITLE II--INCENTIVES FOR PROLIFERATION INTERDICTION COOPERATION SEC. 201. AUTHORITY TO PROVIDE ASSISTANCE. Notwithstanding any other provision of law, the President is authorized to provide, on such terms as he deems appropriate, assistance under section 202 to any country that cooperates with the United States and with other countries allied with the United States to prevent the transport and transshipment of items of proliferation concern in its national territory or airspace or in vessels under its control or registry. SEC. 202. TYPES OF ASSISTANCE. The assistance authorized under section 201 is the following: (1) Assistance under section 23 of the Arms Export Control Act (22 U.S.C. 2763). (2) Assistance under chapter 4 of part II of the Foreign Assistance Act of 1961, notwithstanding section 531(e) or 660(a) of that Act. (3) Drawdown of defense equipment and services under section 516 of the Foreign Assistance Act of 1961. SEC. 203. CONGRESSIONAL NOTIFICATION. Assistance authorized under this title may not be provided until at least 30 days after the date on which the President has provided notice thereof to the appropriate congressional committees, in accordance with the procedures applicable to reprogramming notifications under section 634A(a) of the Foreign Assistance Act of 1961. SEC. 204. LIMITATION. Assistance may be provided to a country under section 201 in no more than 3 fiscal years. SEC. 205. USE OF ASSISTANCE. To the extent practicable, assistance provided under this title shall be used to enhance the capability of the recipient country to prevent the transport and transshipment of items of proliferation concern in its national territory or airspace, or in vessels under its control or registry, including through the development of a legal framework in that country to enhance such capability. SEC. 206. AUTHORIZATION OF APPROPRIATIONS. (a) Appropriations.--There is authorized to be appropriated $250,000,000 to carry out this title. (b) Availability.--Amounts appropriated pursuant to subsection (a) are authorized to remain available until expended. SEC. 207. LIMITATION ON SHIP TRANSFERS. Notwithstanding any other provision of law, the United States may not transfer any excess defense article that is a vessel to a country that has not provided written assurances to the United States that it will support and assist efforts by the United States to interdict items of proliferation concern. SEC. 208. LIMITATION ON AIRCRAFT TRANSFERS. Notwithstanding any other provision of law, the United States may not transfer any excess defense article that is an aircraft to any country that has not provided written assurances to the United States that it will support and assist efforts by the United States to interdict items of proliferation concern. TITLE III--ROLLBACK OF KHAN NUCLEAR PROLIFERATION NETWORK SEC. 301. COOPERATION OF PAKISTAN. (a) Limitation.--Notwithstanding any other provision of law, the President may not provide, in any fiscal year, more than 75 percent of United States assistance to Pakistan unless the President determines and certifies to the appropriate congressional committees that Pakistan-- (1) has verifiably halted any cooperation with any state in the development of nuclear or missile technology, material, or equipment, or any other technology, material, or equipment that is useful for the development of weapons of mass destruction, including exports of such technology, material, or equipment; and (2) is fully sharing with the United States all information relevant to the A.Q. Khan proliferation network, and has provided full access to A.Q. Khan and his associates and any documentation, declarations, affidavits, or other material that bears upon their proliferation network activities and contacts. (b) Waiver.-- (1) Authority.--The President may waive the requirements of subsection (a) in a fiscal year if-- (A) the President has certified to the appropriate congressional committees that-- (i) the waiver is in the vital interest of the national security of the United States; (ii) the waiver will promote Pakistan's cooperation in achieving the conditions set forth in paragraphs (1) and (2) of subsection (a); and (iii) Pakistan's lack of cooperation is not significantly hindering efforts of the United States to investigate and eliminate the Khan proliferation network and any successor networks; and (B) 30 days have elapsed since making the certification under subparagraph (A). (2) Briefing.--Within 5 days after making a certification under paragraph (1), the Secretary of State shall brief the appropriate congressional committees on the degree to which Pakistan has or has not satisfied the conditions set forth in paragraphs (1) and (2) of subsection (a)(1). (3) Limitation.--The waiver authority under paragraph (1) may not be exercised in two successive fiscal years. SEC. 302. IDENTIFICATION OF PROLIFERATION NETWORK HOST COUNTRIES. (a) Report.--Not later than 30 days after the date of the enactment of this Act, the President shall submit a report to the appropriate congressional committees that identifies any country in which manufacturing, brokering, shipment, transshipment, or other significant activity occurs that is related to the transactions carried out by the various elements and entities of the A.Q. Khan nuclear proliferation network. (b) Additional Information.--After the report is submitted under subsection (a), the President shall submit to the appropriate congressional committees any additional information described in subsection (a) with respect to any country, as such information becomes available. SEC. 303. SUSPENSION OF ARMS SALES LICENSES AND DELIVERIES TO PROLIFERATION NETWORK HOST COUNTRIES. (a) Suspension.--Upon submission of the report and any additional information under section 302 to the appropriate congressional committees, the President shall suspend all licenses issued under the Arms Export Control Act, and shall prohibit any licenses to be issued under that Act, to any country identified in the report or additional information, until such time as the President certifies to the appropriate congressional committees that such country-- (1) has-- (A) fully investigated the activities of any person or entity within its territory that has participated in the Khan nuclear proliferation network; and (B) taken effective steps to permanently halt all such activities; (2) is fully cooperating with the United States in investigating and eliminating the Khan nuclear proliferation network and any successor networks operating within its territory; and (3) has enacted new laws, promulgated decrees or regulations, or established practices designed to prevent future such activities from occurring within its territory. (b) Waiver.--The President may waive the requirements of subsection (a) in a fiscal year if-- (1) the President has certified to the appropriate congressional committees that the waiver is in the vital interest of the national security of the United States; and (2) 5 days have elapsed since making the certification under paragraph (1). TITLE IV--GENERAL PROVISIONS SEC. 401. DEFINITIONS. In this Act: (1) Appropriate congressional committees.--The term ``appropriate congressional committees'' means the Committee on International Relations of the House of Representatives and the Committee on Foreign Relations of the Senate. (2) Excess defense article.--The term ``excess defense article'' has the meaning given that term in section 644(g) of the Foreign Assistance Act of 1961 (22 U.S.C. 2403(g)). (3) Foreign person.--The term ``foreign person'' means a person other than a United States person. (4) Items of proliferation concern.--The term ``items of proliferation concern'' means any equipment, materials, or technology that could materially support the research, development, manufacturing, or acquisition by any means of a nuclear explosive device, a chemical or biological weapon, or missile with a payload of 500 kilograms or greater and with a range of 300 kilometers or greater. (5) Non-nuclear weapon state.--The term ``non-nuclear weapon state'' means any state other than the United States, the United Kingdom, France, the Russian Federation, or the People's Republic of China. (6) Person.--The term ``person''-- (A) means a natural person as well as a corporation, business association, partnership, society, trust, any other nongovernmental entity, organization, or group, and any governmental entity, or subsidiary, subunit, or parent entity thereof, and any successor of any such entity; and (B) in the case of a country where it may be impossible to identify a specific governmental entity referred to in subparagraph (A), means all activities of that government relating to the development or production of any nuclear equipment or technology. (7) United states assistance.--The term ``United States assistance'' means assistance under the foreign operations, export financing, and related programs appropriations Act for a fiscal year, and assistance under the Foreign Assistance Act of 1961. (8) United states person.--The term ``United States person'' has the meaning given that term in section 14 of the Iran and Libya Sanctions Act of 1996 (22 U.S.C. 1701 note).

Title: To impose sanctions on foreign entities that engage in certain nuclear proliferation activities, and for other purposes Summary: Nuclear Black-Market Elimination Act - Authorizes the President to prohibit, for at least three years, any transaction or dealing by a U.S. person or within the United States with any foreign person or entity that the President determines sells, transfers, brokers, finances, assists, delivers or otherwise provides or receives, on or after the date of the enactment of this Act: (1) nuclear enrichment or reprocessing equipment, materials, or technology to any country for which an additional protocol with the International Atomic Energy Agency for the application of safeguards is not in force, or to any country that the President determines is developing, constructing, manufacturing, or acquiring a nuclear explosive device; or (2) designs, equipment, or specific information to assist in the development, construction, manufacture, or acquisition of a nuclear explosive device by a non-nuclear weapon state. Authorizes the President to provide assistance for up to three years under the Arms Control Act and the Foreign Assistance Act of 1961, as well as a drawdown of defense equipment and services under the latter Act, to any country that cooperates with the United States and U.S. allies to prevent the transport and transshipment of items of proliferation concern in its national territory or airspace or in vessels under its control or registry. Prohibits the United States from transferring any excess defense article that is a vessel or aircraft to a country that has not provided written assurances that it will support and assist U.S. efforts to interdict items of proliferation concern. Prohibits the President from providing, in any fiscal year, more than 75 percent of U.S. assistance to Pakistan unless Pakistan meets certain requirements, including fully sharing with the United States all information relevant to the A.Q. Khan proliferation network, and providing full access to A.Q. Khan, his associates, and any material that bears upon their activities and contacts. Provides for a national security waiver of such requirements, but prohibits its exercise in two successive fiscal years. Requires the President to: (1) identify proliferation network host countries to appropriate congressional committees; and (2) suspend all arms sales licenses to such countries.

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Summarize: By. Deni Kirkova. PUBLISHED:. 10:13 EST, 23 May 2013. |. UPDATED:. 10:13 EST, 23 May 2013. When someone overweight claims to eat healthily and exercise but sill can't shift the pounds, it can be difficult to believe them. But that is just the situation Londoner Sidone Price found herself in after doctors said her almost permanent bloating and excessive weight was down to IBS. Fortunately Sidoine did not give up the fight to find a diagnosis and, on a colleague's advice, sought a food intolerance test - and found the real reason behind her size. Airport Lounge Manager Sidone, 26, says: 'I had always been quite curvy, but over the last few years I had noticed my weight just kept creeping up, so I decided to do something about it.' She started eating much healthier, sticking. to five small meals a day with a mixture of lean protein and lots of. vegetables and salads. But Sidone struggled to shift much of her uncomfortable 14st weight. Doctors said Sidoine's incessant bloating and excessive weight was down to IBS. 'I also joined a gym and started working out three times a week. I noticed that some of the weight came off, but it just seemed really slow and I wasn't seeing the results I was expecting considering how healthily I was eating and how much effort I was putting in at the gym. 'I was also constantly bloated every time I'd eat which was causing me a lot of pain and discomfort, not to mention left me feeling I felt embarrassed. 'Months of healthy eating and exercise. were proving to have little effect, especially on my stomach area which. remained stubbornly bloated. 'I became quite anxious about the situation and so I went to see my GP. He wasn't very helpful and just told me to carry on as I was, telling me that the bloating was probably just IBS and that the weight would fall off eventually. 'In desperation I tried a radical. diet that involved eating mainly raw carrots and houmous at lunch, but again this proved to have little effect. 'It. was then that a colleague suggested that I might have a food. intolerance and recommended that I try a food intolerance test. Sidoine was able to shed those stubborn excess pounds she'd been trying so hard to get rid of. 'When the results came back they revealed that I had intolerances to a number of the 'healthy' foods I'd been munching for the past few months - including carrots!' 'Cutting these from my diet had an almost instantaneous effect. Within days my stomach was noticeably flatter and my IBS symptoms had greatly reduced.' 'As well as carrots, I also found out. that I am intolerant to cow's milk and yeast. I have cut these. completely from my diet. Three months later I am still sticking to my. new diet. The YorkTest intolerance test made Sidone change her eating habits immediately and while she carries on with her healthy diet from before, she has had to find alternatives to her reactive foods: carrots, cow's milk and yeast. 'I. eat quite a lot of stir-fried vegetables as I find just boiled veg can. be quite dull. Where before I'd always throw in some carrots, now I've. replaced this with other veg such as courgettes. 'As I've completely. cut out cow's milk, I now use almond and soya milk for smoothies and. soups. 'Wine contains yeast so I have also replaced my usual white wine for spirits. I'll go for a G&T now instead of a glass of wine. 'I also steer clear of using dressings and sauces because they can contain yeast too. I've stopped using soya sauce when I make stir-fry as I've struggled to find a yeast-free version.' In the months that followed Sidoine was able to shed the stubborn excess pounds she had been trying so hard to get rid of. 'I don't yet have a boyfriend but I am. dating. I didn't feel confident about dating before but I do much more. now. Also I'm much more confident on. holiday and in summer clothes - I can finally enjoy clothes shopping now whereas. before I would avoid it,' she said. She. now weighs in at a svelte 9st 7lbs, losing well over 4st, and wears a size 12 - three sizes down from her peak at size 18. 'It feels like my entire life has changed', she says. 'I wouldn't go back to my old diet and I'll make sure I never have trigger. foods again... I've gone cold turkey.' Recent research suggests a direct correlation between food intolerance and weight gain. Last year's Obesity and Weight loss Therapy research. entitled: Eliminating Immunologically-Reactive Foods from the Diet and. its Effect on Body Composition and Quality of Life in Overweight Persons. found that by simply cutting trigger foods out of your diet, the battle. of the bulge could be over. 'Cutting carrots, cow's milk and yeast had an almost instant effect and within days my stomach was flatter' Breakfast: porridge or a smoothie for breakfast (strawberry, banana and blueberry with some natural live yogurt) Lunch: carrots and reduced fat hummus (which she later learned was no good for her) Dinner: chicken with stir fried vegetables (onions, mushrooms, bean. sprouts, carrots, peppers), salmon and salad, omelette (usually using. skimmed milk) Breakfast: Smoothie (strawberry, blueberries, banana and soya or almond milk) Lunch: Soup (homemade potato and leek or root vegetable minus carrots and using dairy-free cream) Dinner: omelette minus milk, jacket potato with tuna and sweetcorn, chicken with vegetables (broccoli, cauliflower) With even seemingly healthy foods such as lettuce having the potential to cause a reaction, the study shows it could be beneficial to find out your own personal triggers. The research assessed the effect that IgG food sensitivity tests and the subsequent elimination of trigger foods for a period of 90 days had on the body composition and quality of life of 120 overweight people. The results showed that the subjects who eliminated the foods they reacted to 'had reductions in weight, body mass index, waist and hip circumference… and all indicators of quality of life'. 'Food intolerance is a serious issue and is often misdiagnosed. I meet numerous clients who consistently eat healthy and exercise regularly but still find it hard to shift those last few pounds. 'Food intolerances can cause an immune response in the body which can lead to weight gain or difficulty in losing weight' nutritionist, Alli Godbold. Dr. Gill Hart, Scientific Director at YorkTest, explains: 'A stressed digestive system is less able to process energy and nutrients for the rest of the body. It will also be less able to repel harmful invaders and may be more prone to disorders such as IBS. 'Once the immune response is triggered in the body, it feels that it is under siege thus hoarding supplies, storing fluid and increasing weight.'

Summary: Sidoine Price, 26, ate healthily but was bloated and overweight. GP blamed her 14st weight on IBS and said to carry on as normal. Colleague recommended she try food intolerance testing. Discovered she reacted badly to carrots, yeast and cow's milk. Cut foods from diet and slimmed three dress sizes in three months. Research shows cutting reaction foods result in'reductions in weight, body mass index, waist and hip circumference'

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Write a title and summarize: Home schooling may have been a success for some but in my overall experience - and that of the parents I have spoken to - those successes have been few and far between. Whether at the school gates, or in the counselling arena, mums and dads tell me they cannot cope. I would go so far to say that anyone who thinks working and schooling young children from home has been a success is sadly ill-informed. My husband and I have tried to make the memories of this time more joyful and positive. So we camped in our (courtyard) garden and have had more late nights, cuddled up watching movies than we would have done previously. However, it has inevitably been a very demanding time for everyone. When people ask how I am doing, I often joke that I am "surviving not thriving". When the work calls start, it is very hard to be there for our children. Running feral So many working parents have shared with me stories of having to work on constant Zoom calls while their children "run feral". One single mum called time on her employer after being on yet another conference call when she heard a gushing sound next door. Her three children - aged 10, 7 and 3 - had run a bath and left the water running. The entire kitchen was flooded. Another told me how her four-year-old had left a note to his dad: "This is a storee about a daddy called Androoo, he werced all the time." Parents have spoken of their fears around their children being constantly online. One teacher was in tears to me about the guilt she felt when she was upstairs "looking after other people's children", while her own were downstairs alone, playing video games. To be physically present for our children but not emotionally available - shooing them away while on work calls, sending them downstairs to play while we work upstairs - sends them a very definite message: you're in the way. They feel a deep sense of rejection from what is, to them, a very visible representation that work comes before them. "I can't go on," one mum told me. "It's horrific," said another, for whom the fear of losing her job means she cannot put her children first. Others tell me how inadequate they feel. Some say they are drinking more and shouting too much. Fathers say the pressure of an uncertain future and trying to home-school young children has pushed them to the brink. One dad told me he had never smacked his children but said he had come close during lockdown. A mother admitted that her 10-year-old daughter woke her up to say goodbye to her as she left for school. Frazzled, exhausted and fearful after the loss of her husband's job, and with her own in the balance, she had fallen asleep at the kitchen table over breakfast. Even teachers are struggling. Ed Vainker, executive principal at Reach Academy, Feltham, in west London, shared with me the difficulty he has had. "I don't want to have to be headteacher to my child. Home schooling can challenge the parent-child relationship." We can repair this damage - but only if we collectively acknowledge that the combination of home schooling and working from home is, quite frankly, not working. "We need to think about how to support parents and teachers, with their own feelings of vulnerability, so they feel equipped to manage what often feels so very unmanageable," Dame Benita Refson, founder and president of children's mental health charity Place2Be, says. "As parents and teachers, our own behaviour speaks volumes to children, often leaving a lasting impression on the way the child views their world." Dame Benny says many children are fearful of returning to school. The lack of routine means returning to class is going to be difficult for them. Children from black and Asian communities may be particularly anxious, given the news reports of disproportionate impacts of coronavirus on their communities. Kate's tips to deal with stress at home What helps me is to ensure I have had time to connect with my children each day, to create "magic moments" as I call them. Yesterday, it was a game of skipping where my tripping up was the highlight. The day before we baked. The day before that we made playdough and just sat and created "food". My children are young, but for all our children having our physical presence - watching a movie, or during a meal - is often what they crave the most, even though they might not always articulate it. These are the moments our children will remember of this period - the quality time, without phones, without being hurried, just "being" and laughing. When we carve out that time for our children, to be with them exclusively, it's the equivalent of a big, long hug. It makes them feel safe. And I cannot stress enough how much it helps us as parents too.

Title: Coronavirus: Kate Silverton on her fear for children's mental health Summary: Home schooling is causing stress in millions of homes across the UK. BBC newsreader Kate Silverton, who is also a trainee child psychotherapist and a mother-of-two, has like most parents found lockdown a very challenging time. After speaking to other parents about their own stresses and fears, she warns about the damage the pandemic could do to children's mental health.

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Summarize: These are the chilling selfies a female serial killer and her alleged accomplice took while the pair were on the run from police. Joanna Dennehy, 31, has admitted stabbing three men in the heart and dumping their bodies in ditches. The bodies of her three victims were found in fields near to Peterborough, Cambridgeshire, in March and April last. year. Scroll down for video. Joanna Dennehy, 31, poses for a photo with a terrifying knife while she was on the run for stabbing three men in the heart. Joanna Dennehy poses in another shocking photo with her top lifted, exposing her underwear. The woman has admitted killing three and the attempted murder of two others. Despite killing the three, Dennehy. remained unsatisfied and fled to Hereford in search of more victims, a. jury heard. She cruised the city and selected two other men at random, stabbing Robin Bereza, 64, and John. Rogers, 56, who survived the cold-blooded attacks. Alleged conspirators Gary 'Stretch' Richards, 47, and Leslie Layton, 37, who are now on trial at Cambridge Crown Court, deny helping to cover up the 'terrible truth' of the murders. Pictures of Dennehy and Richards were shown to the court as Mark Lloyd, an associate of the man, gave evidence. Georgina Page, a friend of Dennehy’s, also gave evidence today. The shocking images include one of Dennehy jovially sticking her tongue out as she brandishes a jagged knife with handcuffs attached to her trousers. In another the killer is pictured revealing her underwear and standing in a provocative sexual position in the days after committing her crimes. It shows a series of horrendous scars caused by the mother’s relentless self-harming. Joanna Dennehy's alleged co-conspirator Gary 'Stretch' Richards poses for a photo. He denies three counts of preventing the lawful and decent burial of a body and two counts of attempted murder. In the very next picture, Richards is seen glaring at the camera while pulling his jumper up and standing in another sexual pose. And in another two pictures the defendant and Mr Lloyd are seen sticking their middle fingers up. A final image shows the pair casually leaning on a fence just minutes before Dennehy tried to stab another two men to death. Dennehy,. of Orton Goldhay, Peterborough, Cambridgeshire, pleaded guilty to the. three murders and two attempted murders at the Old Bailey in November. She also admitted preventing the lawful and decent burial of all three. victims and is in custody awaiting sentencing. Her victims were her lover Kevin Lee, 48, his friend Lukasz Slaboszewski, 31, and housemate John Chapman, 56. Mark Lloyd (left) and Gary Richards poses for a photo. Moments later, Dennehy tried to stab another two men to death. Richards of Orton Goldhay,. Peterborough, denies three counts of preventing the lawful and decent. burial of a body and two counts of attempted murder. Leslie. Layton, 37, of Orton Goldhay, denies two counts of preventing the. lawful and decent burial of a body and one count of perverting the. course of justice. A third man Robert Moore, 56, of Peterborough, has admitted assisting an offender and is awaiting sentence. As the pictures were showed to the jury today, Mr Lloyd revealed he had been forced to watch on helplessly as Dennehy tried to kill two strangers in Hereford. Mr Lloyd, who met Dennehy at a friend’s flat in Kington, Herefordshire, on April 1, shortly before the attempted murders, told the court she stank of blood. He said she told him: ‘I’ve killed three people, Gary helped dispose of them and I want to do some more. I want some fun.’ Richards swears at the camera and sticks his tongue out. Mr Lloyd said Dennehy became flirtatious with him before asking him to come in the car when she carried out the attacks. He said: ‘She was caressing my backside and running her hand down my neck. It was like being touched by a rattlesnake.’ Mr Lloyd said he watched helplessly as Dennehy was driven around Hereford looking for people to kill. He. described the two attacks on random dogwalkers as like a scene from the. film Psycho: ‘It was frenzied. She just went absolutely mad. 'She takes a knife out of her. left pocket with her right hand. She strikes him like in the film. Psycho, just like that. Thrusting and putting her whole weight behind. it. 'I just kept saying "what the f*ck is going on, I haven’t signed up for this." 'Gary is seeing exactly what I’m seeing and there’s just no emotion from him at all.' Victims Lukasz Slaboszewski (left) and Kevin Lee - who were stabbed in the heart by Dennehy. Mr Lloyd said Dennehy returned to the car and kissed Stretch on the cheek 'as if to say thanks'. He said the second attack was a 'brutal re-run' of the stabbing just moments earlier. John Chapman was also stabbed to death by the 31-year-old. After the second knife attack Mr Lloyd said Dennehy returned with the victim’s dog in her arms and declared it was her new pet. Mr Lloyd said: 'It was terrified of her. It was physically shaking on her lap. She wanted to take the dog for a walk.' He. added that Dennehy treated 7ft 3in Stretch 'like a puppy' and said the. giant would be dead if the killer hadn’t needed him to drive her around. Georgina Page then told the court how she and Dennehy became friends while serving jail sentences together. She said she next saw her former cell mate when she and Stretch turned up on her doorstep in King’s Lynn, Norfolk, following the murders. She said: 'When I was inside with her she wasn’t a murderer or nothing. At first I didn’t believe her. 'I was very shocked. I was scared, I didn’t know what to do.' The court also heard that during the pair’s stay at Ms Page’s house Dennehy was 'jumping around' in delight at seeing her face on the news. Ms Page said the killer downed whisky and ate a ham sandwich while bragging about her violent acts. She said: 'I was terrified. Stretch turned round and said that if anyone grassed on them he would get his dad to sort them out. Picutred is the knife used by Joanna Dennehy to attack her five victims. The later two men survived the attacks. 'Joanna was talking about something and saying "you know we’re going to get caught and sent to jail for a long time".' She also said Dennehy compared herself and Stretch to 'Bonnie and Clyde' whose gang killed nine policeman in 1930s America. Killer: Joanna Dennehy stabbed three men in a 10-day spree of violence. And. when she asked her why she had killed, Dennehy responded: 'They. shouldn’t have pissed me off, they shouldn’t have flirted with me' A picture of the knife believed to be used in all five attacks was also shown to court and described as a nine-inch fold knife. Mr Lloyd said he saw the same knife in Dennehy’s hands at a flat in Hereford occupied by his friend Moby. It was there that Dennehy took pictures of herself and Stretch as they prepared to drive round the city in a bid to kill again. Mr Lloyd told the jury that the blade 'was as black as the handle' because of all the congealed blood and that it'smelt like copper'. He said Dennehy than told him: 'I’ve killed three people, Gary’s helped dispose of them and I want to do some more. You’ve had your fun Gary, I want my fun.' Mr Lloyd also revealed that Dennehy flirted with him and 'caressed' his neck and backside. He told the court she said: 'I wish I could have met you last year, your my type of guy' He said that Stretch 'was not liking it one bit' before the three of them got into the green Vauxhall Astra that the killer had travelled in from Peterborough. Mr Lloyd then said he tried to call the police while Stretch drove around Hereford looking for victims for Dennehy. But he failed to do so and told a court that 'Gary was the taxi driver, she went around killing people'. The trial continues. Sorry we are not currently accepting comments on this article

Summary: Joanna Dennehy, 31, pictured in a series of shocking selfies. Seen posing with a large knife and in her underwear while on the run. Her alleged accomplice, 7ft 3in Gary 'Stretch' Richards, 47, also pictured. Dennehy has admitted stabbing three men in the heart and dumping bodies. She has also pleaded guilty to attempted murders of two other men. Richards and another alleged accomplice Leslie Layton are currently on trial. Court heard Dennehy compared herself and Stretch to 'Bonnie and Clyde'

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Summarize: By. Ashley Collman. The father of a South Carolina woman who went missing last month, wrote an emotional confessional about the experience of her sudden loss - sharing the last image she sent him the night before she disappeared. Heather Elvis, 20, was last seen after being dropped off from a first date at her condo in Horry County in the early morning of December 18. The next night, a police officer showed up at father Elvis family home, informing Heather's father Terry that her car had been found abandoned at a nearby boat landing. The last photo: Heather Elvis, 20, sent this picture of herself learning to drive a stick-shift while on a first-date to her father Terry the night before she disappeared last month. Tireless: Father Terry Elvis prints missing person posters for his daughter. The last time Mr Elvis had heard from his cosmetology-student daughter was the night of December 17, when she texted him a picture of her driving during her date. Mr Elvis explained in an essay on xoJane that he has a manual transmission truck he had been saving to give to one of his daughters, but that neither of them had ever learned how to drive a stick shift. At the end of her date, the man she was meeting offered to teach her how to drive his manual truck in a mall parking lot and she excitingly sent the picture of her driving to her father to share the accomplishment. The search continues: There is a $25,000 reward for Heather's return. A candlelight vigil will be held for her on Thursday. 'It is the last communication I have had with my daughter, the last picture I have of her on my phone,' Mr Elvis said. 'I didn't talk to Heather about it, but she had a good time,' Mr Elvis added about the date. 'I didn't think much of it.' Heather's disappearance remains a mystery, but Mr Elvis believes'someone who she knew and trusted may have betrayed her'. Mr Elvis never met the man who took his daughter out on a first date, but he has since been ruled out as a suspect after being questioned by police and passing a lie-detector test. His favorite picture: Mr Elvis said this was his favorite picture of Heather, holding the family dog in their kitchen. Heartbroken: The family put Christmas on hold following Heather's mid-December disappearance, but remain optimistic they will find her. He said Heather's car was parked in the condo's driveway when he dropped her off around 2 or 2:30 am on the morning of December 18. Since then, Mr Elvis has worked tirelessly in the police investigation and search for his daughter. According to the Find Heather Elvis Facebook page, there is a $25,000 reward for Heather's return, $10,000 of which is coming from Mr Elvis and his family. Earlier in January, police thought they had made a break in the case when they found a set of skeletal remains while searching for Heather - but the bones ended up belonging to a male and had been in the earth for at least 20 years. Mystery: Police have since ruled out Heather's date as a suspect after he passed a lie-detector test. He says he saw Heather's car parked in her driveway when he dropped her off around 2 or 2:30 on December 18. A prayer vigil will be held for Heather this Thursday as Horry County locals continue to volunteer their time to search for Heather. In his essay, Mr Elvis writes that Heather's disappearance has taught him not to take his loved ones for granted since you never know when you might lose them. 'Hold your loved ones close, tell them how much you love them every day. Never let them doubt how much love you have for them so that if you are ever in the position I am in, you will be able to look in the mirror with less regret,' Mr Elvis advised. Still, Heather's father remains optimistic that he will eventually find Heather. 'It has been 30 days of hell. But I am praying for a miracle. And I refuse to give up hope,' he wrote. 'We will find my daughter. Will will find Heather Elvis.'

Summary: Police are still looking for 20-year-old Heather Elvis who went missing following a first date on December 18. The day following the date, her car was found abandoned at a dock. In an essay on xoJane, her father Terry Ellis shared the last picture she texted him the night before she disappeared. The picture shows Heather learning to drive a stick-shift on the date. Her disappearance remains a mystery but the man who took her out has been ruled out as a suspect after passing a lie detector test. Heather's father remains optimistic that he will find his daughter.

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Summarize: Il 13 maggio del 1999, nei cinema degli Stati Uniti usciva la commedia blockbuster “Notting Hill”, diretta da Roger Michell. La storia è quella di William (Hugh Grant), un uomo divorziato che vive nel famoso quartiere londinese di Notting Hill dove gestisce un negozio di libri di viaggio sulla Portobello Road. Un giorno, entra Anna Scott (Julia Roberts), star del cinema che compra un libro da lui, ci scambia quattro chiacchiere e se ne va. Grazie ad un successivo “incidente”, Will riuscirà ad invitare Anna a casa sua e da quel momento Cupido ci metterà inevitabilmente lo zampino, ma ne succederanno di tutti i colori. Roger Michell e lo sceneggiatore Richard Curtis, autore degli script di altre commedie di successo come “Quattro matrimoni e un funerale” (1994), poi del divertente “Il diario di Bridget Jones” (2001) e “About a boy” (2002), tutti sempre interpretati da Hugh Grant, seppero portare nelle sale una pellicola che già sulla carta profumava di blockbuster. La storia del “Cenerentolo” imbranato e squattrinato che conquista la star del cinema mondiale da 15 milioni di dollari a film non poteva non far sognare i cinefili più romantici e conquistare il cuore di tutti quelli che credono nei sogni e nelle favole a lieto fine. In “Notting Hill” c’è tutto questo e anche di più, grazie anche alla fotografia eccezionale di Michael Coulter e alle scenografie di Stuart Craig che hanno saputo incorniciare alla perfezione gli eventi surreali e i personaggi messi in scena da Michell con estrema maestria e mostrando il famoso quartiere multietnico di Londra come una piccola oasi caotica, poetica, onirica e quasi fuori dal tempo. Il film è quindi già tra i capisaldi della storia del cinema romantico a soli 20 anni dalla sua uscita e rientra nella lista di quei film che non smetteremmo mai di rivedere sempre con piacere. L’alchimia tra Hugh Grant e Julia Roberts è una delle colonne portanti dell’intero progetto, ma lo sono anche tutti gli altri attori “secondari” del film tra i quali spiccano Emma Chambers nei panni di Honey, la stralunata sorella di William; Rhys Ifans nel ruolo del folle e mitico coinquilino Spike e Gina Mckee e Tim McInnerny, rispettivamente Bella e Max, i migliori amici di Will e padroni di casa dove si svolgerà la cena con Anna Scott. Assieme ai due protagonisti sono l’anima del film e danno vita alle gag al fulmicotone, alle situazioni al limite del paradossale e imbarazzanti che ci hanno fatto emozionare e morire dal ridere. Nella pellicola ci sono, però, anche i cameo di altre star come Alec Baldwin, Matthew Modine, Mischa Barton, Sanjeev Bhaskar, Simon Callow e Michael Higgs.

Summary: Il 13 maggio 1999 usciva nei cinema USA la storia tra il libraio squattrinato e timido William (Hugh Grant) e la star del cinema Anna Scott (Julia Roberts) che ha fatto sognare tutto il mondo. Il film ha incassato quasi 364 milioni di dollari ed è stato nominato a 3 Golden Globe ma, soprattutto, è già diventato uno dei più grandi e amati film romantici della storia del cinema.

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Write a title and summarize: Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2) -type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease. Tuberculosis (TB) is a pulmonary disease spread worldwide. It is caused by an infection with Mycobacterium tuberculosis. Only 5–10% of infected individuals develop a primary active disease while the most common outcome of infection is a latent or subclinical disease with no evident symptoms. This latent disease has the inherent ability to reactivate after years or even decades of latency and is therefore a major global threat. The existing vaccine, the Bacille Calmette-Guérin (BCG), is not entirely safe and does not confer protection against latent or reactivated TB. Current antibiotic regimens have started losing their efficacy due to the spread of antibiotic resistance genes [1]. In total, the primary active infections and reactivated infections cause 1–2 million deaths yearly, which makes M. tuberculosis the deadliest bacterium for humans [2]. The dichotomy to a latent and active tuberculosis is an over-simplification, as the infection can actually lead to a wide spectrum of disease states ranging from a well-controlled (or even cleared) latent disease to fulminant, severe forms of TB. Within the latent population, a “sub-spectrum” exists leading to differences in the risk of reactivation [3]. The pathogenesis of tuberculosis has been widely studied for decades, but as it seems that TB is not a single disease but a spectrum of different outcomes, it remains poorly understood. Better understanding on the factors that contribute to the type of TB disease is crucial for the development future treatment strategies. The TB spectrum is likely to arise from genetic variation both in the host and in different pathogen strains as well as from environmental factors. It is known that adaptive immunity and especially T helper (Th) cells are required for controlling the disease. HIV-infected individuals are more susceptible to active and reactivated tuberculosis due to the defective T lymphocyte response [4]. Mice lacking T helper responses are hypersusceptible to TB [5]. Based on the observations that IL-12 or IFN-γ deficient mice are unable to restrict mycobacterial infection, it was initially concluded that Th1 cells are the predominant mediators of protective immunity to M. tuberculosis [6]–[8]. In mice, observations of an early Th1 response (2–3 weeks post infection, wpi) followed by a Th2 response simultaneously with the onset of a chronic phase, have led to a presumption that Th2 response is detrimental to the host by leading to a failure of Th1 response to clear the infection [9], [10]. Subsequently, it has also been shown that the lack of Th2 responses in IL-4, IL-13 or Stat-6 deficient mice does not lead to better resistance to M. tuberculosis infection but, instead, to increased bacterial burdens at later stages of infection [11]. Although the role of humoral immunity in response to mycobacterial infection is still unclear, there is evidence that Th2 responses are needed as well for optimal protective immunity [1], [12]. Despite the emerging understanding of the roles of different Th subtypes in TB immunity, it is still not known what type of Th profiles are needed at different phases of infection to provide optimal protection. In part, this is due to the lack of suitable animal models for studying the full spectrum of disease outcomes, including latency and reactivation. Several animal models have been developed with the aim of understanding the complex pathogenesis of tuberculosis. The murine model of pulmonary TB is well standardized and has made many valuable contributions to the knowledge of the disease pathomechanisms, especially on the role of T cells as mediators of protective immunity [13]. A major constraint of the model is that mice do not develop spontaneous latency although they can restrict the bacterial growth to chronic progressive infection. One of the rare animal models developing true latency is the Cynomolgus macaque. In the macaque, a low-dose M. tuberculosis infection leads to active primary disease in 50% and latent disease in 50% of individuals [14]. In the rabbit model of latent TB, the lung bacterial burdens start declining at 4 wpi following a primary phase with limited bacterial growth. In the rabbit TB model, different outcomes of infection can be induced by using mycobacterial strains with different virulence properties [15], [16]. In addition to the mammalian models of TB, we have previously shown that infection of adult zebrafish with their natural pathogen, Mycobacterium marinum, can be used to model latent TB [17]. M. marinum is a close genetic relative of M. tuberculosis, and typically infects cold-blooded hosts, such as frogs and various freshwater and saltwater fish species [18]. M. marinum infection of zebrafish embryos has been established as an elegant model to dissect the innate mechanisms of protective host responses in active mycobacterial infection [19]–[21]. However, the full spectrum of mycobacterial disease outcomes can be observed only in the adult zebrafish, due to the full maturation of adaptive immune system after the first four weeks post fertilization [21], [22]. In the adult zebrafish model, the injection of a low dose of M. marinum (ATCC 927 type strain) into the abdominal cavity leads to a systemic infection, characterized by an initial 3–4 week phase with rapid bacterial growth, followed in most individuals by a latent phase with stable bacterial burdens. In the latently infected fish, the majority of the mycobacterial population passes into a non-replicative state, dormancy, but can be experimentally reactivated by immunosuppression [17]. The wide disease spectrum typical of mycobacterial disease results from various host- and pathogen-associated factors. It is known that genetic determinants lead to an inherent, stable preference towards either T helper 1 or T helper 2 response that varies between human individuals [23], [24]. As a starting point for our study, we hypothesized that the differences that control the T helper response might be associated with the establishment of the wide spectrum seen in TB patients and that differences in T cell polarity might be related to the progression of the disease. Taking advantage of the heterogeneity of the zebrafish population and the wide spectrum of mycobacterial disease outcomes in the zebrafish model, we set out to look for differences in T helper responses involved in regulating protective response. Finding such differences would 1) allow the use of T helper markers among latently infected individuals to distinguish between those at high or low risk of reactivation and 2) provide understanding on what type of T cell response gives the optimal protection against mycobacterial infection and allow development of novel kinds of therapeutic or preventive approaches. Our previous work [17] provided evidence that functional lymphocyte response is a prerequisite for latency and mycobacterial dormancy in the M. marinum infection of zebrafish. To further demonstrate the significance of lymphocyte responses in the immune defence against mycobacteria in zebrafish, we carried out adoptive transfer experiments on low-dose (21±7 cfu) M. marinum-infected rag1 (−/−) fish. Spleen and kidney marrow cells were transferred from WT or rag1 (−/−) zebrafish immunized with heat-killed M. marinum to rag1 (−/−) recipients at 2 wpi. At 4 wpi, bacterial burdens were significantly lower in the fish that received transplants from immunized WT donors, compared to the fish that received transplants from rag1 (−/−) donors (3. 2×105 vs. 1. 8×106). This indicates that heat-killed M. marinum-induced lymphocytes, rather than NK cells or other innate immune cells, transferred additional immune protection against M. marinum infection to rag1-deficient zebrafish. (Figure 1A). In our current study of ∼150 individuals, a total of 10% of zebrafish were able to clear the bacterial number below the detection limit of M. marinum q-PCR-analysis (∼100 bacteria) (Figure S1B–D). These individuals capable of clearance were not detected in the groups that were collected at 2 wpi, suggesting that the clearance is likely to occur after the activation of adaptive responses. These results attest the significance of adaptive responses in the immune protection against zebrafish mycobacteriosis, and support the view of similarity of human and zebrafish anti-tuberculosis immunity. In the current study, we aimed at further elucidating the details of adaptive immune response leading to a variety of infection outcomes. Unlike many other commonly used laboratory animals, zebrafish populations are genetically heterogeneous. This characteristic causes large variations and standard deviations in most studies utilizing this model, including our studies on mycobacterial infection. On the positive side, the zebrafish population provides starting material for studying the natural differences between individuals. To be able to elucidate the host factors affecting the outcome of infection, the variation in environmental and bacterial factors was first minimized: the bacterial strain, bacterial growth conditions, infection procedure, infection dose and housing of infected fish (water quality and temperature, feeding etc.) were carefully standardized. WT adult zebrafish were infected with a low dose (21±7 cfu) of M. marinum, collected at various time points and divided into three subpopulations, based on their bacterial burdens. The subpopulations were named Low (25% of the population, individuals with the lowest bacterial burdens), Medium (50% of individuals) and High (25% of the population, individuals with the highest bacterial burdens, including the primary-progressive). The Low, Medium and High subpopulations were analyzed at various stages of mycobacterial disease: at 2 wpi (primary active disease), 4 wpi (the onset of latency in the majority of zebrafish) and 5 months post infection (mpi; late stage at which most individuals maintain latency). The reactivation risk of latent mycobacterial infection is thought to increase with increasing bacterial load [25]. The bacterial burdens in the subgroups at different time points are shown in Figure S1. To dissect the differences in the total T lymphocyte numbers between the Low, Medium and High subpopulations, we quantified the cluster of differentiation (cd) 3 levels from internal organs of the zebrafish by q-RT-PCR. Zebrafish cd3 has been shown to be an ortholog of the mammalian T cell marker cd3 [26]. Here, the cd3 expression as a marker for T cell numbers in zebrafish was further validated as described in Figure S2. Induction of T cell expansion was similar in the Low and Medium groups, seen as a 2-fold induction in cd3 expression level already at 2 wpi (Figure 1B) and peaking to 4-fold around 4 wpi (Figure 1C), compared to the cd3 expression levels in non-infected zebrafish. The High group differed from the rest of the population by showing a modest T cell expansion (max. 1. 7±1. 3), which was only seen at 4 wpi (Figure 1C). A similar pattern remained at a late stage of the infection (5 months, Figure 1D). To assess whether the limited T cell expansion is the cause or the consequence of enhanced bacterial growth in the High group, the low-dose-infected fish were compared with a group infected with a high dose of M. marinum (2691±520 cfu). Based on our previous work [17] a high initial dose causes the bacterial load to be significantly higher than with a low initial dose during the first 2 weeks of infection and this difference will even out by 4 wpi. At 4 wpi, cd3 expression levels were significantly lower in the high-dose group (Figure 1E), suggesting that the reduced T cell numbers in the High subpopulation may at least partly be affected by the rapid bacterial growth. Taken together, these results indicate that an early T cell expansion associates with protective response against mycobacterial infection, as the fish with highest cd3 expression levels were always found in the Low and Medium subgroups. However, individuals with modest lymphoproliferative response were found equally in all the three subgroups, suggesting that other factors besides efficient T cell expansion are required for mounting a protective response against mycobacterial infection. As Th cells are potent orchestrators of immune responses during infection, it is reasonable to assume that in addition to total lymphocyte numbers, variation in Th response types may be an important factor underlying the wide spectrum of outcomes in mycobacterial infections. For zebrafish, antibody markers or reporter lines for FACS (fluorescence-activated cell sorting) analysis of different T lymphocyte populations are not available. To assess the Th1/Th2 balance of individuals with different infection outcomes, we measured the levels of master regulator transcription factors for Th1/Th2 lineage development, T-box transcription factor 21 (tbx21) and gata3, from the internal organs of infected zebrafish. Tbx21 is a Th1 cell transcription factor important for Th1 lineage commitment and gata3 is a well-known regulator of Th2 cell differentiation also playing a role in endothelial cell biology [27]. The central T cell transcription factors tbx21, gata3 and foxp3 have been identified in the fish [28]–[30]. The enrichment of tbx21 and gata3 in the zebrafish T cell population was validated as described in Figure S2. During infection, the alterations in the transcript levels of these transcription factors reflect the changes in the numbers of the corresponding T helper cells. The ratio of Th2/Th1 markers was used to assess the balance of T helper cell response. In addition, the induction of a Th2-type cytokine IL-4 (IL4b) [31] and a Th1-type cytokine IFN-γ (ifnγ1-2) was measured and the ratio was calculated. At 2 wpi, the induction of both gata3 and tbx21 was significantly higher in Low and Medium than in the High group (Figure S3A&D). At this time point, there were no significant differences in the gata3/tbx21 ratio between the three groups (Figure 2A). At 4 wpi, gata3 was still significantly more induced in Low and Medium groups compared to High group (Figure S3B). However, the tbx21 levels were similar in all groups (Figure S3E). As determined by the gata3/tbx21 ratio, the Low group had developed a significantly more Th2-biased response than the High group by 4 wpi (Figure 2B), suggesting that insufficiency of Th2 cells is a differentiating factor between the Low and High individuals. The il4/ifnγ ratios generally followed a similar pattern (Figure 2D–E). Also at 2 wpi, the il4/ifnγ ratio was significantly higher in the Low group compared the other two, although there were no significant differences in the gata3/tbx21 ratio at this time point. At 2 weeks, it is likely that the adaptive Th response is in the process of maturation conducted by the cytokines excreted by innate immune cells. Similar patterns were observed at the late time point 5 months post infection (Figure S3C&F, Figure 2C&F). At 4 wpi, the Th2/Th1 balance was also assessed at the protein level by semi-quantitative Western blot analysis of gata3 and CXCR3 (a CXC chemokine receptor preferentially expressed on Th1 cells) from individuals in Low, Medium and High groups (Figure 2J, Figure S4). The results showed a similar trend as seen with q-RT-PCR analyses. To assess the importance of functional, specific lymphocytes for the changes in the levels of the markers used in this study, we also carried out similar infection experiments in rag1 (−/−) mutants. The fish were infected with a low dose (35±18 cfu) and collected 4 wpi. We found that there was some induction of gata3, tbx21, il4 and ifnγ in the infected rag1 (−/−) mutants (Figures S2H&I and 2H&I) showing the proportion of lymphocyte-independent induction of these markers. However, the induction of gata3, tbx21 and il4 was significantly higher in the WT fish (Figures S2H&I and 2H) than in rag1 (−/−) fish at 4 wpi. This clearly demonstrates the major contribution of functional lymphocytes in the changes seen in these markers during mycobacterial infection. The rag1 (−/−) fish were grouped according to bacterial load (Figure S1E) as previously described for the WT fish, and association of the gata3/tbx21 ratio and bacterial load was assessed. In the absence of functional lymphocytes no association was detected, implying that the differences in this ratio relevant to the course of mycobacterial infection seen in WT fish are indeed derived from lymphocytes. However, the expression levels of ifnγ were similar in rag1 (−/−) and WT fish showing that the induction of this Th1-type cytokine in mycobacterial infection might not be as dependent on functional lymphocytes as the other markers used. To assess whether the Th2/Th1 balance is directly influenced by the bacterial burden in the beginning of the infection, low-dose (21±7 cfu) infected fish were compared to fish infected with a high mycobacterial dose (2691±520 cfu) at 4 wpi. Average of gata3/tbx21 ratio was found to be lower in the high-dose infected group (Figure 2K), suggesting that rapid bacterial growth can lead to changes in this ratio. To investigate whether the differences in the disease outcome could result from genetically defined Th1/Th2 preferences, we stimulated healthy WT zebrafish by an i. p. injection of heat-killed M. marinum and 10 days later, analyzed the gata3 and tbx21 transcript levels. The spectrum of individual Th2/Th1 responses was broad, similarly to that seen in humans [23]. The gata3/tbx21 ratio varied from 0. 6 to 12. 8 within a group of 14 zebrafish. 22% of the individuals were substantially Th2-biased (gata3/tbx21>10), whereas 28% had a bias towards Th1 (gata3/tbx21<1) (Figure 2L). This observation of inherent Th1/Th2 phenotypes in healthy fish suggests that genetic Th1/Th2 preferences may in part lead to the development of wide disease spectrum in mycobacterial infections. Based on these results, it seems plausible that both the bacterium and the host can affect the gata3/tbx21 ratio during mycobacterial infection. At any of the time points of the study, the average gata3/tbx21 ratio was never >1. 0 in the progressive High group, whereas Th2 dominant response (average gata3/tbx21 1. 5–2. 7) was seen in the Low group in the primary infection. Altogether, these results show that induction of Th2-type responses during the first four weeks of mycobacterial infection are associated with controlling the bacterial growth. In order to further confirm the reliability of our markers and to characterize the response in the different subgroups, we measured a wider selection of Th2 and Th1 signature genes at 4 wpi: Th2: interleukins il13 and il4, immunoglobulin M (IgM) constant region, V-maf musculoaponeurotic fibrosarcoma oncogene (cmaf), STAT6, St2 (Figure 3A–F) Th1: il12, interferon gamma 1-2 (IFNγ1-2), nitric oxide synthase 2b (Nos2b), tumor necrosis factor alpha (TNFα); (Figure 3G–J). It has been previously shown that the expression levels of the selected Th2 marker genes are upregulated in zebrafish in response to recombinant IL-4 treatment, thus representing a Th2-type response in the zebrafish [31]. In line with gata3 and il4 markers, there was a significantly higher induction of Th2 signature cytokines and other Th2 related genes in the Low group than in the High group (Figure 3A–F). At the same time, Th1 markers showed a somewhat higher induction in the High group (Figure 3G–J). However, the differences in Th2 markers were more indicative of the bacterial burden and the disease state than those seen in Th1 markers. The Low group showed substantial IgM induction at 4 wpi differing significantly from the rest of the population (Figure 3C). Only three antibody classes exist in zebrafish, namely IgM, IgD, and fish-specific IgZ, whereas IgG, IgA and IgE are absent [32]. Secreted tetrameric IgM is the most abundant zebrafish serum immunoglobulin [32], and is induced by Th2-mediated IL-4 signaling [31]. Taken together, these data suggest that Th2-type cytokines participate in the control of bacterial growth during the first four weeks of primary mycobacterial infection. As zebrafish with higher T cell numbers and a Th2-biased response were able to control the infection most efficiently, we next wanted to study whether these protective features were relevant for the ability of maintaining long-term latency. Even though the individuals with a primary progressive disease had already been removed from the experiment, large variations in the bacterial numbers (Figure S1D) and dormancy (Figure 4A) were seen at a late time point, when the population was again divided into subgroups according to their bacterial loads at 5 mpi. The individuals with highest levels of dormancy-associated mycobacterial citrate synthase I (GltA1) expression were found in the Low group, whereas in the High group GltA1 expression levels were significantly lower than in the other groups (Figure 4A). This shows that there is a spectrum even within the “latent” population that has survived the primary infection. The total T cell number still seemed to play a role at this late time point so that the High group had significantly less T cells than the other groups (Figure 1D). However, there was no significant difference in the total T cell number between the Low and Medium group. The protective role of Th2-type responses associated with lower bacterial numbers remained even at this late time point (Figures 2C&F). These results indicate the importance of high T cell numbers and a tendency to Th2-biased responses in determining how well the infection is controlled. It is plausible that the ideal response for inducing stable latency is similar to that required for maintaining long-term latency. As we followed the large population of fish infected with a low initial dose, we saw that from 8 wpi to 5 mpi, there was a 17% overall morbidity in the population (data not shown). Following an eight-week period of asymptomatic infection, until the end of the 5 month follow-up, these spontaneously reactivated individuals were collected as they started showing symptoms of mycobacterial disease. These individuals had a slightly higher bacterial load than the fish in the High group (Figure S1D) but did not differ in terms of dormancy (Figure 4A). Thus, it is likely that the disease is active in the fish in the High group as well, although the fish did not yet show external signs of disease. Based on the gata3/tbx21 ratios, the Th2/Th1 balance was similar in the High and Reactivated groups (Figure 4B), differing significantly from the Low and Medium groups. As direct assessment of the bacterial load within a population of asymptomatic humans is difficult, measuring the Th2/Th1 balance from a blood sample could be used as a clinical biomarker for estimating the activity of the disease. We carried out a ROC-analysis of gata3/tbx21 ratio and found that gata3/tbx21 ratio had a high sensitivity and specificity as a marker in distinguishing the high load individuals (High&Reactivated) from the well-controlling population (Low&Medium) (Figure 4C). We next wanted to study whether there were some differences in the T cell responses between the progressive High and Reactivated groups. In the Reactivated group, half of the individuals showed high induction of cd3. This was surprising, as within all the other groups, high cd3 was generally associated with better disease control. A possibly detrimental role of the induced T cell response in the Reactivated group could be explained by a non-ideal polarization of these cells. The most striking difference between the High and Reactivated group was the significantly higher induction of foxp3 in the Reactivated group (Figure 4E), indicating a pronounced regulatory T helper (Treg) proliferation. When the expression levels of gata3, tbx21 and foxp3 were analyzed relative to cd3 in the Reactivated, only foxp3 was significantly different between the Reactivated and High group (P = 0. 0006,10-fold higher proportion than in the High group, Figure 4E). In fact, the induction of foxp3 was higher in the Reactivated group compared to any of the asymptomatic groups (Figure 4F). This implies that the increase in foxp3 expression is higher than the increase in the total T cell number and thus, the increased proportion of foxp3 cells could be used for distinguishing Reactivated individuals from the rest of the population at a late stage of infection. The role of foxp3 positive cells in reactivation of tuberculosis and its applicability in diagnostics warrants further investigation. It is known that T cell responses are essential in restricting mycobacterial growth in human tuberculosis as well as in various mammalian models for studying tuberculosis. The zebrafish is a newcomer in the field of immunology, and the components of its adaptive immune system have only recently been elucidated in more detail. It has been shown [33] that there are professional antigen presenting cells capable of inducing antigen-specific T cell responses in zebrafish. In our recent publication [34], we have demonstrated the presence of specific and protective immune responses against M. marinum infection in zebrafish. We have shown that vaccination of zebrafish with the Bacillus Calmette-Guérin (BCG) increases survival of adult zebrafish from infection with M. marinum. Furthermore, vaccination of zebrafish with plasmid DNA encoding mycobacterial antigens increases survival, reduces the spreading of bacteria as well as the number of granulomas in M. marinum infection, compared to vaccination with a control pDNA lacking the antigen-encoding sequence. Rag1 (−/−) zebrafish lacking functional adaptive lymphocytes are not protected by the antigen-pDNA vaccine. In the antigen-pDNA -vaccinated zebrafish, interferon gamma expression levels are significantly higher during infection than in the control pDNA vaccinated fish, demonstrating the specificity of the anti-mycobacterial immune response induced in zebrafish. For studies of zebrafish lymphocyte populations, antibody markers for FACS analysis are not available. To characterize the T cell responses of individuals with different infection outcomes, we measured expression levels of marker genes reflecting the total number of T cells and the Th profile from the internal organs of infected zebrafish. We also used the transgenic lck: GFP zebrafish as a tool to isolate T cells and to validate the T cell markers that were used in the study (Figure S2). We showed that the Th marker genes are enriched in sorted lck+ T cells and demonstrated a correlation between the cd3 transcript level and the total number of lck+ cells from lck: GFP reporter fish. These assessments could only be done from uninfected zebrafish, because it is likely that M. marinum infection influences the expression of the GFP reporter gene driven by the lck promoter. It has been previously shown for other pathogenic mycobacteria that one of the mycobacterial virulence strategies is to inhibit host T cell receptor signaling by interfering with the expression and phosphorylation of lck [35]–[37]. Thus, the use of the lck: GFP reporter zebrafish line as a tool to study T cell responses in M. marinum infections would first require careful assessment of the effects of the bacterium on zebrafish lck expression. Instead, we have here relied on the use of several parallel markers reflecting the different Th profiles. In the future, development of new research tools is needed for a more detailed characterization of Th responses and their role in the pathogenesis of mycobacterial infection in the zebrafish model. The results of the current study support the view that both Th1 and Th2 responses are induced in an optimal anti-TB-response. In the well-controlling zebrafish individuals, both Th1 and Th2-type responses were efficiently induced and showed temporal and quantitative differences compared to the Th responses of progressive individuals. The individuals that are not capable of restricting bacterial growth and, subsequently, are likely to develop a progressive disease, maintain a more Th1-biased response at all stages of infection, not reflecting an excessive Th1-type response but, instead, a lack of Th2-type induction. Our study shows that a low-dose M. marinum infection elicits different types of responses in different individuals. It is known that environmental factors, such as nutritional status or infections, influence the differentiation of T helper cells. In addition to environmental factors, it has recently been demonstrated in mice and humans that each mouse strain as well as each human individual has a genetically defined Th1/Th2 bias, and that the characteristic Th phenotype is sustained over the time [23]. Generally, genetic variation in the associated transcription factors, cytokines or cytokine receptors may define the inherent individual Th bias. It is known that there are various single nucleotide polymorphisms in the enhancer regions of human Th differentiation genes, and that these polymorphisms are related to the susceptibility to various disease states [24]. In the studied heterogeneous zebrafish population, the individual outcome of mycobacterial infection can be assumed to be affected by host genetic factors, including the inherent bias in T helper phenotype, as the variation in environmental and bacterial factors is minimal. Based on the results of this study, a Th1-type response is induced equally efficiently in both progressive and well-controlling individuals, but the lack of a Th2-type response causes the disease to progress in the (genetically) susceptible population. However, in a control experiment, in which zebrafish were infected with a high initial dose of M. marinum, we saw that the rapid growth of bacteria may also alter the Th2/Th1 balance tilting it towards Th1. Also, we saw that the high initial dose caused the total cd3 expression levels to remain low suggesting bacterium-induced T cell inhibition. In humans, it is known that M. tuberculosis can cause apoptosis of specific T cells [38] and delayed activation of CD4-positive T cells [39]. It is likely that both the bacteria and the genetic determinants of the host are capable of affecting the T cell responses in mycobacterial infection, and it is challenging to distinguish the contribution of either alone. Also, additional host factors alongside with those related T cell responses are likely to affect the disease outcome. As latent tuberculosis exists in a major part of the human population, its spontaneous reactivation is a serious global threat. Latent tuberculosis, when not initially caused by a resistant strain, can be treated with a 9-month isoniazid monotherapy that reduces the risk of reactivation by 60–90%. However, poor treatment compliance is a common problem in treating this asymptomatic disease, as only half of the patients complete therapy [40]. The poor compliance, in turn, affects the increased antibiotic resistance to isoniazid complicating the treatment of both latent and active tuberculosis. Therefore, it would be of paramount importance to be able to recognize the small population of latently infected individuals with a higher bacterial load and to allocate the treatment to only those who are most likely to benefit from it. At a late time point, at 5 months post infection, there was a zebrafish subpopulation present with a clearly more active disease, as determined by the total bacterial load and mycobacterial dormancy gene (GltA1) expression. The fish with a higher bacterial load had a lower gata3/tbx21 ratio. Based on our results, analysis of the Th1/Th2 ratio from peripheral blood mononuclear cells could provide a correlate of activity of disease among the carriers of latent M. tuberculosis infection. The risk of reactivation is also thought to increase with increasing bacterial loads [25], and thus the Th1/Th2 ratio could have predictive value in evaluating the risk of reactivation of a latent infection. The potential of the Th1/Th2 ratio as a biomarker in the human population warrants further investigation. The high induction of foxp3 expression in spontaneously reactivated individuals is in line with a previous human study showing that quantification of Foxp3 from antigen-induced peripheral blood mononuclear cells can be used to discriminate between latent and active TB [41]. During infection, regulatory T cells (Treg) have an important role in controlling excessive inflammation to prevent tissue damage, but at the same time, their immunosuppressive function can prevent bacterial clearance [42]. The role of Treg cells has been investigated during the early response to TB infection, and there is evidence that M. tuberculosis induces the expansion of antigen-specific Treg cells thus delaying the priming of effector T cells in the lymph nodes and the subsequent arrival of T cells to the infection site [43]. As M. tuberculosis is capable of such exploitation of the Treg response as part of its virulence strategy during the early TB infection, it is plausible that similar pathogen-driven expansion of antigen-specific Treg cells could also play a role in the reactivation of latent TB and the subsequent transmission of the disease. On the whole, the role of Treg cells in reactivation of latent TB is highly interesting and calls for further characterization. The existence of individuals that are able to clear mycobacterial infection illustrates that the optimal immune response to fight TB has already developed during the evolution. Adaptive mechanisms underlying mycobacterial clearance have so far remained enigmatic, and their better understanding will undoubtedly provide valuable knowledge for drug and vaccine development against tuberculosis. The zebrafish model is uniquely suitable for dissecting the natural spectrum of mycobacterial infection in large scale population studies. Analysis of the protective immunity leading to the eradication of bacteria in zebrafish can provide valuable knowledge for the development of new innovative approaches to prevention and treatment of tuberculosis. The importance of Th1-type response in controlling mycobacterial infection is generally recognized because mycobacteria are (facultative) intracellular pathogens. The general – and simplified – paradigm of the reciprocal regulation between Th1 and Th2 responses has led to the idea that Th2 response in tuberculosis might inhibit the bacterial clearance by Th1 immunity. Therefore most tuberculosis vaccines currently under development aim at promoting an efficient Th1 response and inhibiting the induction of a Th2 response [44]. In the studied zebrafish population, 10% of the individuals were able to clear the infection after the activation of adaptive responses (>2 wpi). These clearers had a similar, Th2-biased response as the other individuals in the well-controlling Low and Medium subgroups. On the other hand, inability to induce Th2 responses seems to be a trait that is associated with progressive mycobacterial infection in the zebrafish. Our finding argues against the paradigm of Th2 response not being useful for controlling tuberculosis. If this holds true in human TB, the current therapeutic and preventive approaches promoting Th1 and inhibiting Th2-type response need to be thoroughly reconsidered. For most experiments, adult (5–8 month-old) wild-type AB zebrafish were used. In addition, adult, rag1 (−/−) hu1999 mutant fish and lck: GFP transgenic fish (both from ZIRC) were used. Fish were kept in a flow-through system with a light/dark cycle of 14 h/10 h and were fed with SDS 400 food twice daily. All experiments have been accepted by the Animal Experiment Board in Finland (under the Regional State Administrative Agency for Southern Finland) and were carried out in accordance with the EU-directive 2010/63/EU on the protection of animals used for scientific purposes and with the Finnish Act on Animal Experimentation (62/2006). Permit for the zebrafish facility: LSLH-2007-7254/Ym-23, Permit for experiments: ESAVI/6407/04. 10. 03/2012, PH1267A and ESAVI/733/04. 10. 07/2013. M. marinum (ATCC 927) was cultured as described in [17]. In brief, bacteria were grown at 29°C in standard mycobacterium medium 7H9 (BD) with standard additives to an OD600 of 0. 495–0. 680 Anesthetized fish were intraperitoneally (i. p.) injected with 5 µl of bacteria suspended in sterile PBS using an Omnican 100 30 G insulin needle (Braun, Melsungen, Germany). The bacterial dose was verified by plating on 7H10 (BD) with the standard additives. The low infection dose was 21±7 cfu and the high dose 1783±364 cfu. M. marinum (ATCC 927) was transferred from 7H10 plate into 10 ml of liquid 7H9 medium with standard additives and cultured for 3–4 days at 29°C to an OD600 of 0. 490. Pelleted bacteria were resuspended in PBS corresponding to half of the original culture volume. The bacteria were heat-killed at 100°C for 20 min and thereafter homogenized for 4 min with 4000 rpm using homogenization tubes from Mobio (California, USA) and Mobio PowerLyzer24 bead beater. Samples were plated on 7H10 and LB to verify proper killing. Heat-killed bacteria were injected in a volume of 5 µl i. p using Omnican 100 30 G insulin needles (Braun). Kidney and spleen were collected from a euthanized AB fish in 20 µl of sterile PBS. The organs were gently homogenized by pipetting up and down ∼20 times. 10 µl of this suspension was injected i. p. into an anesthetized recipient rag1 (−/−) fish. For lymphocyte sorting experiments, lck: GFP fish were euthanized and their internal organs collected in ice-cold HBSS supplemented with 2% FBS (both from Life technologies, CA, USA). The tissue was mechanically disrupted by pipetting and passed through a 50 µm cell strainer to prepare single cell suspensions. Cells were washed twice with cold HBSS (+FBS), pelleted at 4°C, 300 g for 5 min and resuspended in 1 ml of the same buffer. 1 ml of Histopaque-1077 (Sigma-Aldrich, MO, USA) was then added under the cell suspension and lymphocytes and other mononuclear cells were enriched by centrifugation at room temperature for 20 min, 400 g. After centrifugation, the middle phase containing the target cells was transferred into a new tube, washed once and resuspended in HBSS (+FBS). Lck+ lymphocytes were sorted with FACSAria I (BD) (purity ≥95% based on GFP expression), collected by centrifugation and RNA was extracted using TRI reagent as described in [17]. The samples for gene expression analysis and mycobacterial quantitation were prepared using TRI reagent for DNA-RNA co-extraction (MRC, OH, USA) as previously described in [17]. RNA samples were treated with DNAse (Fermentas) according to the manufacturer' s protocol. Bacterial loads were measured by q-PCR from DNA samples using SENSIFAST NO-ROX SYBR kit with M. marinum-specific primers as described in [17]. A dilution series of DNA extracted from mycobacterial culture was included in each run to allow absolute quantification. Gene expression was measured by q-RT-PCR using Bio-Rad iScript One-Step RT-PCR Kit with SYBR Green with various primers. Host genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or to elongation factor 1 alpha (Ef1a), and the mycobacterial dormancy gene GltA1 was normalized to the total bacterial load. q-RT-PCR results were analyzed using the ΔCt method. The induction of host genes was compared to a baseline RNA sample extracted from a pool of healthy, non-infected zebrafish and shown as fold induction compared to average normal levels. GltA1 levels are shown in arbitrary units. Gene accession numbers and primer sequences can be found in Text S1. For Western blotting, fish were collected 4 weeks after infection with 21±7 cfu. The peritoneal cavity of the euthanized fish was emptied and the collected organs were homogenized in 1. 5 ml of TRI reagent (MRC, OH, USA) using the PowerLyzer24 bead beater. RNA-DNA co-extraction was carried out as described in [17]. After DNA extraction, the remaining interphase and organic phase were used for protein extraction according to the TRI reagent manufacturer' s protocol. In brief, proteins were precipitated by adding 3 volumes of acetone and pelleted at 12,000 g for 10 min at 4°C. The protein pellet was washed three times with 0. 8 ml of 0. 3 M guanidine hydrochloride in 95% ethanol supplemented with 2. 5% glycerol (v: v) and once with 1 ml of ethanol containing 2. 5% glycerol (v: v). For solubilization of the protein pellet, 0. 1 ml of 1% SDS per 10 mg of tissue sample was used. For Western blotting, 40 µg of total protein was resolved on a 10% SDS-PAGE gel and blotted onto Amersham Hybond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). The following primary antibodies were used: anti-Gata-3 (IN) Z-Fish (AnaSpec, California, USA), anti-CXCR-3. 2 (IN) Z-Fish (AnaSpec), anti-GFP antibody NB600-303 (Novus biological, Colorado, USA). In addition, actin was detected from all the membranes with anti-actin (MAB1501) antibody (Millipore, Temecula, USA) for sample normalization. IRDye infrared secondary antibodies (LI-COR Biosciences, Nebraska, USA) and Odyssey CLx (LI-COR) were used for target protein detection and Image Studio software (LI-COR) was used for protein quantitation. A representative image of the blots showing 10 individuals can be found in the Supplementary material (Figure S4). Statistical analysis was carried out using the GraphPad Prism software (5. 02). For determination of statistical significance of differences between the different groups, a non-parametric one-tailed Mann-Whitney test was used, if not stated otherwise. P-values<0. 05 were considered significant. For estimating the predictive value of gata3/tbx21 and foxp3 expression for activity of the disease a ROC analysis was carried out with a confidence interval of 95%. AUC (area under curve) value of 0. 5 indicates no connection and 1. 0 indicates a perfect marker.

Title: Adequate Th2-Type Response Associates with Restricted Bacterial Growth in Latent Mycobacterial Infection of Zebrafish Summary: Tuberculosis is a common and potentially lethal lung disease spread worldwide. One third of the world' s population is estimated to be infected with Mycobacterium tuberculosis, yet most individuals develop a latent disease which has the potential to reactivate. Some are thought to be able to clear the infection. The current vaccine does not give adequate protection against the disease, and due to incorrect use of antibiotics, resistance to treatment has substantially increased. There is an urgent need for novel treatment approaches, such as modulation of the host' s immune response. However, the ideal immune response against tuberculosis is unknown. In addition, more accurate diagnostic tools are needed for distinguishing the high risk individuals among latent patients so that treatment could be given to those that are most likely to benefit from it. In this study, we used the Mycobacterium marinum-zebrafish model to study the T cell responses in mycobacterial infection. Utilizing the natural heterogeneity of the zebrafish population, we found associations between the disease severity (bacterial load) and the type and magnitude of T cell responses. Our results on typical T cell signatures are useful as diagnostic biomarkers as well as provide new understanding needed for therapeutic approaches based on immunomodulation.

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Write a title and summarize: Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death. Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP) -2 whilst MMP-9 was expressed by neurons. The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE. Japanese encephalitis virus (JEV) continues to be the leading cause of viral encephalitis in Asia and the Western Pacific, where it is a significant cause of mortality and disability. Annually there are estimated to be up to 70,000 cases, with 10,000–15,000 deaths [1]. Although vaccination is the most viable option to prevent the disease, affordable vaccines are still not widely available, and there is no established treatment for JE. Despite the disease' s importance, little is known about the pathogenesis. During in vitro studies neuronal apoptosis was described [2], but its mechanisms and relevance for the disease are still unclear, in particular in relation to the inflammatory response that develops alongside direct viral cytopathology. Opportunities for in depth neuropathogenic studies on JE in humans are very limited, mainly because autopsy tissue from fatal human cases is rarely available due to cultural constraints in many areas where JE occurs. Mouse models of pathogenesis have some similarities to human disease, but there are also differences [3], [4]. The macaque model, developed in the 1990s to test JE vaccines is a useful model for studying human disease, particularly since the macaque immune system closely resembles that of humans [5]. We therefore conducted a retrospective study on the brains of experimentally JEV-infected macaques, to dissect the inflammatory response and the cascade of events that leads to neuronal damage. We were especially interested in apoptotic pathways and inflammatory mediators including cytokines, inducible nitric oxide synthase (iNOS) and matrix metalloproteinases (MMPs), because these may point towards new targeted treatments to control the inflammatory damage, even in the absence of antiviral therapy. The study does not involve animal use as it was conducted on archived paraffin embedded brain tissue of rhesus macaques (Macaca mulatta). The original research on challenge study was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. The original study was approved by the Institutional Animal Care and Use Committee (United States Army Medical Component, Armed Forces Research Institute of Medical Sciences) and by the Animal Use Review Office, United States Army Medical Research and Materiel Command (Permit Number: 93-11). The study was performed on archived paraffin embedded brain tissue of twelve rhesus macaques challenged intranasally with a well characterized wild-type JEV strain (KE93; Genotype Ia, GenBank accession number KF192510. 1) as part of an effort to evaluate second-generation JEV vaccines [5] (Table 1). All archived specimens used in this study are from unvaccinated monkeys. The challenge study had been undertaken in several phases and with different doses, ranging from 7. 5×105 to 2×1010 plaque forming units [6]. Monkeys originating from India and screened negative for both JEV and Dengue virus neutralizing antibodies (aged 3–7years, of both sexes, weighing 4. 0–9. 9 kg) had been intranasally inoculated either with the virus isolate passaged twice in culture (animals 1 and 2) or with an isolate prepared from the brain of animal 2 that was subsequently passaged twice in suckling mice to increase both virus titer and virulence [6]. The monkeys were euthanized at the onset of stupor or coma (10–13 days post inoculation) and JEV infection was confirmed by virus isolation from the brain. Five age-matched uninfected control monkeys from an unrelated study served as negative controls. Immediately after death, brains were exenterated and sections of frontal lobe, thalamus, brainstem and cerebellum fixed in 10% neutral buffered formalin for at least 72 hours. Following routine paraffin wax embedding, 3–5 µm sections were prepared and stained with haematoxylin-eosin (HE) or used for immunohistology. For immunohistological studies, sections of thalamus and brainstem (exhibiting the most consistent histological changes) and, for comparison, the cortex (absence of inflammatory infiltrates) were chosen. These were stained for the presence of JEV antigen, apoptosis and apoptotic pathway markers, glial and inflammatory cell markers, von Willebrand Factor (to confirm blood brain barrier [BBB] breakdown, through the demonstration of plasma protein leakage), and proinflammatory markers. Commercial antibodies to human proteins were selected for this study, especially those known to cross react with Macaca mulatta. Details on the panel of antibodies and the detection methods used are provided in Table S1. Briefly, sections were dewaxed in xylene and hydrated through graded alcohols. To inhibit endogenous peroxidase activity, they were treated with freshly prepared 3% H2O2 for 15 min. Sections underwent heat-induced antigen/epitope retrieval with a laboratory pressure cooker (Decloaking Chamber, Biocare Medical, Concord, USA) using citrate buffer pH 6 or pH 9 [7]. This was followed by incubation with normal serum to block non-specific binding sites in tissues, and the primary antibodies (15–18 hrs at 4°C) (see Table S1-A). Apoptotic cells were also identified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end in situ labelling (TUNEL) method using the Apoptag In Situ Apoptosis Detection kit (Chemicon Inc., Millipore, Billerica, USA) to demonstrate the characteristic DNA changes. Appropriate controls were included for each marker: uninfected control monkey brains as negative controls for JEV and to establish constitutive expression of other markers, sections with known positivity for specific markers as positive controls, and sections incubated with normal mouse/rabbit IgG as isotype controls. Double immunolabeling was performed on selected sections of some monkeys (animals 2,9, 11) to characterize the populations of cells expressing apoptosis markers (TUNEL and caspase-3, -8, and -9) and proinflammatory mediators (cytokines, iNOS and MMPs) and to relate them to the expression of JEV antigen. For this purpose, primary antibodies raised in different species were sequentially localized using non-overlapping secondary reagents and different chromogens (see Table S1-B). Sequential staining was performed on consecutive sections, mainly to detect tumor necrosis factor alpha (TNF-α) expression in inflammatory cells and glial cells and to further characterize JEV-infected cells when primary antibody were used that had been generated in the same species or when the double immunolabeling was difficult to interpret. A confocal laser scanning microscope LSM 700 (Carl Zeiss Micro Imaging, Germany) with solid state laser excitation wavelength 488 nm (for FITC) and 555 nm (for Texas Red) and ZEN 2009 software was used to detect immunofluorescent staining. All other light microscopic assessments were undertaken with conventional microscopes. All JEV-infected animals exhibited mild to moderate, multifocal to diffuse, non-suppurative meningoencephalomyelitis with evidence of neuronal degeneration and death. The inflammatory response was similar in its extent and composition regardless of the dose of inoculum and the day of euthanasia, and was dominated by mononuclear perivascular cuffs (Figure 1A) and meningeal infiltrates. These were accompanied by morphological evidence of endothelial cell activation (represented by a tomb-stone like luminal protrusion of endothelial cells; Figure 1B) and/or vascular damage. The latter was indicated by perivascular haemorrhage and substantial leakage of serum into the parenchyma, as demonstrated by staining for von Willebrand factor (Figure 1C). Neuronal cell death was indicated by morphological neuronal changes suggestive of apoptosis, in association with satellitosis or microglial nodules (Figure 1D, E). Reactive astrogliosis, represented by a multifocal increase in astrocyte numbers (Figure 1F) and evidence of astrocyte activation (presence of gemistocytes) in areas with inflammatory infiltrates was also identified in JEV infective brains. T cells (CD3+) were the predominant leukocytes in both perivascular and meningeal infiltrates. They were also present in small numbers in the adjacent parenchyma (Figure 2A). B cells (CD20+) were sparse and primarily seen in the perivascular infiltrates (Figure 2B), while moderate numbers of macrophage/microglial cells (CD68+) identified in perivascular and meningeal infiltrates and the adjacent brain parenchyma (Figure 2C). Staining for myeloid/histiocyte antigen, reported to stain macrophages [8] and microglial cells [9], identified a substantial number of cells with a morphological appearance of macrophages (Figure 2D), suggesting their recruitment into the tissue. Staining for CD68, which is also expressed by microglial cells, and major histocompatibility complex (MHC) class II antigen (expressed mainly by activated microglial cells) confirmed the presence of microglial nodules but also demonstrated diffuse microgliosis and activation of microglial cells (presence of both reactive and amoeboid microglial cells; Figure 2 C, E). Furthermore, endothelial cells were shown to express MHC II, confirming their activation (Figure 2E). The cells surrounding neurons in satellitosis were also CD68-positive microglial cells (Figure 2F). For comparison, in brain areas without evidence of viral antigen and inflammation (cerebral cortex), only scattered MHCII-positive microglial cells without morphological features of activation were seen. There was no evidence of microglial MHC II expression in control brains. JEV antigen expression, seen as finely granular cytoplasmic staining, was observed in numerous neuronal cell bodies and processes disseminated in the thalamic and brain stem nuclei of all animals and in neuronal cell processes throughout the affected parenchyma (Figure 3A). Most infected neurons appeared morphologically unaltered (Fig. 3A inset), but some were surrounded by microglial cells (satellitosis) and exhibited degenerative changes (Figure 3B). JEV-positive microglial cells were found in some glial nodules, but occasionally as individual cells in affected areas like brainstem and thalamus, as confirmed by sequential staining for CD68 and JEV antigen (Figure 3C). In contrast, there was no evidence of JEV infection of astrocytes (Figure 3D). In one animal with a particularly strong inflammatory response (animal 2), a small percentage of slender perivascular cells (perivascular macrophages) also expressed viral antigen (Figure 3E). There was no evidence of JEV antigen in endothelial cells in any animal. Nor was there any correlation between intensity of viral infection as indicated by immunostaining and degree of inflammatory response. Negative control brain sections did not show any positive reaction. Morphological features of apoptosis were observed in degenerating neurons within glial nodules and in satellitosis, among leukocytes in the perivascular infiltrates and in individual cells with microglial features in the adjacent parenchyma. Cell death by apoptosis was confirmed by the TUNEL method which identified apoptotic JEV-infected neurons in glial nodules and satellitosis as well as apoptotic microglial cells disseminated in the parenchyma, in satellitosis and in microglial nodules (Figure 3F). Occasional lymphocytes in the perivascular infiltrates were also apoptotic (Figure 3G) and the JEV-infected perivascular macrophages were apoptotic in animal 2 (Figure 3E). Key apoptosis molecules, including caspases-8, -9 (both initiator caspases) and cleaved caspase-3 (an executor caspase) were identified by staining to detect cells undergoing early apoptosis and not exhibiting representative morphological features. Small numbers of neurons with normal morphology expressing cleaved caspase-3 and more cells expressing caspase-8 were seen in JEV infected brains. Both caspases were also expressed by some leukocytes in the perivascular infiltrates (Figure 4A, B). Caspase-9, however, was only detected in astrocytes and microglial cells (Figure 4C). Double staining for JEV and the various apoptosis markers confirmed that some JEV-infected neurons were undergoing apoptosis (data not shown). In order to better understand the regulation of apoptotic processes in response to JEV infection, the expression of representative pro- and anti-apoptotic proteins was assessed. While numerous microglial cells and occasional neurons stained positive for the pro-apoptotic protein Bax (Figure 4D), the anti-apoptotic protein Bcl-2 was mainly expressed by lymphocytes in the perivascular infiltrates (Figure 4E). Dual staining showed JEV antigen in some Bax-positive neurons and occasional Bax-positive microglial cells (data not shown). In uninfected control brains TUNEL positive cells were not identified. Caspase and Bcl-2 staining was negligible; weak and infrequent Bax expression was seen in neurons. Having characterized the inflammatory response and the patterns of cell death in the brains for monkeys infected with JEV, we aimed to identify relevant mediators of these processes frequently identified in viral mediated infections. To assess local nitric oxide (NO) production, we investigated the expression of iNOS and nitrotyrosine (NT). We stained for MMP-2 and -9, which are known to cause BBB disruption by degrading collagen IV, its main component [10], interferon (IFN) -α, a potent antiviral cytokine and microglial activator [11], and TNF-α which has been shown to directly activate microglia [12] and induce neuronal apoptosis [13]. Both iNOS and NT were expressed by microglial cells and astrocytes. iNOS expression was also seen in some macrophages in the perivascular infiltrates and the adjacent parenchyma (Figure 5A, B) where staining for NT was only very weak. MMP-2 was expressed in cells with the morphology of reactive astrocytes (Figure 5C) and, to a lesser extent, in microglial cells and in infiltrating macrophages, whereas MMP-9, known to be constitutively expressed in human neurons, was intensely expressed by neurons and relatively weakly by microglial cells (Figure 5D). TNF-α expression was seen in microglial cells, infiltrating macrophages and astrocytes, as confirmed by dual staining with CD68 and sequential staining with GFAP (Figure 5E). It was also occasionally seen in endothelial cells (data not shown). IFN-α expression, however, was seen both in uninfected and infected neurons, as confirmed by dual staining with JEV antigen (data not shown), and in astrocytes and microglial cells (Figure 5F). In control brains, only minimal expression of inflammatory mediators was seen, represented by staining in occasional vascular endothelial cells (iNOS, TNF-α), neurons (MMP-9, iNOS) and vascular smooth muscle cells (TNF-α). The present study used macaques, which have previously been established as a good model for neuropathological studies on JE in humans [5], [6], to evaluate the cytopathic effects of and inflammatory response to JEV in the brain. The apoptosis pathways and the full spectrum of proinflammatory factors have not been fully studied in any previous animal models of JE, or autopsy tissues. This study utilized monkeys challenged with JEV intranasally rather than a route more consistent to natural infections to increase the likelihood of encephalitis. Peripherally challenged monkeys generally do not typically develop encephalitis [14] and with direct intracerebral challenge the encephalitis develops early [15]. The intranasal route was therefore the most useful route in our model and has been reported to provide a useful model for the study of anti-viral compounds and vaccine candidates [5], [15] albeit this unnatural infection route may be a limitation in our study. As in humans, JEV induces a non-suppurative meningoencephalitis with neuronal cell death, microgliosis and astrogliosis in macaques [16], [17]; these classic findings are also common in other viral encephalitides [18]. However, the ‘punched-out’ areas of focal necrosis, often seen in fatal human JE cases [16], [19] were not observed in our experimentally infected monkeys. It is possible that this pathology had not yet developed in the macaques that were euthanized at the onset of stupor or coma in contrast to human infections where histological observations are always made on post mortem material at the end of the disease process [16], [19]. The inflammatory response in macaques even with the chosen challenge route was consistent with the changes seen in humans, characterised by perivascular mononuclear cuffs, with less intense infiltrates in the adjacent parenchyma [16]. While T cells dominated in the perivascular infiltrates and recently recruited macrophages were the largest population in the parenchymal infiltrates, B cells represented a minority and were restricted to the perivascular cuffs. Cytotoxic T cells (CTLs) have been reported to play a key role in mouse models of JE [20], but it remains unclear if these cells are beneficial or deleterious, or both. In the present study, it was not possible to assess the role of CTLs, due to the non-availability of antibodies suitable for macaques. In viral encephalitis, macrophages are known to migrate from the perivascular space into the surrounding parenchyma where they become activated [21]. In addition to microglia, known to cause neuronal death in JE [3], [19], the relative contribution of peripheral macrophages that migrate into the CNS should be elucidated. Our study confirmed neurons as the main targets of JEV, as previously shown in fatal human cases [16], [19], [22]. We also demonstrated viral antigen in microglial cells, mainly within microglial nodules surrounding infected neurons, suggesting virus uptake by phagocytosis. However, productively infected microglial cells cannot be excluded, since they do support viral replication in vitro [23], [24]. Viral antigen was not detected in other glial cell types, despite evidence that astrocytes can become infected in culture systems [23]. There was also no evidence of endothelial cell infection. A similar viral target cell pattern has been reported in human cases, with the exception that some studies found evidence also for endothelial cell infection [16], [19]. Interestingly, we detected JEV antigen in perivascular macrophages in one animal. These cells found at the interface between blood and brain parenchyma are resident macrophages with high phagocytic activity and MHC-II expression [25], which suggests that they had phagocytosed virus that entered the brain via the blood. Viral infection and inflammatory responses were associated with cytopathic changes, and, although not excessive, neuronal death via apoptosis was clearly observed. Apoptosis was shown by the TUNEL assay which has been used in the past to demonstrate apoptosis, although interpretation of the findings can be difficult in the presence of necrosis and autolytic changes [26]; we therefore also confirmed apoptosis by staining for cleaved caspase-3. Apoptotic neurons were often surrounded by microglial cells (satellitosis and formation of microglial nodules) which indicated their impending phagocytosis. Some apoptotic neurons were JEV infected. In addition, several morphologically unaltered, infected neurons were shown to express the pro-apoptotic protein Bax, the initiator caspase-8 or the active effector caspase-3, which indicates that these cells were destined to become apoptotic. These results confirm the in vivo relevance of previous in vitro studies which demonstrated that JEV replication can lead to neuronal apoptotic death [27] and support findings from the mouse model that JEV replication contributes to Bax activation [28]. Taken together, these findings provide clear evidence of a direct, although possibly not rapid, cytopathic effect of JEV on neurons. The demonstration of caspase-8 in affected neurons also indicates that neuronal apoptosis is initiated by the fas-mediated or extrinsic pathway, a mechanism that is central to the process of immune-mediated viral clearance [29] and seen in a number of CNS viral infections including West Nile virus [30]. Importantly, apoptotic cell death or pre-apoptotic caspase-8 expression was also seen in a proportion of JEV antigen-negative neurons, which suggests some degree of bystander neuronal death. In addition, a proportion of microglial cells, often in close proximity to infected neurons but generally not JEV-infected, were apoptotic. Furthermore, the observation of morphologically unaltered microglial cells expressing caspase-9 suggest that microglial apoptosis is initiated by the mitochondria or the intrinsic pathway. A recent in vitro study showed that JEV infection can lead to apoptosis of microglial cells [24]. Our results indicate that in vivo this direct mechanism is probably less relevant and that pro-inflammatory factors are more important; this is also seen in other CNS conditions, such as experimental autoimmune encephalomyelitis (EAE) where microglial apoptosis is considered an important homeostatic mechanism to control microglial activation and proliferation [31]. Apoptotic cell death was also observed in a proportion of infiltrating inflammatory cells in our JEV infected monkeys. Considering that these cells were not JEV-infected, this most likely represents a normal mechanism to eliminate activated leukocytes and thereby limit the inflammatory response in the CNS. On the other hand, infiltrating leukocytes (predominantly T cells) were found to express the anti-apoptotic protein Bcl-2. This supports a murine in vivo study that provides evidence of a critical role of Bcl-2 in the survival of virus-specific CTLs [32]. The occurrence of apoptosis in apparently uninfected neurons suggests that indirect mechanisms (bystander cell death) contribute to neuronal damage in JE, and indeed recent in vitro and in vivo murine studies demonstrated that microglial cells can induce neuronal apoptosis via the release of pro-inflammatory mediators [3], [4]. Also, TNF-α, via its receptor on neurons, has been shown to induce caspase-8 activation in mouse neurons [33]. Indeed, we observed TNF-α upregulation in astrocytes, microglial cells, endothelial cells and infiltrating macrophages in infected macaques. It is likely that these cells were also responsible for the TNF-α upregulation observed in JEV-infected mice [3], [34]. TNF-α related neuronal death is also reported in a recent in vitro study with WNV [35]. The results of our study suggest that JEV might simultaneously trigger, both directly and indirectly, the caspase dependent extrinsic apoptotic pathway in neurons and the intrinsic apoptotic pathway in microglial cells. Further definition of the underlying mechanisms will allow us to understand the processes involved in disease progression and to assess the potential of anti-apoptotic treatment strategies. Alongside the inflammatory infiltration and the cytopathic effects, we found distinct evidence of activation of a range of cells, namely microglial cells, astrocytes and vascular endothelial cells. Microglial activation was confirmed by the demonstration of MHC II antigen, iNOS, NT, TNF-α and MMP expression by microglial cells and has been reported previously in JEV-infected mice [3]. To shed light on the potential mechanism of microglial activation, we assessed the expression of IFN-α (type I IFN); this potent antiviral cytokine is an activator of microglia in response to CNS viral infection [11], and is elevated in the cerebrospinal fluid of patients with JE, where it is associated with a poor outcome [36]. We demonstrated IFN-α expression in neurons which suggests that they might be responsible for microglial activation early after infection; expression by microglia and astrocytes suggests they might be responsible for sustained microglial activation in JE. As described in earlier reports [22], reactive astrogliosis and astrocyte activation was also observed in the present study. Astrocyte activation is considered as a non-specific response to degenerative changes including virus-induced damage in the CNS. However, a recent study provided evidence that this activation might be an effect of TNF-α release from microglial cells [23]. So far, little is known about the role of astrocytes in neuroinflammation caused by JEV, whether they are protective or pathogenic. Nevertheless, the demonstration of TNF-α, IFN-α, iNOS, NT and MMP-2 expression by astrocytes in our study provides the first in vivo evidence that astrocytes may play an important role in the pathogenesis. The same is true for microglial cells and macrophages in the inflammatory infiltrates, through release of the inflammatory mediators, all these cells might actively contribute to the damage of other cells in the brain and in particular induce bystander apoptotic death of neurons [3], [4]. iNOS and NT expression indicate NO production, which is in accordance with results from a mouse study [37]. There, a gradual increase in iNOS activity was observed after intracranial JEV infection, and was considered a consequence of release of cytokines, such as TNF-α or IL-8 which might be beneficial through the inhibition of viral replication and release [37]. However, NO has also been discussed as a potential mediator of pathogenesis in tick-borne encephalitis virus infection [38]. MMP levels have been shown to correlate with the severity of some CNS infections [39]. MMP-9 is known to be constitutively expressed in human neurons. However, it was intensely upregulated in neurons of the JEV-infected macaques and weakly expressed by microglial cells, while glial cells and infiltrating macrophages were sources of MMP-2. MMP release is stimulated by proinflammatory cytokines including TNF-α [40]. In JE, MMPs might play a detrimental role and not only be responsible for BBB disruption through collagen IV degradation, but also contribute to neuronal destruction via stimulation of TNF-α release. We observed endothelial cell expression of MHC II antigen and TNF-α, which confirms that they are activated and suggests they have a role in inflammatory cell recruitment and potential contribution to immune reactions, glial cell activation and neuronal apoptosis. Endothelial cells might also be a source of the increase in serum TNF-α seen in JE patients [36]. Based on our findings we postulate that infection of neurons by JEV triggers a network of inflammatory mediators [41]. Through release of IFN-α, neurons activate microglial cells which, via release of cytokines such as TNF-α, activate astrocytes and endothelial cells. Together, these mediators contribute to BBB breakdown, leukocyte recruitment into the parenchyma and further neuronal apoptosis. Glial cell apoptosis should limit the extent of inflammation. However, the release of further mediators by infiltrating leukocytes, in particular macrophages, results in sustained glial and endothelial cell activation and further leukocyte recruitment, ultimately augmenting the inflammatory response and neuronal cell loss. Although the inflammatory response is intended to be protective, and presumably is so in cases which improve and recover, if uncontrolled it can contribute to disease progression in JE. Our study is mostly descriptive as we used archived materials from a previous challenge study. However it might shed some light on some novel processes mediating pathogenesis which could aid in the experimental design for future studies investigating inflammatory responses to JE. Viral encephalitis is a major cause of morbidity and mortality worldwide. The pathogenesis of flavivirus encephalitis remains incompletely understood but it appears that the immune response is crucial in limiting viral spread to the brain [42]. The cascade of events that we have outlined for JE may also apply to other viral encephalitides. Currently there is no proven efficacious therapy for most viral infections of the CNS including JE. Novel strategies for treating viral CNS infections are urgently needed. Our results from a macaque model indicate that neuronal apoptosis and glial activation are crucial steps in the pathogenesis of JE. They imply that adjunctive therapy with inhibitors of caspases or targeted anti-inflammatory treatments might be a promising therapeutic approach for JE in the future.

Title: Neuropathogenesis of Japanese Encephalitis in a Primate Model Summary: Japanese encephalitis (JE) is one of the most important causes of viral encephalitis worldwide, with no specific antiviral treatment available. Despite some recent successes with widespread vaccination, JE will likely remain an important public health problem; because the virus is mosquito-borne and has natural animal hosts, it will never be eradicated. We have little understanding of what determines the severity and outcome of infection. Data from human post mortem studies is very limited because of cultural constraints on autopsies in areas where JE occurs. Circumstantial evidence suggests that in addition to cytopathology caused directly by infection of neurons, there may be bystander cell death of non-infected neurons, caused by an excessive inflammatory response. Our study used archived brain samples from a prior challenge study in a validated macaque model of JE. We stained for the presence of JEV antigen, apoptosis, and pro-inflammatory markers in affected areas, such as the thalamus and brainstem. We show that bystander neuronal cell death is important, and elucidate the inflammatory and apoptotic mechanisms underlying it. Currently there is no proven efficacious therapy for most viral infections of the central nervous system, including JE. Novel strategies for treating such infections are urgently needed. Our findings suggest new anti-inflammatory and anti-apoptotic therapeutic approaches may be useful in treating this debilitating disease.

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Write a title and summarize: Emmanuel Macron a attendu le 8 janvier 2018 pour aller à la rencontre de la Chine. Sage précaution si l’on en croit la symbolique des chiffres, fort prisée dans l’empire du Milieu : signe de plénitude et de prospérité, le 8 y est considéré comme un porte-bonheur. Pékin n’avait-elle pas mis un point d’honneur, il y a dix ans, à inaugurer « ses » Jeux olympiques le 8 août, autrement dit le 8/8/8… Il sera bien temps de vérifier si 2018 confirme, à Paris, les prédictions de la numérologie chinoise. Une chose est sûre, cependant. Même si elles furent loin de garantir à tout coup réussite et sérénité, les années en 8 ont scandé avec une saisissante régularité notre histoire contemporaine. Prenez 1958, date évidemment inscrite à l’agenda des commémorations officielles des prochains mois. Nul ne doutait, en janvier de cette année-là, que la IVe République était à bout de souffle, minée par la guerre d’Algérie et empêtrée dans l’écheveau de ses intrigues. Mais le jeune Félix Gaillard, président du conseil depuis quelques semaines, tentait encore un énième rafistolage. Hormis chez les gaullistes les plus activistes, l’hypothèse d’en appeler à l’homme du 18 Juin pour sortir le pays de l’ornière était encore évanescente. En peu de temps, pourtant, l’Histoire bascula. Le 13 mai, c’est l’insurrection, à Alger, des partisans de l’Algérie française. Le 15 mai, le général de Gaulle se dit « prêt à assumer les pouvoirs de la République ». Le 1er juin, il est investi président du conseil par l’Assemblée nationale avec, pendant six mois, les pleins pouvoirs. Le 28 septembre, par référendum, les Français approuvent à 83 % la Constitution de la Ve République. Le 21 décembre, de Gaulle est élu président de la République. Beaucoup, alors, croyaient le régime taillé aux seules mesures du « général ». Soixante ans plus tard, consolidé par l’élection présidentielle au suffrage universel, il a démontré une plasticité à toute épreuve. Emmanuel Macron, à son tour, y puise son autorité. 1968 et 1978 ont laissé des traces Dix ans passent. « La France s’ennuie… », écrit Pierre Viansson-Ponté dans ces colonnes, le 15 mars 1968. Et d’un coup, la tornade du mois de mai balaie tout sur son passage, enflamme l’université et la jeunesse, hérisse de barricades les rues de Paris, déborde les syndicats, provoque une grève générale digne du Front populaire, paralyse l’Etat et ébranle son vieux chef. Un ultime sursaut permettra à de Gaulle de reprendre la main. Ultime sursis en réalité : moins d’un an plus tard, après un dernier référendum perdu, il jette l’éponge.

Title: "Les années en 8 ont scandé avec une saisissante régularité notre histoire contemporaine Summary: 1958, 1968, 2008... "Difficile, devant les soubresauts ou les tumultes des années en 8, d'imaginer de quoi 2018 sera porteuse", estime l'éditorialiste au "Monde" Gérard Courtois dans sa chronique hebdomadaire.

mlsum_fr

fr

Summarize: BACKGROUND Unless otherwise indicated herein, the materials described in this section are not prior art to the claims in this application and are not admitted to be prior art by inclusion in this section. A sponge is a common tool used for cleaning, absorbing, drying, filtering, and wiping activities. Sponges are generally constructed out of porous materials designed to effectively absorb liquid solutions. The porous materials consist of a network of interconnected fibers that create holes within the sponge body. When liquid is introduced to the sponge, the holes between the fibers may fill up causing the porous material to swell. The swelling action traps the liquid until active pressure is applied to the sponge, thus releasing the absorbed liquid. Sponges have numerous applications and may be specifically designed to suit the needs of each specific application. In particular, sponges may be constructed out of a number of different porous materials. Examples may include natural sea sponge, cellulose, rubber sponge, viscose sponge, polyester sponge, polyurethane sponge, or polyvinyl alcohol sponge (PVA), among others. The porous material chosen may affect a sponge&#39;s durability, effectiveness, weight, and ability to absorb or retain liquid. SUMMARY In one aspect, a sponge may include: a sponge body, the sponge body comprising one or more side surfaces; and at least one of the one or more side surfaces comprising one or more openings. In another aspect, a sponge may include: a sponge body, the sponge body comprising one or more side surfaces; and at least one of the one or more side surfaces comprising an opening. In a further aspect, a sponge may include: a sponge body, the sponge body comprising one or more side surfaces; and at least one of the one or more side surfaces comprising six openings. These as well as other aspects, advantages, and alternatives, will become apparent to those of ordinary skill in the art by reading the following detailed description, with reference where appropriate to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A shows a perspective view of a first example sponge. FIG. 1B shows a cross-sectional view of the example sponge of FIG. 1A. FIG. 2 shows a perspective view of the example sponge of FIGS. 1A and 1B in use. FIG. 3 shows a perspective view of a second example sponge. DETAILED DESCRIPTION FIG. 1A illustrates a perspective view of a sponge 100 according to an example embodiment. The sponge 100 may include a sponge body 105, side surfaces 110, openings 120 a and 120 b, a perimeter 130, a top surface 140, and a bottom surface 150. The openings 120 a and 120 b may be positioned along the perimeter 130 of the side surfaces 110 on the sponge body 105. Top surface 140 and bottom surface 150 may be located on the sponge body 105 and, in some example embodiments, may not contain any openings 120 a and 120 b. The sponge body 105 may be formed in any of a number of different ways. For example, in some example embodiments, the sponge body may be molded. In other example embodiments, the sponge body may be cut, injection molded, molded by casting or foaming, extruded, or machined. Other examples are possible, as well. In addition, the method of construction may relate to or be determined by the material out of which the sponge is constructed. The sponge body 105 may be formed into any of a number of different shapes. In some example embodiments, as illustrated in FIGS. 1A and 1B, the sponge body 105 may be constructed into a circular shape. Other shapes may be possible as well, including, but not limited to, oval, square, rectangular, triangular, semi-circular, or crescent. The shape may also be irregular or undefined, as well. Additionally, sponges may be produced in many different sizes. For example, a sponge may be sized such that a user can grip the sponge in one hand. Alternatively, a sponge may be large enough to be affixed to a mop or sponge for cleaning purposes. Regardless, the number of different sizes and shapes is limitless. The dimensions of each sponge 100, including the diameter, length, width, and thickness may also vary. For example, some sponges may be manufactured such that they can be held in one hand. In such examples, the dimensions of the sponge body 105 may match the size of, or be slightly larger than, a typical hand. In other examples, the sponge 100 may be larger or smaller than the size of a typical hand. In some cases, the sponge may be used on small surfaces or in small spaces, such as bottles or jars, for example. In such cases, a sponge body 105 with smaller dimensions may be appropriate. In other cases, the sponge may be used on larger surfaces, such as on vehicles or on household surfaces such as counters, shelves, floors, walls, or ceilings. In such cases, a sponge body 105 with larger dimensions may be appropriate. In yet other examples, the sponge 100 may be attached to a handle. In some cases, the sponge may be attached to the end of a very long handle, similar to a mop. Such sponges may also have a sponge body 105 with larger dimensions, especially if the sponge could be used on larger surfaces. Other example uses and applications are possible as well. Moreover, the dimensions of the sponge 100 or sponge body 105 are not necessarily limited or determined by the sponge&#39;s use or application. For example, a sponge with dimensions that match the size of a typical hand may be used with a very long handle, and a handheld sponge may be used on larger surfaces, such as on vehicles, counters, shelves, floors, walls, or ceilings. The sponge body 105 may contain a number of surfaces. According to the example embodiment illustrated in FIGS. 1A and 1B, the sponge body 105 may include one or more side surfaces 110, a top surface 140, and a bottom surface 150. Other surfaces may exist in alternative embodiments. Typically, all surfaces of a sponge may be porous, although in some examples some surfaces may not be porous. In addition, some surfaces may be more porous than others, and some surfaces may be made of different materials from others. In an example embodiment, the side surfaces 110 may create a perimeter 130 around the sponge body 105. The perimeter 130 may be defined as a continuous line formed around the sponge body 105 by the side surfaces 110 to create a boundary of the sponge. As illustrated in the example embodiment in FIGS. 1A and 1B, the perimeter 130 may be located on one or more surfaces comprising the circumference of the sponge body 105. Alternatively, in other example embodiments, the perimeter 130 may be located on one or more surfaces comprising the length and width of the sponge. The perimeter 130 may be centered on the side surfaces 110 or equidistant from the top surface 140 and the bottom surface 150 of the sponge body 105, or may alternatively be located closer to the top surface 140 or bottom surface 150. Additionally, the perimeter 130 may not be located at the same height throughout the same sponge body 105. The side surfaces 110 may exist in various forms. For example, the surfaces may be rounded, angled, or straight. Additionally, the surfaces may vary in height and length depending on the preferred shape. In an example embodiment, the side surface 110 is rounded, allowing the sponge to have a circular shape. Alternatively, all of the surfaces may be straight, thus creating a square or rectangular shape. The sponge 100 may be made out of any of a number of materials. In some examples, the sponge may be made from one material. In other examples, the sponge may be made from more than one material. If the sponge comprises multiple materials, the materials may all be porous or, in some cases, one or more materials may be porous and one or more materials may not be porous. The materials may also have varying degrees of porosity. The sponge body 105 may be constructed from various types of porous materials. These materials may include, but are not limited to, natural sea sponge, polyvinyl alcohol (PVA), cellulose, viscose sponge, rubber latex (synthetic rubber), polyurethane sponge, polyethylene sponge, and polyester sponge. Other materials may be possible as well. The material of the sponge body 105 may impact additional sponge properties, such as ability to retain water, durability, absorption rate, and sponginess, which incorporates compressibility and elasticity. In one example embodiment, the sponge body 105 may be constructed from polyvinyl alcohol (PVA). PVA can form a plastic porous structure that is made from water soluble PVA acetalized with an acid catalyst. Use of PVA may be advantageous because it resembles the properties and qualities of natural sea sponge. PVA may also have a high filtering efficiency and may be reusable after cleaning. It may also exhibit strong chemical resistance and can retain large amounts of liquids. PVA may also resist the growth of mold, mildew, and bacteria. PVA may be manufactured to contain a variety of pore sizes—from very small pores to large pores similar to those of natural sea sponges. Pore size within the sponge body 105 may impact its ability to retain liquids. For example, a sponge body 105 with large pores may allow more liquid to flow through the sponge as compared to a sponge with small pores. In an example embodiment, pore size may range from 60 microns up to and including 1500 microns. Other pore size ranges may be possible as well. In yet another embodiment, the sponge 100 may include an abrasive surface to aid in a cleaning process. For example, the sponge may include an abrasive or resin material, such as melamine. In one example, the abrasive or resin materials may be impregnated into the sponge body 105. In another example, the sponge may contain one or more layers of abrasive or resin on the surfaces of the sponge body 105. Such an abrasive or resin material may act to aid in the cleaning process by providing more friction against the surfaces engaged by the sponge or to be cleaned by the sponge. In addition, fine abrasives may be added to the PVA sponge. The abrasives can be absorbed by the PVA sponge and retained so that the PVA sponge itself becomes abrasive. In other examples, the sponge may include a scouring surface (similar to a scouring pad). The scouring surface may be located on any surface of the sponge, and may provide additional assistance for cleaning purposes. As illustrated in the embodiment in FIGS. 1A and 1B, the sponge body may include one or more openings 120 a and 120 b. One or more of the openings may receive, for example, a finger or thumb. In some embodiments, one or more of the openings may receive, for example, a handle or connection for a handle. Such a handle may be used to, for example, grip the sponge or extend the reach of the sponge. The openings 120 a and 120 b may be located on the side surfaces 110 along the perimeter 130 of the sponge body 105. The number, shape, size, and location of the openings 120 a and 120 b may vary. The number of openings 120 a and 120 b may also vary. In some cases, the number of openings 120 a and 120 b may vary based on, for example, the dimensions of the sponge body 105. In addition, the openings 120 a and 120 b may be in the form of slits, slots, holes, cuts, gaps, crevices, or indentations, for example, and may be formed into the sponge body 105 using various methods. The methods may include, but are not limited to, tooling, water jet cutting, drilling, molding, machining, cutting (with scissors, blades, or other manual or automated cutting implements), die cutting, laser cutting, or router cutting, for example. The openings 120 a and 120 b may also be sized such that a user may place one or more digits, such as one or more fingers or thumbs, into the openings. In an example embodiment, the openings 120 a and 120 b may be approximately ¼ inch to 1¾ inch wide around the perimeter 130 of the sponge body 105. In other embodiments, the openings 120 a and 120 b may be larger or smaller, and may vary in size. The number of openings 120 a and 120 b may vary. In the example embodiment depicted in FIGS. 1A and 1B, the sponge body 105 may contain six openings 120 a and 120 b. The six openings may include four “finger” openings 120 a, and two “thumb” openings 120 b. The two “thumb” openings 120 b may generally oppose the four “finger” openings 120 a. The six openings may allow a user holding the sponge 100 with one hand to place all four fingers into openings 120 a, with two available openings 120 b for a thumb. Such “thumb” openings 120 b can be oriented such that one “thumb” opening may more easily accommodate a thumb on the left hand, while the other “thumb” opening may more easily accommodate a thumb on the right hand. In particular, because a person&#39;s thumb is next to (or closest) to a person&#39;s index finger, a user&#39;s thumb may use whichever “thumb” opening 120 b is closest to the opening used by that user&#39;s index finger. In an example embodiment, each thumb opening 120 b may be oriented approximately 112.5 degrees from the closest finger opening 120 a. In other embodiments, each thumb opening 120 b may be oriented in the range of 90 degrees to 120 degrees from the closest finger opening 120 a. In some embodiments, the thumb openings may be oriented 45 degrees from each other. In other embodiments, each thumb opening 120 b may be oriented in the range of 30 degrees to 90 degrees from each other. In some embodiments, the finger openings may be oriented 22.5 degrees from each other. In other embodiments, each finger opening 120 a may be oriented in the range of 22.5 degrees to 45 degrees away from each other. Other distances any orientations may be possible as well. In yet another example, each thumb opening 120 b may be oriented on the same side surface 110. Likewise, each finger opening 120 a may be oriented on the same side surface 110. In an example embodiment, the side surface 110 comprising the thumb openings 120 b may generally oppose the side surface 110 comprising the finger openings 120 a. In an alternative example embodiment, the openings 120 a and 120 b may be oriented such that they are spaced equally around the perimeter 130 of the sponge body 105. Further, in yet another embodiment, the openings 120 a and 120 b may be placed at varying distances from each other. In an alternative embodiment, the sponge body 105 may contain a single opening that extends around a portion of the perimeter 130 or the entire perimeter 130. In yet a further embodiment, the sponge body 105 may include a sufficient number of openings 120 a and 120 b such that a user may place fingers or thumbs from two hands into the sponge body 105. Such an embodiment may also have openings that comprise finger openings 120 a and thumb openings 120 b, and such openings may be on generally opposing surfaces, or may be oriented to more easily accommodate particular fingers and/or thumbs of a user, as discussed above. The depth of the openings 120 a and 120 b may vary. In an example embodiment, the openings 120 a and 120 b may be tooled approximately ½ inch to 3 inches deep into the sponge body 105. The openings 120 a and 120 b may be sized such that a user may place a portion of one or more digits, such as one or more fingers or thumbs, into the openings. In other embodiments, the openings 120 a and 120 b may be sized such that a user may place one or more digits in their entirety into the openings. In further embodiments, the openings 120 a and 120 b may be larger or smaller, and may vary in size. In one embodiment, the sponge body 105 may be sterilized using a variety of sterilization methods. The sterilization methods may include, but are not limited to, gaseous sterilization, exposure to ethylene oxide (EtO), chlorine or a sterilizing medical liquid, gamma radiation sterilization, autoclave, or exposure to ultraviolet lamps. Further, the sponge body 105 may be impregnated with various types of liquid solutions. The liquid solutions may include an antibacterial solution, which may be used to prevent bacterial growth on the sponge. Alternatively, the solution may include soap, which may be used for cleaning purposes. Other possible liquid solutions may exist as well. FIG. 1B illustrates a cross-sectional view of the sponge 100 according to the embodiment of the sponge constructed in accordance with FIG. 1A. The sponge 100 includes a sponge body 105, a side surface 110, openings 120 a and 120 b and a perimeter 130. As shown in this figure, the openings 120 a and 120 b are located within the sponge body 105 with access through the side surfaces 110. In FIG. 1B, the openings 120 a and 120 b are tooled to a depth that does not reach the center of the sponge. In alternative embodiments, however, the openings 120 a and 120 b may be at a depth that extends through the center of the sponge body 105. Additionally, as depicted in FIG. 1B, the openings 120 a and 120 b may be oriented at different distances from one another. In an alternative embodiment, the openings 120 a and 120 b may be oriented equally around the perimeter 130 of the sponge body 105. As discussed above, the number of openings 120 a and 120 b may vary, ranging from one opening to as many openings as capable of fitting into the sponge. FIG. 2 illustrates a perspective view of the sponge of FIGS. 1A and 1B in use. In operation, the openings 120 a may receive one or more of the user&#39;s fingers. The openings 120 b may receive one or more of the user&#39;s thumb or thumbs. When the openings 120 a and 120 b receive one or more of the user&#39;s fingers and thumbs, protection of the user&#39;s fingers or thumbs, fingernails, or knuckles may occur. The openings may assist the user&#39;s ability to clean tough surfaces, wring out the sponge, and more precisely direct the sponge while cleaning. As mentioned above, the user&#39;s fingers may fit into the individual openings 120 a and 120 b located on the sponge or into the one slit that spans the entire perimeter, as seen in FIG. 3. FIG. 3 illustrates a perspective view of a sponge 300 according to an embodiment of the present invention. The sponge 300 includes a sponge body 305, side surfaces 310, an opening 320, a perimeter 330, a top surface 340, and a bottom surface 350. The opening 320 is positioned around the perimeter 330 of the side surfaces 310 on the sponge body 105. As can be seen in FIG. 3, the opening 320 may extend around the entire perimeter 330 of the sponge body 305. The continuous opening allows a user to place one or more fingers or thumbs into the sponge body 305 at any orientation of the sponge. In an alternative embodiment, the opening 320 may extend around a portion of the perimeter 330. Additionally, while various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are contemplated herein.

Summary: A sponge with a sponge body and at least one opening in the side surface or surfaces to receive one or more fingers and thumbs of a user. The at least one opening may be located along the perimeter of the sponge. The at least one opening is capable of providing protection to a user&#39;s fingernail, knuckles, and other parts of the finger, during use. The sponge body may be formed into any shape or size. Additionally, the openings may be dimensioned such that they are capable of receiving and protecting the fingers and thumbs of a user. The sponge body can be formed from any acceptable material, such as poly vinyl alcohol (PVA).

big_patent

en

Summarize: This fall, restrictive new voting laws in more than a dozen states could keep millions of people from exercising their constitutional right to vote. ID and birth certificate requirements, restrictions on early voting, and shutdowns on election day registration happen to affect non-rich, non-white, non-middle-aged, non-male voters most. This flurry of regulatory activity could confound Jane and John Q. Public: how are citizens supposed to know whether they need an ID, license plate number, proof of insurance, blood sample and baptism certificate in order to cast their vote? The answer might be in the interwebs. The Lawyers Committee for Civil Rights Under Law has partnered with a bunch of other voting rights groups to launch a free downloadable "Election Protection" app that will allow voters to verify their registration status, fill out a voter registration form, look up their polling place, and access info on key rules and dates tailored to their state. Users are also prompted to call Election Protection's hotline to alert the organization to election day abnormalities or concerns. You might be thinking: Mother Jones writer, you are a privileged white girl, oblivious to the fact that poor people don't have fancyphones! Actually, about half of cell phone users in the US have smartphones, and a 2011 Pew study found that blacks and Hispanics are more likely than whites to use their phones for "non-voice" applications. Election Protection is also helpful not just for the poor, but for the scatter-brained, the nomadic, and those who don't have the time to take a day off and parse all the new rules. Since the app launched on August 9, it hasn't gotten much press. So I asked Eric Marshall, legal mobilization manager at the Lawyers Committee, how they and their partners plan to get it out into people's hands. He said they'll do online ads and a social media blitz, but mostly he emphasized "shoe leather": getting partner groups active on civil rights, voter engagement, youth, labor, environmental, and faith issues to spread the message. Good old-fashioned word-of-mouth is especially key for the old folks, Marshall says. "I download it and I check my info, and then there's no reason I can’t then go to my grandmother and get her info," he says, "or go to church and say, 'Hey, do you know your rights on election day? Let's look up your registration status.'" Designed by Revolution Messaging, the app is a mobile-enabled webpage for now, and can be downloaded to any smartphone, but Marshall says they're working on getting it into the iPhone app store. A Spanish version will be out soon. "You look at the landscape this year and all of the restrictive voting changes that have been enacted," Marshall says. "People are concerned about eligible Americans being able to participate in democracy." Not everyone's concerned. Pennsylvania's House Majority leader, Republican Mike Turzai, recently gloated that the state's new voter ID law "is going to allow Governor Romney to win the state of Pennsylvania." Well, whoever wins, this corner of the internet will help make sure that everyone gets to vote. In its first standalone measure of smartphone ownership, the Pew Research Center’s Internet & American Life Project finds that two in five cell owners (42%) own a smartphone as of May 2011. Since 83% of Americans own some kind of mobile phone, this means that one-third of all American adults (35%) are smartphone owners. Measuring smartphone adoption in the context of a telephone survey presents some practical challenges. Smartphones are typically defined as mobile phones with advanced capabilities such as internet access and the ability to download and install applications or “apps”. However, many cell owners—particularly casual users—are unsure of their phones’ capabilities, so measuring smartphone ownership in this way risked overestimating the adoption of this technology. Therefore our definition of a “smartphone user” includes anyone who falls into either or both of the following categories: One-third of cell owners (33%) say that their phone is a smartphone. Just over half (53%) say that their phone is not a smartphone, while the remaining 14% do not know if their phone is a smartphone or not.. Just over half (53%) say that their phone is not a smartphone, while the remaining 14% do not know if their phone is a smartphone or not. Two in five cell owners (39%) say that their phone operates on a smartphone platform common to the US market (these include the iPhone and Blackberry, as well as phones running the Android, Windows or Palm operating systems). One in ten (13%) do not know what type of phone they have, while the remaining responses included those that were not smartphones (i.e. “basic cell phone”, “cheapest phone” or “flip phone”) or that were not easily classified into a particular category (i.e. “Samsung”, “Nokia”, “Verizon phone” or “AT&T”). Taken together, 42% of cell owners said yes to one or both of these questions and are classified as smartphone owners. The remaining 58% of cell owners have some kind of mobile phone other than a smartphone. Age differences in smartphone adoption Smartphone ownership is highest among Americans in their mid-twenties through mid-thirties, as fully 58% of 25-34 year olds own a smartphone. Smartphone ownership begins to tail off at around 45 years of age, before dropping dramatically at around age 65 (just one in ten seniors own a smartphone, and 44% do not have a cell phone of any kind). Income differences in smartphone adoption Smartphone ownership is highly correlated with household income. Respondents from the highest income cohort (those with an annual household income of $150,000 or more) are around three and a half times as likely as those in the lowest income group (with an annual household income of $10,000 or less) to own a smartphone: roughly three quarters of high-income earners do so, compared with one in five low-income earners. A household income of $75,000 is the approximate point at which Americans are more likely to own a smartphone than not—more than half of Americans above this income level are smartphone owners, and cell phone ownership in general is near-ubiquitous (95% or more) past this point in the income distribution. While smartphone ownership is a majority proposition among higher-income earners, those Americans with a household income of less than $30,000 per year primarily own more basic mobile phones. Indeed, members of this lower-income cohort are as likely to lack a cell phone entirely as they are to own a smartphone (22% own a smartphone, while 23% have no cell phone at all). Although low-income Americans as a whole are relatively unlikely to own a smartphone, there is quite a bit of age variation within this group. Among 18-29 year olds earning less than $30,000 per year, 39% own a smartphone (on par with the national average) and just 8% have no cell phone at all. By contrast, fully 57% of low-income seniors do not own a cell phone, and smartphone adoption rates for this group are extremely low at just 4%. Other factors correlated with smartphone ownership For several years, Pew Internet research has found that African-Americans and Latinos are more likely than whites to use their cell phones for non-voice applications such as using the internet, playing games, or accessing multimedia content. These differences extend to smartphone ownership as well, as 44% of black and Latino adults are smartphone owners, compared with 30% of whites. Employment status is also closely linked with smartphone ownership. Nearly half of full-time employees (48%) have a smartphone of some kind, as do 38% of those who are employed part-time. Roughly one quarter of those who are not employed for pay (27%) have this type of device, while just 13% of retirees do so. Smartphone owners and their technology assets Smartphone users own a wide range of devices in addition to their phones. Eight in ten smartphone owners also own a laptop computer, and e-book readers and tablet computers are much more prevalent among smartphone owners than in the general population. In their own words—how smartphone owners describe their phones Along with asking about smartphone adoption and usage, we also included a question in our spring survey that asked cell phone owners to provide the one word that best describes how they feel about their phones. The smartphone owners we surveyed provided an enormous diversity of reactions to this question—the 662 responses we coded included 177 unique descriptors—so few words or phrases stuck out clearly from the pack. The three most common words were “good” (mentioned by 10% of smartphone owners), “great” and “convenient” (each mentioned by 7% of smartphone owners). Overall, 72% of smartphone owners used a positive word (such as “good”, “great”, “excellent” or “convenient”) to describe their phones, 16% used a negative description (such as “expensive” or “frustrated/frustrating”) and 12% offered a neutral word choice (such as “adequate”, “OK”, “fair” or “fine”). How smartphone owners describe their phones (click for a larger version):

Summary: New voting laws could restrict millions of Americans from casting a ballot this fall-but an iPhone app aims to help voters navigate these rules and regs, writes Erika Eichelberger at Mother Jones. Designed by the Lawyers Committee for Civil Rights Under Law and other voting rights groups, the "Election Protection" app provides registration forms and information for each state. It also has a hotline to call for last-minute news on election day snafus and hijinks. "You might be thinking: Mother Jones writer, you are a privileged white girl, oblivious to the fact that poor people don't have fancyphones!" writes Eichelberger. But half of American cell phone owners use smartphones, and according to one study, "blacks and Hispanics are more likely than whites to use their phones for 'non-voice' applications." Eichelberger says the app is not only good for the underprivileged, but "the scatter-brained, the nomadic, and those who don't have the time to take a day off and parse all the new rules."

multi_news

en

Summarize: By. Daniel Mills for Daily Mail Australia. and Leesa Smith for Daily Mail Australia. and Aap. The Boomers' World Cup nightmare has worsened after basketball's governing body opened an investigation into the controversial loss to Angola in which Australia was accused of tanking. A day after a surprise defeat to Turkey sent them out of the tournament in Spain, Australia learned they could face disciplinary action from FIBA over the manner of last week's loss to Angola. The Boomers rested several key players and let a 15-point lead slip in the third quarter against Africa's No.1 nation in their final group game in Gran Canaria to go down by 91-83. The 91-83 loss meant Australia could not face the all-conquering USA team until the semi-finals but Boomers head coach Andrej Lemanis rejected suggestions they lost on purpose. The alleged strategy proved irrelevant after a 65-64 loss to Turkey in their sudden-death round of 16 game on Sunday, but Basketball Australia must now formally defend the performance against Angola or face possible sanctions such as a fine. Scroll down for video. Angola's forward Eduardo Mingas vies with Australia's forward Cameron Bairstow. Australia's Brock Motum fends off Angola's Eduardo Mingas. A number of star players were rested from the Boomers squad in the third round. Angola celebrating winning their game against Australia in the third round of the world cup. 'The on-court behaviour displayed by Australia in that game generated huge disappointment by basketball fans and experts,' FIBA said in a statement on Monday. 'It is widely suspected that Australia lost that game in order to avoid having to face the reigning world champions USA until the semi-finals. 'Basketball Australia has an opportunity to state its case before FIBA decides whether and to what extent disciplinary sanctions shall be imposed.' Slovenian NBA star Goran Dragic was among the fiercest critics of Australia's performance, taking to Twitter after the match to urge FIBA to take action. 'Basketball is a beautiful sport, there is no room for fixing the game like today Australia vs Angola!! @FIBA should do something about that!' he posted. With a place in the knockout stages already secured, Lemanis rested his two best performing players of the tournament, star centre Aron Baynes and sharp-shooting veteran Joe Ingles, while giving fellow starters Matthew Dellavedova and David Andersen less than four minutes each. When challenged post-match on whether his side had contested the match in the spirit of the game, Lemanis was defiant, arguing the heavy tournament schedule demanded he rest his players before the next stage of competition. 'We always, as Australians, compete the right way,' he said. Pressed on how some people would view the loss Lemanis said: 'People will make up their own minds. 'There's always going to be speculators. I can't control what people think.' Injured Australian star Andrew Bogut hit out at FIBA's comments and questioned the governing body's consistency. 'So u can naturalize a plyer who has nver stepped foot in a country, but you cant rest players in a game 2 better a draw 4 youself? Ok @fiba,' Bogut wrote on Twitter. 'Love it or hate it I have seen this and much more occur many times @fiba tournaments I have played in since 2002 (incl. Juniors).' Jubilant Angolan supporters enjoy the eight point win in the game against Australia. A dejected Australian bench watch on as the side comes to terms with losing. Australia were in contention to run away with the game when they led by 15 in the third quarter. Despite missing their two best players, NBA stars Patty Mills and Bogut, Australia had been optimistic of challenging for a first ever World Cup medal. The Boomers were on course for their first quarter-final appearance in 20 years, leading Turkey by six points with just over a minute to play in Barcelona on Sunday night. However, some poor decision-making allowed Turkey back into the game and Emir Preldzic buried a three-pointer with five seconds remaining to give the reigning silver medallists a famous win. Basketball Australia is yet to make comment on FIBA's investigation. Lemanis said suggestions of tanking were ridiculous and defended Australia as a nation that competes in the interest of fair play. 'We always, as Australians, compete the right way,' he said. When asked whether he accepted that some Australians would view the loss with an air of scepticism, Lemanis said: 'People will make up their own minds.' 'There's always going to be speculators. I can't control what people think.' The Boomers were chasing a fourth straight victory for the first time in World Cup history, and for most of the match were cruising before the wheels fell off. Guard Chris Goulding shot a team-high 22 points as the Boomers opened up a 15-point buffer. It was also an opportunity to see highly-rated No.5 NBA draft pick Dante Exum in action, and the 19-year-old didn't disappoint - scoring 12 points and handing out six assists in his 31 minutes on court. Despite fielding a second string team Australia was still the heavily backed favourites at $1.17. Australia's Dante Exum takes on Angola's Edson Ndoniema and Yanik Moreira. Australia's forward Cameron Bairstow shoots from the free foul line. Australia was winning by 15 in the third quarter when the momentum swung in Angola's favour. The loss means Australia will not have to face the US until the semi finals if they win their way there. But an Angolan charge, led by dynamic young star Yanick Moreira's outstanding 38-point display, started midway through the third quarter and they cut the lead to just two at the final break. The onslaught continued thereafter as Australia carelessly gave up the ball, committing 18 turnovers, and played lacklustre defence as the clock wound down in the eight-point loss - prompting the tanking calls. Sports betting agency Sportsbet.com.au refunded $125,500 in bets placed on the Boomers to win the match in light of the allegations. The side was an unbackable $1.17 to win with Angola the $5.20 outsiders. 'Punters bet on the Aussies in good faith not knowing they would be resting a few of their guns and giving their reserves a go, so we think it's only fair that they get their money back to enjoy over the weekend,' the agency's Shaun Anderson said. In good faith the betting agency refunded the amounts wagered on the match in light of the allegations. Former Boomer Shane Heal defended the tactics and said Australia were well within their right to want the best chance of winning a maiden medal. '[It is] un Australian not to give yourself the best chance to medal,' he wrote on Twitter. 'No medals given on pool placings. Pathway to final 4 is the key.' 'There's still games to be played that will determine the outcome of the pool. Australia is chasing its first medal in the history of the world cup. Social media users have called the match 'un Australian' A New Zealander likened the tactics to those employed by Australia's cricket side. A defiant Angola stole the game away in the third quarter. When pressed about allegations of premeditated tanking tactics post match, Lemanis responded:. 'Nobody likes losing. (The loss) doesn't guarantee us third spot. He said the team had 'no control over that game.' 'That was always the case coming in.' Securing third spot would mean Australia would avoid a quarter-final show down against America and conveniently put them in a position to challenge for a maiden medal at the tournament. Former Australian basketball great Shane Heal said if tanking was on the agenda, it was something that he supported in an effort for the Boomers to win a maiden world cup medal. '[It is] un Australian not to give yourself the best chance to medal,' he wrote on Twitter. 'No medals given on pool placings. Pathway to final 4 is the key.' But most shared a different view to Heal on social media - with one Twitter user comparing the match to Australia's infamous underarm bowling scandal. The strategy employed by the then captain Greg Chappel gave New Zealand no hope of winning when his brother Trevor bowled an underarm ball on the final play of the match

Summary: FIBA investigates Australia's controversial loss over Angola. The Boomers were leading until the wheels fell off in the third quarter. The side let a 15-point lead slip to lose by eight and avoid playing the US. Boomers coach Andrej Lemanis said he had 'no control' of the outcome. Betting agency Sportsbet refunded all bets on Australia to win the game. The team went onto lose to Turkey in a sudden death round on Sunday.

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Summarize: Image via Getty. There’s been an unending stream of sexual assault stories hitting us over the last week. Let’s take a break to enjoy a story of women defending women, in which one of those women is Princess Leia. Heather Ross is a screenwriter who was also a close friend of the late, great Carrie Fisher. The Blast reports that Ross called into 94.9 MixFM in Tucson and relayed a story to the hosts of The Morning Mix about how a Sony executive harassed her in 2000. At the time, Ross told Fisher about her harrowing experience, and Fisher decided to give the guy bothering her friend a little gift: “About two weeks later, she sent me a message online and she said, ‘I just saw (blank) at Sony Studios. I knew he would probably be there, so I went to his office and personally delivered a Tiffany box wrapped with a white bow.’” Ross explained. “I asked her what was inside and she said, ‘It was a cow tongue from Jerry’s Famous Deli with a note that said, If you ever touch my darling Heather or any other woman again, the next delivery will be (clears throat) something of yours in a much smaller box!’” A much smaller box. Ross suggests that if people don’t know what a cow tongue looks like, they Google it. Carrie Fisher continues to inspire with her creativity, her supportiveness as a friend, and her ability to scare the crap out of men. Even after her death Carrie Fisher is having none of your bullshit. Heather Ross, a friend of Fisher's, spoke to a local radio station in Tucson, AZ. about her experience with sexual assault in Hollywood. On the 94.9 Morning Mix, Ross recalled inappropriate advances from a big name producer and how Fisher made clear she had a zero-tolerance policy for that kind of behavior. SEE ALSO: Kate Winslet refused to thank Harvey Weinstein in her 2009 Oscars speech After Ross was assaulted by "an Oscar-winning producer" – not Weinstein, but another – Fisher was furious. After the initial shock and fear wore off, Fisher ran into the producer at Sony and delivered him a package. "It was a cow tongue from Jerry’s Famous Deli with a note that said, ‘If you ever touch my darling Heather or any other woman again, the next delivery will be something of yours in a much smaller box.’” The cow tongue was all Fisher's idea, natch, and she made sure to deliver it in person and watch him open the box. "That's just how she was," Ross said. "I miss her dearly. She stood up for people...She put things out there and in your face." Ross met the producer online – their conversations weren't sexual or romantic, she said, but he did want to meet up and get her involved with his latest project. "I felt safe thinking, 'I'm overweight, I'm not attractive to these people, I'm not looking to become an actress,'" she explained. She met with him and he made sexual advances. "When it happened, it happened so quickly that I was ashamed of myself," she said. "I thought I did something wrong. And I thought, 'Well, maybe by meeting up with him...I was asking for it.'" The producer picked her up in his car then made an excuse to pull over. He reached over her to quickly recline the passenger-side seat then pushed himself on top of her. Ross explained she thought she was "protected" by not fitting Hollywood's beauty standards, but her story confirms what most women know and what a piece like Mayim Bialik's New York Times column failed to pick up on: This happens to all women. Ross told her mother and friends but ultimately stayed silent for many years, describing the massive power gap between a "kid from Tucson" and an award-winning producer. h/t Page Six

Summary: With the Harvey Weinstein scandal engulfing Hollywood and people around the film industry wondering what can be done to prevent sexual harassment, the late actress Carrie Fisher may have the best answer yet, even from beyond the grave. Last week, Heather Ross, a screenwriter and friend of Fisher's, told the hosts of the Morning Mix radio show on 94.9 MixFm in Tucson, Arizona, that after she was sexually assaulted by an "Oscar-winning producer" in 2000, the Star Wars star responded by gifting the producer a cow tongue from Jerry's Famous Deli, Mashable reports. The gift included a note, which said, "If you ever touch my darling Heather or any other woman again, the next delivery will be something of yours in a much smaller box." Ross says she met the producer online and that they set up a meeting after he expressed interest in her working on his latest project. After the producer picked her up, he pulled his car over, reached over Ross to recline the passenger-side seat, and got on top of her. Ross told Fisher and two weeks later received an online message from the actress saying she had seen the man at Sony Studios and gone to his office to personally deliver the "gift" in a Tiffany box, complete with a white bow, per Jezebel. "That's who Carrie Fisher was," Ross says. "She put things out there and in your face."

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Summarize: CROSS-REFERENCE TO RELATED APPLICATION [0001] The present patent application claims the benefits of U.S. Provisional Application, Serial No. 62/008,845, filed on Jun. 6, 2014, the disclosures of which are herein incorporated by reference. BACKGROUND [0002] Compared with the traditional thermal processing of juice products, high pressure processing (HPP) is widely recognized as a non-thermal pasteurization method, which is most effective in maintaining the flavor, color, texture and nutritional properties of foods and beverages. HPP is a method of preserving and pasteurizing food, in which a product is processed under a very high pressure, leading to inactivation of certain microorganisms and enzymes in food. HPP utilizes isostatic or hydrostatic pressure which is equal from every direction. Neither the size of a product&#39;s container nor its thickness plays a role in the effectiveness of pasteurization by HPP. The first product pasteurized by high pressure (600 MPa) was commercialized in Japan, since then many inventions used HPP to pasteurize foods. European patent, EP 0480422B1, claims a method for treating fruit juice containing pectin and pectinase with high pressure. The resulting juice featured a superior quality than the juice subjected to the conventional thermal processing. Chinese patent publication CN102429029A provides a production method of low-sugar fruit juice with milk added, wherein the juice with milk is homogenized at 20-25 MPa to pasteurize it. The homogenization was the source of the high pressure. European patent publication 1051087A2 relates to a method for pasteurizing apple juice using HPP to inactivate microorganisms in order to extend the shelf-life of the juice. [0003] HPP will not inactivate the spores of some microorganisms unless the temperature is elevated to 75° C. or higher. As reported in Food Bioprocess Technol (2011) 4:458-468, ultra high pressure (700-900 MPa) combined with elevated temperature (80-100° C.) inactivated the spores of Clostridium sporogenes in milk. It was found that at low temperature, the germination of spore was not possible. Dormant Bacillus coagulans spores are highly resistant to many physical and chemical agents including heat, drying, chemicals such as hydrogen peroxide, radiation and high pressure. [0004] Probiotics are friendly bacteria which can survive in the human digestive system and have been proved to have the ability to balance the intestinal bacteria flora, and to improve the digestive ability. Products containing probiotics available in the market are mainly yogurt or some other dairy products. However, few dairy-free probiotics products have been launched. There are products containing Lactobacillus plantarum 299v, kefir culture, L. plantarum Heal9 in the market. Most of the current probiotics containing products, including dairy products and diary-free products, are thermally pasteurized to inactivate harmful bacteria and enzymes. Thermal process is, however, detrimental to nutrients, color and flavor which are undesirable to consumers. Chinese patent publication CN 101708018A discloses a beverage with Lactobacillus - casei added which is homogenized by high pressure but before the addition of probiotics the beverage is thermally treated at about 100° C. for about 2.5 hours, which causes nutrients loss. European patent publication EP 2432334A1 illustrates a Lactobacillus added citrus fruit juice. [0005] The present inventors discovered that, using HPP to pasteurize juices inoculated with Bacillus coagulans spores, a new product can be attained with superior attributes like long shelf-life, higher nutrient, and more natural. SUMMARY OF THE INVENTION [0006] The invention provides a method of preparing a juice product containing live probiotics, comprising [0007] (1) mixing a juice with probiotics to obtain a juice-probiotics mixture at a concentration of 10 5 to 10 12 cfu of probiotics per ml of the juice mixture, with optional stirring performed at about 10° C. to about 60° C. for about 1 minute to about 30 minutes, and wherein the juice is a fruit juice, vegetable juice or combination thereof; and [0008] (2) subjecting the juice-probiotics mixture to high pressure processing (HPP), wherein the HPP is conducted at a pressure ranging from 100 MPa to 1,000 MPa for about 1 minute to about 30 minutes, preferably for about 1 minute to about 20 minutes, at a temperature of 5° C. to 80° C., or 5° C. to 70° C., or 5° C. to 65° C., or 5° C. to 60° C., or 5° C. to 55° C., or 5° C. to 50° C., or 5° C. to 45° C., or 5° C. tO 40° C., or 5° C. to 35° C., or 5° C. to 30° C., or 5° C. to 25° C., or 5° C. to 20° C., to obtain the juice product containing live probiotics. [0009] Alternatively, in the method of preparing the juice product containing probiotics described above, step (1) is preceded with a step of providing the juice for mixing with the probiotics in step (1). [0010] In one of the embodiments of the method of preparing the juice product, the juice-probiotics mixture is put in a container having a flexible wall before the HPP in step (2). The container can be a plastic container or any other flexible container. DETAILED DESCRIPTION OF THE INVENTION [0011] The juice mixed with probiotics in step (1) of the method of preparing the juice product of the invention is preferably dairy free, or a fresh juice, or a dairy-free fresh juice. The juice product containing live probiotics prepared by the method of the invention is preferably dairy free. The fresh juices are prepared by any conventional fruit juice preparation method, such as extraction, enzymatic hydrolysis, centrifugation and filtration. Probiotics is then added to the prepared juices. The mixture is stirred gently for 1 minute to 30 minutes at a temperature of 10° C-60° C. The final count of probiotics in the juice ranges from 10 5 to 10 12 cfu/mL. The prepared juice with probiotics is filled into a container having flexible wall, e.g., a plastic container such as a plastic bottle or pouch, sealed and subjected to HPP. [0012] The juice used in this invention can be a fruit juice, a mixture of fruit juices, a vegetable juice, a mixture of vegetable juices or a mixture of at least one fruit juice with at least one vegetable juice, with or without addition of extra sugar, sweetener or acid. The juice Brix ranges from 6 to 25, and the pH value ranges from 2.5 to 8. Examples of the juice include, but not limited to, fruit juices, such as apple juice, coconut juice, or orange juice, and vegetable juices, such as carrot juice. [0013] As used herein, the term “probiotics” refer to beneficial bacteria, which can survive in the human digestive system and have the ability to balance the intestinal bacteria flora, and to improve the digestive ability. Probiotics can include bacteria of the Lactobacillus genus and Bifidobacterium genus naturally existing in the intestine of healthy mammals, particularly, humans. Examples of probiotics are Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, and Bacillus coagulans. [0014] Probiotics used in this invention can be a spore forming Bacillus coagulans strain, or a Lactobacillus plantarum strain, or their combination with a number ratio of Bacillus coagulans to Lactobacillus plantarum ranging from 1:99 to 99:1, 1:50 to 50:1, 1:10 to 10:1, 1:5 to 5:1, or approximately 1:1. Before mixing the probiotics with the juice to get a juice having the desired final concentration of the probiotics, probiotics powder can be mixed with the prepared juices at a temperature in the range of 4° C.-100° C. to get a stock with a high count of probiotics in a liquid state for further dilution with the target juice to obtain a juice with an appropriate final concentration of 10 5 to 10 12 cfu/ml for HPP. [0015] HPP is used to pasteurize the juice product from the above step by destroying harmful microorganisms and inactivating most enzymes. Flavor and taste of the juice product are preserved much better by pasteurizing through HPP compared to the traditional thermal pasteurization. In addition, thermal pasteurization will deactivate the probiotics. The pressure and holding time parameters for high pressure pasteurization can range from 100 MPa to 1,000 MPa, 100 MPa to 800 MPa, 100 MPa to 500 MPa, 100 MPa to 300 MPa, 500 MPa to 1,000 MPa, 500 MPa to 800 MPa, 500 MPa to 700 MPa, or 600 MPa to 700 MPa, for 1 minute to 20 minutes. The temperature during the HPP treatment can range from 5° C. to 80° C. [0016] The high pressure pasteurized products can be chilled by any conventional method and stored at a temperature ranging from 0° C. to 40° C. for 1 month to 20 months. Example 1 A. Juice Preparation [0017] One hundred tons of apples were prepared by any conventional methods, such as washing, sanitizing, and rinsing for further processing. Washing was done with water at a temperature ranging from 1° C. to 90° C. Sanitizing agents such as chlorine or its derivatives, peroxyacetic acid, or any organic/inorganic bases were added to the water during the wash or separately added into a second wash before the final rinse. Apple juice was extracted by any conventional method available, including the optional addition of macerating enzyme(s). [0018] Apple juice might or might not be centrifuged at a speed ranging from 100 to 10,000 rpm for 1 to 30 minutes. If centrifuged, the centrifugation temperature was maintained at a temperature ranging from 5° C. to 45° C. The clarified apple juice was prepared for further processing. B. Probiotics Stock Solution Preparation [0019] One gram to 100 grams of probiotics powder, containing 10 2 to 10 12 cfu/g of Bacillus coagulans, Lactobacillus plantarum or a mixture thereof, were mixed with 1 to 100 liters of the juice until complete suspension, wherein the temperature was maintained in the range of 5° C.-60° C. This stock solution was used for the inoculation of the apple juice. C. Stock Solution Application [0020] One liter of the concentrated stock solution was mixed with 10-10 6 liter prepared apple juice at a temperature ranging from 5° C. to 60° C. with stirring at a speed of 1-500 rpm for 1-30 min. [0021] The juice mixture was then filled into a 50 mL to 5 L plastic bottle, cup or pouch and sealed with the headspace volume within 0.1% to 10% of the total bottle or pouch volume and minimizing air or any other gases in the package. [0022] The packaging material could be, but not limited to, polyethylene (PE), polyethylene terephthalate (PET), nylon, ethylene vinyl alcohol (EVOH), or a mixture thereof. [0023] The sealed plastic package was loaded into the chamber of a HPP unit for pasteurization. The processing pressure ranged from 100 MPa to 1 000 MPa, 100 MPa to 800 MPa, 100 MPa to 500 MPa, 100 MPa to 300 MPa, 500 MPa to 1,000 MPa, 500 MPa to 800 MPa or 600 MPa to 700 MPa, The holding time ranged from 1 to 30 minutes, and the temperature ranges from 10° C. to 60° C. [0024] The pasteurized products were dried and chilled using forced air or any other conventional methods of drying/cooling, and stored at the temperature ranged from 1° C. to 30° C. for 1-12 months. Example 2 A. Juice Preparation [0025] One hundred tons of coconuts were washed, sanitized and rinsed for further processing. Washing was done with water at a temperature at 1° C. to 90° C. Sanitizing agents such as chlorine or its derivatives, peroxyacetic acid or any organic/inorganic bases were added to the water during the wash or separately added into a second wash before the final rinse. [0026] The juice was collected in a sanitary condition from the washed coconut for further process. B. Probiotics Stock Solution Preparation [0027] One to 100 grams of probiotics powder containing 10 2 to 10 12 cfu/g of Bacillus coagulans or Lactobacillus plantarum and/or their combination, was added into 1 liter to 100 liters of the prepared coconut juice and mixed to complete dispersion at a temperature in the range of 5° C.-60° C., to get a high concentration probiotics stock in a liquid state for further mixing process. The temperature of the probiotics stock solution was maintained at a temperature in the range of 5° C.-60° C. C. Stock Solution Application [0028] One liter of the concentrated probiotics stock solution was mixed with 1-10 6 liter of the prepared coconut juice The mixing was performed between 1-30 minutes at 1-500 rpm at a temperature of 5° C.-60° C. [0029] The juice mixture was then filled into 50 mL-5 L plastic bottles, cups or plastic bags and sealed with the headspace volume controlled within 1% to 5% of the total bottle, cup or bag volume and minimizing air or any other gases in the package. [0030] The packaging material could be but not limited to polyethylene (PE), polyethylene terephthalate (PET), nylon, ethylene vinyl alcohol (EVOH), or combination of some or all of these materials. [0031] The sealed plastic package was loaded into the chamber of high pressure machine for pasteurization. The pressure for processing ranged from 100 MPa to 800 MPa, and holding time ranging from 1 min to 30 min, and the temperature of the high pressure transmit medium ranges from 5° C. to 90° C. [0032] The pasteurized bottle/cup/bag/any flexible containers of the products were dried and chilled using forced air or any other conventional methods of drying/cooling, and stored at the temperature ranging from 1° C. to 30° C. for 1-10 months. Example 3 A. Juice Preparation [0033] One hundred tons of each oranges, and carrots, were washed, sanitized and rinsed for further processing. The washing was done with water at 1° C. to 90° C. Sanitizing agents such as chlorine or its derivatives, peroxyacetic acid or any organic/inorganic bases were added to the water during the wash or separately added into a second wash before the final rinse. [0034] Oranges and carrots were juiced with a juice extractor respectively at an extraction temperature ranging from 1° C. to 90° C. After extraction, juices were maintained at 20° C. to 50° C. for 30 minutes to 10 hours. Macerating enzyme(s) might or might not be added into orange juice and carrot juice for hydrolysis, if needed, the enzyme(s) concentration range would be from 0.001% to 1% of the total juice volume. [0035] During the juicing process, Vitamin C or other natural antioxidants or mixture of antioxidants were added to the juices to protect juice color or modify the pH. After enzymatic hydrolysis if needed in some cases, both orange and carrot juices were subjected to centrifugation at a speed ranges from 100 to 20,000 rpm/min for 1 minute to 30 minutes, the centrifugation temperature was maintained at temperatures ranging from 5° C. to 45° C. In some cases, centrifugation might not be needed. The orange and carrot juices were mixed with an orange to carrot ratio from 1:3 to 3:1 (v/v). B. Probiotics Stock Solution Preparation [0036] One gram to 100 grams of probiotics powder, containing 10 2 to 10 12 cfu/g of Lactobacillus plantarum or Bacillus coagulans and/or their combination, was combined with 1 liter to 100 liters of the prepared juice mixture until complete mixing to get a high concentration of probiotics stock solution for further dilution. The temperature of mixing process was maintained in the range of 5° C. to 60° C. C. Stock Solution Application [0037] One liter of the high concentration probiotics stock solution was mixed with 1-10 6 liter of the prepared juice mixture maintaining the temperature at 5° C. to 60° C. The mixing process was progressed with stirring at the speed of 1-500 rpm for 1-30 min. The juice mixture was then filled into 50 mL-5 liters plastic bottles, cups or pouches and sealed with the headspace volume within 1%-10% of the total bottle, cup or pouch volume. [0038] The packaging material could be but not limited to polyethylene (PE), polyethylene terephthalate (PET), nylon, ethylene vinyl alcohol (EVOH), or combination of some or all of these materials. [0039] The sealed plastic package was loaded into the chamber of high pressure machine for pasteurization. The pressure for processing ranged from 100 MPa to 800 MPa, the pressure holding time ranged from 1 min to 30 min, and the temperature of pressure transmit medium ranged from 5° C. to 90° C. [0040] The pasteurized bottles/cups/pouches/any other flexible container of the products were dried and chilled using forced air or any other conventional methods of drying/cooling, and stored at the temperature ranging from 1° C. to 40° C. for 1-12 months. REFERENCES [0041] EP 0480422 B1 A method for treating fruit juice with high pressure [0042] CN 102429029 A Production method of low-sugar fruit juice milk [0043] EP 1051087 A2 Method for ultra high pressure inactivation of microorganisms in juice products [0044] CN 101708018 A Lactobacillus - casei probiotics beverage and preparation method thereof [0045] Y Shao, HS Ramaswamy. (2011). Clostridium sporogenes -ATCC 7955 spore destruction kinetics in milk under high pressure and elevated temperature treatment conditions. Food and Bioprocess Technology, 4(3):458-468. [0046] Edwin Ananta, Volker Heinz, Oliver Schluter, and et al. (2001). Kinetic studies on high-pressure inactivation of Bacillus stearothermophilus spores suspended in food matrices. Innovative Food Science &amp; Emerging Technologies, 2(4):261-272. [0047] F.C. Lavinas, M.A.L. Miguel, M.L.M. Lopes, and et al. (2008). Effect of High Hydrostatic Pressure on Cashew Apple ( Anacardium occidentale L.) Juice Preservation. Journal of Food Science, 73(6):273-277. [0048] Daniel N. Silaa,Thomas Duvettera, Ans De Roeck, and et al. (2008). Texture changes of processed fruits and vegetables: potential use of high-pressure processing. Trends in Food Science &amp; Technology, 19(6):309-319.

Summary: The objective of the invention is to provide an improved method of making healthier juice products inoculated with probiotics, wherein the juice includes but is not limited to apple juice, coconut water, coconut milk, orange juice and/or carrot juice. The present invention relates to a method of pressure treating a juice containing probiotics. Vegetative cells of harmful microorganisms and enzymes are inactivated by applying high pressure while maintaining the activity of the probiotics. The juice preferably is dairy-free. The juice could be any dairy-free juice, such as a fruit juice, a vegetable juice, or their combination. Fruit and/or vegetable juice are prepared by any traditional methods, including but not limited to, washing, extracting by optionally treating with enzyme(s), centrifuging and packing. Probiotics, e.g., probiotics from bacterial spores, such as the spores of Bacillus coagulans and/or Lactobacillus plantarum, stay alive and active in the fruit or vegetable juice after high pressure pasteurization, as well as during the product shelf life.

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Summarize: This application claims priority to United States Provisional Application No. 60/083,422, filed Apr. 29, 1998. BACKGROUND OF THE INVENTION This invention relates generally to furniture, and more particularly to furniture used in an office, or home office environment. Today&#39;s businesses rely heavily upon a variety of different electrical apparatus as the primary means by which information is received and disseminated. Thus, it is almost invariable that every desk encountered in a business environment supports one or more of these electronic apparatus. Common to most every office desk is at least a computer and a telephone. However, there are a variety of other devices that are normally positioned atop a business desk. Such other apparatus includes dictation/recorders, computer printers, computer scanners, telephone answering machines, facsimile machines, paper copiers and image scanners. Each of these office tools contain at least one electrical cable and may include additional cables permitting electrical communication with other devices. For example, such additional cables include the cable connecting the computer to the computer printer, the telephone cable between the facsimile machine and the telephone outlet and the connection between an image scanner and a computer. Depending upon the particular arrangement of devices on the desk, these cables are often strewn in a haphazard arrangement on the top of the desk. This arrangement is unacceptable because it decreases the effective area in which a person can work. Also, many of these cables are positioned such that they partially hang over the back of the desk. This arrangement produces a visually unpleasant work environment. Moreover, if the particular desk is in a common area through which people frequently pass, there exists the potential for inadvertent contact with the cables which can damage the cables. In addition, a passing individual may become entangled in these cables and, as a result, pull the dedicated electrical apparatus from the desk causing irreparable damage to the electrical apparatus and personal injury. Additionally, in order to provide maximum space utilization, many offices are arranged such that the individual desks are positioned in an open area. As a result, persons working in this environment do not enjoy a sense of privacy. Furthermore, the typical office desk does not afford the worker any appreciable degree of modesty, i.e. privacy for the area existing below the worksurface of the desk. Another shortcoming with respect to present day office furniture is in the area of conference tables. Normal conference tables comprise a substantially rectangular, horizontal worksurface with a series of legs depending therefrom. During meetings, presentations, and seminars, one or more electrical apparatus are often used to convey information. In this context, such electrical devices include overhead projectors, slide projectors, film projectors, and phone teleconferencing equipment. To use these devices, one must normally attach the electrical cable to the electrical outlet positioned in the wall adjacent to the conference table. Thus, the cable hangs over a side of the conference table and prevents individuals from moving freely about the conference room. Therefore, there exists a need for an office furniture system which effectively eliminate the problems extant in the prior art and is cost effective to manufacture. SUMMARY OF THE INVENTION Accordingly the present invention advances a new and unique office furniture system which successfully eliminates problems unaddressed by the prior art. According to one preferred aspect of the invention, the office furniture system is embodied in a utility desk having a cable channel depending from the back of the worksurface. This cable channel is dimensioned to accept the electrical cables connected to the electrical apparatus positioned on the worksurface. In accepting these electrical cables, the cable channel improves the visual appearance of the work area and prevents inadvertent contact between individuals and the electrical cables. In addition, placement of the electrical cables within the channel increases the effective workspace area. Moreover, the cable channel depends a preselected distance below the worksurface and thereby provides the worker with a degree of modesty. According to another preferred aspect of the invention, the desk includes a vertical back extending from the cable channel. The top of the back may contain a horizontal shelf extending therefrom, thereby providing an additional worksurface. Also, the vertical back may be formed with a window having an adjustable shade positioned thereover. This vertical back in combination with the window provides the worker with a degree of privacy. According to another aspect of the invention, the pair of legs positioned proximate to the back of the worksurface are equipped with rollers. These rollers facilitate movement of the desk when rearrangement of the work area is required. According to still another aspect of the invention, the desk includes a return rotatably attached to a front leg of the desk. The return is equipped with a pair of legs having rollers attached thereto, permitting the return to be rotated about the leg of the desk. Thus, the return provides an additional work surface and can be positioned under the worksurface of the desk when not in use. According to yet another aspect of the invention, a conference table is provided having a generally rectangular worksurface with four legs depending therefrom. The front of the worksurface is formed with a cutout section while the pair of legs positioned proximate to the front of the worksurface are fitted with rollers to thereby provide mobility. In a preferred embodiment, two individual conference tables are juxtaposed such that the front surfaces of the adjacent conference tables are in abutting contact, with the cutout sections of the respective tables in registration. When so positioned, these conference tables create an enlarged cutout section dimensioned to enable electrical cables to extend therethrough. These and other advantages, benefits and objects will be understood by one skilled in the art from the drawings, description and claims which follow. DESCRIPTION OF THE DRAWINGS FIG. 1 is a front perspective view of a utility desk according to the present invention; FIG. 2 a is a rear perspective view of the desk illustrated in FIG. 1; FIG. 2 b is a side view of the desk illustrated in FIGS. 1 and 2 a shown supporting a computer depicted in phantom; FIG. 3 is a front exploded view of the desk shown in FIGS. 1, 2 a and 2 b; FIG. 4 is a rear exploded view of the desk shown in FIG. 3; FIG. 5 is a front view of a utility desk according to the invention illustrating both a return and a window shade; FIG. 6 is a front view of the utility desk of FIG. 5 with the shade shown in the drawn position; FIG. 7 is a front perspective view of a utility desk according to an alternative preferred embodiment of the present invention; FIG. 8 is a rear perspective view of the utility desk illustrated in FIG. 7; FIG. 9 is a front perspective view of a utility desk according to another alternative embodiment of the invention; FIG. 10 is a rear perspective view of the utility desk illustrated in FIG. 9; FIG. 11 is a perspective view of a utility desk according to the invention illustrated attached to a return; FIG. 12 is a top view of the return illustrated in FIG. 11; FIG. 13 is a perspective view of a conference table according to the invention; FIG. 14 is a top view illustrating a pair of conference tables according to the invention shown in the juxtaposed position; FIG. 15 is a front view of the conference tables illustrated in FIG. 14; FIG. 16 is a side view of a utility desk according to an alternative preferred embodiment of the present invention; FIG. 17 is a detailed side view of the cable channel of the utility desk illustrated in FIG. 16; FIG. 18 is a partial cross-sectional front view of a utility desk according to another alternative preferred embodiment of the invention; and FIG. 19 is a cross-sectional side view taken along line I—I of FIG. 18. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is embodied in a unique desk particularly suited for office or home use. Turning now to FIGS. 1, 2 a and 2 b, there is shown a desk according to a preferred embodiment of the present invention, and generally designated by reference numeral 10. Desk 10 contains a worksurface 20 and a plurality of legs 60 depending from bottom surface 22 of worksurface 20. Legs 60 support worksurface 20 a preselected distance above the floor. Worksurface 20 is of a generally rectangular shape having a front 24, opposing sides 26, 28 and a rear 30. Depending from rear 30 of worksurface 20 is a cable channel 32. Cable channel 32 spans from side 26 to side 28 and depends a preselected distance from rear 30 of worksurface 20. Cable channel 32 includes a vertical member 34 attached to rear 30 of worksurface 20 and a vertical back 36. Vertical member 34 and back 36 are joined by a horizontal bridge member 35 and hence defines an interior 37 between vertical member 34 and back 36. As shown in FIG. 2 b, interior 37 is dimensioned to receive one or more electrical cables 152 attached to an electrical apparatus 150 positioned on top 21 of worksurface 20. As illustrated, electrical device 150 is a computer, however, it will be recognized by those with ordinary skill in the art that electrical device 150 may be any electrical device commonly used in a home or business office environment. As shown in FIGS. 9 and 10, top 38 of back 36 may be approximately coplanar with top 21 of work surface 20. In an alternative preferred embodiment as shown in FIGS. 7 and 8, back 36 may extend above the plane defined by top 21 of work surface 20. A shelf 38 extends substantially horizontally from top 37 of back 36. Preferably, shelf 38 extends in a direction towards work surface 20. In the most preferred embodiment, as shown in FIGS. 1, 2 a and 2 b, back 36 extends a greater distance above the plane defined by top 21 of work surface 20 than back 36 of FIGS. 7 and 8. In this embodiment, back 36 is fitted with a window 40. As shown in FIGS. 5 and 6, interior surface 41 of back 36 supports a shade 42 which can be adjustably positioned over window 40. As shown in FIG. 5, shade 42 is illustrated in the open position whereas FIG. 6 illustrates shade 42 in the drawn position, thereby covering window 40. In the most preferred form, window 40 is approximately thirteen and one quarter inches high and six inches wide, but other dimensions may be acceptably utilized. Alternatively, a plurality of windows 40 may be located in back 36. Desk 10 is supported a preselected distance above the floor by a pair of front legs 62 attached proximate to front 24 and a pair of rear legs 64 depending from exterior surface 65 of bridge member 35. As shown, legs 64 are slightly curved, however it will be appreciated by those with ordinary skill in the art that legs 64 may assume any shape without departing from the spirit and scope of the present invention. Preferably, rear legs 64 are fitted with rollers or casters 63 to facilitate the movement of desk 10. Additionally, one or both of front legs 62 may be fitted with glides or levelers (not shown) which serve to adjust legs 62 when desk 10 is positioned on a non-level floor. Turning now to FIGS. 3 and 4, desk 10 is preferably assembled by attaching bottom edge 67 of vertical member 34 to surface 66 of bridge member 35. Attachment can be accomplished by any means commonly employed in the art including, but not limited to, mechanical fasteners and adhesives. In desk 10, the positioning and configuration of cable channel 32 provides the lower region of back 36 with the dual function of forming a component of cable channel 32 as well as forming a modesty panel that depends from work surface 20. Positioning of rear legs 64 on the undersurface of bridge member 35 causes cable channel 32 to also perform a leg support function for a portion of the distance below work surface 20. This reduces the amount of metal utilized in rear legs 64 and thus desk 10. In the most preferred form, cable channel 32 has a thickness or spacing between vertical member 34 and back 36 of approximately two inches, and has a depth or spacing between the upper surfaces of bridge member 35 and work surface 20 of approximately ten and one quarter inches. Other dimensions may be utilized which accommodate cabling for equipment such as computers, modems, ISDNs, telephones, dictating machines, monitors, facsimile machines, photocopiers, image scanners and the like. In a preferred embodiment, as shown in FIGS. 16 and 17, bridge member 35 includes a first pair of vertical uprights 35 ′ and a second pair of uprights 35 ″, dimensioned to receive vertical member 34 and back 36, respectively. Uprights 35 ′ and 35 ″ are formed with teeth 35 ′″ which removably engage mating recesses 36 ′ found in vertical member 34 and back 36. Additionally, bridge member 35 is formed with a cable guide clip 39 dimensioned to accept the electrical cables positioned within bridge member 35. In this embodiment, rear legs 64 are attached to and depend from bridge member 35. Uprights 35 ′ and 35 ″ of bridge member 35 permit facile and secure attachment between vertical member 34 and back 36. Furthermore, the detachable feature of bridge member 35 permits quick dissassembly in the event it is desired to store or transport utility desk 10. In a preferred embodiment, bridge member 35 is made of aluminum. Turning now to FIGS. 5, 6, 11 and 12, desk 10 may also include a rotatable return 70 attached to a leg 62 and extending therefrom below bottom 23 of work surface 20. Return 70 includes a work surface 72 having a bottom surface 73. A pair of legs 74 depend from bottom surface 73 of work surface 72 with each leg 74 having a wheel or caster 75. As shown in FIG. 12, work surface 72 of return 73 is formed with an aperture 76 dimensioned to loosely surround leg 62. Hence, an individual may utilize return 70 as an additional work surface when needed and when not in use, rotate return 70 such that work surface 72 is positioned under bottom 23 of work surface 20. In the most preferred embodiment, legs 62, 64 and 74 of desk 10 and return 70 are made of tubular steel and are powder coated. Also, in the most preferred embodiment, work surface 20, vertical member 34, bridge member 35 and back 36 are made of maple, multi-ply or high grade veneered plywood. However, it will be recognized by one with ordinary skill in the art that other materials can be used without departing from the spirit and scope of the present invention. Turning now to FIGS. 18 and 19, desk 10 may also include a tower 90 dimensioned to receive and support the central processing unit (CPU) of a computer (not shown). Tower 90 is positioned below bottom 23 of work surface 20, and is preferably flush against surface 33 of vertical member 34. As shown in FIG. 18, a vertical shelving unit 94 is positioned against, and preferably attached to, side 28 of work surface 20. In this embodiment, legs 64 (FIG. 16) of bridge member 35 are replaced by legs 95 (FIG. 18) depending from tower 90 and legs 96 of vertical shelving 94. In another aspect, the present invention is embodied in a unique conference table, a preferred embodiment of which is shown in FIGS. 13 through 15, and generally designated by reference numeral 100. Conference table 100 includes a work surface 102 having a plurality of legs 104 depending therefrom which support work surface 102 a preselected distance above the floor. Work surface 102 includes a front 110, opposing sides 112 and 114 and a rear 116. Formed in front 110 of work surface 102 is a cutout section 120. Preferably, cutout section 120 is in the shape of a half circle. Legs 104, positioned proximate to front 110 of work surface 102, are each fitted with a wheel or caster 122 to permit movement of conference table 100. As shown in FIGS. 14 and 15, conference table 100 is preferably used in conjunction with a second conference table 100. In this embodiment, conference tables 100 are juxtaposed such that fronts 110 of work surfaces 102 are placed in abutting contact. When so positioned, cutout sections 120 are positioned in registration, and together form a circular aperture. This circular aperture is dimensioned to permit electrical cables to extend therethrough and thus provides a more convenient method for supporting electrical devices upon top 103 of work surface 102 and subsequently connecting electrical apparatus to an electrical outlet. Alternatively, conference table 100 may be provided with a plurality of circular apertures forming cable access ports through work surfaces 102. A plurality of cutout sections 120 may be provided along fronts 110 in order to form a plurality of cable access openings when work surfaces 120 are mated. Also alternatively, conference tables 100 may be mated with one or more additional spacing sections that have a planar upper surface and opposing facing sides that abuttingly mate with fronts 110 and opposed side edges that conform to the configuration of sides 112 and 114. Such spacing sections may be fitted with support legs or alternatively fasteners that connect to work surfaces 102, and include cutout sections that mate with cutout sections 120. In the most preferred embodiment, front 110 of work surface 102 has a length of approximately 44.38 inches, rear 116 has a length of approximately 36.0 inches and opposing sides 112 and 114 have a length of approximately 60.0 inches. It is to be understood that the foregoing is a description of the preferred embodiments. One skilled in the art will recognize that variations, modifications, and improvements may be made without departing from the spirit and scope of the invention disclosed herein. The scope of protection is to be measured by the claims which follow and the breath of interpretation which the law allows, including the doctrine of equivalents.

Summary: A desk providing a work surface and having a top, a bottom, a perimeter edge, and a U-shaped channel disposed below the top of the work surface adjacent the perimeter edge. The U-shaped channel includes a vertical member extending downwardly from the work surface proximate the perimeter edge, a bridge member, and a back member extending upwardly from the bridge member and spaced from the vertical member. A plurality of legs are attached to the bottom of the work surface to support the work surface a predetermined distance above a floor. The desk may be fitted with a return rotatably attached to at least one of the plurality of legs such that the return can be rotated under the work surface when not in use.

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Summarize: DOHA, Qatar — Now that he is set to become the new emir, the absolute ruler of Qatar, what possibly can Sheik Tamim bin Hamad al-Thani promise to the citizens of a tiny, incredibly rich country that seems to have everything? In Qatar, the unemployment rate flirts with zero (it is 0.1 percent); infants have a per-capita income over $100,000; health, housing, low interest loans and educations are all provided. Qataris have a world-class television network in Al Jazeera, will host the World Cup in 2022, are building an airport that will eclipse the one in nearby Dubai and hope to soon be self-sufficient in food production. But they do not have democracy. Some people here cautiously hope that the surprise decision of the outgoing emir, Sheik Hamad bin Khalifa al-Thani, 61, to hand power to his fourth son, Sheik Tamim, 33, may signal the governing family’s intention to offer Qataris a taste of expanded personal freedoms, even if democracy is not explicitly on the agenda. There are some hints that already have at least a few Qataris excited. (For most, a beneficent feudal monarchy appears just fine, thanks, and they demonstrated their appreciation by lining up by the thousands, on foot and in their Mercedes and other luxury cars, to visit the two emirs, incoming and outgoing, in their palace on Tuesday and pledge their allegiance.) Photo Najeeb al-Nauimi, the lawyer for Qatar’s only political prisoner, Mohammad ibn al-Dheeb al-Ajami, said he was hopeful. His client was jailed for life last year for writing a poem that, in a fairly tame manner, criticized “sheiks playing on their PlayStations.” Mr. Ajami was arrested under a constitutional provision that forbids criticism of the emir, however indirect. Mr. Nauimi, who said he has known the incoming emir since the sheik was 9 years old, said Tuesday that Sheik Tamim had told him that Mr. Ajami would be released within a few days of the new emir’s accession to power. That does not speak to democracy as much as it does to the absolute power of the monarch, but all the same, Mr. Nauimi hoped it would be a signal of openness to come. “There will be a lot of changes, definitely,” Mr. Nauimi said. The outgoing emir already promised parliamentary elections by the end of the year — a constitutional requirement that is long overdue. Mr. Nauimi is among those agitating for amendments to the Constitution that would let that Parliament appoint the prime minister, paving the way for a constitutional monarchy inching closer to the British model and away from the autocratic style of the Persian Gulf states. It would be the first such example in any of the gulf’s monarchies, and one of the few in the Arab world. “I’m optimistic,” Mr. Nauimi said. Optimistic, but not absolutely sure — in part because the governing family has consistently demonstrated that it has no tolerance to being challenged, or even criticized indirectly. In the absence of any sort of public agitation, change will come from the top down, not from the bottom up. Advertisement Continue reading the main story Advertisement Continue reading the main story “I just don’t believe the people who say there won’t be any changes,” he said, offering what may be a case of wishful thinking. But what can the new emir change? Many analysts say that Qatar’s aggressive interventionist foreign policy has become too integral a part of its national character to be rolled back. The betting is that the new foreign minister — expected to be appointed on Thursday with a new cabinet — will be promoted from within. Some here believe it may be Khalid al-Attiyah, the deputy foreign minister, who is close to the emir and a supporter of his many international ventures. “I think you’ll see some very big gestures from Sheik Tamim,” said John Watts, who works here for BLJ Worldwide, a well-paid consulting firm that acts as strategic adviser to the royal family and the Qatari government. The release of Mr. Ajami, the imprisoned poet? “That may well be one of them.” “I haven’t heard anything specific about his policy agenda, but that would fit into his nature and character,” Mr. Watts said. The outgoing emir himself seemed to be priming the pump for change in a televised address to the nation at 8 a.m. on Tuesday. (Oddly, because in the heat of summer few Qataris are actually awake that early). “The time has come to turn a new leaf in the history of our nation, where a new generation steps forward to shoulder the responsibility with their dynamic potential and creative thoughts,” the emir said. That of course could mean anything — or everything. One official close to the royal family said that the new cabinet would include at least one woman, and possibly two — a first here, and a rarity in the Arab countries of the Persian Gulf. “This is hugely exciting,” said Mike Holtzman, the president of BLJ Worldwide. While it is unusual in the region for a ruler to voluntarily step down, he said, “to those who follow Qatar, it is very much in keeping; they have invested heavily in their young people.” The incoming emir is an enigma, even to his own people, because until now his role has been in the background. “I don’t think Tamim and his father are far apart, philosophically,” said one former diplomat who has served in Qatar and who spoke anonymously in keeping with protocol. “But he’s done a good job of playing his cards close to his chest.” That has led to a school of thought here that he is socially conservative — none of his own three wives have anything like the high political profile role enjoyed by his mother, Sheika Mozah. “It’s just not true” that he is socially conservative, Mr. Holtzman said. For years, he was in the wings as his father and his prime minister ran the country, so people had little basis on which to judge him. “Now that he’s got the reigns, he’ll be showing concern for human rights, worker safety,” he said. “He has the wind at his back.” And democracy? “Well, Qatar is already a very open society.” Background on Tuesday's leadership transition in Qatar from the 61-year-old emir to his 33-year-old son, Sheik Tamim bin Hamad Al Thani. A man watches a televised address by Qatar's Emir Sheik Hamad bin Khalifa Al Thani, in Doha, Qatar,Tuesday, June 25, 2013. Qatar's ruler said Tuesday he has transferred power to the 33-year-old crown... (Associated Press) A man prepares to take a picture with his mobile photo of a televised address by Qatar's Emir Sheik Hamad bin Khalifa Al Thani, in Doha, Qatar,Tuesday, June 25, 2013. Qatar's ruler said Tuesday he has... (Associated Press) A man prepares to take a picture with his mobile photo of a televised address by Qatar's Emir Sheik Hamad bin Khalifa Al Thani, in Doha, Qatar,Tuesday, June 25, 2013. Qatar's ruler said Tuesday he has... (Associated Press) ___ WHY IS QATAR IMPORTANT? The Gulf state of Qatar is small _ only about a third the size of Belgium _ but has a carved out a significant global profile in the past decade. Qatar has huge oil and gas riches that feed one of the world's largest and most acquisition-hungry sovereign wealth funds, estimated at more than $100 billion. Its holdings have included stakes in London's capital's Harrods department store, the French luxury conglomerate LVMH Moet Hennessy Louis Vuitton and soccer's Paris Saint-Germain. Qatar also has pledged billions of dollars to help businesses in debt-crippled Greece and Italy. Qatar's political aims are equally ambitious. It has served as mediator for peace efforts in Sudan's Darfur region and among rival Palestinian political factions. It is currently hosting envoys from Afghanistan's Taliban for possible U.S.-led talks seeking to stabilize the country before the American troop withdrawal next year. Qatar has played a central role in the Arab Spring by providing critical aid for Libyan rebels last year and now a leading backer of Syria's opposition. Qatar's government founded the television network Al Jazeera in 1996, which transformed news broadcasting in the Arab-speaking world. The state-run Qatar Airways is among the world's fastest-growing carriers. IS SUCH A TRANSITION UNUSUAL? It is exceedingly rare among the ruling Gulf Arab dynasties. Most leaders remain for life or have been pushed out in palace coups. Qatar's outgoing emir, Sheik Hamad bin Khalifa Al Thani, took control in a bloodless coup against his father in 1995. The change in Qatar was believed to be prompted by health problems with the 61-year-old Sheik Hamad, but Qatar officials have not publicly disclosed any details. Yet it reinforces Qatar's bold political style. The transition to the 33-year-old crown prince, Sheik Tamim bin Hamad Al Thani, appears a direct response to the Arab Spring demands for reforms and its emphasis on giving a stronger political voice to the region's youth. It also upends the ruling hierarchy among neighboring Gulf allies dominated by old guard leaders such as the 90-year-old King Abdullah in Saudi Arabia and Kuwait's 84-year-old emir, Sheik Sabah Al Ahmed Al Sabah. WHAT CHANGES CAN BE EXPECTED? Not many in the short term. The outgoing emir is expected to maintain a guiding hand over Qatar's affairs for years to come. His son also has been involved in most key decisions in recent years as part of the grooming process. The most noticeable changes will likely be among the top government posts. It's expected that Qatar's long-serving prime minister and others could be replaced as Sheik Tamim puts together his own inner circle. Another possible new element could be more social media interaction. The British-educated Sheik Tamim was still a teenager when the Internet age began and is well attuned to its influence. Sheik Tamim also headed up Doha's unsuccessful attempt for the 2020 Olympics. He could give a boost to a possible return bid for the 2024 Games. Sheikh Hamad bin Khalifa Al Thani, the ruler of Qatar for 18 years, has transferred the "reins of power" to his son. The outgoing emir made the announcement to hand over power to Sheikh Tamim bin Hamad Al Thani, 33, in an address to the nation on Tuesday. "I declare that I will hand over the reins of power to Sheikh Tamim bin Hamad Al Thani, and I am fully certain that he is up to the responsibility, deserving the confidence, capable of shouldering the responsibility and fulfilling the mission," Sheikh Hamad, 61, said. Live television footage after the speech finished showed Sheikh Hamad and his son smiling widely, being greeted with traditional embraces by dignitaries and prominent members of the Qatari society. Sheikh Hamad had met royals a day earlier, where he announced the transition plans after weeks of speculation of his abdication. "God Almighty is aware that I had not desired power for the sake of power, nor endeavoured to rule for personal motives," the outgoing emir said. "It has always been the nation’s interest... that we lead through a new chapter." Sheikh Tamim has been groomed for the position since 2003, when his elder brother stepped aside. In the speech, Sheikh Hamad also called on the people of Qatar to protect their identity. "I am confidant that you are fully aware of your loyalty and of your Arab and Muslim identity," he said. "I urge you to preserve our civilised traditional and cultural values, originating from our religion, Arab identity and above all our humanity; as we believe that the Arab World is one human body; one coherent structure; it prospers if all its parts are prosperous." Saudi Arabia was quick to offer congratulations to Sheikh Tamim, who is expected to address the Shura Council, an advisory assembly, on Wednesday in Doha. The kingdom's 90-year-old King Abdullah said: "We are confident that you will continue the journey of your father... and his efforts in serving the state of Qatar and its brotherly people as well as strengthening relations between the two nations." The UAE followed, with President Sheikh Khalifa bin Zayed al-Nahayan, 65, affirming the "deepness of the brotherly relations," in a statement carried by state media. British Foreign Secretary William Hague thanked Sheikh Hamad for friendship and support, and tweeted that Tuesday symbolised an "Historic day for Qatar as the new generation takes over." Handover process Al Jazeera's diplomatic editor, James Bays, said the next 48 hours would be crucial for the new leader. "The way this works is that he needs to see his subjects," he said. "He needs to speak to prominent players in society, and there will effectively be, for some hours today, and some hours on Wednesday, an open house here where he will be meeting all the prominent sheikhs, speaking to them, discussing the future." Sheikh Hamad took power in 1995 while his father was on an overseas trip. Since then Qatar's economy has grown significantly. That growth came largely from natural gas; Qatar possesses some of the largest proven reserves in the world, but produced almost nothing before 1995. He became the ruler of a country with an $8bn economy, a figure which grew to $174bn a decade and half later. During most of Sheikh Hamad's rule, Qatar also adopted a pragmatic policy, cultivating relationships with countries and groups across the political spectrum. "We also know when you speak to diplomats here that [Sheikh Tamim] has been involved very closely with Qatari policy, both domestic and foreign policy for some considerable time," Al Jazeera's Bays said. "Slowly he's been taking more and more of a share of the running of this country. It's not as though he is now going to be sitting at a new desk with a new brief. He has been taking control of many matters, both in terms of their domestic and international policy."

Summary: The emir of Qatar has abdicated and will hand the reins to his 33-year-old son, who in turn becomes the youngest monarch in the region. Sheikh Hamad bin Khalifa al-Thani, 61, announced the news on national TV yesterday, though he didn't say when Sheikh Tamim bin Hamad al-Thani would take over. The younger Sheikh, however, is set to give a speech tomorrow and select a government afterward, the New York Times reports. "I am fully certain that he is up to the responsibility, deserving the confidence, capable of shouldering the responsibility and fulfilling the mission," Sheikh Hamad said. He took over from his own father in 1995, though in a decidedly less friendly manner: He launched a bloodless coup. Indeed, this kind of willing handover of power is extremely unusual in Gulf Arab dynasties, the AP notes. There are unconfirmed murmurs that it's related to Sheikh Hamad's health, but he's likely to continue to play a role in government. Though the country's prime minister is also likely to depart, politics in Qatar-whose influence is growing-aren't likely to change much. Sheikh Tamim "has been involved very closely with Qatari policy, both domestic and foreign... for some considerable time," says an al Jazeera editor. Qatar has a sovereign wealth fund of some $100 billion, one of the biggest in the world, and it's been building its political resume as a mediator of international conflicts, including Darfur; the Taliban also recently opened a political office in the country.

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Summarize: BACKGROUND OF THE INVENTION This invention concerns radiation therapy, especially brachytherapy, for treating tissues which may have diffuse proliferative disease. In brachytherapy, the radiation source is generally placed within a surgically created or naturally occurring cavity in the body. In particular, this invention relates to an applicator for delivering radiation therapy to a vaginal cavity and/or to adjacent tissue, often following surgical treatment of cancer. Radiation therapy of this sort is generally administered over a period of time in partial doses, or fractions, the sum of which comprises a total prescribed dose. This fractional application takes advantage of cell recovery differences between normal and cancerous tissue whereby normal tissue tends to recover between fractions, while cancerous tissue tends not to recover. In brachytherapy, a prescribed dose is selected by the therapist to be administered to a volume of tissue (the target tissue) lying outside the treatment cavity into which the radiation source will be placed. Generally the prescribed dose will include a minimum dose to be delivered at a preferred depth outside the treatment cavity (the prescription depth). Since, in accordance with the laws of physics, radiation intensity falls off with increasing distance from the radiation source, it is desirable to create and maintain a space between the source of radiation and the first tissue surface to be treated (generally the cavity wall since the source is placed within the cavity) in order to moderate the absorbed dose at the cavity surface. Although not always the case, generally the absorbed dose at the prescription depth outside the cavity is to be uniform. In this isotropic case, it is therefore important that the incident radiation on the interior surface of the cavity be the same at all points being treated. To accomplish this objective, it may be necessary to sequentially position a single radiation source through a series of positions (or utilize multiple sources strategically placed) which, in the aggregate, produce a uniform absorbed dose incident on the cavity surface being treated. When this is achieved, the absorbed dose reaching into tissue will be the same at all points being treated, and the minimum prescribed dose can be delivered at the prescription depth as nearly as the treatment plan will allow. Furthermore, by selecting the radiation source intensity (radioisotope emissions or X ray tube output) and controlling treatment time and the distance from the source(s) to the cavity interior surface, the incident radiation can be sufficiently moderated to avoid substantial damage to normal tissue. Rigid applicator cylinders designed to receive radioisotopes have traditionally been used to treat vaginal cancer or malignancies in adjacent tissues. A principal function of an applicator is to establish and maintain distance relationships between the radiation source and the tissues being treated such that the prescribed dose is delivered to a desired prescribed depth of tissue, and yet normal tissues nearest the radiation source are not subjected to absorbed doses sufficient to risk significant necrosis. Applicators of this general type are available, for example, from Varian Medical Systems, Inc., Charlottesville, Va. Such prior art applicator cylinders are sized to the vaginal cavity or adjacent anatomy, but because the tissues should be positioned closely against the exterior surface of the applicator, large applicators must be chosen that are often painful on insertion, and once inserted still fail to provide a good fit. Additionally, prior art cylinders are generally straight, with a central lumen into which radioactive seeds are delivered and later removed after completion of prescribed therapy. As a result, anisotropic treatment plans are difficult to achieve with such symmetrical applicators. Thus conventional applicators are less than ideal in many cases. SUMMARY OF THE INVENTION Although this invention is disclosed with specific reference to therapeutic application of radiation within the vagina, the principles of this invention may be similarly applied to other brachytherapy situations in other natural or surgically created anatomic spaces, or to therapeutic situations other than post-surgical treatment of cancer, and still fall within the bounds of this invention. This invention provides a personalized or customized applicator specific to the individual patient for whom radiotherapy is intended. By this invention, a core portion of the applicator is fashioned within the individual patient&#39;s vagina. A semi-rigid or expandable form or mold is positioned within the vagina serving as a form balloon, and is filled with an amount of material sufficient to fill the vagina to a desired degree and thereby to facilitate delivery of a prescribed radiation dose to adjacent or surrounding tissues. Silicone rubber is one preferable mold material and suitable molds can be fabricated by dip-and-cure or molding processes well known to those skilled in the art. The filler material is preferably capable of undergoing a reaction in which, as examples, a change of state occurs, a foaming process takes place or in which a sufficient change in viscosity occurs such that the cast or molded-in-place applicator retains shape integrity sufficient to allow repeated removal and insertion. Such materials would include reactive gels or polymers, with or without foaming agents, and thermoset materials, all with radiation attenuation and tolerance characteristics within practical limits allowing for delivery of radiation to the target tissues. Within tissue-tolerance limits, thermoset and exothermal filler material reactions can be used, or artificial heating applied, for example from within channels within the mass of filler material being formed, to provide the applicator with sufficient form stability in the desired shape. Furthermore, if adjustment of the general molded-in-place configuration is desired to facilitate isodose manipulation or tissue positioning different from the as-molded shape, thermoplastic materials can be used, such that after removal of the as-molded applicator from the vagina, heat can be used to soften the material, allowing reforming and subsequent cooling in a more desirable configuration. If used, thermoset material can be deliberately cured only partially within the vaginal cavity, and if necessary, further cured to completion of the reaction outside the vagina, and perhaps after any desired shape adjustment. The mold itself can be polymeric and act like a condom responding to internal pressure applied by the pressure of filling, by a fill reaction taking place within the (preferably closed) confines of the mold, or by a combination thereof. If filling is by a reaction within a closed space, the dynamics as well as the temperature and pressure limits of the reaction must be well understood. Pressure relief may be provided if necessary, for example, by conventional valving. Alternately, the mold may be inelastic and resist expansion, thereby tending to shape the cavity to the predetermined and desired shape of the mold. Such a mold is flexible (bendable) but substantially inelastic, as in the properties of a Mylar balloon versus a latex balloon. A channel or channels to position the radiation source or other instrumentation within the applicator can be molded in place within the vagina during the filling (and harden or cure) process by positioning coring within the mold such that the filler material forms around the cores simultaneously with filling of the mold. The coring can be stripped from the filled applicator either from within the vaginal cavity, or after removal. Using this alternative, the coring need not be straight, permitting channels of any configuration consistent with the filling or molding process, and also consistent with the prescribed therapy. Alternatively, the channels can be drilled or otherwise fashioned outside of the vagina as desired after the filling process. In addition to channels created for introduction of radiation sources into the applicator, channels to permit suction, drainage, or infusion of liquid or gaseous agents may be provided by similar form-in-place techniques, and configured to communicate with the outer surface of the applicator in desired locations. Some embodiments may include texturing or channeling of the outer applicator surface in order to facilitate fluid flow between the surface of the applicator and the anatomic or treatment cavity. The portion of the applicator outside the body is connected to the mold balloon, and may optimally comprise a handle which extends outside the vaginal cavity to facilitate insertion and removal of the applicator, or with other manipulation as may be desirable. Such an applicator used by and holding a prefabricated handle external of the anatomy during the molding process such that essentially an extension of the handle is cast or molded to the applicator forming a monolithic structure manipulable via the handle. Conversely, the mold which contains the filler material may be sufficiently long that it extends outside of the body and once filled, serves as a form of handle for manipulation. After such an applicator is fashioned to mimic the interior surface of the vaginal cavity, and after any secondary shaping and coring is complete, the applicator is ready for use. Following a prescription prepared by competent personnel, dose planning may proceed such that, based on the applicator shape and the radiation delivery parameters of the specified radiation source, the source positioning and exposure times are determined, with the aggregate exposure conforming as closely as possible to the prescription. After patient preparation, the applicator is inserted into the vaginal cavity and positioned in a manner consistent with delivery of the therapy to the prescribed plan. The radiation sources can then be introduced into the applicator and manipulated according to the plan. In the case of an isotope source, this may involve therapy being carried out within a bunker with use of an afterloader to comply with safety requirements. With an electronic x-ray source, for example that of the Axxent® system by Xoft, Inc. (Fremont, Calif.), the radiation source can be handled casually and therapy can be administered with comparatively less shielding. If desired and once the isodose characteristics of the source are understood, the applicator may be fitted with a radiation sensor or sensors, for example, of the MOSFET type, in a manner which does not interfere with administration of the prescribed treatment, and sensor feedback can be used to monitor, correct and/or verify proper dose delivery. Correction based on feedback can be applied, for example by manual or automated adjustment during or between fractions, in real time in the sense that the adjustments are made as the procedure progresses. Communication from the sensor(s) can be by conventional hard wiring or may be wireless. DESCRIPTION OF THE DRAWINGS In the following figures, cancerous regions of anatomy are shown shaded. These figures, together with the written description herein, describe the present invention. FIG. 1 depicts an applicator mold in coronal section through the vaginal space prior to filling the mold with filler material. FIG. 2 depicts the mold of FIG. 1 in coronal section after filler material has been placed within the mold and the mold expanded to occupy and conform to the vaginal cavity. FIG. 3 depicts in cross section, the filled applicator of FIG. 2 withdrawn from the vagina and after having been cored to accept a radiation source positioned at the end of the catheter. A radiation source at the end of a catheter is shown adjacent the entry to the cored hole in the applicator, said catheter and source being configured to emit radiation in a substantially radial and distal direction. FIG. 4 depicts in coronal section, the applicator, source and catheter positioned within the vaginal space to irradiate the tip of the cervix. FIG. 5 depicts the apparatus of FIG. 4 in sagittal section, positioned within the vaginal space to irradiate the tip of the cervix. FIG. 6 a depicts the applicator of FIG. 2 in section view corresponding to the sagittal section of FIG. 5, the applicator having been removed from the vaginal space after filling. FIG. 6 b depicts the applicator of FIG. 6 a in section, the applicator having been straightened to facilitate coring. Coring is shown in a manner in which the distal end of the core hole stops short of the distal extremity of the applicator. FIG. 6 c depicts the applicator of FIGS. 6 a and b after coring, having been recurved to the shape of FIG. 6 a to facilitate a proper fit within the vagina when reinserted. FIG. 7 depicts in medial sagittal section, the applicator of FIG. 6 c reinserted into the vaginal space, with a source and catheter positioned within the cored hole of the applicator. The source and catheter are configured to emit radiation laterally in order to irradiate a lesion on the anterior wall of the vagina. FIG. 8 a depicts, in section view, an applicator in which the coring passes through the full length of the applicator so as to communicate with the interior of the vaginal cavity. A source and catheter are shown positioned within the applicator. FIG. 8 b depicts a small section of the wall of the applicator mold showing grooves in the outer surface of the wall. DESCRIPTION OF PREFERRED EMBODIMENTS Applicators of this invention are formed in situ within the patient&#39;s vagina, preferably by insertion of a mold into the vaginal cavity, into which a filler material may be introduced so as to expand the mold, thus filling the vagina. After introduction, the filler may undergo a physical or chemical reaction to create a substantially rigid member customized to and in conformance with the patient&#39;s vaginal cavity, or to facilitate delivery of a radiotherapy prescription. Such a technique seeks to eliminate any air spaces between the applicator and vaginal cavity since it is known that such spaces detract from optimal application of radiation therapy. Filling of a mold in situ is preferable to filling the vagina directly with a filler material in that most fillers are of a form that is introduced at a relatively high temperature and cooled to set their shape, or they undergo a chemical reaction by which they are cured in the desired shape. With proper design, use of a mold can serve to insulate the tissues from undesirable thermal or chemical exposure. Thermal or chemical fillers, directly applied within the vagina, can cause discomfort to the patient, if not outright injury. FIG. 1 shows a mold 10 of an applicator 12 of the invention positioned in an uninflated condition within the vagina 14 of the patient. The mold comprises a hollow distal portion 16, i.e. a mold balloon, capable of being distended in response to application of internal pressure so as to fill the vaginal cavity of the patient. In one preferred embodiment, the mold balloon 16 distends so as to elastically conform generally to the shape of the vaginal cavity, expanding the soft-tissue cavity to a desired degree. In an alternate embodiment, the mold distal portion 16 preferably is flexible but substantially inelastic and, upon application of internal pressure, will expand to a preferred configuration, filling (expanding) and shaping the vaginal cavity, but at the same time bending or curving to follow the anatomy, and in this sense generally conforming to the vaginal anatomy. The applicator 12 preferably further comprises a relatively rigid and tubular proximal portion serving as a handle 18 for the therapist, facilitating manipulation of the applicator 12 within the vagina 14, including its removal and insertion in the course of treatment. The lumen 20 of the handle 18 communicates with the hollow interior of distal portion or mold balloon 16. The material of the mold balloon 16 is preferably thin and polymeric, silicone rubber being an example material for an elastic embodiment, and PET (polyethylene terephthalate) being an example for an inelastic embodiment. The material of the mold balloon 16 can be loaded with a radio-opaque material, for example barium sulfate or bismuth subcarbonate, to facilitate imaging the applicator in the patient&#39;s body by conventional radiographic means, and for treatment planning purposes. The balloon material is lightly doped with radio-opaque additive so as to reveal a “shadow” with external imaging but not so heavily so as to significantly attenuate radiation directed outwardly by a source in the applicator. The material of the proximal handle 18 may be the same material as the distal portion (e.g., integrally formed), but of different geometry so as to provide greater rigidity to the handle portion. Alternatively, the proximal handle 18 may be of a different material, for example polycarbonate to which the distal, distendable portion 16 may be bonded or mechanically fastened using conventional methods. The mold portion 16 and handle 18 may be made by conventional dipping or molding processes, and if not monolithic, may be joined by conventional secondary fastening methods. Such molding, bonding and fastening processes are well known to those skilled in the medical device arts. FIG. 2 shows the mold 10 positioned within vagina 14. The filler 22 has filled the mold of FIG. 2, distending the distal portion of the mold 16 until the mold 10 completely occupies and optionally expands the cavity of the vagina 14. The filler 22 preferably extends at least partially into the handle 18 providing further structural rigidity to the handle. The filler 22 can comprise a settable reactive liquid which can be injected into the mold 10 through the handle lumen 20. Sufficient filler can be injected into the mold 10 under controlled pressure to forcibly distend the mold distal portion 16 until it expands the vaginal cavity to the desired degree. Confirmation of complete filling can be visualized by x-ray or other convention imaging technique. The liquid filler, which may be initially two or more parts that are mixed, for example at injection, will react to form a relatively stable, solid form by any of several conventional methods. For example, a chemical reaction can be initiated, either by waiting an appropriate time, for example by use of polyurea component materials and/or by curing. Alternatively, a catalyst reaction can be initiated, for example by mixing the catalyst (for example, platinum in silicone) during injection and subjecting the injected material to ultraviolet light or to another initiator to start a curing reaction. Other liquid-to-solid chemical reactions known to those of skill in the art may also be used. Alternate chemistry involving foaming materials can also be used. Spontaneous reactions like that described above can be used, as can other reactions requiring an initiator can be used, such as moisture cured polyurethanes. The liquid injected into the mold can cure or set to a soft rubbery consistency, such as a silicone material, or it can be an expanding foam that becomes semi-rigid but somewhat flexible (generally rigid), or it could be another liquid which is injected and does not expand to any appreciable degree but simply is cured to a rigid or semirigid consistency by any of the techniques mentioned above or other known techniques. Note that if the mold balloon is inelastic, the point of complete filling will be easily determined by the pressure rise at injection. If the balloon is elastic, however, the filling can be done by injecting a predetermined volume or injecting the liquid up to a predetermined pressure, or simply injecting liquid until external imaging determines the desired fill has been reached. One class of potential filler materials exhibiting desirable properties suitable for applicator filler material is dental impression material. Physical reactions can also be used to create a solid applicator form. A material having relatively abrupt melting and freezing points with little hysteresis, which is viscous when warmed to temperatures slightly above body temperatures, can be injected into the mold 10, and allowed to cool in situ, forming a rigid applicator 12. An example of such a material would be a low-melting paraffin wax. A form produced in this manner can also be used as a pattern to generate a secondary shape by conventional molding or casting techniques. This secondary shape can then be used as the actual applicator during treatment. FIG. 3 shows a mold filled with a filler material as described above in connection with FIG. 2, having been allowed to assume its relatively rigid shape mimicking the vaginal cavity and then having been removed. The filler 22 is shown having been cored to form an interior lumen 28 within the filler 22. As shown, lumen 28 connects with the handle lumen 20 and extends from within handle 18 distally, but not completely through the filler 22. The purpose of this lumen is to accommodate the radiation source 24 and source catheter 26 for purposes of irradiating the interior of the vaginal cavity. Formation of the interior lumen 28 may be done in situ within the vagina by use of a lumen-forming obturator or core (not shown) held in proper position during the filler material injection and forming process. Alternatively, the cored lumen 28 may be formed by a secondary drilling or similar operation after the filling process (without an obturator positioned in place) has been completed, and after the filled mold 10 has been withdrawn from the vagina 14. If desired, the location of the lumen 28 may be selected to achieve radiation dose profiles which address the specific morbidity of the particular patient for whom the applicator 12 is intended. Alternately, if placed centrally within the filled shape of the applicator, the positioning of the radiation source 24 within the lumen 28 can be programmed to create a uniform dose at the exterior of the applicator 12 and at the surface of the vaginal cavity. Thus, a uniform prescribed dose at the prescription surface (the imaginary locus of all points at the prescription depth, hence an isodose surface) can be achieved. Different positioning of the lumen 28 and the source 24 can be used to create other radiation dose profiles as desired. As noted above, FIG. 3 also shows a radiation source 24 mounted at the tip of a source catheter 26. In this case, the catheter preferably includes a shield (not shown) assuring that the radiation is emitted from the source radially and distally only, not proximally. Without such shielding, the source 24 could be fashioned to emit substantially isotropically, or with different shielding configurations, in other desirable patterns. Shielding is more fully explained in copending U.S. patent application Ser. No. 11/471,277, and preferred radiation sources (miniature x-ray sources) are disclosed in U.S. Pat. No. 6,319,188. These disclosures are incorporated by reference herein in their entirety. Other sources may be used as well, including isotope sources, for example as available from Varian Medical Systems, Inc., Charlottesville, Va. FIG. 4 shows a completed (filled and cored) applicator 12 after insertion into the vagina 14. A source catheter 26 and source 24 are in position within the applicator lumen 28 to commence radiation treatment. As in FIG. 3, the source and catheter preferably are configured and/or shielded to direct the radiation distally and radially in order to treat cancer at the tip of the cervix, as indicated. If desired, a single source may be positioned sequentially through different positions and dwell times within the applicator lumen to create, in the aggregate, a prescribed absorbed dose pattern. Alternatively, the applicator may comprise multiple lumina for source positioning, either at the outer region of the filler at or near the mold balloon surface or more deeply within the body of the applicator. Single or multiple sources may be utilized to create the desired absorbed dose pattern. For clarity, multiple lumina within the applicator structure are not shown, but their incorporation by those of skill in the art will be readily apparent. FIG. 5 shows a sagittal view of the applicator 12, source 24 and catheter 26 of FIG. 4. Again, the radiation preferably is directed radially and distally as in FIG. 4. Note that the applicator is cored with the lumen 28, and the axis of the applicator 12 is curved, conforming to the patient&#39;s anatomy. If the applicator is filled without forming the lumen in situ, a preferred method of providing such a cored lumen having a curved shape is to use a filler 22 which may be formed as described previously, and which is resilient or semi-rigid, or capable of being temporarily reformed after extraction of the applicator 12 from the vagina 14, for example by application of mechanical force. Such reforming might be aided by application of heat as would be the case for a thermoplastic filler material. By such methods, the extracted applicator can be straightened for drilling or other coring operations to create the lumen 28 for the source 24 and the catheter 26. Once cored, the applicator 12 can be again reformed to its original curved shape, again inserted into the vagina 14, and radiation treatment commenced. Such a sequence of coring steps is shown in FIGS. 6 a through 6 c. FIG. 6 a shows the applicator 12 as removed from the vagina after the filler 22 has been placed in the mold 10. FIG. 6 b shows the applicator 12 straightened and cored, with the lumen 28 so formed extending almost through the entire length of the applicator 12. FIG. 6 c shows the applicator 12 once again in a curved configuration, ready for reinsertion into the vagina. FIG. 7 again shows an applicator 12 of the invention inserted in the vagina 14 of a patient similar to that shown in FIG. 5, but this patient presents with a lesion 30 of the anterior formix of the vagina. In FIG. 7, the source 24 and catheter 26 are configured with shielding to direct radiation generally in a radial direction, and the angular orientation is controlled such that the radiation treatment is brought to bear on the lesion in the anterior direction. Again, methods for radiation shielding in the manner described can be found in copending application Ser. No. 11/471,277, referenced and incorporated herein above. FIG. 8 a shows an alternate applicator embodiment 12 with the mold balloon 16 filled and the filler 22 cored generally according to the methods described above. In this embodiment, however, the cored lumen 28 in the filler 22 communicates through a port 32 with the outer surface 34 of the applicator 12 such that fluid may pass through the catheter lumen outside of the catheter 26 and into or out of the vaginal space. In FIG. 8 a, the port 32 opening into the vaginal space is shown as an axial extension of the catheter lumen 28. Alternatively, a port or ports 32 may connect the catheter lumen 28 and outer applicator surface 34 at any position or positions desired, to suit any purpose, as examples, for venting or draining the vaginal space outside the applicator, or for administering liquid agents to the inner surface of the vagina during or in connection with treatment. The handle portion 18 of the applicator 12 of this embodiment includes a conventional seal 36, for example an “0” ring seal, to prevent fluid leakage between the catheter 26 and the handle 18. The handle of this embodiment also comprises hub features including a fluid access port 38 leading to the catheter lumen 28 to complete the communication path from outside the body to the vaginal space when the applicator is positioned within the vagina. FIG. 8 a also shows a radiation sensor 40 affixed to the outer surface 34 of the mold balloon 16 (it could be on the inner surface). If the source 24 is characterized in a manner confirming stable operation and which relates its output at the position of the sensor 40 for each treatment position during therapy, the output of the sensor 40 may be used to verify treatment to plan, and/or to provide feedback for real-time control of the radiation delivered. Real time is intended to mean that radiation is adjusted in some way as the procedure continues, or prior to its conclusion, in response to measurement of radiation received at the sensor. Such sensor output can be communicated through a connecting wire 42 between the sensor 40 and a controller (not shown) which adjusts radiation source position, or in the case of an electronic x-ray source, can also control source output. Communication between the sensor and the controller can alternatively be by wireless methods. If desired, multiple sensors can be employed, and positioned on the surface of the applicator at the vaginal wall, or within or between elements of the applicator. Such an array of sensors can be interrogated sequentially during treatment, or monitored continuously. FIG. 8 b shows in partial section, a portion of the mold 10 of the applicator 12, specifically of the mold balloon 16. On the outer surface 34 of the mold balloon 16 is a pattern of grooves 44. These grooves may cover all or only a portion of the outer surface 34 of the mold balloon, and provide for liquid movement at the interface between the vaginal cavity surface and the applicator 12. Acting together with the port or ports 32, fluids may be administered or withdrawn from the applicator/vagina interface. Furthermore, if the pattern of grooves 44 extends proximally of the vaginal opening onto the handle surface, the grooves can function to vent any fluid trapped between in the applicator/vagina interface without need for ports 32. This invention has been described herein in considerable detail in order to instruct one of skill in the art how to practice the invention. It is to be understood, however, that the invention can also be carried out by other methods and apparatus without departing from the scope of the invention itself.

Summary: A customized gynecological brachytherapy applicator is formed by placing a molded balloon, which is secured to a handle, into a vaginal cavity and then filling the mold balloon with a settable liquid material. The material may be an expanding foam or any other form of curable material that becomes solid, rigid or pliable, when set by chemical action, temperature change, oxidation, a curing means such as a light, or other curing regime. The mold balloon can be elastic or inelastic, depending on the degree to which the applicator is to conform its external surfaces to the vaginal contours. One or more lumina are provided in the set filler material to receive a radiation source, with additional lumina optionally provided for purposes such as drainage or administration of therapeutic agents. The applicator may be withdrawn and reinserted into the vagina during treatment, and selected tissues adjacent to the applicator are irradiated in accordance with a radiation prescription.

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Summarize: Background The purpose of the Endangered Species Act of 1973 is to conserve endangered and threatened species and the ecosystems upon which they depend. The act defines “conservation” as the recovery of endangered and threatened species so that they no longer need the protective measures afforded by the Act. The act defines as endangered any species facing extinction throughout all or a significant portion of its range and defines as threatened any species likely to become endangered in the foreseeable future. The act requires the Secretary of the Interior to publish a list of species it determines are endangered or threatened in the Federal Register and specify any critical habitat of the species with in its range—habitat essential to a species’ conservation. Loss of habitat is often the principal cause of species decline. Additionally, the act establishes a process for federal agencies to consult with the Service about their activities that may affect listed species. Federal agencies must ensure that their activities, or any activities they fund, permit or license, do not jeopardize the continued existence of a listed species or result in the destruction or adverse modification of its critical habitat. There were 1,264 species in the United States listed as endangered or threatened as of September 30, 2004. The Service has responsibility for 1252 of these species. Thirty-two species have been removed from the list: 9 species as a result of recovery efforts, 9 because they have been declared extinct, and 14 species for other reasons, mostly because new information showed that listing was no longer warranted. The Service develops and implements recovery plans, among other things, to reverse the decline of each listed species and ensure its long-term survival. A recovery plan may include a variety of methods and procedures to recover listed species, such as protective measures to prevent extinction or further decline, habitat acquisition and restoration, and other on-the- ground activities for managing and monitoring endangered and threatened species. According to Service officials, it is their policy to issue a recovery plan within two and a half years of the species’ date of listing. The Service exempts species from the plan requirement when it is determined a plan will not promote their conservation. For example, the ivory-billed woodpecker is exempt because the Service thinks it is extirpated from the wild throughout its range. Recovery plans aim to identify the problems threatening the species and the actions needed to resolve them. The act directs the Service, to the maximum extent practicable, to incorporate into each recovery plan (1) a description of site-specific recovery tasks necessary to achieve the plan’s goal for the conservation and survival of the species; (2) objective measurable criteria that will result in a determination that the species can be removed from the list of endangered and threatened species (delisted); and (3) an implementation schedule that estimates the time and cost required to carry out the recovery tasks described in the recovery plan. Service employees, independent scientists, species experts, or a mix of these people can develop recovery plans. According to Service officials, as of September 2004, the Fish and Wildlife Service had 551 approved recovery plans covering more than 1025 species (more than 80 percent of all listed species). The act also requires the Service to report biennially to certain Congressional committees on efforts to develop and implement recovery plans, and on the status of listed species for which plans have been developed. The Service implements this requirement through its biennial Recovery Report to Congress. Additionally, the act requires the Service to submit an annual report to the Congress on federal expenditures for the conservation of endangered or threatened species, as well as expenditures by states receiving federal financial assistance for such conservation activities. As part of its efforts to compile data for this report, the Service collects data on recovery fund expenditures on a species-specific basis, although these data have not been reported separately in published expenditure reports. With regard to Service funds, the Endangered Species program is a small portion of the Service’s overall budget ($132 million of $1.9 billion in fiscal year 2003). Of this amount, about one-half is devoted to the recovery program, $65 million (see fig. 2). This is similar to previous fiscal years. The funds spent on the recovery program, however, are only a portion of the total money spent to recover species. Some of the Service’s other programs, including refuges, contribute funds and staff to species recovery. In addition, according to the Service, other federal and non-federal entities contribute substantial funds to species recovery. In addition to the Service’s Endangered Species Program expenditures to recover species, other programs in the Service as well as other federal and state agencies spend substantial funds on endangered species activities, including land acquisition (see table 1). Congress amended the Endangered Species Act in 1979 to require the Secretaries of the Interior and Commerce to establish, and publish in the Federal Register, agency guidelines that include a priority system for developing and implementing recovery plans. The Service adopted recovery priority guidelines in 1980 and amended them in 1983. The guidelines consist of two parts: Species are assigned a priority ranking between 1 and 18 on the basis of (in descending order of importance) (1) the degree of threat confronting the species, (2) recovery potential (the likelihood for successfully recovering the species), and (3) taxonomy (genetic distinctiveness). (See table 2.) Additionally, a “c” is added to the ranking if there is conflict with economic activities, like development; this gives the species priority over other species with the same ranking but without a “c”. Thus, the highest possible priority ranking is a “1c”. The Service sometimes changes a species’ priority ranking when warranted by a change in the species’ situation. The second part of the priority system ranks the recovery tasks within each recovery plan. Each task is assigned a priority number from 1 to 3, with 1 being the highest. A priority 1 task is “an action that must be taken to prevent extinction or to prevent the species from declining irreversibly.” A priority 2 task is “an action that must be taken to prevent a significant decline in species population/habitat quality or some other significant negative impact short of extinction”, and a priority 3 task is “all other actions necessary to provide for full recovery of the species.” The recovery guidelines emphasize that they should be used only as a guide, not as an inflexible framework for determining funding allocations. Within the Service, responsibility for implementing the act is divided among its three administrative levels: headquarters, regions and field offices. Headquarters officials develop policy and guidance and allocate funding to the regions. Regional directors in the seven regions (shown in figure 3) make most decisions on how to spend endangered species program funds and are responsible for managing their field offices’ program activities. Field offices are responsible for implementing program activities and setting priorities for projects they will undertake. The Service Spends a Significant Portion of Recovery Funds on the Highest Priority Species The Fish and Wildlife Service spent its recovery funds in a manner generally consistent with species priority in fiscal years 2000 through 2003. From fiscal years 2000 to 2003, the Service spent 44 percent of its recovery funds attributable to individual species on those species with the highest priority, the 415 species ranked 1 through 3 on the 18-point priority ranking scale (see fig. 4). However, 25 of these species received no recovery funding at all during fiscal years 2000 through 2003. Additionally, two species with low priority rankings, the bald eagle (with a priority ranking of 14c) and the Canada lynx (with a ranking of 15), received substantial recovery funding during fiscal years 2000-2003. One reason the Service spent 44 percent of its recovery funds attributable to individual species on the highest priority species is that this group accounts for a significant portion of all listed species—one-third (see fig. 5). Similarly, the Service spent almost all (94 percent) of its attributable recovery funds on species ranked 1 through 9 on the 18-point scale, which account for 92 percent of all listed species. As shown in figure 6, analysis of average spending on a per species basis also reveals that more expenditures are made on higher priority species. Additionally, the analysis shows the emphasis the Service placed on species with a high degree of recoverability. The relatively large amount of funding spent on species with low priority rankings (13 through 15) is greatly influenced by spending on the bald eagle (with a priority ranking of 14c) and the Canada lynx (with a ranking of 15). The bald eagle is nearing delisting and the funding was spent on delisting activities. The Canada lynx was embroiled in controversy that required recovery staff to respond to litigation. When spending on these two species is removed, the average amount spent on species in this priority group is significantly lower. In addition to species priority ranking, another obvious measure of priority is whether a species is endangered or threatened. Over three-quarters (78 percent) of species protected under the act are listed as endangered, and most of these have high priority rankings (see fig. 7). We analyzed spending by species status (endangered or threatened) and found that the Service spent a majority (64 percent) of its recovery funds on endangered species during fiscal years 2000 through 2003. Finally, we analyzed spending by the three taxonomic classifications included in the Service’s recovery priority guidelines—monotypic genus, species, and subspecies. As shown in figure 8, an analysis of average spending on a per species basis reveals that more expenditures are made on listed entities classified as monotypic genus. A species that is a monotypic genus is the only remaining species representing the entire genus. The Service Considers Factors Besides Species Priority When Allocating Recovery Funds but Does Not Assess the Results of Its Funding Decisions When Service officials allocate recovery funds, they base these decisions to a significant extent on factors other than a species’ priority ranking. At the headquarters level, a formula that accounts for each region’s workload, but not species’ priority rankings, determines how recovery funds are allocated. Each regional office allocates recovery funds to their field offices differently, but in no case is priority ranking the driving factor. Instead, regional officials focus primarily on partnership opportunities, though regional officials told us they do try to provide funds to species that have a high degree of threat. Although field office staff we spoke with use priority rankings, they also emphasized the importance of having flexibility to allocate funds to develop partnerships. The Service does not know the extent to which these disparate allocation systems yield results consistent with the Service’s priority guidelines because the Service does not have a process to routinely measure the extent to which it is spending its recovery funds on higher priority species. Headquarters Allocates Funds to the Regions Based on Workload In making allocation decisions, headquarters does not consider a species’ priority ranking or any of the factors that go into determining priority rankings. Instead, it allocates recovery funds to its seven regions based primarily on a formula that estimates each region’s workload. The formula estimates the recovery workload for each region by assigning each species a score of between 2 and 7 points, based on the type of species and its habitat needs. Higher points are assigned to those species whose recovery requires higher levels of funding or effort—factors that are not clearly related to a species’ priority ranking. For example, animals are assigned 2 points while plants are assigned 1. Species that occupy habitats larger than 1 million acres or are migratory or aquatic are assigned 5 points whereas species that occupy less than 1,000 acres are assigned 1 point. Recovery funds are then allocated to the regions based on the number of species occurring in each region and the points assigned to those species. Additionally, headquarters uses a workload-based formula to allocate funds to regions to develop recovery plans. Funds are allocated to each region based on the number of species that it is responsible for that have not been exempted from the plan requirement and that have been listed for 4 years or less. If after 4 years there is still no plan, then the region no longer receives recovery-planning money for that species, though the region is still responsible for completing that species’ recovery plan. Service officials in headquarters told us that they use an allocation system based on workload rather than the priority guidelines for a number of reasons. First, this system provides relatively stable funding to each region from year to year. In contrast, priority rankings can change over time, which would add an element of unpredictability to the annual allocations. Stability is important, according to Service officials, because most of a region’s recovery budget supports staff salaries for recovery biologists. These biologists work on a wide variety of recovery activities including helping to develop recovery plans, conducting as well as coordinating on- the-ground actions to implement recovery plans, conducting periodic species status reviews, developing recovery partnerships, and litigation support. Second, although priority rankings indicate which species are higher priority, they do not reflect how much money a species needs. Service officials pointed out that higher priority species are not necessarily more costly to recover than lower priority species. Lastly, Service officials told us that a system based on workload is more objective, and they expressed concern that the subjective nature of priority rankings could create conflict between the regions if allocations were based on these rankings. While Service officials at headquarters told us that recovery funds should be spent according to priority rankings, they believe those decisions should be made at the regional level. Almost all of the regional officials we talked to agreed that the allocation system used by headquarters works well and is fair and equitable, although some of them suggested changes. For example, some regional and field office officials noted that a species’ priority ranking, particularly its degree of threat, could be included, along with the existing workload factors, in headquarters’ formula for allocating recovery funds. Regional Offices Focus on Opportunities for Partnerships When Making Funding Decisions While each region allocates recovery funds to its field offices differently, we found that the most important consideration among the regions is to maintain and develop recovery partnerships, either by funding long- standing arrangements to work with partners to recover specific species or by taking advantage of opportunities to develop new partnerships. For example, officials at the Southwest region told us that for the last 10 years the region has allocated its discretionary recovery funds primarily to four species for which it has long-standing partnerships with other entities—the Kemp’s Ridley sea turtle, the whooping crane, the Mexican wolf and the Attwater’s prairie chicken. The financial support from long-term partners, in concert with expenditures from the Service, provides a stable funding source for recovery projects from year to year, helping to create viable recovery programs for these four species. For example, the Kemp’s Ridley sea turtle population has increased from a low of 270 females to several thousand females in the course of this long-term partnership. Service officials told us that it is important to maintain their yearly contributions to long-standing partnerships, regardless of the species’ priority ranking, because the funds these partners contribute are critical to species’ recovery and the partners could lose interest without the Service’s contributions. Officials at all levels of the Service reported to us that they have insufficient recovery funds. Although it is difficult to develop an accurate estimate of the full cost to recover all listed species (and it is unlikely that some species will ever be recovered), we analyzed the cost data contained in 120 recovery plans covering an estimated 189 listed species. Based on the Service’s estimated recovery costs in these plans, we found that it would cost approximately $98 million dollars to fully fund these plans—plans that cover just 15 percent of listed species—for a single year. This amount is well above the $65 million the Service allocated in fiscal year 2003 to develop and implement recovery plans and does not account for the recovery needs of the remaining 1000 listed species. Even implementing only the highest priority recovery plan tasks for those 120 plans—recovery plan tasks “necessary to avoid extinction,” would cost approximately $57 million, nearly 90 percent of the Services’ total recovery budget in fiscal year 2003. Consequently, the Service is dependent on monetary contributions from partners to facilitate species recovery. Regional officials not only fund long-standing partnerships, but look for opportunities to develop new ones as well. Service officials expressed concern that if they were confined to allocating funds strictly by the priority system, they could alienate potential recovery partners. For example, some regional officials pointed out that land acquisition can take many years, so if willing sellers present themselves, the region will take advantage of that opportunity by allocating recovery funds to acquire those lands even if they do not benefit a species of the highest priority. In another example, officials in a field office in the Pacific region told us they were able to leverage its $20,000 investment into a $60,000 project by developing an agreement with the U.S. Forest Service to jointly fund a study to identify how the California red-legged frog was using suitable habitat. Fish and Wildlife Service officials in the Pacific region also leverage funds with non- federal partners. In 2002, a $10,000 investment in desert tortoise monitoring from the Fish and Wildlife Service was matched by $16,540 from Clark County, Nevada and $5,000 from the Arizona Game and Fish Department. Almost all of the Service officials we talked with stressed the importance of having the flexibility to develop partnerships for recovery, particularly to leverage the Service’s scarce recovery funds. Finding partners and other sources of funds to implement recovery actions is also strongly emphasized in the Service’s course on recovery implementation, which is offered at the National Conservation Training Center in West Virginia and other locations around the country. While a species’ priority ranking is not a primary factor for determining how regions distribute recovery funds, regions do consider priority rankings when making recovery allocations. The two regions responsible for the most species, the Southeast region and the Pacific region, specifically incorporate the priority system into their funding allocations. In the Southeast, field offices and other divisions of the Service, like Refuges, submit proposals to obtain recovery funding to implement recovery plan tasks. Once the regional office receives all the proposals, officials determine which ones to fund that year. In doing so, they consider a number of factors, including the species’ priority ranking. Similarly, the Pacific regional office reserves a portion of the recovery funds it receives and uses them to fund proposals submitted by its field offices to implement recovery plan tasks. One of the factors the region considers when determining which proposals to fund is the species’ priority ranking. Most of the other regions we talked to told us that they consider some aspects of the priority system when making funding decisions, particularly the species’ degree of threat, although they do not directly consider a species’ priority ranking. Sometimes regions will also target funds to lower--priority species if they are nearing recovery. For example, the bald eagle ranked 20th among those species with the highest recovery expenditures from fiscal year 2000 to fiscal year 2003, despite having a priority ranking of 14c. A Service official attributed most of these expenditures to delisting activities for the bald eagle. Many Service officials pointed out that the priority system does not contain a mechanism for funding species that are nearing recovery. Because a species’ priority will decrease as its threats are alleviated and it moves closer to recovery, the priority system would dictate that other more imperiled species be funded before those that are close to delisting. Consequently, species close to recovery might never be delisted because funds would not be allocated to complete the tasks required for delisting. Service officials told us they need flexibility to provide funds that will help get species off the list. Headquarters officials have also recognized this issue and, beginning in fiscal year 2004, created a special fund that directs funding to species close to delisting (as well as those close to extinction) in its “Showing Success, Preventing Extinction” initiative. Field Offices We Talked to Use the Priority System When Making Funding Allocations In the field offices we contacted, we found that species’ priority rankings play an important role in recovery allocations. Service personnel in four of the ten field offices we spoke with told us that a species’ priority ranking is one of the key factors they use to allocate recovery funds. For example, in the Pacific Islands field office, which is responsible for the recovery of over 300 species, officials use the recovery priority system as a “first step,” then overlay other factors, like opportunities to leverage funding. Staff in five of the remaining six offices we spoke with told us that while they do not specifically use the priority system when making recovery allocations, they do consider a species’ degree of threat. Staff in the last field office said they did not use the priority system because most of their funds were spent according to direction provided by the region. Despite their use of the priority system, most of the field office staff we contacted also stressed the importance of having the flexibility to allocate funds to take advantage of unique opportunities when they arise. For example, officials in a field office in California told us they took advantage of an opportunity to leverage recovery funding for the California red-legged frog. A population of this frog was recently discovered in Calaveras County, site of Mark Twain’s famous story The Celebrated Jumping Frog of Calaveras County, which featured the California red-legged frog. The landowner where the population was discovered was eager to work with the Service to build a stock pond to provide habitat for the red-legged frog and eradicate bullfrogs (red-legged frog competitors). The discovery of the frog population was momentous because the species is important to local lore, and a population of the frog had not been found in Calaveras County since the late 1800s (see fig. 9). Even though the field office has 65 species with higher priority rankings than the red-legged frog, officials decided to address this recovery opportunity because of the frog’s importance to the local community. Other unique events also require funding flexibility. In a Utah field office last year, for example, a road expansion threatened the existence of the clay phacelia, an endangered plant. The field office staff responded to this threat by working with partners to collect seeds for future propagation. The Service Does Not Assess and Report on Its Recovery Fund Expenditures The Service does not know the extent to which recovery fund expenditures are consistent with its priority guidelines. All of the Service’s organizational levels participate in funding decisions, often relying on factors other than species priority. Although our analysis shows that the Service generally spent its recovery resources on higher priority species during fiscal years 2000 through 2003, we found that the Service has no process to routinely measure the extent to which it is spending its recovery funds on higher priority species. Without this information, the Service cannot ensure that it is spending its recovery funds on such species, and in cases where it is not, determine whether the funding decisions are appropriate. This is especially problematic as circumstances change—for example, when species are added to the list or priority rankings change for already-listed species. Although the Service is required to report all federal and some state expenditures on listed species, it does not separately report how it spent its recovery funds by species. This lack of separate reporting can make it difficult for Congress and others to determine whether the Service is focusing its recovery resources on the highest-priority species. For example, the species that received the greatest total federal and state expenditures in fiscal year 2003 are substantially different from those we identified as having received the greatest portion of the Service’s recovery fund expenditures. Of the 47 species that the Service reported as having received the greatest total expenditures in fiscal year 2003, the Service has joint or lead responsibility for 20 of them. The list of 20 species is radically different from the list that we identified as having received the greatest portion of the Service’s recovery fund expenditures (see table 3). In the case of the Southwestern willow flycatcher, the Service reported that more funds were expended on the flycatcher in fiscal year 2003 than for all but three other species for which the Service has lead responsibility. However, the information the Service provided to us shows that it spent relatively few recovery funds on the Southwestern willow flycatcher in fiscal year 2003—it ranked 84th in the Service’s recovery expenditures. Total reported expenditures and Service recovery fund expenditures differ substantially because the Service’s recovery priority guidelines do not apply to most of the reported funds—those funds provided by other federal agencies and some funds reported by state agencies. The Service has little control over how other organizations spend their funds. The reported expenditures also include Service expenditures in addition to recovery funds, such as expenditures on listing and consultation, which are also not subject to the Service’s recovery guidelines. In fact, in many instances, the Service does not have discretion over which species should receive these funds. For example, the Service spends consultation funds largely based on projects submitted to it by other federal agencies. Not unexpectedly, the list of 20 species receiving the greatest portion of the Service’s recovery fund expenditures in fiscal year 2003 is also different from the list of species receiving the greatest portion of total federal and state expenditures in fiscal year 2003 (see table 4). For example, the California condor and the Western population of the gray wolf ranked first and third, respectively in recovery fund expenditures but are ranked 25th and 29th, respectively in overall federal and state expenditures. Without a process to measure the extent to which it is spending its recovery funds on the highest-priority species, the Service lacks valuable information that would aid it in making management decisions. For example, while maintaining partnerships to fund certain species may be reasonable, many of these partnerships have been in place for many years, and changes to the species’ status or threat level, as well as changes to the threat level of other species and the addition of newly listed species, could have occurred in that time. As such, perhaps the reasons for creating some of these partnerships may have been superseded by other needs and it may no longer be appropriate for particular species to garner so much funding from the region. Officials in the Southwest region, for instance, told us that most of the region’s discretionary recovery funds are spent on four species (Kemp’s Ridley sea turtle, Whooping crane, Mexican wolf, and Attwater’s prairie chicken). These officials stated that they did not know these species’ recovery priority rankings until after we scheduled a meeting with them, although they did believe the species to be highly ranked. While these four species all have high priority rankings—2c, 2c, 3 and 3c, respectively— the region has lead responsibility for about 80 other species with a priority ranking between 1 and 3. Although many of these species also received funding during fiscal years 2000-2003, more than one-quarter (20 species) had no Service recovery fund expenditures attributable to them. Conclusions The Service faces a very difficult task—recovering more than 1,200 endangered and threatened species to the point that they no longer need the protection of the Endangered Species Act. Many of these species face grave threats and have been imperiled for years. There are few easy solutions. Like many other federal agencies, the Service has limited funds with which to address these challenges. Fortunately, many other organizations contribute resources to help species. The Service maintains that its ability to be flexible in allocating its scarce recovery resources is the key to maximizing those contributions from other organizations. We agree that exercising flexibility in allocating recovery funds under its priority guidelines is important, but this needs to occur within the bounds of a systematic and transparent process. The Service, however, does not have such a process. While the Service acknowledges that it strays from its priority guidelines, it does not routinely analyze its allocation decisions to determine whether it is focusing on the highest priority species and, if not, why. Such an analysis is important to ensure that the Service continues to spend its recovery funds on the highest priority species over the long term. Without this information, the Service cannot show Congress or the public the extent that it is focusing its resources on the highest priority species, or explain, in cases where it is not, that its resource decisions are still appropriate. To this end, we believe the Service’s priority guidelines provide it with the means to create a systematic and transparent allocation process while still allowing it needed flexibility. Because the Service already collects data, on a species by species basis, on how it spends its recovery funds, it would be a simple task to measure the extent to which it is spending its recovery funds on high-priority species. It could then make this information publicly available, thus providing the Congress and the public a yardstick with which to judge the efficacy of the Service’s resource allocation decisions. Recommendations for Executive Action To help ensure that the Service allocates recovery resources consistent with the priority guidelines over the long term and in a transparent fashion, we recommend that the Secretary of the Interior require the Service to take the following two actions: (1) periodically assess the extent to which it is following its recovery priority guidelines and identify how factors other than those in the guidelines are affecting its funding allocation decisions, and (2) report this information publicly, for example, in its biennial recovery report to Congress. Agency Comments and Our Evaluation We received written comments on a draft of this report from the Department of the Interior. In general, the Department agreed with our findings and recommendations but believes that we underestimated the extent to which the Service’s funding decisions are consistent with its recovery priority guidelines. Because we found that the Service spent its recovery funds in a manner generally consistent with species priority, we do not believe this is a significant issue. See appendix II for the Department’s letter and our response to it. Additionally, the Department provided technical comments that we have incorporated into the report, as appropriate. As agreed with your office, unless you publicly announce the contents of this report earlier, we plan no further distribution until 30 days from the report date. At the time, we will send copies of this report to the Secretary of the Interior and other interested parties. We also will make copies available to others upon request. In addition, the report will be available at no charge on the GAO web site at http://www.gao.gov. If you or your staff have any questions, please call me at (202) 512-3841. Key contributors to this report are listed in appendix III. Scope and Methodology In response to a request from the Chairman, House Committee on Resources, we (1) analyzed how the U.S. Fish and Wildlife Service’s allocation of recovery funds compares with its recovery priority guidelines and (2) determined what factors influence the Service’s recovery funding allocation decisions. As agreed with the Chairman’s staff, we evaluated only those funds specifically spent by the Service to implement its recovery program. To address our first objective, we requested recovery expenditure data, on a per species basis, from each of the Service’s seven regions for fiscal years 2000-2003. Because the Service spends most of its recovery funds on salaries that are not allocated on a per species basis, we asked officials in each region to attribute salaries to specific species to the best of their abilities. To assess the reliability of these data, we compared the total estimated expenditures we received from each region for each year to budget documentation provided by headquarters officials, the Department of the Interior’s Budget for fiscal years 2000-2003, and House and Senate committee reports for Department of the Interior appropriations for fiscal years 2000-2003. We also asked the regional officials who provided these data a series of data reliability questions covering issues such as data entry, access, quality control procedures, and the accuracy and completeness of the data, as well as any limitations of the data. All responded that the data were generally accurate, and all but one performed some form of data review to ensure its accuracy. Additionally, officials from all but one region noted, as a limitation to the data, that it is sometimes difficult to link expenditures on activities to specific species. We determined that the expenditure data received from each of the Service’s seven regions were sufficiently reliable for the purposes of this report. We also obtained from the Service data on each species’ priority number for fiscal years 2000 through 2003, as well as other information about each species, such as whether it is threatened or endangered and whether it has a recovery plan. We did not make a judgment about the adequacy or accuracy of the Service’s recovery priority system. The Service also provided us with information on the estimated costs to implement approximately 120 recovery plans. We assessed the reliability of these data by (1) electronically testing required data elements, (2) reviewing existing information about the data and the system that produced them, and (3) interviewing agency officials knowledgeable about the data. In addition, we compared the data set sent to us by the Service to the Service’s publicly available (online) Threatened and Endangered Species System (TESS), which contains data on listed species similar to that we received from the Service. When we identified any difference between these two data sets, we independently corroborated, to the extent possible, which data set was correct by obtaining documentary evidence, either from the Federal Register or the appropriate recovery plan. When appropriate according to this documentary evidence, we made changes to the data sent to us by the Service. For example, the spineless hedgehog cactus was listed in the data set sent to us by the Service but was not found when we compared it to online TESS. We checked the Federal Register and found that this species was removed from the endangered species list in 1993, so we removed it from the data set sent to us by the Service because our time frame of interest is 2000 through 2003. In another instance, the data set sent to us by the Service contained the Berkeley kangaroo rat, but this species was not in TESS. We checked the recovery plan and found that this is a “species of concern,” not an endangered or threatened species. The status field in the data sent to us by the Service was blank, so we re-coded it as a species of concern and then removed it from the data set because species of concern are not part of our review. We also made changes to records that contained errors. For example, the green sea turtle has two different populations. However, the Fish and Wildlife Service reported the total recovery expenditures for these two populations together. When expenditures were merged with species lists, this expenditure total was shown twice. To address that error we removed one expenditure total. All of these types of changes, 5 records with factual errors (or 0.4 percent of the records) and 9 with missing information (0.7 percent of the records), were reviewed and agreed to by all team analysts and supervisors. We also found and removed 14 duplicates and 27 records that were outside our scope (e.g., outside our date range or species managed by the National Marine Fisheries Service, not the Fish and Wildlife Service). On the basis of all of this work, we determined that the data on species and recovery plans we received from Fish and Wildlife Service were sufficiently reliable for the purposes of this report. We then compared the expenditures on each species with the species’ priority ranking for fiscal year 2000 through 2003. We grouped together species with similar rankings to deemphasize minor differences in species’ rankings. Grouping species this way had the effect of eliminating the taxonomic distinction among species found in the recovery priority guidelines. Table 5 shows the groupings. We also assumed that the average cost to implement recovery plans in each group was the same. We made this assumption explicitly because the cost to implement individual recovery plans can vary substantially among species. For example, we analyzed the cost to implement 120 recovery plans (the only plans with these data available electronically) covering an estimated 189 species (or 15 percent of listed species) and found that some plans are very costly—$107,516,000—and some are not—$18,000. However, many plans fall between these two extremes, costing between $1 million and $6 million. We discussed this assumption with the Service, and they agreed to its reasonableness. The number of species in each priority group varied by year (see table 6). In order to analyze overall average spending on a per species bases, we calculated weighted average expenditures per species by priority ranking. To do this we weighted the average expenditure per species for a specific priority group and fiscal year by the proportion: (Number of species in a particular priority group and fiscal year)/ (Number of species in same priority group over all fiscal years). In addressing our second objective, to determine what factors influence the Service’s recovery funding allocation decisions, we interviewed managers and recovery biologists in the Service’s recovery division in headquarters, all seven regions and a nonprobability sample of 10 field offices. We selected at least one field office from each region and selected a second field office from the two regions that collectively have lead responsibility for more than 50 percent of the endangered and threatened species in the United States. Within each region, we selected field offices that have lead responsibility for a high number of species relative to other field offices in that region. The region responsible for the largest number of species, the Pacific region, is operated as two divisions, and we selected a field office from each division. The field office locations in our nonprobability sample were: Hawaii (Pacific Region) Sacramento, California (Pacific Region) Arizona (Southwest Region) Columbia, Missouri (Great Lakes Region) Cookeville, Tennessee (Southeast Region) Vero Beach, Florida (Southeast Region) Virginia (Northeast Region) Utah (Mountain-Prairie Region) Anchorage, Alaska (Alaska Region) Fairbanks, Alaska (Alaska Region) Through our interviews we obtained information on how recovery funds are allocated, the role of the recovery priority system, and suggested improvements to the recovery priority system. We compared the answers we received in these interviews to documents or expenditure data provided by the Service, to the extent this corroborating evidence was available. In addressing both objectives, we reviewed publicly available documents and other information obtained from the Fish and Wildlife Service’s Website. We also reviewed articles in academic and scientific literature related to recovery planning and recovery prioritization, including an extensive study of recovery plans conducted by the Society for Conservation Biology and funded by the Fish and Wildlife Service. We performed our work from February 2004 to January 2005, in accordance with generally accepted government auditing standards. Comments from the Department of the Interior The following are GAO’s comments on the Department of the Interior’s letter dated March 11, 2005. GAO Comments 1. We agree that some of the recovery funds included in our analysis of how recovery fund allocations compare with the Service’s recovery guidelines include funds for which Congress has provided direction that they be spent on particular projects or species. However, we do not believe that by including these funds we have underestimated the degree to which the Service’s funding decisions are consistent with its recovery priority guidelines. First, we found that the Service spent its recovery funds in a manner generally consistent with species priority. Second, we analyzed a list, provided to us by the Service, of congressionally directed funds and associated projects for fiscal years 2000 through 2003. We compared this list with the priority rankings of the species associated with the projects in a way similar to how we compared species’ expenditures and priority rankings in our report. We found that the list of congressionally directed funds resulted in a spending pattern similar to what we identified when we compared species’ expenditures and priority rankings in our report. Thus, by including these funds in our analysis of how recovery funds allocations compare with the Service’s recovery guidelines, we do not believe that we have underestimated the degree to which the Service’s funding decisions are consistent with its recovery priority guidelines. 2. We agree that the Endangered Species Act does not require it to report separately on how it spent its recovery funds by species. However, reporting this information could be part of an effective strategy to help ensure that the Service allocates recovery resources consistent with the priority guidelines over the long term and in a transparent fashion. 3. In our report, we use the term “imperiled” instead of “threatened” to avoid confusion with the distinction the act makes between “threatened species” and “endangered species.” We agree that the act does not state that the purpose for requiring the Service to establish guidelines for prioritizing the development and implementation of recovery plans was to address concerns that recovery funds were not being directed at the most imperiled species. We have modified the report accordingly. However, we disagree with the Department’s contention that its recovery priority guidelines do not provide that funding should be allocated preferentially to species with the highest priority ranking as depicted in table 2 of our report. The Department relies on a table in the guidelines that is virtually identical to table 2 in our report to describe its priority system. Section 4(h)(4) of the act specifically directs the Service to establish guidelines that shall include “a system for developing and implementing, on a priority basis, recovery plans under subsection (f) of this section.” Further, the guidelines state that “the species with the highest degree of threat have the highest priority for preparing and implementing recovery plans.” In addition, the guidelines state that they are to “aid in determining how to make the most appropriate use of resources available to implement the act.” The Department also contends that allocating funding preferentially to species with the highest priority ranking is contrary to section 4(f)(1)(A) of the act. This provision, which was added in a 1982 amendment to the act, states that recovery plans shall, to the maximum extent practicable, give priority to species most likely to benefit from such plans, particularly those that are, or may be, in conflict with construction or other development projects, or other forms of economic activity. The guidelines specifically state that the priority system established by the guidelines “is intended to satisfy the requirements of the amended Act.” Accordingly, the guidelines include likelihood to benefit from recovery plans and conflict as factors. We agree with the Department that focusing on opportunities for partnerships where multiple parties will work to the benefit of the species is consistent with section 4(f)(1)(A) of the act. In fact, we conclude in our report that the Service’s ability to be flexible in allocating its scarce recovery resources is the key to maximizing contributions from other organizations. However, we believe that this flexibility needs to occur within the bounds of a systematic and transparent process and make recommendations to this effect. GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the individual named above, Charles Egan, Jaelith Hall- Rivera, Barry T. Hill, Summer Pachman, Paula Bonin, Judy Pagano, and Cynthia Norris made key contributions to this report.

Summary: Currently there are more than 1,260 species listed as endangered or threatened under the Endangered Species Act of 1973. While few species have gone extinct since 1973, only 9 have been "recovered" or removed from the list because they no longer need the act's protection. This has raised questions about how the U.S. Fish and Wildlife Service (Service) allocates its recovery funds. Proponents of the act believe that the Service's recovery funds are only a small fraction of what is needed to make greater recovery progress. The act and agency guidelines require the Service to prioritize species to guide recovery fund allocation. In fiscal year 2000 through 2003, the Service spent $127 million dollars in recovery funds attributable to individual species. In this report, GAO analyzed (1) the extent to which the Service's allocation of recovery funds compares with its recovery priority guidelines and (2) what factors influence the Service's recovery allocation decisions. The Service spent its recovery funds in a manner generally consistent with species priority in fiscal years 2000 through 2003, spending almost half (44 percent) of the $127 million on the highest priority species (see figure below). Species in the next two highest priority groups received almost all of the remaining recovery funds (51 percent). Species in the three lowest priority groups received very little funding (6 percent). Most listed species (92 percent) are in the top three priority groups. When Service officials allocate recovery funds, they base their decisions to a significant extent on factors other than a species' priority ranking. At the headquarters level, a formula that focuses on each region's workload determines how recovery funds are allocated to regional offices. Each regional office allocates its recovery funds to their field offices differently, but in no case is priority ranking the driving factor. Instead, regional officials focus primarily on opportunities for partnerships, though they told us that they also focus on species facing the gravest threats. Field office staff we spoke with emphasized the importance of pursuing funding partnerships in order to maximize their scarce recovery funds. The Service does not know the effect of these disparate allocation systems because it does not have a process to routinely measure the extent to which it is spending its recovery funds on higher priority species. While we found that for fiscal years 2000 through 2003 the Service spent a majority of its recovery funds on high priority species, without periodically assessing its funding decisions, the Service cannot ensure that it spends its recovery funds on the species that are of the greatest priority and, in cases where it does not, determine whether its funding decisions are appropriate.

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Write a title and summarize: SECTION 1. DEFINITION. For the purposes of this Act, the term ``Cape Fox Corporation'' means the Cape Fox Corporation, an Alaska Native village corporation organized pursuant to the Alaska Native Claims Settlement Act (43 U.S.C. 1601 and following) by the native village of Kassan. SEC. 2. CONVEYANCE AND ELIGIBILITY OF CERTAIN LANDS TO CAPE FOX CORPORATION. (a) Lands Within 6-Mile Radius of Ketchikan.--The following described lands located near Ketchikan, Alaska, shall be treated as lands selected under section 16 of the Alaska Native Claims Settlement Act (43 U.S.C. 1615) by the Cape Fox Corporation, and the Secretary of the Interior shall, within 90 days after the date of the enactment of this Act and subject to valid existing rights, transfer such lands under the terms and conditions of the Alaska Native Claims Settlement Act, notwithstanding section 22(l) of that Act (43 U.S.C. 1621(l)), to the Cape Fox Corporation: T. 74 S., R. 91 E. C.R.M. Section 21 SW\1/4\SW\1/4\. Section 28 W\1/2\W\1/2\. (b) Expansion of Land Selection Area.--In addition to lands made available for selection under the Alaska Native Claims Settlement Act (43 U.S.C. 1601 et seq.), the following described lands, other than any of such lands conveyed to or selected by the State of Alaska under Public Law 85-508 (commonly known as the ``Alaska Statehood Act'', approved July 7, 1958 (72 Stat. 339, 48 U.S.C. note prec. 21), shall be eligible for selection by the Cape Fox Corporation for the 24-month period beginning on the date of the enactment of this Act: T. 73 S., R. 90 E. C.R.M. Sections 13, 14, 23, 24, 25, 26, 27, 34, 35, 36. T. 73 S., R. 91 E. C.R.M. Sections 19, 20, 29, 30, 31, 32. (c) Directed Conveyance of a 1,040-Acre Parcel.--The following described lands located near Ketchikan, Alaska, other than any of such lands conveyed to or selected by the State of Alaska under Public Law 85-508 (commonly known as the ``Alaska Statehood Act'', approved July 7, 1958 (72 Stat. 339, 48 U.S.C. note prec. 21), shall be treated as lands selected under section 16 of the Alaska Native Claims Settlement Act (43 U.S.C. 1615) by the Cape Fox Corporation, an Alaska Native village corporation, and the Secretary of the Interior shall, within 90 days after the date of the enactment of this Act and subject to valid existing rights, transfer such lands under the terms and conditions of the Alaska Native Claims Settlement Act to the Cape Fox Corporation: T. 73 S., R. 90 E. C.R.M. Section 24 E\1/2\E\1/2\. Section 25 NE\1/4\. T. 73 S., R. 91 E. C.R.M. Section 19 SE\1/4\, SE\1/4\SW\1/4\, W\1/2\W\1/2\. Section 29 E\1/2\SW\1/4\, W\1/2\W\1/2\. Section 30 N\1/2\NE\1/4\. Section 32 E\1/2\NW\1/4\, NW\1/4\NW\1/4\. SEC. 3. WAIVER OF CORE TOWNSHIP REQUIREMENT FOR CERTAIN NON-PRODUCTIVE LANDS. The Cape Fox Corporation shall not be required to select up to 200 nonproductive acres of lands within the township in which Cape Fox Corporation is located, notwithstanding the provisions of section 16(b) of the Alaska Native Claims Settlement Act (43 U.S.C. 1615(b)) relating to the selection of lands in the township or townships in which all or part of a Native village is located. SEC. 4. CREDIT FOR RECONVEYANCE OF BEAVER FALLS HYDROPROJECT POWERHOUSE SITE. Within 24 months after the date of the enactment of this Act, the Cape Fox Corporation may transfer all or part of its right, title, and interest in and to the approximately 320-acre parcel that includes Beaver Falls Hydroelectric power-house site to the United States. In exchange for the transfer, the acreage entitlement of the Cape Fox Corporation shall be credited in the amount of the number of acres returned to the United States under this section. SEC. 5. AVAILABILITY OF CERTAIN PARCELS FOR HOMESITE PROGRAM. (a) In General.--The lands described in subsection (b), other than any of such lands conveyed to or selected by the State of Alaska under Public Law 85-508 (commonly known as the ``Alaska Statehood Act'', approved July 7, 1958 (72 Stat. 339, 48 U.S.C. note prec. 21), shall be available to the Cape Fox Corporation for its homesite program under section 21(j) of the Alaska Native Claims Settlement Act (43 U.S.C. 1620(j)). The Secretary shall transfer to the Cape Fox Corporation such portions of the lands as the Cape Fox Corporation requires. (b) Lands Described.--The lands described in this section are the following lands: T. 77 S., R. 91 E. C.R.M. Section 5 S\1/2\SW\1/4\. Section 6 E\1/2\SE\1/4\. Section 31 S\1/2\NE\1/4\. SEC. 6. LIMITATION. (a) No Change in Aggregate Entitlement.--Lands may not be transferred under this Act to the extent that the transfer of such lands would result in the Cape Fox Corporation acquiring a total amount of land under this Act and the Alaska Native Claims Settlement Act in excess of the amount of land to which the Cape Fox Corporation is entitled pursuant to the Alaska Native Claims Settlement Act as modified by sections 2 and 4 of this Act. (b) Relinquishment.--A relinquishment of lands by Cape Fox Corporation under this Act relinquishes the rights of Sealaska Corporation to the subsurface rights to such lands, and Sealaska Corporation is entitled to the subsurface rights in any lands subsequently selected by Cape Fox Corporation to the extent and in the same manner as provided in the Alaska Native Claims Settlement Act.

Title: To resolve certain conveyances under the Alaska Native Claims Settlement Act related to Cape Fox Corporation, and for other purposes Summary: Provides for the conveyance of lands located near Ketchikan, Alaska, to the Cape Fox Corporation (Cape Fox). Makes certain lands eligible for selection by Cape Fox for a 24-month period. Provides for the direct conveyance of a certain parcel of land. Waives the core township requirement for certain non-productive lands. Allows Cape Fox to be credited for reconveyance of a certain parcel of land that includes the Beaver Falls Hydroelectric power-house site. Makes available certain parcels of land to Cape Fox for its homesite program.

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Summarize: In February, the British Prime Minister, David Cameron, delivered a speech on Islamist extremism to the delegates of the forty-seventh Munich Security Conference. After speaking on foreign policy, he shifted to domestic matters. “We will not defeat terrorism simply by the action we take outside our borders,” Cameron said. “Europe needs to wake up to what is happening in our own countries.” Poverty and political injustice, he continued, may contribute to extremism, but many of the perpetrators of Islamist terrorism in the United Kingdom had been well educated and middle class. In Cameron’s view, the “process of radicalization” that prompted someone like Mohammad Sidique Khan, a thirty-year-old primary-school teaching assistant from Dewsbury, to blow up a train was a crisis of identity, precipitated by the erosion of the British character. “Under the doctrine of state multiculturalism, we have encouraged different cultures to live separate lives,” he said. “We have even tolerated these segregated communities behaving in ways that run completely counter to our values.” Britons, fortified by a “much more active, muscular liberalism,” Cameron told the audience, would no longer truckle to politically correct notions of passive tolerance. Repudiating multiculturalism—the notion that varying ethnic, religious, and racial groups can exist separately but peaceably within a society—has lately become obligatory among European leaders. Last October, Germany’s Chancellor, Angela Merkel, reiterated her belief that her countrymen’s attempts to “live side by side” had “failed, utterly failed.” In February, the President of France, Nicolas Sarkozy, announced a nationwide debate on Islam and respect for French secularism, saying, “The truth is that in all our democracies we’ve been too concerned about the identity of the new arrivals and not enough about the identity of the country receiving them.” According to a study commissioned by the German Marshall Fund of the United States, anxiety about immigration is more acute in Britain than in any other European country surveyed. Britain, as Cameron has noted, is experiencing the largest influx of people in the country’s history; in the year ending in September of 2010, five hundred and eighty-six thousand immigrants came to live in Britain. “We’ve seen record levels of immigration in the last ten years,” a Home Office spokesperson told me. “The British public quite rightly want to see those levels drastically reduced. This sort of uncontrolled immigration leads to the loss of public confidence and is a drain on public services.” As in the rest of Europe, in Britain—where the Muslim population has increased by seventy-four per cent, from 1.6 million to an estimated 2.8 million, since 2001—concern about immigration is often a euphemism for concern about Islam. Fifty-two per cent of respondents to a poll of five thousand Britons believed that Muslims create problems in their country. The newspapers are filled with stories about the loss of the British way of life, with halal meat and niqabs as its despoilers. Britons are worried as much about legal immigrants as about illegal ones, and some of the Muslims they regard as foreign are actually second- or third-generation citizens. It did not go unremarked last year that in the West Midlands region Mohammed became the most popular name for baby boys, overtaking Jack. The cultural stresses besetting Britain are profound. Legitimate fears about the allocation of resources and the preservation of liberal democracy mingle—at times indistinguishably—with racism and hysteria. In December, the Sun’s front page warned of an “AL-QAEDA CORRIE THREAT.” It turned out that Al Qaeda did not have plans to bomb the set of “Coronation Street,” Britain’s longest-running soap opera. But this spring four Muslim men were convicted of attacking an East London teacher with a brick and a metal rod; they did not approve of his course on world religions. In May of 2010, Roshonara Choudhry, a former top student at King’s College, London—she had dropped out after watching hours of YouTube sermons by Anwar al-Awlaki, the Al Qaeda leader in Yemen—stabbed Stephen Timms, a Labour M.P., because he had voted for the Iraq war. Timms survived, and Choudhry was sentenced to life imprisonment. After her arrest, Choudhry told the police that she was pleased with what she had done, and that she had not feared for her life. Q: Why? A: I wanted to be a martyr. Q: Why? A: ’Cos, erm, that’s the best way to die. Cameron had maintained a moderate stance on immigration since assuming the leadership of the Tory Party, in 2005. Unlike his predecessor, Michael Howard, he avoided the sort of rhetoric that had given the Tories a reputation as the “Nasty Party,” conjuring the xenophobic nationalism of Enoch Powell and his 1968 “rivers of blood” speech. But in Munich this year Cameron sounded newly combative. Conflating the issues of security and integration, he framed the battle against terrorism as a culture war. In an unseemly coincidence, as Cameron spoke thousands of Britons were preparing to take to the streets in Luton, thirty miles north of London. The occasion was the latest in a series of marches organized by a two-year-old group called the English Defence League. The E.D.L. bills itself as “a human rights organization that exists to protect the inalienable rights of all people to protest against radical Islam’s encroachment into the lives of non-Muslims.” E.D.L. members fancy themselves modern-day knights, Jacks defending Queen and country against those Mohammeds who would turn it into a green and pleasant caliphate. (Last year, Anjem Choudary, one of Britain’s more provocative Islamist leaders, said that he would strive to implement Sharia law “from Land’s End to John o’ Groats.”) To its critics, the E.D.L. is, at best, a nuisance and, at worst, a scourge—a travelling team of troublemakers who descend upon a town on a Saturday afternoon; take it over for a few hours; and scram, leaving behind bruised bodies, shattered glass, and bad feelings. Parading through Leicester in October, E.D.L. members kicked in the door of a halal restaurant and threw smoke bombs at policemen. The papers carried a photograph of a shaven-headed teen-ager in a black hoodie, blood streaming down his face. The E.D.L. is “a dangerous cocktail of football hooligans, far-right activists, and pub racists,” the Labour M.P. Jon Cruddas said; they posed “the biggest danger to community cohesion in Britain today.” The Home Office declared, of the E.D.L., “The Government condemns extremism in all its forms, and those who seek to spread hatred. There is no place for violence, intimidation, and criminal damage, irrespective of how strongly people hold views.” The E.D.L.’s twenty-four demonstrations, in cities across the country, have resulted in hundreds of arrests. According to the police, the Luton march cost taxpayers about three million dollars. Cruddas’s characterization of the group’s membership is accurate but not exhaustive; along with some thugs, the E.D.L. attracts an array of not particularly violent people who either lack access to other outlets of political expression or have nothing more exciting to do with their weekends. Unlike Britain’s far-right movements of years past, such as the British National Party, the E.D.L. is an explicitly non-racist organization. It welcomes, and even solicits, the participation of blacks, Jews, Sikhs, and gays—few of whom have jumped at the chance to hang around people wearing balaclavas and yelling things like “Allah is a Paedo.” On the day of the Luton demonstration, the foot soldiers of the E.D.L. were massing in the city’s pubs, downing pints and shouting chants, in anticipation of their biggest march yet—“Back Where It All Began,” the flyers read. They were an army of rowdies in white sneakers and black jackets. Merchants had boarded up shop windows. According to the Luton Today, citizens were “living in fear.” Tommy Robinson, the E.D.L.’s leader (his real name is Stephen Lennon), stood in the smoky basement of a pub called Charlie Browns. He is twenty-eight, and had dressed for the occasion in a toggle coat, pressed jeans, and a bulletproof vest. He was drinking his usual, a vodka lemonade. “I just really want to get today out of the way,” Robinson said. “My missus has been crying for three weeks.” Someone mentioned the comments that Cameron had made in Germany that morning. “Look at his timing,” Robinson said. “We are the voice of the English people. Whether the politicians like it or not, we are.” That day, the Labour M.P. Sadiq Khan accused Cameron of “writing propaganda for the E.D.L.” Later, the E.D.L.’s leaders bragged on Facebook that they had basically secured an endorsement: “An order has just come in for a hoodie in the name of David Cameron wanting Downing Street Division on the back.” William Hague, Cameron’s Foreign Secretary, was forced to defend his boss’s timing: why, he argued, should Cameron have had to reschedule a speech just because “some people have chosen to march down a street”? Still, the impression remained that Cameron had either pandered to the E.D.L.—whom the year before he had deemed “terrible people”—or had been oblivious of the fact that the group had become a significant enough force in British political life that people would think that he had. Gavin Shuker, a Labour M.P. from Luton, told me that the Prime Minister’s words had exacerbated what had, in his constituency, been extremely difficult days. “My gut feeling is that it was a cock-up, not a conspiracy,” Shuker said. “Having said that, it’s a pretty major cock-up.” At the pub, the din grew to a roar, and Robinson offered some parting words: “David Cameron’s a knob. Tell him I said that.” The Saxons invaded Bedfordshire in the sixth century, establishing farmsteads on sloping hills that line the River Lea. By the time William the Conqueror commissioned the Domesday Book, in 1085, the settlement had become known as Lintone or Loitone, a mill town of seven hundred inhabitants and their pigs and sheep. In the eighteenth and nineteenth centuries, Luton thrived as a center of the straw-hat-making trade. In the twentieth, Vauxhall Motors became the town’s main employer. During the Second World War, the workers of Luton produced ball bearings and Churchill tanks. During the nineteen-seventies, Luton’s fortunes foundered. Vauxhall closed its car plant in 2002, after years of declining revenues. Now Luton ranks sixty-ninth out of three hundred and twenty-six English districts on the government’s Index of Multiple Deprivation. In 2004, the Idler named Luton the United Kingdom’s No. 1 “crap town.” Sarfraz Manzoor, the author of “Greetings from Bury Park,” a memoir of growing up in Luton in the eighties, described it as a sort of English New Jersey. He said, “You could see the shining city—you knew you could be in the heart of metropolitan, sophisticated, glamorous life on a thirty-five-minute train ride, but you never went there.” Luton is a bellwether for the British polity. Until last year, when Gavin Shuker was elected, his seat had been won by the party that went on to form the government in every election since 1951. Luton is home to a large proportion of Muslims—14.6 per cent of its population, versus the nation’s 2.8, according to the 2001 census—and, so, serves as a laboratory for the multicultural experiment. The results have not been heartening. The cleric Sheikh Omar Bakri Mohammed, who praised the September 11th terrorists as the “Magnificent Nineteen,” preached in Luton. He was barred from Britain in 2005, but exponents of Al Muhajiroun—a group that he ran with Anjem Choudary which was banned in 2004—have continued to proselytize in Luton. The 2005 London transport bombers embarked from Luton’s train station; Taimour Abdulwahab al-Abdaly, the Stockholm suicide bomber, received a degree in physical therapy from a university in Luton. Community leaders argue that these events are more significant anecdotally than statistically. Still, they have contributed to Luton’s reputation as a “hotbed of extremism.” A report leaked in 2008 from the intelligence branch of the Ministry of Defence concluded that the main concentrations of extremists were in London and Birmingham, “with significant extremist networks in the South East, notably Luton.” “Fine, ignore me—that’s cool! I’m no stranger to unwanted advances myself.” The E.D.L. traces its origins to March 10, 2009, when Luton hosted a homecoming parade for the soldiers of the 2nd Battalion of the Royal Anglian Regiment, who were returning from a second tour of duty in Iraq. They had lost two men. It was a dry, gray day. Lutonians waved Union Jacks while a brass band played. As the Sunday Times later recounted, the soldiers made their way to St. George’s Square, the heart of the town. On the way, they encountered a group of about a dozen Muslim men, who were holding placards: “BUTCHERS OF BASRA” ; “ANGLIAN SOLDIERS GO TO HELL.” Later, the men started shouting that the British soldiers were baby-killers. (Five of them have since been convicted of using threatening and abusive language.) The crowd shouted back: “Eng-er-land!” “We pay your benefits!” A woman shrieked, “They’re all in their nightdresses. Go ’ome and get dressed!” Someone climbed onto the roof of Luton’s shopping mall, known as the Arndale center, and dumped bacon on the protesters’ heads. After the fracas, local soccer fans— “football casuals”—banded together to make their displeasure known. The men, some of whom have been banned from matches because of hooliganism, were primed for a fight. “The only blokes who had the nerve to take to the streets were proper young lads who could go to the football and shout, who had a bit of courage about them,” Robinson said. Calling themselves United People of Luton, they began recruiting in local pubs. On the Internet, they joined up with similar groups across the country. Within a few months, the various confederations had formed the English Defence League. “We have nothing against Muslims, only those who preach hatred,” Robinson told reporters. “They are traitors who should be hanged, and we’ll keep taking to the streets until the Government kicks them out.” The E.D.L. has attracted more than ninety thousand fans on Facebook, but it does not keep formal rolls. The absence of membership requirements enables the group to take credit for having lots of adherents while conveniently disowning them when they do things, such as burning the Koran, that the group purports to disdain. Officially, the E.D.L. opposes only extremists, but its message boards abound with generalized abuse toward Muslims. Robinson claims that the group relies on donations and the proceeds from hat and T-shirt sales, and his style of leadership is mostly improvisational. “You shoulda chinned him!” I heard his top lieutenant say to a regional organizer, who had called in to complain about a member who was getting out of hand. Robinson has not read much of the Koran. He says that he has not read any books at all. But he is a skilled rhetorician. It is easy to see how, under different circumstances, he might have become a lawyer. He has made several visits to the United Arab Emirates, initially to support England in a soccer tournament. “The women with burkas were so nice, in Dubai, that I didn’t care if they were wearing them,” he told me. But what counts as hospitality in a far-away land constitutes, on native soil, a siege. Although Robinson boasts of the diversity of the E.D.L.’s membership—“Black, Brown, and White Unite”—the group has had only one Muslim member. In the time that I spent with Robinson and his pals, I heard a grab bag of grievances: Muslims want to institute Sharia law in Britain, Muslims marry their cousins, Muslims beat their wives, Muslims are on steroids, Muslims drive Porsches, Christ has been taken out of Christmas, armed robbers are dressing in burkas, mosques are being built higher than castles. “Luton Mall is planning to open a multi faith (mosque) in the Mall centre next to Robbys bakery,” an E.D.L. recruiting pamphlet read. “A mosque in the Arndale spells the end of the Arndale Centre as we know it.” The mosque, which was actually an interfaith prayer room, has not opened. If the E.D.L. had its way, no new mosques would be built in Britain. This winter, I attended a meeting of the E.D.L.’s leadership at a hotel near Birmingham. One of the group’s obsessions has been sexual abuse of British girls by men of Asian origin. (The term “Asian,” in Britain, typically refers to South Asia, rather than to East Asia.) In a highly publicized case in November, two British Pakistani men were jailed for leading a gang that raped and abused teen-age girls. The conviction, in Derby, followed similar prosecutions in Rotherham, Preston, Blackburn, and Rochdale. Jack Straw, the former Home Secretary, told the BBC that there was “a specific problem which involves Pakistani heritage men” targeting “vulnerable young white girls,” whom they saw as “easy meat.” Robinson agreed. “It’s not Asian pimping gangs, it’s Muslim pimping gangs,” he declared, standing at the front of a conference room. He went on, “It’s like having Germans, specifically, raping our kids and saying it’s Europeans.” “Correct, mate!” a man with a football tattoo that read “Wolves Til I Die” yelled. “Bang on, yeah!” “We’ll make leaflets advising everyone, ‘Don’t let your daughters go near young Muslim lads,’ ” Robinson said. He concluded the meeting with a plea for calm in Luton. “We’re going to be on the world stage at this demo,” he said. “This is the biggest thing for us, if we want to become a mainstream organization. Every single one of us is working class. To be honest, we need Middle England.” Unlike the Tea Party in America, the E.D.L. does not want to overthrow the establishment. Its ideology is paradoxical, advocating revolt as a means of preserving the status quo. On matters of class, the group is almost touchingly complacent. “I’m not Oxford-educated,” Robinson told me, when I asked if he planned to run for office. “We’re not idiots. We know who our masters are. We just want them to do their job.” For now, the E.D.L. revels in the adrenal romance of vigilantism. Its sorties are half statecraft, half pure sport. Several days before the Luton march, I visited Robinson at a tanning salon that he owns in Luton. Kevin Carroll, his cousin, and his second-in-command, burst into the salon, wearing a bulky coat, jeans, one Adidas sneaker, and— on his left foot—a sandal with a sock. “Hand on me heart, I thought, Fuck, my head’s gonna come off my face!” he began. The night before, I had got a breathless e-mail from a young E.D.L. member: “SHOOTING IN LUTON!” the subject line read. It continued: A group of Muslim members coaxed EDL member “Kev” out of his house then shot at him with a shotgun, Police have put containment in that area of Luton and moved Kev and his family to safety, Our leader Tommy also has armed guards at his abode. Carroll took off the sock. He had not been shot, it turned out, but he had injured his toes, having stubbed them against a curbstone. He had been running from his attacker, he said, through the front gardens of Luton. “It was the fucking trippiest moment ever!” he said, showing off a row of purple knuckles. Later that afternoon, Robinson and a friend of his, Danny Wilson, were cruising around Luton in a pot-scented plumber’s van. They listened to a CD—“Songs for Our Cause”—that a musician member of the E.D.L. had recorded. Robinson put on a track called “E, E, E-D-L,” and sang, “On the streets of Luton the people stood up to Al Qaeda / And decided to take no more. / And made a stand for this glorious country / Fuck them and Sharia law.” Robinson and Wilson were going to pick up some leaflets, which they planned to distribute at the Arndale center. After a stop at a gas station for a pack of Wotsits cheese puffs and a strawberry milkshake, Wilson eventually pulled up at a residential cul-de-sac. Robinson ran inside a house and returned with a cardboard box of leaflets. Written on its side, graffiti style, was the E.D.L. slogan “No Surrender.” The march was three days away. Robinson decided to make a reconnaissance run into Bury Park, where most of Luton’s Muslims live. Wilson made a turn onto Bury Park Road, the neighborhood’s main thoroughfare. They saw a man standing on the sidewalk outside a chicken joint. They thought he was Sayful Islam, a student of the radical cleric Sheikh Omar Bakri. A Luton native, Islam is about the same age as Robinson. In 2003, he quit his job as an accountant in order to become a full-time proselytizer, living off welfare while promoting the violent overthrow of the British government. He was one of the protesters at the 2009 homecoming march. (“Sharia law should be implemented in the U.K., in the U.S., and all over the world,” he told me last week.) “There he is!” Robinson cried out. “There he is!” Wilson cruised by slowly. “Leave a gap,” Robinson instructed, as if he were a mob don talking to his getaway driver. The man started punching numbers into his cell phone’s keypad. Robinson thought that he was calling for reinforcements. “Get ready to drive, bro!” Robinson yelled to Wilson. “Just ram it if you have to. They’re going to be here in seconds!” Wilson screeched away, wedging the van between two lanes of traffic. “Aw, thank fuck for that, mate,” Robinson said. He was laughing nervously, elated and scared. (Islam was actually in London that day. He told me, “They probably cannot tell one bearded man from another.”) I was wondering how the feud, a Jets vs. Sharks turf war played out against the topography of one town, had metastasized into a national drama. Before leading me from a waiting room to Abdul Qadeer Baksh’s office, Baksh’s assistant informed me that his boss didn’t shake hands with women. (I hadn’t offered.) Baksh is the chairman of the Luton Islamic Center. He is a spokesman for a contingent of Muslims in Luton who feel that Islamist extremists’ relish for riling the E.D.L.—and vice versa—makes their lives in Luton significantly more difficult. (The Islamic Center was firebombed several weeks after the homecoming scuffle.) In 2009, Baksh led a campaign to drive former Al Muhajiroun activists out of Bury Park. But his position as one of Luton’s leading “moderate Muslims”—the phrase is crude, but it persists—was complicated by the revelation, in December, that Taimour Abdulwahab al-Abdaly, the Stockholm suicide bomber, had worshipped at his mosque several years earlier. Baksh told reporters that he had challenged Abdaly’s views on jihad, and that Abdaly had quit the mosque in a huff. Baksh was sitting on a metal chair, wearing a kameez with trousers, a skullcap, and hiking boots. He said that generally Luton was a harmonious place to live, but that, since September 11, 2001, there had been a palpable rise in anti-Muslim harassment—children had been teased, hijab-wearing women had been spat at. He was girding himself for the E.D.L.’s march, which would take place in less than seventy-two hours. He hoped there would be no violence. “If the E.D.L. break our ranks, then the Muslim community will indeed fight back,” he said. “We will defend our blood, our property, our families, like anyone would.” During the course of our conversation, Baksh called Sharia “the best law for the whole of humanity,” but he said that he would “never force it on Muslim or non-Muslim.” He added, “We tolerate all different kinds of people, because we are all part of a multicultural society,” but he described homosexuality as “an abomination.” I asked Baksh whether his beliefs were irreconcilable with those of Robinson and his supporters, and with the British populace at large. He thought that they could find common ground. “Get us round a table with some chicken and chips, and we’ll talk,” he said. But resentments surfaced. “When they say we don’t integrate, what they mean is we don’t assimilate,” he said. “We’re not required to look like you, be like you—especially when we look at the state of you. You get drunk, you beat up your wives, you sell drugs.” Britain’s first mosque was established in 1889, in Liverpool. Muslim migration to Britain on a large scale began after the Second World War, when the loss of able-bodied men, coupled with economic expansion, precipitated a labor shortage. Men from South Asia flocked to industrial cities like Luton. Many of their wives and children followed in the early nineteen-sixties, owing to changes in immigration laws. Between 1961 and 1971, the Muslim population in Britain grew by about three hundred and fifty per cent. In 1989, Ayatollah Khomeini issued a fatwa in which he called for the death of Salman Rushdie, the author of “The Satanic Verses.” According to Ron Geaves, writing in “Muslim Britain: Communities Under Pressure,” the Rushdie affair was a watershed moment for British Muslims, many of whom found the book offensive. It spurred “political mobilisation and the provision of a national voice of protest with remarkable speed.” The next year, the Conservative politician Norman (now Lord) Tebbit proposed a “cricket test” to measure the loyalties of immigrants. “A large proportion of Britain’s Asian population fail to pass the cricket test,” he said. “Which side do they cheer for?” “Mother wouldn’t want us to feel sad—she’d want us to feel guilty.” Relations deteriorated further when, in the spring and summer months of 2001, thousands of young men of Bangladeshi and Pakistani origin fought with white youths in Britain’s northern cities. (More than three hundred police officers were injured in a riot in Bradford.) After 2001, according to a study entitled “Islamophobia and Anti-Muslim Hate Crime,” the “violent extremist nationalist milieu”—encompassing such groups as the B.N.P. and, lately, the E.D.L.—began to shift the focus of its opprobrium from race to religion. Now the onus was cultural rather than biological; the same person might still be bashed, but because he was a Muslim, rather than because he was brown-skinned. (The estimated hundred thousand Britons who have converted to Islam are not exempt from the E.D.L.’s invective.) The recession and the expenses scandal of 2009, in which M.P.s charged British taxpayers for such personal items as the cleaning of a moat, have created a fertile environment for groups like the E.D.L. Roger Eatwell and Matthew J. Goodwin write, in “The New Extremism in 21st Century Britain,” that extremism tends to thrive when government is unresponsive and “civil society fails as a prophylactic.” In January, Baroness Sayeeda Warsi, a Muslim who serves as the co-chairman of the Conservative Party and is a Minister in Cameron’s cabinet, said, in a speech on Islamophobia, that prejudice against Muslims now passes the “dinner-table test,” having become socially acceptable. According to the Telegraph, the speech had not been cleared with Downing Street; Cameron’s spokespeople refused to say whether or not the Prime Minister agreed with Warsi. Of course, even the most discriminating racists sometimes get confused. On the day of the Luton military parade, an eighteen-year-old named Kier McElroy, incensed by the Muslim protesters’ insults to the Armed Forces, ran up behind an older Asian man and launched a flying karate kick to his back. The man was Lakhbir Singh, Luton’s mayor. Singh is a Sikh. It is no accident that the E.D.L.’s name is the English, rather than the British, Defence League. “England” can connote a homogeneous land of tea and cakes, the parson and the village green, in a way that “Britain” does not. This spring, the producer of a popular television show called “Midsomer Murders” was suspended after reflecting on the heartwarming pallor of his cast. “We just don’t have ethnic minorities involved. Because it wouldn’t be the English village with them,” he said. “We’re the last bastion of Englishness, and I want to keep it that way.” The E.D.L. has not achieved the electoral stature of groups like Geert Wilders’ Party for Freedom, in the Netherlands, or France’s National Front, but Tommy Robinson hopes to export its brand of nationalist vigilantism to the rest of Europe, and beyond. Recently, the group has established embryonic divisions in Sweden, Germany, the Netherlands, Australia, and the United States. Robinson plans to travel to New York to commemorate the tenth anniversary of September 11th. (He says that the last time he tried to enter the United States he was turned away at immigration.) In May, he appeared at a rally in Lyon that was organized by the French far-right group Bloc Identitaire, which is known for handing out food that contains pork—“identity soups”—to homeless people. The rally, a protest against halal meat, was billed as La Marche des Cochons. With a guy in a pink polo shirt translating, Robinson told the crowd, “This is not a Western front, but this is a battle against radical Islam.... You are the resistance!” In 2004, Robinson spent several months in jail for assaulting an off-duty police officer who intervened in a domestic dispute between him and his fiancée. He is being investigated for money laundering; he has several public-order and assault charges pending. In May, he was found guilty of a public-order offense for an incident in which he hurdled a metal blockade and ripped a flag out of the hands of a member of a Muslim group that had earlier burned poppies, Britain’s symbol of military remembrance. (He is appealing.) Some of the E.D.L.’s troublemaking has been petty, but a 2010 assault on Big John’s fast-food restaurant, in Leicester, was genuinely terrifying. (You can see it on YouTube, along with Robinson’s strike on the poppy burners.) As women and children take cover in a storeroom amid vats of ketchup, a marauding band of E.D.L. members burst through the door and stream into the dining room, beating their chests. I asked Robinson how he could defend such an attack. “It was bad, wasn’t it?” he said. “That Leicester day was mental. I felt sick when I watched it. It wasn’t fucking clever at all.” Then he launched into a moral-equivalency argument. “But, at the same time, it’s happening all over the country every day to us.” The lead-up to the Luton march was frenzied. Local officials tried to get the E.D.L. banned from coming to town. “All in all, the possibility for serious disorder in Luton on the 5th February is real,” the Borough Council wrote, in a letter to Theresa May, the Home Secretary. When May declined to issue a banning order, community activists railed at the council for ceding St. George’s Square to the E.D.L. as part of its processional route. At an informational meeting, a woman screamed from the back row, “Why are you bending over backward to keep a group of Fascist thugs happy?” Meanwhile, the E.D.L. and its rivals—among them the Muslim Defence League and United Against Fascism—were trading threats, insults, and reprisals. Opponents had a song of their own, by an East London rapper named Naga. It was called “E.D.L., Burn in Hell”: As a family we can stay strong The E.D.L. we can take on Cuss my race, my culture I’ll leave you chavs, head gone. As the leader of the council suggested that law-abiding citizens might want to stay home on February 5th, E.D.L. members offered pump-ups to one another: What else are you gonna do on a Saturday? Sit in your fucking armchair wanking off to Dancing on Ice?... Then go and spunk your wages on kebabs, fruit machines and brasses? Fuck that for a laugh! I know what I would rather do. Luton away, love it! The Saturday before the march, the Luton Borough Council had urged citizens to “turn the town purple” by attending an event called Luton in Harmony, a sort of field day for the community that would counteract the corrosive effects of the looming showdown. “It was freezing cold and people came out and we had a carnival and a little parade, and everyone shared cakes,” Sarah Allen, the Inclusion and Cohesion Manager of the Luton Borough Council, told me. She talked about how there had been face-painting, and salsa dancers, and how stilt walkers had entertained the kids. While acknowledging that the municipality has had its problems, she said, “As far as I’m concerned, Luton is a town that offers a whole range of advantages.” She sounded a little exasperated. “Life in Luton is absolutely fine!” The morning of the march arrived: cold, misting, miserable. By seven-thirty, fifty or so members of the London division of the E.D.L. had gathered at a pub in the concourse of the Liverpool Street railway station. For breakfast, there was beer, tomatoes, bacon, and beans on toast. One member was selling collectible pins—they said things like “Don’t Unpack, You’re Going Back,” and “Enoch Was Right.” An hour later, the members boarded three buses. I was assigned to the first bus, which one of the younger members told me was “the geezer bus”—its passengers included some visiting Germans, who sat quietly in the front, and a few other dignitaries. We pulled out from Liverpool Street. My seatmate was a fortyish man who gave his name only as Merriott Chard, after the British soldier who led the defense of Rorke’s Drift, in the Anglo-Zulu War. Chard, a cabinetmaker, had come with his housemate, who sat in the row behind us, reading aloud obscure passages of the Koran that he had highlighted in yellow. Chard, wearing fatigues and a maroon beret, said that he had once been a member of the Army, and of the B.N.P. “I found it, basically, mundane,” he said, of the latter. “They were just plagiarizing things said fifty years ago. The B.N.P. was just dictation, but with the E.D.L. there’s a sense of spiritual entity. It’s void of all politics and politicians. We only represent ourselves.” Figuring that it was a special occasion, Chard had bought two bottles of Taittinger. “We’re not here for a flower-arranging competition!” someone yelled, as a cork whizzed perilously close to my eye. “Cheers, boys,” Chard said, pouring the warm champagne into red plastic cups. As we approached the outskirts of Luton, the mood tensed. We passed the old Vauxhall recreation club, which had been commandeered as a police staging ground. Hundreds of riot police in fluorescent vests filed out of vans, clutching shields and nightsticks. It looked like a scene from “Star Wars.” Someone on the bus bellowed an “E,” and the passengers began chanting “E-E-E-D-L” in menacing staccato barks. We parked near the Luton train station and got off the bus. The town was deserted. The only sounds were a helicopter buzzing above and a pack of dogs howling from the back of a paddy wagon. The police escorted the E.D.L. members to Midland Road, the beginning of the parade route. For several hours, the E.D.L. rank and file wandered back and forth between “muster pubs.” I saw one man wearing a mask of a pig’s head, and another dressed as a skeleton. In Charlie Browns, the atmosphere was wired. A sort of mosh pit erupted. “Let’s go fucking mental, la la la!” boys yelled—a chant from the football terraces. The police had cordoned off the top of Midland Road. A gusty wind sent trash boomeranging through the street, giving the place an apocalyptic air. The road was bounded by buildings on one side and, on the other, by a brick wall, so that the street turned into a sort of chute, plugged at one end and backing up, toward the west, with increasingly restless men (and a few women). By noon, it was impossible to take a step. At twelve-thirty, the crowd broke loose, with a primal surge. Yelling “Whose streets? Our streets!” and waving whipping flags, they poured down Midland Road and into St. George’s Square. There Robinson delivered a speech under a drizzling sky. The sound system was abysmal, and people milled about. Someone punched someone over something, and gave him a bloody nose. Newspapers reported that fifteen hundred people had turned out for the E.D.L., but it seemed closer to three thousand. The day ended with eight arrests, mostly for fighting. Birmingham who smiled in defiance at English Defence League protester becomes symbol of city standing up to far-right group A photograph showing a young woman smiling bemusedly at an incensed English Defence League protester has been widely shared as a symbol of Birmingham’s defiance in the face of the far right. The image, which has been shared thousands of times on social media, was captured during a demonstration by the far-right group in Birmingham city centre on Saturday. It shows an EDL protester Ian Crossland staring into the eyes of the young woman, who is looking back at him unfazed. A police officer appears to be restraining Crossland. In dark times, this image has a glorious message – resistance is not futile Suzanne Moore Read more The woman pictured has been identified as Saffiyah Khan, who is from Birmingham. Her family has links to Bosnia and Pakistan. She said the picture was taken when she stepped in to defend a woman wearing a hijab, who had been surrounded by a group of the protesters after she called them racists. “She was quite a small woman,” Khan said. “When I realised that nothing was being done [by police] and she was being surrounded 360, that’s when I came in as well.” She described the man who confronted her as “an angry man having a bit of a rant”. Speaking to Radio New Zealand, Khan said the best responses she has had from the photograph were from people who had got in touch to tell her how the picture had personally affected them. “I’ve had lots of stories about [people’s] daughters being affected and how they see me as a role model,” she said. The EDL demonstration attracted about 100 people and was condemned by the Labour, Liberal Democrat and Conservative leaders of Birmingham city council, who said the group was not and never would be welcome in their city. The demonstration attracted a heavy police presence, including riot vans. West Midlands police said two people, thought to be counter-protesters, had been arrested for alleged breaches of the peace. Tweeting the photograph, which was taken by Press Association photographer Joe Giddens, Birmingham MP Jess Phillips wrote: “Who looks like they have power here, the real Brummy on the left or the EDL who migrated for the day to our city and failed to assimilate?” Her tweet had been shared and liked nearly 18,000 times by Sunday evening. Jess Phillips MP (@jessphillips) Who looks like they have power here, the real Brummy on the left or the EDL who migrated for the day to our city and failed to assimilate pic.twitter.com/bu96ALQsOL The journalist and television host Piers Morgan hailed the picture “photo of the week” and shared it on social media with the caption: “Enraged EDL racist stared down by amused, contemptuous Asian woman.” EDL activists claimed on Twitter that scuffles broke out at the demonstration after counter-protesters shouting “Nazi scum” disrupted a silence being held at the rally for victims of terrorism. Writing on Facebook, Crossland described Khan as a “dirty unwashed leftwing scrubber”. He added: “The disrespectful witch chose the minute’s silence for the victims of the terror attack in Stockholm and Westminster. She’s lucky she got any teeth left.” Speaking to Vice News, Khan said video evidence contradicted the EDL’s claim. “Anyone who knows me will agree that I would respect a minute’s silence for the death of innocent people, regardless of who was holding it,” she said. “That is a matter of my conscience, and it is a powerful tool.” Tommy Robinson, the EDL’s former leader, tweeted that the picture was embarrassing. “OK, just had confirmed by a friend who was at EDL demo, this lady was defending a woman in a navy hijab as she said to the papers,” he wrote. “[And] I don’t care how many people don’t like me saying that, the truth is the truth. [And] the picture is embarrassing.” Khan told Vice that she hoped to use her newfound status to “fight against racism on the streets of the UK”. She added: “[I’ve] got big things planned, focusing on the bigger picture. Being viral is worthless if nothing helpful comes of it.” In an event organised to counter the EDL protest, Birmingham central mosque held a “Best of British” tea party, complete with union jack bunting, tea and cake. Addressing the estimated 300 people who attended, the local MP Liam Byrne said the event celebrated “the quiet miracle of a normal life and the things that we love most about our city and our country”. “Getting together as friends, getting together as neighbours, breaking a bit of Victoria sponge and having a cup of tea,” he added. “That is a potent, powerful message that we will send to those who seek to divide us.”

Summary: Around 100 demonstrators representing a far-right UK group gathered Saturday in Birmingham, provoking a presence by law enforcement, counter-protesters, and a photo that's now being shared worldwide. The Guardian reports on the viral image, which was taken when local Saffiyah Khan noticed some English Defence League protesters circling a woman in a hijab and decided to intervene. "I don't like seeing people getting ganged up on in my town," she says. Per BuzzFeed, which notes the protest was railing against last month's London terror attack, Press Association photographer Joe Giddens then snapped a picture of Khan squaring off with a man in an EDL T-shirt, with a police officer appearing to hold him at bay while Khan stared him straight in the eye with a smile. BuzzFeed identifies that man as Ian Crossland, a leader of the group that a Labour MP once called a "dangerous cocktail of football hooligans, far-right activists, and pub racists" for its anti-Muslim rhetoric and often-violent actions, per a 2011 New Yorker article. Khan, who tells BuzzFeed she hails from a Muslim background, says Crossland shoved his finger in her face, but she didn't care. "I didn't feel threatened in the slightest way," she shrugs. Reaction to her stare-down has earned her praise of "amazing," "big respect," and "the most perfect response to EDL hatred; calm, somewhat nonchalant, defiant, and smiling." Giddens himself seems taken aback by the attention given to his handiwork, tweeting, "Sometimes in this job you take a picture and instantly know it'll be popular... that wasn't the case this weekend!"

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Write a title and summarize: We elucidate the mechanisms that lead to population shifts in the conformational states of calcium-loaded calmodulin (Ca2+-CaM). We design extensive molecular dynamics simulations to classify the effects that are responsible for adopting occupied conformations available in the ensemble of NMR structures. Electrostatic interactions amongst the different regions of the protein and with its vicinal water are herein mediated by lowering the ionic strength or the pH. Amino acid E31, which is one of the few charged residues whose ionization state is highly sensitive to pH differences in the physiological range, proves to be distinctive in its control of population shifts. E31A mutation at low ionic strength results in a distinct change from an extended to a compact Ca2+-CaM conformation within tens of nanoseconds, that otherwise occur on the time scales of microseconds. The kinked linker found in this particular compact form is observed in many of the target-bound forms of Ca2+-CaM, increasing the binding affinity. This mutation is unique in controlling C-lobe dynamics by affecting the fluctuations between the EF-hand motif helices. We also monitor the effect of the ionic strength on the conformational multiplicity of Ca2+-CaM. By lowering the ionic strength, the tendency of nonspecific anions in water to accumulate near the protein surface increases, especially in the vicinity of the linker. The change in the distribution of ions in the vicinal layer of water allows N- and C- lobes to span a wide variety of relative orientations that are otherwise not observed at physiological ionic strength. E31 protonation restores the conformations associated with physiological environmental conditions even at low ionic strength. Protein behavior in solution may be manipulated and controlled through tailored structural perturbations [1] and rational control of the solution conditions [2] http: //www. pnas. org/content/109/50/E3454. full. pdfhtml. In the living cell, proteins adapt to particular subcellular compartments which pose different environmental variables such as pH and ionic strength (IS), adapting their biophysical characteristics to tolerate pH fluctuations that are caused by cellular function [3]. Furthermore, proteins interact with many other biological macromolecules while they are transferred from one compartment to another, with subtle control over protonation and pK changes upon binding to other proteins and ligands [4], [5]. Interactions with the environment and other molecules are closely related to local anisotropy and dynamical heterogeneity of proteins [6]. The dynamics may be electrostatically guided, perhaps through long-range electrostatic interactions that select and bring interacting partners together, steering the protein to alternative conformations [7]. The main perturbation effect of the long-range electrostatic interactions is manifested on the acidic/basic groups in the protein which can be charged or neutral in relation to their conformation dependent pKa values [8]. Interacting with other molecules and changes in the environmental variables such as subcellular localization can induce shifts in ionization states of charged groups on a protein by proton uptake/release. Such changes facilitate the protein to span a large conformational space and enable it to participate in diverse interaction scenarios. Any in depth understanding developed through studying the conformational changes in proteins induced by shifts in the charge states of select amino acids would contribute to our knowledge base on diverse functionality observed in promiscuous proteins [9]. In this study, we focus on the conformation-related effects of introducing perturbations on charged group (s) of calmodulin (CaM). CaM is a notorious example among proteins having the ability to change conformation upon binding to diverse ligands [10], [11]. It was shown that negatively charged side chains in calcium loaded CaM (Ca2+-CaM) are attracted to positively charged residues in many of its targets [12]. Another study showed that Ca2+-CaM changes conformation when introduced to a solvent at low pH and low ionic strength [13]. Also, it was proposed that electrostatic interactions between acidic residues in CaM contribute to determining the most populated conformation under varying solution conditions [13]. Previously, we have studied the conformational changes in Ca2+-CaM [14], ferric binding protein [15] and a set of 25 proteins that display a variety of conformational motions upon ligand binding (e. g., shear, hinge, allosteric) [16] using the perturbation response scanning method. This coarse grained methodology is based on the assumption that the equilibrium fluctuations at a given local free energy minimum of the protein possess information on other viable conformations when an external force is applied [15]–[17]. Our study on CaM determined key residues that lead to the experimentally observed conformational changes upon application of force in specific directions [14]. Several different servers (H++ [18], propKa 2. 0 [19], pKd [20] and PHEMTO [21], [22]) showed that the pKa of E31 value is upshifted; furthermore, the equivalent position in Calbindin was measured to have pKa of 6. 5 [23]. In a follow-up study, we focused on residues with upshifted pKa values and we made a systematic study of the dynamics of Ca2+-CaM on time scales up to 200 ns for three separate initial configurations; extended form, compact form and extended structure with 10 protonated residues (9 acidic residues and a histidine) [24]. We found that Ca2+-CaM with 10 protonated residues undergoes a large conformational shift from the extended structure to a relatively compact form on the time scale of tens of nanoseconds. The latter was compatible with other structures reported in a nuclear magnetic resonance (NMR) ensemble of CaM [25]. Experimental work investigating dynamical behavior of Ca2+-CaM has shown that it occupies a number of hierarchical set of substates even in the crystal form [26]. Dynamical information obtained from fluorescence resonance energy transfer (FRET) experiments measuring the distance distributions between labeled sites illustrate that at least two conformations exist in solution under physiological conditions [27]. More recently, pseudo contact shifts and residual dipolar couplings of the C-terminal domain obtained using NMR [28] revealed neither the dumbbell shaped conformation observed in early crystal structures of the molecule [29], nor the compact conformation determined later on [30] exist in significant proportions in solution. Ca2+-CaM is identified as a protein which populates multiple conformations [28], [31]. A shift between the distribution of populations is induced by changing environmental conditions such as pH, Ca2+ concentrations and ionic strength [13], [27]. Each of these manipulated properties has effects on the charged groups of Ca2+-CaM. The presence of multiple conformations is a physical property of Ca2+-CaM, and it is likely that the heterogeneity of structure is at least partially responsible for the ability of Ca2+-CaM to recognize diverse targets. Squier and coworkers have suggested that association of the C-terminal domain of CaM with a target may disrupt a structurally important hydrogen bond involving the central linker, facilitating formation of a compact binding conformation of Ca2+-CaM [32]. More recently, through rather benign mutations such as E47D, they have determined noninterfacial residues important for molecular recognition through indirect effects – an increase in fluctuations stabilizes the bound state [33]. It was further hypothesized that pH and ionic strength dependent shifts in the populations of conformational substates result from changes in electrostatic interactions in the central linker [13], [27]. For example, the shift in favor of the more compact conformation at reduced pH may result from the loss of electrostatic interactions that serve as spacers at neutral pH. This hypothesis is corroborated by inspection of the proximity of side chains of glutamic and aspartic acid residues surrounding the hinge region in the compact Ca2+-CaM crystal structure [30]. In this manuscript, we report extensive molecular dynamics (MD) simulations of fully solvated, extended and compact Ca2+-CaM under different perturbation scenarios, with focus on E31. We have previously shown that E31 is located in a unique position to manipulate the overall structure; it also has an upshifted pKa into the physiological range and there are several experiments implicating its involvement in signaling coordination between the two lobes (see [14] and references cited therein). Structural perturbations are introduced as either E31A mutation or its protonation. We also perturb environmental factors such as pH and IS. We analyze the structural dynamics through identifiers based on reduced degrees of freedom defined specifically for Ca2+-CaM. Key events leading to or preventing conformational change are discussed. We elaborate on the events occurring along the path sampled between different conformational states identified by MD simulations and we evaluate the effect of charge balance on the conformations. The molecular mechanisms that lead to the observed effects, their relationship to the experimental data, and the consequences of the observations that enhance our understanding of the dynamics and function of Ca2+- CaM are outlined. CaM consists of 148 amino acids made up of the N-lobe (residues 1–68), the C-lobe (residues 92 to 148) and a linker which is helical in many, but not all, of the reported structures. Each lobe in CaM has two helix-loop-helix (EF-hand motif) calcium binding sites connected by unstructured sequences. Structured elements include helices I (residues 5–17), II (residues 30–39), III (residues 46–54), IV (residues 69–73), V (residues 83–91), VI (residues 101–110), VII (residues 119–129), and VIII (residues 137–144). Ca2+ coordinating residues in each of the four EF-hands are D20-D22-D24-E31 in loop I, D56-D58-N60-E67 in loop II, D93-D95-N97-E104 in loop III, and D129-D131-D133-E140 in loop IV. All MD simulations reported in this work include the four Ca2+ ions. The existing X-ray structures of Ca2+-loaded, peptide free calmodulin (Ca2+-CaM) are either in an extended or a compact form. There are many examples for the extended form in the protein data bank (PDB) and we utilize that with PDB code 3CLN whereby the coordinates of the first four and the last residue are not reported [29]. The compact form is represented by the 1PRW coded structure [30], and has a bent linker as do many ligand bound Ca+2-CaM conformations present in the PDB. These particular structures have been determined at 2. 2 and 1. 7 Å resolution, respectively, and were both crystallized at low pH conditions in the range of 5–6, by growth in water-organic mixture compounds. We have previously reported the RMSD comparison for the overall structure as well as the N- and C-lobes of various x-ray structures, including 3CLN, 1PRW and five ligand bound forms [14]. An ensemble of Ca2+-CaM structures have also been reported (PDB code 2K0E) [25] by using experimental NMR order parameters (S2) together with interproton distances derived from nuclear Overhauser effects (NOEs) as restraints in MD simulations using RDC-refined solution structure of Ca2+-CaM. The IS of the experimental setup is 10 mM and the pH is 7 (the conditions in ref. [25] are the same as in ref. [34]; personal communication). The ensemble has 160 structures and reveals that Ca2+-CaM state samples multitude of conformations including, but not limited to, the compact and extended ones. In particular, unlike in the X-ray structures, there also exist compact conformers where the linker is not bent, as we pointed out in our previous study [24]. Throughout this work, an efficient approach to distinguish between the different conformations of CaM proves to be useful: We define two low resolution degrees of freedom projecting the 3N-dimensional conformational space into a visually tractable two-dimensional one. These are the linker end-to-end distance (l) and torsion angle (φ). The former is defined as the distance between the Cα atoms of the two outermost residues of the linker, residues 69 and 91. The latter is the torsion angle defined by four points: the center of mass of the N-lobe (residues 5 to 68), linker beginning and end points (Cα atoms of residues 69 and 91), the center of mass of the C-lobe (residues 92 to 147). These points are schematically shown in Figure 1. Six sets of simulations were performed with various initial starting conditions. Simulations are summarized in Table 1; IS values reported correspond to the equilibrated box dimensions. For some systems, we have performed independent runs to check the reproducibility of the results. Each condition has at least 150 ns of total sampling time. We prolong the simulation in case there are substantial changes in the relative positioning of the two lobes and/or the length of the linker, measured by the region sampled on the (l, φ) plane described in the previous subsection. The details of each simulation are as follows; the label for each type of simulation is indicated in parentheses and will be used throughout the text: () Initial coordinates are taken from the extended 3CLN pdb coded structure and all residues are assigned their standard protonation states to study the conformational dynamics of extended form in solution. 45 Na+ and 30 Cl- ions are added to attain IS = 150 mM at the physiological range. There is a one MD run of 150 ns and an additional control run of 50 ns. () Initial coordinates are taken from the compact 1PRW and all residues are assigned their physiological protonation states to study the dynamics of compact structure in solution. The system is neutralized by 15 Na+ ions. Due to the smaller box dimensions formed for this more compact structure, this protocol leads to IS = 161 mM at the physiological range. There are two runs of 200 ns each for this system. () Starting from 3CLN and all residues having the same protonation states as in (), the system is neutralized by 15 Na+ ions. This leads to a low IS of 82 mM. There is a on MD run of 400 ns and an additional control run of 50 ns. () Starting from 3CLN structure, only E31 is protonated. The system is neutralized by 14 Na+ ions leading to IS = 91 mM; there are two runs for this system, one of length 400 ns and the other of 150 ns. () Starting from 3CLN structure, E31A mutation is made. The system is neutralized by 14 Na+ ions leading to IS = 94 mM; there are three runs of 400 ns each for this system. () In all the previously listed simulations, residues are assigned charge states according to pH = 7. 4 using pKa values calculated and listed in ref. [24]. These systems are assumed to be at physiological pH. Acidic residues 11,31,67,84,93,104,122,133 and 140, as well as H109 consistently have pKas shifted from their standard values to ∼5. 5. In the system, these are protonated to mimic the low pH conditions. The reader is referred to ref. [24] for details on the calculation of pKa values. There are two runs for this system, one of length 200 ns and the other of 100 ns. The system is neutralized by 5 Na+ ions leading to IS = 43 mM In addition, control runs of 100 ns duration have been carried out on the extended, low IS proteins, singly or doubly protonating other residues with upshifted pKa values. These are labeled,, and are not separately listed in Table 1; they are neutralized by 14,13 and 13 Na+ ions, respectively. We use the NAMD package to model the dynamics of the protein-water systems [35]. The protein is soaked in a water box with at least 10 Å of water from all directions using VMD 1. 8. 7 program with solvate plug-in version 1. 2 [36]. The CharmM22 force field parameters are used for the protein and water molecules [37]. Water molecules are described by the TIP3P model. Each system is neutralized by using VMD autoionize plug-in. Long-range electrostatic interactions are calculated by the particle mesh Ewald sum method, with a cutoff distance of 12 Å and a switching function of 10 Å [38]. RATTLE algorithm is utilized and a step size of 2 fs is used in the Verlet algorithm [39]. Temperature control is carried out by Langevin dynamics with a damping coefficient of 5/ps. Pressure control is attained by a Langevin piston. Volumetric fluctuations are preset to be isotropic. The system is run in the NPT ensemble at 1 atm and 310 K. Equilibration of the pressure is achieved within 2 ns. The equilibrated box dimensions of each system are listed in Table 1. The coordinate sets are saved at 2 ps intervals for further analysis. Starting from 3CLN which represents the extended Ca+2-CaM structure captured in most X-ray studies, we externally perturb the physiological conditions for which results were displayed in figure 2: (i) We lower the IS while keeping the pH at 7. 4; and (ii) we lower the IS as well as reducing the pH to 5. 0 [24]. These systems are labeled and, respectively (Table 1). The regions sampled by are displayed in figure 3. This is a continuation of the MD simulations from our previous work [24], where the run has now been extended from 200 ns to 400 ns. The RMSD values of the subunits as well as the overall structure are shown in figure 3a. The initial conformer is not stable in solution as confirmed by the protein RMSD change. While the linker and the two lobes each display low intra-domain motions (less than 3±1 Å RMSD), their relative orientations change substantially (up to 13 Å in the overall RMSD). When projected on the reduced degrees of freedom (figure 3b), the trajectories clearly display the three separate sampled states: The two lobes initially point towards each other and within the first 25 ns, the N- and C-lobes complete a ca. 120° torsional motion reaching state II which is then sampled for 195 ns after which a new state is reached (III) by a further torsional motion of 100°. In the last 180 ns of the trajectory, state III is sampled. Snapshots exemplifying these three distinct states are shown in figure 3c. We note that a prompt move into region II also occurs in the supplementary 50 ns run. All the structures that are sampled throughout the MD trajectory are compared with the experimental ones. The protein spends the first 220 ns in intermediate states with linker length (l>31 Å), and φ = [−80°, 100°]. During the MD simulation, the initial (X-ray derived) structure 3CLN is only transient and the conformations sampled in regions I and II do not overlap with any of those from the NMR ensemble. This is consistent with other NMR and single molecule experiments where very low occupancy is assigned to the fully extended structure [31]. After 220 ns, the system eventually relaxes into a region with l = [28], [34] Å, φ = [−210°, −130°] which overlaps with many of the 2K0E NMR ensemble members (figure 3b). These observations imply that there is an energy barrier between the regions with φ = 90° and φ = 150° so that the system must counter-rotate by a large torsional angle, instead of flipping the 60° directly. In the runs, we find that by mimicking low pH, low IS environment, the sampled regions in figure 3b do not change (see figure S1), but the sampling is accelerated. We do not go into the details of these runs since we have already published a detailed account of the conformations sampled and key events leading to the conformational change [24]. However, it suffices to say that the same sequence of states I→II→III are followed in both runs. The shift from state I to II occurs at ca. 20 ns similar to the time scales observed in, but that from II to III occurs at ca. 70 ns. For both the and the systems, the main intra-domain conformational change occurs as a reorientation of helix II in the N-lobe. For example, the nearly right angle between helices I-II, that was always maintained at nearly right angles (80±6°) at physiological IS in the and runs, is now reduced within the first 10–20 ns of the trajectory. It is maintained at a value of 56±7° in and 60±5° in throughout the window of observation. The major event that stabilizes the closed conformation is the formation of salt bridge (s) between the N-lobe and the linker in each case: E7-K77, E11-K77 or E54-K75 in (established in both runs prior to 50 ns) and E7-R74 or E11-K77 in the runs (forming permanently at ca. 40 ns in both samples). Despite being a counterintuitive observation, it was shown as a general result that two negatively charged nano-sized spheres may be put into close contact by utilizing the competition of hydrophobic and Coulombic interactions, provided that the charges are placed discretely along the surface [40]. At physiological IS and pH, there exists a high energy barrier between the extended and compact structures, corroborated by the 100 µs time scale of jumps between them, measured by single molecule experiments [27]. Thus, a direct passage between the black and blue shaded regions in figure 2 is not observed within the time window of observations of the MD simulations. However, once achieved, the compact conformation is stable despite the net repulsions between the two lobes (net charge on the N- and the C-lobes are -8 and -6, respectively). One may argue that the pH of the X-ray experiment (5. 4) may have contributed to the stability of the 1PRW crystal structure, since the acidic residues 7,11,14,120,127 are found to be neutral at this pH [41]. The interface between the two lobes involves E7 and E11 on the N-lobe interacting with E127 on the C-lobe, as well as pairing between E14-E120. Thus, it may well be that the acidic contacts do not repel each other in the crystal due to the loss of the charges. In contrast, our MD simulations starting from the 1PRW structure assigns their usual charges to these residues to mimic the physiological pH conditions. Nevertheless, the interface accommodates the repulsions between the closely located negative charges by slightly expanding around the adjacent helices and rotations in the side chains (figure S2). Thus, the initial state is maintained during the 200 ns window of MD observations, regardless of the charge states of the interface residues. With this robust accommodation of charges in mind, we seek the reasons behind the relaxation of the initial X-ray structure to new conformations when the environmental conditions are perturbed. To be noted is the conformational plasticity in the MD runs at low IS (figure 3b), and the similarity between a subset of the NMR ensemble structures and state III structures. The ionized states of the acidic residues make the electrostatic component dominant and strongly oppose direct inter-domain association on the time scale of the simulations [41]. This fact does not keep the system from sampling a plethora of conformations in the φ space. Thus, to understand how the interfered charge distribution in the environment affects the vicinal solvent layer around the protein, we study the distribution of the solvated non-specific ions around the protein in each case. We display in figure 4a, the radial distribution function (RDF) of the ions in the solvent around the side chain heavy atoms of the protein at low and high IS. The first peak belongs solely to the contact of Na+ ions with O− atoms of the negatively charged residues. The second peak is due to the solvent mediated interactions. Interestingly, although there are plenty of positively charged residues on the surface of the protein, Cl− ions (which only exist in the physiological IS run) rarely interact with them. At low IS, the Na+ ions strongly interact with the negative charges on the protein, thus screening the extreme repulsions between the two lobes and allowing rotational motions around the linker. To achieve physiological IS, Cl- ions as well as additional Na+ ions are added to the system. In the presence of these additional mobile negative charges the Na+ ions mainly reside away from the protein surface and in bulk water where they may also dynamically interact with Cl− ions (we check that there is no permanent ion pairing occurring between Na+ and Cl− ions). More interestingly, lack of salt in the solvent environment reduces the time scale of conformational change by three orders of magnitude, from sub-milliseconds to sub-microseconds. Decomposed into the different regions on the protein at low IS (Figure 4a inset), the most significant interaction of the Na+ ions is with the linker residues, followed by those of the C-lobe and even less so with the N-lobe. We have also monitored the trajectories to find that the cations are mobile and they do not have a preferred position near the linker. These ionic distributions are contrary to expectations from the net charges, with that of the linker being only -1, whereas those of the N- and the C-lobes are -8 and -6, respectively. Thus, the ionic interactions are geometry specific, and designate the smooth surface of the linker (relative to the two lobes) as a region that has a tendency of binding non-specific ions. We conclude that the conformational plasticity of the torsional motions observed in altered charge environments is due to the clustering of the cations around the linker which screens the strong repulsions between the two lobes. We check the effect of the change in the number densities of the ions at different ionic strengths on the values of the RDF peaks. We confirm that the reduction of the peaks exceeds that expected by the 2. 3 fold increase in the number densities of the ions in the system (e. g. the linker peak is reduced 3. 3 fold.) In terms of the absolute values, the average number of Na+ ions within the first coordination shell of the acidic residues of the linker is 0. 5 and 1. 13 for the and systems, respectively. While the removal of Ca2+ ion from EF-hand loop I readily induced compaction of CaM in a previous MD simulation [42], we are interested in revealing its role in CaM dynamics in fully loaded state. We have previously shown that E31located in this loop is unique in that its perturbation in a given direction reproduces the closed form structure with high overlap [14]. In fact, unlike its positional counterparts on the other EF-hand loops of CaM, the role of E31 is not as central in Ca+2 ion coordination as its involvement in signaling coordination between the two lobes. This statement is supported by a series of experimental E→K point mutation studies at the four equivalent EF-hand positions (31,67,104 and 140) [43], [44]. Two results are striking: E31K mutation (i) has wild type activation on four different enzymes while the others do not; (ii) does not lead to apparent binding affinity changes while the rest lead to the loss of Ca+2 binding at one site. It was also shown that proton flux is an important factor affecting conformational changes in CaM and its enzyme targets [45]. We have therefore protonated E31 while keeping the IS low in the set of MD simulations. Since the topology of the residue is the same except for the reduced charge on the side chain, it still interacts with the Ca2+ in the EF hand I. We have monitored this motif throughout the trajectories and ensured there is no loosening in the motif. Strikingly, we find that the net effect of this single point protonation on the sampled conformations is similar to increasing the IS, keeping them near the initial extended structure (compare figures 2b and S3b), with an average l value of 33 Å and torsional angle range φ = [80°, 130°]. Despite the protonation of a single point, the RDFs measured in these runs are also more similar to the high IS system (figure 4b), significantly reducing the density of Na+ ion clustering around the protein, mainly affecting the linker region (value reduced to 3. 1 from 10. 3; see Table 2). E31 is able to significantly reduce the ion density around the protein and the linker at the same time (Table 2). For example, protonation of D122, the second residue with the most significantly upshifted pKa, in a control run also leads to similar values. E31/D122 double protonation further reduces the ion density around the whole protein and the linker; while the E31/H107 double protonation does not bring in this additional effect. However, protonation of 10 residues to mimic the pH 5 environment in is effective in further reducing the charges around the linker environment, while its effect on the overall protein is less apparent. The most drastic change in the extended conformation occurs in the system. As we discuss in detail below, the E31A mutation opens a direct path between the extended conformation and compact structures with a bent linker, accessing conformations not sampled by any of the other systems. runs are characterized by increased mobility of the N-lobe (4 Å RMSD) accompanied by an additional stability in the C-lobe (RMSD<2 Å) as well as the linker. The stability in the latter two regions, not directly perturbed by the E31A mutation, contrasts the simulations discussed in the previous subsection. We emphasize that the Ca+2 ion coordination is never lost in any part of these MD simulations which total 0. 55 µs and 1. 2 µs in and, respectively. By inspecting the MD trajectories, we find that the main direct difference between and runs is that while the calcium binding motif is not disrupted in the former, residue 31 can no longer participate in the motif in the latter due to its short side chain and hydrophobic character. Interestingly, the E31A mutation restores some of the depleted charge distribution around the acidic residues that occurred upon its protonation (Figure 4b and Table 2). The reorientation that takes place in the N-lobe is quantified by an increase of the RMSD value from 2 Å to 4 Å (Figure 5a). The angle between helices III–IV displays a drastic change, with helix III tilting towards helix IV. This is followed by the formation of a salt bridge between residues E47 and R86 at 60 ns which may be traced in the sharp decrease in l from 34 Å to 27 Å (Figure 5b). After the salt bridge formation, at 80 ns, the linker is further bent from residue 81 and l drops to 25 Å bearing a compact conformation. Snapshots are taken before and after transition and shown in Figure 5c. The observed conformational change is reversible, and the extended structure is restored at ca. 160 ns. No significant pKa shift appears for charged residues in any part of the trajectory. We find that the transition state is well defined, occurring through the same point in both forward and reverse steps. Time intervals of the transitions between the extended and compact (forward transition) and between compact and extended conformations (reverse transition) are examined in more detail in figure 6. The positions of structures near the transition state in 200 ps intervals are plotted on the (l, φ) plane. Note that the axes have been zoomed in. The transition between the extended and compact states is also examined via the tool Geometrical Pathways [46], [47]. This tool utilizes geometric targeting (GT) method that has recently been introduced [46] as a rapid way to generate all-atom pathways from one protein structure to some known target structure. GT is based on the philosophy that essential features of protein conformational changes can be captured by solely considering geometric relationships between atoms. The protein is modeled as a geometric system, with constraints established to enforce various aspects of structure quality such as preserving covalent bond geometry, preventing overlap of atoms, avoiding forbidden Ramachandran regions for backbone dihedral angles, avoiding eclipsed side-chain torsional angles, and maintaining hydrogen bonds and hydrophobic contacts. We note GT cannot predict relative timing of events. Using Geometrical Pathways in Biomolecules server [46], we have generated 10 random pathways between representative structures collected structures at 50 ns (extended) and at 80 ns (compact) of the run 1. The RMSD step size is 0. 05 Å. The structures generated in the forward pathway by Geometrical Pathways are also plotted on figure 6 with the median of the pathway and the standard error bars along both axes. The random pathways produced via Geometrical Pathways overlaps with that visited by MD. They are widely distributed along the interdomain torsional angle dimension, but have narrow distribution in end-to-end-linker distance. GT generated pathways take energetics into account indirectly, through geometric factors only. Their overlap with the MD pathway corroborates that the conformational change may be achieved as a series of geometrically viable sequential steps, if the energy barrier between the two states allows them to take place. In fact, a stabilized conformational change between states I and II is observed in only one of the three runs. However, several attempted jumps occur with a kinked linker conformation in all simulations (l<30 Å). Thus, the crucial step stabilizing the bent conformation is not the bending that is facilitated after the mutation, but the formation of the salt bridge between the N-lobe and the linker. Such attempts occur neither in nor in physiological IS runs. The relative positioning of the entering and exiting helices of the EF-hand motifs have been used to characterize the diverse conformations utilized by CaM for target recognition [48]. We therefore focus on the dynamics of the angles between the helices in EF hand motifs to understand how this “rare” conformational change is facilitated by E31A mutation. On the N-lobe, helices I and II in EF hand I, and helices III and IV in EF hand II are both initially posed at about right angles to each other, having values between 80–90°. In all three trajectories, the increase in the RMSD of the N-lobe occurs simultaneously with the loss of this perpendicular arrangement between the former pair of helices. The change in the orientation of the N-lobe helices provides E47 to make the salt bridge with R86. Perhaps most intriguing is the diminished fluctuations between helices V and VII upon E31A mutation. The values of 38±11° in the rest of the simulations of Table 1 are suppressed to 30±5° for all three. The model for inter-lobe communication of CaM is E31 mediated. E31A mutation induces conformational changes between the two EF-hand motifs in the N-lobe, which simultaneously stabilize helix reorientational fluctuations in the C-lobe. No apparent communication pathway between the two lobes is found, thus lending support to the ensemble view of allostery [49]. As a result, the N-lobe performs an intra-lobe conformational search to establish a salt bridge with the linker. The stabilized C-lobe helps maintain the initial contact. The solvent participates in the fluctuations that establish the background for the conformations sampled by the protein. Multiple conformations and cooperative conformational changes are an essential part of many enzyme mechanisms [50]. We explore the role of electrostatics in altering the conformation distributions as well as the dynamics of Ca2+-CaM using extensive MD simulations under physiological and low IS/pH conditions, and by mutating/protonating single residues. While the protein is stable in the initial state at physiological IS, lowering the IS or pH leads to conformational switching to more compact structures on sub-100 ns time scales (Figures 3 and S1). Although the net charges on the N- and C-lobes are significantly higher, at low IS cations approach the linker due to its relatively smooth geometry [51], screening the repulsions between the lobes. This leads to conformational plasticity, enabling large torsional motions around the linker, eventually causing a compact conformation within 200 ns, albeit with a stiff linker. Lowering the pH in addition to the IS, which in effect deletes 10 charges on the protein surface, contributes to the process further, letting the protein achieve similar conformations within 60 ns. At high IS, the Cl− counterions are repelled by the significantly negatively charged protein (figure 4a). Na+ ions are then driven into the bulk solvent, since the counterion interactions are favored over those with acidic residues both energetically and entropically. Repulsion between the two lobes is more pronounced because of ion depletion in the intervening region, restricting the protein conformation near the initial structure (figure 2). It would be interesting to make a systematic study of the effect of ions on the dynamics of CaM, including different ion types and concentrations. However, our scope here is to merely demonstrate that CaM dynamics is sensitive to ionic strength and may be moderated between rigid and very flexible. Such an observation has implications on both tuning and interpreting experimental results and on conditions selected for computer simulations. Perhaps more interestingly, neutralizing the single residue E31 at a key location by protonation has a similar effect on the ion distributions to increasing the IS (figure 4b), confining the sampled conformations near the initial structure (SI figure S3). Alanine mutation of the same residue results in an intermediate distribution of ions, leading to partial mobilization of the protein. This facilitates a bending in the linker near the extended conformation (figure 5). E31A mutation accentuates the existing allosteric interactions, by introducing a change originating on the N-lobe whose action is detected on the C-lobe via the rearrangements of the helices in the EF-hands. The coupling between the two lobes is detected simultaneously, and the stabilizing salt bridge between the N-lobe and the linker is established later on in the simulation, right before the transition state is reached (figures 5c and 6). Thus, no pathway of structural distortions between the allosteric sites is observed, lending support to the ensemble view of allostery [49]. Finally, we note that the sampled structures have representatives in the NMR ensemble of conformations [25]. 40% of the 160 conformers have been visited in the cumulative MD simulations. On the downside, these are predominantly the ones with the straight linker, while the compact conformers (those with l<25 Å) have been rarely observed. The structures are compatible with the FRET results both in terms of the interlobe distances and the fact that lowering the pH to 5. 0 leads to a single stable state as opposed to the presence of at least two distinct forms at pH 7. 4 [13]. Our observations are also compatible with the findings of Bertini and coworkers [31]. Therein, low occupancies are assigned to fully compact structure of Ca2+-CaM (1PRW). Extended conformers similar to 3CLN in general have low occupancies although some other extended conformers have occupancies as high as 35%. This supports our findings that in our cumulative MD trajectories, the initial structures whether starting from compact or extended crystalline structures of Ca2+-CaM relaxes to less extended or less compact forms. Our current view suggests that at physiological IS and pH, there exists a barrier between the extended and compact forms, leading to 100 µs time scales for conformational jumps [27]. Barrier crossing is prevented by the repulsive electrostatic interactions between the two negatively charged lobes. As we show in this work, the crossing may be facilitated by tuning environmental conditions or by perturbing single residues located at key positions. The current study contributes to the knowledge-base in the direction of methods that determine how proteins adapt to changes in their environment or structure.

Title: Designing Molecular Dynamics Simulations to Shift Populations of the Conformational States of Calmodulin Summary: Calmodulin (CaM) is involved in calcium signaling pathways in eukaryotic cells as an intracellular Ca2+ receptor. Exploiting pH differences in the cell, CaM performs a variety of functions by conveniently adopting different conformational states. We aim to reveal pH and ionic strength (IS) dependent shifts in the populations of conformational substates by modulating electrostatic interactions amongst the different regions of the protein and with its vicinal water. For this purpose, we design extensive molecular dynamics simulations to classify the effects that are responsible for adopting different conformations exhibited in the ensemble of NMR structures reported. Lowering the IS or pH, CaM experiences higher inter-lobe orientational flexibility caused by extreme change in the non-specific ion distribution in the vicinal solvent. Amongst the titratable groups sensitive to pH variations, E31 is unique in that its protonation has the same effect on the vicinal layer as increasing the IS. Furthermore, E31A mutation causes a large, reversible conformational change compatible with NMR ensemble structures populating the linker-kinked conformations. The mutation in the N lobe, at a significant distance, both modulates the electrostatic interactions in the central linker and alters the EF-hand helix orientations in the C lobe.

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Write a title and summarize: Snakebites have been recognized as a neglected public health problem in several tropical and subtropical countries. Bothrops snakebites frequently complicate with acute kidney injury (AKI) with relevant morbidity and mortality. To date, the only treatment available for Bothrops envenomation is the intravenous administration of antivenom despite its several limitations. Therefore, the study of novel therapies in Bothrops envenomation is compelling. The aim of this study was to evaluate the protective effect of Allopurinol (Allo) in an experimental model of Bothrops jararaca venom (BJ) -associated AKI. Five groups of Wistar rats were studied: Sham, Allo, BJ, BJ+Allo, BJ+ipAllo. BJ (0. 25 mg/kg) was intravenously injected during 40’. Saline at same dose and infusion rate was administered to Sham and Allo groups. Allo and BJ+Allo groups received Allo (300 mg/L) in the drinking water 7 days prior to Saline or BJ infusion respectively. BJ+ipAllo rats received intraperitoneal Allo (25 mg/Kg) 40’ after BJ infusion. BJ rats showed markedly reduced glomerular filtration rate (GFR, inulin clearance) associated with intense renal vasoconstriction, hemolysis, hemoglobinuria, reduced glutathione and increased systemic and renal markers of nitro-oxidative stress (Nitrotyrosine). Allo ameliorated GFR, renal blood flow (RBF), renal vascular resistance and arterial lactate levels. In addition, Allo was associated with increased serum glutathione as well as reduced levels of plasma and renal Nitrotyrosine. Our data show that Allo attenuated BJ-associated AKI, reduced oxidative stress, improved renal hemodynamics and organ perfusion. It might represent a novel adjuvant approach for Bothrops envenomation, a new use for an old and widely available drug. Snakebites have been recognized as a neglected public health problem in several tropical and subtropical countries [1]. The worldwide incidence of snake envenomation may vary from 421,000 to 1,841,000 cases each year, resulting in up to 94,000 deaths [2]. However, the burden of snakebite envenomation might be underestimated, since many incidence data are derived from hospital admissions and most of the affected individuals do not seek hospital treatment [3]. Despite the nearly universal distribution, venomous snakebites are mainly found among populations living in poverty as it is inversely related to the health care expenditure [4]. These accidents predominantly affect young adults, especially males, yet almost 1/3 of the cases occur in children [5]. Moreover, snake envenomation may also be seen as an occupational hazard whereas it has been associated with significant morbidity and disability mostly in agricultural workers (food producers) [4–6]. Thus, there is a great impact of venomous snakebites in the economy of a community or even a country. Snakes of the genus Bothrops are the major cause of snakebites in Latin America [5]. Over 60 species distributed throughout Central and South America and the Caribbean Basin constitute the genus Bothrops [7,8]. In Latin America, Bothrops asper, Bothrops atrox and Bothrops jararaca are the main medically important Bothrops species [5,9]. Previous studies have reported substantial differences in venom composition among the genus Bothrops which might be explained by variations within species, geographic regions, dietary availability, or even by age difference considering the same animal [8,10–16]. However, some venom biological properties are common to all Bothrops species [8]. Bothrops venoms possess many active enzymes responsible for the local and systemic manifestations [7]. Local edema is a common finding which may be associated with drastic hemorrhagic and necrotic changes [17]. Furthermore, Bothrops venoms exhibit thrombin-like enzyme activity which activates the coagulation cascade and ultimately causes fibrinogen consumption, bleeding and disseminate intravascular coagulation [5,8, 18]. Bothrops envenomation frequently complicates with acute kidney injury (AKI) increasing morbidity and mortality [10,19]. To date, the only treatment available for Bothrops envenomation is the intravenous administration of antivenom (animal hyperimmune immunoglobulin). However, there are several limitations of the antivenom therapy such as high cost, limited availability, (e. g. in remote areas), severe anaphylactic reactions, and inefficiency to treat local manifestations [6,20–22]. Therefore, the study of novel therapies in Bothrops envenomation is a fruitful area for future research. Allopurinol (Allo) is a xanthine oxidase (XO) inhibitor commonly used as uric acid (UA) -lowering agent. Data from several studies suggest that beneficial effects of Allo as a free radical scavenger may transcend the XO inhibition such as reducing lipid peroxidation and heat shock protein expression [23,24]. Additionally, treatment with Allo following rhabdomyolysis-associated AKI showed renal and muscular protective effects by reducing oxidative stress and UA levels [23]. Thus, new therapeutic applications have been accredited to Allo in the last decade particularly due to its potent antioxidant property [23,25–27]. Strapazzon et al. showed marked and persistent increase of several oxidative stress markers in patients hospitalized for Bothrops envenomation [28]. Therefore, the aim of this study was to evaluate the protective effect of Allo, a low-cost and widely available medication, in an experimental model of Bothrops jararaca venom (BJ) -associated AKI. All experimental procedures were approved by the local research ethics committee (“Comissão de Ética no Uso de Animais”–CEUA, 070/17) and developed in strict conformity with local institutional guidelines and with well-established national standards for the manipulation and care of laboratory animals (“Resoluções Normativas do Conselho Nacional de Controle de Experimentação Animal”–CONCEA). Animals were anesthetized intraperitoneally with sodium thiopental (50 mg/kg BW) for all the surgical experiments. After the experimental period, animals were euthanized with excess anesthetic sodium thiopental. A pool of lyophilized venom from specimens of adult B. jararaca snakes was a generous gift from the Institute Butantan, São Paulo, Brazil (lot 01/08–01). B. jararaca snakes are kept in plastic cages in temperature and humidity-controlled rooms, according the Institute Butantan Animal Care and Use Committee (protocol n° 1296/16). The venom is extracted monthly and after centrifugation the supernatant is frozen. Subsequently venoms are pooled, lyophilized, and stored at −20°C until used. On the experiment day, aliquots were dissolved in 10 ml of 0. 9% saline for immediate use. In the present study, we aimed to use doses of Allo well established in other experimental rat studies [29–31]. Nevertheless, in view of the U. S. Food and Drug Administration (FDA) approach to make a dose extrapolation from humans to rats, the dose of a rat would be approximately seven times higher than the human dose (per Kg of BW) [32]. Thus, taking into consideration a daily dose of Allo (300 mg) in an adult human weighting 60 kg (5 mg/Kg/day), the dose of Allo to be administered to the rat would be approximately 35 mg/kg/day. Considering the mean daily water intake of 25 mL/rat/day and mean BW of 272 g, we used a dose of approximately 28 mg/kg/day in the BJ+Allo group. BJ+ipAllo animals received 25 mg/Kg BW of Allo, which was around 5 times the human dose. Male Wistar rats with a mean BW of 272±5 g were obtained from the animal facilities of the University of São Paulo School of Medicine, housed in standard cages, on a 12h light/dark schedules, and given ad libitum access to water and standard diet (Nuvilab CR-1, Curitiba-PR, Brazil). Room temperature was maintained at 23°C. We administered intravenous BJ after the surgical procedure for the inulin clearance study, as previously reported by Martines et al. [6]. Animals were allocated into four groups: In order to study the therapeutic effect of Allo on renal function, we performed an additional group of animals (BJ+ipAllo, n = 5) that received intraperitoneal Allo (Sigma-Aldrich, St. Louis, USA) (25 mg/kg BW) immediately after BJ infusion. Fig 1 illustrates the experimental protocol in the first series of experiments. In separate series of experiments, using the same experimental model described above, we conducted hemodynamic studies after intravenous BJ or saline infusion (five groups of six rats/group). Animals were anesthetized intraperitoneally with sodium thiopental (50 mg/kg BW). The trachea was cannulated with a PE-240 catheter, and spontaneous breathing was maintained. To evaluate the mean arterial pressure (MAP) and allow blood sampling, a PE-60 catheter was inserted into the right carotid artery. MAP was measured 30 minutes after the surgical procedure, using Biopac Systems Inc MP100 (Santa Barbara, CA, USA). Then, a midline incision was made to measure the renal blood flow (RBF). We carefully dissected the left renal pedicle and isolated the renal artery, taking precautions to avoid disturbing the renal nerves. An ultrasonic flow probe was placed around the exposed renal artery. RBF was measured using an ultrasonic flow meter (T402; Transonic Systems, Bethesda, MD, USA) and is expressed as ml/min. Renal vascular resistance (RVR) was calculated by dividing the blood pressure by RBF and is expressed as mmHg/ml/min. To determine the glomerular filtration rate (GFR), we conducted inulin clearance studies. On the day of the experiment, animals were anesthetized and cannulated as above. For infusion of inulin and fluids, another PE-60 catheter was inserted into the left jugular vein. For BJ or 0. 9% saline infusion, a PE-60 catheter was inserted into the right jugular vein. In order to collect urine samples, a suprapubic incision was made, and the urinary bladder was cannulated with a PE-240 catheter. After surgical procedure, a loading dose of inulin (100 mg/kg BW diluted in 0. 9% saline) was administered through the jugular vein. Subsequently, a constant inulin infusion (10 mg/kg BW in 0. 9% saline) was started and was continued at 0. 04 ml/minute throughout the experiment. Blood samples and three urine samples were obtained at the beginning and at the end of the experiment. Blood and urine inulin were determined using the anthrone method. GFR data are expressed as ml/min/100g BW. At the end of either inulin clearance, organs were perfused with PBS solution (0. 15 M NaCl and 0. 01 M sodium phosphate buffer, pH 7. 4). The right kidney was removed and frozen in liquid nitrogen and stored at −80°C. The left kidney was cleaned of connective tissue and fixed in 10% formalin. Kidney sections were homogenized in an ice-cold isolation solution (200 mmol/l mannitol, 80 mmol/l HEPES, 41 mmol/l KOH, pH 7. 5) containing a protease inhibitor cocktail (Sigma, St. Louis, MO, USA), using a Teflon-pestle glass homogenizer (Schmidt and Co., Frankfurt am Main, Germany). The homogenates were centrifuged at low speed (4,000 rpm) for 30 min at 4°C to remove nuclei and cell debris. Pellets were suspended in isolation solution with protease inhibitors. Protein concentrations were determined using the Bradford assay method (Bio-Rad Protein Assay Kit; Bio-Rad Laboratories, Hercules, CA, USA). Plasma and renal nitrotyrosine (NT) levels, which are stable end products of peroxynitrite oxidation, were measured using a commercial ELISA kit (HK: 501–02; Hycult Biotech, Uden, the Netherlands). Reduced GSH, the major endogenous antioxidant in cells, was determined in total blood by the method of Sedlak and Lindsay [33]. Whole blood was processed by addition of four volumes of ice-cold 5% metaphosphoric acid and centrifuged at 4,000 rpm for 10 min at 4°C. This assay consists of the reaction of supernatants of total blood samples with Ellman’s reagent to produce a yellow pigment measured spectrophotometrically at 412 nm. Serum GSH was quantified by means of the standard curve and reported as μmol/mL. Plasma sodium and potassium were measured by flame photometry (CELM, model FC280, São Paulo, SP, Brazil). Hematocrit, serum lactate and bicarbonate were determined with specific electrodes (ABL800Flex–Radiometer, Brønshøj, Denmark). Fibrinogen was assessed using the Clauss modified method (hemostasis coagulation analyzer, Stago Start 4, France). Lactate dehydrogenase (LDH) was determined with a kinetic method using UV absorbency, which measures the conversion from L-lactate in pyruvate (automatized analyzer, Cobas C111, Roche, Switzerland). The enzymatic colorimetric method (Labtest, Lagoa Santa, Brazil) was used to measure plasma creatine phosphokinase (PCPK) and plasma uric acid (UA). Hemoglobinuria was assessed using a dipstick urine test (Cobas, Roche, Switzerland). Four-micrometer histological sections of kidney tissue were stained with hematoxylin–eosin (HE) or Masson’s trichome and examined under light microscopy. For histomorphometry, the images obtained by microscopy were captured on video via an image analyzer (Axiovision; Carl Zeiss, Eching, Germany). We analyzed 30 grid fields (0. 087 mm2 each) per kidney cortex. The interstitial areas were demarcated manually, and the proportion of the field they occupied, excluding the glomeruli, was determined. In 40–60 grid fields (0. 245 mm2 each; magnification, x400), we graded the proportional renal damage (tubular epithelial swelling, vacuolar degeneration, necrosis, and desquamation): 0, < 5%; I, 5–25%; II, 26–50%; III, 51–75%; and IV, > 75%. To minimize bias in the morphometric analysis, the observer was blinded to the treatment groups. The mean scores were calculated by rat and by group. We used monoclonal antibody to 8-isoprostane-PGF2a (F2-IsoP) (1: 500, overnight at 4°C) (Oxford Biomedical Research, Oxford, England). We subjected 4-μm kidney tissue sections to immunohistochemical reaction according to the protocol for the primary antibody. Reaction products were detected by an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA), and the color reaction was developed with 3,3-diaminobenzidine (Sigma Chemical) in the presence of hydrogen peroxide. The sections were counterstained with Harris’ hematoxylin. To evaluate immunoreactivity to F2-IsoP, we analyzed 15 randomly fields of the renal medulla (0. 087 mm2 each). The volume ratios of positive areas of renal medula (%), determined by the color limit, were obtained by image analysis with the program Image-Pro Plus, version 4. 1 (Media Cybernetics, Silver Spring, MD) on a computer coupled to a microscope (Axioskop 40; Carl Zeiss) and a digital camera. Results were expressed as percentages. Data are shown as mean ± SEM. Differences among the means of multiple parameters were analyzed by one-way analysis of variance followed by the Student–Newman–Keuls test. Values of P<0. 05 were considered statistically significant. Data were analyzed using GraphPad Prism software 5. 0. To evaluate the efficacy of prophylactic Allo on BJ-associated AKI, animals received Allo seven days prior to intravenous venom infusion. Renal function, assessed by inulin clearance, was markedly reduced in the BJ group compared to controls. In contrast, Allo pretreated rats exhibited significantly better renal function (Fig 2A). Histopathological examination of kidney tissues showed glomerular microthrombi deposit in the groups that received BJ (Fig 3A). Sham and Allo groups did not show any significant histopathological changes. Tubular injury score was not different between groups (Fig 3B). To assess whether the potent antioxidant effect of Allo would play a role in renal protection in the current experimental model, we evaluated serum reduced GSH (a major endogenous antioxidant), plasma and renal NT (a marker of marker of nitro-oxidative stress). Animals from the BJ group exhibited lower levels of serum reduced GSH compared to control animals. Allo pretreatment improved serum reduced GSH levels (Fig 2B). On the other hand, BJ rats exhibited higher levels of plasma and renal NT, which returned to control levels in the group pretreated with Allo (Fig 2C and 2D). Overall, these data suggest an improvement in the systemic and renal redox balance conferred by Allo following B. jararaca envenomation. Renal ischemia has been considered the hallmark of AKI following B. jararaca envenomation [6,34,35]. Accordingly, we observed severe renal vasoconstriction, associated with increased renal vascular resistance, in BJ animals at 40 and 70 minutes after venom infusion. Allo pretreatment significantly improved RBF and RVR at both time points evaluated. MAP was lower in the BJ group after BJ infusion, albeit not statistically significant. Fig 4A shows a summary of the hemodynamic data. In addition, we found increased expression of F2-IsoP, a potent vasoconstrictor formed during lipid peroxidation, in the renal medulla (Fig 4B). BJ animals showed significant increased expression of F2-IsoP in the renal medulla compared to other groups. Allo pretreatment reduced F2-IsoP medullary renal expression (Fig 4B). Plasma sodium was not different between groups, whereas animals from BJ group showed higher plasma potassium compared to other groups (Fig 5A and 5B). Furthermore, BJ infusion led to a marked decrease in fibrinogen and bicarbonate levels whilst increased serum LDH and lactate levels. BJ+Allo rats exhibited lower arterial lactate, yet fibrinogen, LDH and bicarbonate changes were not prevented by Allo pretreatment (Fig 5C–5F). Moreover, Allo-pretreated rats showed lower plasma UA concentration (Fig 5G). As expected, PCPK was slightly increased, albeit not statistically significant, after intravenous BJ administration (Fig 5H). Although we found hemoglobinuria in animals that received BJ, there were no differences on hematocrit levels between groups (Fig 5I and 5J). We have conducted additional studies using intraperitoneal Allo after BJ infusion in order to assess if therapeutic Allo also improved the functional parameters (renal function, RBF and RVR). Strikingly, therapeutic Allo also improved renal function (0. 74 ± 0. 05 mL/min/100g BW; p<0. 01 vs. BJ group), RBF (4. 4 ± 0. 5 mL/min; p<0. 01 vs. BJ group) and RVR (18. 5 ± 1. 5 mmHg/mL/min; p<0. 001 vs. BJ group) following BJ envenomation. AKI seems to be a major cause of death among patients who survived the early BJ-induced systemic effects [10]. Animal models of AKI following intravenous infusion of BJ have been validated to study the mechanisms involved in this syndrome as well as potential therapeutic agents [6,34,35]. Similar to previous studies, we found that intravenous administration of BJ caused striking reduction of renal function associated with intense renal vasoconstriction, fibrinogen consumption and intravascular hemolysis without systemic blood pressure changes. We also reported that prophylactic and therapeutic Allo attenuated renal dysfunction secondary to BJ infusion. To our knowledge, this is the first demonstration of Allo as both treatment and prevention of BJ-associated AKI. In recent years, there has been an increasing amount of literature on the role of oxidative stress in the pathogenesis of snake envenomation [28,36,37]. Snake venoms present a series of biologically active peptides, such as L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) which might be related to the generation of reactive oxygen species (ROS) [38,39]. LAAOs are flavoenzymes widely distributed among venomous snake families that catalyze the oxidative deamination of L-amino acid to α-keto acids, thus, releasing hydrogen peroxide and ammonia [39]. Venom PLA2, in contrast to endogenous PLA2, has been linked with a wide spectrum of toxic effects, leading to increased systemic oxidative stress [39,40]. Therefore, to evaluate the involvement of the oxidative stress in the pathogenesis of BJ-associated AKI, as well as its inhibition by Allo therapy, we assessed serum reduced GSH, plasma and renal NT. GSH is the most prevalent intracellular thiol, whose main role is to protect cells from oxidative damage [41]. On the other hand, NT is a byproduct of protein tyrosine residues generated from the reaction of nitric oxide (NO) and superoxide (O2•—) [42]. In recent years, it has been demonstrated the important role of NO regulating the redox signaling [43]. When reacted with O2•—, NO is a major substrate to generate reactive nitrogen species which can lead to nitration and nitrosation of several substrates, mostly proteins with various physiological functions [43]. This biological process has been called by some authors as nitro-oxidative stress [43–45]. Furthermore, Walker et al. described renal NT as an early marker of oxidative stress in the setting of renal ischemia reperfusion [46]. We found evidence of redox imbalance in the rats from BJ group. BJ infusion was associated with reduced levels of GSH levels and increased systemic and renal markers of nitro-oxidative stress, whilst Allo pretreatment improved all these markers. Renal vasoconstriction is one of the key elements in the pathogenesis of BJ-associated AKI [6,34,35]. Martines et al. showed a significant reduction of RBF and increase of RVR following BJ infusion [6]. Furthermore, previous studies have highlighted the pivotal role of isoprostanes in oxidative stress-mediated renal vasoconstriction [23,47,48]. Accordingly, we found profound renal vasoconstriction combined with increased expression of F2-IsoP in the renal medulla. We believe that two factors might have contributed to this finding. First, given the high sensitivity of the medulla to decreased O2 supply, it is reasonable to expect that it would be the first portion of the kidney affected by vasoconstriction and oxidative stress [49]. Second, since we performed an early assessment of F2-IsoP (90’ after BJ infusion), we might not have been able to detect cortical alterations at this stage. Allo ameliorated renal hemodynamic changes in this experimental model. We suggest that Allo might have inhibited the lipid peroxidation and consequently reduced F2-IsoP formation. Conversely, F2-IsoP may represent a marker of increased oxidative stress following intense vasoconstriction in the renal medulla. Nevertheless, we could not provide evidence of causality between F2-IsoP and BJ-induced renal vasoconstriction and this is still an important issue for future research. In addition to the renal hemodynamic changes, fibrin thrombi deposits in the glomerular capillary may also play a role in the pathogenesis of BJ-associated AKI contributing to the reduction in GFR and RBF [34]. Hence, data from necropsy kidney samples showed glomerular fibrin thrombi following Bothrops snakebites in individuals with cortical necrosis [50]. We showed glomerular microthrombi deposit in the groups that received BJ. Nevertheless, tubular injury score was not different between groups, which might be due to the renal tissue harvesting in the very early stage of AKI, therefore, too early to observe significant renal histological damage. On the other hand, we did not find significant difference in CPK levels among the study groups, which speaks against rhabdomyolysis as a pathogenic factor for AKI in this experimental model. In fact, Burdmann et al. also reported no increase in CPK levels using similar animal model. We believe that rhabdomyolysis is mainly a local manifestation of BJ envenomation, thus, occurring after subcutaneous or intramuscular inoculation of the venom rather than with intravenous injection. In the past years, numerous studies have reported the pro-oxidant properties of UA [51–53]. Higher UA levels may activate NADPH oxidase, increasing protein nitrosation and lipid peroxidation [54]. Moreover, several lines of evidence suggest an association between UA with hypertension, cardiovascular disease, progression of chronic kidney disease and rhabdomyolysis-associated AKI [23,55–57]. In the present study, we assume that the positive effects of Allo resulted predominantly from free radical scavenging. However, we may not neglect the potential adjuvant effect of UA-lowering therapy. Therefore, further research is warranted to uncover the role of UA in the pathogenesis of BJ-associated AKI. Bothrops venoms are composed of several different substances related to broad range of systemic effects [7]. Intravenous infusion of BJ has been linked with fibrinogen consumption, intravascular hemolysis and shock (reduced organ perfusion). Similarly, our animal model reproduced all the systemic effects of BJ envenomation as described in previous studies [6,34,35]. However, one limitation of our experimental model was the short follow up after BJ infusion. This might be the reason why hemoglobin levels as well as MAP were not significantly lower in the BJ group compared to other groups. In fact, we believe that we might have observed a period of cryptic shock (hyperlactatemia without persistent hypotension) associated with initial features of hemolysis (increased LDH and hemoglobinuria) and hemoconcentration, the latter also described in early stages of hemorrhagic shock [58]. Overall, BJ+Allo rats showed better organ perfusion (i. e. lower arterial lactate levels) which is in accordance with previous studies that reported protective effects of Allo in experimental models of hemorrhagic shock by improving hemodynamics, preserving tissue levels of ATP and ultimately reducing mortality [59,60]. Therefore, the studies discussed here highlighted the role of Allo beyond free radical scavenging in the setting of shock. In conclusion, our study show that Allo attenuated BJ-associated AKI, reduced oxidative stress, improved renal hemodynamics and organ perfusion. We hope that this study encourages further clinical investigation regarding the beneficial effects of Allo on Bothrops envenomation. If supported by clinical studies, it might represent a novel adjuvant approach for Bothrops envenoming, a new use for an old and widely available drug.

Title: Allopurinol attenuates acute kidney injury following Bothrops jararaca envenomation Summary: Snakebites have been recognized as a neglected public health problem in several tropical and subtropical countries. Snakes from the genus Bothrops are the most common snakes found in Latin America. Bothrops snakebites frequently complicate with kidney impairment with increased risk of death. The only treatment currently available for Bothrops envenomation is the infusion of antivenom. Therefore, the study of novel therapies to prevent kidney failure caused by snakebites is compelling. Allopurinol is a drug commonly used to treat gout which also presents beneficial antioxidant properties. The aim of this study was to evaluate the protective effect of Allopurinol in a rat experimental model of Bothrops envenomation. In our study, rats that received the infusion of the snake venom presented with reduced kidney function which was improved by Allopurinol. Overall, Allopurinol might represent a novel approach to prevent kidney failure in victims of Bothrops snakebites. This could be a new use for an old and widely available drug.

lay_plos

en

Summarize: CROSS REFERENCE TO RELATED APPLICATIONS None STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. REFERENCE TO MICROFICHE APPENDIX Not applicable. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to a knife. More specifically, the present invention relates to a field dressing knife for complete field dressing of small and large game and having special application to the severing of the pelvis and brisket bones of small and large game. 2. Background Art Soon after a game animal is killed, field dressing is required to inhibit the growth of bacteria, thereby preventing premature spoilage of meat. Field dressing refers to the process of preparing the carcass of a game animal, often at the approximate location the animal was killed, to minimize spoilage and preserve the meat for later consumption. Once all life functions have ceased, the carcass begins to decay. Unchecked bacterial growth of the internal organs (viscera) and body fluids spreads quickly through the carcass causing the meat to be unfit for human consumption in a matter of hours, depending on the ambient temperature. To prevent this, the process of gutting, or evisceration, is performed. Gutting involves removing all viscera inside the abdomen and thorax. Removing viscera prevents bacterial growth in the viscera from spreading to the meat, aids in cooling the meat, and prevents unpleasant visceral body fluids from spreading to the meat. Heat and moisture further increase the spread of bacteria. The process of gutting necessarily includes severing the brisket and pelvis bones of the game animal carcass in order to remove all the viscera contained under these bones and to open these areas up for better air drying and cooling. Several devices have been used over the years by hunters for field dressing large game, especially for severing of the pelvis and brisket bones. One such tool is a handsaw 100 as illustrated in FIGS. 1 and 2. Handsaws 100 tend to be large and awkward to carry in the field while hunting. They work better on the brisket, where a full pass of the saw blade is possible, than on the pelvis bone. For splitting the pelvis bone, it is natural to attempt to saw from one end to the other as shown in FIG. 1. However, a full pass of the saw blade cannot be made because the area between the pelvis bone and the spine is insufficiently deep. This region also contains both the urethra 110 and colon 120. Sawing the pelvis with vertical strokes, while the carcass is on its back, results in puncturing these waste tubes and contamination of the meat. Placing the handsaw blade across the exposed pelvis bone, parallel to the spine as shown in FIG. 2 is more difficult. In this position, the saw teeth engage a minimum of six inches of bone in a large animal such as an elk. Additionally, sawing from the back end of the elk places the tip of the handsaw 100 directly in line with both the colon 120 and the urine bladder 210. There is, again, danger of meat contamination. Saw blades used for field dressing large game tend to be difficult to clean. As well, bone rapidly dulls the saw 100 which is difficult to sharpen, especially in the field. For complete field dressing of large game, when using a handsaw 100 for severing the pelvis and brisket bones, both the handsaw 100 and a knife must be carried while hunting. Another tool used for splitting the pelvis and brisket bones of large game is a hatchet. A hatchet is heavy for hunters to carry all day. The most convenient way to carry a hatchet is to secure it in a scabbard attached to a belt. The hatchet handle, dangling down below the waist, tends to interfere with tall grass, brush, and trees as the hunter moves through the field or woods. Like the handsaw 100, a hatchet works better on the brisket bone than on the pelvis bone. Caution must be taken when chopping through the pelvis bone because of the proximity of the urethra 110, bladder 210, and colon 120, as mentioned in conjunction with the handsaw 100. Unpleasant results due to using a hatchet for splitting the brisket include jagged and sharp bone splinters left protruding from the split brisket. During the removal of the windpipe and lungs, the hunter&#39;s arm, with a knife in hand, must pass through the chopped brisket opening in order to cut tendons. As with the handsaw 100, two tools—a hatchet and a knife—must be carried while hunting in order to fully field dress game including splitting the pelvis and brisket using the hatchet. Another tool available to hunters for field dressing is a wire saw. Wire saws are compact and light for carrying in the field. However, in use, the end of the wire is difficult to thread under the pelvis bone of a large game carcass to extend out on both ends of the pelvis. A goal is to avoid puncturing the bladder 210 or colon 120. Once threaded, the wire saw ends can be pulled back and forth to cut the pelvis. One drawback of the wire saw is that it kinks with use and becomes unfit for reuse. The wire saw is typically only used for the pelvis bone since the pelvis bone is sufficiently small for the saw to pass completely under it and pulled from the top. The brisket is typically too large for this. The hunter must then carry a knife, wire saw, and another device for splitting the brisket. Knives with saw blades represent another class of tool used for field dressing. These knives are easy to carry but difficult to use to saw through the pelvis and brisket bones of large game animals. The saw blade included in a knife typically does not have sufficient length to be used horizontally across the pelvis bone. Thus, the saw blade is typically oriented vertically to saw through the pelvis bone from one end to the other, aiming the end of the saw blade toward the spine. Again, the urethra 110 and colon 120 are likely to be punctured and the meat contaminated. Knife saw blades tend to have very pointed teeth that dull rapidly. These teeth are difficult to sharpen. Saw blades included with knives, and particularly those having aggressive saw teeth, are difficult to clean. Bone chips, flesh, and fat lodge in-between the teeth, requiring rubbing and/or brushing for removal. An advantage to the use of a knife saw blade is: only one device needs to be carried while hunting. Present day hunting knives typically have a blade that is too long for the purpose of splitting the pelvis or brisket bones of large game. When impacting such a knife with a stone or stick, as is sometimes done, a misalignment can easily break the knife blade. Additionally, a single-edged blade cannot be effectively worked back and forth in the bone to elongate a groove. As shown in FIG. 3, such an attempt will only elongate the groove 310 in the direction of the sharp edge of the blade 300. A skinning and gutting knife was disclosed by Bloch in U.S. Pat. No. 5,033,987. The longitudinal axis of the handle of this knife is not parallel with the longitudinal axis of the blade. The disclosed method for applying a large impact force is to strike the back of the blade (opposite the edge) with a foreign object encountered in the field, such as a rock or piece of wood. Such application has the potential to damage a knife by applying a force that is not parallel to the plane of the blade. Furthermore, there is no guarantee a foreign object such as a rock or piece of wood will be handy to a hunter, especially in conditions of deep snow. A gutting knife is disclosed in U.S. Pat. No. 6,578,271 by Macek. This knife has two forward extensions with a slot between them. In that slot are saw teeth. A method for severing bone is described as applying the slot to the bone and working the handle up and down to apply the saw teeth to the bone. Because the knife blade extends into the abdominal cavity of the carcass, when applying this knife to the severing of a pelvis bone of a large game animal, the probability of cutting or tearing the urethra 110 or the colon 120 is very high. Bone severed by impact of the knife is not disclosed. There is, therefore a need for a single tool by which small and large game may be field dressed, including the expedient and safe severing of the pelvis and brisket bones. BRIEF SUMMARY OF THE INVENTION An object of the present invention is to provide a single tool by which small and large game may be field dressed, including the severing of the pelvis and brisket bones. An additional object is for a tool that is sufficiently light and compact to be carried by a hunter in the field or woods for extended periods. Further, it is an object of this invention to provide a method for conveniently and efficiently splitting the pelvis and brisket bones of small and large game with the same tool used for other field dressing steps. To effect the above objects, a novel field dressing knife is disclosed. Various embodiments of the field dressing knife include the following design aspects: a large surface area at the butt-end of the knife handle, a circumferential groove near the butt-end of the knife handle for engaging the index finger and thumb of the user, further increasing the effective surface area of the butt-end of the handle, a double-edged blade, and a conversion to a hatchet-like device for chopping. The field dressing knife handle butt-end will preferably have a surface area between 1.35 and 10 square inches. In use for splitting the pelvis and brisket bones of large game, the field dressing knife may be held with a gripping hand and the butt-end of the knife handle struck with the user&#39;s free hand to penetrate the cartilage and bone. The great surface area of the butt-end of the field dressing knife, and the additional area provided by the user&#39;s gripping hand, are needed for impacting the butt-end of the knife handle with a hand with sufficient force to sever bone but not so much as to cause a hand injury. After gaining some entrance into the pelvis or brisket bone with the field dressing knife blade, the knife may be worked back and forth to extend the groove made by the knife. Therefore, the blade of the knife is preferably sharp on both edges, so it will cut when worked in both directions. Some hunters have grown accustomed to using a hatchet for splitting the pelvis and brisket bones of their large game. To provide a single tool for both hatchet and knife field dressing operations, the handle of the instant invention may be configured as a knife handle or as a hatchet handle. The handle, when configured for hatchet use, may be angled relative to the blade in a plurality of angles. This feature increases the versatility of the field dressing knife, and permits the user to select the best angle for the instant need. The field dressing knife of the present invention may include a gut hook and a blade suitable for field dressing operations other than splitting bone and severing cartilage. Hence, a single, compact tool may be carried in the field to perform all operations of field dressing. Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS FIG. 1 is a detail view showing a handsaw being used in a vertical orientation for splitting the pelvis bone of a large game carcass; FIG. 2 is a detail view showing a handsaw being used in a horizontal orientation for splitting the pelvis bone of a large game carcass; FIG. 3 is a detail view of a single-edged knife being used for splitting bone; FIG. 4 is a side elevation view of a field dressing knife of the present invention; FIG. 5 is a detail of the field dressing knife showing the handle butt-end groove; FIG. 6 is a plan view showing the field dressing knife being held ready to be struck with a free hand; FIG. 7 is a perspective view of the field dressing knife held in a hand for skinning and cutting; FIG. 8 is a perspective view of the field dressing knife held in position for severing bone by striking with a free hand; FIG. 9 is a side elevation view of a field dressing knife blade, a thumb-push ridge, and finger guard; FIG. 10 is a detail view of a double-edged knife being used for splitting bone; FIG. 11 is a detail of a large animal carcass showing the brisket bone; FIG. 12 is a perspective view of the field dressing knife being used to split the brisket bone of a large animal carcass; FIG. 13 is a side elevation view of a field dressing knife with a handle contracted; FIG. 14 is a side elevation view of a field dressing knife with a handle extended, longitudinal axes of the handle and a blade being parallel; FIG. 15 is a side elevation view of a field dressing knife with a handle extended, longitudinal axes of the handle and a blade being approximately perpendicular; FIG. 16 is a perspective view of a field dressing knife with the handle contracted and held for severing bone; FIG. 17 is a perspective view of a field dressing knife with the handle extended and held for severing bone; FIG. 18 is a perspective view of a field dressing knife with the handle extended and the handle rotated to be used as a hatchet; FIG. 19 a is a detail view of an additional embodiment of the field dressing knife having a telescopic handle, the handle being in a short mode; FIG. 19 b is a detail of a latch for the telescopic handle of the additional embodiment; FIG. 19 c is a cross-sectional shape of the field dressing knife of the additional embodiment; FIG. 20 is a plan view of the additional embodiment of the field dressing knife having a telescopic handle; FIG. 21 is a detail view of an additional embodiment of the field dressing knife having a telescopic handle, the handle being in a long mode; FIG. 22 is an exploded view of a star gear assembly; FIG. 23 a is a perspective view of a first embodiment of a field dressing knife handle attachment; FIG. 23 b is a cutaway view of the first embodiment of the field dressing knife handle attachment; FIG. 24 a is a perspective view of a second embodiment of a field dressing knife handle attachment; FIG. 24 b is a cutaway view of the second embodiment of the field dressing knife handle attachment; FIG. 25 is a side elevation view of the field dressing knife and separate handle for converting the field dressing knife to a hatchet; FIG. 26 is a side elevation view of the field dressing knife and separate handle configured for use as a hatchet; FIG. 27 is a side elevation view of a folding field dressing knife with the blade out for use; FIG. 28 is a side elevation view of a folding field dressing knife with the blade folded in for storage, transport, and safety; FIG. 29 a is a side elevation view of the field dressing knife blade with the star gear; FIG. 29 b is an end view of the field dressing knife blade with the star gear; FIG. 29 c is a plan view of the field dressing knife blade with the star gear; FIG. 30 is a plan view of the field dressing knife with the handle in its contracted mode; FIG. 31 is a side elevation view of the field dressing knife with the handle in its contracted mode; FIG. 32 is a side elevation view of the field dressing knife with the handle in its extended mode; FIG. 33 is a plan view of the field dressing knife with the handle in its extended mode; FIG. 34 is a first side elevation view of the field dressing knife with the handle in its extended mode and its blade in its hatchet mode; FIG. 35 is a first plan view of the field dressing knife with the handle in its extended mode and its blade in its hatchet mode; FIG. 36 is a second side elevation view of the field dressing knife with the handle in its extended mode and its blade in its hatchet mode; FIG. 37 is a second plan view of the field dressing knife with the handle in its extended mode and its blade in its hatchet mode; FIG. 38 is an exploded view of one embodiment of the extensible field dressing knife handle; FIG. 39 is a perspective view of a broad-headed bolt; and FIG. 40 is an exploded view of an expandable field dressing knife handle. DETAILED DESCRIPTION OF THE INVENTION A longitudinal axis of a handle 410 of a field dressing knife 400 is defined as passing through the handle 410 from the butt end 430 of the handle 410 to the guard end 440 of the handle 410 as shown in FIG. 4. The longitudinal axis of the blade 420 is also shown passing from the guard end 440 of the blade to the tip 460 of the blade 420. These axes are substantially parallel in FIGS. 4, 7, 8, 13, 14, 16, 17, 19 a, 20, 30 - 33, and 36. The knife blade 420 will preferably include a gut hook 470, a blade guard 480, and a double edge 490. In one embodiment, near the butt-end 430 of the handle 410, a groove 510, detailed in FIG. 5, is provided into which to engage a user&#39;s thumb 610 and forefinger 620 as shown in FIG. 6 for the purpose of severing a pelvis or brisket bone of a small or large animal carcass 1110 (see FIG. 11 ). A result of the grip arrangement shown in FIG. 6 is a larger effective surface area on which to strike with a free hand 810 (see FIG. 8 ) to provide the force to cause the knife blade 420 to enter the bone or cartilage. In FIG. 7, a user is shown holding the field dressing knife 400 in a position for cutting, slicing, skinning, etc. Another use, already mentioned, for the field dressing knife 400 is that of bone severing. To effect this process, the field dressing knife 400 is turned to an approximately vertical orientation with the blade tip 460 downward in contact with the bone or cartilage 1010 as illustrated in FIGS. 8 and 10. The butt end 430 of the field dressing knife handle 410 is then struck with the user&#39;s free hand 810 to provide the force needed to cause the knife blade tip 460 to enter the bone or cartilage 1010. Once the knife blade tip 460 has entered the bone or cartilage 1010, the field dressing knife 400 is worked back and forth according to the arrows 1020 in FIG. 10. In this way, the slot caused by the knife blade tip 460 is elongated, furthering the bone or cartilage severing process. An example of a knife blade 420 without the knife handle 410 is shown in FIG. 9. In this figure, the blade guard 480 and thumb-push 1320 are shown integral with the knife blade 420. The present invention is not limited to a guard 480 or a thumb-push 1320 integral with the blade 420. Note that the blade guard 480 and the thumb-push 1320 may be made integral with the handle 410 instead of the blade 420 as shown in FIGS. 36 and 37. A large animal carcass 1110 is shown in FIGS. 11 and 12. Preferably, during field dressing, the brisket bone 1120 and the pelvis bone (see FIGS. 1 and 2 ) should be severed to assist in removing viscera and enhance blood drainage and cooling of the carcass. In FIG. 12, the process of severing the brisket bone 1120 is illustrated. The field dressing knife 400 is also shown in operation. A preferred method of splitting the brisket bone 1120 is to drive the field dressing knife 400 off center of the brisket bone 1120 approximately one fourth to one and a half inches. Here, the ribs are softer and connected to the brisket bone 1120 with cartilage, making severing easier. A preferred method of splitting the pelvis bone comprises orienting the knife blade 420 parallel to a center ridge of the carcass pelvis bone and driving the field dressing knife blade tip 460 into the center ridge with the user&#39;s free hand. A novel handle assembly 1310 for the field dressing knife is shown in FIGS. 13-18. The handle assembly 1310 may be used in a contracted position as shown in FIG. 13, or extended as shown in FIGS. 14 and 15. In the extended position, the field dressing knife 400 may be used as a hatchet in which a blade edge 1410 or a tip 460 may be used to strike a bone 1010 for the purpose of severing bone or cartilage 1010. Another aspect of the embodiment of the field dressing knife 400 shown in FIGS. 13-15, 25, 26, 29 a, 31, 32, 34, 36, and 37 is a thumb-push ridge 1320. The location and configuration of this thumb-push ridge 1320 is such that a user&#39;s thumb rests against the thumb-push ridge 1320 during some modes of usage, and provides stability, control, and comfort. In the embodiment shown in FIGS. 13-18, the handle assembly 1310 may engage the blade 420 in either of two positions. In the position shown in FIGS. 13-14 and 16 - 17, the longitudinal axis of the handle 410 is substantially parallel to the longitudinal axis of the blade 420. In this configuration, the field dressing knife 400 may be used to split bone or cartilage, slice, cut, and skin. In the configuration of FIG. 14, the field dressing knife 400 may also be used to chop bone 1010 wherein the edge 1410 is used to strike the bone 1010 and sever bone when gripped as seen in FIGS. 7 and 14. When the field dressing knife 400 is configured as shown in FIGS. 15, 18, 34, 35, and 37, it is typically used as a hatchet wherein the knife blade tip 460 strikes the bone or cartilage 1010. In this configuration, the longitudinal axes of the knife blade 420 and the handle 410 are not substantially parallel, but rather, substantially perpendicular. Preferably, the handle assembly 1310 will extend, giving an overall length of about nine inches for use as a hatchet. The handle assembly 1310 of the present embodiment comprises three sections: the handle 410 and two inner sections 1420 that fit inside the handle 410 section. The inner sections 1420 includes threads 1430 for engaging a knife blade block 1440, being defined for the purposes of this document as the portion between the blade guard 480 and the handle, and is integral with the knife blade 420. The knife blade block 1440 includes two apertures with female threads into which the threads 1430 of the inner sections 1420 are alternately engaged. FIGS. 16-18 show the field dressing knife 400 in various modes of use. The versatility of the field dressing knife 400 is evident in these figures. Extending the handle assembly 1310 for striking the butt end 430 of the handle 410 with the user&#39;s free hand 810 may provide a better working position or angle for accomplishing the requisite bone or cartilage severing process. An additional embodiment of the field dressing knife 400 is shown in FIGS. 19 a - 21. In this embodiment, the included angle, θ 2110, between the longitudinal axis of the blade 420 and the longitudinal axis of the handle 410 is varied by mating two halves of a star gear 1910 in various configurations. The included angle, θ 2110, may take on values from approximately 45 degrees to 315 degrees. A star gear 1910 is defined herein as a construct as detailed in FIG. 22 having two symmetrical, mating halves 2210, 2220. Because of their symmetry, said mating halves 2210, 2220 may be engaged one to another in a plurality of configurations all having a common axis. In order to change the mating configuration, the two halves 2210, 2220 are rotated relative to one another on the common axis of rotation 2230. The shape needs only be symmetric, a multi-pointed star shape is not necessary to this definition. The above definition is used herein, including in the claims. The two symmetric, mating halves 2210, 2220 of the star gear 1910 are held together with a special, T-headed bolt 2240 while the field dressing knife 400 is in use. In the preferred embodiment, the T-headed bolt 2240 is held engaged in the female half 2210 of the star gear 1910 by engaging a snap ring 2250 to a groove 2280 in the T-headed bolt 2240 after inserting the T-headed bolt 2240 through an aperture 2260 in the female half 2210 of the star gear 1910. Said snap ring 2250 has an outer diameter or dimension greater than that of the aperture 2260. The threaded end of the T-headed bolt 2240 is threaded into female threads in an aperture 2270 of the male half 2220 of the star gear 1910. Unthreading the T-headed bolt 2240 draws the female half 2210 of the star gear 1910 away from the male half 2220 because the snap ring 2250 engages an inside surface of the female half 2210 around the aperture 2260. When the T-headed bolt 2240 is tightened down, the mating halves 2210, 2220 of the star gear 1910 are held firmly together so that the field dressing knife 400 may be used for slicing, chopping, piercing, etc. without fear that the included angle, θ 2110, of the longitudinal axis of the blade 420 to that of the handle 410 will change under force. Details of the handle 410 assembly are also shown in FIGS. 19 a - 21. The handle of this embodiment comprises two sections 1920, 1930. At least one inner section 1930 fits slidably inside the outer section 1920 so that the two sections 1920, 1930 may telescope to produce a longer and a shorter handle 410. The cross sectional shape of the sections 1920, 1930 is preferably non-circular such as the oval or ellipse shown in FIG. 19 c. Other non-circular shapes are viable such as polygons. Because of the non-circular cross section, the sections 1920, 1930 do not rotate relative to one another. The two sections 1920, 1930 are held in the shorter arrangement shown in FIGS. 19 a and 20, and secured in the longer arrangement shown in FIG. 21 by a spring lock assembly 1940. The spring lock assembly 1940 comprises a button 1950 (see also FIG. 19 b ) which engages an aperture in the inner handle section 1930 and engages the outer handle section 1920 in two ways to cause the two sections 1920, 1930 to maintain a given configuration with respect to one another. When the handle 410 is in its short mode, the button 1950 engages an aperture toward the butt-end of the outer section 1920. When the handle 410 is in its long mode, the button 1950 engages an edge of the outer section 1920 nearest the blade as shown in FIG. 21. In the long mode, the handle sections 1920, 1930 are held strongly in their respective positions by the matching tapered regions 2120, 2130 engaged to one another. The button 1950, in this case, represents added security. The button 1950 is forced outward by a spring 1960. The spring 1960 may be a coil spring or an elastic material such as rubber. A Z-clip 1970 is made to engage a notch 1980 in the button 1950 when the button 1950 is sufficiently depressed. The thickness of the outer section 1920 wall is sufficiently great in the vicinity of the butt-end aperture to disallow the button 1950 from being adequately depressed to engage the Z-clip 1970. In use, the button 1950 is depressed when the field dressing knife handle 410 is in its short mode as shown in FIGS. 19 a and 20 in order to extend the handle 410 to its long mode as shown in FIG. 21. In this instance, the Z-clip 1970 does not engage in the button notch 1980, but as the outer handle section 1920 is pulled away from the blade 420, the button 1950 slips under the outer section 1920 housing and is thereby disallowed from being forced further outward by the spring 1960. The outer section 1920 of the handle 410 is slid further away from the knife blade 420 until the button 1950 is able to slide through the aperture in the inner section 1930 past the edge of the outer section 1920 closest to the blade 420. At this point, the spring 1960 forces the button 1950 outward to secure the two handle sections 1920, 1930 stationary with respect to one another. To return the handle 410 to its short mode, the button 1950 is again depressed by the operator, this time until the Z-clip engages the notch 1980 in the button 1950. The button 1950 is thereby held in its depressed state while the user uses both hands to return the outer section 1920 of the handle 410 back toward the field dressing knife blade 420 to the short mode. A finger 1990 engages the butt-end of the outer section 1920 when the outer section 1920 has been adequately forced toward the blade 420. The finger 1990 in turn engages an appendage 1995 ( FIG. 19 b ) of the Z-clip 1970, forcing the Z-clip 1970 to disengage from the button notch 1980 and permitting the button 1950 to engage into the aperture of the outer section 1920 nearest the butt-end of the handle 410. The mating tapered regions 2120, 2130 in the handle sections 1920, 1930 are machined or formed accurately to provide firm, rigid engagement of the two sections 1920, 1930 when the handle 410 is in the long mode. The handle 410 is transformed from the short mode to the long mode by depressing the button 1950 while holding the outer section 1920 in a vertical position with the blade 420 downward. When the button is depressed, the handle 410 is raised as the handle 410 extends due to gravity and snaps into place. Due to a small amount of elasticity and the friction between the two handle sections 1920, 1930, the two handle sections 1920, 1930 wedge tightly and rigidly. At this point, the button 1950 returns to its outward position under the force of the spring 1960, to help secure the two handle sections 1920, 1930 in their positions relative to one another. Due to the firm engagement of the two handle sections 1920, 1930, a relatively large, sharp force must be applied to return the handle 410 to its short mode. Therefore, the button 1950 is made to lock down with the engagement of the Z-clip 1970 in the button notch 1980 when the button 1950 is sufficiently depressed. When the button 1950 is in its locked-down position, the user is free to use both hands to apply the large force required to free the tapered regions 2120, 2130 in the two handle sections 1920, 1930 from one another. To apply an adequate force, the blade 420 of the field dressing knife 400 is placed in a sheath or scabbard (not shown). The sheath is firmly gripped and the knife 400 swung by the user&#39;s arm with the handle butt-end 430 downward to strike a safe object such as the ground. With the button 1950 depressed, the outer section 1920 is free to return over the inner section 1930. In this process, it is very helpful that the button 1950 not require continual depressing. An optional aspect of the present invention is shown in FIGS. 19 a and 21. Gage lines 2140 are shown to provide guidance to the user regarding the depth in which the blade 420 has been driven into bone or cartilage in the process of splitting the pelvic bone or the brisket bone of a small or large animal carcass. The gage lines 2140 may be forged, machined, or etched into the blade 420. Any other suitable approach, providing permanent, robust gage lines 2140 may be used. The present invention is not limited to a specific process of providing these gage lines 2140. An additional advantage of the present embodiment over the prior art, as shown in FIG. 4, is the tapering or monotonic increase of the cross sectional area of the field dressing knife handle 410 from the blade 420 to the butt-end. This construction greatly enhances a user&#39;s ability to grip the knife 400 when the knife 400 is in use. Three additional embodiments of the field dressing knife 400 are shown in FIGS. 23 a, 23 b, 24 a, and 24 b. In these embodiments, an existing knife may be enhanced by the addition of a broadened end cap 2310, 2410. In the first embodiment shown in FIGS. 23 a and 23 b, an insert 2320 of the broadened end cap 2310 is inserted into a socket in the end of the knife. The broadened end cap has a butt-end 430 having a large surface area, and a groove 510 for receiving a user&#39;s index finger and thumb, both as discussed for the other embodiments of the field dressing knife 400. In the second embodiment, also shown in FIGS. 23 a and 23 b, the broadened end cap 2310 is made of an elastic material and the narrow section 2320 is stretched over the butt end of the handle of an existing knife. The broadened end cap 2310 is held tightly by the elasticity. The third embodiment of the broadened end cap 2410 is shown in FIGS. 24 a and 24 b. In this embodiment, the broadened end cap 2410 slides over the existing handle of the knife, and is held tight by snugly wrapping a strap 2420 about a base of the broadened end cap 2410. The strap is fitted with hook and loop fasteners. A slot 2430 provides sufficient flexure so the broadened end cap 2410 may be tightened down over the knife handle. The broadened end cap 2410 has a mushroom shape, providing a gripping place 2440 for the user&#39;s index finger and thumb as before. Another valid securing method for the broadened end cap 2310 is threading. Male threads provided on the insert 2320 with corresponding female threads in the handle, or female threads on the first embodiment broadened end cap 2410 with corresponding male threads on the knife handle would be provided. The use of the broadened end caps 2310, 2410 would provide to an existing knife some of the advantages described herein for the field dressing knife 400. It should be noted a separate hatchet handle 2510 for configuring the field dressing knife 400 may be used as clearly shown in FIGS. 25 and 26. In this case, the hatchet handle 2510 is carried by the hunter, in addition to the field dressing knife 400. The hatchet handle 2510 may be extensible, or of fixed length and may be attached at included angles less than or greater than 90 degrees. The field dressing knife 400 and hatchet handle 2510 may attach in a variety of ways, including the threads 2520, shown, wedging, pinning, clipping, etc. The present invention is not limited to a particular attachment method. An additional embodiment of the field dressing knife 400 is illustrated in FIGS. 27 and 28. In this embodiment, the blade 420 of the field dressing knife 400 is foldable into the handle 410 for safety, transport, convenience, portability, etc. A spring clip 2710 provides the security to hold the knife blade 420 in place, both in use and in the folded position as shown in FIG. 28. To release the blade 420, the spring clip 2710 is pressed with a user&#39;s digit at a depression 2720 in the handle against the force of a spring 2730. The spring clip 2710 rotates about a pivot 2740 which lifts the blade-end of the spring clip 2710 to release the blade 420. The handle of the folding field dressing knife 400 has the same broadened butt-end 430 and groove 510 as discussed above. FIGS. 29 a - c show the field dressing knife blade 420 with the star gear 1910, both separated from the remainder of the field dressing knife. Still another embodiment of the extending field dressing knife is shown in FIGS. 30-38. A bolt 3010 with a broad head 3020 and a special circumferential groove 3910 on the shank 3040 side of the broad head 3020 is shown in FIG. 39. The diameter of the shank 3040 of the bolt 3010 is no larger than the minor diameter of the threaded region 3920 for a reason that will become apparent following. The bolt 3010 is inserted into the handle 410 as shown in FIGS. 30-32 so the broad head 3020 of the bolt 3010 makes up the butt-end 430 of the handle 410. The male threaded region 3920 of the bolt 3010 engages either of two female threaded regions 3810, 3820 within the inner section 3030 of the handle depending on whether the handle is in its extended mode ( FIGS. 32-37 ) or its contracted mode ( FIGS. 30-31 ). The bolt 3010 is retained in the field dressing knife handle 410 by, preferably, three or four retaining screws 3050 engaging the special circumferential groove 3910 but generally fitting loosely in the groove 3910 so the bolt 3010 is free to turn. The retaining screws 3050 are preferably threaded into the outer section 3840 of the field dressing knife handle 410. When the handle 410 is in its contracted position, as shown in FIGS. 30-31, the male threads 3920 of the bolt 3010 are engaged in the female threads 3810 of the inner handle section 3830 closest to the knife blade 420. The groove 3910 of the broad bolt head 3020 firmly engages the retaining screws 3050 to hold the two knife handle sections 3830, 3840 together. To extend the field dressing knife handle 410, the broad-headed bolt 3010 is turned by its broad head 3020 by the user to disengage the bolt threads 3920 from the handle threads 3810 closest to the knife blade 420. The outer section 3840 of the knife handle 410 is pulled in a direction away from the knife blade 420 until the bolt threads 3920 can engage the handle threads 3820 nearest the butt-end of the knife handle 410. Therefore, the shank 3040 of the broad-headed bolt 3010 must be able to slide through the handle threads 3820 nearest the butt-end of the knife handle 410. Thus, the shank 3040 must have a diameter no greater than the minor diameter of its threads. The bolt 3010 is turned again in the same direction as before to fully engage the handle threads 3820 nearest the butt-end of the knife handle 410 as shown in FIGS. 32-37. Again, with the retaining screws 3050 engaged in the groove 3910 of the broad bolt head 3020 and the bolt threads 3920 from the handle threads 3820 nearest the butt-end of the knife handle 410, the two knife handle sections 3830, 3840 are rigidly positioned relative to one another. The cross-sectional shape of the two knife handle sections 3830, 3840 is preferably round, however, the present invention is not limited thereto. Comparing FIGS. 32 and 36, it can be seen that the thumb-push 1320 and finger guard 3510 may be part of the knife blade 420 or the knife handle 410. In all of FIGS. 30-38, the cross section of the field dressing knife handle 410 is preferably circular. A structure for securing the two sections 3830, 3840 is clarified in FIG. 40. Again, tapers 4010, 4020 are mated and the slight elasticity and friction between the inner handle section 3830 and the outer handle section 3840 provides a very rigid coupling of the two sections 3830, 3840. The broad-headed bolt 3010 is used to both disengage the tapers 4010, 4020 and also engage the tapers 4010, 4020 by the force the bolt applies via its threads 3920. The above embodiments are the preferred embodiments, but this invention is not limited thereto. It is, therefore, apparent that many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. In the following claims, the word “bone” is herein defined as either bone or cartilage.

Summary: A field dressing knife suitable for all aspects of field dressing small and large game is disclosed. Various novel aspects of the knife include: a large butt-end of the handle and a groove near the butt-end of the handle, both to spread the normal stress over a larger surface area when striking the butt-end of the handle with a free hand for splitting pelvis and brisket bones; and a configurable handle, permitting the double-edged field dressing knife to be used in knife mode or in a hatchet mode.

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Summarize: An unidentified Dakota Access Pipeline protester is arrested inside the Front Line Camp as law enforcement surround the camp to remove the protesters from the property and relocated to the overflow camp... (Associated Press) CANNON BALL, N.D. (AP) — The crowdsourcing goal was modest: $5,000, enough to help a few dozen people camping in North Dakota to protest the nearby construction of the four-state Dakota Access oil pipeline. The fund has since topped a staggering $1 million. The fund is among several cash streams that have provided at least $3 million to help with legal costs, food and other supplies to those opposing the nearly 1,200-mile pipeline. It may also give protesters the ability to prolong their months-long encampments that have attracted thousands of supporters, as the Standing Rock Sioux Tribe pursues the fight in court. And as the number of protest-related arrests increased this week, so did contributions — the funds raked in more than $200,000 between Thursday and Friday alone. But demonstrators are quick to note that the amount of money raised and what they have left isn't the same. "It still feels unreal sometimes because it is such an astronomical figure to me," said Ho Waste Wakiya Wicasa, the protester who set up the GoFundMe account that has raised more than $1 million mostly for operating expenses at the camp, which took root in April. "The money goes as quickly as it comes, but without it having been as much as it is, we certainly wouldn't have been able to be as productive as we have been in the fight," he said. For months now, opponents of the $3.8 billion pipeline — which is slated to move oil from North Dakota through South Dakota and Iowa to a shipping point in Illinois — have been camping near the confluence of the Missouri and Cannonball rivers. They worry the project will disrupt cultural artifacts and hurt drinking water sources on the Standing Rock Sioux's nearby reservation and farther downstream because the pipeline will cross the Missouri River. The Texas-based company building the pipeline, Energy Transfer Partners, insists the project is safe. The tribe is fighting the pipeline's permitting process in federal court. Since the number of protesters soared in August in North Dakota, donations started rolling in more frequently and more than 400 people have been arrested — including more than 140 on Thursday when officers evicted protesters camping on private land recently acquired by Energy Transfer Partners. But running a camp — and readying it for North Dakota's brutal winter — isn't cheap. The account Wicasa set up has only about $100,000 left as of Friday night, according to LaDonna Brave Bull Allard, a tribal historian and preservation employee. She provided family land for use in the original camp, Sacred Stone, in April and still houses demonstrators. The money has been used for grocery store trips every two days that cost about $2,000 each, 20 yurts purchased for $160,000, and around $7,000 for bail money. It has also paid for a storage area, composting toilets, tiny houses, tepees, a medical area and generators powered by solar panels and wind. A bookkeeper and an accountant now keep track of the crowd-sourced money. "I got people to take care of," Brave Bull Allard said. "I got to provide homes for people and blankets, thermal wear, socks, hats and gloves, and food. Right now, we are feeding 670 people." One online legal defense fund has raised more than $655,000 for "the legal defense of warriors protecting land, water and human rights." Meanwhile, much of the money the tribe is using for the legal fight is from at least $1.3 million in direct donations, tribal chairman Dave Archambault recently told The Associated Press. He declined to say how much tribal officials have spent so far, saying that could give their opponents an advantage in the legal case. Energy Transfer Partners also has declined to provide an estimate of its legal expenses. The tribe is pursuing appeals after losing in lower courts. The Standing Rock Sioux didn't solicit money, Archambault said, but asked other tribes for letters of support or formal resolutions. He said it was only after other tribes, including the Red Lake Nation and the Shakopee Mdewakanton Sioux Community in Minnesota, inquired about financial contributions that leaders decided to accept money for legal costs. And as the protests continued, the tribe decided to also use part of the money for waste-management services for protesters, he said. "I know the Standing Rock Sioux Tribe is not alone; we have overwhelming support," Archambault said, adding that his tribe would in return help other tribes "in their fight against corporations." Among the donors to online fundraisers is southern New Hampshire resident Carol DiPirro. She gave $30, motivated in part because of a fight her community waged against a natural gas pipeline. "They are saying the same thing: This is our water supply. You run a pipeline through it and it leaks, you are poisoning us. That's exactly what I spent two years of my life saying," DiPirro said. "This really, really struck a chord with me." ___ Follow Regina Garcia Cano on Twitter at https://twitter.com/reginagarciakNO The Freshet Collective hasn't added a story. On February 22, the Oceti Oyate camp, formerly known as Oceti Sakowin, as well as all other camps on treaty land claimed by the US Army Corps, were ordered to evacuate by 2pm. Hundreds flocked back to Standing Rock to pray as the company resumed drilling under the Missouri River following the granting of the final easement by the US Army Corps on February 7th. Ten unarmed water protectors were arrested and one was badly injured in a violent crackdown. Several of those arrested were journalists and legal observers, clearly targeted for their role in documenting police violence and rights violations. One water protector was tazed Fires kept law enforcement from entering the camps, but they continue to build presence at Standing Rock, preparing to forcibly remove water protectors from unceded territory affirmed in the 1851 and 1868 Ft. Laramie Treaties as sovereign land of the Great Sioux Nation. A contingent of nearly two dozen National Park Service rangers and U.S. Park Police officers arrived at the request of the Standing Rock Sioux Tribe with tactical gear ranging from gas masks and riot gear to rifles, protective vests, and night vision goggles. The Sacred Stone Camp is standing ground, but raids on the remaining camps could happen at any moment. If you witnessed an arrest today at Standing Rock, are arrested, or are concerned about the possible arrest of a relative or loved one, please call the legal jail support line: 605.519.8180. With law enforcement preparing to advance, the legal teams are standing by (Freshet Collective and Water Protector Legal Collective - www.waterprotectorlegal.org ) and will continue to work around the clock to support and stand in solidarity with water protectors. Over 700 water protectors are now fighting criminal cases, and North Dakota is handing down convictions. Six people face federal charges, which carry sentences of up to 15 years. At least one grand jury is active and issuing indictments. Federal agents are knocking on doors at the homes of water protectors and their families. Meanwhile, reactionary legislation targeting water protectors continues to advance through the North Dakota House and Senate. The legal defense of those needing support is going to take a long time, possibly years, and will be very expensive. Although our fundraising has been significant, we likely do not have sufficient resources to meet water protectors’ legal needs. For more information about the legal defense effort and transparent accounting of how the money is being used, please visit freshetcollective.org. We are very grateful for your support. HISTORY The Sacred Stone Camp was established April 1, 2016 as a center of spiritual and cultural opposition to the Dakota Access pipeline, and is determined to stop construction through prayer and non­violent direct action until adequate tribal consultation and environmental review are conducted. The spirit camp is located in between the pipeline’s proposed crossing of the Missouri River and the water intake valves for the Standing Rock Sioux Tribe, just over a mile downstream. The camp has an active facebook page and a crowdfunding page for general operating costs, but urgently needs separate legal defense funds to support the warriors resisting active construction. Funds raised on this page will only be used for the legal support for those involved in direct action. (To save us money on processing fees, send donation via paypal to freshetcollective@gmail.com) On Friday, August 5, 2016, a group of Oceti Sakowin youth from different Dakota and Lakota nations arrived in Washington DC after running ~2000 miles by relay, in spiritual opposition to the pipeline. The runners delivered 140,000 petition signatures to the US Army Corps of Engineers and rallied at the White House to ask President Obama to pressure them to repeal the water crossing permits. Meanwhile, relatives back home set up a tipi and held demonstrations in front of North Dakota’s State Capitol Building during a 3-day special legislative session. The Dakota Access pipeline is proposed to carry 450,000 barrels of fracked crude oil per day, from the Bakken fields of North Dakota, 1,172 miles to Patoka, Illinois. Though originally planned to cross the Missouri River upstream of Bismarck, the crossing was relocated just upstream of the Standing Rock Reservation. The threats this pipeline poses to the environment, public health, and tribal and human rights are strikingly similar to those posed by the Keystone XL. We are not expendable. We have a right to live. Please stand with us as we protect our sacred lands. Water is life, mni wiconi. In North Dakota, winter is coming. This has been a reason to push for more funding for the 700-some protesters remaining at the Standing Rock Reservation near Cannon Ball, North Dakota. The group, which first gathered in April to protest the Dakota Access Pipeline, is increasing its calls for aid as the weather gets colder. The calls for supplies come even after the group has received millions of dollars of donations online, as well as donated supplies from a variety of groups. As the camp has grown in size, area residents have come to see the camp as a drain on local resources. Ranchers have complained that they have had cattle stolen and butchered, and one went on local radio to accuse the protesters of emptying the food banks in Mandan, N.D. from the people who really need the extra help. The protests and the camp are funded through private donations. The bulk of these are funneled through campaigns set up by the camp itself–with one aiming to raise money for a legal defense fund, and another solely dedicated to the camp’s operating costs. On FundRazr, the legal defense fund for the camp has raised some $518,000 out of its goal of $750,000. Meanwhile, a gofundme campaign to cover the camp’s operating costs is seeking to raise $1 million and has already received pledges for more than $800,000. Despite the outpouring of support, an update from October 20 says that the group lacks many cold weather supplies. “We, are lacking much -we (sic) need more wood and shelters and food supplies,” the update reads, before going on to request bales of hay for windbreaks, California sage, and “tarps for the sweat lodge.” In regular updates to the fundraising site, the group stressed that additional funds were still needed to prepare for the coming cold and stressed that it was the only official Standing Rock gofundme campaign. This is true. However, a quick search of the site brings up dozens of other campaigns started by supporters of the movement in order to raise funds for supplies. If anything, the Dakota Access Pipeline protesters are flush, and have received substantial donations both directly and through several different crowdfunding sites. Since April, a series of more than 200 such online campaigns has raised more than $2 million on behalf of the Standing Rock Sioux Tribe and other groups fighting the pipeline project. And this total includes only the funds raised through the gofundme site. The camp also directs supports to give money directly through PayPal, which does not post totals of the donations received. Additionally, the camp asks for and receives gifts of supplies. In September, Greenpeace hosted supply drives for the camp in ten cities. The Los Angeles drive alone sent four carloads of supplies, including blankets, tents, and sleeping bags. A non-profit disaster relief organization known as the Rainbow Rapid Response Fund sent the camp a 12-burner stove it had previously sent to Hurricane Katrina disaster relief efforts. Although the camp pleads want, donors, including the North Dakota’s Homeland Security Division have complained that donated materials are wasted. DHS, which had provided the camp with trailers and water tanks, pulled the materials in late August after vandalism by protesters. Other groups found that donations come in excess and are ruined. NBC reported recently that the camp had been “inundated” with clothing donations, receiving so much clothing that some of it was ruined due to rot and wet without being used at all. Rather than a shortage of funds, the camp’s wants seem to stem, at least in part, from the extensive “earmarks” put on donated dollars. Rather than funding the camp’s general operation, campaign founders instead opted to specialize their requests, leading to such requests as raising $650 to send firewood to the camp, and $1,985 to send a team of acupuncturists from Brooklyn to Standing Rock. As the camp posts repeated pleas for more supplies, it appears to be sitting on a significant amount of cash that may leave many donors questioning the urgency of their funds. Follow Erin on Twitter. on behalf of LaDonna Brave Bull Allard FORT YATES, ND LaDonna Tamakawastewin Allard Hau we are the Inyan wakankagapi otip-Sacred Stone Camp from the Standing Rock Sioux Tribe. We have partnership with the Oectc Sakowin- Seven Council Fires, Indians and Cowboys and anyone who was to stand with us against the Dakota Access Pipeline. This pipeline will cross the Missouri River and Cannon Ball River which is the life line to many tribes and non native, when this pipeline leaks it will destroy the water and land. Water is life! So this pipeline is along the Missouri River and the KL pipeline was along the Ogall aquifer both are important to save. We are asking for financial support for water-propane - food and blankets for the camp. This is a prayer camp movement to save our sacred land and water and has been entirely supported by the people and the campers. We are in great appreciation for all you contributions....Wopila lila tanka-- we are deeply grateful for you contributions thank you! In Spirit Sacred Stone Camp Standing Rock Sioux Reservation, North Dakota (CNN) Ask around and you'll hear stories of pipeline protesters who've traveled great distances. They've come from Japan, Russia and Germany. Australia, Israel and Serbia. And, of course, there are the allies, not exclusively Native American or indigenous, who've flocked here from all corners of the US. Together they stand in opposition to the Dakota Access Pipeline, a $3.7 billion investment to move 470,000 barrels of domestic crude oil a day through four states. They're fighting against what they see as corporate greed, an environmental threat and an assault on sacred land. Demonstrating is their proud daily work. The Standing Rock Sioux call this reservation home, and many are not on the frontlines of this months-long, and at times violent, protest. With no end in sight, what does it mean to them? And are they even united in their support? The answer to that last question: Not even close. Wishing they'd go home No one makes this clearer than Robert Fool Bear Sr., 54, district chairman of Cannon Ball. The town he runs, estimated population of 840, is just a few miles from the action. It's so close that, given the faceoffs with law enforcement, you have to pass through a police checkpoint to reach it. It's about time people heard from folks like him, he says. Robert Fool Bear Sr. Fool Bear has had it with the protesters. He says that more than two years ago, when members of the Standing Rock Sioux Tribe could have attended hearings to make their concerns known, they didn't care. Now, suddenly, the crowds are out of control, and he fears it's just a matter of time before someone gets seriously hurt. Go down to the camps, he says, and you won't see many Standing Rock Sioux. "It irks me. People are here from all over the world," he says. "If they could come from other planets, I think they would." The presence of all these people has become a downright nuisance to his community, he says. Given the roadblocks, residents of Cannon Ball are often forced to go more than 40 miles out of their way. Native Americans march to the site of a sacred burial ground that was disturbed by bulldozers building the Dakota Access Pipeline on September 4, 2016 near Cannon Ball, North Dakota. Not long ago, he found three teenage girls from Ontario, Canada, camped out inside his storage shed. A white woman from Spokane, Washington, came to see him for help, saying she'd come here with nothing and her car had broken down. When he was at the casino recently, someone approached him about two young kids who were on their own because their parents had been arrested. The situation has dissolved to madness, he says, and he wishes Dave Archambault II, the Standing Rock Sioux chairman, would speak up. "If he had any balls, he'd tell [the protesters] to go home," Fool Bear says. And he's not alone in feeling this way. Two women who listen in as he talks keep nodding in agreement, but they don't want to speak. Just look at a recent vote in the community for further proof that Fool Bear's not the only naysayer. When protest organizers presented a request to build a new winter camp in Cannon Ball earlier this month, his community shot it down. Of the 88 people who voted, he says 66 were against the camp, less than 10 were for it and the rest remained undecided. A Standing Rock Sioux flag flies over a protest encampment near Cannon Ball, North Dakota. Even though Fool Bear is against the protests, that doesn't mean he's not preparing to help people out, too. He anticipates opening the community gymnasium for people without beds come winter, and a growing pile of sleeping bags and blankets sits in his office. Those protesters from Arizona, Georgia and California won't know what hit them when the cold rushes in, he says. Photos: North Dakota pipeline protests Fireworks lit the sky at the Oceti Sakowin Camp near Cannon Ball, North Dakota on December 4, 2016, after the Army Corps of Engineers halted the Dakota Access Pipeline route. An executive order by President Donald Trump in January allowed work to resume. Hide Caption 1 of 32 Photos: North Dakota pipeline protests Activists embrace after the December halt of the Dakota Access Pipeline route. The $3.7 billion project that would cross four states and change the landscape of the US crude oil supply. The Standing Rock Sioux tribe says the pipeline would affect its drinking-water supply and destroy its sacred sites. Hide Caption 2 of 32 Photos: North Dakota pipeline protests An activist rides down from a ridge on the edge of the Standing Rock Sioux Reservation on December 4. Hide Caption 3 of 32 Photos: North Dakota pipeline protests Activists celebrate at Oceti Sakowin Camp on December 4. An executive order by President Donald Trump in January allows work to resume on the Dakota Access Pipeline, which the activists oppose. Hide Caption 4 of 32 Photos: North Dakota pipeline protests Dan Nanamkin of the Colville Nez Perce tribe drums a traditional song by the Cannonball River in Cannon Ball, North Dakota, on Thursday, December 1. Hide Caption 5 of 32 Photos: North Dakota pipeline protests A procession makes its way down to the Cannonball River to take part in a Native American water ceremony on December 1. Hide Caption 6 of 32 Photos: North Dakota pipeline protests Snow covers the camp on Wednesday, November 30. Hide Caption 7 of 32 Photos: North Dakota pipeline protests A person walks through snow and wind on Tuesday, November 29. Hide Caption 8 of 32 Photos: North Dakota pipeline protests People against the construction of the Dakota Access Pipeline speak at a news conference near Cannon Ball on Saturday, November 26. Hide Caption 9 of 32 Photos: North Dakota pipeline protests A sculpture stands at an encampment where protesters of the pipeline have been gathered for months. Hide Caption 10 of 32 Photos: North Dakota pipeline protests People protest along Highway 1806 as they walk past a sprawling encampment on Thursday, November 24. Hide Caption 11 of 32 Photos: North Dakota pipeline protests A man stands along Highway 1806 on November 24. Hide Caption 12 of 32 Photos: North Dakota pipeline protests In this image provided by the Morton County Sheriff's Department, law enforcement and protesters clash near the pipeline site on Sunday, November 20. Hide Caption 13 of 32 Photos: North Dakota pipeline protests Tonya Stands recovers after being pepper-sprayed by police on Wednesday, November 2. Stands was pepper-sprayed after swimming across a creek with other protesters hoping to build a new camp to block construction of the Dakota Access Pipeline. Hide Caption 14 of 32 Photos: North Dakota pipeline protests Dozens of protesters wade in cold creek waters as they confront local police on November 2. Hide Caption 15 of 32 Photos: North Dakota pipeline protests Tribe members make their way back to their camp on Saturday, October 29. Hide Caption 16 of 32 Photos: North Dakota pipeline protests Cousins Jessica and Michelle Decoteau take part in a protest outside the North Dakota state capitol in Bismarck on October 29. Hide Caption 17 of 32 Photos: North Dakota pipeline protests The burned hulks of heavy trucks sit on Highway 1806 on Friday, October 28, near a spot where Dakota Access Pipeline protesters were evicted a day earlier. Hide Caption 18 of 32 Photos: North Dakota pipeline protests Pipeline protesters sit in a prayer circle as a line of law enforcement officers make their way across the camp to relocate the protesters a few miles south on Thursday, October 27. Protesters had camped on private property. Hide Caption 19 of 32 Photos: North Dakota pipeline protests A protester is arrested as law enforcement surrounds the camp on October 27. Hide Caption 20 of 32 Photos: North Dakota pipeline protests Tires burn as armed soldiers and law enforcement officers stand in formation to force Dakota Access Pipeline protesters off the private land in Morton County. Hide Caption 21 of 32 Photos: North Dakota pipeline protests A protester shows where he was hit by a bean-bag round fired by officers trying to force protesters off the private land. Hide Caption 22 of 32 Photos: North Dakota pipeline protests JR American Horse leads a march to the pipeline site on Friday, September 9. Hide Caption 23 of 32 Photos: North Dakota pipeline protests Native Americans head to a rally at the state capitol in Denver on Thursday, September 8. They were showing their support for members of the Standing Rock Sioux tribe in North Dakota opposting the Dakota Access Pipeline. Hide Caption 24 of 32 Photos: North Dakota pipeline protests People hang a sign near what they say was sacred burial ground disturbed by bulldozers in Cannon Ball. Hide Caption 25 of 32 Photos: North Dakota pipeline protests Marlo Langdeau of the Lower Brule Sioux Tribe joins hundreds of Native Americans for a march near Cannon Ball on Sunday, September 4. Hide Caption 26 of 32 Photos: North Dakota pipeline protests Protesters march on September 4 in opposition to the Dakota Access Pipeline. Hide Caption 27 of 32 Photos: North Dakota pipeline protests Native Americans march to the site of a sacred burial ground on September 4. Hide Caption 28 of 32 Photos: North Dakota pipeline protests Native Americans ride with raised fists to the sacred burial ground on September 4 to protest the Dakota Access Pipeline. Hide Caption 29 of 32 Photos: North Dakota pipeline protests The Missouri River is seen beyond an encampment near Cannon Ball, where hundreds of people gathered to join the protest on September 4. Hide Caption 30 of 32 Photos: North Dakota pipeline protests Flags of Native American tribes from across the United States and Canada line the entrance to a protest encampment on Saturday, September 3. Hide Caption 31 of 32 Photos: North Dakota pipeline protests Phil Little Thunder Sr. attends an evening gathering at an encampment of Dakota Access Pipeline protesters on September 3. Hide Caption 32 of 32 A fight worth having The tribal headquarters sits in the Sioux County seat of Fort Yates, nearly 30 miles south of the protests. Driving into the small town, population less than 200, a hand-painted sign announces, "Oil & water don't mix!" To hear it from those hanging out in Club Diamond Z, a bar and deli, Standing Rock Sioux support of the protests is universal. They can't fathom how anyone would disagree. Artist Roger Valandra, 61, says he travels north to join the protests once a week. A proud Vietnam Vet, he says the freedom to peacefully demonstrate was something he fought for, just like his nieces and nephews who've served in Iraq. To feel suppressed by the nation he's served offends him. He's baffled by the flood of law enforcement from nearby states and North Dakota counties he's never even heard of. "Don't they have enough to do?" he wonders. Any violence, he says, was provoked by them. Valandra and those around him at the bar begin to rattle off the offenses, many of which they heard about through social media. They talk about tear gas, rubber bullets, sound cannons and concussion grenades. They speak about the elder who was beaten with a club, the horse that had to be put down, the boy whose wrist was broken. (CNN could not confirm these stories.) Roger Valandra The crackdown also maddens Valandra's 25-year-old daughter, Elaina, who works here at "the Z," as locals call it. She grew up believing racism didn't exist; not anymore. She brings up the recent acquittal of Ammon Bundy and six others, who occupied a federal wildlife refuge earlier this year. Ammon Bundy "The Bundys were armed to the teeth, and they're acquitted," she says. She pulls up a Facebook post, showing a photo of a number scrawled on a man's arm after he was arrested Thursday and words likening the treatment to what Nazis once did. Online comments and the stereotyping of her people leave her stunned. She wants people to know this battle isn't about race. "It's about water, not just native people," she says. "We don't get another Earth." Her father doesn't question what will happen to the reservation's water if the pipeline goes in. Just look at the track record of oil companies, he says, and understand that the problem will grow. "When that water gets contaminated, it's going to affect everyone from here all the way to the Gulf of Mexico," he says. He calls the pipeline a "moneyline" for billionaires who'll never live on the land their desecrating. It's just "greed at play," he says, in a world where nothing is ever enough. JUST WATCHED Police remove pipeline protesters Replay More Videos... MUST WATCH Police remove pipeline protesters 01:20 A shrug of indifference Ten miles west of the protests, a man who doesn't want to be named, for fear of retribution, admits he looks forward to the pipeline. It'll mean fewer trucks barreling down these rural highways and fewer trains flying down the tracks. Back at Cannon Ball, however, Carl Bruce, 52, isn't afraid to say his piece. For this Standing Rock Sioux who has lived his life here, the pipeline doesn't matter. If it breaks, he says he'd just work around it. Carl Bruce "Oh hell," he says. "I can move north of the break and get my water over there." The pipeline is coming, like it or not, he says. The world may watch this ongoing battle and believe it's a unifying force for his people, but Bruce just shrugs his shoulders and walks away.

Summary: As the protest in North Dakota against the Dakota Access Pipeline escalates, so has the main crowdfunding page linked to it. The AP reports that since April, a GoFundMe account set up to raise money to aid protesters has far surpassed the $5,000 its organizer had originally hoped it would reach: Over the past six months, more than 21,000 people have contributed to boost the fund to over $1 million. "It still feels unreal sometimes because it is such an astronomical figure to me," says Howaste Wikaya, a protester who set up the fundraising page. But Wikaya adds the money is spent practically the moment it's received: A local tribal historian tells the AP that only about $100,000 from the fund remained as of Friday night for the camp, situated near Cannon Ball where the Missouri and Cannonball rivers meet-and North Dakota's unforgiving winter is coming. Where the money has gone: groceries that run about $2,000 every two days, 20 yurts ($160,000 in total), and bail money (about $7,000 so far) for those arrested for protesting the $3.8 billion pipeline, which protesters say will disturb drinking water and plow through cultural artifacts of the nearby Standing Rock Sioux Reservation. Per InsideSources, however, the protests have benefited from other fundraising as well-one effort has set aside more than $860,000 just for "the legal defense of warriors protecting land, water and human rights"-and some even claim donated supplies have been wasted. But donors continue to send financial support, including one New Hampshire woman from a community that fought its own natural gas pipeline. "This really, really struck a chord with me," she tells the AP.

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Write a title and summarize: Dengue causes 50 million infections per year, posing a large disease and economic burden in tropical and subtropical regions. Only a proportion of dengue cases require hospitalization, and predictive tools to triage dengue patients at greater risk of complications may optimize usage of limited healthcare resources. For severe dengue (SD), proposed by the World Health Organization (WHO) 2009 dengue guidelines, predictive tools are lacking. We undertook a retrospective study of adult dengue patients in Tan Tock Seng Hospital, Singapore, from 2006 to 2008. Demographic, clinical and laboratory variables at presentation from dengue polymerase chain reaction-positive and serology-positive patients were used to predict the development of SD after hospitalization using generalized linear models (GLMs). Predictive tools compatible with well-resourced and resource-limited settings – not requiring laboratory measurements – performed acceptably with optimism-corrected specificities of 29% and 27% respectively for 90% sensitivity. Higher risk of severe dengue (SD) was associated with female gender, lower than normal hematocrit level, abdominal distension, vomiting and fever on admission. Lower risk of SD was associated with more years of age (in a cohort with an interquartile range of 27–47 years of age), leucopenia and fever duration on admission. Among the warning signs proposed by WHO 2009, we found support for abdominal pain or tenderness and vomiting as predictors of combined forms of SD. The application of these predictive tools in the clinical setting may reduce unnecessary admissions by 19% allowing the allocation of scarce public health resources to patients according to the severity of outcomes. After successful eradication programs of the dengue vector Aedes mosquitoes in Latin America in the 1940s, both disease and vector have resurged and disseminated globally since the late 1970s [1], posing substantial disease and economic burdens on tropical and subtropical regions [2], [3]. It is estimated that currently about 40% of the world' s population live at risk of dengue infection and 50–100 million infections occur annually [4], [5]. In Singapore, an equatorial city state in South East Asia, intensive vector control programs initiated in the 1970s led to a substantial decline in dengue notifications [6]. Possibly as a result of reduced herd immunity [7], however, cyclical epidemics have occurred since the 1990s, leading to an estimated average annual economic impacts of US $115 million [8]. Dengue epidemics in Singapore lead to a strong demand for hospital beds and manpower, diverting resources for elective surgery and non-emergency admissions [9] and inflicting large public and private hospitalization costs (e. g. $50 US million in 2005 alone [8]). Despite this, only a small fraction of dengue infections in Singapore develop complications requiring hospitalization – for instance 6% and 21% of patients admitted for dengue into Tan Tock Seng hospital, Singapore, developed dengue hemorrhagic fever [DHF] in 2004 and 2007 respectively [10]. Under the widely adopted and longstanding World Health Organization (WHO) 1997 classification system, some dengue infections with complications were classified as dengue fever (DF), while not all patients classified as having DHF actually required hospitalization. Alternative classification systems were shown to be more sensitive than the WHO 1997 classification system in predicting severity of dengue in Indonesian children [11], showing the need to develop improved classification criteria. In addition, the classification into DF and DHF has been noted to lead to the false perception of low disease burden in the Americas [12]. The need to classify dengue cases according to their clinical severity [11]–[13] led the WHO to issue new classification criteria in 2009 for probable dengue with and without warning signs, and severe dengue (SD) [14]. The new classification criteria has been shown to facilitate dengue case management and surveillance [15] through better classification of severity [16], [17]. This revision makes it urgent to develop predictive tools conforming to the WHO 2009 dengue classification criteria to distinguish patients not likely to develop SD, for whom outpatient management may be appropriate, from those at greatest risk of SD and who require close monitoring and early therapeutic interventions in hospital. Predictive tools to triage dengue patients into those that are most and least likely to develop complications allow better allocation of limited healthcare resources. By identifying risk factors at presentation that are predictive of complications after hospitalization, admissions may be reduced, alleviating pressure on the healthcare system and avoiding unnecessary costs to patients. Statistical procedures have been used to distinguish dengue severity predictors [18], [19] and predictive tools using logistic regression or classification and regression tree models have been developed to triage patients between dengue fever (DF) and other febrile illnesses [20]–[22], between DF and DHF patients [9], [23], between DF and dengue shock syndrome (DSS) or other severe outcomes [22], [24], [25], and, recently, between DF and subtypes of SD including plasma leakage [25] or internal bleeding and plasma leakage [26]. This study addresses the need to identify predictors for the new WHO 2009 dengue classification system by reporting the results of a retrospective analysis of adult dengue cases in Tan Tock Seng hospital, responsible for the treatment of approximately 40% of notified cases in Singapore [27]. The demographic, clinical and laboratory characteristics of confirmed adult dengue patients at presentation to hospital were employed to develop predictive tools of SD after hospital admission. Dengue cases were analyzed with and without laboratory information so that appropriate tools could be developed for both well-resourced and resource-limited settings. GLMs were fitted to six combinations of data and variables: We fitted GLMs to all types of SD combined as a single explanatory variable. The GLMs obtained optimism-corrected 29% specificity for 90% sensitivity (21% specificity for 95% sensitivity) for well-resourced settings with clinical and laboratory variables (Table 2, Figure 2). The GLM to predict SD in well-resourced settings contained 8 explanatory variables (Table 3). Higher risk of SD development was associated with female gender, fever on admission, abdominal distension and vomiting (Table 3). Lower risk of SD development was associated with more years or age, leucopenia and normal levels (36. 1–44. 3% for females and 40. 7–50. 3% for males) of the minimum reading for hematocrit (Table 3). For instance, the mean of minimum hematocrit levels in females that did and did not develop SD was 35. 5 and 36. 6% and for males it was 39. 4 and 41. 6% respectively. Because laboratory tests are not available in some resource-limited settings, we constructed GLMs that excluded laboratory variables. The resulting drop in the specificity of the predictive tools was small and they even obtained lower optimism-corrected prediction errors (RMSPE of 5. 19 compared to 5. 34 in the case of GLMs for resourced settings, Table 2). This could indicate that this model had fewer tendencies to overfitting than a model using laboratory variables and made them comparable to tools reliant on laboratory information. The GLMs obtained 27% specificity for 90% sensitivity (15% specificity for 95% sensitivity. Table 2, Figure 2, implying a reduction of 8–12% in specificity). The GLM to predict SD in resource-limited settings contained 6 explanatory variables (Table 4). Similar to the GLM fitted with laboratory data, higher risk of SD development was associated with abdominal distension, female gender, vomiting and fever on admission; lower risk was associated to older age and fever duration on admission (Table 4). When fitted to data combining PCR and serology confirmed cases, the GLMs obtained increases in specificity compared to GLMs fitted only to PCR data: e. g. 30% optimism corrected specificity for 90% sensitivity for resourced and resource-limited settings (Table 2, Figure 2, increase of 1–3% in specificity with respect to resourced and resource-limited settings using only PCR confirmed data). These models presented an optimism-corrected predictive error lower than the models fitted to PCR data alone (RMSPE of 4. 03–4. 25 versus 5. 19–5. 34). The GLMs to predict SD in resourced and resource-limited settings contained 9 and 8 explanatory variables respectively (Tables S2 and S3). In both models, higher risk of SD development was associated with abdominal distension, female gender, breathlessness, vomiting and fever on admission (Table 4). More years of age and longer fever duration on admission were associated with lower risk of SD development. We fitted GLMs using severe plasma leakage and severe hemorrhage as dependent variables. We could not find robust GLMs for severe organ impairment because of the small number of cases (5 cases). Fitting to severe hemorrhage led to higher specificity than fitting to the development of SD in general (Tables 2). Despite their increase in specificity, GLMs fitted to single forms of SD would not be able to reduce hospital admissions substantially further: cases with lower risk of severe plasma leakage may be at risk of severe hemorrhage and vice-versa, impairing the identification of cases with overall low risk of SD that could be managed as outpatients. These models presented however the highest optimism-corrected predictive errors compared to those predicting SD in general, indicating low robustness in the prediction of new cases (Table 2). Fitting to specific forms of SD was nonetheless useful for comparison with other studies and to increase our understanding of the development of specific forms of SD. We found 6 predictors of severe hemorrhage (Table S4). Less years of age and female gender were common predictors with development of SD in general. High serum urea levels and hemoglobin count were new predictors of severe hemorrhage. Concurrent increase in hematocrit and rapid decrease in platelet count is a warning sign proposed by WHO for SD which was found to be good predictor of severe hemorrhage (Table S4). The prediction of severe plasma leakage presented 7 predictors. Shorter fever duration, vomiting, abdominal distension and fever on admission were predictors shared with any form of SD (Table 3 compared to S5). Several warning signs proposed by WHO 2009 for SD were identified as significant predictors. Abdominal distension – which was a good predictor of SD development – and could be considered related to abdominal pain and vomiting that was significant in models combining PCR and serology data. Concurrent increase in hematocrit and rapid decrease in platelet were found to be statistically significant in predicting severe hemorrhage and severe plasma leakage [14]. Vomiting and abdominal distension were good predictors of severe plasma leakage (Table S5). We used a mean length of hospitalization of 5 days from our data, a mean cost per hospitalized case per day of US 2010 $431 [35], a mean cost per ambulatory visit of $62. 1 and 4. 33 visits per episode [8]. We estimated that the savings per case managed as outpatient in a well-resourced setting like Singapore would be $1886. Using the optimism-corrected sensitivity and specificity of the GLM in a well-resourced setting fitted to PRC confirmed data, reduction in admissions of 19% could be obtained. In the case of Tan Tock Seng hospital in Singapore, incorporating the diagnostic testing costs also to all probable dengue cases from 2006 to 2008 (1920 cases), a total cost to patients of US $0. 69 million could be avoided ($0. 23 million per year). Extrapolating to national level using the proportion of admissions at Tan Tock Seng hospital, US $0. 58 million could be averted annually in Singapore. The new disease severity classification for dengue issued by the WHO in 2009 has necessitated a reassessment of clinical care, management and diagnosis of dengue in many parts of the world. We developed predictive tools to optimize the triage of adult dengue patients in Singapore according to the new guidelines. In the Singapore context, applying these tools would reduce unnecessary admissions by 19%, alleviating demand on scarce hospital beds and healthcare costs to patients. However, these results are based on adopting models with a sensitivity rate of 90%, i. e. 10% of the SD patients would not be hospitalized at presentation. For sensitivities of 95%, reductions of 16% could be instead obtained. It is difficult to compare our current results with studies dealing with the prediction of DHF and DSS according to WHO 1997 classification, given the different WHO 2009 classification that we employ. Good predictors of DHF were not necessarily good predictors of severe dengue. Carlos et al. [19] found a relationship between DHF and restlessness, nose bleeding, abdominal pain and low platelet count but, with the exception of abdominal pain, related to abdominal distension, we did not find these as significant predictors of severe dengue. Lee et al. [23] found that clinical bleeding, serum urea, and serum total protein were good predictors of DHF. Our results concur with respect to serum urea (only in the case of prediction of severe hemorrhage). It was noteworthy that some of the warning signs (abdominal distension, vomiting) proposed by WHO were prognostic of SD when predicted jointly and for specific forms of SD. The apparent low predictive power of other WHO warning signs might respond to the application of intravenous fluid. This application might have eventually prevented the development of SD, particularly shock due to severe plasma leakage and could confound the results. The predictive models are hence trained to predict SD under current fluid therapy conditions and might underperform in resource-limited settings where fluid therapy might not be so prevalent. Another potential reason for the low performance of warning signs is that warning signs tend to appear on the day of defervescence. Given the objective of creating triaging tools, the data were collected at presentation and thus warning signs had not yet been observed in some cases. We further note that two warning signs with slightly different definitions to those provided by the WHO were employed: vomiting during two or more consecutive days and hematocrit change greater than 20%. Using the flexibility to exercise clinical judgments of the WHO guidelines, these warning signs were chosen because they have been shown to be good predictors in the local context [36], [37]. Comparison with other studies focusing on dengue severity prediction, on the other hand, showed some common features. Comparison was not straightforward because several of the studies, contrasting with ours, were based on univariate analyses or specific types of severity only. In relation to abdominal distension as a strong predictor, SD was associated with abdominal pain [22], [25], [26]. We identified below normal serum hematocrit as good predictor of SD. This has been found in previous studies in Thai children [24] and agrees with the prediction of internal bleeding [26] and severe outcomes [22] respectively. Further research would however be needed to unveil the actual mechanisms that could explain this result. In predicting plasma leakage alone, we found that frequent vomiting was associated with plasma leakage, which agrees with previous studies [24], [26], [38], [39]. In contrast with our results, plasma leakage has been associated with older patients and male gender [25] but we could not find such association in our severe plasma leakage specific model. We found however that SD in general was associated with female gender and less years of age. The differences between studies might be due to the further inclusion of laboratory variables and by controlling for the presence of any co-morbidity and by using the Charlson score. This could avoid confounding effects with age in our study. Another potential reason for differences is that our cohort is relatively young (interquartile age of 27–43 years old and only 7% of individuals above 60 years old). Hence the lower risk of SD effect with an extra year of age that we find is mostly representative of young adults and middle age individuals. A precautionary approach should be taken when using the model to predict SD for patients above, for instance, 60 years old as this would involve extrapolating beyond an age for which the cohort was not as representative. We thus contend that further investigation on the relationship between age and SD outcome are thus necessary, preferably with cohorts mainly composed of old age individuals. Interestingly, the variables necessary to predict any form of SD without laboratory variables were easier to measure than those aimed at predicting specific forms of SD such as plasma leakage. These results might derive from the heterogeneity of the different SD types. The predictive tools identify those less exclusive variables that are common to severe plasma leakage, severe bleeding and severe organ impairment. Among these variables, fever on admission, fever duration and vomiting are easy to measure and can lead to rapid and cost-effective triaging of patients. Our study has some limitations. (i) Due to its retrospective nature, it is difficult to ascertain the homogeneity in the level of training of doctors and nurses with regards to the identification of clinical variables that could be subjective (e. g. persistent vomiting, abdominal pain) and how these could be applied to other settings. We have attempted to describe the clinical variables used to minimize ambiguities but different training in other hospitals could lead to variations in their identification. (ii) The GLMs were developed in an overwhelmingly adult cohort, and their performance in pediatric patients more common to other countries in the region needs to be validated. (iii) Not including other febrile illnesses in our study prevented the models from developing discriminatory power against them. This is both one of the strengths of the analysis, because we obtain highly fine-tuned predictive tools for SD, and a caveat, because our predictive tools may need to be supported by dengue diagnostic tests, which might not be easily available in resource-limited settings. Alternatively, published predictive tools to discriminate between DF and other febrile illnesses [20]–[22] can be used as a prior filter to the application of our predictive tools. (iv) Although the dengue epidemics in Singapore in 2006 were predominantly caused by dengue serotype 1, the epidemics in 2007 and 2008 were predominantly caused by dengue serotype 2 [10]. We controlled for serotype via a proxy (year), which was not statistically significant, but further studies in other settings would be needed to verify the validity of our predictive tools for epidemics driven by dengue serotypes 3 and 4. (v) Although serological tests for dengue are less accurate than PCR [28], for comparison we decided to include the results both with PCR and serology tests and PCR combined. Choosing only PCR confirmed cases was initially not preferred as there could be a risk that PCR confirmed cases favored patients that present earlier in the course of the disease and were febrile. If the bias had existed, we would expect fever on admission to be a good predictor in the case of only PCR confirmed cases and not in PCR and serology confirmed cases. This bias appears not relevant and consequently our results on fever on admission are also robust given that fever on admission is significant for both types of datasets (PCR and PCR and serology combined). We note that models fitted to both datasets agreed in a large number of predictors such as age, gender, fever duration, fever on admission, vomiting or abdominal distension. (vi) Our analysis does not control for the exact day of illness. Future research could focus on the prediction of SD one day ahead using the time series of the values of the explanatory variables through time. Although such models would not allow for patient triaging at admission, they will be useful to understand the development of SD and allow for the preparation of treatment at the hospital setting. The easily implementable predictive tools developed in this study do not require laboratory-based variables and may be beneficial in resource-limited settings, where pressure on scarce health resources is especially high. In addition, no considerable reductions in predictive power were observed in resource-limited tools with respect to resourced tools. Their application will allow redistribution of resources to those patients and conditions that need them more urgently without incurring extra screening costs. In conclusion, we have shown a combination of clinical and laboratory data performed well in identifying potential patients with severe dengue.

Title: Predictive Tools for Severe Dengue Conforming to World Health Organization 2009 Criteria Summary: Dengue is a mosquito-borne tropical disease that poses a large economic and health burden to 40% of the world' s population. Some dengue patients can evolve into a more severe form of the illness (severe dengue) and ideally should be hospitalized when this occurs. However, while only a small proportion of patients have severe dengue, many other patients are unnecessarily hospitalized. Being able to identify those patients at the highest risk of developing severe dengue can be very useful to reduce costs to hospitals and patients. In this study, we develop predictive tools to identify those patients that should be hospitalized. We find that some common features such as age, gender, fever duration, fever on admission, vomiting or abdominal distension can be good predictors of severe dengue. This is important because these features are easy to identify even in resource-limited settings where laboratory tests are not widely available.

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