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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Countering Iran in the Western Hemisphere Act of 2012''. SEC. 2. FINDINGS. Congress finds the following: (1) The United States has vital political, economic, and security interests in the Western Hemisphere. (2) Iran is pursuing cooperation with Latin American countries by signing economic and security agreements in order to create a network of diplomatic and economic relationships to lessen the blow of international sanctions and oppose Western attempts to constrict its ambitions. (3) According to the Department of State, Hezbollah, with Iran as its state sponsor, is considered the ``most technically capable terrorist group in the world'' with ``thousands of supporters, several thousand members, and a few hundred terrorist operatives,'' and officials from the Iranian Islamic Revolutionary Guard Corps (IRGC) Qods Force have been working in concert with Hezbollah for many years. (4) The IRGC's Qods Force has a long history of supporting Hezbollah's military, paramilitary, and terrorist activities, providing it with guidance, funding, weapons, intelligence, and logistical support, and in 2007, the Department of the Treasury placed sanctions on the IRGC and its Qods Force for their support of terrorism and proliferation activities. (5) The IRGC's Qods Force stations operatives in foreign embassies, charities, and religious and cultural institutions to foster relationships, often building on existing socioeconomic ties with the well established Shia Diaspora, and recent years have witnessed an increased presence in Latin America. (6) According to the Department of Defense, the IRGC and its Qods Force played a significant role in some of the deadliest terrorist attacks of the past two decades, including the 1994 attack on the AMIA Jewish Community Center in Buenos Aires, by generally directing or supporting the groups that actually executed the attacks. (7) Reports of Iranian intelligence agents being implicated in Hezbollah-linked activities since the early 1990s suggest direct Iranian government support of Hezbollah activities in the Tri- Border Area of Argentina, Brazil, and Paraguay, and in the past decade, Iran has dramatically increased its diplomatic missions to Venezuela, Bolivia, Nicaragua, Ecuador, Argentina, and Brazil. Iran has built 17 cultural centers in Latin America, and it currently maintains 11 embassies, up from 6 in 2005. (8) Hezbollah and other Iranian proxies with a presence in Latin America have raised revenues through illicit activities, including drug and arms trafficking, counterfeiting, money laundering, forging travel documents, pirating software and music, and providing haven and assistance to other terrorists transiting the region. (9) Bolivia, Cuba, Ecuador, Nicaragua, and Venezuela expressed their intention to assist Iran in evading sanctions by signing a statement supporting Iran's nuclear activities and announcing at a 2010 joint press conference in Tehran their determination to ``continue and expand their economic ties to Iran'' with confidence that ``Iran can give a crushing response to the threats and sanctions imposed by the West and imperialism''. (10) The U.S. Drug Enforcement Administration concluded in 2008 that almost one-half of the foreign terrorist organizations in the world are linked to narcotics trade and trafficking, including Hezbollah and Hamas. (11) In October 2011, the United States charged two men, Manssor Arbabsiar, a United States citizen holding both Iranian and United States passports, and Gholam Shakuri, an Iran-based member of Iran's IRGC Qods Force, with conspiracy to murder a foreign official using explosives in an act of terrorism. Arbabsiar traveled to Mexico with the express intent to hire ``someone in the narcotics business'' to carry out the assassination of the Saudi Arabian Ambassador in the United States. While in the end, he only engaged a U.S. Drug Enforcement Agency informant posing as an associate of a drug trafficking cartel, Arbabsiar believed that he was working with a member of a Mexican drug trafficking organization and sought to send money to this individual in installments and not in a single transfer. (12) In February 2011, actions by the Department of the Treasury effectively shut down the Lebanese Canadian Bank. Subsequent actions by the United States Government in connection with the investigation into Lebanese Canadian Bank resulted in the indictment in December 2011 of Ayman Joumaa, an individual of Lebanese nationality, with citizenship in Lebanon and Colombia, and with ties to Hezbollah, for trafficking cocaine to the Los Zetas drug trafficking organization in Mexico City for sale in the United States and for laundering the proceeds. SEC. 3. STATEMENT OF POLICY. It shall be the policy of the United States to use a comprehensive government-wide strategy to counter Iran's growing hostile presence and activity in the Western Hemisphere by working together with United States allies and partners in the region to mutually deter threats to United States interests by the Government of Iran, the Iranian Islamic Revolutionary Guard Corps (IRGC), the IRGC's Qods Force, and Hezbollah. SEC. 4. DEFINITIONS. In this Act: (1) Western hemisphere.--The term ``Western Hemisphere'' means the United States, Canada, Mexico, the Caribbean, South America, and Central America. (2) Relevant congressional committees.--The term ``relevant congressional committees'' means the Committee on Foreign Affairs of the House of Representatives and the Committee on Foreign Relations of the Senate. SEC. 5. REQUIREMENT OF A STRATEGY TO ADDRESS IRAN'S GROWING HOSTILE PRESENCE AND ACTIVITY IN THE WESTERN HEMISPHERE. (a) In General.--Not later than 180 days after the date of the enactment of this Act, the Secretary of State shall conduct an assessment of the threats posed to the United States by Iran's growing presence and activity in the Western Hemisphere and submit to the relevant congressional committees the results of the assessment and a strategy to address Iran's growing hostile presence and activity in the Western Hemisphere. (b) Matters To Be Included.--The strategy described in subsection (a) should include-- (1) a description of the presence, activities, and operations of Iran, the Iranian Islamic Revolutionary Guard Corps (IRGC), its Qods Force, Hezbollah, and other terrorist organizations linked to Iran that may be present in the Western Hemisphere, including information about their leaders, objectives, and areas of influence and information on their financial networks, trafficking activities, and safe havens; (2) a description of the terrain, population, ports, foreign firms, airports, borders, media outlets, financial centers, foreign embassies, charities, religious and cultural centers, and income- generating activities in the Western Hemisphere utilized by Iran, the IRGC, its Qods Force, Hezbollah, and other terrorist organizations linked to Iran that may be present in the Western Hemisphere; (3) a description of the relationship of Iran, the IRGC, its Qods Force, and Hezbollah with transnational criminal organizations linked to Iran and other terrorist organizations in the Western Hemisphere, including information on financial networks and trafficking activities; (4) a description of the relationship of Iran, the IRGC, its Qods Force, Hezbollah, and other terrorist organizations linked to Iran that may be present in the Western Hemisphere with the governments in the Western Hemisphere, including military-to- military relations and diplomatic, economic, and security partnerships and agreements; (5) a description of the Federal law enforcement capabilities, military forces, State and local government institutions, and other critical elements, such as nongovernmental organizations, in the Western Hemisphere that may organize to counter the threat posed by Iran, the IRGC, its Qods Force, Hezbollah, and other terrorist organizations linked to Iran that may be present in the Western Hemisphere; (6) a description of activity by Iran, the IRGC, its Qods Force, Hezbollah, and other terrorist organizations linked to Iran that may be present at the United States borders with Mexico and Canada and at other international borders within the Western Hemisphere, including operations related to drug, human, and arms trafficking, human support networks, financial support, narco- tunneling, and technological advancements that incorporates-- (A) with respect to the United States borders, in coordination with the Governments of Mexico and Canada and the Secretary of Homeland Security, a plan to address resources, technology, and infrastructure to create a secure United States border and strengthen the ability of the United States and its allies to prevent operatives from Iran, the IRGC, its Qods Force, Hezbollah, or any other terrorist organization from entering the United States; and (B) within Latin American countries, a multiagency action plan, in coordination with United States allies and partners in the region, that includes the development of strong rule-of-law institutions to provide security in such countries and a counterterrorism and counter-radicalization plan to isolate Iran, the IRGC, its Qods Force, Hezbollah, and other terrorist organizations linked to Iran that may be present in the Western Hemisphere from their sources of financial support and counter their facilitation of terrorist activity; and (7) a plan-- (A) to address any efforts by foreign persons, entities, and governments in the region to assist Iran in evading United States and international sanctions; (B) to protect United States interests and assets in the Western Hemisphere, including embassies, consulates, businesses, energy pipelines, and cultural organizations, including threats to United States allies; (C) to support United States efforts to designate persons and entities in the Western Hemisphere for proliferation activities and terrorist activities relating to Iran, including affiliates of the IRGC, its Qods Force, and Hezbollah, under applicable law including the International Emergency Economic Powers Act; and (D) to address the vital national security interests of the United States in ensuring energy supplies from the Western Hemisphere that are free from the influence of any foreign government that would attempt to manipulate or disrupt global energy markets. (c) Development.--In developing the strategy under this section, the Secretary of State shall consult with the heads of all appropriate United States departments and agencies, including the Secretary of Defense, the Director of National Intelligence, the Secretary of Homeland Security, the Secretary of the Treasury, the Attorney General, and the United States Trade Representative. (d) Form.--The strategy in this section may be submitted in classified form, but shall include an unclassified summary of policy recommendations to address the growing Iranian threat in the Western Hemisphere. SEC. 6. SENSE OF CONGRESS. It is the sense of Congress that the Secretary of State should keep the relevant congressional committees continually informed on the hostile actions of Iran in the Western Hemisphere. SEC. 7. RULE OF CONSTRUCTION. Nothing in this Act shall be construed to limit the rights or protections enjoyed by United States citizens under the United States Constitution or other Federal law, or to create additional authorities for the Federal Government that are contrary to the United States Constitution and United States law. Speaker of the House of Representatives. Vice President of the United States and President of the Senate.

Title: To provide for a comprehensive strategy to counter Iran's growing hostile presence and activity in the Western Hemisphere, and for other purposes Summary: (This measure has not been amended since it was passed by the Senate on December 12, 2012. The summary of that version is repeated here.) Countering Iran in the Western Hemisphere Act of 2012 - States that it is U.S. policy to use a comprehensive strategy to counter Iran's growing hostile presence in the Western Hemisphere by working together with U.S. allies and partners in the region to deter threats to U.S. interests by Iran, the Iranian Islamic Revolutionary Guard Corps (IRGC), the IRGC's Qods Force, and Hezbollah. Directs the Secretary of State to submit to Congress a strategy to address Iran's growing presence and activity in the Western Hemisphere which should include: (1) descriptions of the presence, activities, and operations of Iran, the IRGC, the IRGC's Qods Force, and Hezbollah; (2) descriptions of the terrain, population, ports, foreign firms, airports, borders, media outlets, financial centers, foreign embassies, charities, religious and cultural centers, and income-generating activities utilized by Iran, the IRGC, the IRGC's Qods Force, and Hezbollah; (3) descriptions of the relationship of Iran, the IRGC, the IRGC's Qods Force, and Hezbollah with transnational criminal organizations; (4) descriptions of the relationship of Iran, the IRGC, the IRGC's Qods Force, and Hezbollah that may be present with governments in the Western Hemisphere; (5) descriptions of federal law enforcement capabilities, military forces, state and local government institutions, and other critical elements, such as nongovernmental organizations that may organize to counter the Iranian threat in the Western Hemisphere; (6) descriptions of activity by Iran, the IRGC, the IRGC's Qods Force, and Hezbollah that may be present at the U.S. borders with Mexico and Canada and at other international borders within the Western Hemisphere; and (7) a plan to address efforts by foreign persons, entities, and governments in the region to assist Iran in evading sanctions, to protect U.S. interests, assets, and allies in the Western Hemisphere, to support U.S. efforts to designate persons and entities in the Western Hemisphere for proliferation and terrorist activities relating to Iran, and to address vital U.S. interests in ensuring energy supplies from the Western Hemisphere. Authorizes such strategy to be submitted in classified form, but requires it to include an unclassified summary of policy recommendations addressing the growing Iranian threat in the Western Hemisphere. Expresses the sense of Congress that the Secretary should keep Congress informed about Iran's hostile actions in the Western Hemisphere.

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Summarize: Last year, former Oklahoma City Department Police Officer Daniel Holtzclaw was accused of abusing women in the community he patrolled. On Thursday, he was found guilty of many of their charges. But the testimonies of these women have never been reported in full, until now. Sue Ogrocki / AP Holtzclaw in September 2014. After four days of deliberation, a jury found former Oklahoma City Police Department Officer Daniel Holtzclaw guilty of multiple counts of rape and sexual assault on Dec. 10. Holtzclaw, who has been on trial since November 2, was accused of targeting black women in the community he patrolled. All 13 women testified against Holtzclaw in the trial. But it wasn’t their first time telling their stories in court. Last November, during a two-day preliminary hearing, each woman told her story in succession — publicly, for the first time ever, in a smaller courtroom than the one in which they delivered their trial testimony. Before the November hearings, these stories had been told only through prosecutors and detectives. This was the first time they explained what Daniel Holtzclaw did to them — how he exerted his authority, and how afterward they felt reporting him would be futile — in their own words. Their testimonies have never been reported altogether, in full, until now. The stories are consistent, from the questions Holtzclaw initially asked them, to the way he exposed himself through the fly of his police uniform, to the remote locations he took some of them to. GPS data from Holtzclaw’s car and various phone records presented in court verify many of the geographical and timeline-related details. According to prosecutors, Holtzclaw targeted these women because they had records and lived in a high-crime neighborhood. He allegedly chose them because they didn’t want any trouble and because they feared the police — because they likely wouldn’t report their assaults to the police. He was the police. According to the defense, these women are drug abusers and sex workers — some convicted felons with histories of lying to the police. Sometimes their testimonies are inconsistent, the defense said; they have “agendas,” they’re lying. Holtzclaw’s attorney built his defense on this approach: focusing on the character of the women and the reliability of their testimony. This is their testimony. S.H. December 2013. “I didn't think that no one would believe me.” S.H. was sitting in a truck outside an apartment complex when she was approached by three Oklahoma City Police Department officers. S.H., 23, said she was high on PCP and having an even stronger reaction after some had spilled on her skin. The officers called an ambulance for her. At a nearby hospital, S.H. was given a drink to help her come down from the drugs. After showering, she changed into a hospital gown and was transferred to various rooms. In the second hospital room, she allegedly found herself alone with Officer Daniel Holtzclaw, who was observing her while she lay down, one arm handcuffed to her bed. S.H. didn’t know yet if she was under arrest. About an hour into Holtzclaw observing her like this, S.H. recalls that he began repeatedly pointing out that her chest was exposed. I'm thinking to myself, is he trying to come on to me? Because I'm like — he knows my condition, so he know that I'm not trying to show it to him. If somebody else was in that situation I wouldn't keep on telling them that, you know, because I wouldn't want them to feel uncomfortable. Holtzclaw allegedly approached her to pull up her hospital gown, but then groped her chest. I'm high, [but] I'm thinking, like, I know I'm not tripping. He just did that. He just went back and sat down at the chair in front of the bed and was talking to me. He was talking nicely to me, as if he was trying to be my friend or wanted me to believe in — believe him. He was asking me, why do I choose the type of baby daddies that I have. Holtzclaw allegedly groped her two more times. She said she thought about trying to run on a bathroom break, "but I was so out of it like I wasn't going to be able to make it.” Holtzclaw allegedly told her he would “fuck the shit” out of her. I just really can't believe it because it's the police. And I thought stuff like that only just really happened on movies. I couldn't believe what was going on was really going on. [He said] that if I cooperate that just give it a month and to trust him that my charges would be off. When he tell me to trust him, I'm saying I never trusted a cop. I never trusted a cop. So he was like, well, I've been straight up front with you all this time. S.H. testified that Holtzclaw then exposed himself and forced her into oral sodomy, telling her to not move too much so that her heart monitor wouldn’t go off. She said that he said to her, "Ha-ha, you've never sucked a white dick before." Afterward, she asked if she was going to jail — he responded that he had to take her to jail. She asked him to call her mom for her. After the call, which he made on his cell phone, Holtzclaw allegedly groped S.H.’s genitalia. I just gasped. Later, he prepared to take S.H. to county jail. When I was talking to the nurse when she was taking my blood pressure and everything she asked me a whole lot of questions and she asked me was I sexually assaulted in the last 24 hours or whatever, and I told her no. He was standing behind her with my files, I guess … And after I said, no, he closed the book up. He told me, he was like, 'I hid your tickets for you and I made your bond super fucking cheap.' About two weeks later after I got out, I had a [Facebook] friend request from him and I accepted it. I remembered his face and I remember seeing his badge with the last name. Holtzclaw and S.H. messaged about her charges. He asked her to call him, giving her his phone number, and said he wanted to meet her again. We was texting and he had told me that he was leaving the Outlet Mall and that he wanted to come see me and talk about the case. And he came and he parked in the driveway next door at the vacant house and waited for me to come outside. My husband just stepped out. I was watching my kids and my little brothers and sisters and the whole time we was in the car we was talking and he was telling me like to — pressuring me to have sex with him. I kept on telling him I couldn’t because the kids was in the house by theyself [sic] and he really wasn't too worried about that. He just was telling me like, ‘Could you just bend over real quick?’ That night, Holtzclaw allegedly exposed himself to her and asked for oral sex. She told him she “couldn't because I had to watch the kids that was in the house.” I just had enough like of him just trying to make me have sex with him, and I was telling him like he don't even have a rubber or the condom and he really didn't care about that. He was asking me like, ‘You don't trust me?' And I was just like, 'I can't do it,' and then I was just like, 'I got to go.' S.H. told her mom and her twin about Holtzclaw. But she didn’t tell the police until they approached her about the ongoing investigation. Because I didn't think that no one would believe me. I feel like all police will work together and I was scared. Update: On Dec. 10, Daniel Holtzclaw was found not guilty of any charges brought forward by S.H. Eric Gay / AP Northeast 23rd Street in Oklahoma City, in a neighborhood where 13 women say they were sexually assaulted by Daniel Holtzclaw. T.B. February through April 2014. “I wanted it to just go away.” Around 11:30 p.m. one night, T.B. was sitting in a car with her two daughters and a friend, warming up the car before going to the store — waiting for midnight so that their food stamps would be ready — when two police cars rolled up. The officers told the women to get out of the car; T.B.’s friend went with one officer, and T.B. went with the other. The kids went inside the house. T.B. said she was put in the backseat of Holtzclaw’s patrol car, where she watched as her friend was allowed to go back into the house after a few minutes and the other officer drove off. She admitted to Holtzclaw she had outstanding tickets. She had been through this routine before, she said. He spoke to me and told me I wasn't going to jail. [He asked did] I have any drugs under my shirt, and I said, no. And he asked could he see, so I lifted my shirt up and let him see. I knew if I didn't I was going to jail. I have been in the streets a very long time. And I know things — it naturally comes. First of all, if they going to search you a woman should be there to search you. Why would a man be asking what's under my shirt and could he see what's under my shirt? He kept telling me, 'You know, you got these warrants. When are you going to take care of them?' Holtzclaw asked T.B. if there were anything under her breasts, and then groped her. He then let T.B. go, and she went back into the house, where her friend and daughters asked what happened. I told them that I had to show him my body to get out of the police car, so I wouldn't go to jail. For real they were shocked. I'm like, 'Do you all believe me?' [They] said, 'Yes.' How come I didn't call the police — I didn't. I wanted it to just go away. The next month, T.B. said she had another encounter with Holtzclaw when she pulled up to her house and found his patrol car in her driveway. Holtzclaw was on the porch — he told T.B. to “come here,” and put her in the backseat of his car, running her name again. She asked him why he was at her house — he asked her where she had been and whether she’d taken care of her tickets yet. She told him he was scaring her daughter, who was on the front porch. We did a little chatting. I can't recall each word, word for word. We all know him by Spike. Because when he knocked on my door and came in my house he always took the mousse and had his hair up and spikes up here at the front. And that's how we identified him. I don't even know what me and him was talking about. I was just ready to get it over with. What I had to do to get out that police car to go in with my babies. I knew what I had to do to get out. I didn't have money to pay my tickets and I knew [what] I had to do with him to get out the car. She testified that she exposed her chest, but Holtzclaw didn’t touch her. He asked her if she had dope on her, and she said no. He asked if she had drugs in her pants. She pulled the waistband of her leggings away from her body. When I was getting out of the car, I turned and I looked at where I was sitting at to see if there was $20 there. And I said, 'Why you got the $20 sitting there?' And he said, 'That's how I set my people up.' I left it at that and I went in the house with my babies. When she got in the house, her boyfriend told her that earlier in the day, while he was sleeping, Holtzclaw had come into the house without permission and woken him up, telling him to come outside so Holtzclaw could run his name. T.B was upset, but she still didn’t want to call the police. I didn't call them. I didn't think anyone would believe the allegations that I was making. To be honest, I don't like the police and I try to stay away from them as far as I can. A month later, she had yet another encounter with Holtzclaw. She was at home, on the phone with her mother when Holtzclaw showed up at her door. He wanted to come in. She told him she was on the phone — she had put the call on speakerphone. Holtzclaw said he wanted to search the house for drugs. She said no, and he got mad. I told him when he go get his search warrant and stop coming by by hisself [sic] and bring other officers I'll freely open my door. T.B.’s mother told T.B she should “pick up and leave.” She followed her mother’s advice, moving her family out of the neighborhood. Update: Holtzclaw was found guilty of sexual battery and procuring lewd exhibition related to T.B.'s testimony, but not stalking or burglary. C.R. March 2014. “It was nobody there but just me and him, so to me, I just took it as my word against his.” C.R. said she was walking alone one night when Holtzclaw stopped her. The officer asked if she had an ID, where she was coming from, and where she was going. He asked if she had anything on her, and she said no. He patted down her front pockets to check. He just kept asking did I have anything on me, he asked me if I had been arrested, and I was asking him why was he stopping me. I mean, for what reason. I don't have any warrants. I had my ID. He said he just wanted to talk. It was just like nonchalant conversation. I don't remember word-for-word, but he was asking me [if] I [had] anything on me, more or less talking about the upper part of my body. He was motioning his hand, like, ‘Do you have anything else on you?’ And I was like, ‘No.’ And he just kept [telling] me that he needed to be sure that I didn't have anything on me. So I just got to the point where I just opened my jacket and raised up my shirt and lifted my bra. Because he kept asking me, talking about making sure that I didn't have anything on me, and I was ready to get away from him. C.R. testified that she had come into contact with officers before that night, but that she had never had been made to raise her shirt like that. Afterward, Holtzclaw put her in the backseat of his patrol car, where she stayed in his for another 30–45 minutes for more “nonchalant talk.” I don't remember exactly what it was [like], because in my mind was just thinking I wanted to get away, you know. And then after he let me out the car, I started walking back down 16th, and then a friend of mine saw me and he picked me up. I spoke only in passing about it maybe once or twice, but I never went into details about it with anybody. I didn't — I just try not to think about it. I didn't think anything would be done. I mean, it was nobody there but just me and him, so to me I just took it as my word against his, so I just blew it off — as best as I could just walked away from it. Update: Holtzclaw was found not guilty of procuring lewd exhibition from C.R. Eric Gay / AP Lincoln Boulevard in Oklahoma City, near the site where a woman says she was sexually assaulted by Holtzclaw. F. April 2014. “He had asked me something about working girls and I told him I didn't know nothing about any working girls.” F. testified that she was walking home one night when Holtzclaw stopped her. Earlier that day, she said, she had been drinking and smoking crack. The officer had stopped the 54-year-old woman before. He asked for her ID, and she showed it to him. He had her empty her pockets — she had a crack pipe on her — and he handcuffed her, sitting her down on the curb. He had asked me something about working girls and I told him I didn't know nothing about any working girls. Holtzclaw also asked F. if she was a working girl. She said she wasn’t. F. had outstanding city warrants, and Holtzclaw told her she needed to take care of them. He crushed her pipe and told her she was free to go. But when he took her cuffs off, Holtzclaw allegedly groped her chest over her clothes. Once she was released, F. walked away from him, across a park. Update: Holtzclaw was found not guilty of sexual battery in F.'s case. R.C. April 2014. “I was just ashamed and I didn't want to face the rape. I didn't want to face it.” R.C. was six months sober at the time of her testimony in November 2014 — but in late April 2014, when she met Daniel Holtzclaw, she had been drinking in a car parked in a vacant lot across from a Family Dollar. She had just given someone a ride home when Holtzclaw pulled her over. He ran her name and told her to get out of her car so that he could search it; he said he smelled alcohol. I told him I didn't want to go to jail. He said he wasn't going to take me to jail. He would take me to detox. After he looked into my car, he told me to pull my pants down. And at this time I was thinking that he was going to search me, but I wondered why he didn't call for a woman police officer, a backup to help him search me to see if I had anything on me. Because I know that police officers can search you, but I didn't think he had a right to search me. She told Holtzclaw she didn’t want to go to detox. He insisted, and she asked if she could first drop her car off at a relative’s house a mile or so away, so that it wouldn’t get towed. He said yes. I knew something was going to happen. I didn't know what. After she parked in front of the house, Holtzclaw led her to the backseat of his police car. He drove away and parked on a street near a bus lot, then told her to get out. [He] was an officer and I was scared and I know that he could hurt me, so I did what he said. R.C. testified that Holtzclaw then raped her for five or ten minutes. He let her go, and she went to her brother’s house. I didn't tell him everything that happened, but I asked him a question. I said, ‘Does a police have the right to ask a woman to pull her clothes down without another officer being there, a woman officer?’ And he told me he didn't know. But she didn’t tell her brother the whole story. Because I was ashamed. I've been a victim. I've been molested as a child by a minister in my church. I've been raped a couple of times. So I was just ashamed and I didn't want to face the rape. I didn't want to face it. She testified that shame stemming from her alcoholism also contributed to her reluctance to come forward — she didn’t want her brother to know she had been stopped for a DUI. Because I'm an alcoholic, but I'm in recovery. I'm an alcoholic and I suffer with that problem since — for years. Since the '80s. And I failed so many times — and I was ashamed, you know. I didn't go to prison until I was 30 years old, but my mother was a Christian, so I was ashamed of what I was doing, you know. Update: Holtzclaw was found guilty of raping R.C. Sue Ogrocki / AP One of the women who accused Holtzclaw of sexual assault, pictured in February 2015. R.G. April 2014. “And he was like ‘This is, you know, better than the county.’” On the night that R.G., 38, testified that she came into contact with Holtzclaw, she was walking by herself; he was in his car. That day, she had been "relapsing and getting high off of crack cocaine,” she said. I was a nervous wreck and so I had — I was relapsing. I told him I was relapsing, you know, I didn't have no business off over here anyways. He searched her bag and found her pipe. I was babbling on and on because, you know, I was still nervous about it. He asked her to throw the pipe on the ground and offered to give her a ride. She gave him the address of the place she was living with her boyfriend. When they got there, she realized she didn’t have her purse — that Holtzclaw had set it on top of his car. He said he’d go back and get it, and she told him not to worry about it. I'm just trying to depart, separate from him. Then I started walking towards the door and he was walking with me. And at first I was — I mean, it was odd, you know, but I wasn't going to question him, you know. R.G. had encountered cops before — even gotten rides from some — but never had them escort her to her door. Now, that's when my spirit was like something is just strange about that. She thought he might have doubts that she really lived in the house, so she started showing him around the house to prove she was familiar with it. He followed her up the stairs, where she showed him her room. Then he told her to sit on the bed. And he was like ‘This is, you know, better than the county.’ Holtzclaw exposed himself to R.G. through his fly, and she was forced into oral sodomy. She said the whole time she noticed his gun close to her face. About 10 minutes later, she testified, Holtzclaw raped her. But after a few minutes, he abruptly stopped, she said. I think because he was kind of looking out because my bedroom is above the front lawn and maybe he was looking out, maybe he got nervous. I don't really know. I could see he was looking like out the window and then back at me at that point. He immediately left. R.G. would tell her boyfriend and father what happened to her, but she didn’t tell the police. She was scared and relapsing, she said. I didn’t really know what to do, to be honest with you. She testified that she saw Holtzclaw one more time, after she’d just relapsed again. She acted like she didn’t see him, and he circled around the block she was walking on before driving away. Update: Holtzclaw was found guilty of forcible oral sodomy related to R.G.'s testimony, but he was found not guilty of raping her. T.M. May 2014. “I know that like I've been in trouble before, so I mean like, who am I to a police officer?” T.M., 44, testified that she was leaving an apartment complex when Holtzclaw pulled up beside her. The officer asked to search T.M.’s purse and if she had anything on her. T.M. told him she had a crack pipe. He put her in his car and ran her name through his system. He asked me a bunch of questions — where was I coming from, where was I going — and I told him all that. We sat there for a few minutes. I guess he was trying to see if I had a warrant … Then he got out. He came to the back door. He opened the door. I thought he was going to let me go. Holtzclaw allegedly told T.M. to pull down her pants and raise up her shirt. After having T.M. expose herself, Holtzclaw told her she could go to jail for the crack pipe. T.M. told him most officers just threw them away — eventually, he gave it back to her. He didn't say I could do anything not [to] go to jail. He just did what he did. That was probably whether I was going [to] or not. Holtzclaw allegedly forced T.M. into oral sodomy, exposing himself. She did it, briefly, and then he let her go. I was scared. About him being an officer. I was nervous and I felt like even though I didn't have no warrants that he might make up something on me and send me into jail any way. I know that like I've been in trouble before, so I mean like, who am I to a police officer? When he let me out he asked me where was I going, I told him I was going to my uncle's house. And he said, I don't think it's safe for you to walk. He said, ‘I wouldn't want anything to happen to you.’ He allegedly told her to get back in the car and then drove her to an open field. We didn't get out. He stopped for a second. I don't know. It was like he was thinking, deciding or something whether — I don't really know, but then I think he was deciding whether to, but I was so hysterical, kind of hollering a little bit. Because I was nervous and scared and I didn't know what was going to happen next. All of the sudden he told me to chill out because I was kind of crying and getting hysterical. And he was like I'm going to take you back. I'm going to take you where you go, and all of the sudden he just turned around and went back up the street and dropped me off where I asked. T.M. said she didn’t want to tell the police at first — that she was too scared. I didn't think nobody was going to believe me anyway. And I'm a drug addict, so the only way I knew to handle it was to go and get high to try to block it out, to make it seem like it didn't happen. I didn't want to because people were telling me... they wasn't going to believe me over a police — and I almost feel like all [officers] are the same. But then one night she and her ex had an argument, and the police were called. When the officers arrived, T.M.’s ex urged her to tell them about her alleged assault. Update: Holtzclaw was found not guilty of any charges brought forward by T.M. Eric Gay / AP S.B. on "Dead Man's Curve." S.B. May 2014. “We hear stories about the police, you know — it's real.” S.B., 48, was out walking when Holtzclaw stopped her, pulling his patrol car alongside her. He stayed in the car while asking her where she was coming from and where she was going. There is a house on the corner, and he asked me did I come from that house. And I was telling him, ‘No.’ And he was saying that it was a drug house. And I didn't know why he was asking me that because that's not where I was coming from. He asked me did I have anything on me or, you know, the usual questions. Any drug paraphernalia, drugs, whatever, weapons, whatever. S.B. said she didn’t have anything on her. Holtzclaw got out of his car, putting her in the backseat, and ran her name for outstanding warrants. She didn’t have any. He said, ‘Well, you got two choices. I can take you to detox or to jail.’ I had been drinking earlier and I guess I had alcohol smell on me or something. She told the officer she’d rather go home. Well, he sat there for a minute and he said, ‘Okay, I'm going to take you home.’ [He said] that he was really trying to get me off the streets and he was going to take me home, you know. Instead Holtzclaw took her to place the neighborhood calls Dead Man’s Curve. He slowed down and told her she had two choices — oral sodomy and rape or jail. I was like, ‘Really?’ … And he said, ‘No, really, I'm serious. You're going to give me head, sex, or you're going to jail.’ S.B. said "Okay." She was forced into oral sodomy and raped. [Afterward] I sat back in the backseat, closed the door. He said, ‘Do you know where you are?’ And I said, ‘Yes, I do.’ He said, ‘Well, it's about time for me to get off duty.’ He said, ‘Can you make it from here?’ I said, ‘Yes, I can.’ He got out the car, he opened the back door and he let me out and he said, ‘I'll see you again.’ S.B. said she saw him "many times" afterward. He would come through the neighborhood, driving through the neighborhood. He would stop, ask me what was I doing, where was I going. As a matter of fact, the day after that I was walking with my boyfriend and he stopped me, asked me what was I doing and where was I going. He stopped me one day in front of a friend. It was a nice day and everyone was out and he asked me did I, you know, tell anybody about the incident that happened and I said, ‘Yes, I did.’ He sped off because there was a lot of people out, and the people that I had told seen him and was looking at him and made remarks, so he sped off real quick. S.B. said that she told her “whole neighborhood” about what happened, but like the others, she never told the police. Well, in my neighborhood it's like, you know, we hear stories about the police, you know — it's real — I mean — doing things. Update: Holtzclaw was found guilty of both forcible oral sodomy and rape in S.B.'s case. S.E. May 2014. “I was very scared. I had never had nothing happen to me like that before.” Just before midnight, S.E. was leaving her house on foot to visit her cousin when Holtzclaw stopped her. I told him my name. And he had me in the back of the police car and he run a check on me, and he found out that I had warrants … He got out of the police car and opened the door. He asked me what was we going to do about the situation. S.E. told Holtzclaw she knew she had warrants from city tickets and that she’d recently gotten out of the penitentiary. After telling her he was going to search her, Holtzclaw groped S.E. under her shirt and pants. I knew it wasn't supposed to happen like that. He exposed himself through his fly, forcing S.E. into oral sodomy. She said she thought if she didn’t do it, she would go to jail. Holtzclaw then drove her to a park next to a shuttered school, parking between a building and a group of overgrown trees. There, she testified, he raped her for about five or ten minutes. I didn't know what was going to happen. I mean, he's a police — I didn't know. I was very scared. I had never had nothing happen to me like that before. Afterward, Holtzclaw said she was free to go. Update: Holtzclaw was found guilty of all charges brought forward by S.E., including rape, forcible oral sodomy, and sexual battery. Sue Ogrocki / AP One of the women who accused Holtzclaw of sexual assault in February 2015. C.J. May 2014. “Who are they going to believe? It's my word against his because I'm a woman and, you know, like I said, he's a police officer.” C.J., 52, was walking to a friend’s house when Holtzclaw stopped her. He asked her what she was doing and where she was going, and told her to take everything out of her pockets. She took her money — $55 in cash — and keys out of her pocket, placing them next to her purse and jacket on the back of the police car. He set my things on the backseat of the police car and he gets me in the backseat and he gets in the front seat and … [gets] to questioning me. So he's asking me questions: have I ever been arrested before, do I have anything stashed on me, you know, have I ever been arrested for prostitution and all that. And then he kept saying. ‘Are you sure you don't have anything stashed on you, because most girls that get arrested, you know, they're always hiding drugs down there close?’ And I told him, ‘No,’ that he can call a female officer to check me if he like, you know. I've been through this before. She heard through his radio that she had no outstanding warrants, and Holtzclaw let her out of his car. So as I'm getting out of the backseat of the car, I notice a $20 bill laying in the backseat of the car. So I go, ‘Oh, I must have dropped my money,’ so I pick my money up and as I'm picking up my money he go, ‘Well, let me check the backseat and make sure you didn't put any drugs back there.’ So by now I'm back at the back end of the car getting my belongings, my purse, and my jacket, and stuff. And then he tells me ‘Oh, yeah, by the way, give me back that $20 you picked up. ‘ And I tell him ‘No, that's my money.’ He go, ‘No, that's mine.’ So I took and pulled my money out — and I'm counting my money, and I see that I actually did still have my money along with an extra $20 bill, and that's when I tell him … ‘I'm sorry. This is yours.’ And he tell me, ‘Well, yeah.’ He said he put that there for a reason. She didn’t ask him what that reason was. She went back on her way to her friend’s house. But a few weeks later, she came into contact with Holtzclaw again while she was out walking, around 2 a.m., after leaving another friend’s house. Holtzclaw’s patrol car nearly hit her as he was turning a corner. He stopped and told her he didn’t see her. He go, 'Haven't I stopped you before? Didn't I arrest you?’ And I told him, no, he stopped me, but, you know, I didn’t get arrested. So anyway we talked and he puts me once again in the backseat of his car and he … calls it in, you know. We're talking and he asked me about drugs being down my pants. She told him there weren’t any drugs down her pants and heard her name come back clear over the radio. So he's getting me out the backseat of the car, so as I'm getting out, he go, ‘Are you sure you don't have anything down your pants?’ And I tell him, ‘No, I didn't.’ That’s when Holtzclaw groped her under her clothes. All I can say was, ‘Sir, you're not supposed to be doing that. Please, sir.’ Because I know that's something that he's not supposed to be doing. He's not supposed to be touching me, not like that. Holtzlcaw released C.J. She walked away, and Holtzclaw began droving away. She was on the phone with her roommate, leaving him a message and telling him what happened, when Holtzclaw’s car approached again. He stops again and he go, ‘By the way, you have a warrant you need to go and get taken care of.’ And I said, ‘A warrant?’ He go, ‘Yeah, I don't know what it's about, but you need to go and get it taken care of,’ and then he drove off. C.J. said she thought about calling the police. But then I thought, then again, you know, who are they going to believe? It's my word against his because I'm a woman and, you know, like I said, he's a police officer. So I just left it alone and just prayed that I never saw this man again, run into him again, you know. Update: Holtzclaw was found guilty of sexual battery in C.J.'s case. K. June 2014. “I was scared … of these police systems.” One night, K. went on a walk to cool off after arguing with her boyfriend. As she walked, a police officer pulled up beside her, his lights flashing. K. was talking to her boyfriend on the phone, and Holtzclaw told her to turn her phone off. She hung up on her boyfriend, telling him she was “fixing to get my name checked.” After putting her in the backseat and running K.'s name, Holtzclaw offered her a ride, she said. She declined. She didn’t want to be seen getting a ride from a cop, fearing people would think she’s a snitch. He wasn't trying to hear it. Like he was still trying to get me to get a ride and saying it was too late at night to be walking. Holtzclaw insisted. But instead of taking her in the direction she wanted, Holtzclaw allegedly drove her to an abandoned school, hopping the curb and sliding between two buildings on the school’s campus. He made her expose herself and allegedly told her, "I bet that pussy is wet,” then forced her into oral sodomy. He then raped her, she testified. Afterward, Holtzclaw told her he wanted to see her the next day. She watched him drive off and she walked home. She told her boyfriend what happened later. He said she should call 911. She didn’t. Because I was scared … of these police systems. I stayed in for a little bit, for a couple of days. K. eventually told her probation officer what happened after seeing news about the Holtzclaw case on TV. Update: Holtzclaw was found not guilty of any charges brought forward by K. AP Daniel Holtzclaw's mugshot. A. June 2014. “‘This is what you're going to have to do.’ That's what he said.” A., then 17, was walking through her neighborhood with two friends, who were arguing, when Holtzclaw pulled up and stopped them. He said he got a call about one of her friends threatening the other, and he questioned each of the three separately. When it was A.’s turn, Holtzclaw searched her purse, ran her name through his system, and told her that she had outstanding warrants. He told her she needed to take care of them, then let her and her two friends go. But later that night — just before dark — A. was alone and walking to her mom’s house when Holtzclaw stopped her again. He says, like, ‘I'm not sure you who you say you are.’ Because I didn't have my ID on me. He asked me where I stayed at and, you know, I told him my mama's house. I was just visiting. So he put me in the car and he took me to my mom's house. He let me out and then he kept on asking me if I had any drugs on me. I said, ‘No, I don't.’ He already had like looked through my purse earlier, so I was wondering — you know, it was kind of suspicious to me. They were on A.’s mom’s porch when Holtzclaw told her he had to search her. He groped her underneath her clothes and inserted his fingers into her genitalia. I was in shock. I was thinking like ‘What's going on? Why would he be doing this?’ He said, ‘You got warrants. I don't want to have to take you to jail. I don't want to make this any harder than it has to be.’ Something like that. I don't remember exactly. I was in trouble. Like this was bad. ‘This is what you're going to have to do.’ That's what he said. She testified that Holtzclaw then exposed his penis through his fly and raped her. I told him I didn't even want to do it before he pulled my drawers down, but it was too late. Afterward Holtzclaw told A., “'I might be back to see you later,'” she said. I just stood there. Why? Why? … I was confused. And I was shocked and I didn't know what to think and I didn't know what to do, like, what am I going to do, call the cops? He was a cop. Later, A. said, she told a friend what happened. It didn’t go over well. She said, like … ‘I wouldn't really tell a lot of people — they would think that you're snitching and it's not like you could tell the cops.’ [Snitches] — they're a waste of life. I was afraid of what could happen to me if I did snitch or if people around my neighborhood thought I was snitching or talking to the police. Update: Holtzclaw was found guilty of sexual battery, rape in the first degree, and rape in the second degree by instrumentation based on A.'s testimony. J. June 2014. “‘Oh, my God, he's going to kill me.’ That's what I kept saying to myself.” J. was driving home from playing cards and dominoes at a friend’s house — “where I usually go to relax,” she said — when she was pulled over by Holtzclaw. Earlier that night, she took a hit of her friend’s joint, she said, but she didn’t feel high. She had left her friend’s house around 2 a.m. — she had to be home to drive her fiancé to work at 4 a.m. [Holtzclaw] had his lights on … He said, he stopped me because I was swerving. And I said, ‘No, I wasn’t swerving.’ And then that's when he asked what did I have in my cup there, [said] that it was alcohol. I said, ‘No, sir, it's Kool-Aid.’ It was sitting in the center of my cup holder. I said, ‘You can taste it.’ Holtzclaw told J. to step out the car. As he led her to his patrol car, he asked why she was nervous. She said she wasn’t. I had my hands on the car and he just started patting me all over and said, ‘Do you have anything illegal on you?’ I said, ‘No, sir.’ He said, ‘You better let me know. If you do I'm going to take you to jail.’ I told him I didn't have any on me. He patted me down, didn't find any, and he told me to go sit in the back of his police car. He went inside my car — apparently, he saw that was Kool-Aid, and I guess he went through my purse. When he came back to the police car, he opened up the back door where I was at and he said to me ‘How do I know you don't have anything in your bra?’ J. testified that Holtzclaw then had her expose her breasts and genitals and shined his flashlight on both. He wasn't touching me, but he was touching himself... had his hand down there touching himself. After he made a comment like he said, ‘Damn, you got a big ass.’ Those are the exact words. And I'm like, ‘Oh, my God, he's going to kill me.’ That's what I kept saying to myself. At this point, J. was still sitting in the backseat of Holtzclaw’s car. He was standing beside her, outside the car, when he exposed his penis through his fly and said “Come on.” I was twirling my hands together I was so afraid. I said, "No, sir. Now, you're not supposed to do this. You're not supposed to do this, sir." He said, ‘Come on.’ He said, ‘I don't have all night.’ He said, ‘ just got off of work.’ And I'm sitting there, ‘No, sir, don't make me do this. Don't make me do this.’ I said, ‘You're going to shoot me,’ and I'm sitting there looking afraid. I try to bend my head down, but I was looking at that gun in his holster and I'm saying to myself when I bend down he's going to shoot me in the head. I was really afraid. I raised my head back up and I said, ‘Sir, please don't make me do this. Don't make me do this. You're going to shoot me.’ Then … he said, ‘I'm not going to shoot you, I promise.’ I said, ‘You promise?’ And he kind of made a snicker sound. A snicker, like a grin. He kind of grinned. A laugh. I held my head back up and I said, ‘Oh, no, sir, I can't do this.’ He backed away and he let me out the car. I thought he was going kill me. I just did. I couldn't see myself getting away with that. J. said that she was forced into oral sodomy, but only a "little bit … Not for very long because I wouldn't allow it.” He backed away from the car, and she got up. I didn't know what else to say. The only thing that would come to my mind because I thought when I walked away he was going to shoot me in the back. The only thing I could say, was ‘Thank you, sir. Thank you, sir, for not taking me to jail.’ J. got in her car, while Holtzclaw got in his car to follow her to her daughter’s house. But right before she turned onto the interstate, Holtzclaw sped right past her. She drove straight to her daughter’s house. I was crying. I woke her up out of her sleep. I said, ‘I need to talk to you. Something just happened to me.’ And she was mad because I woke her up and she jumped out of bed … And I told her what happened and she say, ‘What?’ And she started screaming and she was so upset. So she got her kids out of bed, she got them dressed. Her boyfriend was there. We all got in her car, we went to Springlake Police Division. No one was there. It was dark. I left. I looked around to see if I saw any lights or anything. There was no lights. We left there and went … back towards her house. We saw two police cars [parked] like side-by-side like they were talking. We made a U-turn and went back. And I started telling them what happened, and they called the captain, and the captain came and they took a report, and they took me back to the scene where it happened. Update: Holtzclaw was found guilty of procuring lewd exhibition and the forcible oral sodomy of J. J.’s allegations kicked off an investigation into Holtzclaw, putting him on administrative leave and eventually leading to his arrest. Detectives went through Holtzclaw’s records of running women’s names to interview each suspected victim. Ultimately these 13 women came forward, resulting in these three dozen charges against Holtzclaw, ranging from indecent exposure and stalking to forcible oral sodomy and rape. Of the 36 charges he faced, Holtzclaw was found guilty of 18. OKLAHOMA CITY (Reuters) - A former Oklahoma City police officer was found guilty of crimes including rape and sexual battery by a jury on Thursday in a case where prosecutors said he preyed on women who had trouble with the law, hoping their word would not stand up against his. Rev. T. Sheri Dickerson, of Oklahoma City's Expressions Ministry (L) and Robin Leake of Oklahoma City wait for a verdict in the case of a former city police officer charged with sexually assaulting and raping 13 women, in Oklahoma City, Oklahoma, December 10, 2015. REUTERS/Heide Brandes Daniel Holtzclaw, who turned 29 on Thursday, broke down in tears as the verdict was read. He was charged with sexually assaulting and raping 13 women and found guilty of 18 of the 36 charges, including four of the six rape charges. Sentencing was set for January and he could face life in prison. “I didn’t do it,” said Holtzclaw, before he was led out of the courtroom in handcuffs. The jury has been deliberating since Monday night. Protesters who gathered outside the court earlier this week, demanded that the all-white jury convict the officer who is mixed race Asian and white, based on the physical evidence and the word of the 13 black women, who testified about how they were sexually assaulted. In closing arguments on Monday, prosecutors said Holtzclaw targeted his victims by going after women he came across while on patrol. He ran background checks and went after those who had outstanding warrants, previous arrests or carried drugs or drug paraphernalia. They said he did this because he did not think authorities would take the victims’ word over his if he had to defend himself against sexual assault allegations. “He exercised authority on those society doesn’t care about,” Assistant District Attorney Gayland Gieger said in closing arguments. The defense said the victims provided testimony that was unreliable and dishonest. Defense attorney Scott Adams said Holtzclaw was an honorable police officer whose activities were made to appear evil and suspicious. Thirteen women took the stand in the trial, which began more than a month ago, telling jurors of sexual assaults that ranged from touching over their clothing to forced oral sex and rape. One of the final people to testify was a girl who said she was 17 at the time of the sexual assault and Holtzclaw raped her on her mother’s front porch. Holtzclaw, who did not testify, was fired over the accusations in January 2015 after approximately three years on the job. Daniel Holtzclaw, center, cries as he stands in front of the judge after the verdicts were read in his trial in Oklahoma City, Thursday, Dec. 10, 2015. Holtzclaw, a former Oklahoma City police officer,... (Associated Press) OKLAHOMA CITY (AP) — A former Oklahoma City police officer was convicted Thursday of raping and sexually victimizing eight women on his police beat in a minority, low-income neighborhood. Daniel Holtzclaw, who turned 29 Thursday, sobbed as the verdict was read aloud. Jurors convicted him on 18 counts involving eight of the 13 women who had accused him; the jury acquitted him on another 18 counts. He could spend the rest of his life in prison based on the jury's recommendation that he serve a total of 263 years, including a 30-year sentence on each of four first-degree rape convictions. He was also convicted of forcible oral sodomy, sexual battery, procuring lewd exhibition and second-degree rape. The jury deliberated for about 45 hours over four days. Holtzclaw's sentencing is set for Jan. 21. A judge will decide whether he will have to serve the sentences consecutively. Holtzclaw's father — a police officer in Enid, about 100 miles northwest of Oklahoma City — his mother and sister were in the courtroom as the verdict was read. At least one accuser was present, as well as several black community leaders. Seven armed deputies were stationed around the room. Holtzclaw's defense attorney, Scott Adams, declined to comment after the verdict was read. "Justice was done today, and a criminal wearing a uniform is going to prison now," Oklahoma County District Attorney David Prater said. "In those counts where the not guilty verdicts came back, they determined that we didn't prove those cases beyond a reasonable doubt. It doesn't mean they didn't believe the victims." The lead detective in the case, Kim Davis, said after the verdict: "I feel horrible for his family. It's brutal, but I think justice was served." The allegations against Holtzclaw brought new attention to the problem of sexual misconduct committed by law enforcement officers, something police chiefs have studied for years. During a monthlong trial, jurors heard from 13 women who said Holtzclaw sexually victimized them. Most of them said Holtzclaw stopped them while out on patrol, searched them for outstanding warrants or checked to see if they were carrying drug paraphernalia, then forced himself on them. Holtzclaw's attorney, meanwhile, described him as a model police officer whose attempts to help the drug addicts and prostitutes he came in contact with were distorted. Among the eight women Holtzclaw was convicted of attacking was a grandmother in her 50s, who launched the police investigation and who was in the courtroom Thursday. She said she was driving home after 2 a.m. when Holtzclaw pulled her over. He first asked her if she had been drinking, then ordered her out of the car and into the backseat of his squad car. He then stood over her and ordered her to perform oral sex. The woman was tearful after the verdict and prayed with supporters outside the courtroom. She was the first victim to testify. The last was a teenager who was 17 when Holtzclaw attacked her. Holtzclaw was convicted of three charges related to her case: first-degree rape, second-degree rape and sexual battery. The teenager recalled Holtzclaw pulling up in his police car as she walked home one night in June 2014. Holtzclaw drove her home and walked her to her door, where he told her he had to search her. She said he grabbed her breasts, then pulled down her shorts before forcing them off and raping her. Despite the number of victims, the case presented prosecutors with several challenges. Many of the women had arrest records or histories of drug abuse. Holtzclaw's attorney made those issues a cornerstone of his defense strategy. Adams questioned several women at length about whether they were high when they allegedly encountered Holtzclaw. He also pointed out that most did not come forward until police identified them as possible victims after launching their investigation. Ultimately, that approach did not sway the jury to dismiss all the women's stories. Holtzclaw was convicted of one of two charges related to a woman who testified he gave her a ride home, then followed her into her bedroom where he forced himself on her and raped her, telling her, "This is better than county jail." That woman testified in orange scrubs and handcuffs because she had been jailed on drug charges hours before appearing in court. But the jury still convicted Holtzclaw of forcible oral sodomy in her case. All of the accusers were black. Holtzclaw is half-white, half-Japanese. The jury appeared to all be white, though Oklahoma court officials said they did not have race information for jurors. Some supporters of the women questioned whether the jury would fairly judge their allegations. A former college football star, Holtzclaw joined law enforcement after a brief attempt at pursuing an NFL career. Oklahoma City police chief Bill Citty fired Holtzclaw before the trial began. Citty said in a statement Thursday night that the department was satisfied with the outcome of the "long and difficult trial and deliberation process." "We are satisfied with the jury's decision and firmly believe justice was served," the statement said. Holtzclaw's case was among those examined in an Associated Press investigation of sexual misconduct by law enforcement. The AP's yearlong probe revealed about 1,000 officers had lost their licenses for sex crimes or other sexual misconduct over a six-year period. Holtzclaw was not included in that count because he has not yet lost his license. The AP's finding is undoubtedly an undercount of the problem of sexual abuse in law enforcement. Not every state has a process for banning problem officers from re-entering law enforcement, known as decertification. And of those states that do, great variations exist in whether officers are prosecuted or reported to their state licensing boards. The mother of the 17-year-old victim told The Associated Press on Thursday night that she feels like justice has been served. The Associated Press generally does not identify victims of sex crimes and is not using the mother's name so as not to identify her daughter. The mother said she believes the type of police crime brought to light by the Holtzclaw case "isn't just a problem in Oklahoma — it's a problem for the nation." ___ Associated Press National Writer Matt Sedensky in West Palm Beach, Florida, and AP writer Nomaan Merchant in Dallas contributed to this report. (CNN) A jury found former Oklahoma City police officer Daniel Holtzclaw guilty Thursday of some of the most serious charges against him, including sexual battery, forcible oral sodomy and rape. Holtzclaw faced 36 counts. He was found guilty on 18. The former officer cried openly in the courtroom and rocked in his chair as the verdict was being read. Jurors deliberated for more than 40 hours over four days. The Oklahoma City Police Department welcomed the verdict. "We are satisfied with the jury's decision and firmly believe justice was served," it said. Sentencing is set for next month. His trial touched upon the explosive intersection of race, policing and justice in America. Holtzclaw, whose father is white and mother Japanese, was accused of assaulting or raping 13 women, all black, while he was on the job. Court records identify his race as "Asian or Pacific Islander." The jury was all-white, composed of eight men and four women. These racial dimensions energized civil rights and women's activists to draw attention to the case. Before the verdict was read, Oklahoma City NAACP President Garland Pruitt was concerned about the jury in the case, he told CNN affiliate KOCO. "We're very disappointed, very, very disappointed, that we don't have any minorities on there," Pruitt said. "We're not saying justice can't prevail, but we can be suspicious of it being (run) in a manner." Benjamin Crump, president of the National Bar Association, was also monitoring the case. He represented the families of Trayvon Martin and Michael Brown. Martin, a black teenager, was fatally shot by George Zimmerman, who was acquitted of charges in 2013. Brown was killed by an officer in Ferguson, Missouri, in 2014. "We will be here to make sure that this is not swept under the rug," Crump told reporters after attending a portion of the trial last month. "We come here to stand with these 13 victims of rape, who happen to be African-American women, to say that their lives matter, too." From #DanielHoltzclaw to #RKelly: For Black Girls When Their (Alleged) Rapes Are Not Enough" -- My latest @TheRoot: https://t.co/IKikdLuoU2 — Kirsten West Savali (@KWestSavali) November 30, 2015 In the first pool of 24 potential jurors, there were three black men, but they were not picked for the jury. Two alternates have Spanish surnames. About 70 potential jurors were initially called in for the case, the affiliate reported. Prosecutors would not comment on the jury composition. At the center of the case was how all victims, ages 17 to 50s, had criminal histories of drug use or prostitution, according to The Oklahoman newspaper. #BlackWomenMatter I saw these great pics from downtown from the protest for the rape victims of Daniel Holtzclaw pic.twitter.com/qS1NCuIDHM — BIG SAM (@SammieSawce) November 17, 2015 Prosecutors said Holtzclaw preyed upon the women in one of the state's poorest neighborhoods and used his badge and uniform during traffic stops to force the victims to submit to an escalating level of crimes, from groping to rape. Holtzclaw was accused of manipulating the victims by promising to drop a drug charge against them if they didn't report the purported assaults. "I didn't think anyone would believe me. I'm a black female," one accuser testified in the trial, according to the newspaper. She alleged Holtzclaw touched her bare breasts and later went to her home to force her to expose her genitals and breasts. But defense attorneys Scott Adams and Robert Gray painted a different picture of the accusers, saying the women were "street-smart like you can't imagine" according to CNN affiliate KFOR. The defense argued some of the alleged victims were high when the purported assaults took place. During the trial, one accuser was removed from the stand until she sobered up after she told the court, "I'm not going to lie. Before I came here, I smoked some marijuana and a blunt stick laced with PCP," The Oklahoman reported. She alleged that Holtzclaw touched her bare breasts and forced her to expose her genitals in 2014. Holtzclaw, who turned 29 on Thursday, had his own campaign for justice, with a Facebook page posted by his family. Holtzclaw was a star middle linebacker on the Eastern Michigan University football team and graduated with a degree in criminal justice. His father is a lieutenant on the Enid Police Department, the family said. He is also the brother-in-law of a law enforcement officer, The Oklahoman reported. In all, Holtzclaw faced 36 charges, including burglary, stalking, indecent exposure, sexual battery, forcible oral sodomy and rape. The case began after one woman came forward and accused Holtzclaw of "sexual impropriety" during a traffic stop, Oklahoma City police Capt. Dexter Nelson told CNN last year. It snowballed from there. Holtzclaw was fired from the force after allegations surfaced in 2014, which led to an internal investigation. In a termination letter obtained by KFOR, his former boss, Chief William Citty, wrote of the alleged offenses against women, "The greatest abuse of police authority I have witnessed in my 37 years as a member of this agency."

Summary: An Oklahoma City ex-cop who preyed on women in the communities he was supposed to be protecting has been found guilty on a slew of sex charges and could be going to prison for life. Prosecutors said Daniel Holtzclaw-whose victims ranged in age from 17 to 57-targeted women he encountered in low-income neighborhoods and raped or sexually assaulted those who had previous convictions or outstanding warrants because he thought they wouldn't be believed if they complained, reports Reuters. Holtzclaw, who had faced 36 charges involving 13 women, wept in court Thursday as he was found guilty on 18 counts relating to eight women, including four rape charges, the AP reports. It was his 29th birthday. Holtzclaw-who was arrested after forcing a woman to perform oral sex during a June 2014 traffic stop and was fired by the force in January this year-has a white father and Japanese mother, while all of his victims were black, CNN reports. BuzzFeed has testimony from the 13 accusers, including a woman who was 17 when he raped her on her mother's front porch. "I didn't know what to do, like, what am I going to do, call the cops?" she said. "He was a cop." "Justice was done today, and a criminal wearing a uniform is going to prison now," Oklahoma County District Attorney David Prater told reporters. "In those counts where the not guilty verdicts came back, they determined that we didn't prove those cases beyond a reasonable doubt," he added. "It doesn't mean they didn't believe the victims."

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Summarize: RELATED APPLICATIONS [0001] The present application is a Continuation of co-pending PCT Application No. PCT/ES02/00043, filed Jan. 30, 2002 which in turn, claims priority from Spanish Application Serial No. 200100266, filed on Jan. 30, 2001. Applicants claim the benefits of 35 U.S.C. §120 as to the PCT application and priority under 35 U.S.C. §119 as to said Spanish application, and the entire disclosures of both applications are incorporated herein in their entireties. TECHNICAL FIELD OF THE INVENTION [0002] The present invention is set within the agricultural sector, particularly in the field of process for combating insects. More specifically, it refers to a carrier vehicle of spores of at least one entomopathogenic microorganism adsorbed on a granular porous support or in powder form; to a device which includes said composition or said vehicle; to an incorporated attractant semiochemical component and to a method for combating insects by means of the use of said device. BACKGROUND OF THE INVENTION [0003] The use of insecticides in controlling plagues of insects presents problems such as toxicity; the lack of selectivity which leads to the destruction of beneficial insects or of natural predators of the plague it is sought to combat; and the resistance developed by the insects which causes an increase in the dose needed to maintain its efficacy. [0004] The use of semiochemical substances has developed substantially and, in particular, techniques such as surveillance in order to anticipate the appearance of plagues and extinguish them, sexual confusion in order to prevent reproduction of the insects, and mass capture in order to decrease the population of them. [0005] Semiochemicals display low toxicity and very high selectivity, since they act on a single species of insect. Also, they do not provoke any resistance among insects or contaminant effects for the environment. [0006] As well as the use of semiochemicals in a process for combating insects, the entomopathogenic properties of certain types of microorganisms, such as fungi, can also be exploited. These properties are known, but their effectiveness in biological control of insect plagues depends largely on their method of application. [0007] For the specific and directed application of entomopathogenic microorganisms, supports are needed for the spores that will keep them viable and without germinating for long periods of time in the field and, moreover, combining them with specific semiochemicals that will attract the particular species to be combated, towards the contaminant support. [0008] For the use of semiochemicals to be effective, it is necessary to have physical supports complying with requisites such as: [0000] providing an adequate emission rate of the semiochemical, [0000] permitting prolonged duration of the emission, [0000] avoiding degradation of the semiochemical, [0000] not producing contaminant waste, and [0000] to be economical and to permit easy application of the semiochemical. [0009] Of the emitter supports existing on the market, such as, for example, rubber septa, polyethylene pipes, porous plastic sheets, etc., none of them complies with the stated requisites. [0010] Moreover, the methods so far used for the application of entomopathogenic microorganisms present problems of survival which diminish their efficacy. [0011] Application WO-9101736 claims a gel of natural polymers to which attractant entomopathogens, feeding stimulants and protectors against UV are incorporated. It also claims several forms of gelling the polymers by means of metal cations and the insects ingest the entomopathogen with the gel. [0012] The differences with the present process are that what is claimed is the adsorption of entomopathogenic fungi spores in adsorbent materials selected among silicates, silicoaluminates, phosphoaluminates and ion exchange resins, which further incorporate the insect-specific volatile attractant compounds (pheromones and other natural or synthetic attractants) in such a way that a slow, controlled and lasting emission of the same takes place (Patent UPV No. P 9701077) and they maintain a suitable degree of humidity for the survival of the spores. [0013] WO-A-9208355 claims a process for drying microbial cultures, mixing them with a carrier like those used for formulating insecticides for dusting and drying, them with air. Its application is by means of coating seeds with microbial dust and is of no use for protecting aerial parts. [0014] Application WO-9211356 claims some particular strains of entomopathogenic fungi. It also claims a formulation wherein the spores are incorporated into an emulsified bait containing cotton seed flour (proteins and carbohydrates), extracts of parts of cotton plants, oil and an emulgent, all these as a “feeding stimulant”. This bait is sprayed in liquid form or it is added to a solid carrier in powder form, in the usual form of insecticide formulations and is applied as a wettable powder or in dusting. [0015] WO-9324013 claims a storage chamber for entomopathogenic fungi for their conservation until they are used for controlling insects such as cockroaches, flies, ants and plagues of larvae, plagues on lawns and caterpillars. In these chambers, the temperature, humidity and oxygen are regulated. An emulgent can also be added for applying the fungus in aqueous suspension or in powder form. It cites two patents for cockroach traps. [0016] WO-9510597 claims a formulation of entomopathogenic fungi wherein the conidia are suspended in a mineral oil. This formulation is used for being applied by spraying on crops. It can be made emulsionable for dispersion in water by the addition of an emulgent or adding an inert carrier to it for its application in the form of a suspendible powder as is usual in insecticide formulations. [0017] EP-A-0406103 claims the culture of an entomopathogenic fungus on an inert solid support such as montmorillonite or atapulgite and the use of the resulting mass for spraying against plagues or for fermenting in the soil under sporulation conditions. [0018] The use of adsorbent materials as controlled rate emitters of insect attractants—generally sexual pheromones—is described WO-A-9944420-A and WO-A-0000446. Nevertheless, the process described in these patent applications does not include the use of an entomopathogenic agent. [0019] As is revealed from the above paragraphs, both the actual existing physical supports and, in general, the methods for combating insects based on the use of entomopathogenic microorganisms still display serious deficiencies in several basic aspects, such as the period of duration of the composition or vehicle used and the demonstrated efficacy, for which reason there exists a demand for the development of new insect control systems. DESCRIPTION OF THE INVENTION [0020] The present invention aims to overcome the drawbacks of the state of the art by means of a carrier vehicle for spores of at least one entomopathogenic microorganism, consisting of an adsorbent support selected among a granular form, a powder form and mixtures of them, capable of retaining the spores and maintaining their viability and a attractant semiochemical component, wherein: [0021] the adsorbent support is a material selected among silicates, silicoaluminates, phosphoaluminates, ion exchange resins and combinations of them; [0022] the spores are adsorbed onto the mineral support; [0023] and the semiochemical component, selected among attractant semiochemical substances of insects susceptible of suffering the entomopathogenic effects of the spores, is also adsorbed on that support or on another analogous support. [0024] The advantage of this invention is that it combines three different effects: a) The fixing of the biological material on a support which adsorbs it on its surface in an adequate way for contaminating insects by contact and for maintaining its viability during a long period of time. b) This support maintains by adsorption a degree of humidity that is adequate for preventing the biological material from drying out yet is insufficient for its germination, giving a prolonged life. c) The same or another support, or a mixture of them, adsorbs the attractant producing a controlled and lasting emission thereof and ensuring the specificity. d) An adhesive fixes the adsorbent support, in powder or granular form, on a solid surface (plates, spheres, etc.). This adhesive can be an organic polymer or an aqueous gel which contributes to maintain a constant and regulated supply of humidity to the adsorbent support located thereon. e) The local form of application is more specific and ecological than general spraying. f) The carrier support of spores and/or of attractants is adhered to a solid surface (plates, spheres, etc.) by means of a natural or synthetic polymeric adhesive, and the device is located in the field in the usual way for traps. [0031] A first additional object of the present invention is a composition for combating insects which incorporates that vehicle comprising spores of at least one entomopathogenic microorganism, at least one adsorbent support selected from a granular form or in powder form and an attractant semiochemical component. [0032] A second additional object of the present invention is a device for combating insects, characterized in that it comprises a receptacle comprising, in a way that is accessible for insects, a carrier vehicle of spores of at least one entomopathogenic microorganism, at least one adsorbent support in granular or powder form, and an attractant semiochemical component. [0033] A third additional object of the present invention is a method for combating insects by means of infection of them with spores of at least one entomopathogenic microorganism, characterized in that an efficacious quantity of the vehicle or of the composition obtained, according to the invention, is made available to the insects. [0034] The entomopathogenic microorganism can be any fungus or bacterium capable of contaminating the insects. In a preferred manner, said microorganism is a fungus, for example Metarhizium anisopliae, Paecilomyces fumosoroseus, Beauveria bassiana, etc. The spores of the entomopathogenic microorganism are present in a quantity between 1×10 3 and 1×10 12 spores per gram of adsorbent support. [0035] The support is a natural or synthetic adsorbent material selected from among silicates, silicoaluminates, phosphoaluminates, ion exchange resins, or any combination of them. In a preferred manner, said support is a zeolite and more preferably still it is a sepiolite. [0036] The size of the structural channels of the adsorbent support must be adequate for housing the spores yet preventing them from penetrating to the interior of the structure of the support when exposed and accessible to insects. This size of particle lies between 230-450 nm, preferably between 240 nm and 420 nm (FIG. 1). The quantity of adsorbent support used is between 50 and 60 mg per cm 3. [0037] The device of the present invention can furthermore include an intermediate base for fixing the adsorbent support on a flat or curved solid surface. This intermediate base can be any organic adherent polymer, such as for example an elastomeric adhesive, or an emulsion of them, or an aqueous adherent gel obtained with one or more natural or synthetic gelling agents, such as for example agar, alginates or other polymers of algae and fungi, carboxymethylcellulose, crystalline cellulose, quitosanes and derivatives, methylcellulose and methylbutylcellulose. [0038] When this adherent intermediate base is an aqueous gel, a wetting agent can also be added to it, such as sorbitol, glycerol, manitol, xylitol and combinations thereof. Desiccation is thereby avoided and this intermediate base also helps to maintain the humidity of the adsorbent support in powder or granular form for the spores. [0039] In a preferred embodiment, methylbutylcellulose is used. In another preferred embodiment, a mixture is used of carboxymethylcellulose (CMC) and methylcellulose (MC) in a proportion of CMC and MC of between 5 and 40% by weight. The moistening agent is selected from among one or more polyalcohols, in a preferred way it is selected from among sorbitol, glycerol, manitol, xylitol and combinations thereof. In a still more preferred way, sorbitol or glycerol is used. [0040] The quantity of said moistening agent in the device is between 20% and 96% by dry weight. It is preferably in a proportion of from 25% to 85% by dry weight of the device. [0041] The attractant semiochemical component is specific for the species being dealt with and can be a pheromone or other natural or synthetic attractant that produces an adequate response. Specific examples of semiochemicals are trimedlure and 1-4-tetramethylenediamine, specific attractants of Ceratitis capitata, or methyl-eugenol, an attractant of Bactrocera dorsalis. The semiochemical component is present in the adsorbent support in a proportion between 0.005-1.0 gram per gram of adsorbent support, preferably in a proportion between 0.02-0.7 grams per gram of said adsorbent support. [0042] The adsorbent support of the present invention can furthermore comprise an oil component selected among mineral oils, vegetable oils, animal oils and mixtures thereof, which contributes to fixing the attractant semiochemical component and protecting the spores. The function of the oil component is to help in the retention of the semiochemical and its slow and controlled emission and to maintain the spores in an oily medium in order to increase their protection and extend their life-time. Said oil component is present in the vehicle by an amount between 20% and 75%. [0043] The device of the present invention can adopt various forms according to the arrangement of its components, thereby ensuring the maximum duration of the attraction and contamination of the insect. So, the surface coated with the support can be flat (plates of different dimensions, folded or unfolded) or curved (spheres imitating fruits, cones, cylinders and other shapes). [0044] In a first form, an adsorbent support containing the spores and the semiochemical component with or without oil is adhered to a solid surface by means of organic adhesives or aqueous gels. [0045] In a second form, the spores and the semiochemical component are adsorbed onto different adsorbent supports. The mixture of the two supports is adhered to a solid surface by means of organic adhesives or aqueous gels. [0046] In a third form, the adsorbent support containing the semiochemical component is incorporated into the adhesive base layer and the adsorbent support containing the spores is spread on the surface. [0047] In a fourth form, the adsorbent support containing the semiochemical component is in the form of a pill, located in the centre of the adhesive base layer and surrounded by the adsorbent support containing the spores. [0048] The vehicle of the present invention can include a UV ray photoprotector in its composition. [0049] For its transportation and use, the vehicle of the present invention is located on an object, for example, plates or spheres, the surface of which contains an adherent component and the adsorbent. These plates are located in the field underneath the traps, which protect them from the sun and rain. [0050] An additional object of the present invention is a composition that includes a carrier vehicle for spores of at least one entomopathogenic microorganism adsorbed on a support, as specified earlier, the application of which can be done in a suspension of the vehicle in a fluid, such as water for example, or by dusting, in an manner analogous to insecticide formulations. [0051] A second additional object of the present invention is a device for combating insects, comprising a receptacle which, in a manner accessible for the insects, comprises a vehicle or a composition which incorporates spores of at least one entomopathogenic microorganism adsorbed on an adsorbent support, as has been specified earlier. [0052] By means of the present invention, a long period of activity of the spores is achieved since the device maintains the necessary humidity in the adsorbent material for their survival and permits the degree of humidity to be maintained for prolonged periods, of the order of 2 to 5 months of exposure in the field. [0053] Another advantage over the prior art is the selective effect that is achieved, thanks to the use of an attractant semiochemical, specific to the species to be dealt with. [0054] Another advantage of the present invention consists of the use of particles of adsorbent material as support for the spores, which grants efficacy to the method of combating insects since it ensures its contact with the insect and contamination of it, as well as acting as an attractant vehicle and so that the humidity can reach the spores. EXAMPLES [0055] The following examples serve to illustrate the different aspects of the invention. Example 1 [0056] An adherent layer of 2 mm of polyisobutylene is deposited on a plastic plate, and on this another layer of MCM-41 zeolite carrier of adsorbed spores of Metarhizium anisopliae suspended in mineral oil is deposited. The zeolitic support is also impregnated with specific attractants of Ceratitis capitata, for example, with trimedlure or 2-4-tetramethylenediamine in a proportion of 0.5 and 0.005 g per gram of zeolite. [0057] Table 1 shows the result obtained with the spore carrier vehicle, exposed in the field for three months. In particular, the effect of ageing is shown on the physical and biological properties of the attractant-contaminant plates of Ceratitis capitata, along with the results of its action on the insects. TABLE 1 Power of Attraction Mortality Loss of Time (%) a) (%) b) humidity (days) (mean ± SD) (mean ± SD) (%) 0 2.10 ± 1.5 15.0 ± 0.9 0 15 8.2 ± 0.5 45.8 ± 1.6 35.4 ± 1.1 30 30.2 ± 2.6 68.4 ± 2.7 45.1 ± 2.7 45 37.4 ± 2.1 70.3 ± 2.7 59.7 ± 3.6 60 46.7 ± 1.7 76.4 ± 3.4 65.4 ± 2.6 75 40.9 ± 0.9 70.8 ± 3.2 69.3 ± 1.8 90 27.5 ± 2.5 67.6 ± 1.8 72.3 ± 3.1 a) Mean count of males alighting on the plate, every 5 minutes, for 3 hours. b) Number of dead flies as a result of mycosis following a 24 hour exposure period to the plate. Example 2 [0058] A plate is prepared with a gelled adherent base of methybutylcellulose in a proportion that can vary from 10 to 45%. Glycerol is added as a moistening agent in a proprtion of 20 to 75% by dry weight. Deposited on the is a layer of sepiolite of particle size between 240-420 nm, which is impregnated with spores of Paecilomyces fumosoroseus, suspended in mineral oil. The attractant semiochemical used is methyl-eugenol, an attractant of Bactrocera dorsalis, which is adsorbed on another portion of sepiolite in a proportion between of 0.8 gram of sepolite and is compacted to form a pill, which is partially submerged in the centre of the plate carrying the gel component. [0059] The plates are located in the field underneath the traps, which protect them from the sun and rain. [0060] Table 2 shows the effect of ageing in the physical and biological properties of the attractant-contaminant plates of Bactrocera dorsalis, along with the results of its action on the insects. TABLE 2 Power of Attraction Mortality Loss of Time (%) a) (%) b) humidity (days) (mean ± SD) (mean ± SD) (%) 0 6.10 ± 5 30.0 ± 0.7 0 15 11.2 ± 1.3 40.3 ± 1.1 30.1 ± 1.6 30 35.4 ± 1.9 78.0 ± 2.8 45.1 ± 2.7 45 49.8 ± 3.4 84.7 ± 3.4 43.4 ± 2.4 60 55.9 ± 2.4 90.3 ± 2.1 46.3 ± 2.6 75 50.2 ± 1.3 85.7 ± 1.4 50.1 ± 2.2 90 38.3 ± 2.2 70.4 ± 0.9 62.3 ± 1.3 a) Mean count of males alighting on the plate, every 5 minutes, for 3 hours. b) Number of dead flies as a result of mycosis following a 24 hour exposure period to the plate.

Summary: The patent describes a method of selective application of entomopathogenic fungi, characterized by employing an attractant-contaminant device in which the spores of said fungus are fixed on an adsorbent material; this same adsorbent material or another, depending on the case, incorporates a specific attractant and is located on an adherent material. This adherent material can, in certain cases, incorporate a gelling agent and different additives, which maintain the adequate level of humidity for the survival of the spores. Standing out among the advantages of this ecological method of application of entomopathogenic fungi is the selectivity resulting from the use of specific attractants and the long duration of the attractant-contaminant effect thanks to the use of the controlled rate emitter (adsorbent substance) and to the fact that a greater persistence of the spores is achieved with control of the humidity.

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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a safety ski binding having lateral retention wings and adjustment screws for adjusting the position of the wings to accommodate boots of different widths. 2. Description of the Prior Art Safety ski bindings known as "front abutment" bindings, immobilize of the front portion of a ski boot. This type of front abutment generally comprises a support element integral with the ski. The abutment also includes a unit comprising a body and a retention jaw. The retention jaw holds the boot, and the unit is adapted to laterally pivot, to the right or the left around the support element, against the bias of an elastic energization mechanism which defines the release threshold of the binding. In order to adapt the retention jaw of such a front abutment to different shoe widths, the jaw is composed of two lateral retention wings, each journalled on the body around their respective pivoting axes. Adjustment elements such as screws are provided to independently adjust the position of each lateral retention wing. Such an arrangement is illustrated, for example, in French Pat. No. 1,336,704, the disclosure of which is hereby incorporated by reference. In the binding described therein, each wing comprises an adjustment screw located on either side of the binding. One adjusts the configuration and position of the jaw by manipulating the two screws using a screwdriver on either side of the binding to bring the two retention wings into contact with the boot. The necessity of using a screwdriver on both sides of the binding is both time consuming and awkward. To overcome this disadvantage, other binding constructions have been proposed. For example, French Pat. No. 1,480,207 describes a front abutment having a body which pivots around a vertical axis integral with the ski. The body comprises two lateral retention wings whose angular positions with respect to the body are adjusted by means of a single screw which is screwed into the central portion of the body. Although this binding only requires one adjustment, such an adjustment apparatus has the disadvantage of having a complex and cumbersome structure. In addition, such a vertically disposed screw also has the disadvantage of being easily confused with the screw which adjusts the binding height and which is most often, itself, positioned vertically on the binding. The skier who is not knowledgeable can thus misadjust the width of the jaw of the binding while believing that he has adjusted the height, and vice versa. SUMMARY OF THE INVENTION The present invention overcomes the disadvantages of the prior art by providing a ski binding having journalled lateral retention wings and an apparatus for the adjustment of the wings which is of a particularly simple design and which allows for the simultaneous adjustment of the two wings in a single maneuver. To achieve this objective, the binding of the present invention comprises a jaw adapted to maintain the boot. This jaw comprises two lateral retention wings journalled on a body around respective axes, and two adjustment screws for adjusting the position of the lateral retention wings. The two screws are adapted to be disposed in a substantially transverse direction, respectively, in the two wings. The adjustment screws each include a head which is accessible from the exterior so that they can be rotated by means of a tool such as a screwdriver. The internal end of each screw is immobilized against translational movement on a central portion of the body. The openings in the binding in which the screws are engaged are threaded so as to receive the correspondingly threaded portions of the two adjustment screws. The internal ends of these screws are connected to one another by a coupling apparatus which couples the rotation of these screws, while allowing for an angular movement or pivoting of the axes of the screw. This ski binding of the present invention has the advantage that to adjust the spacing of the lateral retention wings, it suffices to rotate only one of the adjustment screws so that it is displaced into one of the wings. This rotational movement of this screw is transmitted by the coupling apparatus to the other screw, such that the two adjustment screws rotate at the same time and concomitantly pivot the two lateral retention wings around their respective axes. The adjustment of the spacing of the wings, so that they can be adapted to boots of different widths, is thus considerably simplified. According to one embodiment, the invention comprises a safety ski binding for holding a boot on a ski. The binding comprises a jaw which includes first and second displaceable elements, each adapted to hold at least a portion of the boot. The binding also includes first and second adjustment means for adjusting the displacement of the first and second jaw elements, respectively. Also, a coupling means is provided for coupling the first and second adjustment means so that adjustment of the displacement of one of the jaw elements also adjusts the displacement of the other of the jaw elements. These jaw elements are adapted to be displaced in a direction transverse to the longitudinal axis of the binding. In this embodiment, the first and second adjustment means each comprise adjustment screws adapted to rotate about an axis transverse to the binding in one of the jaw elements. In addition, each adjustment screw comprises means for adjusting the transverse position of one of the elements, in response to rotation of the adjustment screw. The coupling means couples the rotation of one of the screws with the rotation of the other of the screws. In one embodiment, the coupling means may comprise a universal joint, and the coupling means may be adapted to permit pivoting of the adjustment screws with respect to a horizontal longitudinal axis and with respect to a vertical axis. In addition, the horizontal longitudinal axis and this vertical axis may intersect at a point intersected by the longitudinal plane of symmetry of the binding and the longitudinal axis of the screws. In one embodiment, the jaw elements comprise lateral retention wings journalled around a substantially vertical axis. In addition, the binding may further include a body attached to the ski, on which the lateral retention wings are journalled. In this embodiment, each adjustment screw comprises means for journalling one of the wings on the body. The binding may also include elastic means for biasing each of the wings toward the interior of the body. In one embodiment, these elastic means comprise two elastically deformable extensions, each integral with one of the wings. The extensions are so positioned that they abut the body, regardless of the position of the wings. At least one of the screws comprises an outer head, accessible from the exterior of the binding, which is adapted to engage a tool, such as a screwdriver, for rotating the head. In another embodiment, both screws comprise such an outer head. The body comprises a central portion adapted to receive an internal portion of the screw. This internal portion of the screw is immobilized against translational movement in the central portion of the body, as will be described herein below. Each adjustment screw comprises a threaded portion having threads thereon such that the threads on one screw are oriented in the opposite direction from the threads on the other screw. In addition, each wing further comprises two openings, adapted to receive one of the screws. Each opening comprises internal threads adapted to mate with the threads on one of the screws. In one embodiment, the longitudinal axes of the openings are horizontal. In another embodiment, the longitudinal axes of the openings in the wings are inclined with respect to a horizontal axis from the bottom to the top of the wing and from the exterior to the interior of the wing. The coupling means comprises a male element integral with one of the screws, and a female element integral with the other of the screws. In addition, the body further comprises two notches opening toward the rear of the binding, and a stirrup disposed in the notches. The stirrup opens toward the rear of the binding and comprises two lateral arms each having a notch therein. Each screw further comprises a shaft having a smaller diameter than the its threaded portion. This shaft connects the threaded portion of the screw to one of the male and female elements. Each shaft extends through one of the notches in the body and extends through one of the notches in the lateral arms of the stirrup. The notches and the body together comprise a substantially U-shaped central opening, open toward the rear of the binding. In addition, the stirrup is also substantially U-shaped, and further comprises a core, positioned in the anterior portion of the central space such that the lateral arms are attached to either end of this core. The lateral arms extend toward the rear of the binding, and the coupling means is housed in this central space. The male and female elements further comprise an expanded head having a diameter greater than the width of the notches in the body and the stirrup. The male element further comprises a flattened portion, integral with the expanded head and having a width substantially greater than its height. The flattened portion includes two flat surfaces on opposite sides of the flattened portion and parallel to the longitudinal axis of the male element. The expanded head of the female element comprises a transverse opening, open in the direction of the male element. The transverse opening is defined by two spaced apart planar surfaces on opposite sides of the female element and on the internal surface of the transverse opening. The two flat surfaces of the male element are adapted to engage the two planar surfaces of the female element, respectively. The transverse opening has a width substantially greater than its height, and the internal surfaces of the opening are flared outwardly toward the exterior of the female element. The flattened portion of the male element further comprises two converging surfaces each integral with one of the flat surfaces, and extending from the flat surfaces toward the female element. Each converging surface converges toward the longitudinal axis of the male element. The male element further comprises a conical projection extending from the converging sections toward the female element. In addition, the female element further comprises an opening in the expanded head and at one end of the transverse opening, adapted to receive this conical projection therein. The screws are adapted to be displaced between a first and a second position in response to rotation of the screws. In the first position, the longitudinal axes of the screws are positioned in a single transverse plane. In the second position, the longitudinal axes of the screws form an obtuse angle open toward the front of the binding. The longitudinal axes of the screws are inclined symmetrically with respect to the longitudinal and vertical plane of symmetry of the binding. According to another embodiment of the invention, the invention comprises an adjustment apparatus for a ski binding having first and second jaw elements adapted to hold a ski boot on a ski. These jaw elements are adapted to be displaced. The apparatus comprises first and second adjustment means for adjusting the displacement of the first and second jaw elements, respectively. The apparatus also includes a coupling means for coupling the first and second adjustment means so that adjustment of the displacement of one of the jaw elements adjusts the displacement of the other of the jaw elements. The first jaw element comprises a first lateral retention wing, and the second jaw element comprises a second lateral retention wing. These first and second lateral retention wings are adapted to be displaced in a direction transverse to the longitudinal axis of the ski, and the first and second adjustment means adjust the transverse position of the wings. Each wing is journalled on a body attached to the ski, around a substantially vertical axis. In addition, the first and second adjustment means each comprises adjustment screws adapted to be rotated around an axis transverse to the longitudinal axis of the ski in one of the wings. Each screw comprises means for journalling the wing on the body and the coupling means comprises means for coupling the rotation of one of the screws with the rotation of other of the screws. The coupling means is also adapted to permit pivoting of the screws with respect to a horizontal longitudinal axis of the binding and a vertical axis. In one embodiment, the coupling means may comprise a universal joint. At least one of the screws comprises an outer head, accessible from the exterior of the binding, and adapted to engage a tool for rotating the head. In another embodiment, both screws comprise such an outer head. In addition, the body comprises two notches therein. Each screw comprises a threaded portion having an exterior end to which the head is attached, and an interior end. Each wing comprises an opening adapted to receive one of the screws and comprising an internally threaded portion. The internally threaded portion of each screw is adapted to mate with the internally threaded portion of the wing. The threaded portions of the first screw and the first wing have opposite orientation from the threaded portions of the second screw and the second wing. In addition, the coupling means comprises two elements, each integral with the interior end of one of the threaded portions of the screws. Each element of the coupling means comprises an expanded head abutting the notch in the body, and having a diameter greater than the width of the notch. In one embodiment, one of the elements of the coupling means comprises a male element and the other of the elements of the coupling means comprises a female element. In addition, the body comprises a stirrup having two lateral arms, and each having a notch therein. Each screw further comprises a shaft having a smaller diameter than the threaded portion, and connecting the threaded portion to the expanded head. Each expanded head abuts a notch in the stirrup, and each shaft extends through one notch in the stirrup and in the body. The male element further comprises a flattened portion integral with the expanded head, and having a width substantially greater than its height. The flattened portion includes two flat surfaces on opposite sides of the flattened portion and parallel to the longitudinal axis of the male element. The expanded head of the female element further comprises a transverse opening, open in the direction of the male element. The transverse opening is defined by two spaced apart planar surfaces on opposite sides of the female element and on the internal surfaces of the transverse opening. The two flat surfaces of the male element are adapted to engage the two planar surfaces of the female element, respectively. The internal surfaces of the openings are flared outwardly toward the exterior of the female element. In addition, the flattened portion further comprises two converging surfaces, each integral with one of the flat surfaces, and extending from the flat surfaces toward the female element. These converging surfaces converge toward the longitudinal axis of the male element. The male element further comprises a conical projection extending from the converging sections toward the female element. The female element further comprises an opening in the expanded head and at one end of the the transverse opening, adapted to receive the conical projection therein. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described by way of nonlimiting example with reference to the attached drawings in which: FIG. 1 is a planar view, partially broken away, of a safety ski binding of the present invention, the lateral retention wings being shown in the contracted, tightened position corresponding to a minimum shoe width; FIG. 2 is a vertical and transverse cross-sectional view along line II--II of FIG. 1; FIG. 3 is a partial planar view of the binding, the lateral retention wings being shown in a spaced apart position corresponding to a shoe having a width larger than the width of the shoe held by the binding in FIG. 1; FIG. 4 is a planar view, partially in horizontal cross-section, of an embodiment of an apparatus for coupling the two adjustment screws, in a coaxial position; FIG. 5 is a cross-sectional view, on an enlarged scale, along line V--V of FIG. 4; FIG. 6 is a longitudinal cross-sectional view, on a magnified scale, of the apparatus for the coupling of the two adjustment screws, wherein the axes of these two screws form an angle therebetween; and FIGS. 7 and 8 are perspective views, respectively, illustrating the male and female elements of the coupling apparatus. DESCRIPTION OF PREFERRED EMBODIMENTS The safety binding shown in FIGS. 1 and 3 is adapted to maintain the end of a ski boot 1 shown in dashed lines. This safety binding comprises, in a known manner, an assembly pivotably mounted on a support element 2 attached to the ski. This assembly comprises a body 3 at the front end thereof. The rear end of the assembly comprises two lateral retention wings 4 and 5 forming, with a central support 6, a maintenance jaw for maintaining the front of the boot. Central support 6 forms a single element with body 3. The two lateral retention wings 4 and 5 are journalled on the body around respective substantially vertical axes 7 and 8. Body 3 is pivotably mounted on support element 2, in a known manner, around two parallel or converging lateral support lines, of which one, i.e., the right support line A, appears in FIG. 1. Body 3 is biased under pressure against support element 2 by an elastic energization mechanism which is not shown in detail and which is indicated in its entirety by reference numeral 9 in FIG. 1. In FIG. 1, wings 4 and 5 are shown in their contracted or tightened position, corresponding to the maintenance of a boot 1 of small width, while in FIG. 3, wings 4 and 5 are spaced further apart towards the exterior, to maintain a boot 1 of greater width. Two adjustment screws 11 and 12 are used to adjust the position of the two lateral retention wings 4 and 5, respectively. More specifically, screws 11 and 12 are adapted to displace wings 4 and 5 in a direction transverse to the longitudinal axis of the binding, to accommodate boots of different widths. This is accomplished by journalling wings 4 and 5 around axes 7 and 8, respectively, as will be discussed below. Screws 11 and 12 are positioned symmetrically with respect to the longitudinal plane of symmetry P of the binding. In addition, screws 11 and 12 engage housings or openings 13 and 14, respectively, which extend substantially transversely in each wing 4 and 5. The longitudinal axes of these openings are either horizontal or slightly inclined with respect to a horizontal axis, from bottom to top of the binding and from the exterior of the binding towards the longitudinal plane of symmetry P as is shown in FIG. 2. Openings 13 and 14 communicate with the vertical external surfaces 4a and 5a of wings 4 and 5 by means of recesses. Screws 11 and 12 comprise, respectively, outer heads 11a and 12a which are adapted to be positioned in these recesses. These heads are, therefore, accessible from the exterior and are adapted to be rotatably manipulated by means of a tool such as a screwdriver. It is within the scope of the invention to provide only one screw with a head. Thus, in this alternative embodiment, screw 11 may have head 11a, but screw 12 will not include an outer head. Such an embodiment is seen in FIG. 9. Alternately, screw 12 may include head 12a, but screw 11 may not have head 11a. Screws 11 and 12 further comprise threaded portions 11b and 12b, respectively, which are integral with heads 11a and 12a, respectively. Threaded portions 11b and 12b have identical threads but are oriented in the opposite direction. These threaded portions 11b and 12b are screwed into internal threads of corresponding threadings provided in the wall of openings 13 and 14. Screws 11 and 12 also include shafts 11c and 12c, respectively, which are integral with threaded portions 11b and 12b, respectively. Shafts 11c and 12c extend away from threaded portions 11b and 12b, in the direction of the longitudinal plane of symmetry P of the binding and the ski. Shafts 11c and 12c are substantially cylindrical and have a smaller diameter than threaded portions 11b and 12b, respectively. In addition, shafts 11c and 12c extend towards one another and traverse notches 15 and 16 provided in the rear portion of body 3 and opening substantially horizontally towards the rear. Notches 15 and 16 define a central space in body 3 having a substantially U-shaped configuration which opens rearwardly and which is adapted to receive a stirrup 17 which is lodged therein. Stirrup 17 comprises a core 17a positioned in the anterior end of the central space, and two lateral arms 17b and 17c extending towards the rear. Lateral arms 17b and 17c both comprise, respectively, notches 17d and 17e. These notches 17d and 17e of stirrup 17 are superimposed, as seen in the transverse direction, on notches 15 and 16 of body 3. Shafts 11c and 12c also extend through notches 17d and 17e, respectively. According to the invention, screws 11 and 12 are coupled to one another by means of a coupling apparatus 18 which links the internal ends of the adjustment screws 11 and 12. Coupling apparatus 18, as seen in FIGS. 1-6, is housed in the central space defined between lateral arms 17b and 17c of stirrup 17. This coupling apparatus 18 comprises any universal journal apparatus or joint which is known in the art. Coupling apparatus 18 couples screws 11 and 12 so that adjustment of the displacement of one of the wings adjusts the displacement of the other wing. More specifically, coupling apparatus 18 is adapted to transmit the rotation of one of the adjustment screws 11 and 12 to the other screw, and is also adapted to permit an angular movement or pivoting of screws 11 and 12 with respect to two concurrent axes, i.e., a horizontal, longitudinal axis of said binding and ski, and a vertical axis, such that these two axes intersect at the center of the journal point O. Journal point O is defined by the intersection of the longitudinal plane of symmetry P of the binding and ski, and the longitudinal axes of screws 11 and 12. Coupling apparatus 18, illustrated by way of example in FIGS. 1-8, comprises two elements which are nested within one another, i.e., a male element 19 integral with screw 12 and a female element 21 integral with screw 11. Each of the male and female elements, 19 and 21 comprise respectively, an expanded head 19a and 21a. Head 19a has a diameter greater than the width of the notches 16 and 17e, and head 21a has a diameter greater than the width of notches 15 and 17e. As a result, elements 19 and 21 of coupling apparatus 18 are retained within stirrup 17, as can be seen in FIGS. 1 and 2, and they cannot, as is the case for screws 11 and 12, be displaced to the exterior of the binding. Furthermore, because expanded heads 19a and 21a abut notches 17e and 17d, respectively, when screws 11 and 12 are rotated, they cannot be displaced to any substantial extent in the transverse direction. As a result, when screws 11 and 12 are rotated, wings 4 and 5 will pivot around axes 7 and 8, respectively. Heads 19a and 21a of elements 19 and 21 are, respectively, connected to shafts 11c and 12c by truncated cone-shaped projections 19f and 21f supported against lateral arms 17b and 17c of stirrup 17. Cylindrical head 19a of the male element includes a flattened portion 19b integral with head 19a and which extends from head 19 toward the female element. Flattened portion 19 has a width that is substantially greater than its height and which is substantially greater than the height of opening 21b in female member 21. As a result, when male member 19 is rotated in opening 21b, female member 21 also rotates. Flattened portion 19b is defined by and comprises two surfaces 19c which extend in the substantially longitudinal direction of the male member and are disposed on diametrically opposite sides of head 19a from one another. Each surface 19c comprises two portions, i.e., a first portion and surface 190c substantially parallel to the longitudinal axis of head 19 and a second portion and surface 191c which extends from first portion 190c toward the female element and which converges towards the longitudinal axis of head 19. Flattened portion 19b also includes a central conical projection 19d integral with head 19 and which extends from portion 19b toward the female element. Head 21a of the female element comprises a transverse opening 21b which opens in the direction of male element 19 and which is adapted to receive male element 19 therein. The transverse opening is defined by two opposite planar surfaces 21c which extend towards one another and which are flared outward towards the exterior. In addition, an opening 21e is provided along the longitudinal axis of head 21a, in the end of transverse opening 21b. Opening 21e is adapted to receive the extreme conical projection 19d of male element 19. In addition, flattened portion 19b of male element 19 is adapted to engage transverse opening 21b of female element 21 and surfaces 190c and 191c are adapted to engage and be disposed between planar surfaces 21c. The coupling of screws 11 and 12 occurs because the cooperation between projecting flattened portion 19b and surfaces 21c of opening 21b. As can be seen in FIG. 5, the distance between the two opposed planar surfaces 21d of female member 21 is slightly greater than the thickness of the male element 19 between the two opposed flattened portions 19c. It is evident from the description which has preceded that when one of the adjustment screws, for example screw 11, is rotated to adjust the spacing of lateral retention wings 4 and 5, the rotation of screw 11 is transmitted, by means of coupling apparatus 18, to adjustment screw 12, and vice versa. As a result, it is evident that coupling apparatus 18 is a universal journal. Furthermore, coupling apparatus 18 permits screws 11 and 12 to move from their position illustrated in FIG. 1, in which their respective longitudinal axes are positioned in a single transverse plane (a position corresponding to the minimal shoe width) to the position illustrated in FIG. 3 in which the longitudinal axes of screws 11 and 12 form, in a plane, an obtuse angle which is open towards the front (a position corresponding to the maximum width of the boot). It should also be noted that in a vertical and transverse plane, as seen in FIG. 2, the longitudinal axes of the two screws can also be symmetrically inclined with respect to the vertical and longitudinal plane of symmetry P of the binding and the ski. The outwardly flared portion 21c of head 21a of female element 21 is adapted to permit a certain angular movement or pivoting between the longitudinal axes of screws 11 and 12 to prevent the locking of the coupling apparatus, as can be seen in FIG. 6. In addition, conical portion 19d engages opening 21e to assure linkage between the two screws during the assembly of the jaw. By virtue of this projection 19d there can be no sliding or uncoupling in the plane of the flattened portion 19b. To prevent floating of wings 4 and 5, these wings are preferably provided with means for permanently elastically biasing them towards the interior of the binding. These elastic biasing means can comprise extensions 22 which are attached to or are integral with each wind and extend towards the front. Extensions 22 permanently abut a fixed abutment 23 of body 3, regardless of the position of wings 4 and 5. Extensions 22 which are elastically deformable, act as return springs which bias, respective, wings 4 and 5 in the direction of the longitudinal plane of symmetry P of the binding and the ski. As a result, each wing 4 and 5 is maintained firmly against body 3, regardless of its angular position with respect to body 3. Extensions 22 also cause male element 19 and female element 21 of coupling apparatus 18 to be firmly engaged with each other, thereby avoiding the necessity of providing a transverse return spring housed in stirrup 17 to bias screws 11 and 12 towards the exterior. As was previously noted, linkage apparatus 18 could comprise different means. For example, apparatus 18 could comprise, for example, a universal or cardan joint or could further comprise two conjugated male and female elements adapted to be nested to each other, which are different from those which have been illustrated in the drawings. In addition, the adjustment apparatus according to the present invention can be utilized on a front abutment as well as on a heel clamp or any other apparatus of the boot binding type. Additionally, although the invention has been described with respect to particular means, materials and embodiments, it is to be understood that the invention is not limited to the particulars disclosed and extends to all equivalents within the scope of the claims.

Summary: A safety binding for a ski including a body and a jaw having two lateral retention wings adapted to hold the boot and which are journalled on the body. Also included are two rotatable adjustment screws each adapted to adjust the position of one of the lateral retention wings, to accommodate boots of different widths. A coupling apparatus is also provided for coupling the rotation of the adjustment screws. As a result, when the skier adjusts the position of one of the wings by rotating one of the screws, the position of the other wing is automatically adjusted. In order to accommodate the journalling of the wings on the body, the coupling apparatus permits the orientation of the longitudinal axes of the screws to be changed with respect to each other, as the wings journal on the body in response to rotation of the screws.

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Summarize: Officers filled an elderly man's pantry with enough groceries to last him a month. (Source: Facebook) Saturday night, a call came in at the Mount Pleasant Police Department that tugged at the heartstrings of officers that were on duty. An elderly man called dispatch saying that he had not eaten in two days. Officers quickly jumped into action. Officers Brian Gray, Nathan Bolton, Buddy Odom and Adam Runions went grocery shopping and bought enough food to fill the man's pantry for a month. The officers delivered the food to the man and even put the groceries in the cabinets for him. Copyright 2016 WAFF. All rights reserved. Report an Error Submit a Tip to WAFF 48 MT. PLEASANT, Tenn. (WKRN) – A group of Mt. Pleasant police officers are being recognized for going out of their way to help an elderly man over the weekend. Saturday night, the 79-year-old disabled man called dispatchers and said he had not eaten in two days. A short time later, a group of officers showed up at his door with several bags full of groceries. The officers had spent $160 out of their own pockets to make sure the man had enough food in his kitchen to last him a month. “I think he was shocked at the amount of food that we bought and just the fact that it was there without question,” said Nathan Bolton. A picture of the officers stocking the man’s cabinets was posted on Facebook and the good deed has been shared by people across the country. “It’s difficult for us to see as police officers. We’re out here to take care of the public at large and that doesn’t always mean stopping a car. Sometimes it’s us doing little things like this,” said Mark Billions. The police officers have since been praised for going above and beyond the call of duty. The elderly man, who asked News 2 not to use his name, said he appreciates everything they did to help him. The man is on a fixed income and relies on his social security benefits each month. He said he didn’t have any money to buy food because a former caretaker stole his debit card last weekend. She was arrested by the Mt. Pleasant Police Department Sunday night and has been identified as Tammy Brooks, 36, of Mt. Pleasant. Police say they have surveillance video showing her using the stolen debit card at various retailers. She is charged with theft and fraudulent use of a debit card. She was booked in the Maury County Jail. Mt. Pleasant police have started a food pantry to help citizens in situations like these. They are accepting canned food donations at the police department.

Summary: The Tennessee town of Mount Pleasant is living up to its name through its police officers, who responded to a call from an elderly disabled man in the best way possible. WKRN reports that the PD received a call Saturday evening from the 79-year-old resident, who said he lived on a fixed Social Security income and that he hadn't had a bite to eat in two days. A caretaker had stolen his debit card the weekend before, he noted. So the officers showed up at his door with $160 worth of food (enough to last a month, per WAFF) that they paid for themselves-and they even put it all away for him. "We would like to commend these fine Mount Pleasant Police Officers for going above and beyond the call of duty," a post put up Monday on the police department's Facebook page reads. They also arrested a suspect in the debit card theft: 36-year-old Tammy Brooks, per WKRN. And the officers decided to take their good deed further: They've since started a food pantry and are now asking for donated canned goods to help others in similar situations, per the station.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Homeland Security Information Sharing Enhancement Act of 2006''. SEC. 2. FINDINGS ON DISSEMINATION OF HOMELAND SECURITY-RELATED INFORMATION. Congress finds the following: (1) Section 201(d)(1) of the Homeland Security Act of 2002 gives the Department of Homeland Security authority to access, receive, and analyze law enforcement information, intelligence information, and other information from Federal, State, and local government agencies--including law enforcement agencies-- and to integrate such information in order to detect, identify, and assess terrorist threats to the homeland. (2) Section 201(d)(4) of the Homeland Security Act of 2002 likewise gives the Department the power to ensure ``timely and efficient access'' to these categories of information in order to effectively discharge its information sharing responsibilities. (3) Section 102A(f)(1)(B)(iii) of the National Security Act of 1947 (50 U.S.C. 403-1(f)(1)(B)(iii)), as amended by section 1011 of the Intelligence Reform and Terrorism Prevention Act of 2004, prohibits the Director of National Intelligence from disseminating information directly to State and local government officials. (4) Under section 119(f)(1)(E) of the National Security Act of 1947 (50 U.S.C. 404o(f)(1)(E)), as amended, the Director of the National Counterterrorism Center supports the responsibilities of the Department of Homeland Security to disseminate terrorism information. (5) Section 201(d)(9) of the Homeland Security Act of 2002 gives the Department of Homeland Security the responsibility to disseminate information analyzed by the Department to other Federal, State, and local agencies with responsibilities relating to homeland security ``in order to assist in the deterrence, prevention, preemption of, or response to, terrorist attacks...''. (6) Section 201(d)(11) of the Homeland Security Act of 2002 (6 U.S.C. 121(d)(11)) explicitly gives the Department the responsibility to ensure ``appropriate exchanges of information, including law enforcement-related information, relating to threats of terrorism against the United States''. (7) Section 201(d)(14) of the Homeland Security Act of 2002 gives the Department the responsibility ``to establish and utilize... a secure communications and information technology infrastructure... in order to access, receive, and analyze data'' and to disseminate that data to State, local, and tribal law enforcement agencies as appropriate. SEC. 3. HOMELAND SECURITY ADVISORY SYSTEM. (a) In General.--Subtitle A of title II of the Homeland Security Act of 2002 is amended by adding at the end the following: ``SEC. 203. HOMELAND SECURITY ADVISORY SYSTEM. ``(a) Requirement.--The Under Secretary for Information and Analysis shall implement a Homeland Security Advisory System in accordance with this section to provide public advisories and alerts regarding threats to homeland security, including national, regional, local, and economic sector advisories and alerts, as appropriate. ``(b) Required Elements.--The Under Secretary, under the System-- ``(1) shall include, in each advisory and alert regarding a threat, information on appropriate protective measures and countermeasures that may be taken in response to the threat; ``(2) shall, whenever possible, limit the scope of each advisory and alert to a specific region, locality, or economic sector believed to be at risk; and ``(3) shall not, in issuing any advisory or alert, use color designations as the exclusive means of specifying the homeland security threat conditions that are the subject of the advisory or alert.''. (b) Clerical Amendment.--The table of contents in section 1(b) of such Act is amended by adding at the end of the items relating to subtitle A of title II the following: ``Sec. 203. Homeland Security Advisory System.''. SEC. 4. HOMELAND SECURITY INFORMATION SHARING. (a) In General.--Subtitle A of title II of the Homeland Security Act of 2002 (6 U.S.C. 121 et seq.), as amended by section 3, is further amended by adding at the end the following: ``SEC. 204. HOMELAND SECURITY INFORMATION SHARING. ``(a) Information Sharing Environment.--Consistent with section 1016 of the National Intelligence Reform and Terrorism Prevention Act of 2004 (6 U.S.C. 485), the Secretary shall integrate and standardize the information of the intelligence components of the Department into a Department information sharing environment, to be administered by the Under Secretary for Intelligence and Analysis. ``(b) Information Sharing and Knowledge Management Officers.--For each intelligence component of the Department, the Secretary shall designate an information sharing and knowledge management officer who shall report to the Under Secretary for Intelligence and Analysis with respect to coordinating the different systems used in the Department to gather and disseminate homeland security information. ``(c) State, Local, and Private-Sector Sources of Information.-- ``(1) Establishment of business processes.--The Under Secretary for Intelligence and Analysis shall establish Department-wide procedures for the review and analysis of information gathered from State, local, tribal, and private- sector sources and, as appropriate, integrate such information into the information gathered by the Department and other department and agencies of the Federal Government. ``(2) Feedback.--The Secretary shall develop mechanisms to provide analytical and operational feedback to any State, local, tribal, and private-sector entities that gather information and provide such information to the Secretary. ``(d) Training and Evaluation of Employees.-- ``(1) Training.--The Under Secretary shall provide to employees of the Department opportunities for training and education to develop an understanding of the definition of homeland security information, how information available to them as part of their duties might qualify as homeland security information, and how information available to them is relevant to the Office of Intelligence and Analysis. ``(2) Evaluations.--The Under Secretary shall, on an ongoing basis, evaluate how employees of the Office of Intelligence and Analysis and the intelligence components of the Department are utilizing homeland security information and participating in the Department information sharing environment.''. (b) Clerical Amendment.--The table of contents in section 1(b) of such Act is further amended by adding at the end of the items relating to such subtitle the following: ``Sec. 204. Homeland security information sharing.''. (c) Establishment of Comprehensive Information Technology Network Architecture.-- (1) In general.--Subtitle A of title II of the Homeland Security Act of 2002 (6 U.S.C. 121 et seq.) is amended by adding at the end the following new section: ``SEC. 205. COMPREHENSIVE INFORMATION TECHNOLOGY NETWORK ARCHITECTURE. ``(a) Establishment.--The Secretary, acting through the Chief Intelligence Officer, shall establish a comprehensive information technology network architecture for the Office of Intelligence and Analysis. ``(b) Network Model.--The comprehensive information technology network architecture established under subsection (a) shall, to the extent possible, incorporate the approaches, features, and functions of the network proposed by the Markle Foundation in reports issued in October 2002 and December 2003, known as the System-wide Homeland Security Analysis and Resource Exchange (SHARE) Network. ``(c) Comprehensive Information Technology Network Architecture Defined.--the term `comprehensive information technology network architecture' means an integrated framework for evolving or maintaining existing information technology and acquiring new information technology to achieve the strategic goals and information resources management goals of the Office of Information and Analysis.''. (2) Clerical amendment.--The table of contents in section 1(b) of such Act is amended by adding at the end of the items relating to such subtitle the following: ``Sec. 205. Comprehensive information technology network architecture.''. (3) Reports.-- (A) Report on implementation of plan.--Not later than 360 days after the date of the enactment of this Act, the Secretary of Homeland Security shall submit to the Committee on Homeland Security and Governmental Affairs of the Senate and the Committee on Homeland Security of the House of Representatives a report containing a plan to implement the comprehensive information technology network architecture for the Office of Intelligence and Analysis of the Department of Homeland Security required under section 209 of the Homeland Security Act of 2002, as added by paragraph (1). Such report shall include the following: (i) Priorities for the development of the comprehensive information technology network architecture and a rationale for such priorities. (ii) An explanation of how the various components of the comprehensive information technology network architecture will work together and interconnect. (iii) A description of the technology challenges that the Office of Intelligence and Analysis will face in implementing the comprehensive information technology network architecture. (iv) A description of technology options that are available or are in development that may be incorporated into the comprehensive technology network architecture, the feasibility of incorporating such options, and the advantages and disadvantages of doing so. (v) An explanation of any security protections to be developed as part of the comprehensive information technology network architecture. (vi) A description of any safeguards for civil liberties and privacy to be built into the comprehensive information technology network architecture. (vii) An operational best practices plan. (B) Progress report.--Not later than 180 days after the date on which the report is submitted under subparagraph (A), the Secretary of Homeland Security shall submit to the Committee on Homeland Security and Governmental Affairs of the Senate and the Committee on Homeland Security of the House of Representatives a report on the progress of the Secretary in developing the comprehensive information technology network architecture required under section 209 of the Homeland Security Act of 2002, as added by paragraph (1). (d) Intelligence Component Defined.--Section 2 of the Homeland Security Act of 2002 (6 U.S.C. 101) is amended by adding at the end the following new paragraph: ``(17) The term `intelligence component of the Department' means any directorate, agency, or element of the Department that gathers, receives, analyzes, produces, or disseminates homeland security information except-- ``(A) a directorate, agency, or element of the Department that is required to be maintained as a distinct entity under this Act; or ``(B) any personnel security, physical security, document security, or communications security program within any directorate, agency, or element of the Department.''. SEC. 5. AUTHORITY FOR DISSEMINATING HOMELAND SECURITY-RELATED INFORMATION. (a) In General.--Title I of the Homeland Security Act of 2002 (6 U.S.C. 111 et seq.) is amended by adding at the end the following: ``SEC. 104. AUTHORITY FOR DISSEMINATING HOMELAND SECURITY-RELATED INFORMATION. ``(a) Primary Authority.--Except as provided in subsection (b), the Secretary or the Secretary's designee shall be the executive branch official responsible for disseminating homeland security-related terrorist threat information to State and local government and tribal officials and the private sector. ``(b) Prior Approval Required.--No Federal official may issue any homeland security-related analysis, advisory, or alert without the Secretary's prior approval, except-- ``(1) in exigent circumstances under which it is essential that the information be communicated immediately; or ``(2) when such analysis advisory or alert is issued to Federal, State, local, or tribal law enforcement officials for the purpose of assisting them in any aspect of the administration of criminal justice.''. (b) Clerical Amendment.--The table of contents in section 1(b) of such Act is amended by adding at the end of the items relating to such title the following: ``Sec. 104. Authority for disseminating homeland security-related information.''.

Title: To amend the Homeland Security Act of 2002 to enhance homeland security information sharing, and for other purposes Summary: Homeland Security Information Sharing Enhancement Act of 2006 - Amends the Homeland Security Act of 2002 to require the Under Secretary for Information and Analysis to implement a Homeland Security Advisory System to provide advisories and alerts regarding threats to homeland security. Requires such an advisory or alert to: (1) include information on protective measures and countermeasures; (2) be limited in scope to a specific region, locality, or economic sector; and (3) not use color designations as the exclusive means of specifying threat conditions. Directs the Secretary of the Department of Homeland Security (DHS) to: (1) integrate and standardize the information of the Department's intelligence components into a Department information-sharing environment; and (2) designate, for each such component, an information-sharing and knowledge management officer. Requires the Under Secretary to: (1) establish Department-wide procedures for the review and analysis of information gathered from state, local, tribal, and private-sector sources; (2) develop mechanisms to provide analytical and operational feedback; (3) provide Department employees training and educational opportunities; and (4) evaluate how employees of the Office of Intelligence and Analysis and the Department's intelligence components are utilizing homeland security information. Directs the Secretary, acting through the Chief Intelligence Officer, to establish a comprehensive information technology architecture for such Office. Makes the Secretary the executive branch official responsible for disseminating homeland security-related terrorist threat information to state and local government and tribal officials and the private sector. Prohibits any federal official from issuing a homeland security-related analysis, advisory, or alert without the Secretary's approval, with exceptions.

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Summarize: Luis Suarez has revealed he is receiving psychological help in a bid to overcome the problems that have seen him suspended twice in 15 months for biting opposition players. The Uruguay international's most recent offence occurred during the World Cup, where he was caught sinking his teeth into Italy defender Giorgio Chiellini's shoulder. That incident saw him handed a four-month suspension from all football, after he had been banned for 10 games for doing the same thing to Chelsea defender Branislav Ivanovic in a Premier League match against Chelsea in April 2013. VIDEO scroll down for Luis Suarez: I'll be smarter on the pitch after controversies. Luis Suarez caused huge controversy at the World Cup in June after biting Italy defender Giorgio Chiellini. The controversial forward was banned for 10 games in April 2013 for this bite on Chelsea's Branislav Ivanovic. Suarez was labelled 'fat' by some sections of the press prior to his Barcelona debut against Real Madrid. Now a Barca player following his £75million summer move from Liverpool, the 27-year-old is keen to prove that he is a changed man by talking about the steps he has taken to banish his disciplinary problems once and for all. He told El Observador: 'After the suspension I had some really hard times. I'm getting psychological help and the club have been a constant support.' As he moved ever closer to his return to football against Real Madrid on October 25, Suarez was also accused of being fat by sections of the press, but he has now spoken out against those who questioned his fitness. Suarez scored against Costa Rica, but Uruguay were denied by a late equaliser and then in a shootout. The £75m Barcelona forward equalised for Uruguay, but his side drew 3-3 and then lost 7-6 on penalties. 'I was never overweight. It was just as Luis Enrique said on one occasion: "Tell me when you see Luis Suárez thin". 'The doctor who carries out the medicals at the club said that I was one of the six players at Barcelona who had the lowest percentage of fat and lowest weight. It was all part of the newspapers' game.' His inability to control his temper has seemingly been the factor behind Suarez's omission from the list of candidates for this year's Ballon d'Or, but rather than protest against the snub, the former Ajax man is remaining philosophical, while targeting his first league goals for his new club. Suarez is yet to open his account for new side Barcelona after making four appearances so far. The former Liverpool striker shakes hands with old boss Kenny Dalglish after winning the Golden Shoe. 'I'm sure I haven't been nominated because of what I did at the World Cup,' he added. 'But in that case, they should take into account all those who did nothing at the World Cup. 'It hurts you, because I did a lot of things at Liverpool last year. But in football there's always another chance. It'll come. 'I'm not worried. If I don't score goals and we win, I'll be happy all the same. I really want to celebrate a goal in the shirt of the team I've always dreamed of - but we all have droughts.'

Summary: Luis Suarez is yet to open his account for new side Barcelona. The 27-year-old has revealed he is receiving psychological help. Suarez has been suspended on two occasions in the past 15 months for first biting Branislav Ivanovic, and then Giorgio Chiellini at the World Cup. The former Liverpool forward accepts he needs to change his ways. Suarez rejects claims he has ever been overweight in his entire career after being labelled fat from some sectors of the press in Spain.

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Summarize: Image caption This image shows the first waters of the tsunami breaching the Fukushima power plant's buildings Japan was unprepared for a nuclear accident on the scale of the one at the Fukushima plant, the government said in a report to be submitted to the IAEA. The report says poor oversight may also have contributed to the crisis. The authorities have pledged to make the country's nuclear regulator (Nisa) independent of the industry ministry, which also promotes nuclear power. It comes after Nisa doubled its initial estimate of leaked radiation in the first week after the disaster. The nuclear safety agency now says 770,000 terabecquerels escaped into the atmosphere following the 11 March disaster - more than double its earlier estimate of 370,000 terabecquerels. Although the amount is just 15% of the total released at Chernobyl in Ukraine in 1986 - the world's worst nuclear disaster - it suggests the contamination of the area around the plant is worse than first thought. More than 80,000 local residents living within a 20km (12 mile) radius of the plant have been evacuated from their homes. A voluntary evacuation policy is operating in the area 20-30km from the plant. Nearly three months into the crisis, the Fukushima Daiichi plant is still leaking radioactive material. Chief Cabinet Secretary Yukio Edano says more evacuations are being considered. Monitoring shows the lie of the land and wind patterns may be causing a build-up of radiation in other areas. Lessons learned The government admitted that it was unprepared for a severe accident, in a report by Japan's nuclear emergency taskforce, to be handed in to the IAEA later this month. ''We are taking very seriously the fact... that consistent preparation for severe accidents was insufficient. "In light of the lessons learned from the accident, Japan has recognised that a fundamental revision of its nuclear safety preparedness and response is inevitable,'' the taskforce said in an outline of the report. The report also confirms that three reactors went into meltdown earlier than previously thought when the earthquake and tsunami knocked out cooling systems and back-ups. Earlier, Nisa said that in reactor No 1, molten nuclear fuel dropped to the bottom of the pressure vessel within five hours of the quake - 10 hours earlier than initially estimated by operator Tepco. The safety agency also said a meltdown damaged the No 2 reactor after 80 hours, and the No 3 reactor after 79 hours of the twin natural disasters. But the government says it is still on track to bring the reactors at Fukushima to a cold shutdown by January at the latest. The government report comes as an independent 10-member expert panel begins an investigation into the causes of the nuclear accident. An investigation by the UN's nuclear watchdog, the IAEA, has already pointed out a key failure - admitted by Japan - to plan for the risk of waves crashing over the sea wall and knocking out the plant's back-up generators. Even though a major faultline lies just offshore, the sea wall at Fukushima was less than 6m (20ft) high. The height of the tsunami wave was about 14m. In its draft report, the IAEA said continued monitoring of the health and safety of the nuclear workers and the general public was necessary. The report also emphasised the importance of independent regulators in the nuclear industry. Facing widespread criticism for its handling of the crisis at the Fukushima Daiichi plant, the Japanese government on Tuesday announced its intention to create an independent nuclear agency, breaking up the ministry that both promotes and regulates atomic energy. The decision to separate the regulator (the Nuclear and Industrial Safety Agency, or NISA) from the promoter (the Ministry of Economy, Trade and Industry, or METI) came as part of a government report that calls for several major overhauls in the way Japan operates its nuclear plants and provides information about the ongoing crisis after the earthquake and tsunami in March. Previously, NISA was a subdivision of METI, an arrangement that critics say contributed to lax oversight of nuclear safety in Japan. The report, to be submitted this month to the International Atomic Energy Agency, also serves as a reminder of the challenges at the disaster-stricken facility. In it, Japan cites the “possibility” that melted fuel has penetrated the reactor pressure vessels in units 1, 2 and 3 and dropped on to the floors of the primary containment vessel. That acknowledgment came a day after the government doubled its estimate of the radiation released so far during the crisis. The report is at once a pointed self-critique and a pledge to learn from mistakes. Many of its admissions are familiar, echoing assessments by international agencies and outside experts. They include acknowledgments that Fukushima Daiichi was unprepared for a tsunami and that the government was too slow to provide information on radiation to people in the disaster zone. But the report also lends insight into the jumbled initial response, which was characterized by poor communication among the government and the plant operator, Tokyo Electric Power, as well as government agencies. It was unclear, the report says, exactly who had ultimate authority for nuclear safety. NISA is a regulator, but it was tucked within METI. And Japan’s Nuclear Safety Commission was responsible for part of METI. Local governments were in charge of environmental monitoring. “This is why it was not clear who has the primary responsibility for ensuring citizens’ safety in an emergency,” the report says. “Also, we cannot deny that the existing organizations and structures made mobilization of capabilities difficult to promptly respond to such a large-scale nuclear accident.” As the disaster unfolded, Japanese authorities relied on computer models and uncertain data while trying to assess the full scope of the damage to the reactor cores. Even now, it will probably be years before guesses about units 1, 2 and 3 turn into facts. But the government’s admission Tuesday of a possible melt-through reaffirmed assumptions held by outside scientists. “It’s much like the Japanese government conceding that gravity is a possibility,” David Lochbaum, an independent nuclear power expert, wrote in an e-mail. “Studies for decades have consistently concluded that a reactor core without cooling and makeup will overheat, melt, slump to the bottom of the reactor vessel, and burn through the vessel wall and drop on to the drywell floor.” Goshi Hosono, the lawmaker in charge of the latest report, said that the government often found itself torn over whether to release unconfirmed — and constantly changing — information to the public. Too often, he said, officials preferred to wait, opening themselves to accusations that they were hiding information. On March 18, for instance, Japan raised its assessment of the Fukushima accident to a Level 5, using the International Nuclear and Radiological Event Scale. On April 12, it revised that assessment, giving Fukushima a Level 7 rating — on par with Chernobyl. The government should have been more forthcoming in March, Hosono said during an interview in his office. “We could have — and should have — told the public even then [on March 18] that there was the possibility of a full meltdown and a level 7 event,” Hosono said. Special correspondent Akiko Yamamoto contributed to this report.

Summary: The closer investigators look, the worse the news gets out of Japan's Fukushima Dai-ichi plant. A new government report doubles the estimated radioactive fallout from 370,000 to 770,000 terabecquerels so far, reports the BBC. If a terabecquerel sounds a little obscure, it may be easier to fathom that the total is about 15% of the Chernobyl fallout. The government also acknowledged it was woefully unprepared to handle a disaster of this scope and promised to create an independent nuclear agency, one that doesn't also serve to promote the industry as under the current set-up, notes the Washington Post. "In light of the lessons learned from the accident, Japan has recognized that a fundamental revision of its nuclear safety preparedness and response is inevitable,'' said a report outline. Investigators also said three reactors at the plant went into meltdown earlier than thought and conceded the possibility that nuclear fuel melted through inner containment vessels, not just the core, notes AP. Ground contamination could be far worse as a result.

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Summarize: Center Director [email protected] My job at the Center is to steer our team to successful projects that will improve the lives of people suffering from complicated grief. As Founder and Director of the Center I am involved in all aspects of our work. I oversee the website, supervise all of our staff, lead our training initiatives and spearhead our fund-raising efforts. You might think this is a full time job, but in fact I have one of those. I serve as the Marion E. Kenworthy Professor of Psychiatry at Columbia School of Social Work so I am a Senior member of an academic faculty at a large university. If you are interested – that means research, teaching, writing, presenting and administrative responsibilities at the School. I have a lot of energy and I feel very lucky to be able to contribute in this way to the Center, the School of Social Work and Columbia University. I began working with complicated grief in the mid 1990’s, when colleagues at the University of Pittsburgh asked me to help figure out how to treat people suffering in this way. To do so I reached out to grief counselors, studied the science of attachment, loss and grief, enlisted the help of friends and colleagues and supervised clinicians in a pilot project. The approach that grew out of this early work was influenced by my inherent optimism and faith in human creativity and resilience. CGT now has the strongest evidence-base of any grief treatment in the world and hundreds of therapists are now using it both nationally and internationally. This is enormously gratifying. Also gratifying is the feedback from clinicians who are attending our Center workshops in growing numbers. They report that my enthusiasm for sharing what I have learned about bereavement is infectious. Highly satisfied workshop participants have come from around the world to learn our treatment method. In addition to teaching, I have now authored more than 400 peer-reviewed papers and book chapters and have successfully completed 13 grants from the National Institute of Mental Health totaling more than $16 million, among them three separate randomized controlled clinical trials testing complicated grief treatment. I stay sane by playing with 3 year olds and dogs, sharing ideas and stories with my psychologist daughter, reading novels, playing tennis, bike riding, hanging out with my wonderful husband who does a great job taking care of me and letting me take care of him, and of course with a little help from my friends. Workwise, at the end of the day, I am a clinical researcher and my heart is in clinical practice. It is deeply gratifying that CGT has helped so many people rediscover enthusiasm for life while finding a way to accept and honor their loss. ​The pain and sorrow of bereavement is supposed to get easier to bear as time passes. But what if it doesn’t? Psychiatrists call it "complicated grief" – and it can be treated. After Stephanie Muldberg’s 13-year-old son Eric died of Ewing’s sarcoma in 2004, she was lost in a sea of grief. Her days were long, unstructured, monotonous. She barely left her New Jersey home. When she did leave, she planned her routes carefully to avoid driving past the hospital, just a few miles away, where Eric had been treated during the 16 months of his illness, or the fields where he had played baseball. Grocery shopping was a minefield, because it was painful to contemplate buying Eric’s favourite foods without him. To enjoy anything when he could not felt wrong. And Muldberg never thought she would be able to return to the temple where he had celebrated his bar mitzvah – and where his funeral was held. Looking back, she describes herself as not knowing how to grieve after Eric died. “I didn’t know what to do, how to act in front of people – what I needed to do privately, who I could reach out to. I was fearful of making people more emotional, too emotional, and having to comfort them,” she tells me, by Skype. “I didn’t know how to talk about what I was thinking.” Muldberg’s long dark hair is pulled back and she’s wearing a white T-shirt. One of the things she says is that she thought if she stopped grieving, her memories of Eric would fade, and she’d lose her connection to her son for ever. The passage of time often seems the only remedy for grief, but time didn’t help Muldberg. In the years following Eric’s death, she says, she felt consumed by grief. Then a family physician heard a talk by Columbia University psychiatrist Katherine Shear about treating chronic and unremitting grief and thought Shear might be able to help her. Four years after Eric died, Muldberg arrived at the New York State Psychiatric Institute in Manhattan, for her first meeting with Shear. She answered Shear’s questions with as few words as possible. It was as if she were barely present in the small, windowless room. Her face was drawn and clouded; she sat crumpled in her chair, arms crossed tightly around her, as if the weight of her loss made it impossible to sit up straight. It felt to her as if Eric had died just the day before. Shear diagnosed Muldberg with complicated grief, the unusually intense and persistent form of grief she has been researching and treating for almost 20 years. Clinicians are just beginning to acknowledge how debilitating this form of grief can be. Grief, by definition, is the deep, wrenching sorrow of loss. The initial intense anguish, what Shear calls acute grief, usually abates with time. Shear says that complicated grief is more chronic and more emotionally intense than more typical courses through grief, and it stays at acute levels for longer. Women are more vulnerable to complicated grief than men. It often follows particularly difficult losses that test a person’s emotional and social resources, and where the mourner was deeply attached to the person they are grieving. Researchers estimate complicated grief affects approximately 2 to 3 per cent of the population worldwide. It affects 10 to 20 per cent of people after the death of a spouse or romantic partner, or when the death of a loved one is sudden or violent, and it is even more common among parents who have lost a child. Clinicians are just beginning to acknowledge how debilitating this form of grief can be. But it can be treated. I first learned about complicated grief while riding the subway in Boston, where I read an advertisement recruiting participants for a study at the Massachusetts General Hospital, which I later discovered was related to Shear’s research. By then, I’d been a widow for about a decade. I was 33 when my husband died and it was fast – just six weeks from when he was diagnosed with pancreatic cancer. My grief had a different kind of complication: I was pregnant, and our son was born seven months after his father’s death. By the time I read that subway ad, he was in elementary school, and I was holding my own. I gradually went back to work. Single parenting was overwhelming, but it kept me focused on what was right in front of me. Having a young child is filled with small pleasures and motherhood enlarged my sense of community. I fell in love again. But it still felt like I walked with a limp, and that limp was grief. Often, I felt that the course of my grief – as it slowed or accelerated – wasn’t within my control. Sometimes I’d buckle, and wait it out. Sometimes I’d push back. Somehow, I knew it was going to take as long as it took. There wasn’t anything to do about it except live. Freud, writing in Mourning and Melancholia, one of the first psychological essays on grief, saw it this way, too: “Although mourning involves grave departures from the normal attitude of life, it never occurs to us to regard it as a pathological condition and to refer it to a medical treatment. We rely on its being overcome after a certain lapse of time, and we look upon any interference with it as useless or even harmful.” That’s how it went for me. I’d be the first to say that my path through grief has been intellectual. I’ve spent years contemplating what grief is. That subway ad made me wonder: Was my grief a disease? To be diagnosed with an illness is to seek – or wish for – a cure. But conceiving of grief as a disease with a cure raises questions about what is normal – and abnormal – about an experience that is universal. Is grief a condition that modern psychology, with its list of symptoms and disorders and an ample medicine cabinet, should treat, as if it were an illness rather than an essential part of being human? A little more than a year ago I began sitting in on clinical training workshops at Columbia’s Center for Complicated Grief, which Shear directs. The first workshop was both a challenge and a relief. It was strangely comforting to be in the company of so many people – grief counsellors, social workers and therapists – who spent their time thinking about what it meant to grieve. It would be almost another year until I called Stephanie Muldberg to see if she’d be willing to talk at length about what her treatment was like. Sometimes I can feel in our conversations how deliberately she chooses her words. She is, she tells me, a very private person. At times her desire to talk about her experience of complicated grief feels in tension with her natural inclination to be more self-contained. “I think the problem is people don’t talk about grief, and I want to normalize the fact that people can talk about it, and make it easier, and not so taboo,” she tells me. § For something so fundamental to being human, there’s still a great deal we don’t know about the grieving process. It wasn’t until the 20th century that psychologists and psychiatrists claimed expertise over our emotions, including grief. The conventional wisdom about grieving is that it’s something to be worked through in a series of stages. Lingering on any stage too long, or not completing them within a certain window of time, might be dysfunctional. Clinicians disagree about how long is too long to grieve, about whether the grieving person should wait for her grief to shift on its own or do something to initiate that process, and about what to do, and what it means, if grief is slow or stalled. The idea of grief as something we need to actively work through started with Freud. John Bean, a psychoanalyst who has trained extensively with Shear and worked with her to treat patients in her research studies, explains to me that because Freud believed we have a limited supply of psychological energy, he viewed the central emotional “task of grieving” to be separating ourselves emotionally from the person who died so that we can regain that energy and direct it elsewhere. Freud thought this would take time and effort and it would hurt. His theory of “grief work” persists, often in tandem with newer theories of grief. There’s still a great deal we don’t know about the grieving process. If grief is work, then Elisabeth Kübler-Ross provided the directions for how to do it. Kübler-Ross first proposed the five-stage model in 1969 as a way to understand the psychology of the dying, and it quickly became a popular way to understand bereavement. Today, those stages – denial, anger, bargaining, depression and, finally, acceptance – are practically folklore. But it turns out grief doesn’t work this way. In the past several decades, more rigorous empirical research in psychology has challenged the most widely held myths about loss and grief. When George Bonanno, professor of clinical psychology at Columbia University’s Teachers College, researched the paths people take through grief, he discovered there’s more variation to how we grieve than psychologists thought. His office, in a massive gothic brick building in New York City’s Morningside Heights, is crammed with books and lined with Chinese sculptures. On a rainy afternoon he outlines the three common paths he identified. Some people, whom he terms “resilient”, begin to rebound from loss in a matter of weeks. Others adapt more gradually, following a “recovery” path. The intensity of those first days, weeks and months of mourning subsides. They “slowly pick up the pieces and begin putting their lives back together”, typically a year or two after losing someone close to them. People with complicated grief, like Muldberg, struggle to recover. Their grief becomes what Bonanno calls “chronic”, staying at a high level of intensity for years. One school of thought that has influenced Shear is called the dual-process model: grief is stressful, so we alternate between confronting the emotional pain of our loss and setting it aside. Even grieving people, research has shown, have moments of positive emotion in their lives. Hope returns gradually. If the stage model maps a single, clear path through grief, then the dual-process model could be seen as a charting a wave pattern through grief. It’s now an axiom of grief counseling that there’s no one right way to grieve. That seems like a good thing, but it’s also a problem. If everyone grieves differently, and there’s no single theory of how grief works, then who’s to say that someone like Muldberg isn’t making her way through grief in her own way, on her own clock? Even though it was clear to her and to those around her that, four years after her son’s death, she was still suffering, bereavement researchers don’t agree about how to explain why her grief was so prolonged – or what to do about it. § Shear, who is in her early 70s, is the warmest shrink you’ll ever meet. Everything about her conveys equanimity, especially the way she can sit with the stories of patients whose grief is unrelenting. It wasn’t always that way. “At the beginning,” she tells me, she was “afraid to sit in the room with someone who was really intensely grieving because I was still a little bit uneasy with death and dying, but also because it makes you feel so helpless – because you feel like there’s nothing you can do”. The grieving person, she says, “feels like the only thing that’s going to help” is bringing back the person they are grieving – “and you agree”. “Grief is not one thing,” Shear says. “When it’s new, it crowds out everything else, including even people and things that are actually very important to us. It stomps out our sense of ourselves, too, and our feelings of competence. We think of grief as the great disconnector, but over time, it usually settles down and finds its own place in our lives. It lets us live in a meaningful way again. It lets us have some happiness again.” Two weeks later, I’m jammed into a hard plastic desk in an overheated university classroom listening as Shear, who is professor of psychiatry at Columbia’s School of Social Work, explains the underlying principle of her work, which is that “grief is a form of love”. She quotes me C S Lewis’s A Grief Observed to explain what she means: “Bereavement is an integral and universal part of our experience of love. It is not the truncation of the process but one of its phases; not the interruption of the dance, but the next figure.” This is called an attachment approach to grief. It’s shared by many grief researchers and counsellors, and it can be traced back to the British psychiatrist John Bowlby. Attachment is what gives our lives security and meaning. When an attachment is severed by death, Shear says, grief is the response to the lost attachment. Peel back the psychological theory, and what you’ll find is something that anyone who has experienced grief knows intuitively: “Nature is so exact, it hurts exactly as much as it is worth, so in a way one relishes the pain, I think. If it didn’t matter, it wouldn’t matter,” writes the novelist Julian Barnes in Levels of Life, his extended essay on grief following the death of his wife. Shear explains that it’s our close bonds to those dearest to us that also help us want to care for other people and confidently explore the world. These attachments are woven into our neurobiology. The longing and yearning of acute grief, and the feeling of unreality that comes with it, she says, are symptoms of just how much grief short-circuits our bio-behavioral wiring. The effects of complicated grief are symptoms of just how much grief short-circuits our bio-behavioral wiring Shear agrees with Bonanno that over time most grieving people integrate their loss into their lives. But people with chronic grief face some complicating factor. Complicated grievers tend to be women. They are often excellent carers but not so good at taking care of themselves or accepting help. Often, their emotional reserves of self-compassion and self-motivation have been drained. Shear says that “we don’t grieve well alone”, but frequently people with complicated grief become isolated because their grief has remained at high levels for so long; the people around them may feel that they “should have gotten over it by now”. Shear believes that adapting to grief and loss is “a normal, natural process”, she says. “We’re not talking about grief itself being abnormal. We’re talking about an impedance in some problem of adaptation.” Think of it this way: her therapy jump-starts a stalled process, the way a defibrillator restarts a stopped heart. § Shear’s office, with its striped beige wallpaper and mahogany furniture, is so spotless it would feel like a hotel room it if weren’t for the picture of her grandson as a chub-cheeked toddler on her panoramic Apple monitor. It’s a sticky day in July, and she’s telling me how she came to study and treat grief. In the 1990s, Shear was researching anxiety and panic disorders at the Western Pennsylvania Psychiatric Institute and Clinic when she became involved with research on depression and anxiety in elderly people. One of the common triggers for depression in the elderly is the death of a spouse, and the team she was working with identified a cluster of symptoms in depressed patients that weren’t depression. They expressed deep yearning, were often driven to distraction by thoughts of their deceased spouse, and had great difficulty accepting death, to the point that persistent, acute grief became a risk to their physical and mental health. To differentiate grief-related symptoms from depression and anxiety, Shear worked with a research team that included psychiatric epidemiologist Holly Prigerson. It was Prigerson who, in 1995, had published a questionnaire that identified complicated grief as a specific syndrome and could accurately assess its symptoms. Shear has relied on it as a diagnostic and assessment tool in her research ever since. Shear and her colleagues also used it to design a new treatment, complicated grief therapy. Prigerson, who now holds an endowed professorship at Weill Cornell Medicine in New York City, and directs Cornell’s Center for Research on End-of-Life Care, continues to work on the epidemiology of prolonged grief. § In their first meeting, Shear asked Stephanie Muldberg to keep a daily grief diary, recording and rating her highest and lowest levels of grief. Muldberg kept this diary for the duration of the therapy. Every day for almost half a year she was paying such close attention to her grief that it became inscribed in her daily life. Not that her grief wasn’t already a pronounced everyday presence, but now, with Shear’s help, she was facing it head-on rather than avoiding it. The diary was one of several techniques Shear used to help Muldberg look her grief in the eye. Muldberg says that the grief diary helped her pay attention to herself in a way she hadn’t been able to do in the four years after Eric’s death. Using the diary, she began to see that she had some happy moments interspersed with some low times of grief. “There were always going to be hard times during the day for me, but I wasn’t only focusing on the hard times, I was starting to learn how to move forward.” Complicated grief therapy (CGT) takes place over 16 sessions, structured, Shear says, by techniques adapted from approaches used to treat anxiety disorders, including cognitive behavioral therapy, a well-researched approach to psychotherapy, and exposure therapy, used to treat avoidance and fear in anxiety disorders. The structure itself is part of the therapy, she says, because structure is reassuring to people who are feeling intense emotions. Shear has been testing CGT since the mid 1990s. In 2001, she and her colleagues published a small pilot study that showed promising results. Since then, they have published several randomized controlled studies supported by the National Institute of Mental Health, demonstrating that CGT helps patients who have complicated grief to reduce their symptoms better than conventional supportive grief-focused psychotherapy. Shear is a pioneer, but she’s not an outlier. Currently a group therapy version of CGT is being studied at the University of Utah. Researchers in the Netherlands and Germany are also exploring variations on cognitive behavioral therapy and exposure therapy to treat traumatic and prolonged grief. And a recent study in Wales confirms one of Shear’s main findings, which is that the techniques in her treatment are more effective together than separately. § A few sessions into her treatment, Shear asked Muldberg to do something she had never done, which was to tell the story of the day Eric died. It’s a technique Shear adapted from prolonged exposure therapy that she calls “imaginal revisiting”. At first, Muldberg says, she was apprehensive because she wasn’t sure if she could remember what had happened. Over the course of three weekly sessions, Muldberg told the story of Eric’s death, rating her levels of emotional distress as she did. The purpose of this technique is to “help people connect with the reality of the death in the presence of a supportive person who is bearing witness to it,” Shear explains. “We want to keep grief centre stage,” she says. “If you do let yourself go there, paradoxically your mind finds a way to ace that reality and to reflect on it.” Then, as with the grief diary, Muldberg had “homework”: listening to a tape of herself telling the story every day between sessions. At first, this was distressing, but she gradually learned how to manage her emotions, recognising, she tells me, that she wasn’t going to forget Eric. The intensity of her feelings began to lessen, so that by about halfway through the therapy she began to feel better. Muldberg admits she was sometimes skeptical of what Shear was asking her to do, and she says sometimes she pushed back. Part of CGT includes psychoeducation, in which the therapist explains to the patient the premise and purpose of the therapy. Shear’s explanations, Muldberg says, helped her understand that “there was a reason I was feeling this way”. She describes Shear’s approach as “I don’t want to push you but we’re going to figure out ways that you can accomplish these things, feel good about them, and do them.” A few weeks after Muldberg started revisiting the story of Eric’s death, she worked with Shear to make a list of the places and activities she had been avoiding since he died, and gradually started trying to face them. Shear calls this “situational revisiting”, a form of prolonged exposure therapy. “We do this to provide people with an opportunity to confront the reality of the loss and actually understand its consequences, because being there without the person is going to be different than being there with the person. We want people to start to reflect on that,” she tells me. For Muldberg, many of the things she had avoided were the everyday parts of being a mother, such as going to the grocery store, but she says, “I didn’t realise how much harder avoidance was than doing some of these things.” Together with Shear, she broke down tasks, such as driving past the baseball field where Eric had played, into smaller steps until she could do them again. § Sitting in that classroom listening to Shear explain these exercises makes my chest tighten until my heart aches. I can’t imagine doing them myself, let alone how anyone with complicated grief could withstand them. It seems like a wrenching exercise in repeatedly tearing a scab off a wound. When I ask Shear about this she acknowledges that her approaches are counter-intuitive because they “ask people to go toward their grief”. She tells me it’s by explicitly detailing and describing their grief that people with complicated grief become unstuck, as they learn to shift back and forth between the pain of grief and restoring their lives. Shear is more interested in having patients engage with the therapy techniques than she is with getting them to reach a certain point. To her workshop audience, she puts it this way: “We do not try to lower grief intensity. I’m just trying to turn the Titanic one degree.” In one of my conversations with Muldberg, I remark that CGT seems counter-intuitive, almost confrontational, and that these exercises seem extremely emotionally demanding. She is quick to correct me. Therapy was challenging, she says, but it came as a great relief to finally feel understood and have the support to face Eric’s death. “When I started to do things, I started to feel better,” she tells me. For Shear, “feeling better” is a sign that our natural adaptive abilities are kicking in, allowing a person who is suffering from complicated grief to begin the emotional learning process that ultimately helps grief subside. This also creates an opening for the person to begin to reimagine their life after a devastating loss. At the same time that Shear was helping Muldberg come to terms with the reality of Eric’s death, she was also helping her begin to envision the future. Part of losing someone very close, Shear says, is that we lose our sense of identity. Part of grieving is regaining it. In another CGT exercise, the therapist asks a scripted question: “If someone could wave a magic wand and your grief was at a manageable level, what would you want for yourself? What would you be doing?” Someone with complicated grief can’t imagine a future without the person they’ve lost, or without the unrelenting, intense grief that’s taken up residence in their life. It’s a future-oriented question for someone who has lost sight of the future. Just asking the question, Shear says, can activate our innate exploratory system and spark hope. One way to think of the therapist’s role in CGT is that she’s teaching her patient what grief is. “Loss is a learning process. The problem is, it’s unwanted information,” says therapist Bonnie Gorscak, one of Shear’s long-time collaborators and a clinical supervisor at the Center for Complicated Grief. Learning from loss, Gorscak says, means being able to “stand in a different place and look at grief”, to approach the pain it causes, experience it, and have some respite from it. It’s a counter-intuitive approach for therapists, too. Sitting with someone with complicated grief, Gorscak says, “is some of the worst pain I’ve ever sat with”. § CGT is challenging, but it works. Still, Shear’s therapy has sparked controversy, starting with the very idea that there is a form of grief so severe and debilitating that it meets the definition of a mental illness. In recent years, Shear and a group of colleagues have advocated for a grief disorder to be included in the Diagnostic and Statistical Manual (DSM), psychology’s diagnostic bible, because they believe complicated grief is a clear-cut, diagnosable syndrome, separate from depression, anxiety or post-traumatic stress disorder. (Shear and Prigerson, once collaborators, now disagree about the best way to diagnose complicated grief, but they agree it should be viewed as a mental disorder.) Without sanction by a DSM diagnosis, psychotherapy in the US is not covered by health insurance. Without insurance reimbursement, CGT is out of most people’s reach. In 2013 the DSM-5 listed Persistent Complex Bereavement Disorder as a “condition for further study”, calling for more research on the issue. The major issue therapists have with complicated grief is that they believe it pathologizes a fundamental human experience. Leeat Granek, a health psychologist at Israel’s Ben-Gurion University, is concerned that including a grief disorder in the DSM could narrow the spectrum of acceptable ways to grieve and create a narrative that would distort the ways people understand their own grief. She believes that this would lead to “a lot of shame and embarrassment for the mourner because the expectations around grief are no longer realistic”. Donna Schuurman, senior director of advocacy and training at Portland’s Dougy Center, which supports grieving children and families, questions the idea of a grief disorder. She rejects the use of terms such as “complicated”, “debilitating” or “persistent” to describe grief reactions and as the basis for constructing a diagnosable syndrome. Schuurman agrees that “grieving people may have chronic issues or chronic problems related to what has happened after someone dies”, but says that “often those issues were already there before the death”, and that “chronic issues ought not to be framed as mental disorders of grief”. The major issue therapists have with complicated grief is that they believe it pathologizes a fundamental human experience. “Medicalizing or pathologizing the experience of someone who is having difficulty after a death does not do justice to the full social and cultural context in which he or she is grieving,” she writes. “Grief is not a medical disease, it is a human response to loss. Many people who are experiencing severe challenges after a loss are doing so because the social expectations around them are not supporting them.” Instead of labelling complications of grief as symptoms that define a disorder, Schuurman says she would focus on the experiences and behaviors that were contributing to any “serious challenges” a grieving person was facing. “We can label it depression, drug or alcohol abuse, etc., as any good therapist should do,” and “try to look at underlying issues, and not just symptoms, to be of help,” she explains. Good professional help, she believes, “could take a variety of forms and theoretical backgrounds”. New scientific research on grief, Shear’s among it, is challenging some of the foundational premises of grief counseling as it has been practiced, often in community settings. As George Bonanno discovered, there are several common trajectories through grief, meaning that there are some commonalities among grieving people as they adapt to loss. Still, Shear says, “each experience of grief is unique, just as each love experience is unique”. CGT, she says, “helps people find their pathway to adapting to loss”. § One way to answer the question of whether or not grief is a disease is to ask if the treatment provides a cure. Stephanie Muldberg describes her grief as “a wound that wasn’t healing”, but CGT isn’t a cure the way antibiotics cure an infection. Grief doesn’t end, it just changes form. Muldberg says CGT taught her how to live with grief as part of her life. She still carries her grief for Eric with her, but she is also back in the world. She travels with her husband and daughter. She volunteers for the Valerie Fund, an organization that supports families of children with cancer and blood disorders, and that helped Eric and their family when he was sick. § I ask Shear when her fear of sitting with intensely grieving people had subsided. “Well,” she says, “there’s this entire field of study called terror management.” I was expecting her to tell me about her feelings but she answers by telling me how research explicated them – exactly what she’s done in designing a therapy for complicated grief. I look up terror management: it’s the theory that in order to deal with the fear of our own mortality, we find ways to find meaning and value in our lives – like helping people. In that sense, what Shear has done with CGT is to create a form of evidence-based compassion. It’s compensation, perhaps, for the existential helplessness of the therapist, but it also compensates for many of our communal failures helping people grieve. We are too busy, too secular, too scared to deal with grief. It’s hard for Western culture – American culture in particular – to sit with something that can’t be fixed. § The more I thought back over my conversations with Stephanie Muldberg, the more I thought about how her therapy with Shear helped her put Eric’s death in context of her life story. The idea that a story needs a beginning, middle, and end goes back to Aristotle. People with complicated grief can’t see the arc of their own stories. They can’t get to what classic plot theory calls denouement – resolution. Most of us, when faced with a loss, find a way of putting what happened into the form of a story: this is what happened, this is who I was, this is what the person who died meant to me, and this is who I am now. But people who have complicated grief can’t do this. Grief is a problem of narrative. A story, in order to be told, needs a narrator with a point of view who offers a perspective on what happened. But you can’t narrate if you don’t know who you are. Many of Shear’s therapy techniques are about learning to narrate in the face of great pain and devastating losses. Start with the grief diary, which records the emotional story of your everyday life. Follow that by imaginal revisiting, akin to a wide-angle shot in cinema, which helps organize a story arc amidst intense emotion. Plotting out the story restores the narrator and the narrative. Then, you can begin to imagine a new story, a new plot for yourself. It’s not a choice between grief or living, remembering or forgetting, the way Muldberg once worried it was. The book of life is a multi-volume set. A sequel can only start when the first volume is brought to a close and when the narrator knows she’s going to be all right. This article first appeared on Mosaic and is republished here under a Creative Commons license.

Summary: The grieving process is never a simple one, but for some, it becomes never-ending and debilitating-what psychiatrists call "complicated grief." Andrea Volpe delves for Digg into this more emotionally intense form of bereavement: a deep, unceasing sorrow affecting no more than 3% of the population, overtaking women more than men, and usually following the death of a romantic partner, the loss of a child, or the abrupt or violent death of a loved one. Volpe explores the various ways society has so far suggested we deal with a great loss, from Freud's theory that grief is hard psychological work that needs dedicated time, to Elisabeth Kubler-Ross' "five stages of grief" mode (the default resource of sorts) for people in emotional pain, to the dual-process paradigm, in which people alternate between dealing with their grief and putting it aside, so hope "returns gradually." But Volpe notes that because everyone grieves differently, and for different lengths of time, it can be hard to treat complicated grief. Enter Katherine Shear, a CGT (complicated grief therapy) pioneer. CGT, which Shear has been using since the mid-'90s, is a highly structured, intense method that combines cognitive behavior therapy with "exposure therapy." That includes the hard stuff like taking part in activities that remind you of a lost loved one or recounting the day that person died-i.e., asking patients to "go toward the grief." As Volpe puts it, grief is a "problem of narrative," where people get frozen in one part of the story. CGT helps the patient get "unstuck" and take control of plotting the tale so he or she can "begin to imagine a new story" and resume living. Read more about this unusually deep type of sorrow, including why Shear wants it categorized as a grief disorder in the DSM-and why others don't.

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Summarize: FIELD OF THE INVENTION [0001] The invention relates to kneepads and shin pads and means for alleviating pressure and discomfort of the knees and legs of workers when they are working on inclined surfaces such as roofs. BACKGROUND TO THE INVENTION [0002] Kneeling to work on either a sloped surface such as a roof, or a horizontal surface such as a floor, presents an ergonomically challenging task that stresses a worker's legs and feet. When a worker is kneeling on any surface, either with both knees or partially kneeling on one knee, the front of the worker's knee makes contact and supports the bulk of the worker's weight. Contact with the surface is made, through the skin of course, by the upper part of the shin bone, also called the tibia, and sometimes but not necessarily the kneecap, also called the patella. This contact surface has no established name, and will here be called the “kneefront”. However the worker's toes must typically apply balancing pressure that varies with upper body posture. Each of the worker's legs forms an open triangle over the work surface comprised of the kneefront and unsupported along the triangle's top side, the foot and toe oriented roughly at a right angle to the shin and forming its second side, with the floor or roof or other kneeling surface closing the triangle along its bottom side between the toe and kneefront. This unsupported leg geometry continuously strains a kneeling worker's ankle and toe muscles as they counterbalance fore and aft motions of the upper body, regardless of whether a task is being performed on a flat or inclined surface. It also puts strain on the worker's back. [0003] These kneeling strains are considerably exacerbated when working on an inclined surface such as a roof because gravity imposes a relentless shear force onto all points where the worker contacts the sloped surface. The shear force results in an unpleasant feeling, as the worker's skin, whether through clothing or not, stretches to stop sliding down the roof. The worker's ankles and toes also experience increased stress due to force exerted by the body's weight tending to move down the slope, which the feet must be continuously counteracting, regardless of upper body posture. The strain on the back is also worse in this position. [0004] A wide variety of kneepads exists in the prior art. They could generally be classed as simple “kneefront protectors”. In general, the prior art kneepads do not alleviate the foot muscle strain caused by unsupported shins, and none provide relief from the shear forces experienced when working on an inclined work surface. [0005] A few more advanced kneepads attempt to exploit the users shin, defined as the front part of the leg below the knee and above the ankle, in order to spread the kneeling forces onto a larger area and thereby improve comfort. U.S. Pat. No. 6,795,974 to Howell discloses a kneepad that extends over the shin, but does not have any significant thickness to raise the feet partly or fully off the kneeling surface. U.S. Pat. No. 6,637,034 to Worden discloses a kneepad that extends along the shin and raises the knee from the kneeling surface, but leaves the foot as the other point of support, so that the main result is that the knee is less bent than it would be without that kneepad. [0006] U.S. Pat. No. 4,438,754 to Canney discloses a knee protector that does not make contact with the kneefront, but straps to the shin and leaves the kneefront in mid-air. This creates an uncomfortable, or uneasy feeling in the wearer, and undue pressure on the knee joint because there is no support directly under the thigh which is putting downward pressure on the knee joint. [0007] U.S. Pat. No. 6,845,515 to Sveilich discloses a shin rest that is relatively short, at about 5 to 10 inches, and is intended to be worn low on the leg and used in association with a separate kneepad. Sveilich discloses one embodiment that joins the kneepad to the shin rest, at an articulated joint, which would be altogether quite long and massive and that is undesirable for the wearer when standing up while still wearing the device. [0008] All of these kneepads and shin rests in the prior art have essentially constant thickness, and are best suited for working on a horizontal surface. The present invention is intended for working on a sloped surface, such as a roof. The present invention comprises a wedge shape that is not found in the prior art. SUMMARY OF THE INVENTION [0009] It is an object of the present invention to overcome the deficiencies noted in the prior art concerning kneepads, particularly as they pertain to working on an inclined roof. It is another object of the present invention to provide kneepads that reduce or eliminate pressure on its user's feet. [0010] It is another object of the present invention to provide shin wedges that reduce or eliminate shear forces on a user's body when working on an inclined surface. [0011] It is another object of the present invention to increase the safety of workers on steeply inclined surfaces by increasing their frictional contact onto it. [0012] These objects are attained by a shin wedge comprising a body that is wedge shaped, having an upper surface that is dimensioned and configured to support a user's kneefront and to supportingly hold a portion of a user's shin, and having a lower surface that comprises a substantial area that is substantially flat. The upper surface extends from a user's kneefront distally to more than one-quarter of the distance along the user's shin but less than one-half of the distance along the user's shin. There is a third surface that faces in the direction of a user's foot. One or more harness straps are connected to that body, and are dimensioned and configured to secure the shin wedge to a user's leg. [0013] The dihedral angle included between the upper surface and the lower surface is called here the “wedge angle”. [0014] The user of the present invention is more comfortable because the effect of the incline is substantially eliminated, and the user feels as if he is kneeling on a nearly horizontal surface. This reduces strain on the back, ankle, and toes. The shearing force on the skin of the user, whether felt through clothing or directly on the skin, is completely eliminated if the wedge angle is the same as the pitch of the roof. More generally, that shearing force is at least substantially reduced in proportion to how closely the wedge angle equals the pitch of the roof. [0015] Furthermore, the user is safer because the contact with the roof is the entire bottom surface of the shin wedge, which can easily be made much larger than the combined area of a person's kneefronts and toes, even if the kneefront is supplemented by the sort of kneepad existing in the prior art. [0016] The force of gravity is vertical and in the customary notation of physics is m*g, where m is the mass (in this case, of the worker plus the shin wedge) and g is the acceleration of gravity. When the mass is resting on an inclined plane (such as a roof) having pitch angle P, measured from horizontal, the force of gravity can be resolved into m*g*sin(P) pointing along the roof, and m*g*cos(P) perpendicular to the roof. The vector m*g*cos(P) is not relevant to the following discussion. It is the force vector m*g*sin(P) that causes the worker to slide down the roof. The frictional force that resists sliding of the shin wedge is a vector pointing along the roof in the opposite direction to m*g*sin(P). Undesired sliding occurs when the force vector m*g*sin(P) exceeds the frictional force. As the value of P increases, sin(P) increases, and more frictional force is needed. The wedge angle does not come into this calculation directly. The only way to increase frictional force is to increase the area of the shin wedge in contact with the roof, or to enhance its frictional properties. A larger wedge angle for a given length of the shin wedge along the user's shin will result in a longer surface, that being the hypotenuse of the triangle in which the length along the shin is adjacent to the wedge angle. Therefore, the wedge angle comes indirectly into the calculations for resisting sliding, by increasing the area providing the friction. The sine for P=45 degrees is approximately twice the sine for P=20 degrees, so the area of frictional contact, and the frictional force (although that also depends on the friction material) would be approximately double for a shin wedge with wedge angle 45 degrees compared to a shin wedge with wedge angle 20 degrees. No knee or shin protector in the prior art has this beneficial effect that increases safety of the user of the present invention. [0017] These and other objects, features, and characteristics of the present invention will be more apparent upon consideration of the following detailed description and appended claims with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The preferred embodiments of the invention will now be described in detail with reference to the following drawings, in which: [0019] FIG. 1 illustrates a shin wedge according to the present invention, in the environment in which it is used. [0020] FIG. 2 is a large-scale illustration of one of the shin wedges shown in FIG. 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT [0021] Referring to FIG. 1, roofer 1 is shown kneeling comfortably on inclined roof surface 3 using a pair of shin wedges 2 configured according to the present invention. The shape of the shin wedges is generally that of a wedge, having two non-parallel plane surfaces meeting at the thin side of the solid figure but not necessarily forming a sharp edge there. This shape could be described as a blunt wedge, but here is simply called “wedge”. The top surface may be not a flat plane, but rather (as will be described below) it may have a lengthwise depression to conform to the shin of the user. Opposite the thin blunt side is a thicker side to complete the solid triangle. The shin wedges are always worn so that the thin end is near the users knee (the proximal end) and the thick end is towards the foot (the distal end). [0022] The shin wedge also usefully provides leverage, allowing the user to lever a knee up by rotation of the shin wedge about its distal edge. [0023] In FIG. 1, for illustration, the wedge angle, is the same angle as the pitch of the roof, so that the upper plane is horizontal, and therefore the user's kneefronts and shins rest on a horizontal surface, which is the position of least discomfort. In general, the closer the wedge angle is to the pitch of the roof, the greater the comfort of the roofer, because drag on the skin is reduced. However, the wedge angle and the pitch will not be equal in all cases, and any particular shin wedge can be used on roofs with various pitches. The wedge angle may be greater than the pitch of the roof or less than the pitch of the roof, circumstances called here “over-wedging” and “under-wedging” respectively. [0024] In a case of over-wedging, the user's shin will be sloping down towards the kneefront, and this is reasonably comfortable as that is the angle of the shin when a person is kneeling on a horizontal surface without any support other than the kneefronts and feet. The tendency, although small, is to slide forward, and that is less distressing and more easily managed than a tendency to slide backward. [0025] In a case of under-wedging, the user's shin will be sloping down towards the foot, and that resembles the situation of working without any support other than the kneefronts and feet on a sloping roof of lesser pitch. The present invention then has the effect of reducing the apparent pitch of the roof, which is beneficial to the comfort and safety of the roofer. In cases of pitches of approximately 30 degrees or more, the user should choose a shin wedge with a wedge angle moderately close to the pitch. For steep pitches, usually thought of as 45 degrees or more, the present invention will still be useful, but should not be relied on without other safety measures such as safety ropes. [0026] In FIG. 1, the roofer's kneefronts 4 and at least a portion of the roofer's shins 5 rest upon the shin wedges 2 and are secured in place by means of harness straps 6 and 8. The roofer's feet 7 are thereby held at least partly suspended above the roof. If the toes of the feet or the toe of the user's boot are touching the roof and the roofer does not want that, a small turn of the foot sideways, with either the toes going inward or outward in relation to the body, would lift the toes clear of the roof. In this way, the roofer can relieve muscle stresses that would normally be needed to restrain him from sliding down the roof 3. Since the upper surface of each shin wedge is approximately horizontal, little or no shear force is applied to the user's kneefront or shin at any time. All forces between the anatomy of user 1 and the shin wedges 2 are widely spread compressive loads onto the shin bone and kneefront, which is strong and well adapted to bear loads, rather than shearing loads onto skin of the kneefront, thereby minimizing discomfort. [0027] FIG. 2 shows one of the shin wedges 2 shown in FIG. 1. Each shin wedge 2 comprises wedge body 11 having an upper surface 12 for engagement to a user such as a kneeling roofer, a lower surface 13 for engagement to a work surface such as an inclined roof, triangular wedge sides 14 and 15, and rear face 16. The wedge angle 17 between upper and lower surfaces 12 and 13 is approximately equal to the pitch of the inclined surface upon which the user will kneel. [0028] Wedge body 11 is typically made of a lightweight plastic material such as a thermoplastic elastomer or expanded polystyrene, thereby providing sufficient strength to support a user while minimizing weight that would be an encumbrance when the user is walking upright while wearing the shin wedges. The width of each wedge body 11 is typically in the range 4 to 7 inches, with about 5 inches preferred. Its length is in the range 6 to 16 inches, with about 9 inches preferred, and in any case should stop well above the ankle. The longer it would be, the heavier it would be, and since it begins at the kneefront, it typically extends from one-third to one-half of the distance from the kneefront to the ankle. That length gives sufficient support to the shin, and a longer supporting surface is not a good trade-off for the added weight and bulk. Lengths in the stated range give sufficient leverage. That range of width and length gives sufficient contact area with an inclined surface so that a useful coefficient of friction may be obtained with suitable material on the lower surface 13. The coefficient of friction between two surfaces depends on the nature of both surfaces. The roofs on which this may be used may be of a variety of materials, including asphalt shingles, wood, copper, steel, tile, slate, and waterproofing membrane. The metal roofs contribute little to the friction, so the material on the lower surface 13 must accommodate that fact. [0029] The height of its rear face 16, which faces the foot, varies with the angle 17 used to compensate for roof pitch. The angle from a typical user's kneefront to the toe of a boot is in the range 15 degrees to 20 degrees. For such a user, if the wedge angle is greater than that angle, a toe will not make contact with the roof. That is not the preferred situation, but individual users will decide for themselves on whether they will tolerate that situation, or tolerate significant under-wedging. A small wedge angle of about 10 degrees might be preferred by some users. [0030] The dimensions stated here are rough approximations and may vary as the general wedge shape is rounded or sculpted for either ergonomic or stylistic reasons. Rounded edges 25, 26 and 27 at the lower surface 13 allow the user easier movement when crawling in any direction on the roof, because relatively sharp edges would tend to catch on the roof. [0031] Each shin wedge 2 includes an attachment harness comprising one or more adjustable straps 6, each configured for securing the user's shin against the upper surface 12 of the shin wedge 2. Optionally, there may be a harness element 8 that can encircle the user's leg at or above the knee, which would be attached to the front of the wedge body 11 by a garter strap 18. [0032] Adjustable straps 6 and 8 may be somewhat elastic to aid in achieving a secure fit. Various well-known length adjustment and strap positioning fixtures may be supplied to insure proper fit. FIG. 2 shows a hook-and-loop fastener (as sold under the trademark Velcro®) patch 20 affixed to each side of wedge body 11 permits straps 6 having corresponding hook-and-loop fastener patches to be adjusted in both length and location. Buckles and snaps may also be used to provide similar functionality. A strap may be a single unit that is detachable only at one or both sides of the wedge body 11, or it may be a two-piece unit that is fastened to each side of the wedge body 11 and joined by a buckle or other type of fastener [0033] To provide a more secure leg fixation, the harness may also comprise a garter strap 18 that is affixed to the front of wedge body 11 and to a leg encircling strap 8 that can be secured above the user's knee as shown attached to in FIG. 1. [0034] The upper surface 12 of wedge body 11 may include an ergonomic indentation 22 that conforms in a general way to a user's kneefront and shin. This would be a groove running from the distal end, where it is open, either to all the way to the proximal end where it is open, or to near the proximal end where it is closed and shaped to receive the user's kneefront in a semi-globular hollow. Since the human leg is rarely a simple cylinder, and since the shin wedge should be usable by persons with a range of leg sizes, the groove should be a portion of an elliptical cylinder, that is, it should be wider than it is deep. [0035] The wedge body 11 may be made of more than one piece. The upper surface 12 may comprise a layer of material that is different than the bulk of the wedge body 11. The distal end 28 of the upper surface 12 may extend beyond the rear face 16, in case longer support of the shin is desired while adequate gripping of the inclined surface can be achieved with a shorter length of the lower surface 13. [0036] To further improve comfort, a resilient covering 23 of plastic foam or gel may be formed onto upper surface 12, either just where the kneefront makes contact, as illustrated, or along the full length of the upper surface. This feature can apply to either a flat upper surface or an upper surface that has an ergonomic indentation. [0037] In its simplest embodiment, the lower surface 13 of wedge body 11 directly contacts the inclined roof on which it is deployed. More typically though, a somewhat denser plastic bottom pad 24 a is affixed to the bottom of wedge body 11 to form the lower surface 13, so as to provide better durability than the lighter material, such as expanded polystyrene or thermoplastic elastomer, that is preferred for wedge body 11. The bottom pad 24 a should have a large coefficient of friction to resist sliding. The bottom pad 24 a may have a flat, convex, or concave shape when not in use, but when the user is kneeling the bottom pad 24 a should conform to the surface on which the user is working in order to maximize contact with that surface. The surface of the bottom pad 24 a may have knobs or ridges to enhance its coefficient of friction. [0038] Bottom pad 24 a may be permanently bonded to the bottom of wedge body 11. Alternatively, for added flexibility, bottom pad 24 a may be made replaceable by providing temporary fixation means such as the hook-and-loop fastener (as sold under the trademark Velcro®) strips 25 shown on replaceable pad 24 b. However, the fastening system must resist sideways motion as much as the coefficient of friction on the bottom pad resists sliding down the roof, for it is pointless for the bottom pad to grip well to the roof if it does not stick well to the wedge body 11. If the bottom pad is replaceable, worn pads can be replaced with new pads with the economy of retaining the wedge body 11 and its harness. [0039] The bottom pad may have a constant thickness as shown as 24 a or else have varying degrees of wedge-shape such as shown by replaceable pads 24 b and 24 c. If wedge-shaped, the replaceable pads would provide the user with a means of adapting a single shin wedge 2 to work on different roof pitches. [0040] In normal use, a user will have a pair of matching shin wedges 2, one on each leg. There is no necessity for the shin wedges to be configured differently for the left and right legs, but in embodiments that have rounded edges 26, there can be a benefit from having the inside edge (that is nearer the centreline of the body) more rounded than the outside edge. In embodiments that have a resilient covering 23 on the upper surface, that covering may optionally be shaped differently for the left leg and right leg. [0041] The user must be facing substantially up the slope of the roof while wearing the shin wedges, and can easily crawl up the slope. The user also can easily move sideways while facing up the slope, and can move down the slope by crawling backwards. What the user cannot do is remain kneeling while facing sideways on the roof, or facing down the slope. However, the user can easily move about by standing up and walking on the slope while still wearing the shin wedges. [0042] If the user wants to ascend or descend by ladder, some caution is required, especially if he is wearing shin wedges having a large wedge angle. The shin wedges will collide with the rungs. One reason why the shin wedges are not as long as the entire shin is to provide better clearance from the rungs of a ladder. Also, the attachment harness in most forms will permit the shin wedges to be swung away from the front of the shin, and so out of the way of the ladder. [0043] It should be understood that where this description refers to user's shins and knees and other body parts, it envisages a person within the range of common adult sizes. Many modifications and variations besides those mentioned herein may be made in the techniques and structures described and depicted herein, resulting in other embodiments of the present invention without departing from the concept of the present invention. The foregoing disclosures should not be construed in any limited sense other than the limits of the claims that follow. Thus the scope of the invention should be determined by the appended claims and their legal equivalents rather than by the examples given.

Summary: The invention is a wedge-shaped support adapted to be fastened to a user's lower leg to provide comfort when the user is kneeling on an inclined roof, used in pairs. Each shin wedge comprises a wedge of lightweight material capable of supporting the user's weight. The acute dihedral angle of each wedge is approximately equal to the pitch angle of the roof on which the wedge kneepads are being used, thereby providing a substantially horizontal upper kneeling surface when the wedge is oriented towards the peak of the roof. The wedge's lower surface typically comprises a durable pad that prevents premature abrasion of the lightweight material and improves grip onto the inclined surface. Each wedge's upper surface typically includes a soft layer that serves as an ergonomic kneeling pad for the user. An attachment harness secures the wedge kneepad to the user's shin and thigh or knee.

big_patent

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Summarize: Floyd Mayweather's astonishing run of form betting on the NFL has continued after he picked up a bumper $1.4million (£895,000) payout as the Denver Broncos beat the New York Jets 31-17, taking his winnings to $3,267,637.36 (£2.03million) in three weeks. Mayweather, the world's highest paid sportsman, had already banked $1,825,714.28 (£1,13m) after successfully betting on both the Seattle Seahawks and the Indianapolis Colts to win by more than 7.5 points over the past few weeks. And the American boxer's luck was in again as he successfully gambled $815,000 (£506,000) on the Denver Broncos to beat the New York Jets by more than 7.5 points on Sunday. VIDEO Scroll down to watch Floyd Mayweather shows off his NFL winnings. Naturally Floyd Mayweather took to Instagram to show off his huge $1.4m winnings from his latest NFL bet. New York Jets' Eric Decker (87) talks with Denver Broncos' Jacob Tamme (84) after the Broncos beat the Jets. Mayweather also posted the betting slips from his Seahawks and Colts wins to his Instagram account. Sure enough, the Broncos came through to win 31-17 as Peyton Manning's three touchdown passes helped his side to victory. The margin of victory required for Mayweather was only secured in the last minute though as Broncos cornerback Aqib Talib picked off Jets quarterback Geno Smith and returned the ball 22 yards for a touchdown. Naturally the Money Man took to Instagram to celebrate his success - posting his betting slip - as he did with his Seahawks and Colts successes. Mayweather laid his latest bet with CG Technology at the M Resort in Las Vegas, and the company's vice president of race and sports, Jason Simbal, confirmed the bet to ESPN. 'Floyd didn't watch the game here,' Simbal said, 'but he certainly showed up shortly thereafter to cash.' Mayweather is the world's highest paid sportsman and recently posed in bed with stacks of money. The undefeated champion displayed his considerable wealth on his Instagram account again last week. VIDEO Maidana outclasses Maidana to retain title

Summary: Floyd Mayweather bet $815,000 (£506,000) on the Denver Broncos to beat the New York Jets by more than 7.5 points. The Broncos won 31-17, handing Mayweather a $1.4m (£895,000) payout. The Boxer had already collected almost $2million (£1,130,000) betting on NFL matches since September 21. Mayweather staked $940,000 (£580,000) on Indianapolis Colts and Seattle Seahawks in two separate bets and both sides won comfortably. World's highest paid sportsman recently posed in bed with wads of cash.

cnn_dailymail

en

Summarize: Mikey Welsh 1971-2011 Dear friends and fans, It saddens me and the guys in Weezer so much to say that our beautiful, creative, hilarious and sweet friend Mikey Welsh has passed away at the very young age of 40. A unique talent, a deeply loving friend and father, and a great artist is gone, but we will never forget him. His chapter in the weezer story ('98 - '01) was vital, essential, wild, and amazing. Mikey was never one to shy away from the absurd, dangerous or strange, and he did so with a gusto few others had. No one had quite the stage presence of Mikey, nor have there been many who pulled the types of shenanigans he did at shows. If it rocked, he had to try it - and he always found a way to pull it off. When he emerged from his nervous breakdown that spelled his exit from the rock n roll world, he took on a new role as an astonishing and pure visual artist. It was a glorious flowering of a talent he always possessed, but he had chosen to rock out first, paint later. Our more recent meet-ups at weezer shows, including recently at the Weezer/Flaming Lips show at Jones Beach were always a great time. We will miss him terribly. Our deepest heartfelt condolences go out to his family and friends. The world has lost a truly one of a kind guy....Tomorrow we play the RIOTfest in Chicago as planned - Mikey was planning on attending this show and we were looking forward to seeing him again. As sad as it is to think about, we know Mikey would never want the rock stopped on his account - quite the contrary in fact. While we wont see him, we know he will be there rocking out with us! Mikey's website: see his art. Mikey's Facebook page - see more art, and his writings - including some wild weez related stories from back in the day... Mikeys Twitter page. "...hello hello.. so i'm going on my first vacation in quite a long time.. very stoked.. first i'm off to nashville, to be photographed working on a big painting by a very good friend of mine [we'll get some shots up here eventually].. and then off to chicago, to see the mighty =w= rock out.. i'm excited to see the boys, hang out and have some fun.. so everyone take care.. i'll be posting some weird random sh*t up here [as usual] over the next 9 days or so..... love--m." ---from Mikeys Facebook page, 10/01/11 Photo above taken in Nashville October 3rd by Mikey's friend Jordan Vittitow. Authorities say a drug overdose is suspected in the death of former Weezer bass player Mikey Welsh, who was found in a Chicago hotel room Saturday afternoon. Welsh, 40, was found unresponsive on the floor after failing to check out of his room at the Raffaello Hotel, 201 East Delaware Place at 1 p.m. Saturday, according to Chicago Police News Affairs Officer Laura Kubiak. Hotel staff found him. Police said a cleaning crew tried to open the door to Welsh's hotel room and was unable to get in, thinking maybe luggage was blocking the entrance. Another hotel employee then pushed the door open and saw Welsh lying unresponsive near the door, police said. Hotel staffers called 911 shortly before Welsh was pronounced dead. Welsh was pronounced dead at the scene at 2:50 p.m., according to the Cook County medical examiner's office. The cause of death was pending toxicology results, but authorities said narcotics are suspected. Police said prescription drugs were found in Welsh's hotel room along with a ziplock-type bag containing white powder, which was suspected to be heroin. Weezer is in town for the Chicago RiotFest tonight, according to the band's web page, and Welsh was expected to catch their show. Welsh, 40, of Burlington, Vt., performed with Weezer from 1998 to 2001, leaving after suffering a nervous breakdown, according to the band's website. He eventually established himself in a second career as a painter. "I'm taking a break from music," he told MetroWest Daily News in 2002. "I really feel the need to reinvent myself and move on, and I couldn't be happier painting. Music is still an important part of my life, but I really have no desire to actually play it." The group's "Weezerpedia" page carried a note this morning that read, "As many of you may have heard, Mikey Welsh has passed away. "The news was announced via his official Facebook page earlier in the evening. Understanding that many here are grieving, Weezerpedia has created a digital eulogy page for Mikey Welsh where fans can post stories, pictures, or thoughts. R.I.P. Mikey." Scott Shriner, the band's current bassist, posted a short note to his Twitter account at about 12 a.m. Sunday. "Really bummed about Mikey. My heart goes out to his family and friends. Such a talent... he made a special mark on the world with his art. [email protected]

Summary: Former Weezer bassist Mikey Welsh has been found dead in a Chicago hotel room at age 40, reports the Chicago Tribune. Police suspect a drug overdose is involved. Welsh was found unresponsive yesterday afternoon after he failed to check out of his room at 1pm; he was reportedly in town to watch his former band play tonight. Current bassist Scott Shriner tweeted this morning: "Really bummed about Mikey. My heart goes out to his family and friends. Such a talent... he made a special mark on the world with his art." "As many of you may have heard, Mikey Welsh has passed away," the band posted online. "Understanding that many here are grieving, Weezerpedia has created a digital eulogy page for Mikey Welsh where fans can post stories, pictures, or thoughts. R.I.P. Mikey."

multi_news

en

Summarize: BACKGROUND OF THE INVENTION Generally this invention relates to the field of areaways, the enclosures for basement windows. More specifically, the invention relates to modular areaway escape systems. The invention concerns improvements to areaway designs which allow for simple assembly and replacement of sections. These improvements make the present areaway system useful and appealing to residential and commercial users. For more than a century the technique of allowing light in through a basement window has existed in order to make the space more desirable and made it meet other requirements. In large part the technique has been accomplished through the use of a monolithic areaway to surround the bottom and sides of the basement window to hold earth away from the window so that light can be admitted. An early effort in this regard is shown in U.S. Pat. No. 300,654 to Smith for an "area window protector" first patented in 1884. Many other areaway designs and improvements have been patented since that date. In almost every one of these designs, the focus has been to provide a design which admits light and which excludes earth. The latter of these goals has been met with varying degrees of success. Often elaborate designs have been proposed, including some which are formed as an integral part of the foundation surrounding the basement window. Perhaps because of this focus designers of areaway systems have failed to see the value of a modular design with independent sections. Even with the most elaborate designs, none have addressed the need for making a system which may be assembled "on-site" or the need for a system which allows damaged or worn sections to be replaced. In recent years it has been discovered that the areaway was also useful as an escape avenue in the event of fire or some other catastrophe U.S. Pat. No. 3,999,334 to Webb describes an areaway entitled "Webb Basement Window Escape." Although not a true areaway but an extension of basement space beyond the foundation wall, this 1976 patent appears to be the first to recognize that the basement window could be useful as a means of escape. Although--with the benefit of hindsight--it may at first glance appear surprising that it took almost 92 years to improve a product similar to an areaway to allow it to become a means of escape, this delay makes sense when it is understood that those skilled in the art of areaway construction tended to improve existing designs in small degrees rather than to freshly innovate to overcome undesirable limitations. This is why until U.S. Pat. No. 4,876,833 to the inventors of the present invention the seemingly simple combination of allowing for an escape system and utilizing the space within the areaway for aesthetic and practical purposes has not been proposed. The present invention accomplishes this goal as well. Those skilled in the art were usually primarily practical people who sought to overcome one perceived problem rather than people who completely re-thought areaway systems. The present invention focuses on the desirability to allow not only additional safety features to be incorporated within an areaway, but also to provide some designs which, rather than requiring complete installation of a new system when damage or wear begins to show allows replacement of just the damaged or worn sections. In addition to achieving these goals, embodiments of the present invention have been designed with features that accommodate the perspectives of not only the consumer, but also the supplier, the installer, and the manufacturer. In addressing each of the various perspectives of those involved with the product from its manufacture through its replacement, various independent desires have been especially accommodated by the modular design. With respect to the consumer, the present invention allows for a cost effective areaway system by permitting replacement of less expensive sections. In addition, the design still avoids the difficulties of maintaining the space and providing for drainage inside the areaway as described in the Applicants' previous patent. With respect to both the supplier and the manufacturer, the design allows for a construction which is not only easily manufactured, but which allows individual sections to be nested together for shipping and storage. With respect to the installer, the design avoids any need to integrate the areaway with the foundation so that simple installation and, perhaps more commercially significant, simple replacement of components can be easily accomplished. In this fashion the design is adaptable to existing structures, and is especially suited for replacement of existing areaways. Prior to the present invention, no solution to these various goals was accomplished by any one areaway design. Another key element of some embodiments of the present invention was the recognition that in earlier unitary bodied inventions retrofitting to existing mounts was sometimes difficult if widths differed greatly. The present invention permits a variety of widths to be accomplished by altering the manufactured dimensions of a minimum number of sections. Larger or smaller areaway systems could then be accomplished by the installer by replacing the key sections. This aspect presents a significant effort and cost savings to the consumer, installer and manufacturer. Attempts by those skilled in the art were simply inadequate because they focused on other problems in the art, or were willing to cope with the particular problems addressed by the present invention. The degree to which these seemingly simple recognitions are significant seems apparent when one considers that the present invention, although unexpectedly simple in achieving these goals and overcoming the limitations of the prior art, has not been available even though areaways have existed for over one hundred years. U.S. Pat. No. 4,876,833, issued Oct. 31, 1989, to the same inventors is hereby incorporated by reference. SUMMARY OF THE INVENTION It is broadly an object of the present invention to provide a design which allows for simplistic construction of an areaway. It is therefore a further object to provide a modular design with individual sections designed to be easily connected together to form the desired areaway system. An object is thus to provide a design with uncomplicated connecting means. A further object of the modular design is to allow components to be changed, replaced, or up-graded with only a minimal amount of time, cost and effort. It is thus another object of the design to allow individual sections to be detached from the areaway to allow for replacement. It is also an object of the present invention to accommodate the needs of retailers by minimizing storage and shipment requirements such as space and inventory. It is a further object of the present invention to allow the modular device to be fitted to many varying sizes and shapes of basement windows. It is thus an object of the present invention to allow various sized and shaped sections to be interchangeable with each other. A further object of the present invention is to allow for nesting of each of the individual sections together with similar sections to simplify storage and transportation of the areaways prior to installation. It is a further object of the present invention to provide a design which allows for escape features in an areaway which can be easily retrofitted to existing structures. An object is also to provide a design which can be easily installed in new structures. An object of the design is not only to provide for escape, but also to provide for access by emergency personnel through the areaway. Such access is provided in a useful fashion which accommodates the inherent equipment and needs of such emergency personnel. It is a further object of the present invention to provide for designs which accommodate the various perspectives of those persons coming in contact with the areaway. Such perspectives include the perspectives of manufacturer, supplier, builder, installer, building owner, occupant and emergency personnel. An object of the present invention is to accommodate each of these perspectives in one simple design which balances the various competing interests. It is a further object of the present invention to provide for an areaway design which allows access to the earth surrounding the areaway. An object of the present invention is to provide a design which allows planting within the areaway. It is further object of the present invention to simplify the installation of areaway escape systems. An object is thus to accommodate new structures and to allow for easy replacement of existing areaways. An object is also to simplify installation by allowing for access to the earth surrounding the areaway from within the areaway itself. An object is thus to avoid having to install the areaway and backfill from behind the areaway after it has been attached to the structure. Another object is to provide a design which does not require modification to the existing structure in order to accomplish installation of the areaway escape system. It is thus an object to allow for installation which is easy enough to be accomplished by a homeowner in residential applications. It is further object of the present invention to provide an escape system which is outside the living space of a building. An object is to provide an areaway which need not be integrated into the foundation of a building and which may separated from the interior space of the building through existing windows and the like. A further object is to accommodate conventional grading and slopes away from the structure. Similarly an object is to provide for a device which may be simply manufactured which also achieves the various aims mentioned. Naturally, further objects of the invention are disclosed throughout other area of this specification and claims. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a perspective view of an embodiment of the invention. FIG. 2 is a top view of the embodiment shown in FIG. 1. FIG. 3a is an enlarged view of the connecting means between the side wall members and the step facings as shown by the circle in FIG. 1. FIG. 3b is an enlarged view of another possible connecting means between the side wall members and step facings. FIG. 4 is a cross-sectional side view of the embodiment shown in FIG. 1 as it would look when attached to a foundation. FIG. 5 is an exploded view of the embodiment shown in FIG. 1. FIG. 6 is a perspective view of another embodiment which includes a substantially vertical retaining member. FIG. 7 is a top view of the embodiment shown in FIG. 6. FIG. 8 is an enlarged view of the connecting means between the vertical retaining members. FIG. 9 is a cross-sectional side view of the embodiment shown in FIG. 6 as it would look when attached to a foundation. FIG. 10 is an exploded view of the embodiment shown in FIG. 6. FIG. 11 is a perspective view of another embodiment which includes additional features. FIG. 12a is a perspective view of two areaways connected in series. FIG. 12b is an enlarged view of the "dovetail" connectors used for linking units in series. FIG. 13 is an perspective view of an embodiment having a railing attached. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As can be seen from the drawings, the basic concepts of the present invention may be embodied in many different ways. FIG. 1 shows one embodiment of modular areaway system (10) in perspective. The modular or sectional design of the present invention includes independent first and second side wall members (11 and 12, respectively), and independent step facings (13). By "modular" and "sectional" it is meant that the invention is designed in sections which are assembled to produce the desired product. Likewise, by the term "independent" it is meant that each section operates separately until connected in some manner to other sections. By providing a modular assembly, manufacturing and cost efficiencies are gained allowing the invention to meet the objects of an efficient, cost effective design from various perspectives. Not only is manufacturing more easily accomplished, but it can be accomplished in a less expensive manner. Equally important, installation, replacement of individual sections, and transportation and storage are greatly facilitated. Some aspects of the present invention are disclosed in the incorporated reference U.S. Pat. No. 4,876,833 to the same inventors. The manner in which most of these aspects are accomplished by the invention disclosed in U.S. Pat. No. 4,876,833--a unitary body design--is very different from the modular design of the present invention. FIGS. 3a and 5 show a more detailed view of first and second side wall members (11 and 12) and step facings (13) and the means for detachably connecting. Each side wall member (11 and 12) is designed to have an integral male connector (15) located on external face (20) of each tier (25). Similarly, each step facing (13) is designed having female receptors (16) located on each end of internal face (30). FIG. 3a shows a design in which male connector (15) is a tab-like projection, while female receptor (16) is an L-shaped recess. FIG. 3b shows a possible alternate design. To assemble, female receptors (16) are attached onto male connectors (15), a single step facing (13) for each tier (25). Naturally alternative designs may have connector (15) and receptors (16) switched between step facing (13) and side wall members (11 and 12). Other alternative designs may modify this means for detachably connecting in any number of ways to facilitate the attachment between side wall members (11 and 12) and step facings (13). Such variations include, but are not limited to, sliding interlocking connectors (as shown in FIG. 3b), interlocking connectors, and conventional bolts, or screws. Also shown in FIG. 3a is notch (17) adjacent to each male connector (15) of side wall members (11 and 12). Notch (17) tapers from its open end towards its closed end to perform a two-fold function. First, it aids connection by allowing step facing (13) to be inserted into notch (17) at an angle and pivoted to engage female receptor (16) with male connector (15). Second it, together with the backfill to be discussed later, serves to retain step facings (13) in the desired attachment position. The taper of notch (17) is preferably designed so that it frictionally engages step facing (13) when vertical. In the event that other connecting means are used to attach step facings (13), notch (17) may be omitted from construction. The unique design, interrelation and function of male connector (15) and female receptor (16)--the preferable means for detachably connecting--is beneficial in many ways besides its quick and easy use. It does not need metal nuts, bolts, or such which are very prone to rust, especially under the conditions present underground. This also negates the need for any tools for assembling areaway (10). Aesthetically, the design shows a single seam formed between the connection of side wall members (11 and 12) and step facings (13). The present design may also inhibit the infiltration of dirt from the exterior into the means for connecting, as this could detract from the appearance of areaway (10) and the function of male connector (15) and female receptor (16). An important feature of areaway (10) is that it be designed to attach to the exterior of the foundation of a building without significant alteration of the foundation. This is accomplished through foundation flanges (14). Although some designs for an areaway escape system have required significant alteration of the foundation, the present invention affords easy attachment to the foundation by providing numerous holes in foundation flanges (14) so that height and attachment may be easily adjusted and accomplished. This allows utilization of the present invention in situations where prior areaways are replaced. Unitary body construction areaways do not allow for adjustment to wider or narrower window sizes in cases of replacement. This limitation is addressed in the present invention. When areaway system (10) is assembled, the area between foundation flanges (14) forms a vertical opening which is adjacent to foundation face (42) of side wall members (11 and 12) when installed. This opening may be adjusted in a manner which will be discussed later to accommodate existing window structures of various widths. Foundation flanges (14) may be outward facing as shown in FIG. 1, or inward facing (not shown). This aspect is possible due to the modular design of the present invention. Side wall members and 12) are interchangeable, and therefore may be positioned on either the left or right side of areaway (10) when assembled, permitting inward or outward facing flanges (14). A feature of the present embodiment is the fact that bottom edge (18) of side wall members (11 and 12) creates floor opening (22). Floor opening (22) may serve as a drain for areaway (10). In providing an open area rather than a fixed drain design, users and installers of areaway (10) can incorporate any particular type of drain mechanism they desire. For instance in FIG. 2, a "french drain" design is shown. Additional drainage is created by offsetting each tier (25) of side wall members (11 and 12) to enhance drainage into step openings (36). These horizontal areas are typically called the "tread" of the step by those skilled in the art. In addition, concrete floors even with plumbed drains or planting are certainly possible through this feature of the present embodiment, the essence of such a feature being that it can accommodate a broad variety of drain designs. In addition to the floor drain, it can be seen in FIG. 4 that step facings (13) form the "riser" of steps (31) within the areaway. This aspect serves as an escape means. Step opening (36) is located perpendicularly to the riser. Opening (36) serves not only as an additional drain mechanism, but also as a means for accessing the earth below the areaway and as a means for planting within the areaway. Referring again to FIG. 2, a top view of the embodiment shown in FIG. 1, it can be seen that openings (36) form a substantial amount of the area of each horizontal step (31). Although simple provision of a small drain tube could be utilized, by providing opening (36) which is larger than that merely necessary to allow water drainage, the present invention accommodates several needs. The provisions for such openings throughout areaway (10) are discussed in U.S. Pat. No. 4,876,833, to the same inventors. Those skilled in the art typically felt that in order to be commercially acceptable areaways must exclude earth to the largest extent possible. As an example, U.S. Pat. No. 2,453,609 for an "Areaway Wall" states that it relates to walls which define an areaway and which prevents substantially all infiltration of earth into the areaway. Although at first it might seem like a simple modification to depart from this preconception, those skilled in the art had simply not questioned the need and thus were limited in their designs. By leaving the tread of each step (31) open in the described manner the modular design allows for the desired removal of individual sections. Perhaps more importantly, it allows for the replacement of individual sections without total removal of areaway (10). The step openings (36) not only serve as a means for draining areaway (10), or a means to allow proper installation and replacement of individual sections, but they also permit planting within areaway (10). As alluded to earlier by allowing access to the earth behind areaway (10) through openings (36), installation and replacement is greatly facilitated. While excavation of the earth surrounding the foundation of a house is necessary, once areaway (10) is installed the problem of backfilling the earth was difficult for areaways which were not simply vertical walls. Since support of the areaway by the earth is a very beneficial structural aspect with the modular design, backfilling under the areaway was a problem for some designs. The present invention solves this problem through step openings (36). After attachment of assembled areaway (10) to the foundation through foundation flanges (14), the installer may then simply backfill areaway (10) through step openings (36). This allows for proper compaction and enhances the structural and support needs of areaway (10) thus minimizing the torsion on foundation flanges (14). This is significant because it allows the implementation of a simple escape device in situations where prior art had required involved measures including even reconstruction of edges of the foundation. When replacement of an individual section is warranted the backfilling ma be simply removed through opening (36). Removal allows for the detachment of each step facing (13), if side wall member (11 or 12) is to be replaced, or just a single step facing (13). An ancillary benefit of the backfilling process is that the dirt, or other material piled against step facings (13) serves to aid in their retention to side wall members (11 and 12). In addition, step openings (36) also allow the capability of planting within areaway (10). Although most areaways serve solely to admit light and in so doing create a volume of space which is basically undesirable, by allowing planting within areaway (10), the present embodiment enhances the aesthetic appeal of areaway (10) and even integrates the volume of space within areaway (10) into the aesthetic surroundings of the interior living area. In the present invention this volume of space is not integrated as part of the interior living area. This is significant in that it allows areaway (10) to be incorporated in existing designs where basement windows are utilized with minimal or no modification. As can be seen in FIGS. 1 and 5, a great benefit of the modular system is their potential for maximum space utilization during storage, transportation and packaging. Side wall members (11 and 12) are designed with offset tiers which allow side wall members (11 and 12) to be nested together. Although each side wall member (11 and 12) is shown to be straight it may be desirable to design members (11 and 12) to curve. This may be done for mere aesthetics, or to further facilitate the nesting together of these sections. Similarly, step facings (13) are easily and compactly stored when disassembled from areaway (10). The benefits of this aspect affect the manufacturing of areaway (10) and the inventory requirements of the supplier of areaway (10) in a positive manner. Not only is manufacturing of smaller sections now possible, but each smaller section can be stored in a smaller area. Certainly a variety of nesting aspects could be included such as tapers on other surfaces or curved sections and still be within the scope and spirit of the present invention. Referring to FIG. 4, additional features of the areaway can be understood. Areaway (10) is attached to the foundation at foundation flanges (14; not shown). Such attachment may be through any number of means including bolting the areaway directly to the foundation. Since present areaways often utilize similar flanges, attachment is easily accomplished to existing mounts when an old areaway is replaced. However, because of the modular design of the preferred embodiments, only damaged or worn sections of areaway (10) requiring replacement need be replaced. This provides a great economics benefit to the consumer. A further feature of this embodiment is the fact that the lowest horizontal step facing (13) may be designed to be lower than the bottom edge of a basement window after installation. This allows additional space for opening the window in the event casement or other such designs are utilized. Such a window is shown in the open position in FIG. 4. In providing a series of open steps, the present invention allows a positive means for escape through areaway (10). In addition, by providing a relatively wide floor opening (22) and by providing a large upper opening (27), areaway (10) allows not only for egress from the building but also for ingress as may be necessary for emergency personnel. A proposed change to uniform building codes states that areaways shall supply "sufficient horizontal dimensions to inscribe a circle with a diameter not less than 30 inches", this horizontal area extending "the full vertical height" of the areaway. This aspect is significant because the majority of areaway designs are not sufficiently wide to allow rescue operations through the opening. Certainly this would include the ability to wear oxygen tanks and carry similar equipment into the structure through areaway (10). The 30 inch inscribed circle is just a proposed code change. Certainly dimensions of less than 30 inches are also possible and are considered to fall within the scope and spirit of the present invention. As mentioned earlier, in contrast to the inventors' previous design, a modular or sectional construction is preferred. In the preferred embodiments, manufacture can be easily accomplished through the rotational molding process. Each individual section could be mass produced to satisfy inventory requirements, or upon consumer demand. The molded sections may be of a hollow double-walled construction, but may be filled for added weight and strength. A chemical foam filler is one example of a filler that may be used with the present invention. Others include recyclable materials, cement, and the like. Additional supports, commonly called "kiss-offs" in the art, are molded within each section connecting exterior surface (A) to interior surface (B). Naturally, color and other aspects can be included to customize areaway (10). Certainly other manufacture methods well-known to those skilled in the art are possible. Injection molding, blow molding, resin transfer to name a few. Solid construction and single-walled construction are also possible alternatives when utilizing some of these other manufacturing techniques commonly known. Each of these alternative manners of construction and manufacture are considered to fall within the scope and spirit of the present invention. Referring to FIG. 6, another embodiment of the present invention can be seen. FIG. 6 presents a simplified modular embodiment which provides for a means of escape in an areaway which may be easily retrofitted to existing designs without modification of the foundation. Such attachment is accomplished as before through foundation flanges (19). The means for escape comprises a vertical series of horizontal steps (32) which are molded into a middle vertical retaining member (41). End vertical retaining members (23 and 24) are adjoined to retaining member (41) in much the same manner as described in the first embodiment. FIG. 8 shows one possible design for connecting retaining members (23, 24 and 41). Certainly other designs, as described earlier, are possible to effect this connection and would still fall within the scope and spirit of the present invention. As a means for customizing the present embodiment to existing window sizes vertical retaining member (41) may be designed in various widths. Certainly, any number of vertical retaining members (41) may be used to accomplish the desired width as well. A possible alternative to the embodiment of FIG. 6 may be a design which excludes vertical retaining member (41). In this instance vertical retaining members (23 and 24) would connect together, and steps (32) would be molded into members (23 and 24). The integrally molded steps (32) may be positioned anywhere on retaining members (23 and 24) and may naturally be either positive steps as shown, or negative inserts. Naturally, because each retaining member (23 and 24) might contain only half of step (32) the retaining members should be designed so that the portions of steps align to create full steps (32). Since molded steps (32) extend either little or no distance beyond retaining members (23 and 24), backfill underneath each step may or may not be necessary. As with the previous embodiment, floor opening (21) may serve as a planting means. The embodiment may also be specifically designed to modify current areaway structures to the smallest extent possible to address users accustomed to existing designs or who prefer the features of such existing designs. In designing molded steps (32), certainly various shaping differences could be provided and yet would still fall within the scope and spirit of the present invention. Handles could be included through different molding of the steps, however, such have not been included in the present design as a most simplified version has been sought. In like fashion, retaining members (23 and 24) can have different shapes and textures. FIG. 7 shows a top view of the embodiment shown in FIG. 6. As can be seen, molded steps (32) need only comprise a sufficient amount of area to provide a positive means for escaping. The horizontal dimensions of this embodiment may conform to the above mentioned building code by providing an area capable of being inscribed with a circle having a diameter of not less than 30 inches. As shown in FIG. 9, this embodiment may also be mounted to the foundation by bolting through foundation flanges (19). The bottom of the areaway creates floor opening (21) which serves as a drain and may be utilized in any number of fashions as mentioned earlier. Again, a french drain is shown with gravel inserted. Referring to FIG. 11, an additional embodiment incorporating several separate features is shown. First, an areaway cover (26) is shown over upper opening (27). Areaway cover (26) not only serves the conventional purpose of excluding the elements while admitting light to the areaway, but it also enhances embodiments of the present invention which allow planting by creating a greenhouse effect. Cover (26) is preferably detachably connected by a "snap-on/snap-off" design. Hinging or some other attachment technique may be utilized, but areaway cover (26) should not be permanently fixed to upper flange (37) as both ingress and egress in emergency situations must be allowed. As an enhancement to the means for escape, handles (28) may be added to the upper area of either or both side wall members (11 and 12). Handles (28) would be angled to assist a person in accessing areaway (10) and escaping from the interior space. Since the vast majority of basement windows are raised somewhat from floor level, handles would assist the person in extricating themselves through the basement window. In addition, a ladder (29) could be provided. While certainly ladder (29) could be stored externally and attached to areaway (10) or the foundation through some attachment means, ladder (29) such as a rope ladder could be integral to areaway (10) through the use of some compartment (33). Naturally compartment (33) could be a variety of designs, one possibility being the openable compartment as shown in FIG. 11. In FIG. 12a two areaways connected in series are shown. Since it may be desirable to have several basement windows, the embodiment shown lends itself to a means for connecting areaways (10) in a series. One of side wall members (11 or 12) becomes a center connecting support. Through the use of interlocking connectors on step facings (13) two units may be connected using only 3 side wall members (11, 12 and 42). FIG. 12b shows opposite ends of a step facing (13) having "dovetail" connectors as one type of interlocking connector. Although this method provides a simple connection technique, certainly other connections are possible. This type of connection has been chosen based upon the goal of providing a simple device at the outset. The use of single center side wall member (42) as shown in FIG. 12a serves to integrate the two units and is a simple device. More units may be similarly attached in series to create a longer integrated unit if necessary. Since certain building codes require the use of railings to protect any fall into an areaway, a means to accommodate a railing is shown in FIG. 13. Through well known manufacturing techniques, a mounting guide such as a railing sleeve can be attached to either side wall member (11 and 12) as shown. The railing sleeve could naturally be a metal tube or any other kind of receptacle for railing (39). As shown in FIG. 13, railing could be bolted to the modular side wall members (11 or 12) underneath upper flange (37). Naturally, other structural enhancements could be added including additional sleeves lower on side wall member (11 or 12) and the like. Railing (39) could also be designed to be detachable without bolting it from below upper flange (37). The foregoing discussion and the claims which follow describe the preferred embodiment of the present invention. Particularly with respect to the claims, it should be understood that changes may be made to the invention without departing from its essence. In this regard it is intended that such changes will still fall within the scope of the present invention. It simply is not practical to describe and claim all possible revisions to the present invention which may be accomplished. To the extent such revisions utilize the essence of the present invention, each would naturally fall within the breadth of protection encompassed by this patent.

Summary: A modular areaway system is disclosed having independent side wall members and independent step facings. The modular system allows quick and easy construction of units without the use of extra tools. The independent sections are detachably connectable to one another to form the final single unit assemblage. The means for detachably connecting is integral in part to each independent section of the areaway system. Male and female connectors are disposed on each section to make assembly of each unit by most any person simple and easy, as no tools are necessary. The modular system allows for replacement of sections rather than requiring replacement of entire units. In the event that any individual section is damaged or worn replacement of that section is easily replaced with a new section by detaching the damaged or worn section and reattaching the new section in its place. The modular system allows for varied widths of the areaway system by replacing one size step facings--as the step facings dictate the width of each unit--with a smaller or larger sized step facing. The modular system allows for conservation of space during transportation and storage. Each of the sections may be nested together with similar sections (i.e., the side wall members together, and the step facings together) in a space much less than that required for storing and transporting complete, assembled units.

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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is directed to a medical examination system including means for generating an internal image of a patient, and means for generating an image of the external surface of the patient, and in particular to such a system wherein signals from the latter means are used to control selected components in the former means. 2. Description of the Prior Art Medical examination systems are known wherein internal images of a patient are produced using an x-ray diagnostics installation for positioning the patient, even if the examination system is not itself used to generate an x-ray image. Such systems include those for generating conventional x-ray shadow images, computer tomography, magnetic resonance imaging, and ultrasound imaging. The diagnosis made from an image assumes that a precise and accurate positioning of the patient has been undertaken prior to creating the image. It is also known to produce x-ray transillumination images and surface images of an examination subject using two video cameras. SUMMARY OF THE INVENTION It is an object of the present invention to provide a medical examination system having means for generating an internal image of a patient, and means for generating an external, surface image of the patient, wherein the signals obtained from the surface scan of the patient are used to control the positioning of selected components in the means for generating the internal image. The above and other objects are achieved in accordance with the principles of the present invention in a medical examination system wherein the means for generating an internal image has a memory to which the surface images of the patient are supplied, obtained from the surface scan. The means for generating an internal image also includes a comparator. The comparator generates an output signal for driving an adjustable component, such as the primary radiation diaphragm and/or the patient support. The output of the comparator is dependent upon the difference between the stored image of the patient, and a current patient image. In addition to being used to obtain information about the patient for the examining personnel to complete the internal image of the patient, the surface image can also be used as a three-dimensional mask for re-positioning the patient in routine follow-up examinations. In medical technology, especially in radiology, such follow-up examinations are often necessary, which require that the patient be placed in precisely the same position as in a preceding examination. The surface image can be used to adjust the patient and the components, such as the patient support, to achieve such position repetition. Precise reproduction of the same patient position as was used in a preceding exposure is required, for example, in subsequent exposures which are taken for the purpose of measuring the calcium content of bone substance in osteoporosis. This is because the percentage change of calcium content over time, i.e., over successive examinations, is on the order of magnitude of the reproduction precision of the measuring position. Although computer tomography x-ray images have been used as a positioning aid for this purpose, the use of a surface image in accordance with the principles of the present invention makes further improvement possible. Another problem in the generation of internal patient images is that of blooming. Blooming occurs when the radiation is not properly gated onto the subject. This has two disadvantages. The first disadvantage is that the patient is exposed to higher than necessary radiation levels, due to scattered radiation. The second disadvantage is a loss in image quality. The surface image generated in accordance with the principals of the present invention can be used to control the positioning of the primary radiation diaphragm to achieve an exact gating of the x-ray beam, thereby avoiding blooming. DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic block diagram of a medical examination system constructed in accordance with the principles of the present invention. FIG. 2 is a schematic block diagram of another embodiment of a medical examination system constructed in accordance with the principles of the present invention. FIG. 3 is a schematic block diagram of a further embodiment of a medical examination system constructed in accordance with the principles of the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS As shown in FIG. 1, a medical examination system constructed in accordance with the principles of the present invention includes an x-ray source 1 which generates an x-ray beam which transirradiates a patient 2 (shown in cross-section) through a primary radiation diaphragm 3. The patient 2 lies on a patient support 4. Radiation attenuated by the patient 2 is incident on the input screen of an x-ray image intensifier 5, having an output screen connected to a video chain 6 in a known manner for producing x-ray shadow images. A patient surface scanner 7, which may be a laser scanner, is disposed facing the patient 2 for producing a geometrical or topological scan of the surface of the patient 2. Various types of surface scanners suitable for use as the scanner 7 are known in the art. Electrical signals corresponding to the shape of the surface of the patient are formed from information identifying the scan direction and the scan angle. A surface image generated by the scanner 7 is stored in a memory 8 of a computer 9. The computer 9 includes a further memory 10, which receives and stores the x-ray image from the video chain 6, via an analog-to-digital converter 11. An image computer 12 produces a contour image from the surface image in the memory 8, and supplies electrical signals corresponding to this contour image to a comparator 13. The comparator 13 also receives signals corresponding to the contours of the x-ray image stored in the memory 10. The comparator 13 generates a superposition image at its output 14. This superposition image permits a better diagnosis than is possible only on the basis of the x-ray shadow image from the video chain 6. The video chain 6 generates an internal image of the patient. Such an image may also be acquired using another type of image-generating system, for example, a computer tomography system, a magnetic resonance imaging system, or an ultrasound imaging system. The scanner 7, instead of being a laser scanner, may be any suitable type of means for scanning the surface of the patient, for example, a system which produces such a surface image by acoustic emission. A further embodiment of the system is shown in FIG. 2, wherein components identical to those already described in FIG. 1 are provided with the same reference symbols. In this embodiment, the memory 8 is contained in a computer 9a, which also contains a memory 10a for a three-dimensional surface image of the patient 2, obtained in an earlier examination. A comparator 13a compares the current surface image in the memory 8 to the earlier surface image in the memory 10a. The output of the comparator 13a controls a motor 15 via an amplifier 6, which adjusts the position of the patient support 4 until the two surface images coincide. At this point, the patient 2 is positioned as he or she was positioned in the earlier examination. Additionally, the computer 9a controls the size of the opening of the primary radiation diaphragm 3 via an amplifier 17 so that the x-ray beam from the x-ray source 1 is gated to be as small as possible without degrading the resulting image, i.e., the boundary or size of the radiation field is optimally approximated (matched) to the examined region of the patient 2. Blooming is substantially prevented by such control. In addition to controlling the position of the patient 2 on the basis of the comparison of the current image in the memory 8 and the earlier image in the memory 10a, the position of the patient 2 via the motor 15 can also be undertaken by comparing the current image in the memory 8 with the current image from the output of the analog-to-digital converter 11. In this case, the motor 15 then adjusts the patient support 4 until the current surface image in the memory 8 substantially coincides with the earlier surface image in the memory 10a and, with respect to the contour of the patient 2, substantially coincides with the current image from the analog-to-digital converter 11. It is also possible to combine the comparison of the surface images (old and current) with the comparison of the x-ray images (old and current), as is done in the embodiment of FIG. 3. In this embodiment, a further memory 10b is provided in a computer 9b for the earlier x-ray image. The outputs of both of the comparators are supplied to an evaluation stage 20 which supplies a positioning signal for the patient support 4 via the amplifier 16 and the motor 15, and a positioning signal for the primary radiation diaphragm 3 via the amplifier 17. The evaluation stage 20 can provide a drive signal based on various evaluation criteria. For example, it can be determined in the evaluation circuit 20 which of the two comparisons has resulted in a higher correlation value, and that comparison signal is then assigned priority and is used exclusively, or primarily, to generate the positioning signal. Another possibility is to use the mean value of both comparison results. It is also possible to use different sets of criteria for generating the respective drive signals, i.e., one set for generating the patient support drive signal and another set for generating the primary radiation diaphragm drive signal. Gating of the x-ray beam is usually set on the basis of a desired x-ray attenuation value of the tissue being examined. Control of the opening of the primary radiation diaphragm 3 in the manner described above, can be undertaken by adjusting the extent to which the contour of the patient is overlapped by the radiation shadow of the primary radiation diaphragm 3. As in the embodiment of FIG. 1, the embodiments shown in FIGS. 2 and 3 may use other image generating systems, such as a magnetic resonance imaging system or an ultrasound imaging system to generate the internal image of the patient. Any suitable type of surface scanner can be used as the surface scanner 7 in the embodiments of FIGS. 2 and 3 as well. In all of the above embodiments, the computer 9, 9a or 9b receives electrical signals at an input 18 which correspond to the apparatus geometry, which are generated in a known manner, to assist in the exact positioning of the patient 2. The output signal from the computer 9, 9a or 9b may also be used to drive an ink printer 19 for applying markings on the patient to identify a field. Coordinates for setting the ink printer can be acquired from the image from the analog-to-digital converter 11, and from the current surface image in the image memory 8. In addition to portraying a surface image, it is also possible in the embodiment of FIG. 1 to portray the contour of the patient 2 in a defined sectional plane or slice, for example, the section A--A shown in FIG. 1. Both the three-dimensional surface image and the contour image can be acquired by turning the surface scanner 7 relative to the patient 2. In the embodiment of FIG. 1, the superposition of the surface image in the memory 8 with the image of the memory 10 ensues with reference to the contours of the patient 2. It is also possible to apply a suitable reference member to the patient, for example, a lead block, which is visible in both images. When superimposing the images, this reference member is then brought into coincidence in the images. A suitable system and technique for generating the surface images, which can be used in the subject matter of the present application, is described, for example, in the periodical "Automatisierungstechnische Praxis," Vol. 2, 1988, at page 99. Although modifications and changes may be suggested by those skilled in the art, it is the intention of the inventors to embody within the patent warranted hereon all changes and modifications as reasonably and properly come within the scope of their contribution to the art.

Summary: A medical examination system includes components for generating an internal image of a patient, such as an x-ray image, and components for generating an image of the external surface of at least a portion of the patient. The signals obtained from the surface scan of the patient are used to control the positioning of other components, such as the patient support and/or the primary radiation diaphragm. The internal and surface images can be compared to achieve precise positioning of the patient and/or a setting of the primary radiation diaphragm to avoid blooming.

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Summarize: [0001] This application claims the benefit of U.S. provisional application No. 60/482,097, filed Jun. 24rd, 2003, the entire content of which is hereby incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to a novel method of creating an immunogen and using it to produce antibodies against nonenveloped and enveloped viruses, bacterial pathogens, fungal pathogens, other microbial pathogens, and proteins. [0003] The invention relates generally to agents and methods for preventing a viral outbreak and, more specifically, to compositions containing a β-cyclodextrin (β-CD) and methods of using such compositions to decrease the probability and/or reduce the severity of a viral outbreak. [0004] The present invention also relates to a pharmaceutical composition, which includes 1) β-CD, which is in a sufficient amount to block viral passage through lipid rafts in the membrane of nerve cells. [0005] The present invention further relates to a composition, comprising a solid substrate that contains an effective amount of β-CD useful for reducing viral release. BACKGROUND OF THE INVENTION [0006] The plasma membrane of immune and non-immune cells is composed of detergent-insoluble domains called lipid rafts, which are membrane compartments enriched in cholesterol and sphingolipids. In some tissues these specialized domains are referred to as caveolae. The initiation and propagation of intracellular signaling events occurs in these specialized membrane regions. Lipid rafts also contain many lipid-modified signaling proteins, and restrict their diffusion. Some examples of proteins associated with lipid rafts are tyrosine kinases of the Src family, glycophosphatidylinositol (GPI)-linked proteins, as well as adaptor proteins. [0007] The confinement of signaling molecules to membrane subdomains suggests that lipid rafts are platforms for the formation of multicomponent transduction complexes. When immune receptors binds to their ligand, they become associated with lipid rafts. Additional components of the receptor signaling pathways are subsequently recruited to the rafts and form macromolecular signaling complexes. The initial translocation of immune receptors into lipid rafts is an important step in regulating cell activation. [0008] Numerous experiments have provided substantial evidence that the integrity of lipid rafts is crucial for the initiation and maintenance of intracellular signals. Depletion of cholesterol, a component of lipid rafts, has been shown to inhibit HIV infection and illustrates the importance of lipid rafts in viral infection. Virus fusion and entry involves sequential interactions between viral proteins and proteins of the cell surface. These fusion and entry interactions proceed via three-dimensional rearrangements of viral and cell-surface proteins, thus giving rise to novel, but transient antigenic features. The present invention exploits those unique antigenic features to create novel anti-viral antibodies. [0009] Virus entry into host cells involves the specific interaction of virus with receptor molecules contained within. Virus assembly and increases in viral concentration also occur in lipid rafts. Thus, the interaction between virus particles and lipid rafts presents an environment in which novel viral epitopes may be exposed. The interaction of proteins and lipid rafts generates novel configurations of the proteins that may be exploited to produce novel antibodies against the protein. [0010] The significance of detergent-insoluble, glycolipid-enriched membrane domains (“lipid rafts”) has been demonstrated, particularly in regard to activation and signaling in T lymphocytes. Lipid rafts can be viewed as floating rafts comprised of sphingolipids and cholesterol that sequester glycosylphosphatidylinositol (GPI)-linked proteins such as Thy-1 and CD59. CD45, a 200 kDa transmembrane phosphatase protein, is excluded from these domains. Human immunodeficiency virus type 1 (HIV-1) particles produced by infected T cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 is poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC16 (see below), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that ( 3 H)-myristic acid-labeled HIV Gag protein showed a nine-to-one enrichment in lipid rafts. As disclosed herein, the budding of HIV virions through lipid rafts is associated with the presence of host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains in the viral envelope, indicating preferential sorting of HIV Gag to lipid rafts (see Example 1). [0011] Glycolipid-enriched membrane (GEM) domains are organized areas on the cell surface enriched in cholesterol, sphingolipids, and GPI-linked proteins. These domains have been described as “rafts” that serve as moving platforms on the cell surface (Shaw and Dustin, Immunity. 6:361-369, 1997). The domains, now referred to as “lipid rafts,” exist in a more ordered state, conferring resistance to Triton X-100 detergent treatment at 4° C. (Schroeder et al., J. Biol. Chem. 273:1150-1157, 1998). Many proteins are associated with lipid rafts, including GPI-linked proteins, Src family kinases, protein kinase C, actin and actin-binding proteins, heterotrimeric and small G proteins, and caveolin (see, for example, Arni et al., Biochem. Biophys. Res. Commun. 225:8001-807, 1996; Cinek and Horejsi, J. Immunol. 149:2262-2270, 1992; Robbins et al., Mol. Cell. Biol. 15:3507-3515, 1995; and Sargiacomo et al., J. Cell. Biol. 122:789-807, 1993). Saturated acyl chains of the GPI anchor have been shown to be a determinant for the association of GPI-linked proteins with lipid rafts (Rodgers et al., Mol. Cell. Biol. 14:5384-5391, 1994; Schroeder et al., Proc. Natl. Acad. Sci., USA. 91:12130-12134, 1994). Lipid rafts exclude certain transmembrane molecules, specifically the membrane phosphatase CD45 (Arne et al., supra, 1996; Rodgers and Rose, J. Cell. Biol. 135:1515-1523, 1996). Exclusion of CD45 results in the accumulation of phosphorylated signaling molecules in lipid rafts, and T cell activation requires clustering of signaling molecules in these membrane domains (Lanzavecchia et al., Cell. 96:1-4, 1999). [0012] The role of lipid rafts in viral infection can further be extended to viruses other than HIV. For example, selective budding occurs for a virus of the influenza family, fowl plague virus, from ordered lipid domains (Scheiffele et al., J. Biol. Chem. 274:2038-2044, 1999, which is incorporated herein by reference). The requirement for cholesterol and sphingolipids in target membranes for Semliki Forest virus fusion also has been established (Nieva et al., EMBO J. 13:2797-2804, 1994; Phalen and Kielian, J. Cell Biol. 112:615-623, 1991, each of which is incorporated herein by reference). The interactions of lipid rafts with accessory HIV-1 molecules such as Vif and Nef can have important roles in virus budding, since interactions of myristylated HIV and simian immunodeficiency virus Nef with Lck, which is present in lipid rafts, and its incorporation into virions have been established (see, for example, Collette et al., J. Biol. Chem. 271:6333-6341, 1996; Flahertyet al., AIDS Res. Hum. Retrovir. 14:163-170, 1998). [0013] Example 1 describes the interaction of HIV virus with lipid raft resident molecules such as GM1. The results disclosed in Example 1 indicate that HIV-1 buds through lipid rafts. During the course of infection, the cell becomes activated and polarization occurs, capping normally dispersed lipid rafts along with GPI-linked proteins and associated intracellular signaling molecules, and membrane areas containing CD45 can be cleared out of the cap site. The newly translated viral Gag precursor protein associated with lipid rafts then can be directed to the capped pole, where assembly and budding occurs. Palmitylated gp41 (gp160) is also directed into lipid rafts, and the interaction of its cytoplasmic tail with Gag protein in lipid rafts can prevent its internalization, allowing for the incorporation of gp160 into virions only at the site of budding (see Egan et al., J. Virol. 70:6547-6556, 1996; Yu et al., J. Virol. 66:4966-4971, 1992). Individual targeting of Gag and Env to the same site at the membrane can be an important means for delivering these proteins to the site of budding, since Gag and Env are processed and transported in different pathways within the cell. The host membrane then can become the new viral coat, resulting in the incorporation of cholesterol, sphingolipids, Thy-1, and CD59 and in the exclusion of CD45. HIV-1 also acquires functional adhesion molecules from host cells (Orentas and Hildreth, supra, 1993). These host-acquired proteins can significantly affect the biology of HIV-1 (see, for example, Fortin et al., J. Virol. 71:3588-3596, 1997). BRIEF SUMMARY OF INVENTION [0014] The present invention relates to a novel method of designing an immunogen and producing antibodies to nonenveloped and enveloped viruses, and proteins. Specifically, it uses the co-culture of purified lipid rafts and viral particles as an immunogen. [0015] The present invention also relates to a novel therapeutic method of preventing viral outbreaks (budding), using β-cyclodextrins. [0016] Viral entry into cells involves unfolding of the virus and penetration into the cell at specific lipid raft sites. Antibodies created against the lipid raft/virus co-culture exploit viral unfolding to reveal novel epitopes in the virus that may be exploited as immunogens to create novel neutralizing antibodies. Purified preparations of lipid rafts are easily prepared from primary lymphoctes or transformed lymphocytes such as the Jurkat cell line. Purified isolates of HIV obtained from infected cell supernatants can be co-cultured with purified fractions of lipid rafts. These co-cultures can be used intact as immunogens or partially proteolysed to create viral/raft fragments. Additionally, the viral/raft co-cultures can remain co-cultured intact or fixed while co-cultured in mild fixative such as paraformaldehyde or glutaraldehyde. The steps involve mixing purified raft fractions with isolated virus. This admixture of raft/virus serves as the novel immunogen. Antibodies created against the lipid raft/virus co-culture will recognize antigenic determinants of the virus unique to the lipid raft/virus interaction. The method employs lipid raft/virus or lipid raft/protein suspensions as immunogens to develop polyclonal or monoclonal antibodies. Alternatively, lipid raft fractions may be made from infected cells such as lymphocytes or an immune cell line such as Jurkat. Lipid rafts produced in this fashion would already contain interactive virus and would be ready for use as a raft/virus immunogen. A lipid raft/virus or lipid raft/protein co-cultured immunogen that will generate an antibody response able to neutralize a broad spectrum of primary viral isolates and generate immune responses is created in this fashion. Antibodies to novel epitopes in virus and proteins are also created in this fashion. [0017] The use of lipid raft terminology in this disclosure also includes use of caveolae (i.e., co-cultures of caveolae/virus or co-cultures of caveolae/protein) as immunogens. This method can also be applied to the co-culture of lipid rafts and any pathogen. As such, the pathogen can be an enveloped virus, including but not limited to an immunodeficiency virus such as human immunodeficiency virus, a T lymphocytic virus such as human T lymphocytic virus (HTLV), a herpes virus such as herpes simplex virus (HSV), a measles virus, or an influenza virus. The pathogen also can be a microbial pathogen, for example a bacterium, a yeast such as Candida, a mycoplasma, a protozoan such as Trichomonas, or a Chlamydia. [0018] Lipid raft preparations are easily obtained from a variety of cell sources. Immune cells susceptible to viral infection represent good source for raft preparation. Whole immune cells exposed to virus could also be used as a source of lipid raft/virus immunogen. Co-culture combinations of lipid rafts, viral proteins and various peptides (e.g., the HIV envelope glycoprotein gp120) would also be used as immunogens. Thus, this method is also applicable to generating antibodies to novel conformations of viral, microbial, fungal, and animal proteins when co-cultured and interacting with lipid rafts. [0019] Many proteins translocate into lipid rafts following stimulation. This translocation involves modifications such as palmitylation and/or myristylation. Antibodies raised against such lipid raft/protein immunogens may recognize novel epitopes in the translocated protein. Natural lipid raft/virus co-cultures [define this term/idea/concept better] will serve as immunogens, but lipid raft/virus co-cultures or whole cell/virus co-cultures fixed with low concentrations of fixative such as formalin [glutaraldehyde, or methanol] may also be used. Lipid raft preparations can also be modified to include or exclude selected proteins in order to vary the immunogenic effect of the raft/virus, raft/protein co-culture. In a preferred embodiment, immunizations are performed in mice engineered to be transgenic for human antigens, thus reducing the possibility that the antibodies generated would recognize human proteins. [0020] This novel method of immunogen production may prove useful in the generation of anti-viral vaccines. In the case of human immunodeficiency virus type 1 (HIV-1), success has been gauged by the ability of candidate immunogens to generate measurable immune responses in human volunteers and animal models. The two crucial responses have been the generation of virus-specific CD8 + cytotoxic T lymphocytes (CTLs), which attack and destroy infected cells, and production of neutralizing antibodies, which bind to the virus and prevent infection of new cells. For HIV-1, an effective anti-viral vaccine has remained elusive. [0021] A number of studies published in recent years have shown that neutralizing monoclonal antibodies of the IgG class alone can be effective in blocking the infection of non-human primates by mucosal challenge with SHIV. Such studies provided a rationale for testing groups of monoclonal antibodies with synergistic neutralizing antibodies in vitro as immediate postexposure prophylaxis, modeling for perinatal exposure in infants. Cocktails of human IgG1b12, 2G12, 2F5, and 4E10 neutralizing monoclonal antibodies prevented disease in newborn macaques and prevented the establishment of SHIV89.6P infection in half of the animals when given within an hour of exposure (Ferrantelli et al., AIDS; 17: 301-309,2003). Studies in in recently infected HIV patients indicated that neutralizing antibodies are indeed involved in controlling viral replication during the first months after infection, and that the pressure they exert on the virus is significant (Richman et al., Proc Natl Acad Sci USA; 100:4144-4149, 2003). [0022] Budding of nascent virus also occurs from lipid rafts. Thus, in addition to preventing new infection, the present invention is applicable to preventing the spread of infection or re-infection. Clinical application of antibodies created by this method may also prevent outbreaks of virus in infected individuals (e.g., herpes outbreaks). [0023] Beta-cyclodextrins deplete cholesterol and disrupt lipid rafts. A novel use of β-cyclodextrins is extended to applications (e.g., topical cream) to prevent recurrent herpes zoster, herpes oral or genital outbreaks. Topical use of β-cyclodextrins may also reduce the severity of outbreaks as well as shorten their duration. Many pathogens exploit lipid rafts for cell infection as well as cell outbreak. This use of β-cyclodextrins and the disruption of raft structure as a portal for entry or exit are applicable to any pathogen outbreak, which involves lipid rafts. As such, pathogen release from infected cells or neurons may be prevented by the disruption of raft structure by β-cyclodextrins. [0024] The present invention relates to methods of reducing the risk of virus budding or diminishing the severity of outbreak of viral infections. It also may be used to diminish pain and associated symptoms of post-outbreak neuralgia. In one embodiment, a method of the invention is performed by contacting area of viral release (e.g., dermatomes in shingles) with a β-cyclodextrin (β-CD). The afflicted dermatomes may be identified by a tingling sensation (a prodrome), which signals the onset of viral release. Examples of said releasable viruses include but are not limited to: an enveloped virus, for example, an immunodeficiency virus such as human immunodeficiency virus (HIV); a T lymphotrophic virus such as human T lymphotrophic virus (HTLV); a herpes virus such as a herpes simplex virus (HSV); a measles virus; a chicken pox virus or an influenza virus. The β-CD can be any β-CD derivative, for example, 2-hydroxypropyl-β-cyclodextrin. In the case of herpes zoster or any outbreak resulting in an outbreak-induced neuralgia, the pain and associated symptoms may be amenable to topical treatment of β-CD. [0025] The present invention also relates to a pharmaceutical composition, which includes: 1) β-CD, which is in a sufficient amount to block viral release through lipid rafts in the membrane of a nerve cell. [0026] The present invention further relates to a composition, comprising a solid substrate that contains an effective amount of β-CD useful for reducing the risk of viral release and the severity of viral outbreak. The pharmaceutical composition can be formulated in a solution, a gel, a foam, an ointment, a cream, a paste, a spray, or the like. DETAILED DESCRIPTION OF INVENTION [0027] To isolate detergent resistant membranes (DRMs) from a cell type including but not limited to primary or transformed lymphocytes. Cells are washed in Buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidizole, pH 6.8), then in TKM buffer (50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl, and 1 mM EGTA). To reduce proteolysis, the following protease inhibitors are included in Buffer A: 2 mg/ml of leupeptin (Calbiochem Novabiochem Corp., La Jolla, Calif.); 5 mM Pefa-Bloc (Roche Molecular Biochemicals, Indianapolis, Ind.); 1% aproptinin (Sigma); 1% pepstatin A (Roche Molecular Biochemicals); and 100 nM benzamidine (Sigma). DRMs were prepared using a discontinuous sucrose density gradient. DRMs are located at the interface between 5 and 36% sucrose. [0028] Alternatively, isolation of low-density, Triton X-100-insoluble membrane complexes is easily performed. Briefly, cells were homogenized in 2-morpholinoethanesulfonic acid (MES)-buffered saline containing 1% Triton X-100 (unless otherwise indicated), and sucrose was added to a final concentration of 40%. A 5 to 30% discontinuous sucrose gradient was layered on top of this detergent extract followed by ultracentrifugation [54,000 rpm in a rotor (Beckman Coulter, Fullerton, Calif.)] for 18 to 24 h at 4° C. in a TL-100 ultracentrifuge (Beckman Coulter). Successive gradient fractions were collected from the top and subjected to SDS-PAGE and Western blot analysis. [0029] HIV-1.sub.RF viral supernatant from an infected Jurkat cell line can be collected and clarified through a 0.45.mu.m filter. Virus supernatant (10 ml) can be co-cultured with purified lipid raft fractions as described above. These lipid raft/virus co-cultures serve as immunogens for the creation of novel antibodies. Following hybridoma fusion to create monoclonal expressing immortalized B-cells, antibodies produced in this fashion can be mass screened to determine their effectiveness as neutralizing antibodies. The capacity of purified IgG as well as whole serum, to neutralize HIV can be tested in an assay with phytohemagglutinin-stimulated peripheral blood mononuclear cells. Briefly, antibodies or sera were incubated for 1 h at 37° C. with diluted tissue culture supernatant of virus-infected peripheral blood mononuclear cells (40 to 100 50% tissue culture infective doses, 100 μl). Peripheral blood mononuclear cells (10 5 in 50 μl) were added to the virus-antibody reaction mixture, and the mixture was incubated overnight. All dilutions were performed with RPMI 1640 medium (GIBCO, Life Technologies Ltd., Paisley, Scotland) supplemented with 10% fetal calf serum, 3 mM glutamine, 20 IU of interleukin-2, and antibiotics. Medium changes were performed on days 1 and 4. Seven days after infection, supernatants were collected and analyzed for HIV antigen by a capture ELISA. The neutralization titer was defined as the reciprocal of the last dilution step that showed an 80% or greater reduction in the OD at 490 nm of the culture supernatant compared to that of HIV antibody-negative serum. [0030] Beta-cyclodextrins (β-CDs) are widely used as solubilizing agents, stabilizers, and inert excipients in pharmaceutical compositions (see U.S. Pat. Nos. 6,194,430; 6,194,395; and 6,191,137, each of which is incorporated herein by reference). Beta-CDs are cyclic compounds containing seven units of α-(1,4) linked D-glucopyranose units, and act as complexing agents that can form inclusion complexes and have concomitant solubilizing properties (see U.S. Pat. No. 6,194,395; see also, Szejtli, J. Cyclodextrin Technol. 1988). [0031] The compositions and methods of the invention are exemplified using 2-hydroxypropyl-β-CD (2-OH-β-CD). However, any β-CD derivative can be used in a composition or method of the invention, provided the β-CD derivative disrupts lipid rafts in the membranes of nerve cells. Beta-CDs act, in part, by removing cholesterol from cell membranes, and different β-CDs are variably effective in such removal. For example, methyl-β-CD removes cholesterol from cell membranes very efficiently and quickly and, as a result, can be toxic to cells, which require cholesterol for membrane integrity and viability. In comparison, a β-CD derivative such as 2-OH—β-CD can effectively remove cholesterol from cells without producing undue toxicity. Thus, it will be recognized that a β-CD useful in a composition or method of the invention is one that removes cholesterol in an amount that disrupts lipid rafts, without substantially reducing cell viability (see, for example, Rothblat and Phillips, J. Biol. Chem. 257:4775-4782 (1982), which is incorporated herein by reference). [0032] Beta-CDs useful in the present invention include, but are not limited to, β-CD derivatives wherein one or more of the hydroxy groups is substituted by an alkyl, hydroxyalkyl, carboxyalkyl, alkylcarbonyl, carboxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkoxycarbonylalkyl or hydroxy-(mono or polyalkoxy)alkyl group or the like; and wherein each alkyl or alkylene moiety contains up to about six carbons. Substituted β-CDs that can be used in the present invention include, for example, polyethers (see, for example, U.S. Pat. No. 3,459,731, which is incorporated herein by reference); ethers, wherein the hydrogen of one or more β-CD hydroxy groups is replaced by C1 to C6 alkyl, hydroxy-C1-C6-alkyl, carboxy-C1-C6 alkyl, C1-C6 alkyloxycarbonyl-C1-C6 alkyl groups, or mixed ethers thereof. In such substituted β-CDs, the hydrogen of one or more β-CD hydroxy group can be replaced by C1-C3 alkyl, hydroxy-C2-C4 alkyl, or carboxy-C1-C2 alkyl, for example, by methyl, ethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, carboxymethyl or carboxyethyl. It should be recognized that the term “C1-C6 alkyl” includes straight and branched saturated hydrocarbon radicals, having from 1 to 6 carbon atoms. Examples of β-CD ethers include dimethyl-β-CD. Examples of β-CD polyethers include hydroxypropyl-p-β-CD and hydroxyethyl-β-CD (see, for example, Nogradi, “Drugs of the Future” 9(8):577-578, 1984 ; Chemical and Pharmaceutical Bulletin. 28:1552-1558 (1980); Yakugyo Jiho No. 6452 (Mar. 28, 1983); Angew. Chem. Int. Ed. Engl. 19:344-362 (1980); U.S. Pat. No. 3,459,731; EP-A-0,149,197; EP-A-0,197,571; U.S. Pat. No. 4,535,152; WO-90/12035; GB-2,189,245; Szejtli, “Cyclodextrin Technology” (Kluwer Academic Publ. 1988); Bender et al., “Cyclodextrin Chemistry” (Springer-Verlag, Berlin 1978); French, Adv. Carb. Chem. 12:189-260; Croft and Bartsch, Tetrahedron 39:1417-1474, 1983; Irie et al., Pharm. Res. 5:713-716, 1988; Pitha et al., Internat'l. J. Pharm. 29:73, 1986; U.S. Pat. No. 5,134,127 A; U.S. Pat. Nos. 4,659,696 and 4,383,992, each of which is incorporated herein by reference; see, also, U.S. Pat. No. 6,194,395). [0033] A method of the invention is performed, for example, by contacting an area of skin susceptible to viral release with a β-CD. As used herein, the term “contacting,” when used in reference to a β-CD and the pathogen or cells susceptible to a sexually transmitted pathogen, means that the β-CD is applied to the susceptible area such that it prevents viral budding through lipid rafts at nerve terminals. [0034] As described above, budding of HIV-1 particles occurs at lipid rafts, which are characterized by a distinct lipid composition that includes high concentrations of cholesterol, sphingolipids, and glycolipids. Since cholesterol plays a key role in the entry of some other viruses, the role in HIV-1 entry of cholesterol and lipid rafts in the plasma membrane of susceptible cells was investigated. Example 2 demonstrates that intact lipid rafts are necessary for viral infection. A β-CD derivative, 2-hydroxypropyl-β-cyclodextrin (2-OH-β-CD), was used to deplete cellular cholesterol and disperse lipid rafts. As disclosed herein, removal of cellular cholesterol rendered primary cells and cell lines highly resistant to HIV-1-mediated syncytium formation and to infection by both CXCR4- and CCR5-specific strains of HIV-1 virus. 2-OH-β-CD treatment of the virus or cells partially reduced HIV-1 binding, while rendering chemokine receptors highly sensitive to antibody-mediated internalization, but had no effect on CD4 expression. These effects were readily reversed by incubating cholesterol-depleted cells with low concentrations of cholesterol-loaded 2-OH-β-CD to restore cholesterol levels. Cholesterol depletion also made cells resistant to SDF-1-induced binding to ICAM-1 through LFA-1. This may have contributed to the reduction in HIV-1 binding to cells after treatment with the β-CD, since LFA-1 contributes significantly to cell binding by HIV-1 which, like SDF-1.alpha, can trigger CXCR4 function through gp120. These results indicate that cholesterol is involved in the HIV-1 co-receptor function of chemokine receptors and is required for infection of cells by HIV-1 (Example 2). [0035] As discussed above, cholesterol, sphingolipids, and GPI-anchored proteins are enriched in lipid rafts (see Simons and Ikonen, Nature. 387:569-572, 1997). The high concentration of cholesterol and sphingolipids in lipid rafts results in a tightly packed, ordered lipid domain that is resistant to non-ionic detergents at low temperature. The structural protein caveolin causes formation of flask-shaped invaginations (caveolae) in the cell membrane with a lipid composition very similar to that of lipid rafts (Schnitzer et al., Science 269:1435-1439, 1995). Signaling molecules, including Lck, LAT, NOS, and G protein a subunit, are localized to rafts on the intracellular side of the membrane, and are targeted by lipid modifications such as palmitylation, myristylation, or both. In comparison, many other transmembrane proteins do not show a preference for lipid rafts; for example, CD45 and E cadherin are excluded from these areas. Certain lipid modified transmembrane proteins such as the HA molecule of influenza virus localize to lipid rafts. [0036] As disclosed herein, HIV-1 buds selectively from lipid rafts of infected T cells (Example 1). In addition, Semliki Forest Virus (SFV), measles viruses, influenza viruses, and polioviruses all assemble by raft association and, in the case of influenza virus, bud from lipid rafts (see, for example, Marquardt et al., J. Cell Biol. 123:57-65, 1993; Manie et al., J. Virol. 74:305-311, 2000; Zhang et al., J. Virol. 74:4634-4644, 2000, each of which is incorporated herein by reference). The involvement of lipid rafts in HIV-1 biology beyond its role in virus budding has been further examined. As further disclosed herein, partial depletion of cholesterol from cell membranes using a β-CD inhibited HIV-1-induced syncytium formation in cell lines and primary T cells (Example 2). β-CD treatment of cells also increased CR internalization induced by monoclonal antibody (MAb) binding. Primary cells and cell lines were rendered resistant to infection CXCR4-specific and CCR5-specific HIV-1 strains by treatment with 2-OH-β-CD (Example 2). The effects observed were not due to loss of cell viability after treatment with the β-CD, and demonstrate that intact lipid rafts and cholesterol are required for HIV-1 infection and syncytium formation. [0037] The present invention also provides compositions useful for reducing the risk of transmission of sexually transmitted disease. A composition of the invention contains a β-CD, which can be in a form suitable for topical administration to a subject, particularly intravaginal or intrarectal use, including a suppository or a bioadhesive polymer, which can provide timed release of the β-CD (see, for example, U.S. Pat. Nos. 5,958,461 and 5,667,492, each of which is incorporated herein by reference); or can be formulated in combination with a solid substrate to produce a condom, diaphragm, sponge, tampon, a glove or the like (see, for example, U.S. Pat. Nos. 6,182,661 and 6,175,962, each of which is incorporated herein by reference), which can be composed, for example, of an organic polymer such as polyvinyl chloride, latex, polyurethane, polyacrylate, polyester, polyethylene terephthalate, polymethacrylate, silicone rubber, a silicon elastomer, polystyrene, polycarbonate, a polysulfone, or the like (see, for example, U.S. Pat. No. 6,183,764, which is incorporated herein by reference). [0038] For topical administration, the β-CD can be formulated in any pharmaceutically acceptable carrier, provided that the carrier does not affect the activity of the β-CD in an undesirable manner. Thus, the composition can be, for example, in the form of a cream, a foam, a jelly, a lotion, an ointment, a solution, a spray, or a gel (see U.S. Pat. No. 5,958,461, which is incorporated herein by reference). In addition, the composition can contain one or more additional agents, for example, an antimicrobial agent such as an antibiotic or an antimicrobial dye such as methylene blue or gentian violet (U.S. Pat. No. 6,183,764); an antiviral agent such as a nucleoside analog (e.g., azacytidine), a zinc salt (see U.S. Pat. No. 5,980,477, which is incorporated herein by reference), or a cellulose phthalate such as cellulose acetate phthalate or a hydroxypropyl methylcellulose phthalate (see U.S. Pat. No. 5,985,313, which is incorporated herein by reference); a contraceptive (see U.S. Pat. No. 5,778,886, which is incorporated herein by reference); a lubricant, or any agent generally useful to a sexually active individual, provided the additional agent, either alone or in combination, does not affect the activity of the β-CD or, if it affects the activity of the β-CD, does so in a predictable way such that an amount of β-CD that is effective for reducing viral outbreak can be determined. [0039] A pharmaceutically acceptable carrier useful in a composition of the invention can be aqueous or non-aqueous, for example alcoholic or oleaginous, or a mixture thereof, and can contain a surfactant, emollient, lubricant, stabilizer, dye, perfume, preservative, acid or base for adjustment of pH, a solvent, emulsifier, gelling agent, moisturizer, stabilizer, wetting agent, time release agent, humectant, or other component commonly included in a particular form of pharmaceutical composition. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the β-CD, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. [0040] The pharmaceutical composition also can comprise an admixture with an organic or inorganic carrier or excipient suitable for intravaginal or intrarectal administration, and can be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use. The carriers, in addition to those disclosed above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see, for example, U.S. Pat. No. 5,314,695). [0041] The β-CD also can be incorporated within an encapsulating material such as into an oil-in-water emulsion, a microemulsion, micelle, mixed micelle, liposome, microsphere or other polymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981), each of which is incorporated herein by reference). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. “Stealth” liposomes (see U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of such encapsulating materials particularly useful for preparing a pharmaceutical composition of the invention, and other “masked” liposomes similarly can be used, such liposomes extending the time that the β-CD remains at the site of administration. [0042] The amount a β-CD in a composition can be varied, depending on the type of composition, such that the amount present is sufficient to reduce viral outbreak or reduce severity of outbreak. An example of such an amount is about 1 to 100 mM, generally about 5 to 30 mM, when administered in an ointment, gel, foam, spray or the like, our about 0.1 to 2 grams, generally about 0.25 to 0.75 grams, when administered as a suppository or in combination with a solid substrate. An effective amount of a β-CD also can be measured in a weight:weight (w:w) or weight:volume (w:v) amount, for example, about 0.1% to 3% w:w with respect to a solid substrate or about 0.1% to 3% w:v with respect to a pharmaceutically acceptable carrier. In addition, an amount of a β-CD sufficient to reduce viral outbreak or decrease outbreak severity can be determined using routine clinical methods, including Phase I, II and III clinical trials. [0043] Currently, several HIV-1 vaccine approaches are being developed, each with its own relative strengths and weaknesses. These approaches include the development of live attenuated vaccines, inactivated viruses with adjuvant peptides and subunit vaccines, live vector-based vaccines, and DNA vaccines. Envelope glycoproteins were considered as the prime antigen in the vaccine regimen due to their surface-exposure, until it became evident that they are not ideal immunogens. This is an expected consequence of the immunological selective forces that drive the evolution of these viruses: it appears that the same features of envelope glycoproteins that dictate poor immunogenicity in natural infections have hampered vaccine development. However, modification of the vaccine recipe through the use of raft/virus co-cultures to expose novel viral epitopes may overcome these problems. [0044] Accordingly, there is a need in the art for new effective methods of identifying candidate sequences for vaccine development to prevent and treat HIV infection. The present invention fulfills this and other needs. [0045] “Antibody” refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, that specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. [0046] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain has a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. [0047] Antibodies exist, for example, as intact immunoglobulins or as a number of well characterized antigen-binding fragments produced by digestion with various peptidases. For example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce an F(ab′).sub.2 fragment, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab′).sub.2 fragment can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab′).sub.2 dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, Third Edition, W. E. Paul (ed.), Raven Press, N.Y. (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments, such as a single chain antibody, an antigen binding F(ab′).sub.2 fragment, an antigen binding Fab′ fragment, an antigen binding Fab fragment, an antigen binding Fv fragment, a single heavy chain or a chimeric antibody. Such antibodies can be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. [0048] Thus, an immunogenic composition to this subtype B ancestor protein will elicit broad neutralizing antibody against HIV-1 isolates of the same subtype. An immunogenic composition to this subtype B ancestor protein will also elicit a broad cellular response mediated by antigen-specific T-cells. [0049] Monoclonal antibodies (Mabs) have been available for over 25 years and have revolutionalized biomedical research, especially in the areas of disease diagnosis and the treatment of infection and diseases. [0050] The conventional method for the production of monoclonal antibodies involves hybridomas (Kohler & Milstein, Nature 256:495-7, 1975). In this method, splenic or lymphocyte cells from a mammal which has been injected with antigen are fused with a tumor cell line, thus producing hybrid cells. These hybrid cells, or “hybridomas”, are both immortal and capable of producing the genetically coded antibody of a B cell. To select a hybridoma producing a single antibody, the hybridomas made by cell fusion are segregated by selection, dilution, and regrowth until a single genetically pure antibody-expressing cell line is selected. Because hybridomas produce homogeneous antibodies against a desired antigen, they are called “monoclonal” antibodies. Hybridoma technology has primarily been focused on the fusion of murine lines, but also human-human hybridomas, human-murine hybridomas, rabbit-rabbit hybridomas and other xenogenic hybrid combinations have been made. EXAMPLES Example 1 [heading-0051] HIV-1 Selectively Buds from Lipid Rafts [0052] This example demonstrates that HIV-1 budding occurs through lipid rafts, thereby accounting for the cholesterol-rich, sphingolipid-rich virus membrane, which bears GPI-linked proteins such as Thy-1 and CD59, but lacks CD45. [0053] The relative incorporation of GM1, a ganglioside marker specific for lipid rafts, also was examined. Using a soluble CTB binding assay, as much as 75% of HIV-1 was precipitated using goat anti-CTB and SaC after treating the virus with GM 1-specific CTB). The CTB binding to virus was specific and dose dependent, and no virus was precipitated in the absence of CTB as measured by p24 ELISA. These results demonstrate that the majority of HIV-1 particles incorporate the lipid raft-specific marker GM1. [0054] Thy-1, CD59, and GM1 colocalized with Hw-1 proteins on infected cell uropods, which excluded CD45. To determine the distribution of HIV-I proteins relative to GPI-linked proteins that serve as lipid raft markers, infected cells were subjected to immunofluorescence staining followed by confocal microscopy. Expression of HIV-1 proteins was localized to uropods projecting from one end of the cell. This capping pattern was seen on most cells in the infected cell culture. Uropods protruding from HIV-1-infected cells have been described for adherent T cells. Thy-1 and CD59 both colocalized with cell surface HIV-1 proteins, as shown by a superimposed green (Thy-1 or CD59) and red (HIV-1 proteins) fluorescence (see Nguyen and Hildreth, supra, 2000; FIG. 4 ). Cells that were prefixed with 2% paraformaldehyde before staining showed a similar appearance, indicating that the colocalization was not due to antibody crosslinking of viral and GPI-linked proteins. Since the cells were not permeabilized before staining, the HIV proteins seen in these studies are likely gp41 and gp120. This was confirmed in studies with anti-gp41 MAb T32 in the colocalization studies. Uninfected cells showed no capping of Thy-1 or CD59. CD45 did not localize to areas of HIV-1 protein expression and was excluded from uropods. The distribution of CD45 was unaffected by HIV-1 infection, and the molecule remained evenly dispersed in patches all over the cell surface. These results confirm those obtained using the virus phenotyping studies. The ability of GM1 to colocalize on the cell surface with HIV-1 proteins was examined to confirm the finding that GM1 was present on virions. GM1 staining was relatively faint with rabbit anti-GM1 antibody, but confocal microscopy showed colocalization of this molecule with HIV-1 labeled cells. [0055] HIV-1 proteins were detected in isolated lipid raft fractions. Lipid rafts were purified by cell lysis and equilibrium centrifugation in order to confirm the presence of HIV-1 proteins in these membrane structures. The fractions were assayed for the presence of viral and host proteins by immunoblot analysis. The separation of detergent-resistant lipid rafts was confirmed by the abundance of Thy-1 and CD59 in fractions 3 through 5, while CD45 was present only in the bottom fractions 9 and 10 (see Nguyen and Hildreth, supra, 2000; FIG. 6 ). Immunoblot detection of membrane fractions revealed that the HIV MA protein, p17, and gp41 were both present in the detergent-insoluble lipid rafts of infected cells. Example 2 [heading-0056] Host Membrane Cholesterol is Required for HIV-1 Infection [0057] By removing cholesterol, 2-OH-.beta.CD is believed to partially perturb organized lipid rafts, resulting in dispersal of their components (Ilangumaran and Hoessli, Biochem. J. 335:433-440, 1998). The capture of HIV-1 by MAbs against CD59 and gp41 decreased substantially after treating cells with 2-OH-.beta.CD, as measured by the percentage of total p24. CD45 capture remained unaffected. The effects on virus precipitation through gp41 indicate that intact lipid rafts are required for efficient gp41 incorporation into virions, since the overall cellular release of p24 actually increased after 2-OH-.beta.CD treatment. [0058] Results 2-OH-betaCD treatment blocked syncytium formation of primary cells and cell lines. The role of lipid rafts in the HIV-1 fusion process was examined by treating CD4+HIV-susceptible target cells with 2-OH-betaCD to deplete membrane cholesterol and disperse lipid rafts. Treatment of cells with 10 to 20 mM 2-OH-betaCD for 1 hr at 37 degree ° C., followed by washing to remove free 2-OH-.beta.CD, depleted greater than 70% of total cellular cholesterol without any loss in cell viability as measured by Trypan Blue exclusion. Furthermore, treated cells continued to grow normally after 2-OH-betaCD treatment when placed back into culture in cholesterol-containing medium. The non-toxicity of betaCD treatment was further demonstrated by finding 2-OH-.beta.CD treated Jurkat cells still showed Ca 2+ flux responses to anti-CD3 MAb.

Summary: This invention provides a novel application for β-cyclodextrin, a cholesterol depletor, as an inhibitor of viral outbreak. Topical applications of β-cyclodextrin are recommended to inhibit or reduce the severity of viral outbreaks such as oral or genital herpes. This invention also provides a novel technique for the creation of immunogens. Viral entry and outbreak also occurs at specialized lipid raft domains and disruption of rafts with cholesterol depletors blocks viral entry and outbreak (budding). Lipid microdomains (lipid rafts) are mobile regions of the plasma membrane and exist in all mammalian cell membranes. They are produced by the preferential packing of cholesterol and sphingolipids into the plasma membrane and are identified by their low solubility in detergents and enrichment with gangliosides such as G M1. The size and composition of rafts can be dynamically altered during transmembrane signaling. Depletion of membrane cholesterol disrupts lipid rafts and inhibits viral entry and outbreak. Virus entry into cells involves virus/lipid raft interaction wherein the virus unfolds to enter the cell via the lipid raft. I provide a technique for the creation of an immunogen with novel viral epitopes based on the virus/lipid raft interaction and viral unfolding. Viral unfolding only occurs in lipid rafts. This fact can be exploited to create novel immunogens based on viral interaction with lipid rafts. The virus/lipid raft co-culture technique will create novel immunogens which will be used to create novel neutralizing monoclonal and polyclonal antibodies to fight viral disease such as HIV infection.

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Summarize: CROSS REFERENCE TO RELATED APPLICATIONS This Application is a continuation application of U.S. patent application Ser. No. 14/148,374 filed Jan. 6, 2014, entitled “Convection Recirculating Fryer for Cooking Foods,” by David R. Highnote, pending, which is a continuation application and claims the benefit of and priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 12/435,722 filed May 5, 2009, entitled “Convection Recirculating Fryer for Cooking Foods,” by David R. Highnote, now patented as U.S. Pat. No. 8,646,382 issued Feb. 11, 2014, which is hereby incorporated by reference herein as if set forth herein in its entirety. TECHNICAL FIELD The present invention is generally related to a convection frying apparatus for cooking foods within a recirculating bath of cooking liquid. BACKGROUND OF THE INVENTION Although there are many ways to prepare food for consumption, one common method of preparing foods is to cook the food by frying. Additionally, one method of frying food is to “deep fry” the food by placing the uncooked food in a quantity of-cooking liquid, in most deep frying situations, the cooking liquid typically comprises a cooking oil, such as vegetable oil or animal fat. The food product is immersed in the cooking oil. The cooking oil is typically at a high temperature, such as above 350 degrees Fahrenheit. Devices for deep frying are common in commercial food preparation environments. They are also becoming increasingly common in the home environment. Although a commercial frying apparatus and a home frying apparatus may he constructed on different scales, these two types of fryers have some of the same basic features. The primary feature of a typical fryer, whether commercial or residential, is a cooking tank housing the heated bath of cooking oil The cooking tank is usually designed so that it may receive a metal basket into the tank. Food is placed in the metal basket and lowered into the cooking oil so that the food is at least partially submersed in the oil. A heating device is typically used to maintain the oil in the tank at a substantially constant temperature. This heating device usually comprises a gas burner placed below the tank. The typical fryers used in commercial and residential sailings do not remove the oil from the tank during operation. The cooking oil simply remains in the tank and the temperature of the oil is regulated by heating the oil while the oil remains in the tank. In contrast a recirculating fryer removes oil from the tank, adjusts the heat energy in the oil, and then returns the oil to the tank. There have been previous attempts to develop a commercial recirculating fryer. However, these recirculating fryer designs have all suffered from a number of limitations. For example, some recirculating fryer designs exhibited problems with leaking seals in the pump. The pump seals became worn with use and began to leak. Other designs, while not necessarily having a problem with leaking seals, experienced failure of the pump bearings. This was usually a result of the arrangement of the pump. In general, the prior recirculating fryers were far too expensive to maintain in order to be feasible for commercial use. Thus, a heretofore unaddressed need exists in the industry to develop a convection recirculating fryer that is efficient, cost-effective, and having reasonable maintenance costs. BRIEF DESCRIPTION OF THE DRAWINGS Many aspects of the invention can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views. FIG. 1 is a side view of the convection recirculating fryer. FIG. 1A is section view of the gas injectors of the convection recirculating fryer of FIG. 1. FIG. 2A is a top view of the convection recirculating fryer of FIG. 1 FIG. 2 B is a front view of the convection recirculating fryer of FIG. 1 FIG. 3 is cut away view of the magnetic pump of the convection recirculating fryer of FIG. 1. FIG. 4 is a cut away side view of a magnetic pump to be used in the convection recirculating fryer of FIG. 1. FIG. 5 is an exploded part view of a magnetic pump used in the convection recirculating fryer of FIG. 1. DETAILED DESCRIPTION A convection recirculating fryer of one exemplary embodiment comprises, generally, a fryer housing forming a tank, and encasing a pump and a heat exchanger. Although all of these elements are not required by the invention disclosed herein, these three elements are the basic components of an exemplary embodiment of a convection recirculating fryer. PARTS LIST 10. Convection fryer 11. Free standing housing 13. Fry tank 16. Oil inlet orifice 17. Oil outlet orifice 18. Filter screen 19. Bottom of tank 20. Oil dispersement pipe 21. Magnetic pump 22. Pump inlet line 26. Motor 27. Motor casing 28. Motor shaft 29. Driving magnet assembly 31. Impeller housing 32. Driven magnet 34. Impeller 35. Pump housing 46. Hot oil supply line 50. Blower 52. Fuel injector tube 54. Glow plug with flame sensor 56. A-D Fuel injectors 58. Gas manifold 60. Perforated burner sleeve 62. Control 64. Ceramic pump shaft 66. Magnet 67. Seal 68. Filter pump An exemplary embodiment of a convection recirculating fryer 10 is depicted in FIG. 1. FIG. 1 is not to scale and is merely presented to provide a general understanding of the components of the convection recirculating fryer 10. The arrangement of the components of the convection recirculating fryer 10, as discussed below, is only one exemplary arrangement. FIG. 1 depicts the convection recirculating fryer 10 having a housing 11. The housing 11 of the fryer 10 can be comprised of a metal material. The housing 11 of the preferred embodiment 10 is a free-standing housing 11. FIG. 2A is a top view of the convection recirculating fryer 10. Alternatively, the housing 11 of the convection recirculating fryer 10 could comprise a smaller, counter-top model such as for home use. The principles of the invention described herein are not changed by scaling the fryer 10 up or down in size. The fryer 10 also has a fry tank 13. See FIG. 1. The fry tank 13 of the preferred embodiment 10 is adapted to receive and hold cooking liquid (not depicted), such as cooking oil (e.g. animal fat shortening, vegetable-oil, or the like). The tank 13 of the preferred embodiment 10 has an open top, an oil inlet orifice 10 and an oil outlet orifice 17. Basically, the preferred tank 13 resembles a deep tub, or retangularly-cubic bucket The tank 13 is also preferably designed to receive a basket (not depicted). The basket typically houses a food product to be cooked in the cooking liquid. Other implementations of placing food to be cooked in the cooking liquid are, of course, possible. The present invention is not limited to any particular method and apparatus for exposing food products to a cooking liquid. The tank 13 of the preferred convection recirculating fryer 10 may also comprise a filter apparatus 18, as depicted in FIG. 3. As depicted in FIG. 3, the filter apparatus is a screen 18 positioned along the bottom 19 of the tank 13. This apparatus maybe of any appropriate type; however, the preferred filtering apparatus is an active filtering device that pulls cooking liquid into the apparatus 18, removes foreign matter, and then deposits the cooking liquid back into the main compartment of the tank 13. The preferred filtering apparatus 18 is comprised of a frame that supports the filter material or “filter sock,” and a gear pump. The filter sock is placed over the frame; which has a pipe connection at a bottom portion. The gear pump draws the cooking liquid from the tank 13, through the filter sock, through an oil dispersement pipe 20, a filter pump 68, and back into the tank 13. Contaminants in the cooking liquid are deposited on the filter sock where they become imbedded and remain there. The fryer 10 also employs a passive filter (not depicted) at the oil outlet orifice 17 of the tank 13. The convection recirculating fryer 10 includes a magnetic pump 21 that draws cooking liquid from the tank 13. For this reason, a passive filtering apparatus could include a screen-type assembly releasably mounted in the cooking tank 13, and substantially covering the oil outlet orifice 17 of the cooking tank 13, for prohibiting larger foreign matter from entering into the magnetic pump 21 and disturbing beat exchanger 41. As noted above, the tank 13 has an oil outlet orifice 17. This orifice 17 is preferably near a lower portion of the tank 13. This orifice 17 of the tank 13 is connected to pump inlet line 22. This inlet line 22 is designed to carry the cooking liquid to an inlet of the pump 21. The magnetic pump 21 is depicted in FIG. 4. The magnetic pump 21 comprises a motor 26 in a motor casing 27. A motor shaft 28 to be driven by the motor 26 protrudes from the motor casing 27 and is attached to a driving magnet assembly 29 comprising a driving magnet. The driving magnet assembly 29 of the preferred embodiment is cylindrical in shape. A magnet such as the driving magnet 29 can be manufactured from a Samarium Cobalt material which will withstand the high temperature of the cooking oil, Inside the cylindrical driving magnet assembly 29, with a plurality of magnets 66, is an impeller magnet housing, or casing 31. The impeller magnet housing 31 of the magnetic pump 21 is hermetically sealed so that any fluid in the impeller magnet housing 31 will not escape to an area exterior to the impeller magnet housing 31. Inside the impeller magnet housing 31 is a driving magnet 32 and an impeller 34. As depicted, the driven magnet 32 and the impeller 34 may be a single unit, in an alternative embodiment, the impeller 34 and the driven magnet 32 may be separate elements connected by a shaft. As is understood by one with ordinary skill in the art, the impeller 34 is the actual device that moves fluid through the magnetic pump 21. The impeller magnet housing 31 is preferably connected to a pump housing 35. The pump housing 35, in combination with the impeller magnet housing 31, encases the impeller 34. The pump housing 35 has a pump inlet 36 and a pump outlet 37. The cooking liquid is drawn from the tank 13, though the pump inlet line 22, into pump inlet 36 and into the pump housing 35 by the action of the impeller 34. The impeller 34 also, through its motion, ejects the cooking liquid from the pump housing 35 of the magnetic pump 21. The cooking liquid is ejected though the pump outlet 37 and into pump outlet line 38. It is essential that the shaft 64 of the pump about which the impeller 34 turns be constructed of ceramic material to withstand the heat of the oil. A seal 67 prevents the leakage of oil from the pump. The preferred motor 26 of the magnetic pump 21 has approximately 1.0 horsepower and will perform at approximately 3450 revolutions per minute. Of course, the motor 26 may be sized differently depending on the particular design of the convection reciprocating fryer 10. One having ordinary skill in the art will readily be able to size the motor 26 for a particular fryer. FIG. 5 depicts an exploded part diagram of the magnetic pump to be used with the present preferred embodiment. FIG. 5 depicts that pump 21 comprises a motor 26 and a housing 27, a driving magnet assembly 29, an impeller magnet housing 31, an impeller 34, with driven magnets 32 and a pump housing 35. Of course, this is only one possible magnetic pump that may be used with the present invention. As depicted in FIG. 2A, the magnetic pump 21 is preferably situated vertically within the fryer housing 11 in order to minimize the possibility of a steam look in the pump, which would prevent the circulation of the oil through the fryer. Although preferred in the exemplary embodiment 10, the pump is not required to be situated vertically. Also, the pump outlet 37 of the pump housing 35 is connected by the pump outlet line 38 to a heat exchanger 41. The preferred heat exchanger 41 comprises a series of tubing (not depicted) with a heat source near, or even within, the tubing. In the preferred embodiment 10, the heat exchanger 41 comprises a cylindrical heat exchanger as is conventionally known in the art. The heat exchanger has a heat exchanger exhaust 42. The heat exchanger exhaust 42 of the heat exchanger 41 is in fluid communication with the pump outlet 37 of the pump 21 via the pump outlet line 38. The heating element of the heat exchanger 41 preferably comprises a burner positioned along the axis of the heat exchanger 41. The heating element could be electric or gas powered, for example. It is preferred that the heating element, comprise an LP or natural gas powered burner. The heating element, of course, could also be equipped with a blower 50 in order to more evenly distribute heat throughout the heat exchanger 41. Gas can be distributed through a gas manifold 58 as shown in FIG. 1A to a number of fuel injectors 56 A-D to a fuel injector tube 52. Several fuel injectors are preferred for the even burning of the gas. It has been found that four injectors are preferred. The gas is ignited by a glow plug with a flame sensor 54 to make sure the gas is turned off if it does not ignite in a specified time. The flame extends from the fuel injector tube 52 into the heat exchanger 41 through a perforated burner sleeve 60. The convection recirculating fryer 10 is controlled by a controller 60. The heat exchanger 41 is designed such that the cooking oil travels through heat exchanger tubing within the heat exchanger 41. The internal heat exchanger tubing is configured to permit the passage of the cooking oil back and forth across the burner within the heat exchanger. The internal tubing also includes fins for facilitating the absorption of heat from the burner. The heated cooking oil is ejected from the heat exchanger 41 at the hot oil supply line 46. Preferably, the cooking oil is moved from the heat exchanger 41 at a constant predetermined temperature (which is usually around 350 degrees Fahrenheit). A control system (not depleted) operates in conjunction with a temperature sensor (not depicted) mounted on the outside of the hot oil supply line 46 to ensure that the cooking oil outlet from the heat exchanger 41 remains at the predetermined temperature. Obviously, if the temperature of the cooking oil drops below the target value, or range, the heating element is instructed by the control system to emit more heat energy into the cooking oil. Conversely, if the temperature of the cooking oil increases above the target value, or range, the heating element is caused to emit less heat energy. The hot oil supply line 46 of the heat exchanger 41 is connected to the oil inlet orifice 16 of the tank 13. Thus, the cooking oil completes its journey from the tank 13, to the pump 21, to heat exchanger 41, and back, to the tank 13. As noted above, the magnetic pump 21 of the fryer 10 is the device that actually causes the cooking oil to flow from the tank 13, to the pump 21, to heat exchanger 41, and back to the tank 13. The appropriate rate of flow of the oil can be determined by one of ordinary skill in the art and is not important to the present invention. It should be emphasized that the above-described embodiments of the present invention, particularly, any “preferred” embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the invention. Many variations and modifications may be made to the above-described embodiment(s), of the invention without departing substantially from the spirit and principles of the invention. All such modifications and variations are intended to be included herein within the scope of this disclosure.

Summary: A convection recirculating food product fryer with a fry tank with and inlet tube connected to a heat exchanger and an outlet connected to a magnetic pump with an outlet tube to the heat exchanger, the pump having a driving magnet assembly housing an impeller and a driven magnet, with a ceramic shaft extending through the impeller about which the impeller rotates when pumping oil; an electric motor magnetically coupled to the magnetic pump; a burner to heat the oil in the heat exchanger; a controller to control the ignition and running of the burner.

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Summarize: Police rush to Sinead O’Connor’s home after serious online suicide bid Sinead O'Connor After reading Sinead O’Connor’s online suicide pleas on Wednesday night Irish police went to her home to ensure her personal safety. Online the Irish singer had admitted that she was heartbroken by the breakdown of her marriage to Steve Cooney last April. She also admitted that she had been seeing a psychiatrist. The mother-of-four left a string of messages on her website which showed her desperate fragile state. _________________ READ MORE: Sinead O'Connor a big hit at Lisdoonvarna matchmaking festival Sinead O’Connor announces winner of her perfect man contest Sinead O’Connor says she is ‘tired of being labeled as crazy’ _________________ She said the only reason she had launched an online search for a partner was due to her deep unhappiness. She explained “Truth is I only did all this for the last month cuz I was so depressed about my marriage breaking up and I got tired of crying.” She continued “And tired of thinking I'm a sh*t person all day. So I thought I'd turn it into something funny. And it worked! And I had more fun last four weeks than I had for years. And had self-esteem.” It seems that on Tuesday O’Connor had had a bad experience which tipped her over the edge. She said “Now I wish I was dead. No hope…Anyway…If anyone knows how I can kill myself without my kids finding out I did it deliberately please tell me.” At 6.30pm she posted “I want to go to heaven so bad. Have for years…But I don't want to abandon my kids. But if I could die without them knowing I did it myself, I would.” The alarm was raised by her Twitter followers were receiving a live feed of her posts. Local police in Bray, County Wicklow were contacted by a concerned fan and a squad car was sent over. On fan on Twitter wrote “So much love out there for you.” Another wrote “If anyone knows the lovely, clever, funny [Sinead O'Connor] can they go and give her the hugest hug.” See more: Sinead O'Connor, Irish celebrity news Sinead O’Connor may not be in the best of spirits as of late but she would like her fans to know that, despite openly flirting with such thoughts, she doesn’t actually plan on killing herself. The assurance, written in the form of an open letter on her official website, came three days after she issued a series of red-flag tweets expressing suicidal thoughts. In a succession of tweets sent on Sept. 14, O’Connor wrote, in part, "All this shit we’re not supposed to say. Including suicidal feelings, sex, etc. U just get treated like a crazy person. I want to go to heaven SO bad. Have for yrs... Can’t manage any more. Badly wish cud die without it ruining my kids lives." While the open letter that she published on Saturday (Sept. 17) is intended to reassure concerned fans that her life is not actually in danger, she makes it clear that she wasn’t exactly crying wolf, either. In the letter, she says that while she does "believe that suicide is a sin," she also states her belief that it is healthy to express suicidal thoughts when you’re feeling that way. "People who express suicidal feelings are least likely to act on them," she writes. "Anyone who gives u the remotest bit of shit for expressing suicidal feelings is a wanker and is to be politely asked to permanently vacate your precious company. Even if its ur mudda-fuggin Mama." O’Connor closes the letter by soliciting email advice and suggestions for local "support services in Ireland other than nut-houses and psychiatrists and drugs (DO NOT STOP MEDS BCUZ I SAID THAT.) where people can maybe have a cup of tea..." These developments come in the midst of a renewed campaign to help her find a new boyfriend. She has been sorting through applications from eligible bachelors and, on Friday, posted an updated set of prerequisites and criteria on her website. 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.

Summary: Police turned up at Sinead O'Connor's door to check on her after the irreverent Irish singer tweeted a succession of disturbing thoughts about suicide. "I'm so tired. 24 years of being treated like a crazy person. I want to go to heaven SO bad. Have for years. Can't manage any more. Badly wish could die without it ruining my kids lives," worried fans read last week. But now O'Connor is reassuring her public... sort of. In an open letter to fans on her blog, which also appeared in the Independent, she says her life isn't in danger just now. O'Connor says she believes "suicide is a sin," but also thinks that expressing suicidal thoughts can be healthy. "People who express suicidal feelings are least likely to act on them," she writes. She asks for email suggestions for "support services" in Ireland, other than "nut houses and psychiatrists and drugs." O'Connor has long battled bipolar disorder, and the media has recently piled on her for packing on some pounds. The downer talk comes amid O'Connor's new search for a boyfriend, and she's been poring over eligible bachelor applications being sent to her, notes Rolling Stone.

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Summarize: When Australian actor Damon Gameau found out he was going to be a father, he put his body on the line to find out the effects sugar can have on a person's health and well-being. The question of what he was going to feed his little girl in her school lunch box prompted him to embark on a radical diet to measure the impact sugars in so-called 'healthy foods' can have on the body. The Underbelly star only ate foods equivalent to 40 teaspoons of sugar a day - the average amount most Australians eat each day - for two months. He stuck to low fat foods and drinks perceived to be healthy, including muesli bars, low-fat yoghurt, cereal and juices. Damon Gameau embarked on a radical diet to measure the impact sugars in so-called 'healthy foods' can have on the body when his girlfriend Zoe Tuckwell-Smith was pregnant with their daughter Velvet. But the outcome, captured in That Sugar Film, shocked Gameau, his then pregnant Winners & Losers girlfriend Zoe Tuckwell-Smith and the number of medical professionals involved in the experiment. The 38-year-old stacked on 8.5kg in 60 days and 10cm of visceral fat around his stomach. 'Within a month I was on the verge of being diabetic… I was very close to my liver getting cirrhosis. I was on the verge of obesity for my size. I hit that risk point,' he told Daily Mail Australia. 'It was a complete shock, I had no knowledge of nutrition. I didn't think I'd have these results.' The 38-year-old actor, who had 40 teaspoons of sugar a day for 60 days, stacked on 8.5kg in 60 days and 10cm of visceral fat around his stomach. He stuck to low fat foods and drinks perceived to be healthy, including muesli bars, low-fat yoghurt, cereal and juices and documented the results in his new film That Sugar Film. Within a month, the Underbelly actor says he was on the verge of being diabetic and obese and was very close to getting liver cirrhosis. He was shocked to find drinks like ice tea, vitamin waters, smoothies and sports drinks often had as much sugar as a can of coke. Low fat mayonnaise, BBQ sauce and baked beans were also a major shock. When Gameau started dating Tuckwell-Smith, 32, in 2008, he gave up alcohol, cigarettes and sugar in a bid to impress her. So when he re-introduced refined sugar after three years, Gameau said he immediately noticed the impact it had on his mental and physical well-being - he was moody, lethargic and temperamental. But after the 60 days were up and he went back to his whole food diet, Gameau said 90 percent of the weight fell right off just from cutting out the refined sugar. Gameau said he noticed the impact sugar had on his mental and physical well-being - he was moody, lethargic and temperamental - while girlfriend Tuckwell-Smith was pregnant. He was shocked to find drinks like ice tea, vitamin waters, smoothies and sports drinks often had as much sugar as a can of coke. Sugar high foods like low fat mayonnaise, BBQ sauce and baked beans were also a major shock. When Gameau started dating Tuckwell-Smith, 32, in 2008, he gave up alcohol, cigarettes and sugar in a bid to impress her. He said he immediately felt the effects of the refined sugar. Gameau and Tuckwell-Smith, who welcomed their baby girl Velvet midway through the sugar experiment, now regularly draw on the experience when the family prepares meals. The couple rarely feed Velvet products with refined sugar and hope to move her away from sweet foods. 'We don’t want to give her an eating disorder,' he said. 'Zoe and I get the sugar we need from fruits… I think from my experience the palate does adjust. If you teach them the definition of sweet it doesn’t need to come from a Kit Kat. 'I hope with our daughter the sweetness (cravings) can come from fruit. 'We have a strong focus on being clean and leading a healthy life. She could learn that by copying us rather than us telling her.' Gameau and Tuckwell-Smith, who welcomed their baby girl Velvet midway through the sugar experiment, now regularly draw on the experience when the family prepares meals. When it comes time for Gameau and Tuckwell-Smith to pack 14-month-old Velvet's lunch box, the pair are loading it with boiled eggs, carrot sticks, cheese and fruit. Gameau said he hopes his daughter Velvet will copy her parent's healthy lifestyle as they try to steer her away from foods with added sugar. In his film, Gameau made a point of consuming his 40 teaspoons of sugar in one sitting just from foods often found in a child's lunch box - museli bars, fruit juice and yoghurt were all on the menu. But when it comes time for Gameau and Tuckwell-Smith to pack 14-month-old Velvet's lunch box, the pair are adamant they'll steer clear of such foods. 'Boiled eggs, carrot sticks with hommos, cheese – there are so many options. It’s just about being creative and not believing the marketing ploys,' he said. That Sugar Film opens nationally on March 1. For details, visit their website

Summary: Damon Gameau, 38, embarked on a radical diet to measure the impact sugars in so-called 'healthy foods' can have on the body. Underbelly actor wanted to make sure he was feeding his now 14-month-old daughter Velvet the right foods. He only ate foods equivalent to 40 teaspoons of sugar a day for 60 days with the outcome captured in his documentary That Sugar Film. He stacked on 8.5kg and 10cm of visceral fat around his stomach. Gameau and his girlfriend actress Zoe Tuckwell-Smith are now trying to steer their daughter away from sweet foods with refined sugar.

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Summarize: Parts of a sickening video released by Islamic State militants that shows members of the terror group beheading 21 Coptic Christians have been faked, experts have claimed. The footage, which lasts five minutes, shows the Egyptian Christians dressed in jumpsuits being marched one by one along a lonely beach, each held by a fighter clad in black. The captives, their faces uncovered, are made to kneel before being forced to lie down. The masked jihadists then behead them simultaneously. While experts believe the men were killed by the terrorists, questions have been raised over whether some scenes - including one where the militants appear to be 7ft tall - have been manipulated. Scroll down for video. Doctored? The executioners appear to be seven-foot tall in this still taken from the sickening footage. Experts believe that the scene of ISIS terrorists marching 21 Coptic Christians to their death was faked. It is believed the actual murders were filmed in a different location and the sea was added at a later stage. There are also doubts as to whether the men were all murdered on the beach and whether they were all killed at the same time. It is thought sections of the footage might have been shot on a 'green screen' in a studio - a technique used in Hollywood blockbusters - and that the beach background was added at a later stage. Veryan Khan, of the Florida-based Terrorism Research and Analysis Consortium, told Fox News that there are several technical mistakes in the video that show it was manipulated. She said that in the shot of the terrorists marching their prisoners along the beach, the jihadis appear to be 7ft tall - towering as much as two feet above their victims. This observation was supported by Hollywood director Mary Lambert who described it as the shot with the'really tall Jihadists and the dwarf Christians.' Ms Khan added that the terrorist who speaks in the clip - dubbed 'Jihad Joseph' - appears much bigger than the sea in some shots, while his head looks out of proportion to his body. The sickening image of blood in the sea is believed to have been created using special effects. Ms Khan explained that this meant it was likely he had been filmed indoors and the sea scene, believed to be in northern Libya, had been placed behind him at a later stage. Meanwhile, Lambert said that the scene that apparently shows the sea turning red with the blood of the beheaded men 'was obviously special effects'. The experts claimed that only a few Jihadis would have been on the beach along with a 'less talented crew' than the ones responsible for some of the group's more high-production videos. Earlier this week, local media reported that militants from the Islamic State and Ansar Al-Sharia were understood to have rounded up dozens of farm workers in the wake of bombings by Cairo. It raises the chilling prospect of yet another mass execution in what is being seen as ISIS's bid to announce its presence in a new region where it is gaining influence. Initial reports said seven men had been seized, but that figure had risen to more than 35 by mid-afternoon yesterday, according to The Libya Herald. It came as Egypt blitzed ISIS training camps, weapons stockpiles and fighters in two waves of air strikes following the gruesome murder of captured Egyptian workers. Meanwhile, the Egyptian government called for the US-led coalition to also target ISIS in Libya

Summary: Isis released a video last week showing the murder of 21 Coptic Christians. The five-minute film showed the men being marched along a beach. The victims are then shown lined up and beheaded in sequence. Experts believe the Egyptian victims were not murdered at the beach. Instead, they were murdered elsewhere with the background added later. The shocking scene of the sea turning red is believed to be special effects. The scene where the sea turns red with blood is also thought to be faked.

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Summarize: Despite Sharing E Umbrella’s initial setback, the company’s founder still plans to roll out 30 million umbrellas across China by the end of the year In China the sharing economy seems to have met its match after a startup that rented umbrellas in 30-minute blocks lost nearly all of its stock in just three months. The company Sharing E Umbrella required a deposit of £2.20 per umbrella and charged about 6p for every half an hour of use, deducted from a minimum top-up of £1. Each GPS-enabled umbrella cost the company about £6.85. Uber for bikes: how 'dockless' cycles flooded China – and are heading overseas Read more In theory users would scan a code with their smartphone and receive a code to unlock a combination lock built into the handle. There was no penalty for an unreturned brolly, a factor that could explain the high incidence of missing umbrellas. Despite Sharing E Umbrella’s initial setback, the company’s founder still plans to roll out 30 million umbrellas across China by the end of the year, according to local media. “We were all baffled by the model of dockless bike sharing; it made users think anything on the street can be shared now,” said founder Zhao Shuping, according to The Paper, a state media website. “But umbrellas and bicycles are not the same: a bike you can park anywhere but an umbrella needs a stand.” Sharing E Umbrellas investors are sure to be disappointed – in May the company received £1.1m in funding. And it is not the only firm pushing the brolly-sharing revolution, with at least three other Chinese startups launching around the country. Chinese versions of Uber and AirBnb have exploded in popularity over the past few years, and the country pioneered dockless bike sharing schemes where people could unlock bikes with their smartphones and return them anywhere. A few startups have even started sharing mobile phone charging banks. As the first city to run Mobike, one of China’s top bike-sharing companies, Shanghai attracts tech startups looking to strike oil by dealing in shareable commodities, from electric cars and washing machines to batteries and basketballs. But none has been more controversial than shared umbrellas, which came to town in late May. About 100 such umbrellas, code-locked to curbside railings, first appeared in the Lujiazui financial district on May 23. Within a few days, though, they were nowhere to be seen. Some believe city management officials ordered their removal, since it’s illegal to tie the umbrellas to public infrastructure without the prior consent of the government. A representative from an unnamed company, though, said they had not received any official reprimand, nor had they retrieved the umbrellas voluntarily. “Our customers took them all and haven’t returned them yet,” he said. According to the company’s current promotion, customers can rent an umbrella free of charge as long as they pay 20 yuan ($3) as a refundable deposit. However, it seems most people have opted to keep the umbrellas rather than go out of their way to return them. Lujiazui’s experience strikes a chord with Shanghai Metro, the company that runs the city’s vast subway network, and which had deployed about 100,000 shared umbrellas years ago at its stations for passengers who find themselves caught in the rain. The service was canceled, however, since most umbrellas were never returned. The same went for large-scale city malls and hotels, as customers gallivanted off with their “complimentary” umbrellas. Currently, there are about 15 players in China’s shared umbrella industry. Some Shanghai-based startups, particularly OTO and Molisan, have exercised more caution in developing their rentable umbrella businesses. Unlike its peers in southern cities like Fuzhou and Shenzhen, OTO and Molisan have been around for four and three years, respectively. The latter company test-ran its business by deploying rental stations in malls and subways in Fuzhou in the east and Guangzhou in the south. Molisan’s chief concern today is somewhat more prosaic: Wet floors from drippy umbrellas pose a hazard to passersby. OTO, meanwhile, targets large outdoor landmarks in Beijing and Shanghai, though its success will largely depend on whether it can fulfill its promise not to disturb public order as it rolls out its products in areas where big crowds gather — for government scrutiny will be sure to follow. But the real challenge is how companies like OTO can turn a profit from a business that hinges upon a variable as fickle as weather. Rainfall is largely seasonal — most of the aforementioned cities are wettest in the summer — and regional, with the southern and northeastern parts of the country receiving the most rain. This constrains the business in terms of scope. In addition, residents of places prone to precipitation often carry umbrellas with them, which means the business must grow in a niche populated by the few who find themselves ill-prepared. A further issue is that rentable umbrellas no longer lie in the conventional camp of the shared economy, which is usually defined as the redistribution of otherwise idle resources at bargain prices. This is the philosophy driving ride-hailing apps like Uber and Didi Chuxing, as well as room rental site Airbnb and its Chinese counterpart, Tujia. The fact that shared umbrella companies are betting on such unpredictable situations means their clientele is unlikely to grow substantially. - Lu Hongyong, editor In the wake of China’s bike-sharing craze, however, the commodities being shared are no longer privately owned secondhand items, but rentable corporate items. No matter where you borrow them from, shared umbrellas require a deposit — between 19 and 99 yuan per registered user. That’s pretty much the price of an average umbrella, leading some to suspect that manufacturers have merely thought of a more innovative way to sell their product. Esan, a Shenzhen-based operator, charges the highest deposit, on the grounds that its umbrellas are more than a just shield from the sun and rain — the company markets them as canes for the elderly or even music players, given a plan for each product to come with an embedded music player. A more feasible extension of the business is for Esan to turn its umbrellas into mobile advertisements. The company has already made inroads in that direction: It is reportedly negotiating the placement of 1.75 million public umbrellas, each of which will bring 10 yuan in ad revenue. Molisan, too, has courted advertisers for its umbrellas in Guangzhou. Most umbrellas last no more than two years, and with such a short life span, there are hopes that a combination of rental and advertising income may provide the most sustainable business model for such companies. The venture capitalists who sunk huge amounts of money into bike-rental companies have been lukewarm at best toward suppliers of rentable umbrellas. So far, only three players — Chunsun, Esan, and JJsan — have received angel funding in excess of 5 million yuan. In contrast,, investors that missed out on the shared bike bonanza have poured their enthusiasm, and their yuan, into shared batteries for charging cellphones and other devices — so much so that earlier this year, the new industry raised 1.2 billion yuan in a little more than a month. Like shared batteries, which have limited practical use, rentable umbrellas also provide solutions to uncommon scenarios, many of which hinge on whether people own cars, whether they checked the weather report, or whether they left their own umbrella at the restaurant where they had lunch. The fact that shared umbrella companies are betting on such unpredictable situations means their clientele is unlikely to grow to truly substantial numbers. It’s fair to say the chilly investor sentiment toward rental umbrellas marks an apex in the growth of the shared economy. Investors are no longer blind to the now-familiar model’s potential pitfalls; instead, they have become more circumspect when it comes to parting with their cash. Without a truly viable business model — in this case, a tremendously difficult prospect — shared umbrella companies will do little more than burn their investors’ funds before going belly-up themselves. Editor: Matthew Walsh. (Header image: Several shared umbrellas hang on a fence in Shenzhen, Guangdong province, May 19, 2017. Zou Bixiong/IC) The sharing economy has made it easier for communities to peer exchange anything from a spare bedroom in your house to car rides and bicycles. But the model doesn’t work for everything, as one umbrella-sharing startup learned the hard way. Just under three months after launch, China-based E Umbrella has reportedly lost almost all of its 300,000 umbrellas available to rent across 11 Chinese cities. "Umbrellas are different from bicycles" E Umbrella had an investment of 10 million yuan (approximately $1.47 million USD) when it launched in April, and charged customers 19 yuan ($2.90 USD) per umbrella deposit and an additional half yuan ($0.07 USD) per 30 minutes. Stands were typically scattered across the cities near train and bus stations (some of which were outside... in the rain?) and users receive a code to unlock the umbrella after paying for it on an app. However, the startup apparently didn’t provide enough information on how customers can return the umbrellas when they’re done. "Umbrellas are different from bicycles," E Umbrella founder Zhao Shuping told South China Morning Post. "Bikes can be parked anywhere, but with an umbrella you need railings or a fence to hang it on." The founder discloses that an umbrella costs the company 60 yuan ($8.82 USD) each to replace, but despite the losses he plans to add 30 million more available across China by the end of the year. E Umbrella (and other startups just like it) has a questionable business model to start, as unlike bikes, an umbrella-sharing startup would typically see higher demands and steady profits during the rainy season, reports Shanghaiist. Umbrellas are also an inexpensive investment. E Umbrella doesn’t appear to charge users an unreturned umbrella fee so most users just end up keeping their rentals. The idea isn’t new either; over in the US, a startup named BrellaBox pitched a similar concept on Shark Tank and was called “the worst idea ever heard” on the show by one of the panelists.

Summary: If you have a habit of regularly misplacing your umbrella, Sharing E Umbrella feels your pain. The startup intends to do what its name suggests: provide shareable umbrellas-like a rain-themed version of Citi Bike-in China. Except not even three months after launch, it has a big problem in the 11 cities it has a presence in: Most of the 300,000 umbrellas it started out with have now been lost. The Guardian suggests the fault lies with the company's founders: While a would-be umbrella user must pay a roughly $2.80 deposit and about $0.07 per half-hour of use, the paper reports no additional penalty is levied for failing to return an umbrella. The Verge suggests another problem, that "the startup apparently didn't provide enough information on how customers can return the umbrellas when they're done." It gets even more confusing from there, with the South China Morning Post noting the original report on Sharing E Umbrella's woes didn't specify the return policy but quoted CEO Zhao Shuping as saying, "Bikes can be parked anywhere, but with an umbrella you need railings or a fence to hang it on"-and so taking the umbrella home for safekeeping is likely the "best" route. But Zhao sounds undaunted, saying his company intends to spread 30 million umbrellas throughout the country by the end of the year. Sixth Tone notes there are roughly 15 Chinese umbrella startups, and the deposits they charge get as high as $14.50-raising the possibility this is just a clever way to sell, not rent, umbrellas. As for whether we'll see this stateside, the Verge points out the idea was called the "worst... ever heard" when it was pitched on Shark Tank.

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Summarize: RELATED APPLICATIONS [0001] Not Applicable FEDERALLY SPONSORED RESEARCH [0002] Not Applicable SEQUENCE LISTING [0003] Not Applicable BACKGROUND OF THE INVENTION [0004] The invention relates to support of the head or occipital region of patients with possible spinal trauma and in particular to a support system particularly suitable to the needs of emergency responders. [0005] Emergency response procedures for patients with suspected-spinal trauma usually involve immobilizing the patient on to a carrier device, such as a backboard, gurney, rescue basket, stretcher, litter and the like, in a manner that is intended to maintain neutral alignment of the head and spine. Ideally, no further twisting or displacing of the head, neck and spine are done until the patient is delivered to a care facility. Thus the goal of spinal trauma procedure is to restrain the patient so that alignment is preserved and no pressure is put on the spinal/head region during initial restraint and transport of the patient. Unfortunately, in practice it is difficult to accomplish this, in large part due to the configuration of the restraint and support tools currently available to emergency responders. [0006] Often a cervical collar is put on the patient to maintain head/neck/spine alignment. After a cervical collar is in place, most procedures call for the patient's head and torso to be restrained on a carrier. Typically, some type of head/occipital support is placed under the patient and the patient is strapped or taped in place, usually with at least one restraint over the head (or cervical collar). Due to the limitations involved with current emergency response tools, this process has several serious drawbacks. [0007] Current emergency response head supports are designed to restrain the head from moving laterally, vertically, inline and from rotating. Currently available supports are typically some sort of block or fixed wedge designed to secure the head firmly when strapped down. They offer little or no height adjustment. This is a problem because when the head is strapped down, even with a cervical collar, strain is applied because bodies of different sizes and/or spine/neck shape will experience different amounts of loading and dynamic forces during packaging and transport unless they happen to fit the support structure exactly. Basically most patients will either experience undesirable upward or downward leverage of their head relative to their spine when the straps are tightened (Secured/Attached/fastened) due to the lack of adjustability of the height of the occipital/head support needed to keep the head in alignment as well as unwanted forces that can produce distraction on the cervical spinal region due to the movement of the torso while the head has been completely secured to the carrying device. Moreover many of the currently available supports are bulky, with dimensions on the order of several inches in thickness, height and length. Space in emergency response vehicles is at a premium, so storage of such large devices is problematic, and often limits the number of devices that a rescue team can carry, therefore minimizing the number of patients that can be provided with timely treatment. And in fact in many cases multiple response teams may have to respond to the scene of an accident for this reason, when it would be so much better to provide adequate timely treatment as soon as possible. [0008] In addition, the direct strapping of the head is extremely stiff longitudinally. This is a drawback when the patient is carried up and down ramps or stairs, or when the emergency vehicle experiences sudden accelerations or decelerations or other vehicle motions. Both loading due to gravity or movement of the body can cause further strain, distraction and potentially further damage at the site of the injury if the head is too rigidly attached to the carrier. Also, some of the larger, bulkier head restraints use straps for securing the patient, but these units are expensive and not disposable requiring decontamination after each and every use. Other more compact, disposable units typically require adhesive tape as a restraint, which can fail to adhere to wet patient carrying equipment, stick to gloves as well as pick up dirt or debris, often further complicating an already difficult situation for emergency responders. Thus it is the object of this invention to provide a support system that is space efficient when stowed, provides for a large degree of height adjustment, secures the patient for vertical and lateral movement only in a manner that reduces the patient's unwanted manipulation due to loading forces created during transport, is disposable, inexpensive and doesn't involve require the use of adhesive tape. BRIEF SUMMARY OF THE INVENTION [0009] The invention includes a head/occipital support adapted to store as a relatively thin, flat piece consisting of sections which may be stacked during use to provide variable height depending on number of sections stacked. The sections may be stacked in a variety of ways, including breaking the support into sections along perforations, folding the section along foldlines, or removing the sections attached, such as by Velcro® or adhesive, to a backing. Preferably the sections include adhesive, Velcro® or the like to facilitate attachment to themselves and to the carrier [0010] The invention also includes at least one adjustable securing strap, the strap(s) adapted to connect at least two opposing regions of the carrier to attachment points on each side of the patient's head. The strap(s) are adjustable. In one version, the carrier end may be a Velcro® closable loop intended to attach to the openings commonly found on the sides of backboards or litters, and adjustable by the closure length of the loop. The attachment to the cervical collar may also be by Velcro®, or by buckles including adjustable strap retainers [0011] In preferred embodiments, the sections are substantially rectangular or square, with possible cut-outs for storage and handling. Preferable dimensions range from sections being on the order of a few inches in length and width, and around a half inch thick. Three to four sections per support are preferred and the total length should be less than typical carrier width, about 18 inches. The material should be moisture resistant, stiff enough to provide support but still flexible, such as common closed cell foams. The perforated version is also preferred, with adhesive strips covered by removable film disposed on the top of over the adhesive and on the bottom of the sections. Such a support is sufficiently stowable that it can be included with an emergency type cervical collar at little or no space penalty [0012] For patients where the planar, sectionable support does not provide enough height adjustment, the invention includes a novel wedge support as an accessory, including two wedge shaped pieces with corresponding, fingers and slots cut into the angled wedge section. These wedges are disposed such that the fingers/slots are interlaced when the pieces are placed together. They may be fully interlaced for storage and the two pieces then slid apart during use to provide a height adjustable v-shaped support. Relatively few of these wedge accessories are need to be carried by the response vehicle as the planar, sectionable support will accommodate most patients. BRIEF DESCRIPTION OF THE DRAWINGS [0013] The invention will be better understood by referring to the following figures. [0014] FIG. 1 illustrates the basic features of the novel support. [0015] FIG. 2 illustrates the stacking of sections of the support. [0016] FIG. 3 illustrates one embodiment for fixing the support to the carrier. [0017] FIG. 4 illustrates the novel system including straps adapted to attach to a cervical collar, and one embodiment for strap attachment to the carrier. [0018] FIG. 5 illustrates one embodiment for attaching the straps to the collar. [0019] FIG. 6 illustrates an important advantage of the novel system. [0020] FIG. 7 illustrates an optional wedge support for cases where more height adjustment is required. [0021] FIG. 8 illustrate the use of the novel wedge support. DETAILED DESCRIPTION OF THE INVENTION [0022] The invention is a novel sectional head support for use in patient head restraint on a patient carrier, such as a backboard, gurney, rescue basket, stretcher, litter and the like, for emergency response applications. The support is highly stowable while still providing, during deployment, a wide degree of head to carrier height adjustment to minimize loading and unwanted movement on the head-spine axis for a range of patient size and body types. The support may be made out of inexpensive material such as closed cell foam, so it is disposable and inexpensive. The novel support is used with securing straps, rather than directly taping the head as in current disposable systems. In the novel system, the straps attach the patient carrier to attachment points on each side of the head. In the preferred embodiment, the attachment points are on each side of the cervical collar placed on the patient. Thus the invention preferably requires that the cervical collar used with the invention include means for strap attachment. However, other accessories which provide attachment points on each side of the head are conceivable and fall within the broad interpretation of the invention. A suitable collar is the X-Collar, produced by Emegear, the assignee of the current invention. [0023] The stowable support portion of the invention is illustrated in FIG. 1. The support 1 is preferably a substantially planar structure, consisting of multiple sections. In the exemplary case shown in the figure, three sections, 1 a, 1 b, and 1 c are shown, but within reason, other numbers such as 4 or more may be used. Two sections are possible but would be limited in adjustability. The sections are separable and stackable. A variety of separation approaches are possible. One preferred approach is to perforate support 1 along separation boundaries 2 allowing for sections to be broken off as needed. The support is placed between head 3, with cervical collar 4 in place, and patient carrier 5. As shown in FIG. 2, section 1 a has been broken off and stacked on support 1. Thus depending on how many sections are broken off and stacked, the gap between head 3 and carrier 5 may be filled for a variety of patient size and body shapes with out adding strain to the spine/neck and maintaining proper cervical spine alignment. [0024] In stowable form, the support is a flat piece, easily stacked or packaged with a cervical collar, for very convenient and space efficient stowage in an emergency vehicle. Yet despite the space efficiency, the support still has far more patient height adjustability then any currently available emergency response head restraint support. Obviously many variations on the shape and section arrangement are possible. For instance instead of perforated section boundaries, fold lines may be employed to stack the sections by folding. Another possible arrangement is the sections may be precut. In preferred embodiments the support is basically rectangular, with possible handling cutouts. Although specific dimensions obviously may vary, the sections should be at least on the order of a few inches on a side to provide adequate area for occipital support. The overall length of the support before sectioning should fit well within the width of carriers, so should be preferably be no more then 12-18 inches. Thickness is a trade-off between stowability and height adjustment range. The inventors have found that a four section piece, a half inch thick provides a one half to two inch range, with half inch increments. Thus is adequate for most patient body types. The material should be inexpensive, moisture resistant, firm enough to provide support while flexible enough for comfort and to allow for folding or breaking. Many materials fit these requirements such as common closed cell foams. Also as shown in FIG. 3, to facilitate placement and to hold the support in place, adhesive with removable cover 10 may be placed on the bottom of the support 1 both to fix to the carrier and between stacked sections. [0025] The invention also includes novel provision for securing the head using head attachment means, preferably attachment points on a cervical collar, to facilitate a superior strapping arrangement compared to current emergency support systems. Referring to FIG. 4, two securing straps 6, one on each side of the head 3, supported by adjustable support 1, connect the carrier to the cervical collar 4 as shown. This bi-lateral arrangement both stabilizes the collar laterally as well as vertically onto the carrier. Straps need to be adjustable to provide some tension and readjustment as needed. A variety of attachment/adjustment means are possible. Since many patient carriers 5, such as backboards and litters have openings along the sides, a preferred attachment 7 is a Velcro® closable loop, which may be looped through the opening, and the slack taken up by where the loop is closed. Other fastening arrangements within the scope of the invention will suggest themselves to skilled practitioners. As shown in FIG. 5, the straps 6 also attach on each side of collar 4. A preferred simple solution is shown at 8 where a Velcro® pad on strap 6 attaches to an existing Velcro® pad on collar 4. Locking buckles are also possible, and these could include adjustable strap retainers. Although two straps are shown in the Figure, one skilled in the art will readily appreciate that the use of two straps is not required. The goal is to connect a strap attachment point on one side of a patient's head with a strap attachment point on the same side of the carrier and to do the same thing on the other side of the head/carrier. This could be done with one strap, for example, that had provision for attachment to the head attachment point with a service loop in between that went over or around the head. As long as the slack could be taken up at the carrier attachment points, such an arrangement would be equivalent to the two strap example in the figure. Alternatively more than one strap on each side could also be employed. Thus the two straps shown in the figures are by way of example only. [0026] A critical advantage of the novel support system is shown in FIG. 6. Securing Straps 6 are connected between carrier 5 and collar 4 as a relatively long arm with endpoints capable of a degree of pivoting. This arrangement has the effect that although it stabilizes the head laterally and vertically, it is not particularly stiff along the body axis. In Emergency situations, once the patient is secured to the carrier, during movement up and down ramps or stairs, in and out of the vehicle, and during vehicle motion, significant loads can be placed along the body axis. The strap attachment scheme clearly shown in the figure allows for some compliance along this axis, resulting in avoiding further trauma to the spine/neck when exposed to loads along the body axis than compared to existing systems. [0027] As clearly shown, the novel support and strap system is inexpensive and disposable, space efficient during stowage, has a great degree of height adjustability, and provides a superior, less traumatic securing arrangement. In the preferred approach the support/straps are packaged with the collar, ensuring all of the needed tools are available to the responder in a convenient manner. However, even with the wide range of adjustability provided, some patients will fall outside the range covered by the support. Thus the invention optionally includes another item, which may be carried in smaller numbers for the out of range cases. This item is shown in FIG. 7. It is basically a wedge support 9 consisting of two pieces with slots and fingers cut into an angled surface as shown. The slots and fingers are sized to allow for interlacing as shown. Thus in storage, the pieces are pushed all the way together forming a compact block. During use they may be pulled apart to provide the desired height of support. The V-shape is advantageous for head support as well. Again adhesive with removable covering or Velcro® may be used to facilitate placement of the support. Such a wedge has several advantages over other wedge block configurations. For a suspended object, such as a head held at the proper alignment, the two interlaced wedges may be slid into place to the desired height with no forces applied to the suspended object of any kind, yet still providing lateral and vertical support once placed. Such a wedge may have application in many areas beyond head support for emergency response. [0028] Use of the wedge is illustrated in FIG. 8. The planar support 1 is shown with the wedge 9 suitably placed to support head 3 in alignment with, the spine for a patient with a large vertical alignment gap between the head and spine. [0029] It will be apparent to the skilled artisan that there are numerous changes that may be made in embodiments described herein without departing from the spirit and scope of the invention. Other features not mentioned in the specification, but known to one skilled in the art may be integrated as well without departing from the spirit and scope of the present invention. There are, for example, a wide array of materials, apparatuses, and methods which may be interchangeably used and there are many changes that may be made in dimensions and so forth to accommodate different needs which may be used, all within the scope of the invention. The methods, system, and apparatuses of the present invention should therefore be afforded the broadest possible scope under examination. As such, the invention taught herein by specific examples is limited only by the scope of the claims that follow.

Summary: A head/occipital support system for emergency response use, intended to be employed in conjunction with a cervical collar. The system achieves very favorable stowabilty while still providing a wide range of height adjustability to accommodate a range of patients with decreased loading on the head/spine alignment. The system also includes at least one strap(s) to secure the patient by bi-laterally connecting each side of the cervical collar to the patient carrier. An accessory wedge is utilizes for patients requiring a wider range of head support.

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Summarize: "Oh, more or less." I fancy my smile was pale. "Not absolutely. We shouldn't like that!" I went on. "No--I suppose we shouldn't. Of course we have the others." "We have the others--we have indeed the others," I concurred. "Yet even though we have them," he returned, still with his hands in his pockets and planted there in front of me, "they don't much count, do they?" I made the best of it, but I felt wan. "It depends on what you call'much'!" "Yes"--with all accommodation--"everything depends!" On this, however, he faced to the window again and presently reached it with his vague, restless, cogitating step. He remained there awhile, with his forehead against the glass, in contemplation of the stupid shrubs I knew and the dull things of November. I had always my hypocrisy of "work," behind which, now, I gained the sofa. Steadying myself with it there as I had repeatedly done at those moments of torment that I have described as the moments of my knowing the children to be given to something from which I was barred, I sufficiently obeyed my habit of being prepared for the worst. But an extraordinary impression dropped on me as I extracted a meaning from the boy's embarrassed back--none other than the impression that I was not barred now. This inference grew in a few minutes to sharp intensity and seemed bound up with the direct perception that it was positively HE who was. The frames and squares of the great window were a kind of image, for him, of a kind of failure. I felt that I saw him, at any rate, shut in or shut out. He was admirable, but not comfortable: I took it in with a throb of hope. Wasn't he looking, through the haunted pane, for something he couldn't see?--and wasn't it the first time in the whole business that he had known such a lapse? The first, the very first: I found it a splendid portent. It made him anxious, though he watched himself; he had been anxious all day and, even while in his usual sweet little manner he sat at table, had needed all his small strange genius to give it a gloss. When he at last turned round to meet me, it was almost as if this genius had succumbed. "Well, I think I'm glad Bly agrees with ME!" "You would certainly seem to have seen, these twenty-four hours, a good deal more of it than for some time before. I hope," I went on bravely, "that you've been enjoying yourself." "Oh, yes, I've been ever so far; all round about--miles and miles away. I've never been so free." He had really a manner of his own, and I could only try to keep up with him. "Well, do you like it?" He stood there smiling; then at last he put into two words--"Do YOU?"--more discrimination than I had ever heard two words contain. Before I had time to deal with that, however, he continued as if with the sense that this was an impertinence to be softened. "Nothing could be more charming than the way you take it, for of course if we're alone together now it's you that are alone most. But I hope," he threw in, "you don't particularly mind!" "Having to do with you?" I asked. "My dear child, how can I help minding? Though I've renounced all claim to your company--you're so beyond me--I at least greatly enjoy it. What else should I stay on for?" He looked at me more directly, and the expression of his face, graver now, struck me as the most beautiful I had ever found in it. "You stay on just for THAT?" "Certainly. I stay on as your friend and from the tremendous interest I take in you till something can be done for you that may be more worth your while. That needn't surprise you." My voice trembled so that I felt it impossible to suppress the shake. "Don't you remember how I told you, when I came and sat on your bed the night of the storm, that there was nothing in the world I wouldn't do for you?" "Yes, yes!" He, on his side, more and more visibly nervous, had a tone to master; but he was so much more successful than I that, laughing out through his gravity, he could pretend we were pleasantly jesting. "Only that, I think, was to get me to do something for YOU!" "It was partly to get you to do something," I conceded. "But, you know, you didn't do it." "Oh, yes," he said with the brightest superficial eagerness, "you wanted me to tell you something." "That's it. Out, straight out. What you have on your mind, you know." "Ah, then, is THAT what you've stayed over for?" He spoke with a gaiety through which I could still catch the finest little quiver of resentful passion; but I can't begin to express the effect upon me of an implication of surrender even so faint. It was as if what I had yearned for had come at last only to astonish me. "Well, yes--I may as well make a clean breast of it, it was precisely for that." He waited so long that I supposed it for the purpose of repudiating the assumption on which my action had been founded; but what he finally said was: "Do you mean now--here?" "There couldn't be a better place or time." He looked round him uneasily, and I had the rare--oh, the queer!--impression of the very first symptom I had seen in him of the approach of immediate fear. It was as if he were suddenly afraid of me--which struck me indeed as perhaps the best thing to make him. Yet in the very pang of the effort I felt it vain to try sternness, and I heard myself the next instant so gentle as to be almost grotesque. "You want so to go out again?" "Awfully!" He smiled at me heroically, and the touching little bravery of it was enhanced by his actually flushing with pain. He had picked up his hat, which he had brought in, and stood twirling it in a way that gave me, even as I was just nearly reaching port, a perverse horror of what I was doing. To do it in ANY way was an act of violence, for what did it consist of but the obtrusion of the idea of grossness and guilt on a small helpless creature who had been for me a revelation of the possibilities of beautiful intercourse? Wasn't it base to create for a being so exquisite a mere alien awkwardness? I suppose I now read into our situation a clearness it couldn't have had at the time, for I seem to see our poor eyes already lighted with some spark of a prevision of the anguish that was to come. So we circled about, with terrors and scruples, like fighters not daring to close. But it was for each other we feared! That kept us a little longer suspended and unbruised. "I'll tell you everything," Miles said--"I mean I'll tell you anything you like. You'll stay on with me, and we shall both be all right, and I WILL tell you--I WILL. But not now." "Why not now?" My insistence turned him from me and kept him once more at his window in a silence during which, between us, you might have heard a pin drop. Then he was before me again with the air of a person for whom, outside, someone who had frankly to be reckoned with was waiting. "I have to see Luke." I had not yet reduced him to quite so vulgar a lie, and I felt proportionately ashamed. But, horrible as it was, his lies made up my truth. I achieved thoughtfully a few loops of my knitting. "Well, then, go to Luke, and I'll wait for what you promise. Only, in return for that, satisfy, before you leave me, one very much smaller request." He looked as if he felt he had succeeded enough to be able still a little to bargain. "Very much smaller--?" "Yes, a mere fraction of the whole. Tell me"--oh, my work preoccupied me, and I was offhand!--"if, yesterday afternoon, from the table in the hall, you took, you know, my letter."

Summary: The Governess and Miles, alone at last, speak cagily about Miles's explorations of the grounds over the past day or so, when he's been left to his own devices. Finally, it's time to get everything out in the open. The Governess insists that she's only there to help Miles and to be with him - and he sees that she wants him to tell her everything. As he prepares to confess, she sees for the first time that he's afraid - of her. Miles nervously tells the Governess that he'll tell her anything, but not just now; he says he has to go see Luke. Before she lets him go, the Governess just asks him one thing: did he take the letter she left in the hall yesterday?

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Summarize: BACKGROUND OF THE INVENTION This invention relates to a cat litter box with a mechanism for screening and draining reusable cat litter. Conventional cat litter boxes comprise an open container into which one or two inches of granular absorbent material is placed. Periodically the litter material is removed, discarded and replaced with new material. Optionally, special tools are provided to scoop and remove fecal matter to extend the life of the litter material. However, after being impregnated with cat urine, the absorbent litter material produces odor that requires the litter to be removed because it has become repugnant to either the cat or the cat's owner, or both. Certain conveniences have been devised for assisting in the use and operation of a cat litter box. For example, in the patent of Ellis, U.S. Pat. No. 3,831,557, entitled CAT LITTER BOX, a molded container is disclosed that includes an impervious liner. Periodically, the liner and contained litter may be removed and discarded. The discarded liner is replaced with a new liner and new litter material is placed over the liner in the box. In the patents of Rigney, U.S. Pat. No. 3,809,013, entitled DISPOSAL INSERT FOR LITTER BOX, and Harrington, U.S. Pat. No. 4,312,295, entitled CAT BOX LITTER SCREENING DEVICE, a series of stacked liners with holes or apertures are provided to allow litter placed on top of the liners in a cat litter box to be screened such that bulk material is removed and the life of the remaining litter is extended. While certain odor absorbing or scent masking chemicals may be applied to the cat litter to further extend the life of the litter material, the material must usually be discarded well before it becomes saturated with urine. The requirement to periodically change and remove the cat litter is a necessary nuisance in the ownership of a cat. The cat owner must be continually conscious of the existing supply of absorbent cat litter and must always be attentive to the current state of the litter box. The mechanical cat litter box of this invention is designed to minimize the task of preparing a cat litter box and maintaining it in a condition that is usable and inoffensive to both cat and owner. SUMMARY OF THE INVENTION The mechanical cat litter box of this invention comprises a container for holding a nonabsorbent litter material that is periodically screened and drained by a mechanism actuated by simple push-pull hand bars. Instead of using a conventional absorbent litter material, the mechanical litter box of this invention operates by use of an impervious granular material that does not absorb the urine and moisture but allows such to drain through the material to the bottom of a container basin for collection and periodic removal. The litter box is equipped with a mechanism for straining the fecal matter from the container and draining the liquid matter from the basin. The mechanical litter box of this invention includes a frame and a base pad. Positioned on the base pad and connected to the frame is a litter container having an inner and outer compartment. The litter is contained in the inner compartment. The outer compartment comprises a basin for collection of liquid that passes through holes in the bottom of the inner compartment. The litter container has side walls and a top with a front open portion and a rearward covered portion. Located in the forward portion of the container on the bottom of the inner compartment is a screen. The screen is sized and constructed to pass special granular litter material while retaining larger fecal material when the screen is drawn through the liquid impervious cat litter. In a two stage actuation process initiated by first and second plunger bars, the screen is lifted through the container and the container and displaced screen are raised and tipped to deposit the liquids and solids into a collection box. Once the liquids are drained and the solids deposited in the box, the actuating mechanism can be dropping the screen to the bottom of the container and redistributing the displaced impervious litter material over the screen as the litter container first shifts to a downward tilted position before dropping back onto the base pad. The collection box can be emptied in any sanitary facility such as a toilet. When the self draining litter box of this invention is used, a non absorbent litter material can be used over and over. The litter material may comprise the colorful enamel coated pebble material sold in tropical fish stores, may comprise a coated expanded polymer that is light in weight, or any similar liquid-impervious granular material. During use of the mechanical litter box unit, the litter material can be repeatedly sprayed with a deodorized flush liquid from a conventional squeeze bottle. After draining through the impervious litter material the deodorizing liquid mixes with the collected urine in the under compartment or basin and is discharged with the urine during the mechanical flushing operation. The actuating mechanism detailed in the detailed description of the preferred embodiment mechanically accomplishes the various motions required to screen the litter and dispose of the solid and liquid effluent. Other equivalent mechanisms may of course be utilized to achieve the objects of this invention. The principal object of this invention as detailed hereafter is to provide a self-draining litter box having liquid-impervious litter that can be periodically screened of solid matter and drained of liquid matter by an easily operable actuating mechanism. The actuating mechanism may be electrical, hydraulic or mechanical. The description herein is directed to a mechanical means as a preferred embodiment. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a side elevational view of the mechanical litter box unit. FIG. 2 is a fragmented partial cross sectional view taken along the lines 2--2 in FIG. 1. FIG. 3 is a front elevational view partially and cross section of the litter box unit of FIG. 1. FIG. 4 is a cross sectional side view taken along the lines 4--4 in FIG. 3. FIG. 5 is a cross sectional detailed view taken along lines 5--5 in FIG. 4. FIG. 6 is a cross sectional detailed view taken on the lines 6--6 in FIG. 1. FIG. 7 is a cross sectional top view taken on the lines 7--7 in FIG. 1. FIG. 8 is a schematic view of the box unit in a first sequence of operation. FIG. 9 is the box unit in a second sequence of operation. FIG. 10 is the box unit in a third sequence of operation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIGS. 1-7 of the drawings, the mechanical litter box unit, designated generally by the reference numeral 10 is shown in various views detailing its construction. The litter unit as shown in FIG. 1 is constructed with a pair of side frames 12 interconnected by thin back wall 14 and a base pad 16 which together form a housing 18 for a litter container 20 and an actuating mechanism 22. The actuating mechanism 22 includes several sub mechanisms for accomplishing the operations described hereinafter. In addition to the litter container 20 is a refuse collection box 24 mounted against the back wall 14 of the housing 18. The refuse collection box 24 has a handle 26 for removing the box through a contoured aperture 28 in one of the side frames 12 for withdrawing the refuse collection box 24 for periodic disposal of collected waste materials. The litter container 20 as shown most clearly in FIG. 4 is constructed with an upper or inner compartment 30 and a lower or outer compartment 32 which forms a basin for collection of liquids that pass through a series of holes 34 in the bottom of the inner compartment 30. The inner compartment 30 contains a deep layer of non absorbent granular material 36 which is sized slightly larger then the holes 34 to prevent their passage from the inner compartment to the outer compartment. The litter container 20 is constructed with an open portion 38 and a covered portion 40. Seated in the open portion 38 is a screen strainer 42 which has an open end wall 44, side walls 46, as shown in FIGS. 3 and 7, and a bottom screen 48 formed by a series of elongated rods 50 connected to a cross rod 52 at the open end of the strainer 42. The rods 50 are spaced sufficiently close together to collect solid fecal matter while allowing the granular material 36 to pass between adjacent rods 50. In its normal position for use, the litter container 20 appears to a cat to be substantially similar to the conventional litter box. The open portion 38 is sufficiently large to allow the cat to comfortably enter the litter container for deposit or of liquid or solid wastes. Operation of the actuating mechanism 22 is a two step operation. In a first step depression of a plunger 54 by manual pushing on handle 56 rotates pinion gear 58 which engages a tooth rack 60 on the plunger shaft 62 as shown in FIGS. 3 and 4. Pinion gear 58 is connected to spindle 64 on which is wound a wire 66. Wire 66 (displaced for clarity in FIG. 4)branches at ring 68 to wires 70 which pass through guide 72 and over guides 74 to connect to the central portion of lever arm 78. Lever arm 78 has a slide connection to pivot 80 at one end and a pivotal connection to link 82 which is pivotally connected to the top of side wall 46 at pivot 84. When the plunger 54 is depressed, the wire 66 wraps on the spindle 64 and raises lever arm 78, thereby raising the screen strainer 42 until a detent pin 86 engages a dimple 88, shown in phantom in FIG. 4 in the alternate position of the strainer 42. The detent pin 86 maintains the strainer in a position in which the end of the bottom screen 48 is juxtaposed to the top cover 90 over the covered portion of the container 20. The detent pin 86 is shown in cross section in FIG. 5 being positioned in a housing 92 on the container 20, with the detent pin being biased by a small compression spring 94. Once the screen strainer 42 has been raised to the position shown in phantom in FIG. 4, the litter container 20 can be tipped by use of a cross bar mechanism 94. The cross bar mechanism 94 has a manually operated cross bar 96 connected to two vertical link members 98 positioned proximate the side frames 12 inside the housing 18. The vertical link members 98 connect by pivotal connections 100 and 102 to intermediate link 104 and elongated end length 106. The end link 106 connects to a pivot bracket 108 that has a cross member 110 under the litter container 20 having a similar pivot bracket 108 on the other side of the litter container for connection to an identical link assemblage. The litter container 20 has two projecting bosses 112 mounted on the upper back corner of the side walls 114 of the outer compartment 32. Each boss 112 engages a track groove 116 in the side frames 12. The bosses 112 act both as pivots when positioned at the base of the track groove and as guides when the litter container 20 is displaced such that the bosses 112 are transported up the grooves during the return operation. When the cross bar 96 is depressed, the vertical link members 98, which are retained by straps 117 and restricted to vertical motion, imparts the linear motion to the end of inter connecting link 104. End link 106 has an intermediate connection to a pivot 118 on the side frames 12 such that the end link acts as a lever arm to lift the outer end of the litter container 20 when the cross bar 96 is depressed. The litter container 20 swings upward as shown in phantom in FIG. 1 whereupon the fecal matter retained on the bottom screen 48 slides across the screen and cover 90 and is deposited into the collection box 24. To insure that the collection box is in place when the cross bar mechanism 94 is actuated, a simple locking mechanism 120 as shown in FIG. 2 is provided. The collection box 24 has its handle 26 connected to a false front 122 to match side frames 12 when the box is in place. Spacers 124 connect the false front 122 to the side wall 126 of box 24. The side wall 126, contacts a biased plate 128 loosely connected at one end by bolt 130 and alignment bolt 132 having a compression spring 133 to bias the plate 128 toward the side frame 12. Neither bolt 130 nor bolt 132 interfere with motion of the interconnecting link 104 as shown in FIG. 1. However, bolted on the plate 128 is a machine screw 136 which is adjusted and positioned to interfere with end link 106 when the box is removed and the plate 128 is displaced toward the side frame 12. In this manner depression of the cross bar 96 will be inhibited. The screw and washer assembly 138 mounted on the false front 122 of the collection box 24 provides a stop for inserting the collection box as the washer assembly will in part contact the side frame 12, As shown in FIGS. 1 and 2. The cross bar mechanism 94 operates in conjunction with a delay mechanism 140 which comes into operation when it is desired to return the litter box in it to its operable position. As shown in the schematic views of FIGS. 8-10 when the litter box has been raised to the position that results in discharge of the solid material collected by the screen strainer 42, the granular material 36 shifts to the back of the upper compartment 30 such that all of the liquid drains to the bottom and back of the lower or outer compartment 32. When the final upright position is reached as shown in FIG. 9 the back edge 142 of the lower or outer compartment 32 is positioned over the collection box 24 the collected liquid waste pours into the collection box. Concurrently, the top of the side walls 46 of the screen strainer 42 contacts the cross shaft 144 of the delay mechanism 140. This contact forces the detent pin 86 to be released from the dimple 88 such that the screen strainer 44 will drop back against the inner compartment 30 when the litter container begins to be returned to its operational position. It is to be noted that the granular material 36 has shifted out of the way such that the screen strainer 42 can reseat on the bottom of the inner compartment. The delay mechanism 140 includes a pair of spaced cams mounted on the ends of the cross shaft 144 with support wires having end connectors 150 and 152 for connecting to the top of the cam 146 and the bottom part of the litter container 20. The cams 146 have a grooved, arcuate edge 154 which allow the support wires 148 to wrap and unwrap from the cams when the litter container 20 is raised or lowered. The cams 146 as shown in FIG. 1, are connected to a tension spring 156 connected at one end to a pin 158 in side frame 12 and mounted at its other end to a pin 160 on a crank member 162 connected to the shaft 144 by a nut and washer assembly 164. The tension spring 156 compensates for the weight of the litter container 20 and contained granular material 36 such that operation of the cross bar mechanism 94 requires minimal application of force. The lever member 162 also includes an extension 166 connected to an air cylinder 168 having a connection to the outside of one of the side frames 12 by pin 170. The air cylinder 168 coacts with the tension spring 156 to provide a smooth cushioned motion to the actuation of the litter container, on its descent to the base pad 16. When the litter container 20 is raised to the position shown in FIG. 9 by actuation of the cross bar mechanism 94, the tension spring causes the cam 146 to rotate maintaining tension on the support wires 148. In the upright position a detent cam 172 has rotated together with the support wire cams 146 such that a detent 174 engages a notch 176 on a locking arm 178. The locking arm 178 shown in cross section in FIG. 6 is pivotally mounted to one of the side frames 12 by a pin assembly that is constructed with a bolt 180 having a thick shaft 181 and a thinner threaded end 182 for seating a washer 194 and wing nut 186 with spacers 188 and 190 allowing free pivot of the locking arm 178. The locking arm 178 is biased by a tension spring 192 connected to pin 194 on the side frame 12 and hole 196 on an extension 198 of the locking arm 178. When the support wires 148 are wrapped on the cams 146 when the container is lifted, detent 174 engages the notch 176 to lock the delay mechanism 140 in its wrapped position. The litter container will shift its position when the cross bar mechanism 94 is raised and the front end of the container is dropped to the base pad as shown in phantom in FIG. 10. In this tilted position the granular material 36 has spread evenly back across the inner compartment 30 covering the bottom and the screen strainer 42. With the litter container 20 in the rear raised position shown in phantom in FIG. 10, the locking arm 178 can be automatically released as the container 20 is pulled forward and down tracking the bosses 112 up grooves 116. At the time the front edge of the container 20 reaches the pad 16, one of the bosses 112 contacts and displaces the tip 179 of locking arm 178 allowing the cams 146 to rotate and the wires 148 to unwrap such that the back end of the litter container 20 gently seats onto the base pad 16 in the position shown in FIG. 10. Since the litter container 20 is only seated on the cross member 110 of the cross bar mechanism it can be removed from the housing 118 by disconnecting the looped wire on the end connectors for the support wires 148 and displacing the extension 198 of the locking arm 178 into a notch 200 in the side frame 12 clearing the track groove 116 such that the litter container 20 can be lifted up and out the housing 118. With the litter container removed the container can be easily washed and the gravel replaced if desired. Generally, the gravel is coated with a non-previous material such that even after long use the gravel can be used, rinsed and replaced without emitting offensive odors. As noted, the gravel can be periodically sprayed with a odor mask and disinfectant while seated in the inner or upper compartment of the container. The liquid rinse will simply seep through the granular material 36 and be collected in the lower compartment of the liquid container along with any liquid waste from the household pets. While in the foregoing, embodiments of the present invention have been set forth in considerable detail for the purposes of making a complete disclosure of the invention, it may be apparent to those of skill in the art that numerous changes may be made in such detail without departing from the spirit and principles of the invention.

Summary: A cat litter box with a mechanism for screening and draining reuseable, liquid-impervious granular material contained in the box, the box having a housing with a container, the container having an inner compartment with holes for holding the granular material and draining liquids, and an outer compartment for receiving drained liquids, the inner compartment having a screen, wherein the mechanism both raises the screen through the granular material to separate solid wastes, and tips the container to drain the liquid wastes and separated solids into a collection box for removal, and lowers the container, returning the screen to the inner compartment and redistributing the granular material over the screen before the container seats in the housing for reuse.

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Summarize: Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more Sen. Lindsey Graham said Friday that President Trump deserves the Nobel Peace Prize if both Koreas actually end hostilities and work toward denuclearization. “It wouldn’t have happened without Trump. It may not happened, but it’s the biggest change since the end of the hostilities,” Mr. Graham, South Carolina Republican, said on Fox News. “What happened? Donald Trump convinced North Korea and China he was serious about bringing about change.” “We’re not there yet, but if this happens, President Trump deserves the Nobel Peace Prize,” he added. Mr. Graham warned North Korea leader Kim Jong-un not to “play” Mr. Trump and to follow through on his promise to officially end the Korean War and work toward peace. “A word of warning: The worst thing Kim could do is play Trump — to go through all these motions and to go back to the old way of doing business — Donald Trump will not tolerate being played,” he said. Mr. Trump is set to meet with Mr. Kim sometime in the coming weeks, but details on the location and timing have not been released. Mr. Kim met with South Korean President Moon Jae-in at the demilitarized zone and walked together into the Peace House, a conference room on the southern side. Copyright © 2018 The Washington Times, LLC. Click here for reprint permission.

Summary: Friday's developments between North and South Korea had President Trump making an all-caps statement: "KOREAN WAR TO END!" he wrote triumphantly. Now others are making bold statements of their own, calling for Trump to get the Nobel Peace Prize for his role in bringing the adversaries together. A typical sentiment among supporters comes from Laura Ingraham of Fox News, who tweets, "When will we see the headline: 'Trump Ends the Korean War'? Unlike Obama, he actually deserves the Nobel Peace Prize." She wasn't the only one making the assertion. Details on that and other related developments: 2 lawmakers: Perhaps the most prominent voice on the subject is that of GOP Sen. Lindsey Graham, who said the Korean pledges for denuclearization and peace "wouldn't have happened without Trump," per the Washington Times. "We're not there yet, but if this happens, President Trump deserves the Nobel Peace Prize." Meanwhile, GOP Rep. Luke Messer of Indiana is trying to drum up congressional support for a Trump Nobel nomination. "We are seeing unprecedented progress toward peace, and it's a direct result of President Trump's strong leadership," he said.

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Write a title and summarize: Der Wiener Kongress, der vom 18. September 1814 bis zum 9. Juni 1815 stattfand, ordnete nach der Niederlage Napoleon Bonapartes in den Koalitionskriegen Europa neu. Nachdem sich die politische Landkarte des Kontinentes als Nachwirkung der Französischen Revolution erheblich verändert hatte, legte der Kongress wiederum zahlreiche Grenzen neu fest und schuf neue Staaten. Unter der Leitung des österreichischen Außenministers Fürst von Metternich berieten politisch bevollmächtigte Vertreter aus rund 200 europäischen Staaten, Herrschaften, Körperschaften und Städten, darunter alle bedeutenden Mächte Europas mit Ausnahme des Osmanischen Reiches. Die führende Rolle spielten Russland, das Vereinigte Königreich, Österreich und Preußen sowie das wiederhergestellte Königreich Frankreich und der Kirchenstaat. Die deutschen Fragen wurden angesichts ihrer Komplexität und ihres Umfangs getrennt von den übrigen europäischen Angelegenheiten beraten. == Vorgeschichte und Beginn Nach dem Sturz Napoleons im Frühjahr 1814 beendete der Erste Pariser Frieden den Krieg zwischen den Mächten der Sechsten Koalition und der französischen Regierung, der restaurierten Bourbonenmonarchie unter Ludwig XVIII. Nach Artikel 32 dieses Friedensvertrages sollte in Wien ein Kongress zusammentreten, um eine dauerhafte europäische Nachkriegsordnung zu beschließen. Dazu waren alle am Krieg beteiligten Staaten eingeladen. Die siegreichen Könige und ihre führenden Minister trafen sich zunächst in London. Im Herbst 1814 begann in Wien der Kongress, zu dem sich Delegationen fast aller Staaten und Mächte Europas einfanden. Von September 1814 bis Juni 1815 wurde Wien und vor allem der Tagungsort, das Außenministerium (später auch die Staatskanzlei) im Palais am Ballhausplatz, der Amtssitz von Metternich, zum politischen Zentrum des Kontinents. Gastgeber war Kaiser Franz I. von Österreich. Die Gastgeber bemühten sich, den Aufenthalt der Kongressteilnehmer möglichst angenehm zu gestalten. Die Abfolge geselliger Ereignisse, Bälle und sonstiger Vergnügungen veranlasste Charles Joseph Fürst von Ligne in einem Brief an den französischen Staatsmann und Diplomaten Talleyrand vom 1. November 1814 zu der Äußerung: Auch andere Zeitgenossen zeigten sich, obwohl sie die politische Unbeweglichkeit beklagten, von der Prachtentfaltung beeindruckt. Der Generalsekretär der Versammlung Friedrich von Gentz schrieb in einem Brief vom 27. September 1814: Ob der Kongress bei allen Vergnügungen seine eigentliche Aufgabe - den Rahmen für eine dauerhafte europäische Friedensordnung zu schaffen - vernachlässigte oder nicht, wird bis heute kontrovers diskutiert. Marschall Blücher charakterisierte die Verhandlungen so: == Die Verhandlungen Der Wiener Kongress erarbeitete, dies war eine verhandlungstechnische Neuheit, seine Ergebnisse in Kommissionen. Es gab unter anderem einen Ausschuss für die Deutschen, einen für die europäischen Angelegenheiten, einen für Gebietsfragen, einen für die Flussschifffahrt und einen für den Sklavenhandel. Zu einer formellen Vollversammlung kam es nie, die Ergebnisse wurden meist in bilateralen Verträgen festgehalten. Die Schlussakte des Kongresses (Kongressakte) trägt nur die Unterschriften der acht Hauptmächte Österreich, Spanien, Frankreich, Großbritannien, Portugal, Preußen, Russland und Schweden (in dieser auf Französisch alphabetischen Reihenfolge). Die Deutsche Bundesakte, deren Allgemeine Bestimmungen (Artikel 1 bis 11) in die Kongressakte aufgenommen sind, wurde separat von den Bevollmächtigten der deutschen Staaten unterzeichnet. Der wichtigste Gegenspieler Metternichs war Zar Alexander I. Daneben spielten auch der britische Gesandte Castlereagh und der Vertreter des besiegten Frankreich, Talleyrand, der sowohl unter dem alten wie dem neuen französischen Regime erheblichen Einfluss hatte, die wichtigsten Rollen. Preußen wurde durch Karl August von Hardenberg und Wilhelm von Humboldt vertreten und konnte erhebliche Zugewinne an Land (vor allem im Rheinland und gegenüber Sachsen) verzeichnen und seine politische Stellung ausbauen. === Prinzipien und Interessenkonflikte Das häufig gezeichnete Bild der Harmonie existiert so nicht: Tatsächlich verschärften sich die Interessengegensätze der (Haupt-)Verhandlungspartner im Verlauf des Kongresses deutlich. Der Kongress arbeitete nach fünf übergeordneten Prinzipien, die allerdings teilweise die nachträgliche Konstruktion der Historiker sind. Der Begriff der Legitimität bezeichnet in diesem Zusammenhang die Liquidierung des napoleonischen Staatensystems und die Wiedereinsetzung der alten Dynastien (Bourbonen, Welfen usw.). Wenn ausgerechnet Talleyrand das Legitimitätsprinzip betonte, ging es ihm vor allem um die Anerkennung Frankreichs als gleichberechtigter Macht und damit die Überwindung des Status als Kriegsverlierer. In diesen Zusammenhang gehört auch der Grundsatz der Restauration der vorrevolutionären politischen und gesellschaftlichen Verhältnisse. Die Restauration sollte zwar nicht so weit gehen, dass alle seit 1789 eingetretenen Veränderungen wieder rückgängig gemacht werden sollten, sehr wohl sollte aber allen zukünftigen revolutionären Bestrebungen ein Riegel vorgeschoben werden. Dazu zählten nicht nur die freiheitlichen, sondern auch die nationalen Bewegungen der Zeit. Zur Sicherung und Durchsetzung dieses Ziels setzten die Delegationen einerseits auf eine starke monarchische Autorität nach innen und andererseits auf die zwischenstaatliche Solidarität der Länder nach außen. Einig war man sich in der Schaffung eines europäischen Gleichgewichtssystems zur Verhinderung zukünftiger Kriege. Die praktische Umsetzung vor allem des letztgenannten Ziels kollidierte dabei zunächst jedoch mit den unterschiedlichen machtpolitischen Interessen. Metternichs Ziel etwa war ein österreichisch geführtes Mitteleuropa, das ein Gegengewicht zu den Flügelmächten Frankreich und Russland bilden sollte. Das russische Hauptziel war es dagegen, den größten Teil Polens zu gewinnen. Der Zar spielte dabei mit dem Gedanken, Polen zu einem Muster eines konstitutionellen Staates zu machen. Der britische Gesandte strebte, ähnlich wie Metternich, ein konservativ bestimmtes Europa an und wollte gleichzeitig eine weitere Machtausdehnung Russlands möglichst verhindern. Zum Schutz seiner Großmachtstellung bekämpfte die französische Delegation auch die Einigungsbestrebungen in Deutschland. Preußen dagegen wollte eine Stärkung der eigenen Position durch den Erwerb ganz Sachsens und eine preußisch-österreichische Hegemonie in Deutschland erreichen. Dem entgegen standen allerdings die Interessen der kleineren deutschen Staaten und Österreichs. === Polen, Sachsen und neue Konstellationen Bei aller Solidarität der Monarchien sah es zeitweise so aus, als ob der Kongress ohne Ergebnis enden könnte. Hauptgrund war der Interessengegensatz zwischen Österreich, Preußen und Russland um Polen. In diesem diplomatischen Konflikt, der sich auf verschiedenen Ebenen abspielte, kam es zu neuen Bündnissen der beteiligten Staaten. Der Plan Alexanders I., auf dem Gebiet des Herzogtums Warschau ein polnisches Königreich unter russischer Herrschaft zu errichten, fand zunächst wenig Zustimmung. Als im November 1814 die preußische Delegation auf Weisung Friedrich Wilhelms III. die russische Position ohne Vorbehalt unterstützte, entstand ein Bündnis zwischen Großbritannien und Österreich, dem sich auch Frankreich annäherte. Die damit verbundene Anerkennung Frankreichs als Großmacht, bei gleichzeitiger Zuspitzung der Interessengegensätze unter den Alliierten, wurde an der Jahreswende 1814/1815 zu einem Triumph des Verhandlungsgeschicks Talleyrands. Der Konflikt verlagerte sich dabei von Polen weg auf die sächsische Frage. Man spricht auch von der Polnisch-Sächsischen Frage, da der König von Sachsen in Personalunion auch Herzog von Warschau war und damit Staatsoberhaupt in dem Gebiet, auf das es Alexander I. abgesehen hatte. Der Fortbestand Sachsens als Staat war durch die Inhaftierung des Königs Friedrich August I., dem die Alliierten Kollaboration mit Napoleon vorwarfen, mehr als unsicher. Nur über Mittelsmänner konnte der Wettiner Einfluss auf die Diskussionen nehmen. Zeitweise lag sogar ein Krieg zwischen den ehemaligen Verbündeten in der Luft, und Preußen begann bereits mit militärischen Vorbereitungen. Gegen Preußen und Russland kam es am 3. Januar 1815 zu einem Geheimabkommen zwischen Großbritannien, Österreich und Frankreich, dem auch die Niederlande, Bayern und Hannover beitraten. Damit zerschlugen sich die seit Friedrich II. gehegten preußischen Hoffnungen auf einen vollständigen Erwerb des Nachbarstaates Sachsen. Die weiterhin existierenden Unstimmigkeiten über territoriale Fragen wurden in verschiedenen Kommissionssitzungen relativ problemlos ausgeräumt. Die Verhandlungen wurden auch fortgeführt, als Napoleon Bonaparte aus dem Exil zurückkehrte und seine Macht in Frankreich im März 1815 wiederherstellte. Die Schlussakte des Kongresses wurde neun Tage vor Napoleons endgültiger Niederlage bei Waterloo unterzeichnet. == Territoriale Neuordnung Den Entscheidungen darüber, welcher Staat welche Territorien abzugeben hatte bzw. welche Territorien ihm zugeschlagen wurden, lagen Vorarbeiten einer "Statistischen Kommission" zugrunde. In dieser Kommission hatten Fachleute, darunter Geographen, Ökonomen und Bevölkerungsstatistiker in aufwendiger Kleinarbeit den jeweiligen "Territorialwert" veranschlagt, in den vor allem die Größe des Territoriums, seine Einwohnerzahl und dessen Ertragskraft einflossen. So ließen sich abgehende und gewonnene Territorien, Forderungen und Zugeständnisse näherungsweise miteinander verrechnen. Das Territorium Frankreichs war bereits vor Beginn des Kongresses im Ersten Pariser Frieden auf die Grenzen von 1792 zurückgeführt worden. === Österreich und Luxemburg Österreich musste auf seine ehemaligen Besitzungen am Oberrhein verzichten. Insgesamt zog sich Österreich aus dem deutschen Westen tendenziell zurück. Dafür bekam es erneut Galizien (samt dem Tarnopoler Kreis), während Krakau und Umgebung zu einer von den drei Teilungsmächten garantierten Republik Krakau wurde. Auch Illyrien fiel an Österreich zurück. Mit dem Besitz der ehemaligen Republik Venedig und der Lombardei, zusammengeschlossen im Königreich Lombardo-Venetien, sowie der Zuweisung der Toskana an Erzherzog Ferdinand und der Stadt Parma an die österreichische Ehefrau Napoleons Marie-Louise hatten die Habsburger in Oberitalien eine noch stärkere Stellung als vor der Revolution. Im Norden kamen Salzburg und das Innviertel hinzu. Im Vergleich zu den territorialen Zugewinnen von Preußen und Russland erschien der Gebietszuwachs Österreichs allerdings begrenzt. Insbesondere blieben die ehemals Österreichischen Niederlande (aus denen später Belgien hervorgehen sollte) verloren. Diese Gebiete fielen an die Niederlande, und es entstand das Vereinigte Königreich der Niederlande. In Personalunion stellte das Haus Oranien-Nassau nicht nur den niederländischen König, sondern auch den Großherzog von Luxemburg. Insgesamt ist Österreich aus Deutschland geografisch "hinausgewachsen", politisch aber im ebenfalls durch den Wiener Kongress gebildeten Deutschen Bund Führungsmacht geworden, während für Preußen zunächst der umgekehrte Weg gilt. === Preußen Preußen erhielt entgegen den ursprünglichen Plänen und Erwartungen nicht ganz Sachsen, sondern nur den nördlichen Teil, der zum Teil der neuen Provinz Sachsen zugeschlagen wurde. Dafür erzielte es im rohstoffreichen Westen erhebliche Gebietszuwächse und konnte die Provinzen Jülich-Kleve-Berg, Großherzogtum Niederrhein und Westfalen errichten. Im Osten kamen Posen und die Stadt Danzig wieder hinzu, dafür musste Preußen endgültig auf die schon 1807 verlorenen Erwerbungen aus der dritten und zum Teil auch aus der zweiten Teilung Polens verzichten. An Bayern gab es Ansbach und Bayreuth, an das Königreich Hannover Ostfriesland, Hildesheim, Goslar und den größeren Teil des Untereichsfelds ab und erhielt dafür Schwedisch-Pommern mit Rügen von Dänemark im Tausch gegen das Herzogtum Lauenburg. Die Zuteilung der Rheinlande und Westfalens an Preußen entsprach sowohl den Zielsetzungen von Talleyrand, der Frankreich in Wien vertrat, als auch den Wünschen von Castlereagh, des britischen Gesandten, wenn auch aus unterschiedlichen außenpolitischen Erwägungen. Während Frankreich erwartete, dass es Preußen nicht gelingen werde, sich in den Rheinlanden dauerhaft zu verankern, so dass damit die Chance eröffnet werden könnte, die französische Westgrenze wieder an den Rhein vorzuschieben, ging das Vereinigte Königreich davon aus, dass das militärisch starke Preußen französischen Expansionsbestrebungen wirksam einen Riegel vorschieben werde. Mit dem Erwerb der rheinischen Gebiete wurde Preußen zum Schutzwall gegen Frankreich, das noch immer die Rheingrenze anstrebte, was auch für die linksrheinische Pfalz und Rheinhessen von größter sicherheitspolitischer Bedeutung war. Die Schutzwallfunktion gegen Frankreich wurde später auch volkstümlich kultiviert, etwa durch das Lied Die Wacht am Rhein. Durch die Expansion und Zweiteilung seines Staatsgebietes in ein östliches "Altpreußen" und ein "Neupreußen" im Westen war Preußen gezwungen, in Deutschland hineinzuwachsen, und wurde so zum Motor der wirtschaftlichen und politischen Einigung. Der Historiker Thomas Nipperdey geht sogar so weit, in dieser Schwerpunktverlagerung eine Vorentscheidung über den späteren deutschen Einigungsprozess zu sehen: "Die Versetzung Preußens an den Rhein ist eine der fundamentalsten Tatsachen der deutschen Geschichte, eine der Grundlagen der Reichsgründung von 1866/1871." === Bayern Bayern, dem mit dem Vertrag von Ried gerade noch rechtzeitig der Absprung vom Bündnis mit Napoleon gelungen war, gewann zwar im Tausch gegen Tirol den größten Teil Frankens sowie die nach schwierigen Verhandlungen neugeschaffene linksrheinische Pfalz mit Teilen der alten Pfalz hinzu, konnte seine territorialen Ambitionen aber nicht ganz verwirklichen. Erst im Vertrag von München wurden 1816 die endgültigen Grenzen des nachnapoleonischen Bayerns bestimmt. Der badisch-bayerische Grenzstreit über die rechtsrheinische Pfalz mit Mannheim und Heidelberg wurde dann 1818 auf dem Aachener Kongress zugunsten Badens entschieden. Für die Reichsgründung von 1866/1871 wurde die Tatsache bedeutsam, dass die nördlichen Regionen Franken und die Pfalz sie mehrheitlich begrüßten, und so zusätzlichen Zugzwang auf die Bayerische Regierung ausübten. Wie Preußen war Bayern, anders als Österreich, 1815/16 nach Deutschland "hineingewachsen". === Sachsen Auf der Verliererseite des Kongresses stand das Königreich Sachsen. Gleichsam als Strafe für sein zu spätes Abrücken vom Bündnis mit Frankreich - in der Völkerschlacht bei Leipzig hatte es noch auf der Seite Napoleons gekämpft - verlor das Königreich durch Abtretung an Preußen etwa 60 % seiner Fläche mit etwa 40 % seiner Einwohner in den nördlichen und östlichen Gebieten sowie in Thüringen an Preußen, das aber einen Teil dieser thüringischen Gebiete dann an das Großherzogtum Sachsen-Weimar-Eisenach weiterreichte. === Übrige deutsche Staaten Das Königreich Württemberg, die Großherzogtümer Baden und Hessen sowie das Herzogtum Nassau konnten ihren Territorialbestand aus der Rheinbundzeit behaupten, es fanden bis 1825 nur kleine Grenzkorrekturen statt. Als souveräne Staaten wiedererrichtet wurden das in Personalunion mit dem Vereinigten Königreich verbundene ehemalige Kurfürstentum Braunschweig-Lüneburg (nun zum Königreich Hannover erhoben), Braunschweig, Oldenburg, Hessen-Kassel, Hessen-Homburg und die freien Städte Lübeck, Frankfurt, Bremen und Hamburg. Allerdings wurde die Mediatisierung der zurückliegenden Jahre, trotz der Proteste der betroffenen Fürsten, nicht rückgängig gemacht, genauso wenig die Säkularisation der Geistlichen Territorien. Insofern blieb die Zahl der Staaten deutlich geringer als in vorrevolutionärer Zeit. === Schweiz Die Schweiz musste das Veltlin, Chiavenna und Bormio sowie die Stadt Mülhausen im Elsass endgültig aufgeben. Als Ausgleich wurden ihr jedoch das ehemalige Hochstift Basel, das Fricktal, die Herrschaften Rhäzüns und Tarasp sowie einige Gemeinden in der Umgebung von Genf zugesprochen. Der Wiener Kongress erkannte die inneren und äußeren Grenzen der Schweiz und ihrer Kantone wie auch die Zugehörigkeit des Wallis, des Fürstentums Neuenburg (Hohenzollern) und Genfs als neue Kantone an. Nordsavoyen wurde neutralisiert und sollte im Kriegsfall von Schweizer Truppen besetzt werden, blieb aber beim Königreich Sardinien. Die von Schweizer Politikern angestrebte Abrundung der Grenzen gegen das Großherzogtum Baden bei Schaffhausen und die Gewinnung der Stadt Konstanz sowie die Rückkehr des Veltlins, Chiavennas und Bormios zu Graubünden konnten nicht erreicht werden. Einen bis heute entscheidenden Einfluss auf die weitere Geschichte der Schweiz hatte die Anerkennung der immerwährenden bewaffneten Neutralität sowie ihrer Unabhängigkeit von jedem fremden Einfluss durch die europäischen Großmächte. Diese internationale Anerkennung bzw. Verpflichtung der Schweiz auf die Neutralität bildet bis heute die maßgebende Grundlage für die schweizerische Außenpolitik (= Schweizerische Neutralität). === Übrige europäische Staaten Der ehemalige Kriegsgegner der Alliierten, Frankreich, musste, wie angesichts des von Talleyrand selbst vertretenen Legitimitätsprinzips zu erwarten war, die zwischen 1795 und 1810 durchgeführten Annexionen rückgängig machen. Ein großer Erfolg war allerdings die gleichberechtigte Rückkehr in die europäische Völkerfamilie und die Anerkennung als Großmacht. Dänemark musste wegen seiner Unterstützung für Napoleon Norwegen an Schweden abgeben (= Kieler Frieden). Es erhielt aber als Ausgleich Schwedisch-Pommern. Diese Territorien wurden schnell an Preußen abgetreten. Als Kompensation dafür erhielt Dänemark das Herzogtum Lauenburg (das Preußen zuvor mit Hannover gegen Ostfriesland getauscht hatte) und Geld. In Spanien, Portugal und in Neapel wurden die alten Dynastien wiederhergestellt. Ebenso in Sardinien, das Savoyen, Piemont und Nizza zurückbekam und zusätzlich Genua erhielt. Auch der Kirchenstaat wurde restauriert und bekam einen Großteil seiner ehemaligen Gebiete zurück. Metternich hatte für die italienischen Staaten einen dem Deutschen Bund ähnlichen italienischen Bund unter dem Vorsitz Österreichs geplant, konnte sich aber mit dieser Idee nicht bei Kaiser Franz I. und den italienischen Fürsten durchsetzen. Damit und durch die erheblichen österreichischen Zugewinne in Oberitalien blieb Italien zersplittert und seine Vereinigung zu einem Nationalstaat auf Jahrzehnte verwehrt. Großbritanniens Erwerbungen aus dem Britisch-Französischen Kolonialkonflikt wurden ebenfalls bestätigt. Malta und Helgoland blieben somit bei Großbritannien. Die Ionischen Inseln im Mittelmeer fielen unter britisches Protektorat. Im Osten fand sich Zar Alexander I. mit einer vierten Teilung Polens ab. Allerdings wurde Russland mit dem sogenannten Kongresspolen der größte Teil zugesprochen, und es sicherte sich durch die Anerkennung seiner territorialen Gewinne in Finnland (1808/09) und Bessarabien die bisherige Ausweitung nach Westen. Die nördlichen Niederlande (bis 1795 Republik der Sieben Vereinigten Provinzen, später Batavische Republik und Königreich Holland) wurden mit den südlichen, ehemals habsburgisch-österreichischen Niederlanden sowie dem ehemaligen Hochstift Lüttich im Vereinigten Königreich der Niederlande vereint. == Der Deutsche Bund Grundlage für die Verhandlungen über eine staatliche Neuordnung der Länder des vormaligen Heiligen Römischen Reiches (Deutscher Nation) während des Wiener Kongresses war der Artikel VI des Ersten Pariser Friedens vom 30. Mai 1814. Dort wurde den deutschen Staaten ihre Unabhängigkeit und die Vereinigung durch ein föderatives Band zugesichert. Der Ausschuss zu den Beratungen der deutschen Angelegenheiten, das sogenannte "Deutsche Komitee", tagte unter dem Vorsitz von Preußen, Österreich, Hannover, Bayern und Württemberg. In der Folge öffnete sich das Gremium allen deutschen Staaten und freien Städten. Auch wenn der Kongress das Legitimitätsprinzip verfocht und im Kern auf eine Restauration der vorrevolutionären Verhältnisse abzielte, hatten diese Grundsätze doch auch ihre Grenzen. Die mit dem Reichsdeputationshauptschluss 1803 eingeleitete Mediatisierung wurde nicht wieder rückgängig gemacht. Dasselbe gilt auch für die Säkularisation und das Ende der geistlichen Staaten, für deren Wiederherstellung sich der päpstliche Gesandte Ercole Consalvi vergebens einsetzte. Ebenso wurde die Souveränität der ehemaligen Rheinbundstaaten anerkannt. Eine Rekonstruktion des Heiligen Römischen Reiches wurde von den Kongressteilnehmern nicht ernsthaft erwogen, auch nicht von Freiherr vom Stein, der als russischer Gesandter am Kongress teilnahm und die Wiederherstellung der Kaiserwürde befürwortete. Gleichwohl wurde die Suche nach einem funktionalen Ersatz für die 41 deutschen Staaten und freien Städte eine der zentralen Fragen des Kongresses. Zu Beginn der Verhandlungen gingen sowohl Metternich als auch die preußischen Gesandten von einer vergleichsweise stark zentralistischen Lösung aus. Zwar kursierten zahlreiche Vorschläge, aber einflussreich wurden nur Hardenbergs "41 Artikel" und der daraus in Zusammenarbeit mit Metternich hervorgegangene "12-Punkte-Plan". Beide gingen von einer im Kern bundesstaatlichen Ordnung mit starken Zentralorganen aus. Dazu gehörte eine kollektive Exekutive, der "Rat der Kreisobersten", aus Vertretern der größeren Staaten. Dieses Gremium sollte so angelegt werden, dass Preußen und Österreich die anderen Staaten majorisieren konnten. Das Bundesgebiet sollte in sieben Kreise eingeteilt werden, die für die Umsetzung der Bundesbeschlüsse und für das Kriegs- und letztinstanzliche Gerichtswesen zuständig sein sollten. Dadurch wären die de jure weiter bestehenden kleinen Territorien de facto mediatisiert worden. Gescheitert ist dieses Projekt nicht so sehr an der heftigen Gegenwehr der kleinen Staaten, sondern am oben geschilderten sächsisch-polnischen Konflikt. Die dort offen zu Tage tretenden Expansionsbestrebungen Preußens führten auf österreichischer Seite zur Aufgabe des Plans, eine Doppelhegemonie der beiden Staaten anzustreben. Geschaffen wurde schließlich der lose Deutsche Bund souveräner Staaten mit Österreich als Präsidialmacht. Als Verfassung wurde die Deutsche Bundesakte am 8. Juni 1815, einen Tag vor der Unterzeichnung der Wiener Kongressakte, verabschiedet. Die ersten elf Artikel der Bundesakte wurden in die Wiener Kongressakte aufgenommen und dadurch vermeintlich unter den Schutz bzw. die Garantie der Signatarmächte gestellt. Aufgegeben wurde eine starke Exekutive ebenso wie ein oberstes Bundesgericht. Aus den ursprünglichen Überlegungen erhalten blieb die Bestimmung, dass sich jeder Bundesstaat eine landständische Verfassung geben müsse. Eine ganze Reihe von Ländern kam dieser Forderung auch rasch nach. Aber ausgerechnet die beiden Großmächte innerhalb des Deutschen Bundes, Preußen und Österreich, verfügten bis 1848 über keine geschriebene Verfassung. Ausdrücklich wurde erklärt, dass der Deutsche Bund nicht der Rechtsnachfolger des alten Deutschen Reiches sei. Ebenso wurde hervorgehoben, dass der Bund rein defensiven Charakter habe und nur der äußeren und inneren Sicherheit Deutschlands diene. Der Deutsche Bund wurde damit, auch wenn eine gemeinsame aktive Außenpolitik unmöglich war, ein notwendiger Teil im System des europäischen Gleichgewichts. Zum Deutschen Bund gehörten Preußen und Österreich nur mit ihren ehemaligen Reichsländern. Das heißt Österreich ohne die polnischen, ungarischen, südosteuropäischen und italienischen Gebietsteile, Preußen ohne West- und Ostpreußen und Posen. Als ausländische Monarchen waren der König von Großbritannien als König von Hannover, der König der Niederlande als Großherzog von Luxemburg und der König von Dänemark als Herzog von Holstein und Lauenburg, Bundesfürsten mit Sitz und Stimme in der Bundesversammlung. == Ächtung des Sklavenhandels Auf britischen Druck hin wurde in Artikel 118 der Kongressakte die Ächtung des Sklavenhandels ("Die Declaration der Mächte über die Abschaffung des Negerhandels, vom 8. Februar 1815") festgelegt. Das Übereinkommen verzichtete auf ein konkretes Umsetzungsdatum. Mit dem Beschluss der europäischen Grossmächte wurde das Ende eines der ältesten und unmenschlichsten Geschäftszweige der Geschichte eingeleitet. Es dauerte noch einige Jahrzehnte, bis die letzten Länder auf den Sklavenhandel verzichteten. Nach den USA im Jahr 1865 schaffte Brasilien 1888 als letzter Staat der Neuen Welt die Sklavenhaltung ab. == Unterzeichnung und Ratifikation Die Beschlüsse des Kongresses wurden in der Wiener Kongressakte, auch Schlussakte des Wiener Kongresses (Acte final) genannt, schriftlich fixiert. Sie umfasste 121 Artikel und enthielt auch sämtliche in Wien abgeschlossenen Verträge. Am 9. Juni 1815 wurde die Kongressakte unterzeichnet. Die Signatarmächte Österreich, Russland, Preußen, Großbritannien, Frankreich, Portugal, Spanien und Schweden garantierten damit die Ratifikation der Beschlüsse. Allerdings trat Baden erst am 26. Juli und Württemberg am 1. September 1815 dem Vertrag bei. Frankreich unter Ludwig XVIII. bestätigte den Vertrag am 7. Dezember 1815. Auch der Signatarstaat Spanien, der unzufrieden darüber war, dass der Sohn der Königin von Etrurien keine Entschädigung in Italien erhalten hatte, schloss sich erst am 7. Mai 1817 diesem Abkommen an. == Die Heilige Allianz Die Gründung der Heiligen Allianz, die am 26. September 1815 geschlossen wurde, war zwar nicht Bestandteil der offiziellen Verhandlungsergebnisse des Kongresses, steht aber inhaltlich in einem engen Zusammenhang mit diesem und bildet einen entscheidenden Bestandteil des 1815 entstehenden Metternich'schen Systems in der ersten Hälfte des 19. Jahrhunderts. Zur Heiligen Allianz gehörten zunächst Preußen, Österreich und Russland. Dieses Manifest der drei Monarchen rief zur christlichen Brüderlichkeit auf und stand damit im direkten Gegensatz zur revolutionären Brüderlichkeit der Völker. Metternich, der diesem Bund äußerst skeptisch gegenüberstand, hat dabei aus dem ursprünglichen Entwurf, der von einem Bündnis der "Völker und Heere" sprach, in seiner endgültigen Fassung ein "Bündnis der Herrscher" gemacht, die über den "Völkern und Heeren" stünden. Ziel der Vereinbarung war einerseits die Aufrechterhaltung der Balance zwischen den Fürsten und andererseits etwa bei revolutionären Bewegungen die Intervention bei den Völkern. Der Heiligen Allianz traten außer Großbritannien (dort verweigerte das Parlament einen Beitritt) und dem durch den Kongress wiederhergestellten Kirchenstaat unter Papst Pius VII., der das überkonfessionelle Konzept ablehnte, fast alle europäischen Staaten bei. == Fazit und Folgen Der Wiener Kongress hatte für die Verhältnisse der damaligen Zeit, zumal auf übernationaler Ebene, durchaus zukunftsweisende Beschlüsse gefasst. So wurde auf britischen Druck die Ächtung der Sklaverei im Artikel 118 der Kongressakte durchgesetzt. Außerdem wurde eine Übereinkunft über die Freiheit der internationalen Flussschifffahrt getroffen und eine Zentralkommission für die Rheinschifffahrt eingesetzt. Eine verbindliche Regelung des Gesandtschaftsrechts setzte den bis dato üblichen Rangstreitigkeiten unter Diplomaten ein Ende. Den Vorrang hatte nicht mehr derjenige, der den vermeintlich angesehensten Staat vertrat (denn an der Frage, welchem Staat diese Würde zukam, hatte sich der Zank immer wieder entzündet). Der Wiener Kongress bestimmte, dass Botschaftern der erste Rang gebührt, Gesandten der zweite, Geschäftsträgern der dritte. Innerhalb dieser Kategorien hat derjenige Diplomat den Vorrang, der am Dienstort länger akkreditiert bzw. im Dienst ist (Grundsatz der "lokalen Anciennität"). Diese Regelung gilt bis heute. Der Kongress hatte mit der Rückgängigmachung der Eroberungen des revolutionären und napoleonischen Frankreichs sein Hauptziel erreicht. Auf Kosten Frankreichs und durch die erneute Teilung Polens wurden die Großmächte Preußen, Österreich und Russland gestärkt. Zusammen mit Großbritannien und dem besiegten, aber wieder in das Konzert der Großmächte aufgenommenen Frankreich entstand das auf Gleichgewicht ausgerichtete System der Pentarchie. Nach der vorangegangenen jahrzehntelangen Zeit der Koalitionskriege war es ein wesentliches Ziel des Wiener Kongresses, dem zerrütteten Kontinent eine neue Ordnung zu geben, dabei zwischenstaatliche Gewalt zu vermeiden und mögliche Konflikte künftig diplomatisch zu lösen. Dies bedeutete eine historisch neue politische Qualität. Bis zum Krimkrieg in den frühen 1850er Jahren blieb Europa von Kriegen zwischen den Großmächten verschont. Der Sardinische Krieg, die Italienischen Unabhängigkeitskriege und die Schleswig-Holsteinische Erhebung standen im Zusammenhang mit den Revolutionen von 1848/49. Allerdings hatte der Konflikt um Polen und Sachsen im Verlauf des Kongresses gezeigt, dass die Politik des Ausgleichs auch ihre Grenzen hatte. Was die Gestaltung der inneren staatlichen Zustände angeht, war der Kongress eher von restaurativen Grundsätzen und einer grundsätzlichen Skepsis gegenüber allen revolutionären, liberalen und nationalen Bestrebungen geprägt. Für die deutschen Staaten war die Schaffung des Deutschen Bundes das zentrale Ergebnis des Kongresses. Der Deutsche Bund war aber in den Augen vieler Zeitgenossen primär ein Instrument zur Unterdrückung nationaler und liberaler Bewegungen. Es gelang allerdings nicht, die liberal bürgerlichen Bewegungen auszuschalten. Diese forderten den Nationalstaat, statt ein Bündnis von monarchischen Einzelstaaten. Die verordnete Ruhe in Europa durch den Wiener Kongress, die im Grunde eine Rückbesinnung auf die Zustände vor Napoleon und vor der Französischen Revolution von 1789 war, blieb langfristig ohne Änderungen nicht haltbar. Die dem Kongress folgende Restauration, die Unterdrückung nationaler und liberaler sowie demokratischer Bestrebungen, konnte nicht verhindern, dass sich die Ideen von bürgerlichen Rechten und nationaler Eigenständigkeit im Bürgertum weiter verbreiteten. Vor allem das Jahr 1830 wurde in dieser Hinsicht zu einer Zäsur: Die Idee eines gesamtdeutschen Staates etablierte sich trotz Niederschlagung der Märzrevolution im Jahre 1849 auch in konservativen Kreisen. Im Anschluss an den Deutsch-Dänischen Krieg 1864 und den Deutschen Krieg 1866 entstand mit der Verfassungsgebung des Norddeutschen Bundes 1867 der erste Bundesstaat, der die deutschen Länder nördlich der Mainlinie umfasste. Nach dem Deutsch-Französischen Krieg 1870/1871 wurde 1871 das deutsche Kaiserreich unter preußischer Führung als Kleindeutsche Lösung (d. h. ohne Österreich) ausgerufen. In den italienischen Staaten und Provinzen flammten nach 1815 bis 1870 immer wieder verschiedene Aufstände des Risorgimento (deutsch: Wiedererstehung) mit dem Ziel einer Einigung Italiens auf, die endgültig zwischen 1861 und 1870 auch in Kriegen gegen Österreich erkämpft wurde (= Italienische Unabhängigkeitskriege). Die italienischen Nationalrevolutionäre lehnten sich gegen die Vorherrschaft der österreichischen Habsburger in Norditalien und der spanischen Bourbonen in Süditalien auf. In der Schweiz folgte die Restauration mit dem Bundesvertrag durch die an der Existenz der Schweiz interessierten Mächte. Dieses sehr einfache Grundgesetz sollte bis 1847 die staatsrechtliche Basis der Schweizerischen Eidgenossenschaft bilden. == Delegationen und bedeutende Teilnehmer == Quellen Die vollständigen Dokumente des Wiener Kongresses wurden in den Jahren 1815 bis 1835 von Johann Ludwig Klüber unter dem Titel Acten des Wiener Congresses in den Jahren 1814 und 1815 in neun Bänden im Verlag J. J. Palm und Ernst Enke in Erlangen herausgegeben. Die ersten acht Bände erschienen zwischen 1815 und 1818, Nachträge als neunter Band 1835. Die Bände enthalten - in Auswahl - als wichtigste Aktenstücke (mit den Digitalisaten der Bayerischen Staatsbibliothek für die Bände 1 bis 8): == Literatur Sammelbesprechung von mehreren aktuellen Publikationen zum Wiener Kongress bei H-Soz-Kult.

Title: Wiener Kongress Summary: Der Wiener Kongress fand in den Jahren 1814 und 1815 statt. Damals trafen sich wichtige Politiker aus Europa. Wien war schon damals die Hauptstadt von Österreich. In den Jahren davor hatte es oft Krieg gegeben: Napoleon Bonaparte aus Frankreich wollte über ganz Europa herrschen, er wurde aber besiegt. Nun sprachen die Politiker darüber, was aus den einzelnen Ländern werden sollte. Wichtige Staaten damals waren Russland und Grossbritannien, Österreich und Preussen. Russland hatte die grösste Armee und Grossbritannien besonders viele Kriegsschiffe. Österreich war noch ein sehr grosses Land in der Mitte von Europa. Preussen war ein Staat im Nordosten von Deutschland. Aber auch kleinere Länder waren in Wien vertreten, ebenso das besiegte Frankreich. Die Politiker in Wien haben über viele Gebiete bestimmt, wem sie gehören sollten. Die deutschen Staaten gründeten den Deutschen Bund. Das war ein Verein von Staaten, kein Staat. In den kommenden Jahren arbeiteten Russland, Grossbritannien, Österreich, Preussen und Frankreich zusammen. Sie wollten weitere Kriege, Aufstände und Revolutionen verhindern.

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Write a title and summarize: The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome. In Drosophila, Male Specific Lethal (MSL) complex binds to the single male X chromosome to increase transcription approximately two-fold, in order to equalize the output of both female X chromosomes [2]–[4]. We have proposed that MSL complex locates its target binding sites using a two-step mechanism [5]. First, the complex distinguishes X from autosomes by binding a subset of 200–300 sites on X known as “chromatin entry sites” (CES) [6]–[8] or “high affinity sites” (HAS) [9], [10]. Recognition of CES is a sequence-dependent step, as these sites share a GA-rich motif [8], [10] designated the “MSL recognition element”, or MRE, whose function has been demonstrated by site-directed mutagenesis [8]. In contrast, the second targeting step lacks a consensus sequence but is strongly linked to transcription [11]–[14], with the complex locating active genes on the same chromosome [15]. CES were first identified in msl3 mutant embryos, in which the initial, sequence-specific step of MSL binding occurs but the second, sequence-independent step does not [7], [8]. The MRE sequence motif was discovered based on the first 150 mapped CES (Figure 1A). CES function was tested in transgenes for the ability to attract MSL complex to autosomal insertion sites, and found to be dependent upon the intact MRE motif [8]. 150 is likely an underestimate of the total number of CES and functional MREs on X, as subsequent analysis of high occupancy MSL binding sites in wild type cells has revealed 309 peaks containing 379 MREs [8]. However, a conservative set of 150 should be sufficient to test for predictive features. The MRE motif is only modestly enriched on the X chromosome compared to the autosomes. At a stringency where 137 of 150 CES contain the consensus motif (p-value of 10−5), there is a 1. 8 fold higher MRE density on X compared to autosomes (on average 1 per 6 Kb on X, and 1 per 11 Kb on autosomes; Figure S1A), [8]. These average densities correspond to 12,481 total MREs in the genome, of which only 1 in 91 correspond to the set of CES considered here. Even if we restrict our attention to chromosome X, only 1 in 28 MREs maps to the CES set. Therefore, a key question is how functional MREs within CES are somehow recognized amongst a vast excess of un-utilized sites. That the MSL complex targets only a fraction of potential MRE sites for initial binding is a characteristic it shares with many sequence-specific binding factors whose predicted target motifs are often in vast excess to the sites actually utilized [16], [17]. Here, we investigate whether chromatin features influence binding site selection, using the MSL complex and a large compendium of genome-wide ChIP-chip profiles generated by the NHGRI modENCODE project as a model [18]. Our results support a model in which active chromatin composition and intrinsic GC content help define the initial binding sites of the MSL complex. To search for chromatin features that can distinguish functional MREs from those that do not recruit MSL complex (i. e., non-functional MREs), we defined five classes of MREs in the Drosophila genome. The first set consists of 137 MREs that were experimentally defined by MSL complex binding [8], as discussed above. We called this set “Functional MREs”. The remaining four sets of 150 sequences each consist of the MREs that have the best consensus motif matches on either X or a control autosomal arm (2L) (“Best on X” and “Best on 2L” respectively), and 150 MREs chosen at random from either X or 2L (“Random X” and “Random 2L” respectively). We note that, in general, functional MREs display a broad range of motif binding specificity rather than being the best matches to the MRE consensus motif (Figure S1B). The result of this analysis is not affected by the choice of chromosome arm or the choice of random MREs (data not shown). In each of the five classes of MREs, their locations are distributed along the length of the chromosome arm with no obvious clustering (Figure S1C). Within each set of sequences, we calculated the average profiles of various chromatin marks mapped by the modENCODE Drosophila Chromatin Consortium using genome-wide ChIP-chip. The average ChIP enrichment profiles for 10 kb regions centered around the motif in two male cell lines (S2 and BG3) are shown in Figure 1B–1C. We found that a number of chromatin marks associated with active transcription are strikingly enriched near functional MREs in CES, and not in the best or random MRE classes on X or autosomes. These include RNA pol II, H3K36me3, H3K9ac, and H2B-ubiquitin. In addition, functional sites are relatively depleted for core histone H4 and linker histone H1. Consistent binding of the sequence-dependent GAGA factor [19] across categories serves as an important control to demonstrate that GA-rich elements are broadly represented in each group of MREs. Another notable feature of these profiles is the enhancement of H4K16ac on the X chromosome as a whole [20]–[22], with additional enrichment of H4K16ac on true CES (Figure 1B–1C). Since enrichment of H4K16 acetylation is a known consequence of MSL targeting, we proceeded to ask whether the observed difference in chromatin context for other chromatin marks at CES might simply be a consequence of MSL binding rather than a contributing factor. To test whether the observed difference in chromatin context at CES is a consequence of MSL binding rather than a contributing factor, we examined the profiles of the same subset of chromatin marks in female Kc cells. Interestingly, we found that the set of marks that correlates with MRE function in male S2 cells were likewise informative in female Kc cells, in the absence of MSL complex (Figure 1D). The enrichment of H4K16 acetylation on the male X is notably less pronounced in female Kc cells lacking MSL complex. Still, this mark is enriched over functional MREs, consistent with previous observations of MSL-independent H4K16 acetylation at active genes on all chromosomes in males and females [21], [22]. Most strikingly, H3K36me3 and JIL-1, are enriched in functional MREs in both male and female cells, suggesting that these marks are independent of MSL binding at CES. We also examined MREs for intrinsic sequence composition to search for correlations with function (Figure 1E). Surprisingly, we found a marked elevation of GC content in the 10 Kb flanking region surrounding functional MREs, coupled with a decrease in GC content in the 1 Kb nearest the functional MREs. Taken together, our results support a model in which local sequence characteristics and the active chromatin context of functional MREs may facilitate their initial selection. Next, we wanted to directly test the potential of MREs in an active chromatin environment in females to recruit MSL complex. The key female sex determination protein, SXL, represses dosage compensation by inhibiting MSL2 translation [23], [24]. Loss of SXL results in the expression, stabilization, and targeting of the MSL complex in female cells [25]. Therefore, we depleted SXL by RNA interference (RNAi) in Kc cells [26]. Upon treatment, we observed a general, MSL2-dependent increase in transcription from the female X chromosomes, consistent with a partial induction of dosage compensation (Figure 2A–2B). The increase in X-linked gene expression did not reach the maximum theoretical amount for perfect compensation (log22 = 1), consistent with observations that MSL-independent ploidy effects can also contribute to overall compensation [27], [28]. The induction of MSL complex in Kc cells allowed us to ask whether ectopic MSL complex in female cells recognized the same functional subset of MREs on X as in male cells, by examining the distribution of H4K16 acetylation as a mark of MSL function. We found that Sxl RNAi induces high levels of this modification preferentially at the same MREs that are functional in males (Figure 2C), supporting the idea that MSL complex recognizes MREs in an active chromatin context. Our findings with MSL complex parallel recent results for the heat shock transcription factor HSF [29], suggesting that sequence-specific DNA binding factors may generally utilize chromatin context to facilitate selective targeting within a complex eukaryotic genome. Since specific chromatin marks are enriched near MREs that are utilized compared with those that are not, we next asked whether these marks provide enough information, either individually or in combination, to explain the MSL entry site binding pattern on X. To address this question, we systematically investigated the predictive power of the chromatin marks for functional MREs in Kc cells, which have the potential for MSL targeting but do not express the MSL complex. We asked whether we could build a simple prediction model based on individual or a combinations of chromatin features in Kc cells that would distinguish functional MREs from non-functional ones in male S2 and BG3 cells, where MSL complex is expressed. We first tested whether individual chromatin features could discriminate functional MREs from non-functional ones. A chromatin feature is defined by its average ChIP enrichment within the 10 kb region surrounding each MRE. In addition, we defined two features to represent the average GC content near the MRE (center 1 kb) or in its flanking regions (10 kb excluding the center 1 kb). To test whether individual or combinations of features could distinguish the functional MREs from the non-functional ones, we used support vector machine (SVM), a classification algorithm demonstrated to have excellent performance in a wide range of problems [30], [31]. Briefly, the set of ChIP enrichment at each MRE is treated as a feature vector of that MRE. Given a set of training samples, SVM calculates an optimal hyperplane that can separate non-functional MREs from functional MREs in the feature space. Here we used a SVM with a radial basis kernel that is implemented in the R package e1071 (See Methods and Materials). To accurately estimate predictive power, and to avoid the potential bias due to using the same set of CES and non-functional MRE genomic locations in S2, BG3 and Kc cell lines, we evaluated the predictive power of a feature using 10-fold cross-validation. In this scheme, we withhold a random 10% of the MREs, build a model based on the remaining 90%, measure how well the model predicted the functionality of the withheld MREs, and repeat this process multiple times to obtain the average performance. The cross-validation result is presented using a standard measure called the Area Under Curve (AUC) of Receiver Operating Characteristic [32] curve. The ROC curve (examples are shown in Figure 3C) quantifies the sensitivity and specificity of classification by estimating true and false positive rates over all threshold values, and the AUC summarizes the curve with a single number. A random predictor receives an AUC of 0. 5, and a perfect predictor achieves an AUC of 1 [32]. By comparing the AUC of each individual chromatin feature in Kc, S2 and BG3 cells, we observed that many active chromatin marks could distinguish functional from non-functional MREs (Figure 3A). Among all the features tested, H3K36me3 was the best predictor in all three cell lines (mean AUC of 0. 884), followed by JIL-1 (mean AUC of 0. 864). Interestingly, we had previously speculated that H3K36me3 might be involved, based on MSL affinity for this mark in active genes [8], [12], [14]. Since H3K36me3 and JIL-1 are enriched in Kc cells at predicted CES even without MSL binding, they could prime functional MREs for sequence-specific MSL binding, or be coincident with true causative factors. Nearly all putative CES in Kc cells are embedded in a chromatin environment enriched for H3K36me3 (Figure 3B), however, we previously determined that when H3K36me3 is depleted, there are still enough features for the MSL complex to distinguish MREs [12]. Since no single feature may be sufficient to drive MSL recognition, we next asked whether combinations of marks and local sequence composition might further improve predictive power for CES. On average, the GC content of CES is similar to the random or best MREs in the 1 Kb of sequence immediately surrounding the motif, but the GC content consistently rises in flanking sequences, to produce a distinctive average profile (Figure 1E). Examination of the individual heatmaps confirms that this is a broadly consistent characteristic of CES (Figure S2). We found that the relative GC content in D. melanogaster is elevated in genes compared to intergenic sequence (44% vs. 41%) [33] so it is not surprising to see this characteristic in conjunction with the active gene clusters where CES are found. However, the distinctive shape of the profile, with low GC immediately surrounding the CES, is unexpected and not seen with autosomal MREs, even when associated with H3K36me3 and thus presumably analogous active gene clusters (Figure S2). The significance of this intrinsic feature clearly merits future experimental analysis. However, GC profile alone does not appear to provide enough information to predict functional MREs with high accuracy (Figure 3C). To search for combinatorial marks that might distinguish functional MREs, we compared the predictive power of the SVM generated using every possible combination of features in our dataset (Figure S3A–S3B). We found that the best individual features (H3K36me3 and JIL-1) performed very well, similarly to the best combinations of features when SVM trained using Kc data, and tested on S2 or BG3 data (Figure 3C). There are many combinations of features that predict functional MREs with high accuracy. In general, the best performing combinations included the following: (1) H3K36me3 or JIL-1; (2) H2B-ubiq or H3K9ac; (3) a core histone; and (4) GC content (Figure S3A–S3B). The excellent performance of this combination is consistent with the identification of these factors as core features by feature correlation analysis (Figure S3C). The cross-validation results are summarized in the ROC plots in Figure 3C and Figure S3D. Although not perfect, the best combination separates the functional and non-functional MREs with high accuracy (mean AUC = 0. 931), as visualized in principal component space in Figure 3D. Each MRE can be considered as a point in multi-dimensions, with each axis as the enrichment level for a mark; to show the data in two-dimensions, we define new axes, called principal components, which are combinations of the original variables satisfying certain desirable properties. We can indeed observe a good separation of the functional from non-functional MREs in this view (Figure 3D). The analyses presented so far focused on only a subset of clearly functional and non-functional MREs. This allowed us to effectively identify chromatin features that can distinguish functional MREs from non-functional ones, and to build a predictive SVM model for functional MREs. Here we extend our analysis to test whether our model could accurately select additional functional MREs genome-wide. We trained an SVM model with Kc cell chromatin features and then tested its ability to eliminate non-functional MREs on autosomes and chromosome X using S2 data. The SVM algorithm selects the decision threshold that optimally separates functional from non-functional MREs, as confirmed in Figure S3E, and the overall AUC of this prediction is about 0. 84 (Figure S3F). We specifically tested different individual and combinations of features. Using the best combination of features, our SVM model can eliminate over 75% of candidate MRE sites on X, and ∼85% of candidate sites on autosomes, while retaining almost all of the functional MREs on the X chromosome (up to 94%) (Table S2). Almost 10,000 non-functional sites are eliminated using our model, with retention of approximately 1600 MREs genome-wide. Approximately half (763) of those remaining MREs map to the X chromosome but are not included in the conservative set of 137 CES. We suspect that the large number of remaining MREs on the X chromosome indicates that there may be more true MSL binding sites than the set of 137 CES we used in this study. Therefore, we asked how many of these additional 763 MREs overlap with previously mapped MSL binding sites identified by MSL3 ChIP-seq [8]. Of the 763 SVM-predicted functional MREs on X, 503 overlapped with an MSL binding site (Figure 3E). This suggests that the actual false positive rate on the X chromosome may be as low as 260/3343 = 7. 8%. This is slightly lower than the false positive rates for the autosomes (895/8144 = 11%) (Table S2), likely due to the fact that even though most strong peaks identified in the MSL3 ChIP-seq data with stringent criteria are CES [8], some may be sites to which MSL complex spreads. In addition to enrichment for marks associated with active genes, we also saw relative depletion of histone H1 over functional MREs (Figure 1), and, from previous work, depletion of H3 and thus presumably nucleosomes themselves [8], [10]. Therefore, we examined modENCODE data from S2 and Kc cells on the release of nucleosomes following salt extraction to determine whether functional MREs are packaged into more labile chromatin when compared to non-utilized MREs [34]. We found that in male S2 cells functional MREs are depleted for histones in general, and enriched for nucleosomes that are extracted in low salt or remain in the pellet after high salt extraction (Figure 4), both fractions that were previously characterized as enriched in regulatory regions [35]. In addition, the two sets of non-functional MREs on the X chromosome appeared to have a milder, but discernable increase in nucleosome lability when compared to the MREs on autosomes, consistent with the observation that X chromosome in male S2 cells generally adopts a more open chromatin conformation [36]. In contrast, CES MREs in female Kc cells exhibit a modest average decrease in nucleosomal occupancy (Figure 4), but intriguingly, this appears to be a difference at a subset of sites rather than the entire set (Figure S4). Notably, the entire X chromosome in Kc cells does not appear to be packaged in a more open chromatin state compared to autosomes (Figure 4). We conclude that strong nucleosome depletion and a more open chromatin conformation on X are mainly consequences rather than causes of MSL binding. Once MSL complex identifies the male X, we have proposed that it spreads to affect the active genes on the chromosome as a whole [5]. In addition to the core MSL complex consisting of proteins that are essential in males but not females (MSL1, MSL2, MSL3, MOF, and MLE), the JIL-1 kinase is known to be enriched on the male X [37]. JIL-1 is essential in both males and females and binds interband regions on all chromosomes in both sexes [38]. Since we observed that JIL-1 is enriched at functional MREs (Figure 1 and Figure 3A), we tested whether it is also associated with active gene bodies. We constructed average scaled profiles of JIL-1 binding (meta-gene profiles) of all genes greater than 2 kb in length based on the FlyBase dm3 gene annotation [39]. Gene expression in the three cell lines was determined by RNA-seq data (Figure S5) [34]. We found that JIL-1 binds active gene bodies, with a bias towards 3′ ends, on all chromosome arms (Figure 5A). On the male X, this pattern is increased in its intensity above the level on autosomes and correlates strongly with binding of the MSL complex since the increased occupancy is not seen on female X chromosomes (Figure 5B). These results are consistent with previous polytene immunostaining [37] and ChIP analyses [40]. In contrast to JIL-1, the linker histone H1 shows depletion on active genes (Figure 5C). Interestingly, in male cells this depletion is more prominent on X-linked gene bodies than on autosomal gene bodies, whereas X and autosomes show no obvious difference in female cells (Figure 5D), consistent with previous polytene immunostaining results [41]. These two results further underscore the distinct character of the Drosophila male X chromosome once MSL complex is bound to active genes. When examining our modENCODE data, we noticed that many additional data sets are slightly enriched on the entire X chromosome compared to autosomes in male S2 cells, but not in female Kc cells (Figure S6, and Table S3). X-chromosome-wide enrichment of nucleoporins, Megator and Nup153 have also been observed in male S2 cells, but not in female Kc cells [42]. These results are unlikely to simply reflect increased access of antibodies to X chromatin in the ChIP procedure, because histone H1 shows the opposite trend. Therefore, these enrichments may be related to dosage compensation of the male X, either directly, as is the case for H4K16ac, JIL-1, and possibly RNA pol II, or indirectly, as a consequence of the more open chromatin environment created by the dosage compensation mechanism. MSL-dependent dosage compensation is thought to be an organism-wide phenomenon in Drosophila males, excluded from the germline [43], but otherwise not restricted to particular tissues or developmental time points. Classical mitotic recombination experiments support a requirement for MSL proteins throughout development in dividing tissues [44]. In addition, comparison of gene expression in males and females demonstrates that the vast majority of X-linked genes are up-regulated in males [45]. However, stable MSL binding appears to be more restricted than its functional consequence, H4K16 acetylation [22], so we wondered whether binding favored particular types of genes. To examine this, we plotted the chromatin marks and MSL binding along active X linked genes in S2 cells (>2 Kb long), asking if clustering might define genes of particular structure, expression level, or gene ontogeny category (Figure 6). We found that MSL1 and H4K16 acetylation cover the vast majority (86%) of active X linked genes. Apparent exceptions (green cluster) are interesting as those genes also lack H3K36me3 (Figure 6) and H2B-ubiquitin (data not shown), two prominent marks of transcription [18]. While MSL1 and H4K16ac are associated with the bodies of virtually all active X linked genes in male S2 cells, the MOF H4K16 histone acetyltransferase was notably absent from a subset (dark brown cluster at bottom of Figure 6). Most of these genes still showed limited MOF enrichment around the promoter region, but not within the gene bodies as is characteristic of MSL complex. We were unable to identify any feature, such as relative expression level or chromatin state, that would distinguish these genes from the genes that showed MOF enrichment. Since functional MSL chromatin entry sites are also preferentially associated with active gene marks, we asked where CES map relative to the set of clustered genes on X, indicating the location of CES by red dots on the gene structures in Figure 6 (H3K36me3 column). Our results demonstrate that CES are located at variable positions relative to active genes, with a bias towards their 3′-ends. Interestingly, the subset of genes lacking strong MOF binding within gene bodies also lack nearby entry sites, in agreement with the observation that MSL binding is strongest in the close vicinity of mapped chromatin entry sites [10], [46]. Genetic, genomic, and biochemical analyses in eukaryotes have revealed that DNA binding motifs alone are insufficient to explain the selective occupancy or specificity of regulatory factor function [16], [17]. The number of predicted binding sites is often vastly greater than the number of sites actually utilized. Therefore, a very important question in transcriptional regulation is how to identify additional parameters that must govern accurate binding site selection. In this study, we considered the roles of chromatin environment and flanking sequence composition in selection of functional binding sites by a sequence-specific protein complex. It is generally not clear whether the chromatin features that are often observed at the binding sites of proteins contribute directly to binding selectivity or are simply a consequence of binding. In the dosage compensation system of the X chromosome in Drosophila, we had a unique opportunity to address this question because we can compare the chromatin environment of MSL binding sites in female cells, in the absence of the complex, to male cells, where the functional sites are bound. We also utilized binding data from an RNAi experiment in which we knocked down a component of the sex determination pathway in females to induce dosage compensation. Our bioinformatic analysis of a large number of profiles from the modENCODE project suggests that a pre-existing active chromatin context plays a critical role in establishing the initial binding of the MSL complex on the X. We also made the surprising discovery that GC content in the DNA surrounding functional binding sites has a characteristic profile. In summary, our results strongly support a model in which an active chromatin composition helps define the initial entry sites selected by the MSL complex (Figure 7). Functional MSL binding results in increased lability of local nucleosomal composition, and H4K16 acetylation and JIL-1 binding along the bodies of virtually all active X-linked genes. Our work provides key insights into the order of events leading to dosage compensation in Drosophila, and can also serve as a model for using genome-wide data sets to understand how sequence-specific factors find their ultimate targets. The majority of the ChIP-chip data are from the modENOCODE project [18]. Genomic DNA Tiling Arrays v2. 0 (Affymetrix) were used to hybridize both ChIP and input DNA. We obtained the log-intensity ratio values (M-values) for all perfect match (PM) probes: M = log2 (ChIP intensity) −log2 (input intensity), and performed a whole-genome baseline shift so that the mean of M in each microarray is equal to 0. The smoothed log intensity ratios were calculated using LOWESS with a smoothing span corresponding to 500 bp, combining normalized data from two replicate experiments. All data are publicly accessible online through the modENCODE project (URL listed in Table S1). Data analysis was performed in R statistical programming environment (http: //www. r-project. org). For the visualization of the heatmap (e. g., Figure 1), the +/−5 kb region surrounding each MRE was separated into non-overlapping bins of 200 bp. The smoothed probe value within each bin is averaged to obtain the enrichment value for that bin. The GC content around each nucleotide is defined as the proportion of G or C in the closest 101 bp (ie, the target nucleotide, 50 bp upstream and 50 bp downstream). Similar to the ChIP-chip data, we separated the +/−5 kb region surrounding each MRE into non-overlapping bins of 200 bp. The average GC content in each bin represents the average of the GC content of the 200 bp within that bin. We generated double-stranded RNA (dsRNA) to target GFP (negative control) or Sxl transcripts (amplicons designed by the Drosophila RNAi Screening Center (www. flyrnai. org), as described previously [22]. The following primer sets were utilized to amplify PCR products to template dsRNA synthesis: GFP F 5′-TAATACGACTCACTATAGGGAGAGGTGAGCAAGGGCGAGGAGCT-3′ R 5′-TAATACGACTCACTATAGGGAGATCTTGAAGTTCACCTTGATGCCG-3′ Sxl (DRSC21490) F 5′-TAATACGACTCACTATAGGGAGAGATCACAGCCGCTGTCC-3′ R 5′-TAATACGACTCACTATAGGGAGATACCGAATTAAGAGCAAATAATAA-3′ Sxl (DRSC28896) F 5′-TAATACGACTCACTATAGGGAGACCCTATTCAGAGCCATTGGA-3′ R 5′-TAATACGACTCACTATAGGGAGAGTTATGGTACGCGGCAGATT-3′ For expression analyses, GFP and Sxl DRSC21490 RNAi was performed in Kc cells using 6-well plates as described [47]. For ChIP-chip, RNAi using GFP, Sxl DRSC 21490, and Sxl DRSC28896 amplicons in Kc cells was scaled up to T225 flasks and chromatin preparation, and H4K16ac ChIP was performed using anti-H4K16ac antibody (Millipore, 07-329) and custom Nimblegen tiling arrays as described [22]. Kc GFP H4K16ac ChIP-chip datasets were published previously [22]: Gene Expression Omnibus accession numbers: GSM372470 (replicate #1) and GSM372471 (replicate #2). We used 10-fold cross-validation to estimate the predictive power of a classification model based on a training dataset (e. g., chromatin feature in Kc cells) and a test dataset (e. g., chromatin feature in S2 cells). Each sample in a dataset is an MRE, a feature is a histone modification (e. g., H3K36me3) or a chromatin binding protein (RNA Pol II), and the label for each sample is either “Functional MRE” (positive class) or “Non-functional MRE” (negative class). The aim is to train a prediction model that can distinguish functional from non-functional MREs based on the chromatin features. In a 10-fold cross-validation, the training data are randomly divided into 10 equal-sized portions in which the same proportion of positive and negative samples are preserved in each portion. In each of the 10 iterations, the data from nine portions are used to train a predictive model, while the remaining one portion is used to test the performance of the prediction model. Performance is measured by true positive rate (sensitivity) and false positive rate (1-specificity). The tradeoff between true and false positive rates are often represented by a receiver operation characteristic curve, and the Area Under the ROC Curve (AUC) is a measure of the prediction accuracy that takes into account both sensitivity and specificity of the prediction model. A random predictor receives an AUC of 0. 5, and a perfect predictor achieves an AUC of 1. Using 10-fold cross-validation, an AUC is calculated for each fold (one iteration), and the mean and standard deviation of the 10 AUC values are recorded. Calculation and visualization of the ROC curves were performed by the ROCR package [32]. SVM is a supervised classification algorithm that separates two classes of data based on a set of features [30], [31]. In this study, the set of ChIP enrichment at each MRE is treated as a feature vector of that MRE. Given a set of feature vectors from functional MREs and a set of feature vector from non-functional MREs, the SVM algorithm calculates an optimally-separating hyperplane by maximizing the distance (called margin) between the hyperplane and the nearest points from the two classes. This hyperplane effectively divides the space of feature vectors into two regions, one for each class, with the idea that the larger margin lowers the error of the classifier. To make a prediction, the feature vector corresponding to a MRE from the test set is compared to this hyperplane to determine on which side of the separating boundary this sample is located. We used the SVM implementation in the R package e1071, which is optimized for the radial basis function kernel and uses an Sequential Minimal Optimization-type algorithm [48] using default parameters for training the SVM. We used the MSL3-TAP ChIP-seq data from Alekseyenko et al [8]. The raw sequence reads were aligned to the Drosophila melanogaster genome assembly dm3 using bowtie with default options [49]. We only allowed uniquely mapped reads to be reported. This procedure resulted in 2. 8 and 2. 4 million mapped reads for ChIP and input DNA samples. The aligned reads were then analyzed with SPP [50] to identify ChIP-enriched regions (FDR threshold of 0. 05). Gene expression level estimates in S2, BG3 and Kc cells were obtained from the modENCODE project [34]. The expression of each gene is quantified in terms of RPKM (reads per million reads per kilobase). The distribution of gene expression in each cell line was assessed and a cut-off of RPKM = 3 was determined to be a good threshold to separate active vs. inactive genes (Figure S5A). This definition of active vs. inactive genes was used in the construction of meta-gene profiles. We used the gene annotation from FlyBase [39] to define transcription start and end sites (TSS and TES respectively). We only included genes with a minimum length of 2 kb (7,231 of 15,186 genes) to exclude short genes from our analysis. The ChIP enrichment in the 2 kb region centered on the TSS and TES, as well as the ChIP enrichment within the gene body scaled to 1 kb, were calculated and averaged for the active and inactive genes in X and autosomes. The definitions of active vs. inactive genes were defined by RNA-seq data. All ChIP-chip and RNA-seq data are available from modENCODE, and the URL for individual datasets is listed in Table S1. The ChIP-chip and microarray gene expression data pertaining to the Sxl RNAi experiments are accessible from GEO (Accession number: GSE34859).

Title: Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context Summary: The genomes of complex organisms encompass hundreds of millions of base pairs of DNA, and regulatory molecules must distinguish specific targets within this vast landscape. In general, regulatory factors find target genes through sequence-specific interactions with the underlying DNA. However, sequence-specific factors typically bind only a fraction of the candidate genomic regions containing their specific target sequence motif. Here we identify potential roles for chromatin environment and flanking sequence composition in helping regulatory factors find their appropriate binding sites, using targeting of the Drosophila dosage compensation complex as a model. The initial stage of dosage compensation involves binding of the Male Specific Lethal (MSL) complex to a sequence motif called the MSL recognition element [1]. Using data from a large chromatin mapping effort (the modENCODE project), we successfully identify an active chromatin environment as predictive of selective MRE binding by the MSL complex. Our study provides a framework for using genome-wide datasets to analyze and predict functional protein-DNA binding site selection.

lay_plos

en

Summarize: The earliest known colour footage of the Beatles showing the Fab Four backstage at a concert in Blackpool has surfaced for the very first time. The three-and-a-half minute film, that has never before been seen, shows John, Paul, George and Ringo at the ABC Theatre in August 1963. The footage, that has no sound, was shot on an 8mm film by Chas McDevitt of the Chas McDevitt Skiffle Group, which was one of the support acts for the Beatles that night. A still from the earliest known colour footage of the Beatles showing the Fab Four backstage at a concert in Blackpool. The footage features all four members of the group, including John Lennon, pictured. It was filmed by Chas McDevitt of the Chas McDevitt Skiffle Group, which was one of the support acts for the Beatles that night. Some of the film shows the band performing live and was recorded in the wings of the stage by Mr McDevitt. The footage has now been made available for sale with copyright at auction alongside more than 100 photos of the Beatles that have also never been seen before. The black and white snaps were taken by Eve Bowen during the band’s tour of America in 1965 at the height of Beatlemania. The photographer followed the band as they travelled by train from Washington to New York where they appeared and performed on the Ed Sullivan Show. Her photos show one of superstar John Lennon having to wait behind the buffet trolley as it wheels its way down the narrow train aisle while passengers sit either looking on. A sign on the trolley advertising cheese and pickle sandwiches and a can of Coca Cola for 60 cents shows the snacks the group might have enjoyed on the journey. John Lennon, pictured waiting behind a buffet trolley, while the Beatles travelled by train from Washington New York as part of their US tour in 1965. George Harrison listening to music on a pair of headphones, left, and John Lennon holding a newspaper, right. The pictures of the Fab Four were taken by photographer Eve Bowen, and the 109 original negatives she took are expected to fetch £12,000 at auction. There is also a snap of George Harrison relaxing while listening to a pair of white earphones, not too dissimilar to the standard Apple ones used today. While another shot shows the hoards of screaming fans which greeted the Beatles as well as a snaps of them performing on the Ed Sullivan Show taken by Ms Bowen from the audience. The images, that are also being sold with full copyright, are all previously unseen and unreleased. The 109 original negatives are valued at £12,000 while the colour footage is estimated at £9,000. Both lots were acquired by Mark Hayward, a well-known Beatles collector, who is now selling them at Ewbank’s Auctioneers of Woking, Surrey. Alistair McCrea, of Ewbank’s, said: 'The film footage is the earliest known colour footage of the Beatles. Ms Bowen also took snaps of the group from the audience while they performed on the Ed Sullivan TV show. The pictures are being auctioned at Ewbank’s Auctioneers of Woking, Surrey next month. John Lennon, singing, left, and Ringo Starr playing the drums, right, while performing on the Ed Sullivan show. The hoards of screaming fans which greeted the Beatles everywhere they went as the toured across America. 'There might be some earlier film still stuffed in someone’s drawer but we don’t know of any earlier colour footage than this. 'If you think about it, this was 1963 and not that many people had access to colour cameras at that time. 'The film shows the Beatles messing about back stage, chatting and laughing and reading fan letters. There is some footage of them on stage that was shot from the wings. 'Unfortunately it is silent and is a little bit eerie in a way. The full collection of footage and documentation, which is up for sale, which includes a letter of provenance from Chas McDevitt, who took the footage. 'The same owner who has consigned the negatives has recently had half-a-dozen 6ins by 4ins print made to show the market what they are like. 'They are unusual because they are quite informal and intimate.' The colour 8mm footage also comes with a DVD version of it and a letter of provenance from Mr McDevitt, who is still alive. The sale takes place on February 5

Summary: Three and a half minute clip was filmed at the ABC Theatre in Blackpool. Shows the Fab Four backstage on the night they performed in August 1963. Was shot by a member of one of the support acts for the band that night. New pictures have also emerged of the Beatles tour of the U.S. in 1965. Black and white snaps show group on train from Washington to New York. Rare images also show them performing on the Ed Sullivan TV Show.

cnn_dailymail

en

Write a title and summarize: Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR) which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR) analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses). The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators) were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5α-androstan-3β-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches. Promiscuous proteins generally bind a large array of diverse ligand structures. These proteins include enzymes like cytochrome P450s (e. g. CYP3A4, EC 14. 13. 97), transporters such as P-glycoprotein (ABCB1), the human ether-a-go-go related gene (hERG, Kv11. 1) potassium channel and nuclear hormone receptors (NHRs) such as the pregnane X receptor (PXR; NR1I2; also known as SXR or PAR) [1]. This promiscuous binding may be facilitated by a very large binding site, multiple (overlapping) binding sites, or a flexible binding site that can adjust to the size of the ligand. Intrinsic disorder in the protein may also have a role [2], [3]. These proteins described above are also particularly important as xenobiotic sensors and represent key mechanisms to respond to toxic stress. The human PXR [4]–[6] transcriptionally regulates genes involved in xenobiotic metabolism and excretion, as well as other cellular processes such as apoptosis [7]–[11]. Human PXR has a very broad specificity for ligands as exemplified by the structurally diverse array of activators including endogenous (bile acids, steroid hormones, fat-soluble vitamins) and exogenous (prescription and herbal drugs, and environmental chemicals) compounds. Activation of human PXR can cause drug-drug interactions [4], [5] or result in physiological effects ranging from ameliorating cholestatic injury to the liver, altering bone homeostasis, and causing cell proliferation [12]. As PXR represents a potential target for pharmacologic modulation in disease, it is therefore becoming even more important to develop methods that can identify whether a molecule is likely to be a PXR agonist [13]. Currently there are five high-resolution crystal structures of human PXR [14]–[18] available in the Protein Data Bank (PDB) (and another structure to be deposited [19]). The structures have provided atomic level details that have led to a greater understanding of the ligand binding domain (LBD) and the structural features involved in ligand-receptor interactions [9], [10], [12]–[15]. The co-crystallized ligands include the natural products hyperforin (active component of the herbal anti-depressant St. John' s wort) and colupulone (from hops), the steroid 17β-estradiol, the synthetic compounds SR12813, T1317 and the antibiotic rifampicin. These ligands span a range of molecular sizes (M. Wt range 272. 38 – 713. 81Da, mean 487. 58±147. 25Da,) and are predicted as generally hydrophobic (calculated ALogP [20] 3. 54–10. 11, mean 5. 54±2. 41). The cavernous ligand binding pocket (LBP) with a volume >1350 Å3 accepts molecules of these widely varying dimensions and chemical properties, and is likely capable of binding small molecules in multiple orientations [21]. This complicates overall prediction of whether a small molecule is likely to be classified as a PXR agonist using traditional structure-based virtual screening methods like docking [13], [22]. With regard to this, we have previously shown that the widely used structure-based docking methods FlexX and GOLD performed relatively poorly in predicting human PXR agonists [7], [16] and this is perhaps not surprising based on the observations described above. An alternative method, which has been found to be valuable elsewhere in drug discovery, particularly when there may not be an available crystal structure of the target protein, uses a ligand-based approach. In this case a series of small molecule structures with PXR agonist activity data can be used to facilitate a structure activity relationship (SAR). When the biological activity data is continuous this will enable a quantitative structure activity relationship (QSAR) [23]–[25]. One widely used computational technology produces pharmacophores [20]–[23], which represent models that encode the key chemical features important for biological activity. Human PXR agonist pharmacophore models have been shown to possess hydrophobic, hydrogen bond acceptor and hydrogen bond donor features, consistent with the crystallographic structures of human PXR ligand-receptor complexes [26]–[29]. These pharmacophore models have predominantly used structurally diverse ligands in the training set and have the limitation in most cases of compiling data from multiple laboratories using different experimental protocols, ultimately forcing binary classifications of ligands for the training sets (i. e., activating versus non-activating). Most of the models so far use EC50 data, a measure of receptor transactivation. Although binding assays have been done with human PXR, they are problematic given the low affinity of most PXR activators. As a result, there is little radioligand binding data in the literature other than competition experiments with radiolabeled SR12813. To date there have been few attempts to build ligand-based models around a large structurally narrow set of PXR activators. The absence of large sets of quantitative data for PXR agonists has restricted QSAR models to a relatively small universe of molecules compared to the known drugs, drug-like molecules, endobiotics and xenobiotics in general [30]. The PXR data limitation has resulted in the use of various machine learning methods (e. g support vector machine, recursive partitioning etc.) when the biological data is binary in nature (e. g. activating or binding versus non-activating / non-binding) [13], [22], [25], [30]. As part of an ongoing analysis of NHRs [25]–[28], we have generated a large cadre of experimental data for classes of steroidal compounds, namely androstanes, estratrienes, pregnanes and bile acids/salts [31]. The advantages of using steroidal compounds for QSAR are that they are amenable to common alignments based on the steroidal backbone. For, example steroids represented the first datasets used for comparative molecular fields analysis (CoMFA) [32] and have been widely used as a benchmark for other methods such as comparative molecular similarity analysis (CoMSIA) [33]. Pharmacophore methods, in contrast, generally do not require the rigid alignment methods and have found use with more diverse structure sets [28], [31]. Using this large quantitative data set of PXR activators, we applied various ligand-based computational methods including Bayesian modeling with 2D fingerprints. We also compared the results from QSAR approaches to molecular docking into the six available human PXR crystal structures. Modeling of a broad specificity receptor such as PXR represents a challenge for in silico modeling and it is invaluable to know what approaches prove successful, if any. Ideally, these methods will also translate to modeling approaches for other broad specificity enzymes, transporters and ion channels [1], or other promiscuous proteins [34]. We are not aware of any similar studies using a comparative approach to predicting ligand-protein interactions for promiscuous proteins. This study also provides further insights into PXR-steroid interactions which have not been well studied [19] and is clinically relevant due to the widespread use of steroidal compounds and steroid mimics (e. g. oral contraceptives [35], for inflammation and as cancer treatments etc.) in clinical medicine [36], as well as the increasing problem of environmental contamination by endocrine disruptors [24]. All compounds shown in Table S2 were docked to the six human PXR crystal structures using GOLD which we have used previously for docking diverse compounds into the human PXR structure [22]. All six crystal structures superimposed with a backbone root mean squared deviation of 0. 5 Å suggesting that they had very similar structures and their co-crystallized ligands bound to the same binding pocket (Figure S1). The docking scores for all the compounds (Table S2) were in the range of 36 to 77 for all the crystal structures and their corresponding Tanimoto similarity scores to 5α-androstan-3β-ol and the crystal ligand 17β-estradiol using MDL public keys were between 0. 4 and 1. To evaluate docking results, we compared docking scores for classifying compounds as activators or non-activators of PXR. Using an EC50 value of 10 µM as a cutoff the compounds listed in Table S2 were classified as activators (30 compounds) and non-activators (89 compounds). These results were compared to the classification obtained from the docking studies. The overall accuracy (Q values) were in the range of 35 to 55 % for models that used 5α-androstan-3β-ol based similarity scores as weights to the goldscore, while the Q values were in the range of 47 to 58% for models that were generated with goldscores weighted with 17β-estradiol based similarity scores (Table 1). The Matthews coefficient C showed a modest prediction rate with the best score for docking of compounds to PXR crystal structure 1M13. Further changing the cutoff values to either 100 µM or 40 µM did not improve the prediction rates. The Q value for a model computed by averaging all the models with 5α-androstan-3β-ol weighted goldscore was 46% and for the average model with 17β-estradiol the weighted goldscore was 51%. Although the overall performance of docking produced rather modest results for classification the results for individual classes of compounds was better than average. In the best classification model (compounds docked to crystal structure 1M13 and weighted with 17β-estradiol based similarity scores), 20 out of 30 PXR activators and 49 out of 89 non-activators were predicted correctly. Among the androstanes, 6 out of 11 compounds were predicted correctly as activators and 9 out of 14 compounds were classified as non-activators (Table S2). Among the bile salts, all 4 activators and 22 out of 46 non-activators were predicted correctly. Among the estratrienes, 5 out of 7 activators were predicted correctly, while the 4 non-activators were predicted as activators (Table S2). The reason for this mis-classification was due to the high similarity scores of the estrogens with 17β-estradiol. In the pregnane class, 4 out of 7 activators and 16 out of 20 non-activators were correctly classified (Table S2). Some examples of molecules in their binding modes with PXR structure 1M13 are shown in Figure 1. All 115 compounds shown in Table S2 were used to generate a Bayesian classification model [37], using a definition of active as a compound having an EC50 for PXR activation of less than 10 µM. Using molecular function class fingerprints of maximum diameter 6 (FCFP_6) and 8 interpretable descriptors (AlogP, molecular weight, rotatable bonds, number of rings, number of aromatic rings, hydrogen bond acceptor, hydrogen bond donor and polar surface area) a model was developed with a receiver operator curve (ROC) statistic for leave one out cross validation of 0. 84. In addition to the leave one out cross validation, further validation methods were undertaken. After leaving 20% of the compounds out 100 times the ROC is 0. 84±0. 08, concordance 73. 2 %±8. 94, specificity 69. 14%±12. 12, and sensitivity 84. 11%±18. 04. The Bayesian method appears to have good model statistics for internal cross validation of steroids. These statistics suggest the model is stable and not over-trained as the ROC values are essentially identical to that obtained with leave one out cross validation. We have additionally used this model to classify a previously used diverse molecule test set [13], [22]. After removing the steroids from the test set, the Bayesian PXR model was used to rank 123 molecules (65 activators and 58 non activators). Out of the top 30 molecules scored and ranked with this model 20 (75%) were classified as activators (EC50 <100 µM) (Table S3). Even though the cutoff for activity for the Bayesian model is more stringent it still appears to be able to predominantly pick out the key molecular features that contribute to activity in non-steroidal compounds. The Bayesian model with FCFP_6 descriptors also enabled the visualization of substructure fingerprints (Figure 2) that either contributed positively or negatively to the activity classification. It appears that all positive contributing substructures are essentially hydrophobic, while negatively contributing features possess hydroxyl or other substitutions which are likely not optimally placed to facilitate interactions with hydrogen bonding features in PXR. Therefore possession of these hydrogen bond acceptor and donor features indicated in the steroidal substructures appears to be related to loss of PXR activation. The method does not readily identify where these groups should be added in contrast to methods like docking [13], [38]. A major challenge in CoMFA and CoMSIA modeling is alignment of molecules, which must be defined by the user. As described in Text S1, multiple alignment approaches were attempted. Despite the use of multiple alignments, the best CoMFA and CoMSIA models consistently showed a large difference between the correlation R2 and cross-validated (XV-R2), whether modeling the entire set of steroidal compounds or the various subsets (androstanes, bile salts, pregnanes). This suggests that the CoMFA and CoMSIA models do not generalize beyond the molecules in the training set, even for a subset of steroidal compounds (Text S1, Tables S4, S5, S6, S7 and Figures S2, S3, S4, S5, S6, S7). Using the pharmacophore approach for the individual steroids, the training set r values were quite low but increased upon inclusion of excluded volumes with variable weight and tolerances (0. 81–0. 93) (Table S8). All PXR pharmacophores (Figure S8) had at least 2 hydrophobes and a hydrogen bond acceptor in common (Text S1). Using the pharmacophores derived from training sets based on subsets of steroidal compounds (e. g., androstanes only) to predict the other respective subsets did not result in reliable correlations (data not shown), suggesting that highly specific pharmacophores were generated or this may be due to the addition of the excluded volumes which limits the chemical space of molecules mapping to the features. These class-specific pharmacophores may therefore only be useful for making predictions of very closely related molecules and even crossing steroidal classes may be extrapolating too far beyond the training sets. 4D-QSAR performed somewhat better than CoMSIA and CoMFA in modeling the compounds in the training sets using three atom alignments (Table S9). One potential advantage of 4D-QSAR relative to standard 3D-QSAR methods is the ability to consider an ensemble of different ligand conformations, theoretically increasing the chances of defining the active conformation. The best 4D-QSAR models are found in Table S10 and Figure S9, and generally predict steric/non-polar interactions between ligand and receptor. Although the XV-R2 for the best 4D-QSAR models are better than for CoMFA and CoMSIA models of the same training sets, the 4D-QSAR were poorly predictive of the activity of compounds in the test set (Table S10). 4D- and 5D-QSAR have the advantage of being able to select the bioactive conformation from a pool of possible binding modes in parallel to the QSAR modeling stage. We have tested three different alignment protocols in conjunction with the 5D-QSAR technique Raptor. In the alignment protocols (2) and (3) the protein crystal structure was used as a forbidden (excluded) region. A penalty was added to the similarity score for alignment solutions that overlapped with the protein, thus physically impossible solutions were removed from the alignment. As significant protein flexibility is observed on the side chain level, all crystal structures were aligned using PyMol [39]. Side chains that have different rotamer states for different co-crystallized ligands were removed from the forbidden region definition. Our multidimensional QSAR study (software Raptor [40]) was based on the same set of 115 molecules as described in the CoMFA and CoMSIA studies. The dataset was split into 95 training set compounds, and 20 test set compounds identical to the separation used in the CoMFA and CoMSIA studies. For 33 compounds only an upper limit for their Ki values has been experimentally determined. These molecules defined the “threshold class” (26 training, 7 test). A threshold value of 100 µM was chosen considering that the lowest affinities were measured for this dataset at approximately this value. To allow for topological and physicochemical variation at the true biological receptor with different ligands bound, the Raptor results were averaged over 10 individual models defining a surrogate conformational family. For alignment (1) we were not able to derive QSAR models with predictive models for leave-5-groups-out (r2CV-5) or cross-validation values (i. e. >0. 3). This is not surprising, as the identification of bioactive binding modes using docking is difficult for this system (see docking results). If we use an alignment with only the top-1 or top-2 solutions, we most probably end up with an alignment containing incorrect binding modes. Using the top-10 or top-20 binding modes generates too large a variety of contacts between ligand and binding site model that the QSAR algorithm is not able to extract the critical interactions throughout the binding site modeling phase. For alignment (2) a QSAR model with a r2CV-5 value of 0. 55 could be generated, but with no observed correlation for the test set. For alignment (3) a QSAR model with an r2CV-5 of 0. 56 was derived with a predictive r2 for the test set of 0. 45. The superior model based on alignment (3) was due to the focused class-based alignment process (Figure 3). The maximum deviation of predicted from experimentally measured EC50 is 5. 6 and 3. 0 fold for training and test set, respectively. Significantly higher regression coefficients can hardly be expected for this dataset considering the fact that the threshold compounds have to be removed from the calculation of the regression coefficients yielding a rather small range in EC50 of 2. 2 log units (Figure S10, Table S11). This is in contrast to the CoMFA and CoMSIA simulations where the threshold compounds have been assigned an EC50 value of 10,000 µM yielding a range of 4. 1 log units. All except one of the 33 threshold compounds have been predicted with an EC50 value lower than the given threshold or maximally a factor of 6. 6 fold higher. Only 5α-Androstane was predicted to have a 46 fold higher value than the threshold. Thus, the model was able to predict the affinity of compounds accurately and at the same time was able to classify weak- or non-binding molecules correctly. It has been suggested that PXR forms a heterotetramer and exhibits a range of motions which are key for its functioning and preparing for coactivator binding at the Activator Function (AF-2) site [41]. The large and promiscuous ligand binding pocket of PXR accepts molecules of widely varying sizes (Table S1), and is likely capable of binding small molecules in multiple orientations. Furthermore, movement of regions of this pocket may be translated elsewhere in the protein to influence protein-protein interactions. Thus, the identification of the bioactive conformation of a ligand binding to PXR (and the effect it might have as an agonist, antagonist or allosteric antagonist [10]) and development of a ligand alignment based on these conformations represents a challenge for any computational technique. A realistic ligand alignment, however, is the basis for a reliable 3D-QSAR model. Computational methods including QSAR (3D, 4D and 5D), pharmacophores and machine learning classification models for PXR can assist in rapid prediction of whether a compound is likely to be an agonist (activator), however each method has its limitations and advantages (Table 2). For example a previous study used human PXR activation data for 30 steroidal compounds (including 9 bile acids) to create a pharmacophore with four hydrophobic features and one hydrogen bond acceptor [27]. This pharmacophore contained 5α-androstan-3β-ol (EC50 0. 8 µM) which contains one hydrogen bond acceptor, indicating that in contrast to the crystal structure of 17β-estradiol (published EC50 20 µM) bound to human PXR with two hydrogen bonding interactions [19], hydrophobic interactions may therefore be more important for increased affinity [27]. This and other pharmacophores have been used to predict PXR interactions for antibiotics [35] which were verified in vitro, suggesting one use for computational approaches in combination with experimental methods. To our knowledge there has been no comparative analysis of the steroidal classes with respect to their use as PXR agonists. The use of the Bayesian classification with 2D fingerprints represents a low computational cost approach [42] which has been used frequently with large molecule datasets [43]–[46]. Using 2D-molecular fingerprint descriptors identified regions in the training set molecules that were predominantly hydrophobic and that were important for PXR activation. Substructures with free hydroxyls as hydrogen bonding features were associated with compounds that were not activators. This is in general agreement with other studies which have used docking to try to help design out PXR activation [38]. This model was able to successfully rank a large test set (Table S3) of non-steroidal molecules, indicative that the molecular descriptors adequately captured the global properties of PXR agonists and suggests some utility. The current study suggests that while it is generally possible to create 3D-QSAR (CoMFA, CoMSIA, Catalyst) and 4D-QSAR models that can be cross-validated, these models perform poorly when used to predict external molecules. Only the 5D-QSAR model generated displays some success in predicting external test set steroidal compounds. Three main differences between the 5D-QSAR and the 3D-QSAR studies that might contribute to the difference in performance are the less rigid alignment using Symposar [40], the possibility to present a ligand in more than one binding pose and the better treatment of weak or non-binding compounds. Pharmacophore models for the 4 classes of steroidal compounds possessed some of the features in the published human PXR crystal structures, however the models contained two or three hydrophobic regions (rather than four as shown previously) [27], [28], [31] and one to two hydrogen bond acceptors or a hydrogen bond acceptor and hydrogen bond donor (compared to one hydrogen bond acceptor as shown previously). This might suggest that the steroids evaluated occupy just a part of the ligand binding pocket while larger molecules like rifampicin occupy most of the binding pocket and have subsequently many more interactions with the protein [17]. The addition of the excluded volumes to the pharmacophores was shown to improve the correlation for the training sets and likely acts in a similar manner to using the crystal structures in 5D-QSAR. Consistent with the QSAR findings were those from docking studies that though modest in success overall, fare much better with individual classes of compounds. The classification was performed using two similarity weighted scoring schemes: one based on a highly potent compound 5α-androstan-3β-ol and the other based on a structurally relevant compound 17β-estradiol. The goal was to test the utility of biasing the scoring scheme with either a structurally relevant compound or a functionally significant compound. However, in this case 17β-estradiol and 5α-androstan-3β-ol share nearly 75% structural similarity (using MDL Keys and Tanimoto similarity coefficient). The results from the classification studies showed that biasing the scoring scheme with a structurally relevant compound (17β-estradiol) produced classification rates with sensitivity and specificity values averaging at 52% and 50% respectively with slightly better prediction accuracy (Table 1). These results unfortunately cannot be compared with our recent docking study [47] as a different co-crystal ligand was used for the scoring scheme. Although the structure biased scoring scheme performed better among all the compounds, both the scoring schemes performed equally well when individual classes were considered. In the case of androstanes, 6 out of 11 compounds were correctly predicted as activators in docking studies. 5α-Androstan-3β-ol that had the lowest EC50 value (described earlier) was predicted to be an activator in all structures. 5α-Androstan-3β-ol binds with very high docking scores and has a hydrogen bond interaction with His407, a key interaction of PXR (Figure 1A). This interaction was consistent among all the androstane activators. However, epitestosterone sulfate has an EC50 of 3. 39 µM and was misclassified in the combined model using predictions from all structures as a non-activator. Docking studies show that epitestosterone sulfate has a consistently reversed docking pose (when compared with 5α-Androstan-3β-ol) in all the models and the sulfate group is predicted to make a hydrogen bond interaction with His407, as opposed to the steroid ester in 1M13 structure (Figure 1B). A few other misclassified activators were docked in reversed poses and often had favorable hydrogen bonding partners such as sulfates that probably influence the binding mode of these steroids. This is a surprising and novel finding of this study and other researchers should be aware of this when docking similar compounds with this functional group. Among the bile salts, all four activators were correctly predicted and the ligands bind in a conserved mode with the steroid esters participating in favorable interactions with the side chain of His407 and Arg410, and the steroid rings with hydrophobic groups such as Leu411, Leu239 and Phe281 (Figure 1C). The pregnanes had similar activation patterns as the bile salts and docking studies could predict 4 out of the 9 compounds correctly. Among the misclassified compounds, levonorgestrol was predicted to be an activator in three models, and a non-activator in three models and hence could not be classified with high confidence. Levonorgestrol has an EC50 of 4. 30 µM and is predicted to have favorable interactions with hPXR as shown in Figure 1D. Despite this, the similarity weighted scoring functions generally performed well in classifying activators as described in the examples above and by the sensitivity values in Table 1. The paucity of available PXR binding data may limit some of the insights from docking experiments performed to date. It is not surprising that CoMFA and CoMSIA do not perform well as they use rigid alignments of the molecules. This is potentially a seriously limitation given that the binding pocket of PXR may accommodate multiple orientations of the steroids (Figure 1A vs. Figure 1B). Theoretically, 4D- and 5-QSAR should perform better by considering an ensemble of ligand conformations and in fact 4D-QSAR does well within subsets (especially androstanes) but like all methods extrapolates poorly. 5D-QSAR appears to perform the best with the test set. Alignment independent methods like Catalyst which can deal with structurally diverse molecules can generate pharmacophores for the individual classes of compounds but their inter-class predictivity is limited. Another alignment independent method such as using 2D fingerprints and descriptors with the Bayesian classification approach may represent a fast approach to screen for potential PXR agonists, but like all methods their applicability domain [48], [49] is dependent on the training set. In this case the set of steroids would be expected to limit the utility of such models to a relatively narrow class of compounds, although it may be picking up key features in more diverse molecules (Table S3) suggesting overlap in the chemical space. This study shows the inherent difficulty of producing predictive ligand or structure-based computational models for PXR. Some of the methods used are ligand alignment dependent while others are alignment independent, and each has limitations when used with flexible proteins. These computational models also confirm some of the molecular features (hydrophobicity and hydrogen bond acceptors) identified in previous models and structures, while using a large quantitative dataset to create new QSAR, classification and pharmacophore models to test docking and scoring. The study represents an initial step comparing multiple methods focused on steroidal compounds rather than a more diverse series of drug-like molecules. Using a more diverse series of molecules would have been expected to present even more difficulty for the alignment dependent methods such as CoMFA and CoMSIA. There are also many more commercial computational methods that could be evaluated and compared, although we have used several 3D, 4D, 5D-QSAR methods, machine learning with 2D descriptors, pharmacophore and GOLD docking and scoring methods in this study. The results from these methods could be used in combination as part of a consensus approach or Pareto optimization [50]. The provision of the 115 molecule human PXR dataset is potentially useful as a benchmark PXR set for testing further methods in future. For example, flexible docking methods [51] could be used as well as algorithms that could differentiate multiple binding mechanisms [52]. In conclusion, there are many promiscuous proteins [34] where the modeling of ligand-protein interactions is complicated by a large binding site, multiple binding pockets, protein flexibility or all of the preceding. We have applied several different computational approaches which could also be applied to other proteins like CYPs, transporters and ion channels. This work is therefore more broadly applicable in an attempt to predict whether molecules bind in such flexible proteins, and which methods perform the best. Depending on the desired use of such information, different modeling methods may be appropriate and required. While 2D methods do not encode 3D information like shape [53] they are fast and they can highlight important features likely interacting with the protein. 3D-5D methods provide more shape based information but they are fragile, with a narrow applicability domain and may not be able to differentiate close analogs. Docking is also limited unless key interactions with the protein are already known. Our results suggest that even in the presence of multiple crystal structures, the full range of protein motions may not be captured. As we have previously shown, when docking classification predictions are correct the binding conformation information alone may be instructive [13]. This current analysis indicates that using many different computational approaches (both alignment dependent and alignment independent) may be necessary and expectations should be scaled accordingly if some do not work with such promiscuous proteins. Even with their respective limitations, these methods have provided some useful information of general interest that could be applicable beyond PXR. Human PXR activation was determined by a luciferase-based reporter assay as has been previously described [21], [33], [34]. The datasets modeled in this study were collected by a consistent protocol and have been previously published [31], [54]. Experimental data for four classes of steroidal compounds, namely androstanes, estratrienes, pregnanes and bile acids/ salts are shown in Table S2. All molecules described in Table S2 were used for docking experiments. The molecules were docked into these six crystallized structures of human PXR (PDB IDs 1M13,1NRL, 1SKX, 2O9I, 2QNV and one structure co-crystallized with 17β-estradiol that is not in the PDB identified here as EST). In all cases, the crystal structure ligand was removed, and hydrogen atoms were added to the amino acids. All amino acids within 6 Å of the co-crystallized ligand were identified as the binding site. The docking program GOLD (ver 4 [55]) was used for docking all compounds to the binding sites of each PXR crystal structure. GOLD uses genetic algorithm to explore the various conformations of ligands and flexible receptor side chains in the binding pocket. Further, 20 independent docking runs were performed for each ligand. The docked complexes were scored with goldscore [55] and then rescored using similarity weighted scoring scheme (SWscore). For each ligand, the best ranking conformation' s goldscore denoted by Si was used to derive the SWscore shown in equation 1. The similarity scores Wi were computed based on 2D similarity encoded in MDL fingerprint keys calculated using Discovery Studio 2. 1 (Accelrys, San Diego, CA, USA). The Tanimoto coefficient was used as the metric to compare the molecular fingerprints. The coefficients varied between 0 and 1, where 0 meant maximally dissimilar and 1 coded for maximally similar. The Tanimoto coefficient between fingerprints X and Y has been defined to be: [number of features in intersect (A, B) ]/[number of features in union (A, B) ], where A and B are two compounds. So the SWscore is given by, SWscore = Wi*Si, where Wi was the similarity score of compound i against 5α-Androstan-3β-ol which had the best EC50 value of 0. 8 µM for PXR or 17β-estradiol which had a steroid core that was present in most of the compounds. Further, the quality of the scoring function was assessed using standard statistical indicators namely sensitivity (SE), specificity (SP), overall prediction accuracy (Q) and Matthews correlation coefficient (C) (Table 1) and were derived as described previously [22]. Bayesian models were generated using Discovery Studio 2. 1 (Accelrys, San Diego, CA) Laplacian-corrected Bayesian classifier [37], [42], [43], [45], [56]. FCFP_6 fingerprints, AlogP, molecular weight, number of rotatable bonds, number of rings, number of aromatic rings, number of hydrogen bond acceptors, number of hydrogen bond donors and molecular fractional polar surface area were calculated from the input sdf file using the “calculate molecular properties protocol”. The “create Bayesian model protocol” was used for model generation and a custom protocol for validation (leave out 20% 100 times) was used. 5D-QSAR studies were performed using Raptor [40]. Raptor includes the possibility of representing each ligand molecule as an ensemble of conformations, orientations, stereoisomers and protonation states (4D-QSAR), thereby reducing the bias in identifying the bioactive conformer. In addition, it explicitly allows for induced fit by a dual-shell representation of the three-dimensional binding-site model, onto which the physicochemical properties (hydrophobicity and hydrogen-bonding propensity) are mapped (5D-QSAR). The inner shell is tailored using the most potent ligand of the training set, the outer shell accommodates the topology of all molecules from the training set. The adaptation of both field and topology of the receptor surrogate to each ligand is achieved by combining a steric adjustment to the topology of every ligand and a term due to the attraction or repulsion between ligand and receptor model. The latter is obtained by correlating their physicochemical properties (hydrophobicity and hydrogen-bond propensity) in 3D space. Since the mapping of properties onto the shells is not unambiguously determinable, different models with similar predictive power can be identified. Raptor generates a family of receptor models. Such model families may be interpreted to represent the various configuration states of the true biological receptor. The obtained binding affinities are averaged over the individual models. The underlying scoring function for evaluating ligand-protein interactions includes directional terms for hydrogen bonding (ΔGHbond), hydrophobicity (ΔGHphob) as well as terms for the cost of the topological adaptation (ΔGIF) and the changes in entropy (TΔS) upon ligand binding: ΔGbinding = ΔGconstant + ΔGHbond + ΔGHPhob − TΔS + ΔGIF. Experimental determination of binding affinity for weak inhibitors is often prevented due to limited solubility or limited sensitivity. Thus, only an upper limit (‘threshold’) for Ki values is accessible. To prevent artificial assignment of affinities in a QSAR study including weak binders, the Raptor concept allows the use of a threshold option: the optimization algorithm forces the model to reproduce the binding affinity of the weak- and non-binding ligand molecules to be lower than the experimental limit. Obviously, compounds which are experimentally measured to bind weaker than a threshold Ki (t) and are correctly classified during the model optimization, no penalty is added to the lack-of-fit value, if, on the other hand, the binding affinity of the ligand is predicted higher than the threshold, the lack-of-fit function applies a penalty proportional to ΔGbinding (t) − ΔGbinding. 4D sets of alternative conformations for each ligand as input for Raptor were performed with Symposar [57]. In Symposar the ligand molecules are superimposed onto one or several template molecules, first, on the basis of fuzzy-like 2D substructure similarities and, subsequently, in 3D space with respect to their similarity of physicochemical fields. This two-step process combines the speed of a 2D similarity search with the accuracy and authenticity of protein-ligand interactions in 3D space. The molecules are thereby treated as flexible and are fully relaxed at the end of the alignment process.

Title: Challenges Predicting Ligand-Receptor Interactions of Promiscuous Proteins: The Nuclear Receptor PXR Summary: Promiscuous proteins generally bind a large array of diverse ligand structures. This may be facilitated by a very large binding site, multiple binding sites, or a flexible binding site that can adjust to the size of the ligand. These aspects also increase the complexity of predicting whether a molecule will bind or not to such proteins which frequently function as exogenous compound sensors to respond to toxic stress. For example, transporters may prevent absorption of some molecules, and enzymes may convert them to more readily excretable compounds (or alternatively activate them prior to further clearance by other detoxification enzymes). Nuclear hormone receptors may respond to ligands and then affect downstream gene expression to upregulate both enzymes and transporters to increase the clearance for the same or different molecules. We have assessed the ability of many different ligand-based and structure-based computational approaches to model and predict the activation of human PXR by steroidal compounds. We find the most effective computational approach to identify potential steroidal PXR agonists which are clinically relevant due to their widespread use in clinical medicine and the presence of mimics in the environment.

lay_plos

en

Summarize: Unlike traditional speed cameras, the new gantry devices, pictured, are painted grey, making them harder to spot. Motorists risk heavy fines following the introduction of a new generation of speed cameras on Britain’s busiest motorway. Digital technology has been introduced to catch drivers breaking the 70mph speed limit on the M25 in Kent. It was reported last night that the devices – dubbed ‘stealth cameras’ by critics – have caught almost 700 drivers in just two months. Unlike traditional yellow speed cameras, the gantry devices are painted grey, making them harder to spot for drivers. The same technology is being introduced on a northern section of the M25 and also parts of the M1, M3, M60 and M6. Front and rear-facing cameras are used to verify a vehicle’s speed. And, while conventional devices have to be trained on only one lane at a time, the digital cameras can scan four. They are similar to cameras used during roadworks but do not work on the basis of calculating an average speed over a fixed distance. Motoring groups claim the devices will see thousands of drivers facing at least £100 in fines and points on their licence for straying marginally over the 70mph limit. The Association of Chief Police Officers recommends drivers are not charged unless they exceed 79mph in a 70mph limit zone. Hugh Bladon, one of the founder members of the Alliance of British Drivers, said they did not believe targeting drivers on the motorways was the best way to improve safety and that punishing drivers for exceeding the 70mph limit was often unnecessary. 'The 70mph limit is not a speed that a lot of people bother to observe any more,' he said. 'It was originally brought in as an experiment and was made permanent without any real testing. It was brought in at a time when the stopping power of cars was a bit like stopping an oil tanker, and the maximum speed of most cars was 74mph. We've moved on now, some 50 years later we have cars that stop much more quickly. 'The amount of traffic that exceeds the 70mph limit is enormous. Most people are driving at 80mph on motorways, and these are our safest roads in the country.' Mr Bladon said he did not think using speed cameras on the M25 was appropriate because there are many times, particularly late at night or when there is little traffic, when it is safe to driver faster than 70mph. He also said these should be made more visible, rather than disguised to try and catch drivers out. In the two months since they have been installed on the M25, pictured, almost 700 drivers have been caught breaking the 70mph limit. Where the new cameras will be placed: Several stretches of road are being upgraded to become'smart motorways', with new speed cameras a part of improvements. Above, a map detailing which will be affected. He added: 'We seem to want to punish drivers rather than help them. Our whole philosophy about speed cameras in this country is wrong. 'In France, for example, if they need to slow traffic down for a particular junction or nasty bend they bit up a great big sign, miles before the camera. We just whack cameras up for no reason whatsoever. 'The idea of a camera should be that you want people to see it and slow down.' Mr Bladon said he believed cameras only looked at a small fraction of the problem and could not test how tired people were or if they had taken drugs or been drinking. He said the best way to make roads safer was to increase the number of police on the roads. In 2013 the number of people fined for speeding peaked at more than 115,000 - the highest level since 2009. Ministers said the rise was largely due to the increased number of speed cameras that were in operation for 24 hours a day. In total 115,549 motorists were fined more than £100 that year. It was also announced last year that the maximum fines for motorway speeding that could be imposed by magistrates would rise from £2,500 to £10,000. In 2012-13 the Government collected £284million in speeding fines. Rupert Lipton, managing director of the National Motorists Action Group, said he was'shocked' officials had decided to use this type of speeding enforcement. He said: 'This is a another missed opportunity. The Highways Agency is introducing so called ‘smart motorways’ but relying on dumb enforcement. It is policing by numbers, by remote control. 'The completely ignored key to road safety is driver training. If drivers who had competed some advanced training ‘earned’ the right to drive at a higher speed on motorways, many millions would take such training, learn proper observation, anticipation and planning in their driving and accident figures would plummet. Drivers would even be prepared to pay out of their own pockets and the benefits would affect all roads not just motorways.' Professor Stephen Glaister, director of the RAC Foundation, told The Times: ‘The law is the law but it needs to be applied consistently. Many constabularies have followed the ACPO guidance. ‘If the new approach is one of zero tolerance then it needs to be equally applied across the network and understood by motorists and police forces.’ The Highways Agency said signs warning about speed cameras should be displayed on every gantry of the motorway where they are used. Studies have shown that nine out of ten drivers admit to breaking the motorway speed limit. The Government had planned to raise the limit to 80mph before the proposals were shelved by transport secretary Patrick McLoughlin two years ago. Brake, the road safety group, has previously said that raising the speed limit would cause more accidents and deaths on motorways. The first cameras were installed between junctions five and six of the M25. Kent police figures showed that 668 speeding offences were logged in just over two months. Until now, cameras have mainly been used to keep drivers below 50mph in sections of the network undergoing roadworks. The Highways Agency, which is responsible for the nation’s main roads, is creating so-called ‘smart’ motorways where the hard shoulder can be used as an additional lane to help ease heavy traffic flow at peak times. A spokesman said: 'Variable speed limits on smart motorways are primarily there to smooth traffic flow, reduce congestion and make journeys more reliable. 'Hundreds of thousands of motorists use this stretch of the M25 every day. The vast majority are sticking to the speed limits and are experiencing better journeys as a result of smart motorways. 'There are clear signs where cameras are in place and the new cameras are more visible than the previous versions.'

Summary: A new generation of speed cameras being introduced on the M25 in Kent. They have been installed to catch drivers breaking the 70mph speed limit. Unlike traditional yellow cameras, they are painted grey and harder to spot. Have already caught almost 700 motorists on the M25 in just two months. Same technology is now set to be introduced on the M1, M3, M60 and M6. Association of British Drivers said they were not appropriate and were being introduced to punish drivers rather than make roads safer.

cnn_dailymail

en

Summarize: FIELD OF THE INVENTION [0001] The present invention relates to an arrangement comprising at least two medical treatment units and at least two peripheral devices, wherein one medical treatment unit of the at least two medical treatment units and one peripheral device of the at least two peripheral devices is to be allocated to a patient and the peripheral device to be allocated to the patient is to be allocated to the medical treatment unit to be allocated to the patient. Moreover, the present invention relates to a peripheral device and a treatment unit for use in such an arrangement. BACKGROUND [0002] In medical technology, various treatment units, to which patients are connected or which are connected to patients, are used for the treatment of patients. The connection between patient and machine generally takes place via tubes and/or lines. The known treatment units include, for example, dialysis machines with an extracorporeal blood circuit. [0003] It is known to operate medical treatment units together with one or more peripheral devices. Such satellites serve, for example, to monitor the patient during the treatment, the ascertained patient-specific data being transmitted to the treatment unit. [0004] During dialysis, for example, physiological data of the dialysis patient are detected with various sensors. An attempt is made to ensure that the connection between patient and dialysis machine and the peripheral device consists only of the necessary blood tubes of the extracorporeal blood circuit and as few other lines as possible. [0005] Whereas a fixed connection exists between the treatment unit and the patient via the tubes, the data transmission between the peripheral device and the treatment unit can take place wirelessly, for example by radio or light signals. Consequently, the operative can immediately detect the allocation between the treatment unit and the patient on the basis of the fixed connection, but not the allocation between the peripheral device and the treatment unit. [0006] If a plurality of treatment units and peripheral devices are operated together in a treatment area, it is necessary to allocate in each case one treatment unit and one peripheral device to a patient and to produce a connection from the respective peripheral device to the respective treatment unit. [0007] An incorrect allocation between the peripheral device and the treatment unit on the one hand and the peripheral device and the patient on the other hand can in the extreme case lead to life-threatening complications during the treatment. This is especially problematic when the incorrect allocation is not detected immediately on account of a cableless connection. [0008] If a plurality of devices which are communicating with one another are operated at the same time, use is generally made of so-called identification (ID) signals, with which it can be detected whether the signals of the one or the other satellite are being received. U.S. Pat. No. 6,332,094 B1, for example, describes a pulsometer, which transmits pulse signals together with an identification signal wirelessly to a receiver. [0009] International Application Publication No. WO 20041056263 A1 describes a method for the wireless transmission of signals between a plurality of peripheral devices and a plurality of treatment units. Allocated to the treatment units is a plurality of receivers, which receive the signals of the peripheral devices. The readiness of the receiver for receiving the signals of the peripheral devices is produced by the fact that the peripheral device notifies the receiver. Only in the state of readiness for reception does the receiver convert the signals of the peripheral device which are transmitted to the respective treatment unit. [0010] There is known from U.S. Pat. No. 6,870,475 B2 a charging station for charging the battery of a portable medical monitoring unit, which serves for the monitoring of patient-specific data. The patient monitoring system provides a large number of charging stations at different points, into which the patient can insert the monitoring unit in order to charge the battery. Patient-specific data of the portable monitoring units can be transmitted wirelessly to a central monitoring unit. [0011] U.S. Pat. No. 6,184,65 1 B1 and U.S. Pat. No. 5,455,466 describe in general terms charging stations for charging the battery of electrical devices, wherein an inductive coupling takes place between the electrical device and the charging station. [0012] The problem underlying the invention is to increase the reliability and flexibility during the operation of at least two medical treatment units and peripheral devices. SUMMARY [0013] The reliability and flexibility during the operation of a plurality of medical treatment units and peripheral devices is increased with the arrangement according to example embodiments of the invention by the fact that the operative acknowledges, after allocation of a treatment unit to a patient and a peripheral device to a patient, that the patient to whom the peripheral device has been allocated is the patient to whom the treatment unit, to which the peripheral device has been allocated, has been allocated. The peripheral device is released only on condition that the correct allocation of peripheral device and patient has been acknowledged. This thus eliminates the situation where, although the correct peripheral device is allocated to the correct treatment unit, the peripheral device allocated to the treatment unit is allocated to the wrong patient. [0014] The means for releasing the peripheral device can be designed in such a way that the patient-specific data is not transmitted to the treatment unit or received or evaluated by the treatment unit until the correct allocation of the peripheral device and patient has been acknowledged. The means for releasing the peripheral device can for example also be designed in such way that the medical treatment cannot be started until the correct allocation of the peripheral device and patient has been acknowledged. [0015] In a preferred embodiment of the invention, the means for allocating a peripheral device to a treatment unit comprises means for sending an address for identification of the peripheral device to the medical treatment unit and means for receiving the address for identification of the peripheral device by the treatment unit. For example, the address can be an MAC address (media access control), which is used for the unequivocal identification of the peripheral device in the network. [0016] A further preferred embodiment makes provision such that the means for allocating the peripheral device and the treatment unit comprises means for accommodating a peripheral device and means for detecting a peripheral device accommodated in the means for accommodating a peripheral device. The means for accommodating a peripheral device can be unequivocally allocated to a treatment unit, this allocation being able to be immediately detected by the operative. For example, the means for accommodating a peripheral device is arranged in close proximity to the treatment unit or is a component of the treatment unit. The peripheral device is unequivocally allocated to the treatment unit when the peripheral device is located in the means allocated to the treatment unit for accommodating a peripheral device. [0017] The means for accommodating a peripheral device can be designed in different ways. For example, the means for accommodating a peripheral device can be an accommodation unit into which the peripheral device is placed. The peripheral device can be placed loosely into the accommodation unit or also be secured in the accommodation unit. [0018] The means for detecting the peripheral device in the accommodation unit can also be designed differently. For example, the means for detecting the peripheral device can comprise mechanical contact makers, with which it can be detected whether the peripheral device is located in the accommodation unit. Alternatively, the means for detecting the peripheral device can comprise optical or inductive sensors. [0019] A particularly advantageous embodiment, which offers great advantages in practice, makes provision such that the means for accommodating a peripheral device comprises means for charging a battery for the power supply of the peripheral device. The accommodation units can be designed as charging stations for the peripheral devices. The accommodation units thus ensure not only the allocation of the peripheral device and the treatment unit, but also the permanent operation of the peripheral devices. [0020] In a further particularly preferred embodiment, the peripheral device sends the address for identification to the treatment unit when the peripheral device has been detected in the accommodation unit. The identification of the peripheral device therefore takes place fully automatically upon insertion of the peripheral device into the accommodation unit. It is however also possible for the peripheral device to send the identification address continuously and for the treatment unit to evaluate the identification address only after detection of the peripheral device in the accommodation unit. [0021] The successful allocation of the peripheral device and the patient is preferably signaled by the peripheral device to the treatment unit by the fact that the peripheral device sends a corresponding signal to the treatment unit. For this purpose, the means for checking the allocation of the peripheral device and patient preferably comprise means for sending and receiving signals, in particular means for the wireless transmission of signals, for example a radio transmitter and a radio receiver. Other wireless transmission links, for example an optical data transmission, are however also possible. [0022] It is concluded that there is a successful allocation of the peripheral device and patient preferably when the peripheral device receives patient-specific data for the monitoring of the patient, which presupposes that the peripheral device is connected to the patient. It is, however, also possible to provide additional sensors which detect the position of the peripheral device, for example contact makers on the tubes and/or lines. [0023] The reliability can, furthermore, be increased by the fact that the operative is prompted, within a preset time interval, to allocate to the patient the peripheral device which is allocated to the treatment unit and removed from the accommodation unit. A particularly preferred embodiment makes provision such that the means for checking the allocation of the peripheral device and patient cooperate with means for measuring the time after removal of the peripheral device from the means for accommodating the peripheral device. The means for checking the allocation of the peripheral device and patient preferably cooperate with means for displaying the remaining time in the preset time interval, in order to signal to the operative how much time still remains in order to connect the peripheral device to the patient. [0024] If the peripheral device is allocated to the patient only after the lapse of the preset time interval, the successful allocation of the peripheral device and patient is not signaled to the treatment unit. The operative is therefore forced to place the peripheral device back into the accommodation unit, in order to be able to carry out again the allocation of the peripheral device, treatment unit and patient within the preset time interval. This prevents the peripheral device, removed from the accommodation unit, being carried around by the operative for an indeterminate time, because in this case there is a particularly great risk of an incorrect allocation of the peripheral device and patient. [0025] The prompting after the lapse of the preset time interval to insert the peripheral device back into the accommodation unit preferably takes place with means for emitting an optical and/or acoustic signal, which the means for checking the allocation for the peripheral device and patient preferably comprise. [0026] The means for acknowledging the allocation of the treatment unit, peripheral device and patient preferably comprise means for manual input, for example switches, pushbuttons etc. The means for the manual input can also be designed as sensors which operate in a contactless manner, for example inductive switches etc. [0027] The invention is based on the fact that the allocation of the treatment unit and patient requires a fixed connection of patient and treatment unit, so that this allocation is unequivocal for the operative. In the case where the medical treatment unit is, for example, a blood treatment apparatus, the fixed allocation of the treatment unit and patient is provided by the blood tube system, so that mistakes are ruled out in practice. [0028] The peripheral devices may be designed differently and have different functions. For example, the peripheral devices may comprise one or more sensors for detecting patient-specific data, for example for measuring physiological measured values, such as blood pressure or pulse etc. [0029] An example embodiment of the invention is explained below in greater detail by reference to the drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0030] FIG. 1 shows a schematic representation of a correct allocation between two peripheral devices and treatment units to the respective patient. [0031] FIG. 2 shows the arrangement of FIG. 1, wherein the peripheral devices allocated to the respective treatment unit have been allocated to the wrong patient. [0032] FIG. 3 shows the main components of a medical treatment unit and a peripheral device in a schematic representation. [0033] FIG. 4A and 4B show a flow diagram, which describes the sequence of the allocation of the peripheral device and the treatment unit as well as the patient. DETAILED DESCRIPTION [0034] FIG. 1 shows two medical treatment units 1, 1 ′, for example, extracorporeal blood treatment apparatuses with an extracorporeal blood circuit. A patient 2, 2 ′ is connected to each treatment unit 1, 1 ′. In the case of an extracorporeal blood treatment, patient 2, 2 ′ is connected to blood treatment apparatus 1, 1 ′ via a venous and arterial tube line 3, 4 ; 3 ′, 4 ′. A fixed allocation between the patient and the treatment unit is thus made. Moreover, a peripheral unit or device 5, 5 ′, for example a blood pressure monitor, is allocated in each case to the two patients 2, 2 ′. [0035] Peripheral devices 5, 5 ′ monitor the patient&#39;s bodily function, for example the blood pressure or pulse, and transmit the physiological data to the respective treatment unit, to which the patient is connected. [0036] FIG. 1 shows the correct allocation of the peripheral device and patient on the one hand and the peripheral device and treatment unit on the other hand, while FIG. 2 shows an incorrect allocation of the peripheral device and patient and, respectively, the peripheral device and treatment unit. In the case of an incorrect allocation, treatment unit 1 receives the data of peripheral device 5 ′ of patient 2 ′, who is not connected to treatment unit 1, but rather to treatment unit 1 ′. [0037] The arrangement according to example embodiments of the invention comprises at least two medical treatment units and peripheral devices. The individual components of a treatment unit and a peripheral device as well as the individual steps for the allocation of the treatment unit, the peripheral device and the patient are described below by reference to FIGS. 3, 4 A, and 4 B. [0038] Peripheral device 5, for example a blood pressure monitor, has various sensors 6 shown solely schematically for monitoring the patient, for example sensors for measuring the blood pressure or pulse. For bi-directional communication with respective treatment unit 1, peripheral device 5 has means 7 for sending and receiving signals, in particular a radio transmitter/receiver 7. The data transmission may, however, also take place solely directionally from the peripheral device to the treatment unit. Rechargeable battery 8 (accumulator) serves as the power supply of the peripheral device. [0039] Furthermore, peripheral device 5 has means 19, with which the operative can acknowledge that the patient to whom the peripheral device has been allocated is the patient to whom the treatment unit has been allocated to which the peripheral device has been allocated. This means is preferably means for manual input, in particular a pushbutton 19. [0040] Treatment unit 1, for example a blood treatment apparatus with an extracorporeal blood circuit, has various components 9, represented only schematically, for the treatment of patient 2, for example a dialyser, pumps etc. Patient 2 is fixedly connected to blood treatment apparatus 1, for example via a venous and arterial blood line 3, 4. A fixed allocation between patient and treatment unit is thus produced. [0041] For the communication with respective peripheral device 5, treatment unit 1 has means 10 for sending and receiving signals, in particular a radio transmitter/receiver 10 for a bi-directional data transmission. The data transmission between peripheral device 5 and treatment unit 1 thus takes place by radio, whereby the transmitter and receiver, as well as the interfaces required for this, are generally known to the person skilled in the art. [0042] Treatment unit 1, moreover, has an optical and/or acoustic alarm emitter 12 and a display unit 13. [0043] An accommodation unit 14 is fixedly allocated to each treatment unit 1, so that the operative can immediately detect that accommodation unit 14 belongs to blood treatment unit 1. Accommodation unit 14 is designed as a charging station. It comprises means 15 for the power supply, in particular a power pack, with which battery 8 of the peripheral device can be charged when the peripheral device is inserted into the accommodation unit. The electrical connection between power pack 15 and battery 8 can take place via suitable plug-in connections 11 or an inductive coupling. [0044] Peripheral device 5 and/or accommodation unit 14 have means 16 for detecting whether peripheral device is inserted into the accommodation unit. This means may, for example, be electrical contact makers, which are provided either on the peripheral device or the accommodation unit or on both devices. [0045] The function of the treatment units and peripheral devices is described in detail below by reference to FIGS. 3, 4 A, and 4 B. [0046] It is assumed that the patients are connected to treatment units 1, for example extracorporeal blood treatment apparatuses, via venous and arterial blood lines 3, 4, so that in each case a fixed allocation of patient and treatment unit is produced, which can immediately be seen by the operative. The operative now has the task of connecting the peripheral device belonging to the treatment unit to the respective patients. [0047] Respective peripheral device 5 is first inserted into accommodation unit 14, which is allocated to respective treatment unit 1. The operative has thus allocated the peripheral device to be treatment unit. [0048] Means 16 detects that the peripheral device is located in accommodation unit 14. This is signalled by means 16 with a status signal. While peripheral device 5 is located in accommodation unit 14, an electrical connection is produced between power pack 15 and battery 8, so that the battery is charged. [0049] Treatment unit 1 continuously inquires whether the status signal, indicating that the peripheral device is located in the accommodation unit, has been generated. If this is the case, radio transmitter/receiver 7 of peripheral device 5 sends an address for the identification of the peripheral device, for example an MAC address, to treatment unit 1, which receives the MAC address via radio transmitter/receiver 10. Treatment unit 1 has thus identified peripheral device 5. [0050] For the identification of the peripheral devices by the exchange of MAC addresses, use is preferably made of transmission/reception means with a relatively short range in the case of a wireless transmission of the signals. If the range of the transmission/reception means is not greater than the distance between the peripheral device and the treatment unit with the respective accommodation unit, it can be ensured that the peripheral device communicates solely with its “own charging shell”. For this purpose, the transmission means can also limit the range themselves. It is, however, also possible to provide separate transmission/reception means for the transmission of the patient-specific signals on the one hand and the MAC addresses for the identification of the peripheral device on the other hand. For example, transmission/reception means for inductive data transmission can also be used for the identification, said means being part of an inductive battery charging device provided in the charging shell. [0051] After identification of the peripheral device, the operative must allocate the identified peripheral device 5 to the patient, who is connected to treatment unit 1 which has identified the peripheral device. For this purpose, the operative must take the peripheral device out of the accommodation unit. [0052] Constant monitoring is carried out to check whether the peripheral device is still located in the accommodation unit. When the operative takes the peripheral device out of the accommodation unit, this is immediately detected by means 16, which generates a status signal indicating that the operative has removed the peripheral device from the accommodation unit. This status signal is sent to the treatment unit, which receives the status signal. With the reception of the status signal for the removal of the peripheral device from the accommodation unit, the treatment unit starts up means (timer) 18 for measuring the time that has passed after the removal of the peripheral device. [0053] The operative must now connect the peripheral device to the patient within a preset time interval. The remaining time in the preset time interval is displayed to the operative on a display unit 13 of treatment unit 1. [0054] When the operative has fitted the peripheral device to the patient and has activated the peripheral device, sensors 6 of the peripheral device receive the physiological measured values, such as the patient&#39;s blood pressure and pulse. The signals received from sensors 6 are evaluated by means 20 for checking the allocation of a peripheral device to a patient. This event is signaled by means 20 of peripheral device 5 to treatment unit 1 by the fact that the peripheral device sends to the treatment unit a status signal for the successful allocation of the peripheral device to the patient. [0055] The treatment unit continuously checks whether the status signal for the successful allocation of the peripheral device and patient is received during the preset time interval. If the preset time interval has lapsed before reception of the status signal for the successful allocation of the peripheral device and patient, the treatment unit emits with alarm emitter 12 an optical and/or acoustic alarm signal, with which the operative is prompted to place the peripheral device back into the accommodation unit. At the same time, or alternatively, a corresponding display can also take place on display unit 13. The routine described above then begins afresh. [0056] If, however, the status signal for the successful allocation of the peripheral device and patient is received, there appears on display unit 13 (display) of the treatment unit an indication which prompts the operative to press an acknowledge button 19. The treatment unit waits until button 19 is pressed. [0057] The operative must now check whether patient 2, to whom peripheral device 5 is connected, is the patient to whom treatment unit 1 is connected to which the peripheral device has been allocated by accommodation unit 14. If this is the case, the operative presses acknowledge button 19, which is provided on the peripheral device. A signal for the acknowledgement by the operative is then sent to the treatment unit, which receives the status signal. [0058] It is, however, also possible for the acknowledge button to be located on the treatment unit. It is then not necessary to send the status signal for the acknowledgement of the allocation of the peripheral device and patient to the treatment unit. [0059] Treatment unit 1 now checks whether the operative has acknowledged the correct allocation of the peripheral device and patient by pressing button 19 within the preset time interval. [0060] Treatment unit 1 has means 17, with which the peripheral device is released. Only in the case where acknowledge button 19 has been pressed within the preset time interval do means 17 release the peripheral device, so that the patient-specific data are transmitted from the peripheral device to the treatment unit with radio transmitter/receiver 7, 10. Means 17 must also release the treatment. The treatment program can now be started. [0061] The means described above can form separate modules, but in practice the means are a component of a microprocessor, which is generally provided anyway in the treatment units and peripheral devices. [0062] In the embodiment described by reference to the figures, individual means are allocated in each case to the blood treatment units and individual means are allocated in each case to the peripheral devices. The means which are allocated to the blood treatment units, however, may also be allocated to the peripheral devices. For example, the means for the acknowledgement of the allocation of the patient and treatment unit may be provided not on the peripheral device, but on the treatment unit. The means for emitting an acoustic and/or optical alarm can also be provided on the peripheral device instead of on the treatment unit. [0063] Even though the figures show an arrangement of peripheral devices and treatment units, it can be seen from the description that the peripheral devices and the treatment units are independent devices which have the described modules.

Summary: An assembly includes at least two medical treatment units, each of which can be allocated to a patient and at least two peripheral devices, each of which can be allocated to a patient. The peripheral devices and the treatment units have means for allocating a peripheral device to a treatment unit and means for verifying the allocation of a peripheral device to a patient. To allocate a peripheral device to a treatment unit, the peripheral device is placed by operators in a receiving unit belonging to the treatment unit. Said receiving unit is preferably designed as a charging station for the battery of the peripheral device. In the event of a successful allocation of peripheral device and patient, confirmed by the receipt of physiological data of the patient, the operators verify the correct allocation of a peripheral device to the patient and must confirm the correct allocation by confirmation means. The peripheral device is only released if the operator has confirmed the correct allocation, preferably within a predefined time period.

big_patent

en

Summarize: Background The H-1B program enables companies in the United States to hire foreign workers for work in specialty occupations on a temporary basis. A specialty occupation is defined as one requiring theoretical and practical application of a body of highly specialized knowledge and the attainment of a bachelor’s degree or higher (or its equivalent) in the field of specialty. The law originally capped the number of H-1B visas at 65,000 per year; the cap was raised twice pursuant to legislation, but in fiscal year 2004, the cap reverted to its original level of 65,000. Statutory changes also allowed for certain categories of individuals and companies to be exempt from or to receive special treatment under the cap. The American Competitiveness in the Twenty-First Century Act of 2000 exempted from the cap all individuals being hired by institutions of higher education and also nonprofit and government-research organizations. More recently, the H-1B Visa Reform Act of 2004 allowed for an additional 20,000 visas each year for foreign workers holding a master’s degree or higher from an American institution of higher education to be exempted from the numerical cap limitation. In 2004, consistent with free trade agreements, up to 6,800 of the 65,000 H-1B visas may be set aside for workers from Chile and Singapore. While the H-1B visa is not considered a permanent visa, H-1B workers can apply for extensions and pursue permanent residence in the United States. Initial petitions are those filed for a foreign national’s first-time employment as an H-1B worker and are valid for a period of up to 3 years. Generally, initial petitions are counted against the annual cap. Extensions—technically referred to as continuing employment petitions— may be filed to extend the initial petitions for up to an additional 3 years. Extensions do not count against the cap. While working under an H-1B visa, a worker may apply for legal permanent residence in the United States. After filing an application for permanent residence, H-1B workers are generally eligible to obtain additional 1-year visa extensions until their U.S. Permanent Resident Cards, commonly referred to as “green cards,” are issued. The Departments of Labor (Labor), Homeland Security (Homeland Security), and State (State) each play a role in administering the application process for an H-1B visa. Labor’s Employment and Training Administration (Employment and Training) receives and approves an initial application, known as the Labor Condition Application (LCA), from employers. The LCA, which Labor reviews as part of the application process, requires employers to make various attestations designed to protect the jobs of domestic workers and the rights and working conditions of temporary workers. Homeland Security’s U.S. Citizenship and Immigration Services (USCIS) reviews an additional employer application, known as the I-129 petition, and ultimately approves H-1B visa petitions. For prospective H-1B workers residing outside the United States, State interviews approved applicants and compares information obtained during the interview against each individual’s visa application and supporting documents, and ultimately issues the visa. For prospective H-1B workers already residing in the United States, USCIS updates the workers’ visa status without involvement from State. USCIS has primary responsibility for administering the H-1B cap. Generally, it accepts H-1B petitions in the order in which they are received. However, for those years in which USCIS anticipates that the number of I-129 petitions filed will exceed the cap, USCIS holds a “lottery” to determine which of the petitions will be accepted for review. For the lottery, USCIS uses a computer-generated random selection process to select the number of petitions necessary to reach the cap. With regard to enforcement, Labor, the Department of Justice (Justice), and Homeland Security each have specific responsibilities. Labor’s Wage and Hour Division (Wage and Hour) is responsible for enforcing program rules by investigating complaints made against employers by H-1B workers or their representatives and assessing penalties when employers are not in compliance with the requirements of the program. Justice is responsible for investigating complaints made by U.S. workers who allege that they have been displaced or otherwise harmed by the H-1B visa program. Finally, USCIS’s Directorate of Fraud Detection and National Security (FDNS) collaborates with its Immigration and Customs Enforcement Office to investigate fraud and abuse in the program. Demand for H-1B Workers Exceeded the Cap in Most Years and Was Driven by a Small Number of Employers Over the past decade, demand for H-1B workers tended to exceed the cap, as measured by the number of initial petitions submitted by employers, one of several proxies used to measure demand since a precise measure does not exist. As shown in figure 1, from 2000 to 2009, initial petitions for new H-1B workers submitted by employers who are subject to the cap exceeded the cap in all but 3 fiscal years. However, the number of initial petitions subject to the cap is likely to be an underestimate of demand since, once the cap has been reached, employers subject to the cap may stop submitting petitions and Homeland Security stops accepting petitions. If initial petitions submitted by employers exempt from the cap are also included in this measure (also shown in figure 1), the demand for new H- 1B workers is even higher, since over 14 percent of all initial petitions across the decade were submitted by employers who are not subject to the cap. In addition to initial requests for H-1B workers, employers requested an average of 148,000 visa extensions per year, for an average of over 280,000 annual requests for H-1B workers. Over the decade, the majority (over 68 percent) of employers were approved to hire only one H-1B worker, while fewer than 1 percent of employers were approved to hire almost 30 percent of all H-1B workers. Among these latter employers are those that function as “staffing companies” that contract out H-1B workers to other companies. The prevalence of such companies participating in the H-1B visa program is difficult to determine. There are no disclosure requirements and Homeland Security does not track such information. However, using publicly available data, we learned that at least 10 of the top 85 H-1B-hiring employers in fiscal year 2009 participated in staffing arrangements, of which at least 6 have headquarters or operations located in India. Together, in fiscal year 2009, these 10 employers garnered nearly 11,456 approvals, or about 6 percent of all H-1B approvals. Further, 3 of these employers were among the top 5 H-1B-hiring companies, receiving 8,431 approvals among them. Most Interviewed Companies Said the H-1B Cap Was Not a Key Factor in Their Decisions to Move Operations Overseas but Cited Other Program Burdens To better understand the impact of the H-1B program and cap on H-1B employers, GAO spoke with 34 companies across a range of industries about how the H-1B program affects their research and development (R&D) activities, their decisions about whether to locate work overseas, and their costs of doing business. Although several firms reported that their H-1B workers were essential to conducting R&D within the U.S., most companies we interviewed said that the H-1B cap had little effect on their R&D or decisions to locate work offshore. Instead, they cited other reasons to expand overseas including access to pools of skilled labor abroad, the pursuit of new markets, the cost of labor, access to a workforce in a variety of time zones, language and culture, and tax law. The exception to this came from executives at some information technology services companies, two of which rely heavily on the H-1B program. Some of these executives reported that they had either opened an offshore location to access labor from overseas or were considering doing so as result of the H-1B cap or changes in the administration of the H-1B program. Many employers we interviewed cited costs and burdens associated with the H-1B cap and program. The majority of the firms we spoke with had H- 1B petitions denied due to the cap in years when the cap was reached early in the filing season. In these years, the firms did not know which, if any, of their H-1B candidates would obtain a visa, and several firms said that this created uncertainty that interfered with both project planning and candidate recruitment. In these instances, most large firms we interviewed reported finding other (sometimes more costly) ways to hire their preferred job candidates. For example, several large firms we spoke with were able to hire their preferred candidates in an overseas office temporarily, later bringing the candidate into the United States, sometimes on a different type of visa. On the other hand, small firms were sometimes unable to afford these options, and were more likely to fill their positions with different candidates, which they said resulted in delays and sometimes economic losses, particularly for firms in rapidly changing technology fields. Interviewed employers also cited costs with the adjudication and lottery process and suggested a variety of reforms: The majority of the 34 firms we spoke with maintained that the review and adjudication process had become increasingly burdensome in recent years, citing large amounts of paperwork required as part of the adjudication process. Some experts we interviewed suggested that to minimize paperwork and costs, USCIS should create a risk-based adjudication process that would permit employers with a strong track- record of regulatory compliance in the H-1B program to access a streamlined process for petition approval. In addition, several industry representatives told us that because the lottery process does not allow employers to rank their top choices, firms do not necessarily receive approval for the most desired H-1B candidates. Some experts suggested revising the system to permit employers to rank their applications so that they are able to hire the best qualified worker for the job in highest need. Finally, entrepreneurs and venture capital firms we interviewed said that program rules can inhibit many emerging technology companies and other small firms from using the H-1B program to bring in the talent they need, constraining the ability of these companies to grow and innovate in the United States. Some suggested that, to promote the ability of entrepreneurs to start businesses in the United States, Congress should consider creating a visa category for entrepreneurs, available to persons with U.S. venture backing. In our report, we recommended that USCIS should, to the extent permitted by its existing statutory authority, explore options for increasing the flexibility of the application process for H-1B employers. In commenting on our report, Homeland Security and Labor officials expressed reservations about the feasibility of our suggested options, but Homeland Security officials also noted efforts under way to streamline the application process for prospective H-1B employers. For example, Homeland Security is currently testing a system to obtain and update some company data directly from a private data vendor, which could reduce the filing burden on H-1B petitioners in the future. In addition, Homeland Security recently proposed a rule that would provide for employers to register and learn whether they will be eligible to file petitions with USCIS prior to filing an LCA, which could reduce workloads for Labor and reduce some filing burden for companies. Limitations in Agency Data and Systems Hinder Tracking the Cap and H-1B Workers Over Time The total number of H-1B workers in the United States at any one point in time—and information about the length of their stay—is unknown due to data and system limitations. First, data systems among the various agencies that process H-1B applications are not easily linked, which makes it impossible to track individuals as they move through the application and entry process. Second, H-1B workers are not assigned a unique identifier that would allow agencies to track them over time or across agency databases—particularly if and when their visa status changes. Consequently, USCIS is not able to track the H-1B population with regard to: (1) how many approved H-1B workers living abroad have actually received an H-1B visa and/or ultimately entered the country; (2) whether and when H-1B workers have applied for or were granted legal permanent residency, leave the country, or remain in the country on an expired visa; and (3) the number of H-1B workers currently in the country or who have converted to legal permanent residency. Limitations in USCIS’s ability to track H-1B applications also hinder it from knowing precisely when and whether the annual cap has been reached each year—although the Immigration and Nationality Act requires the department to do so. According to USCIS officials, its current processes do not allow them to determine precisely when the cap on initial petitions is reached. To deal with this problem, USCIS estimates when the number of approvals has reached the statutory limit and stops accepting new petitions. Although USCIS is taking steps to improve its tracking of approved petitions and of the H-1B workforce, progress has been slow to date. Through its “Transformation Program,” USCIS is developing an electronic I-129 application system and is working with other agencies to create a cross-reference table of agency identifiers for individuals applying for visas that would serve as a unique person-centric identifier. When this occurs, it will be possible to identify who is in the United States at any one point in time under any and all visa programs. However, the agency faces challenges with finalizing and implementing the Transformation Program. We recommended that Homeland Security, through its Transformation Program, take steps to (1) ensure that linkages to State’s tracking system will provide Homeland Security with timely access to data on visa issuances, and (2) that mechanisms for tracking petitions and visas against the cap be incorporated into business rules to be developed for USCIS’s new electronic petition system. While a complete picture of the H-1B workforce is lacking, data on approved H-1B workers provides some information about the H-1B workforce. Between fiscal year 2000 and fiscal year 2009, the top four countries of birth for approved H-1B workers (i.e., approved initial and extension petitions from employers both subject to the cap and cap- exempt) were India, China, Canada, and the Philippines. Over 40 percent of all such workers were for positions in system analysis and programming. As compared to fiscal year 2000, in fiscal year 2009, approved H-1B workers were more likely to be living in the United States than abroad at the time of their initial application, to have an advanced degree, and to have obtained their graduate degrees in the United States. Finally, data on a cohort of approved H-1B workers whose petitions were submitted between January 2004 and September 2007, indicate that at least 18 percent of these workers subsequently applied for permanent residence in the United States—for which about half were approved, 45 percent were pending, and 3 percent were denied by 2010. Restricted Agency Oversight and Statutory Changes Weaken Protections for U.S. Workers The provisions of the H-1B program designed to protect U.S. workers— such as the requirement to pay prevailing wages, the visa’s temporary status, and the cap on the number of visas issued—are weakened by several factors. First, H-1B program oversight is shared by four federal agencies and their roles and abilities to coordinate are restricted by law. As a result, there is only nominal sharing of the kind of information that would allow for better employer screening or more active and targeted pursuit of program abuses. For example, the review of employer applications for H-1B workers is divided between Labor and USCIS, and the thoroughness of both these reviews is constrained by law. In reviewing the employer’s LCA, Labor is restricted to looking for missing information and obvious inaccuracies, such as an employer’s failure to checkmark all required boxes on a form denoting compliance. USCIS’s review of the visa petition, the I-129, is not informed by any information that Labor’s Employment and Training Administration may possess on suspicious or problematic employers. With regard to enforcement of the H-1B worker protections, Wage and Hour investigations are constrained, first, by the fact that its investigators do not receive from USCIS any information regarding suspicious or problematic employers. They also do not have access to the Employment and Training’s database of employer LCAs. Second, in contrast to its authority with respect to other labor protection programs, Wage and Hour lacks subpoena authority to obtain employer records for H-1B cases. According to investigators, it can take months, therefore, to pursue time-sensitive investigations when an employer is not cooperative. To improve Labor’s oversight over the H-1B program, we recommended that its Employment and Training Administration grant Wage and Hour searchable access to the LCA database. Further, we asked Congress to consider granting Labor subpoena power to obtain employer records during investigations under the H-1B program. To reduce duplication and fragmentation in the administration and oversight of the application process, consistent with past GAO matters for Congressional consideration, we asked Congress to consider streamlining the H-1B approval process by eliminating the separate requirement that employers first submit an LCA to Labor for review and certification, since another agency (USCIS) subsequently conducts a similar review of the LCA. Another factor that weakens protection for U.S. workers is the fact that the H-1B program lacks a legal provision to hold employers accountable to program requirements when they obtain H-1B workers through staffing companies. As previously noted, staffing companies contract H-1B workers out to other employers. At times, those employers may contract the H-1B worker out again, creating multiple middlemen, according to Wage and Hour officials (see fig. 2). They explained that the contractual relationship, however, does not transfer the obligations of the contractor for worker protection to subsequent employers. Wage and Hour investigators reported that a large number of the complaints they receive about H-1B employers were related to the activities of staffing companies. Investigators from the Northeast region—the region that receives the highest number of H-1B complaints—said that nearly all of the complaints they receive involve staffing companies and that the number of complaints are growing. H-1B worker complaints about these companies frequently pertained to unpaid “benching”—when a staffing company does not have a job placement for the H-1B worker and does not pay them. In January 2010, Homeland Security issued a memo—commonly referred to as the “Neufeld Memo”—on determining when there is a valid employer- employee relationship between a staffing company and an H-1B worker for whom it has obtained a visa; however officials indicated that it is too early to know if the memo has improved program compliance. To help ensure the full protection of H-1B workers employed through staffing companies, in our report we asked that Congress consider holding the employer where an H-1B visa holder performs work accountable for meeting program requirements to the same extent as the employer that submitted the LCA form. Finally, changes to program legislation have diluted program provisions for protecting U.S. workers by allowing visa holders to seek permanent residency, broadening the job and skill categories for H-1B eligibility, and establishing exemptions to the cap. The Immigration Act of 1990 removed the requirement that H-1B visa applicants have a residence in a foreign country that they have no intention of abandoning. Consequently, H-1B workers are able to pursue permanent residency in the United States and remain in the country for an unlimited period of time while their residency application is pending. The same law also broadened the job and skill categories for which employers could seek H-1B visas. Labor’s LCA data show that between June 2009 and July 2010, over 50 percent of the wage levels reported on approved LCAs were categorized as entry-level (i.e. paid the lowest prevailing wage levels). However, such data do not, by themselves, indicate whether these H-1B workers were generally less skilled than their U.S. counterparts, or whether they were younger or more likely to accept lower wages. Finally, exemptions to the H-1B cap have increased the number of H-1B workers beyond the cap. For example, 87,519 workers in 2009 were approved for visas (including both initial and extensions) to work for 6,034 cap-exempt companies. Conclusions Taken together, the multifaceted challenges identified in our work show that the H-1B program, as currently structured, may not be used to its full potential and may be detrimental in some cases. Although we have recommended steps that executive agencies overseeing the program may take to improve tracking, administration, and enforcement, the data we present raise difficult policy questions about key program provisions that are beyond the jurisdiction of these agencies. The H-1B program presents a difficult challenge in balancing the need for high-skilled foreign labor with sufficient protections for U.S. workers. As Congress considers immigration reform in consultation with diverse stakeholders and experts—and while Homeland Security moves forward with its modernization efforts—this is an opportune time to re-examine the merits and shortcomings of key program provisions and make appropriate changes as needed. Such a review may include, but would not necessarily be limited to the qualifications required for workers eligible under the H-1B program, exemptions from the cap, the appropriateness of H-1B hiring by staffing companies, the level of the cap, and the role the program should play in the U.S. immigration system in relationship to permanent residency. GAO Contact and Staff Acknowledgments If you or your staffs have any questions about this statement, please contact Andrew Sherrill at (202) 512-7215 or [email protected]. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this statement. In addition to Andrew Sherrill (Director), Michele Grgich (Assistant Director) and Erin Godtland (Economist-in-Charge) led this engagement with writing and technical assistance from Nisha Hazra, Melissa Jaynes, Jennifer McDonald, Susan Bernstein (Education, Workforce and Income Security); and Rhiannon Patterson (Applied Research and Methods). Stakeholders included: Barbara Bovbjerg (Education, Workforce, and Income Security); Tom McCool (Applied Research and Methods); Ronald Fecso (Chief Statistician); Sheila McCoy and Craig Winslow (General Counsel); Hiwotte Amare and Shana Wallace (Applied Research and Methods); Richard Stana and Mike Dino (Homeland Security and Justice); Jess Ford (International Affairs and Trade). Barbara Steel-Lowney referenced the report. This is a work of the U.S. government and is not subject to copyright protection in the United States. The published product 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: This testimony comments on the H-1B program. Congress created the current H-1B program in 1990 to enable U.S. employers to hire temporary, foreign workers in specialty occupations. The law capped the number of H-1B visas issued per fiscal year at 65,000, although the cap has fluctuated over time with legislative changes. The H-1B cap and the program itself have been a subject of continued controversy. Proponents of the program argue that it allows companies to fill important and growing gaps in the supply of U.S. workers, especially in the science and technology fields. Opponents of the program argue that there is no skill shortage and that the H-1B program displaces U.S. workers and undercuts their pay. Others argue that the eligibility criteria for the H-1B visa should be revised to better target foreign nationals whose skills are undersupplied in the domestic workforce. Our comments in this statement for the record are based on the results of our recent examination of the H-1B program, highlighting the key challenges it presents for H-1B employers, H-1B and U.S. workers, and federal agencies. Specifically, this statement presents information on (1) employer demand for H-1B workers; (2) how the H-1B cap impacts employers' costs and whether they move operations overseas; (3) the government's ability to track the cap and H-1B workers over time; and (4) how well the provisions of the H-1B program protect U.S. workers. From 2000 to 2009, the demand for new H-1B workers tended to exceed the cap, as measured by the numbers of initial petitions submitted by employers who are subject to the cap. While the majority (68 percent) of employers was approved for one H-1B worker, demand was driven to a great extent by a small number (fewer than 1 percent) of H-1B employers garnering over one quarter of all H-1B approvals. Cap-exempt employers, such as universities and research institutions, submitted over 14 percent of the initial petitions filed during this period. Most of the 34 H-1B employers GAO interviewed reported that the H-1B program and cap created additional costs for them, such as delays in hiring and projects, but said the global marketplace and access to skilled labor--not the cap--drive their decisions on whether to move activities overseas. Limitations in agency data and systems hinder tracking the cap and H-1B workers over time. For example, data systems among the various agencies that process these individuals are not linked so it is difficult to track H-1B workers as they move through the immigration system. System limitations also prevent the Department of Homeland Security from knowing precisely when and whether the annual cap has been reached each year. Provisions of the H-1B program that could serve to protect U.S. workers--such as the requirement to pay prevailing wages, the visa's temporary status, and the cap itself--are weakened by several factors. First, program oversight is fragmented between four agencies and restricted by law. Second, the H-1B program lacks a legal provision for holding employers accountable to program requirements when they obtain H-1B workers through a staffing company--a company that contracts out H-1B workers to other companies. Third, statutory changes made to the H-1B program over time--i.e. that broadened job and skill categories for H-1B eligibility, increased exceptions to the cap, and allowed unlimited H-1B visa extensions while holders applied for permanent residency--have in effect increased the pool of H-1B workers beyond the cap and lowered the bar for eligibility.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Airline Passenger Bill of Rights Act of 2009''. SEC. 2. AIRLINE CUSTOMER SERVICE COMMITMENT. (a) In General.--Chapter 417 of title 49, United States Code, is amended by adding at the end the following: ``SUBCHAPTER IV--AIRLINE CUSTOMER SERVICE ``Sec. 41781. Air carrier and airport contingency plans for long on- board tarmac delays ``(a) Definition of Tarmac Delay.--The term `tarmac delay' means the holding of an aircraft on the ground before taking off or after landing with no opportunity for its passengers to deplane. ``(b) Submission of Air Carrier and Airport Plans.--Not later than 60 days after the date of the enactment of the Airline Passenger Bill of Rights Act of 2009, each air carrier and airport operator shall submit, in accordance with the requirements under this section, a proposed contingency plan to the Secretary of Transportation for review and approval. ``(c) Minimum Standards.--The Secretary of Transportation shall establish minimum standards for elements in contingency plans required to be submitted under this section to ensure that such plans effectively address long on-board tarmac delays and provide for the health and safety of passengers and crew. ``(d) Air Carrier Plans.--The plan shall require each air carrier to implement at a minimum the following: ``(1) Provision of essential services.--Each air carrier shall provide for the essential needs of passengers on board an aircraft at an airport in any case in which the departure of a flight is delayed or disembarkation of passengers on an arriving flight that has landed is substantially delayed, including-- ``(A) adequate food and potable water; ``(B) adequate restroom facilities; ``(C) cabin ventilation and comfortable cabin temperatures; and ``(D) access to necessary medical treatment. ``(2) Right to deplane.-- ``(A) In general.--Each air carrier shall submit a proposed contingency plan to the Secretary of Transportation that identifies a clear time frame under which passengers would be permitted to deplane a delayed aircraft. After the Secretary has reviewed and approved the proposed plan, the air carrier shall make the plan available to the public. ``(B) Delays.-- ``(i) In general.--As part of the plan, except as provided under clause (iii), an air carrier shall provide passengers with the option of deplaning and returning to the terminal at which such deplaning could be safely completed, or deplaning at the terminal if-- ``(I) 3 hours have elapsed after passengers have boarded the aircraft, the aircraft doors are closed, and the aircraft has not departed; or ``(II) 3 hours have elapsed after the aircraft has landed and the passengers on the aircraft have been unable to deplane. ``(ii) Frequency.--The option described in clause (i) shall be offered to passengers at a minimum not less often than once during each successive 3-hour period that the plane remains on the ground. ``(iii) Exceptions.--This subparagraph shall not apply if-- ``(I) the pilot of such aircraft reasonably determines that the aircraft will depart or be unloaded at the terminal not later than 30 minutes after the 3 hour delay; or ``(II) the pilot of such aircraft reasonably determines that permitting a passenger to deplane would jeopardize passenger safety or security. ``(C) Application to diverted flights.--This section applies to aircraft without regard to whether they have been diverted to an airport other than the original destination. ``(D) Reports.--Not later than 30 days after any flight experiences a tarmac delay lasting at least 3 hours, the air carrier responsible for such flight shall submit a written description of the incident and its resolution to the Aviation Consumer Protection Office of the Department of Transportation. ``(e) Airport Plans.--Each airport operator shall submit a proposed contingency plan under subsection (b) that contains a description of-- ``(1) how the airport operator will provide for the deplanement of passengers following a long tarmac delay; and ``(2) how, to the maximum extent practicable, the airport operator will provide for the sharing of facilities and make gates available at the airport for use by aircraft experiencing such delays. ``(f) Updates.--The Secretary shall require periodic reviews and updates of the plans as necessary. ``(g) Approval.-- ``(1) In general.--Not later than 6 months after the date of the enactment of this section, the Secretary of Transportation shall-- ``(A) review the initial contingency plans submitted under subsection (b); and ``(B) approve plans that closely adhere to the standards described in subsections (d) or (e), whichever is applicable. ``(2) Updates.--Not later than 60 days after the submission of an update under subsection (f) or an initial contingency plan by a new air carrier or airport, the Secretary shall-- ``(A) review the plan; and ``(B) approve the plan if it closely adheres to the standards described in subsections (d) or (e), which ever is applicable. ``(h) Civil Penalties.--The Secretary may assess a civil penalty under section 46301 against any air carrier or airport operator that does not submit, obtain approval of, or adhere to a contingency plan submitted under this section. ``(i) Public Access.--Each air carrier and airport operator required to submit a contingency plan under this section shall ensure public access to an approved plan under this section by-- ``(1) including the plan on the Internet Web site of the carrier or airport; or ``(2) disseminating the plan by other means, as determined by the Secretary. ``Sec. 41782. Air passenger complaints hotline and information ``(a) Air Passenger Complaints Hotline Telephone Number.--The Secretary of Transportation shall establish a consumer complaints hotline telephone number for the use of air passengers. ``(b) Public Notice.--The Secretary shall notify the public of the telephone number established under subsection (a). ``(c) Authorization of Appropriations.--There are authorized to be appropriated such sums as may be necessary to carry out this section, which sums shall remain available until expended.''. (b) Conforming Amendment.--The chapter analysis for chapter 417 of title 49, United States Code, is amended by adding at the end the following: ``subchapter iv--airline customer service ``41781. Air carrier and airport contingency plans for long on-board tarmac delays. ``41782. Air passenger complaints hotline and information.''.

Title: To amend title 49, United States Code, to ensure air passengers have access to necessary services while on a grounded air carrier, and for other purposes Summary: Airline Passenger Bill of Rights Act of 2009 - Requires each air carrier and airport operator to submit for approval by the Secretary of Transportation a proposed contingency plan meeting minimum standards established by the Secretary. Requires an air carrier to provide passengers on a departure- or arrival-delayed grounded aircraft with: (1) adequate food, water, restrooms, ventilation, and medical services; as well as (2) a time frame under which passengers may deplane a delayed aircraft after three hours, except in specified circumstances. Requires an airport operator plan to describe: (1) how passengers will be deplaned following a long tarmac delay; and (2) how facilities will be shared and gates made available to aircraft that experience such delays. Authorizes the Secretary to assess a civil penalty against air carriers and airport operators that fail to submit, obtain approval of, or adhere to a contingency plan. Requires public access to such plans. Directs the Secretary to establish a consumer hotline telephone number for air passenger complaints.

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Summarize: It was also a hard slap to a former colleague and member of the chamber. Asked about the Senate vote during an online “fireside hangout,” Obama said that he expects that Hagel will be confirmed. But he slammed Senate Republicans for their “unprecedented filibuster” of a defense chief nominee. “What seems to be happening, and this has been growing over time, is the Republican minority in the Senate seems to think that the rule now is that you need to have 60 votes for everything,” Obama said. “Well, that’s not the rule.” He added that “it’s just unfortunate that this kind of politics intrudes at a time when I’m still presiding over a war in Afghanistan and I need a secretary of defense who is coordinating with our allies” on U.S. strategy in the region. Republicans predicted they would relent to a simple majority vote, guaranteeing confirmation, later this month -- but only if they see more information about Hagel’s post-Senate foreign policy speeches and his work in private investment groups. Senior Republicans initially scoffed at those demands, first raised by freshman Sen. Ted Cruz (R-Tex.), as unnecessary, but now party leaders hold them up as the main cause for delay. Even if Hagel is eventually confirmed, the process marked another escalation in long-running nomination wars dating to the 1980s, now crossing into an area that has long been the most bipartisan on Capitol Hill: national security. The Hagel fight also demonstrated the Republican fixation on the events surrounding the Sept. 11 attack on the U.S. consulate in Libya that killed four American diplomats. GOP senators have clung to the tragedy as cause for holding Hagel’s nomination hostage in exchange for more details about the attack. Such demands are commonplace in the Senate but are usually reserved for lower-level Cabinet posts or for deputy-secretary positions, not for the person who is in charge of overseeing more than 2 million service members — 66,000 of them in battle in Afghanistan. “This isn’t high school, getting ready for a football game or some play that’s being produced at high school,” Senate Majority Leader Harry M. Reid (D-Nev.) said during an angry floor speech Thursday morning. “This is - we’re trying to confirm somebody to run the defense of our country, the military of our country.” The final tally Thursday was 58 votes to end the filibuster to 40 against, but actually 59 backed Hagel because Reid changed his vote to no so that he could use parliamentary rules to quickly reconsider the nomination when the Senate returns from its Presidents Day break Feb 25. Republicans have gained another 12 days in which to beat up on Chuck Hagel. And even though they may not ultimately stop him from taking over the Pentagon, they relished the opportunity to keep trying. “The fight goes on,” said conservative editor Bill Kristol, who marshaled opposition research, media buys and op-eds against Hagel. Kristol vowed that he would “continue to work to convince a majority of senators of the undeniable truth that we can do much, much better than Mr. Hagel.” Text Size - + reset Reid: Hagel filibuster 'tragic' PHOTOS: Hagel's confirmation hearing Play Slideshow POLITICO LIVE: Hagel, Brennan fight Obama's battles (Also on POLITICO: Reid sets stage for Hagel showdown) And one Republican aide promised the long Presidents’ Day recess would represent for Hagel “one more week of additional questions on top of the questions they refuse to answer.” Just the same, Democrats said they remained confident they would finally make Hagel secretary of Defense when they try again on Feb. 26. In that sense, Thursday’s 58-40 vote to cut off Senate debate was as much an attempt to advance him as Potomac jiu-jitsu by Senate Majority Leader Harry Reid (D-Nev.), who moved it up from Friday knowing it would fail in a bid to get Republicans on the record blocking him. Sixty votes were needed to move the nomination forward. Reid and the White House seized the opportunity to slam the GOP for what they called needless obstructionism that might prove dangerous given the appearance that the Defense Department would be headless. Its incumbent boss, Leon Panetta, was set to stay on until Hagel is finally confirmed, the Pentagon confirmed, but Panetta flew home to California on Thursday to help sell the narrative that Republicans were leaving his office in the E-Ring vacant. (PHOTOS: What they’re saying about Hagel) White House Press Secretary Jay Carney charged Republicans with putting “political posturing ahead of our nation’s security.” “A clear majority in the United States Senate supports Senator Hagel’s confirmation, so today’s action runs against both the majority will of the Senate and our nation’s interest,” Carney said. “Allow this war hero an up or down vote, and let our troops have the Secretary of Defense they deserve.” (Also on POLITICO: McCain 'largely satisfied' on Hagel) Republicans weren’t buying it – Senate Minority Whip John Cornyn said Reid had moved up the Hagel vote just “to get a story in the newspaper,” and he defended the validity of the sticking points over which Republicans had chosen to make their stand: Hagel’s financial disclosures and the Sept. 11 attack on the U.S. Consulate in Benghazi. Republicans said they needed to be confident Hagel hadn’t taken payments from “foreign sources,” and they said the White House owed them more detail about its actions in the immediate aftermath of the Benghazi attack. Democrats fumed, arguing the issues were unconnected and that Hagel had satisfied the Senate Armed Services Committee’s disclosure requirements, but they could also not muster the 60 votes to break Republicans’ barricade. The White House attempted to mollify at least two key Republican opponents, John McCain of Arizona and Lindsey Graham of South Carolina, with a letter that explained that President Barack Obama had called his Libyan counterpart on the day after the attack, but it wasn’t enough. Senate Republicans in a 58-40 vote Thursday blocked former Sen. Chuck Hagel’s (R-Neb.) nomination as Defense secretary from proceeding to a final up-or-down vote. Four Republicans — Sens. Susan Collins (Maine), Thad Cochran (Miss.), Lisa Murkowski (Alaska) and Mike Johanns (Neb.)— joined 55 Democrats and Independents in supporting the nomination. Sixty votes were needed to cut off debate, leaving Democrats one vote short. ADVERTISEMENT The final 58-40 tally reflected a no vote from Senate Majority Leader Harry Reid (D-Nev.), who switched his vote from yes to preserve his ability to bring up the nomination again. Sen. Orrin Hatch (R-Utah) voted present and Sen. David Vitter (R-La.) missed the vote. Republicans said it was too early to clear Hagel’s nomination, but that they would consider allowing an up-or-down vote after the Senate returns to business on Feb. 25. They blamed Democrats for rushing the vote and the White House for not providing additional information about Hagel’s compensation for paid speeches. Reid scolded Republicans for holding up Hagel, saying it was the first filibuster of a Defense nominee in history. Hagel seems likely to win confirmation eventually, but the delay highlighted the contentiousness of his nomination. “I think it’s appropriate to wait until we come back,” said Sen. John McCain (R-Ariz.). “I think there’s plenty of time to have any further questions answered and I intend to vote for cloture then. … He’d certainly get mine and a number of others.” Collins said after the vote she did not try to lobby her Republican colleagues to vote for cloture, but she did not want to filibuster his nomination because she believes the president should have deference in picking his Cabinet. Collins plans to vote against Hagel for Defense secretary. Reid and the White House blasted Republicans for holding up the nomination, accusing them of playing politics at a time when a Defense secretary is sorely needed. The current secretary, Leon Panetta, is headed back home for California on Thursday, though he will remain on as Pentagon chief until a new one is in place. “These delaying tactics are unconscionable, and they should end right away,” White House deputy press secretary Josh Earnest told reporters aboard Air Force One Thursday. A White House official said that the delay would not stop Hagel’s confirmation. “Senator Hagel is going to be confirmed, if not tomorrow then when the Senate returns from recess,” the aide told reporters. President Obama, while participating in a Google Plus hangout, said the vote was “unfortunate.” He noted that Hagel had been consistently praised by Republicans as a senator and was “imminently qualified” to be Defense secretary. Democrats were seeking to finish Hagel’s confirmation this week after he cleared the Senate Armed Services Committee Tuesday. Republicans have demanded more information about speeches the nominee gave and his compensation for them. Sen. Ted Cruz (R-Texas) at a hearing this week suggested the speeches were given to extreme or radical groups, a statement some Democrats have criticized. Other Republicans, including McCain and Sen. Lindsey Graham (R-S.C.), had threatened to block Hagel because the White House wasn’t giving them the information they were looking for about the terrorist attack last year on a U.S. consulate in Benghazi, Libya. Democrats had hoped that a White House letter sent to Graham, McCain and Sen. Kelly Ayotte (R-N.H.) on Wednesday might convince them to vote for cloture, but the senators stuck with their party. “There’s a good many of us who believe tomorrow is ridiculous because he just came out of committee two days ago,” Graham told reporters. “But when we come back, I’d feel very comfortable, unless something really stunning comes out, to go to vote.” Graham also blamed Democrats for forcing the vote on Hagel this week, saying they had delayed votes in the past on Bush administration appointees. “Lousy of them — what a double standard,” Graham said. “I’m highly confident if the Democrats were in our shoes and you had a controversial nominee like this with outstanding information, that they would do at least what we’ve done, probably more.” Sen. Bob Corker (R-Tenn.) suggested that a cloture vote might not even be necessary after the recess, if no Republicans objected going straight to a final up-or-down confirmation vote. He also felt that the White House would provide the “legitimate information” that GOP senators have been asking for. “I think the legitimate information that’s been asked for will come,” Corker said. “Some people may have asked for things that are over the top — I don’t know that, by the way — but I think the legitimate requests will be answered.” After a party lunch Thursday, GOP senators were nearly united in saying that the Senate was moving too quickly to confirm a controversial nominee. “The bottom line is it’s premature for Sen. Reid to cut off debate today,” said Sen. Lamar Alexander (R-Tenn.). “I have a little personal experience with this — I was nominated and it took 87 days between the time I was nominated and the time I was confirmed.” Republicans have bristled at the notion they are filibustering Hagel’s nomination, and say he will almost surely be confirmed after the recess. But Democrats say that Republicans are in fact taking the unprecedented step of filibustering a Defense secretary nominee. “It is shocking that our Republicans colleagues would leave our nation without a secretary of Defense with all the things going on and when we’re in a war,” Reid said Thursday. The White House had hoped Hagel would be in place after this week to attend a NATO meeting of defense ministers in Brussels next week. Now Panetta may take one more trip abroad before he retires back to California. "We'll cross that bridge if we come to it," a senior defense official told The Hill. This story was posted at 4:59 p.m. and updated at 5:51 p.m.

Summary: The Chuck Hagel drama continued on Capitol Hill today, as Republicans refused to allow a final vote on his confirmation to become defense secretary, reports the Hill. Hagel is expected to prevail eventually-but now it can't happen until lawmakers return from the Presidents' Day recess. Expect the next vote on Feb. 26, reports Politico. One of the sticking points is that Republicans want more information about Hagel's compensation for speeches he gave after leaving the Senate in 2008. "I think it's appropriate to wait until we come back," said John McCain. "I think there's plenty of time to have any further questions answered and I intend to vote for cloture then." Harry Reid needed 60 votes to force an up-or-down vote today, but he came up with only 58. The White House, noting earlier that Hagel would miss a NATO meeting in Brussels next week, called the GOP tactics "unconscionable." The move marks the first time a nominee for defense secretary has faced a filibuster, reports the Washington Post.

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Write a title and summarize: The Venus Kinase Receptor (VKR) is a single transmembrane molecule composed of an intracellular tyrosine kinase domain close to that of insulin receptor and an extracellular Venus Flytrap (VFT) structure similar to the ligand binding domain of many class C G Protein Coupled Receptors. This receptor tyrosine kinase (RTK) was first discovered in the platyhelminth parasite Schistosoma mansoni, then in a large variety of invertebrates. A single vkr gene is found in most genomes, except in S. mansoni in which two genes Smvkr1 and Smvkr2 exist. VKRs form a unique family of RTKs present only in invertebrates and their biological functions are still to be discovered. In this work, we show that SmVKRs are expressed in the reproductive organs of S. mansoni, particularly in the ovaries of female worms. By transcriptional analyses evidence was obtained that both SmVKRs fulfill different roles during oocyte maturation. Suppression of Smvkr expression by RNA interference induced spectacular morphological changes in female worms with a strong disorganization of the ovary, which was dominated by the presence of primary oocytes, and a defect of egg formation. Following expression in Xenopus oocytes, SmVKR1 and SmVKR2 receptors were shown to be activated by distinct ligands which are L-Arginine and calcium ions, respectively. Signalling analysis in Xenopus oocytes revealed the capacity of SmVKRs to activate the PI3K/Akt/p70S6K and Erk MAPK pathways involved in cellular growth and proliferation. Additionally, SmVKR1 induced phosphorylation of JNK (c-Jun N-terminal kinase). Activation of JNK by SmVKR1 was supported by the results of yeast two-hybrid experiments identifying several components of the JNK pathway as specific interacting partners of SmVKR1. In conclusion, these results demonstrate the functions of SmVKR in gametogenesis, and particularly in oogenesis and egg formation. By eliciting signalling pathways potentially involved in oocyte proliferation, growth and migration, these receptors control parasite reproduction and can therefore be considered as potential targets for anti-schistosome therapies. Trematode parasites of the Schistosoma genus are responsible for schistosomiasis or bilharzia, one of the most important parasitic endemias worldwide in terms of mortality and morbidity. According to the World Health Organisation, more than 240 million people are currently infected by schistosomes, with about 200 000 deaths per year [1]. The pathology of schistosomiasis mostly results from the accumulation of parasite eggs in host tissues. Indeed, among the several hundreds of eggs laid daily by each female schistosome, a large part gets trapped into host tissues and elicits immune responses, such as inflammation and granuloma formation, causing severe disorders, particularly hepatosplenomegaly, hepatic fibrosis and bladder cancer [2]. Praziquantel (PZQ) is the only drug currently used to cure schistosomiasis. This drug is active against the three main species infecting humans (S. mansoni, S. haematobium, S. japonicum). However, its widespread use for mass treatment since the early 80' s, has already led to the emergence/of drug-insensitive Schistosoma strains. Moreover, a limit of PZQ is that it does not affect the larval parasites and, therefore, does not provide a total clearance of the infection [3]–[6]. In the absence of a vaccine much efforts are currently made to characterize molecules that control survival, growth and reproduction of schistosomes in order to identify targets for novel drugs against these parasites [7]–[9]. In this context, several schistosome protein kinases (PK) have been studied during the last decade and some of them were shown to be involved in gametogenesis and egg formation in the parasite S. mansoni [10]–[20]. Among these kinases are cellular PKs such as Polo-like kinases (Plks), which regulate cell-cycle progression during M-phase by activating cyclin-dependent PKs. Two Plks, SmPlk1 and SmSak, have been characterized in S. mansoni and their role in the control of parasite reproduction has been demonstrated [17]–[19]. Besides these mitotic kinases, tyrosine kinases (TKs) were also shown to play essential roles in schistosome reproduction [11], [16]. TKs constitute a large family of receptor and cytosolic molecules that regulate development, cell division, differentiation and metabolism in many organisms, and they actually represent major targets in drug discovery programs against cancer and metabolic disorders [21], [22]. Recent work indicated that S. mansoni TKs can be as well considered as interesting targets against schistosomes. By inhibiting the kinase activities of schistosome Src, Abl, and Syk cytosolic TKs with the commercial TK inhibitors Herbimycin, Imatinib, Piceatannol, respectively, dramatic changes were observed in the reproductive organs of in vitro-cultured adult S. mansoni, affecting gametogenesis and egg production [11]–[16]. Also, the harmful effect on schistosomes of other TK inhibitors (tyrphostins) directed against human EGF and insulin receptors revealed the importance of RTK signalling in major processes of embryogenesis and reproduction in these parasites [23]–[25]. Few years ago, we discovered in S. mansoni an atypical receptor tyrosine kinase (RTK) named Venus Kinase Receptor [26]. VKR is a transmembrane molecule composed of an intracellular TK domain close to that of Insulin Receptors (IRs), and an extracellular ligand binding domain (LBD) with a Venus Flytrap (VFT) structure similar to that of many class C G Protein Coupled Receptors (GPCR) [27]. Such a VFT-TK fusion was described for the first time in S. mansoni, but recent analysis of genomic data has allowed us to extend the presence of VKRs to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum [28], [29]. We showed that VKR kinase activity is inducible upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site [28]. Preliminary experiments indicated that VKRs were preferentially expressed in larvae and in gonads of several species [28], suggesting a role in development and reproduction. A single vkr gene was usually found in each invertebrate genome but exceptionally the genome of S. mansoni, as that of another trematode Clonorchis, contains two genes vkr1 and vkr2 with a similar organization but encoding distinct proteins [29], [30]. The expression of Smvkr1 and Smvkr2 was shown to be variable in the different parasitic stages, likely indicating their independent regulation [30]. In this work, we investigated the function of SmVKR in the reproduction of schistosomes. We first showed that Smvkr1 and Smvkr2 are expressed in adult female organs at different sites, and the results of RNAi experiments suggest that SmVKR1 and SmVKR2 participate in major pathways along the maturation of oocytes in female schistosomes. The two VKRs are activated by distinct ligands, and the search for interacting partners by yeast two-hybrid screening coupled to the analysis of signalling pathways induced by SmVKRs in Xenopus oocytes support the hypothesis that SmVKR1 and SmVKR2 exert different roles in gametogenesis and reproduction processes in schistosomes. We have shown previously that SmVKRs were expressed in all developmental stages of the parasite S. mansoni and that the expression of SmVKR2 was globally higher than that of SmVKR1, except in the sporocyst stage [30]. Using as a reference α-tubulin transcripts, we show here that both Smvkr1 and Smvkr2 transcripts are more abundant in female than in male adult worms, and we confirm that Smvkr2 is transcribed more actively than Smvkr1 in each gender (Figure 1, A). Former studies have already indicated that SmVKR1 (firstly named SmRTK-1) was expressed in the parenchyma of male worms and in the ovary of female worms [26]. In this work, we have localized Smvkr1 and Smvkr2 transcripts on sections of adult worm pairs by in situ hybridization. Results (Figure 1, B) demonstrate the presence of abundant Smvkr1 transcripts in the female ovary. From the staining intensity, indications were obtained for a more intense staining of the big, mature oocytes which are contained in the posterior part of the ovary whereas the immature oocytes (oogonia) are located within the smaller, anterior part of the ovary. Smvkr1 labelling is also observed around the ootype (a female structure which receives fertilized oocytes and vitelline cells, and constitutes the egg-forming organ) and in the parenchyma of males. Smvkr2 transcripts are found also in the ovary but, unlike Smvkr1 transcripts, their abundance seemed to be higher within the anterior part of the ovary containing immature oocytes. An intense Smvkr2 labelling was also obtained in the region of the ootype and its surrounding area, which could correspond to the Mehlis gland and/or the oviduct (Figure 1, B). In male testes, a specific labelling could be observed for Smvkr1 as well as for Smvkr2, which was more evident following longer exposure (Figure S1). Hahnel et al [31] have recently established a protocol to isolate schistosome reproductive organs usable for morphological and structural studies, and as sources of proteins and RNA. Q-PCR experiments performed with RNA extracted from isolated ovaries have shown that Smvkr1 and Smvkr2 are expressed at the same level in the ovaries of sexually mature females recovered from bisexual parasite infections (Figure S2, A). However, within the ovaries isolated from female worms grown in the absence of males and which are known to contain only immature and undifferentiated cells (oogonia) [32], we observed that the Smvkr2 gene was more actively transcribed than the Smvkr1gene (2. 6 fold more) (Figure S2, A). This result is in accordance with the detection of Smvkr2 transcripts preferentially found within the part of the ovary containing oogonia (Figure 1, B). Moreover, qPCR data indicated that both Smvkr1 and Smvkr2 transcripts were up-regulated strongly (13. 5 fold and 5. 4 fold respectively) in the ovaries of sexually-developed females as compared to the organs from virgin females issued from unisexual infections (Figure S2, B). This indicated the importance of SmVKR receptors during development and maturation of reproductive organs. The up-regulation of Smvkr1 could be related to primary oocytes, which dominate within the posterior part of the ovary (Figure 1, B). Q-PCR analyses of isolated testes confirmed the presence of Smvkr1 and Smvkr2 transcripts in male reproductive organs and showed their up-regulation in testes from males issued from bisexual infections (data not shown). Smvkr gene expression was targeted for suppression by introducing via electroporation Smvkr1 or Smvkr2 dsRNA in couples of adult parasites in vitro. Figure 2 shows that a 5 day-treatment of worm couples with dsSmvkr1 led to a decrease of about 80% of the amount of Smvkr1 transcripts in total parasites with a non-significative reduction of the level of Smvkr2 transcripts, indicating a priority targeting of the Smvkr1 gene by dsSmvkr1. Similar treatment with dsSmvkr2 gave comparable results, showing a reduction of more than 60% of Smvkr2 transcripts but only a small and non-significative decrease of Smvkr1 transcripts. Concomitant introduction of both dsSmvkr1 and dsSmvkr2 induced a simultaneous and significant reduction of both transcripts (70% and 55% for Smvkr1 and Smvkr2 respectively). The suppression of Smvkr gene expression did not result in any detectable changes in worm behavior and male-female pairing. To investigate whether RNAi-mediated silencing of Smvkr genes could have any impact on the morphology of parasite reproductive organs, we examined the worm couples after 5 days of dsRNA treatment by using CLSM (Figure 3). In male worms we did not observe any significant changes in the morphology of testicular lobes of both dsSmvkr1- and dsSmvkr2-treated parasites for which we noted the presence of sperm in seminal vesicle (Figure 3B, D). However, in double-targeted worms, we observed frequently a reduced diameter of the testicular lobes accompanied by a reduction of cell density in testes as well as empty seminal vesicles, compared to single dsSmvkr-treated worms or to control worms treated with dsLucRNA (Figure 3F). In female worms, drastic changes in the structure and size of the ovary appeared when parasites were treated with dsSmvkr1 or dsSmvkr2 or both of them, compared to control dsLucRNA (Figure 3A, C, E, F). Structure and content of the ovary, normally composed of differentially matured oocytes, i. e., small oogonia in the anterior part and primary oocytes within the posterior part, were drastically changed in Smvkr-suppressed parasites. A strong disorganization of the ovary containing predominantly big cells was observed following dsSmvkr1 or dsSmvkr2 treatment (Figure 3 A, C), and this phenotype was associated to a strong reduction of the ovary size in dsSmvkr2-treated females (Figure 3C). When both genes were targeted, we observed an addition of the two phenotypes (Figure 3E). The bottom insert (Figure 3, G) shows the presence of an egg formed by a fertilized oocyte and 30–40 vitellocytes in the ootype of dsLuc-treated control worms. Within the ootype of dsSmvkr1-targeted females, clusters of unorganized vitelline cells and the absence of a regular egg shell were identified indicating problems in egg formation (upper insert Figure 3, A). These results confirmed that SmVKR1 and SmVKR2 are important for gametogenesis, oogenesis and perhaps also for egg formation and thus are implicated in the reproductive functions of S. mansoni. Moreover, obtaining distinct phenotypes in dsSmvkr1 and dsSmvkr2-treated females could corroborate the different localization of the receptors in the ovary, and suggested possible different functions in this organ. The mode of activation and the signalling pathways potentially elicited by SmVKR1 and SmVKR2 respectively, were further studied. VKRs form a family of structurally conserved transmembrane receptors, composed of a TK intracellular domain and an extracellular VFT domain that serves for ligand binding and subsequent receptor activation. It was shown previously that VKR from the insect Apis mellifera was activated by amino acids, particularly by arginine [28] and the capacity of L-Arg to activate SmVKR1 was then demonstrated. Indeed, following receptor expression in Xenopus oocytes, binding of L-Arg induced SmVKR1 autophosphorylation and the activation of pathways leading to resumption of meiosis in the oocytes ([30], Figure 4). In former studies, we showed that meiosis resumption was dependent on kinase activation [17], [18], [30] leading to Germinal Vesicle BreakDown (GVBD), a process easily detectable by the formation of a white spot at the animal pole of the oocyte. According to this, we used this assay system to test the ability of all proteinogenic L-amino acids to behave as ligands and to activate SmVKR1 or SmVKR2 respectively. Results showed that seven L-amino acids (Arg, Ser, Gly, Ala, Cys, Thr and Glu) had the potential to activate SmVKR1 at a 1 mM concentration (Table S1) and also confirmed that L-Arg was the most potent agonist for SmVKR1 (Figure 4, A). When added at 1 µM in the oocyte incubation medium, L-Arg was able to induce GVBD in 80% of the SmVKR1-expressing oocytes. Surprisingly, expression of SmVKR2 in oocytes induced spontaneously their maturation in the usual ND96 medium, a minimally buffered saline solution complemented with CaCl2 (2. 8 mM) and MgCl2 (1 mM). This observation suggested that bivalent ions could be responsible for the spontaneous activation of SmVKR2 and the use of salt-depleted media further indicated that Ca ions (at a 1 mM minimal concentration) were effectively responsible for the activation of SmVKR2 (Figure 4, B). To assess the specific action of Ca2+, we tested the effect of other bivalent ions (Ni2+, Fe2+, Mn2+ and Cu2+) on SmVKR2-expressing oocytes, but none of these ions were able to induce GVBD, even when they were used at a 10 mM dose in the Ca2+-depleted medium (data not shown). Additional experiments performed in ND96 medium without Ca2+ demonstrated that the activation of SmVKR1 by 1 µM L-Arg was not affected by the absence of Ca2+. They also showed that L-Arg itself was able to activate SmVKR2 but at a minimal concentration of 1 mM, thus 1000- fold higher than that required for SmVKR1 (Figure 4, C). Three other L- amino acids (Thr, Trp and Cys) also activated SmVKR2 at 1 mM (Table S1). SmVKR1 and SmVKR2 receptors share similar VFT modules (48% residue identity). VFT modules constitute the binding pocket of various receptors activated by small molecules [27]. In most class C GPCRs, they are the binding sites for natural amino acids or derivatives and ligand binding depends on a consensus motif of 8 residues implied in the recognition of the α-amino acid group (i. e. primary amine and carboxylic acid) [33]. Particularly, the Ser165 residue which binds the COOH group of glutamate in the metabotropic glutamate receptor (mGluR1) and is the most conserved residue in class C GPCRs, is highly conserved in all VKRs [28], [29]. In order to confirm the implication of this Ser residue in amino acid binding to SmVKR1 and SmVKR2, we mutated the Ser466 and Ser410 of SmVKR1 and SmVKR2 respectively in Ala, and the mutated receptors were expressed in oocytes. GVBD assays were performed using L-Arg as a ligand. We found that a 1000-fold higher concentration of L-Arg (1 mM) was required to activate the mutated SmVKR1S466A and to induce GVBD. Similarly, SmVKR2S410A failed to respond to L-Arg (Figure 4, C). Western blot results confirmed that such differences in L-Arg sensivity were not due to a lower level of expression of the mutant receptors in oocytes. Wild type and mutated forms of SmVKR1 and SmVKR2 were detected with the same intensity in oocyte membrane extracts by anti-V5 antibodies. On the same blots, anti-phosphotyrosine (PY) antibodies revealed that phosphorylation of SmVKR1 and SmVKR2 perfectly correlates to their ability to induce GVBD in oocytes (Figure 4, D). These data confirmed the importance of the conserved Ser residue in amino acid binding. Among all amino acids, Arg is uniquely containing a guanidino group. Therefore, we investigated the importance of this group in the affinity of L-Arg to SmVKR1 by testing a panel of Arg derivatives devoid either of the guanidino group (ornithine), or of the α-amino acid function (creatine, agmatine) as well as two Arg analogs (D-Arg, Canavanine). Results (not shown) indicated that all these compounds were inactive on SmVKR1 and that all of them had the potential to compete and to block totally the inducing effect of L-Arg when added with a 10-fold excess ratio. Such data are in favor of a specific activating effect of L-Arg on SmVKR1, which very likely implies a precise recognition of the amino groups of the Arg molecule together with specific constraints for a correct positioning of the ligand. Further studies of structure-activity relationships are needed to elucidate the nature of the residues involved in the interaction between SmVKR1 and L-Arg. RTK activation is known to require homo- or heterodimerization of receptor molecules and similarly, it has been clearly demonstrated that the VFT modules of class C GPCRs also function as dimers [27]. A three dimensional model of the VKR of A. mellifera was previously built which suggested that a VFT dimer interface was present in VKR proteins similar to that in GPCRs. It was shown further that AmVKR proteins effectively form dimers at the cell surface [28]. In this work, we have investigated the capacity of SmVKR proteins to homo- or heterodimerize. Results in Figure 5 demonstrate that SmVKR1 and SmVKR2 are active as dimers when they are expressed in Xenopus oocyte. Using two versions of SmVKR1 differentially tagged with V5 or Myc epitopes, we showed by co-immunoprecipitation and Western blot analysis that SmVKR1 proteins can form homodimers at the oocyte membrane. The interaction between SmVKR1-V5 and SmVKR1-Myc molecules required the presence of Ca2+ or of L-Arg. However, as shown by labelling with anti-PY antibodies, SmVKR1 phosphorylation and thus kinase activation were dependent on the addition of L-Arg and could not be induced by Ca2+ alone. Similarly, analyses performed with SmVKR2 showed the effect of Ca2+ on receptor dimerization but also on its activation, both processes being independent on the addition of L-Arg (Figure 5). These data thus confirmed the previous demonstration that SmVKR1 and SmVKR2 were activated respectively by L-Arg and Ca2+. Even if in the developing ovary, the detection of SmVKR1 and SmVKR2 transcripts at different sites is not in favor of their possible interaction in this organ, we cannot exclude that in certain tissues (for example in testes where they are equivalently distributed in the whole organ) the two receptors can co-interact and co-activate at the cell membrane, similarly to many other RTKs. Following co-expression of SmVKR1 and SmVKR2 in Xenopus oocytes, we showed effectively that they were able to interact and that this interaction occurred only when both the ligands L-Arg and Ca2+ were added. In these conditions, two bands were revealed by anti-PY antibodies in the precipitated heterocomplexes, corresponding respectively to activated SmVKR1 (150 kDa) and SmVKR2 (170 kDa) (Figure 5). When only Ca2+ or only L-Arg was added, a single band was revealed by anti-PY antibodies in immune complexes precipitated either by anti-V5 or by anti-Myc antibodies. These bands likely represent activated homodimers of SmVKR2-V5 and SmVKR1-Myc, respectively. The identification of potential SmVKR signalling partners was performed by yeast two-hybrid (Y2H) screening of a S. mansoni cDNA library [34], using the intracellular domains (ICD) of SmVKR1 or of SmVKR2 as baits. ICD mutants exhibiting constitutive kinase activity due to the introduction of a negatively charged glutamic residue next to the YY autophosphorylation site [30], [35] (SmVKR1ICDYYRE and SmVKR2ICDYYRE) were used as baits in order to facilitate the capture of partners interacting with the receptors in their activated/phosphorylated state. About 400 clones positive for β-galactosidase expression were selected. Prey plasmids were isolated and their sequences determined. Among these, we could identify by BLASTn analyses 55 and 15 potential protein partners for SmVKR1 and SmVKR2 respectively, which were classified according to their putative functions (Table 1). Several proteins were selected by both SmVKR1 and SmVKR2, like actin and prefoldin involved in cytoskeleton functions and coatomer subunit with roles in membrane trafficking. The NAD-dependent deacetylase sirtuin-7 involved in chromatin remodelling was also shown to interact with both ICDs. Concerning proteins involved in phosphosignalling, we noted the selection of two important molecules Cbl and Shb, already known for their direct interaction with RTK. The E3 ubiquitin-protein ligase Cbl was only trapped by SmVKR2 and this fact might be corroborated by the prediction using UbPred program [36] of more ubiquitination sites in SmVKR2 than in SmVKR1. Secondly, an SH2 domain-containing adapter of the Shb protein family was demonstrated to bind specifically to SmVKR1 in its phosphorylated form and the role of the SH2 domain of SmShb in this interaction was recently demonstrated (results not shown). Two other proteins (XM_002575792. 1 and XM_002574592. 1) were selected with SmVKR1 but not with SmVKR2. Using phylogenetic analyses, the first one was shown to belong to the subfamily of protein phosphatase PP2C/PPM1G gamma subfamily (Figure S4) and the second one was shown to belong to the MEK7 subfamily of the MAPK kinase family (Figure S5). As protein phosphatases PP2C are known to be regulators of the activity of stress-induced MAPK pathway components [37] and as MEK7 proteins are responsible for JNK activation [38], it was interesting to further analyse the processes of signalling by these two receptors in order to potentially discriminate distinct pathways for each VKR. From the indications that SmVKR1 and SmVKR2 i) were expressed differentially in the tissues of S. mansoni, ii) were activated by distinct ligands, and iii) were able to interact with different partners in the yeast expression system, we explored the possibility that these receptors could mediate distinct signalling pathways in the parasite. The Xenopus oocyte is a convenient model system to study signalling pathways initiated by extracellular inducers. Thus, we analyzed in oocytes expressing SmVKR1 and SmVKR2, the effect of L-Arg or of Ca2+ on the phosphorylation of key cellular proteins already known to be involved in RTK signalling, and particularly those belonging to PI3K/Akt/mTOR and MAPK pathways. In controls, we treated oocytes with progesterone (PG), a natural stimulus known to induce activation of these pathways in stage VI Xenopus oocytes [39]. Analyses of phosphoproteins in oocyte extracts were performed 5 hours after the addition of L-Arg and Ca2+ ligands and prior to oocyte maturation, in order to anticipate the phosphorylation loops which will set up following the activation of MPF (Maturation Promoting Factor). Results in Figure 6 show that activation and phosphorylation of both SmVKR1 and SmVKR2 elicit in the oocyte the activation of the PI3K pathway resulting in the phosphorylation of the kinase Akt/PKB by its activating kinases PDK (phosphoinositide-dependent kinase) and mTORC2 (mammalian target of rapamycin complex 2) respectively on Thr308 and Ser473 residues. Akt is known to activate mTOR leading to the activation of p70S6K, a mitogen activated kinase responsible for the phosphorylation of the S6 protein of the 40S ribosomal subunit, and involved in the translational control of ribosomal proteins and elongation factors [40]. The phosphorylation of p70S6K on Thr389, a residue critical for its kinase activity, was detected in the oocytes following activation of SmVKR1 and SmVKR2 by their respective ligands. Such profiles were similar to those obtained in PG-stimulated oocytes, confirming the activation of the PI3K/Akt/S6K pathway by SmVKR1 and SmVKR2. No activation of the PI3K pathway was observed in the absence of ligand in SmVKR-expressing oocytes. Activated RTKs constitute platforms for the recognition and recruitment of adaptor proteins with SH2 domains linking extracellular signals for RTK activation to downstream signal transduction pathways. Among these, the MAP kinase signalling cascade (Ras-Raf-MEK-ERK) plays a pivotal role and is essential for a variety of processes such as growth, differentiation, proliferation, survival and apoptosis in all eukaryotes. Using anti-phospho ERK2 antibodies, we showed that both SmVKR1 and SmVKR2 can activate downstream the canonical ERK/MAPK cascade. JNK/MAPK pathway activation is also a classical component of RTK signalling [41], [42]. Our results show that SmVKR1 when activated by L-Arg induces the phosphorylation of JNK, exactly as does the activation of Xenopus receptors by PG or by insulin in control oocytes (see Figure 6 A and S3). However, JNK phosphorylation is not detected in the case of SmVKR2 activated by Ca2+ in spite of the potential of oocytes to undergo GVBD, and this is in agreement with the fact that JNK plays a limited role in cell-cycle progression [42]. The activation of JNK by SmVKR1 corroborated the identification of JNK pathway actors, like MEK7 and PP2C, as potential interacting partners of SmVKR1 but not of SmVKR2 (Table 1), providing evidence that each VKR can elicit common but also distinct pathways. Additional data (Figure S3) also indicated that the autophosphorylation of JNK was inhibited by its specific inhibitor SP600125 [43] but not by the addition of purvanalol, the inhibitor of the cyclin-dependent kinase CDK1 that is responsible for MPF activation and meiosis resumption in the oocyte. Therefore, JNK activation, which is elicited by SmVKR1 but not by SmVKR2, follows a pathway parallel to meiotic processes and this confirms the potential of SmVKR1 in specific functions. In higher eukaryotes, activation of p38 MAPK usually correlates with cell cycle arrest. However, in Xenopus oocytes, p38γ/SAPK3 activation has been shown to be important for the meiotic G2/M progression [44]. Following activation by its upstream kinase MKK6, it phosphorylates and activates Cdc25C, the phosphatase directly responsible for MPF activity. Active mutants of MKK6 accelerate PG-induced maturation in oocytes whereas kinase-dead mutants of MKK6 or p38γ inhibit this meiotic progression [45]. Results in Figure 6 A confirm this activation of p38 MAPK in PG-stimulated oocytes but they clearly indicate that SmVKR1 and SmVKR2 kinases are not able to induce this pathway. VKR is an uncommon RTK that bears a VFT ligand-binding domain not found in any other RTK [26]. Since its first discovery ten years ago in S. mansoni, VKR has been found in a large variety of organisms. It is preferentially expressed in larval stages and in gonads of several organisms, suggesting its role in development and reproduction [28]. This work is centered on the functions of VKR in S. mansoni, and specially on the demonstration of its role in reproduction. We performed a comparative analysis of the two members SmVKR1 and SmVKR2 expressed in S. mansoni, studying their respective tissue distribution, the morphological and physiological impact of their knock-down expression in parasites, molecular aspects of receptor activation and cellular signalling pathways. Firstly, we showed by in situ detection of transcripts that SmVKRs were expressed in parasite reproductive organs, more intensively in female ovaries than in testes, in which only a diffuse labelling was obtained. These data were in agreement with the previous observation that VKRs were massively expressed in female gonads of insects and sea urchin, and thus corroborated the hypothesis of a role of VKR in reproductive processes. Additionally, the hints towards distinct localizations of SmVKR1 and SmVKR2 inside of the schistosome ovary suggested that each receptor could potentially initiate different processes in gametogenesis and egg production. Within the vitellarium, a tissue devoted to intense mitotic activities and in which different PKs known to be involved in mitosis are largely expressed (SmPlk1and SmSak polo-like kinases [17], [18], SmTK3 Src kinase [13] and TGFβ receptor SmTR1 [20]), Smvkr transcripts were not detected. This indicated that SmVKRs could play specific functions in gonads, particularly in gamete differentiation. Recent data have shown that the well-known IR inhibitor, tyrphostin AG1024, is able to inhibit with a similar efficiency the kinase activities of SmIR1 and SmIR2 but also those of SmVKR1 and SmVKR2, hence confirming the IR-like TK properties of the four receptors [24]. Following AG1024 treatment of female parasites, a remarkable reduction of the size of the ovary was observed with a dominance of primary oocytes within the whole ovary. Additionally, the drug had a visible impact on the formation of the egg. In male schistosomes, AG1024 also affected spermatogenesis and differentiation of sperm [24]. These results already indicated that AG1024–sensitive IR-like receptor signalling played essential functions in gametogenesis. In the present study, we used RNA interference to target specifically SmVKR1 or/and SmVKR2 and to demonstrate that SmVKR were actually important actors for gamete production, oocyte maturation and egg formation. In male worms, targeting of SmVKR1 or SmVKR2 was not sufficient to clearly affect the production of sperm but when both SmVKRs were knocked down simultaneously, a modification of the cell density in testes was noticed in a large proportion of worms, which contained no more sperm in their seminal vesicles. From this result, it can be hypothesized that SmVKR1 and SmVKR2 may be co-expressed in male germinal cells, exerting redundant cellular functions in spermatocytes. However, we cannot fully exclude that subtle differences exist which are not detectable by this approach since the affected structures are minor compared to their clearly visible counterparts within the ovary. In female worms, the consequences of the suppression of SmVKR are spectacular, with important morphological changes in the ovary, following treatment with dsSmvkr1 or dsSmvkr2 or both. In all conditions, we observed a strong disorganization of the ovary which was dominated by the presence of primary oocytes. Additionally, in the case of dsSmvkr2 a reduction of the ovary size was found and in Smvkr1-suppressed worms an abortion of egg formation. These data confirm the essential roles of SmVKRs for female gametogenesis and egg production. Furthermore, the differences between the phenotypes observed in dsSmvkr1 or dsSmvkr2-treated parasites suggest that SmVKR1 and SmVKR2, could exert different functions at different steps of oocyte maturation (Figure 7). Structural and functional differences between the two members of the VKR family in S. mansoni have been highlighted. Both SmVKR1 and SmVKR2 were shown to function as homodimers, as other insect VKRs [28] and we have shown that receptor dimerisation occurs for both SmVKR in the presence of Ca2+. The bivalent cation is sufficient to obtain dimerisation and activation of the kinase receptor in the case of SmVKR2 but not in the case of SmVKR1, which required to be activated that an amino acid, preferentially L-Arg, binds to its VFT domain. This property was found as common to all the insect VKRs studied ([28], not published). Thus, Ca2+ appears to be the main activator (and/or ligand) of SmVKR2 whereas L-Arg (or other amino acids with a lower efficiency), represents the ligand of SmVKR1. An important aspect of these studies concerns the difference in L-Arg-dependence for each SmVKR that is probably related to their different distribution inside the ovary and thus to the availability of ligands for oocytes along their maturation in reproductive organs. It is tempting to speculate that functional activity of SmVKR1, which is mainly present in mature, primary oocytes ready to be transported to the oviduct to be fertilized by sperm in the receptaculum seminis, is regulated by L-Arg since this amino-acid is largely known to be a major sperm constituent in many organisms [45], [46]. Considering as unlikely the formation of SmVKR1/SmVKR2 heterocomplexes in the ovary, their formation may be possible in the male gonad. Here, heterodimers could be active owing to the presence of L-Arg and Ca2+, which are both required to form active heterodimers, as shown in this study. Following the demonstration by RNAi studies of the importance of SmVKR1 in schistosome reproduction, it was interesting to investigate the use, as an alternative to TK inhibitors, of antagonist ligands to block SmVKR1 activity. In this context, the fact that DmXR, a Drosophila GPCR with a VFT similar to that of all VKRs, could bind and be activated by the L-Arg analog, L-Canavanine [47], prompted us to check for the potential of various Arg derivatives, including L-Cana, to activate SmVKR1. Results indicated that none of the Arg derivatives tested was able to activate SmVKR1 and more importantly that all of them blocked in a competitive manner the effect of L-Arg on SmVKR1 activation. Preliminary experiments in which worms were incubated in the presence of L-Cana indicated that this L-Arg antagonist exerts a deleterious effect on reproductive organs comparable to that obtained with dsSmvkr1 treatment by RNAi (unpublished). This reinforces the idea that ligand mimics/antagonists could be, together with TK enzyme inhibitors (like tyrphostins) [24], efficient drugs to inhibit SmVKR activity and control reproduction in schistosomes. Of course, a better knowledge of the VFT structure of SmVKR1 is still required for the design of such antagonists and further SAR studies are currently in progress to identify the residues implied in L-Arg specific binding. Not much is known about VKR signalling in schistosomes except that there is recent evidence for a cooperation of SmVKR1 with a kinase complex consisting of –in part- unusual kinases [16]. To enlarge the knowledge about binding partners of SmVKR1 but also SmVKR2, a cDNA library screening was performed using the Y2H protocol and intracellular parts of SmVKR1 and SmVKR2 with constitutive kinase activity in order to capture proteins interacting with the active receptors, and particularly those recognizing phosphotyrosines and containing SH2 or PTB domains. A large number of potential partners for SmVKR1 and SmVKR2 were trapped and they were classified in five groups according to their putative functions. Proteins involved in cytoskeleton reorganization, in vesicular trafficking, kinase signalling, gene expression or protein synthesis were captured by both SmVKR proteins, and they were more numerous in the case of SmVKR1. Several of these interacting partners possess known functions in reproduction. Among the specific SmVKR1 partners, we found SmRho1 (the RhoA GTPase already shown to be expressed in schistosome reproductive organs [34]), the kinase Mek7 and a phosphatase PP2C, three proteins involved in the JNK activation pathway [37], [38], [48]. Several evidences have pointed out diverse functions of the JNK pathway in germline homeostasis, meiosis progression and spindle assembly [49]–[51]. Additionally, a PP2C isoform (PPM1A) is synthesized during oocyte maturation in mammals [52] and the RhoA GTPase is involved in ovulation in Caenorhabditis elegans [53]. Another interacting partner of SmVKR1 is the transmembrane protein Notch, which is known to be involved in germline proliferation and meiosis progression [54]–[57]. Distinct SH2-containing adaptor proteins interacted with SmVKR1 and SmVKR2, these were SmShb and SmCbl, respectively. Interestingly, Shb was described to regulate oocyte meiosis I progression in mice and to be involved in ERK and RSK pathways [58]. Cbl is a E3 Ubiquitin-ligase expressed in Drosophila oocytes and responsible for dorso-ventral patterning of the ovary and germline proliferation [59], [60]. Finally, the SmVKR1 interacting partner Zyxin is a cytoskeletal protein that interacts with germline RNA helicases GLH in P granules in C. elegans [61]. From these data showing for each SmVKR different expression, ligand-activation and partner interactions, it seemed that SmVKR1 and SmVKR2 could elicit different cellular signalling pathways. In Xenopus oocytes, ligand-activated RTKs, and particularly insulin receptor, activate the Erk MAPK as well the PI3K/Akt/mTOR pathways. Using this cellular model, we observed that similarly to the insulin receptor, ligand-activated SmVKR1 and SmVKR2 both induce the phosphorylation of Erk1/2, Akt and p70S6K, indicating a potential role of SmVKRs in protein synthesis and cellular growth. Hyperphosphorylation of p70S6K in the case of SmVKR2 could be related to the predominant function in growth of SmVKR2 (Figure 7). Concerning the activation of the two other MAPK pathways, p38 and JNK, no phosphorylation of p38γ/SAPK3 was observed for SmVKR1 and SmVKR2 and JNK was only phosphorylated in SmVKR1-expressing oocytes, corroborating the results of Y2H screening and the finding that SmVKR1 interacted with Rho1, Mek7 and PP2C. In conclusion, all these data give evidence of the roles of SmVKR in schistosome reproduction. RNAi results, together with previous results obtained with the use of TK inhibitors [24] confirm the functions of SmVKR in gametogenesis, and particularly in oogenesis and egg formation and the importance to consider these RTKs as potential targets against schistosomes in novel anti-fecundity therapies. Each receptor could be susceptible to act at different steps of oocyte maturation (Figure 7). SmVKR1, which is more expressed in mature ovocytes, would activate JNK, a pathway which is known to be involved in cell migration and meiotic progression. By this way, SmVKR1 might be responsible for meiosis resumption and/or ovocyte migration under activation by a male stimulus, L-Arg present in seminal fluid. SmVKR2 present in immature and dividing cells might be implied in priority in proliferation and growth of oocytes, thus explaining its preferential activation of the Akt/mTORC2/p70S6K pathway. As a conclusion, we propose that VKR receptors might represent privileged targets to combat schistosomes, not only because of their role in parasite reproduction and thus their impact on pathogenesis and parasite transmission, but also because they have the main advantage over other parasite molecules to be absent from the host kinase panel. Moreover, as VKRs are also expressed in the gonads of disease-transmitting insects (like Anopheles vector of malaria or Aedes vector of filaria and viruses) [28], [29], and are probably involved in similar physiological processes of reproduction in these organisms, VKRs might also represent interesting drug targets for novel strategies to control other vector-borne infectious diseases besides schistosomiasis. All experiments involving hamsters within this study have been performed in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No 123; revised Appendix A) and have been approved by the committee for ethics in animal experimentation of the region Nord Pas de Calais France (authorisation No. AF/2009) in the local animal house of the Pasteur Institute of Lille (Agreement No. A59-35009). A Puerto-Rican strain of S. mansoni was maintained by passage through albino Biomphalaria glabrata snails and Mesocricetus auratus golden hamsters. Adult schistosomes were collected by portal perfusion from infected hamsters at 42–45 days p. i, then maintained in complete M199 medium (supplemented with 10% SVF, 10 mM HEPES pH 7. 4,60 µg/ml Rifampicin and 50 U penicillin/streptomycin) at 37°C under a 5% CO2 atmosphere. Unisexual worm populations were generated by monomiracidial snail infection as described elsewhere [62]. Adult worm pairs were fixed in Bouin' s solution (picric acid/acetic acid/formaldehyde; 15/1/5) and embedded in paraplast (Paraplast plus, Sigma). Sections (5 µm) were incubated in xylol to remove paraplast. Following rehydration, slides were treated with proteinase K (1 µg/ml) and sections dehydrated. For hybridization, in vitro transcripts (corresponding respectively to nt 4057–4379 and 2617–3027 sequences of SmVKR1 (Genbank Acc N° AF101194) and SmVKR2 (Genbank Acc N° GU270860) ) were synthesized and labeled with digoxigenin following the protocol of the manufacturer (Roche). The correct size of labeled sense and antisense transcripts was controlled by gel electrophoresis and the quality of RNA probes was checked by blotting and detection of digoxigenin using alkaline phosphatase-conjugated anti-digoxigenin antibodies, naphtol-AS-phosphatase, and Fast Red TR (Sigma). In situ hybridization was performed at 57°C for 16 h as previously described [34]. Sections were washed in 0,5×SSC (75 mM NaCl, 7,5 mM sodium citrate, pH 7) and detection of alkaline phosphatase on slides was achieved as described above. Ovaries were isolated from female worms issued either from unisexual or bisexual schistosome infections according to the procedure described by Hahnel et al [31], and total RNA was purified from the gonad tissues using the PepGOLD TriFast reagent (Peplab) according to the manufacturer' s protocol. For this 50 to 250 ovaries were incubated in 500 ml TriFast-solution frozen in liquid nitrogen and thawed on ice three times to support tissue disruption. Precipitation of total RNA in 2-propanol was driven by adding of 35 µg glycogen (RNase-free PeqGOLD glycogen, Peqlab). RNA quality and quantity were checked by electropherogram analysis employing the BioAnalyzer 2100 (Agilent Technologies). Synthesis of cDNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen) containing a genomic DNA wipe-out step and 200 ng of total RNA per reaction. The obtained cDNA was diluted 1∶20 and used in subsequent qPCR analyses. The detection of synthesized DNA double strands was based on the incorporation of SYBRGreen using PerfeCTa SYBR Green Super Mix (Quanta). All qPCR experiments were performed with a Rotor Gene Q Cycler (Qiagen) under the following conditions: 95°C for 3 min followed by 45 cycles of 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec. SmVKR1qPCRF/SmVKR1qPCRR and SmVKR2qPCRF2/SmVKR2qPCRR2 primer couples (Table S2) were used to amplify SmVKR1 and SmVKR2 transcripts, respectively. All primers were designed to have melting points at 60°C and were commercially synthesized (Biolegio, Netherlands). To distinguish between specific amplification products and unspecific primer dimers following each qPCR analysis, a melting point analysis was done. Amplification reactions occurred in technical triplicates, and analyses were performed using a relative quantification against the reference gene actin (Smp_161930) with the ΔΔCt method [63]. Worms fixed in AFA (ethanol 95%, formalin 3% and glacial acetic acid 2%) were stained for 30 min in 2,5% Hydrochloric Carmin red (Certistain®, Merck), and then destained in acidic 70% ethanol. Following dehydration in 70%, 90% and 100% ethanol, 5 min each, worms were preserved as whole-mounts in Canada balsam (Merck) on glass slides [11], [32], [64]. Confocal Laser Scanning Microscopy (CLSM) images were taken using a Leica LSM710 microscope with a 488 nm He/Ne laser and a 470 nm long-pass-filter under reflection mode. SmVKR1 (640 bp) or SmVKR2 (410 bp) cDNA fragments were generated as dsRNA templates by PCR amplification using gene specific primers containing T7 promoter sequences (see Table S2). A luciferase dsRNA template of similar size was generated using the pGL3-basic plasmid (Promega) as template. dsRNA was prepared and purified using the Megascript RNAi kit (Ambion) according to the manufacturer' s instructions, and quantified spectrophotometrically (NanoVue PlusTM, GE Healthcare). 20 µg of dsRNA were delivered to 8 worm couples in 100 µL M199 medium by electroporation using the square-wave protocol already described (125 V, 20 ms impulse at room temperature in a 4 mm cuvette) [65]. Worms were then cultured in complete M199 medium. After 5 days, microscopic examination was performed by CLSM on 4 worm couples, as described above. Gene knock-down was monitored in the remaining worms by quantitative RT-PCR. RNA was extracted using the Dynabeads® mRNA DIRECT™ Micro Kit following manufacturer' s instructions (Invitrogen). Reverse transcription was performed for 1 h at 55°C using the ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis (Invitrogen) and cDNA was used as template for PCR amplification using KAPA SYBR® FAST Universal 2× qPCR Master Mix kit (Clontech) and the ABI PRISM 7300 detection system (Applied Biosystems, Foster City, CA, USA). SmVKR1qPCRF/SmVKR1qPCRR and SmVKR2qPCRF/SmVKR2qPCRR primer couples (Table S2) were used to amplify respectively SmVKR1 and SmVKR2 transcripts in triplicate assays. Tubulin gene (GenBank Acc N°M80214) was used as internal control. For graphical representation, the delta-delta Ct (ΔΔCt) method was applied [63] to compare interfered worms to control dsLuc-treated parasites. The statistical significance of the levels of SmVKR1 and SmVKR2 transcript knock-down was evaluated using Student' s t-test in the GraphPad Prism programme (GraphPad Software Inc.). Full-length SmVKR1 and SmVKR2 sequences inserted in frame into the pcDNA3. 1-V5/His expression vector (Invitrogen) [30] were mutated in their respective VFT domain by replacement of Ser466 and Ser410 residues into Ala using the QuickChange Site-Directed Mutagenesis Kit (Stratagene), and the SmVKR1S466AF and SmVKR2S410AF primers (Table S2) and their respective reverse complement sequences. cRNA encoding SmVKR proteins was synthesised in vitro using the T7 mMessage mMachine Kit (Ambion, USA) and PmeI-linearised SmVKR-pcDNA plasmids as templates, and injected in stage VI Xenopus laevis oocytes according to the procedure previously described [66]. Each oocyte was injected with 60 nl (60 ng) of cRNA in the equatorial region and incubated at 19°C in ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2,1, 8 mM CaCl2,5 mM Hepes pH 7. 4 supplemented with 50 µg. ml−1 Streptomycin/Penicillin, 225 µg. ml−1 sodium pyruvate, 30 µg. ml-1 trypsin inhibitor). Kinase activation was obtained by adding external ligands (amino-acids, Ca) in the incubation medium and Germinal vesicle breakdown (GVBD) was detected by the appearance of a white spot at the centre of the animal pole after 15 h. Expression of SmVKR proteins in oocytes was confirmed by immunoprecipitation of membrane extracts according to the procedure described previously [66]. Following 24 h of expression, oocytes were lysed in buffer A (50 mM Hepes pH 7. 4,500 mM NaCl, 0. 05% SDS, 5 mM MgCl2,1 mg ml−1 bovine serum albumin, 10 µg ml−1 leupeptin, 10 10 µg ml−1 aprotinin, 10 µg ml−1 soybean trypsin inhibitor, 10 µg ml−1 benzamidine, 1 mM PMSF, 1 mM sodium vanadate) and centrifuged at 4°C for 15 min at 10,000 g. Membrane pellets were resuspended and incubated for 15 min at 4°C in buffer A containing 1% Triton X-100 and then centrifuged under the same conditions. Supernatants were incubated with anti-V5 or anti-Myc antibodies (1∶100; Invitrogen) overnight at 4°C. Protein A-Sepharose beads (5 mg; Amersham Biosciences) were added for 1 h at 4°C. Beads were washed three times and resuspended in Laemmli sample buffer. Eluted immune complexes were subjected to a 7. 5% SDS–PAGE, then analyzed by Western blotting using anti-V5 (1∶50,000), anti-Myc (1∶50,000) or PY20 (1∶10,000; anti-phosphotyrosine, BD Biosciences) antibodies and the advanced ECL detection system (Amersham Biosciences). Oocyte proteins phosphorylated during the activation of signalling cascades by SmVKR, were detected by Western blot analyses of oocyte lysates [67]. Oocytes were lysed in buffer A containing 0. 5% Triton X-100 and centrifuged at 12,000 g for 15 min at 4°C. The following primary antibodies were used to detect total or phosphorylated Akt, S6K, ERK2, JNK and P38 oocyte kinases: anti-Akt1 (C-20) (1∶5000), anti-p70 S6 Kinase alpha (H160) (1∶10000) and anti-ERK2 (1∶10000) were from Santa Cruz Biotechnology; anti-phospho Akt (Thr308) and anti-phospho Akt (Ser 473) (1∶5000) from Upstate Biotechnology; anti-phospho p44/p42 MAPK (ERK1/2) (Thr 202/Tyr 204) (1∶10000), anti-phospho p70 S6 Kinase (Thr 389) (1∶5000) and anti-phospho p38 MAPK (Thr 180/Tyr 182) from Cell Signalling Technology; anti-c-jun N-terminal kinase JNK (1∶10000) from Sigma; anti-active JNK polyclonal antibodies (1∶8000) from Promega; p38 (Xp38γ/SAPK3) antiserum prepared by immunizing rabbits with purifed GST-Xp38γ [44] was kindly provided by Prof. A. R. Nebreda (Barcelona, Spain). Mouse, rabbit or goat Trueblot® secondary antibodies (eBioscience) were used as secondary antibodies and chemoluminescence was revealed using the advanced ECL detection system (Amersham Biosciences). Intracellular domains (ICD) of SmVKR1YYRE and SmVKR2YYRE [30] were amplified by PCR using respectively SmVKR1YYREICDF/SmVKR1YYREICDR and SmVKR2YYREICDF/SmVKR2YYREICDR as primers (Table S2). Insertion of SmVKR1YYREICD and SmVKR2YYREICD in pGBKT7 plasmid (Gal4-BD-containing vector) was performed by directional cloning using EcoRI/PstI and BamHI/NcoI restriction sites respectively. Fusion of bait proteins in frame with Gal4-BD was checked by sequencing. Y187 yeasts were transformed by pGBKT7 bait constructs using the Lithium acetate method and plated on selective growth media, as described in the Yeast Protocols Handbook (Clontech). Transformed Y187 cells were then mated with the S. mansoni library formed by pGAD (Gal4-AD-containing vector) -transformed AH109 cells, as described before [34]. Diploid yeasts were selected on the quadruple dropout medium, SD-Leu/-Trp/-His/-Ade. Positive clones were submitted to the beta-galactosidase filter assay using X-Gal substrate, following manufacturer' s instructions (Yeast Protocols Handbook, Clontech). Confirmed clones were amplified and plasmid DNA was extracted using the NucleoSpin® Plasmid QuickPure kit (Macherey Nagel), after lysis of yeasts with glass beads (Sigma). Plasmid DNA was used to transform chemically competent DH5α bacteria (Invitrogen). Two successive rounds of plating on ampicilline-containing plates were performed to select bacteria containing pGADT7-Rec plasmids. Insert sequences were determined and the nature of preys identified using the BLASTn tool. MEK and PP2C protein sequences were aligned using ClustalW algorithm in the BioEdit v7. 1 software, and manually corrected. Maximum likelihood trees were built using MEGA5 [68] under the JTT model, with 1000 bootstrap repetitions. Sequences were analyzed using the LASERGENE package (DNAStar, Madison, WI, USA). BLASTn analyses of sequences obtained from the library screening were performed using the NCBI databank http: //blast. ncbi. nlm. nih. gov. gate2. inist. fr/Blast. cgi.

Title: Venus Kinase Receptors Control Reproduction in the Platyhelminth Parasite Schistosoma mansoni Summary: Schistosomiasis is a chronic, debilitating disease affecting more than 200 million people in the world caused by parasitic flatworms of the genus Schistosoma. Pathology is mainly due to massive egg production by parasites and formation of granulomas around the eggs trapped in liver and different organs. Therefore, targeting the molecular processes responsible for gonad development or egg production in schistosomes appears as a valuable strategy to reduce pathogenesis and dissemination of schistosomiasis. In the present study, we investigated the importance of Venus Kinase Receptors (VKRs) which are unusual receptor tyrosine kinases (RTKs) with an extracellular Venus Flytrap (VFT) ligand-binding domain in the control of reproduction of schistosomes. SmVKRs are expressed in female ovaries of Schistosoma mansoni and the knock-down of their expression provoked dramatic alterations of the oocyte content in ovaries and reduction of egg formation. SmVKRs were also shown to activate different signalling pathways potentially involved in oocyte proliferation, growth and migration. Therefore our results demonstrate that VKRs are essential actors of oogenesis and egg formation in S. mansoni. Moreover, their presence in a large variety of invertebrate species including other helminth parasites and insect parasite vectors can open new perspectives in the control of various vector-borne infectious diseases.

lay_plos

en

Summarize: A smartphone game that encourages girls to dress sexily and go on dates with 'hot guys' in the hope of becoming models has come under fire for promoting the wrong values to girls. Star Girl, which has 1.3million likes on Facebook and was until recently aimed at children aged four+ on iTunes, is now rated 12+ but is played by thousands of young children. It invites children to step 'into the high heels of an aspiring celebrity who is setting out to build her career as a superstar while having a ton of fun along the way'. Controversial: Previously age-rated four+ but now upgraded to 12+, Star Girl encourages children to dress sexily and meet men in nightclubs. Values: Gamers are told they must dress well and date sexy celebrities in order to further their careers. Gamers are encouraged to dress their celebrity avatar, or on-screen character, in racy clothing from lingerie shops and to visit nightclubs to flirt with 'hot celebrities' in exchange for gifts. The app, which is free to download but lets players make in-app purchases, tells girls: 'You've got the looks, but it takes more than than to be a star! 'Just remember that first impressions are the most important so make sure to wear only the trendiest outfit.' 'Hang out with your friends and go on dates with the hottest guys in town to find your perfect match.' Players are told they have 'three main career options: singer, actress, model', and encourages them to 'Shop til you drop! Flirt and go on dates to find the man of your dreams.' They even compete in flirting challenges to win the affections of grown male avatars. Melinda Liszweski from Collective Shout, a movement against the objectification of women and sexualisation of girls, said: 'The game teaches girls that the way to succeed in life is to be sexually appealing to men. 'Very inappropriate': Players of the Star Girl app are encouraged to invite men home with them. Critics of the game, which is free to download, say it promotes irresponsible values to young girls. 'Having had a rating of 4+ this app will likely have slipped under the radar of many parents who have taken steps to make sure their children aren’t exposed to inappropriate content.' Reviewers took to Amazon to air their disgust with the Star Girl app, with one describing it as'stupid and vapid' and another saying'very inappropriate - flirting, girls wearing underwear - I deleted it after a day.' One reviewer, Sarah, wrote: 'This app is simply horrid. How can someone actually enjoy this? 'All this is is about are: guys, clothes, competition and nothing else... And, to top it off, after you seduce a male (for this occurs in many of the games) you take him home with you like a sad little puppy who can flirt. Screengrab: The game has more than 1.3million likes on Facebook and is popular with school-age children. 'I may be a seventh grader, but I can tell this is just wrong.' One child wrote on Amazon: 'I do not have this, but my nine-year-old little sister does. The clothes are revealing and the avatars have very unrealistic shapes. 'This game teaches her terrible morals and values. Once I even heard her say, "I have all my gifts. I need a new boyfriend." 'I don't think you would like your daughter to say that.' Animoca, the company behind Star Girl, were unavailable for comment

Summary: Star Girl app lets girls pick one of three careers: singer, model or actress. The game was age-rated four plus, but has now been upgraded to 12 plus. Popular with young girls, the app has more than 1.3million likes on Facebook. Players are encouraged to 'Shop til you drop! Go on dates with hot guys!' Children choose avatars which they can dress with provocative clothes. Critics say it teaches girls 'they have to be sexually appealing to succeed'

cnn_dailymail

en

Summarize: PRIORITY [0001] This application is related to and claims the priority benefit of U.S. Provisional Application Ser. No. 62/025,490 to Johnson, filed Jul. 17, 2014, the contents of which are hereby expressly incorporated by reference in its entirety into this disclosure. BACKGROUND [0002] Headbands are devices worn in the hair to hold a wearer&#39;s hair back and away from the face and eyes. Conventional headbands typically include a flexible or semi-flexible band configured to fit the head of the wearer. For example, headbands may comprise a loop of elastic material dimensioned to encircle the head of a wearer or, alternatively, a horseshoe-shaped piece of firm or flexible plastic or metal configured to sit atop the wearer&#39;s head. In all cases, conventional headbands make continuous contact with and, thus, apply equal pressure across, the top and sides of the wearer&#39;s head in order to securely hold the wearer&#39;s hair in place. [0003] One issue commonly seen with conventional headbands relates to the tendency of headbands to slip out of place while being worn. This is especially true when a wearer has thicker and/or a large volume of hair that requires a firmer hold to keep the headband in place. In addition to the pressure conventional headbands apply across the wearer&#39;s head, it is also known to increase the friction between the hair and the headband in order to prevent slippage. For example, some headbands comprise tapered prongs, combs or teeth-like elements configured to engage the wearer&#39;s hair. Alternatively, other conventional designs include layers of rubber and/or synthetic products to increase the friction between the headband and the wearer&#39;s hair. These friction increasing elements are typically positioned along a substantial portion of the headband that is configured for contact with the top and sides of the wearer&#39;s head to achieve the most effect with respect to maximizing friction. [0004] In addition to utilitarian purposes (i.e. keeping a wearer&#39;s hair back and away from the face and eyes), headbands are also worn as fashion accessories. Unfortunately, however, the line of pressure applied by the bands across the top and sides of the head tends to flatten out the wearer&#39;s hair, thereby creating a severe “pulled-back” look. This is especially problematic from an aesthetic perspective for those individuals who have thicker/fuller hair, as it causes the hair in front of the headband to lie flat, while the remainder of the hair retains its natural voluminous appearance. Additionally, due to the continuous line of contact conventional headbands apply across the top and sides of the head and/or the use of friction increasing components to prevent slippage, after the headband is removed, the wearer&#39;s hair will retain a crimped or indented appearance resulting from the pressure of the headband. [0005] Accordingly, conventional headbands do not satisfy the aesthetic needs of consumers of all hair types. Hair fashion consumers continue to seek a device for their hair that effectively pulls the hair back and remains fixed and secured in the position it was originally placed, while also providing a look that is full of body and not severe. BRIEF SUMMARY [0006] In at least one exemplary embodiment of a headband of the present disclosure, the headband comprises an elongated band having at least a top portion and two side portions. The top portion of the headband defines at least two humps separated by a lowered restriction component, with each of the humps comprising a height and defining a space thereunder. In at least one embodiment, the top portion of the elongated band comprises at least three humps, with each of the humps separated from each other by a lowered restriction component. Additionally or alternatively, at least one of the lowered restriction components of the top may comprise a length between about 0.25 inches and about 1 inch. [0007] Each lowered restriction component may be configured to securely engage a head of a wearer and/or hair. In at least one additional embodiment, each of the humps of the top portion is configured to secure hair back from a face of a wearer without smashing or unmoveably compressing the hair against the head. Accordingly, in at least one embodiment, the top portion of the elongated band is configured such that a continuous pressure is not applied against the user&#39;s head when the headband is positioned thereon. Each of the humps may additionally comprise a length. In such cases, the height of a hump may be between about 0.5 inches and about 2.5 inches and the length of a hump may be between about two and about three inches. In yet another embodiment of the headband, at least one of the humps may comprise dimensions that are unique to that hump as compared to the other humps of the headband. [0008] As previously indicated, a lowered restriction component of the top portion may be configured to securely engage a head of a wearer and/or hair. In at least one embodiment, the restriction component of the elongated band comprises one or more friction enhancing components configured to facilitate secure engagement with the head and/or hair. For example, at least one friction enhancing component may comprise a comb, tapered teeth, and/or a rubber or synthetic material (or any combination of the aforementioned). Still further, the elongated band may further comprise a setting configured to receive one or more decorative ornaments. [0009] The elongated band may be flexible, semi-flexible, or stiff. Additionally or alternatively, the different portions of the elongated band may comprise different properties. For example, in at least one embodiment, the top portion of the elongated band may comprise a semi-flexible material, while the side portions of the elongated band comprise a stretchable material. In yet another embodiment, the elongated band may comprise a continuous loop or band of stretchable material. [0010] In additional embodiments of the headband of the present disclosure, the top portion of the elongated band is configured such that, when the elongated band is positioned on a head of a wearer, the top portion does not apply a continuous pressure against the head. Additionally or alternatively, each of the side portions of the headband may further comprise a restriction component comprising a first length and defined, at least in part, by a hump, and the lowered restriction component of the top may comprise a second length, with the first length being shorter than the second length. [0011] Methods for loosely restricting hair are also provided. In at least one exemplary embodiment, a method for loosely restricting hair comprises the steps of: applying a headband to a head having hair, the headband comprising an elongated band having a top portion and two side portions, wherein the top portion defines at least two humps separated by a lowered restriction component, each of the humps having a height and defining a space thereunder; and loosely restricting the hair against the head within each space while concurrently flattening the hair against the head under each of the lowered restriction components. In at least one additional embodiment, each of the side portions of the headband further comprises a restriction component comprising a first length and is defined, at least in part, by a hump. Additionally, there, the lowered restriction component of the top comprises a second length, the first length being longer than the second length. Still further, in yet another exemplary embodiment of the method, when the headband is applied to a head, the hair that is loosely restricted within each space is not indented or crimped due to the heights of the lumps. [0012] At least one exemplary embodiment of the present disclosure comprises a band. In such embodiments, the band comprises at least a top portion and two side portions defining a length of the band, as well as one or more settings positioned at least along the top portion of the band, each setting configured to receive one or more decorative ornaments. Here, the top portion of the band defines at least two humps separated by a lowered restriction component and each of the humps has a height and defines a space thereunder. Furthermore, the band may be flexible or semi-flexible and when the band is positioned on a head, the top portion does not apply a continuous pressure against the head along the length of the band. BRIEF DESCRIPTION OF THE DRAWINGS [0013] FIG. 1 is a front view of an exemplary headband of the present disclosure; and [0014] FIG. 2 illustrates application of the exemplary headband of FIG. 1 as applied to a person&#39;s head; and [0015] FIG. 3 illustrates a front view of an exemplary headband of the present disclosure. [0016] An overview of the features, functions and/or configurations of the components depicted in the various figures will now be presented. It should be appreciated that not all of the features of the components of the figures are necessarily described. Some of these non-discussed features, such as various tissues, etc., as well as other discussed features are inherent from the figures themselves. Other non-discussed features may be inherent in component geometry and/or configuration. DETAILED DESCRIPTION [0017] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended, with any additional alterations and modifications and further applications of the principles of this disclosure being contemplated hereby as would normally occur to one skilled in the art. [0018] The disclosure of the present application provides novel devices and methods for holding hair back and away from the face and eyes while not significantly restricting the hair. Unlike conventional headbands, the inventive headband of this disclosure is capable of not only maintaining a secure albeit non-severe look, but doing so without creasing the underlying hair and while maintaining hair volume. Because of these unique and advantageous properties, and as will be described herein in further detail, the headbands of the present disclosure are particularly well suited for individuals with long, thick and/or voluminous hair and can create a pulled-together look in seconds. [0019] Referring now to FIGS. 1 and 2, a headband 10 of the present disclosure is shown. More specifically, FIG. 1 shows a front view of the headband 10 and FIG. 2 shows a front view of the headband 10 positioned on a person&#39;s head 20. [0020] As shown in FIG. 1, headband 10 comprises a band 12 adapted for being snuggly fitted about the top and side portions of a person&#39;s head 20. In at least one embodiment, the band 12 is a relatively flat band comprising two or more humps 14, each defined by a lowered restriction component 16 positioned therebetween, which in at least one embodiment may comprise a lowered apex. [0021] The band 12 may be formed from a variety of materials suitable for the intended application. Various metals and/or plastics are particularly well adapted for use in this manner, including those that are durable and flexible or semi-flexible. Additionally or alternatively, the band 12 may be formed of various fabrics, including those which are moisture resistant and/or comprise an elastic or semi-elastic material (or combinations thereof). [0022] The humps 14 of the band 12 provide at least one of the unique properties to the inventive headband 10 disclosed herein. Perhaps more specifically, when the headband 12 is positioned on a person&#39;s head 20, the each hump 14 defines a space between the person&#39;s head and the band 12 such that hair can flow therethrough without undue restriction. In this manner, the humps 14 enable the headband 12 to securely keep the hair out of a person&#39;s face, without overly smashing or compressing the hair to the scalp. Indeed, the hair is prevented from falling into the person&#39;s face, but allowed to naturally drape under the humps 14 such that it flows down the back of the wearer&#39;s head 20. [0023] Each of the humps 14 may comprise any height or shape and it will be appreciated that these dimensions may be modified to achieve the desired headband 10 sizing. For example, in at least one embodiment, the two or more humps 14 may each comprise a height of between about 0.5-0.75 inches (indicated as line H in FIG. 1 ) and a length of between about 2-3 inches (indicated as line L in FIG. 1 ). Perhaps more specifically, in at least one exemplary embodiment, the two or more humps 14 each comprise a height of about 0.5 inches and a length of about 2.5 inches. Furthermore, each of the humps 14 need not be configured as having the same length and height dimensions. Indeed, the number of humps 14 (and thus lowered apexes—i.e. restriction components 16 —positioned therebetween) and the heights and lengths thereof are customizable pursuant to user preference and/or head size. [0024] As previously stated, every two humps 14 of the band 12 are connected by a restriction component 16 (lowered or otherwise). The one or more restriction components 16 of the band 12 are configured to come into contact with a person&#39;s scalp and/or engage or be coupled with hair and/or the head 20 when the headband 10 is positioned on a person&#39;s head 20. In at least one embodiment, a lowered restriction component 16 may further comprise a comb, teeth or other friction enhancing component (e.g., rubber and/or synthetic products) to facilitate its ability to engage the underlying hair and prevent slippage of the headband 10 when positioned on an individual&#39;s head 20. The dimensions of a restriction component 16 may also be customized pursuant to the desired hold and/or fit of the headband 10. For example, the restriction component 16 may have a short length (such that the restriction component 16 forms substantially a point as shown in FIGS. 1 and 2 ), or may comprise a longer length (not shown), provided that a single restriction component 16 does not comprise a majority of the top portion of the band 12. For example, in at least one exemplary embodiment, at least one of the restriction components 16 of a band 12 hereof may comprise a length of between about 0.25 inches and about 1 inch. [0025] As shown in FIG. 1, in at least one exemplary embodiment, the headband 10 comprises two humps 14 and one lowered restriction component 16 positioned on the top portion of the band 12 such that the headband 10 comprises a substantially heart-shaped configuration. Furthermore, while the band 12 shown in FIG. 1 is depicted in an open-ended configuration (see open-end 18 ), it will be appreciated that alternative embodiments of the band 12 may be configured as a closed loop. Additionally or alternatively, the band 12 may comprise multiple restriction components 16 (lowered or comprising an alternative configuration), with at least one having a different length and/or with one or more restriction components 16 positioned on one or more side portions of the band 12. Indeed, in the at least one embodiment shown in FIG. 3, the medially positioned restriction component(s) 16 of the top portion of the band 12 may comprise a shorter length, while one or more restriction components 16 located more laterally on the side portions of the band 12 may comprise longer lengths. In this manner, the laterally-positioned restriction components 16 facilitate the security of the band 12 when applied to an individual&#39;s head 20, while the shorter length of the medially-positioned restriction component(s) 16 minimizes the band&#39;s 12 restriction (and thus flattening) of the underlying hair. It will be that restriction components 16 may be positioned on the top and/or side portions of the band 12 as desired. [0026] In at least one embodiment, ornamental items may be attached to the top and/or sides of the band 12 to make the headband 10 more attractive and/or to enable a wearer to express his or herself. For example, in at least one embodiment, a wearer may attach jewelry, ribbons, charms, or other embellishments to the band 12 (not shown). Alternatively, the elongated band 12 may be configured with such embellishments as an integral component of the elongated band 12 itself, either through the inclusion of settings or otherwise (not shown). For example, the elongated band 12 may comprise one or more bezel or prong settings positioned on the top and/or side portions of the elongated band 12, or a channel setting that extends along a portion or the entirety of the length thereof. In such “bejeweled” embodiments of the headband 10, each of the settings is configured to receive embellishments such as crystals, gemstones, rhinestones, etc. Additionally or alternatively, the band 12 may comprise one or more colors. [0027] In an exemplary embodiment of the headband 10 described above, when the headband 10 is in use, it is placed on a person&#39;s head 20 in a general manner known in the art. Unlike conventional headbands, the headband 10 does not apply downward pressure to the wearer&#39;s hair continuously along the length of the elongated band 12 ; instead, the humps 14 enable the person&#39;s hair to maintain its natural lift by loosely gathering the hair along a substantial portion of the top of the headband 10. Indeed, only the one or more lowered restriction components 16 of the band 12 contact the top of the person&#39;s head, thereby minimizing the application of downward pressure across the top of the hair. Because of this, while the person&#39;s hair is pulled back and secured within the humps 14, the hair is not overly constricted by the band 12 nor smashed against the person&#39;s scalp per conventional techniques. Furthermore, the side portions of the elongated band 12 may also contact and/or apply pressure to the sides of the wearer&#39;s head 20 in order to further facilitate the secure placement of the headband 10. [0028] While embodiments of headbands and methods for using the same have been described in considerable detail herein, the embodiments are merely offered by way of non-limiting examples. It will therefore be understood that various changes and modifications may be made, and equivalents may be substituted for elements thereof, without departing from the scope of the disclosure. Indeed, this disclosure is not intended to be exhaustive or to limit the scope of the disclosure. [0029] Further, in describing representative embodiments, the disclosure may have presented a method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. Other sequences of steps may be possible. Therefore, the particular order of the steps disclosed herein should not be construed as limitations of the present disclosure. In addition, disclosure directed to a method and/or process should not be limited to the performance of their steps in the order written. Such sequences may be varied and still remain within the scope of the present disclosure.

Summary: A headband adapted for effectively holding hair back from a person&#39;s face without smashing or crimping a substantial portion of the hair at the top of a person&#39;s head. The headband comprises an elongated band having at least a top portion and two side portions, with the top portion defining at least two humps separated by a lowered restrictive component. In application, the lowered restrictive component engages the wearer&#39;s head and/or hair to hold the headband in place, while the majority of the wearer&#39;s hair is allowed to flow freely down the back of the wearer&#39;s head. In this manner, a secure hold is achieved, without applying significant pressure to the hair and reducing hair volume.

big_patent

en

Write a title and summarize: There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited. Radiation damage often limits the resolution and accuracy of macromolecular crystal structures (Garman, 2010; Zeldin et al., 2013). Femtosecond X-ray free electron laser (XFEL) pulses enable the possibility of visualizing molecular structures before the onset of radiation damage, and allow the dynamics of chemical processes to be captured (Solem, 1986; Neutze et al., 2000). Thus, from the first XFEL operation at the Linac Coherent Light Source (LCLS) in 2009, there has been considerable effort dedicated to the development of methods to utilize this rapid succession of bright pulses for macromolecular crystallography, with the aim of obtaining damage-free, chemically accurate structures. Most of the structures reported from XFELs to date use a liquid jet to inject small crystals into the beam (DePonte et al., 2008; Sierra et al., 2012; Weierstall et al., 2014), but diffraction data have also been measured from crystals placed in the beam with a standard goniometer setup (Cohen et al., 2014; Hirata et al., 2014). In both cases, the illuminated volume diffracts before suffering damage by a single XFEL pulse. Because the crystal is effectively stationary during the 10–50 fs exposure, ‘still’ diffraction patterns are obtained, in contrast to standard diffraction data collection where the sample is rotated through a small angle during the exposure. Extracting accurate Bragg peak intensities from XFEL diffraction data is a substantial challenge. An XFEL data set comprises ‘still’ diffraction patterns generally containing only partially recorded reflections, typically from randomly oriented crystals. The full intensity then has to be estimated from the observed partial intensity observations. Most XFEL diffraction data processing approaches reported to date have approximated the full intensity by the so-called “Monte Carlo” method, in which thousands of partial intensity observations of a given reflection are summed and normalized by the number of observations, which assumes that these observations sample the full 3D Bragg volume. Because a single diffraction image—in which each observed reflection samples only part of each reflection intensity–contains much less information than a small continuous wedge of diffraction data (as used in conventional crystallography), this method requires a very large number of crystals to ensure convergence of the averaged partial reflection intensities to the full intensity value (Kirian et al., 2010). Moreover, shot-to-shot differences in pulse intensity and energy spectrum that arise from the self-amplified stimulated emission (SASE) process (Kondratenko and Saldin, 1979; Bonifacio et al., 1984), along with differences in illuminated crystal volume, mosaicity, and unit-cell dimensions, contribute to intensity variation of the equivalent reflections observed on different images. These differences are assumed to be averaged out by the Monte Carlo method (Hattne et al., 2014). Thus, accurate determination of these parameters for each diffraction image should, in principle, provide more accurate integrated intensities, and converge with fewer measurements. Furthermore, it is desirable to assemble a data set from as few diffraction images as possible, since the potential of XFELs has been limited by the very large amounts of sample required for the Monte Carlo method, compounded by severe limitations in the availability of beamtime. In the 1970' s, the Harrison and Rossmann groups developed ‘post-refinement’ methods (Rossmann et al., 1979; Winkler et al., 1979), in which the parameters that determine the location and volume of the Bragg peaks are ‘post’-refined against a reference set of fully recorded reflections following initial indexing and integration of rotation data. Accurate estimation of these parameters, including the unit-cell lengths and angles, crystal orientation, mosaic spread, and beam divergence enables accurate calculation of what fraction of the reflection intensity was recorded on the image, i. e., its ‘partiality’, which is then used to correct the measurement to its fully recorded equivalent. Applied to virus crystals, for which only a few images can typically be collected before radiation damage becomes significant, post-refinement made it possible to obtain high-quality diffraction data sets collected from many crystals (Rossmann et al., 1979; Winkler et al., 1979). The implementation of post-refinement for XFEL diffraction data poses unique challenges. Firstly, since XFEL diffraction data generally do not contain fully recorded reflections, the initial scaling and merging of images is difficult. Secondly, since the XFEL diffraction images are stills rather than rotation data, different approaches are required for the correction of measurements to determine the full spot equivalent. Other schemes for implementing post-refinement of XFEL diffraction data have been described previously, but thus far they have been only applied to simulated XFEL data (White, 2014), and to pseudo-still images collected using monochromatic synchrotron radiation (Kabsch, 2014). We have developed a new post-refinement procedure specifically designed for diffraction data from still images collected from crystals in random orientations. We implemented our method in a new computer program, prime (post-refinement and merging), that post-refines the parameters needed for calculating the partiality of reflections recorded on each still image. We describe here our method and demonstrate that post-refinement greatly improves the quality of the diffraction data from XFEL diffraction experiments with crystals of three different proteins. We show that our post-refinement procedure allows complete data sets to be extracted from a much smaller number of diffraction images than that necessary when using the Monte Carlo method. Thus, this development will help make XFEL crystallography accessible to many challenging problems in biology, including those for which sample quantity is a major limiting factor. Units are arbitrary unless specified in parenthesis. Iobs, observed intensity. Iref, reference intensity. w, weighting term (inverse variance of the observed intensity). G, function of linear scale (G0) and resolution-dependent (B) factors that scales the different diffraction images to the reference set. Eoc, Ewald-offset correction function. rh, offset reciprocal-space distance from the center of the reflection to the Ewald sphere (Å−1). rp, radius of the disc of intersection between the reciprocal lattice point and the Ewald sphere (Å−1). rs, radius of the reciprocal lattice point (Å−1). θx, θy, θz, crystal rotation angles (see Figure 1A; °). 10. 7554/eLife. 05421. 003Figure 1. Geometry of the diffraction experiment and calculation of the Ewald-offset distance, rh. (A) A reciprocal lattice point intersects the Ewald sphere. The inset shows the coordinate system used in cctbx. xfel and prime. The vector S0 represents the direction of the incident beam (–z-axis) and forms the radius of the Ewald sphere of length 1/λ. The reciprocal lattice point i is expressed in reciprocal lab coordinates using Equation 5 as represented by the vector xi. The Ewald-offset distance, rh, is the difference between the distance from the Ewald-sphere center to the reciprocal lattice point (length of Si) and 1/λ. The inset shows the definition of the crystal rotation axes; they are applied in the following order: θz, θy, θx. (B) Shown is the volume of a reciprocal lattice point with radius rs. The offset rh defines the Ewald-offset correction Eocarea, which is the ratio between the area intersecting the Ewald sphere, Ap, and the area at the center of the volume, As. DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 003 γ0, parameter for Equation 3 (Å−1). γe, energy spread and unit-cell variation (see Equation 3; Å−1). γx and γy, beam divergence (see Equation 4; Å−1). {uc}, unit-cell dimensions (a, b, c (Å), α, β, and γ (°) ). Vc, reciprocal-lattice volume correction function (Å−3). xobs and xcalc, observed and predicted spot positions on the detector (mm). x, position of the reciprocal lattice point (Å−1). S, displacement vector from the center of the Ewald sphere to x (Å−1). S0, incident beam vector with length 1/wavelength (Å−1). O, orthogonalization matrix. R, rotation matrix. fL and fLN, Lorentzian function and its normalized counterpart. Γ, full width at half maximum (FWHM) of the Lorentzian function. Partiality can be modeled by describing the full reflection as a sphere (Figure 1A). In a still diffraction pattern, assuming a monochromatic photon source, the observed intensity Iobs, h for Miller index h is a thin slice through a three-dimensional reflection. To calculate partiality, we assume that the measurement is an areal (i. e., infinitely thin) sample of the volume (Figure 1B). The maximum partial intensity that can be recorded for a given reflection will occur when its center lies exactly on the Ewald sphere. By definition, the center of the reflection will be offset from the Ewald sphere by rh, and the corresponding disc will have a radius rp. The offset rh is determined by various experimental parameters, including the crystal orientation, unit-cell dimensions, and X-ray photon energy. The offset distance is used to calculate the Ewald offset correction, Eocarea, defined as the ratio between the areas defined by rp and rs (implemented as a smoothed correction function Eoch as defined in ‘Materials and methods’). The Ewald-offset corrected intensity is then converted to the full intensity in 3D by applying a volume correction factor, Vc. We define the target Tpr for the post-refinement of a partiality and scaling model by: (1) Tpr= ∑hwh (Iobs, h−G (G0, B) Eoch (θx, θy, γ0, γe, γx, γy, {uc}) Vc, h−1 × Iref, h) 2, which minimizes the difference between the observed reflections Iobs and a scaled and Ewald-offset corrected full intensity ‘reference set’ Iref using a least-squares method. The sum is over all observed reflections with Miller indices h. In alternate refinement cycles, we also minimize the deviations between predicted (xcalc) and observed (xobs) spot positions on the detector using a subset of strong spots as has been suggested previously (Hattne et al., 2014; Kabsch, 2014): (2) Txy=∑h (xobs−xcalc) 2. Sets of parameters associated with each diffraction image, i. e., G0, B, θx, θy, γ0, γe, γx, γy and the unit-cell constants, are iteratively refined in a series of ‘microcycles’ against the current reference set (Figure 2). 10. 7554/eLife. 05421. 004Figure 2. Post-refinement protocol. The flowchart illustrates the iterative post-refinement protocol, broken up into ‘microcycles’ that refine groups of parameters iteratively (blue boxes), and ‘macrocycles’. At the beginning of first macrocycle, a reference diffraction data set is generated. At the end of each macrocycle, the reference diffraction data set is updated. Both the micro- and macrocycles terminate either when the refinement converges or when a user-specified maximum number of cycles is reached. DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 004 Procedures for generating the initial reference set Iref (initial) are described below. After convergence of the microcycles, scaled full intensities are calculated from the observed partial intensities Iobs by multiplication of the inverse of the Ewald-offset correction and the scale factor G, along with the volume correction factor Vc. These scaled full reflections are then merged for each unique Miller index, taking into account estimated errors of the observed intensities, σ (Iobs), and propagation of error estimates for the refined parameters. This merged and scaled set of full reflections is then used as the new reference set in the next round of post-refinement using the target functions (Equations 1 and 2, for details see ‘Materials and methods’). These ‘macrocycles’ are repeated until convergence is achieved, after which the merged and scaled set of full intensities is provided to the user. The prime program controls post-refinement of specified parameters in a particular microcycle (Figure 2). One can refine all parameters together, or selectively refine groups of parameters iteratively, starting from (1) a linear scale factor and a B-factor, (2) crystal orientations, (3) crystal mosaicity, beam divergence, and spectral dispersion, and (4) unit-cell dimensions. Space-group-specific constraints are used to limit the number of free parameters for the unit-cell refinement. A particular microcycle is completed when the target functions converge or when a specified number of iterations is reached; the program then generates the new reference intensity set to replace the current reference set for the next macrocycle. Finally, the program exits and outputs the latest merged reflection set either when the macrocycles converge or when a user-specified maximum number of cycles has been reached. The starting point for our post-refinement method is a set of indexed and integrated partial intensities, along with their estimated errors, obtained from still images. For this study, diffraction data and their estimated errors were obtained from the cctbx. xfel package (Sauter et al., 2013; Hattne et al., 2014), although in principle integrated diffraction data from any other program can be used. Observed intensities on the diffraction image were classified as ‘spots’ by the program Spotfinder (Zhang et al., 2006), which identifies Bragg spots by considering connected pixels with area and signal height greater than user-defined thresholds. By trial and error, we accepted reflections larger than 25 pixels with individual-pixel intensity more than 5 σ over background for myoglobin and hydrogenase (collected on a Rayonix MX325HE detector with pixel size of 0. 08 mm and beam diameter [FWHM] of 50 μm). For thermolysin (collected on a Cornell-SLAC pixel array detector with pixel size of 0. 1 mm and beam size of 2. 25 μm2), where reflections are generally smaller, these values were 1 pixel and 5 σ. A full list of parameters is available on the cctbx. xfel wiki (http: //cci. lbl. gov/xfel). Separate resolution cutoffs for each image were applied by cctbx. xfel, at resolutions where the average I/σ (I) fell below 0. 5 (Hattne et al., 2014). Prior to post-refinement, the experimentally observed partial intensities need to be corrected by a polarization factor. The primary XFEL beam at LCLS is strongly polarized in the horizontal plane, and we calculate the correction factor as a function of the Bragg angle (θ) and the angle ϕ between the sample reflection and the laboratory horizontal planes (Kahn et al., 1982; see ‘Materials and methods’). For a stationary crystal and a monochromatic beam, a Lorentz factor correction is not applicable; the spectral dispersion of the SASE beam (δE/E ∼ 3 × 10−3 for the data sets studied here) is accounted for by the γe term (see ‘Materials and methods’). An essential step to initiate post-refinement is the generation of the initial reference set Iref (initial). This reference set has to be estimated from the available unmerged and unscaled partial reflection intensities after application of the polarization correction. For the results presented here, linear scale factors for each diffraction image were chosen to make the mean intensities of each diffraction image equal. Since this procedure can be affected by outliers in the observed intensities, we select a subset of reflections with user-specified resolution range and signal-to-noise ratio (I/σ (I) ) cutoffs. From this selection, we calculate the mean intensity on each diffraction image and then scale each image to make the mean intensity of all images equal. We correct the scaled observed reflections to their Ewald-offset corrected equivalents using the starting parameters, and then merge the observations, taking into account the experimental σ (Iobs), to generate the initial reference set. The initial values for crystal orientation, unit-cell dimensions, crystal-to-detector distance, and spot position on the detector were obtained from the refinement of these parameters by cctbx. xfel. The photon energy was that provided by the LCLS endstation system and is not refined. Initial values for the parameters of the reflection width model are described in the ‘Materials and methods’ section. In order to separately assess the effects of scaling, the Ewald offset correction (Equation 1), and post-refinement, we refer to three alternative schemes for processing the diffraction data sets: (1) ‘Averaged merged’, in which intensities were generated by averaging all observed partial intensities from equivalent reflections without Ewald-offset correction and scaling; (2) ‘Mean-intensity partiality corrected’, in which intensities were generated by scaling the reflections to the mean intensity and also applying the Ewald-offset correction determined from the initial parameters obtained from the indexing and integration program, followed by merging; and (3) ‘Post-refined’, in which intensities were from the final set of scaled and merged full reflections after the convergence of post-refinement. We note that although the ‘averaged merged’ process is similar to the original Monte Carlo method (Kirian et al., 2010), the integrated, unmerged partial intensities used in our tests were obtained from the program cctbx. xfel (Hattne et al., 2014), which also refines various parameters on an image-by-image basis (Sauter et al., 2014). We tested our post-refinement method on experimental XFEL diffraction data sets from three different crystallized proteins of known structure: myoglobin, hydrogenase, and thermolysin (Table 1). For quality assessment, we performed molecular replacement (MR) with Phaser (McCoy et al., 2007) using models with selected parts of the known structures omitted, followed by atomic model refinement with phenix. refine (Afonine et al., 2012), and inspection of (mFo-DFc) omit maps. We further used three different metrics: CC1/2, and the crystallographic Rwork and Rfree of the fully refined atomic model. We then compared changes in the three quality metrics between merged XFEL diffraction data sets after scaling, partiality correction, and post-refinement. We also investigated the effect of reducing the number of images used by randomly selecting a subset from the full set of diffraction images and repeating the entire post-refinement, merging, MR and refinement processes using this subset. 10. 7554/eLife. 05421. 005Table 1. XFEL diffraction data sets used in this studyDOI: http: //dx. doi. org/10. 7554/eLife. 05421. 005MyoglobinClostridium pasteurianum hydrogenaseThermolysinSpace groupP6P42212P6122Resolution used (Å) 20. 0–1. 3545. 0–1. 6050. 0–2. 10Unit cell dimensions (Å) a = b = 90. 8, c = 45. 6a = b = 111. 2, c = 103. 8a = b = 92. 7, c = 130. 5No. of unique reflections46,55585,27319,995No. of images* indexed75717712,692No. of images with spots to resolution used307751957Average no. of spots on an image (to resolution used) 16283640352Energy spectrumSASE†SASE†SASE†DetectorRayonix MX325HERayonix MX325HECSPAD‡Sample delivery methodfixed targetfixed targetElectrospun jet*This is the number of images indexed using cctbx. xfel program, and in the case of thermolysin it is the number of images indexed for one of the two wavelengths. †SASE: self-amplified spontaneous emission. ‡CSPAD: Cornell-SLAC pixel array detector. Diffraction data for both myoglobin and hydrogenase were collected from frozen crystals mounted on a standard goniometer setup (Cohen et al., 2014), whereas the thermolysin data were collected using an electrospun liquid jet to inject nanocystals into a vacuum chamber (Sierra et al., 2012; Bogan, 2013). The completeness of each data set was better than 90% at the limiting resolution used in our tests (Tables 2,3, 4). Each diffraction data set involved a different number of images due the differing diffraction quality of the crystals. 10. 7554/eLife. 05421. 006Table 2. Statistics of post-refinement and atomic model refinement for myoglobinDOI: http: //dx. doi. org/10. 7554/eLife. 05421. 006No. images100757Resolutiona (Å) 20. 0–1. 35 (1. 40–1. 35) 20. 0–1. 35 (1. 40–1. 35) Completenessa (%) 80. 0 (22. 2) 97. 7 (79. 8) Average no. observations per unique hkla4. 0 (1. 2) 25. 7 (2. 0) Averaged-mergedMean-scaled partiality correctedPost-refinedAveraged mergedMean-scaled partiality correctedPost-refinedPost-refinement parametersb Linear scale factor G01. 00 (0. 00) 2. 79 (5. 02) 1. 00 (1. 04) 1. 00 (0. 00) 2. 19 (3. 83) 0. 89 (1. 07) B0. 0 (0. 0) 0. 0 (0. 0) 3. 2 (7. 8) 0. 0 (0. 0) 0. 0 (0. 0) 6. 2 (8. 3) γ0 (Å−1) NA0. 00135 (0. 00028) 0. 00128 (0. 00022) NA0. 00147 (0. 00042) 0. 00132 (0. 00034) γy (Å−1) NA0. 00 (0. 00) 0. 00007 (0. 00080) NA0. 00 (0. 00) 0. 00007 (0. 00009) γx (Å−1) NA0. 00 (0. 00) 0. 00010 (0. 00011) NA0. 00 (0. 00) 0. 00008 (0. 00010) γe (Å−1) NA0. 00200 (0. 00) 0. 00344 (0. 00266) NA0. 00200 (0. 00) 0. 00423 (0. 00323) Unit cell a (Å): 90. 4 (0. 4) 90. 4 (0. 4) 90. 5 (0. 4) 90. 4 (0. 4) 90. 4 (0. 4) 90. 5 (0. 3) c (Å) 45. 3 (0. 4) 45. 3 (0. 4) 45. 3 (0. 3) 45. 3 (0. 3) 45. 3 (0. 3) 45. 3 (0. 3) Average Tpr Start/EndNANA19. 39 (7. 68) /7. 17 (3. 38) NANA19. 83 (7. 54) /6. 02 (2. 59) Average Txy (mm2) Start/EndNANA169. 74 (132. 56) /132. 02 (104. 08) NANA170. 66 (144. 52) /133. 42 (109. 58) CC1/2 (%) 81. 379. 686. 591. 895. 798. 2Molecular replacement scoresc LLG2837. 5043. 5291. 8264. 8364. 9320. TFZ10. 513. 013. 413. 713. 814. 0Structure-refinement parameters R (%) 39. 428. 023. 521. 120. 317. 8 Rfree (%) 42. 129. 424. 823. 122. 519. 7 Bond r. m. s. d. 0. 0060. 0060. 0040. 0060. 0060. 006 Angle r. m. s. d. 1. 140. 980. 791. 031. 350. 86 Ramachandran statistics Favored (%) 98. 098. 098. 098. 098. 098. 0 Outliers (%) 0. 00. 00. 00. 00. 00. 0aValues in parentheses correspond to highest resolution shell. bPost-refined parameters are shown as the mean value, with the standard deviation in parentheses. cMolecular replacement scores reported by Phaser (McCoy et al., 2007): log-likelihood gain (LLG) and translation function (TFZ). 10. 7554/eLife. 05421. 007Table 3. Statistics of post-refinement and atomic model refinement for hydrogenaseDOI: http: //dx. doi. org/10. 7554/eLife. 05421. 007No. images100177Resolutiona (Å) 45. 0–1. 60 (1. 66–1. 60) 45. 0–1. 60 (1. 66–1. 60) Completenessa (%) 83. 0 (47. 7) 91. 2 (63. 5) Average no. observations per unique hkla4. 4 (1. 7) 7. 13 (2. 3) Averaged-mergedPost-refinedAveraged-mergedPost-refinedPost-refinement parametersb Linear scale factor G01. 00 (0. 00) 0. 56 (1. 27) 1. 00 (0. 00) 0. 53 (1. 22) B0. 0 (0. 0) 10. 0 (7. 0) 0. 0 (0. 0) 10. 5 (6. 9) γ0 (Å−1) NA0. 00132 (0. 00042) NA0. 00126 (0. 00041) γy (Å−1) NA0. 00002 (0. 00004) NA0. 00002 (0. 00004) γx (Å−1) NA0. 00008 (0. 00009) NA0. 00008 (0. 00011) γe (Å−1) NA0. 00269 (0. 00138) NA0. 00288 (0. 00160) Unit cell a (Å): 110. 1 (0. 4) 110. 4 (0. 3) 110. 1 (0. 4) 110. 3 (0. 4) c (Å) 103. 1 (0. 4) 103. 1 (0. 2) 103. 0 (0. 4) 103. 0 (0. 2) Average Tpr Start/EndNA28. 20 (10. 86) /5. 92 (2. 35) NA26. 47 (12. 70) /5. 22 (2. 72) Average Txy (mm2) Start/EndNA623. 36 (314. 57) /381. 23 (198. 44) NA564. 30 (267. 45) /372. 28 (202. 28) CC1/2 (%) 62. 077. 371. 784. 8Molecular replacement scoresc LLG53,352. 9612. 7229. 11774. TFZ69. 275. 975. 079. 0Structure-refinement parameters R (%) 33. 425. 329. 122. 0 Rfree (%) 36. 728. 931. 325. 0 Bond r. m. s. d. 0. 0060. 0070. 0070. 007 Angle r. m. s. d. 1. 431. 501. 681. 97 Ramachandran statistics Favored (%) 96. 397. 097. 096. 7 Outliers (%) 0. 00. 00. 00. 0aValues in parentheses correspond to highest resolution shell. bPost-refined parameters are shown as the mean value, with the standard deviation in parentheses. cMolecular replacement scores reported by Phaser (McCoy et al., 2007): log-likelihood gain (LLG) and translation function (TFZ). 10. 7554/eLife. 05421. 008Table 4. Statistics of post-refinement and atomic model refinement for thermolysinDOI: http: //dx. doi. org/10. 7554/eLife. 05421. 008No. images200012,692Resolutiona (Å) 50. 0–2. 10 (2. 18–2. 10) 50. 0–2. 10 (2. 18–2. 10) Completenessa (%) 81. 3 (24. 3) 96. 5 (74. 8) Average no. observations per unique hkla32. 8 (1. 2) 176. 6 (2. 4) Averaged-mergedPost-refinedAveraged-mergedPost-refinedPost-refinement parametersb Linear scale factor G01. 00 (0. 00) 1. 65 (1. 66) 1. 00 (0. 00) 2. 26 (75. 12) B0. 0 (0. 0) 23. 0 (33. 8) 0. 0 (0. 0) 30. 1 (59. 8) γ0 (Å−1) NA0. 00052 (0. 00040) NA0. 00051 (0. 00039) γy (Å−1) NA0. 00001 (0. 00003) NA0. 00001 (0. 00003) γx (Å−1) NA0. 00002 (0. 00004) NA0. 00002 (0. 00004) γe (Å−1) NA0. 00110 (0. 00129) NA0. 00103 (0. 00128) Unit cell a (Å): 92. 9 (0. 3) 92. 9 (0. 2) 92. 9 (0. 3) 92. 9 (0. 3) c (Å) 130. 5 (0. 5) 130. 4 (0. 4) 130. 5 (0. 5) 130. 4 (0. 4) Average Tpr Start/EndNA1. 15 (0. 49) /0. 55 (0. 23) NA1. 15 (0. 52) /0. 28 (0. 13) Average Txy (mm2) Start/EndNA168. 13 (117. 29) /167. 72 (106. 14) NA169. 01 (122. 20) /170. 00 (122. 57) CC1/2 (%) 77. 793. 594. 398. 8Molecular replacement scoresc LLG3590. 4491. 5477. 6022. TFZ8. 99. 724. 124. 6Structure-refinement parameters R (%) 25. 219. 520. 718. 4 Rfree (%) 29. 124. 023. 921. 1 Bond r. m. s. d. 0. 0040. 0020. 0020. 002 Angle r. m. s. d. 0. 750. 580. 590. 62 Ramachandran statistics Favored (%) 95. 994. 695. 294. 9 Outliers (%) 0. 00. 00. 00. 0 Zinc peak height Zn (1) (σ) 14. 016. 014. 320. 9 Zn (2) (σ) 3. 65. 17. 77. 1 Average peak height for calcium ions (σ) 9. 711. 314. 216. 1aValues in parentheses correspond to highest resolution shell. bPost-refined parameters are shown as the mean value, with the standard deviation in parentheses. cMolecular replacement scores reported by Phaser (McCoy et al., 2007): log-likelihood gain (LLG) and translation function (TFZ). For myoglobin, we used both an XFEL diffraction data set consisting of 757 diffraction images (Table 1) collected by the SSRL-SMB group using a goniometer-mounted fixed-target grid (Cohen et al., 2014), and a randomly selected subset of 100 diffraction images. The diffraction images were from crystals in random orientations, with a single still image collected from each crystal. XFEL diffraction data for Clostridium pasteurianum hydrogenase were measured from eight crystals by the Peters (University of Montana) and SSRL-SMB groups using a goniometer-mounted fixed-target grid (Cohen et al., 2014). This experiment generated 177 diffraction images that could be merged to a completeness of 91%, with more than half of the diffraction images containing reflections to 1. 6 Å (each diffraction image typically has approximately 3000 spots). We also used a randomly selected subset of 100 diffraction images to assess the effect of post-refinement on a smaller number of images. The CC1/2 value improved significantly with post-refinement (Table 3). For quality assessment, the Fe-S cluster was omitted from both the molecular replacement search model (PDB ID 3C8Y) and subsequent atomic model refinement. The omit map densities for the post-refined diffraction data sets using the complete set of 177 diffraction images and the randomly selected subset of 100 diffraction images (83% complete) clearly show the entire Fe-S cluster whereas the densities using the averaged merged data sets are much poorer (Figure 8A). Upon atomic model refinement with the Fe-S clusters and water molecules included, the R and Rfree values for both post-refined data sets were significantly better than the averaged merged case (Figure 8B). 10. 7554/eLife. 05421. 014Figure 8. Impact of post-refinement on the hydrogenase diffraction data set. (A) Difference Fourier (mFo-DFc) omit maps of one of the four Fe-S clusters (which were omitted in molecular replacement and atomic model refinement) for the averaged merged and the post-refined hydrogenase XFEL diffraction data sets consisting of all 177 diffraction images (Table 1) and a randomly selected subset of 100 diffraction images. The maps are contoured at 3 σ. (B) Crystallographic R and Rfree values vs resolution after atomic model refinement using the specified diffraction data sets with inclusion of the three Fe-S clusters and water molecules. DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 014 For thermolysin, we tested the entire deposited XFEL diffraction data set consisting of 12,692 diffraction images (Table 1) (Hattne et al., 2014; the diffraction data are publicly archived in the Coherent X-ray Imaging Data Bank, accession ID 23, http: //cxidb. org), as well as a randomly selected subset of 2000 diffraction images. In this experiment, the crystal-to-detector distance gave a maximum resolution of 2. 6 Å at the edge and 2. 1 Å at the corners of the detector. Thus, a large number of diffraction images were required to achieve reasonable completeness of the merged data set for reflections in the 2. 1—2. 6 Å resolution range. As in the other two cases, post-refinement significantly improved the CC1/2 value (Table 4). For quality assessment, zinc and calcium ions were omitted from the thermolysin molecular replacement search model (PDB ID: 2TLI) and subsequent atomic model refinement. Post-refinement improved the peak heights of both the zinc and calcium ions (Table 4). The completeness of the merged data sets has a direct impact on the overall quality of the diffraction data set (CC1/2), quality of the electron density maps and the refined structures (Tables 2–4, and Figure 6). When completeness is high, adding more images to increase the multiplicity of observations has only a modest impact on the quality of the final refined structures using the post-refined diffraction data. For example, when subsets ranging from 2000 to 12,000 thermolysin diffraction images (all subsets 100% complete at 2. 6 Å) were post-refined the peak height in the omit map for the larger of the two anomalous sites (Figure 11C), the CC1/2 values, and the R values of the refined structures did not improve significantly when more than 8000 images were used. 10. 7554/eLife. 05421. 017Figure 11. Convergence of structure refinements for the post-refined thermolysin XFEL data set at 2. 6 Å resolution, using increasing numbers of diffraction images. (A) Average number of observations per unique hkl. (B) CC1/2 for merged subsets using 2000–12,000 images (100% completeness for all subsets). (C) Peak height (σ) in the omit map for the largest peak. (D) Rwork and Rfree after refining the thermolysin model without zinc and calcium ions against the corresponding post-refined diffraction data sets. DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 017 Diffraction data collection using conventional x-ray sources typically employs the rotation method, in which a single crystal is rotated through a contiguous set of angles, and the diffraction patterns are recorded on a 2-D detector. If a full data set can be collected from a single crystal without a prohibitive level of radiation damage, diffraction data processing is a well-established and reliable process. In contrast, processing of XFEL diffraction data, which are collected from crystals in random orientations as ‘still’ diffraction images, requires new methods and implementations such as those described here. Improved data collection and processing methods, particularly those that can significantly reduce the amount of sample needed to assemble a complete and accurate diffraction data set, are important for making XFELs useful for certain challenging investigations in structural biology. We developed a post-refinement method for still diffraction images, such as those obtained at XFELs, and implemented it in new computer program, prime, that applies a least-squares minimization method to refine parameters as defined in our partiality model. Other post-refinement methods for XFEL diffraction data have been described recently (Kabsch, 2014; White, 2014), but our implementation differs from these reports. Kabsch uses a partiality model in which an Ewald offset correction is defined as a Gaussian function of angular distance from the Ewald sphere. White used the intersecting volume between the reflection and the limiting-energy Ewald spheres defined by the energy spectrum for the partiality calculation, and calculates the initial reference data set by averaging all observations without scaling. Neither report describes an application to experimental XFEL diffraction data, so we cannot compare these methods to the results presented here. We have demonstrated here that our implementation of post-refinement substantially improves the quality of the diffraction data from three different XFEL experiments. Moreover, the resulting structures can be refined to significantly lower Rfree and R values, with electron density maps that reveal novel features more clearly, than those using non-post-refined XFEL data sets. A key feature of our method is that the parameters that define the diffracted spot are iteratively refined against the reference set. This approach is superior to methods that only consider each diffraction image individually. Moreover, our post-refinement procedure allows accurate diffraction data sets to be extracted from a much smaller number of images (average number of observations) than that necessary without post-refinement. Thus, this development will make XFEL crystallography accessible to many challenging problems in biology for which sample quantity is a major limiting factor. At present, it is difficult to assess the relative quality of post-refined XFEL data studied here with conventional rotation data measured at a synchrotron. The comparison of myoglobin omit maps (Figure 7) suggests that the SR data are perhaps somewhat better, but more systematic studies will be needed to understand the relative merits of the different data sets. We suspect that rotation data would be better due to the ability to directly measure full reflections (at least by summation of partials) without modeling partiality, which is still a relatively crude process (see below). However, a comparison between still data sets measured at a synchrotron and an XFEL is needed to deconvolute the effect of rotation vs other differences between these sources. Our formulation of post-refinement employs the simplifying assumption that reflections are spherical volumes. More sophisticated models consider crystal mosaicity to have three components, each with a distinct effect on the reciprocal lattice point (Juers et al., 2007; Nave, 1998,2014). First, the domain size (the average size of the coherently scattering mosaic blocks) produces reciprocal lattice points of constant, finite size: small domains produce large-sized spots, while large domains produce small spots, as there is an inverse (Fourier) relation between spot size and domain size. Second, unit-cell variation among domains produces reflections that are spheres whose radii increase with distance from the origin. In cctbx. xfel, mosaicity (modeled as isotropic parameter) and effective domain size are taken into account when predicting which reflections are in diffracting position prior to integration (Sauter et al., 2014; Sauter, 2015). Third, orientational spread among mosaic domains produces spots shaped like spherical caps. Each cap subtends a solid angle that depends on the magnitude of the spread. In addition, anisotropy in crystal mosaicity is not considered; this would require refining separate parameters along each lattice direction. Finally, the rugged energy spectrum that results from the SASE process of the XFEL is not yet considered in our current model. These issues will require future investigation. The observed intensity Ih (i) for observation i of Miller index h is a thin slice through a three-dimensional reflection. To calculate partiality, we assume that the measurement is an infinitely thin, circular sample of a spherical volume (Figure 1B). We assume a monochromatic beam as the starting point to define the Ewald offset correction Eocarea. The Eocarea of any reflection centered on the Ewald sphere is defined as 1; this position corresponds to the maximum partial intensity that could be measured for the reflection. The Eocarea for any other position is defined as a function of the normal distance from the Ewald sphere to the center of the reciprocal lattice point (the offset distance, rh), and of the reciprocal-lattice radius of the spot rs, which is a function of the crystal mosaicity and spectral dispersion (Figure 1B). The Eocarea can be described by the ratio of the observed area (Ap) with a radius rp to the Ewald-offset corrected area (As) with a radius rs (Figure 1B). The SASE spectrum emitted by the XFEL is broad and varies from shot-to-shot (Zhu et al., 2012). To calculate the Ewald sphere, we set the wavelength to be the centroid of the SASE spectrum recorded with each shot. For XFEL data measured with a seeded beam (Amann et al., 2012), the spectrum is narrow and constant from shot-to-shot, and this single value can be used in this case. In order to model spectral dispersion and the possible effects of asymmetric beam divergence, we adapt the rocking curve model described in Winkler et al. (1979). The four-parameter function used for the rocking curve is rs (γ0, γe, γx, γy) = rs (θ) + rs (α), where the first term includes the contribution by spectral dispersion and the second term models beam anisotropy. Specifically, (3) rs (θ) =γ0+γe tan θ, where γ0 is a parameter that is initially set to the r. m. s. d. of the Ewald offset calculated for all the reflections on a given image, γe represents the width of the energy spread and the unit-cell variation (the initial value of γe is calculated from the average energy spread), and θ is the Bragg angle. The second term is provided by: (4) rs (α) =[ (γy cos α) 2+ (γx sin α) 2]1/2, where α is the azimuthal angle going from meridional (α = 0) to equatorial (α = π/2). The values of γy and γx are initially set to 0. The distribution of rh values for the myoglobin case with 757 images after post-refinement is shown in Figure 12. The parameters γe, γy, γx, γ0 are refined within a microcycle (Figure 2). 10. 7554/eLife. 05421. 018Figure 12. Distribution of the Ewald sphere offset rh. The histogram shows the distribution of rh calculated after post-refinement for myoglobin using 757 diffraction images. The number of observations after applying the reflection selection criteria for merging and outlier rejections for this 1. 35 Å data set is 1,136,447 (∼96% of the total observed reflections). The standard deviation is 0. 0016 1/Å or approximately 0. 12° (when calculated with the mean of the energy distribution). DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 018 The crystal orientation is described in a right-handed coordinate system with the z-axis pointing to the source of the incident beam and the y-axis vertical (Figure 1A). We define the crystal orientation by rotations in the order θz, θy, θx about these axes. For each Miller index h (i), the reciprocal lattice point vector x (i) is obtained by applying orthogonalization and rotation matrixes O and R: (5) x (i) =ROh (i), wherex (i) = (x (i), y (i), z (i) ), h (i) = (h (i), k (i), l (i) ), O= (a∗b∗ cos γ∗c∗ cos β∗0b∗ sin γ∗c∗ (cos α∗− cos γ∗) /sin γ∗00c∗ cos (c∗, c) ), R=RθxRθyRθz, where Rθi is the rotation matrix for a rotation around the i-th axis, a∗, b∗, c∗, α∗, β∗, γ∗ are the reciprocal unit-cell parameters, and cos (c∗, c) = (1+2 cos α∗ cos β∗ cos γ∗−cos2α∗−cos2β∗−cos2γ∗) 1/2/sin γ∗. As shown in Figure 1A, the displacement to x (i) from the center of the Ewald sphere is given by: (6) S (i) = x (i) +S0, where S0 = (0,0, −1/λ). The offset distance is thus the difference between the length of S (i) and the Ewald-sphere radius, (7) rh= |S (i) |−1/λ. We introduce a smooth approximation of the area ratio Eocarea (see ‘Results’) in order to circumvent the undefined first derivative when the ratio is zero. We use a Lorentzian function (fL) to model the radius as function of distance from the Ewald sphere: (8) fL= 1π12Γ (rh) 2+ (12Γ) 2. The function is normalized so that fL (rh = 0) = 1. 0 when the reciprocal-lattice point is centered on the Ewald sphere, so that (9) fLn= πΓ2fL. We then use the ratio of the observed area (Ap) with a radius rp to the Ewald-offset corrected area (As) with a radius rs (Figure 1B) that corresponds to the full width at half maximum (FWHM), Γ, in the Lorentzian function. Using the Lorentzian function to describe the falloff in radius as we move away from the Ewald sphere makes the Eoc function differentiable at rh = rs. For the reciprocal lattice volume being bound by a sphere of radius rs centered on the reciprocal lattice point, the intersecting area of the volume is given by: (10) Ap=πrp2, whererp= (rs2−rh2) 1/2. The Eoc is then given by the ratio of this intersecting area to the area when this reflection is centered on the Ewald sphere (As), (11) Eocarea=ApAs=πrp2πrs2=1−rh2rs2. By setting the FWHM of Γ proportional to the radius, rs, at half Eocarea, (12) Eocarea=1−rh2rs2=0. 5, (13) Γ=rsat 0. 5 Eocarea=2rh, we arrive at the Ewald-offset correction function (Figure 13A) (14) Eoc=rs22rh2+rs2. 10. 7554/eLife. 05421. 019Figure 13. The Ewald-offset correction function. (A) Ewald-offset correction Eoc (Equation 14) viewed as a function of the reciprocal-lattice radius (rs) and the offset distance (rh). (B) A slice through Eoc at rs = 0. 003, comparing Eoc (Equation 14) and Eocarea (Equation 11). DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 019 The use of this Lorentzian approximation to derive the Eoc function vs an actual sphere function, Eocarea, is illustrated in Figure 13B. To adjust the observed still intensity to its equivalent at zero offset, we apply the Ewald-offset correction to the observed intensity, (15) IEoc, h (i) =Ih (i) Eoch (i) Gm, where Ih (i) is the observed partial intensity i of Miller index h on image m, Eoch (i) is the Ewald-offset correction, and Gm is a scale function for image m. We then convert this maximum partial intensity to a full intensity estimate by correcting for the volume of the spot, a factor of 43πrs3πrs2 =43rs: (16) Ifull, h (i) =Vc, h (i) IEoc, h (i), whereVc, h (i) =43rs, h (i). Note that Ifull, h (i) will be on an arbitrary scale, and appropriate scaling methods may be applied to place the data on a quasi-absolute scale prior to structure determination and refinement, as is done for conventional rotation data. We refine image m by first minimizing the target function: (17) Tpr=∑h∑iWh (i) (Ih (i) −GmEoch (i) Vc, h−1 (i) Ih) 2, where1/Wh (i) =σh2 (i), and the scale function Gm comprises a linear scale factor G0 and a B-factor: (18) Gm=G0, me−2Bm (sin θh (i) /λm) 2. We apply a spot position restraint as a second target function in subsequent steps during a microcycle using the x, y positions determined by the spot-finding step of data processing (Hattne et al., 2014; Kabsch, 2014). (19) Txy=∑h∑i (xhobs (i) −xhcalc (i) ) 2, where xhobs (i) and xhcalc (i) are the observed and calculated spot centroids, respectively. The Levenberg–Marquardt (LM) algorithm from the scipy python library (Oliphant, 2007), which is a combination of the gradient descent and the Gauss–Newton iteration, is used to minimize the target function residuals. The refinement of the unit-cell parameters (a, b, c, α, β, γ) takes crystal symmetry constraints into account to make the procedure more robust. After these iterative refinement cycles are complete, we apply the refined parameters to the reflection intensities of each still, and then merge the same reduced Miller indices (from all stills) to obtain the zero-offset still intensities, which are used for the new reference intensity set (see next section). At each step in a microcycle, the user can select reflections that are used for post-refinement of a parameter group using the following criteria: resolution range, signal strength (I/σ (I) ), and the Ewald offset correction value. In addition to these selection criteria, deviations from the target unit-cell dimensions (specified as a fraction of each dimension) can also be used in the merging step so that only diffraction patterns with acceptable unit-cell dimension values are included in the merged reflection set. Each post-refinement parameter group can have its own separate set of reflection selection criteria. Starting from the observed intensities, we obtain the full-volume intensity, Ifull, h (i), from IEoc, h (i) by first applying the Ewald offset correction (Equation 15) and then the full-intensity correction (Equation 16). Prior to merging equivalent observations, we detect outliers using an iterative rejection scheme, discarding reflections with intensity more or less than a user-specified cutoff (3 σ default, where σ is defined as the standard deviation of the distribution of the full reflections Ifull, h). Finally, in order to obtain the merged reflection set, we calculate 〈Ih〉 from the intensity of reflections with the same reduced Miller indices using the sigma-weighted average: (20) 〈Ih〉=∑iWh (i) I full, h (i) ∑iWh (i), whereWh (i) =1σ2 (i) [I full, h (i) ], and σ (i) [I full, h (i) ] is derived from the calculation of error: (21) (ΔIfull, h (i) Ifull, h (i) ) 2= (ΔIh (i) Ih (i) ) 2+ (ΔGG) 2+ (ΔEocEoc) 2. Since G is a function of G0 and B, and Eoc is a function of crystal orientation, mosaicity, and unit-cell parameters, the error estimates for G can be further calculated as: (22) ΔG2= (∂G∂G0) 2ΔG02+ (∂G∂B) 2ΔB2, and ΔEoc2 can be calculated similarly by summing all over products of partial derivatives and errors estimated for each parameter in the Eoc function (square root of the diagonal elements of the covariance matrix). We use CC1/2 as a quality indicator for the diffraction data sets (Diederichs and Karplus, 2013). We calculate CC1/2 by randomly partitioning all (partial) intensity observations of a given reflection into two groups. We reject any reflections with fewer than four observations; for all other reflections, we merge the observations in each group using Equation 20. CC1/2 is then calculated as the correlation between these two independently merged diffraction data sets. Let (23) g=1σ (I−G0e−2B (sin θ/λ) 2EocVc−1I), for observed partial intensity i of miller index h. The XFEL beam is nearly 100% polarized in the horizontal direction. The optics at both the LCLS XPP and CXI stations do not introduce additional polarization. To account for the polarization of the primary beam, for a given reflection, we consider the angle ϕ between the sample reflection plane formed by the h vector and the -z-axis, and the laboratory horizontal (Figure 14). 10. 7554/eLife. 05421. 020Figure 14. Geometry of the incident and diffracted beam for polarization correction. The diagram shows a reflection on a plane formed by its reciprocal-space vector and the -z-axis at angle ϕ. This reflection is affected by the polarization of the incoming primary beam in both the horizontal (x) and vertical (y) directions. DOI: http: //dx. doi. org/10. 7554/eLife. 05421. 020 As described in Kahn et al. (1982), the beam I0 incident on the sample crystal can be described in terms of two components, one parallel (σ) and the other perpendicular (π) to the plane of reflection: (29) I0=Iσ+Iπ. Each of these components is affected by the polarization of the primary beam in both the horizontal (x) and vertical (y) directions. Using fx and fy as the fractions horizontal and vertical in the laboratory frame (fx + fy = 1), (30) Iσ= (fx cos2 ϕ+fy sin2 ϕ) I0, and (31) Iπ= (fx sin2 ϕ+fy cos2 ϕ) I0, where fx and fy are the polarization fractions in the x and y directions. After reflection, only Iσ is attenuated: (32) I′=I′π+ I′σ= |F|2 (Iπ+Iσ cos22θ). By substituting Iσ and Iπ from Equations 30 and 31 in Equation 32, we arrive at (33) I′=|F|2[fx (sin2 ϕ+cos2 ϕ cos22θ) +fy (cos2 ϕ+sin2 ϕ cos22θ) ]I0, where the bracketed expression is P (Kahn et al., 1982). To ensure atomic model refinements against the various diffraction data sets were as comparable as possible, we used a standard semi-automated solution and refinement protocol. First, we performed molecular replacement phasing with known structures as search models (PDB ID 3U3E for myoglobin, 3C8Y for hydrogenase, and 2TLI for thermolysin) with all heteroatoms, water molecules, and ligands removed. Molecular replacement was carried out with Phaser (McCoy et al., 2007) using default settings, with r. m. s. d. set to 0. 8. The resulting solutions were then refined using phenix. refine (Afonine et al., 2012) in two cycles. In the first cycle, we carried out rigid body refinement, positional (xyz) refinement with automatic correction of Asn, Gln and His sidechain orientations, and atomic displacement parameter (ADP) refinement. We then used the difference density maps for missing ligands and heteroatoms obtained from this cycle to calculate real-space correlation coefficients using phenix. get_cc_mtz_pdb from the PHENIX software suite (Adams et al., 2010) for myoglobin and thermolysin and the program ‘Map Correlation’ from the CCP4 software (Winn et al., 2011) for hydrogenase. These omit difference density maps are shown in Figures 6,7, 8,10. In the second cycle, all ligands and heteroatoms were placed in the difference density maps and combined with the refined structure from the first cycle using Coot (Emsley et al., 2010). The second cycle employed positional and ADP refinement with target weights optimization and water update was carried out with these complete models. The structures were validated by MolProbity (Chen et al., 2010). Final refinement statistics (Tables 2,3, 4) were analyzed with phenix. polygon (Urzhumtseva et al., 2009) and found to be within acceptable range for other structures at similar resolutions. For the thermolysin structure obtained from anomalous diffraction data (processed keeping Friedel pairs separate), only one cycle of atomic model refinement was carried out. All figures were made in PyMOL (The PyMOL Molecular Graphics System, Version 1. 5. 0. 4 Schrödinger, LLC.). The computer program, prime, is implemented as a part of the cctbx computational crystallography toolbox (Grosse-Kunstleve et al., 2002). Download and installation instructions are available on the cctbx website (http: //cctbx. sourceforge. net). Subsequent to acceptance of this article, a paper was published by Ginn et al. (2015) describing an alternative method for orientation refinement as compared to the method of Sauter et al. (2014), and partiality estimation for each individual image, but without post-refinement.

Title: Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals Summary: Large biological molecules (or macromolecules) have intricate three-dimensional structures. X-ray crystallography is a technique that is commonly used to determine these structures and involves directing a beam of X-rays at a crystal that was grown from the macromolecule of interest. The macromolecules in the crystal scatter the X-rays to produce a diffraction pattern, and the crystal is rotated to provide further diffraction images. It is then possible to work backwards from these images and elucidate the structure of the macromolecule in three dimensions. X-ray beams are powerful enough to damage crystals, and scientists are developing new approaches to overcome this problem. One recent development uses 'X-ray free electron lasers' to circumvent the damage caused to crystals. However, early applications of this approach required many crystals and thousands to millions of diffraction patterns to be collected-largely because methods to process the diffraction data were far from optimal. Uervirojnangkoorn et al. have now developed a new data-processing procedure that is specifically designed for diffraction data obtained using X-ray free electron lasers. This method was applied to diffraction data collected from crystals of three different macromolecules (which in this case were three different proteins). For all three, the new method required many fewer diffraction images to determine the structure, and in one case revealed more details about the structure than the existing methods. This new method is now expected to allow a wider range of macromolecules to be studied using crystallography with X-ray free electron lasers, including cases where very few crystals are available.

lay_elife

en

Summarize: A Queensland mother who received an anonymous note from neighbours complaining about her crying baby has urged them to come forward and hit out at her detractors for being'so critical'. Trish LaForty, 26, found the letter in her mailbox last Thursday after a day out with her 11-month-old son Will, which read: 'You may be able to ignore your baby crying but we are tired of listening to him crying non-stop all day.' The mother, who lives in a Sunshine Coast apartment block with her son and partner Wil, posted a response and a copy of the note to a local community Facebook page and has since received global media attention. Trish LaForty, 26, found a 'rude' note in her mailbox last week after a day out with her 11-month-old son Will. The Sunshine Coast mother posted it to a community Facebook page and has since received global attention. 'I had no idea that it was this worldwide until this morning my dad said that I should Google my name, and it came up in all these different languages and I just thought "Wow that's crazy",' she told Daily Mail Australia. 'I definitely wasn't expecting that when I put the little rant on Facebook.' Ms LaForty wrote the post on the Sunny Coast Community Board in the hope the letter's authors would see it. 'Like being a parent isn't tough enough... You didn't even have the guts to come knock on our door,' she posted on the Facebook page. 'FYI Our baby doesn't cry "all day", he cries when he doesn't want do go to sleep, when he's getting his nappy changed and when I say "no" because I refuse to let him be spoiled and run around like he owns the joint. 'If you'd like to chat more, feel free to pop in. You obviously know where we live.' Ms LaForty who lives in an apartment block with her son and partner Wil (pictured right), denied that her baby cried 'for hours on end' The 26-year-old her has urged her neighbours to come forward and told detractors to stop being'so critical' Ms LaForty and her partner still don't know who sent the note, but they have their suspicions. 'We've talked to a couple of other neighbours and another lady in our block had received notes from somebody as well,' she said. 'Just about petty little things as well, like that they could hear their birds. 'I don't know if they're going to come forward now because they might be shamed.' But Ms LaForty still wants the people who sent the note to come talk to her face-to-face. 'I guess I kind of hope that they would do that so maybe we could resolve whatever issues they seem to have,' she said. 'But if they don't that's not really our problem, it's them that are going to have to put up with our baby.' Many have jumped to Ms LaForty's defence but others have accused her of having a'superiority complex' Ms LaForty said after she posted the note on Facebook many people jumped to her defence, and she thought the issue had struck a nerve because of the way the situation was handled by her neighbours. 'People really believe that kids should be kids and it's part of growing up,' she said. 'But more so because of the way it was handled. 'The writer could have given more information or at least said who they were if they're really that concerned about our baby, instead of writing a rude note.' And while many have jumped to Ms LaForty's defence, others have accused her of having a'superiority complex'. 'Eurgh, I'd write that letter. Nothing worse than non stop barking dogs and screeching children. Sick of the "everyone should bow down to those who procreate". Shut em up!,' one reader wrote. Ms LaForty said the issue struck a nerve because of the way the situation was handled by her neighbours. Another reader said: 'I don't see anything "insulting" about the letter. It may be seen as impatient or unsympathetic, but it's definitely not insulting. 'She sounds like one of those mothers who think that because she's got a baby, everyone else in the world must go out of their way to accommodate her.' But Ms LaForty just dismisses her critics as 'trolls' and denies her son Will is as noisy as her neighbours say. 'If they're going to have children they're going to find out what it's like eventually,' she said. 'Maybe instead of being so critical they can offer a helping hand.' Ms LaForty added: 'Will is not upset all the time or crying for hours on end. 'I'd be the first to admit it if my child was a terror.'

Summary: Trish LaForty, 26, found a handwritten note in her mailbox on Thursday. Her neighbours accused her of ignoring her crying 11-month-old son Will. She posted about the letter on a Queensland community Facebook page. Ms LaForty's story has since received worldwide media attention. She still doesn't know who wrote the note and wants them to come forward. The Sunshine Coast mum also told her detractors to stop being'so critical'

cnn_dailymail

en

Summarize: Background Energy audits typically identify information on projects that could address the consumption of fossil fuel and electricity as well as projects that could reduce emissions from other sources, such as leaks in refrigeration equipment. Energy audits also include information on the cost- effectiveness of projects and on the extent to which the projects could reduce emissions. This information can then be used to evaluate and select projects. The audits generally fall into three categories— preliminary, targeted, and comprehensive—and are distinguished by the level of detail and analysis required. Preliminary audits are the least detailed and provide quick evaluations to determine a project’s potential. These audits typically do not provide sufficiently detailed information to justify investments but may prove useful in identifying opportunities for more detailed evaluations. Targeted audits are detailed analyses of specific systems, such as lighting. Comprehensive audits are detailed analyses of all major energy-using systems. Both targeted and comprehensive audits provide sufficiently detailed information to justify investing in projects. Energy-saving projects that fall outside the scope of energy audits include efforts to enhance outreach and education efforts to curtail energy use by building occupants and purchasing high-efficiency appliances. Outreach and education efforts include providing information on how employees can conserve energy, such as AOC’s “how-to guides” that detail cost- effective methods to save energy in the workplace. Efforts to curtail energy use include purchasing energy-efficient computer equipment and appliances, using information available from the Environmental Protection Agency’s Energy Star program or the Federal Energy Management Program (FEMP). Energy Star-qualified and FEMP-designated products meet energy-efficiency guidelines set by the Environmental Protection Agency and the Department of Energy and, in general, represent the top 30 percent most energy-efficient products in their class of products. These products cover a wide range of categories, including appliances and office equipment. According to the Energy Star program, office products that have earned the Energy Star rating use about half as much electricity as standard equipment and generally cost the same as equipment that is not Energy Star qualified. AOC Has Made Some Progress toward Implementing GAO’s Recommendations on Energy Audits and Implementing Projects that Decrease Emissions AOC has made some progress toward implementing the two recommendations in our April 2007 report. First, AOC has taken steps to address our recommendation that it develop a schedule for routinely conducting energy audits by developing a prioritized list of buildings for which it plans to conduct comprehensive energy audits (see App. 1). Specifically, AOC is currently undertaking a comprehensive energy audit of the U.S. Capitol Police Buildings and Grounds and obtained a draft submission in May 2008 from the private contractor performing the audit. AOC also plans to use $400,000 of fiscal year 2008 funds to perform comprehensive energy audits of the Capitol Building and the Ford House Office Building, and says it will direct any remaining fiscal year 2008 funds to an audit of the Hart Senate Office Building. Additionally, AOC has contracted with a private firm to conduct a preliminary energy audit of the Senate Office Buildings that could prove useful in identifying opportunities for more comprehensive and targeted evaluations. AOC officials said that they developed the prioritized list of buildings to audit by comparing the amount of energy used per square foot of space in each building (referred to as energy intensity) and then placing the buildings that use relatively higher levels of energy at the top of the list. However, AOC’s prioritized list does not provide information on the energy intensity of each building, an explanation of its prioritization scheme, or cost estimates. Furthermore, AOC has not developed a schedule for routinely conducting audits as we recommended in our April 2007 report. AOC officials said that they cannot complete a more comprehensive schedule because of uncertainty about the extent to which AOC will receive future appropriations to conduct the audits. We believe that developing a more detailed schedule for future audits along with an explanation of its prioritization scheme and cost estimates would assist the Congress in its appropriations decisions and facilitate the completion of additional audits. Second, AOC can do more to fully address the second recommendation in our April 2007 report that it implement selected projects as part of an overall plan to reduce emissions that considers cost-effectiveness, the extent to which the projects reduce emissions, and funding options. In recent years, AOC has undertaken numerous projects throughout the Capitol Hill Complex to reduce energy use and related emissions, but these projects were not identified through the process we recommended. Projects completed or underway include upgrading lighting systems, conducting education and outreach, purchasing energy-efficient equipment and appliances, and installing new windows in the Ford building. Examples of projects in Senate office buildings include upgrading the lighting in 11 offices with daylight and occupancy sensors, installing energy efficiency ceiling tiles in the Hart building, and replacing steam system components. According to AOC, these efforts have already decreased the energy intensity throughout the Capitol Hill Complex. Specifically, AOC said that it decreased its energy intensity—the amount of energy used per square foot of space within a facility—by 6.5 percent in fiscal year 2006 and 6.7 percent in fiscal year 2007. As AOC moves forward with identifying and selecting projects that could decrease energy use and related emissions, it could further respond to our recommendation by developing a plan that identifies the potential benefits and costs of each option based on the results of energy audits. Such a plan could build on AOC’s existing Sustainability Framework Plan and its Comprehensive Emissions Reduction Plan for the Capitol Complex, which identify measures that could lead to improvements in energy efficiency and reductions in greenhouse gas emissions. Complementing these plans with information on projects identified through energy audits would further assist AOC in using the resources devoted to energy efficiency enhancements as effectively as possible. Efforts to Further Reduce Energy Consumption May Prove More Cost- Effective Than Other Measures in Decreasing Emissions The Senate has three primary options for decreasing greenhouse gas emissions and related environmental impacts associated with its operations. These include (1) implementing additional projects to decrease the demand for electricity and steam derived from fossil fuel; (2) adjusting the Capitol Power Plant’s fuel mix to rely more heavily on natural gas, which produces smaller quantities of greenhouse gas emissions for each unit of energy input than the coal and oil also burned in the plant; and (3) purchasing renewable electricity or carbon offsets from external providers. Each option involves economic and environmental tradeoffs and the first option is likely to be the most cost-effective because the projects could lead to recurring cost savings through reductions in energy expenditures. Regarding the first option, as we reported in April 2007, conducting energy audits would assist AOC in addressing the largest sources of emissions because the audits would help identify cost-effective energy-efficiency projects. In general, energy projects are deemed cost-effective if it is determined through an energy audit that they will generate sufficient savings to pay for their capital costs. These projects may require up-front capital investments that the Senate could finance through direct appropriations or contracts with utility or energy service companies, under which the company initially pays for the work and the Senate later repays the company with the resulting savings. Until AOC exhausts its opportunities for identifying energy-efficiency projects that will pay for themselves over a reasonable time horizon, this option is likely to be more cost-effective than the second two options, both of which would involve recurring expenditures. In pursuing energy audits, AOC faces a significant challenge collecting reliable data on the baseline level of energy use within each Senate office building. Such data would help identify inefficient systems and provide a baseline against which AOC could measure potential or actual energy- efficiency improvements. First, while AOC has meters that track electricity use in each building, the meters that track the steam and chilled water used by each building no longer work or provide unreliable data. AOC officials said that they have purchased but not installed new meters to track the use of chilled water and are evaluating options for acquiring new steam meters. Installing these meters and collecting reliable data would enhance any efforts to identify potential energy saving measures. Second, AOC does not have submeters within each building to track the electricity use within different sections of each building. Such submetering would further assist in targeting aspects of the Senate buildings’ operations that consume relatively high quantities of energy. AOC said that it plans to install submeters for electricity, chilled water, and steam by February 2009. A second option for decreasing greenhouse gas emissions would involve directing AOC to further adjust the fuel mix at the Capitol Power Plant to rely more heavily on the combustion of natural gas in generating steam for space heating. The plant currently produces steam using a combination of seven boilers—two that primarily burn coal, but could also burn natural gas, and five boilers that burn fuel oil or natural gas. The total capacity of these boilers is over 40 percent higher than the maximum capacity required at any given time, and the plant has the flexibility to switch among the three fuels or burn a combination of fuels. The percentage of energy input from each fuel has varied from year to year, with an average fuel mix of 43 percent natural gas, 47 percent coal, and 10 percent fuel oil between 2001 and 2007. In June 2007, the Chief Administrative Officer of the House of Representatives released the Green the Capitol Initiative, which directed AOC to operate the plant with natural gas instead of coal to meet the needs of the House. The House Appropriations Committee subsequently directed GAO to determine the expected increase in natural gas use for House operations and the associated costs at the power plant that would result from the initiative. In May 2008, we reported that the fuel-switching directive should lead to a 38 percent increase in natural gas use over the average annual quantity consumed between 2001 and 2007. We also estimated that the fuel switching should cost about $1.4 million in fiscal year 2008 and could range from between $1.0 and $1.8 million depending on actual fuel costs, among other factors. We also estimated that the costs would range from between $4.7 million and $8.3 million over the 2008 through 2012 period, depending on fuel prices, the plant’s output, and other factors. While we have not analyzed the potential costs of fuel switching at the Capitol Power Plant to meet the needs of the Senate, our estimates for the House may provide some indication of the potential costs of such a directive. Additionally, AOC officials said that further directives to increase its reliance on natural gas in the plant could require equipment upgrades and related capital expenditures. Our May 2008 report also found that decreasing the plant’s reliance on coal could decrease greenhouse gas emissions by about 9,970 metric tons per year at an average cost of $139 per ton and could yield other environmental and health benefits by decreasing emissions of nitrogen oxides, particulate matter, and pollutants that cause acid rain. While fuel switching could decrease emissions of carbon dioxide and other harmful substances, it would also impose recurring costs because natural gas costs about four times as much as coal for an equal amount of energy input. Thus, fuel switching may prove less cost-effective than decreasing the demand for energy. Finally, a third option for the Senate to decrease greenhouse gas emissions and related environmental impacts includes purchasing electricity that is derived from renewable sources and paying external parties for carbon offsets. Neither of these activities would involve modifications to the Capitol Complex or its operations but could nonetheless lead to offsite reductions in emissions and related environmental impacts. Both options, if sustained, would result in recurring costs that should be considered in the context of other options for decreasing emissions that may prove more cost-effective. Madam Chairman, this completes my prepared statement. I would be pleased to answer any questions that you or Members of the Committee may have. For further information about this testimony, please contact Terrell Dorn at (202) 512-6923. Other key contributors to this testimony include Daniel Cain, Janice Ceperich, Elizabeth R. Eisenstadt, Michael Hix, Frank Rusco, and Sara Vermillion. Appendix I: Prioritized List of Buildings for Comprehensive Energy Audits Senate Employees Child Care Center CPP Storage (Butler) Building East and West Underground Garages Capitol Police Courier Acceptance Site BG Production Facility (Greenhouse) Construction Management Division Supreme Court Building This is a work of the U.S. government and is not subject to copyright protection in the United States. The published product 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: In April 2007, GAO reported that 96 percent of the greenhouse gas emissions from the Capitol Hill Complex facilities--managed by the Architect of the Capitol (AOC)--resulted from electricity use throughout the complex and combustion of fossil fuels in the Capitol Power Plant. The report concluded that AOC and other legislative branch agencies could benefit from conducting energy audits to identify projects that would reduce greenhouse gas emissions. GAO also recommended that AOC and the other agencies establish a schedule for conducting these audits and implement selected projects as part of an overall plan that considers cost-effectiveness, the extent to which the projects reduce emissions, and funding options. AOC and the other agencies agreed with our recommendations. This statement focuses on (1) the status of AOC's efforts to implement the recommendations in our April 2007 report and (2) opportunities for the Senate to decrease greenhouse gas emissions and associated environmental impacts. The statement is based on GAO's prior work, analysis of AOC documents, and discussions with AOC management. AOC has made some progress toward implementing the recommendations in GAO's April 2007 report, but opportunities remain. For example, AOC has prioritized a list of Capitol Hill buildings that need energy audits but has not developed a schedule for conducting the audits that explains the prioritization scheme or provides information on the anticipated costs. AOC prioritized the order of energy audits based on each building's energy use and has begun conducting the first of the audits. In addition, AOC has contracted with a private firm to conduct preliminary audits of the Senate office buildings that could lead to more targeted audits and eventually identify cost-effective projects that would decrease energy use and related greenhouse gas emissions. We believe that developing a more detailed schedule for future audits that includes an explanation of the prioritization scheme and cost estimates would assist the Congress in its appropriations decisions and facilitate the completion of additional audits. With respect to our recommendation that AOC implement selected projects as part of an overall plan to reduce emissions, AOC has implemented projects to reduce energy use and related emissions, but the projects were not identified through the processes we recommended. AOC could more fully respond to our recommendation by first completing the energy audits and then evaluating the cost-effectiveness and relative merits of projects that could further decrease the demand for energy. The Senate's options for decreasing the greenhouse gas emissions and related environmental impacts associated with its operations fall into three main categories--implementing projects to decrease the demand for electricity and steam derived from fossil fuels, adjusting the Capitol Power Plant's fuel mix, and purchasing carbon offsets or renewable electricity from external providers. Of these options, efforts to decrease the demand for energy could lead to recurring cost savings through reductions in energy expenditures while the other options may prove less cost-effective and involve recurring expenses. However, a key challenge in identifying energy-saving opportunities results from limited data on the baseline level of energy use within each Senate building. Specifically, the meters for steam and chilled water no longer function or do not provide reliable data. In addition, the buildings are not equipped with submeters for electricity that, if installed, could enhance efforts to identify sections of the buildings that consume relatively high levels of energy. AOC has purchased but not installed new chilled water meters, is evaluating options for acquiring new steam meters, and plans to install submeters by February 2009.

gov_report

en

Summarize: FIELD OF THE INVENTION The invention relates to a knee joint prosthesis and particularly to a type thereof having two components with a knob and cavity relationship therebetween wherein relatively wide contact surfaces are provided for direct transmission of load from one component to the other and wherein the screws fastening together the parts defining the cavity are normally subjected only to shear stress. BACKGROUND OF THE INVENTION In the recent accelerating activity with respect to metal, plastics, and composite prostheses for various joints in the human body, a great deal of attention has been paid to the knee joint. As is well known, this joint must absorb substantial stress, primarily loadings directed axially of the adjacent leg portions, but also some flexing stresses. In fact, the vertical loading on the knee joint of a 200-pound man often reaches values as high as at least 800 pounds per square inch. There have in the past been designed a number of prosthetic knee joints intended for manufacture from plastics materials, or from plastics and metal materials, wherein large surfaces have been provided as bearing surfaces for the support of these loads and to reduce the unit stress thereon and these designs have had many good features. However, as for example in the patent to Lagrange, U.S. Pat. No. 3,918,101, wherein this basic concept is developed, there is also provided a centrally split housing to define the joint cavity. In this case, the two parts of such housing are held together by screws and hence in conditions of use involving tension through the joint such screws will be subjected to tension forces. Since these forces must be resisted by the thread screws in the plastics material, this presents a point of weakness. In other proposals, such as Goldberg U.S. Pat. No. 3,765,033, wherein the broad concept of providing wide support for the joint loading is suggested, the components are fastened together by a transversely positioned pin in an oversize bore. This not only requires quite precise original dimensioning but also inevitably provides for looseness in the joint, particularly when the user&#39;s leg is under tension and it would be unsatisfactory. Various other disclosures recognize the problem and attempt to solve it by providing wide support surfaces as above mentioned but they all have other features involving either looseness, undesirable concentration of forces in certain use situations with resulting points of weakness or other undesirable constructional and/or operational features. Accordingly, the objects of the invention include: 1. To provide a prosthetic knee joint which can be formed from plastics materials and/or plastics-metal materials as desired, which will provide heavy load capabilities in the normal condition of use as in walking by the user, but which will provide firm and smooth bending of the knee under all circumstances of use and permit no looseness therein even under conditions of lateral flexure or leg tension. 2. To provide a prosthesis, as above mentioned, wherein the load in all directions of major magnitudes is at least partially carried by a solid piece of material and wherein the rest of such load is carried by shear stress on the screws holding component parts together, and only very minor loadings will apply tension stress to such screws. 3. To provide a prosthesis, as above mentioned, wherein the joint will be firm and solid and wherein particularly there will be no free play present. 4. To provide a prosthesis, as aforesaid, which will be of sufficiently simple construction as to be formable in relatively large masses by molding and will be free from complex parts and consequent difficulties in molding. Other objects and purposes will be apparent to persons acquainted with devices of this general type upon reading the following specification and inspection of the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS In the drawings: FIG. 1 is an oblique exploded view of an embodiment of the invention. FIG. 2 is an end view of the socket and cylinder portions in assembled relationship taken from the left end as appearing in FIG. 1. FIG. 3 is a central section taken on the line III--III of FIG. 2 with, however, the cap portion in place. FIG. 4 is an elevation view of the cap taken from the righthand end as appearing in FIG. 1. DETAILED DESCRIPTION Turning now to the drawings in more detail, there is shown a recess portion 1 having a conventional prong 2 thereon for insertion into the femur, a cylinder portion 3 having a prong 4 thereon for insertion into the user&#39;s tibia and a cap portion 6 for closing the end of the cavity provided within the recess portion 1. Said recess portion 1 comprises a central zone 7 and an end portion 8. Said central zone 7 is of generally cylindrical shape with a portion of such cylinder removed to provide a concentric, substantially hemicylindrical, recess 9, a surface 10 extending radially from the exterior of said cylindrical body 7 to the hemispherical surface defining the recess 9 and a further surface 11 extending from another portion on the exterior surface of said cylindrical body 7 to the surface defining the hemispherical recess 9 and intersecting therewith. The surface 11 is, as best seen in FIG. 2, substantially parallel with the center line of the prong 2 and positioned on the side of the recess 9 remote from both said prong 2 and said surface 10. The surface 10 defines an angle with respect to said center line and is angularly spaced from said surface 11 an angular distance dependent upon the number of degrees of flexure desired for the prosthesis, here approximately 120°. The end portion 8 comprises in its outer surface a substantial continuation of the correspondingly adjacent portions of the central section 7 and is provided internally with a blind cylindrical opening 12, a part of whose walls are a continuation of the wall defining the hemispherical recess 9. The end cap 6 forms with the central section 7 a mirror image of the end portion 8 and is fastened thereto by a convenient means such as suitable screws 18 positioned in the screw holes 13. Said screw holes are in register when the parts are assembled with threaded openings 16 by which screws 18 may be utilized to hold the parts together. The inner surface 21 of the end cap 6 is flat throughout an arcuate portion 22 thereof, said portion being that portion which bears against the end surface 23 of said central portion 7. A further portion of said end cap 6 comprises a circular zone 26 which is concentric with and opposed to the end 27 of the blind opening 12, the broken line A indicating that portion of the boundary of the area 26 which lies against the edge 28 of the hemispherical surface 9. Said surfaces 21 and 26 are perpendicular to the axis of the recess 9 and blind opening 12. A projecting portion 31 extends centrally past the surface 23 whereby to define in effect when the parts are assembled a blind opening between the central part 7 and the part 6 corresponding to and identical (in mirror image) with the blind opening 12. Upon reference to FIGS. 2 and 4, it will be seen that the lower surface of the socket part 1, including the central portion 7, the end portion 8 and the cap 6 all cooperate to define an arcuate cylindrical external surface 32 which is concentric with the center of the hemispherical recess 9. A flat portion 33 may be provided if desired for use in affixing a knee cap thereto but at least that portion of said surface 32 contacted by the cylinder part 3 of the prosthesis between its extreme pivoted positions, namely between the positions 32A and 32B of the socket part 1, are arcuate as above stated for reasons appearing below. Turning now to cylinder part 3, there is provided as above mentioned the prong 4 for reception into the tibia of the user. At the upper end of said prong is positioned a platform 34 whose upper surface 36 is arcuate on the same radius as the surface 32. Projecting further upwardly in a substantial continuation of the prong 4 is a neck 37 which is of length substantially equal to (or a clearance distance greater than) the dimension 38 of the cap 6 and the equal dimension 38A of the flange 41 of the end portion 8. At the upper end of the neck 37 is a cylindrical head 39 which is of diameter and length to fit snugly into the blind opening 12, against the hemicylindrical surface of the recess 9 and into the blind opening formed between the central portion 7 and the end cap 6 when said two portions are assembled as shown in FIG. 3. A slide unit 42 is shown for positioning between, and cooperation with, both the normal knee cap K (which usually does not need replacing) and the joint prosthesis of the invention. Same is shaved by the surgeon to present the flat surface K1 on the side facing the joint and the small pilot hole K2 drilled centrally therein. Said slide 42 has a contact portion 43 and a locating pin 44. The locating pin is received into the pilot opening K2 and the contact portion 43 is then positioned to bear against the flat 33 of the prosthesis. As the knee flexes, said contact portion will ride as needed on the outer (leftward as appearing in FIG. 2) surface of the recess portion 1. The muscles and tendons normally attached to the natural knee cap will in most instances remain attached thereto and will continue after the prosthesis is installed to operate in a normal manner with the muscles and tendons controlling the prosthetic knee joint. Since the parts are large and provided with simple planar or arcuately cylindrical surfaces, same can be and are molded to provide close tolerances therebetween whereby with the head 39 in place the joint will be firm and without looseness but wherein it can still pivot easily and smoothly. The limits of said pivoting are determined by the surfaces 10 and 11 as will be apparent from FIG. 2. OPERATION It will be apparent from the drawings that the parts are assembled by placing the head 39 against hemicylindrical surface of the recess 9 and moving same axially until the rightward (as seen in FIG. 1) end 39A of said head enters fully into the blind opening 12. This brings the end 39B of said cylinder substantially flush with the end surface 23 of the recess part 1. The end cap 6 is then put in place with its projecting portion 31 located between said head 39 and the platform 34. The screws 18 are put into place through the openings 13 and then into the openings 16 and suitably tightened. The head 39 is thus firmly captured within the recess provided by the recess part 1 and only under exceptional conditions will any axial stress be applied to the screws 18. When the user is standing, the load is connected directly from the recess portion 1 through the surface 32 thereof against the surface 36 and to the prong 4. If there is simultaneous contact between the upper end of the head 39 and the surface cooperating therewith of the hemispherical recess 9, the plastic parts have sufficient resilience to permit the pressure to become substantially equalized between said parts and the surfaces 32 and 36. Likewise under such conditions, the entire length of the head 39 bears against the single molding comprising the recess part 1 and no pressure at all is applied to any part of the screws 18. With the user in a crouched or sitting position, the load is similarly transmitted from the recess portion 1 to the cylinder portion 3 and no stress whatever is placed upon said screws. When, however, the user&#39;s foot is lifted so that tension force is placed upon his leg, then a portion of such load is placed upon the flange 41 of the end portion 8 which it will be noted is molded as an integral part of the recess portion 1 and only a portion of such tension load is placed upon the flange 31 of the end cap 6. Even this, however, is applied through the screws 18 as shear stress and will be adequately held by even relatively small screws. The only way in which an axial stress may be applied to said screws is when there is a side thrust placed upon one portion of the user&#39;s leg which is resisted by the other portion thereof and this will be of only minor magnitudes in all normal situations. Accordingly, the objects of the invention as above set forth are adequately accomplished by the prosthesis as above described and illustrated by the accompanying drawings and the loading on the prosthesis under any normal use will seldom, if ever, exceed 300 psi. Although a particular preferred embodiment of the invention has been disclosed in detail for illustrative purposes, it will be recognized that variations or modifications of the disclosed apparatus, including the rearrangement of parts, lie within the scope of the present invention.

Summary: Prosthetic knee joint. A prosthetic knee joint is proposed which incorporates into a single product a variety of previously recognized desirable features. The joint in question has a generally cylindrically shaped knob on one component thereof and a receptacle for receiving said knob in the other component thereof. Said receptacle is designed for full engagement of said cylindrical knob less only the clearance required to permit the desired pivotal motion of said joint and the components defining said receptacle are arranged so that only shear stress is normally imposed on the screws by which the components defining said receptacle are assembled and the parts are further arranged in order that wide contact surfaces between the components of said prosthesis are provided for direct transmission of load between said components without a major portion thereof being transmitted through said joint.

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Write a title and summarize: Xi Jinping abogó en Davos por el libre comercio. El lunes pasado, el presidente estadounidense, Donald Trump, dio al traste con este tratado comercial que fue negociado durante siete años por el gobierno de su antecesor. Durante ese tiempo, el exmandatario hizo todo lo posible para recordar que no era casual que China no estuviera incluida en esa negociación, en la que participaron otros 11 países de la región, incluyendo a tres latinoamericanos: México, Perú y Chile. Los miembros restantes son: Japón, Malasia, Vietnam, Singapur, Brunei, Australia y Nueva Zelanda. Washington buscaba así aumentar su presencia en una de las zonas económicas más dinámicas del mundo y, al mismo tiempo, prevenir un vacío regional que pudiera ser llenado por China. El TPP fue suscrito en febrero de 2016. El TPP era una parte esencial del llamado "giro estratégico" hacia Asia del gobierno de Obama. Pero, esa estrategia ya es historia y, paradójicamente, tras la decisión de Trump de retirarse del TPP parece estarse abriendo la oportunidad para que Pekín se integre en ese acuerdo, del que expresamente Washington quería mantenerle fuera. ¿Es esto posible? El TPP menos uno Aunque algunos expertos consideran que la salida de Estados Unidos implica el fin del TPP, toda vez que el mayor incentivo para los 11 miembros restantes del acuerdo era la posibilidad de acceder con mayores facilidades al mercado estadounidense, algunos países han dado señales de querer seguir adelante. Los gobiernos de Australia y Nueva Zelanda anunciaron su deseo de impulsar un acuerdo con los otros estados participantes. Australia, incluso, llegó a proponer una fórmula: "TPP 12 menos 1". El ministro de Comercio de ese país, Steve Ciobo, afirmó que no piensan abandonar el tratado solo porque este requiera un poco de esfuerzo para mantenerlo con vida. Ciobo acudió la semana pasada al Foro Económico Mundial de Davos, en Suiza, donde mantuvo contacto con varios países miembros del TPP. ¿Permitirá Japón que China se una al TPP? "Conversé con Canadá, México, Japón, Nueva Zelanda, Singapur y Malasia", dijo en declaraciones a la Corporación Australiana de Radiodifusión (ABC, por sus siglas en inglés). "Sé que ha habido conversaciones con Chile y Perú, así que hay varios países que tienen interés en ver si podemos hacer funcionar el TPP 12 menos 1", agregó. Ciobo destacó que la arquitectura original del acuerdo fue diseñada para permitir el ingreso de otros países. "Ciertamente, sé que Indonesia manifestó su posible interés y habría margen para China si somos capaces de reformular el acuerdo para ser un TPP 12 menos 1 para que países como Indonesia, China u otros consideren sumarse con el fin de obtener los beneficios que se derivan de ello", apuntó. Por su parte, el primer ministro de Nueva Zelanda, Bill English, dijo que tiene esperanzas de mantener vivo el acuerdo con los miembros restantes del TPP. El ministro de Comercio de ese país, Todd Clay, dijo a medios locales que espera reunirse en los próximos meses con sus colegas del resto de países miembros para buscar la fórmula para seguir adelante. El presidente de Perú, Pedro Pablo Kuczynski, ha sido un defensor de la incorporación de China a los acuerdos comerciales. La fórmula de incorporar a China ha sido respaldada desde hace meses por el presidente de Perú, Pedro Pablo Kuczynski, quien criticaba al TPP por excluir a esa gran potencia asiática que es en la actualidad el principal socio comercial de su país. Este martes Kuczynski propuso trabajar en un acuerdo comercial alternativo al TPP para poder incorporar a Pekín. "Deberíamos trabajar con China, los países de Asia, India, Australia y Nueva Zelanda", dijo este martes en una entrevista en televisión. "Deberíamos hacer un grupo de la Asociación de Países de Asia Pacífico (APEC) que llegue tan lejos como hasta India, así incluiremos a todos. Vamos a tener las mejores partes del TPP y nos deshacemos de las partes menos buenas", agregó. La esperanza de Japón Cualquier posibilidad de mantener vivo este acuerdo comercial dependerá de Japón, tercera economía del mundo y primera del TPP tras la salida de Estados Unidos. Por lo pronto, el primer ministro japonés, Shinzo Abe, se ha limitado a señalar su esperanza de lograr persuadir al nuevo gobierno estadounidense sobre continuar el diálogo comercial. "Creo que el presidente Trump entiende la importancia del comercio libre y justo, así que me gustaría continuar buscando su comprensión sobre la importancia estratégica y económica del TPP", dijo Abe el lunes durante una sesión del Parlamento japonés. Tras la victoria de Donald Trump, muchos críticos del TPP dieron por muerto el acuerdo. El ministro de Finanzas de ese país, Taro Aso, anunció este martes que trabajan en preparar una visita de Abe a Estados Unidos para reunirse con Trump. Un cambio de posición de Washington sería bienvenido por Canadá, país que señaló que el TPP no puede seguir adelante sin Estados Unidos. "Este acuerdo fue diseñado de tal forma que solo puede entrar en vigor si es ratificado por Estados Unidos, así que no puede hacerse realidad sin él", dijo la ministra de Exteriores, Chrystia Freeland. Canadá, sin embargo, no parece que se quedará a esperar por Trump. Este martes, el primer ministro canadiense, Justin Trudeau, anunció que tras la retirada de Estados Unidos del TPP su foco en materia comercial se dirigirá hacia Japón y China. Destacó que el año pasado iniciaron un diálogo con Tokio con miras a mejorar las relaciones comerciales y que el próximo mes se realizarán los primeros diálogos exploratorios sobre libre comercio con Pekín. Trudeau, sin embargo, evitó responder las preguntas sobre si Canadá se uniría a un TPP más pequeño sin la participación de Estados Unidos. ¿Y China? Aunque ha resultado claramente favorecido por la decisión de Estados Unidos de retirarse del TPP, el gobierno de China ha evitado hasta ahora manifestarse sobre su posible adhesión a ese acuerdo. En una rueda de prensa realizada este martes, un portavoz chino evitó responder las preguntas sobre ese asunto. Dijo que su país ha estado abogando por rutas comerciales abiertas en la región del Pacífico "junto a soluciones en las que ganen todos". El primer ministro de Japón, Shinzo Abe, espera persuadir a Donald Trump sobre las virtudes del acuerdo. "Creemos en la integración regional. Estamos abiertos a acuerdos económicos regionales transparentes. Las economías de Asia Pacífico son diversas. Es importante comportarse de una manera abierta. Estamos listos para trabajar con todas las partes para darle ímpetu a la economía global y a la de Asia Pacífico", agregó. Pekín ha estado abogando por el Área de Libre Comercio de Asia y el Pacífico (FTAAP en inglés) y ha dado impulso a la Asociación Económica Integral Regional (RCEP en inglés), de lo cual podría surgir un tratado comercial que incluya a países como Australia, China, India, Japón, Corea del Sur y Nueva Zelanda. La semana pasada, durante la primera participación de un líder chino en el Foro Económico Mundial de Davos, el presidente Xi Jinping defendió el libre comercio y comparó el proteccionismo con "encerrarse solo en una habitación oscura". Los comentarios del dirigente chino fueron interpretados como una referencia al discurso y las políticas de "Estados Unidos primero" de Donald Trump. También como una señal de que Pekín está viendo una oportunidad para jugar un papel más significativo en el comercio mundial, llenando el vacío que está dejando Estados Unidos.

Title: ¿Washington fuera, Pekín dentro? El inesperado giro que puede dar el TPP tras la salida de Estados Unidos por orden de Donald Trump Summary: Aunque fue diseñado como parte de la estrategia del expresidente de EE.UU. Barack Obama para hacer frente al desafío de China en la región de Asia-Pacífico, ahora es posible que el Acuerdo de Comercio Transpacífico (TPP, por sus siglas en inglés) termine convertido en una herramienta para la consolidación de la presencia de Pekín en la zona.

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Write a title and summarize: Interactions between an organism and its environment can significantly influence phenotypic evolution. A first step toward understanding this process is to characterize phenotypic diversity within and between populations. We explored the phenotypic variation in stress sensitivity and genomic expression in a large panel of Saccharomyces strains collected from diverse environments. We measured the sensitivity of 52 strains to 14 environmental conditions, compared genomic expression in 18 strains, and identified gene copy-number variations in six of these isolates. Our results demonstrate a large degree of phenotypic variation in stress sensitivity and gene expression. Analysis of these datasets reveals relationships between strains from similar niches, suggests common and unique features of yeast habitats, and implicates genes whose variable expression is linked to stress resistance. Using a simple metric to suggest cases of selection, we found that strains collected from oak exudates are phenotypically more similar than expected based on their genetic diversity, while sake and vineyard isolates display more diverse phenotypes than expected under a neutral model. We also show that the laboratory strain S288c is phenotypically distinct from all of the other strains studied here, in terms of stress sensitivity, gene expression, Ty copy number, mitochondrial content, and gene-dosage control. These results highlight the value of understanding the genetic basis of phenotypic variation and raise caution about using laboratory strains for comparative genomics. A major focus of genetic study is to elucidate the effects of genetic variation on phenotypic diversity. The evolution of phenotypes is often driven by environmental factors and the interactions between each organism and its environment. Recently, there has been a renewed interest in characterizing the diversity and ecology of organisms long used in the laboratory as models for biological study. Yeast, worms, flies, and mice have been studied on a molecular level for decades and have provided many insights into basic biology. However, most of our knowledge base exists for only a handful of domesticated lines. Little is known about the natural ecology of these organisms or the degree to which individuals of each species vary within and between natural populations. The budding yeast Saccharomyces cerevisiae exists in diverse niches across the world and can be found in natural habitats associated with fruits, tree soil, and insects, in connection with human societies (namely through brewing and baking), and in facultative infections of immuno-compromised individuals [1]. These yeasts are transported by insect vectors and likely through association with human societies. Recent population-genetic studies have begun to explore the genetic diversity of S. cerevisiae strains [2]–[5]. These studies have demonstrated little geographic structure in natural yeast populations and relatively low sequence diversity, particularly within vineyard strains. It has been proposed that low sequence diversity in this species may be due to a more recent common ancestor compared to other yeasts [6]. Genomic comparisons also suggest low rates of outcrossing between strains [7], which may limit the fixation of genetic differences under selection by reducing effective population sizes [8]. Although the genetic diversity of S. cerevisiae populations is emerging from large-scale sequencing projects, the phenotypic diversity within and between yeast populations has been less systematically studied. Myriad studies have characterized strain-specific differences in specific phenotypes to identify the genetic basis for phenotypes of interest (for example, those related to wine making [9], thermotolerance [10]–[12], sporulation efficiency [13]–[16], drug sensitivity [17]–[19], and others [20]–[25]). The degree to which these phenotypes vary across diverse strains has not been systematically explored. Other genomic studies have investigated variation in genomic expression across strains, with the goal of investigating the mode and consequence of gene-expression evolution [26]–[30]. These studies demonstrated significant variation in gene expression between strains, and in some cases pointed to the genetic basis for those differences [27], [31]–[35]. However, each study investigated only a few strains, typically vineyard strains. The broader phenotypic variation across diverse yeast strains and populations, particularly natural isolates, is largely uncharacterized. Here we investigated the variation in stress sensitivity and genomic expression in a large panel of Saccharomyces strains. We quantified the sensitivity of 52 strains collected from diverse niches to 14 environmental conditions and measured genomic expression in 18 of these strains growing in standard medium. We observe a large amount of phenotypic variation, both in terms of stress sensitivity and gene expression. Associations among phenotypes revealed relationships between environmental conditions and among yeast strains. One case in particular suggests that genetically diverse strains collected from oak soil have undergone selection for growth in a common niche. This study provides a representative description of expression variation and stress sensitivity within and across yeast populations, particularly non-laboratory strains, setting the stage for elucidating the genetic basis of this variation. Fay and Benavides conducted a population-genetic study of 81 Saccharomyces strains by analyzing ∼7 kb of coding and non-coding sequence from each isolate [2]. We characterized the phenotypic diversity of 52 of these strains, shown in Figure 1. This set included natural isolates from European vineyards, yeasts collected from African palm-wine fermentations, commercial wine- and sake-producing strains, clinical yeasts, natural isolates collected from African and Asian fruit substrates, strains from oak-tree soil and exudates from the Northeastern United States, three common lab strains, and other isolates (see Table S1 and [2] for references). We also characterized two haploid S. cerevisiae strains (RM11-1a and YJM789) and three other Saccharomyces species (S. paradoxus, S. mikatae, and S. bayanus) for which whole-genome sequence is available [36], [37]. Each strain was grown under 31 different conditions representing 14 unique environments, chosen to provoke diverse physiological responses. These environments varied in nutrient composition, growth temperature, and presence of toxic drugs, heavy metals, oxidizing agents, and osmotic/ionic stress. Cells were grown on solid medium in the presence of each environmental variable, and viability was scored relative to a no-stress control for each strain (see Materials and Methods for details). The results reveal a tremendous amount of phenotypic diversity in environmental sensitivity (Figure 2). Although there were similarities between strains, no two strains were exactly alike in phenotypic profile. Each displayed a propensity for growth under at least one environment and sensitivity to one or more conditions. Some strains were generally tolerant to stressful environments across the board. For example, strain Y2, originally collected from a Trinidadian rum distillery, and clinical isolates YJM454 and YJM440 were tolerant of most of these conditions, while the S. bayanus strain used in our study was sensitive to nearly all stresses tested. Several strains, including commercial sake-producing strains, showed a wide standard deviation of growth scores across the stresses, reflecting that they were either highly sensitive or highly resistant to different stresses. In contrast, most vineyard isolates grew moderately well in most of the environments examined (see Discussion). Exploration of the range of strain sensitivities measured for each environment also suggested common and unique features of Saccharomyces' habitats. Collectively, this set of strains showed the greatest variation in copper sulfate tolerance, sodium chloride resistance, and freeze-thaw survival, implicating these as niche-specific features not generally experienced by yeast. In contrast, strains showed the least variation (but some variability nonetheless) for growth on non-fermentable acetate, in minimal medium lacking supplemental amino acids, and at 37°C. Presumably, defects in respiration, prototrophy, and growth at physiological temperature represent a significant selective disadvantage, regardless of the particular niche. Hierarchical clustering of the phenotype data revealed interesting relationships between groups of strains. In particular, several groups of strains displayed similar profiles of stress sensitivity across the environments tested (Figure 2). As a group, the sake-producing strains were extremely resistant to lithium chloride but sensitive to copper sulfate, calcium chloride, cadmium chloride, and SDS detergent (p<0. 005 based on 10,000 permutations, see Materials and Methods); indeed, this group was slightly more sensitive to stress in general. Many of the vineyard strains shared specific phenotypes, including resistance to copper sulfate, as previously noted for other vineyard strains [26], [38], [29]. The group of laboratory strains was also highly resistant to copper sulfate as well as sodium and lithium chloride. In contrast, strains collected from oak soil were particularly sensitive to copper sulfate and sodium chloride but highly resistant to freeze-thaw stress (p<0. 005,10,000 permutations). The similarities in phenotypic profiles could arise through selection (either directional or purifying) due to shared selective pressures across strains living in the same environment. Alternatively, phenotypic similarity could result simply if the strains are genetically related due to a recent common ancestor. For example, many of the lab strains are closely related, since a large fraction of their genomes is derived from a common progenitor [39], [40]. We wished to distinguish between these possibilities for other strain groups. Natural selection can be inferred by comparing the population genetic structure (FST) to an analogous measure of phenotypic structure (QST) [41], [42]. A deviation from unity suggests that either divergent (QST/FST>1) or purifying (QST/FST<1) selection has occurred across populations. We wished to analyze each subpopulation separately, and therefore we devised a simple alternative approach to identify deviations from neutral phenotypic variation. We calculated the average pairwise phenotypic distance over the average pairwise genetic distance for pairs of strains collected from the same environment (‘sake’, ‘vineyard’, ‘oak’, ‘clinical’, ‘natural’ or ‘other fermentation’). This ratio was compared to the ratio of distances calculated for pairs of strains between niche groups, generating the parameter P/G. A P/G ratio = 1 is expected under neutrality, where the phenotypic to genetic distance is equal for within-group versus between-group comparisons. In contrast, a value of P/G<1 suggests that the strains within the group are more similar in phenotype than would be expected under the neutral model, whereas a ratio >1 indicates that the strains are phenotypically more variable than expected based on their genetic relatedness. The results provide evidence of both selection and shared ancestry for different groups of strains. First, the P/G ratio did not deviate significantly from unity for strains in the ‘clinical’, ‘natural’, or ‘other fermentation’ groups (average P/G = 1. 02+/−0. 22), nor did it deviate significantly for randomized simulations (data not shown). In contrast, P/G was 4. 2 and 3. 0 for sake strains and vineyard strains, respectively. Thus, the similarity in their phenotypes likely arises due to their recent divergence from a common ancestor. Interestingly, these P/G values were significantly higher than expected by chance (p<0. 0001 from 10,000 permutations), suggesting that the strains show more phenotypic variation than expected. This could arise if strains have experienced diversifying selection for disparate phenotypes, although it could also result if genetic distances are underrepresented or skewed due to limited sequence data. In contrast, strains collected from oak-tree exudates and soil are phenotypically more similar than would be expected under a neutral model. We observed a P/G ratio of 0. 31 (p = 0. 0013 from 10,000 permutations), indicating that phenotypic variation within this group is lower than expected based on the strains' genetic relatedness. This suggests that the strains have undergone selection for growth in a common environment (see Discussion). Consistent with this model, the S. paradoxus strain YPS125, also collected from Northeastern oak flux [6], is phenotypically more similar to S. cerevisiae strains collected from that environment (pairwise R of 0. 61,0. 66, and 0. 77 to YPS1000, YPS1009, and YPS163, respectively) than to the other S. paradoxus strain in our collection (R = 0. 51). At least some of the phenotypes shared by these strains are likely important for their ability to thrive in their niche (see Discussion). Numerous studies have characterized differences in genomic expression between individual strains of yeast, typically vineyard and lab strains [13], [26]–[31], [34], [43]. To more broadly survey the variation in genomic expression across populations, we measured whole-genome expression in 17 non-laboratory strains compared to that in the diploid S288c-derived strain DBY8268, using 70mer oligonucleotide arrays designed against the S288c genome. The long oligos used to probe each gene minimize hybridization defects due to sequence differences from S288c. We verified this by hybridizing genomic DNA from 6 strains of varying genetic distance from S288c: indeed, fewer than 5% of the observed gene expression differences described below could be explained by defective hybridization to the arrays (see Materials and Methods). Therefore the vast majority of measured expression differences are due to differences in transcript abundance. A striking number of yeast genes showed differential expression from the laboratory strain in at least one other strain (Figure 3A). Of the ∼5,700 predicted S. cerevisiae open reading frames, 2680 (∼47%) were statistically significantly altered in expression (false discovery rate, FDR = 0. 01) in at least one non-laboratory strain compared to S288c, with an average of 480 genes per strain. At an FDR of 0. 05, over 70% of genes were significantly altered in expression in at least one non-lab strain (Table 1). The number of expression differences is comparable to that observed by Brem et al., who reported over half of yeast genes differentially expressed between the vineyard strain RM11-1a and S288c [27]. However, closer inspection revealed that many of these expression differences were common to all of the non-laboratory strains (Figure 3A), revealing that these expression patterns were unique to S288c. This group was enriched for functionally related genes, including those involved in ergosterol synthesis, mitochondrial function, respiration, cell wall synthesis, transposition, and other functions (Table 2). Many of these functional groups were also reported by Brem et al., who noted that multiple categories (including ergosterol synthesis and mitochondrial function) can be linked to a known polymorphism in the Hap1p transcription factor [44]. Indeed, the expression differences specific to S288c were enriched for targets of Hap1p (p<10−11, hypergeometric distribution) as well as targets of Hap4p (p<10−6) [45], which regulates genes involved in respiration. Hence, many of the observed expression differences may result because of S288c-specific physiology (see Discussion). For a more representative description of expression variation in non-laboratory strains, we sought to represent the expression differences in a way that was not obscured by S288c. First, we identified genes whose expression varied significantly from the oak strain YPS163. Second, we identified transcripts whose abundance varied from the mean of all non-laboratory strains (see Materials and Methods). Although the mean expression value of each gene is merely an arbitrary reference point, this data transformation serves to remove the effect of S288c from each array while maintaining the statistical power to identify expression differences. Roughly 1330 (23%) of yeast genes varied in expression in at least one non-laboratory strain relative to the mean of all strains, while 953 (17%) of genes varied significantly from YPS163 (FDR = 0. 01). In both cases, two thirds of significant expression differences were specific to only one strain (Figure 3B and 3C). The number of genes with statistically significant expression differences from the mean ranged from 30 (in vineyard strain I14) to nearly 600 (in clinical isolate YJM789), with a median of 88 expression differences per strain. The number of expression differences did not correlate strongly with the genetic distances of the strains (R2 = 0. 16). However, this is not surprising since many of the observed expression differences are likely linked in trans to the same genetic loci [27], [31], [34], [35], [43]. Consistent with this interpretation, we found that the genes affected in each strain were enriched for specific functional categories (Table S4), revealing that altered expression of pathways of genes was a common occurrence in our study. We noticed that some functional categories were repeatedly affected in different strains. To further explore this, we identified individual genes whose expression differed from the mean in at least 3 of the 17 non-laboratory strains. This group of 219 genes was strongly enriched for genes involved in amino acid metabolism (p<10−14), sulfur metabolism (p<10−14), and transposition (p<10−47), revealing that genes involved in these functions had a higher frequency of expression variation. Differential expression of some of these categories was also observed for a different set of vineyard strains [26], [28], and the genetic basis for differential expression of amino acid biosynthetic genes in one vineyard strain has recently been linked to a polymorphism in an amino acid sensory protein [35]. We also noted that the 1330 genes with statistically variable expression in at least one non-laboratory strain were enriched for genes that contained upstream TATA elements [46] (p = 10−16) and genes with paralogs (p = 10−6) but under-enriched for essential genes [47] (p = 10−25). The trends and statistical significance were similar using 953 genes that varied significantly from YPS163. Thus, genes with specific functional and regulatory features are more likely to vary in expression under the conditions examined here, consistent with reports of other recent studies [30], [43], [48], [49] (see Discussion). Expression from transposable Ty elements was highly variable across strains. However, Ty copy number is known to vary widely in different genetic backgrounds [50], [51], suggesting that these and other observed expression differences could be due to copy number variations in particular strains. Indeed, numerous expression differences could be linked to known gene amplifications in S288c, such as ASP3, ENA1, CUP1, and hexose transporters [52], [51]. We quantified the contribution of increased copy number to the observed increases in gene expression relative to S288c in 6 of our strains. In general, ∼2–5% of expression differences could be wholly or partially explained by differences in gene copy number (see Materials and Methods). YPS1009 was an exception to the trend, since nearly 20% of genes with higher expression could be attributed to increased copy number - most of these genes reside on Chromosome XII. In fact, more than 80% of genes on Chromosome XII met our criteria for increased copy number (Figure S1A), indicating that the entire chromosome is duplicated in this strain. Another example of chromosomal aneuploidy is evident in strain K9, for which Chromosome IX appears amplified (Figure S1B). Whole-chromosome aneuploidy has been frequently observed in strains growing under severe selective pressure (for example [53]–[56]. Interestingly, the majority of genes on these duplicated chromosomes do not show elevated transcript abundance in the respective strains. In fact, only ∼25% of genes with increased copy number in each strain showed elevated expression (defined at FDR = 0. 01 or as genes whose expression is >1. 5× over S288c). This is in stark contrast to previous studies demonstrating little dosage compensation in S288c in response to gene amplification and chromosomal aneuploidy, leading to the conclusion that yeast does not have a mechanism for dosage compensation. [53], [54], [57]. Instead, our results suggest that some form of feedback control acts to normalize the dosage of most genes in non-laboratory yeast strains. The remaining quarter of amplified genes may be inherently exempt from this feedback mechanism. Alternatively, relaxed feedback may occur for specific amplifications if the resulting transcript increase provides a selective advantage to the strain in question. Indeed, 15–40% (depending on the strain) of genes lacking feedback control show at least 1. 5× higher expression beyond what can be accounted for by gene amplification alone, indicating that the expression differences are affected by both gene dosage and regulatory variation. These genes are excellent candidates for future studies of adaptive changes. As observed for gene expression, we found that some genomic amplifications were common across all 6 strains compared to S288c. All strains showed decreased Ty1 copy number, ranging from 2–15× lower than S288c. This is consistent with previous studies that showed higher Ty1 copy number (including active and partial Ty elements) in S288c compared to wine strains and natural isolates [50], [51], [58]. Most strains also showed even lower Ty1 transcript abundance, beyond what could be explained by copy number variations. Thus, in addition to a higher Ty content, S288c also shows higher expression from Ty genes, perhaps reflecting elevated rates of retrotransposition under the conditions studied here. In contrast, all strains showed higher copy number of the mitochondrial genome compared to S288c, typically elevated 2–3× but nearly 7× higher in clinical strain YJM789. The most likely explanation is that these strains harbor more mitochondria than S288c, a fact confirmed in vineyard strain RM11-1a by mitochondrial staining [25]. In addition to revealing phenotypic diversity within and between yeast populations, natural variation can also uncover new insights into the effects of each environment on cellular physiology. For example, we noted correlations between environments based on the distribution of strain-sensitivity scores. The most likely explanation is that these stresses have similar effects on cellular function, and thus strains display similar sensitivities to them. Resistance to sodium chloride and lithium chloride or tolerance of ethanol and elevated temperature were highly correlated (R = 0. 66 at p<0. 0001 and R = 0. 51 at p<0. 0006, respectively, based on 10,000 permutations), consistent with the known effects of these stress pairs on ion concentrations or membrane fluidity/protein structure, respectively. Other relationships were not previously known, including the correlation between sensitivity to SDS detergent and the heavy metal cadmium (R = 0. 64, p<0. 0001) and between ethanol and caffeine tolerance (R = 0. 59, p<0. 0001). In contrast, resistance to freeze-thaw stress was anticorrelated to sodium chloride resistance (R = −0. 35, p = 0. 006), suggesting antagonistic outcomes of the same underlying physiology. These relationships point to commonalities in the cellular consequences inflicted by these environments that will be the subject of future investigations of stress-defense mechanisms. We also conducted an associative study to identify gene expression patterns correlated with environmental sensitivity across the 17 non-laboratory strains (see Materials and Methods for details). As basal expression differences could significantly contribute to the inherent ability of cells to survive a sudden dose of stress, the results point to genes whose expression is related to, and perhaps causes, the phenotypes in question. Among the top genes associated with copper sulfate resistance was the metallotheionein CUP1, important for copper resistance and known to have undergone tandem duplications in copper-resistant strains [59], [60]. Of the genes whose expression was correlated to sodium chloride tolerance, nearly 20% are known to function in Na+ homeostasis and/or osmolarity maintenance (including RHR2, COS3, SIS2 identified through genetic studies [61]–[63] and JHD2, SRO7, YML079W, YOL159C, TPO4, UTH1 implicated in high-throughput fitness experiments in S288c [64]). Thus, these and likely other genes whose expression is highly correlated with each stress-sensitivity profile play a functional role in surviving that condition. Other correlations were not expected. Ethanol and caffeine tolerance were both correlated to the expression of genes encoding transmembrane proteins (p<0. 003, hypergeometric distribution), perhaps related to the effect of these drugs on membrane fluidity. Sensitivity to the cell-wall damaging drug Congo Red was significantly correlated to the expression of genes involved in mitochondrial function and translation, respiration, and ATP synthesis (p<10−13), revealing a link between mitochondria/respiration and the cell wall. Although these connections will require further characterization, they demonstrate the power of using natural diversity to uncover previously unknown relationships between stresses and cellular processes. This study demonstrates the vast amount of phenotypic variation in Saccharomyces strains collected from diverse natural habitats, used in industrial processes, and associated with human illness. Considering the phenotypic responses to the conditions studied here provides insights into the relationships between specific strains and their niches. For example, the wide variance in growth scores of sake-producing strains indicates that they are either highly resistant or sensitive to the different environments studied here, suggesting that they may be specialized for growth in the defined conditions of sake fermentation. In contrast, many of the vineyard isolates survived relatively well in most of the conditions tested. This may reflect their ability to thrive in more variable, natural environments and may also have facilitated their dispersal into new environments in a manner associated with human interactions [5]. Geographic dispersal might also explain the higher-than-expected phenotypic diversity of vineyard strains, which might be driven by diversifying selection (suggested by our analysis) due to unique pressures imposed after expansion into new environments. Although many of the phenotypic differences we observed are probably neutral, providing no benefit or disadvantage to the strains in question, some are likely to provide a selective advantage. Copper-sulfate resistance in European vineyard strains may have arisen through positive selection, since copper has long been used as an antimicrobial agent in vineyards and orchards [1], [65]. Another example may apply to the oak strains studied here. Our simple metric comparing phenotypic to genetic diversity in strains collected from similar environments suggests that oak strains are phenotypically more similar than expected based on their genetic relationship. Formally, this could arise if multiple traits are evolving neutrally (but slower than the genetic drift represented by the sequences used here) since the strains diverged from a distant, common progenitor. However, the fact that S. paradoxus oak isolate YPS125 is phenotypically more similar to S. cerevisiae oak strains than the other S. paradoxus isolate in our analysis instead supports that these strains have undergone selection for growth in a common environment. One intriguing phenotype is freeze-thaw resistance, which may be important to survive the wintry niche from where these strains were collected. Consistent with this hypothesis, we have recently isolated numerous Sacharomycete strains (including S. cerevisiae) from Wisconsin oak exudates, of which 86% (19/22) are freeze-thaw tolerant (DJK and APG, unpublished data). Ongoing studies in our lab are dissecting the genetic basis for this phenotypic difference. In addition to stress sensitivity, gene expression also varies significantly across yeast populations. More than a quarter of yeast genes varied in expression in at least one non-laboratory strain under the conditions studied here. Consistent with other recent reports [30], [48], [49], [66], we find that genes with specific structural or functional characteristics (including nonessential genes and those with upstream TATA elements and paralogs) show higher levels of expression variation across strains. This has previously been interpreted as a higher rate of regulatory divergence for genes with these features, either in response to selection [48] or mutation accumulation [49]. However, these features are also common to genes whose expression is highly variable within the S288c lab strain grown under different conditions ([67] and data not shown), particularly those induced by stressful conditions [46], [68]. It is also notable that genes with TATA elements show higher ‘noise’ in gene expression within cultures of the same strain [69], [70]. Thus, an alternative, but not necessarily mutually exclusive, hypothesis is that the expression of these genes is more responsive to environmental or genetic perturbations, again consistent with previous studies [66], [30], [48], [49]. We have conducted our experiments under ‘common garden’ lab conditions in attempt to minimize environmental contributions to expression phenotypes. However, because each strain may have evolved for growth in a unique environment, each may in fact respond differently to the same growth conditions used here. Indeed, this may explain the prevalence of metabolic genes in our set of genes showing variable expression in multiple strains, since many of these strains have not evolved for growth in highly artificial laboratory media. Emerging from our analysis is the fact that S288c is phenotypically distinct from the other non-laboratory strains studied here. This strain displays extreme resistance to specific stresses, harbors fewer mitochondria, contains more transposable elements, and shows unique expression of many genes compared to all other strains investigated (a direct comparison of the number of differentially expressed genes in S288c is difficult due to the different statistical power in calling these genes). We have also found that this strain has an aberrant response to ethanol, since it is unable to acquire alcohol tolerance after a mild ethanol pretreatment, unlike natural strains [71]. It is likely that additional responses found in natural strains have been lost or altered in this domesticated line. The progenitor of S288c was originally isolated from a fallen fig in Merced, California, and sequence analysis indicates that S288c is genetically similar to other natural isolates [1]–[3]. A recent study by Ronald et al. counters the proposal that S288c has undergone accelerated divergence during its time in the laboratory [72]. Instead, our results suggest that the strain has evolved unique characteristics through inadvertent selection for specific traits (such as growth on artificial media) and population bottlenecks. Thus, the laboratory strain of yeast may not present an accurate depiction of natural yeast physiology. Indeed, no single strain can be used to accurately represent the species, a note especially important for comparing phenotypes across species. Complete exploration of an organism' s biology necessitates the study of multiple genetic backgrounds to survey physiology across populations. Despite its limitations, the lab strain offers nearly a century of detailed characterization, along with powerful genetic and genomic tools. A useful approach is to complement studies on laboratory strains with investigations of natural variation. By characterizing stress sensitivity in a large set of strains, we have leveraged the power of natural diversity to uncover new relationships between stresses and to reveal previously unknown connections between genes, stresses, and cellular processes. These connections lead to hypotheses about stress defense mechanisms that can often be dissected using the valuable tools provided by the lab strain. Application of genomic techniques to characterize natural yeast strains will foster such studies while revealing additional insights into genetic and phenotypic variation in Saccharomyces. Strains used in this study and references are found in Table S1. In addition to sequence data from [2], an additional 5,305 bp of noncoding DNA was sequenced for 41 S. cerevisiae strains over 8 intergenic sequences (GENBANK accession numbers EU845779 - EU846095) for a total of 13,016 bp over 13 loci. Phylogenetic analysis shown in Figure 1 was performed on the combined sequence set using the program MrBayes [73]. Evolutionary distances were estimated using the Jukes-Cantor (JC) model based on 2,056 bp noncoding sequence data present in all strains; results and significance were very similar when the distance was based on 9,334 bp of noncoding sequence excluding only pairwise-deletion data [74]. Strains with evolutionary distances equal to zero over this subsequence (but clearly non-zero when all sequence was assayed) were set to 0. 00001 to facilitate permutation calculations. Paralogs were defined as genes with a BLAST E-value score <10−100. Yeast strains were grown in YPD medium at 30°C to an optical density of ∼0. 3 in 96-well plates. Three 10-fold serial dilutions were spotted onto YPD agar plates containing the appropriate stress, as well as a YPD plate for a no-stress control. Cells were also plated onto minimal medium [75] or YP-acetate. In the case of freeze-thaw stress, 200 µl cells was frozen in a dry ice/ethanol bath for two hours or left on ice as a control before spotting onto YPD plates. Cells were grown for 2–3 days at 30°C unless otherwise noted, and viability of each dilution was scored relative to the no-stress control for each strain. All experiments were done in at least duplicate over 2–3 doses of most stresses (see Table S2 for raw data and stress doses). Final resistance scores were summed over the 3 serial dilutions then averaged over replicates and stress doses, providing a single score ranging from 0 (no growth) to 6 (complete growth) for each strain and each stress condition. For Figure 2, strains were clustered based on phenotypes using the Pearson correlation and UPGMA clustering [76]. Correlations between stresses were calculated based on the Pearson correlation between strains, excluding 14 strains of highly similar genetic distance (JC<0. 0008). Phenotypes specific to groups of strains collected from similar environments (see Table S1 for groupings) were calculated based on the median growth score of strains in that group. Significance was estimated by 10,000 permutations of strain-group labels, scoring the frequency of observing a median growth score equal to or greater than that observed. A parameter, P/G, was calculated to compare the similarity in phenotype to the similarity in genotype for strains within and between niche groups. The average pairwise phenotypic distance, taken as the Pearson distance (1 – Pearson correlation) between phenotype vectors, was divided by the average pairwise JC distance for strains within a niche group. This value was divided by the same ratio calculated for all pairs of strains between niche groups (see Table S1 for niche groupings). Significance was estimated based on 10,000 random permutations of strain-group labels. The distribution of P/G ratios from randomized trials was centered on 0. 99; furthermore P/G was ∼1. 0 for strains in the ‘clinical’, ‘natural’, and ‘other fermentation’ groups, reflecting either neutral drift for these groups or that these strains were inappropriately grouped together into somewhat amorphous categories. Seventeen strains (including B1, I14, M22, M8, PR, RM11-1a, K1, K9, YJM308, YJM789, YJM269, Y12, SB, Y1, Y10, YPS1009, and YPS163) were chosen for whole-genome expression analysis. Cells were grown 2–3 doublings in YPD medium to early log-phase in at least biological triplicate. Cell collection, RNA isolation, and microarray labeling and scanning were done as previously described [77], using cyanine dyes (Flownamics, Madison, WI) and spotted DNA microarrays consisting of 70mer oligos representing each yeast ORF (Qiagen). For all arrays, RNA collected from the denoted strain was compared directly to that collected from the diploid S288c lab strain DBY8268, with inverse dye labeling used in replicates to control for dye-specific effects. At least three biological replicates were performed for all comparisons. Data were filtered (retaining unflagged spots with R2>0. 1) and normalized by regional mean-centering [78]. Genes with significant expression differences (compared to the S288c control, strain YPS163, or the mean expression across all strains) were identified separately for each strain with a paired t-test (or unpaired t-test in reference to YPS163) using the BioConductor package Limma v. 2. 9. 8 [79] and FDR correction [80], taking p<0. 01 as significant unless otherwise noted (see Table S3 for limma output and Figure S2 for a comparison of the statistical power for each strain). All microarray data are available through the NIH Gene Expression Omnibus (GEO) database under accession number GSE10269. Array-based comparative genomic hybridization (aCGH) was performed in duplicate on six strains (K9, M22, RM11-1a, Y10, YJM789, and YPS1009) relative to the DBY8268 control as previously described [81], using amino-allyl dUTP (Ambion), Klenow exo-polymerase (New England Biolabs), and random hexamers. Post-synthesis coupling to cyanine dyes (Flownamics) was performed using inverse dye labeling in replicate experiments. Technical variation in hybridization was defined as the mean+2 standard deviations (a log2 value of 0. 3) of all spot ratios, based on triplicate comparisons of DBY8268 to DBY8268 genomic DNA. For non-lab strains compared to DBY8268, genes with negative aCGH ratios outside the range of technical variation on both duplicates were defined as those affected by copy number and/or hybridization defects. Transcript levels within 0. 45 (3 standard deviations of technical variation) of the aCGH ratio were identified as those largely explained by copy number and/or hybridization defects – on average, fewer than 5% of genes with statistically significant (FDR = 0. 01) differential expression compared to DBY8268 fell into this class. Genes with a positive aCGH ratio >0. 7 in log2 space were defined as genes with increased copy number in each non-lab strain. All microarray data are available through the NIH Gene Expression Omnibus (GEO) database under accession number GSE10269. A vector of relative phenotype scores was generated by dividing scores from Figure 2 by the score measured for DBY8268. The Pearson correlation between this vector and the measured expression vector for each strain relative to DBY8268 was calculated for all genes in the dataset. Genes whose expression was correlated above or below what was expected by chance (p<0. 01) were defined based on 100 permutations of each of the ∼6,000 expression vectors.

Title: Variations in Stress Sensitivity and Genomic Expression in Diverse S. cerevisiae Isolates Summary: Much attention has been given to the ways in which organisms evolve new phenotypes and the influence of the environment on this process. A major focus of study is defining the genetic basis for phenotypes important for organismal fitness. As a first step toward this goal, we surveyed phenotypic variation in diverse yeast strains collected from different environments by characterizing variations in stress resistance and genomic expression. We uncovered many phenotypic differences across yeast strains, both in stress tolerance and gene expression. The similarities and differences of the strains analyzed uncovered phenotypes shared by strains that live in similar environments, suggesting common features of yeast niches as well as mechanisms that different strains use to thrive in those conditions. We provide evidence that some characteristics of strains isolated from oak tree soil have been selected for, perhaps because of the shared selective pressures imposed by their environment. One theme emerging from our studies is that the laboratory strain of yeast, long used as a model for yeast physiology and basic biology, is aberrant compared to all other strains. This result raises caution about making general conclusions about yeast biology based on a single strain with a specific genetic makeup.

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Summarize: DAVID J. PHILLIP/AP Judge Susan Criss, who presided over Robert Durst’s 2003 trial, said she’s convinced he left a dead cat’s head on her doorstep. Courtesy of HBO Durst with his first wife, Kathie. It has long been suspected he was involved in her disappearance. Previous Next Enlarge A Texas judge thinks there's probable claws to believe Robert Durst left a cat’s head on her doorstep. Judge Susan Criss said she found the head three years after Durst was acquitted of killing neighbor Morris Black in Galveston, Tex. — even after admitting at trial that he chopped him up. “It was June 29, 2006,” she told the Daily News. “I came home from work about 5 p.m., and you can imagine how hot it was. I saw something on the walk that leads straight to my front door.” Criss said she “thought it was a rat at first.” “But it was a little gray cat that had been cut behind the shoulder,” she said. “It was a head with its two front legs, cut right behind the shoulders. It was not just thrown there. It was placed there.” Criss described the cut a “surgical, done somewhere else.” “I believe it was Robert Durst,” she said. “I knew this guy had a history of cutting up his dogs.” Criss, who presided of the creepy multimillionaire’s 2003 trial, was referring to reports that Durst butchered seven of his pet Alaskan malamutes — all named Igor. Gerald Herbert/AP Robert Durst is transported to prison in New Orleans on Tuesday. Courtesy of HBO Durst seen with Susan Berman, who Robert Durstis accused of killing in 2000. MIKE SEGAR/REUTERS Real estate heir Robert Durst appears in a criminal courtroom in December. Previous Next Enlarge “My fear was, 'Oh my God, did he do anything to my dogs?'” she recalled. “My heart was beating so fast. I rushed inside and found the dogs were fine, thank God. But after that, I was afraid to leave my home. I was pretty shaken up for a good while.” Criss said Galveston police “came out and did a necropsy.” “They sent (the cat head) off to see if there was any DNA attached,” she said. “They didn't find anything.” The judge she believes Durst sent her the head as payback for her testifying against him at a parole hearing. The 71-year-old scion of a Manhattan real estate empire is now in a New Orleans jail and charged with the 2000 killing of his old friend Susan Berman. Durst has also long been suspected in the disappearance of his first wife, Kathie Durst. His lawyers deny Durst killed anyone. [email protected] Judge in Robert Durst's Previous Murder Case: 'I Hope the Court Doesn't Drop the Ball This Time' AP Judge Susan Criss was sitting on the bench when Robert Durst was acquitted of the brutal murder and dismemberment of his Galveston, Texas, neighbor, Morris Black, in 2003.The jury's decision that day has haunted Criss for the past 12 years – and she continues to blame what she believes were mistakes made by the court for contributing to the eccentric millionaire's acquittal.Criss tells PEOPLE she's "very relieved" about Durst's arrest in a New Orleans hotel on Saturday, adding, "I hope this helps bring results and closure to the courts that have been waiting for it for three decades."Durst, 71, was charged Monday with first-degree murder in the December 2000 killing of Susan Berman in Los Angeles, and, prosecutors say, he could face the death penalty if found guilty.Despite Criss's sense of cautious relief, no one knows better than the former judge how easily a high-profile murder case can fall apart – no matter how solid the evidence against the accused may appear to be."I hope that everyone's got their act together and nobody drops the ball this time," Criss says bluntly, adding, "There's a lot riding on this."Not just for the killing of Morris Black, but for the two other people Criss believes he killed, as well.Durst first avoided arrest in the disappearance of his wife Kathleen in 1982. Then he escaped charges in the shooting of his longtime friend Berman. And finally, Durst avoided a murder conviction in the death of Black, whom he admitted to killing and dismembering in what was ruled self-defense."He doesn't seem to fit into any sort of stereotype that we have about a serial killer," Criss says of the alleged murderer, adding, "He seems to have genuinely cared about the people he killed."Her only explanation for Durst's bizarre personality – which was chronicled in the just-conculded HBO's documentary series The Jinx – is that he "began to thrive on the publicity.""He's watching television also and he's reading what's in the media, and I think he was getting ready to run because of all the speculation of an arrest coming up," she says.Criss also believes that Durst "really has enjoyed tormenting his family. He hates his brothers. He resents them so much, and he has forever, and they've been estranged from him. I think he enjoyed tormenting them."That hatred stems, at least in part, from legal battles over the family fortune, which Forbes most recently listed at more than $4 billion. In 1994, family patriarch Seymour Durst appointed Robert's younger brother, Douglas, as heir to the family business. Feeling slighted, Robert left the company and later sued his family in an effort to gain a larger piece of the fortune. While the brothers eventually reached a business settlement (Seymour Durst died in 1995), their personal relationship continued to deteriorate."I think it's so fun to him to aggravate them and worry them and stress them out that he thought he could avoid arrest doing this," Criss speculates.But now Criss wants to make sure the court hearing the Berman case doesn't repeat old mistakes. "No matter how sure you think your case is, work it as hard as you can to make sure you've done everything possible, because if there's a mistake made, his lawyer's going to be good enough to catch it," she says."At our trial, they had so much that was said and the state never challenged it," Criss admits, "the state didn't do everything they could do, they thought they had a leg up and came in without preparing."She adds, "I don’t care if you're the richest person in the world or the poorest person in the world, if you've got a murder case – whether you're the prosecution or the defense – you prepare! You do your job as well as you can and you cover all your bases. No matter if you think it's a strong case or weak case, you give it your all."Durst will be returning for trial in L.A. after waiving his right to fight extradition from New Orleans, where he is being held without bail, although it is being reported that there may be a delay in New Orleans as Durst may first face potential weapons-possession charges.Says Criss, "I'm glad he's locked up, and I hope he stays there."

Summary: Eccentric heir Robert Durst is behind bars, and the judge who oversaw his 2003 murder trial says she hopes he stays there. Texas Judge Susan Criss tells the New York Daily News that she believes Durst, holding a grudge after she testified against him at a parole hearing, was the person who placed a cat's severed head on her doorstep three years after the trial. "It was a head with its two front legs, cut right behind the shoulders," she tells the Daily News. Police couldn't find any human DNA on the head, but the judge says she's sure it was Durst, who admitted dismembering neighbor Morris Black in the 2003 case and also allegedly butchered seven pet dogs. In 2003, the jury decided Durst had killed Black in self-defense, and Criss blames the acquittal on state prosecutors failing to do their homework. With Durst now facing a murder charge in California, "I hope that everyone's got their act together and nobody drops the ball this time," the judge tells People. "There's a lot riding on this." She tells People that Durst's bizarre behavior may come from a desire for publicity, and to torment estranged family members. "He doesn't seem to fit into any sort of stereotype that we have about a serial killer," she says. "He seems to have genuinely cared about the people he killed."

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Summarize: This application is a Continuation of application Ser. No. 07/843,595, filed Feb. 28, 1992 now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/373,264, filed Jun. 27, 1989 U.S. Pat. No. 5,158,071 which is incorporated by reference. BACKGROUND OF THE INVENTION The present invention relates to an ultrasound therapeutic system suitable for therapy for malignant tumors, therapy for calculi, etc. It has been proposed by U.S. Pat. No. 4,821,730 to add aiming and monitoring function by ultrasound to a lithotriptor with ultrasound or shockwave, which makes more suitable treatment possible. According to this U.S. Patent, an ultrasonic probe, which is relatively small with respect to a transmission aperture for ultrasound or shockwave, is integrated therein to effect aiming and monitoring by ultrasound. Since acoustic wave is used both for producing therapeutic effects and for aiming and monitoring, this system has advantages in principle that it has a self-matching property and that it is possible to suppress aiming errors due to influences of refraction, etc. in a living body to a low level. SUMMARY OF THE INVENTION The technique described above has made therapy by focused ultrasound wave with small aiming errors possible. However, in the case where it is considered from the view point of the aiming and the monitoring, one cannot help but say that only the technique described above is insufficient. That is, in the case of lithotomic therapy, since it is possible to judge whether shockwave hits a calculus or not by monitoring on an ultrasound image that the calculus subjected to the shockwave is moved by radiation force, it is possible to judge whether a target position is correctly irradiated or not. However, in the case of therapy for malignant tumors, since differences in acoustic impedance between the tumors and surrounding tissue are slight, it is difficult to monitor on the ultrasound image movements of a part to be treated by radiation force. In addition, in the case of therapy for malignant tumors, the therapy is effected by producing cavitations at the part to be treated by the shockwave. However, from the view point of preventing side effect on the surrounding tissue, it is important to monitor and suppress the cavitations produced at parts other than the target part to be treated. This is extremely difficult only by the technique described above, because differences in acoustic impedance among the target part to be treated, the surrounding tissue and the cavitations are slight. The object of the present invention is to solve such a problem and to realize a system capable of monitoring precisely that focused ultrasound for therapy hits the target part to be treated. In this way therapy by focused ultrasound producing scarcely side effect at parts other than the target part to be treated can be realized. In order to achieve the above object according to the present invention, a monitoring ultrasound transmitter/receiver having a directivity, by which scanning is possible, is added for aiming and monitoring to a focused ultrasound generating transmitter, as described in the U.S. Pat. No. 4,821,730 described above. The monitoring ultrasound transmitter/receiver is so constructed that not only it receives echo of pulsed ultrasound transmitted by itself, but also it can receive even order harmonic wave signals, which ultrasound transmitted by the focused ultrasound generating transmitter produces at the part to be treated. Further, according to the present invention, disposing a plurality of groups of monitoring ultrasound transmitters/receivers at different positions, it is possible to monitor continuously a plurality of two dimensional pulse echo images by receiving echos of the pulsed ultrasound transmitted by themselves, and a plurality of images representing orientation and intensity in which even order harmonic wave signals, which ultrasounds transmitted by the focused ultrasound generating transmitter described above produce at the part to be treated, are received by the plurality of groups of monitoring ultrasound transmitters/receivers, by constructing the system so as to display them superimposed on each other. In addition, according to the present invention, disposing only one monitoring ultrasound transmitter/receiver, sampling-like monitoring utilizing interruption is possible by constructing the system so as to display a pulse echo image obtained by receiving echos of the pulsed ultrasound transmitted by itself, and an image representing orientation and intensity, in which interruptions of even order harmonic wave signals produced at the part to be treated, by interrupting generation of the focused ultrasound in the focused ultrasound generating transmitter, superimposed on each other. For example, as described in Ser. No. 07/373,264 (filed Jun. 27, 1989), which is the parent application now U.S. Pat. No. 5,158,071 of the present application, sounds of even order harmonics of the focused ultrasound are produced, in many cases, from the region where calculi are destructed or from the region where destruction of tumor tissue accompanied by collapse of cavitations takes place. According to the invention of the present application these even order harmonics are received by the focused ultrasound generating transmitter to be utilized. That is, not only aiming, but also continuous or sampling-like monitoring of the irradiated position during irradiation with ultrasound can be effected owing to imaging by means of the monitoring ultrasound transmitter/receiver having a directivity, by which scanning is possible, and to imaging by receiving even order harmonics produced by irradiation with the focused ultrasound by means of the focused ultrasound generating transmitter. BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a cross-sectional view of an applicator, which is an embodiment of the present invention; FIG. 2 is a diagram showing the circuit construction of a whole therapeutic system representing a first embodiment of the present invention; FIG. 3 is a block diagram showing the circuit construction of a whole therapeutic system representing a second embodiment of the present invention; FIG. 4(a)-4(f) are waveform diagrams representing timing relation at focused ultrasound transmission in the second embodiment of the present invention; and FIG. 5 is a circuit diagram showing an example of a variable resonance circuit used in the second embodiment of the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinbelow the present invention will be explained, referring to two embodiments. FIGS. 1 and 2 are a cross-sectional view of an applicator and a diagram showing the circuit construction of a whole therapeutic system representing a first embodiment of the present invention. A focused ultrasound generating transmitter used for the applicator consists of a base plate 1 made of light metal serving as an acoustic matching material, a ground electrode and a heat sink in common, to one surface of which a number of piezoelectric elements 2 made of piezoelectric ceramics (these are so selected that the resonance frequency is 750 kHz) are adhered. Further, on the other surface of the light metal base plate 1, a water bag 3 filled with degassed water is mounted as an acoustical coupler for the body 4 of a patient to be treated. A concave spherical curvature is given to the light metal base plate 1 in order to make it possible to focus acoustic wave on an affected part to be treated and to move the focal point within a region to be treated by using a necessary number of transducer elements as small as possible. On the applicator two monitoring ultrasound transmitters/receivers 5-1 and 5-2 are mounted, in order to make aiming with focused ultrasound on the affected part to be treated and continuous monitoring during 20 irradiation with the focused ultrasound by means of an ultrasound image possible. Each of the monitoring ultrasound transmitters/receivers is composed of an array-shaped ultrasound transducer consisting of e.g. 100 piezoelectric elements 2&#39;, by means of which ultrasonic imaging by the electronic scanning pulse echographic method is possible. Toothed wheels 6-1 and 6-2 are mounted over the monitoring ultrasound transmitters/receivers 5-1 and 5-2, respectively, and they are so constructed that they can be rotated through toothed wheels 8-1 and 8-2 mounted on the extremities of rotating motors 7-1 and 7-2, respectively. The rotational angles thereof are detected by rotary encoders 9-1 and 9-2, respectively. Linear motors 10-1 and 10-2 are mounted on the whole monitoring ultrasound transmitters/receivers 5-1 and 5-2, respectively, so that they can be moved up- and down-wards. Amounts of the up and down displacement thereof are detected by linear encoders 11-1 and 11-2, respectively. The whole applicator is mounted on a holding mechanism (not shown in the figure) disposed outside through an arm 12. The applicator described above, which is the first embodiment of the present invention, is controlled by a circuit of the whole therapeutic system indicated in FIG. 2, as described below. The rotation and the translation of the monitoring ultrasound transmitters/receivers 5-1 and 5-2 are effected by driving the rotating motors 7-1 and 7-2 and the linear motors 10-1 and 10-2 by means of control circuits 16-1 and 16-2 connected to a main control circuit 20 through operating signals, which an operator gives to the main control circuit 20. The rotational angles and the amounts of up- and down-ward displacement are detected and fed back to the main control circuit 20. Monitoring ultrasound is emitted by the monitoring ultrasound transmitters/receivers 5-1 and 5-2 controlled by an ultrasound pulse transmission control circuit 25 triggered by the main control circuit 20 through an ultrasound transmission/reception amplifier 24. Reflected waves of the transmitted monitoring ultrasound are received by the monitoring ultrasound transmitters/receivers 5-1 and 5-2 and converted into imaged ultrasound pulse echographic images by a received wave beam scanning and focus circuit 26 triggered by the main control circuit 20 through the ultrasound transmission/reception amplifier 24. The ultrasound pulse echographic images by the monitoring ultrasound transmitters/receivers 5-1 and 5-2 are once recorded in image memories 27-1 and 27-2 and displayed on a displaying screen of a display device 28 as two-dimensional tomographic images 31-1 and 31-2, respectively, as indicated by hatching lines having different directions, superimposed on each other, as shown in the figure. Since transmission/reception of the monitoring ultrasound, conversion itself thereof into the ultrasound pulse echographic images and display thereof on the displaying device are well-known, explanation thereof will be omitted. Further, when the monitoring ultrasound transmitters/receivers 5-1 and 5-2 are rotated, while transmitting the monitoring ultrasounds, it is possible to form three-dimensional images within conical regions determined by respective directive beam scanning angles. In a region where these two conical regions are superposed on each other, a three-dimensional image doubly superimposed is obtained. In the case where therapy using this applicator is effected, a doctor moves the applicator up to a position suitable for therapy for an affected part, while observing the displayed image, and fixes it there. Thereafter he gives necessary operation signals thereto through the main control circuit 20, aims at the affected part with the focal point of the focused ultrasounds, and transmits the focused ultrasounds on a trial or experimental basic by means of a drive signal producing circuit 21, an element drive signal generating circuit 22 and a drive amplifier 23. At this time, in the neighborhood of the focal point of the focused ultrasound harmonics and in particular even order harmonics are produced by non-linear effects. This harmonic signal passes through the same path and it is subjected to the same processing as the ultrasonic pulse echo signal. Then it is displayed, superimposed on the pulse echographic image. That is, it is received by the transmitters/receivers 5-1 and 52, passes through the ultrasound transmission/reception amplifier 24 and the received wave beam scanning and focus circuit 26, is recorded once in the image memories 27-1 and 27-2, and is displayed on the display screen 30 as linear line band-shaped images 32-1 and 32-2, respectively, superimposed on each other. It is because transmission of the focused ultrasounds is effected not in a pulsed shape but continuously and generation of harmonics by them is also continuous that they are displayed as liner band-shaped images. By an imaging system using a single monitoring ultrasound transmitter/receiver it is impossible to detect the position in the depth direction of the generation of the harmonics. On the contrary, in the first embodiment, since it is provided with an imaging system using two monitoring ultrasound transmitters/receivers, the generation point of the harmonics, i.e. the focal point of the focused ultrasounds, is displayed and detected as a position, where the two band-shaped images 32-1 and 32-2 intersect each other in the two-dimensional tomographic image. The doctor, who effects the therapy by means of this system, carries out the main irradiation with the focused ultrasound, after having confirmed that the position, where harmonics are generated, is in accordance with the affected part. In the case where the position, where harmonics are generated, is deviated from the affected part, the operation signal given through the main control circuit 20 is corrected and the irradiation is carried out again, after having corrected the focal point of the focused ultrasound in an electronic manner by means of the drive signal circuit 21. Further, in the case where it is judged that the degree of the generation of the harmonics is not appropriate, too small or too great, the magnitude of the output of the drive signal producing circuit 21 acting as the focused ultrasound intensity signal is displayed on the display screen 30 and the irradiation is carried out again, after having corrected the operation signal, referring thereto. Since the focal point of the focused ultrasound thus obtained is displayed continuously during the transmission of the focused ultrasound, the doctor can take a measure to interrupt the transmission of the focused ultrasound as soon as the focal point of the focused ultrasound is deviated or becomes otherwise inappropriate, which makes a safer therapy possible. Here, although it is well known how to determine the focal point of the focused ultrasound, it will be briefly explained. That is, denoting the center of each of the piezoelectric elements constituting the array type focused ultrasound generator by Pn (n=1, - - -, N) (here N being e.g. 100), the target focal point by F; the length of a segment PnF by dn; an approximate value of the sound velocity in a living body by c; and the frequency of the focused ultrasound by f, the ultrasound can be focused at the target focal point F by driving the different piezoelectric elements by using a drive signal having a phase φn given by a following equation at a point of time t: ##EQU1## Here the difference between the sound velocity in the water bag and the sound speed in the living body as well as the sound speed distribution in the living body are neglected. A part or a major part of deviations of the focal point F due to the refractive effect of sonic wave produced by the fact that these are not zero can be corrected selfconsistently by effecting determination of the phase at the transmission and the reception of the monitoring ultrasound in the same way as described above. That is, there is essentially no problem, because errors are produced in a similar manner for the irradiation for therapy and for the transmission and the reception for monitoring. Calculations for the drive phase φn as described above are carried out in the drive signal producing circuit 21. A result thus obtained is sent to the element drive signal generating circuit 22 and the different piezoelectric elements are driven by the drive amplifier 23. The electronic focal point scanning with the focused ultrasound is realized in this way. Consequently, when it is desired to vary the depth of the focal point, it is sufficient e.g. to give operation signals for varying dn in the above equation. In the present embodiment the system was so constructed that the two transmitters/receivers were disposed inside of the water bag. This is a construction efficient for detecting cavitations produced inside of the water bag at the same time. However, in the case where it is aimed to detect exclusively cavitations produced within the body of the patient to be treated, it is desirable to adopt a construction, in which the transmitters/receivers are placed outside of the water bag. Further, in this case, there may be another method, by which any water bag, which is apt to be a cavitation generating source, is not used at all. Furthermore, in order to display an image obtained by a plurality of ultrasound transmitters/receivers 5 so as to be more easily observed, it is preferable to use a color display and to make different color tones correspond to the image signals from the different ultrasound transmitters/receivers. Next the second embodiment of the present invention will be explained. The applicator in this embodiment includes only one imaging system using a monitoring ultrasound transmitter/receiver. An example of the construction is essentially identical to that indicated in FIG. 1 and it is omitted to show it in the figure. That is, between the monitoring ultrasound transmitters/receivers 5-1 and 5-2 in the applicator indicated in FIG. 1 the parts relating to that indicated by 5-2 are removed and that indicated by 5-1 is located at the central portion of the focused ultrasound generating transmitter in the applicator. Also in the present embodiment, the focused ultrasound generating transmitter consists similarly of a base plate 1 made of light metal serving as an acoustic matching material, a ground electrode and a heat sink in common, to one surface of which a number of piezoelectric elements 2 made of piezoelectric ceramics (these are so selected that the resonance frequency is 750 kHz) are adhered. FIG. 3 is a block diagram showing the circuit construction of a whole therapeutic system representing a second embodiment. In FIG. 3, the parts having same functions as those shown in FIG. 2 are indicated by the same reference numerals. As clearly seen by comparing the two, in the second embodiment there is only one imaging system by the monitoring ultrasound transmitter/receiver. Therefore the imaging system consisting of the ultrasound pulse transmission control circuit 25 triggered by the main control circuit 20; the monitoring ultrasound transmitter/receiver 5 transmitting monitoring ultrasound through the ultrasound transmission/reception amplifier 24 by means thereof; the received wave beam scanning and focus circuit 26, which receives reflected waves of the transmitted monitoring ultrasound by means of the monitoring ultrasound transmitter/receiver 5, and converts them into an ultrasound pulse echographic image, triggered by the ultrasound transmission/reception amplifier 24 and the main control circuit 20, can be simplified. Similarly to the first embodiment, an image obtained by receiving reflected waves of the monitoring ultrasound is displayed on the screen 30 of the display device 28, as indicated by the hatching 31. On the other hand, necessary operation signals are given through the main control circuit 20 to aim at the affected part with the focal point of the focused ultrasound and a variable resonance circuit 14 is added to the focused ultrasound irradiation system consisting of piezoelectric elements 2 emitting focused ultrasound, triggered by the drive signal producing circuit 21, the element drive signal generating circuit 22 and the drive amplifier 23. As described later,these are disposed for transmitting the focused ultrasound in a pulsed shape; receiving even order harmonics generated in the neighborhood of the focal point of the focused ultrasound by the monitoring ultrasound transmitter/receiver 5; displaying an image thus obtained, superimposed on the pulse echographic image; and detecting the position of the focal point in the depth direction. FIG. 4 is a diagram indicating waveform and timing at the focused ultrasound transmission in the second embodiment. As indicated in the timing chart shown by (a) in the figure, imaging and aiming by the pulse-shaped ultrasound are effected during a period of time from a point of time t1 to another point of time t2. Thereafter the focused ultrasound is transmitted continuously from a point of time t3 to another point of time t4. Then, thereafter, imaging and aiming by the pulse-shaped ultrasound are effected again from a-point of time t5 to another point of time t6. Then, thereafter, continuous transmission of the focused ultrasound is started again at a point of time t7. Subsequently the same process is repeated for a required irradiation period of time. That is, before the continuous transmission of the focused ultrasound, the focal point of the focused ultrasound in the depth direction is confirmed in the two-dimensional tomographic image by transmitting and receiving the pulse-shaped focused ultrasound. When the focal point is inappropriate, the focal point of the focused ultrasound is confirmed again by transmitting and receiving the pulse-shaped focused ultrasound after having given operation signals for adjusting the focal point to recalculate the drive phase by means of the drive signal producing circuit 21 in order to correct the focal point. After the start of the continuous transmission, the continuous transmission is once interrupted after having been continued the continuous transmission for a predetermined period of time, in order to confirm the focal point of the focused ultrasound by transmitting and receiving the pulse-shaped focused ultrasound. When it is confirmed at thus stage that the focal point is inappropriate, similarly to the start of the transmission, the focal point of the focused ultrasound is confirmed again by transmitting and receiving the pulse-shaped focused ultrasound, after having given operation signals for adjusting the focal point to correct it. The imaging by transmitting and receiving the pulse-shaped ultrasound as described above is performed, based on a principle basically identical to the imaging principle of the pulse echo method widely used for ultrasound therapeutic systems. However, it is characterized in that the system is so constructed that the pulse-shaped ultrasound is transmitted also from the focused ultrasound generator in synchronism with the transmission of the pulse-shaped ultrasound similar to that transmitted by an ultrasound therapeutic system where it is transmitted from the monitoring ultrasound transmitter/receiver 5, in order to make the aiming with the focused ultrasound possible. This aspect will be explained in detail, referring to FIGS. 4(a)-4(f). The timing charts indicated by FIGS. 4(d)-(f) represent transmission timing of the pulse-shaped ultrasound from the focused ultrasound generator and the monitoring ultrasound transmitter/receiver, respectively, and 4(d) and 4(f) represent transmission waveforms (acoustic pressures) in respective timing enlarged in the time scale. Since the transmitting fronts of the focused ultrasound generator and the monitoring ultrasound transmitter/receiver are, in general, not coplanar, as indicated in FIG. 1, the pulse-shaped ultrasound transmitted by the focused ultrasound generator passes through the position corresponding to the transmitting front of the monitoring ultrasound transmitter/receiver with a predetermined delay time td, as indicated by FIG. 4(e). Since this delay td can be determined, based on the construction of the system, the imaging pulse-shaped ultrasound of relatively high frequency is transmitted by the monitoring ultrasound transmitter/receiver, taking this timing into account, as indicated by FIG.(f). Here, as clearly seen from comparing 4(e) and 4(f), it is desirable that the acoustic pressures of the pulse-shaped ultrasound transmitted by the focused ultrasound generator and the imaging pulse-shaped ultrasound transmitted by the monitoring ultrasound transmitter/receiver are greatest with a same timing. In the case where the transmission/reception direction of the imaging pulse-shaped ultrasound is in accordance with the direction of the focal point of the focused ultrasound, the transmitted pulse-shaped focused ultrasound and the imaging pulse-shaped ultrasound arrive almost simultaneously at the neighborhood of the focal point of the focused acoustic wave and a part of energy of the pulse-shaped ultrasound, which has been focused to have a high intensity, is converted into harmonics in the same frequency band as the imaging ultrasound by a non-linear phenomenon. These harmonics have the highest intensity in the proximity of the focal point of the focused ultrasound and they are scattered by scatterers in the neighborhood of the focal point of the focused ultrasound approximately at the same time as the imaging pulse-shaped ultrasound. Scattered ultrasounds are received further similarly almost simultaneously by the monitoring ultrasound transmitter/receiver 5 and inputted to the received wave beam scanning and focus circuit 26, where they are treated similarly to the imaging pulse echo signal. Consequently a received signal is obtained, in which a signal due to the harmonics of the pulse-shaped focused ultrasound is superimposed on the usual imaging pulse echo signal due to the transmission/reception by the monitoring ultrasound transmitter/receiver at the position corresponding to the focal length of the focused ultrasound. In this way, an image indicating the position, where the harmonics are generated by the focused ultrasound, is obtained in a state, where it is superimposed on the pulse echographic image 31, as indicated in FIG. 3. A line 41 in FIG. 3 indicates the position of the rotational axis of the monitoring ultrasound transmitter/receiver 5. Since it can be easily realized by giving a signal corresponding to the position thereof to construct it on an imaging screen, it is not specifically explained. Now, when the scanning plane by the transmission/reception of the imaging pulse-shaped ultrasound doesn&#39;t include the position, where the harmonics are generated by the focused ultrasound, the harmonics of the pulse-shaped focused ultrasound generated in the neighborhood of the focal point of the focused ultrasound don&#39;t appear on the imaging screen. At this time, the operator may vary the position of the rotational axis of the monitoring ultrasound transmitter/receiver or rotate the last so that the harmonics of the pulse-shaped focused ultrasound appear on the imaging screen, to know the correct position of the irradiation. Further the image accompanied by the generation of the harmonics by the focused ultrasound is in a band-shaped, as indicated by 32-1 or 32-2 in FIG. 2, during the continuous transmission of the focused ultrasound and it is not possible to know the position, where the harmonics are generated, in the depth direction. In the present second embodiment, it is possible to know the position, where the harmonics are generated by the focused ultrasound, in the depth direction by making the transmission of the focused ultrasound, which should be transmitted originally continuously, in a pulse shape in a sampling-like manner. For this reason it is necessary to make the transmission of the focused ultrasound by the piezoelectric elements 2 either in a pulse shape or continuous and the device therefor is the addition of the variable resonance circuit 14. FIG. 5 is a partial diagram, in which only the part of the variable resonance circuit 14 is taken out from the circuit construction of the whole therapeutic system shown in FIG. 3 to be indicated. The variable resonance circuit 14 consists of a resonance circuit composed of an inductance 50 and a capacitance 51, a short-circuiting circuit 55 bypassing this resonance circuit, and switches 52 and 53 for selecting them. The switches 52 and 53 are changed over by a signal from the drive signal calculating circuit 21. That is, the switches are connected on the terminal aside at the transmission of the pulse-shaped focused ultrasound for a time period indicated by t1-t2 or t5-t6 to exclude the resonance circuit, attaching importance to pulse characteristics. The switches are connected on the terminal b side at the continuous transmission of the focused ultrasound to include the resonance circuit 50, 51, attaching importance to the efficiency. Consequently the drive of the piezoelectric elements 2 is effected with characteristics suitable for either one of the cases, transmission of the pulsed focused ultrasound and continuous transmission of the focused ultrasound. As described above, according to the present invention, it is possible to realize a therapeutic system, by which it can be easily confirmed on an image that the focused ultrasound is correctly focused on an affected part and as the result a safer therapy can be made possible.

Summary: In an ultrasound therapeutic system provided with an ultrasound transmitter having a focusing mechanism and a plurality of groups of ultrasound transmitters/receivers, each of which has a controllable directivity, each of the transmitters/receivers is constructed so as to be able to receive both echo of pulse-shaped ultrasound transmitted by itself and even order harmonic signals of the ultrasound transmitted by the transmitter, and a plurality of two-dimensional pulse echographical images constructed by ultrasound signals obtained by transmitting/receiving beams, while controlling the directivity of the beam emitted by each of the plurality of groups of ultrasound transmitters/receivers and a plurality of images indicating orientation and intensity, in which an even order harmonic wave signal due to the ultrasound transmitted by the transmitter is received by each of the plurality of groups of ultrasound transmitters/receivers, are displayed, superimposed on each other.

big_patent

en

Summarize: FIELD OF THE INVENTION [0001] The present invention is directed to the implantation of intracorporal leads, such as leads for cardiac sensing/pacing that are usually associated with “active implantable medical devices” as such devices are defined by the Jun. 20, 1990 directive of the Counsel of European Communities, and more specifically to intracorporal leads for implantable devices for heart pacing, resynchronization, cardioversion and/or defibrillation. BACKGROUND OF THE INVENTION [0002] Cardiac leads may be endocardial leads, such as leads for sensing/pacing in right heart cavities, or leads introduced in the coronary network, notably leads comprising an electrode positioned in front of a left cavity of the myocardium. [0003] The insertion of the latter type of leads, carried out through endocavitary approaches, is a particularly tricky intervention, taking into account the difficult access to the coronary sinus entrance via the right atrium, and also the required accuracy for the pacing sites once the lead is guided to its desired location and immobilized within the coronary network. [0004] One of the implantation techniques of such a lead requires an accessory known as “guide-catheter”. This accessory comprises a hollow tubular sheath reinforced by a wire mesh and with an inner surface presenting a low coefficient of friction (for example, a surface made with PTFE, extruded or co-molded with the rest of the sheath). In addition, the sheath is designed to present a flexibility allowing a stiffness in torsion high enough to allow transmission of a rotational movement applied at one end to the other end, so as to allow guiding the lead tip within the myocardium during the procedure. [0005] Once in place, the guide-catheter serves as a direct “tunnel” between the “external world” and the coronary sinus, a tunnel that can be utilized by the surgeon for sliding the lead therethrough to its final target site. [0006] Once the lead is in place, the guide-catheter needs to be extracted. The extraction procedure is tricky because the lead must not be displaced, or its position or orientation altered as a result of the extraction. [0007] One other difficulty of this extraction step is due to the presence of the electrical connector, at the proximal end of the lead: the diameter of this connector being greater than that of the internal lumen of the guide-catheter, it prevents the guide-catheter from being withdrawn by being simply slid backwardly along the lead. [0008] The step of extracting the guide-catheter therefore usually requires it to be cut, starting from its proximal end and along a generatrix line by means of a slitting tool, also known as “slitter,” for slitting the proximal end of the catheter and reinforcement wire mesh forming the frame of the hollow sheath. [0009] With one hand, the surgeon then pulls the guide-catheter towards him with a continuous gesture, while firmly maintaining the lead and the slitting tool with the other hand, allowing the slitting tool to simultaneously slit the sheath as it is being thus extracted. [0010] It has already been proposed in the prior art, in order to avoid resorting to a sharp cutting tool, to use a non-reinforced sheath that is simply strippable; however, cutting of such a sheath along its whole length leads to a weakness and a lower rigidity of the guide-catheter, with a risk of folding and lower transmission of efforts (forces) during its setting up preparatory to an intervention event. It has also been proposed, notably by European patent EP 1,155,710 and its U.S. counterpart U.S. Pat. No. 6,625,496 (commonly assigned herewith to ELA Medical), to provide the lead with a removable connector, which avoids having to cut the guide-catheter. This method however leads to an increasing number of steps required for setting up and assembling the different elements, and also renders the intervention procedure more complicated. [0011] Therefore slitting/cutting of the guide-catheter is, in practice, the most usual way to proceed. [0012] One additional difficulty of this type of intervention comes from the presence, at the proximal end of the guide-catheter, of a hemostatic valve allowing to obdurate at will the internal lumen emerging from the proximal end, opened, of the guide-catheter. [0013] This valve is usually provided with a lateral way (i.e., a passageway) able to communicate with the inner lumen of the guide-catheter, so as to allow purging the guide-catheter after its setting in place, and also eventually injecting therein an antithrombotic agent or radio contrast medium. [0014] This hemostatic valve must meet several requirements, adding to the difficulties explained above. Firstly, the hemostatic valve must be able to allow the lead to pass through it, in order to make the lead penetrate into the guide-catheter then guide it up to its final position. Secondly, once the lead has been set in place, the valve has to be able to be dissociated from the guide-catheter, notably in order to allow the surgeon to initiate the cutting of the guide-catheter sheath so as to extract the latter. [0015] Various configurations of guide-catheter/hemostatic valve sets have been already proposed in the prior art. [0016] In U.S. Pat. No. 6,159,198 (Gardeski), the valve is a removable valve, fit into a coupling bell or “hub” through a Luer Lock type assembly, the hub being formed at the proximal end of the guide-catheter. Closing of the valve is ensured by a screwing mechanism located in the rear area of the valve and allowing to create at will a passage to the inner lumen in order to allow lead insertion. In order to allow cutting of the guide-catheter at the end of the intervention after removal of the valve, the coupling bell is also divisible (cuttable) following a thinner part formed along a generatrix and continued by a transition toward the reinforced sheath. [0017] U.S. published patent application 2005/0,010,238 (Potter) discloses a hemostatic valve mounted at the tip of a guide-catheter on a coupling bell by means of a clipped assembly system. Differently from the Gardeski device, the coupling bell is not divisible (i.e., a bladed tool is required for cutting it), but frangible (i.e. it can be broken into two parts through a force exerted by the fingers of the surgeon, without the need for any additional tool). Once the coupling bell has been thus removed, the proximal end of the guide-catheter is directly accessible, allowing the cutting thereof by means of a usual method using a cutting tool. [0018] U.S. published patent application 2004/0,176,781 (Lindstrom) discloses a toolkit for implanting a lead for a guide-catheter that is not reinforced, but simply strippable, and therefore does not require any cutting tool for its extraction. However, the procedure is rendered more complicated due to the need for resorting to an additional accessory known as TVI or dilator, in order to open the filling element of the valve and protect the lead during the passing through thereof. The dilator also allows increasing the rigidity of the strippable sheath, but of course it also has to be extracted after being used, which requires an additional step in the procedure, the dilator having to also be provided with a strippable structure. [0019] U.S. Pat. No. 6,966,996 (Kurth) describes a toolkit for implanting a lead wherein the valve itself, instead of the coupling bell, is frangible. This allows separating the valve in two parts—the valve initially forming a single block with the guide-catheter—in order to let the proximal tip of the guide-catheter appear, and allow its cutting and extraction. [0020] U.S. published patent application 2007/0123825 describes a toolkit implementing an introducer provided with a valve which can be dissociated in two halves, which are then able to be separated in order to allow peeling the introducer (which can therefore be extracted without the need for a cutting tool). The valve further comprises a means allowing, through pinching the two winglets of the valve, to open, level with the proximal end, an axial hole giving access to the lumen of an axial tubular part that is continuing the introducer. The catheter, provided with the lead, can then be inserted into this hole of the introducer. [0021] All the various devices that have been proposed so far, however, suffer from several difficulties, particularly among them are the following: [0022] there is always an existing significant risk, during the intervention, of a lead displacement at the moment when the valve is removed and the guide-catheter extracted, especially with those leads that are provided with an electrical connector having a large diameter, as all the accessories that are part of the introducing toolkit have to be removed over this connector; [0023] the valves that are driven by a screw system require that the surgeon has a very accurate dexterity in order to prevent from damaging the lead by squeezing it too much and, reversely, from creating a lack of tightness during the intervention, due to an insufficient squeezing; [0024] in the case of a divisible coupling bell, cutting thereof by means of a standard slitter is difficult, due to the brutal variation of the cutting force during the transition between the material of the bell and the reinforced sheath, with a risk of displacing the lead if the forces exerted by the surgeon are not well accurately balanced; [0025] due to their simplicity, the frangible systems allow an easy removal of the valve, but have the drawback of not comprising any practical mechanism for driving the valve, thus oblige resorting to a dilator for introducing the lead in the valve; this accessory is “floating around” along the lead conductor during the intervention, and anyhow will have to be removed by means of peeling at the end of the intervention; [0026] the frangible valves that are made as a single block with the guide-catheter, do not allow the surgeon to turn the lateral way around the guide-catheter; [0027] all the solutions proposed up to now require a certain number of accessories or separate elements, which renders the surgeon&#39;s operation more complicated, during the critical step of the guide-catheter extraction, when the surgeon has to be focused on maintaining the position of the lead; [0028] as the lead has to pass through all these accessories, notably the dilator, the lead electrodes may become polluted, especially by the lubricant usually utilized with the valve. OBJECTS AND SUMMARY OF THE INVENTION [0029] It is therefore an object of the present invention to palliate one or more of the aforementioned difficulties, by proposing a toolkit comprising a hemostatic valve and a combined guide-catheter, which in combination present one or more of the following advantages: [0030] a hemostatic valve of the frangible type, reducing the risk of contacting the lead and displacing it during the removal of the valve at the end of the intervention; [0031] after removal of the valve, obtaining a guide-catheter having a tip free from any element along the cutting direction of the cutting tool, which can in turn be handled with a constant effort (force) during the entire guide-catheter extraction step; [0032] a simple and effective driving (opening and closing) of the valve, for example, through a simple translation movement, without resorting to any dilator or other type of insertion tool, in order to notably allow direct and free introduction of the lead in the lumen of the guide-catheter; [0033] direct insertion of the lead in the guide-catheter, without contacting any other element, thus minimizing any risk of polluting the lead during its introduction and passing through the valve; [0034] obviating any need for an accessory (other than the cutting tool), neither for introducing the lead in the guide-catheter, nor for the removal of the valve and extraction of the guide-catheter; [0035] providing a valve to freely turn around the guide-catheter, so as to allow orienting the lateral way along any radial direction relating to the guide-catheter hold in position; [0036] “On-Off”-type driving of the valve between opened and closed positions, with no risk for a leak nor damaging the lead, in contrast to known existing systems such as screwing systems or other mechanisms requiring an accurate dexterity. [0037] Broadly, the present invention is for a toolkit of the general type as described in US 2007/0123825 cited above. One aspect of the invention is directed to a toolkit for setting in place an intracorporal lead, including: [0038] a guide-catheter, having a sheath with an internal lumen opened at its respective distal and proximal ends, and a generatrix, said sheath being able to be opened along the generatrix so as to allow removal of the guide-catheter after use thereof; [0039] a hemostatic valve able to selectively fill the internal lumen of the guide-catheter at the input end, said valve comprising an element that is mobile relative to the guide-catheter between an opened position, where the proximal end of the guide-catheter freely emerges out of the valve to provide an access to the internal lumen of the guide-catheter, and a closed position, where said proximal end of the guide-catheter is filled in a tight manner, wherein the valve is frangible, separating into at least two parts that are dissociable from each other along a median axial plane, wherein: [0040] the sheath comprises a rigidified reinforced sheath, adapted for being cut along said generatrix by means of a cutting tool having a blade; [0041] the guide-catheter presents a constant diameter in its region receiving the valve; [0042] the frangible valve is mounted at the proximal end of the guide-catheter, the proximal end being fitted between said at least two parts of the frangible valve; and [0043] said at least two parts of the frangible valve are further each dissociable from the guide-catheter, so as to be able to be separated and moved away from each other to provide free access to the guide-catheter. [0044] Further advantageous characteristics of the present invention include the following. The guide-catheter is essentially devoid of a bell for coupling with its proximal end. The valve comprises a mobile body sliding on the guide-catheter between said extreme opened and closed positions. Preferably, the valve body has a central inner cavity able to receive the proximal end of the guide-catheter, and also has at the distal side of said inner cavity, a material that provides a circumferential tight coupling to the sheath of the guide-catheter. [0045] In yet another embodiment, the valve body has, at the proximal side of the inner cavity, a closure deformable between a first non-stressed position where said closure fills said inner cavity at the proximal side, and a second distended position where the closure is penetrable to be crossed by the sheath of the guide-catheter while ensuring a circumferential tightness with the sheath. The non-stressed position corresponds to said closed position, and the distended position corresponds to said opened position. Further, the valve body preferably embeds an internal part made of an elastically deformable material, said internal part including said circumferential tight coupling at its distal side, and said deformable closure at its proximal side. [0046] The valve preferably comprises a lateral way communicating with a radial derivation of the internal cavity, and is freely mobile in axial rotation around the guide-catheter. In addition, the valve preferably comprises at least one prehension winglet spreading in an axial plane. Similarly, the guide-catheter preferably comprises at least one prehension winglet spreading in an axial plane. [0047] In one preferred embodiment, the valve comprises means for locking said valve in at least one position selected from among an opened and a closed position. Such a locking means can include cooperating fitting means respectively provided on the valve body and on an axial extension of the guide-catheter BRIEF DESCRIPTION OF THE DRAWINGS [0048] Further features, advantages and characteristics of the present invention will become apparent to a person of ordinary skill in the art in view of the following detailed description of preferred embodiments of the invention, made with reference to the drawings annexed in which like reference characters refer to like elements, and in which: [0049] FIG. 1 is a perspective view of the guide-catheter/hemostatic valve set, according to a preferred embodiment of the present invention, in the closed position; [0050] FIG. 2 is a perspective view of the guide-catheter/hemostatic valve set of FIG. 1, in the opened position; [0051] FIG. 3 shows the step of removal of the hemostatic valve after breaking thereof, and placing the cutting tool facing the guide-catheter so as to allow slitting the sheath and extraction of the guide-catheter; [0052] FIG. 4 is a section view in an axial plane, showing the details of the internal structure of the hemostatic valve in its closed position, and showing the way the tightness is ensured at different levels of this valve; [0053] FIG. 5 is a partial view, in section in an axial plane, showing the stop mechanisms of the valve in a closed position, and locking mechanisms of the valve on the guide-catheter in an opened position; and [0054] FIGS. 6 and 7 are schematics, in section in an axial plane, respectively showing the way the different elements of the guide-catheter and the valve communicate with each other, in the closed and opened positions of the valve. DETAILED DESCRIPTION OF THE INVENTION [0055] One will now describe an example embodiment of a toolkit in accordance with the present invention. With reference to the drawings, FIGS. 1 to 7 show the different aspects of an exemplary set comprising a guide-catheter and associated hemostatic valve, the valve being represented either in “closed” position ( FIGS. 1 and 7 ) or “open” position ( FIGS. 2, 4, 5 and 6 ), or after separating the valve from the guide-catheter, right after cutting thereof ( FIG. 3 ). [0056] The guide-catheter 10 comprises a hollow sheath 12 reinforced with a wire mesh in order to improve its rigidity; once the guide-catheter is in place, its proximal end (the one that is visible on the figures) is emerging from the introduction site, the surgeon using this emerging part for introducing, driving and positioning the lead at its final target site. [0057] The proximal end of the guide-catheter 10 is provided with a hemostatic valve 14 allowing to selectively fill the internal lumen of sheath 12 at its input end. [0058] In a characteristic manner of this invention, valve 14 comprises a mobile body 16 sliding on the guide-catheter 10 between two extreme positions defining the “opened” and “closed” states of the valve, the valve being driven between these two states through a very simple movement of the “push-pull” type. [0059] The body 16 of the valve 14 comprises all along its length, along two diametrically opposite generatrices, an area with a thinner thickness 18, forming a fracture initiation score and thus rendering the valve frangible, i.e., able to be broken apart in two dissociable parts at both sides around a median plane comprising the fracture initiation scores 18. [0060] In order to facilitate its driving, the valve is provided with two opposite lateral winglets 20, 22 spreading in the illustrated embodiment in an axial plane. These winglets also allow, as it will be described below, to exert on the valve an effort sufficient for breaking it into two parts, when it comes time to remove it at the end of the intervention. It should be understood that the winglets need not be in a common plane. [0061] In addition, in a manner that is already known per se, the valve is provided with a lateral way 24 able to come and communicate with the internal lumen of the guide-catheter so as to allow its purge, and eventually injecting a radio contrast medium or an antithrombotic agent. [0062] In order to facilitate its handling, the guide-catheter 10 is also provided with a prehension winglet 26 preferably spreading in an axial plane, and for instance constituting the continuation of a longitudinal reinforcement 28, integral of sheath 12 and spreading circumferentially on a part thereof (so as to allow ulterior cutting without any interference from element 28 ). [0063] It is easy for the surgeon to drive the valve by holding the winglet 26 of the guide-catheter in one hand, and in the other hand one of the winglets 20 or 22 of the valve, for example by moving these winglets away from each other (arrow 30 ) to put the valve in its “closed” position. It shall be further noticed that the valve is freely mobile in rotation around the guide-catheter 10 (arrow 32 ), which allows orienting the lateral way 24 along any possible direction providing as little as possible constraint for the surgeon. [0064] With an opposite movement (arrow 34 in FIG. 2 ), the surgeon will put the valve in its “opened” position by moving the winglets 20, 22 of the valve and winglet 26 of the guide-catheter closer to each other. The valve will be hold in this position by a lock mechanism using a neck 36 formed on the body 16 of the valve in the most distal area thereof, this neck 36 being fitting in a recess 38 formed in the winglet 26 of the guide-catheter. [0065] It should be understood that the valve as implemented in the present invention gives immediate tactile information to the practitioner as to the effective position, opened or closed, of the valve, in contrast to other existing devices such as valves with a screw element. It is further possible to add a visual marking appropriate for rendering this indication on the valve&#39;s position even more apparent. [0066] It should be understood by a person of ordinary skill in the art that, in the opened position of the valve, the emerging end 40 of sheath 12 of the guide-catheter 10 emerges from the most proximal part of body 16 of the valve, thus giving access to the internal lumen 42 of the guide-catheter (see FIG. 2 ), in order to preferably allow introducing the lead in this lumen. [0067] After placing the lead at its final position, it is required to extract the guide-catheter, no longer utilized, by taking all required precautions so as not to displace the lead during the extraction procedure. [0068] The first step consists of removing the valve to allow direct access to the end of the catheter. [0069] As disclosed above, valve 14 is a frangible valve, which can be broken in two parts 14 a, 14 b that are roughly symmetrical (see FIG. 3 ) around an axial median plane, through a bending stress exerted by the surgeon on the two opposite winglets 20, 22. The two halves 14 a, 14 b can then be separated and moved away from each other (arrows 44, 46 ). This grants access to the guide-catheter, no longer fitted between the two halves 48 a, 48 b of the valve body. [0070] The guide-catheter can then be cut, in a manner already known per se, by means of a cutting tool or “slitter” 50 comprising a blade 52 allowing to slit the reinforced sheath of the guide-catheter. The tool 50 is immobilized by a surgeon&#39;s hand, and with his/her other hand, the surgeon pulls the guide-catheter toward him/her, preferably with help from winglet 26, so as to slit the sheath 12 along a generatrix 54. This movement of cutting and extracting the guide-catheter is pursued until complete extraction thereof (arrow 56 ). [0071] FIGS. 4 to 7 are section views showing the internal configuration of the elements of valve 14, mounted on the guide-catheter 10. The valve body embeds an internal part 58 that is made of a material that is elastically deformable, for example, a silicone material co-assembled or co-molded with the valve body, which is itself made of a relatively rigid material such as polypropylene or polyamide. [0072] In its distal area 60, the elastic part 58 defines a central cavity 62 of cylindrical shape, in which the sheath 12 of guide-catheter 10 penetrates. The tightness between the elastic part 58 and sheath 12 is ensured by one or more internal circumferential reliefs 64 filling the cavity 62, and thus the internal lumen 42 of the guide-catheter, in a tight manner vis-à-vis the external environment. [0073] At its proximal end 66, the elastic part 58 is closed, i.e., the cavity 62 is a one-eyed cavity only opened toward its distal direction. The wall closing the elastic part 58 however is a penetrable closure, thanks to a piercing or a slot 68. In the free or non-stressed position, thanks to the elasticity of the material of part 58, this closure closes the cavity 62 in a tight manner, but it can be penetrated and crossed through an external stress such as by an axially driven element, that is, in the present case, the tip 40 of the sheath of the guide-catheter 10 which crosses the closure when the valve is moved (arrow 34 ) to the “opened” position. As shown in FIG. 7, the closure is then dilated and crossed by the tip 40 of the guide-catheter, which freely emerges from the valve through a hole 70 formed in the proximal area of the valve body. [0074] Also, the internal cavity 62 is provided with a radial hole 72 allowing to make this cavity 62, and therefore the internal lumen 42 of the guide-catheter 10, communicate with the lateral way 24 of the valve. [0075] With reference to FIG. 5, further details of the stop and locking mechanism of the valve on the guide-catheter are shown. [0076] Firstly, when the valve is moved toward its closed position (arrow 30 ), a skirt 74, formed at the end of reinforcement part 28 integral of the guide-catheter, bumps against a neck 76 formed at the distal tip of the valve body. In addition to acting as a stop, allowing to geometrically define the “closed” position of the valve, these cooperating elements ensure the mutual integration of the guide-catheter and the valve, while allowing a degree of freedom in axial rotation of the valve around the guide-catheter, as explained above. The guide-catheter can only be separated from the valve by breaking the valve, as described above in reference to FIG. 3, the skirt 74 of the guide-catheter being thereafter simply retained by the neck 76 of the valve. [0077] FIG. 5 also shows the elements allowing to define the “open” position of the valve and locking in that position, thanks to a neck 36 of the valve fitting in a recess 38 of the winglet 26 integral with the guide-catheter. [0078] FIGS. 4 and 6 show the respective “closed” and “opened” positions of the guide-catheter and the valve, respectively. As explained above, switching from one position to the other is achieved simply by a “push-pull” driving of the valve relative to the guide-catheter (arrows 30, 34 ). This driving is of the “On-Off” type and does not require any specific handling or balancing of efforts (driving forces), in contrast to the known screwing mechanisms used in the prior art. [0079] The easy and fast driving of the valve between the two positions further dramatically reduces the risks of air ingress (and embolism) or blood leak during the intervention, therefore enhances the safety of setting up the lead. [0080] It should be understood that in opened position (see FIG. 7 ), the proximal end 40 of the guide-catheter freely emerges from the valve body, which allows to directly insert the lead in the guide-catheter, with no risk of polluting the lead, for the guide-catheter itself acts as a dilator for opening the closure 68 of the valve. This further prevents from resorting to any specific tool, thus eliminates all the drawbacks listed associated therewith referenced above. [0081] It also should be understood that the emergence of the guide-catheter at the proximal end of the valve provides a visual indication, simple and unambiguous, of the opened position of the valve. [0082] One skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments which are presented for purposes of illustration and not of limitation.

Summary: A toolkit for implanting an intracorporal lead, preferably a cardiac sensing/pacing lead. This toolkit includes a guide-catheter ( 10 ), having a sheath ( 12 ) with an internal lumen ( 42 ) opened at its distal and proximal ends, and cuttable along a generatrix in order to allow extracting of the guide-catheter after use. A hemostatic valve ( 14 ) is mounted at the proximal end ( 40 ) of the guide-catheter, for selectively filling, or not, the internal lumen of the guide-catheter at its input end. The valve is frangible in at least two parts, each dissociable from the guide-catheter at both sides around a median axial plane. The valve comprising a mobile element sliding on the guide-catheter between two extreme positions, with an opened position where the proximal end ( 40 ) of the guide-catheter ( 10 ) freely emerges out of the valve so as to allow access to said internal lumen, and a closed position where this proximal end of the guide-catheter is filled in a tight manner.

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Summarize: NOTHING can be more uniform and undiversified than the life of the Typees; one tranquil day of ease and happiness follows another in quiet succession; and with these unsophisicated savages the history of a day is the history of a life. I will, therefore, as briefly as I can, describe one of our days in the valley. To begin with the morning. We were not very early risers--the sun would be shooting his golden spikes above the Happar mountain, ere I threw aside my tappa robe, and girding my long tunic about my waist, sallied out with Fayaway and Kory-Kory, and the rest of the household, and bent my steps towards the stream. Here we found congregated all those who dwelt in our section of the valley; and here we bathed with them. The fresh morning air and the cool flowing waters put both soul and body in a glow, and after a half-hour employed in this recreation, we sauntered back to the house--Tinor and Marheyo gathering dry sticks by the way for fire-wood; some of the young men laying the cocoanut trees under contribution as they passed beneath them; while Kory-Kory played his outlandish pranks for my particular diversion, and Fayaway and I, not arm in arm to be sure, but sometimes hand in hand, strolled along, with feelings of perfect charity for all the world, and especial good-will towards each other. Our morning meal was soon prepared. The islanders are somewhat abstemious at this repast; reserving the more powerful efforts of their appetite to a later period of the day. For my own part, with the assistance of my valet, who, as I have before stated, always officiated as spoon on these occasions, I ate sparingly from one of Tinor's trenchers, of poee-poee; which was devoted exclusively for my own use, being mixed with the milky meat of ripe cocoanut. A section of a roasted bread-fruit, a small cake of 'Amar', or a mess of 'Cokoo,' two or three bananas, or a mammee-apple; an annuee, or some other agreeable and nutritious fruit served from day to day to diversify the meal, which was finished by tossing off the liquid contents of a young cocoanut or two. While partaking of this simple repast, the inmates of Marheyo's house, after the style of the ancient Romans, reclined in sociable groups upon the divan of mats, and digestion was promoted by cheerful conversation. After the morning meal was concluded, pipes were lighted; and among them my own especial pipe, a present from the noble Mehevi. The islanders, who only smoke a whiff or two at a time, and at long intervals, and who keep their pipes going from hand to hand continually, regarded my systematic smoking of four or five pipefuls of tobacco in succession, as something quite wonderful. When two or three pipes had circulated freely, the company gradually broke up. Marheyo went to the little hut he was forever building. Tinor began to inspect her rolls of tappa, or employed her busy fingers in plaiting grass-mats. The girls anointed themselves with their fragrant oils, dressed their hair, or looked over their curious finery, and compared together their ivory trinkets, fashioned out of boar's tusks or whale's teeth. The young men and warriors produced their spears, paddles, canoe-gear, battle-clubs, and war-conchs, and occupied themselves in carving, all sorts of figures upon them with pointed bits of shell or flint, and adorning them, especially the war-conchs, with tassels of braided bark and tufts of human hair. Some, immediately after eating, threw themselves once more upon the inviting mats, and resumed the employment of the previous night, sleeping as soundly as if they had not closed their eyes for a week. Others sallied out into the groves, for the purpose of gathering fruit or fibres of bark and leaves; the last two being in constant requisition, and applied to a hundred uses. A few, perhaps, among the girls, would slip into the woods after flowers, or repair to the stream will; small calabashes and cocoanut shells, in order to polish them by friction with a smooth stone in the water. In truth these innocent people seemed to be at no loss for something to occupy their time; and it would be no light task to enumerate all their employments, or rather pleasures. My own mornings I spent in a variety of ways. Sometimes I rambled about from house to house, sure of receiving a cordial welcome wherever I went; or from grove to grove, and from one shady place to another, in company with Kory-Kory and Fayaway, and a rabble rout of merry young idlers. Sometimes I was too indolent for exercise, and accepting one of the many invitations I was continually receiving, stretched myself out on the mats of some hospitable dwelling, and occupied myself pleasantly either in watching the proceedings of those around me or taking part in them myself. Whenever I chose to do the latter, the delight of the islanders was boundless; and there was always a throng of competitors for the honour of instructing me in any particular craft. I soon became quite an accomplished hand at making tappa--could braid a grass sling as well as the best of them--and once, with my knife, carved the handle of a javelin so exquisitely, that I have no doubt, to this day, Karnoonoo, its owner, preserves it as a surprising specimen of my skill. As noon approached, all those who had wandered forth from our habitation, began to return; and when midday was fairly come scarcely a sound was to be heard in the valley: a deep sleep fell upon all. The luxurious siesta was hardly ever omitted, except by old Marheyo, who was so eccentric a character, that he seemed to be governed by no fixed principles whatever; but acting just according to the humour of the moment, slept, ate, or tinkered away at his little hut, without regard to the proprieties of time or place. Frequently he might have been seen taking a nap in the sun at noon-day, or a bath in the stream of mid-night. Once I beheld him perched eighty feet from the ground, in the tuft of a cocoanut tree, smoking; and often I saw him standing up to the waist in water, engaged in plucking out the stray hairs of his beard, using a piece of muscle-shell for tweezers. The noon-tide slumber lasted generally an hour and a half: very often longer; and after the sleepers had arisen from their mats they again had recourse to their pipes, and then made preparations for the most important meal of the day. I, however, like those gentlemen of leisure who breakfast at home and dine at their club, almost invariably, during my intervals of health, enjoyed the afternoon repast with the bachelor chiefs of the Ti, who were always rejoiced to see me, and lavishly spread before me all the good things which their larder afforded. Mehevi generally introduced among other dainties a baked pig, an article which I have every reason to suppose was provided for my sole gratification. The Ti was a right jovial place. It did my heart, as well as my body, good to visit it. Secure from female intrusion, there was no restraint upon the hilarity of the warriors, who, like the gentlemen of Europe after the cloth is drawn and the ladies retire, freely indulged their mirth. After spending a considerable portion of the afternoon at the Ti, I usually found myself, as the cool of the evening came on, either sailing on the little lake with Fayaway, or bathing in the waters of the stream with a number of the savages, who, at this hour, always repaired thither. As the shadows of night approached Marheyo's household were once more assembled under his roof: tapers were lit, long curious chants were raised, interminable stories were told (for which one present was little the wiser), and all sorts of social festivities served to while away the time. The young girls very often danced by moonlight in front of their dwellings. There are a great variety of these dances, in which, however, I never saw the men take part. They all consist of active, romping, mischievous evolutions, in which every limb is brought into requisition. Indeed, the Marquesan girls dance all over, as it were; not only do their feet dance, but their arms, hands, fingers, ay, their very eyes, seem to dance in their heads. The damsels wear nothing but flowers and their compendious gala tunics; and when they plume themselves for the dance, they look like a band of olive-coloured Sylphides on the point of taking wing. In good sooth, they so sway their floating forms, arch their necks, toss aloft their naked arms, and glide, and swim, and whirl, that it was almost too much for a quiet, sober-minded, modest young man like myself. Unless some particular festivity was going forward, the inmates of Marheyo's house retired to their mats rather early in the evening; but not for the night, since, after slumbering lightly for a while, they rose again, relit their tapers, partook of the third and last meal of the day, at which poee-poee alone was eaten, and then, after inhaling a narcotic whiff from a pipe of tobacco, disposed themselves for the great business of night, sleep. With the Marquesans it might almost most be styled the great business of life, for they pass a large portion of their time in the arms of Somnus. The native strength of their constitution is no way shown more emphatically than in the quantity of sleep they can endure. To many of them, indeed, life is little else than an often interrupted and luxurious nap.

Summary: Tommo, understanding that in the valley most days are like every other day, explains his daily schedule: He wakes after sunrise, then bathes with Fayaway and Kory-Kory while Tinor and Marheyo take half an hour to build the day's fire. Then they eat a morning meal and smoke a pipe. Post-breakfast there are naps or minor chores, after which Tommo heads to the Ti to eat with Mehevi. After, Tommo sails around the lake with Fayaway, or goes swimming, then watches the young women dance. At last, he returns to the house of Marheyo, to eat a bit, smoke a bit, and go back to sleep. It sounds like a real grind.

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Write a title and summarize: Microglia are the immune cells of the brain. In the absence of pathological insult, their highly motile processes continually survey the brain parenchyma and transiently contact synaptic elements. Aside from monitoring, their physiological roles at synapses are not known. To gain insight into possible roles of microglia in the modification of synaptic structures, we used immunocytochemical electron microscopy, serial section electron microscopy with three-dimensional reconstructions, and two-photon in vivo imaging to characterize microglial interactions with synapses during normal and altered sensory experience, in the visual cortex of juvenile mice. During normal visual experience, most microglial processes displayed direct apposition with multiple synapse-associated elements, including synaptic clefts. Microglial processes were also distinctively surrounded by pockets of extracellular space. In terms of dynamics, microglial processes localized to the vicinity of small and transiently growing dendritic spines, which were typically lost over 2 d. When experience was manipulated through light deprivation and reexposure, microglial processes changed their morphology, showed altered distributions of extracellular space, displayed phagocytic structures, apposed synaptic clefts more frequently, and enveloped synapse-associated elements more extensively. While light deprivation induced microglia to become less motile and changed their preference of localization to the vicinity of a subset of larger dendritic spines that persistently shrank, light reexposure reversed these behaviors. Taken together, these findings reveal different modalities of microglial interactions with synapses that are subtly altered by sensory experience. These findings suggest that microglia may actively contribute to the experience-dependent modification or elimination of a specific subset of synapses in the healthy brain. Upon invasion of the central nervous system during embryonic and early postnatal development, bone-marrow-derived microglia become involved in apoptosis and phagocytic elimination of supernumerary neurons [1]–[3]. As they complete their differentiation, microglia change their morphology from amoeboid to ramified and are thought to become quiescent [4]. In the event of pathological insult, microglia rapidly become activated, thicken and retract their processes, migrate to the site of injury, proliferate, and participate in the presentation of antigens, phagocytosis of cellular debris, and secretion of proteases that promote microglial motility, as well as myelin and extracellular matrix degradation [5]–[7]. Additionally, activated microglia can separate presynaptic axon terminals from postsynaptic neuronal perikarya or dendrites in a process known as synaptic stripping [8]. Even though microglia are quiescent under non-pathological conditions, their highly motile processes continually survey the local environment and make transient contacts with astrocytes, neuronal perikarya, axon terminals, and dendritic spines in vivo [9]–[11]. Microglial apposition with astrocytic and neuronal elements has also been observed with electron microscopy (EM) in situ [9], but a detailed analysis of microglial ultrastructural relationships is still lacking. Reports of spontaneous engulfment of cellular debris [10] suggest that resting microglia may exert phagocytic roles in the healthy brain. Because changes in the level of neuronal activity can also modify the volume of neuropil that microglia sample [10], as well as their frequency of contact with axon terminals [9], Wake et al. [9] proposed that resting microglia could monitor the functional state of synapses. However, aside from immune surveillance, the fate of synaptic architecture under the care of microglia remains poorly understood. The dynamic nature of microglial processes and their interaction with synapses suggest that microglia could effect structural changes at synapses, which are crucial to circuit remodeling and brain plasticity. To begin to investigate this possible task of quiescent microglia at synapses, we verified whether microglial interactions with synapses occur at random or coincide with structural synaptic changes and alterations in sensory experience. Specifically, we characterized the ultrastructural and structural/dynamic interactions between microglia and synapse-associated elements during normal sensory experience, sensory deprivation, and subsequent light exposure in the primary visual cortex (V1) of juvenile mice. In addition to revealing the three-dimensional (3-D) geometry of cell–cell contacts between microglia and all synapse-associated elements (dendritic spines, axon terminals, perisynaptic astrocytic processes, and synaptic clefts), our observations uncovered new modes of microglia–synapse interaction under non-pathological conditions, particularly the regulation of the perisynaptic extracellular space and the phagocytosis of synaptic elements. Moreover, we found that microglia specifically localize to the vicinity of a subset of synaptic elements in vivo, in particular the structurally dynamic and transient dendritic spines. Lastly, we demonstrate that several modalities of microglia–synapse interactions are regulated by sensory experience. Thus, our findings indicate that microglia are not activated only during early brain development or pathological conditions; rather, they also subtly change their behavior toward synapses in correspondence with sensory experience. This raises the intriguing possibility that microglia may contribute to fine-tuning the plastic capacities of individual synapses in the healthy brain. To provide a detailed view of the modes of interaction between microglia and excitatory synapses, we analyzed their ultrastructural relationships in layer II of mouse V1 on postnatal day (P) 28, around the peak of the critical period for experience-dependent plasticity [12]. Using immunocytochemical EM with an antibody against the microglia-specific marker IBA1 [13] (see Figure S1 for IBA1 immunostaining at the light microscopic level), we found that microglial cell bodies, as well as proximal and distal processes, juxtaposed synapse-associated elements including synaptic clefts (Figures 1A–1C), an area generally thought to be exclusively reserved for astrocytic processes. Quantitative analysis revealed that the vast majority of microglial process profiles directly contacted at least one of the synapse-associated elements (synaptic index: 94%±0. 6%; ∼1,000 µm2 of neuropil in each of three animals). Axon terminals, dendritic spines, perisynaptic astrocytic processes, and synaptic clefts were contacted by microglial processes, in decreasing order of frequency (n = 150 IBA1-positive microglial processes; three animals; see Table S1 for detailed analysis), and more than one synapse-associated element was generally contacted by each process (68%±4%; see Table S2). To uncover the 3-D relationships between microglia and synapse-associated elements, we used serial section EM (SSEM) with 3-D reconstructions (Figures 2 and S2) in layer II of mouse V1 at P28. The 3-D reconstructions showed that proximal and distal microglial processes simultaneously contacted synapses of different shape and size, with typically more than one subcellular element contacted at individual synapses (Figures 2B, 2D, 2E, S2A, and S2B). Whereas most contacts between microglia and synapses occurred en passant along microglial processes, without morphologic evidence of specialization, strikingly, the reconstructed distal microglial process displayed finger-like protrusions that wrapped around a dendritic spine making a synapse with an axon terminal (Figure 2B, 2D, and 2E). Interestingly, the proximal microglial process was found to engulf cellular components (see SSEM images in Figure S4, but also the reconstructed cellular inclusion in Figures 2B, 2C, and S2A, S2B, S2C), suggesting microglial involvement in phagocytosis during normal sensory experience, along with our immunocytochemical EM observations that microglial perikarya and large processes sometimes contained cellular inclusions. Lastly, analysis of the series also showed occasional coated pits at the sites of cell–cell contact between microglia and dendritic spines, axon terminals, or astrocytic processes, either inside microglia or inside synapse-associated elements (see Figures 1D and S3 for examples). Immunocytochemical EM and SSEM also revealed the appearance of large electron-lucent extracellular spaces nearby microglia, with both acrolein (Figures 1A–1C and 2A) and glutaraldehyde (Figure 1D) fixatives. Pockets of extracellular space, which consists of interstitial fluid supplemented with various extracellular matrix proteins, were otherwise rarely observed around any other structural elements in juvenile mice, in clear contrast with early postnatal stages of development [14]–[16]. Areas of extracellular space apposing IBA1-positive microglia were found to be significantly larger (985±41 nm2; n = 3 animals; ∼500 µm2 of neuropil in each) than areas not associated with IBA1-positive processes (382±18 nm2; p<0. 0002; Figure 1E). This analysis likely underestimated the association of extracellular space with microglial processes because of the partial penetration of antibodies under these stringent immunocytochemical conditions. In fact, most extracellular spaces were associated with unlabeled structural elements that resembled microglia morphologically (see Materials and Methods for identification criteria). In addition, areas of a microglial process and associated extracellular space were tightly correlated (r = 0. 48; p<0. 0001; n = 150 processes; three animals; Figure 1F), suggesting that microglia may exert a large influence in the creation of such space. SSEM with 3-D reconstructions also showed that microglia-associated spaces exhibited highly complex and varied morphologies (Figures 2B, 2C, and S2C). Quantitative analysis of the series revealed that the 3-D pockets of extracellular space varied in volume by two orders of magnitude: from 20,223 to 7,048,921 nm3 (mean: 905,834 nm3; median: 150,225 nm3; n = 15 extracellular spaces; Table S3). In light of these results, when considering the interactions of synapses and microglia, it is important to take into account the complex organization of extensive contacts between a single microglia and every synapse-associated element at multiple synapses, interrupted by many pockets of geometrically complex microglia-specific extracellular spaces, simultaneously throughout the neuropil. Taken together, our findings from immunocytochemical EM and SSEM with 3-D reconstructions indicate that microglia are uniquely positioned to play several physiological roles at synapses: through cell–cell communication with perisynaptic astrocytic processes, dendritic spines, and axon terminals simultaneously, as well as through the regulation of the extracellular environment intervening between them. To characterize the structural dynamics of microglia–synapse interactions, we used two-photon in vivo imaging of layers I/II of V1 in juvenile CX3CR1-GFP/Thy1-YFP mice [17], [18], in which both microglia and layer V neurons are fluorescently labeled. We utilized a thinned-skull preparation, which minimizes brain injury and allows long-term tracking of microglial dynamics without causing microglial activation [19] (Figure S5). Even though the resolution of two-photon microscopy (see Materials and Methods for details on measurement of the experimental point spread function) prevents visualization of direct contacts between fluorescently labeled elements, it enables the study of their structural dynamics and determination of close proximity (the apparent colocalization of fluorescence for microglial and neuronal elements was considered putative contact). A recent study demonstrated a relatively constant duration (4. 60±0. 08 min) of putative contacts between microglia and synaptic elements in layer II/III of juvenile mouse V1 [9]. In contrast, our time-lapse imaging (every 5 min for 30–120 min) revealed similar contacts between microglial processes and a subset of the YFP-labeled dendritic spines and axon terminals (n = 37 putative contacts with spines and 29 putative contacts with terminals in eight animals; Figures S7A and 3A; see Videos S1 and S4) that varied between 5 and 50 min. The cortical layers examined or the identity of synapses imaged (GFP-M and YFP-H mice label different subsets of pyramidal cells) [20]–[23] might explain this discrepancy. Nevertheless, these differences in contact duration reveal a new dimension in microglial interactions with synapses in the healthy brain. To verify whether microglial processes target specific subsets of synapses, we measured the size of dendritic spines and axon terminals in the presence and absence of a putative microglial contact. In this analysis, we noticed that dendritic spines that were in close proximity to microglial processes at any point during the imaging session were generally smaller than the rest of the spine population. We quantitatively recorded spine size at the beginning of imaging and found that dendritic spines receiving putative contact during imaging were significantly smaller than spines remaining non-contacted (p<0. 001; n = 31 contacted spines in five animals and 45 non-contacted spines in three animals, with infrequent stubby spines excluded; see Materials and Methods). When microglia came into close proximity with these synaptic structures, individual pre- and postsynaptic elements both expanded and shrank: 38% of axon terminals grew, 55% shrank, and 7% remained stable (Figure S7B and S7C). Average axon terminal sizes were not significantly different with and without putative microglial contact (size differential: −1%±3%; p>0. 9; n = 24 terminals and 29 putative contacts in three animals; Figure S7B and S7C), nor correlated to initial terminal size or putative microglial contact duration (p>0. 5 and p>0. 2, respectively; Figure S7D and S7E). In contrast, 62% of dendritic spines grew, 32% shrank, and 6% remained stable during putative contact; additionally, we observed a significant increase in average spine sizes in the presence versus in the absence of putative microglial contact (size differential: 9%±3%; p<0. 03; n = 31 spines and 37 contacts in five animals; Figure 3B). These changes were generally transient, since dendritic spine size was not significantly different between before and after the contact (p>0. 9; Figure 3C). No correlation between size change and putative contact duration was noted (p>0. 9; Figure S8A), but the size change and initial spine size were significantly correlated (r = 0. 28; p<0. 001; Figure 3D), with the smallest spines undergoing the largest size changes during contact. Indeed, the comparison of small and large dendritic spines revealed a significant difference in their structural changes during putative microglial contact (average size differential for large spines: 1%±3%; for small spines: 17%±5%; p<0. 01; Figures 3E, 3F, S8B, and S8C). Therefore, these observations indicate that microglial processes preferentially localize to small and structurally dynamic dendritic spines. To determine whether spines targeted by microglia exhibit a different long-term fate with respect to their longevity, we carried out chronic in vivo imaging experiments tracking individual dendritic spines over a period of 2 d (Figure 3G and 3H). Surprisingly, we found that dendritic spines that received putative microglial contact during the first imaging session were more frequently eliminated (24%±6%; n = 30 spines in four animals) than non-contacted spines (7%±3%; p<0. 05; n = 56 spines in four animals). Interestingly, among contacted dendritic spines, only small spines were lost (8/18 small spines and 0/12 large spines lost). We conclude that the subset of small and dynamic dendritic spines contacted by microglia also have an increased rate of elimination over a span of 2 d. Our two-photon imaging results raise the intriguing possibility that microglia may not only monitor the functional status of synapses, but also exert control on structural changes or spine elimination either through direct contact or indirect signaling that requires close microglia–synapse proximity. To investigate whether microglial behavior towards synapses is regulated by sensory experience, we altered visual experience by housing juvenile mice in complete darkness (dark adaptation [DA]) for 6 d, from the beginning to the peak of the critical period [12]. Binocular deprivation increases dendritic spine motility and turnover in mouse V1 in vivo [24], [25]; this provides an excellent model to correlate microglial behavior with the experience-dependent modification and elimination of synapses under non-pathological conditions. While microglial processes were generally larger in layer II of V1, they exhibited two morphological phenotypes at the ultrastructural level: some processes appeared very large and thick (“bulky”; Figure S10D and S10E), while others exhibited many short, thin fingers and appeared “spindly” (Figures 4C and S10F). Both types of microglial processes made multiple contacts with synapse-associated elements, including synaptic clefts (Figures 4C and S10D, S10E, S10F). Microglial perikarya and bulky processes often contained cellular inclusions (p<0. 0001; n = 3 control and 3 DA animals; 50 IBA1-immunopositive microglial processes each; Figures 4A, 4B, 4E, S9A, S10D, and S10E), which sometimes resembled dendritic spines or axon terminals, suggesting their phagocytic engulfment by microglia. Spindly processes typically ensheathed dendritic spines or axon terminals and displayed extended extracellular space areas (Figures 4C and S10F). Microglial process area (p<0. 01; n = 3 animals per condition; 50 IBA1-immunopositive processes each; Figure 4F; Table S1) and extracellular space area (p<0. 05; n = 3 animals; Figure 4G; Table S1) were significantly increased during DA, with the correlation between microglial process area and extracellular space area remaining significant (r = 0. 44; p<0. 0001; n = 150 processes in three animals; Figure S9B). In contrast, microglial process density (DA: 131±6; control: 142±6 processes per 1,000 µm2 of neuropil; n = 3 animals; 1,000 µm2 of neuropil each; Figure S9C) and synaptic index (DA: 95%±0. 6%; control: 94%±0. 6%; n = 3 animals; Figure S9D) were unchanged by sensory experience (p>0. 3 and p>0. 1, respectively). Surprisingly, contacts with synaptic clefts were more frequent in DA animals (p<0. 05; n = 3 animals per condition; total for 50 processes each; Figure 4H; Table S1) than controls, while contact frequency with dendritic spines, axon terminals, and astrocytic processes was unchanged (Figures 4H and S9E; Tables S1 and S2). Furthermore, as expected from extended microglial processes, their average perimeter of contact with every synapse-associated element was also increased during visual deprivation (spine: p<0. 01; astrocyte: p<0. 02; terminal: p<0. 05; n = 3 animals per condition; 50 processes each; Figure 4I; Table S1). Thus, our ultrastructural observations reveal subtle experience-dependent changes in the behavior of microglia, most notably an expansion of their processes and associated extracellular space, an increased occurrence of cellular inclusions, an increased frequency of contact with synaptic clefts, and an increased apposition with every synapse-associated element. To determine whether these changes in behavior could be reversed by reexposure to daylight, we subjected mice to DA for 6 d followed by a fixed 12-h light/dark cycle for 2 d (DA+light), as a few hours of reexposure to light after dark rearing (i. e., housing in complete darkness from birth) can cause rapid molecular changes at synapses, cause structural changes of dendritic spines, and reverse the effects of dark rearing on synaptic transmission and plasticity [26]–[29]. At the ultrastructural level in layer II of V1, most microglial process profiles appeared bulky during light reexposure, contained many inclusions, and were surrounded by small pockets of extracellular space (see Figures 4D and S10G, S10H, S10I for examples). Quantitative analysis revealed that microglial process area returned to control levels (p>0. 2 versus control; p>0. 09 versus DA; n = 3 animals per condition; Figure 4F) while microglia-associated extracellular space area was significantly reduced following light reexposure (p<0. 004 versus control; p<0. 02 versus DA; n = 3 animals per condition; Figure 4G). In contrast, the number of cellular inclusions, many of which contained elements resembling synaptic profiles (Figure S10G, S10H, S10I), remained significantly higher than in control animals (p<0. 008 versus control; p>0. 2 versus DA; n = 3 animals per condition; Figure 4E). Similarly, contacts with synaptic clefts (p<0. 03 versus control; p>0. 9 versus DA; n = 3 animals per condition; Figure 4H) and perimeters of contact with dendritic spines (p<0. 01 versus control; p>0. 4 versus DA; Figure 4I) and axon terminals (p<0. 02 versus control; p>0. 6 versus DA), but not with perisynaptic astrocytes (p>0. 2 versus control and DA), remained extended. Future experiments with longer light exposures after deprivation will be needed to determine whether these phenomena can be reversed with further light reexposure. Taken together, these observations reveal a complex interaction between sensory-driven activity and microglial behavior. While the expansion of microglial processes and associated extracellular spaces reversed after brief reexposure to daylight, microglial ensheathment of dendritic spines and axon terminals, as well as their phagocytic inclusion, were still increased. To assess the dynamic changes in microglia–synapse interactions during visual deprivation, we used two-photon imaging of layers I/II of V1 in juvenile mice that were subjected to DA for 8–10 d, from the beginning to the peak of the critical period [12]. Microglial processes appeared thickened and sparse, and more often terminated into crown-like structures resembling phagocytic cups than in control animals [30] (Figure 5A and 5B; Video S2, as well as Figure S12 and Videos S4 and S5 for comparison of microglial morphology in control and DA animals). We also found that the average motility of microglial processes was significantly reduced (Figure 5C and 5D) when assayed in two ways: comparing morphology over a 5-min interval (motility index; control: 8%±0. 8%; DA: 6%±0. 5%; p<0. 05; n = 10 microglia in four control animals and 8 microglia in four DA animals) and over a 25-min interval, where the difference between control and DA animals was more pronounced (control: 11%±0. 8%; DA: 7%±0. 6%; p<0. 01). Quantitative analysis of dendritic spines showed that spines not receiving putative microglial contact were significantly smaller in DA animals than in non-light-deprived animals (p<0. 001; n = 45 spines in three animals per experimental condition), whereas contacted spines were equally small in both DA and control animals (p>0. 5; Figure 5E), in agreement with an observed reduction in synaptic strength during binocular deprivation [31], [32] and the smaller sizes of dendritic spines during synaptic depression [33], [34]. Surprisingly, dendritic spines putatively contacted by microglia in DA animals were significantly bigger than non-contacted spines (p<0. 05), revealing that microglia no longer localize to smaller spines in this condition. The average duration of putative microglial contact with dendritic spines was slightly increased in DA animals (10±2 min; n = 13 contacts in three animals) compared with controls (9±1 min; n = 37 contacts in five animals; Figure S11A), but the difference was not significant (p>0. 8), suggesting that increased coverage of synaptic elements by microglia is not a result of longer duration of contact. Similarly, the frequency of putative contacts with individual dendritic spines was unchanged by sensory experience (DA: 1±0. 08; control: 1±0. 07 contacts per 40 min; p>0. 6; Figure S11B). Lastly, most dendritic spines shrank during microglial contact (29% grew, 57% shrank, and 14% remained stable; n = 12 spines and 14 putative contacts in three animals), unlike in the control condition, where most spines grew. While average size changes during contact were not significant (size differential: −4%±4%; p>0. 3; Figure S11C), interestingly, dendritic spine shrinkage persisted after microglial contact, with a significant difference in size between before and after the contact (p<0. 02; Figure 5F), a phenomenon which may contribute to the reduction in size of the spine population. These results indicate that sensory-deprived microglia undergo subtle behavioral changes reminiscent of activation, including reduced motility, thickened processes, and phagocytic specializations. Intriguingly, despite unaltered duration and frequency of microglial contacts with synaptic elements, their preference of localization did change, from a subset of smaller dendritic spines that transiently grow to a subset of bigger spines that persistently shrink. To further investigate these experience-dependent changes in microglial behavior, juvenile animals that were subjected to DA for 6–8 d were reexposed to daylight for 2 d before two-photon imaging of layers I/II of V1. Microglial morphologies resembled those in control animals, with generally thinner and more abundant processes within the neuropil (Figure S12; Video S6), but microglial processes still displayed phagocytic structures (Video S3). Microglial motility in animals reexposed to light was similar to that in control animals and was significantly increased compared with DA animals, when assessed during a 5-min interval (motility index = 10%±0. 8%; p>0. 2 versus control; p<0. 001 versus DA; eight microglia in three DA+light animals; Figure 5D) and a 25-min interval (motility index = 13%±1. 3%; p>0. 1 versus control; p<0. 0007 versus DA). Quantitative analysis of dendritic spines showed that spines receiving putative microglial contacts were of similar sizes to those in non-light-deprived and light-deprived animals (p>0. 1 versus control and DA; n = 14 contacted spines in three DA+light animals; Figure 5E), while non-contacted spines were significantly bigger in animals reexposed to light (p<0. 01 versus control; p<0. 0001 versus DA; n = 45 spines in three animals per condition). The sizes of contacted and non-contacted spines were not significantly different in animals reexposed to light (p>0. 9; Figure 5E), indicating that microglia no longer localize to specific spine types as in control or DA animals. Lastly, microglia–synapse interactions showed similar structural effects on dendritic spines as in control conditions. Most dendritic spines increased in size during putative microglial contact (67% grew, 20% shrank, and 13% remained stable; average size differential: 13%±5%; p<0. 01 comparing with/without and before/during contact; n = 14 spines and 15 putative contacts in three animals; Figures 5F and S11C), and this growth was transient (p>0. 8 comparing size differential before/after contact; Figure 5F). These results reveal additional changes in microglial behavior that can be reversed by brief reexposure to daylight, particularly their motility and preference of contact for subsets of dendritic spines. One of the most striking findings of our study was the demonstration of distinctive extracellular spaces closely correlated with the presence of microglial processes. Our EM observations revealed large electron-lucent pockets of extracellular space surrounding microglia. To our further surprise, we also observed changes in the distribution of these microglia-associated extracellular spaces during alterations in visual experience: an expansion during light deprivation and shrinkage during light reexposure. Although alteration of these spaces by brain fixation and embedding for EM may warrant further investigation, we observed them under all conditions tested. In future experiments, it will be important to determine whether microglial processes create this extracellular space themselves or move in to fill space that is created by an unknown mechanism. In any case, our findings of microglia-specific extracellular spaces suggest that microglia have an intimate relationship with their extracellular milieu and may even regulate their surrounding environment in a unique and specific way that is determined by physiological conditions. If microglia directly modulate the extracellular space, they may do this through the secretion of various proteases that degrade extracellular matrix proteins, including cathepsins, metalloproteases, and tissue-type plasminogen activator [35]. This proteolytic degradation of specific matrix proteins could, in turn, facilitate microglial motility, as suggested by the finding that the migratory behavior of cathepsin S–deficient microglia is severely impaired in vitro [35]. In line with this, the volume of extracellular space greatly decreases during postnatal cortical development, concomitant with changes in extracellular matrix composition and reductions in cell migration and process elongation [14]–[16], [36], [37]. The appearance of extracellular spaces specifically associated with microglial processes could therefore reflect their highly motile behavior, relative to other structural elements of neuropil in juvenile cortex. Additionally, regulation of the extracellular matrix composition by microglia-derived proteases could contribute to dendritic spine motility and pruning, as well as activity-dependent and experience-dependent plasticity, which are profoundly affected in vitro and in vivo by treatments with proteases that degrade extracellular matrix proteins [38]–[43]. Immunocytochemical EM and SSEM with 3-D reconstructions enabled us to analyze the morphology of microglial processes and their ultrastructural relationships with the other subcellular compartments of neuropil—astrocytic processes, axon terminals, and dendritic spines—in situ at high spatial resolution. Building on previous EM observations that microglia contact axon terminals and dendritic spines [9], [44], [45], our quantitative analysis revealed that most microglial processes directly appose not only axon terminals and dendritic spines, but also perisynaptic astrocytic processes and synaptic clefts. Our SSEM with 3-D reconstructions also uncovered the 3-D relationships between microglia and synapses, revealing that microglial processes contact multiple synapse-associated elements at multiple synapses simultaneously. Additionally, we found clathrin-coated pits at interfaces between microglia and dendritic spines, axon terminals, or perisynaptic astrocytic processes, suggesting clathrin-mediated endocytosis of membrane-bound receptors and their ligands, a phenomenon known to initiate various cellular signaling events [46]. Since clathrin-coated pits also occur at the tips of most spinules undergoing invagination, as previously observed in small dendritic spines, axon terminals, and perisynaptic astrocytic processes of mouse hippocampus [47], this may suggest trans-endocytosis of membrane-bound receptors and their ligands, in addition to a direct exchange of cytoplasm, between microglia and synapse-associated elements. To better understand the functional significance of these forms of molecular communication between microglia and synapse-associated elements, it will be important to identify the molecules that are being internalized during their dynamic interactions. Following visual deprivation and reexposure to daylight, our EM results revealed that microglial processes change their morphology, appose synaptic clefts more frequently, and envelop synapse-associated elements more extensively. Since glial presence at the synaptic cleft was classically restricted to astrocytic processes regulating synaptic function through modification of the extracellular space geometry and bidirectional communication with synaptic elements [48], [49], this novel finding suggests that microglia may also contribute uniquely to synaptic transmission and plasticity in the healthy brain. Additionally, the ensheathment of synaptic elements and synaptic clefts by microglial processes may imply their participation in activity-dependent synapse elimination [50], through a separation of pre- and postsynaptic elements reminiscent of synaptic stripping, as previously reported between axon terminals and neuronal cell bodies during immune responses [8]. Lastly, our EM and two-photon in vivo imaging observations revealed that a subpopulation of microglial processes displays phagocytic structures, with an increasing prevalence during alterations in visual experience, thus providing evidence for a microglial role in phagocytic engulfment under non-pathological conditions. This is supported by observations that quiescent microglia can spontaneously engulf tissue components in vivo [10] and that activated microglia play an essential role in phagocytosis of cellular debris [1]–[3], [5], [6]. At the ultrastructural level, we also found cellular inclusions that resembled dendritic spines or axon terminals, suggesting that quiescent microglia can phagocytose synaptic elements in juvenile cortex. In line with this, classical complement proteins C1q and C3 were recently shown to be involved in the pruning of inappropriate retino-geniculate connections during early postnatal development [51]. Since C1q and downstream complement protein C3 can trigger a proteolytic cascade leading to microglial phagocytosis, this finding supports a model in which microglia may contribute to synaptic pruning under non-pathological conditions [52]. Taken together, our findings indicate that distinct modes of microglial interactions with synapses, most notably apposition, ensheathment, and phagocytosis, are subtly regulated by sensory experience. Two-photon visualization of microglia and synaptic elements with two different colors in CX3CR1-GFP/Thy1-YFP mice enabled clear distinction of the separate structures, which facilitated identification of their putative contacts. This approach revealed transient localization of microglial processes to the vicinity of dendritic spines and axon terminals, in agreement with observations from a recent study [9]. In our study, clear distinction of the separate structures also allowed, for the first time, measurement of dendritic spine and axon terminal morphological changes during episodes of proximity with microglial processes. Our results revealed a specificity of these putative microglial contacts for a subset of small, transiently growing, and frequently eliminated dendritic spines. This is consistent with previous findings showing that in mouse visual and somatosensory cortical areas in vivo, small dendritic spines are more motile and more frequently eliminated than their larger counterparts [20], [22], [24], [53], [54]. Dendritic spines undergoing long-term potentiation transiently or persistently enlarge in vitro [33], [34], [55], [56], but since dendritic spine structural changes can occur independently of changes in synaptic strength [57], a connection between putative microglial contact and synaptic plasticity remains to be determined. During light deprivation, microglia preferentially localized to larger dendritic spines that persistently shrank, akin to spines undergoing long-term depression in vitro [33], [34] or shrinking before elimination in vivo [58], while during reexposure, microglia reversed to contacting spines that transiently grew, as in control animals. This uncovers a specificity of microglial interaction for subsets of structurally dynamic and transient dendritic spines, a specificity that, surprisingly, changes with sensory experience. In future experiments, correlating the temporal dynamics of microglial contact with dendritic spine activity could be achieved by in vivo labeling of neurons with electroporated calcium indicators [59] or viral delivery of genetic calcium indicators [60]. A challenge with such techniques will be their invasive nature; the mechanical disruption of the brain alone during indicator delivery can result in microglial activation. Additionally, it will be important to determine whether microglial contacts occur in response to structural changes in dendritic spines and whether such contacts instruct subsequent spine elimination. This will, however, require new technical advances enabling interference with microglial contacts while preserving physiological conditions. Importantly, whether direct microglial contacts, such as observed with EM, are required for these structural changes or whether microglia can exert effects on synapses without close apposition will need to be explored. Several lines of evidence, including those presented here, suggest that microglial motility may be regulated by neuronal activity. A pioneer study reported an increase in the volume sampled by quiescent microglia in vivo over a period of 1 h after application of the ionotropic GABA receptor blocker bicuculline, whereas the sodium channel blocker tetrodotoxin had no significant effects [10]. More recently, microglia were shown to retract their processes and reduce their frequency of contact with axon terminals in vivo, over a period of 4–6 h after binocular enucleation or intraocular injection of tetrodotoxin [9]. In our study, two-photon analysis revealed a global decrease in microglial motility during light deprivation without correlated changes in the duration or frequency of microglial contact with individual dendritic spines. This may highlight differences between short-term (1–6 h) and long-term (8–10 d) microglial responses to sensory deprivation or distinguish between more invasive manipulations, which approximate nervous system injury, and more physiological paradigms such as DA. Our observations further suggest that subsets of microglial processes may have different behavior: those in contact with dendritic spines may be highly motile, while others (for example bulky microglial processes displaying phagocytic structures) may become less motile. It is also possible that spindly microglial processes (<100 nm), which were typically devoid of cellular inclusions and surrounded by extended extracellular space, were undetected with two-photon imaging and therefore not accounted for in our motility analysis. This could explain the finding that microglial motility decreased during light deprivation while microglia-associated extracellular spaces expanded. However, as we found an increase in microglial motility during light reexposure that was accompanied by a decrease in microglia-associated extracellular space areas, it is likely that microglial motility and extracellular space areas are regulated independently. Using several technical approaches, we were able to provide a qualitative and quantitative characterization of the ultrastructural and structural/dynamic interactions between microglia and synapses under non-pathological conditions. This characterization revealed specific modalities of microglia–synapse interactions that are subtly altered by sensory experience, supporting the exciting possibility that microglial influence on synaptic plasticity is not restricted physiologically to an immune response to brain injury and disease. Animals were treated in strict accordance with the University of Rochester Committee on Animal Resources and the United States National Institutes of Health standards. Light-reared animals were housed under a fixed 12-h light/dark cycle; DA animals were placed in complete darkness for 6–10 d, from P20–P22 until P28–P32 (the onset of experimentation); DA+light animals were housed under a fixed 12-h light/dark cycle for 2 d following DA until P29–P32 (the onset of experimentation). For immunocytochemical EM and SSEM, eight C57Bl/6 mice (P28) were anesthetized with sodium pentobarbital (80 mg/kg, i. p.) and perfused through the aortic arch with 3. 5% acrolein followed by 4% paraformaldehyde as previously described [14], [61] or with 2. 75% glutaraldehyde in 2% paraformaldehyde to compare extracellular space areas surrounding microglia between both types of fixatives. For two-photon imaging [17], 21 CX3CR1-GFP/Thy1-YFP mice (P28–P39), in which microglia and cortical layer V neurons are respectively GFP- and YFP-labeled [17], [18], were anesthetized with a mixture of fentanyl (0. 05 mg/kg, i. p.), midazolam (5. 0 mg/kg), and metatomadin (0. 5 mg/kg) [62] and kept at 37°C with a heating pad. Stereotaxical coordinates encompassing a 2- to 3-mm-diameter region (A +0. 16 to A +0. 64, between 2 and 3 mm from the midline) [63] were used to identify V1. In all cases, DA animals were anesthetized in the dark using infrared goggles or a darkroom red light before undergoing perfusion or surgery in the light. After acute (30 min to 2 h) and chronic (two imaging sessions over 2 d, 30 min to 2 h each) two-photon imaging, eight mice were perfused with 4% paraformaldehyde to confirm that microglia are not activated by the surgical procedure and imaging paradigm (Figure S4). Transverse sections of the brain (50 µm thick) were cut in ice-cooled PBS (0. 9% NaCl in 50 mM phosphate buffer [pH 7. 4]) with a vibratome. Sections were immersed in 0. 1% sodium borohydride for 30 min at room temperature (RT), washed in PBS, and processed freely floating following a pre-embedding immunoperoxidase protocol previously described [14], [61], [64], [65]. Briefly, sections were rinsed in PBS, followed by a 2-h pre-incubation at RT in a blocking solution of PBS containing 5% normal goat serum and 0. 5% gelatin. They were incubated for 48 h at RT in rabbit anti-IBA1 antibody (1∶1,000 in blocking solution; Wako Pure Chemical Industries) and rinsed in PBS. After incubation for 2 h at RT in goat anti-rabbit IgGs conjugated to biotin (Jackson Immunoresearch) and with streptavidin-horseradish peroxidase (Jackson Immunoresearch) for 1 h at RT in blocking solution, labeling was revealed with diaminobenzidine (0. 05 mg/ml) and hydrogen peroxide (0. 03%) in buffer solution (DAB Peroxidase Substrate Kit; Vector Laboratories). Sections for light microscopy were mounted onto microscope slides, dehydrated in ascending concentrations of ethanol, cleared in xylene, and coverslipped with DPX (Electron Microscopy Sciences). Sections for EM were post-fixed flat in 1% osmium tetroxide and dehydrated in ascending concentrations of ethanol. They were treated with propylene oxide, impregnated in Durcupan (Electron Microscopy Sciences) overnight at RT, mounted between ACLAR embedding films (Electron Microscopy Sciences), and cured at 55°C for 48 h. Areas of V1, at a level approximating the transverse planes A +0. 16 to A +0. 72 [63], were excised from the embedding films and re-embedded at the tip of resin blocks. Ultrathin (65–80 nm) sections were cut with an ultramicrotome (Reichert Ultracut E), collected on bare square-mesh grids, stained with lead citrate, and examined with a Hitachi 7650 electron microscope. Light microscope pictures of IBA1 immunostaining were taken at 20× in layer II of V1, using a Spot RT color digital camera (Diagnostic Instruments). Eighty pictures were randomly taken at 40,000× in layer II of V1 in each animal (n = 3 control, 3 DA, and 3 DA+light), corresponding to a total surface of ∼1,000 µm2 of neuropil per animal. Cellular profiles were identified according to criteria previously defined [14], [44], [66], [67]. In addition to their IBA1 immunoreactivity, microglial cell bodies were recognized by their small size and the clumps of chromatin beneath their nuclear envelope and throughout their nucleoplasm. Microglial processes displayed irregular contours with obtuse angles, dense cytoplasm, numerous large vesicles, occasional multivesicular bodies, vacuoles or cellular inclusions (large lipidic vesicles, profiles of cellular membranes, and profiles of other structural elements including dendritic spines and axon terminals), and distinctive long stretches of endoplasmic reticulum. These morphological characteristics enabled identification of microglial processes on an ultrastructural level in non-immunocytochemical SSEM and non-immunocytochemical glutaraldehyde-fixed material. In each of three control and three DA animals (∼1,000 µm2 of neuropil per animal), IBA1-immunopositive microglial process profiles were counted and their contacting structural elements identified. A synaptic index was calculated based on the percentage of IBA1-positive microglial processes contacting synapse-associated elements divided by the total number of IBA1-positive microglial processes (Figure S9). It is important to note that since analysis was performed on single sections, different microglial process profiles could be part of a single microglial process, which may lead to an overestimation of the synaptic index in this analysis. In 39 pictures in each of three control animals (∼500 µm2 of neuropil per animal), all extracellular space areas wider than synaptic clefts (10–20 nm) [66] were measured and determined to contact or not contact IBA1-stained microglial processes (Figure 1; Table S3). To characterize the ultrastructural relationships between microglia and synapse-associated elements, 50 randomly selected IBA1-positive microglial processes per animal were analyzed in more detail with Image J software (United States National Institutes of Health). For measurement of microglial process and extracellular space areas, individual microglial processes and all their adjacent extracellular spaces wider than synaptic clefts were traced with the freehand line tool. Their area was quantified in pixels and converted into nanometers (Figures 1,4, and S9; Table S1). For quantification of contacts between microglial processes and synapse-associated elements, we first counted all direct juxtapositions between individual microglial processes and dendritic spines, axon terminals, synaptic clefts, and perisynaptic astrocytic processes (Figure 4; Table S1). For example, microglial processes contacting synaptic clefts were also contacting dendritic spines and axon terminals. Since most microglial processes contacted multiple synapse-associated elements simultaneously, we performed a second analysis where we categorized individual microglial processes based on their contacting partners: dendritic spine only, axon terminal only, perisynaptic astrocytic process only, spine+terminal, spine+astrocytic process, terminal+astrocytic process, or spine+astrocytic process+terminal. Microglial contacts with synaptic clefts were included in the spine+terminal or spine+astrocytic process+terminal categories (Table S2). For measurement of perimeters of contact between microglia and dendritic spines, axon terminals, or perisynaptic astrocytic processes, all microglial cell membranes in direct juxtaposition with these structural elements were traced with the freehand line tool (Figure 4; Table S1). Their length was quantified in pixels and converted into nanometers. A series of 100 ultrathin sections (65 nm) was cut, collected on pioloform-coated grids, stained with lead citrate, and examined with a JEOL JEM-1230 electron microscope. Fifty serial images of a microglial process were taken at 10,000×. The alignment of images, tracing of structural elements, and 3-D reconstructions were performed with the Reconstruct software [68]. To calculate the volume of 15 extracellular spaces, we measured their area in all sections in which they appeared and multiplied their total area by the number and thickness of sections. The skull above V1 was exposed, cleaned, glued to a thin metal plate, and carefully thinned to an approximately 20- to 30-µm thickness, using a high-speed dental drill (Fine Science Tools) and a microsurgical blade [19], [24], [69]. Drilling was interrupted periodically, and sterile saline was applied on the skull to prevent heat-induced damage. A custom-made two-photon microscope [70] with a Ti∶Sapphire laser (Mai Tai; Spectra Physics) tuned to 920 nm was used for transcranial imaging. Fluorescence was detected using two photomultiplier tubes in whole-field detection mode and a 506-nm dichroic mirror with 580/180 (green channel: GFP) and 534/34 (yellow channel: GFP and YFP) emission filters. A 20× water-immersion lens (0. 95 N. A. ; Olympus) was used throughout the imaging session. Dendrites and axons near microglia, located at least 50 µm below the pial surface, were imaged under a digital zoom of 4–6×, using the FluoView software. Z stacks taken 1 µm apart were acquired every 5 min for 30 min to 2 h. To measure the resolution of two-photon imaging under our experimental conditions, we imaged subresolution fluorescent beads (0. 1–0. 2 µm diameter; Molecular Probes) to obtain the experimental point spread function. The measured 1/e radius was 0. 35 µm radially and 1. 3 µm axially, corresponding to the X/Y and Z resolution, respectively. Analysis of microglial contacts with dendritic spines (n = 5 control animals, 3 DA animals, and 3 DA+light animals) and axon terminals (n = 3 control animals), changes in synaptic structures (n = 3 and 5 control animals with terminals and spines, respectively, 3 DA animals with spines, and 3 DA+light animals with spines), dendritic spine turnover (n = 4 control animals), and microglial motility (n = 4 control animals, 4 DA animals, and 3 DA+light animals) was performed with Image J software. Axon terminals were identified as finger-like protrusions (>0. 2 µm) from the axon or bead-like structures along the axon (at least two times the axon diameter), as previously described [54]. While dendritic spine morphology exists as a continuum, different behaviors have been assigned to spines with different structures [71]. Therefore we classified dendritic spines into mushroom, thin, and stubby types based on their length and spine head volume, as previously described [24]. The only three stubby spines contacted by microglia were excluded from the analysis of spine size because of their rarity and the difficulty of measuring their fluorescence intensity. Consequently, we also removed stubby spines from the analysis of size for non-contacted and contacted dendritic spines. Among thin and mushroom spines, we determined a range of normalized dendritic spine sizes. The spines belonging to the first quarter of this range were considered “small, ” and those in the other three-quarters were identified as “large. ” Filopodia were rarely observed at these ages and were excluded from the analysis. For visualization of microglial contacts with dendritic spines and axon terminals, the green channel was arbitrarily assigned the color red and the yellow channel assigned the color green, enabling the visualization of microglia in yellow and neuronal elements in green (see Figure S6). Microglial contacts were identified manually by stepping through the Z stack without projection. All microglial contacts (colocalization of fluorescence for microglia and synaptic elements) that started and ended during imaging were included in the analysis. For analysis of changes in synaptic structures, the background was subtracted from each of the two channels and the green channel bleedthrough was subtracted from the yellow channel (see Figure S6 for uncorrected and corrected images). In the stacks unadjusted for brightness or contrast, dendritic spines and axon terminals were analyzed for fluorescence at the Z level where they appeared brightest. A line was traced through each element, and a fluorescence plot profile was created, which was then fitted to a Gaussian. Because the majority of dendritic spines are below the resolution of our two-photon microscope [72], the maximal fluorescence (amplitude of the Gaussian fit) was used to assess the relative spine size (see Figure S8C for sizes of large spines assessed with the width of the fluorescence profile [1/e1/2 radius of the Gaussian fit] to rule out underestimation of their size changes during putative contact). Since axon terminals are generally much larger than dendritic spines, the width of the Gaussian fit to the fluorescence profile was used to determine their relative size. To rule out contamination of neuronal measurements by any unsubtracted bleedthrough of microglial fluorescence, we analyzed thin fluorescent axons (n = 1 axon in each of 14 animals) that were contacted by microglia, using the height of the Gaussian fit to assess relative size. Axon size measured in this manner was relatively unchanged between populations with and without microglial contact (average size differential of −0. 1%±1%; see Figure S6D and S6E), as expected for these structurally stable elements. Size differentials of terminals and spines were calculated as the ratio of size difference with and without contact over the size without contact. In order to compare spine size between animals, we normalized spine fluorescence by the maximal fluorescence in the adjacent dendrite. Axon terminals and dendritic spines with size differentials under 1% were considered stable. For presentation purposes, we normalized the size of terminals and spines by dividing each individual structure' s average size in the presence of microglial contact (with or during) by its average size in the absence of microglial contact (without, or before or after). We also normalized the size of terminals and spines by dividing each individual structure' s average size by the size of the largest terminal or spine. To determine the turnover of dendritic spines that were or were not contacted by microglia in the first imaging session, the position of individual dendritic spines was compared between time points separated by 2 d. The proportion of eliminated dendritic spines was defined as the proportion of spines from the original population not observed on the second day of imaging. Spines located more than 0. 8 µm laterally from their previous location were considered to be new spines. For measurement of microglial motility, images centered on the cell body from five consecutive Z levels were projected into two dimensions, for each microglia and each time point analyzed (0,5, and 25 min). For each microglia, the images were aligned and grouped into a stack. Stacks were adjusted for brightness and contrast, and then binarized. For each microglia, the difference between images taken at the 0- and 5- or 25-min time points was calculated. A motility index was determined, as the proportion of the pixels that differed between the two images. Analyses were performed with Prism 5 software (GraphPad Software). All values reported in the text are mean ± standard error of the mean (SEM). For all statistical tests, significance was set to p<0. 05. Two-tailed unpaired Student' s t tests and linear regressions were used for both EM and two-photon analyses. For two-photon analyses, two-tailed paired Student' s t tests were also used to compare the size of the same dendritic spines or axon terminals in the presence versus in the absence of microglial contact. Sample size (n) represents individual animals for EM (except for correlation analysis, where n represents individual extracellular space and microglial process areas) and synaptic elements or microglial contacts for two-photon analyses (except for analysis of dendritic spine turnover, where n represents individual animals).

Title: Microglial Interactions with Synapses Are Modulated by Visual Experience Summary: Microglia are important players in immune responses to brain injury. In the event of pathological insults, microglia rapidly become activated and acquire the ability to release various inflammatory molecules that influence neuronal survival as well as synaptic function and plasticity. Similarly to macrophages in other areas of the body, activated microglia can engulf, or phagocytose, cellular debris and are believed to eliminate synapses. In the absence of pathological insult, microglia are more quiescent, but still, these immune surveillants continually sample their surrounding environment and contact neighboring cells and synapses. To further explore the roles of microglia at synapses under non-pathological conditions, we used quantitative electron microscopy and two-photon in vivo imaging to characterize the interactions between quiescent microglia and synaptic elements in the visual cortex of juvenile mice. We also examined the "activity-dependent" processes involved by preventing light exposure in a group of mice. We show surprising changes in microglial behavior during alterations in visual experience, such as increased phagocytosis of synaptic elements and interaction with subsets of structurally dynamic and transient synapses. These observations suggest that microglia may participate in the modification or elimination of synaptic structures, and therefore actively contribute to learning and memory in the healthy brain.

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Summarize: NARRATIVE CONTINUED BY THE DOCTOR--END OF THE FIRST DAY'S FIGHTING We made our best speed across the strip of wood that now divided us from the stockade, and at every step we took the voices of the buccaneers rang nearer. Soon we could hear their footfalls as they ran, and the cracking of the branches as they breasted across a bit of thicket. I began to see we should have a brush for it in earnest, and looked to my priming. "Captain," said I, "Trelawney is the dead shot. Give him your gun; his own is useless." They exchanged guns, and Trelawney, silent and cool, as he had been since the beginning of the bustle, hung a moment on his heel to see that all was fit for service. At the same time, observing Gray to be unarmed, I handed him my cutlass. It did all our hearts good to see him spit in his hand, knit his brows, and make the blade sing through the air. It was plain from every line of his body that our new hand was worth his salt. Forty paces farther we came to the edge of the wood and saw the stockade in front of us. We struck the inclosure about the middle of the south side, and, almost at the same time, seven mutineers--Job Anderson, the boatswain, at their head--appeared in full cry at the southwestern corner. They paused, as if taken aback, and before they recovered, not only the squire and I, but Hunter and Joyce from the blockhouse, had time to fire. The four shots came in rather a scattering volley, but they did the business; one of the enemy actually fell, and the rest, without hesitation, turned and plunged into the trees. After reloading we walked down the outside of the palisade to see to the fallen enemy. He was stone dead--shot through the heart. We began to rejoice over our good success, when just at that moment a pistol cracked in the bush, a ball whistled close past my ear and poor Tom Redruth stumbled and fell his length on the ground. Both the squire and I returned the shot, but as we had nothing to aim at, it is probable we only wasted powder. Then we reloaded and turned our attention to poor Tom. The captain and Gray were already examining him, and I saw with half an eye that all was over. I believe the readiness of our return volley had scattered the mutineers once more, for we were suffered without further molestation to get the poor old gamekeeper hoisted over the stockade, and carried, groaning and bleeding, into the log-house. Poor old fellow, he had not uttered one word of surprise, complaint, fear, or even acquiescence, from the very beginning of our troubles till now, when we had laid him down in the log-house to die! He had lain like a Trojan behind his mattress in the gallery; he had followed every order silently, doggedly, and well; he was the oldest of our party by a score of years; and now, sullen, old, serviceable servant, it was he that was to die. The squire dropped down beside him on his knees and kissed his hand, crying like a child. "Be I going, doctor?" he asked. "Tom, my man," said I, "you're going home." "I wish I had had a lick at them with the gun first," he replied. "Tom," said the squire, "say you forgive me, won't you?" "Would that be respectful like, from me to you, squire?" was the answer. "Howsoever, so be it, amen!" After a little while of silence he said he thought somebody might read a prayer. "It's the custom, sir," he added, apologetically. And not long after, without another word, he passed away. In the meantime the captain, whom I had observed to be wonderfully swollen about the chest and pockets, had turned out a great many various stores--the British colors, a Bible, a coil of stoutish rope, pen, ink, the log-book, and pounds of tobacco. He had found a longish fir tree lying felled and cleared in the inclosure, and, with the help of Hunter, he had set it up at the corner of the log-house, where the trunks crossed and made an angle. Then, climbing on the roof, he had with his own hand bent and run up the colors. This seemed mightily to relieve him. He re-entered the log-house and set about counting up the stores, as if nothing else existed. But he had an eye on Tom's passage for all that, and as soon as all was over came forward with another flag and reverently spread it on the body. "Don't you take on, sir," he said, shaking the squire's hand. "All's well with him; no fear for a hand that's been shot down in his duty to captain and owner. It mayn't be good divinity, but it's a fact." Then he pulled me aside. "Doctor Livesey," he said, "in how many weeks do you and squire expect the consort?" I told him it was a question, not of weeks, but of months; that if we were not back by the end of August Blandly was to send to find us, but neither sooner nor later. "You can calculate for yourself," I said. "Why, yes," returned the captain, scratching his head, "and making a large allowance, sir, for all the gifts of Providence, I should say we were pretty close hauled." "How do you mean?" I asked. "It's a pity, sir, we lost that second load. That's what I mean," replied the captain. "As for powder and shot, we'll do. But the rations are short, very short--so short, Doctor Livesey, that we're perhaps as well without that extra mouth." And he pointed to the dead body under the flag. Just then, with a roar and a whistle, a round shot passed high above the roof of the log-house and plumped far beyond us in the wood. "Oho!" said the captain. "Blaze away! You've little enough powder already, my lads." At the second trial the aim was better and the ball descended inside the stockade, scattering a cloud of sand, but doing no further damage. "Captain," said the squire, "the house is quite invisible from the ship. It must be the flag they are aiming at. Would it not be wiser to take it in?" "Strike my colors!" cried the captain. "No, sir, not I," and as soon as he had said the words I think we all agreed with him. For it was not only a piece of stout, seamanly good feeling; it was good policy besides, and showed our enemies that we despised their cannonade. All through the evening they kept thundering away. Ball after ball flew over or fell short, or kicked up the sand in the inclosure; but they had to fire so high that the shot fell dead and buried itself in the soft sand. We had no ricochet to fear; and though one popped in through the roof of the log-house and out again through the floor, we soon got used to that sort of horse-play and minded it no more than cricket. "There is one thing good about all this," observed the captain; "the wood in front of us is likely clear. The ebb has made a good while; our stores should be uncovered. Volunteers to go and bring in pork." Gray and Hunter were the first to come forward. Well armed, they stole out of the stockade, but it proved a useless mission. The mutineers were bolder than we fancied, or they put more trust in Israel's gunnery, for four or five of them were busy carrying off our stores and wading out with them to one of the gigs that lay close by, pulling an oar or so to hold her steady against the current. Silver was in the stern-sheets in command, and every man of them was now provided with a musket from some secret magazine of their own. The captain sat down to his log, and here is the beginning of the entry: "Alexander Smollett, master; David Livesey, ship's doctor; Abraham Gray, carpenter's mate; John Trelawney, owner; John Hunter and Richard Joyce, owner's servants, landsmen--being all that is left faithful of the ship's company--with stores for ten days at short rations, came ashore this day and flew British colors on the log-house in Treasure Island. Thomas Redruth, owner's servant, landsman, shot by the mutineers; James Hawkins, cabin-boy--" And at the same time I was wondering over poor Jim Hawkins' fate. A hail on the land side. "Somebody hailing us," said Hunter, who was on guard. "Doctor! squire! captain! Hallo, Hunter, is that you?" came the cries. And I ran to the door in time to see Jim Hawkins, safe and sound, come climbing over the stockade. [Illustration]

Summary: The landing party reaches the stockade just as a group of seven mutineers appear, led by Job Anderson. The mutineers seem really surprised, so Squire Trelawney, Doctor Livesey, Hunter, and Joyce all have time to fire first. One of the mutineers drops and the rest of them scatter at the gunfire. The landing party is really happy - until a bullet flies out of the trees and hits poor Tom Redruth, Squire Trelawney's loyal gamekeeper. Doctor Livesey can see at once that it's curtains for Redruth. Trelawney is crying like a baby. He asks Redruth for forgiveness. Redruth says there's no need, and that what's meant to happen will happen. Redruth asks them to read a prayer over him, then he passes away. Meanwhile, Captain Smollett is unpacking all the things he brought with him stuffed in his coat: a British flag, a Bible, a coil of rope, pen, ink, the ship's log, and lots of tobacco. Captain Smollett raises the British flag above the fort. He also has an extra flag to spread over Redruth's body. Captain Smollett then pulls Doctor Livesey aside and asks how long they should expect to have to hole up in this fort. Doctor Livesey admits that no one's going to come after them until August Captain Smollett notes that they're in a tough spot - in fact, one good thing about Redruth's death is that they have one fewer mouth to feed. A couple of cannons go over the stockade, but the pirates can't get good aim from the Hispaniola.. Squire Trelawney points out that the pirates are probably aiming for the flag, which is the only thing visible in the forest from the ship. Captain Smollett refuses to lower the flag, and everyone is comforted by his bravery. They start ignoring the cannons going overhead. Captain Smollett says the forest is probably clear of pirates, since they're willing to fire at the trees like this. Captain Smollett suggests that a couple of them go out at night to find what can be recovered of their supplies. Now that the tide has gone out, Captain Smollett hopes that the tiny rowboat will be visible again above the water. Gray and Hunter steal out of the fort to take a look. They find, unfortunately, that the pirates have gotten there first. All the pirates also seem to be armed - apparently, they brought their own weapons aboard the ship, which Captain Smollett was unaware of. Captain Smollett sits down to write a diary entry about the day, including the death of Tom Redruth. Doctor Livesey wonders what has happened to Jim Hawkins. Just then, they hear a voice calling out a greeting. It's none other than Jim, climbing over the fence of the fort.

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Summarize: "What in the world are you going to do now, Jo?" asked Meg one snowy afternoon, as her sister came tramping through the hall, in rubber boots, old sack, and hood, with a broom in one hand and a shovel in the other. "Going out for exercise," answered Jo with a mischievous twinkle in her eyes. "I should think two long walks this morning would have been enough! It's cold and dull out, and I advise you to stay warm and dry by the fire, as I do," said Meg with a shiver. "Never take advice! Can't keep still all day, and not being a pussycat, I don't like to doze by the fire. I like adventures, and I'm going to find some." Meg went back to toast her feet and read _Ivanhoe_, and Jo began to dig paths with great energy. The snow was light, and with her broom she soon swept a path all round the garden, for Beth to walk in when the sun came out and the invalid dolls needed air. Now, the garden separated the Marches' house from that of Mr. Laurence. Both stood in a suburb of the city, which was still country-like, with groves and lawns, large gardens, and quiet streets. A low hedge parted the two estates. On one side was an old, brown house, looking rather bare and shabby, robbed of the vines that in summer covered its walls and the flowers, which then surrounded it. On the other side was a stately stone mansion, plainly betokening every sort of comfort and luxury, from the big coach house and well-kept grounds to the conservatory and the glimpses of lovely things one caught between the rich curtains. Yet it seemed a lonely, lifeless sort of house, for no children frolicked on the lawn, no motherly face ever smiled at the windows, and few people went in and out, except the old gentleman and his grandson. To Jo's lively fancy, this fine house seemed a kind of enchanted palace, full of splendors and delights which no one enjoyed. She had long wanted to behold these hidden glories, and to know the Laurence boy, who looked as if he would like to be known, if he only knew how to begin. Since the party, she had been more eager than ever, and had planned many ways of making friends with him, but he had not been seen lately, and Jo began to think he had gone away, when she one day spied a brown face at an upper window, looking wistfully down into their garden, where Beth and Amy were snow-balling one another. "That boy is suffering for society and fun," she said to herself. "His grandpa does not know what's good for him, and keeps him shut up all alone. He needs a party of jolly boys to play with, or somebody young and lively. I've a great mind to go over and tell the old gentleman so!" The idea amused Jo, who liked to do daring things and was always scandalizing Meg by her queer performances. The plan of 'going over' was not forgotten. And when the snowy afternoon came, Jo resolved to try what could be done. She saw Mr. Lawrence drive off, and then sallied out to dig her way down to the hedge, where she paused and took a survey. All quiet, curtains down at the lower windows, servants out of sight, and nothing human visible but a curly black head leaning on a thin hand at the upper window. "There he is," thought Jo, "Poor boy! All alone and sick this dismal day. It's a shame! I'll toss up a snowball and make him look out, and then say a kind word to him." Up went a handful of soft snow, and the head turned at once, showing a face which lost its listless look in a minute, as the big eyes brightened and the mouth began to smile. Jo nodded and laughed, and flourished her broom as she called out... "How do you do? Are you sick?" Laurie opened the window, and croaked out as hoarsely as a raven... "Better, thank you. I've had a bad cold, and been shut up a week." "I'm sorry. What do you amuse yourself with?" "Nothing. It's dull as tombs up here." "Don't you read?" "Not much. They won't let me." "Can't somebody read to you?" "Grandpa does sometimes, but my books don't interest him, and I hate to ask Brooke all the time." "Have someone come and see you then." "There isn't anyone I'd like to see. Boys make such a row, and my head is weak." "Isn't there some nice girl who'd read and amuse you? Girls are quiet and like to play nurse." "Don't know any." "You know us," began Jo, then laughed and stopped. "So I do! Will you come, please?" cried Laurie. "I'm not quiet and nice, but I'll come, if Mother will let me. I'll go ask her. Shut the window, like a good boy, and wait till I come." With that, Jo shouldered her broom and marched into the house, wondering what they would all say to her. Laurie was in a flutter of excitement at the idea of having company, and flew about to get ready, for as Mrs. March said, he was 'a little gentleman', and did honor to the coming guest by brushing his curly pate, putting on a fresh collar, and trying to tidy up the room, which in spite of half a dozen servants, was anything but neat. Presently there came a loud ring, than a decided voice, asking for 'Mr. Laurie', and a surprised-looking servant came running up to announce a young lady. "All right, show her up, it's Miss Jo," said Laurie, going to the door of his little parlor to meet Jo, who appeared, looking rosy and quite at her ease, with a covered dish in one hand and Beth's three kittens in the other. "Here I am, bag and baggage," she said briskly. "Mother sent her love, and was glad if I could do anything for you. Meg wanted me to bring some of her blanc mange, she makes it very nicely, and Beth thought her cats would be comforting. I knew you'd laugh at them, but I couldn't refuse, she was so anxious to do something." It so happened that Beth's funny loan was just the thing, for in laughing over the kits, Laurie forgot his bashfulness, and grew sociable at once. "That looks too pretty to eat," he said, smiling with pleasure, as Jo uncovered the dish, and showed the blanc mange, surrounded by a garland of green leaves, and the scarlet flowers of Amy's pet geranium. "It isn't anything, only they all felt kindly and wanted to show it. Tell the girl to put it away for your tea. It's so simple you can eat it, and being soft, it will slip down without hurting your sore throat. What a cozy room this is!" "It might be if it was kept nice, but the maids are lazy, and I don't know how to make them mind. It worries me though." "I'll right it up in two minutes, for it only needs to have the hearth brushed, so--and the things made straight on the mantelpiece, so--and the books put here, and the bottles there, and your sofa turned from the light, and the pillows plumped up a bit. Now then, you're fixed." And so he was, for, as she laughed and talked, Jo had whisked things into place and given quite a different air to the room. Laurie watched her in respectful silence, and when she beckoned him to his sofa, he sat down with a sigh of satisfaction, saying gratefully... "How kind you are! Yes, that's what it wanted. Now please take the big chair and let me do something to amuse my company." "No, I came to amuse you. Shall I read aloud?" and Jo looked affectionately toward some inviting books near by. "Thank you! I've read all those, and if you don't mind, I'd rather talk," answered Laurie. "Not a bit. I'll talk all day if you'll only set me going. Beth says I never know when to stop." "Is Beth the rosy one, who stays at home good deal and sometimes goes out with a little basket?" asked Laurie with interest. "Yes, that's Beth. She's my girl, and a regular good one she is, too." "The pretty one is Meg, and the curly-haired one is Amy, I believe?" "How did you find that out?" Laurie colored up, but answered frankly, "Why, you see I often hear you calling to one another, and when I'm alone up here, I can't help looking over at your house, you always seem to be having such good times. I beg your pardon for being so rude, but sometimes you forget to put down the curtain at the window where the flowers are. And when the lamps are lighted, it's like looking at a picture to see the fire, and you all around the table with your mother. Her face is right opposite, and it looks so sweet behind the flowers, I can't help watching it. I haven't got any mother, you know." And Laurie poked the fire to hide a little twitching of the lips that he could not control. The solitary, hungry look in his eyes went straight to Jo's warm heart. She had been so simply taught that there was no nonsense in her head, and at fifteen she was as innocent and frank as any child. Laurie was sick and lonely, and feeling how rich she was in home and happiness, she gladly tried to share it with him. Her face was very friendly and her sharp voice unusually gentle as she said... "We'll never draw that curtain any more, and I give you leave to look as much as you like. I just wish, though, instead of peeping, you'd come over and see us. Mother is so splendid, she'd do you heaps of good, and Beth would sing to you if I begged her to, and Amy would dance. Meg and I would make you laugh over our funny stage properties, and we'd have jolly times. Wouldn't your grandpa let you?" "I think he would, if your mother asked him. He's very kind, though he does not look so, and he lets me do what I like, pretty much, only he's afraid I might be a bother to strangers," began Laurie, brightening more and more. "We are not strangers, we are neighbors, and you needn't think you'd be a bother. We want to know you, and I've been trying to do it this ever so long. We haven't been here a great while, you know, but we have got acquainted with all our neighbors but you." "You see, Grandpa lives among his books, and doesn't mind much what happens outside. Mr. Brooke, my tutor, doesn't stay here, you know, and I have no one to go about with me, so I just stop at home and get on as I can." "That's bad. You ought to make an effort and go visiting everywhere you are asked, then you'll have plenty of friends, and pleasant places to go to. Never mind being bashful. It won't last long if you keep going." Laurie turned red again, but wasn't offended at being accused of bashfulness, for there was so much good will in Jo it was impossible not to take her blunt speeches as kindly as they were meant. "Do you like your school?" asked the boy, changing the subject, after a little pause, during which he stared at the fire and Jo looked about her, well pleased. "Don't go to school, I'm a businessman--girl, I mean. I go to wait on my great-aunt, and a dear, cross old soul she is, too," answered Jo. Laurie opened his mouth to ask another question, but remembering just in time that it wasn't manners to make too many inquiries into people's affairs, he shut it again, and looked uncomfortable. Jo liked his good breeding, and didn't mind having a laugh at Aunt March, so she gave him a lively description of the fidgety old lady, her fat poodle, the parrot that talked Spanish, and the library where she reveled. Laurie enjoyed that immensely, and when she told about the prim old gentleman who came once to woo Aunt March, and in the middle of a fine speech, how Poll had tweaked his wig off to his great dismay, the boy lay back and laughed till the tears ran down his cheeks, and a maid popped her head in to see what was the matter. "Oh! That does me no end of good. Tell on, please," he said, taking his face out of the sofa cushion, red and shining with merriment. Much elated with her success, Jo did 'tell on', all about their plays and plans, their hopes and fears for Father, and the most interesting events of the little world in which the sisters lived. Then they got to talking about books, and to Jo's delight, she found that Laurie loved them as well as she did, and had read even more than herself. "If you like them so much, come down and see ours. Grandfather is out, so you needn't be afraid," said Laurie, getting up. "I'm not afraid of anything," returned Jo, with a toss of the head. "I don't believe you are!" exclaimed the boy, looking at her with much admiration, though he privately thought she would have good reason to be a trifle afraid of the old gentleman, if she met him in some of his moods. The atmosphere of the whole house being summerlike, Laurie led the way from room to room, letting Jo stop to examine whatever struck her fancy. And so, at last they came to the library, where she clapped her hands and pranced, as she always did when especially delighted. It was lined with books, and there were pictures and statues, and distracting little cabinets full of coins and curiosities, and Sleepy Hollow chairs, and queer tables, and bronzes, and best of all, a great open fireplace with quaint tiles all round it. "What richness!" sighed Jo, sinking into the depth of a velour chair and gazing about her with an air of intense satisfaction. "Theodore Laurence, you ought to be the happiest boy in the world," she added impressively. "A fellow can't live on books," said Laurie, shaking his head as he perched on a table opposite. Before he could more, a bell rang, and Jo flew up, exclaiming with alarm, "Mercy me! It's your grandpa!" "Well, what if it is? You are not afraid of anything, you know," returned the boy, looking wicked. "I think I am a little bit afraid of him, but I don't know why I should be. Marmee said I might come, and I don't think you're any the worse for it," said Jo, composing herself, though she kept her eyes on the door. "I'm a great deal better for it, and ever so much obliged. I'm only afraid you are very tired of talking to me. It was so pleasant, I couldn't bear to stop," said Laurie gratefully. "The doctor to see you, sir," and the maid beckoned as she spoke. "Would you mind if I left you for a minute? I suppose I must see him," said Laurie. "Don't mind me. I'm happy as a cricket here," answered Jo. Laurie went away, and his guest amused herself in her own way. She was standing before a fine portrait of the old gentleman when the door opened again, and without turning, she said decidedly, "I'm sure now that I shouldn't be afraid of him, for he's got kind eyes, though his mouth is grim, and he looks as if he had a tremendous will of his own. He isn't as handsome as my grandfather, but I like him." "Thank you, ma'am," said a gruff voice behind her, and there, to her great dismay, stood old Mr. Laurence. Poor Jo blushed till she couldn't blush any redder, and her heart began to beat uncomfortably fast as she thought what she had said. For a minute a wild desire to run away possessed her, but that was cowardly, and the girls would laugh at her, so she resolved to stay and get out of the scrape as she could. A second look showed her that the living eyes, under the bushy eyebrows, were kinder even than the painted ones, and there was a sly twinkle in them, which lessened her fear a good deal. The gruff voice was gruffer than ever, as the old gentleman said abruptly, after the dreadful pause, "So you're not afraid of me, hey?" "Not much, sir." "And you don't think me as handsome as your grandfather?" "Not quite, sir." "And I've got a tremendous will, have I?" "I only said I thought so." "But you like me in spite of it?" "Yes, I do, sir." That answer pleased the old gentleman. He gave a short laugh, shook hands with her, and, putting his finger under her chin, turned up her face, examined it gravely, and let it go, saying with a nod, "You've got your grandfather's spirit, if you haven't his face. He was a fine man, my dear, but what is better, he was a brave and an honest one, and I was proud to be his friend." "Thank you, sir," And Jo was quite comfortable after that, for it suited her exactly. "What have you been doing to this boy of mine, hey?" was the next question, sharply put. "Only trying to be neighborly, sir." And Jo told how her visit came about. "You think he needs cheering up a bit, do you?" "Yes, sir, he seems a little lonely, and young folks would do him good perhaps. We are only girls, but we should be glad to help if we could, for we don't forget the splendid Christmas present you sent us," said Jo eagerly. "Tut, tut, tut! That was the boy's affair. How is the poor woman?" "Doing nicely, sir." And off went Jo, talking very fast, as she told all about the Hummels, in whom her mother had interested richer friends than they were. "Just her father's way of doing good. I shall come and see your mother some fine day. Tell her so. There's the tea bell, we have it early on the boy's account. Come down and go on being neighborly." "If you'd like to have me, sir." "Shouldn't ask you, if I didn't." And Mr. Laurence offered her his arm with old-fashioned courtesy. "What would Meg say to this?" thought Jo, as she was marched away, while her eyes danced with fun as she imagined herself telling the story at home. "Hey! Why, what the dickens has come to the fellow?" said the old gentleman, as Laurie came running downstairs and brought up with a start of surprise at the astounding sight of Jo arm in arm with his redoubtable grandfather. "I didn't know you'd come, sir," he began, as Jo gave him a triumphant little glance. "That's evident, by the way you racket downstairs. Come to your tea, sir, and behave like a gentleman." And having pulled the boy's hair by way of a caress, Mr. Laurence walked on, while Laurie went through a series of comic evolutions behind their backs, which nearly produced an explosion of laughter from Jo. The old gentleman did not say much as he drank his four cups of tea, but he watched the young people, who soon chatted away like old friends, and the change in his grandson did not escape him. There was color, light, and life in the boy's face now, vivacity in his manner, and genuine merriment in his laugh. "She's right, the lad is lonely. I'll see what these little girls can do for him," thought Mr. Laurence, as he looked and listened. He liked Jo, for her odd, blunt ways suited him, and she seemed to understand the boy almost as well as if she had been one herself. If the Laurences had been what Jo called 'prim and poky', she would not have got on at all, for such people always made her shy and awkward. But finding them free and easy, she was so herself, and made a good impression. When they rose she proposed to go, but Laurie said he had something more to show her, and took her away to the conservatory, which had been lighted for her benefit. It seemed quite fairylike to Jo, as she went up and down the walks, enjoying the blooming walls on either side, the soft light, the damp sweet air, and the wonderful vines and trees that hung about her, while her new friend cut the finest flowers till his hands were full. Then he tied them up, saying, with the happy look Jo liked to see, "Please give these to your mother, and tell her I like the medicine she sent me very much." They found Mr. Laurence standing before the fire in the great drawing room, but Jo's attention was entirely absorbed by a grand piano, which stood open. "Do you play?" she asked, turning to Laurie with a respectful expression. "Sometimes," he answered modestly. "Please do now. I want to hear it, so I can tell Beth." "Won't you first?" "Don't know how. Too stupid to learn, but I love music dearly." So Laurie played and Jo listened, with her nose luxuriously buried in heliotrope and tea roses. Her respect and regard for the 'Laurence' boy increased very much, for he played remarkably well and didn't put on any airs. She wished Beth could hear him, but she did not say so, only praised him till he was quite abashed, and his grandfather came to his rescue. "That will do, that will do, young lady. Too many sugarplums are not good for him. His music isn't bad, but I hope he will do as well in more important things. Going? well, I'm much obliged to you, and I hope you'll come again. My respects to your mother. Good night, Doctor Jo." He shook hands kindly, but looked as if something did not please him. When they got into the hall, Jo asked Laurie if she had said something amiss. He shook his head. "No, it was me. He doesn't like to hear me play." "Why not?" "I'll tell you some day. John is going home with you, as I can't." "No need of that. I am not a young lady, and it's only a step. Take care of yourself, won't you?" "Yes, but you will come again, I hope?" "If you promise to come and see us after you are well." "I will." "Good night, Laurie!" "Good night, Jo, good night!" When all the afternoon's adventures had been told, the family felt inclined to go visiting in a body, for each found something very attractive in the big house on the other side of the hedge. Mrs. March wanted to talk of her father with the old man who had not forgotten him, Meg longed to walk in the conservatory, Beth sighed for the grand piano, and Amy was eager to see the fine pictures and statues. "Mother, why didn't Mr. Laurence like to have Laurie play?" asked Jo, who was of an inquiring disposition. "I am not sure, but I think it was because his son, Laurie's father, married an Italian lady, a musician, which displeased the old man, who is very proud. The lady was good and lovely and accomplished, but he did not like her, and never saw his son after he married. They both died when Laurie was a little child, and then his grandfather took him home. I fancy the boy, who was born in Italy, is not very strong, and the old man is afraid of losing him, which makes him so careful. Laurie comes naturally by his love of music, for he is like his mother, and I dare say his grandfather fears that he may want to be a musician. At any rate, his skill reminds him of the woman he did not like, and so he 'glowered' as Jo said." "Dear me, how romantic!" exclaimed Meg. "How silly!" said Jo. "Let him be a musician if he wants to, and not plague his life out sending him to college, when he hates to go." "That's why he has such handsome black eyes and pretty manners, I suppose. Italians are always nice," said Meg, who was a little sentimental. "What do you know about his eyes and his manners? You never spoke to him, hardly," cried Jo, who was not sentimental. "I saw him at the party, and what you tell shows that he knows how to behave. That was a nice little speech about the medicine Mother sent him." "He meant the blanc mange, I suppose." "How stupid you are, child! He meant you, of course." "Did he?" And Jo opened her eyes as if it had never occurred to her before. "I never saw such a girl! You don't know a compliment when you get it," said Meg, with the air of a young lady who knew all about the matter. "I think they are great nonsense, and I'll thank you not to be silly and spoil my fun. Laurie's a nice boy and I like him, and I won't have any sentimental stuff about compliments and such rubbish. We'll all be good to him because he hasn't got any mother, and he may come over and see us, mayn't he, Marmee?" "Yes, Jo, your little friend is very welcome, and I hope Meg will remember that children should be children as long as they can." "I don't call myself a child, and I'm not in my teens yet," observed Amy. "What do you say, Beth?" "I was thinking about our '_Pilgrim's Progress_'," answered Beth, who had not heard a word. "How we got out of the Slough and through the Wicket Gate by resolving to be good, and up the steep hill by trying, and that maybe the house over there, full of splendid things, is going to be our Palace Beautiful." "We have got to get by the lions first," said Jo, as if she rather liked the prospect.

Summary: Jo visits Laurie one snowy afternoon when she has nothing else to do and Laurie is shut up in his house with a cold. The two teens spend a while getting acquainted. Laurie reveals that he knows quite a bit about the sisters including their names as he often watches them through his windows. Laurie describes his grandfather as kind and generous even though he doesnt look it. He takes Jo on a tour of the house ending in the library where she is enchanted by all the books. Lauries opinion is that one cant live on books. His doctor calls, so Laurie leaves Jo in the library to wait his return. While waiting for Laurie, Jo examines the picture of his grandfather that is hanging on the wall. She talks aloud to herself, saying that she doesnt think she would be afraid of him even though he looks strong willed, for he has kind eyes. She decides that she likes him although he isnt as handsome as her own grandfather. Unknown to Jo, Mr. Laurence has come into the house and is standing in the doorway to the library when Jo makes her comments. He tells her that he knew and was a friend to her grandfather, and inquires as to her intentions in visiting his grandson. She explains that she was just being neighborly as Laurie seemed lonely. Mr. Laurence is surprised at her effect on Laurie and asks her to stay for tea. After tea, the two explore more of the house whereupon Jo finds a gand piano. Laurie plays a bit for her, which seems to displease his grandfather. When Jo returns home, her mother explains that Mr. Laurences objection to Lauries music is related to an old disagreement between Mr. Laurence and his son, Lauries father. That man had married an Italian musician whom Mr. Laurence did not like. He never saw his son again after they were married, and he fears losing Laurie if he should learn to like music too well. After Jos descriptions, Marmee decides that it would be okay to pursue a friendship with the Laurences. Beth suggests, upon recalling their Pilgrims Progress play-acting, that perhaps the big house with all of its beautiful things will be a Palace Beautiful for them.

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Summarize: FIELD OF THE INVENTION [0001] A novel synergistic herbal formulation as a brain tonic, cognition, improvement of memory and treatment of amnesia and in recalling of thoughts. BACKGROUND INFORMATION [0002] A major discovery of the past two decades in the field of neurosciences has been the elucidation of behavioral, neurobiological and cellular basis of learning and memory processes. The brain is an assembly of interrelated neural systems that regulates their owns and each other&#39;s activity in a dynamic, complex fashion. Morphological properties of central neurons have been very useful for the description of the functional characteristics. Learning is defined as the acquisition of information and skills, and subsequent retention of that information is called memory. The subsequently deterioration of retention of information which in medical term is known as “amnesia”. Accordingly, effect of a wide variety of pharmacological agents or brain lesion on cognitive behavior have been studied and most validly interpreted as “enhancement or impairment” of learning and memory process. Learning and memory can be conceived as both psychological process as well as a change in synaptic neural connectivity. The development of scientifically validated models of ischemia induced-amnesia is vital to the analysis of the functional consequences of ischemic damage and to testing the behavioral efficacy of potentially therapeutic drugs. The role of medicinal plants in increasing the memory and acting as a brain tonic is still much underestimated. Besides this, certain oils have been found to be used as sedatives, central nervous system stimulants, adaptogens, bronchodilators, anti-stress and muscle relaxants (Singh et al, 2000). During late prenatal and early postnatal brain development, the cholinergic system in the central nervous system plays an important role in learning and memory function and that brain cholinergic hypofunction causes dementia with symptoms such as memory loss and disorientation in cerebrovascular or alzheimers disease (Coyle et al 1983). Following cerebral ischemia, a reduction in the cerebral blood flow and blood oxygen occur. It has also been reported that hypoxia induces a reduction of memory and judgement that is associated with a decrease in acetylcholine synthesis (Gibson and Duffy, 1981). Principally, main characteristic of memory formation in animals, as well as in human being, is its progression from a short-lived labile form to a long-lasting stable form. During this period of consolidation, memory can be disrupted by administration of a wide variety of amnesia-inducing agents. Electroconvulsive shock, hypothermia and hypoxia are non-invasive procedures that can render the animal unconscious, inducing retrograde amnesia through mechanisms correlated to the practical utility to the clinical drugs. The retrieval hypothesis postulates that amnesic agents disrupt memory recall rather than storage, as the effect of some agents diminish over time resulting in the reappearance of normal memory retention. The consolidation of information is mediated by limbic structures, with the hippocampal formation particularly playing a key role in memory processing. The major pathways have been proposed in the limbic system and cortical structures as being responsible for the neuronal interconnection of information processing. Drugs like amphetamine, caffeine-containing substances which has a stimulant activity on memory. Accordingly, studies shown that the herbal formulation(s) having the property of improving the memory and used in treatment of amnesia as a brain tonic and acting as a central antioxidant. OBJECT OF THE INVENTION [0003] The main object of the present invention provides a synergistic herbal formulation as a brain tonic, cognition, recalling of thoughts and as an antioxiadant capable of treating or preventing amnesia and having property for improving memory. [0004] Another object of the present invention provides a method of preparing a synergistic herbal formulation as a brain tonic, cognition, recalling of thoughts and as an antioxiadant capable of treating or preventing amnesia and having property for improving memory. [0005] Yet another object of the present invention provides a use of synergistic herbal formulation as a brain tonic, cognition, recalling of thoughts and as an antioxiadant capable of treating or preventing amnesia and having property for improving memory. SUMMARY OF THE INVENTION [0006] The present invention provides a herbal formulation useful in the treatment of herbal dosage form from the seed oil of Sesamum indicum used as a brain tonic and cognition. The herbal oil comprising of sesamin, sesamolin, sesamol (a phenolic antioxidant) vitamins, proteins and aminoacids. Sesame oil varies from light to deep reddish yellow in colour. It is used as nourishing food and flavoring agent. Sesamum seeds are considered as emollient, diuretic, lactagogue and a nourishing tonic and said to be useful in curing bleeding piles and also from the fresh leaves extract of centilla asiatica that is having a potential memory enhanching role and also we have found to produce transquilizing effects. The extracts comprising of centoic acid, centellic acid, oleic acid linolic acid, linolenic and lingocericacid. It is used as acures for leucoderma, bronchitis, kapha, enlargement of spleen (Ayurveda). It is also used as a cardio tonic diuretic and also used to improve appetite (Yunani). It was shown that it produce a significant improvement in general ability and behavioural pattern. DETAILED DESCRIPTION OF THE INVENTION [0000] Sesamum indicum Linn. Family: Pedaliacae [0007] Botanical description: A genus of annual or perennial herbs or occasionally shrubs found in the warmer regions of Africa, Asia and Australia. About six species are recorded in India of which Sesamum indicum is widely cultivated. An erect, branched or un branched annual 60-180 cm high, cultivated throughout the plains of India and upto an altitude of 1,200 m. Leaves 7.5-12.5 cm simple (or) when variable, with upper ones narrowly oblong, middle ones ovate and toothed and the lower ones lob ate or pedatisect. Flowers white, pink or mauve pink with darker markings, borne in racemes in the leaf axils, fruit capsular, oblong. Quadrangular, slightly compressed, deeply 4-grooved, 1.5-5 cm long, seeds black, brown or white 2.5-3 mm long and 1.5 mm broad. (Wealth of India, 1992) [0008] Medicinal uses: seasame seed is used as a nourishing food and also as flavouring agent. It is invariably dehulled for use of food. The method of dehulling consists in soaking the seed in cold water overnight, followed by partial drying and rubbing against a rough surface. Sesamin and sesamolin exhibit little antioxidant activity. (Wealth of India, 1992) [0009] Phytochemistry: The oleaginous edible seeds of Sesamum indicum esteemed for their oil, have acquired, in recent years, additional importance as a source of protein for human nutrition. It varies in colour from white, through brown, to black. Analyses of seeds grown in various parts of the world for their proximate composition gave values which lies within the ranges (in g/100 g, of dry seed): moisture, 4.1-6.5; ether extr., 43.0-56.8; protein 17.6-26.4; crude fibre, 2.9-8.6; carbohydrates, 9.1-25.3; it also contain vitamins, fairly rich in thiamine and niacin. It also contains other vitamins like riboflavin, nicotinic acid, 80.0; pantothenic acid, 9.5; and ascorbic acid in trace amount. It also contains carbohydrates from the alcoholic extraction of deffated seed meal (% dry-matter basis) like glucose, 2.6; sucrose, 0.57; galactose, 1.1; and raffinose in trace amount. I contain the principal protein globulin (alpha and beta globulin). Sesame oil is rich in oleic and linoleic acids, which together constitute account for 85 percent of total fatty acids. The main constituent of studying its minor constituents of the oil contain two constitutes, sesamin and sesamolin, which are not found in any vegetable oil and responsible for the synergistic effect on the action of insecticides. Another compound, sesamol, aphenolic antioxidant, is usually present in traces. [0010] Pharmacology: The sesamum oil having the antioxidant activity. Sesamum seeds are considered emollient, diuretic, lactagogue and a nourishing tonic. They are said to be helpful in piles, a paste of seeds mixed with butter being used in bleeding piles. A decoction of seeds is said to be an emmenagogue and also use in cough. Combined with linseed, the decoction of seeds is used as an aphrodisiac. A plaster made of ground seeds are applied to burns, scalds, and etc. and a poultice of the seeds is applied to ulcers. Powdered seeds are used in amenorrhoea and dysmenorrhoea (Kirt, &amp; Basu, II, 1859; Nadkarni, I, 1128). [0000] Centella asiatica Family: Umbelliferae [0011] Botanical description: A slender herbaceous creeping; stem long, prostate coming off from the leaf-axils of a vertical rootstock, filiform, often reddish, and with long internodes, rooting at the nodes. Leaves 1.3-6.3 cm in diameter., several from the rootstock which often have much elongated petioles, and 1-3 from each nodes of the stems, orbulicar, reniform, rather broader than long, more or less cupped, entire or shallowly crenate, glabrous on both sides, and with numerous slender nerves from a deeply cordate base; petioles very variable in length 7.5-15 cm long or more, channelled, glabrous or nearly so; stipule short, adnate to the petioles forming a sheating base. Flower in fasicicled umbel consisting of 34 pink, sessile (rarelt pedicelled) flowers; [0012] peduncles pubescent or glabrous, short, pink bracts ovate, acute, concave, 2 beneath each umbel. Calyx-teeth 0. petals minute, pink, ovate acute. Fruit 4 mm. Long, longer than broad, ovoid, hard, with thickened pericarp, reticulate-rugose, often crowned by the persistent petals, the peimary and secondary ridges distinct. Distributed through India, Ceylon and also in tropical and subtropical region of the world. [0013] Medicinal uses: The plant is Acrid, bitter, digestible, laxative, cooling effect, tonic, and antipyretic, improve appetite (Yunani), cures leucoderma anaemia, urinary discharge, disease of blood, use in insanity (Ayurveda). The plant has bad taste; soporific, sedatives to the nerves, acts as a cardiotonic clears the voice and the brain; cures hiccough, headache. The plant is considered as a useful alernative and tonic in diseases of skin, nerves. In some part of India, the people are in the habit of taking the powdered dried leaves with milk for improving their general intelligence the leaves are said to be useful in syphilitic skin diseases, both externally and internally; and on the malabar coast, the plant is one of the remedies for leprosy. It is also a popular remedy for slight dysenteric derangement of bowls to which children are subject: three or four leaves are given with cumin and sugar, and the pounded leaves are applied to navel. In konkan, one or two leaves are given every morning to cure stuttering; and the juice is applied (generally as a lep with Cadamba bark, and black cumin) to skin eruption supposed to arise from heat of blood. [0014] Phytochemistry: The alcoholic extract of herb an essential oil, green in colour and possing the strong odour of the herb, fatty oil, sitosterol and a resinous substance have been obtained. The fatty oil consists of the glyceride, linolic, lignoceric, palmitic and stearic acid. An alkaloid hydrocortylin has been obtained from the dried plant. Vellarine, pectic acids are present in the leaves and roots. The plants also contain ascorbic acid in a conc. Of 13.8 mg %. A glycoside asiaticoside has been isolated from the plant. The major componant of the triterpine mixture is centoic acid. [0015] Pharmacology: The ususal dose for the oral administration is 5-10 grains of the plant powder thrice daily. In larger doses, the drug is a simplifying narcotic, producing giddiness and some times coma. The alcoholic extract produce tranquillising effect in rats. It was found non-toxic up to a dose of 350-mg/kg i.p. The alcoholic and aqueous extracts antagonise spontaneous contraction and also caused relaxation of musculature of isolated ileum of rat. The alcoholic extract was found to have depressant effect in rat in toxic doses. The glycosidal fractions have a sedative action in rats. It decreases the tone and diminished the amplitude of contractions of isolated ileum of rabbit and albino rat. In anaesthetised dogs, it produces slight respiratory stimulation, hypotension and bradycardia. The alcoholic extract of entire plant was found to possess anti-protozoal activity against E. histolytica. (Wealth of India, 1992, 115-118; Kirtikar and Basu, Indian Medicinal Plant, Vol 5, 2001 p. 219). Accordingly, the main embodiment of the present invention relates to a synergistic herbal formulation as a brain tonic, cognition, recalling of thoughts and as an antioxiadant capable of treating or preventing amnesia and having property for improving memory, said formulation comprising pharmaceutically acceptable amounts of extracts from plants Centella asiatica and Sesamum indiucm optionally along with acceptable salt/s, carrier/s or dilutent/s. [0016] Another embodiment of the present invention relates to a method of preparing a synergistic herbal formulation as a brain tonic, cognition, recalling of thoughts and as an antioxiadant capable of treating or preventing amnesia and having property for improving memory as a brain tonic and as an antioxiadant capable of treating or preventing amnesia and having property for improving memory, said method comprising steps of: (a) extracting the powdered material obtained from seeds of Sesamum indicum and leaves of Centella asiatica in aqueous alcohol, (b) filtering the extract of step (a) to remove the debris, (c) concentrating and lyophislizing the filtrate obtained from step (b) at a temperature of less than about 55° C., and (d) mixing the plant extracts obtained in step (c) with carbohydrates of about 70% and alcohol of about 12% to make a volume of 100 ml to obtain the formulation [0021] Yet another embodiment of the present invention relates to the aqueous alcohol in steps (a) and (d), wherein the aqueous alcohol is ethanol. [0022] One more embodiment of the present invention relates to the aqueous alcohol in step (a) wherein the aqueous alcohol is about 60%. [0023] Another embodiment of the present invention relates to the aqueous alcohol in step (a) wherein the aqueous alcohol is about 50%. [0024] One more embodiment of the present invention relates to the temperature wherein the temperature in the step (b) is about 50° C. [0025] Yet another embodiment of the present invention relates to the carbohydrates, wherein the carbohydrates in step (d) are selected from sucrose or lactose. [0026] In one more embodiment of the present invention relates to the carbohydrates, wherein carbohydrate concentration is about 66%. [0027] Another embodiment of the present invention relates to the method of treating and or preventing amnesia and improving memory in mammals, particularly humans said method comprising administering synergistic herbal formulation of extracts from plants Centella asiatica and Seasmum indicum optionally along with pharamaceutically acceptable salt/s, carrier/s or dilutent/s to a subject. [0028] Another embodiment of the present invention relates to Sesamum indicum oil and Centella asiatica oil wherein Sesamum indicum oil is in the range of about 2-20% and Centella asiatica oil is in the range of about 1-15%. [0029] One more embodiment of the present invention relates to Sesamum indicum oil and Centella asiatica oil wherein Sesamum indicum oil is about 10% and Centella asiatica oil is 5%. [0030] In another embodiment of the present invention relates to the extract of the formulation wherein the said formulation comprises Sesamum indicum oil is about 4% and Centella asiatica oil is about 2%. [0031] Another embodiment of the present invention relates to the pharmaceutically acceptable dilutent/s, carrier/s, salt/s, wherein said pharmaceutically acceptable dilutent/s, carrier/s, salt/s are selected from group comprising of lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, sodium chloride or dicalcium phosphate. [0032] Still another embodiment of the present invention relates to the formulation, wherein the said formulation has a high antioxidant, cooling, oleaginous, diuretic and nerve relaxant properties. [0033] Yet another embodiment of the present invention relates to the formulation wherein said formulation may be delivered in form of capsule, tablet, syrup, suspension, pills or elixirs. [0034] Another embodiment of the present invention relates to the extract of the formulation wherein said extract of the formulation is obtained from leaves of Centella asiatica and seeds of Sesaumum indicum. [0035] One more embodiment of the present invention relates to the plant parts, wherein plant parts are selected from a group consisting of seeds of white and black varieties and leaves. [0036] In another embodiment of the present invention relates to the use of formulation wherein said formulation is used for curing migraine, vertigo, leucoderma, anaemia and improve appetite. [0037] Yet another embodiment of the present invention relates to the formulation, wherein formulation may be used for curing wounds, fractures, syphilitic skin diseases, both externally and internally and also in treatment of leprosy and to ameliorate the symptoms of disease and to improve the general health of the patient. [0038] Still another embodiment of the present invention relates to the formulation wherein the said formulation is used to reduce the pain of piles, stomachic, and enlargement of spleen. [0039] Another embodiment of the present invention relates to the dosage of the formulation wherein said dosage of the formulation in the range of about 20-110 mg/kg does not show abnormality of the locomotor activity, on passive avoidance test showed significant and dose dependent activity, showed significant and dose dependent antioxidant activity of the frontal cortex and of striatum regions of the brain. [0040] Another embodiment of the present invention relates to the dosage of the formulation wherein said dosage of the formulation in the range of about 25-100 mg/kg does not show abnormality of the locomotor activity, on passive avoidance test shows significant and dose dependent activity and shows significant and dose dependent antioxidant activity of the frontal cortex and of striatum regions of the brain. [0041] Still another embodiment of the present invention relates to the formulation wherein formulation reduces the latency period in the range of about 0.05 to 2.0 seconds. [0042] Still another embodiment of the present invention relates to the formulation wherein formulation reduces the latency period in the range of about 0.18 to 1.22 seconds. [0043] Another embodiment of the present invention relates to the formulation wherein said formulation lowers the number of mistakes in the range of about 1 to 35. [0044] Another embodiment of the present invention relates to the formulation wherein said formulation lowers the number of mistakes in the range of about 6.1 to 27. [0045] One more embodiment of the present invention relates to the formulation, wherein the said formulation enhances the body weight in the range of about 140 to 170 gms. [0046] One more embodiment of the present invention relates to the formulation, wherein the said formulation enhances the body weight in the range of about 141.6 to 168.7 gms. [0047] Still another embodiment of the present invention relates to the formulation, wherein the said formulation enhances the kidney weight in the range of about 0.80 to 1.5 gms. [0048] Still another embodiment of the present invention relates to the formulation, wherein said formulation enhances the kidney weight in the range of about 0.82 to 1.03 gms. [0049] Yet another embodiment of the present invention relates to the formulation wherein said formulation enhances the liver weight in the range of about 4 to 7 gms. [0050] Yet another embodiment of the present invention relates to the formulation wherein said formulation enhances the liver weight in the range of about 5.26 to 6.42 gms. [0051] One more embodiment of the present invention relates to the formulation wherein said formulation enhances the spleen weight in the range of about 0.60 to 0.80 gms. [0052] One more embodiment of the present invention relates to the formulation wherein said formulation enhances the spleen weight in the range of about 0.63 to 0.76 gms. [0053] Another embodiment of the present invention relates to the formulation wherein said formulation under non-stress conditions lowers the lipid peroxidase (LPO) activity in the frontal cortex and stratium regions of the brain in the range of 1.0 to 5.0. [0054] Another embodiment of the present invention relates to the formulation wherein said formulation under non-stress conditions lower the lipid peroxidase (LPO) activity in the frontal cortex and stratium regions of the brain in the range of 0.74 to 3.48. [0055] Still another embodiment of the present invention relates to the formulation wherein said formulation under non-stress conditions enhances the catalase (CAT) activity in the frontal cortex and stratium regions of the brain in the range of 22 to 40. [0056] Still another embodiment of the present invention relates to the formulation wherein the said formulation under non-stress conditions enhances the catalase (CAT) activity in the frontal cortex and stratium regions of the brain in the range of 24.5 to 35.3. [0057] Another embodiment of the present invention relates to the formulation wherein said formulation under non-stress conditions enhances the superoxide dismutase (SOD) in the frontal cortex and stratium regions of the brain activity in the range of 22 to 40. [0058] Another embodiment of the present invention relates to the formulation wherein said formulation non-stress conditions enhance the superoxide dismutase (SOD) activity in the frontal cortex and stratium regions of the brain in the range of 23.2 to 30.3. [0059] Yet another embodiment of the present invention relates to the formulation wherein the said formulation under stress conditions lower the LPO activity in the frontal cortex and stratium regions of the brain in the range of about 1 to 7. [0060] Yet another embodiment of the present invention relates to the formulation wherein the said formulation under stress conditions lower the LPO activity in the range of about 2.8 to 4.86. [0061] One more embodiment of the present invention relates tot the formulation wherein the said formulation under chronic stress conditions enhance CAT activity in the frontal cortex and stratium regions of the brain in the range of 10 to 25. [0062] One more embodiment of the present invention relates tot the formulation wherein the said formulation under chronic stress conditions enhance CAT activity in the frontal cortex and stratium regions of the brain in the range of 12.4 to 22.5. [0063] Yet another embodiment of the present invention relates to the formulation wherein the said formulation under chronic stress conditions lower the SOD activity in the frontal cortex and stratium regions of the brain in the range of 20 to 35. [0064] Yet another embodiment of the present invention relates to the formulation wherein the said formulation under chronic stress conditions lower SOD activity in the frontal cortex and stratium regions of the brain in the range of 21 to 33. [0065] The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. EXAMPLES Example 1 [0066] The invention is further illustrated by the following non-limiting examples. Formulation (F1) Sesamum indicum 2 wt. % Sucrose/Lactose 66.7 g/1.2 g Alcohol 10 wt. % Water q.s. to make 100 ml [0067] Formulation (F2) Centella asiatica 2 wt. % Sucrose/Lactose 66.7 g/1.2 g Alcohol 10 wt. % Water q.s. to make 100 ml [0068] Formulation (F3) Sesamum indicum 4 wt. % Centella asiatica 2 wt. % Sucrose/Lactose 66.7 g/1.2 g Alcohol 10 wt. % Water q.s. to make 100 ml Sesamum indicum, and Centella asiatica were collected and dried in shade. The dried material (1 Kg) is then powdered and extracted with 50% aqueous alcohol (3 L) for 5 days. At the end of this, the solvent is decanted and filtered if necessary to remove the plant debris. The extract is then concentrated under vacuum at less than 50° C. Then the extract is lyophilised to obtain the extract in powder form. Mix the plant extracts and dissolve them in 500 ml 10% alcohol, filter the solution and add specified quantity of sugar and heat the until the sugar dissolves and then cool and make up the volume with required amount of water to make 100 ml. [0069] The formulation is useful to a brain tonic and cognition. Accordingly, the investigation deals with the oral dosage form has been described in detail giving the formula of the ingredients along with the method and mode of usage of the standardzed edible oil. [0000] Locomotor Activity: [0070] The locomotor activity was measured by an open-field method. The apparatus put in the soundproof, darkened room was a round open field (bottom diameter, 60 cm; height, 50 cm). The bottom was divided into 19 parts that were equal in area. A 100-W lamp was positioned 80 cm above the bottom each rat was placed at the center of the open field and the spontaneous activity (ambulation and rearing) was recorded for 5 min. [0000] Passive Avoidance Task (Step-Down Test): [0071] A step-down passive avoidance was examined using apparatus consisted of a box (25×25×40 cm), a floor with stainless-steel grid 2 mm in diameter at 8-mm intervals, and a rubber platform (4 cm diameter, 4 cm height) set on the grid in one corner. Electric stimulation was given through the grid connected with a scrambled shock generator. After 24 hr of cerebral ischemia/scopolamine (0.4 mg/kg, i.p.), an acquisition trail was performed. For this trial, each rat was placed gently on the platform and allowed to habituate freely for 3 min, and then electric shock (0.4 mA) were delivered to the grid. If the rat stepped down from the platform, the electric shock was delivered to the rat on the grid floor. The cut off time was 2 min. A retention trail was performed 24 hr after the acquisition trail. Each rat was again placed on the platform. The time (step-down latency) that elapsed until the rats stepped down from the electric grid of the platform to shock free zone was recorded. If the rat did not step down from the platform within 2 min&#39;s, the retention trail was terminated and the maximal step down latency of 2 min was recorded. An error was counted when ever the rat stepped down from the platform and the number of error made in 2 min was recorded (Tables 1 to 4). [0000] Foot Shock-Induced Chronic Stress: [0072] The rats were subjected to daily 1 hr footshock through a grid floor in a Perspex box for 21 days. The duration of each shock (2 mA) and the intervals between the shock was randomly programmed between 3-5 sec and 10-110 sec, respectively and brain tissue was separated for the detailed central antioxidant enzymes (Tables 5 to 6). TABLE 1 Effect of formulation F1 on impairment of memory acquisition in step-down test in mice Treatment Memory parameters (Mg/kg) Latency (sec) No of mistakes Control 3.81 ± 0.01 20.2 ± 3.5 Scopalamine 0.4 7.7 ± 0.04 c 66.8 ± 8.9 c F1 25 7.2 ± 0.03 y 45.3 ± 7.6 F1 50 6.9 ± 0.03 y 31.5 ± 3.4 x F1 100 3.26 ± 0.02 y 10.0 ± 2.9 y P: c &lt;0.001 compared to control group. P: x &lt;0.01 and y &lt;0.001 compared to scopolamine group. Note: There is no mortality/gross abnormality was observed in the animals during the treatment of Sesamum indicum oil. [0073] The formulation F1 contains the Sesamum oil only [0074] The results of the table 1 represent a dose response decrease in the number of mistakes done by the animals. Whereas the scopolamine treated group showed a significant increase in the number of mistakes. Therefore latency period was increased with F1 formulation and showed a significant result. TABLE 2 Effect of formulation F2 on impairment of memory acquisition in step-down test in mice Treatment Memory parameters (Mg/kg) Latency (sec) No of mistakes Control 4.32 ± 0.02 21.2 ± 3.6 Scopalamine 0.4 7.8 ± 0.03 c 65.3 ± 8.6 c F2 25 6.3 ± 0.02 x 53.2 ± 7.1 F2 50 5.9 ± 0.02 x 46.2 ± 7.3 x F2 100 4.2 ± 0.01 x 32.1 ± 4.1 y P: c &lt;0.001 compared to control group. P: x &lt;0.05 and y &lt;0.01 compared to scopolamine group. Note: There is no mortality/gross abnormality was observed in the animals during the treatment of without Sesamum indicum oil containing formulation. [0075] The formulation F2 contains Centella asiatica only. The result showed a significant decrease in number of mistakes but when we see the table 1 the number of mistakes done with F1 formulation is less than F2 formulation. Whereas the scopolamine showed a significant increase in number of mistakes. TABLE 3 Effect of formulation F3 on impairment of memory acquisition in step-down test in mice Treatment Memory parameters (Mg/kg) Latency (Sec) No of mistakes Control 3.89 ± 0.02 20.9 ± 3.2 Scopalamine 0.4 7.8 ± 0.04 c 69.8 ± 8.9 c F3 25 1.2 ± 0.02 x 20.3 ± 6.9 x F3 50 0.8 ± 0.03 x 10.5 ± 3.4 x F3 100 0.2 ± 0.02 x 2.9 ± 3.2 x Tacrine 1 0.1 ± 0.02 x 2.4 ± 2.6 x P: c &lt;0.001 compared to control group. P: x &lt;0.05 and y &lt;0.01 compared to scopolamine group. Note: There is no mortality/gross abnormality was observed in the animals during the treatment of Sesamum indicum oil containing formulation. [0076] F3 formulation contains Sesamum indicum plus Centella asiatica. [0077] The results of Table 3 represents a highly significant effect with the dose. Whereas Tacrine is a positive control showed a better result but as a synthetic drug the side effect on saturation of various receptors cannot be ignored. The scopolamine treated animals showed negative results of losing the memory and increased the number of mistakes. Therefore F3 formulation showed a synergetic effect than that of F1 (Table 1) and F2 (Table 2) formulations. [0078] Tacrine (1,2,3,4-tetrahydro-5-aminoacridine or THA) (Summers et al, Clinical Tox 1980; 16(3): 269-281) is more effective in improving memory in Alzheimer&#39;s patients and used to treat the symptoms of Alzheimer&#39;s disease, but it does not cure the disease and it also upset the stomach, vomiting, diarrhea, heartburn, muscle aches, headache, loss of appetite etc. TABLE 4 Effect of formulation (F3) on relative mean ± SEM organ weights of rats (n = 6) Type of Treatment treatment group Body weight (g) Kidney (g) Liver (g) Spleen (g) 6 days Control 150.8 ± 10.1 0.93 ± 0.05 5.81 ± 0.44 0.64 ± 0.05 oral F3 25 154.2 ± 11.6 0.98 ± 0.05 5.85 ± 0.59 0.67 ± 0.04 treatment F3 50 152.5 ± 10.9 0.91 ± 0.09 5.97 ± 0.45 0.74 ± 0.2 F3 100 157.2 ± 11.5 0.97 ± 0.07 5.88 ± 0.62 0.68 ± 0.04 [0079] F3 formulation contains mixure of Sesamum indicum and Centella asiatica. [0080] The results of the table 4 shows there is no significant changes in body weight of various vital organs in the body in toxicity studies. [0081] Therefore the formulation F3 is highly effective (Table 3) and it is safe (Table 4). [0082] Note: No mortality/gross abnormality was observed in the animals during the treatment of Sesamum indicum oil containing formulation. TABLE 5 Effect of formulation F3 in chronic stress (CS) induced perturbations in frontal cortex of brain region and the levels of superoxide dismutase (SOD), catalase (CAT), and lipid peroxidase (LPO) Treatment Groups (mg/kg) n LPO CAT SOD I Normal 10 2.94 ± 0.5 21.4 ± 2.2 19.3 ± 2.1 II F3 50 8 1.71 ± 0.3 25.2 ± 0.7 a 24.6 ± 1.4 III F3 100 8 1.44 ± 0.7 30.1 ± 1.2 b 27.1 ± 1.2 a IV Normal + CS 10 5.62 ± 0.8 9.8 ± 0.9 39.7 ± 2.6 V F3 50 + CS 8 3.91 ± 0.6 13.2 ± 0.8 y 24.2 ± 2.9 y VI F3 100 + CS 8 2.81 ± 0.8 x 16.1 ± 0.7 z 29.6 ± 2.8 z P: a &lt;0.05 and b &lt;0.01 compared to group I. P: x &lt;0.05, y &lt;0.01 and z &lt;0.001 compared to group IV. Values are mean±SEM for six mice in each Group II and Group III compare with Group I Group V and Group VI compare with Group IV [0086] The F3 formulation contains Sesamum indicum and Centella asiatica. [0087] The results of table 5 represents a significant antioxidant activity by increasing the levels of catalase (CAT) and superoxide dismutase (SOD) in frontal cortex of brain region as such with F3 formulation and also in chronic stress (CS) with F3 formulation. The lipid peroxidase (LPO) product was scavenged in higher dose with F3 formulation and the levels were lowered. Therefore the F3 shows antioxidant activity in frontal cortex in brain. [0088] Note: There is no mortality/gross abnormality was observed in the animals during the treatment of Sesamum indicum oil. TABLE 6 Effect of formulation (F3) on chronic stress (CS) induced perturbations in stratium of brain region and the levels of superoxide dismutase (SOD), catalase (CAT), and lipid peroxidase (LPO). Treatment Groups (mg/kg) n LPO CAT SOD I Normal 10 3.91 ± 0.9 26.4 ± 1.4 23.2 ± 1.2 II F3 50 8 2.68 ± 0.8 32.2 ± 0.9 28.9 ± 1.4 III F3 100 8 1.95 ± 0.7** 34.1 ± 1.2* 34.7 ± 1.4* IV Normal + CS 10 6.64 ± 0.8 13.8 ± 0.7 43.5 ± 1.7 V F 3 50 + CS 8 3.96 ± 0.9 17.8 ± 0.6* 27.1 ± 0.9** VI F 3 100 + CS 8 3.78 ± 0.8* 21.6 ± 0.9** 21.8 ± 0.8** P: *&lt;0.05 and ** &lt;0.01 compared to Group I. P: *&lt;0.05 and ** &lt;0.001 compared to Group V and Group VI. Values are mean±SEM for six mice Group II and Group III compare with Group I Group V and Group VI compare with Group IV [0092] The results showed with the F3 formulation contains Sesamum indicum and Centella asiatica a significant antioxidant activity such as with F3 formulation (Group II and III) and also with chronic stress (CS) in F3 formulation (Group V and VI) showed significant antioxidant activity by scavenging free radicals lipid peroxide (LPO) and increased the levels of catalase (CAT) and SOD (super oxide dismutase). [0000] Note: There is no mortality/gross abnormality was observed in the animals during the treatment of Sesamum indicum oil containing formulation. REFERENCES CITED [0000] 1. U.S. Pat. No. 6,187,313, February, 2001, Segelman 2. U.S. Pat. No. 5,728,384, March, 1998, Tokuyama 3. Amani E. Khalifa, J Of Ethnopharmacology, 76, pp. 49-57, 2001. 4. Coyle et al, Science, 219, pp 1184-1189, 1983. 5. Eichenbaum, H., Stewart, C. and Morris, R. G., 1990. Journal of Neurosciences 10, pp. 3531-3542 1990. 6. Gibson et al J of Neuro Chemistry, 36, pp 28-33, 1981 7. Gogte. Ayurvedic Pharmacology and therapeutic use of medicinal plants. Bharatiya vidya Bhavan, Mumbia, India 2000. 8. Jinghua Xu et al, J of Ethnopharmacology, 73, pp. 405-413, 2000. 9. Mahajan et al. Int J Food Sci Nutr. 53(6) pp. 455-463, 2002. 10. Robert W. Flint, Jr, Behavioural Brain Research, 142, pp. 217-228, 2003 11. Sheila M. Mihalik, neurobiology of learning and memory, 80, pp. 55-62, 2003. 12. Singh et al J of Medicinal and Aromatic Plant sciences 22, pp 732-738, 2000. 13. So Ra Kim et al, Cognitive Brain Research, 17, pp. 454-461, 2003. 14. Tzen et al. Plant Physiol, 101(1), pp. 267-276, 1993. 15. Zaghloul and Prakash. Nahrung, 46 (5), pp. 364-369, 2002. 16. Wealth of India, pp 116-118, 1994. 17. Kirtikar &amp; Basu. Indian medicinal plants, vol 5, pp 1658-1663. 18. Satyavati. Medicinal plants of India, vol 1, pp 216-220. 19. Veerendra kumar M H, Gupta Y K. Clin Exp Pharmacol Physiol. May-June; 30(5-6), pp 336-42.2003 20. Veerendra kumar M H, Gupta Y K. J Ethnopharmacol, February; 79(2), pp 253-260. 21. K. Nalini et al. Fitotherpia vol LXIII, No 3, pp 232-237. 1992. 22. “Charaka Samhita Chikitsa Sthana”, Ist Chapter, 3 rd pada, 30 th stanza. 23. Mukerji B, “Indian Pharmaceutical Codex”, New Delhi, India, 1953, pp 60.

Summary: The invention provides a novel herbal formulation used to improve the memory and in treatment of amnesia as a brain tonic. Formulation(s) comprises of oleaginous oil of Sesamum indicum and the alcoholic extract of Centella asiatica. Conventionally used as emulsion or as a soft gelatin capsule for oral dosage forms. Sesamum indicum used in paralysis, aphrodisiac and dysmenorrhoea. The plant of Centella asiatica is considered as a useful alternative and tonic in diseases of the skin, nerves and blood.

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Write a title and summarize: Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms. Host pattern-recognition receptors (PRRs) detect nucleic acid from invading viruses or necrotic cells and trigger a series of signaling events that lead to the induction of type I interferons (IFNs), which plays a central role in autoimmune diseases as well as protective immune responses against viruses, respectively [1], [2]. Much progress has been made to characterize viral nucleic acid-triggered signaling pathways that result in transcriptional activation of type I IFN genes. A family of DExD/H box RNA helicases consisting of retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and LGP2 are RNA sensors and recruit the adaptors VISA (also called MAVS, IPS-1 and Cardif) [3]–[6] and MITA (also known as STING, MPYS and ERIS) [7]–[10]to activate the transcription factors NF-κB and interferon regulatory factor (IRF) 3 or IRF7, leading to transcriptional induction of the genes encoding type I IFNs and other antiviral effectors [2]. A number of DNA sensors have been identified, including DAI, IFI16, DDX41 and MRE11 which may function in a ligand- and/or cell-type-specific manner [11]–[14]. In addition, cyclic GMP-AMP synthase (cGAS) has been recently characterized as a viral DNA sensor in almost all types of cells [15]. These sensors depend exclusively on the adaptor MITA to activate NF-κB and IRF3/7 and lead to subsequent induction of type I IFNs [16]. MITA is localized to the endoplasmic reticulum (ER), mitochondria-associated membrane and mitochondria [7], [8], [10], [17]. Upon viral infection, MITA translocates to intracellular membrane-containing compartments to form punctate aggregates and acts as a scaffold protein to facilitate the phosphorylation of IRF3 and STAT6 by the kinases TBK1 and IKKε [18]–[21]. Recent studies also suggest that MITA is a direct sensor that recognizes cyclic dinucleotides such as c-di-AMP, c-di-GMP and cGAMP generated from self and viral DNA infection [22]–[24]. It has been demonstrated that MITA undergoes various post-translational modifications and such modifications are key to the activity and stability of MITA [7], [21], [25]–[27]. MITA is phosphorylated at Ser358 by TBK1 which is critical for phosphorylation and activation of IRF3 [7], while UNC-51-like kinase (ULK1) phosphorylates MITA at Ser366 which impairs MITA-IRF3 interaction and subsequent activation of IRF3 [21]. In addition, RNF5 catalyzes K48-linked polyubiquitination of MITA and targets MITA for degradation [25], whereas TRIM56 and TRIM32 promote K63-linked polyubiquitination of MITA and positively regulates virus-triggered type I IFN induction [26], [27]. Whether and how additional proteins mediate other types of modifications of MITA is unknown. In the present study, we identified an E3 ubiquitin ligase, RING finger protein 26 (RNF26) that targeted MITA for K11-linked polyubiquitination upon viral infection. MITA with K11-linked polyubiquitin chains remained as reservoir of MITA and was protected from RNF5-mediated K48-linked polyubiquitination and degradation. However, overexpression of RNF26 indirectly induced degradation of IRF3 through an autophagy pathway. As a result, knockdown of RNF26 promoted degradation of MITA after viral infection and prevented degradation of IRF3. Virus-triggered phosphorylation of IRF3 and induction of IFN-β were inhibited at the early time points and potentiated at late time points in the absence of RNF26, respectively. Our findings suggest that RNF26 temporally regulates virus-triggered induction of type I IFNs by two distinct mechanisms. Because post-translational modifications of MITA are critical for mediating viral nucleic acid-triggered type I IFN induction, we assumed there are additional proteins that interact with and target MITA for various modifications and thereby regulate innate antiviral signaling. We attempted to unambiguously identify E3 ubiquitin ligases that regulate MITA ubiquitination and function [27]. This effort led to the identification of RNF26 which could promote polyubiquitination of MITA [27]. Analysis of the NCBI EST profile database indicates that RNF26 is ubiquitously expressed in most examined cells and tissues. In overexpression experiments, RNF26 dose-dependently promoted polyubiquitination of MITA (Figure 1A). In contrast, the enzymatic inactive mutants RNF26 (C395S), RNF26 (C399S) or RNF26 (C401S) failed to mediate polyubiquitination of MITA (Figure 1B). Results from in vitro ubiquitination assays demonstrated that RNF26 catalyzed polyubiquitination of MITA in vitro, which depended on the enzymatic activity of RNF26 (Figure 1C–D). These data suggest that RNF26 is an E3 ubiquitin ligase targeting MITA for polyubiquitination. Since RNF26 caused polyubiquitination of MITA, we examined whether RNF26 interacted with MITA. Transient transfection and coimmunoprecipitation experiments indicated that RNF26 was associated with MITA in 293 cells (Figure 2A). In untransfected THP-1 cells, endogenous RNF26 constitutively interacted with MITA. This interaction was enhanced at 6 hours and decreased at 12–24 hours after SeV or HSV-1 infection (Figure 2B). It has been reported that MITA is localized to the ER, mitochondria-associated membrane and mitochondria [7], [8], [10], [17]. The subcellular localization of RNF26 was examined. Fluorescent confocal microscopy and cellular fractionation analysis suggested that RNF26 was mainly localized to the ER and a minor fraction of RNF26 was found to be colocalized with the mitochondria marker (Figure 2C and D). In contrast, RNF26 was not colocalized with the Golgi marker (Figure 2C). RNF26 was colocalized with MITA mostly at the ER and formed punctate dots with MITA after SeV or HSV-1 infection (Figure 2E). In this context, MITA was reported to translocate to microsomes to form punctate aggregates after viral infection or transfection of poly (dA: dT) or interferon stimulatory DNA (ISD) [17], [18], [20], [21]. These data suggest that RNF26 is physically associated with MITA. Previously, we have demonstrated that the N-terminal transmembrane domains of MITA are critical for its subcellular localization and function [7]. Interestingly, sequence analysis indicated that RNF26 contained five transmembrane domains at the N-terminus and a RING domain at the C-terminus (Figure S1A). Domain mapping experiments indicated that the association of RNF26 and MITA depended on their respective transmembrane domains (Figure S1A and B). These data collectively suggest that RNF26 is physically associated with MITA and the interaction is dependent on their transmembrane domains. To map the residue (s) of MITA that are targeted by RNF26, we examined RNF26-mediated polyubiquitination of MITA mutants in which all the lysine residues of MITA were individually substituted by arginine. As shown in Figure 3A, mutation of K150 to arginine impaired its polyubiquitination by RNF26. In addition, RNF26 could not induce polyubiquitination of MITA (K150R) in in vitro ubiquitination assays (Figure 3B). These data suggest that RNF26 targets K150 of MITA for polyubiquitination. Having demonstrated that RNF26 is a MITA-interacting E3 ubiquitin ligase targeting MITA for polyubiquitination, the types of polyubiquitin chains conjugated to MITA by RNF26 were next examined. Ubiquitin mutants were constructed in which all but one lysine residues were simultaneously mutated to arginines (K-O) or all seven lysine residues were individually mutated to arginine (K-R). These mutants were examined for their abilities to be conjugated to MITA by RNF26. As shown in Figure 4A, the ubiquitin mutant retaining only lysine 11 (Ub-K11O) but not other lysine residues could be conjugated to MITA. Conversely, mutation of lysine 11 to arginine (Ub-K11R) markedly reduced its ability to be conjugated to MITA by RNF26 (Figure 4B). These data indicate that RNF26 induces K11-linked polyubiquitination of MITA. Since an antibody against K11-linkage of polyubiquitin chains was not available to us, THP-1 cells stably transfected with Flag-Ub-K11O (THP-1-Flag-Ub-K11O) together with a control or RNF26-RNAi plasmids (Figure 4C and D) were established to examine viral infection-induced K11-linked polyubiquitination of MITA and the role of RNF26 in this ubiquitination. As shown in Figure 4E, MITA was modified with K11-linked polyubiquitin chains at 6–12 hours after SeV or HSV-1 infection, and such polyubiquitination was substantially impaired by knockdown of RNF26. Similar results were obtained in 293 cells stably transfected with HA-Ub-K11O plasmids (293-HA-Ub-K11O) together with a control or RNF26-RNAi plasmids (Figure 4F). In similar experiments, K11-linked polyubiquitination of VISA after viral infection was not affected by RNF26 knockdown (Figure S2). These data suggest that RNF26 mediates K11-linked polyubiquitination of MITA upon viral infection. Because multiple E3s have been reported to target polyubiquitin chains of distinct linkages (K48-linked or K63-linked) to K150 of MITA [25]–[27], we speculated that RNF26-mediated K11-linked polyubiquitination at K150 might compete with K48- or K63-linked polyubiquitination at the same lysine residue. Thus, virus-induced K48- or K63-linked polyubiquitination of MITA in THP-1-RNF26-RNAi and control cells was examined. As shown in Figure 5A, SeV or HSV-1 infection triggered K48- and K63-linked polyubiquitination of MITA. Knockdown of RNF26 potentiated K48- but not K63-linked polyubiquitination of MITA after viral infection (Figure 5A). RNF5 has been shown to induce K48-linked polyubiquitination of MITA at K150 [25]. Interestingly, we found that knockdown of RNF5 greatly enhanced SeV-induced K11-linked polyubiquitination of MITA in 293-HA-Ub-K11O cells (Figure 5B). In addition, RNF26-mediated K11-linked polyubiquitination of MITA was diminished by co-expression of RNF5, and RNF5-mediated K48-linked polyubiquitination and degradation of MITA was partially inhibited by co-expression of RNF26 (Figure 5C). Consistent with these observations, SeV- or HSV-1-induced degradation of MITA was accelerated by knockdown of RNF26 (Figure 5D). These data suggest that RNF26 catalyzes K11-linked polyubiquitination of MITA which protects MITA from RNF5-mediated K48-linked polyubiquitination. Since RNF26-mediated K11-linked polyubiquitination of MITA protected its degradation after viral infection, whether RNF26 regulates virus-triggered induction of type I IFNs was determined. Luciferase reporter assays suggested that knockdown of RNF26 inhibited SeV-induced activation of NF-κB, ISRE and IFN-β promoter but had no marked effects on TNFα- or IL-1β-induced activation of NF-κB (Figure 6A and S3A). The expression of IFNB1 gene was impaired at 6 hours after SeV infection by knockdown of RNF26 in 293 cells (Figure S3B). However, we unexpectedly observed that SeV-induced expression of IFNB1 was potentiated in RNF26 knockdown cells compared to control cells at 12–24 hours after SeV infection (Figure S3B). In a mouse macrophage cell line Raw264. 7 cells, SeV- or HSV-1-induced expression of Ifnb1 gene was also inhibited and potentiated at the early and late time points by knockdown of murine Rnf26, respectively (Figure S3C and D). It should be noted that the degrees of inhibition or potentiation of expression of IFNB1 or Ifnb1 genes were correlated with the knockdown efficiencies of the RNF26-RNAi or Rnf26-RNAi plasmids, indicating that RNF26 is involved in regulating RNA and DNA virus-triggered induction of type I IFNs. To further confirm this notion, RNAi-transduced stable THP-1 cell lines were established and the expression of IFNB1 in these cells was examined after stimulation with SeV or HSV-1. As shown in Figure 6B and C, the mRNA and protein levels of IFN-β were impaired at 6 hours and potentiated at 12–24 hours in THP-1-RNF26-RNAi compared to control cells after viral infection, respectively. Similar results were obtained with various RNA (VSV and EMCV) or DNA (ECTV) viruses (Figure 6D) as well as virus-induced expression of CCL5 (Figure S3E). Interestingly, SeV- or HSV-1-induced expression of the proinflammatory cytokines TNFα and IL-6 was inhibited by RNF26 knockdown at all examined time points after viral infection (Figure 6E, 6F, S4A and B). In similar experiments, RNF26 knockdown did not affect IFN-β-induced expression of ISG15 or ISG56 genes (Figure S4C). Consistent with the gene induction experiments, we found that although SeV- or HSV-1-induced phosphorylation of TBK1 and IκBα was inhibited in THP-1-RNF26-RNAi cells, phosphorylation of IRF3 was inhibited at the early time points and increased at the late time points in THP-1-RNF26-RNAi stable cells compared to that in control cells after viral infection (Figure 6G). Thus, we conclude that RNF26 temporally regulates virus-triggered induction of type I IFNs by two distinct mechanisms. When examining virus-triggered activation of IRF3 in RNF26 knockdown and control cells, we observed that the level of IRF3 protein was raised in THP-1-RNF26-RNAi compared to control cells (Figure 6G and 7A), and the mRNA levels of IRF3 were comparable in these cells (Figure 7A), indicating that RNF26 regulates IRF3 at the protein level. In support of this notion, we found that overexpression of RNF26 but not RNF26 (C395S) promoted degradation of IRF3 and inhibited SeV-induced activation of the IFN-β promoter (Figure 7B and C). However, we failed to observe an interaction between IRF3 and RNF26 (Figure 2A) or polyubiquitination of IRF3 by RNF26 (Figure 7D). Interestingly, RNF26-mediated degradation of IRF3 was blocked by the autophagy inhibitor 3-methyladenine (3-MA) but not the lysosome inhibitor ammonium chloride (NH4Cl) or the proteasome inhibitor MG132 (Figure 7E). To further determine whether autophagic degradation system is responsible for RNF26-mediated degradation of IRF3, we determined the effect of knockdown of ATG12, an important component of the autophagic degradation system [28], on RNF26-mediated IRF3 degradation. The results indicated that RNF26-mediated IRF3 degradation was markedly inhibited in ATG12 knockdown cells (Figure 7F). Thus, RNF26 might temporally regulate virus-triggered type I IFN induction through regulating K11-linked polyubiquitination of MITA at the early phase and autophagy-dependent degradation of IRF3 at late phase, respectively. Since RNF26 regulates virus-induced expression of IFN-β and other downstream genes, we examined its roles in cellular antiviral response. We found that knockdown of RNF26 inhibited VSV and HSV-1 replication in plaque assays (Figure S5), suggesting that RNF26 functions as a negative regulator in cellular antiviral response. MITA plays critical roles in virus-triggered type I IFN induction and innate antiviral immune response [7], [8], [10], [15], [17], [22]–[24]. Various studies have shown that post-translational modifications of MITA are essential for its function [7], [21], [25]–[27]. In this study, we demonstrated that RNF26 but not the enzymatic inactive mutants induced polyubiquitination of MITA. RNF26 mediated K11-linked polyubiquitination of MITA and modulated expression of type I IFN triggered by viral infection. RNF26 was localized mainly at the ER and constitutively interacted with MITA through their respective transmembrane domains. Viral infection potentiated this association and induced RNF26 to form punctate dots with MITA. Our studies suggest that RNF26 is a MITA-interacting E3 ubiquitin ligase which targets MITA for K11-linked polyubiquitination. Polyubiquitination of MITA catalyzed by RNF26 was mapped to K150, which is also targeted by RNF5 for K48-linked polyubiquitination and by TRIM56 or TRIM32 for K63-linked polyubiquitination [25]–[27]. These observations prompted us to hypothesize that polyubiquitin chains of distinct linkages might compete with each other at the same residue of MITA. Interestingly, virus-triggered K48- but not K63-linked polyubiquitination of MITA was enhanced by knockdown of RNF26. Previously, it has been demonstrated that TRIM32 targets not only K150 but also K20,224 and 236, whereas RNF5 targets only K150 of MITA [27]. This is consistent with our observations that RNF26 impaired K48- but not K63-linked polyubiquitination of MITA. RNF26-mediated K11-linked polyubiquitination of MITA protected it from RNF5-mediated K48-linked polyubiquitination and degradation. Consistently, degradation of MITA was accelerated in RNF26 knockdown cells compared to control cells after viral infection. These results suggest that RNF26-meidated K11-linked polyubiquitination competes with RNF5-mediated K48-linked polyubiquitination of MITA at K150. These results are consistent with our observations that knockdown of RNF26 inhibited induction of type I IFNs at early phase of viral infection. The functions of K11-linked polyubiquitination are not well understood so far [29]–[33]. To our knowledge, our study represents the first report on the function of K11-linked polyubiquitination in virus-triggered signaling and innate antiviral response. Unexpected, although knockdown of RNF26 inhibited virus-triggered induction of type I IFNs at the early phase of viral infection, it had opposite effect at the late phase of viral infection. This led us to hypothesize that RNF26 temporally regulates virus-triggered type I IFN induction by distinct mechanisms. In this context, we observed that overexpression of RNF26 promoted degradation of IRF3, whereas knockdown of RNF26 increased the level of IRF3. Interestingly, RNF26-mediated degradation of IRF3 was blocked by the autophagy inhibitor 3-MA but not the lysosome inhibitor NH4Cl or the proteasome inhibitor MG132. In addition, RNF26-induced degradation of IRF3 was markedly inhibited by knockdown of ATG12, an essential component in the autophagic degradation pathway. These results indicate that RNF26 indirectly regulates stability of IRF3 protein in an autophagy-dependent manner. The exact mechanism on how RNF26 mediates autophagy-dependent degradation of IRF3 is currently unknown. Based on our findings, we come to a working model on how RNF26 temporally regulates virus-triggered type I IFN induction (Figure 8). At the early phase of infection, RNF26 mediates K11-linked polyubiquitination of MITA, which protects it from K48-linked polyubiquitination and degradation to facilitate the fast induction of type I IFN genes. In addition to its early phase function through K11-linked polyubiquitination of MITA, RNF26 constitutively down-regulates IRF3 level by autophagic degradation. This may contribute to the termination of type I IFN induction at the late phase of viral infection. Interestingly, since IRF3 activation is not required for virus-triggered induction of the proinflammatory cytokines such as TNFα and IL-6, RNF26 positively regulates virus-triggered induction of the proinflammatory cytokines in a constitutive but not temporal manner. Therefore, our findings not only reveal the mechanisms on how RNF26 temporally modulate virus-triggered type I IFN induction, but also provide an explanation on how virus-triggered induction of type I IFNs and proinflammatory cytokines can be distinctly regulated. Recombinant IFN-β, TNFα and IL-1β (R&D Systems); mouse monoclonal antibodies against FLAG (Sigma), HA (Covance), β-actin (Sigma), AIF, KDEL (Santa Cruz Biotechnology), β-tubulin (Invitrogen), HSV-1 ICP27, ATG12 (Abcam), TBK1, p-TBK1 and p-IκBα (CST); rabbit polyclonal antibodies against ubiquitin, IRF3, p-IRF3 (Santa Cruz Biotechnology), polyubiquitin K48-linkage and K63-linkage (Millipore) were purchased from the indicated manufacturers. SeV, HSV-1, VSV, EMCV, ECTV, anti-SeV, anti-RIG-I, anti-VISA, anti-MITA anti-RNF5 and anti-IκBα sera were previously described [7], [25], [27], [34]. Rabbit anti-RNF26 was raised against recombinant human RNF26 (241–433). IFN-β, ISRE and NF-κB luciferase reporter plasmids, mammalian expression plasmids for HA-, Flag-, or GFP-tagged MITA and its mutants, ubiquitin, RIG-I, VISA, TRAF3, TRAF6, TBK1, IRF3, IRF7, Sec61-β, GALT, RNF5 and β-actin were previously described [7], [25], [27], [34], [35]. Mammalian expression plasmids for human Flag-, GFP-, Cherry- or CFP-tagged RNF26 and its mutants were constructed by standard molecular biology techniques. The cells were seeded and transfected the following day by standard calcium phosphate precipitation method or by FuGENE (Roche). Empty control plasmids were added to ensure that each transfection receives the same amount of total DNA. To normalize for transfection efficiency, pRL-TK Renilla luciferase reporter plasmids were added to each transfection. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega). Firefly luciferase activities were normalized on the basis of Renilla luciferase activities. The cells were lysed in lysis buffer containing 1% SDS and denatured by heating for 5 minutes. The supernatants were diluted with regular lysis buffer until the concentration of SDS was decreased to 0. 1%. The diluted supernatants were subjected for immunoprecipitation as described [7], [25], [27], and the immunoprecipitates and whole cell lysates were analyzed by immunoblots with the indicated antibodies. The tested proteins were expressed with a TNT Quick-coupled Transcription/Translation Systems kit (Promega) following instructions of the manufacturer. Ubiquitination was analyzed with an ubiquitination kit (Enzo Life Science) following protocols recommended by the manufacturer. The transfected cells were incubated with the ER-Tracker Blue/White or Mito-Tracker Red (Invitrogen) following protocols recommended by the manufacturer. The cells were then fixed with 4% paraformaldehyde for 10 minutes and observed with an Olympus confocal microscope under a ×60 oil objective. The cells were washed with PBS and lysed by douncing 30 times in 2 mL of homogenization buffer (10 mM Tris-HCl pH 7. 4,2 mM MgCl2,10 mM KCl and 250 mM sucrose) on wet ice. The homogenate was centrifuged at 500×g for 10 minutes twice. The supernatant (S5) was centrifuged at 5,000×g for 30 minutes to precipitate crude mitochondria (P5K). The supernatant (S5K) was further centrifuged at 50,000×g for 60 minutes to generate S50K and P50K. Double-stranded oligonucleotides corresponding to the target sequences were cloned into the pSuper. Retro RNAi plasmids (oligoengine Inc.). The following sequences were targeted for human RNF26 cDNA: #1: 5′-GAGCAAGAGGAGCGGAAGA-3′; #2: 5′-GAGAGGATGTCATGCGGCT-3′. The following sequences were targeted for murine Rnf26 cDNA: #1: 5′-GAGCGGAAGAAGTGTGTTA-3′; #2: 5′-GATCAACAGTCTAGTCAAC-3′. Total RNA was isolated from cells using TRIzol reagent (Takara) and subjected to real-time PCR analysis to measure expression of mRNA. Gene-specific primer sequences were as described [27] or as follow: Human RNF26 (Forward: 5′-CAGGACCATCAGAGTGACACCT-3′; Reverse: 5′-GCAACACTGTCTTGCTCTGGTC-3′). Murine Rnf26 (Forward: 5′-TGGCTGCTTTCCTCGCTCACAT-3′; Reverse: 5′-GCAACACCAATCCAGTGAGATGG-3′). Human IRF3 (Forward: 5′-TCTGCCCTCAACCGCAAAGAAG-3′; Reverse: 5′-TACTGCCTCCACCATTGGTGTC-3′). Murine Irf3 (Forward: 5′-CGGAAAGAAGTGTTGCGGTTAGC-3′; Reverse: 5′-CAGGCTGCTTTTGCCATTGGTG-3′). The supernatants of cell culture medium were analyzed with a human IFN-β (PBL), human TNFα or human IL-6 (Boster) ELISA kit following protocols recommended by the manufacturers. The 293 cells were transfected with two packaging plasmids (pGAG-Pol and pVSV-G) together with pMSCV-GFP-Flag-Ub-K11O retroviral plasmids by calcium phosphate precipitation. Twenty-four hours after transfection, cells were incubated with new medium without antibiotics for another twenty-four hours. The recombinant virus-containing medium was filtered with 0. 22 µm filter (Millex) and then added into cultured THP-1 cells in the presence of polybrene (4 µg/mL). The infected cells were cultured for at least seven days and sorted by a flow cell sorter before additional experiments were performed. The 293 cells were transfected with pRK7-Neo-HA-Ub-K11O plasmid. Twenty-four hours after transfection, cells were selected with G418 (0. 8 µg/mL). Single cell colonies were picked and identified by immunoblot analysis. The 293 cells were transfected with two packaging plasmids (pGAG-Pol and pVSV-G) together with a control, RNF26-RNAi or Rnf26-RNAi retroviral plasmids respectively by calcium phosphate precipitation. Twenty-four hours after transfection, cells were incubated with new medium without antibiotics for another twenty-four hours. The recombinant virus-containing medium was filtered with 0. 22 µm filter (Millex) and then added into cultured 293, Raw264. 7 or THP-1 cells in the presence of polybrene (4 µg/mL). The infected cells were selected with puromycin (1 µg/mL for 293 and Raw264. 7 cells or 0. 5 µg/mL for THP-1 cells) for at least seven days before additional experiments were performed. UniProtKB/Swiss-Prot accession numbers (parentheses) are indicated for proteins mentioned in text: RNF26 (Q9BY78), MITA (Q86WV6), RNF5 (Q99942), VISA (Q7Z434).

Title: RNF26 Temporally Regulates Virus-Triggered Type I Interferon Induction by Two Distinct Mechanisms Summary: Virus infection induces the host cells to produce type I interferons, which are secreted proteins important for the host to clear viruses. Previously, we identified a cellular protein called MITA, which is essential for virus-triggered induction of interferons. In this study, we found an enzyme called RNF26 could covalently modify MITA with one type of polypeptide, called polyubiquitin. This modification caused increased stability of MITA after viral infection. RNF26 also caused disability of IRF3, another important component required for virus-triggered interferon induction. Thus, RNF26 could temporally regulate virus-triggered interferon induction by two distinct mechanisms. This discovery helps to understand how the antiviral response is delicately regulated.

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Summarize: Hiker Gregg Hein of Clovis "entertained the idea" of possibly dying in the high Sierra of Sequoia and Kings Canyon National Parks as he lay severely injured for six days. But he said the thought only crossed his mind a few times. The 33-year-old experienced hiker, rock climber and rafting guide was determined to live -- and he did. From a wheelchair Tuesday at Community Regional Medical Center in Fresno, Hein talked about breaking his leg on July 5, hiking down from 13,600-foot Mount Goddard, and his eventual rescue Thursday by a National Park Service helicopter. Hiking down the mountain, he dislodged a boulder that plowed into the back of his right calf, breaking bones in three spots. The impact forced bone to protrude about an inch and a half through the skin, Hein said. His foot was soon "dangling," Hein said. "I had to grab it so hopefully it wouldn't rip off." After sliding down several ice fields, he was without food for six days -- except for a few crickets and moths -- but he managed to drink a little melted ice and later, from a stream. Rescue crews starting combing the high Sierra on Wednesday, the day Hein's dad, Doug Hein, called the Fresno County Sheriff's Office. Gregory Hein had been expected home two days earlier. "I can't commend them enough for the efforts and the energies that they put out to try and save one person's life," Gregg Hein said of the rescue teams from Fresno County and the Park Service -- about 80 people in all -- who helped look for him. Hein said Tuesday he has at least two more surgeries on his right leg to combat infection and repair bones, and that he could be in the hospital into next week. The eventual goal is to install a metal rod into his calf. Hein has family ties to Clovis Unified: His mother is Randy Hein, principal of Temperance-Kutner Elementary School, and his grandfather is Floyd "Doc" Buchanan, former longtime district superintendent. Hein's perilous trek began July 3. He parked his car at Florence Lake in Fresno County and within two days, had hiked alone more than 20 miles -- much of it cross-country -- to the summit of Mount Goddard. After summiting on July 5, he hoped to make it to Blayney Hot Springs that evening, but the goal was soon shattered with his leg. Hein wasn't due home until July 7, and he said he realized he had at least three days to survive before anyone would start looking for him. In an attempt to avoid potential rockfall, and knowing he had to get further down the mountain to find help, Hein left his backpack on the side of Mount Goddard. He grabbed only a few things from the pack: A poncho, pocket knife, cords, whistle and a bivvy sack -- a small, lightweight shelter. He didn't take more because he miscalculated, believing he was closer to Evolution Valley, where he hoped he'd see hikers. As he lay bleeding, Hein contemplated applying a makeshift tourniquet, a device used to tightly clench blood vessels connected to an injury. It was a big decision: If he did, "I would have lost a limb." By the time night fell, Hein wasn't feeling light-headed. So he took a chance and didn't cinch his leg. By the next morning, the bleeding had slowed significantly. For four days, he lay near the edge of a small glacier, nursing his injury with ice. Hein stabilized his leg with hiking poles, wrapping them with a belt and some cord, and on Wednesday, headed for Davis Lake -- crawling about a mile and dropping about 1,000 feet. He hoped the new location might increase his chance of being found. On Thursday, he saw helicopters -- but they didn't see him. Two flew over him several times, he said. "It was kind of wrenching." But around 7:30 p.m. Thursday, a Park Service helicopter landed about 50 feet from him at Davis Lake. The pilot was dropping off a search-and-rescue crew in the area when Hein came into view. After seeing the crew spot him, "I laid down on my back for a while, and breathed a deep sigh of relief." Alive and safe at Community Regional Medical Center, Hein has a lot to look forward to. He just finished his undergraduate degree in environmental studies at Humboldt State and hopes to attend the San Joaquin College of Law in Clovis. The next time Hein treks into the wilderness -- which he says will "definitely" happen again -- he plans to be "more cautious about my own life." In the future, Hein plans to carry a reflective mirror, which can be used to signal rescue aircraft; a satellite-linked device, which can be used to alert rescuers about a location; and more medical supplies and gear. Hein said backpackers should also apply for a wilderness permit so there is documentation of a proposed route, and make sure loved ones are aware of that route, too. As for Hein's parents, there's just a "lot of relief" to see their son "alive, breathing, talking." "I've been amazed at the outpouring of support from friends and family," father Doug Hein said. "It took a huge team, not just the searchers, but the prayer network, that helped bring him back." The reporter can be reached at (559) 441-6386, [email protected] or @CarmenGeorge on Twitter. FRESNO, Calif. (AP) — A hiker who was stranded for six days in California's Sierra Nevada with a badly broken leg says survival mode kicked in when he treated his own injury and sought sustenance by eating crickets and moths, and drinking melting ice. Gregg Hein, who broke his leg on a solo hike in the Sierra Nevada mountains, recovers at the Community Regional Medical Center in Fresno, Calif. on Wednesday, July 16, 2014. The 33-year-old hiker from... (Associated Press) Recovering at a Fresno hospital, Gregg Hein, 33, said Wednesday that he was a couple days into a solo hike high in the Sequoia and Kings Canyon national parks northeast of Fresno when a large rock crushed his right leg above the ankle. After letting out a yelp, the Clovis man said his first thought was treating his dangling leg and protruding bone to boost his chances of making it out alive. "I have to get these next moments right," said Hein, an avid outdoorsman. "What do I do to make sure I have the best chance for a positive outcome?" He briefly considered applying a tourniquet to stop the bleeding, a move that he knew would end with an amputation. Rather, Hein said he used hiking gear to wrap and secure his leg, and then he scooted to a flat clearing with a good vantage point to wait for rescuers. He had left his heavy pack behind, and the few insects he could scour at arm's reach hardly filled him up. He blew a whistle, hoping its echoes would catch somebody's attention. Back home, Doug Hein reported his son missing two days after he didn't return home as planned. Rescuers searched on foot and from the air. A helicopter crew eventually spotted the hiker July 10 and lifted him to safety. Hein underwent two surgeries and expects two more in a healing process likely to take months. Five pins hold his bones in place, and his legs are covered with scrapes from the 150-foot fall he took in the accident. Hein's father said he has warned his son against hiking alone, but that didn't keep him from two major expeditions, one covering 165 miles of wilderness. He's waiting for his son to recover to have another heart-to-heart conversation. "I've got a long time to get him back home and get him cornered and say, 'Hopefully you've learned from this,'" Doug Hein said. Gregg Hein said his risky days of hiking alone are behind him, but not his love of the outdoors. "As soon as I can get back to trail running and hiking, I'll be out there," he said. "It's my community."

Summary: Hiking alone has its disadvantages, and experienced climber, rafter, and trail runner Gregg Hein got up close and personal with most of them earlier this month. Two days into a solo hike in the Sequoia and Kings Canyon National Parks near Fresno, Calif., his footing set loose a boulder that caused the 33-year-old to fall 150 feet and broke his right leg in three places, leaving his foot "dangling" and his bone protruding out of his skin more than an inch. Knowing that a tourniquet would later lead to amputation, he took his chances and went without it; the bleeding eventually slowed. "I have to get these next moments right," Hein tells the AP. "What do I do to make sure I have the best chance for a positive outcome?" Part of that meant surviving for at least three days; he wasn't expected home for another two, so that's how long it would take for a search party to be dispatched. Hein abandoned his heavy pack on Mount Goddard, taking a poncho, pocket knife, whistle, and bivvy sack with him as he scooted to a glacier; there, he nursed his injury with ice for four days, surviving on melted ice, moths, and crickets. He then decided he'd have a better chance of being spotted elsewhere; held his leg together using hiking poles, a belt, and a cord; and crawled for about a mile. On day six-July 10-two helicopters flew above him several times. "It was kind of wrenching," he tells the Fresno Bee. Around 7:30pm, a "fortuitous" moment arrived: A crew was dropped off just 50 feet from him, and when he saw the searchers spot him, he rolled onto his back and "breathed a deep sigh of relief." Full recovery is expected to take months. Hein plans to get back in the wilderness-though next time not alone, he says.

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Summarize: Nearly two centuries old, the historic St. Joe Lighthouse is not only a symbol of the town of Saint Joseph, Michigan but also an icon of the Great Lakes. The original St. Joseph, MI lighthouse was constructed in 1832, making it the second lighthouse on Lake Michigan. The first St. Joe lighthouse was a single-story dwelling made of stone. In 1859, a new, two-story lighthouse was built on the bluff of the city of St. Joseph, MI and is considered St. Joe’s first Federal lighthouse. The Red Cross took over the original St. Joe lighthouse in 1924, and from that point until 1955, the structure served as the headquarters for the Red Cross. The St. Joseph, MI Lighthouse Today The St. Joe lighthouse that tourists and locals alike enjoy today was constructed at the end of a wooden pier extending out into lake Michigan in 1846. In 1907 and 1919 the city built the north and south piers that enabled boats easier access into the mouth of the St. Joe River. The lights on these piers are still operating today as one of the Great Lake’s last remaining pier range light systems. The catwalk, which was originally built for access to the lighthouse by the keepers during rough seas, is still accessed by walkers, runners and bikers today. As noted previously, it was cold as hell this weekend. In a meteorological pattern similar to the horrible Polar Vortex of 2014, things got frigid across the country. Temperatures hit well below -20 with the wind chill in parts of Minnesota and the Upper Midwest. A video of a lighthouse in St. Joseph, Michigan is showcasing exactly what the weekend felt like where the weather was the worst. The lighthouse is on the southeast shore of Lake Michigan, not too far down the road from Kalamazoo and it's entirely caked in ice. A coating of ice turned the lighthouse into a spindly spire that looks more at home in a fantasy film than real life. The Great Lakes Drone Company did a spectacular job in the above video capturing the incredible sight. As did photographer Joshua Nowicki, who created the below video of the same lighthouse and posted it to Facebook. Rating is available when the video has been rented. This feature is not available right now. Please try again later. It's been really, really cold in some parts of the country this week (don't say we didn't warn you) but one side effect has been some incredible images like this lighthouse in St. Joseph, Michigan, along the southwest shores of Lake Michigan. Another terrific video, captured by photographer Joshua Nowicki gives you an even closer look at the frozen castle that looks straight out of a fairy tale. This mystical sight has occurred in the past, with footage and photos also emerging in 2013 and 2014. Temperatures remained in the teens on Sunday in the area with wind chills that made it feel like several degrees below zero, so you might want to enjoy these images from warm confines of your home instead of venturing outside for an up-close look. Video credit: Great Lakes Drone Company via Storyful

Summary: The addition of a smokestack to a lighthouse in St. Joseph, Mich., led some to describe the building as "unsightly," per the AP. It was anything but on Thursday, as the area was hit with chilly weather. In a video captured by photographer Joshua Nowicki, the outer portion of the lighthouse is seen entirely caked in ice as furious waves spit water in all directions on the southeastern shore of Lake Michigan. The video has now been viewed almost 1 million times, reports Mashable, which notes the lighthouse "looks straight out of a fairy tale," though it was ice-free a day earlier. "It's so beautiful and unbelievable that it's hard to believe there's no CGI involved," adds Thrillist. "If it weren't for the seagulls flying by, it could be mistaken for a scale model," notes a Facebook user. Nowicki, meanwhile, says the lighthouse resembled "an ice monster" with "one gnarly ice beard." The detail of the ice is more visible in a separate video shot on Friday by the Great Lakes Drone Company. It shows the ice stretching all the way to the inner lighthouse, where that "unsightly" smokestack was added this summer to improve air circulation. An original smokestack for coal-fired boilers had stood from 1907 to 1949 following the lighthouse's construction in 1859. It is now "one of the Great Lake's last remaining pier range light systems," according to its website.

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Write a title and summarize: SECTION 1. FINDINGS AND DECLARATIONS. The Congress finds and declares that-- (1) Crime, particularly crime involving drugs and guns, is a pervasive, nationwide problem. (2) Problems with crime at the local level are exacerbated by the interstate movement of drugs, funds, and criminal gangs. (3) Firearms and ammunition, and handguns in particular, move easily in interstate commerce, as documented in numerous hearings in both the Judiciary Committee of the House of Representatives and Judiciary Committee of the Senate. (4) In fact, even before the sale of a handgun, the gun, its component parts, ammunition, and the raw materials from which they are made have considerably moved in interstate commerce. (5) While criminals freely move from State to State, ordinary citizens may fear to travel to or through certain parts of the country due to the concern that violent crime is not under control, and foreigners may decline to travel in the United States for the same reason. (6) Just as the hardened drug kingpins begin their life in the illicit drug culture by exposure to drugs at a young age, violent criminals often start their criminal careers on streets where the ready availability of guns to young people results in the acceptability of their random use. (7) Violent crime and the use of illicit drugs go hand-in- hand, and attempts to control one without controlling the other may be fruitless. (8) Individual States and localities find it impossible to handle the problem by themselves; even States and localities that have made a strong effort to prevent, detect, and punish crime find their effort unavailing due in part to the failure or inability of other States and localities to take strong measures. (9) Inasmuch as illicit drug activity and related violent crime overflow State lines and national boundaries, the Congress has power, under the interstate commerce clause and other provisions of the Constitution, to enact measures to combat these problems. (10) The Congress finds that it is necessary and appropriate to assist the States in controlling crime by stopping the commerce in handguns with juveniles nationwide, and allowing the possession of handguns by juveniles only when handguns are possessed and used for legitimate purposes under appropriate conditions. SEC. 2. PROHIBITION OF THE POSSESSION OF A HANDGUN OR AMMUNITION BY, OR THE PRIVATE TRANSFER OF A HANDGUN OR AMMUNITION TO, A JUVENILE. (a) Definition.--Section 921(a) of title 18, United States Code, is amended by adding at the end the following new paragraph: ``(29) The term `handgun' means-- ``(A) a firearm that has a short stock and is designed to be held and fired by the use of a single hand; and ``(B) any combination of parts from which a firearm described in subparagraph (A) can be assembled.''. (b) Offense.--Section 922 of title 18, United States Code, is amended by adding at the end the following new subsection: ``(s)(1) It shall be unlawful for a person to sell, deliver, or otherwise transfer to a juvenile, or to a person who the transferor knows or has reasonable cause to believe is a juvenile-- ``(A) a handgun; or ``(B) ammunition that is suitable for use only in a handgun. ``(2) It shall be unlawful for any person who is a juvenile to knowingly possess-- ``(A) a handgun; or ``(B) ammunition that is suitable for use only in a handgun. ``(3) This subsection does not apply-- ``(A) to a temporary transfer of a handgun or ammunition to a juvenile, or to the possession or use of a handgun or ammunition by a juvenile, if the handgun and ammunition are possessed and used by the juvenile-- ``(i) in the course of employment, in the course of ranching or farming related to activities at the residence of the juvenile (or on property used for ranching or farming at which the juvenile, with the permission of the property owner or lessee, is performing activities related to the operation of the farm or ranch), target practice, hunting, or a course of instruction in the safe and lawful use of a handgun; ``(ii) with the prior written consent of the juvenile's parent or guardian who is not prohibited by Federal, State, or local law from possessing a firearm; ``(iii) with the prior written consent in the juvenile's possession at all times when a handgun is in the possession of the juvenile; and ``(iv) in accordance with State and local law; ``(B) during transportation by the juvenile of an unloaded handgun in a locked container directly from the place of transfer to a place at which an activity described in subparagraph (A)(i) is to take place, and transportation by the juvenile of that handgun, unloaded and in a locked container, directly from the place at which such an activity took place to the transferor; ``(C) to a juvenile who is a member of the Armed Forces of the United States or the National Guard who possesses or is armed with a handgun in the line duty; ``(D) to a transfer by inheritance of title (but not possession) of a handgun or ammunition to a juvenile; or ``(E) to the possession of a handgun or ammunition by a juvenile taken in defense of the juvenile or other persons against an intruder into the residence of the juvenile or a residence in which the juvenile is an invited guest. ``(4) A handgun or ammunition, the possession of which is transferred to a juvenile in circumstances in which the transferor is not in violation of this subsection shall not be subject to permanent confiscation by the Government if its possession by the juvenile subsequently becomes unlawful because of the conduct of the juvenile, but shall be returned to the lawful owner when such handgun or ammunition is no longer required by the Government for the purposes of investigation or prosecution. ``(5) For purposes of this subsection, the term `juvenile' means a person who is less than 18 years of age. ``(6)(A) In a prosecution of a violation of this subsection, the court shall require the presence of a juvenile defendant's parent or legal guardian at all proceedings. ``(B) The court may use the contempt power to enforce subparagraph (A). ``(C) The court may excuse attendance of a parent or legal guardian of a juvenile defendant at a proceeding in a prosecution of a violation of this subsection for good cause shown.''. (c) Penalties.--Section 924(a) of title 18, United State Code, is amended-- (1) in paragraph (1) by striking ``paragraph (2) or (3) of''; and (2) by adding at the end the following new paragraph: ``(5)(A)(i) A juvenile who violates section 922(s) shall be fined under this title, imprisoned not more than 1 year, or both, except that a juvenile described in clause (ii) shall be sentenced to probation on appropriate conditions and shall not be incarcerated unless the juvenile fails to comply with a condition of probation. ``(ii) A juvenile is described in this clause if-- ``(I) the offense of which the juvenile is charged is possession of a handgun or ammunition in violation of section 922(s)(2); and ``(II) the juvenile has not been convicted in any court of an offense (including an offense under section 922(s) or a similar State law, but not including any other offense consisting of conduct that if engaged in by an adult would not constitute an offense) or adjudicated as a juvenile delinquent for conduct that if engaged in by an adult would constitute an offense. ``(B) A person other than a juvenile who knowingly violates section 922(s)-- ``(i) shall be fined under this title, imprisoned not more than 1 year, or both; and ``(ii) if the person sold, delivered, or otherwise transferred a handgun or ammunition to a juvenile knowing or having reasonable cause to know that the juvenile intended to carry or otherwise possess or discharge or otherwise use the handgun or ammunition in the commission of a crime of violence, shall be fined under this title, imprisoned not more than 10 years, or both.''. (d) Technical Amendment of Juvenile Delinquency Provisions in Title 18, United States Code.-- (1) Section 5031.--Section 5031 of title 18, United States Code, is amended by inserting ``or a violation by such person of section 922(s)'' before the period at the end. (2) Section 5032.--Section 5032 of title 18, United States Code, is amended-- (A) in the first undesignated paragraph by inserting ``or(s)'' after ``922(p)''; and (B) in the fourth undesignated paragraph by inserting ``or section 922(s) of this title,'' before ``criminal prosecution on the basis''. (e) Technical Amendment of the Juvenile Justice and Delinquency Prevention Act of 1974.--Section 223(a)(12)(A) of the Juvenile Justice and Delinquency Prevention Act of 1974 (42 U.S.C. 5633(a)(12)(A)) is amended by striking ``which do not constitute violations of valid court orders'' and inserting ``(other than an offense that constitutes a violation of a valid court order or a violation of section 922(s) of title 18, United States Code, or a similar State law)''. (f) Model Law.--The Attorney General, acting through the Director of the National Institute for Juvenile Justice and Delinquency Prevention, shall-- (1) evaluate existing and proposed juvenile handgun legislation in each State; (2) develop model juvenile handgun legislation that is constitutional and enforceable; (3) prepare and disseminate to State authorities the findings made as the result of the evaluation; and (4) report to Congress by December 31, 1994, findings and recommendations concerning the need or appropriateness of further action by the Federal Government. Passed the House of Representatives November 20, 1993. Attest: DONNALD K. ANDERSON, Clerk.

Title: Youth Handgun Safety Act of 1993 Summary: Amends the Federal criminal code to prohibit: (1) the sale, delivery, or transfer to a juvenile, or to a person who the transferor knows or has reasonable cause to believe is a juvenile, of a handgun or ammunition that is suitable for use only in a handgun; and (2) the possession by a juvenile of a handgun or such ammunition. Makes exceptions with respect to: (1) certain temporary transfers of a handgun or ammunition to a juvenile, or to possession or use of a handgun or ammunition by a juvenile, if the handgun and ammunition are possessed and used by the juvenile in the course of employment or ranching or farming related to activities at the juvenile's residence, target practice, hunting, or a course of instruction in the safe and lawful use of a handgun, with the prior written consent of the juvenile's parent or guardian, subject to specified requirements, and in accordance with State and local law; (2) transportation by the juvenile of an unloaded handgun in a locked container, under specified circumstances; (3) a juvenile who is a member of the U.S. armed forces or the National Guard who possesses or is armed with a handgun in the line of duty; (4) a transfer by inheritance of title (but not possession) of a handgun or ammunition to a juvenile; or (5) the possession of a handgun or ammunition by a juvenile taken in defense of the juvenile or other persons against an intruder into the residence of the juvenile or a residence in which the juvenile is an invited guest. Directs the court to require the presence of a juvenile defendant's parent or legal guardian at all proceedings for violations of this Act, except for good cause shown. Sets: (1) limits on the permanent confiscation by the Government of a handgun or ammunition from a juvenile; and (2) penalties for violations of this Act. Directs the Attorney General to: (1) evaluate existing and proposed juvenile handgun legislation in each State; (2) develop model juvenile handgun legislation that is constitutional and enforceable; (3) prepare and disseminate to State authorities the findings made as the result of the evaluation; and (4) report to the Congress regarding the need or appropriateness of further Government action.

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Summarize: By. Kieran Corcoran. PUBLISHED:. 14:20 EST, 6 November 2013. |. UPDATED:. 19:10 EST, 6 November 2013. A dressmaker made famous by the TV show Big Fat Gypsy Weddings has been accused of treating one of her staff 'like an animal', and almost attacking her head designer after a shouting match. Thelma Madine, 61, who makes dresses which have featured on the hit Channel 4 show series exploring the raucous marriage ceremonies of Britain's travelling community, was accused of mistreating her staff today during an employment tribunal. Pauline Wooley, whose daughter Leanne Phillips, 31, was head designer at Nico Bridal Company until she was dismissed after the incident last year, gave evidence suggesting Ms Madine routinely mistreated her staff. Tribunal: Thelma Madine, 61, (left) is being sued for. unfair dismissal and £1,200 in wages by former employee Leanne Phillips,. 31, (right) after she was fired last Christmas. Collaborators: Ms Phillips, left, and Ms Madine, right, posing with a dress made of hair. Ms Wooley, who is currently suspended from her role as Assistant Manager at the company, told the court hearing in Liverpool that she was 'treated like a dog' by her employer, who had also been a close friend for more than 20 years. She claimed that she had to physically restrain Ms Madine after she advanced angrily on her daughter after she intervened in an argument by calling Ms Madine a 'horrible b****'. The dispute, on 18 October last year, led to Miss Phillips being dismissed from her job. She is now pursuing her former employer for unfair dismissal and £1,200 of outstanding wages. Ms Wooley, who says she has been suspended from her job for nine weeks without pay, told the tribunal: 'Thelma treated me like an animal. She intimidated me and she bullied me. Leanne was just defending her mother. 'Thelma started the whole argument - she had treated me really badly. She was screaming so loud her face went red and her veins were sticking out of her neck. 'Thelma advanced aggressively at Leanne, and I had to pull Thelma away. The way she reacted was disgusting.' The court also heard that Miss Phillips had known her employer from the age of four and said she saw Ms Madine 'as like an aunt'. Ms Madine shot to fame after her dressmaking. business Nico was featured on Channel 4's My Big Fat Gypsy Wedding for. making elaborate wedding gowns for the travelling community. Ms Madine was said to have treated her like a member of the family - even giving her a lavish sports car for her 30th birthday. Ms Wooley said: 'Leanne thought the world of Thelma, and Thelma treated her like a daughter.' However, Peter Maratos, an employment expert speaking on behalf of Ms Madine, said that Miss Phillips was'spoiled' and 'over-reacted' after the publicity from featuring on the TV programme had 'gone to her head'. 'Like family': The tribunal heard how Miss Phillips, pictured at court earlier this week, looked up to Ms Madine and thought of her like an aunt. Ms Madine was also accused of botching a disciplinary process launched after Miss Phillips tried to appeal her sacking. The court heard that she sought legal advice from her company lawyer Neil Gouldson, who then led the investigation, which accused her of encouraging her mother to act insubordinately, bullying co-workers, and making racist comments. Miss Phillips' solicitor, Steve Pinder, said: 'Ms Madine made a number of very serious errors. Her approach to the whole disciplinary process was flawed. Ms Madine made it clear that she would not have taken Miss Phillips back, even after the appeal.' Mr Maratos defended the set-up, saying: 'In this case family relationships had deteriorated. Thelma's position had been undermined - the position was untenable. 'However Ms Madine handled the process, it would have still resulted in a dismissal. The investigation gave the respondent reasonable belief of misconduct.' Judge Vincent Ryan reserved judgement on the case at the request of Mr Maratos, and an outcome is expected to be delivered by the end of the month. He said: 'It is extremely sad when relationships of over 20 years come to this. 'No one will be happy with the argument - it is sad.' The court case comes one year after Ms Madine published her autobiography, Tales of The Gypsy. Dressmaker, in which she praised Miss Phillips. She. wrote: 'Leanne is now the head designer at Nico and is starting to. oversee parts of the process that I had to do, which is great, because. it means that I can concentrate on other parts of the business. Over the top: Nico Bridal Company produced wedding dresses for Big Fat Gypsy Weddings, such as the one pictured. Extreme: The show became famous for the lavish wedding outfits it featured. 'I. used to feel that every one of my dresses was like my baby and that no. one could do them as well as me but I know now that the future of Nico. is in Leanne's hands. 'Since. she started working for me, Leanne has taken Nico to another level. Not. only that, she also inherited her mum Pauline's determination and. loyalty. 'And I have to admit that she reminds me of me at her age, full of imaginative ideas and passion for the task in hand.' Ms Madine has denied the claims, while Miss Phillips maintains that she was treated unfairly and denies any wrongdoing. Sorry we are unable to accept comments for legal reasons

Summary: Thelma Madine, 61, who starred in the hit Channel 4 show, is being pursued for unfair dismissal and unpaid wages. She sacked Leanne Phillips, 31, her head designer, after a heated argument. The tribunal heard that Miss Phillips called Ms Madine 'a horrible b****' Pauline Wooley, Miss Phillips's mother, said that she too was bullied, intimidated and 'treated like an animal' by Ms Madine.

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Summarize: Background The Forest Service and Interior collectively manage about 700 million acres of federal land, much of which is considered to be at high risk of fire. Federal researchers estimate that from 90 million to 200 million acres of federal lands in the contiguous United States are at an elevated risk of fire because of abnormally dense accumulations of vegetation, and that these conditions also exist on many nonfederal lands. Addressing this fire risk has become a priority for the federal government, which in recent years has significantly increased funding for fuels reduction. Fuels reduction is generally done through prescribed burning, in which fires are deliberately lit in order to burn excess vegetation, and mechanical treatments, in which mechanical equipment is used to cut vegetation. Although prescribed burning is generally less expensive on a per-acre basis than mechanical treatment, prescribed fire may not always be the most appropriate method for accomplishing land management objectives—and in many locations it is not an option, because of concerns about smoke pollution, for example, or because vegetation is so dense that agency officials fear a prescribed fire could escape and burn out of control. In such situations, mechanical treatments are required, generating large amounts of wood—particularly small-diameter trees, limbs, brush, and other material that serve as fuel for wildland fires. Woody biomass can be used in many ways. Small logs can be peeled and used as fence posts, or can be joined together with specialized hardware to construct pole-frame buildings. Trees also can be milled into structural lumber or made into other wood products, such as furniture, flooring, and paneling. Woody biomass also can be chipped for use in paper pulp production and for other uses—for example, a New Mexico company combines juniper chips with plastic to create a composite material used to make road signs—and can be converted into other products such as ethanol and adhesives. Finally, woody biomass can be chipped or ground for energy production in power plants and other applications. Citing biomass’s potential to serve as a source of electricity, fuel, chemicals, and other materials, the President and the Congress have encouraged federal activities regarding biomass utilization—but until recently, woody biomass received relatively little emphasis. Major congressional direction includes the Biomass Research and Development Act of 2000, the Farm Security and Rural Investment Act of 2002, the Healthy Forests Restoration Act of 2003, and the American Jobs Creation Act of 2004. Utilization of woody biomass also is emphasized in the federal government’s National Fire Plan, a strategy for planning and implementing agency activities related to wildland fire management. For example, a National Fire Plan strategy document cites biomass utilization as one of its guiding principles, recommending that the agencies “employ all appropriate means to stimulate industries that will utilize small-diameter woody material resulting from hazardous fuel reduction activities.” Federal agencies also are carrying out research concerning the utilization of small-diameter wood products as part of the Healthy Forests Initiative, the administration’s initiative for wildland fire prevention. Most Woody Biomass Utilization Activities Are Implemented by the Departments of Agriculture, Energy, and the Interior and Include Grants, Research, and Education Most of the federal government’s woody biomass utilization efforts are being undertaken by USDA, DOE, and Interior. While some activities are performed jointly, each department also conducts its own activities, which generally involve grants for small-scale woody biomass projects; research on woody biomass uses; and education, outreach, and technical assistance aimed at woody biomass users. Some Woody Biomass Activities Are Performed Jointly by Multiple Agencies USDA, DOE, and Interior have undertaken a number of joint efforts related to woody biomass. In June 2003, the three departments signed a memorandum of understanding on woody biomass utilization, and the departments sponsored a 3-day conference on woody biomass in January 2004. The departments also have established an interagency Woody Biomass Utilization Group, which meets quarterly to discuss relevant developments and to coordinate departmental efforts. Another interdepartmental collaboration effort is the Joint Biomass Research and Development Initiative, a grant program conducted by USDA and DOE and authorized under the Biomass Research and Development Act of 2000. The program provides funds for research on biobased products. DOE also has collaborated with both USDA and BLM on assessment of biomass availability, while USDA and Interior have entered into a cooperative agreement with the National Association of Conservation Districts to promote woody biomass utilization. USDA, DOE, and Interior also participate in joint activities at the field level. For example, DOE’s National Renewable Energy Laboratory (NREL) and the Forest Service have collaborated in developing and demonstrating small power generators that use woody biomass for fuel. The Forest Service also collaborates with Interior in funding and awarding grants under the Fuels Utilization and Marketing program, which targets woody biomass utilization efforts in the Pacific Northwest. The agencies also collaborate with state and local governments to promote the use of woody biomass—for example, the Forest Service, NREL, and BLM entered into a memorandum of understanding with Jefferson County, Colorado, to study the feasibility of developing an electricity-generating facility that would use woody biomass. USDA’s Efforts Related to Woody Biomass Utilization Are Concentrated in the Forest Service, with Some Efforts Under Way in Other USDA Agencies Most of USDA’s woody biomass utilization activities are undertaken by the Forest Service and involve grants, research and development, and education, outreach, and technical assistance. The Forest Service provides grants through its Economic Action Programs, created to help rural communities and businesses dependent on natural resources become sustainable and self-sufficient. The Forest Service also has created a grant program in response to a provision in the Consolidated Appropriations Act for Fiscal Year 2005, which authorized up to $5 million for grants to create incentives for increased use of biomass from national forest lands. Two other USDA agencies—the Cooperative State Research, Education and Extension Service (CSREES) and USDA Rural Development—maintain programs that could include woody biomass utilization activities. CSREES oversees the Biobased Products and Bioenergy Production Research grant program and the McIntyre-Stennis grant program, which provides grants to states for research into forestry issues under the McIntyre-Stennis Act of 1962. Within USDA Rural Development, the Rural Business-Cooperative Service oversees a grant program emphasizing renewable energy systems and energy efficiency among rural small businesses, farmers, and ranchers, and the Rural Utilities Service maintains a loan program for renewable energy projects. Forest Service researchers are conducting research into a variety of woody biomass issues. Researchers have conducted assessments of the woody biomass potentially available through land management projects and have developed models of the costs and revenues associated with thinning projects. Researchers also are studying the economics of woody biomass use in other ways; one researcher, for example, is beginning an assessment of the economic, environmental, and energy-related impacts of using woody biomass for power generation. The Forest Service also conducts extensive research, primarily at its Forest Products Laboratory, into uses for woody biomass, including wood-plastic composites and water filtration systems that use woody biomass fibers, as well as less expensive ways of converting woody biomass to liquid fuels. In addition, the Forest Service conducts extensive education, outreach, and technical assistance activities. Much of this activity is conducted by the Technology Marketing Unit (TMU) at the Forest Products Laboratory, which provides woody biomass users with technical assistance and expertise in wood products utilization and marketing. Forest Service field office staff also provide education, outreach, and technical assistance, and each Forest Service region has an Economic Action Program coordinator who has involvement in woody biomass issues. For example, one such coordinator organized a “Sawmill Improvement Short Course” designed to provide information to small-sawmill owners regarding how to better handle and use small-diameter material. The Forest Service also has partnerships with state and regional entities that provide a link between scientific and institutional knowledge and local users. DOE Is Engaged Primarily in Biomass Research and Development Activities Most of DOE’s woody biomass activities are overseen by its Office of the Biomass Program and focus primarily on research and development, although the department does have some grant and technical assistance activities. DOE’s research and development activities generally address the conversion of biomass, including woody biomass, to liquid fuels, power, chemicals, or heat. Much of this work is carried out by NREL, where DOE recently opened the Biomass Surface Characterization Laboratory. DOE also supports research into woody biomass through partnerships with industry and academia. Program management activities for these partnerships are conducted by DOE headquarters, with project management provided by DOE field offices. In addition to its research activities, DOE provides information and guidance to industry, stakeholder groups, and users through presentations, lectures, and DOE’s Web site, according to DOE officials. DOE also provides outreach and technical assistance through its State and Regional Partnership, Federal Energy Management Program (FEMP), and Tribal Energy Program. FEMP provides assistance to federal agencies seeking to implement renewable energy and energy efficiency projects, while the Tribal Energy Program provides technical assistance to tribes, including strategic planning and energy options analysis. DOE’s grant programs include (1) the National Biomass State and Regional Partnership, which provides grants to states for biomass-related activities through five regional partners; and (2) the State Energy Program, which provides grants to states to design and carry out their own renewable energy and energy efficiency programs. In addition, DOE’s Tribal Energy Program provides funds to promote energy sufficiency, economic development, and employment on tribal lands through renewable energy and energy efficiency technologies. Interior’s Woody Biomass Activities Include Education, Outreach, and Some Grant Programs Interior’s activities include providing education and outreach and conducting grant programs, but they do not include research into woody biomass utilization issues. Four Interior agencies—BLM, the Bureau of Indian Affairs (BIA), Fish and Wildlife Service (FWS), and National Park Service (NPS)—conduct activities related to woody biomass. These agencies conduct education, outreach, and technical assistance, but not to the same degree as the Forest Service. For example, BIA provides technical assistance to tribes seeking to implement renewable energy projects, and while FWS and NPS conduct relatively few woody biomass utilization activities, in some cases the agencies will work to find a woody biomass user nearby if a market exists for the material. Interior plans to expand its outreach efforts by using the National Association of Conservation Districts, with which it signed a cooperative agreement, to conduct outreach activities related to woody biomass. And while Interior’s grant programs generally do not target woody biomass, BIA has provided some grants to Indian tribes, including a 2004 grant to the Confederated Tribes of the Warm Springs Reservation in Oregon to conduct a feasibility study for updating and expanding a woody biomass-fueled power plant. Several Other Federal Agencies Participate in Woody Biomass Activities Several other federal agencies are engaged in limited woody biomass activities through their advisory or research activities. The Environmental Protection Agency provides technical assistance, through its Combined Heat and Power Partnership, to power plants that generate combined heat and power from various sources, including woody biomass. Three other agencies—the National Science Foundation, Office of Science and Technology Development, and Office of the Federal Environmental Executive—also are involved in woody biomass activities through their membership on the Biomass Research and Development Board, which is responsible for coordinating federal activities for the purpose of promoting the use of biobased industrial products. Woody Biomass Coordination Efforts among and within Federal Agencies Include Both Formal and Informal Mechanisms, and the Forest Service, DOE, and Interior Have Assigned Responsibility for Overseeing Woody Biomass Activities Two groups serve as formal vehicles for coordinating federal agency activities related to woody biomass utilization. One, the Woody Biomass Utilization Group, is a multiagency group that meets quarterly on woody biomass utilization issues and is open to all national, regional, and field- level staff across numerous agencies. The other, the Biomass Research and Development Board, is responsible for coordinating federal activities to promote the use of biobased industrial products. The board consists of representatives from USDA, DOE, and Interior, as well as EPA, the National Science Foundation, Office of the Federal Environmental Executive, and Office of Science and Technology Policy. When discussing coordination among agencies, however, agency officials more frequently cited using informal mechanisms for coordination—through telephone discussions, e-mails, participation in conferences, and other means— rather than the formal groups described above. Several officials told us that informal communication among networks of individuals was essential to coordination among agencies. Officials also described other forms of coordination, including joint review teams for interagency grant programs and multiagency working groups examining woody biomass at the regional or state level. The Forest Service—the USDA agency with the most woody biomass activities—developed a woody biomass policy in January 2005, and, in March 2005, in response to a recommendation in our draft report, the agency assigned responsibility for overseeing and coordinating its woody biomass activities to an official within the Forest Service’s Forest Management branch. In addition, the agency has created the Biomass Utilization Steering Committee, consisting of the staff directors of various Forest Service branches, to provide direction and support for agency biomass utilization. DOE coordinates its woody biomass utilization activities through its Office of Energy Efficiency and Renewable Energy. Within this office, the Office of the Biomass Program directs biomass research at DOE national laboratories and contract research organizations, while the Federal Energy Management Program and the Tribal Energy Program conduct a small number of other woody biomass activities. Interior has appointed a single official to oversee its woody biomass activities and is operating under a woody biomass policy adopting the principles of the June 2003 memorandum of understanding among USDA, DOE, and Interior. Interior also has appointed a Renewable Energy Ombudsman to coordinate all of the department’s renewable energy activities, including those related to woody biomass, and has worked with its land management agencies to develop woody biomass policies allowing service and timber contractors to remove woody biomass where ecologically appropriate. Similarly, BLM has appointed a single official to oversee woody biomass efforts and has developed a woody biomass utilization strategy to guide its activities that contains overall goals related to increasing the utilization of biomass from treatments on BLM lands. Most Officials Cited Economic Obstacles to Woody Biomass Utilization, and While Agencies Generally Targeted These Obstacles, Some Officials Believe Additional Steps beyond the Agencies’ Authority Are Needed Agency officials cited two principal obstacles to increasing the use of woody biomass: the difficulty in using woody biomass cost-effectively and the lack of a reliable supply of the material. Agency activities are generally targeted toward the obstacles identified by agency officials, but some officials told us that their agencies are limited in their ability to fully address these obstacles and that additional steps beyond the agencies’ authority to implement are needed. However, not all agree that such steps are appropriate. Most Officials Noted the Difficulty Involved in Using Woody Biomass Cost-Effectively, and Many Also Cited the Lack of a Reliable Woody Biomass Supply The obstacle most commonly cited by officials we spoke with is the difficulty of using woody biomass cost-effectively. Officials told us the products that can be created from woody biomass—whether wood products, liquid fuels, or energy—often do not generate sufficient income to overcome the costs of acquiring and processing the raw material. One factor contributing to the difficulty in using woody biomass cost- effectively is the cost incurred in harvesting and transporting woody biomass. Numerous officials told us that even if cost-effective means of using woody biomass were found, the lack of a reliable supply of woody biomass from federal lands presents an obstacle because business owners or investors will not establish businesses without assurances of a dependable supply of material. Officials identified several factors contributing to the lack of a reliable supply, including the lack of widely available long-term contracts for forest products, environmental groups’ opposition to federal projects, and the shortage of agency staff to conduct activities. A few officials cited internal barriers that hamper agency effectiveness in promoting woody biomass utilization, including limited agency expertise related to woody biomass and limited agency commitment to the issue. A variety of other obstacles were noted as well, including the lack of a local infrastructure for handling woody biomass, consisting of loggers, mills, and equipment capable of treating small- diameter material. Agency Efforts Are Generally Targeted toward the Obstacles Identified, but Officials Cited the Need for Additional Actions Such as Subsidies and Tax Credits Agency activities related to woody biomass were generally aimed at overcoming the obstacles agency officials identified, including many aimed at overcoming economic obstacles. For example, Forest Service staff have worked with potential users of woody biomass to develop products whose value is sufficient to overcome the costs of harvesting and transporting the material; Economic Action Program coordinators have worked with potential woody biomass users to overcome economic obstacles; and Forest Products Laboratory researchers are working with NREL to make wood-to-ethanol conversion more cost-effective. Despite ongoing agency activities, however, numerous officials believe that additional steps beyond the agencies’ authority are need to fully address obstacles to woody biomass utilization. Among these steps are subsidies and tax credits, which officials told us are necessary to develop a market for woody biomass but which are beyond the agencies’ authority. According to several officials, the obstacles to using woody biomass cost- effectively are simply too great to overcome by using the tools—grants, outreach and education, and so forth—currently at the agencies’ disposal. One official stated that “in many areas, the economic return from smaller- diameter trees is less than production costs. Without some form of market intervention, such as tax incentives or other forms of subsidy, there is little short-term opportunity to increase utilization of such material.” Some officials stated that subsidies have the potential to create an important benefit—reduced fire risk through hazardous fuels reduction—if they promote additional thinning activities by stimulating the woody biomass market. Rather than incentives or subsidies, some officials noted the potential for increased use of woody biomass through state requirements—known as renewable portfolio standards—that utilities procure or generate a portion of their electricity by using renewable resources, which could include woody biomass. But not all officials believe these additional steps are efficient or appropriate. One official told us that, although he supports these activities, tax incentives and subsidies would create enormous administrative and monitoring requirements. Another official stated that although increased subsidies could address obstacles to woody biomass utilization, he does not believe they should be implemented, preferring instead to allow research and development efforts and market forces to establish the extent of woody biomass utilization. Further, not all agree that the market for woody biomass should be expanded. One agency official told us he is concerned that developing a market for woody biomass could result in overuse of mechanical treatment (rather than prescribed burning) as the market begins to drive the preferred treatment, and representatives of one national environmental group told us that relying on woody biomass as a renewable energy source will lead to overthinning, as demand exceeds the supply that is generated through responsible thinning. Conclusions The amount of woody biomass resulting from increased thinning activities could be substantial, adding importance to the search for ways to use the material cost-effectively rather than simply disposing of it. However, the use of woody biomass will become commonplace only when doing so becomes economically advantageous for users—whether small forest businesses or large utilities. Federal agencies are targeting their activities toward overcoming economic and other obstacles, but some agency officials believe that these efforts alone will not be sufficient to stimulate a market that can accommodate the vast quantities of material expected— and that additional action may be necessary at the federal and state levels. Nevertheless, we believe the agencies will continue to play an important role in stimulating woody biomass use. The Forest Service took a significant step recently by designating an agency lead for woody biomass activities, responding to a need we had identified in our draft report and enhancing the agency’s ability to ensure that its multiple activities contribute to its overall objectives. Given the magnitude of the woody biomass issue and the finite nature of agency budgets, it is essential that federal agencies appropriately coordinate their woody biomass activities—both within and across agencies—to maximize their potential for addressing the issue. Mr. Chairman, this concludes my prepared statement. I would be pleased to answer any questions that you or other Members of the Subcommittee may have at this time. GAO Contacts and Staff Acknowledgments For further information about this testimony, please contact me at (202) 512-3841 or at [email protected]. David P. Bixler, James Espinoza, Steve Gaty, Richard Johnson, and Judy Pagano 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: In an effort to reduce the risk of wildland fires, many federal land managers--including the Forest Service and the Bureau of Land Management--are placing greater emphasis on thinning forests and rangelands to help reduce the buildup of potentially hazardous fuels. These thinning efforts generate considerable quantities of woody material, including many smaller trees, limbs, and brush--referred to as woody biomass--that currently have little or no commercial value. GAO was asked to determine (1) which federal agencies are involved in efforts to promote the use of woody biomass, and the actions they are undertaking; (2) how these agencies coordinate their activities; and (3) what the agencies see as obstacles to increasing the use of woody biomass, and the extent to which they are addressing the obstacles. This testimony is based on GAO's report Natural Resources: Federal Agencies Are Engaged in Various Efforts to Promote the Utilization of Woody Biomass, but Significant Obstacles to Its Use Remain (GAO- 05-373), being released today. Most woody biomass utilization activities are implemented by the Departments of Agriculture (USDA), Energy (DOE), and the Interior and include awarding grants to businesses, schools, Indian tribes, and others; conducting research; and providing education. Most of USDA's woody biomass utilization activities are undertaken by the Forest Service and include grants for woody biomass utilization, research into the use of woody biomass in wood products, and education on potential uses for woody biomass. DOE's woody biomass activities focus on research into using the material for renewable energy, while Interior's efforts consist primarily of education and outreach. Other agencies also provide technical assistance or fund research activities. Federal agencies coordinate their woody biomass activities through formal and informal mechanisms. Although the agencies have established two interagency groups to coordinate their activities, most officials we spoke with emphasized informal communication--through e-mails, participation in conferences, and other means--as the primary vehicle for interagency coordination. Internally, DOE coordinates its woody biomass activities through its Office of Energy Efficiency and Renewable Energy, while Interior and the Forest Service--the USDA agency with the most woody biomass activities--have appointed officials to oversee, and have issued guidance on, their woody biomass activities. The obstacles to using woody biomass cited most often by agency officials were the difficulty of using woody biomass cost-effectively and the lack of a reliable supply of the material; agency activities generally are targeted toward addressing these obstacles. Some officials told us their agencies are limited in their ability to address these obstacles and that incentives--such as subsidies and tax credits--beyond the agencies' authority are needed. However, others disagreed with this approach for a variety of reasons, including the concern that expanding the market for woody biomass could lead to adverse ecological consequences if the demand for woody biomass leads to excessive thinning.

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Write a title and summarize: The current standard of care for hepatitis C virus (HCV) infection – combination therapy with pegylated interferon and ribavirin – elicits sustained responses in only ∼50% of the patients treated. No alternatives exist for patients who do not respond to combination therapy. Addition of ribavirin substantially improves response rates to interferon and lowers relapse rates following the cessation of therapy, suggesting that increasing ribavirin exposure may further improve treatment response. A key limitation, however, is the toxic side-effect of ribavirin, hemolytic anemia, which often necessitates a reduction of ribavirin dosage and compromises treatment response. Maximizing treatment response thus requires striking a balance between the antiviral and hemolytic activities of ribavirin. Current models of viral kinetics describe the enhancement of treatment response due to ribavirin. Ribavirin-induced anemia, however, remains poorly understood and precludes rational optimization of combination therapy. Here, we develop a new mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia in HCV patients. Based on the assumption that ribavirin accumulation decreases erythrocyte lifespan in a dose-dependent manner, model predictions capture several independent experimental observations of the accumulation of ribavirin in erythrocytes and the resulting decline of hemoglobin in HCV patients undergoing combination therapy, estimate the reduced erythrocyte lifespan during therapy, and describe inter-patient variations in the severity of ribavirin-induced anemia. Further, model predictions estimate the threshold ribavirin exposure beyond which anemia becomes intolerable and suggest guidelines for the usage of growth hormones, such as erythropoietin, that stimulate erythrocyte production and avert the reduction of ribavirin dosage, thereby improving treatment response. Our model thus facilitates, in conjunction with models of viral kinetics, the rational identification of treatment protocols that maximize treatment response while curtailing side effects. 130–170 million people worldwide are currently infected with hepatitis C virus (HCV) [1]. Over 70% of HCV infections become chronic and if untreated may lead to cirrhosis and hepatocellular carcinoma, necessitating liver transplantation [1]. The standard of care for HCV infection involves combination therapy with pegylated interferon and ribavirin [2]. Ribavirin alone does not elicit a lasting antiviral response [3]–[6], yet it substantially improves treatment response in combination with interferon [7]–[11]. For instance, whereas ∼29% of the patients treated with interferon exhibited a sustained virological response (SVR), the response rate increased to ∼56% upon addition of ribavirin [8]. Ribavirin, however, is associated with the side-effect, hemolytic anemia, which often renders therapy intolerable [4], [12]–[15]. With the standard ribavirin dosage of 1000–1200 mg/day, 54% of the patients treated experienced a decline in the hemoglobin (Hb) level of over 3 g/dL, and 10% of the men and 7% of the women treated experienced an Hb decline of over 5 g/dL (normal Hb range: 14–16 g/dL) [15]. This drop in Hb often necessitates a reduction of ribavirin dosage, which significantly compromises treatment response [7], [13], [14], [16]. The probability of achieving SVR is estimated to decrease from ∼65% to ∼45% when ribavirin dosage is reduced from ∼15 mg/kg to ∼7 mg/kg of body weight, in combination with pegylated interferon at 1. 5 µg/kg of body weight [7]. Patients receiving fewer than 60% of the planned ribavirin doses had lower response rates [14], indicating that lower cumulative ribavirin exposure results in poorer treatment response [13], [16]. The rates of relapse of infection following the end of treatment also increased upon lowering ribavirin dosage [14], [16]. In a recent clinical trial where interferon was employed with telaprevir, a promising new inhibitor of HCV protease, response rates were lowest in patients who were not administered ribavirin [17], underscoring the importance of ribavirin in achieving SVR. Alternatives for patients who do not respond to combination therapy do not exist yet [2], [18]. Significant efforts are underway therefore to identify treatment protocols that maximize response rates to combination therapy while curtailing side-effects [16], [19]–[25]. A particularly promising strategy is to supplement combination therapy with growth hormones, such as erythropoietin, that stimulate erythropoiesis and thus avert the reduction of ribavirin dosage, potentially improving treatment response [26]–[31]. The predominant mechanism (s) of the anti-HCV activity of ribavirin remain to be established [32]–[34]. Mathematical models of viral kinetics have been developed that describe the antiviral activity of interferon and the enhancement of treatment response rates due to ribavirin, and are being extended to predict the impact of new antiviral drugs [34]–[42]. Ribavirin-induced anemia, on the other hand, remains poorly understood [13], [24], [25], [43]–[47] and precludes rational optimization of combination therapy. Here, we construct a mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia and informs future strategies for improving outcomes of combination therapy. Model predictions capture experimental observations of the accumulation of ribavirin in erythrocytes and the ensuing Hb decline in HCV patients following the onset of combination therapy, estimate the enhanced turnover rate of erythrocytes during therapy and the threshold ribavirin exposure beyond which anemia is intolerable, present guidelines for the optimal usage of growth hormone supplements, and provide a framework, in conjunction with models of viral kinetics, for rational optimization of combination therapy. Prior to the onset of treatment with ribavirin, the population of erythrocytes (RBCs) in an HCV infected individual is constant; a balance exists between RBC production and death (Fig. 1). Following the onset of treatment, ribavirin administered orally gets rapidly transported from the plasma to RBCs, where it is phosphorylated to its mono-, di- and tri-phosphate analogs (RMP, RDP, and RTP) [48]. Phosphorylated analogs are neither easily metabolized nor transported out of RBCs [48]. Consequently, ribavirin accumulates inside RBCs in the form of its phosphorylated analogs; the total intracellular concentration of ribavirin can be >100-fold its extracellular concentration [47]. This dramatic accumulation of ribavirin may induce oxidative damage and result in enhanced extra vascular death of RBCs [12]. Indeed, RBC lifespan decreased from 107±22 d in HCV patients not exposed to ribavirin to 39±13 d in HCV patients undergoing treatment with ribavirin [49], [50]. The shortened RBC lifespan creates an imbalance between RBC production and death and results in a decline in the RBC population. Accordingly, Hb levels drop and patients become anemic. We construct a mathematical model to describe this dynamics of ribavirin-induced anemia (Methods). We consider a recent study of the time-evolution of and in 19 Japanese patients following the onset of combination therapy [47]. In this latter study, no reduction of ribavirin dosage is reported. The patients were divided into two groups based on whether <1000 µM (7 patients) or >1000 µM (12 patients); the data are reported as the average within each group. We fit model predictions of and to the data of the former 7 patients using,, and as adjustable parameters. (Interferon may also induce anemia, but does so to a much smaller extent than ribavirin [13]. We therefore assume that the Hb decline in patients undergoing combination therapy is primarily due to ribavirin.) We fix the remaining parameters based on previous studies or from analysis of independent experiments (Methods). Model predictions provide good fits to the data and yield estimates of,, and (Fig. 5A). The fits suggest that our model is able to describe the underlying dynamics of ribavirin-induced anemia in HCV patients. Interestingly, with the same parameter values, our model captures changes in Hb and from the other 12 Japanese patients, as well as an independent data set of Hb decline in another group of HCV patients undergoing combination therapy [29] (Fig. 5B), validating our best-fit parameter estimates. Further, with the same parameter values, we estimate that the RBC lifespan is 38 days (95% CI: 19–55 days) in Japanese patients with <1000 µM and 33 days (95% CI: 14–53 days) in Japanese patients with >1000 µM. These estimates of the RBC lifespan are in close agreement with independent estimates, 39±13 days, from measurements of alveolar carbon monoxide [49], [50], presenting another successful test of our model. Finally, we find that our predictions of the dependence of on and using the same parameters above are also in agreement with observations in the Japanese patients [47] (Fig. 5C, D). Our model thus presents a robust description of ribavirin-induced anemia in HCV patients undergoing combination therapy. Our model has several clinical implications. First, it enables estimation of the threshold ribavirin exposure beyond which anemia is intolerable. Current treatment guidelines recommend a reduction of ribavirin dosage when Hb decreases below 10 g/dL. We apply our model to predict as a function of. We find that on average (when = 14. 4 g/dL) <10 g/dL when >13 µM (Fig. 6A). Thus, steady state plasma concentrations above 13 µM would render ribavirin therapy intolerable. While the dependence of the peak plasma concentration on dosage following a single ribavirin dose has been determined [48], the dependence of on dosage remains to be established. A description of the multiple dose pharmacokinetics of ribavirin, which also remains elusive [6], [34], [48], [52], [53], would establish the dosage corresponding to of 13 µM that would render ribavirin intolerable. Second, when is above the threshold, our model allows estimation of the increase in RBC production, which may be achieved by administration of exogenous growth hormones such as recombinant erythropoietin, necessary to avert the currently recommended reduction of dosage. Because growth hormones also have side-effects [29], [54], one strategy is to use them at levels just enough to increase to 10–12 g/dL (rather than the pretreatment level), which renders ribavirin tolerable [16]. We apply our model to predict the level of RBC production necessary for achieving of 10–12 g/dL for different values of (Fig. 6B). Thus, when = 15 µM, RBC production rates of 8. 44 and 10. 2 million cells s−1 are necessary for ensuring of 10 and 12 g/dL, respectively. Increase in endogenous erythropoietin levels during therapy, also observed experimentally [45], [55], results in an enhanced production rate of 8. 1 million cells s−1, which is 3. 5-fold higher than the basal production rate (here 2. 3 million cells s−1 in the absence of ribavirin) but inadequate to achieve the desired. Hormone supplements may be employed to provide the balance of 0. 34 or 2. 1 million cells s−1 increase in the RBC production rate to ensure of 10 or 12 g/dL, respectively. This deficiency in RBC production that hormone supplements must compensate increases with ribavirin exposure (Fig. 6B). The ability to enhance treatment response rates renders ribavirin central to the treatment of HCV infection. Maximizing the benefit of ribavirin to patients requires striking the right balance between its antiviral activity and its treatment-limiting side-effect, hemolytic anemia. Rational approaches to therapy optimization thus rely on quantitative descriptions of both the antiviral and the hemolytic activities of ribavirin. Extant mathematical models predict the enhancement in treatment response due to ribavirin [34]–[42]. Ribavirin-induced anemia, however, remains poorly described and limits our ability to maximize treatment response. Here, we fill this gap by constructing a model of the population dynamics of RBCs that quantitatively describes ribavirin-induced anemia. By assuming that intracellular accumulation of ribavirin enhances RBC death rate in a dose-dependent manner, our model captures several independent observations of ribavirin-induced anemia in HCV patients undergoing combination therapy. In particular, our model predicts the dynamics of the accumulation of ribavirin in RBCs and the resulting decline of Hb in patients following the onset of therapy, estimates the reduced lifespan of RBCs during therapy, and describes inter-patient variations in the severity of anemia, thus presenting a robust description of ribavirin-induced anemia, which, in conjunction with models of viral kinetics, may facilitate identification of treatment protocols that maximize the impact of ribavirin in the treatment of HCV infection. Our model has clinical implications. First, it allows estimation of the threshold ribavirin exposure beyond which ribavirin-induced anemia becomes intolerable. For instance, with model parameters that describe ribavirin-induced anemia in the patients we considered (Fig. 5), we estimate that steady state plasma ribavirin concentrations above 13 µM would render ribavirin therapy intolerable. Determining dosage levels corresponding to this steady state plasma concentration requires knowledge of the pharmacokinetics of ribavirin, which is currently lacking [6], [34], [48], [52], [53]. Ribavirin pharmacokinetics is peculiar because of an unusually long elimination phase that follows rapid absorption and distribution phases upon oral dosing [48]. Standard absorption-elimination models of drug pharmacokinetics are unable to describe this long elimination phase. Models that include additional compartments have been proposed to capture the three-phase pharmacokinetics of ribavirin [52], but the biological origin of these compartments remains unclear. An additional complication is that the half-life of the elimination phase increases from 79 h following a single dose to 274–298 h following multiple dosing [48], suggesting that parameters that describe single dose pharmacokinetics may not apply to multiple dose pharmacokinetics. In the absence of rigorous models of ribavirin pharmacokinetics, one may have to rely on empirical relationships between the dosage and the resulting steady state plasma concentration following multiple dosing (e. g., [56]) to establish the dosage that would ensure tolerability of ribavirin while maximizing treatment response. Second, our model suggests guidelines for the usage of hormone supplements, such as erythropoietin, which enhance RBC production and improve the tolerability of ribavirin. For instance, we predict that when ribavirin accumulates to a plasma concentration of 15 µM, the associated enhanced RBC death rate elicits a natural response that increases RBC production 3. 5-fold, from 2. 3 to 8. 1 million cells s−1. This response, however, is inadequate to suppress ribavirin-induced anemia adequately and renders ribavirin intolerable. We estimate then that growth hormone supplements must increase RBC production rate by an additional 0. 34–2. 1 million cells s−1 to render ribavirin tolerable. This compensation that hormone supplements must provide increases with ribavirin accumulation. Identifying the dosage of the growth hormones that induces the necessary RBC production requires knowledge of the dose-response relationships and of the pharmacokinetics of the growth hormones, which are yet to be fully elucidated [26]–[31]. Third, genetic variations that resulted in a deficiency in the enzyme inosine triphosphatase (ITPA) were recently found to protect HCV patients against ribavirin-induced anemia [51]. Deficiency in ITPA causes an increase in inosine triphosphate levels in RBCs, which is thought to interfere with RTP activity and thereby suppress the hemolytic potential of ribavirin. Because deficiency in ITPA is a clinically benign condition, therapeutic intervention to suppress ITPA presents a promising new strategy to curtail ribavirin-induced anemia without compromising the antiviral activity of ribavirin [51]. Our model may be adapted to inform the development of such an intervention strategy. In our model, the dependence of the death rate of RBCs on ribavirin accumulation, determined by Eq. (2) (Methods), would now be a function of the ITPA level. Thus, experiments that determine how variations in the ITPA level both in the absence and in the presence of ribavirin influence RBC lifespan would provide the necessary inputs for our model to account explicitly for the role of ITPA in ribavirin-induced anemia. The resulting model would enable determination of the minimal inhibition of ITPA necessary to maintain ribavirin-induced anemia within tolerable limits. Conversely, using information of the ITPA level intrinsic to a patient, the model can be applied to predict the maximum ribavirin dosage that the patient can tolerate, thus presenting an avenue for personalizing the treatment of HCV infection. We consider the RBC population in an individual at time t following the onset of treatment with ribavirin (t = 0) (Fig. 1). RBCs produced at different times in the interval from 0 to t will have been exposed to ribavirin for different durations and accordingly have different intracellular levels of ribavirin. We define as the population of RBCs that contain ribavirin phosphorylated analogs, RXP, which comprises RMP, RDP, and RTP, at concentrations between and at time. is thus the number density of RBCs containing RXP at concentration C at time t. The time evolution of is governed by the following equation (Text S1) (1) The first term on the right-hand-side in Eq. (1) represents the change in due to intracellular phosphorylation of ribavirin. is the net rate of increase of C due to phosphorylation, is the intracellular concentration of (unphosphorylated) ribavirin, is the phosphorylation rate and is the rate of loss, including by possible slow dephosphorylation, of RXP. In vitro studies of ribavirin uptake by RBCs observe rapid (<10 min) equilibration of intracellular and extracellular ribavirin [53], [57]. We assume therefore that, the concentration of ribavirin in plasma. With twice daily oral administration of ribavirin, rises from zero at and reaches an asymptotic maximum,, so that, where is the characteristic timescale of the accumulation of ribavirin in plasma [6], [38]. The second term on the right-hand side of Eq. (1) accounts for the loss of RBCs due to their death. We assume that the death rate, D, of RBCs increases with as follows (2) where is the death rate of RBCs in the absence of ribavirin, is that value of at which the death rate doubles (or the lifespan halves) compared to that in the absence of ribavirin, and, analogous to the Hill coefficient, determines the sensitivity of to changes in. (A saturable form for D (C) appears inconsistent with available data; see Text S2, Fig. S1.) Equation (1) is constrained by the initial condition that in all cells at the start of therapy, so that, where N0 is the population of RBCs at t = 0, and is the Dirac delta function, which satisfies and. In other words, the Dirac delta function ensures that no cells have RXP at non-zero concentrations at t = 0. A second constraint on Eq. (1) is imposed by the boundary condition that when >0, newborn cells contain no RXP so that (Text S1) where (3) is the rate of production of RBCs at time t. The production of RBCs by the bone marrow is regulated by a negative feedback mechanism involving the hormone erythropoietin [58]. Recent studies on modeling erythropoiesis elucidate the complexities involved in a quantitative description of this feedback mechanism [59]–[65]. Here, we employ Eq. (3) to capture the essential features of this negative feedback: As the population of RBCs,, decreases, P increases. is the maximum production rate of RBCs, which occurs when N is vanishingly small, is that value of the RBC population per unit volume of blood () at which, is the volume of blood, and, analogous to the Hill coefficient, determines the sensitivity of to changes in. Eq. (3) provides good fits to independent measurements of the dynamics of the recovery of RBCs following phlebotomy (Text S3, Fig. S2). Equations (1) – (3) present a model of the population dynamics of RBCs in individuals undergoing treatment with ribavirin. We solve the equations (see below) and obtain the population density,, and the corresponding cumulative population,, using which we predict the time-evolution of the hemoglobin level in blood, (where is the volume of a single erythrocyte); the average concentration of ribavirin in RBCs, ; and the average RBC lifespan,, where is the average death rate of RBCs. Equation (1) along with the initial and boundary conditions is equivalent to the following set of differential equations obtained using the method of characteristics (Text S4) (4) where Si (t) is the subpopulation of cells born within an interval of that survive at time t. Ci (t) is the concentration of RXP in the latter cells at time t. We solve Eq. (4) along with Eqs. (2) and (3) with d using a program written in MATLAB (Text S5). We validate our solution methodology against an analytical solution that can be obtained in the limiting case when the RBC death rate is independent of RXP accumulation (Text S6, Fig. S3). We also ensure that d allows accurate integration of Eq. (4) without compromising computational efficiency (Fig. S4). From the solution, we calculate the quantities of interest, viz.,,,, and. We employ the following values of the model parameters unless stated otherwise. The average RBC lifespan in normal man is ∼120 days [49], [66], which corresponds to d−1. We let b = 7 following earlier studies [59] and obtain cells d−1 from an independent analysis of blood loss experiments (Text S3). We fix and [67]. Using [47], we get. We obtain from the initial steady state. Further, we let [47] and because ribavirin accumulates in plasma to its maximum concentration in ∼4 weeks, we set [6], [38]. The remaining parameter values,,, and are obtained from best-fits of our model predictions to experimental data (Fig. 5A). We summarize model parameters and their values in Table 1. We fit model predictions to experimental data (Fig. 5A) using the nonlinear regression tool NLINFIT in MATLAB.

Title: Ribavirin-Induced Anemia in Hepatitis C Virus Patients Undergoing Combination Therapy Summary: The treatment of HCV infection poses a major global health-care challenge today. The current standard of care, combination therapy with interferon and ribavirin, works in only about half of the patients treated. Because no alternatives are available yet for patients in whom combination therapy fails, identifying ways to improve response to combination therapy is critical. Increasing exposure to ribavirin does improve response but is associated with the severe side-effect, anemia. One way to maximize treatment response therefore is to increase ribavirin exposure to levels just below where anemia becomes intolerable. A second way is to supplement combination therapy with growth hormones, such as erythropoietin, that increase the production of red blood cells (erythrocytes) and compensate for ribavirin-induced anemia. Rational optimization of combination therapy thus relies on a quantitative description of ribavirin-induced anemia, which is currently lacking. Here, we develop a model of the population dynamics of erythrocytes in individuals exposed to ribavirin that quantitatively describes ribavirin-induced anemia. Model predictions capture several independent observations of ribavirin-induced anemia in HCV patients undergoing combination therapy, estimate the threshold ribavirin exposure beyond which anemia becomes intolerable, suggest guidelines for the usage of growth hormones, and facilitate rational optimization of therapy.

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Summarize: ACT III. SCENE I. Rome. A street Enter the JUDGES, TRIBUNES, and SENATORS, with TITUS' two sons MARTIUS and QUINTUS bound, passing on the stage to the place of execution, and TITUS going before, pleading TITUS. Hear me, grave fathers; noble Tribunes, stay! For pity of mine age, whose youth was spent In dangerous wars whilst you securely slept; For all my blood in Rome's great quarrel shed, For all the frosty nights that I have watch'd, And for these bitter tears, which now you see Filling the aged wrinkles in my cheeks, Be pitiful to my condemned sons, Whose souls are not corrupted as 'tis thought. For two and twenty sons I never wept, Because they died in honour's lofty bed. [ANDRONICUS lieth down, and the judges pass by him with the prisoners, and exeunt] For these, Tribunes, in the dust I write My heart's deep languor and my soul's sad tears. Let my tears stanch the earth's dry appetite; My sons' sweet blood will make it shame and blush. O earth, I will befriend thee more with rain That shall distil from these two ancient urns, Than youthful April shall with all his show'rs. In summer's drought I'll drop upon thee still; In winter with warm tears I'll melt the snow And keep eternal spring-time on thy face, So thou refuse to drink my dear sons' blood. Enter Lucius with his weapon drawn O reverend Tribunes! O gentle aged men! Unbind my sons, reverse the doom of death, And let me say, that never wept before, My tears are now prevailing orators. LUCIUS. O noble father, you lament in vain; The Tribunes hear you not, no man is by, And you recount your sorrows to a stone. TITUS. Ah, Lucius, for thy brothers let me plead! Grave Tribunes, once more I entreat of you. LUCIUS. My gracious lord, no tribune hears you speak. TITUS. Why, 'tis no matter, man: if they did hear, They would not mark me; if they did mark, They would not pity me; yet plead I must, And bootless unto them. Therefore I tell my sorrows to the stones; Who though they cannot answer my distress, Yet in some sort they are better than the Tribunes, For that they will not intercept my tale. When I do weep, they humbly at my feet Receive my tears, and seem to weep with me; And were they but attired in grave weeds, Rome could afford no tribunes like to these. A stone is soft as wax: tribunes more hard than stones. A stone is silent and offendeth not, And tribunes with their tongues doom men to death. [Rises] But wherefore stand'st thou with thy weapon drawn? LUCIUS. To rescue my two brothers from their death; For which attempt the judges have pronounc'd My everlasting doom of banishment. TITUS. O happy man! they have befriended thee. Why, foolish Lucius, dost thou not perceive That Rome is but a wilderness of tigers? Tigers must prey, and Rome affords no prey But me and mine; how happy art thou then From these devourers to be banished! But who comes with our brother Marcus here? Enter MARCUS with LAVINIA MARCUS. Titus, prepare thy aged eyes to weep, Or if not so, thy noble heart to break. I bring consuming sorrow to thine age. TITUS. Will it consume me? Let me see it then. MARCUS. This was thy daughter. TITUS. Why, Marcus, so she is. LUCIUS. Ay me! this object kills me. TITUS. Faint-hearted boy, arise, and look upon her. Speak, Lavinia, what accursed hand Hath made thee handless in thy father's sight? What fool hath added water to the sea, Or brought a fagot to bright-burning Troy? My grief was at the height before thou cam'st, And now like Nilus it disdaineth bounds. Give me a sword, I'll chop off my hands too, For they have fought for Rome, and all in vain; And they have nurs'd this woe in feeding life; In bootless prayer have they been held up, And they have serv'd me to effectless use. Now all the service I require of them Is that the one will help to cut the other. 'Tis well, Lavinia, that thou hast no hands; For hands to do Rome service is but vain. LUCIUS. Speak, gentle sister, who hath martyr'd thee? MARCUS. O, that delightful engine of her thoughts That blabb'd them with such pleasing eloquence Is torn from forth that pretty hollow cage, Where like a sweet melodious bird it sung Sweet varied notes, enchanting every ear! LUCIUS. O, say thou for her, who hath done this deed? MARCUS. O, thus I found her straying in the park, Seeking to hide herself as doth the deer That hath receiv'd some unrecuring wound. TITUS. It was my dear, and he that wounded her Hath hurt me more than had he kill'd me dead; For now I stand as one upon a rock, Environ'd with a wilderness of sea, Who marks the waxing tide grow wave by wave, Expecting ever when some envious surge Will in his brinish bowels swallow him. This way to death my wretched sons are gone; Here stands my other son, a banish'd man, And here my brother, weeping at my woes. But that which gives my soul the greatest spurn Is dear Lavinia, dearer than my soul. Had I but seen thy picture in this plight, It would have madded me; what shall I do Now I behold thy lively body so? Thou hast no hands to wipe away thy tears, Nor tongue to tell me who hath martyr'd thee; Thy husband he is dead, and for his death Thy brothers are condemn'd, and dead by this. Look, Marcus! Ah, son Lucius, look on her! When I did name her brothers, then fresh tears Stood on her cheeks, as doth the honey dew Upon a gath'red lily almost withered. MARCUS. Perchance she weeps because they kill'd her husband; Perchance because she knows them innocent. TITUS. If they did kill thy husband, then be joyful, Because the law hath ta'en revenge on them. No, no, they would not do so foul a deed; Witness the sorrow that their sister makes. Gentle Lavinia, let me kiss thy lips, Or make some sign how I may do thee ease. Shall thy good uncle and thy brother Lucius And thou and I sit round about some fountain, Looking all downwards to behold our cheeks How they are stain'd, like meadows yet not dry With miry slime left on them by a flood? And in the fountain shall we gaze so long, Till the fresh taste be taken from that clearness, And made a brine-pit with our bitter tears? Or shall we cut away our hands like thine? Or shall we bite our tongues, and in dumb shows Pass the remainder of our hateful days? What shall we do? Let us that have our tongues Plot some device of further misery To make us wonder'd at in time to come. LUCIUS. Sweet father, cease your tears; for at your grief See how my wretched sister sobs and weeps. MARCUS. Patience, dear niece. Good Titus, dry thine eyes. TITUS. Ah, Marcus, Marcus! Brother, well I wot Thy napkin cannot drink a tear of mine, For thou, poor man, hast drown'd it with thine own. LUCIUS. Ah, my Lavinia, I will wipe thy cheeks. TITUS. Mark, Marcus, mark! I understand her signs. Had she a tongue to speak, now would she say That to her brother which I said to thee: His napkin, with his true tears all bewet, Can do no service on her sorrowful cheeks. O, what a sympathy of woe is this As far from help as Limbo is from bliss! Enter AARON the Moor AARON. Titus Andronicus, my lord the Emperor Sends thee this word, that, if thou love thy sons, Let Marcus, Lucius, or thyself, old Titus, Or any one of you, chop off your hand And send it to the King: he for the same Will send thee hither both thy sons alive, And that shall be the ransom for their fault. TITUS. O gracious Emperor! O gentle Aaron! Did ever raven sing so like a lark That gives sweet tidings of the sun's uprise? With all my heart I'll send the Emperor my hand. Good Aaron, wilt thou help to chop it off? LUCIUS. Stay, father! for that noble hand of thine, That hath thrown down so many enemies, Shall not be sent. My hand will serve the turn, My youth can better spare my blood than you, And therefore mine shall save my brothers' lives. MARCUS. Which of your hands hath not defended Rome And rear'd aloft the bloody battle-axe, Writing destruction on the enemy's castle? O, none of both but are of high desert! My hand hath been but idle; let it serve To ransom my two nephews from their death; Then have I kept it to a worthy end. AARON. Nay, come, agree whose hand shall go along, For fear they die before their pardon come. MARCUS. My hand shall go. LUCIUS. By heaven, it shall not go! TITUS. Sirs, strive no more; such with'red herbs as these Are meet for plucking up, and therefore mine. LUCIUS. Sweet father, if I shall be thought thy son, Let me redeem my brothers both from death. MARCUS. And for our father's sake and mother's care, Now let me show a brother's love to thee. TITUS. Agree between you; I will spare my hand. LUCIUS. Then I'll go fetch an axe. MARCUS. But I will use the axe. Exeunt LUCIUS and MARCUS TITUS. Come hither, Aaron, I'll deceive them both; Lend me thy hand, and I will give thee mine. AARON. [Aside] If that be call'd deceit, I will be honest, And never whilst I live deceive men so; But I'll deceive you in another sort, And that you'll say ere half an hour pass. [He cuts off TITUS' hand] Re-enter LUCIUS and MARCUS TITUS. Now stay your strife. What shall be is dispatch'd. Good Aaron, give his Majesty my hand; Tell him it was a hand that warded him From thousand dangers; bid him bury it. More hath it merited- that let it have. As for my sons, say I account of them As jewels purchas'd at an easy price; And yet dear too, because I bought mine own. AARON. I go, Andronicus; and for thy hand Look by and by to have thy sons with thee. [Aside] Their heads I mean. O, how this villainy Doth fat me with the very thoughts of it! Let fools do good, and fair men call for grace: Aaron will have his soul black like his face. Exit TITUS. O, here I lift this one hand up to heaven, And bow this feeble ruin to the earth; If any power pities wretched tears, To that I call! [To LAVINIA] What, would'st thou kneel with me? Do, then, dear heart; for heaven shall hear our prayers, Or with our sighs we'll breathe the welkin dim And stain the sun with fog, as sometime clouds When they do hug him in their melting bosoms. MARCUS. O brother, speak with possibility, And do not break into these deep extremes. TITUS. Is not my sorrow deep, having no bottom? Then be my passions bottomless with them. MARCUS. But yet let reason govern thy lament. TITUS. If there were reason for these miseries, Then into limits could I bind my woes. When heaven doth weep, doth not the earth o'erflow? If the winds rage, doth not the sea wax mad, Threat'ning the welkin with his big-swol'n face? And wilt thou have a reason for this coil? I am the sea; hark how her sighs do blow. She is the weeping welkin, I the earth; Then must my sea be moved with her sighs; Then must my earth with her continual tears Become a deluge, overflow'd and drown'd; For why my bowels cannot hide her woes, But like a drunkard must I vomit them. Then give me leave; for losers will have leave To ease their stomachs with their bitter tongues. Enter a MESSENGER, with two heads and a hand MESSENGER. Worthy Andronicus, ill art thou repaid For that good hand thou sent'st the Emperor. Here are the heads of thy two noble sons; And here's thy hand, in scorn to thee sent back- Thy grief their sports, thy resolution mock'd, That woe is me to think upon thy woes, More than remembrance of my father's death. Exit MARCUS. Now let hot Aetna cool in Sicily, And be my heart an ever-burning hell! These miseries are more than may be borne. To weep with them that weep doth ease some deal, But sorrow flouted at is double death. LUCIUS. Ah, that this sight should make so deep a wound, And yet detested life not shrink thereat! That ever death should let life bear his name, Where life hath no more interest but to breathe! [LAVINIA kisses TITUS] MARCUS. Alas, poor heart, that kiss is comfortless As frozen water to a starved snake. TITUS. When will this fearful slumber have an end? MARCUS. Now farewell, flatt'ry; die, Andronicus. Thou dost not slumber: see thy two sons' heads, Thy warlike hand, thy mangled daughter here; Thy other banish'd son with this dear sight Struck pale and bloodless; and thy brother, I, Even like a stony image, cold and numb. Ah! now no more will I control thy griefs. Rent off thy silver hair, thy other hand Gnawing with thy teeth; and be this dismal sight The closing up of our most wretched eyes. Now is a time to storm; why art thou still? TITUS. Ha, ha, ha! MARCUS. Why dost thou laugh? It fits not with this hour. TITUS. Why, I have not another tear to shed; Besides, this sorrow is an enemy, And would usurp upon my wat'ry eyes And make them blind with tributary tears. Then which way shall I find Revenge's cave? For these two heads do seem to speak to me, And threat me I shall never come to bliss Till all these mischiefs be return'd again Even in their throats that have committed them. Come, let me see what task I have to do. You heavy people, circle me about, That I may turn me to each one of you And swear unto my soul to right your wrongs. The vow is made. Come, brother, take a head, And in this hand the other will I bear. And, Lavinia, thou shalt be employ'd in this; Bear thou my hand, sweet wench, between thy teeth. As for thee, boy, go, get thee from my sight; Thou art an exile, and thou must not stay. Hie to the Goths and raise an army there; And if ye love me, as I think you do, Let's kiss and part, for we have much to do. Exeunt all but Lucius LUCIUS. Farewell, Andronicus, my noble father, The woefull'st man that ever liv'd in Rome. Farewell, proud Rome; till Lucius come again, He leaves his pledges dearer than his life. Farewell, Lavinia, my noble sister; O, would thou wert as thou tofore hast been! But now nor Lucius nor Lavinia lives But in oblivion and hateful griefs. If Lucius live, he will requite your wrongs And make proud Saturnine and his empress Beg at the gates like Tarquin and his queen. Now will I to the Goths, and raise a pow'r To be reveng'd on Rome and Saturnine. Exit

Summary: On a street in Rome, a group of judges, tribunes, and senators troop along with the prisoners, Martius and Quintus, who have been framed for Bassianus's murder. Titus confronts them and plays the "I'm a war hero" card, pleading for mercy on behalf of his sons. Titus lies down on the ground in protest of his sons' imprisonment, but the judges just walk past him. With nobody left to plead to, Titus begs the "earth" not to drink his sons' blood and promises to cry enough tears to "staunch" the "earth's appetite." Lucius shows up with his sword drawn and tells his dad to quit with the begging and crying since nobody's around to hear it. Lucius announces that he just attempted a dramatic rescue of his falsely accused brothers. As punishment, the judges pronounced an "everlasting doom of banishment." Titus laments that Rome is "a wilderness of tigers," and the Andronicus family is its "prey." Enter Marcus and Lavinia. Marcus approaches and warns Titus to brace himself because he's about to reveal something awful. Lucius takes one look at his sister and says the sight of Lavinia "kills" him. Titus orders Lucius not to be a wimp and asks Lavinia to reveal who chopped off her hands. Titus declares that this is the worst thing that has ever happened to him. Marcus explains that he found Lavinia wandering around the forest like a wounded deer, and Titus repeats that Lavinia's assault is the worst thing that could possibly happen to him. As Lavinia stands crying before her family, Titus states the obvious: Lavinia has no tongue to tell them what happened to her. Marcus speculates that Lavinia is crying because 1) she thinks her brothers killed her husband, or 2) Lavinia knows her brothers didn't kill Bassianus and she can't do anything about it. Titus wonders aloud what he, Lucius, and Marcus should do next. Should they cut off their hands or bite off their tongues to commiserate with Lavinia? Nah, he decides. Marcus and Titus should use their tongues to talk about how they're going to get revenge for what happened to Lavinia. As if on cue, Aaron enters and announces that Saturninus is willing to make a deal with the Andronicus family. If one of them will cut off his hand and send it to the emperor, then Saturninus will let Quintus and Martius go free. Titus immediately asks Aaron if he'll help him chop off his hand. Before Aaron can agree, Lucius and Marcus both offer to cut off one of theirs and proceed to bicker over who gets to mutilate himself to save Quintus and Martius. While Lucius and Marcus run off to fetch the ax, Aaron lends Titus a hand and chops off Titus's, promising to deliver it as ransom for Titus's sons. As Aaron runs off with Titus's hand, he gleefully announces that he loves being a villain. "Let fools do good, and fair men call for grace," he says. "Aaron will have his soul black like his face." As Titus laments his suffering, a messenger arrives with a bag containing - you guessed it - the heads of Martius and Quintus, along with Titus's hand. We interrupt this program for a brain snack: in Elizabethan England, criminals were often punished by getting their hands lopped off. Just saying. The Messenger declares that Titus is being mocked for his loyalty to Rome. Marcus announces that things just couldn't be any worse than they are now, and Titus begins to laugh like a madman. Titus vows to get revenge and starts ordering his family around like a general. Lucius should go to the Goths and raise an army against Saturninus and Tamora. Everyone else is to head home. Marcus will carry one head while Titus carries the other. Lavinia will carry Titus's hand in her teeth, since she doesn't have any hands of her own.

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Summarize: Background Prior to 1970 most road construction and maintenance was financed and carried out by the territorial governments. Locally levied fuel taxes supported road projects. The most significant federal involvement in road construction in the territories was limited to the construction of military roads (especially significant for Guam) and provision of disaster relief. During the 1960s, rapid growth in population and motor vehicle use led to increasing traffic congestion which in turn led to interest in establishing a federally financed territorial road program. The Federal Aid Highway Act of 1968 (P.L. 90-495) directed the Department of Transportation (DOT) to carry out a highway study of Guam, American Samoa, and the Virgin Islands. The report, Territorial Highway Study: Guam, American Samoa, Virgin Islands, released January 7, 1970, revealed that "concentrations of people and motor vehicles within the compact areas of these island territories produce large volumes of traffic compelled to move over substandard highways at slower speeds and at greater hazard than drivers ordinarily encounter in the states. Traffic growth is currently several times more rapid than the national average." The study, however, recommended against any new federal highway grant programs for the three territories. Instead, the report recommended that the territorial governments should themselves finance an expanded highway development program by raising the territorial gasoline tax rates by five cents. It recommended that additional studies be conducted over the following years in order to develop a suitable highway improvement program for each territory, based on its individual needs. The report also recommended that DOT provide technical guidance for each territorial government. Part of the report authors' reluctance to recommend spending significant amounts of federal funds on territorial roads grew out of the constrained fiscal environment of the time. The most significant restraint, however, had to do with the nature of the financing of the federal highway trust fund (HTF) that supports spending on highways in the United States. The HTF is supported by excise tax revenues (mostly from excise taxes on gasoline and diesel fuel) paid by highway users in the states. The territories, however, do not pay these federal taxes and therefore do not provide revenues to the highway trust fund. Territorial Highway Program (THP) Founding Legislation Despite the DOT recommendation against the establishment of a federally financed highway program for the territories, the Federal-Aid Highway Act of 1970 (FAHA70)(P.L. 91-605), section 112, established the Territorial Highway Program(THP). The Act authorized DOT to assist each of the three territories (the Virgin Islands, Guam, and American Samoa) in a program for the construction and improvement of a system of arterial highways and inter-island connectors (envisioned as road links from arterial highways to airports or seaports) to be designated by each territory's Governor. DOT was also directed to provide technical assistance to each territory to assist in the creation of an appropriate agency to administer the program. Provisions of Title 23 applicable to Federal-aid primary highway funds (other than formula provisions) applied to funds authorized to carry out the THP. No funds so authorized were to be used for maintenance of the highway system (i.e. the funds were to be used for building and improving roads only). FAHA'70 authorized $2 million annually for Guam and the Virgin Islands and $500,000 annually for American Samoa for fiscal years 1971 through 1973. Federal assistance was granted to the territories based on a federal contribution of 70% of total project cost. Federal-Aid Highway Acts of 1973 (P.L. 93-87) and 1976 (P.L. 94-280) These authorization acts changed little in the THP other than the authorization levels. The 1973 Act authorized $5 million for the Virgin Islands, $2 million for Guam, and $1 million for American Samoa, annually for FY1974-FY1976. The 1976 Act provided $1.25 million each for the Virgin Islands and Guam and $250,000 for American Samoa for the 1976 special fiscal quarter, ending Sept 31, 1976. The Act then authorized $5 million annually for both the Virgin Islands and Guam and $1 million for American Samoa, for FY1977 and FY1978. Surface Transportation Assistance Act of 1978 (STAA78) (P.L. 95-599) STAA78 made two significant changes to THP. First, the bill added the Northern Mariana Islands to the THP and provided the Islands with $1 million annually for the life of the authorization, FY1979-FY1982. Second, the Act raised the federal contribution under THP from 70% to 100%. The Virgin Islands, Guam, and American Samoa annual authorizations were continued for the life of the authorization at the $5 million, $5 million, and $1 million levels respectively. Surface Transportation Assistance Act of 1982 (STAA82) (P.L. 97-424) STAA82 made a major change in the funding for THP. The Act authorized that the four territories be treated together as a state for Federal-Aid Primary (FAP) funds. This meant that the territories got at least ½ % of FAP funds, in contract authority, from the highway trust fund, each year of the authorization. This also made THP funds subject to reduction under the obligation limitation. The Formula Controversy The apportionment for the territories was allocated to the individual territories under a formula that was based: 1/3 on urban population greater than 5000, 1/3 on rural population, 1/6 on public road mileage, and 1/6 on area. According to FHWA, several territories contested the figures based on population. In September 1983, FHWA decided to infer congressional intent from the funding set forth in STAA78 (i.e. $1 million for American Samoa, $5 million for Guam, $5 million for the Virgin Islands, and $1 million for the Northern Marianas) and derive ratios based on this division of funds. Consequently, from FY1984 through FY1992, THP funds were divided among the territories as follows: 1/12 to American Samoa, 5/12 to the Virgin Islands, 5/12 to Guam, and 1/12 to the Northern Mariana Islands. Surface Transportation and Uniform Relocation Assistance Act of 1987 (STURAA) (P.L. 100-17) STURAA extended the authorizations set forth in STAA82. The THP continued to receive ½ % of each year's federal-aid primary funding under contract authority and the federal share continued at 100%. Intermodal Surface Transportation Efficiency Act of 1991 (ISTEA) (P.L. 102-240) ISTEA made no changes to statutory language of the THP (23 U.S.C. 215) but did change the source of funding. The Act defined the four territories as a state under the National Highway System (NHS) Program and set aside 1% of NHS funds for the territories. Formula Controversy: the Second Round According to FHWA, in 1992 the agency reviewed the 1-5-5-1 allocation, at the request of one of the territories. The agency concluded that American Samoa and the Northern Mariana Islands were not getting their fair share based on road mileage, area, population, or any combination of these factors. FHWA developed and analyzed over twenty possible formulas based on census information and 1990 highway statistics. Beginning in 1993 the administrative allocation was changed to a new 1-4-4-1 formula. This raised the ratio received by American Samoa and the Northern Mariana Islands to 1/10 and reduced the ratio received by the Virgin Islands and Guam to 4/10. ISTEA required each territory to classify their highways. Since then, the territories have each established, with FHWA approval, a combination of arterial and collector highways and inter-island connectors. Called the Federal-Aid Territorial Highway System, these routes are eligible for Federal-aid funds under THP. Transportation Equity Act for the Twenty First Century (TEA-21)(P.L. 105-178) TEA-21 changed the set-aside for THP from 1% of NHS funds to a set amount of $36.4 million per fiscal year. During the debate on TEA-21 the House voted to continue the 1% set-aside from NHS, but the Senate voted not to fund the THP. The $36.4 million set-aside was agreed to in conference (H. Rept.105-550). Because the funds are subject to the annual obligation limitation, only the amount of the $36.4 million for which obligation authority is made available may be distributed to the territories. TEA-21 also expanded project eligibility to include any project eligible for assistance under the Surface Transportation Program, as well as any airport or seaport in the four territories. FHWA continues to divide these funds according to the 1-4-4-1 administrative formula. THP Funding History Federal funding of transportation projects under the THP has changed over the years in accordance with the relevant provisions of the surface transportation authorization acts discussed in the previous section. Table 1 sets forth the actual amounts distributed to the territories under the THP. Other than the founding year, the years which initiated significant THP funding increases were FY1974, FY1977, and FY1992. In FY1974, funding for American Samoa doubled and funding for the Virgin Islands more than doubled. Guam's funding was increased to $5 million for FY1977 which brought Guam into parity with the Virgin Islands. This rough parity continues to this day. The drop in funding in FY1983 reflects the shift to ½ % of federal aid primary contract authority and also the impact of THP being made subject to obligation limitation. The next major increase in funding occurred during FY1992-FY1993 under ISTEA. By FY1993 the funding for American Samoa and the Northern Marianas had more than tripled (due in part to the change in the distribution formula in 1993, mentioned earlier). Funding for Guam and the Virgin Islands more than doubled. Under TEA-21 funding for THP has remained at roughly the ISTEA level. Non-THP Highway Funding for the Territories The territories may receive highway funding from sources other than the THP, including the Emergency Relief Program, the High Priority Projects Program, and certain highway safety programs. The territories are not, however, eligible for Federal Highway Administration discretionary programs. The eligibility for these programs is limited by statute to the states and the District of Columbia, usually with Puerto Rico as the only exception. Emergency Relief The largest other source of federal highway funding is the Emergency Relief Federal-Aid Highway program (ER). The territories are subject to storm damage and other natural disasters, and may request ER assistance to restore damaged roads to their pre-disaster condition. From FY1990 to FY2000 the territories received $72.9 million in ER funds, American Samoa received $22.9 million, Guam received $39.8 million, and the Virgin Islands received $10.2 million. High Priority Projects American Samoa and the Virgin Islands have also participated in the High Priority Project Program (HPP) and its ISTEA predecessor, the Demonstration, Priority, and Special Interest Projects Program. Projects under these programs are designated in the text of the authorizing legislation. According to FHWA, since October 1991, a total of $48.6 million has been authorized and $44.1 million has been allocated for projects in these same two territories. Safety Programs Under chapter 4 (Highway Safety) of Title 23 of the U.S. Code the territories are defined as "states" and therefore receive Section 402 formula grants each year and may apply for incentive grants under sections 405 (occupant protection incentive grants), section 410 (alcohol-impaired driving countermeasures), section 411 (state highway safety data improvements), and TEA-21 Section 2003 (b) (child passenger protection education grants). Under these sections the territories together received $2.1 million for FY2000 and $2.3 million for FY2001. The National Highway Traffic Safety Administration (NHTSA) administers these sections. As mentioned earlier, the territories are not considered as states under FHWA programs. This makes them ineligible for FHWA safety grants under sections 157 (safety incentive grants for the use of seat belts) and 163 (safety incentives to prevent operation of motor vehicles by intoxicated persons). The territories participate in the Motor Carrier Safety Assistance Program (MCSAP). MCSAP provides grants to help the territories enforce their truck and bus safety regulations. As of FY2001, each of the territories began receiving a fixed amount of $350,000 annually for MCSAP. Congressional Issues and Options The arguments for and against federal-aid highway assistance to the territories have changed little since the debate of the 1960s that culminated in the founding of the THP. With the approaching reauthorization of TEA-21 the overall arguments are worth reviewing. Pro The arguments generally made in support of federal aid to the territories, including highway aid, are usually framed in terms of the transportation and development needs of the territories, the benefits the territories provide to the United States, and their limited enfranchisement. In general, population growth and an increase in the number of vehicles on territorial roads has increased traffic congestion in all the territories. Although significant improvements have been made under the THP, some territorial roads continue to be under stress from inadequate capacity while other roads continue to be substandard and hazardous. Because the territories are all islands, much of the road construction is more expensive than in the 50 U.S. states. Remoteness from suppliers is especially a problem for the Pacific territories. In addition, the territories are significantly less well off than the states, have a lower per capita domestic product, and pay higher prices for many basic goods. Advocates of highway assistance to the territories argue that the territories are not wealthy enough to provide for their road infrastructure needs without federal assistance. Supporters also argue that the goal of bringing the territories into economic parity with the states is an established principle of the federal government. Federal highway assistance is seen as falling under this principle. Advocates of assistance also point out that all the territories were acquired for strategic purposes and contend that they continue to be of current or potential strategic value to the United States. Three of the territories, Guam, American Samoa, and the Virgin Islands are each represented in Congress by a single non-voting representative in the U.S. House of Representatives. The Commonwealth of the Northern Mariana Islands has no congressional representative. Some advocates of assistance to the territories see a link between this limited representation and what they see as low levels of federal assistance. Con The main objection to full or expanded territorial participation in federal-aid highway program (FAHP) is that the territories do not pay the taxes that support the program. The FAHP is funded from the highway account of the HTF. The HTF is supported by highway user taxes (mostly fuel taxes) paid for by highway users in the 50 states. In addition, under TEA-21, each state's percentage share of the aggregate spending on the "core" highway programs is adjusted every fiscal year to ensure that each state's share of the apportionments for these programs is at least a 90.5% return on its estimated contributions to the highway account of the HTF. The link that this "minimum guarantee" makes between the amount of taxes paid and the federal assistance received, according to some, strengthens the arguments that most FAHP programs should be restricted to the states whose user taxes support the HTF. Opponents of spending HTF money on territorial roads argue that the territories should raise their own territorial fuel taxes to provide for their highway funding needs. Their small size, both in territory and population, compared with the smallest of the states also forms the basis of arguments against expanded program participation for the territories. All four territories' populations combined are less than the population of the state with the least population, Wyoming, whose land area is much greater. Their combined land areas together are only 532 square miles larger than Rhode Island, whose population is more than 2 ½ times as large. If the territories were allowed to participate in all or most of FAHP programs, they would qualify for the various program minimums which, because of the territories small size, would actually, some argue, be excessively generous. The final argument is simply that the FAHP is based on the assumption of a federal-state partnership that is different than the federal relationship with the territories. From this perspective, federal highway assistance to the territories should be accomplished outside the existing FAHP programs and should not draw from the HTF for funding. TEA-21 Reauthorization Options As the debate over the reauthorization of federal surface transportation programs continues, Congress may address a number of options concerning federal highway funding for the territories. The overall issue for the territories is the appropriate extent of their participation in the FAHP. Options range from elimination of the THP to expanding territorial participation in FAHP programs to equal that of the states. Elimination of the THP is unlikely since the federal commitments to economic development of the territories would probably be strong enough to prevent elimination of the THP. On the other hand, the fact that the territories do not pay the taxes that provide revenue to the HTF will probably preclude them from full participation in the programs that the HTF pays for. Any reauthorization debate concerning federal highway assistance to the territories will probably be over more modest changes. In a legislative sense, it is the territories' non-state status that excludes them from most federal highway programs. Because the definition of a "state" in Title 23 U.S.C. section 101 does not include the territories, the only programs that the territories are eligible for are programs whose legislative language specifically includes them or defines them as a state for the purposes of the particular program. Expanding the Territories' Eligibility to Other FAHP Programs As mentioned earlier, because of the link the minimum guarantee makes between a state's federal highway tax revenues and the amount a state receives under the core highway programs, it is doubtful that the territories will be granted participation in the core highway programs beyond their existing participation in NHS. However, territorial projects could be made eligible for other FAHP programs, referred to as the allocated or discretionary programs, that are under the control of the FHWA. These programs are much smaller than the main core highway programs and their grants are competed for on a project by project basis or are designated by congressional earmarking. To make the territories eligible, Congress could pass amending language that would define the four territories collectively as a state under one or more of the program sections in Title 23 U.S.C. Even if the territories are made eligible for any of these programs, however, there is no guarantee any grants will be awarded to territorial projects. Changing the Funding of the THP As mentioned earlier, TEA-21 changed the THP's funding from 1% of National Highway System funding (set under ISTEA) to a fixed annual amount of $36.4 million through FY2003. This amount is significantly lower than the THP would have received had the program retained the 1% NHS set-aside the program had under ISTEA. The issue of restoring the 1% set-aside could be raised during the reauthorization debate. Another possibility would be to define the four territories as a state for allocation purposes under NHS. This would qualify THP for the NHS minimum allocation equal to ½% of NHS and Interstate Maintenance allocations combined. Another option would be to argue for a continued dollar amount set-aside but at a higher level, perhaps one that would more closely approximate the outcome of a 1% NHS set-aside. Since early 2001 the federal revenue outlook has become increasingly constrained. This may be the greatest obstacle to increasing federal aid to the territories. The Bush Administration Reauthorization Proposal: the Safe, Accountable, Flexible, and Efficient Transportation Equity Act of 2003 (SAFETEA) (H.R. 2088) SAFETEA, which was introduced by request on May 14, 2003, would make a number of changes to the THP. Section 1808 proposes a number of changes to the various parts of Title 23 of the U.S. Code that are relevant to the THP. Some of the changes are technical and can be seen as adding clarity to the statute, while other changes have significant program effects. Overall the bill could be seen as broadening the project eligibility for THP road projects. It does this specifying the eligibility of "collector roads," by allowing the Secretary of Transportation to apply any Federal-Aid Highway eligibility provision to THP projects, and by allowing THP participation in ferry and ferry facility projects. The bill also ends the existing prohibition on tolls on territorial roads. On the other hand the bill does add some limitations including dropping the TEA21 eligibility language that made seaports and airports eligible, as well as specifically prohibiting THP spending on local roads and for routine maintenance. Funding for THP is held at the annual TEA21 level of $36.4 million for the life of the reauthorization and the federal share is maintained at 100%. The bill also requires each of the territories to negotiate a new THP agreement with the Secretary of Transportation within 12 months of enactment.

Summary: The United States Territories—American Samoa, Guam, Northern Mariana Islands, and the U. S. Virgin Islands—all receive federal funding for their roads and highways. The Territories' treatment under the Federal-Aid Highway Program (FAHP), however, differs significantly from that of the 50 states and the District of Columbia. Prior to 1970 most of the road construction and maintenance activity was financed and carried out by the territorial governments. In 1970, Congress established the Territorial Highway Program (THP) in the Federal-Aid Highway Act of 1970 (P.L. 91-605). The Act authorized the Department of Transportation (DOT) to assist each of the three territories, the Virgin Islands, Guam, and American Samoa (the Northern Mariana Islands were added to the program in 1978), in a program for the construction and improvement of a system of arterial highways to be designated by each territory's Governor. DOT was also directed to provide technical assistance to each territory to assist in the creation of an appropriate agency to administer the program. During the more than 30 years the program has existed the THP's enacting language (23 U.S.C. 215) has basically remained the same but the level of financing varied with each reauthorization. Program funding varied from a low of $4.5 million annually, in FY1970-FY1973 to a high of $36.2 million in FY2003. Some funding from other program sources has also gone to the territories. The Emergency Relief Program, the High Priority Project Program, and certain safety programs have provided funding for territorial highways over the years. The arguments generally made in support of federal aid to the territories, including highway aid, are usually framed in terms of the transportation and development needs of the territories and the benefits the territories provide to the United States. The arguments against expanded territorial eligibility under federal-aid highway programs are framed by the territories' exemption from the taxes that support the highway account of the Highway Trust Fund (HTF), the implications of the territories limited land area, and their non-state status. As reauthorization of the Transportation Equity Act for the 21 st Century (TEA-21) ( P.L. 105-178 ), Congress may address a number of options concerning federal highway funding for the territories. The overall issue for the territories is the appropriate extent of their participation in the FAHP. The options range from elimination of the THP to expanding territorial participation in FAHP programs to equal that of the states. Elimination of the THP appears unlikely given the federal commitments to economic development of the territories. On the other hand, the fact that the territories do not pay the taxes that provide revenue to the HTF will probably preclude them from full participation in HTF programs. The Bush Administration proposal, the Safe, Accountable, Flexible, and Efficient Transportation Equity Act (SAFETEA)( H.R. 2088 ), would make a number of changes in the THP and retain its authorization at the TEA21 annual level of $36.4 million. This report will be updated as warranted by events.

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Summarize: By. Chris Pleasance. Tahir Alam, Muslim Council of Britain activist, could now be removed from his post as Ofsted is expected to impose special measures on six Birmingham schools, three of which are run by him. Six secular schools at the centre of a plot to introduce hardline Islamic teaching into classrooms are to be placed in special measures. It has been reported that Ofsted inspectors will rate the schools 'inadequate' after snap inspections this week, which usually leads to a school being placed in special measures, allowing officials to remove senior managers or close the school. Inspectors were ordered in to the Birmingham schools after a 'Trojan Horse' letter was uncovered last month detailing a plot by hardline Muslims to force moderate and secular headteachers from their posts and install extreme Islamic teachers in their place. Today a report published in The Telegraph alleges that Park View, Golden Hillock, Nansen, Oldknow and Saltley schools will be rated inadequate, the lowest possible score, while Alston school is already in special measures. If the new. Ofsted investigation does place the schools in special measures, Tahir. Alam, who is also governor of Highfield school, is almost certain to be. removed from his post. Mr. Alam, an activist for the hardline Muslim Council of Britain, is alleged to be at the centre of the trjan Horse plot, but has. denied the allegations, calling them a 'fabrication'. According to the Ofsted report, a further nine schools Springfield, Adderley, Regents Park, Highfield, Gracelands, Ladypool, Marlborough, Montgomery and Waverley will be given the second lowest rank of'requiring improvement'. A letter from Adderley school to parents yesterday confirmed the school had been subjected to a'malicious and targeted' attempt to oust senior staff. In its statement to parents, Adderley Primary's governing body confirmed that several headteachers in the city had faced pressure to resign from their posts. The school, which is also embroiled in a fraud inquiry into allegedly fake resignation letters, said: 'Many of you will now be aware that a number of headteachers across Birmingham have come forward and confirmed that they have experienced malicious and targeted campaigns to remove them and that this issue is not unique to the leadership of Adderley Primary School. 'The seriousness of this issue has now been taken up by the Department for Education who have appointed a new education adviser responsible for investigating this matter thoroughly. 'Birmingham City Council have also appointed a former head teacher, Ian Kershaw, as Birmingham's chief adviser to assist in a wider investigation.' In a picture taken inside Park View school in 2008, boys and girls appear to have been segregated in an assembly. The school has always maintained segregation was voluntary, but an investigation found it was enforced by teachers. Park View school, along with its two sister schools Golden Hillock and Nansen, have bee rated 'inadequate' by Ofsted inspectors, along with Oldknow and Statley. The update for parents concluded: 'Attempts have been made at Adderley to destabilise the school by a very small but well organised group of individuals. 'The governors, headteacher, senior leadership team and staff have been robust in ensuring that Adderley remains a multi-cultural school providing a safe and positive learning environment for all of our 630 children.' Four former staff members at the primary school were arrested on Thursday and have been bailed pending further inquiries into allegedly forged resignation letters. Of the 17 schools investigated by Ofsted in connection with the plot, so far only one has been given a clean bill of health though another report is still to be completed. Meanwhile a Department for Education (DfE) probe, lead by Peter Clarke, the former head of Scotland Yard's anti-terrorism unit, is thought to be looking into 25 schools in the area. Yesterday The Telegraph revealed the DfE investigation found boys and girls had been forcibly segregated during lessons, Christian students had been left to teach themselves religious education, and any discussion of intimacy or the menstrual cycle had been banned in some classes. The probe into Park View School, Golden Hillock and Nansen primary school found that Shiekh Shady al-Suleiman, a known al-Qaeda sympathiser who has previously said gay people should be stoned to death and espoused anti-semetic views, was also invited to speak to children. A report from the Department for Education leaked yesterday found that pupils at Park View were forcibly segregated and Christian students were left to tech themselves. The headteacher, Lindsey Clark (pictured) was sidelined and eventually resigned. Pupils at two of the schools said the theory of evolution was only covered 'briefly' in biology, sex education was banned, and lessons on the arts and literature severely restricted or abandoned. Despite these restrictions, Arabic lessons were compulsory for all students. Pupils were also encouraged to begin and end lessons with prayer, and loudspeakers were used to broadcast calls to prayer. The report said headteachers at the schools were marginalised, with Park View, Golden Hillock and Nansen in reality being run by Tahir Alam, who ran the board of governors. The headteacher of Park View, Lindsey Clark, resigned last month after being reduced to a 'figurehead' within the school, despite gaining the school an 'outstanding' Ofsted rating. In a statement to Mail Online, the Park View Education Trust said: 'Just two years ago Park View was the first Academy to be rated 'Outstanding' in all categories under the new Ofsted inspection regime. 'It took more than 10 years to build the school to its Outstanding levels, from a situation where children were being failed and the school was in special measures. 'Park View is now the most successful school in England serving its social group - that is, with 72% of pupils on free school meals. 'If the inspectors who carried out both inspections at Park View Academy have ignored these achievements then we have to conclude that they are working to a different set of criteria than is normally used for an Ofsted inspection.' However, the spokesman refused to comment on the current allegations, saying only that they did not recongise reports that some of the schools would close. A spokesman for Ofsted refused to comment on the report, but yesterday a spokesman for the Department for Education said: 'The allegations made in relation to some schools in Birmingham are very serious and we are investigating all evidence put to us in conjunction with Ofsted, Birmingham City Council and the police. 'It is absolutely vital these investigations are carried out impartially, without pre-judgment. Birmingham City Council is also investigating 15 schools, though the DfE is thought to be frustrated with attempts by the council to play down what is happening in the schools

Summary: Schools investigated by Department for Education, Ofsted and Birmingham City Council over alleged plot to introduce extreme Islamic teaching. Ofsted report expected to place six schools into specials measures. Officials will then have powers to remove senior staff or close schools. Another nine schools to be ranked as 'needing improvement' Tahir Alam, governor of four schools, expected to be removed from post. DfE investigation into three of his schools found pupils being segregated. Sex education was also banned and evolution taught only 'briefly'

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Biomass Thermal Utilization Act of 2015'' or the ``BTU Act of 2015''. SEC. 2. RESIDENTIAL ENERGY-EFFICIENT PROPERTY CREDIT FOR BIOMASS FUEL PROPERTY EXPENDITURES. (a) Allowance of Credit.--Subsection (a) of section 25D of the Internal Revenue Code of 1986 is amended-- (1) by striking ``and'' at the end of paragraph (4), (2) by striking the period at the end of paragraph (5) and inserting ``, and'', and (3) by adding at the end the following new paragraph: ``(6) in the case of taxable years beginning before January 1, 2021, 30 percent of the qualified biomass fuel property expenditures made by the taxpayer during such year.''. (b) Qualified Biomass Fuel Property Expenditures.--Subsection (d) of section 25D of the Internal Revenue Code of 1986 is amended by adding at the end the following new paragraph: ``(6) Qualified biomass fuel property expenditure.-- ``(A) In general.--The term `qualified biomass fuel property expenditure' means an expenditure for property-- ``(i) which uses the burning of biomass fuel to heat a dwelling unit located in the United States and used as a residence by the taxpayer, or to heat water for use in such a dwelling unit, and ``(ii) which has a thermal efficiency rating of at least 75 percent (measured by the higher heating value of the fuel). ``(B) Biomass fuel.--For purposes of this section, the term `biomass fuel' means any plant-derived fuel available on a renewable or recurring basis, including agricultural crops and trees, wood and wood waste and residues, plants (including aquatic plants), grasses, residues, and fibers. Such term includes densified biomass fuels such as wood pellets.''. (c) Effective Date.--The amendments made by this section shall apply to expenditures paid or incurred in taxable years beginning after December 31, 2015. SEC. 3. INVESTMENT TAX CREDIT FOR BIOMASS HEATING PROPERTY. (a) In General.--Subparagraph (A) of section 48(a)(3) of the Internal Revenue Code of 1986 is amended by striking ``or'' at the end of clause (vi), by inserting ``or'' at the end of clause (vii), and by inserting after clause (vii) the following new clause: ``(viii) open-loop biomass (within the meaning of section 45(c)(3)) heating property, including boilers or furnaces which operate at thermal output efficiencies of not less than 65 percent (measured by the higher heating value of the fuel) and which provide thermal energy in the form of heat, hot water, or steam for space heating, air conditioning, domestic hot water, or industrial process heat,''. (b) 30-Percent and 15-Percent Credits.-- (1) Energy percentage.-- (A) In general.--Subparagraph (A) of section 48(a)(2) of the Internal Revenue Code of 1986 is amended by redesignating clause (ii) as clause (iii) and by inserting after clause (i) the following new clause: ``(ii) except as provided in clause (i)(V), 15 percent in the case of energy property described in paragraph (3)(A)(viii), but only with respect to periods ending before January 1, 2021, and''. (B) Conforming amendment.--Subparagraph of section 48(a)(2)(A)(iii) of such Code, as so redesignated, is amended by inserting ``or (ii)'' after ``clause (i)''. (2) Increased credit for greater efficiency.--Clause (i) of section 48(a)(2)(A) of such Code is amended by striking ``and'' at the end of subclause (III) and by inserting after subclause (IV) the following new subclause: ``(V) energy property described in paragraph (3)(A)(viii) which operates at a thermal output efficiency of not less than 80 percent (measured by the higher heating value of the fuel), but only with respect to periods ending before January 1, 2021,''. (c) Effective Date.--The amendments made by this section shall apply to periods after December 31, 2015, in taxable years ending after such date, 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: BTU Act of 2015 Summary: Biomass Thermal Utilization Act of 2015 or the BTU Act of 2015 Amends the Internal Revenue Code to include 30% of qualified biomass fuel property expenditures made in taxable years beginning before 2021 in the residential energy efficient property tax credit. Defines &quot;qualified biomass fuel property expenditure&quot; as an expenditure for property which uses the burning of biomass fuel (a plant-derived fuel available on a renewable or recurring basis) to heat a dwelling used as a residence, or to heat water for use in such dwelling, and which has a thermal efficiency rating of at least 75%. Allows: (1) a 15% energy tax credit until 2021 for investment in open-loop biomass heating property, and (2) a 30% credit for boilers or furnaces that operate at thermal output efficiencies of at least 80% and provide thermal energy.

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Summarize: As mentioned earlier, TMZ had been holding their tongue to report on the now widely circulated rumor that Brangelina were going to cause the earth's core to melt by separating. They've now weighed in, and the decision goes like this: Brad Pitt and Angeline Jolie are not pulling the plug on their relationship, multiple people directly connected with the couple tell TMZ. There's a report out that Brad and Angie are calling it quits. One source — who should know — says, "It's B.S." It is now safe to step back from the ledge. Perez Hilton and Just Jared have also gone with the PR deflection, but—as it bears repeating—when you're a publicist playing on the level of Brad Pitt and Angelina Jolie, you're going to do anything to put ice on a rumor while you work a press strategy to go about working it. Also, as Maura Johnston noted earlier today, TMZ totally screwed the pooch on that Axl Rose story they reported earlier. Also, they're far "better" at matters of dead celebrities (Michael Jackson, Brittney Murphy: two beats they've done early, spot-on reporting with) than anything else. So if one of them were reportedly dead, and TMZ reported it, I wouldn't question it nearly as much as this, which is something that's been buzzed about in the celebrity press particularly hard over the last few months. The fact that I have any perspective to offer on this just struck me as, at the very least, sad. In recent years Broadway's stages have been littered with dim performances from bright screen stars, including Julia Roberts and Katie Holmes. Film actresses as famous as Ms. Johansson tend to create their own discomfort zones onstage, defined by the mixed expectations of fans and skeptics. I was definitely aware of that zone when I saw Keira Knightley in The Misanthrope in London recently. BRAD PITT's brother begged the Hollywood star to leave ANGELINA JOLIE because their family was being torn apart, The Sun can reveal. The couple have struggled during the last 12 months of their five-year relationship but are still trying to make it work for their six children. Brad, 46, spent the weekend in Los Angeles to take part in GEORGE CLOONEY's Haiti telethon - where he went to great lengths to avoid ex JENNIFER ANISTON - while Angelina, 34, was on a New York photoshoot. Have Brad and Ange split? The couple have been together for 5 years News Click here to download the latest flash player. Despite having never wed, reports yesterday claimed the couple have visited a top Beverly Hills divorce lawyer and signed a �205million split deal to carve up their assets. Role... pair display togetherness in February 2009 The report claimed they will also share custody of their kids who will live full time with Angelina. Brother Doug, 41, visited Brad in New York over Christmas and urged him to pull the plug on the relationship because it was making the actor and his family unhappy. The couple have been putting on a united front but a source said: "It's no secret they have been in a pretty loveless relationship for about a year. "They barely spend time together and when they do it is very fraught. They want different things from life. Ex... Brad with Jennifer Aniston "Brad's family are being more vocal with their doubts over the relationship. The only thing they still have in common is their kids. That's keeping them together at the moment." Brad and Angelina had a miserable Christmas and Thanksgiving after Angelina refused once again to spend any time with Brad's family. Tomb Raider star Angelina is also reported to have become close with a Russian teacher while filming her latest movie Salt. Brad and Ang have also feuded for months over the issue of adoption. Strain... Brad hides behind beard in December 2009 She wants a baby from Syria and now Haiti while he thinks the six kids they have are more than enough. Blamed Other thorny issues are his desires to relocate to New Orleans and to spend more time having fun with pals. Meanwhile Brad's mum Jane has never warmed to Angelina and is still in touch with Friends star Jen. Advertisement But sources close to the couple called the split reports "totally false." One source told a US website: "Everything is fine. It's business as usual." The couple were recently spotted at a New York restaurant looking "in love". The report blamed work schedules for the time they spend apart. Brad and Angelina have three adopted children Maddox, eight, Pax, six, and Zahara, five. They also have three biological children Shiloh, three, and twins Knox and Vivienne, 17 months. [email protected] Family, friends can't seem to agree on whether Angelina Jolie and Brad Pitt split rumor is true Poujoulat/Getty Despite rumors of a split, Brad Pitt and Angelina Jolie have remained mum on the state of their relationship. Don't write off Brangelina so fast. Several sources close to Brad Pitt and Angelina Jolie yesterday denied reports they are splitting up - even as the Hollywood power couple remained mum. "I was told it's not true," a source close to the couple told the Daily News. "[Brad and Angelina] are very secretive. But I was told it's not true." "Everything is fine," another source told People magazine's Web site. The Sun of London reported today that Pitt's brother, Doug, recently urged him to end the relationship. "They barely spend time together and when they do it is very fraught. They want different things from life," a source close to Pitt's family told the paper. The breakup drama started when London's News of the World reported the pair have met with California divorce lawyers - even though they're not married - to hash out an amicable division of their vast fortune and custody of their six kids. A close friend of Pitt told author Ian Halperin that the hunky actor has been preparing to bail from his five-year relationship with the pillow-lipped Jolie for at least six months. "His relationship with Angie is over," the pal told Halperin, author of the book "Brangelina: The Untold Story of Brad Pitt and Angelina Jolie." Buzz about a Brangelina breakup has been circulating for months. The National Enquirer reported that Pitt and Jolie had a heated argument Jan. 6 at Alto restaurant in midtown Manhattan. But just three days earlier, a source told The News, the duo shared a "pleasant" meal of roasted chicken at Le Perigord on 52nd St. "They looked happy - very, very happy," said a manager at the restaurant. Reps for Pitt and Jolie did not return calls for comment yesterday. Ruthie Richardson, an aunt of Jolie's who lives in Texas, said she believed her niece and Pitt were still a couple.

Summary: As rumors of a Brangelina break-up threaten to overtake even the Tiger Woods drama, everyone is weighing in-and Brad Pitt's family is firmly on Team Jen. Sources tell the Sun Pitt's brother Doug has been urging Brad to end the non-marriage, which has been "loveless" for a year. Brad's mother is reportedly still close to Jennifer Aniston, and Angelina Jolie didn't score any points by refusing to spend time with her not-quite-in-laws over the holidays. Meanwhile, tell-all author Ian Halperin says one of Pitt's friends confirmed the relationship "is over," he tells the New York Daily News-and that's enough to make Gawker believe the rumors aren't true. Halperin "is always getting stuff wrong," Maureen O'Connor writes. But, Foster Kamer points out, even TMZ has jumped on the they're-not-breaking-up bandwagon, and they've "screwed the pooch" on stories that aren't about dead celebrities before. Who knows what to believe?

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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention A rack loaded, radiant heated, cantilevered deck baking oven to be used for baking or roasting. 2. Description of the Related Art Radiant heated baking ovens are shown in U.S. Pat. No. 4,215,266 and U.S. Pat. No. 4,503,837. Shown in those patents are discrete deck radiant heated ovens. Baking at point of purchase markets is performed by one of two technologies, (1) Convection baking, accomplished by passing large volumes of heated air across proofed dough, and (2) Radiant baking, accomplished by baking proofed dough in a stationary thermal gradient between two heated decks. Baking by convection, is faster and requires less handling than does baking by radiant heating. A typical convection oven is large enough to accept a caster mounted rack on which pans of bread are mounted. The rack is placed in the oven, and the rack, with its pans of proofed dough, is rotated about a vertical axis in front of jets of heated air. The large volume of heated air required, removes moisture from the bread, shortening the bread&#39;s shelf life and storage life. Baking by radiation, requires establishing a stable thermal gradient through the bread to be baked and then maintaining that thermal gradient until the bread is baked. The temperature at the top of the bread must be high enough to brown the bread. Baking temperature must be maintained through the body of the bread. Drying is not a problem with radiant baking. Adding moisture is not necessary. Shelf life of radiant baked bread is longer than shelf life of bread baked by convection. A typical radiant baking oven comprises discrete heated decks, mounted one above the other, each deck having its own door or group of doors. The decks have back, sides, top and bottom. Access to the heated deck is through a single door or through group of doors. Trays of proofed bread dough are placed on the heated deck. The trays remain in the oven, within an established thermal gradient until the bread is baked and browned. Baking in such an oven requires multiple handling of trays and opening and closing of oven doors. This invention is a rack loading, radiant heated, deck oven with cantilevered baking decks. Open sided baking chambers are used to bake the bread. By leaving a space between the sides of the decks and the oven wall, the oven can be loaded by a rack loaded with pans of bread. It is an object of the invention to provide a baking oven that can use racks for loading pans of bread for baking, using radiant baking technology. It is an object of the invention to provide a baking oven that does not require rotation of the rack during baking. It is an object of the invention to provide a rack loading oven of substantially smaller external dimensions than convection ovens. It is an object of the invention to provide a radiant heated baking oven, that can be loaded with single baking pans and can bake product on a single hand loaded baking pan on a single deck, or back a rack of multiple pans per deck. This oven can be hand loaded or rack loaded. It is an object of the invention to mount the heat sensors inside the heated decks, to eliminate breakage of the sensors. It is an object of invention to locate a sensor within each deck to control the amount of heat provided by each deck. It is an object of the invention to provide a radiant heated baking oven sensitive to internal air currents. BRIEF DESCRIPTION OF THE DRAWINGS Drawing 1 is a perspective view of the radiant deck oven with a section through the side and top of the oven. Drawing 2 is a perspective disassembly view, showing structure of the cantilevered decks. Drawing 3 is a section view taken through the left side of a foreshortened intermediate deck showing location of heat sensor, and construction of the deck. Drawing 4 is a section view taken through the left side of a foreshortened intermediate deck showing the attachment of the deck to the back of the oven. Drawing 5 is a front view of the deck with cover plate off, with a partial rotation on the drawing showing the upside down L shape of the front structural member, and showing location of the heat sensor. Drawing 6 is a top view of a deck showing placement of heating element, placement of heat absorption pattern, placement of sensor shield and location of sensor. Drawing 7 is a perspective view showing a rack inserted into the radiant heated oven. Drawing 8 is a section view showing pitch of deck and complementary pitch of rack. Drawing 9 is a partial view showing guidance system for rack. Drawing 10 is an electrical schematic of the device. DESCRIPTION OF THE PREFERRED EMBODIMENT Description of Apparatus The elements of the baking oven are: Radiant Heated Deck Oven 12 Cabinet 14 Baking Chamber 16 Cabinet Top 18 Baking Chamber Top 20 Insulation in top of Oven 22 Moisture Vent Top 23 Moisture Vent Right Side 24 Moisture Vent Left Side 25 Cabinet Left Side 26 Baking Chamber Left Side 28 Insulation in Wall 30 Cabinet Right Side 32 Baking Chamber Right Side 34 Insulation in Wall 36 Baking Compartments 38 lighting glass (7) 41 Thermostats (8) 39 Cabinet Back 40 Baking Chamber Back 42 Insulation 44 Vertical Support Member 46 Vertical Support Member 48 Cabinet Bottom 50 Baking Chamber Bottom 52 Deck Oven Front 54 Access Opening 56 Door 58 Gasket 59 Hinges 60 Guide Opening 62 Open Sided Baking Compartment 64 Open Sided Baking Compartment 66 Open Sided Baking Compartment 68 Open Sided Baking Compartment 70 Open Sided Baking Compartment 72 Open Sided Baking Compartment 74 Open Sided Baking Compartment 76 Top Deck 80 Intermediate Deck 82 Intermediate Deck 84 Intermediate Deck 86 Intermediate Deck 88 Intermediate Deck 90 Intermediate Deck 92 Bottom Deck 94 A PARTICULAR DECK Intermediate Deck 82 Slots 142 and 142 Top Plate 96 Top Plate Flange 104 Top Plate Flare at Rear of Plate 106 Cantilever arm left 98 Cantilever arm right 100 Cantilever arm cross member 102 Top Plate Retaining Screw 108 Heat Sensor 112 Heat Sensor Tip 114 Heat Sensor Radiant Energy Shield 116 Shield Retaining Rivets 118 and 120 Bottom Plate 122 Upper Side Bottom Plate 124 Vent 126 Heat absorptive coating 128 Heating Element 130 Break of Bottom Plate 132 Space Between Plates 134 Side of Deck 136 Side of Deck 138 Side of Baking Compartment 139 Side of Baking Compartment 140 Spacing between Side of Deck and Sidewall 142 Spacing Between Side of Deck and Sidewall 142&#39; Rolling Baking Rack 144 Wheels 146, 148, 150, and 152 Axles 154, 156, 158, and 160 Pivot Mount 162, 164, 166, and 168 Spacing Between Wheels--Width 170 Spacing Between Wheels depth 172 Height of Rack 174 Width of Rack 176 Depth of Rack 178 Shelves on Rack (7) 180, 182, 184, 186, 188, 190, 192 Pitch of Shelves on rack 194 Pitch of Deck 196 Handle 198 Baking Pans 200 Product 202 Proofer 204 Proofing Compartment 206 ELECTRICAL Heat Sensor Leads (8) Baking Chamber Lights (7) Baking Chamber Power Source (8) Heating Elements (8) Referring to the drawings, FIG. 1 shows in perspective, a rack loaded, radiant heated, cantilevered deck, baking oven, 12, with door removed. The oven comprises an insulated cabinet 14, enclosing an internal baking chamber 16. Cabinet top 18, and baking chamber top 20, are separated by a layer of insulation 22. Cabinet side 26, and baking chamber side 28, are separated by a layer of insulation 30. Cabinet right side 32, and baking chamber right side 34, are separated by insulation layer 36. Between left side 26, and right side 34, are mounted seven parallel open sided baking compartments 38. Back 40, and baking compartment back 42, are separated by insulation layer 44. Cabinet bottom 50, and baking compartment bottom 52, have no insulation between them. Bottom 50, and bottom 52, are simply the two sides of a single piece of metal. Insulation is not placed in the cabinet bottom. The temperature below chamber 16, i.e. below the lowest cooking deck, is cooler than is chamber 16. The front 54, of the baking cabinet 14, has an access opening 56, gasketed by gasket 59, and closed by insulated glass paneled door 58. Door 58 is supported by hinges 60. Attached through the back of the cabinet 40 are structural members 46 and 48. The back of cabinet 40, and the back of the baking chamber 42, have holes formed therethrough to allow electrical connection to the heating elements. The left cabinet side 26, and the left side of the baking chamber 28, are pierced by moisture vent 25. Vent 25 allows outside air to enter the bottom of the oven. The right side of the cabinet 32 and the baking chamber right side 34 are pierced for vent 24. At the top of the oven, between cabinet top 18, above the baking chamber, an adjustable vent 23 is mounted. This adjustable vent is used to adjust air flow through the oven, 12. Air flow removes moisture that passes into the oven as the bread is baked. In discrete deck, radiant heated ovens, this moisture is vented during the regular insertion and removal of baking pans. In a rack loaded, radiant baking oven, the moisture must be vented with the least disturbance to thermal gradients between decks. On the right side of the oven, lights are mounted. The baking chamber right side 34 is cut for seven circular windows. The outside of the cabinet 32 is also cut for seven circular windows. Between the inside and outside windows are mounted light bulbs. The light from the bulbs, lights the cooking decks so that baking can be observed. Mounting of the lighting is best seen in FIG. 1. Eight Thermostats, 39, are mounted on the front right side of the oven. Each thermostat controls the temperature of a single deck. Baking chamber 16, in the oven shown, comprises seven open sided baking compartments 38, individually numbered here as 64, 66, 68, 70, 72, 74 and 76. The baking chambers are disposed vertically over each other and are separated by intermediate decks 82, 84, 86, 88, 90 and 92. There is also a bottom deck 94. Each intermediate deck as for example, deck 82 as best shown in FIG. 2 and in FIG. 3, comprises a top plate 96 supported by cantilever arm 98 and 100 and cantilever arm cross member 102 attached to and passing through the back of the cabinet 42; arms 98 and 100 are welded to vertical supports 46 and 48; slots 95 and 95&#39; form access opening 56 from the front of the oven to the back of the oven 42, along the sides of the plates. Top plate 96 has a depending flange 104 extending around the perimeter of the deck; bottom plate 122, of deck 82, is spaced apart from the top plate 96. Top plate 96 extends substantially across the width and depth of the baking compartment, except for the slots between the sides of the deck and the sides of the baking compartment; 142 and 142&#39; extend from the front of the oven to the back of the oven. The depending flange 104 of top plate 96 extends laterally across the oven. The bottom plate 122 extends substantially across the width of the baking compartment, except for the slots 142 and 142&#39;. The bottom plate extends forwardly a predetermined distance short of flange 104 of the top plate 96, to provide a vent 126 of predetermined width extending across the width of the deck 82. Bottom plate 122 being shorter than the top plate 96, leaves a passage or vent 126 into the interior space between top plate 96 and bottom plate 122 as best shown in FIG. 3. As best shown in FIG. 6, bottom plate 122 has its upper side 124 blackened by a heat absorptive coating 128 in a pattern preferably in the form of a rectangle with extended legs, shown in stippling in FIG. 6. The coating is located a predetermined distance from the front and rear of bottom plate 122. Between top plate 96 and bottom plate 122 is electrical heating element 130 as shown in FIG. 6; element 130 extends substantially across the surface of the bottom plate 122, above the blackened coating 128 and rearwardly beyond the plane section thereof. Heating element 130 is located substantially midway of top plate 96 and bottom plate 122. Element 130 is electrically connected to a plug built into the back 40 of the oven cabinet. Within space 134, between plates 96 and 122 is mounted a fluid filled heating sensing bulb 112. Alternately a thermocouple can be used. As shown in FIG. 3, sensor 112, is located so tha tip 114, thereof is located substantially within vent 126 and in close proximity to vent 126 as shown in FIG. 3, and is supported by a bracket attached thereto as best shown in FIG. 6. Sensor 112 is connected to thermostat 39 in the usual manner. Intermediate deck 84 is identical to intermediate deck 82 described above, and is controlled by thermostat 39 shown in FIG. 3. Upper baking compartment 64 top deck 80 is identical to deck 82 described above, except that the cabinet ceiling is used as the upper plate of the deck. Preferably the cabinet ceiling is provided with a depending flange (not shown) which cooperates with the lower plate to provide a vent to the heat sensor. Bottom deck 94 of baking chamber 76 the lowest baking compartment is similar to deck 82 but differs in that the bottom deck of the lowest baking compartment is not blackened and is insulated to limit heat transfer to the area below deck 94. In bottom deck 94 the flange on the upper plate cooperates with bottom wall of the oven chamber to provide a suitable vent. Bottom deck 94 is controlled by its own thermostat as are the other decks. FIG. 10 shows an electrical wiring diagram, a ladder diagram, of the oven. N is neutral with lines 1, 2 and 3 marked L1, L2 and L3 respectively. The Neutral line is connected to a buzzer, to the contractor coils and to the light bulbs that light the inside of the oven. Running off line 1, is contactor, single pole switch, fuse to timer, fuse to high limit thermostat and to the other side of the light bulbs. The high limit thermostat turns the oven off if the oven gets too hot. In this example, the high limit thermostat turns the oven off at 550 DEG F. When power is on, the switch and the timer must be closed, to complete the circuit and power the oven. As can be seen from the diagram, fluid operated thermostats operate to stop power supply to each heating element if the element reaches 550 DEG Fahrenheit. A pilot light is wired across each element and when the element is on, that pilot light is lit. Each deck is controlled by a thermostat connected in series with the heating element, across 208 volt or 240 volt leads. A pilot light, as stated, is connected across the heating element to indicate if the heating coil is energized. A 15 amp single toggle switch is provided across line 1 for energizing or de-energizing the oven. ROLL-IN-RACK FIG. 7 shows the insertion of a baking rack 144 into the cantilevered deck oven 12. Rack 144 is slightly shorter than the height of the baking chamber 16, measured between the baking chamber top 20 and the baking chamber bottom 52. At the bottom of the chamber 52, guide openings 62 serve to guide rack 144 into the baking compartment as best shown in FIG. 9. The width of the rack 144 is such as to allow the rack 144 to enter the baking compartment. The depth of the rack 144 is such as to allow the door 58 to close once the rack is inserted. The spacing between the sides of the decks and the sidewalls of the baking compartment which allows the rack 144 to be inserted is shown in FIG. 6 and FIG. 7 as 142 and 142&#39;. DESCRIPTION OF OPERATION Referring again to FIG. 1, intermediate deck 82 is heats the lower half of the upper compartment and the upper half of the lower baking compartment. The bottom deck 94 heats the lower half of the baking compartment above it and the top deck above heats the upper half of the baking compartment below it. Seven sets of centilever arms support the decks. Cantilever mounting of the decks from the back allows a rack to be used to load the baking oven. The cantilever arms are mounted with a slight pitch, with the end of the arm closest to the door, slightly lower than the end of the arm at the back of the chamber. Fourteen support arms are mounted in two vertical rows to support the seven decks. One inch by inch square tubing of 14 gauge thickness, forty one and one half inches long are used to make the support arms. The capillary tube from the sensor to the thermostat passes through the hollow center section of the tubing. Top plate 84 and bottom plate 103 are affixed to the support arms with heating element 108 sandwiched between. The heating elements are 3000 watt elements, except for the bottom element which is a 3500 watt element. Top plate 84 is formed of a bright polished high temperature aluminum sheet 0.190 inch in thickness having the commercial designation 3003 H 14 Aluminum. The top plate can also be coated with a non-stick coating such as a suitable high temperature solid fluorinated hydrocarbon. The bottom plate, 103, is formed of a 0.125 inch, high temperature aluminum sheet having the commercial designation 3003 H14 Aluminium. Top and bottom plate are preferably spaced about two inches apart. The bottom of the top deck is formed of the same is 0.125 inch, high temperature aluminum sheet; the bottom of the bottom baking compartment is 14 gauge aluminized steel, with 0.063 Aluminum sheet 3003 H 14 Aluminum used as a reflector plate. The aluminized steel is used as an insulator below the aluminum sheet which is a reflector. This is to keep the bottom of the oven below the baking chamber, in the area where the rack wheels are located, cooler. The bottom plate 122, on all decks except for the lowest deck bottom plate, is coated with a heat absorptive coating 128 to enable it to transmit heat from the heating element 130 between the plates at a higher rate than is transmitted through the top plate, so that the bottom plate 122 on each deck, except the bottom deck, is heated to a temperature of about 100 degrees Fahrenheit higher than the top plate so that bread can browned. The paint pattern allows varying the temperature in the browning gradient. In the preferred embodiment, the pattern is in the form of a rectangle 27 inches by 221/2 inches with extended legs. A rectangular section, 10 inches by 16 inches, is left bare at the back of the deck. The paint pattern stops 7 inches back from the back of the oven. The object of the pattern is to establish a uniform thermal gradient. The back of the baking chamber loses less heat because of insulation and less air flow. The browning gradient can be varied in temperature by varying the thickness of the lower plate and by varying the pattern of the heat absorption layer. The inside of the baking compartment is formed of 18 gauge aluminized steel as an oven liner. Between the liner and the external sides of the baking oven are 2 and 1/2 inches of fibreglass insulation. Six inches of insulation are in the top of the device. The decks are 26 inches wide with a depth of 36 inches. A standard baking pan is 18 inches wide by 26 inches deep. This deck width and the individual heating elements, allow the oven to be used as a discrete deck oven by heating only two decks and baking in the thermal gradient established in the open sided baking compartment between the two heated decks. The rack to be inserted in the oven as shown in FIGS. 7, 8 and 9 uses 5 inch high temperature plastic wheels. The rack itself is aluminum and is dimensioned to accept two baking trays per level. Pitch of the rack is 3/8 of an inch over 37 inches. The rack width is 30 inches outside and 27 and 1/2 inches internal with a depth of 36 inches. An object of the dimensioning and pitch of the deck and the pitch of the rack is to attempt to attain a uniform one-half inch clearance between the bottom of the pan holding the bread and the surface of the deck. A cantilevered deck, radiant baking oven must maintain stable thermal gradients in baking compartments open on three sides and must allow removal of moisture given off by bread being baked. If the moisture is not removed, the bread doesn&#39;t bake or brown. If the moisture is not removed without creating excessive convection currents the goods are not baked uniformly. The baking chamber is vented in both lower corners and at the top. Venting is not necessary in discrete deck ovens because the continuing opening and closing of the doors to each deck, as pans are inserted and removed, removes moisture. In this rack loaded device, for example, a rack loaded with 84 pounds of bread containing 2 ounces per pound of moisture to be baked off, 168 ounces of moisture must be vented through one baking cycle. The moisture must be vented without creating internal air currents that would disturb the desired thermal gradients. Vents at the bottom of the oven, one inch in diameter, fitted with adjustable dampers allow air to infiltrate into the bottom of the oven, be preheated, and then pass along the sides of the chamber. A vent 2.25 inch diameter at the top of the oven with an adjustable damper, allows air to exhaust. Air circulation through these vents serves to carry off the moisture generated during the baking cycle. To calibrate the oven to establish the desired thermal gradients in the baking components, a temperature of 375 DEG F at a point midway between the decks as for example of the baking space between decks 82 and 84 is assumed. All of the heating elements in the decks are energized and heated to the same temperature to establish the baking temperature midway of the baking compartment at 375 DEG, as determined by a pyrometer. In the instant example this results in a top plate temperature of 400 DEG F as determined by a contact pyrometer. By reason of the construction of the deck, the bottom plate is heated to a temperature of 500 DEG. The thermostat is then set to read 375 DEG F. Other baking compartment temperatures are then selected to calibrate the thermostat. The other thermostats are calibrated in a similar manner. Each thermostat dial is used to establish the baking temperature midway of the decks. Preferably the thermostats are adjusted to cycle or de-energize the heating elements at a bottom plate temperature of 500 DEG. If the entire surface of the bottom plate is blackened, the top areas of the bread loaves will burn or blacken. It has also been found that uniform baking and browning of the bread loaves is accomplished by blackening the bottom plate in the rectangular pattern described above. The dimensions of this blackened surface is determined experimentally and empirically. A suitable coating material is the commercial coating known as Bar-B-Que Black sold by Rust-Oleum Corporation. It has also been found that the top plate provides uniform heating of the lower portion of the upper baking compartment without any surface modification of portions thereof. As shown in FIG. 6, the heating element is located forwardly of the rear of the oven compartment to improve uniform heating since the rear portions of the baking compartments are better insulated than the forward portions due to the relatively poor insulating capabilities of the glass paneled door, if one is used, the heat loss due to opening and closing of the door and heat loss from air used to carry away the moisture generated by baking. The establishment of the top and bottom plate temperatures as above indicated results in a desired temperature of about 375 DEG F vertically midway of the baking compartment with the upper portion of each baking compartment having a temperature gradient ranging from about 375 DEG to 500 DEG in an upward direction to provide a suitable browning temperature and with the lower half having a temperature gradient ranging from about 400 DEG F at the bottom thereof to about 375 DEG F midway of the compartment to provide a suitable baking temperature. A feature of the invention is the location of the tip is the temperature sensing bulb within or in close proximity as for example within one-fourth inch of the vent portion between the upper and lower plates and in the vent air flow path. When the oven door is opened or the intake and exhaust vents are opened cool air is immediately drawn into the vent into the enclosed space between the decks and impinges on the bulb tip causing heating element 108 to be energized and compensate for heat loss due to air infiltration and opening of the door. The sensing tip is spaced from the wall as shown in FIG. 3 so that it senses the air temperature rather than wall temperature and is located close to the top plate so that it is not overheated. Preferably the thickness or transverse dimension of the vent into the enclosed space is about one inch. The width of the vent into the enclosed space is determined empirically so as to permit the bulb tip to sense both the deck temperature and the air flow from through the vent when the door is opened and as air is allowed into the oven to carry off moisture. If the vent is too narrow the bulb tip will sense predominately the deck temperature and if too wide it will sense predominately the ambient air and in either case the thermostat control will not provide the desired temperature gradients. The location of the temperature sensing bulb within the deck and behind a protective shielf protects it from physical damage or mislocation in the use of the oven. While the use of a fluid filled bulb is preferred as the sensing means, other devices such as a thermocouple may be used. A protective shield is placed between the sensor and the heating element to prevent radiant heat from causing the element to cycle. While this invention is primarily concerned with multiple baking compartment baking ovens, it is also applicable to discrete deck ovens using a single baking compartment or chamber. In such embodiment, only two decks are heated, one above the other, the top deck provides the upper portion of the baking chamber with a browning temperature gradient and the bottom deck provides the lower portion of the baking temperature gradient.

Summary: A hot deck baking oven, which is an insulated cabinet enclosing internally heated decks, mounted one above the other; the bottom of one deck and the top of an adjacent deck form a baking chamber; baking is accomplished by holding bread dough in a stable thermal gradient in the baking chamber; each of such decks is cantilever supported from the rear and is spaced away from the sidewalls of the cabinet; the vertical supports of a bread rack inserted into the oven are guided into the oven by the opening between the sidewalls and the decks; the bakery product on the rack is held within each baking chamber; the sidewall/deck separation, allows moisture generated at each baking chamber to pass up the sidewalls of the chamber, to be vented from the top of the oven through an exhaust vent, an intake vent located below the lowest baking chamber allows air to preheat before entering the baking chamber.

big_patent

en

Summarize: Tra i primi farmaci a essere impiegati in uso compassionevole per combattere l'infezione provocata dal coronavirus SARS-CoV-2 (COVID-19) vi è stato il Remdesivir, un antivirale inizialmente progettato dalla casa biofarmaceutica americana Gilead Sciences contro Ebola e virus Marburg. Poiché aveva dato risultati positivi in laboratorio anche contro SARS e MERS, malattie provocate da betacoronavirus affini al patogeno pandemico, il farmaco è stato rapidamente coinvolto nella lotta al SARS-CoV-2, ma con risultati contrastanti. Alcuni studi hanno dimostrato una certa efficacia nel ridurre la mortalità per la polmonite da COVID-19, mentre altri non hanno evidenziato benefici, tanto da spingere l'Organizzazione Mondiale della Sanità (OMS) a non raccomandarlo. Ciò nonostante, il Remdesivir viene ancora ampiamente impiegato negli Stati Uniti, dove rappresenta un vero e proprio standard di cura. Pur essendo considerato sicuro, un nuovo studio ha dimostrato che l'antivirale può indurre una severa bradicardia (rallentamento del battito cardiaco) nei pazienti Covid trattati, che va opportunamente e rapidamente trattata. A descrivere il primo caso di questo grave effetto collaterale indotto dall'antivirale è stato un team di medici e ricercatori americani dell'HCA Healthcare – Morsani College of Medicine dell'Università della Florida Meridionale (USF), coordinati dal professor Jomel Patrick Jacinto. Gli studiosi hanno descritto il caso di una paziente di 78 anni giunta in ospedale con gravi difficoltà respiratorie (saturazione all'89 percento), dolori muscolari e tosse a causa dell'infezione da SARS-CoV-2 (confermata da tampone oro-rinofaringeo). La donna, ex fumatrice affetta da ipertensione e prediabete, è stata immediatamente trasferita nel reparto di terapia intensiva dove ha ricevuto un trattamento anti Covid che includeva il Remdesivir. La terapia prevedeva una dose iniziale da 200 milligrammi per via endovenosa (EV) seguita da una dose di mantenimento di 100 milligrammi per quattro giorni, oltre a ceftriaxone, azitromicina, desametasone, zinco, vitamina C e vitamina D. A 20 ore dalla somministrazione dell'antivirale, la paziente ha iniziato a sperimentare un crollo della pressione sanguigna, shock cardiogeno e una frequenza cardiaca che è arrivata ad appena 38 battiti al minuto. Dall'elettrocardiogramma è stata diagnosticata una severa bradicardia sinusale o, più specificatamente, una “disfunzione del nodo del seno emodinamicamente significativa”.

Summary: A 20 ore dalla somministrazione del Remdesivir, una paziente di 78 anni colpita da COVID-19 ha sperimentato crollo della pressione sanguigna, shock cardiogeno e netta riduzione della frequenza cardiaca, arrivata ad appena 38 battiti al minuto. Dall'elettrocardiogramma i medici hanno diagnosticato una severa bradicardia sinusale indotta dal farmaco. Trattata con dopamina, al termine della somministrazione dell'antivirale la paziente ha ripreso il suo normale ritmo cardiaco.

fanpage

it

Summarize: The Nevada desert is normally associated with enormous casinos and lavish hotels. But it could soon become a focal point of the motoring industry - as Tesla's $5billion Gigafactory takes shape. The huge project is part of co-founder and CEO Elon Musk's plans to drive down the cost of electric cars. In September, the South-African born entrepreneur announced that the site near Reno, Nevada, was the chosen location for the lithium-ion battery plant, which is expected to reduce the price of the Tesla Model 3 by $2,800. Growing: Tesla's $5billion Gigafactory - which will be able to produce 500,000 lithium-ion batteries every year - takes shape in the desert near Reno, Nevada. Once completed it will be between 5 and 6 million square feet - or the size of the Pentagon. Ambitious: The project is part of co-founder Elon Musk's plans to drive down the cost of electric cars. The South African-born entrepreneur has said the cheaper production that will come with the site will bring down the cost of the Tesla Model 3 by almost $3,000. Site preparation began in July at an industrial park along U.S. Interstate 80 15 miles east of Sparks, a Reno suburb. Since then the building has grown and looks set to be ready by the time partial production begins in 2017. The aim is to produce enough batteries to power 500,000 vehicles a year when it is fully completed in 2020 and produce a power source that can also be installed in gadgets such as toys and drones. Production is currently based in Freemont, California, but the plant does not have the capacity to serve the company's future production needs. The facility is expected to bring 6,500 jobs to Nevada and $100 billion in economic benefits, according to CNBC. In return, the state offered Tesla up to $1.3 billion in tax breaks. Economic benefits have already started coming in with 700 construction jobs being created. Projections also suggest the factory will lead to a surge in the local population - with an additional 35,000 residents moving to the area by 2032. It is set to be completed in 2020 and will be around 10 million square feet - as big as the Pentagon or 174 football fields. The venture is partnered by Panasonic, who are believed to have invested billions in the motor company. Even though Reno has attracted big businesses such as Apple and Amazon, there is concern the city's infrastructure will not be able to cope with the increase in demand. Nevada's governor Brian Sandoval said it's further proof the state, that has also landed huge Switch data center expansion, is emerging as a leader in new technology innovation. 'This announcement demonstrates the possibilities within our state if we continue to recruit the growing industries of the 21st century,' he said. Nevada overall is expected to add 528,000 people over the next two decades, for a population of 3.3 million. That's led by an estimated 328,000-person bump in Clark County and an estimated 147,000-person gain in Washoe County. But northern Nevada, where the gigafactory will be built, is expected to feel the most direct impact from Tesla. Location: An image taken from the hills surrounding the complex reveal its isolation and scale. Development: Construction has moved along at a rapid rate since November (pictured). The project is set to last for another five years but partial production is set to begin in 2017. Future: An artist's impression shows what the site could look like when it is finished in 2020. Nevada will also benefit from an investment in roads leading to the facility and potential to surround it with other ventures. Entrepreneur: The huge project is part of co-founder and CEO Elon Musk's plans to drive down the cost of electric car

Summary: Huge construction project which started last year is underway near Reno. It is set to be completed by 2020. Founder says the new plant will reduced the price of the Tesla Model 3 by almost $3,0000. When finished the building will be 10 million square feet - as big as the Pentagon or 174 football fields. It is also set to reduce the cost of production which means the range of electric cars will be cheaper.

cnn_dailymail

en

Write a title and summarize: Mutational correlation patterns found in population-level sequence data for the Human Immunodeficiency Virus (HIV) and the Hepatitis C Virus (HCV) have been demonstrated to be informative of viral fitness. Such patterns can be seen as footprints of the intrinsic functional constraints placed on viral evolution under diverse selective pressures. Here, considering multiple HIV and HCV proteins, we demonstrate that these mutational correlations encode a modular co-evolutionary structure that is tightly linked to the structural and functional properties of the respective proteins. Specifically, by introducing a robust statistical method based on sparse principal component analysis, we identify near-disjoint sets of collectively-correlated residues (sectors) having mostly a one-to-one association to largely distinct structural or functional domains. This suggests that the distinct phenotypic properties of HIV/HCV proteins often give rise to quasi-independent modes of evolution, with each mode involving a sparse and localized network of mutational interactions. Moreover, individual inferred sectors of HIV are shown to carry immunological significance, providing insight for guiding targeted vaccine strategies. HIV and HCV are the cause of devastating infectious diseases that continue to wreak havoc worldwide. Both viruses are highly variable, possessing an extraordinary ability to tolerate mutations while remaining functionally fit. While individual residue mutations may be deleterious, these are often compensated by changes elsewhere in the protein which restore fitness [1,2]. These interacting residues form compensatory networks which provide mutational escape pathways against immune-mediated defense mechanisms, presenting a major challenge for the design of effective vaccines [3]. The compensatory interaction networks that exist for HIV and HCV—and for other viruses more generally—are complicated and far from being well understood. Resolving these by experimentation is difficult, due largely to the overwhelming number of possible mutational patterns which must be examined. An alternative approach is to employ computational methods to study the statistical properties of sequence data, under the basic premise that the residue interactions which mediate viral fitness manifest as observable mutational correlation patterns. For HIV, recent analytical, numerical and experimental studies [4–7] provide support for this premise, indicating that these patterns may be seen as population-averaged evolutionary “footprints” of viral escape during the host-pathogen combat in individual patients. This idea has been applied to propose quantitative fitness landscapes for both HIV [8,9] and HCV [10] which are predictive of relative viral fitness, as verified through experimentation and clinical data. Fitness is a broad concept that is ultimately mediated through underlying biochemical activity. For both HIV and HCV, experimental efforts have provided increased biochemical understanding of the constituent proteins, leading in particular to the discovery of small and often distinct groups of residues with functional or structural specificity (Fig 1 and S1 File). These include, for example, sets of protein residues lying at important structural interfaces, those involved with key virus-host protein-protein interactions, or those found experimentally to directly affect functional efficacy. An important open question is how these biochemically important groups relate to the interaction networks formed during viral evolution. Some insights have been provided for a specific protein of HIV [11] and HCV [12]. The main objective of these investigations was to identify potential groups of co-evolving residues (referred to as “sectors”) which may be most susceptible to immune targeting. In each case, a sector with potential immunological vulnerability was inferred, and this was found to embrace some residues with functional or structural significance. It is noteworthy that the employed inference methods were designed to produce strictly non-overlapping groups of co-evolving residues, which may hinder identification of inherent co-evolutionary structure and associated biochemical interpretations. In a parallel line of work, computational methods have been used to understand co-evolution networks for various protein families, with compelling results (reviewed in [13]). Notably, for the family of S1A serine proteases [14], a correlation-based method termed statistical coupling analysis (SCA) uncovered a striking modular co-evolutionary structure comprising a small number of near-independent groups of co-evolving residues (again referred to as sectors), each bearing a clear and distinct biochemical association, in addition to other qualitative properties. Sectors have also been obtained for other protein families using SCA, and the functional relevance of these has been confirmed through experimentation [14–17]. A natural question is to what extent such modular, biochemically-linked co-evolutionary organization exists for the viral proteins of HIV and HCV? This is not obvious, particularly when one considers the evolutionary dynamics of these viruses, which are complex and very distinct to those of protein families. Specifically, they involve greatly accelerated mutation rates, and are shaped by effects including intrinsic fitness, host-specific but population-diverse immunity, recombination, reversion, genetic drift, etc. The sampling process is also complicated and subject to potential biases, making inference of co-evolutionary structure difficult. In this paper, considering multiple proteins of HIV and HCV, we identify in each case a sparse and modular co-evolutionary structure, involving near-independent sectors. This is established by introducing a statistical method, which we refer to as “robust co-evolutionary analysis (RoCA) ”, that learns the inherent co-evolutionary structure by providing resilience to the statistical noise caused by limited data. Strikingly, the sectors are shown to distinctly associate with often unique functional or structural domains of the respective viral protein, indicating clear and well-resolved linkages between the evolutionary dynamics of HIV and HCV viral proteins and their underlying biochemical properties. Our results suggest that distinct functional or structural domains associated with each of the viral proteins give rise to quasi-independent modes of evolution. This, in turn, points to the existence of simplified networks of sparse interactions used by both HIV and HCV to facilitate immune escape, with these networks being quite localized with respect to specific biochemical domains. The insights provided by the inferred sectors also carry potential importance from the viewpoint of immunology and vaccine design, which we demonstrate for a specific protein of HIV. By employing available sequence data, we investigated the co-evolutionary interaction networks for multiple HIV and HCV proteins. Our proposed approach RoCA resolves co-evolutionary structures by applying a spectral analysis to the mutational (Pearson) correlation matrices and identifying inherent structure embedded within the principal components (PCs), which are representative of strong modes of correlation in the data. The RoCA algorithm is designed to be robust against statistical noise, which is a significant issue since the number of available sequences for each protein is rather limited, being comparable to the protein size (Fig 2A). The RoCA method comprises two main steps. Similar to the previous PCA-based co-evolutionary methods [11,12,14], the first step involves isolating PCs which carry correlation information from those which are supposedly dominated by statistical noise (Fig 2B, top panel). The second and most significant step of RoCA provides robust estimates of the PCs (Fig 2B, bottom panel) selected in the first step. We developed an automated and suitably adapted version of a sparse PCA technique [19], which is based on the standard orthogonal iteration procedure used to obtain the PCs of a matrix [20]. To summarize, this step (Fig 2B, bottom panel) involves: (i) a data-driven thresholding procedure applied to the PCs—designed based on ideas from random matrix theory—that distinguishes, for each PC, the significantly correlated residues from those residues whose correlations are consistent with statistical noise, and (ii) an iterative procedure that tries to robustly estimate the correlation structure between the selected residues across different PCs (see Materials and methods for details). Based on the resulting PCs, the RoCA algorithm directly infers co-evolutionary sectors, representing groups of residues whose mutations are collectively coupled (Fig 2C). We note that while many sparse PCA methods have been developed [19,21–24] and applied previously to problems in different fields (e. g., senate voting and finance [25], network systems [26], image processing [27], and genome-wide association studies in cancer [28]), to our knowledge, RoCA is the first application of sparse PCA techniques to the co-evolutionary analysis of proteins. The automated robust estimation of PCs produced by RoCA (Fig 2B, bottom panel) bears an important distinction from previously proposed sectoring methods [11,12,14] which (indirectly) attempted to reduce the effect of statistical noise in the PCs using either visual inspection or an ad hoc thresholding procedure. Moreover, other than applying a suitable data-driven thresholding step to remove statistical noise, RoCA makes no structural imposition on the inferred sectors (e. g., enforcing the sectors to be non-overlapping as in [11,12,15]), and it is therefore designed to reveal inherent co-evolutionary networks as reflected by the data. We used RoCA for inferring the co-evolutionary structure of two proteins each of HIV and HCV which represent a good mix of structural (HIV Gag), accessory (HIV Nef), and non-structural (HCV NS3-4A and NS4B) proteins. For each viral protein, the RoCA method identified a small number of sectors (Fig 3A and S1 Fig) which together embraced a rather sparse set of residues (i. e., between 35%–60% of the protein; see S2 File for the complete list). In some cases the sector residues were localized in the primary sequence, while in others they were quite well spread (Fig 3B and S1 Fig). Importantly, while each sector was identified from a distinct PC, they were found to be largely disjoint (Fig 3A and S1 Fig). This suggests that the co-evolutionary structures are highly modular, with the different modules (sectors) being nearly uncorrelated to each other. In fact, further statistical tests demonstrated that the inferred sectors are nearly independent (Fig 3C and S1 Fig). This identified modular co-evolutionary structure is in fact reminiscent of ‘community structure’ that has been observed in numerous complex networks, e. g., metabolic, webpage, and social networks [29]. In such applications, the identified modules or communities have been shown to represent dense sub-networks which perform different functions with some degree of autonomy. For our co-evolutionary sectors, in line with previous studies on the fitness landscape of HIV [6–8] and HCV [10], they appear to represent groups of epistatically-linked residues which work together to restore or maintain viral fitness when subjected to strong selective pressures during evolution (e. g., as a consequence of immune pressure). In light of this, one anticipates that the co-evolutionary sectors should afford an even deeper interpretation in terms of the underlying biochemical properties of the viral proteins, which fundamentally mediate viral fitness. To explore potential correspondences between the identified RoCA sectors and basic biochemical properties, we compiled information determined by experimental studies for each of the viral proteins. This consists of residue groups having prescribed functional or structural specificity (see Fig 1; also S1 File for a more extensive list including small groups). These groups, which are seen to occupy sparse and largely distinct regions of the primary structure (Fig 1), are collectively referred to as “biochemical domains”. (This should not be confused with the term “domain”, as classically used for a folding unit in structural biology and biochemistry.) For each viral protein, structural domains were defined based on spatial proximity of residues in the available protein crystal structure; they include, for example, residues which lie on critical interfaces needed to form stable viral complexes, or those involved in essential virus-host protein-protein interactions. Functional domains, on the other hand, were typically identified using site-directed mutagenesis or truncation experiments, and they include groups of residues found to have a direct influence on the efficacy of specific protein functions. It is important to note, however, that while structural domains are typically clearly specified, functional domains are expected to be less so, due to experimental limitations. Results reported based on truncation experiments, for example, may comprise false positives. This is because the truncation experiment (see [30,31] for specific examples) typically involves a coarse procedure to predict the functionally important residues by removing different groups of contiguous residues from the protein, and studying the effect of each truncation on the protein function. This procedure may suggest a particular group of residues to be important for a protein function when only one or two residues may be critical. Thus, the remaining residues in the reported important group would be false positives. Despite potential limitations of the compiled biochemical domains, contrasting these domains with the RoCA sectors (for all four viral proteins) revealed a striking pattern, with most sectors showing a clear and highly significant association to a unique biochemical domain (Fig 4). This is most marked for the HIV Gag protein, where there is a one-to-one correspondence. These observations carry important evolutionary insights. Not only are the co-evolutionary networks of both HIV and HCV proteins modular, but the modules (sectors) seem to be intimately connected to distinct biological phenotype. Our results suggest that the fundamental structural or functional domains of these viral proteins spawn quasi-independent co-evolutionary modes, each involving a simplified sparse network of largely localized mutational interactions. The observed phenomenon is seemingly a natural manifestation of immune targeting against residues within the biochemical domains, since escape mutations at these residues likely lead to structural instability or functional degradation, necessitating the formation of compensatory mutations to restore fitness. We investigated whether our main findings could also be revealed by other sectoring methods. We first considered a method that we proposed previously based on classical PCA [12], that sought to identify groups of collectively-correlated viral residues which may be susceptible to immune targeting. Note that this PCA-based method [12] is very similar to the method introduced in [11], mainly differing in the procedure to form sectors from PCs; specifically, the method in [11] formed sectors from visual inspection of PC biplots, whereas an automated procedure was applied in [12] (see S1 Text for implementation details). Moreover, while the PCA-based method [12] was used to study only a specific HCV protein, it is general in its application (similar to the method presented in [11] and the proposed RoCA method) and can be used to infer co-evolutionary sectors for the viral proteins under study (S1 Text). An important feature of the PCA method [12] (and also [11]) was the imposition of a structural constraint in the inferred sectors, enforced to be disjoint, which may compromise its ability to infer natural co-evolutionary structure (S1 Text). Despite the imposed zero inter-sector-overlap constraint, sectors produced by the PCA-based method [12] for the studied viral proteins tended to be larger than RoCA sectors (S3 Fig), and they collectively embraced a larger set of residues (covering 40%–80% of the protein). Comparing the sectors inferred by this method with those obtained using the RoCA method revealed that they included a mix of residues from multiple RoCA sectors (Fig 5A), a fact that was also reflected in the biochemical associations of the sectors, where much of the resolved (unique) sector/domain associations shown by RoCA (Fig 4) were indeed no longer revealed (Fig 5C). We found that these key differences were attributed to the sensitivity of the PCA-based approach to sampling noise, as reflected by the noisy and significantly overlapping principal components (Fig 5B). This was corroborated with a ground-truth simulation study, through which the ability to infer co-evolutionary structure was tested in synthetic model constructions (S2 Text). The RoCA method resolved all the individual (true) sectors with high accuracy, whereas our previous method [12] inferred comparatively large sectors, which often included false positives and merged residues from different true sectors (S3 Fig). We also tested other co-evolutionary methods, which tended to return very different results to RoCA, and generally revealed little biochemical association for the studied viral proteins (S5 Fig). Most notable is the limited biochemical association of sectors identified using the benchmark SCA method [14] (S5 Fig) which has shown much success in resolving co-evolutionary structure for certain protein families [15,32]. Aside from the noise sensitivities shared by both SCA and classical PCA-based methods (S5 Fig), the surprising disparity in this case appears due to the weighted covariance construction employed by SCA (as opposed to the Pearson correlation) which, while apparently suited to the analysis of certain protein families data [14–17], does not seem suitable for identifying the co-evolutionary structure in the considered HIV and HCV proteins (see S3 Text for details). In the following, we provide details on the biochemical associations of the identified RoCA sectors for each of the four viral proteins. Our main results carry potential immunological significance, which may provide useful input for vaccine design. To demonstrate this, we considered the HIV Gag protein, and contrasted the inferred RoCA sectors with the epitope residues targeted by T cells of HIV “long-term non-progressors” (LTNP) and “rapid progressors” (RP). LTNP correspond to rare individuals who keep the virus in check without drugs, whereas RP are individuals who tend to progress to AIDS in less than 5 years (compared to the population average of 10 years [56]). Clinical studies of HIV-infected cohorts have demonstrated a high correlation between possession of specific human leukocyte antigen (HLA) alleles with the disease outcome (LTNP or RP) [57,58]. The information of the epitopes targeted by the T cells in HIV-infected individuals with these specific HLA alleles was obtained from the Los Alamos HIV Molecular Immunology Database (http: //www. hiv. lanl. gov/content/immunology) and is presented in S1 Table. Our analysis revealed that LTNP elicit immune responses strongly directed towards residues in sector 3, whereas RP elicit responses against residues in sector 2 (Fig 7). Recalling the sector biochemical associations (Figs 4 and 6), these observations seem to promote the design of T-cell vaccine strategies which target sector residues lying on the p24 intra hexamer interfaces, while avoiding targeting residues on the p24-SP1 interface. In the former case, such targeting seemingly compromises viral fitness by disrupting the formation of stable HIV capsid [35], which appears quite difficult to restore through compensatory mutations. In contrast, restoring fitness costs associated with destabilization of the p24-SP1 interface appears less difficult. These results were contrasted against a previous analysis of HIV Gag [11], in which an inferred sector based on a classical PCA approach (a slight variant of the approach [12], discussed earlier) was also found to associate with LTNP. The residues defining this immunologically important sector were directly extracted from [11]. Analyzing the biochemical association of the residues in this sector (similar to the analysis done in Fig 4) revealed a significant association (P < 0. 05, Fisher’s exact test) with the p24 intra-hexamer interface (as pointed out in [11]), but also with the p24-SP1 interface (see S6 Fig for details). Hence, while reaffirming the importance of targeting interfaces within p24 hexamers, different conclusions were established regarding p24-SP1, suggesting that this interface should be targeted, rather than avoided. This important distinction arises as a consequence of the methodological differences between RoCA and the previous PCA-based methods [11,12], as discussed previously. By integrating our observations with population-specific HLA allele and haplotype information, candidate HIV immunogens eliciting potentially robust T cell responses can be proposed [11,12]. A more detailed investigation along these lines, as well as broadening the analysis to other viral proteins, is planned to be carried out in future work. Characterizing the co-evolutionary interactions employed by HIV and HCV is an important problem. These interactions reflect the mutational pathways used by each virus to maintain fitness while evading host immunity. However, they are not well understood and pose a significant challenge for vaccine development. By applying statistical analysis to the available cross-sectional sequence data, we showed that for multiple HIV/HCV proteins the interaction networks possess notable simplicity, involving mainly distinct and sparse groups of interacting residues, which bear a strikingly modular association with biochemical function and structure. Essential to unraveling this phenomena was the introduction of a robust inference method. Our approach is particularly well suited for the “internal” proteins of chronic viruses such as HIV and HCV that are subjected to broadly directed T cell responses. For such proteins, and for HIV in particular, recent experimental and computational work has provided evidence that the population-averaged mutational correlations are reflective of intrinsic interactions governing viral fitness. This was shown to be a consequence of multiple factors which influence the complex evolutionary dynamics of HIV, including the extraordinary diversity of HLA genes in the human population which place selective pressure on diverse regions of the protein, thereby promoting wide exploration of sequence space, in addition to the tendency of mutations to revert upon transmission between hosts [4–6]. An additional important evolutionary factor is that of recombination, which introduces diversity through template switching during viral replication. A consequence of recombination is that it breaks mutational correlations between residues that are distant in the primary structure. That is, higher rates of recombination should lead to shorter-range correlations and vice-versa. Thus, the recombination involved in HIV and HCV evolution [59] may consequently distort the mapping between biochemical domains and the inferred sectors, and possibly result in inference of multiple distinct sectors associated with the same biochemical domain. However, such an effect of recombination is not evident from our results. Nonetheless, the effect of high recombination rate of HIV as compared to HCV [59] seems to be reflected in the separation among residues involved in the inferred sectors. Specifically, the HIV protein sectors are quite localized, with a median separation in the primary structure of up to 140 residues (sector 6 of HIV Gag), while those of the HCV proteins are well separated with a median separation of up to 480 residues (sector 1 of HCV NS3-4A). In general, the predicted sectors primarily comprise residues within the corresponding biochemical domains and a few other residues which are close in either primary or tertiary structure. However, these sectors also include a small proportion of residues which are distant from those within the respective biochemical domains (S7 Fig) and thus, appear to influence the associated structure or function by an allosteric mechanism. Such long-range interactions have been reported to play a role in maintaining viral fitness and facilitating immune evasion [60–62]. Allosteric interactions have also been observed in the co-evolutionary sectors of different protein families obtained previously with the SCA method [14–17]. The identified sectors for each viral protein together comprise between 35%–60% of the total residues in the protein (Fig 3A and S1 Fig). This is consistent with the sparse sectors of co-evolving residues observed in different protein families using the SCA method [14–17]. However, one may ask about the role of non-sector residues, i. e., those not allocated to any sector. Similar to the observations in other proteins [14–17], our analysis suggests that non-sector residues evolve nearly independently, with associated biochemical domains being impacted only by individual mutations at these residues. Our co-evolutionary analysis is based on a binary approximation of the amino acid sequences, with mutants distinguished from the most frequent amino acid at each position (see Materials and methods for details). This is a reasonable approximation due to the high conservation of the studied internal viral proteins (S8 Fig). For other viral proteins which are comparatively less conserved, like surface proteins HIV Env and HCV E1/E2, a similar co-evolutionary analysis may require refinement of such approximation by incorporating the information of amino acid identities of different mutants. This is a worthwhile problem to be pursued in a future study, in particular given the relevance for vaccine design of surface proteins, as these are a major target of neutralizing antibodies. We point out however that multiple HIV and HCV clinical studies have also demonstrated strong correlation of a broadly-directed cellular (T-cell-based) immune response against internal proteins with HIV control [57,58] and spontaneous HCV clearance [63,64]. These reports suggest that, for HIV and HCV, the immune response against internal proteins (similar to those studied in this work) may be just as important as—if not more important than—the antibody response against surface proteins. While our analysis has focused primarily on viral proteins, the proposed RoCA approach is general and may be applied more broadly, provided that the studied proteins are reasonably conserved. As an example, we computed sectors for the S1A family of serine proteases and compared these with results obtained previously with the SCA algorithm [14,15]. Similar to SCA, RoCA yielded three co-evolutionary sectors which had statistically-significant associations with distinct phenotypic properties; namely thermal stability, enzymic activity, and catalytic specificity (S9 Fig). We point out however that the very notion of a “sector” as defined previously for SCA [14–17] has some differences to that of the RoCA sectors. Specifically, while for the HIV/HCV proteins in this work we employed an unweighted Pearson correlation measure and the inferred sectors were interpreted as simply groups of correlated residues; for the protein families [14–17], SCA involved a conservation-weighted correlation measure and thus the inferred sectors represented groups of not only correlated but also conserved residues (see S3 Text for details). For serine proteases, the relatively higher statistical significance of biochemical association for SCA sectors (S9 Fig) suggests that using a measure that also incorporates conservation may be useful for identifying biologically important residues in this case. Nonetheless, the RoCA sectors produced for the serine proteases, based on an unweighted Pearson correlation measure, further attest to the importance of residue interactions in mediating fundamental protein functions. For the HIV/HCV viral proteins under study, the relation between the biologically important residues (reflected by the biochemical domains) and conservation was not clearly apparent (S10 Fig). In fact, a significant and particularly surprising aspect of our analysis is the substantial extent to which the correlation patterns, with no regard for conservation, encode information regarding qualitatively distinct phenotypes including structural units—virus-host and virus-virus protein interactions—and functional domains. The identified sectors may therefore also be seen as predictors of important biochemical domains. For each of the four viral proteins under study, there is at least one sector with unknown biochemical significance. Subsequent experimentation, such as mutagenesis experiments targeted at the identified sector residues, could therefore provide new insight which furthers the current understanding of HIV and HCV. Particularly interesting is the poorly understood NS4B protein of HCV, for which any biochemical activity underpinning the leading two sectors—representing the strongest co-evolutionary modes—have yet to be resolved. The sequence data for HIV-1 clade B Gag and Nef was obtained from the Los Alamos National Laboratory HIV database, http: //www. hiv. lanl. gov/. We restricted our analysis to drug-naive sequences and any sequence marked as problematic on the database was excluded. To avoid any patient-bias, only one sequence per patient was selected. After aligning the sequences based on the HXB2 reference, they were converted to a N × M amino acid multiple sequence alignment (MSA) matrix, where N denotes the number of sequences and M denotes the number of amino acid sites (residues) in the protein. The downloaded sequences may include a few outliers due to mis-classification (e. g., sequences assigned to an incorrect subtype or clade) in the database. Such outlying sequences were identified and removed using a standard PCA clustering approach (see [11] for details). This yielded N = 1897 and N = 2805 sequences for HIV Gag and Nef, respectively. Moreover, the fully conserved and problematic residues (with blanks or gaps greater than 12. 5%) were eliminated, resulting in M = 451 variable residues for Gag and M = 202 for Nef. Similarly, the sequence data for HCV subtype 1a NS3-4A and NS4B was downloaded from the Los Alamos National Laboratory HCV database, http: //www. hcv. lanl. gov/. The downloaded HCV sequences were then aligned based on the H77 reference and converted to the amino acid MSA. Applying the above-mentioned pre-processing resulted in N = 2832 sequences for NS3-4A and N = 675 sequences for NS4B, with an effective length of M = 482 for NS3-4A and M = 190 for NS4B. The processed amino acid MSA matrix A = (Aij) was converted into a binary matrix B, with (i, j) th entry B i j = { 0 if A i j is the consensus amino acid at residue j, 1 otherwise. (1) Thus, ‘0’ represents the most prevalent amino acid at a given residue and ‘1’ represents a mutant (substitution). This is a reasonable approximation of the amino acid MSA, given the high conservation of the internal viral proteins under study (S8 Fig). The binary sequences in B are generally corrupted by the so-called phylogenetic effect, which represents ancestral correlations. A comparatively large eigenvalue is observed in the associated correlation matrix due to these phylogenetic correlations [11,12]. Following previous ideas (for details, see Sections 3 and 4 in the Supporting Information of [11] and Section 2 in the Appendix of [12]), such effects are reduced using standard linear regression. The resulting data matrix, denoted by B ^, was the base for computing the correlations used to infer sectors. Specifically, we computed the M × M sample correlation matrix, along with its spectral decomposition, given by C ≜ V - 1 2 S V - 1 2 = ∑ k = 1 M λ k q k q k ⊺. (2) Here, S is the sample covariance matrix with entries S i j = 1 N ∑ k = 1 N (B ^ k i - B ¯ i) (B ^ k j - B ¯ j) where B ¯ i = 1 N ∑ k = 1 N B ^ k i is the sample mean, while V is a diagonal matrix containing the sample variances, i. e., Vii = Sii, and λk and qk are the kth-largest eigenvalue of C and its corresponding eigenvector, respectively. The superscript ⊺ denotes vector transposition. We introduced an approach based on robust PCA methods to accurately estimate the PCs (i. e., the leading eigenvectors) of the correlation matrix, which were then directly used to identify sectors. In particular, we considered the iterative thresholding sparse PCA (ITSPCA) method which, in short, is a combination of the standard orthogonal iteration method [20], used to compute the eigenvectors of a given matrix, and an intermediate thresholding step which filters out noise in the estimated PCs. However, the original ITSPCA method was not directly applicable to our correlation-based sectoring problem, since it was designed primarily for covariance matrices, and it involved a variance-dependent coordinate pre-selection algorithm which is no longer suitable. As such, for RoCA, we developed a version (called Corr-ITSPCA, see Algorithm 1) which is appropriately adapted to operate on correlation matrices, and we designed automated methods for tuning the relevant parameters; specifically, the number of significant PCs α and the noise threshold γk. Algorithm 1 Corr-ITSPCA Method Inputs: 1. Correlation matrix of size M × M, C; 2. Number of PCs to be estimated, α; 3. Noise threshold, γk, k = 1,2, ⋯, α. Output: Robust estimates of the PCs, pk, given as columns of the M × α matrix P = Q (∞), where Q (∞) denotes Q (i) at convergence. 1: Initialization: i = 1; 2: Initial orthonormal matrix of size M × α, Q (0) = Qα; here Qα is a matrix whose columns are the α leading eigenvectors of C, i. e., Qα = [q1 q2 ⋯ qα]. 3: repeat 4: Multiplication: T (i) = (T ℓ k (i) ) = C Q (i - 1); 5: Thresholding: T ^ (i) = (T ^ ℓ k (i) ), with T ^ ℓ k (i) = T ℓ k (i) 1 { | T ℓ k (i) | > γ k }, where 1{E} is the indicator function of an event E; 6: QR Factorization: Q (i) R (i) = T ^ (i); 7: i = i + 1; 8: until convergence Such automated design is crucial to obtain accurate results, as these parameters control respectively the number of sectors that we infer and the number of residues included in each sector. Note that this is a principled design approach, as opposed to an ad hoc approach considered previously by the authors to uncover vaccine targets against the NS5B protein of HCV [65]. These parameters are designed as follows: In Figs 3 and 6, we used heat maps to illustrate the computed sample correlation matrix C. As discussed above, the sample correlations were generally corrupted by statistical noise due to the finite number of available sequences. Thus, for a better visualization and, in particular, to appreciate the strong correlations within the inferred sectors, the sample correlation matrix was cleaned from statistical noise by thresholding the sample eigenvalues in such a way that the significant α spectral modes (Eq (3) ) were kept unaltered, while the remaining eigenvalues (which do not appear to contribute genuine correlations) were collapsed to a constant. Specifically, the cleaned sample correlation matrix was obtained as C ^ * = ∑ k = 1 M λ k * q k q k T, (11) where λ k * = { λ k if k ≤ α, ζ otherwise, with ζ a constant value such that the trace of C ^ * remained normalized (equal to M). Note that C ^ * is not a standard correlation matrix as C ^ k k * ≠ 1. A standardized version was then computed as C ^ = D - 1 2 C ^ * D - 1 2, (12) where D is a diagonal matrix with D k k = C ^ k k *, and used to depict the cleaned correlations as a heat map. We introduced a metric called “normalized entropy deviation (NED) ” to quantify the extent to which two groups of residues are statistically independent of each other. The NED between two sectors i and j is defined as NED inter (i, j) = (H s i + H s j) - H s i ∪ s j H s i ∪ s j, (13) where si is a set comprising the five residues with largest correlation magnitude of sector i and H s i is the entropy of si computed from the binary MSA matrix. Specifically, this entropy is computed over all κ = 1,2, ⋯, 2 # (s i) combinations of the residues in set si as follows H s i = - ∑ κ = 1 2 # (s i) f κ ln f κ, f κ > 0 (14) where fκ is the frequency of the combination κ in the MSA and # (si) is the cardinality of set si. In theory, if two given sectors are perfectly independent, the sum of the entropies of the individual sectors must be equal to the entropy of both sectors taken together, resulting in NEDinter = 0. In practice however, a small non-zero value of NEDinter is expected due to finite-sampling noise, even if the sectors are independent. We obtain an estimate of it by constructing a null case, where the entries of the MSA corresponding to the sets si and sj are randomly shuffled in such a way that any correlation between the two sets is essentially eliminated, while the correlations between residues in an individual set remain unaltered. Using Eq (13), NEDinter is computed for 500 such randomly shuffled realizations of the MSA and the average value (referred to as NEDrandom) represents the null (lower) reference value for NEDinter which is expected if the two sectors are independent. Substantial deviations from NEDrandom should reflect correlation between the sectors. In order to quantify the extent of such deviations in a clearly correlated case, we computed an upper reference NEDintra, obtained when the residues in both sets si and sj belong to the same sector. It is defined as NED intra (i, j) = max ( (H s i + H s i ′) - H s i ∪ s i ′ H s i ∪ s i ′, (H s j + H s j ′) - H s j ∪ s j ′ H s j ∪ s j ′), (15) where s i ′ is the set comprising the five residues with largest correlation magnitude of sector i with the residues in si excluded. The HLA alleles associated with LTNP and RP were reported in [58]. A list of HIV Gag epitopes that are presented by HLA alleles and targeted by T cells of either HIV LTNP or RP was compiled using the data from the Los Alamos HIV Molecular Immunology Database (http: //www. hiv. lanl. gov/content/immunology) and is presented in S1 Table.

Title: Co-evolution networks of HIV/HCV are modular with direct association to structure and function Summary: HIV and HCV cause devastating infectious diseases for which no functional vaccine exists. A key problem is that while individual mutations in viral epitopes under immune pressure may substantially compromise viral fitness, immune escape is typically facilitated by other "compensatory" mutations that restore fitness. These compensatory pathways are complicated and remain poorly understood. They do, however, leave co-evolutionary markers which may be inferred from measured sequence data. Here, by introducing a new robust statistical method, we demonstrated that the compensatory networks employed by both viruses exhibit a remarkably simple decomposition involving small and near-distinct groups of protein residues, with most groups having a clear association to biological function or structure. This provides insights that can be harnessed for the purpose of vaccine design.

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Summarize: WASHINGTON (October 1, 2018) — Simulations with animal models meant to mirror galactic cosmic radiation exposure to astronauts are raising red flags for investigators at Georgetown University Medical Center (GUMC) about the health of astronauts during long voyages, such as to Mars. Their most recent study, published in the Proceedings of the National Academy of Sciences of the United States of America (PNAS), suggests that deep space bombardment by galactic cosmic radiation (GCR) could significantly damage gastrointestinal (GI) tissue leading to long-term functional alterations. The study also raises concern about high risk of tumor development in the stomach and colon. Their previous work has highlighted potential impairment to brain tissue, as well as accelerated aging, on long space trips due to the effect of energetic heavy ions, which don’t affect Earthlings due to the protective global magnetosphere. “Heavy ions such as iron and silicon are damaging because of their greater mass compared to no-mass photons such as x-rays and gamma (g)-rays prevalent on Earth, as well as low mass protons in outer space,” says the study’s senior investigator, Kamal Datta, MD, an associate professor in the Department of Biochemistry and a project leader of the NASA Specialized Center of Research (NSCOR) at GUMC. “With the current shielding technology, it is difficult to protect astronauts from the adverse effects of heavy ion radiation. Although there may be a way to use medicines to counter these effects, no such agent has been developed yet,” says Datta, also a member of Georgetown Lombardi Comprehensive Cancer Center. “While short trips, like the times astronauts traveled to the Moon, may not expose them to this level of damage, the real concern is lasting injury from a long trip, such as a Mars or other deep space missions which would be much longer” he says. The GI tract is a self-renewing tissue with continuous cell division/proliferation, the researchers say. The mucosal (top) layer of cells is replaced every three to five days through coordinated migration of new cells from the bottom of a flask-shaped structure called the crypt toward the lumen of the gut. “Any disturbance of this replacement mechanism leads to malfunctioning of physiologic processes such as nutrient absorption and starts pathologic processes such as cancer,” says co-author Georgetown’s Albert Fornace Jr., MD, director of the NSCOR. To investigate the effect of heavy ions on the GI tract, the scientists used mouse small intestine as a model system. Mice were exposed to a low dose of iron radiation at the NASA Space Radiation Laboratory (NSRL) in Brookhaven National Laboratory on Long Island, New York, and the animals were then examined at Georgetown. Researchers compared the group of mice that received heavy ions to mice exposed to gamma rays, which are comparable to X-rays, and to a third, unexposed control group. The scientists found that intestinal cells in the heavy ion group did not adequately absorb nutrients and that they formed cancerous polyps. Additionally, there was evidence that iron radiation induced DNA damage that increased the number of senescent cells. Senescent cells are incapable of normal cell division but they are not “quiet,” says Datta. “They generate oxidative stress and inflammatory molecules that induce more damage. This greatly affected migration of cells that are needed to replace the intestinal lining, which slowed down GI functioning,” he says. Even though a very low dose was delivered over the equivalent of a months-long period in deep space, the effects of heavy ion radiation appeared to be permanent, says Fornace. “We have documented the effects of deep space radiation on some vital organs, but we believe that similar damage responses may occur in many organs,” says Datta. “It is important to understand these effects in advance so we can do everything we can to protect our future space travelers.” ### Study co-authors also include Santosh Kumar, PhD, a postdoctoral fellow; and Shubhankar Suman, PhD, an assistant professor, both in the department of oncology at Georgetown University Medical Center. Funding for the study came from NASA grants (NNX13AD58G and NNX15A121G). About Georgetown Lombardi Comprehensive Cancer Center Georgetown Lombardi Comprehensive Cancer Center is designated by the National Cancer Institute as a comprehensive cancer center — the only cancer center of its kind in the Washington, D.C. area. A part of Georgetown University Medical Center and MedStar Georgetown University Hospital, Georgetown Lombardi seeks to improve the diagnosis, treatment, and prevention of cancer through innovative basic and clinical research, patient care, community education and outreach, and the training of cancer specialists of the future. Connect with Georgetown Lombardi on Facebook (Facebook.com/GeorgetownLombardi) and Twitter (@LombardiCancer). About Georgetown University Medical Center Georgetown University Medical Center (GUMC) is an internationally recognized academic medical center with a three-part mission of research, teaching and patient care (through MedStar Health). GUMC’s mission is carried out with a strong emphasis on public service and a dedication to the Catholic, Jesuit principle of cura personalis — or “care of the whole person.” The Medical Center includes the School of Medicine and the School of Nursing & Health Studies, both nationally ranked; Georgetown Lombardi Comprehensive Cancer Center, designated as a comprehensive cancer center by the National Cancer Institute; and the Biomedical Graduate Research Organization, which accounts for the majority of externally funded research at GUMC including a Clinical and Translational Science Award from the National Institutes of Health. Connect with GUMC on Facebook (Facebook.com/GUMCUpdate) and Twitter (@gumedcenter). Connect with Georgetown University School of Medicine on Facebook (Facebook.com/somgeorgetown), Twitter (@gumedicine) and Instagram (@GeorgetownMedicine). (CNN) Astronauts may not be able to stomach long voyages into space -- literally speaking. A new NASA-funded study reveals that exposure to space radiation on long trips, like a voyage to Mars, could permanently harm astronauts' intestines and lead to stomach and colon cancer. The study, published by cancer researchers at Georgetown University Medical Center, used mice to test exposure to heavy ion radiation, which mimics the galactic cosmic radiation found in deep space. If that sounds complicated, essentially researchers compared "space" radiation to X-ray radiation and found its effects to be much more dangerous. A NASA illustration of the Phoenix Mars Lander on the Red Planet. After long exposures to a low dose of galactic radiation, mice had permanent damage to their gastrointestinal tracts and could no longer absorb nutrients in food. The mice also developed cancerous growths in their intestines -- raising concerns that astronauts who venture far into space would face the same deadly health issues. "While short trips, like the times astronauts traveled to the moon, may not expose them to this level of damage, the real concern is lasting injury from a long trip," said Kamal Datta, head of Georgetown's NASA Specialized Center of Research, in a press release. Read More Scientists are figuring out how we will eat on Mars, govern on Mars, and even pray on Mars, but one crucial question persists: How will we deal with the effects of space radiation on Mars? On Earth, we’re protected by our atmosphere and magnetic field, but on Mars future colonists will be naked against cosmic rays. We’d better how to figure out how to protect ourselves from it soon, as new research suggests that we won’t last long without getting sick. In a paper published Monday in the journal Proceedings of the National Academy of Sciences, a team of researchers from Georgetown University show that deep space radiation can have significant damaging effects on the small intestines of mice, increasing the risk of cancer. This damage persisted for a year after exposure to the radiation. In particular, they studied the effects of ionizing radiation, a type of radiation found in space. Ionizing radiation consists of beams of high-energy particles with so much energy they can knock the electrons and protons off of atoms; in this case, the researchers used heavy iron (56Fe) ions in their experiments at the NASA Space Radiation Laboratory, bombarding mice with a low dose. The team used a dose of radiation comparable to what would be experienced on a trip to Mars. “Since the estimated radiation dose for a 1,000-d Mars mission is about 0.42 Gy, with an estimate of an 860-d Mars mission dose equivalent of ∼1.01 Sv so doses of 0.5 Gy or less are more relevant, we have used 0.5 Gy to study [intestinal epithelial cell] migration, which is important for intestinal homeostasis,” the team writes. Significant DNA damage in the small intestines of the mice and slowed migration of intestinal epithelial cells occurred as a result. The effect of DNA damage is associated with an increased risk of cancer, and indeed, the study’s authors observed cell growth in the presence of this DNA damage that suggested a higher risk of cancer. INVERSE LOOT DEALS Stop Overpaying for Wireless Earbuds $39.99 We get it. No one likes the cord anymore but that's no reason you should pay hundreds of dollars for your earbuds. These Touchwave True Wireless Earbuds feature noise cancelling, sweat reistance and up to 12 hours of music for under $40 dollars. Buy Now But the effect of ionizing radiation on the migration of epithelial cells, while a little less obvious, is also significant. Epithelial cell migration describes the process by which the cells lining the intestine replace themselves, ensuring that nutrient absorption, immune response, and all other intestinal functions go as expected. Slowed epithelial cell migration throws this all out of wack. “Epithelial cell turnover is important for maintaining overall GI health, and its perturbation by space radiation, as well as by heavy-ion radiotherapy, raises concerns that require an understanding of its molecular underpinnings,” wrote the study’s authors, led by Santosh Kumar, Ph.D., a postdoctoral fellow at Georgetown. “Altered migration of intestinal epithelial cells could not only compromise barrier function and nutrient absorption but could also prolong exposure of cells to luminal content and initiate stress responses with pathological consequences, including colon cancer.” This study raises new, serious questions about how ionizing radiation could affect astronauts on long, crewed flights to Mars or the people who eventually settle there. As Inverse previously reported, “a crew going to Mars would probably be exposed to about one gray of radiation — over 277 times the dose of normal year’s worth of radiation exposure on Earth.” Longer missions would increase exposure and, in turn, increase cancer risk, not to mention the risk of acute radiation poisoning. Scientists have proposed shielding mechanisms, as well as protective drugs, though so far scientists haven’t come up with anything that can defend humans against this cosmic bombardment.

Summary: A study funded by NASA in a bid to determine what effects a journey into deep space might have on the health of astronauts has uncovered some startling possibilities for those brave souls who might one day venture to Mars. Per CNN, the findings out of Georgetown University Medical Center suggest that the sort of radiation to which such travelers would be exposed has the potential to cause major intestinal issues, including cancer. It's called galactic cosmic radiation, or GCR, and consists of so-called heavy ions that we're protected from by the magnetosphere that surrounds Earth. Deep in space, as well as on Mars, there is no such protection and scientists say there is no way yet known to prevent exposure with shielding or medications. Per Inverse, the study bombarded mice with doses of ionizing radiation that mimicked what's found in deep space. Researchers found that long exposure permanently altered the gastrointestinal tracts of the mice in a way that caused them to lose their ability to absorb nutrients. What's more, the mice also developed cancerous growths in their intestines, leading researchers to further worry about the effects of space radiation after previous findings suggested it poses a danger to the brain. "We have documented the effects of deep space radiation on some vital organs, but we believe that similar damage responses may occur in many organs," wrote senior investigator Kamal Datta. "It is important to understand these effects in advance so we can do everything we can to protect our future space travelers."

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Poison Control Center Enhancement and Awareness Act Amendments of 2003''. SEC. 2. FINDINGS. The Congress finds the following: (1) Poison control centers are our Nation's primary defense against injury and deaths from poisoning. Twenty-four hours a day, the general public as well as health care practitioners contact their local poison centers for help in diagnosing and treating victims of poisoning and other toxic exposures. (2) Poisoning is the third most common form of unintentional death in the United States. In any given year, there will be between 2,000,000 and 4,000,000 poison exposures. More than 50 percent of these exposures will involve children under the age of 6 who are exposed to toxic substances in their home. Poisoning accounts for 285,000 hospitalizations, 1,200,000 days of acute hospital care, and 13,000 fatalities annually. (3) Stabilizing the funding structure and increasing accessibility to poison control centers will promote the utilization of poison control centers, and reduce the inappropriate use of emergency medical services and other more costly health care services. (4) The tragic events of September 11, 2001, and the anthrax cases of October 2001, have dramatically changed our Nation. During this time period, poison centers in many areas of the country were answering thousands of additional calls from concerned residents. Many poison centers were relied upon as a source for accurate medical information about the disease and the complications resulting from prophylactic antibiotic therapy. (5) The 2001 Presidential Task Force on Citizen Preparedness in the War on Terrorism recommended that the Poison Control Centers be used as a source of public information and public education regarding potential biological, chemical, and nuclear domestic terrorism. (6) The increased demand placed upon poison centers to provide emergency information in the event of a terrorist event involving a biological, chemical, or nuclear toxin will dramatically increase call volume. SEC. 3. AMENDMENT TO PUBLIC HEALTH SERVICE ACT. Title XII of the Public Health Service Act (42 U.S.C. 300d et seq.) is amended by adding at the end the following: ``Part G--Poison Control ``SEC. 1271. MAINTENANCE OF A NATIONAL TOLL-FREE NUMBER. ``(a) In General.--The Secretary shall provide coordination and assistance to regional poison control centers for the establishment of a nationwide toll-free phone number to be used to access such centers. ``(b) Rule of Construction.--Nothing in this section shall be construed as prohibiting the establishment or continued operation of any privately funded nationwide toll-free phone number used to provide advice and other assistance for poisonings or accidental exposures. ``(c) Authorization of Appropriations.--There is authorized to be appropriated to carry out this section $2,000,000 for each of the fiscal years 2000 through 2009. Funds appropriated under this subsection shall not be used to fund any toll-free phone number described in subsection (b). ``SEC. 1272. NATIONWIDE MEDIA CAMPAIGN TO PROMOTE POISON CONTROL CENTER UTILIZATION. ``(a) In General.--The Secretary shall establish a national media campaign to educate the public and health care providers about poison prevention and the availability of poison control resources in local communities and to conduct advertising campaigns concerning the nationwide toll-free number established under section 1271. ``(b) Contract With Entity.--The Secretary may carry out subsection (a) by entering into contracts with one or more nationally recognized media firms for the development and distribution of monthly television, radio, and newspaper public service announcements. ``(c) Evaluation.--The Secretary shall-- ``(1) establish baseline measures and benchmarks to quantitatively evaluate the impact of the nationwide media campaign established under this section; and ``(2) prepare and submit to the appropriate congressional committees an evaluation of the nationwide media campaign on an annual basis. ``(d) Authorization of Appropriations.--There are authorized to be appropriated to carry out this section $600,000 for each of fiscal years 2000 through 2005 and such sums as may be necessary for each of fiscal years 2006 through 2009. ``SEC. 1273. MAINTENANCE OF THE POISON CONTROL CENTER GRANT PROGRAM. ``(a) Regional Poison Control Centers.--The Secretary shall award grants to certified regional poison control centers for the purposes of achieving the financial stability of such centers, and for preventing and providing treatment recommendations for poisonings. ``(b) Other Improvements.--The Secretary shall also use amounts received under this section to-- ``(1) develop standardized poison prevention and poison control promotion programs; ``(2) develop standard patient management guidelines for commonly encountered toxic exposures; ``(3) improve and expand the poison control data collection systems, including, at the Secretary's discretion, by assisting the poison control centers to improve data collection activities; ``(4) improve national toxic exposure surveillance by enhancing activities at the Centers for Disease Control and Prevention and the Agency for Toxic Substances and Disease Registry; ``(5) expand the toxicologic expertise within poison control centers; and ``(6) improve the capacity of poison control centers to answer high volumes of calls during times of national crisis. ``(c) Certification.--Except as provided in subsection (d), the Secretary may make a grant to a center under subsection (a) only if-- ``(1) the center has been certified by a professional organization in the field of poison control, and the Secretary has approved the organization as having in effect standards for certification that reasonably provide for the protection of the public health with respect to poisoning; or ``(2) the center has been certified by a State government, and the Secretary has approved the State government as having in effect standards for certification that reasonably provide for the protection of the public health with respect to poisoning. ``(d) Waiver of Certification Requirements.-- ``(1) In general.--The Secretary may grant a waiver of the certification requirement of subsection (c) with respect to a noncertified poison control center or a newly established center that applies for a grant under this section if such center can reasonably demonstrate that the center will obtain such a certification within a reasonable period of time as determined appropriate by the Secretary. ``(2) Renewal.--The Secretary may renew a waiver under paragraph (1). ``(3) Limitation.--In no instance may the sum of the number of years for a waiver under paragraph (1) and a renewal under paragraph (2) exceed 5 years. The preceding sentence shall take effect as if enacted on February 25, 2000. ``(e) Supplement Not Supplant.--Amounts made available to a poison control center under this section shall be used to supplement and not supplant other Federal, State, or local funds provided for such center. ``(f) Maintenance of Effort.--A poison control center, in utilizing the proceeds of a grant under this section, shall maintain the expenditures of the center for activities of the center at a level that is not less than the level of such expenditures maintained by the center for the fiscal year preceding the fiscal year for which the grant is received. ``(g) Matching Requirement.--The Secretary may impose a matching requirement with respect to amounts provided under a grant under this section if the Secretary determines appropriate. ``(h) Authorization of Appropriations.--There are authorized to be appropriated to carry out this section $25,000,000 for each of the fiscal years 2000 through 2004 and $27,500,000 for each of fiscal years 2005 through 2009. ``SEC. 1274. RULE OF CONSTRUCTION. ``Nothing in this part may be construed to ease any restriction in Federal law applicable to the amount or percentage of funds appropriated to carry out this part that may be used to prepare or submit a report.''. SEC. 4. CONFORMING AMENDMENT. The Poison Control Center Enhancement and Awareness Act (42 U.S.C. 14801 et seq.) is hereby repealed. Speaker of the House of Representatives. Vice President of the United States and President of the Senate.

Title: A bill to provide assistance for poison prevention and to stabilize the funding of regional poison control centers Summary: Poison Control Center Enhancement and Awareness Act Amendments of 2003 - (Sec. 3) Amends the Public Health Service Act to direct the Secretary of Health and Human Services to provide coordination and assistance to regional poison control centers for a nationwide toll-free phone number. Authorizes appropriations through FY 2009. Directs the Secretary to establish a national media campaign to educate the public and health care providers about poison prevention and the availability of local poison control resources, and to conduct advertising campaigns concerning such nationwide toll-free number. Authorizes the Secretary to enter into contracts with one or more nationally recognized media firms for the development and distribution of related monthly television, radio, and newspaper public service announcements. Authorizes appropriations through FY 2009. Directs the Secretary to award grants to certified (with a discretionary five-year maximum certification waiver) regional poison control centers for: (1) center financial stability; and (2) poison prevention and treatment. Directs the Secretary to: (1) develop standardized poison prevention and poison control promotion programs; (2) develop standard patient management guidelines for commonly encountered toxic exposures; (3) improve and expand poison control data collection systems; (4) improve national toxic exposure surveillance by enhancing activities at the Centers for Disease Control and Prevention and the Agency for Toxic Substances and Disease Registry; (5) expand toxicologic expertise within poison control centers; and (6) improve poison control center capacity to answer high call volumes during a national crisis. Authorizes appropriations through FY 2009. (Sec. 4) Repeals the Poison Control Center Enhancement and Awareness Act.

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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 60/428,672 entitled FOOT OPERATED TOILET SEAT filed in the name of Steve Stewart on Nov. 25, 2002, the entirety of which is incorporated herein by reference. FIELD OF THE INVENTION [0002] The present disclosure relates to a toilet seat adjusting mechanism for residential or commercial toilets, and more specifically relates to a foot-operated mechanism for raising a toilet seat that results in improved sanitation and convenience to all users of the toilet. BACKGROUND OF THE INVENTION [0003] Most toilets in the developed western world have a seat. The typical seat is a moveable device, to be manually lifted when using the toilet as a urinal. Seat design, however, has never favored this fact, and it is very common to this day to fumble for a finger-hold on the bottom edge of toilet seats in both public and residential bathrooms in order to lift them to the raised position. This is both unsanitary and inconvenient. [0004] Other numerous attempts to produce a commercially viable toilet seat lifting device have encompassed designs that are ungainly or involve mechanisms that require considerable manufacturing cost and complexity and user maintenance. U.S. Pat. No. 4,103,371 to Wilson, U.S. Pat. No. 5,014,367 to Gamblin, U.S. Pat. No. 5,448,782 to Ratajac and U.S. Pat. No. 6,112,335 to Gaston all involve hydraulic and pneumatic cylinders and complicated levers and linkages necessitating manufacturing complexity and undue expense, along with user assembly and maintenance issues associated with hydraulic and pneumatic designs. U.S. Pat. No. 5,404,595 to Carmel involves two levers, a floor-mounted base and numerous linkages, as well as an electrical motor option to lift the seat. This too, is overly complex to manufacture and difficult for the end user to install and maintain. SUMMARY OF THE INVENTION [0005] The present disclosure provides a simple, easy to manufacture, and cost effective solution to the age-old problem of lifting the toilet seat in order to use the toilet as a urinal in a sanitary and convenient manner. The present disclosure will overcome this problem by utilizing a very simple foot-operated mechanism to lift the seat while using the toilet as a urinal. It can be inexpensively mass produced and can be easily fitted to new and existing popular toilet models. [0006] Instead of fumbling for a finger-hold around the bottom of the toilet seat, a foot-operated device is more convenient and keeps a user&#39;s hands away from the toilet bowl. Using a robust two geared shaft design and a single lever, this invention can be installed quickly on new toilets, and can easily replace or supplement conventional toilet seats on popular existing toilet models. [0007] The present disclosure is operated by means of a mechanical assembly near the rear base of the toilet seat, mounted near the rear edge of the toilet bowl, in front of the toilet tank, and consisting of a pair of geared shafts, one attached to the toilet seat and the other attached to an adjustable lever that extends down one side of the toilet bowl, at an angle, toward the floor, terminating in a foot pedal a few inches above the floor itself Downward pressure on the foot pedal causes the rear shaft to rotate toward the front of the toilet bowl, with the gearing then forcing the front shaft to rotate in the opposite direction. This front shaft is connected to the toilet seat at its rear base, causing it to lift up from the toilet bowl as the shaft is rotated. Its maximum travel is almost 90 degrees from the closed position, resulting in unfettered access to the toilet bowl. Releasing pressure on the pedal reverses the process, with gravity causing the seat to lower back into the horizontal position, resting on the toilet bowl. A set of friction bushings on each end of the front shaft can be adjusted to control the amount of resistance necessary for smooth operation in both raising and lowering the seat, and will eliminate the seat from being dropped too quickly into the lowered position. A toilet seat cover will ride on top of the toilet seat and will rise with the toilet seat from the pressure of the toilet seat rising beneath it and lower by force of gravity, resting on top of the toilet seat. The toilet seat cover may also be left in the open (upright) position by rotating it beyond 90 degrees from the toilet bowl (so it rests against the toilet tank) if so desired, with the toilet seat moving up and down independently. [0008] The present disclosure will increase sanitation when using the toilet by eliminating the need to touch the toilet seat to raise it. It would also make it easier for young male children, the elderly, handicapped or those with bad backs to raise the toilet seat and will eliminate the need for men to hold the toilet seat in a raised position while urinating. The present disclosure would also serve to eliminate the common problem of male household members forgetting to put the toilet seat down after use. [0009] The present disclosure overcomes the problems associated with prior technologies by retaining a simple design for both manufacturing and use. This simplicity equates to lower production costs (and thus, lower retail prices) and ease of installation and use by the consumer. The robust structure of the mechanism and the small number of moving parts involved translates into a high level of durability for the end user. The present disclosure is also easily retrofitted to existing toilets, and doesn&#39;t require special tools, electricity, drilling, floor-mounted brackets or floor-mounted pedals to install or use. BRIEF DESCRIPTION OF THE DRAWINGS [0010] [0010]FIG. 1 is a side view of a foot-operated lever mounted on a typical toilet with the toilet seat in a lowered position. [0011] [0011]FIG. 2 is a side view of the foot-operated lever depressed to adjust the toilet seat to a raised position. [0012] [0012]FIG. 3 is a perspective view of a mechanism for raising the toilet seat in response to a depression of the foot-operated lever. [0013] [0013]FIG. 4 is a second perspective view of the mechanism of FIG. 3. [0014] [0014]FIG. 5 is a top view of the mechanism of FIG. 3. [0015] [0015]FIG. 6 is a perspective view of a decorative plastic cover for the mechanism of FIG. 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0016] Referring to FIG. 1, a foot-operated mechanism 10 is shown in a side perspective, mounted on a typical toilet, just in front of the tank, on the rear edge of the toilet bowl. In a lowered position, the toilet seat 11 rests on the edge of the toilet bowl, with the foot pedal 12 in a normal position above the floor. The lower lever 13 to which the foot pedal 12 is attached is slightly smaller in diameter than an upper lever 14, allowing for an adjusting friction screw 15 to be used to adjust the extension length of the lower lever 13, and thus the distance from the foot pedal 12 distance above the floor. A toilet seat cover 16 is mounted on a hinge 17 just to the rear of the toilet seat and on top of a metal flange 18. [0017] [0017]FIG. 2 depicts the foot-operated mechanism 10 shown in a side perspective with the toilet seat 11 in a raised position. Note that as the foot pedal 12 is depressed, the toilet seat 11 and toilet seat cover 16 are lifted into the raised position. [0018] [0018]FIG. 3 depicts a front, right perspective view of the foot-operated mechanism 10 with the toilet seat 11 and toilet seat cover 16 in the lowered position. FIG. 4 depicts a front, left perspective view of the foot operated mechanism 10 with the toilet seat 11 and toilet seat cover 16 in the lowered position. [0019] [0019]FIG. 5 depicts a top perspective view of the foot-operated mechanism 10. The foot-operated mechanism 10 includes a base plate 20 that may be constructed of a metal or other durable material. The base plate 20 includes a left mounting bracket 21 and a right mounting bracket 22 that may also be constructed of a metal or other durable material. The base plate 20 is secured to the toilet between the bowl and the tank with two lock nuts 23 and 24, or other useful fasteners. This positioning of the base plate allows the mechanism 10 to be positioned where it is less likely to be damaged and does not interfere with normal use of the toilet, than for example previous devices that are disposed on the floor or a side of the bowl of the toilet. [0020] A metal rear geared shaft 25 is mounted between the left 21 and right 22 mounting brackets with a steel rear threaded bolt 26 running through the left mounting bracket 21 and a left rear bearing 27. The rear threaded bolt 26 continues through the center of the rear geared shaft 25 and then through a right rear bearing 28 and right mounting bracket 22, threading into the upper lever&#39;s 14 threaded recess. A left rear bearing 27 is recessed in the left mounting bracket 21 and a right rear bearing 28 is recessed into the right mounting bracket 22 for supporting the rear geared shaft 25. The rear geared shaft 25 features a female geared recess on the right side, where a male end of the upper lever 14 fits into it, such that a depression of the foot pedal 12 provides torque to the rear geared shaft 25. [0021] The rear geared shaft 25 meshes with a metal front geared shaft 29, such that a rotation of the rear geared shaft 25 causes rotation of the front geared shaft 29. The front geared shaft 29 is mounted in front of the rear geared shaft 25 and between the left and right mounting brackets 21 and 22 by a steel front threaded bolt 30. The front threaded bolt 30 runs through the left mounting bracket 21 and a left front bearing 31, then through a left friction bushing 32. The front threaded bolt 30 then continues through the front geared shaft 29, through a right friction bushing 33, a right front bearing 34 and then the right mounting bracket 22, terminating in a lock nut 35. The left front bearing 31 is recessed in the left mounting bracket 21 and the right front bearing 34 is recessed in the right mounting bracket 22 for supporting the front geared shaft 29. The left 32 and right 33 friction bushings can be constructed of plastic or polyurethane, or any compressible and durable substance or other known apparatus that will provide a damping effect to or friction against the rotation of the metal front geared shaft 29. In certain embodiments, the friction is adjustable by tightening or loosening the front threaded bolt 30. [0022] The toilet seat 11 is connected to the front geared shaft 29 by the metal flange 18. The metal flange 18 is secured to the front geared shaft 29 in a position that does not interfere with the rear geared shaft 25. The toilet seat 11 is mounted on top of the flange 18, which is welded to a position on the front geared shaft 29. The flange 18 is secured by providing two self-tapping screws 37 and 38, or other useful fasteners or attachment methods, into the bottom of the toilet seat 11. The toilet seat cover&#39;s hinge 17 is secured to the top of the flange 18 with two machined screws 39 and 40, or otherwise fastened or attached thereto. [0023] The described components cooperate together in the following manner to facilitate raising and lowering the toilet seat. As the user applies foot pressure to the foot pedal 12, the lower lever 13 and upper lever 14 cooperatively provide torque to cause the rear geared shaft 25 to rotate in a direction toward the front geared shaft 29. Due to the engagement of the rear geared shaft 25 with the front geared shaft 29, this rotation causes the front geared shaft 29 to rotate in the opposite direction toward the rear geared shaft 25, thus lifting the metal flange 18 and the toilet seat 11 into the raised position, substantially 90 degrees from the toilet bowl. When the user releases foot pressure to the foot pedal 12, gravity causes the toilet seat 11 to move back to its lowered position, dampened by the left friction bushing 32 and the right friction bushing 33 on both sides of the front geared shaft 29. The toilet seat cover 16, rides on the toilet seat as it is lifted into the raised position and lowered back to the lowered position. It may also be left in a raised position by lifting it beyond 90 degrees from the toilet seat 11. With the toilet seat cover 16 in this position, the toilet seat 11 will raise and lower independently of the toilet seat cover 16. [0024] [0024]FIG. 6 depicts a decorative plastic cover 41 that fits over the foot operated mechanism 10 with adequate apertures for the upper lever and toilet seat 11 and toilet seat cover 16 to operate. The cover is constructed to fit snuggly over the left 21 and right 22 mounting brackets, utilizing a pair of plastic clips 42 and 43 built into the underside of the top and aligned with the top centers of the left 21 and right 22 mounting brackets to hold it in place. [0025] The foot operated mechanism 10 may be manufactured in a left side version where the foot pedal 12 is disposed on the left side of the toilet, as opposed to the right side as shown in FIGS. 1 - 5. In addition, a foot pedal 10, lower lever 13 and upper lever 14 may simultaneously be provided on both the left and right sides of a toilet. [0026] The rear geared shaft 25 and front geared shaft 29 may be provided in a 1:1 gear ratio, or in certain embodiments may be provided in a 2:1 ratio, or other useful gear ratio, to lessen the force needed to raise the toilet seat 16 with the foot pedal 12. [0027] Various alternate embodiments are readily contemplated. For example, it is contemplated that bearings 27, 28, 31, 34 and/or screws 38, 39 may be omitted, or that alternate equivalent components may be substituted therefore. Also, a single lever may be provided in place of lower lever 13 and upper lever 14. [0028] In a further alternate embodiment, the rear geared shaft 25 and/or the front geared shaft 29 may not be geared continuously across its length as shown in FIGS. 3 - 5. In such embodiments, one or more individual gears may, for example, be cooperatively disposed along the length of the threaded bolts 26, 30. Such individual gears may have a threaded opening in the center thereof for engaging the threaded bolts 26, 30, thus allowing the gears to be spun along the length of a bolt 26, 30 to a desired position. The flange may then be welded or otherwise secured to the individual gear or gears disposed in place of the front geared shaft 29. [0029] In another alternate embodiment, the rear threaded bolt 26 may be disposed in the opposite direction than as shown in FIGS. 4 and 5, while the front threaded bolt 30 remains in the orientation shown. In this exemplary embodiment, the upper lever 14 may be welded or otherwise secured directly to the head of the rear threaded bolt 26. [0030] The components described herein can be constructed of any material that is both strong and durable. The base plate 20, flange 18, threaded bolts 26, 30, two geared shafts 25, 29, screws 37, 38, nuts 24, 35, bearings 28, 34 and upper lever 14 should be constructed of metal, as these parts support the weight of the toilet seat and will require strength. The lower lever 13 and foot pedal 12 may be constructed of metal or a composite material with high strength. The toilet seat 11 and seat cover 16 may be constructed of plastic or any other lightweight high strength material. The decorative cover 41 may be constructed of plastic or other lightweight material. The present disclosure may be finished in any color or powder coat finish to match any décor. Brushed metal and chromed finishes are also possible. Other minor decorative changes and derivatives that do not affect the operation of the present disclosure are possible are well. [0031] Although the best methodologies of the invention have been particularly described in the foregoing disclosure, it is to be understood that such descriptions have been provided for purposes of illustration only, and that other variations both in form and in detail can be made thereupon by those skilled in the art without departing from the spirit and scope of the present invention, which is defined first and foremost by the appended claims.

Summary: A mechanism for raising a toilet seat includes two geared shafts that are cooperatively engaged while mounted on a toilet. A foot-operated lever attached to a first of the geared shafts provides torque thereto, causing a rotation of the first geared shaft upon depressing the lever. This, in turn, causes a rotation of the second of the geared shafts. The second geared shaft includes a metal flange or other connector that secures a toilet seat thereto over the bowl of the toilet. As the two geared shafts rotate, the toilet seat raises and lowers with the movement of the metal flange on the second geared shaft.

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Summarize: Jonny Wilkinson says Owen Farrell is the man into take England into next year’s World Cup in the pivotal fly-half position. Saracens No10 Farrell, sidelined with a leg injury but expected to be fit ‘within a fortnight’ according to club coach Mark McCall, faces stiff competition for his international place from Bath’s George Ford, Sale’s Danny Cipriani and Leicester’s Freddie Burns. But World Cup winner Wilkinson, who retired last season, insists that with 25 caps and 271 points already to his name, 23-year-old Farrell is the man for the job. Owen Farrell is the man to play fly-half for England at the World Cup, says Jonny Wilkinson. Saracens playmaker Farrell is due to return from injury within a fortnight. England Rugby World Cup 2015 ambassador Jonny Wilkinson poses with future stars in Newcastle. ‘Owen Farrell has done his bit, he’s served his time and done his work; he’s shown that he’s the guy in charge of the shirt,’ said Wilkinson. ‘You can’t say he’s really put a foot wrong. He’s a young guy who’s taken on huge amounts of responsibility and is happy to put his neck on the line in a sport that can be fairly ruthless. 'A young guy who’s willing to do that and has already shown he’s very capable of doing it is a bit of Godsend — you don’t want to throw that away, believe me.’ Cipriani, who faces former club Wasps in the Aviva Premiership on Sunday, has been in outstanding form this season after forcing his way back into the England reckoning on their summer tour to New Zealand. ‘Danny Cipriani is a great talent,’ said Wilkinson. ‘Everybody knows he’s got the ability and mentally he’s probably as strong as he’s ever been.’ Wilkinson was speaking at a World Cup event at his former club Newcastle, who host Exeter in the Aviva Premiership on Sunday. Danny Cipriani has been in terrific form for Sale, but Wilkinson still thinks Farrell has proven himself. Freddie Burns (left) has struggled to shine with out-of-form Leicester in the Aviva Premiership but George Ford (right) has been firing for Bath and is the other lead contender for the England fly-half role

Summary: Jonny Wilkinson says Owen Farrell should be England's fly-half. The Saracens playmaker will return from injury within a fortnight. Bath's George Ford, Sale's Danny Cipriani and Leicester's Freddie Burns all covet incumbent Farrell's position. Wilkinson said Farrell is in charge of the shirt after proving himself. He said Cipriani is 'a great talent' and that he's mentally stronger than ever.

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Summarize: Italian Runner Wins Venice Marathon After All The Favorites Take A Wrong Turn Updated at 4 p.m. ET It's fair to say things were not supposed to go this way at the Venice Marathon. It would've been little surprise if Kenya's Gilbert Kipleting Chumba had won the race Sunday — or maybe it could have been his countryman David Kiprono Metto. And, in fact, both of those favorites were among the leaders roughly 16 miles into the marathon. Then, they took a wrong turn. A cluster of motorcycles and cars that had been in front of the runners left the planned route — as they were supposed to, Enrico Jacomini says. A longtime president of the Venice Marathon Club and co-founder of the race, Jacomini tells NPR that "Venice is not a city for cars or motorcycles," and for that reason, the vehicles have always separated from the runners before the marathon's final leg. But the small group of runners leading the race followed them anyway, straying more than 100 hundred meters off course and apparently onto a main thoroughfare. A video captured the crucial, crushing moment the runners learned of the error and turned back. euronews via YouTube Delayed by about 2 minutes, according to The Associated Press, the favorites ultimately lost out to Eyob Ghebrehiwet Faniel, a local who was reportedly running in only his second marathon. Faniel, who has been referred to by both surnames, has run for the Venice Marathon Club for several years. And the Italian made his official marathon debut in Florence last year, as noted by the International Association of Athletics Federations. Now, he is the first Italian man to win the Venice Marathon in 22 years. He won the race in 2:12:16 — which is better than all but two times set by Americans this year, according to Sports Illustrated. "He was doing well," Jacomini says, "and this was just a lucky circumstance." "Today's race shows that the work is paying off," Faniel said after the marathon, according to the IAAF. "It was not an easy race as I had to run alone on the Ponte della Libertà. I dedicate the win to myself as I have always believed in my work despite all the difficulties." "I've been following athletics for 55 years and I've been a part of the international federations, I've been manager of many organizations," Jacomini adds. "And I've never seen anything like this." Eyob Gebrehiwet improved his PB by three minutes to win the 32nd edition of the Huawei Venice Marathon in 2:12:16 on Sunday (22), becoming the first Italian man since 1995 to win the IAAF Bronze Label road race. In what was just his second marathon to date, following a 2:15:39 debut in Florence last year, Gebrehiwet’s winning mark is the second-fastest time by an Italian man this year behind Daniele Meucci’s 2:10:56 at the World Championships in London. But the race was not without controversy as Gebrehiwet capitalised on a moment just after the half-way mark when the lead pack of seven men followed the lead motorcycle who had taken a wrong turn. Having built a lead of almost one minute at half way, reached in 1:05:30, the seven leaders – Bernard Bett Kiplangat, Moses Kemei Kipngetich, Robert Kiplimo, Abdulah Shami, Mutai Kipkemei, Gilbert Chumba and David Metto Kiprono – unintentionally ran off course. They later rejoined the race, but at 25 kilometres they trailed the then lead pack – which had earlier been the chase pack – by a minute. Gebrehiwet, Mogos Shumay, Muhammed Mussa and Rodgers Maiyo reached 25 kilometres in 1:18:32. Maiyo then dropped out and Mussa began to struggle, leading Shumay and Gebrehiwet alone in front. Gebrehiwet then pushed the pace after passing the famous Ponte della Libertà, reaching the 35-kilometre mark in 1:49.23. By 40 kilometres, reached in 2:05:13, his lead over Mussa had grown to more than two minutes. The Italian runner of Eritrean origin, who competes for the local Venice Marathon Club, crossed the finish line in Riva dei sette Martiri in 2:12:16 to take the first win of his career after missing this race in 2015 and 2016 due to injury problems. Mussa finshed runner-up in 2:15:14 ahead of Tariq Bamaarouf, who held off Chumba in the final stages of the race. Gebrehiwet became Italian citizen in October 2015 and was introduced to athletics by Marco Maddalon. He was previously coached by Giancarlo Chittolini (the trainer of 3000m steeplechase Alessandro Lambruschini). He is now trained by Ruggero Pertile, who has recently a coaching career after hanging up his running shoes last year. “On the eve of the race I felt pressure but I managed to stay calm as I knew that I had worked well,” said Gebrehiwet, who is coached by Ruggero Pertile, the fourth-place finisher in the marathon at the 2015 IAAF World Championships. “Ruggero is not only my coach, but also a good friend. He dedicated a lot of his time to train me. Today he gave me a lot of support along the course. “Today’s race shows that the work is paying off,” added Gebrehiwet, whose next big goal is the 2018 European Championships. “It was not an easy race as I had to run alone on the Ponte della Libertà. I dedicate the win to myself as I have always believed in my work despite all the difficulties.” Pertile, who retired from international racing last year, was delighted with Gebrehiwet’s run. “Eyob is a determined athlete,” said Pertile. “We have set a long-term goal building towards the 2020 Olympic Games in Tokyo. I knew that he felt well. He could have run faster but I am very happy with his race.” Utara the clear winner Misfortune also struck in the women’s race as defending champion Priscah Jepleting Cherono suffered from stomach cramps during the race and was unable to mount a challenge to Sule Utura of Ethiopia. Utura, the 2008 world U20 5000m champion, was joined by Cherono in the first half. They passed through 10 kilometres in 35:02 and 15 kilometres in 52:35, building up a gap of 16 seconds over Aynalem Woldemichael. The lead duo reached the halfway mark in 1:14:10 and increased their gap to more than five minutes at 25 kilometres. Utura broke away soon after, though, and had a 53-second lead over Cherono by 30 kilometres. The Ethiopian continued increasing her lead and eventually went on to win in 2:29:04, smashing her PB by five minutes. Despite fading dramatically in the final kilometres, Cherono held on to second place and crossed the line in 2:41:08. Diego Sampaolo for the IAAF Leading results Men 1 Eyob Faniel Gebrehiwet (ITA) 2:12:16 2 Mohammed Mussa (ERI) 2:15:14 3 Tarik Bamaarouf (MAR) 2:16:41 Women 1 Gedo Sule Utura (ETH) 2:29:04 2 Priscah Cherono Jepleting (KEN) 2:41:08 3 Aynalem Woldemichael (ETH) 2:42:12

Summary: Sunday's Venice Marathon was just the second Eyob Ghebrehiwet Faniel ever ran-and yet the Eritrean-born Italian runner won the race, thanks to a wrong turn. A guide motorcycle led the pack of seven runners who were in the lead several hundred meters off course before they realized the mistake and turned back, costing them about two minutes. The incident, which happened about halfway through the race per the International Association of Athletics Federations, ultimately helped to make Faniel the first Italian man to win the marathon in 22 years, NPR reports.

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Summarize: BACKGROUND OF THE INVENTION This invention is directed to an amusement toy capable of producing an output wherein the output is of an entertaining nature for a small child. Most specifically, the invention is directed to a toy which includes a first member capable of producing a rotational output and a second member associated with the first member wherein in response to linear movement of the second member, the first member is rotated. A variety of amusement toys are known. Included are tops and other spinning type devices. These date back to antiquity. The simplest of these are nothing more than a carved wooded device which can be rapidly spun by winding a string around the device and pulling the string from the device. The toy tops described in the preceeding paragraph, of course, require certain manual dexterity to operate. This precludes the use of these type of devices by small children. A device more suitable for use by a small child includes the common pump type rotating top toy. This toy utilizes a central shaft which the child pumps up and down in order to impart a rotational momentum to a flywheel portion of the toy. These types of toys have been equipped with music boxes and the like, so as to emit a noise in response to spinning of the rotational member of the toy. The toys described in the preceeding paragraph have been quite popular for several decades because of the play value inherent therein. They, however, require use upon a hard surface because the housing of the toy itself spins. If these types of toys were utilized on a soft surface, such as a rug or the like, the momentum of the toy is easily dissapated because of friction between the housing and the softer surface. BRIEF DESCRIPTION OF THE INVENTION In view of the above, it is a broad object of this invention to provide a top-type of toy wherein the outer housing itself is not rotated, but includes further elements formed as a portion of the toy which are rotated so as to produce an enjoyable and amusing output for the user of the device. It is a further object of this invention to provide a toy which is easily utilized by small children without adult supervision, thus allowing the small child to enjoy the toy by himself. It is a further object of this invention to provide a toy which, because of the engineering principles inherent therein, is capable of a long and useful lifetime by a small child, yet is also capable of being manufactured at an economical price for the consumer. These and other objects, as will become evident from the remainder of the specification, are achieved in a toy which comprises a housing; first and second members located on said housing; said first member shaped as a surface of rotation having a hollow interior and open ends opening into said hollow interior, said first member rotatably mounted on said housing; said second member being elongated in shape and located on said housing so as at least a portion of said second member is located within the hollow interior of said first member with at least a portion of said second member extending out of one of said ends of said first member, said second member linearly movable in and out of one of said ends of said first member; first gear means located on said first member; second gear means located on said second member; connecting gear means located on said housing in operative association with both said first and said second gear means so as to transfer linear motion of said second member into rotational motion of said first member; output means independent of said first member, said output means movably located on said housing in operative association with said first member whereby rotational motion of said first member moves said output means on said housing. In the illustrative embodiment of the invention, the connecting gear means includes a clutch means whereby motion of the second member is only transferred to the first member when the second member moves in a first direction, not in a second direction. Further, in the illustrative embodiment, a biasing means is associated with the second member whereby when the second member is pushed from an initial position to a subsequent position, the biasing means is energized such that it then pushes the member from the subsequent position back to the initial position. Further, in the illustrative embodiment, the housing includes an upstanding element shaped as a surface of rotation mimicking the shape of the first member whereby the first member fits around the upstanding element and it is rotated thereon in response to movement of the second member. The connecting gear means is journalled in the upstanding member, so as to hold it in position to engage both the first member and the second member. BRIEF DESCRIPTION OF THE DRAWINGS This invention will be better understood when taken in conjunction with the drawings wherein: FIG. 1 is an isometric view of an embodiment of the invention; FIG. 2 is a side elevational view in section of the embodiment shown in FIG. 1 with certain of the components in a first spacial configuration; FIG. 3 is a side elevational view similar to FIG. 2 except that certain of the components are shown in a second spacial configuration; FIG. 4 is an exploded isometric view of certain of the components generally located in the central portions of FIGS. 2 and 3; FIG. 5 is a plan view in partial section about the line 5--5 of FIG. 2; FIG. 6 is a sectional view about the line 6--6 of FIG. 3; and FIG. 7 is a sectional view about the line 7--7 of FIG. 3. This invention utilizes certain principles and/or concepts as are set forth in the claims appended hereto. Those skilled in the toy arts will realize that these principles and/or concepts are capable of being expressed in a variety of embodiments which may differ from the exact embodiment utilized for illustrative purposes. For this reason this invention is not to be construed as being limited solely to the illustrative embodiment, but should only be construed in view of the claims. DETAILED DESCRIPTION OF THE INVENTION FIG. 1 shows an amusement toy 10 which is primarily designed for use by very small children. It requires a minimum of manipulative skills in order to &#34;play&#34; with the toy. As such, it is suitable for toddler age children. However, it also has a certain asthetic appeal and, as such, is further suitable as a collector type item for older children and even adults. To activate the amusement toy 10 one only needs to depress downwardly on the large button 12 which is located on the top of the toy. This in turn transfers downward movement to a shaft 14 which is attached to the bottom of the button 12. Pushins on the button 12 pushes the shaft 14 into the interior of the housing 16. The housing 16 and other componenets located thereon are viewable through a transparent cover 18 which in turn attaches to a base 20. Located on the base 20 so as to rotate on the base 20, is a small rotating flower-shaped disk 22. Further, a bell 24 is located on the base 20 in association with a bell clanger 26. Formed as a part of the interior housing 16 is a first rotating element 28 which includes a plurality of second rotating elements collectively identified by the numeral 30. When the button 12 is depressed, this activates certain mechanisms within the interior of the toy as hereinafter described. Upon release of the button 12, the shaft 14 and the button 12 slowly move upwardly and, in doing so, activate mechanisms within the interior of the toy 10 causing rotation of the first rotating element 28 and the second rotating elements 30 attached thereto, rotation of the flower-shaped disk 22 and movement of the clanger 26 against the bell 24 so as to produce an audio output from the bell 24. The button 12 and the shaft 14 can be moved into the interior of the housing 16 very fast. However, movement of the shaft 14 upwardly out of the housing 16 is much slower, such that the outputs of the toy 10, i.e. the rotation of the components 22, 26, and 28, and the noise emitted from the bell 24, are evident over an extended time period. As is evident from FIGS. 2 and 3, the base 20 is hollow. It is composed of lower base member 20a and upper base member 20b which fit together. The upper base member 20b includes a surface 32 located thereon. A bell support member 34 extends upwardly from the lower base member 20a through an opening not separately identified or numbered in the surface 32. The bell 22 is attached via a screw 36 to the bell support member 34. Extending upwardly centrally from the surface 32 is the housing 16. On the upper periphery of the housing 16 is a collar 38 whose function will be described below. Directly above the collar 38 is the first rotating element 28. The first rotating element 28 includes a central top opening through which the shaft 14 passes. Referring briefly to FIG. 5, located within the housing 16 is a further interior housing 40. The interior housing 40 is shaped as a cylindrical surface of rotation and includes a hollow interior. The interior housing 40 is fixed to the lower base member 20a. The interior housing 40 is hollow and is open at its upper end. Located within the hollow interior of interior housing 40 is a guide 42. Guide 42 is square in shape in cross section as seen in FIG. 5, and contains a compression spring 44 within its bottom most portion. The shaft 14 is formed as a upper portion of a first moving member 46. The lower portion of the first moving member 46, as is best seen in FIG. 4, is formed as an open sided elongated parallelopiped. Within the interior of this portion of the first moving member 46 is a gear rack 48. The gear rack is on one of the solid faces, with the open faces exposing both the left and right hand side of the gear rack. The square shape in cross section of the bottom portion of the first moving member 46 allows it to fit within the guide 42 with the square shape of the guide 42 limiting the movement of the first moving member 46 linearly upperwardly and downwardly within it. A shaft 50 is journalled across the top of the interior housing 40 directly above the guide 42. Fixedly located onto the shaft 50 is a spur gear 52. The spur gear 52 includes a reentrant gear 54 formed as an intrical part thereof. A further reentrant gear 56 mates with reentrant gear 54. Formed as a portion of reentrant gear 56 is a pinion 58. The pinion 58 and reentrant gear 56 are free to rotate on the shaft 50 but are biased by a compression spring 60 which is positioned between a bushing 62 and the pinion 58. The compression spring 60, pushing against the pinion 58, engages reentrant gear 56 with the reentrant gear 54. The engagement of the reentrant gears 54 and 56 with one another, serve as a clutch. The shape of these reentrant gears is such that they include helical extending surfaces in one direction and a locking tooth in the other direction. As viewed in FIG. 4, if the pinion 58 is rotated counterclockwise, this rotation will be tranferred to the spur gear 52 so as to rotate it counterclockwise. However, if the pinion 58 is rotated counterclockwise, and for any reason the spur gear 52 is inhibited from easily rotating, the reentrant gear 56 will slip across the helical surface of the reentrant gear 54 such that rotation of the pinion 58 in a clockwise direction is not transferred to the spur gear 52. The pinion 58 is in mesh with the gear rack 48. Upon depression of the button 12, downward movement of the first moving member 46 within the guide 42 causes clockwise rotation of the pinion 58 such that the reentrant gear 56 slips against the surface of the reentrant gear 54, and the pinion 52 is not rotated. This, however, does compress the compression spring 44. When the button 12 is released, the bias induced in the compression spring 44 pushes the first moving member 46 upwardly, thus causing the gear rack 48 to rotate the pinion 58 counterclockwise. This rotation is transferred via the reentrant gears 54 and 56 to the spur gear 52 so as to rotate the spur gear 52 counterclockwise as seen in FIG. 4. A second moving member 64 includes a cylindrical surface of rotation which fits around the interior housing 40. This allows the second moving member 64 to rotate about the interior housing 40. An interior radially extending flange 66 has a set of crown teeth 68 located on its under side. These are positioned so as to engage with the spur gear 52. Rotation of the spur gear 52 is therefore transferred to the crown teeth 68 rotating the second moving member 64. Thus, the linear movement of the first moving member 46 is transferred via the pinion 58 and spur gear 52 to the second moving member 64 to rotate it. The second moving member 64 includes a cutout area 70 which extends around 180° of the top edge of the second moving member 64. This engages with interior webs 72 formed within the interior of the first rotating element 28. Rotation of the second moving member 64 is thus directly transferred to the first rotating element 28. A large spur gear 74 is formed on the bottom of the second moving member 64. Formed directly above the spur gear 74 is a set of ratchet teeth 76. Since each of these is intrically formed with the second moving member 64, they, of course, rotate in conjunction with rotation of the second moving member 64. A shaft 78 is journalled between the upper and lower base members 20a &amp; 20b so as to freely rotate. Fixedly located on the shaft 78 is a small pinion 80, and intrically formed with the pinion 80 is a large spur gear 82. The pinion 80 meshes with the spur gear 74 formed on the bottom of the second moving member 64. Located next to the outer periphery of the spur gear 82 is a further shaft 84 which is also journalled between the upper and lower base members 20a &amp; 20b. A pinion 86 and a spur gear 88, intrically formed with pinion 86, are located on the shaft 84. The pinion 84 meshes with the spur gear 82 so as to ultimately be rotated in response to rotation of the second moving member 64. The disk 22 is fixed to a shaft 90 which is journalled in the upper base 20b. The shaft 90 passes through the surface 32 and includes a pinion 92 fixed to the shaft 90 and located below the surface 32. Both the pinion 92 and the disk 22 are fixed to the shaft 90 such that rotation of the pinion 92 is transferred to the disk 22. The pinion 92 meshes with the spur gear 88. Because of this, ultimately the disk 22 is rotated in response to rotation of the second moving member 64. The clanger 26 is fixed to a shaft 94. As seen in FIG. 5, the shaft 94 passes through the surface 32 on the upper base 20b. Located on the shaft 94 below the surface 32 is a lever 96. The lever 96 is fixed to the shaft 94 such that movement of the lever 96 is transferred via the shaft 94 to the clanger 26. Attached to one end of the lever 96 is a spring 98. The spring 98 stretches between the lever 96 and a small peg 100 which extends downwardly from the bottom of the surface 32. The spring 98 has a small amount of tension incorporated therein such that the other end of the lever 96 is biased towards and meshes with the ratchet teeth 76. As the second moving member 64 rotates, each of the ratchet teeth 76 in turn contact the lever 96 to rotate the lever 96. In doing so, this stretches the spring 98 and pulls the clanger 26 away from the bell 24. As seen in FIG. 5, when the lever 98 has cleared the particular ratchet tooth which it was engaged with, the tension within the spring 98 rotates the lever 96 clockwise to bring the end of the clanger 26 against the bell 24 to ring the bell 24. As noted above, located on top of the housing 16 is a collar 38. The collar 38 includes a plurality of small webs collectively identified by the numeral 102 which extends radially from it. Each of the second rotating elements 30 also include a small web 104 which projects downwardly. The webs 104 are positioned whereby they are engaged by the webs 102 as the first rotating element 28 rotates on top of the housing 16. Each time one of the webs 104 on one of the second rotating elements 30 contacts one of the webs 102, the second rotating element 30 is rotated on the first rotating element 28. As soon as the web 104 passes over the particular web 102, the second rotating element 30 rotates in the opposite direction. Because of this, as the first rotating element 28 rotates on the housing 16, the second rotating element 30 rotates on the first rotating element 28. In FIG. 2, the button 12 is being depressed and the reentrant gear 56 is slipping on the reentrant gear 54. Depressing of the button 12 moves the first moving member 46 downwardly to compress the spring 44. When the button 12 is released, the tension created in the spring 44 moves the first moving member 46 upwardly. However, at this time the reentrant gear 56 engages reentrant gear 54 so as to transfering the upward linear motion of the first moving member 46 to rotary motion of the second moving member 64 via the gears described previously. Since the second moving member 64 includes the large spur gear 74 on its periphery which engages a small pinion 80, and since the pinion 80 is intrically formed with large spur gear 82 which engages a small pinion 86, and further since the pinion 86 is intrically formed with large spur gear 88 which engages a small pinion 92, a small increment of rotation of the second moving member 64 is compounded to a large increment movement of the disk 22. In compounding the rotation from the second moving member 64 to the disk 22, the gear train between the disk 22 and the second moving member 64 serves as a speed govenor for the speed of rotation of the second moving member 64. In view of this, the bias created within the spring 44 is slowly released as the second moving member 64 slowly rotates. The disk 22, however, is rapidly rotating. The clanger 26, since it is driven by the ratchet teeth 76 which are on the second moving member, and the first and second rotating elements 28 and 30, since they also are in direct contact with the rotating member 64, move at a rate equal to the rate of rotation of the second moving member 64.

Summary: An amusement toy includes a first member which is cylindrical in shape and is located within a housing. This first member is capable of rotating on the housing. A second elongated member is located within the center of the cylindrical shaped first member and moves up and down within the first member. A first gear located on the first member engages a connecting gear which in turn engages a gear located on the second member so as to transfer linear motion of the second member into rotational motion of the first member. Rotation of the first member transferred to at least one output member whereby the output member is rotated ultimately in response to linear motion of the second member.

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Write a title and summarize: Des prostituées au bois de Boulogne, en mars 2012. AFP/THOMAS SAMSON Lire (édition abonnés) : "Vers la création d'un délit de recours à la prostitution" Benoît : Si l'on met à part les cas bien évidemment condamnables de prostitution forcée, en quoi un rapport tarifé entre deux personnes majeures et consentantes est-il une menace pour la société? Maud Olivier : On n'est pas du tout sur ce registre. Nous sommes sur le registre du respect de la personne humaine, du respect des droits humains, et pas du tout sur une question de menace. Ce respect porte évidemment sur le fait de lutter contre tout ce qui est contrainte. Vous me parlez de choix, là, pas de souci pour la société. Nous ne sommes pas liberticides. Mon rapport ne prône pas de supprimer des libertés à qui que ce soit, mais il ne faut pas accepter dans notre société que quelqu'un puisse contraindre, pour des raisons économiques ou financières, à lui accorder des services sexuels. Sur le plan général de la prostitution, il faut recadrer sa réalité en France. 90 % des personnes prostituées en France sont des victimes de la traite et des réseaux. Tortue : Ce sont les pays les plus développés et les plus respectueux des droits de l'homme (Allemagne, Australie, Nouvelle-Zélande, etc.) qui ont les lois les plus libérales en matière de prostitution, avec par exemple la légalité des maisons closes, etc. A l'inverse, de très nombreux pays, de l'Arabie saoudite au Zimbabwe, interdisent purement et simplement la prostitution. N'est-ce pas là un signe qu'une approche libérale de ce problème est préférable à une approche liberticide? Ce n'est pas notre propos d'être liberticide. Le rapport recommande 40 propositions, à aucun moment vous ne verrez une atteinte à la liberté. Se prostituer pour des raisons économiques, sous la contrainte, sous la violence, est-ce une liberté de choix? Sur la liberté, puisque le mot revient, qu'est-ce que c'est que le respect de la liberté? Ce n'est pas d'instrumentaliser le sexe. Le droit et le choix du plaisir, ce n'est pas de faire que le sexe soit instrumentalisé. Ce n'est pas le sexe, le plaisir ou la liberté qui font problème dans la prostitution, c'est l'argent, la violence, l'oppression des femmes et le trafic d'êtres humains. Sur les pays dits libéraux, vous savez que l'Allemagne, que vous citez, est considérée comme un pays réglementariste, qui organise la prostitution dans des maisons closes, des Eros centers. Elle a signé la main sur le cœur la disposition européenne de lutte contre la traite des êtres humains en vue d'exploitation sexuelle. Mais en revanche, une fois que les personnes victimes de cette traite sont sur son territoire, cela ne la concerne plus, alors que quand on s'engage à appliquer cette disposition européenne, il faudrait faire en sorte que les victimes puissent être protégées, et non pas encouragées à rapporter de l'argent au système de prostitution allemand, par exemple. Et il faut savoir aussi, sur le côté libéral de l'Allemagne, que seulement 4 % des prostituées dans ce pays travaillent dans un cadre légal. Pascale : Pensez-vous sérieusement que parce qu'un client risque une sanction, il va renoncer à une passe? Le but n'est pas de sanctionner, ce n'est pas notre démarche. L'idée est de faire prendre conscience que participer à l'exploitation sexuelle des personnes prostituées, c'est quelque chose qui ne respecte pas nos droits fondamentaux, les droits de l'homme, les droits humains en général. Et donc qu'il participe à l'exploitation sexuelle de ces personnes. Ensuite, oui, je crois que le fait que ce soit posé comme un interdit dans la loi peut lui faire comprendre cela, et surtout, lui faire prendre conscience que, comme dans toutes les lois, quand on transgresse un interdit, il peut y avoir sanction à la clé. Simon : Votre mesure semble efficace pour éradiquer la prostitution visible, celle des rues, mais totalement inefficace pour la prostitution en intérieur, celle des sites Internet et des escorts. Prévoyez-vous des mesures complémentaires pour pénaliser la prostitution "cachée"? De toute façon, la prostitution cachée est déjà largement développée. De plus en plus, les rendez-vous se prennent par Internet au travers des réseaux, par des moyens de communication et de publicité. Nous devons évidemment amplifier notre lutte contre ces phénomènes. Nous prévoyons dans une des recommandations de pouvoir interdire aux fournisseurs d'accès de faire de la publicité sur leurs sites pour des sites qui appellent à des activités de prostitution organisées par des réseaux de traite. Donc c'est une proposition qui découle d'une de nos auditions, de ce qu'on appelle les cybergendarmes, qui travaillent déjà sur cette possibilité en ce qui concerne par exemple les jeux d'argent ou la pédopornographie. Nous pourrons, par le biais de ces publicités, numéros de téléphone, faire en sorte que la prostitution ne puisse plus s'exercer. Mais aujourd'hui, la prostitution dite de rue et la prostitution cachée se passent souvent dans des lieux clos, dans une chambre d'hôtel, dans un appartement. On va lutter contre ce type de prostitution avec la même efficacité. Fauchmanne : Comment résoudre le problème de la misère sexuelle de beaucoup d'hommes, sans la prostitution? 50 % des clients de la prostitution sont en couple, donc ne parlons pas de misère sexuelle. Et il faut se retirer de l'esprit cette idée reçue qu'il y a des besoins particuliers chez les hommes. Il n'y a pas d'instinct sexuel, c'est un apprentissage global, de la société, qui fait que les hommes, comme les femmes, ont un besoin sexuel. Ce sont des besoins qui sont créés de toutes pièces, qui n'ont pas d'existence réelle. Les pulsions sexuelles peuvent être contrôlées, c'est une question d'éducation, d'apprentissage de la relation entre filles et garçons. On doit apprendre à réguler, à organiser son envie de relations sexuelles en fonction, déjà, des préférences de sa partenaire, et aussi des contraintes de la vie sociale. Florent : Une régularisation de toutes les personnes prostituées sans papiers est-elle prévue? Car comment sortir de la prostitution sans avoir aucun droit sur le sol national? C'est la question clé. Cela fait partie des 25 recommandations que nous faisons dans le rapport. Sur ces recommandations, il y a l'obtention d'une possibilité de rester sur le sol, mais il n'est pas question de donner à toutes les personnes en situation irrégulière sur notre sol la possibilité de rester. Ce n'est pas l'objet. Nous allons créer une commission départementale comprenant l'autorité administrative, la police, des élus, les associations, commission qui pourra apprécier les besoins de la personne qui souhaite se sortir de la prostitution en termes d'autorisations, de financement, etc. Et qui pourra être présent pour l'accompagner jusqu'à sa sortie de la prostitution. Tofu : Pour aller au bout de votre raisonnement (que je ne partage pas), ne faudrait-il pas interdire aussi les films pornographiques, qui sont une autre forme de relation sexuelle tarifée et en définitive une exploitation sexuelle de la précarité des acteurs X? Je pense que cela relève pour l'instant de l'éducation. Je crois qu'effectivement, d'après une enquête réalisée par le mouvement du Nid, les jeunes gens qui regardent beaucoup de films pornographiques sont ceux qui envisagent de recourir à la prostitution. Donc il y a un lien entre prostitution et films pornographiques. Ce rapport va déjà assez loin, je pense, pour qu'on arrive à faire en sorte de diminuer la demande. Il est fondamental pour moi d'éduquer – et c'est prévu dans notre rapport – à la sexualité les filles et les garçons dans le respect du corps de l'autre, dans l'idée du plaisir partagé, de construire une relation basée sur la liberté de l'autre. Florent : Un problème central dans le fait de faire sortir les personnes prostituées de leur situation est l'accompagnement social, la réinsertion dans le monde du travail. Quel moyens nouveaux seront effectivement alloués à cet objectif? Les personnes qui seront accompagnées dans le cadre de cette commission pourront se voir proposer une formation qui soit professionnalisante, bien sûr, et un titre de séjour. La situation sanitaire de ces personnes est très grave, nous avons auditionné l'Inspection générale des affaires sociales sur la santé de ces personnes, il y a aussi beaucoup à faire en termes de santé. John : Avez-vous conscience que votre discours va à l'encontre des positions défendues par les associations de prostituées, lors par exemple d'événements comme les assises de la prostitution? Ces personnes réclament un vrai droit du travail et la reconnaissance d'une profession. Je me souviens, il y a vingt ans, d'Ulla, qui était la première à Lyon à avoir mené une marche pour demander la régularisation de ce "travail". Aujourd'hui, Ulla nous dit : "Comment avez-vous pu me croire à l'époque? Comment avez-vous pu croire que je voulais faire de cette activité toute ma vie, que c'était quelque chose dans lequel je me réalisais pleinement?" J'ai auditionné deux personnes qui chacune ont écrit un livre qui sont sorties de la prostitution, qui racontent comment elles y sont tombées, et ce qu'elles ont vécu. Et je peux vous dire que considérer que c'est un travail, on ne peut pas être d'accord avec cette façon de voir les choses. Et cette notion de légalisation, d'organisation, de réglementation de la prostitution augmente considérablement la présence des réseaux dans des pays comme l'Allemagne ou les Pays-Bas, où la traite à de très beaux jours devant elle. Nous estimons qu'en France il y a entre 20 000 et 40 000 personnes qui se prostituent. En Allemagne, maintenant, avec ces lois, on estime ce nombre à 300 000 personnes. Cela veut tout dire. Antoine : Donc si je comprends bien votre référence anecdotique à Ulla, vous considérez qu'en réclamant des droits, les travailleuses du sexe se mentent à elles-mêmes, qu'elles ne sont pas lucides sur leur situation? Je ne me permettrai pas de les juger ou de leur faire des procès d'intention. Je considère que quand on n'a pas la liberté de son corps, quand on subit une contrainte, qu'elle soit économique ou de tout autre nature, notre pays des droits de l'homme, du respect, doit agir pour faire en sorte que ces personnes vivent dignement. Mais encore une fois, ce sera leur choix, nous ne sommes pas prohibitionnistes. Gérard : Quels moyens aura la police pour faire appliquer vos propositions? Les personnes prostituées pour se faire connaître doivent communiquer sur un numéro de téléphone, une adresse. Donc la police, qui n'aura plus à poursuivre les personnes prostituées pour délit de racolage, pourra bien évidemment reporter ses forces sur la lutte contre la prostitution. La personne ayant communiqué un numéro de téléphone, il sera facile à la police de remonter vers elle pour faire son enquête, comme cela se fait depuis quatorze ans en Suède. Quel est, à votre avis, l'avenir de votre proposition? Ce texte a-t-il une chance d'être voté? Si non, d'où pourraient venir les oppositions? Il s'agit d'un rapport avec 40 recommandations. Mais évidemment ce rapport a été construit pendant une année pour arriver à une proposition de loi. Nous avons travaillé pour arriver à ce que cette loi passe si possible aux alentours du 25 novembre, Journée internationale de lutte contre les violences faites aux femmes. Cette loi est donc programmée dans l'agenda pour être présentée dans l'Hémicycle à cette date-là. J'ajoute que cette proposition traverse tous les partis politiques qui sont à l'Assemblée nationale et au Sénat. Je ne suis pas naïve sur le fait qu'il va falloir convaincre, parce que forcément, il y aura à faire de la pédagogie, mais j'ai bon espoir dans l'intelligence de mes collègues parlementaires.

Title: "Prostitution:" "Quand on transgresse un interdit, il peut y avoir sanction" " Summary: Dans un chat sur Le Monde.fr, Maud Olivier, députée PS de l'Essonne et rapporteuse du texte proposant de pénaliser le recours à la prostitution tout en abrogeant le délit de racolage public à l'encontre des prostituées, estime que ses quarante propositions ne sont ni "liberticides" ni "prohibitionnistes".

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Summarize: Introduction and Background Leading up to the passage of the Homeland Security Act of 2002 ( P.L. 107-296 ), a debate ensued regarding the exemption of critical infrastructure information from the Freedom of Information Act, 5 U.S.C. § 552. Both the House and Senate versions of the Homeland Security Act ( H.R. 5005 and S. 2452, respectively) contained language exempting such information, but the two versions were significantly different. Final passage of the Act included the House language (sections 211 - 215 of P.L. 107-296 ). This report discusses the differences in language and some of the arguments and concerns expressed by both supporters and critics of the exemption. Certain socio-economic activities are vital to the day-to-day functioning and security of the country; for example, transportation of goods and people, communications, banking and finance, and the supply of electricity and water. These activities and services have been referred to as components of the nation's critical infrastructure. Domestic security and our ability to monitor, deter, and respond to outside hostile acts also depend on some of these activities as well as other more specialized activities like intelligence gathering, law enforcement, and military forces. Serious disruption in these activities and capabilities could have a major impact on the country's well-being. In July 1996, President Clinton established the President's Commission on Critical Infrastructure Protection (PCCIP). The Commission was tasked with assessing the vulnerabilities of the country's critical infrastructures and proposing a strategy for protecting them. In its final 1997 report, the Commission stated that the "... two-way sharing [of] information is indispensable to infrastructure assurance," and that "increasing the sharing of strategic information within each infrastructure, across different sectors, and between sectors and the government will greatly assist efforts of owners and operators to identify their vulnerabilities and acquire tools needed for protection." According to the Commission, the exchange of information is also necessary to develop an analytic capability to examine information about incidents, vulnerabilities, and other intelligence information to determine whether events are related and can be used possibly to recognize or predict an attack. The Commission also noted that there is a reluctance on the part of the private sector and the government to share information related to vulnerabilities or incidents needed to plan for and effect adequate protections. The private sector is reluctant to submit information to the government related to vulnerabilities or incidents that might damage its reputation, weaken its competitive position, lead to costly investigations, be used inappropriately, or expose it to liability as a result of disclosure by the government of confidential business information. The government is reluctant to disclose threat information that might compromise intelligence activities or investigations. The first objective of the Commission's recommended Strategy for Action was to promote a partnership between government and infrastructure owners and operators that would increase the sharing of information relating to infrastructure threats, vulnerabilities, and interdependencies. The Commission proposed developing an Information Sharing and Analysis Center (ISAC) that would consist of government and private sector representatives working together to receive information from all sources, analyze it, draw conclusions about vulnerabilities or incidents within the infrastructures, and inform government and private sector users. It also recognized that, in order to facilitate the exchange of information, the private sector would need assurances that its confidential information would be protected. The Commission noted that this might require that a legal vehicle be established within the critical infrastructure information sharing mechanism that would protect confidential information, and examined the ramifications of different approaches and strategies related to the federal government's protection of private sector information. It briefly discussed some pros and cons associated with the creation of a FOIA exemption 3 statute for critical infrastructure information. Under exemption 3 of the Freedom of Information Act (FOIA), 5 U.S.C. 552, information protected from disclosure under other statutes is also exempt from public disclosure under FOIA. In response to the Commission's report, President Clinton released Presidential Decision Directive No. 63 (PDD-63). The Directive instructed the National Coordinator for Security, Infrastructure Protection and Counter-Terrorism and other government officials to consult with private sector owners and operators of critical infrastructures, and encourage the creation of a private sector information analysis and sharing center as envisaged by the PCCIP. Although the Directive did not address FOIA explicitly, it did direct the National Coordinator to undertake studies to examine: liability issues arising from participation by private sector companies in the information sharing process; existing legislative impediments to information sharing with an eye toward removing those impediments; and the improved protection, including secure dissemination of industry trade secrets, of other confidential business data, law enforcement information and evidentiary material, classified national security information, unclassified material disclosing vulnerabilities of privately owned infrastructures and apparently innocuous information that, in the aggregate, would be imprudent to disclose. The Clinton Administration, however, never adopted a formal position on the desirability of an exemption to FOIA or the necessity for any additional confidentiality protections. In connection with the implementation of PDD-63, a number of industrial sectors which own and/or operate critical infrastructures formed ISACs, and entered into arrangements with the federal government to share information. However, the General Accounting Office reported in April 2001, that very little or no formalized flow of information has occurred from the private sector to the federal government. According to the Director of the National Infrastructure Protection Center, the organization with which industry is to share information, one of the reasons for this is the uncertainty regarding FOIA exemptions. Similarly, the Partnership for Critical Infrastructure Security, a cross-industry group formed to facilitate communication among industry sectors, has stated that it is not clear that any of the existing FOIA exemptions provide the certainty of protection that many companies require before disclosing threat and vulnerability information to the government. In the 106 th Congress, both H.R. 4246 (Davis/Moran) and S. 3188 (Kyl) included an exemption from FOIA for cyber security information voluntarily provided to the federal government, and prohibited the information from being used, by either the federal government or a third party, in any civil action. Neither bill was reported out of committee. During the 107 th Congress, two bills were introduced with many of the same provisions: H.R. 2435 (Davis) and S. 1456 (Bennett/Kyl) would have exempted information voluntarily submitted to the federal government in connection with critical infrastructure protection from FOIA, and provided protection against civil action. Both bills remained in committee. In an effort to reconcile the two bills, S. 1456 was modified, taking some of the House language. The rewritten bill, however, was never introduced. The Bush Administration offered qualified support for both bills. In President Bush's initial proposal to establish a new Department of Homeland Security, part of which proposed establishing a critical infrastructure protection function, a FOIA exemption was included for information held by the Department. Subsequently, both the House and Senate bills establishing the new Department ( H.R. 5005 and S. 2452, respectively) included more detailed language exempting critical infrastructure information from FOIA. The House language also offered more extensive protections: see " Legislative Responses," below. Freedom of Information Act In 1966, during floor debate on passage of the Freedom of Information Act (FOIA), Representative Rumsfeld quoted James Madison when he said, Knowledge will forever govern ignorance. And a people who mean to be their own governors, must arm themselves with the power knowledge gives. A popular government without popular information or the means of acquiring it, is but a prologue to a farce or a tragedy, or perhaps both. The sentiments expressed by Madison in 1822 are prescient today. The populace desires knowledge about the activities of its government in order to ensure accountability and oversight. The government desires information from owners and operators of critical infrastructures in order to protect persons and assets in the war on terrorism. The terrorist attacks of September 11 have prompted a reevaluation of how to balance public access to information with the need for safety and security. The federal government, since its beginnings, has delegated to agency heads the basic authority to control the papers and documents of their departments. Through the Housekeeping Statute of 1789, federal agencies have kept control of the disclosure of their files. The Administrative Procedure Act (APA) of 1946 had a slight impact upon departmental control of agency information. Instances were documented, however, where both the Housekeeping Statute and the Administrative Procedure Act had been used as excuses for withholding information, and concern mounted that the APA had become a loophole for agency secrecy permitting agency heads to exercise broad, unrestrained powers of a discretionary nature. The Housekeeping Statute was amended to clarify that it does not authorize withholding information from the public or limiting the availability of records to the public. The amendment of the Housekeeping Statute did not produce the results sought by advocates of greater public access to public information. The House Government Information Subcommittee proposed a freedom of information bill that created a right of any person to use the courts to enforce the right of access to federal information. Although the proposal was well received by the press, federal agencies were resistant. The Senate passed S. 1160 in 1965, the House in 1966, and the Freedom of Information Act (FOIA) was signed into law by President Johnson on July 4, 1966. The FOIA was subsequently amended in 1974, 1986, and 1996 for several reasons: ambiguity in the text and legislative history; agency and Department of Justice resistance to broader disclosure; increased oversight by Congress; court interpretations of the statute and its procedural requirements and exemptions; time delays by agencies in responding to requests for access to information and delaying tactics by agencies in litigation; to clarify the scope of the exemptions in response to Supreme Court decisions interpreting the Act's provisions; and to accommodate technological advances related to the methods prescribed for public access. The purpose of the Freedom of Information Act (FOIA) was to ensure by statute citizen access to government information. The FOIA establishes for any person—corporate or individual, regardless of nationality—presumptive access to existing, unpublished agency records on any topic. The law specifies nine categories of information that may be exempted from the rule of disclosure. The exemptions permit, rather than require, the withholding of the requested information. Records which are not exempt under one or more of the Act's nine exemptions must be made available. If a record has some exempt material, the Act provides that any reasonably segregable portion of the record must be provided to any person requesting such record after deletion of the portions which are exempt. Disputes over the accessibility of requested records may be reviewed in federal court. Fees for search, review, or copying of materials may be imposed; also, for some types of requesters, fees may be reduced or waived. The FOIA was amended in 1996 to provide for public access to information in an electronic form or format. In 2001, agency annual reports indicated that they received approximately 1.9 million FOIA requests. With respect to the Freedom of Information Act, three of the nine exemptions from public disclosure provide possible protections against the release of homeland security and critical infrastructure information: exemption 1 (national security information), exemption 3 (information exempted by statute), and exemption 4 (confidential business information). FOIA Exemption 1—National Security Information Exemption 1 of the FOIA protects from disclosure national security information concerning the national defense or foreign policy, provided that it has been properly classified in accordance with the substantive and procedural requirements of an executive order. As of October 14, 1995, the executive order in effect is Executive Order 12,958 issued by President Clinton ( and amended in 1999 by Executive Order 13,142). Section 1.5 of the order specifies the types of information that may be considered for classification: military plans, weapons systems, or operations; foreign government information; intelligence activities, sources or methods, or cryptology; foreign relations or foreign activities, including confidential sources; scientific, technological, or economic matters relating to national security; U.S. government programs for safeguarding nuclear materials and facilities; or vulnerabilities or capabilities of systems, installations, projects, or plans relating to national security. The categories of information that may be classified seemingly appear broad enough to include homeland security information concerning critical infrastructures. Under E.O. 12,958 information may not be classified unless "its disclosure reasonably could be expected to cause damage to the national security." On March 19, 2002, the White House Chief of Staff issued a directive to the heads of all federal agencies addressing the need to protect information concerning weapons of mass destruction and other sensitive homeland security-related information. The implementing guidance for the directive concerns sensitive homeland security information that is currently classified, and previously unclassified or declassified information. The guidance provides that with respect to such information currently classified, the classified status of such information should be maintained in accordance with Executive Order 12,958. This includes extending the duration of classification as well as exempting such information from automatic declassification as appropriate. With respect to previously unclassified or declassified information concerning weapons of mass destruction and other sensitive homeland security-related information, the implementing guidance provides that, to the extent it has never been publicly disclosed under proper authority, it may be classified or reclassified pursuant to Executive Order 12,958. If the information has been subject to a previous request for access, such as a FOIA request, classification or reclassification is subject to the special requirements of the executive order. Section 792 of H.R. 5005, as passed by the House, directed the President to prescribe and implement procedures applicable to all federal agencies to share relevant, appropriate homeland security information among federal agencies, including the Department of Homeland Security, and with appropriate state and local personnel; to identify and safeguard sensitive, unclassified homeland security information; to determine whether, how, and to what extent to remove classified homeland security information, and to determine with whom such homeland security information should be shared after such classified information is removed. H.R. 5005 specifically stated that the substantive requirements for classification are not changed. S. 2452, agreed to by the Senate Governmental Affairs Committee on July 25, 2002, did not have a parallel provision. The House language prevailed (in Section 982 of P.L. 107-296 ). FOIA Exemption 3—Information Exempt by Statute Under exemption 3 of the FOIA, information protected from disclosure under other statutes is also exempt from public disclosure. Exemption 3 provides that the FOIA does not apply to matters that are: specifically exempted from disclosure by statute... provided that such statute (A) requires that the matters be withheld from the public in such a manner as to leave no discretion on the issue, or (B) establishes particular criteria for withholding or refers to particular types of matters to be withheld. Exemption 3 allows the withholding of information prohibited from disclosure by another statute only if the other statute meets any one of the three criteria: (1) it requires that the records be withheld ( i.e., no agency discretion); (2) grants discretion on whether to withhold but provides specific criteria to guide the exercise of that discretion; or (3) describes with sufficient specificity the types of records to be withheld. To support an exemption 3 claim, the information requested must fit within a category of information that the statute authorizes to be withheld. As with all FOIA exemptions, the government bears the burden of proving that requested records are properly withheld. Numerous statutes have been held to qualify as exemption 3 statutes under the exemption's first subpart – statutes that require information to be withheld and leave the agency no discretion. Several statutes have failed to qualify under exemption 3 because too much discretion was vested in the agency, or because the statute lacked specificity regarding the records to be withheld. Unlike other FOIA exemptions, if the information requested under FOIA meets the withholding criteria of exemption 3, the information must be withheld. Congress has considered a number of proposals that address the disclosure under FOIA of cyber security information, of information maintained by the Department of Homeland Security, and of critical infrastructure information voluntarily submitted to the Department of Homeland Security. Generally, the legislation has specifically exempted the covered information from disclosure under FOIA, in effect creating an exemption 3 statute for purposes of FOIA. FOIA Exemption 4—Confidential Business Information Exemption 4 of FOIA exempts from disclosure "trade secrets and commercial or financial information obtained from a person and privileged or confidential." The latter category of information (commercial information that is privileged or confidential) is relevant to the issue of the federal government's protection of private sector critical infrastructures information. To fall within this second category of exemption 4, the information must satisfy three criteria. It must be: a) commercial or financial; b) obtained from a person; and c) confidential or privileged. The D.C. Circuit has held that the terms "commercial or financial" should be given their ordinary meaning, and that records are commercial if the submitter has a "commercial interest" in them. The second criteria, "obtained from a person," refers to a wide range of entities. However, information generated by the federal government is not "obtained from a person," and as a result is excluded from exemption 4's coverage. Most exemption 4 cases have involved a dispute over whether the information was "confidential." In 1974, the D.C. Circuit in National Parks and Conservation Association v. Morton, held that the test for confidentiality was an objective one. It held that neither the fact that a submitter would not customarily make the information public, nor an agency's promises of confidentiality were enough to justify confidentiality. National Parks enunciated a two-part test: commercial information is confidential "if disclosure of the information is likely to have either of the following effects: (1) to impair the government's ability to obtain necessary information in the future; or (2) to cause substantial harm to the competitive position of the person from whom the information was obtained." These criteria are commonly referred to as Test 1 and Test 2. In 1992, in Critical Mass Energy Project v. NRC, after examining arguments in favor of overturning National Parks, the D.C. Circuit reaffirmed application of the National Parks test based on the principle of stare decisis – which counsels against overruling established precedent. The plaintiff was seeking reports which a utility industry group prepared and gave voluntarily to the NRC. The agency did, however, have the authority to compel submission. The full Circuit Court of Appeals clarified the scope and application of the National Parks test. The court limited its application "to the category of cases to which [they were] first applied; namely those in which a FOIA request is made for commercial or financial information a person was obliged to furnish to the Government." The court established a new test for confidentiality when the information is submitted voluntarily; the information is exempt from disclosure if the submitter can show that it does not customarily release the information to the public. Under the Critical Mass decision, one standard (the traditional National Parks tests) applies to any information that a submitter "is required to supply," while a broader exemption 4 standard (a new "customary treatment" test) applies to any information that is submitted to an agency on a voluntary basis. The burden of establishing the submitter's custom remains with the agency seeking to withhold the records. Applying the customary treatment test to the information at issue (utility industry group reports voluntarily submitted), the D.C. Circuit agreed with the district court's conclusion that the reports were commercial; that they were provided to the agency on a voluntary basis; and that the submitter did not customarily release them to the public. Thus, the reports were found to be confidential and exempt from disclosure under exemption 4. The key issue raised by Critical Mass is the distinction between "required" and "voluntary" information submissions. In its decision, the court did not expressly define the two terms. The Department of Justice has issued policy guidance on the distinction between information required and information voluntarily submitted under Critical Mass, and has taken the position that the submission of records in instances such as the bidding on government contracts is mandatory rather than voluntary. The basic principles developed by the Justice Department are that a submitter's voluntary participation in an activity does not determine whether any information submission made in connection with that activity is "voluntary;" that Critical Mass determinations should be made according to the circumstances of information submission; that information submissions can be "required" by a range of legal authorities, including informal mandates that call for the submission of information as a condition of dealing with the government or of obtaining a government benefit; and that the existence of agency authority to require an information submission does not automatically mean that the submission is "required." The decision in Critical Mass has generated a great deal of commentary. In addition, there are many cases where courts have applied the Critical Mass distinction between voluntary and required submissions. Nonetheless, the Critical Mass voluntary vs. required standard has not been widely adopted by the other circuits that have endorsed the National Parks test. Executive Order 12,600 ( Predisclosure Notification Procedures for Confidential Commercial Information ), issued in 1987, requires each federal agency to establish procedures to notify submitters of confidential commercial information whenever an agency "determines that it may be required to disclose" such information under the FOIA. The submitter is provided an opportunity to submit objections to the proposed disclosure. If the agency decides to release the information over the objections of the submitter, the submitter may seek judicial review of the propriety of the release, and the courts will entertain a "reverse FOIA" suit to consider the confidentiality rights of the submitter. Another area of concern under exemption 4 jurisprudence is the so-called mosaic effect which recognizes that an individual piece of information, which in and of itself may not qualify as confidential business information, may be combined with other information to cause substantial competitive harm. Private information hawkers routinely engage in the business of assembling all of the pieces of information. Courts have applied the mosaic effect to prevent the disclosure of confidential business information. As previously noted with regard to critical infrastructure information, the federal government seeks to ensure that it is able to obtain information from the private sector on a voluntary basis. S. 2452, the Senate version of National Homeland Security and Combating Terrorism Act of 2002, would have essentially codified the voluntary/required rule from the D.C. Circuit's decision in Critical Mass v. NRC, and applies it to critical infrastructure information voluntarily submitted by the private sector, and not customarily available to the public, to the new Department of Homeland Security. Codification of the Critical Mass standard could eliminate differences in treatment in the federal courts of confidential business information related to critical infrastructure. Legislative Responses FOIA Exemption in the Administration's Initial Proposal for Homeland Security The Bush Administration's initial legislative proposal establishing the new Department of Homeland Security proposed to exempt from disclosure under FOIA critical infrastructure information voluntarily submitted to the government by non-federal entities. Section 204 of the proposal stated: Information provided voluntarily by non-federal entities or individuals that relates to infrastructure vulnerabilities or other vulnerabilities to terrorism and is or has been in the possession of the Department [of Homeland Security] shall not be subject to section 552 of title 5, United States Code. This proposed language did not provide additional specificity, and was criticized by the FOIA requester community as "cast[ing] a shroud of secrecy over one of the Department of Homeland Security's critical functions, critical infrastructure protection." FOIA Exemptions in Homeland Security Proposals When the President's legislative proposal was reported out of the House Select Committee on Homeland Security as H.R. 5005 (Armey), the Administration's FOIA exemption was modified and included in a separate subtitle (Title VII, Subtitle C, sections 721 - 724). The Senate Government Affairs Committee, too, voted to add a FOIA exemption to its bill S. 2452 (Lieberman, section 198) establishing a Department of Homeland Security. The House language prevailed as Title II, Subtitle B, Section 214, in P.L. 107-296. A brief discussion of the FOIA exemptions in these two homeland security bills follows. A comparison of the language regarding FOIA exemptions is included in the CRS Report RL31513, Homeland Security: Side-By-Side Comparison of H.R. 5005 and S. 2452, 107 th Congress. P.L. 107-296, Title II, Subtitle B Section 214 of the Homeland Security Act of 2002 ( P.L. 107-269 ) exempted from disclosure under FOIA "critical infrastructure information (including the identity of the submitting person or entity) that is voluntarily submitted to a covered agency for use by that agency regarding the security of critical infrastructure (as defined in the USA PATRIOT Act)..., when accompanied by an express statement.... " The Homeland Security Act defines critical infrastructure information to mean "information not customarily in the public domain and related to the security of critical infrastructure or protected systems— (A) actual, potential, or threatened interference with, attack on, compromise of, or incapacitation of critical infrastructure or protected systems by either physical or computer-based attack or other similar conduct (including misuse of or unauthorized access to all types of communications and data transmission systems) that violates federal, state, or local law, harms interstate commerce of the United States, or threatens public health and safety; (B) the ability of critical infrastructures or protected systems to resist such interference, compromise, or incapacitation, including any planned or past assessment, projection or estimate of the vulnerability of critical infrastructure or a protected system, including security testing, risk evaluation thereto, risk management planning, or risk audit; or, (C)any planned or past operational problem or solution regarding critical infrastructure... including repair, recovery, reconstruction, insurance, or continuity to the extent it relates to such interference, compromise, or incapacitation." A "covered agency" is defined as the Department of Homeland Security. The submission of critical infrastructure information is considered voluntary if done in the absence of the Department of Homeland Security exercising its legal authority to compel access to or submission of such information. Information submitted to the Securities and Exchange Commission pursuant to section 12 (i) of the Securities and Exchange Act of 1934 is explicitly not protected by this provision. Nor is information disclosed or written when accompanying the solicitation of an offer or a sale of securities, nor if the information is submitted or relied upon as the basis for licensing or permitting determinations, or during regulatory proceedings. Besides exempting from FOIA critical infrastructure information which has been submitted voluntarily with the appropriate express statement to the Department of Homeland Security, the Homeland Security Act also states that the information shall not be subject to any agency rules or judicial doctrine regarding ex parte communications with decision making officials. The Act also prohibits such information, without the written consent of the person or entity submitting such information in good faith, from being used directly by the Department of Homeland Security, any other federal, state, or local authority or any third party, in any civil action. Nor may the information, without the written consent of the person or entity submitting such information, be used or disclosed by any officer or employee of the United States for any purpose other than the purposes of the subtitle, except, in the furtherance of a criminal investigation or prosecution, or when disclosed to either House of Congress, or to the Comptroller General or other authorized General Accounting Office official, in the conduct of official business. Furthermore, any federal official or employee who knowingly publishes, divulges, discloses, or makes known in any manner or to any extent not authorized by law, any protected information, is subject to removal, imprisonment up to one year, and fines. If the information is disclosed to state or local officials, it may not be used for any purpose other than the protection of critical infrastructures, and it may not be disclosed under state disclosure laws. The protections afforded protected information do not result in waiver of any privileges or protections provided elsewhere in law. Finally, no communication of critical infrastructure information to the Department of Homeland Security shall be considered to be an action subject to the requirements of the Federal Advisory Committee Act. For information to be considered protected, it must be accompanied with a written marking to the effect that "this information is voluntarily submitted to the federal government in expectation of protection from disclosure as provided by the Critical Infrastructure Information Act of 2002 [the name given to Subtitle B]." The Secretary of the Department of Homeland Security is to establish procedures for handling the information once it is received. Only those agency components or bureaus, designated by the President or the Secretary of Homeland Security, as having a Critical Infrastructure Program may receive critical infrastructure information from the Department. The above protections for information voluntarily submitted by a person or entity to the Department of Homeland Security do not limit or otherwise affect the ability of a state, local, or federal government entity, agency or authority, or any third party, under applicable law, to obtain critical infrastructure information (including any information lawfully and properly disclosed generally and broadly to the public) and to use that information in any manner permitted by law. Submittal to the government of information or records that are protected from disclosure is not to be construed as compliance with any requirement to submit such information to a federal agency under any other provision of law. Finally, the Act does not expressly create a private right of action for enforcement of any provision of the Act. S. 2452, Section 198 (107th Congress) S. 2452, National Homeland Security and Combating Terrorism Act of 2002, as agreed to by the Senate Governmental Affairs Committee on July 25, 2002, exempted a "record" pertaining to the vulnerability of and threats to critical infrastructure (as defined in the USA PATRIOT Act) furnished voluntarily to the Department of Homeland Security from being made available under FOIA. A record was covered by the bill if the provider would not customarily make the record available to the public. It also required the provider to designate and certify, in a manner specified by the Department of Homeland Security, that the record is confidential and not customarily made available to the public. Unlike the Homeland Security Act ( P.L. 107-296 ), the Senate bill did not include a definition of "critical infrastructure information." However, the bill covered "records pertaining to the vulnerability of and threats to critical infrastructure (such as attacks, response, and recovery efforts)." Under S. 2452 a record is submitted voluntarily if it was submitted to the Department of Homeland Security "in the absence of authority of the Department requiring that record to be submitted," and it is not submitted or used to satisfy any legal requirement or obligation or to obtain any grant, permit, benefit, or other approval from the federal government. Agencies with which the Department of Homeland Security shares protected records were to be bound by the FOIA exemption. FOIA requests for protected information were to be referred back to the Department of Homeland Security, and the Department was permitted to provide any portion of the record that is reasonably segregable from that part of the record which is exempt from disclosure, after deleting the protected information. The bill also allowed the provider of a record that is furnished voluntarily to the Department of Homeland Security to withdraw the confidential designation at any time in a manner specified by the Department. S. 2452 allowed an agency which had received independently of the Department a record "similar or identical" to that received by the Department, to disclose the record under FOIA. The Senate bill did not preempt state or local disclosure laws if the state or local authority received the information independent of the Department of Homeland Security, nor did it contain any civil liability immunity, or criminal penalties. The Secretary of the Department of Homeland Security was directed to prescribe procedures for: acknowledging the receipt of records furnished voluntarily; the certification of records furnished voluntarily as confidential and not customarily made available to the public; the care and storage of records furnished voluntarily; and the protection and maintenance of the confidentiality of records furnished voluntarily. Finally, the Senate bill required the Comptroller General to report to Congress on the implementation and use of the above protections. The report was to include the number of persons in the private sector and the number of state and local agencies that furnished records voluntarily under these provisions, the number of requests for access granted or denied under these provisions, and any recommendations regarding improvements in the collection and analysis of sensitive information related to the vulnerabilities of and threats to critical infrastructures. In sum, significant differences existed between H.R. 5005 (enacted into law as P.L. 107-296 ) and S. 2452. These differences included the scope of the information protection; the type of information covered and exempted from FOIA; the definition of a voluntary submission; the other purposes authorized for use or disclosure of the information; the disclosure of information with the consent of the submitter; the permissibility of disclosures of related information by other agencies; immunity from civil liability; preemption; and criminal penalties. Issues and Concerns The general concerns of the owners and operators of critical infrastructure are that the type and breadth of information they are being asked to submit on vulnerabilities, incidents, remedies, etc., if made available to competitors or to the general public, could harm their public relations, compromise their competitive position, expose them to liability, or disclose sensitive information to terrorists and others who might wish to disrupt the function of their infrastructure. It was their position that crafting a specific exemption to FOIA in statute (i.e., a (b)(3) exemption) would provide the greatest legal protections for the information they share. They believed that a narrowly tailored (b)(3) exemption would eliminate agency discretion to disclose protected information in response to a FOIA request. In addition, given the federal government's need to share sensitive business information for homeland security purposes with state and local officials, owners and operators also sought federal preemption of state and local disclosure laws. Owners and operators were concerned that some of this information could make them subject to liability in unforeseen ways. A number of public interest groups have expressed (and continue to express) their opposition to the protections being applied, particularly those contained in the House version. The primary concern is that the type of information exempted from FOIA was too broadly defined, and could allow any company claiming to be an owner or operator of a critical infrastructure to voluntarily submit almost any kind of information in order to protect the information from disclosure under the FOIA. Critics also believe the definition of critical infrastructure adopted from the USA PATRIOT Act is too broad. The Act also covers information regarding an attack, or similar conduct, that violates law or harms interstate commerce. According to one critique, the language "or similar conduct" and "harms interstate commerce" is broad and could include non-criminal or inadvertent incidents that cause temporary interruption of normal business operations. The criticism goes on to state that the purposes for which the information may be used (and therefore contributing to the definition of what kind of information may be protected) includes analysis, warning, interdependency study, recovery, reconstitution, or "other informational purposes." According to the critique, "other informational purposes" covers untold amounts of information, some of which may have been previously available to the public. These groups also are concerned that information currently collected by various agencies and available to the public could now be protected from disclosure if submitted to the Department of Homeland Security initially as critical infrastructure information. This is particularly an issue in the area of environmental law relating to a community's right to know. Both bills stated that the protections are granted "notwithstanding any other provisions of law." Under current law (the Emergency Planning and Community Right-to-Know Act, P.L. 99-499, 42 USC 11001-11050), facilities handling certain toxic substances in excess of a threshold amount annually must report to the Environmental Protection Agency and local officials the maximum and average daily amounts of such substances that they had on hand during the previous year; the location of such chemicals within the facility; and estimates of how much was released into the environment as part of normal handling and processing. In addition, in the event of an accidental release above a threshold amount, facilities immediately must report the amount released to local officials. The 1990 amendments to the Clean Air Act (which were passed in P.L. 101-549, Section 301, amending 42 USC 7412) made it the duty of owners and operators of facilities producing, processing, handling, or storing certain extremely hazardous substances: to identify hazards that may result from releases; to design and maintain a safe facility; and to minimize the consequences of accidental releases which do occur. To prevent accidental releases, the Clean Air Act requires facilities handling such substances to develop "risk management plans." Among the items included in these plans are an accounting of any accidental releases of those substances over the previous five years; estimates of the quantities of chemicals that might be released in the event of an accident, including a worst-case accident; estimates of the potential exposures to affected downwind populations; a program for preventing releases; and an emergency response program to protect public health and the environment in the event of a release. Under the 1990 law, public disclosure of most of this information (which also could be released in response to FOIA requests) is required, but the details of the off-site consequence analyses (OCA) for hypothetical accidents are not required to be disclosed. In addition, companies may claim confidentiality for some submitted information, provided they can support that claim. Security concerns arose about the potential utility to terrorists of risk management planning data, just as EPA was planning to make the plans widely available to the public via the Internet. Convinced of the need for caution, EPA agreed not to post OCA data on its website. Nevertheless, the information could be obtained electronically using FOIA, and several public interest groups announced that they would do so and post the data. In 1999, Congress responded by again amending the Clean Air Act. The amended Act exempts OCA data from disclosure under FOIA, and directs EPA to limit public disclosure as necessary to reduce risks. EPA issued a final regulation on data access on August 4, 2000. It allows the public to see paper copies of sensitive OCA information through federal reading rooms, approximately one per state, and provides Internet access to the OCA data elements that pose the least serious criminal risk. State and local agencies are encouraged to provide the public with read-only access to OCA information on local facilities. At the federal reading rooms, members of the public may read OCA information for up to 10 facilities per calendar month and for all facilities with potential effects in the jurisdiction of the local emergency planning committee. State and local officials and other members of the public may share OCA information as long as the data are not conveyed in the format of sensitive portions of the RMP or any electronic database developed by EPA from those sections. A Clinton Administration proposal to implement the final rule (66 Federal Register 4021, Jan. 17, 2001) would have allowed people to view plans of facilities outside their local area and enhanced access for "qualified researchers." The draft plan was rescinded by the Bush Administration (66 Federal Register 15254, Mar. 16, 2001). No further regulatory action has been taken to date. Critics of the FOIA exemption for critical infrastructure information submitted voluntarily with the appropriate express statement are concerned that the "notwithstanding any other provision of law" clause could possibly exempt from FOIA information about facilities handling potentially dangerous chemicals that is currently available under the Emergency Planning and Community Right-to-Know Act and the Clean Air Act. Some public interest groups are concerned that the breadth of information that could be exempted from disclosure, combined with the prohibition on use of critical infrastructure information in any civil suit, could give owners or operators of critical infrastructures an "unprecedented immunity" from complying with a variety of laws (i.e., antitrust, tort, tax, civil rights, environmental, labor, consumer protection, and health and safety laws). Another concern centers on a perceived lack of clarity on whether information obtained independently by subpoena, for example, could be used to bring civil suit (e.g., would a victim of chemical exposure be precluded from suing if information previously submitted to the Department of Homeland Security was obtained independently from the company by subpoena). Another argument made by the public interest groups is that existing FOIA exemptions and case law offer sufficient protections to owner/operators. They cite exemption (b)(4), which allows agencies to withhold commercial information that is privileged or confidential, if by disclosing that information, the competitive position of the provider is harmed or the ability of the government to continue receiving that information is impaired. An exemption from FOIA for critical infrastructure information, they argue, would promote government secrecy and harm public access. These groups are also concerned about a provision they say gives the private sector the power to determine what information is to be protected, simply by including an express statement of protection from disclosure on the submission to the federal government. The criminal penalties provided for the unauthorized disclosure of protected information are viewed by some groups as essentially an anti-whistleblower provision designed to stifle government accountability. Another issue raised by the groups is whether a submission of information to the government will be treated as voluntary in situations where an agency has not exercised its authority to compel submission. Finally, the groups take issue with the provision that preempts state and local freedom of information laws. The public interest groups concerned with granting specific FOIA exemptions have expressed a guarded acceptance of the Senate version. They feel it basically puts into statute recent FOIA case law regarding the protections afforded confidential information submitted to government agencies under FOIA exemption 4. Representatives from industry responded to some of these concerns by stating that it was not their intent to evade current laws and regulations, but that the extra protections are needed before they are willing to voluntarily submit information that might be used against them later, either legally or competitively. Under the existing law, companies had no assurance that information they share with a government agency will be treated confidentially, and agencies are not required to commit to confidentiality at the time of disclosure. Agencies are not required to initiate the FOIA exemption process until a FOIA request is received. When it is received, the agency is asked to defend the information's confidentiality, and is not required to inform the originator if it believes it has enough information to proceed. Industry is generally in favor of legislation that accomplishes the goal of encouraging it to submit security-related information without fear of public disclosure. Representatives from owners and operators have also stated that they favor a narrow exemption so as to cover only infrastructure threat and vulnerability information. Conclusion Compelling arguments existed on both sides of the debate for and against exempting critical infrastructure information from the Freedom of Information Act. However, the Senate bill, S. 2452, never made it to the Senate floor. After the November 2002 election, sentiment to pass a Homeland Security Act led to the adoption by the Senate of large portions of the House-passed bill. The provisions regarding the exemption of Critical Infrastructure Information from FOIA adopted the House language in total. Public interest groups continue to criticize the language. S. 6 introduced January 7, 2003, in the 108 th Congress, and sent to the Senate Judiciary Committee, resurrects S. 2452 (107 th Congress) language (Title VIII, Subtitle B).

Summary: Critical infrastructures have been defined as those systems and assets so vital to the United States that the incapacity of such systems and assets would have a debilitating impact on the United States. One of the findings of the President's Commission on Critical Infrastructure Protection, established by President Clinton in 1996, was the need for the federal government and owners and operators of the nation's critical infrastructures to share information on vulnerabilities and threats. However, the Commission noted that owners and operators are reluctant to share confidential business information, and the government is reluctant to share information that might compromise intelligence sources or investigations. Among the strategies to promote information sharing was a proposal to exempt critical infrastructure information from disclosure under the Freedom of Information Act. The Freedom of Information Act (FOIA) was passed to ensure by citizen access to government information. Nine categories of information may be exempted from disclosure. Three of the nine exemptions provide possible protection against the release of critical infrastructure information: exemption 1 (national security information); exemption 3 (information exempted by statute); and exemption 4 (confidential business information). Congress has considered several proposals to exempt critical infrastructure information from FOIA. Generally, the legislation has created an exemption 3 statute, or adopted the exemption 4 D.C. Circuit standard. Prior to passage of the Homeland Security Act (P.L. 107-296), the House (H.R. 5005) and Senate (S. 2452) bills differed significantly on language providing a FOIA exemption. Differences included the type of information covered and exempted from FOIA; the scope of the protections provided; the authorized uses or disclosures; the permissibility of disclosures of related information by other agencies; immunity from civil liability; preemption; and criminal penalties. The Homeland Security Act (P.L. 107-296, section 214 ) provisions regarding the exemption of critical infrastructure information from FOIA adopted the House language in its entirety. Public interest groups question the necessity of a FOIA exemption suggesting that existing FOIA exemptions provide sufficient protections.. They also argued that the House language (which passed) was too broad and would allow a wider range of information to be protected (including information previously available under FOIA). They favored the more limited protections proposed in the S. 2452. Public interest groups also expressed concern that the provision which bars use of the protected information in civil actions would shield owners and operators from liability under antitrust, tort, tax, civil rights, environmental, labor, consumer protection, and health and safety laws. Owners and operators of critical infrastructures insisted that current law did not provide the certainty of protection needed. While they viewed the Senate language as a workable compromise, they favored the protections in H.R. 5005. Compelling arguments existed on both sides of the debate for and against exempting critical infrastructure information from the Freedom of Information Act. S. 6, introduced in the 108th Congress, resurrects S. 2452 (107th Congress). This report will be updated as warranted.

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Write a title and summarize: The pathogenic fungus Cryptococcus neoformans uses the Bwc1-Bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. How the complex controls these functions is unclear. Here, we identify and characterize a gene in Cryptococcus, UVE1, whose mutation leads to a UV hypersensitive phenotype. The homologous gene in fission yeast Schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the UVDE-dependent excision repair (UVER) pathway. C. neoformans UVE1 complements a S. pombe uvde knockout strain. UVE1 is photoregulated in a Bwc1-dependent manner in Cryptococcus, and in Neurospora crassa and Phycomyces blakesleeanus that are species that represent two other major lineages in the fungi. Overexpression of UVE1 in bwc1 mutants rescues their UV sensitivity phenotype and gel mobility shift experiments show binding of Bwc2 to the UVE1 promoter, indicating that UVE1 is a direct downstream target for the Bwc1-Bwc2 complex. Uve1-GFP fusions localize to the mitochondria. Repair of UV-induced damage to the mitochondria is delayed in the uve1 mutant strain. Thus, in C. neoformans UVE1 is a key gene regulated in response to light that is responsible for tolerance to UV stress for protection of the mitochondrial genome. The ability to sense light provides well-known advantages to organisms, such as adapting to photosynthetic light sources in plants and for vision in animals, yet the benefits of light-sensing in non-photosynthetic and non-motile organisms are less established. The fungi contain a suite of potential photoreceptor proteins, with the White Collar complex (WCC) being found throughout the fungal kingdom, except for species in which the two genes encoding the complex were lost. Light influences different responses in different fungi, including phototropism, induction of pigmentation, asexual and sexual sporulation, changes is primary and secondary metabolism, and regulating the circadian clock: most of which are controlled, where established, by the WCC [1]–[3]. A major question is what advantage is provided in using light as an environmental signal to regulate these processes. One compelling hypothesis is that protecting DNA from damage provides a selective pressure, and that wavelengths in the visible spectrum are sensed to indicate the presence of deleterious ultraviolet radiation. However, a DNA repair system common to light-sensing fungi and that acts directly downstream of the WCC is unknown to date. The White collar-1 and White collar-2 proteins interact to form a complex (WCC) capable of sensing blue and near UV light. Both proteins were originally characterized in the ascomycete fungus Neurospora crassa [4]–[7] where WC-1 acts as a photoreceptor. In N. crassa, WCC has roles to play both in light and dark environments. In the dark the WCC has a major function as a circadian clock component, via regulation of FRQ gene expression [8]–[10]. Light-dependent functions of WCC include conidiation, carotenoid production and mating [11], [12]. The photons and the signal are transduced via the chromophore flavin adenine dinucleotide (FAD) bound within an N-terminal specialized type of PAS domain (from the Per, Arnt, Sim proteins), named the LOV (light, oxygen and voltage) domain [13]. The two proteins interact using other PAS domains, to form the transcription factor complex that regulates transcription of target genes via their GATA-type zinc finger DNA-binding domains [4], [5], [7], [14]. The human pathogen Cryptococcus neoformans is a member of the phylum Basidiomycota, a distant relative to N. crassa. The fungus grows vegetatively as a budding yeast and during mating in a dikaryotic filamentous form. C. neoformans is divided into two varieties: var. grubii is the most prevalent in the clinic and var. neoformans is less common but was the most amenable to experimental methods until the discovery of an opposite mating partner for var. grubii about a decade ago [15]. C. neoformans primarily causes disease in immunocompromised people. The closest relative to C. neoformans is C. gattii, which is also a human pathogen but with a greater tendency to infect immunocompetent individuals [16]. Homologs of N. crassa wc-1 and wc-2 are present in Cryptococcus species, designated BWC1/CWC1 and BWC2/CWC2, and have been characterized in strains of both var. grubii and var. neoformans [17], [18]. The Bwc1-Bwc2 complex has three known functions in C. neoformans: it represses mating in the light, promotes virulence, and provides protection against UV light. The downstream targets of Bwc1-Bwc2 that control these functions remain to be elucidated. Several genes have been identified from C. neoformans that are regulated by light. These include SXI1α and MFα1, found within the mating type locus and encoding a transcription factor and pre-pheromone protein required for sexual reproduction [17]. Their expression could explain the repression of mating by light. However, the regulation of these transcripts was compared after a long time exposure of 24 h in the light or constant darkness, such that this time point likely reflects indirect regulation by Bwc1-Bwc2. More recently a microarray experiment was carried out in C. neoformans to detect light-regulated transcripts with an hour of exposure to light. The HEM15 gene, encoding ferrochetalase that is the last step in the heme biosynthetic pathway, was identified as a gene under control of Bwc1-Bwc2 [19]. However, the phenotype of the knockout hem15 strain differs significantly from mutation of BWC1 or BWC2. For example, HEM15 is essential for viability, and yet the bwc1 and bwc2 mutants do not show any growth defects in the light or dark. Another light-regulated gene is CFT1, required for iron uptake and virulence [20], yet no iron-dependent phenotype of bwc1 and bwc2 mutants is known. Thus, while CFT1 and HEM15 may be targets of the Bwc1-Bwc2 complex, they likely play minimal roles in the physiological response of C. neoformans to light. Moreover these two genes are also under the control of other transcription factors in addition to WCC. As a transcription factor complex, it was puzzling that more light-regulated genes were not identified by microarray analysis that could potentially explain the phenotypes of deleting the WCC from C. neoformans. The microarray results of C. neoformans contrast to ascomycete species. For instance, in N. crassa a microarray study in wild type, wc-1Δ, and wc-2Δ strains at different time periods identified 314 light-regulated genes, constituting 5. 6% of the total detectable transcripts in the genome [11]. These genes were grouped into two broad categories, the early or late light responsive genes (ELRGs and LLRGs). Some of these ELRGs are involved in the synthesis of vitamins, photo-protective pigments, prosthetic groups and cofactors, cellular signaling, DNA processing, circadian rhythm and secondary metabolism. Many of the LLRGs are implicated in carbohydrate metabolism, fatty acid oxidation and free radical detoxification. In N. crassa there is also a link between the WCC and DNA repair, for example through the clock gene prd-4, which is a cell cycle checkpoint kinase 2 [21]–[23]. As the White Collar complex acts as an UV/blue light photoreceptor and mutation of the complex causes an increase in sensitivity to UV light, potential targets are hypothesized to be genes involved in repairing DNA damage for survival under UV stress. The UVE1 gene of C. neoformans was previously identified in a UV sensitive strain in a collection of insertional mutants [24]. The product of UVE1 is a homolog of an apurinic/apyrimidinic endonuclease that is best characterized in fission yeast Schizosaccharomyces pombe. In S. pombe the gene was called UVDE for UV damage endonuclease, and renamed uve1 for consistency with nomenclature [25], [26]. The mus-18/UVE-1 gene is the homolog characterized from N. crassa [27]. The protein removes UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, acting in its own pathway termed the UVDE-dependent excision repair (UVER) pathway. UVDE recognizes single-stranded DNA nicks, apurinic/apyrimidinic sites, and nucleotide mismatches [26], [28]–[30], a suite of DNA lesions that also extends a possible role for UVDE in repairing the equivalent types of DNA damage caused by reactive oxygen species [31]. In S. pombe, UVDE is localized and functional in both the nucleus and mitochondria, and was suggested to act as a reserve mechanism for repairing UV-induced DNA damage in the mitochondria [32]. Homologs of UVE1 are present in a subset of species in the Archaea, Bacteria and Eukaryotes [25], [33]–[37], with one exception being humans where there is no homolog. A preliminary northern blot experiment suggested that UVE1 in C. neoformans var. neoformans is a light-regulated gene with two isoforms, triggering this investigation. We hypothesized that UVE1 is a downstream target of the WCC that functions in repairing UV-induced damage, and tested this hypothesis in the experiments described below. A T-DNA insertion mutant within the promoter of the UVE1 gene was identified previously [14]. Under UV stress conditions the mutant showed negligible survival as compared to the wild type (KN99α) and the unexposed strains (Figure 1A). Transformations derived from Agrobacterium T-DNA delivery can have phenotypes that are not due to the insertion of the T-DNA into the host genome. The UV sensitivity phenotype of the original mutant was verified by constructing UVE1 gene replacement strains for both C. n. var. neoformans and C. n. var. grubii. The knockout strains in both varieties had reduced survival after exposure to UV (Figure 1A, B). To confirm that the UV sensitivity phenotype was because of the absence of UVE1, an uve1Δ strain was complemented with a wild type copy of UVE1. The UVE1 complemented strain completely rescued the UV sensitivity phenotype (Figure 1). These results show that in C. neoformans the UVE1 gene is required for survival under UV stress. The responses of the uve1 mutant to stresses other than UV light were tested, showing no other phenotypes (Figure S1). The gene also plays no major role in the formation of mating filaments (Figure S2A, B). A prior analysis, using comparative growth in a pool of 48 strains in the mouse lung, suggested the UVE1 gene has no role in virulence [38]. To examine the uve1Δ strain in isolation, its virulence was tested in an insect model. While the bwc1Δ strain is less pathogenic than wild type in this model, the uve1Δ strain is equally virulent as wild type (Figure S2C). Thus, the only established function of Uve1 in C. neoformans is in response to UV stress. Since UVE1 is required for surviving exposure to UV light, regulation of UVE1 was tested in response to light. Wild type strains of C. n. var. neoformans, C. n. var. grubii and C. gattii were grown in the dark, and one replicate provided a one hour exposure to white light. Expression of the UVE1 gene was examined by northern blot analysis on polyA RNA purified from these cultures. The UVE1 transcript levels were higher in light grown conditions for all wild type strains (Figure 2). Two transcripts were observed for var. neoformans strain JEC21, one longer (L, for light) induced specifically under light conditions and another shorter (D, dark) expressed in the dark. For var. grubii and C. gattii one isoform of UVE1 was expressed in response to light with negligible expression in dark grown conditions. The role for the Bwc1 photoreceptor in UVE1 induction by light was examined in the bwc1Δ deletion strains of C. neoformans. In the var. neoformans deletion there was loss of induction of the longer isoform by light, and residual expression of this isoform in both light and darkness. There was no observed effect of bwc1Δ on the shorter isoform in dark grown conditions. Dark isoforms expressed equally well in both light and dark conditions in some replicates, possibly reflecting the age of the culture or shading by other cells. In the var. grubii bwc1Δ mutant, the UVE1 transcript was barely detectable under either illumination regime (Figure 2). These analyses in the bwc1 mutant backgrounds indicate that UVE1 expression is controlled by the Bwc1-Bwc2 complex. Rapid amplification of cDNA ends (RACE) was used in the var. neoformans strain to define the 5′ and 3′ ends of the two transcripts that were produced in the light and the dark. The UVE1 dark isoform is part of the UVE1 light isoform, with the dark isoform starting in the middle of the light isoform and both sharing a common 3′ end (GenBank accessions KF234405 and KF234406; Figure 3). Alignment of the Uve1 homologs from various fungi indicated that the dark isoform of C. neoformans is truncated and missing key residues in the active site of this protein (Figure 3; Figure S3). The light-dependent regulation of UVE1 in wild type strains suggested that induction of the gene prior to UV exposure would correlate with increased UV resistance. The wild type, bwc1Δ and the complemented strains were grown in complete darkness overnight. One set was kept in the dark and the other set exposed to light for 2 hours, prior to treatment of both sets with UV light (Figure S4). All three strains grown in darkness showed similar levels of sensitivity to UV light. Exposure of the strains with the wild type copy of BWC1 to light before UV stress increased their resistance to UV, a property not seen for the bwc1Δ strain. Hence, light signaled by Bwc1 promotes UV resistance. Two isoforms of UVE1 were observed in the var. neoformans strain, raising the possibility that differential transcript sizes may be a common feature for DNA repair genes in C. neoformans. To identify other genes involved in protecting the fungus against UV damage, a collection of 1200 defined knock out mutants in the var. grubii background [38] was screened for those sensitive to UV light. 13 strains were identified, including the uve1Δ strain in the collection (Figure S5A). For the corresponding genes, northern analysis were performed for var. neoformans and var. grubii cultured under light and dark conditions (Figure S5B). No altered size or induction in response to light was observed, as had been for the UVE1 gene. To examine if UVE1 photoregulation is common among fungi, northern blot analysis of UVE1 was performed in two fungi, Neurospora crassa and Phycomyces blakesleeanus (Figure 4). The species are light-sensing models in the phylum Ascomycota and subphylum Mucoromycotina, respectively. In N. crassa the expression of UVE1 has already been reported in a microarray study of the light-induced genes [11]. For the N. crassa wild type strain we observed by northern blot analysis the induction of one transcript in light grown conditions and minimal expression of the UVE1 transcript in dark grown mycelia, confirming the previous microarray data. For the P. blakesleeanus wild type strain two transcripts were present in light grown conditions that were both absent in samples grown in the dark. Based on the expressed sequence tag information in the genome database, the 5′ end of the UVE1 homolog is shared between the two transcripts and the 3′ end differs, the reverse of the situation in C. neoformans var. neoformans. The longer isoform in P. blakesleeanus has a 3′ extension due to transcriptional read-through into the 3′ neighboring gene. We also examined the transcript profile of UVE1 in a white collar-1 mutant (wc-1) of N. crassa and a madA-madB mutant of P. blakesleeanus (madA and madB are functional wc-1 and wc-2 homologs in this species [39]). We observed complete loss of light-dependent induction of UVE1 in these mutant strains of N. crassa and P. blakesleeanus. As an additional control to show that UVE1 induction was not an indirect effect of light on the media, UVE1 expression was examined in S. pombe, a “blind” species because it encodes no homologs of the WCC. We observed equal transcript levels of the S. pombe UVE1 homolog in cultures grown under light and dark conditions (Figure 4). These studies demonstrate that UVE1 is photoregulated among highly-diverged fungal species, and substantiates the WC-1 dependent light-induction of UVE1 in the fungal kingdom. The protein sequences predicted for the two isoforms in var. neoformans from RACE were examined by bioinformatic approaches for their subcellular localization. PSORT II and MitoProt analysis of UVE1 light and dark isoforms predicts the longer form to be most likely mitochondrial and no specific localization pattern for the dark isoform. To confirm these predictions, light and dark isoforms of UVE1 were fused to the N-terminal end of GFP and expressed in the uve1Δ strain AI191. To assess mitochondrial localization we used MitoTracker red, which specifically stains respiring mitochondria. Confocal fluorescence microscopy for the GFP-fused light form of Uve1 showed co-localization of Uve1-GFP with MitoTracker red, giving a yellow fluorescence in merged images (Figure 5A). No expression of Uve1-GFP light form was observed in the nucleus, confirmed by co-staining with Hoechst (Figure S6). For the dark isoform of Uve1-GFP, GFP localization was throughout the cell, but clearly excluded from the mitochondria (Figure 5B; Figure S6). Transformation of the UVE1-GFP constructs into a uve1Δ genetic background enabled a test of their functionality in complementing the UV sensitive phenotype. Expression of the light form of UVE1 rescued in part the UV sensitive phenotype of strain AI191; however, the dark isoform fused to GFP did not. These experiments suggest that the light isoform of UVE1 is localized solely to the mitochondria, and as such it protects the mitochondrial genome from lethal effects of UV-induced DNA damage in C. neoformans. The function of Uve1 in the fungi at a biochemical level is best characterized in S. pombe [25], [29], [40], [41]. Alignment of the two homologs suggests that they are similar, sharing residues within the active site of the enzyme (Figure S3). To infer functional similarity, a cross-species complementation test was performed. An uve1Δ knockout was generated in S. pombe by replacing the gene via homologous recombination with the KanMX cassette that confers resistance to G-418. We then expressed in this S. pombe knockout strain the cDNA clones of light or dark isoforms of UVE1 from C. neoformans var. neoformans. The UV sensitivity of the S. pombe strains was tested (Figure 6A, B). The uve1 deletion strain was highly sensitive to UV irradiation, as was the control strain transformed with the empty vector. In the strain expressing the light isoform of UVE1, UV sensitivity was rescued as the strain survived UV doses equivalent to the wild type strain. For the uve1: : KanMX+C. neoformans dark isoform, no rescue in the UV sensitive phenotype was observed. These observations suggest that the C. neoformans light isoform of UVE1 is functionally active and has the equivalent biochemical functions of Uvde from S. pombe required for repair of DNA damaged by UV exposure. It also suggests that the dark isoform of C. neoformans Uve1 may not have any function in conferring protection against UV. C. n. var neoformans Uve1 was localized in S. pombe as a GFP fusion to see if the rescue of UV sensitivity phenotype in S. pombe is due to complementing mitochondrial or nuclear genome repair by Uve1 (Figure 6C, D). The Uve1-GFP construct was functional, complementing the UV sensitive phenotype of the S. pombe uve1 mutation (Figure 6A, B). The localization of Uve1 (L) -GFP is in part nuclear, as confirmed by co-localization with the nuclear Hoechst stain (Figure 6C). No localization in mitochondria was observed (Figure 6D). These results suggest that the Cryptococcus Uve1 protein, which seems to be important for mitochondrial DNA repair in C. neoformans, also plays a role in nuclear DNA repair in S. pombe. This observed localization pattern conforms to previous reports where Uve1 in S. pombe repairs nuclear DNA after UV stress, rather than mitochondrial DNA [32]. If Uve1 localizes to mitochondria in C. neoformans, it is expected to play a role in mitochondrial DNA repair in consequence of DNA damage due to UV stress. As no nuclear localization was observed for Uve1, a negligible role of this endonuclease is expected in nuclear DNA damage repair. We performed a PCR-based DNA damage assay to assess the role of Uve1 in mitochondrial and nuclear DNA repair post-UV stress. The assay is based on the principle that damaged DNA impedes the progression of Taq polymerase on the template DNA in a PCR reaction [42]. Hence, there is an inverse relationship between the amount of DNA damage and PCR amplification products. Long template size increases the sensitivity of assay, as the longer the DNA the more chances of encountering damage (dimers). Small template amplification serves as a control, minimizing chances of encountering a damaged DNA strand, and is used for normalization of the amount of starting DNA or mitochondrial DNA copy number. We compared DNA damage between the nuclear genome and mitochondrial genome of UV treated samples for both wild type and uve1Δ (Figure 7). After one hour there is delayed repair for both nuclear and mitochondrial genomes in the uve1Δ strain compared to wild type, probably reflecting retrograde signaling between the mitochondria and nucleus or that repair pathways of the nuclear genome can be ATP dependent [43]–[48]. The key observation is that in the uve1Δ strain there is lag in the mitochondrial repair. For later time points of 4 hour and 6 hours, the uve1Δ strain repair of mitochondrial genome is delayed as compared to wild type strain, which returned to normal (Figure 7). At 6 hours recovery, amplification of the mitochondrial genome in the wild type is as it was prior to DNA damage, but for uve1Δ the lesion damage still persists. However, the uve1Δ strain shows more efficient repair of lesions in the nuclear genome. For the 4 hour and the 6 hour time points, as the repair process of the mitochondrial genome initiates in uve1Δ by some unknown mitochondrial DNA repair enzymes, the repair of the nuclear genome is faster and comparable to wild type levels. These data implicate Uve1 in the efficient repair of UV-damaged mitochondrial DNA, with evidence for a complex interplay between mitochondrial and nuclear repair and the contributions of other repair pathways. No induction of the UVE1 transcript encoding the functional form of the protein was observed under light conditions in bwc1Δ mutants (Figure 2). We examined if UVE1 is a direct target of the Bwc1-Bwc2 complex through two approaches. First, we tested if overexpression of Uve1 could rescue the UV hypersensitive phenotype of the bwc1Δ mutant in C. neoformans. Uve1 from var. neoformans was expressed under a galactose-inducible promoter (PGAL7) in the bwc1 deletion mutants of var. neoformans and var. grubii. The strains were grown overnight in media with glucose or galactose as the primary carbon source, serial diluted and plated, and UV sensitivity tests performed. Results from the var. neoformans strains are illustrated in Figure 8, and var. grubii in Figure S7. Uve1 overexpression rescued the UV sensitive phenotype of bwc1Δ as comparable to wild type when induced by galactose. In contrast, there was only slight rescue in strains grown in non-inducing glucose (Figure 8). For the bwc1Δ+PGAL7-UVE1 overexpression strains and wild type strains we performed northern analysis to check the levels of UVE1 induction (Figure S8). The levels of UVE1 transcripts were comparable between the galactose-induced PGAL7-UVE1 and the light-induced wild type strains. These results provide one piece of evidence for the Bwc1-Bwc2 complex directly controlling UV resistance in C. neoformans through regulation of the effector protein Uve1. The second piece of evidence that UVE1 is a direct target of Bwc1-Bwc2 comes from gel mobility shift assays. Bwc2 has a C-terminal GATA-type zinc finger whose binding target sites are not known in C. neoformans. We searched the promoter of UVE1 for putative Bwc2 binding sites based on those used by the WCC of N. crassa [11], [14], [49]. For instance, one light regulated element (LRE) found in the frq promoter is TCGATCCGCTCGATCCCCT, with the underlined nucleotides similar to a TCGATCTTCATCTCGATCTCCA sequence found in the promoter of C. neoformans UVE1. We amplified and radiolabeled the UVE1 promoter region with this site and performed gel mobility shift assays with recombinant Bwc2 (amino acids 26 to 383) expressed and purified from Escherichia coli (Figure 9A). A retardation in gel migration of the UVE1 promoter DNA was observed, that increased with higher Bwc2 concentration or by adding zinc which is expected for a zinc finger protein (Figure 9B). The nature of the higher mobility forms is unknown, but may represent aggregation of Bwc2 monomers. Control interactions using a non-specific DNA fragment confirmed the specificity of Bwc2 for the UVE1 promoter. These observations indicate that the UVE1 promoter is a direct target for Bwc2 binding. Light influences diverse aspects of fungal biology, presumably by acting on unique pathways in specific species. However, the potential for conserved regulation also exists, and this is predicted to reflect the original selective pressure (s) and current maintenance of light-sensing in extant fungi. Some responses to light in ascomycete fungi relate to protection against damage caused by light. For instance, expression of the DNA repair enzyme photolyase and genes for biosynthesis of carotenoid pigments are often induced by light. There are previous reports of links between light via its input in circadian rhythms with DNA repair in fungi, as well as in mice and humans. For instance PRD-4 is a checkpoint kinase 2 homolog in N. crassa that is regulated by the WCC and contributes to DNA repair [21]. Another DNA repair protein, XPA, has been shown to have circadian rhythm dependent oscillations in mouse brain [50], [51]. However, between 2. 8–6% of genes are regulated at the transcript level in response to a light exposure in ascomycete species [11], [52], [53], yielding a long list of candidate genes for further analysis. In contrast, the basidiomycete C. neoformans may serve as a simpler model for understanding the evolution of light-sensing in fungi, because (a) it does not encode a photolyase gene and pigmentation is not induced by light, (b) few genes are induced in response to light at the transcript level [19], (c) there is no evidence of photoadaptation [17], a trait that influences the intensity of the response to light, and (d) the White collar complex contains only one protein with a zinc finger DNA binding domain [17], [18]. Here, we identify the UVE1 gene as a downstream target of the WCC in C. neoformans, and show that homologs are also light regulated in species that represent two other major branches in the fungal kingdom. We suggest that UVE1 acts as the key factor controlling the UV sensitive phenotype caused by mutating BWC1 or BWC2 in C. neoformans (Figure 1, Figure 10, Figure S4), and likely plays similar roles in other fungi to survive the deleterious effects of sunlight, which has UV wavelengths as an inevitable DNA damaging component. Northern blot analysis and characterization of 5′ and 3′ ends of C. neoformans var. neoformans UVE1 showed two transcripts of differing size for the UVE1 gene. The functional complementation experiments (Figure 6) in S. pombe uve1Δ by the homologous UVE1 long isoform implies that the C. neoformans protein has similar DNA repair activities; such as against cyclobutane pyrimidine dimers, 6-4 photoproducts, apurinic/apyrimidinic sites, and stretches of single-stranded DNA nicks or gaps. Moreover the localization of C. neoformans Uve1-GFP in S. pombe is in part nuclear rather than mitochondrial (Figure 6). This explains the functional complementation of the UV stress tolerance phenotype in S. pombe uve1Δ strains, as in S. pombe the nuclear UVER pathway is attributed for survival under UV stress [32]. The UVE1 short isoform did not show any functional complementation. Bioinformatic analysis to identify the active domain of UVE1 (pfam03851) provides a possible explanation for the inactivity of the short isoform, because it does not encode the complete conserved region (Figure 3, Figure S3). Another possibility may be the absence of subcellular localization signals on the dark form, such that the protein is rendered inactive due to improper compartmentalization. Correct subcellular localization of proteins involved in DNA repair has been implicated in countering genotoxic stress [54], as improper localization can result in loss-of-function and may even lead to disease development in humans [55], [56]. The light isoform of Uve1 in Cryptococcus localizes to the mitochondria, as shown by Uve1-GFP fusion studies. C. neoformans is an obligate aerobe: it cannot survive loss of mitochondrial function from something like unrepaired DNA damage. Based on the following evidence: (a) no observed localization of Uve1 in nucleus (Figure S6); (b) localization of Uve1 to mitochondria (Figure 5); (c) strains with the UVE1 gene deleted exhibited reduced survival under UV stress and Uve1-GFP partially complements the UV sensitive phenotype of the uve1Δ strain; and (d) reduced mitochondrial DNA damage repair in uve1Δ strain comparatively to the wild type strain (Figure 7) suggest that Uve1 in Cryptococcus is required for protection of mitochondrial DNA for survival under UV stress. Overexpression of UVE1 in bwc1 mutants of either var. grubii or var. neoformans restores UV sensitivity to the wild type level (Figure 8), and recombinant Bwc2 physically binds to the promoter of UVE1 to cause a gel mobility shift (Figure 9B). These data further corroborate the hypothesis that UVE1 is a direct downstream target of Bwc1. The light-induced genes in C. neoformans were previously sought by a whole genome microarray expression analysis in var. neoformans [19]. The UVE1 gene was not identified in that study although UVE1 transcript data are present for five of the six biological replicates, with an average 1. 15 fold difference between dark and light treatment. By contrast, quantification by ImageJ of our northern blot data normalized to actin levels indicates that the longer isoform is upregulated about 16 fold in the light (Dataset S1). UVE1 remained undetected in microarray experiments because the 70-mer probe on the array is common to both light and dark transcript isoforms (Figure 3). The dark isoform must be under control of another transcription factor, using elements within the light isoform to drive transcription. These findings demonstrate one limitation of the microarray technique in comparison to more comprehensive transcript analysis techniques like tiling arrays or RNA-seq, or to conventional hybridization techniques like northern blotting that can detect alternative transcripts. Two other phenotypes associated with mutation of BWC1 or BWC2 in C. neoformans are loss of the inhibition of mating by light and reduced virulence. To further verify the role of Uve1 in other BWC related phenotypes, we examined the role of Uve1 in C. neoformans mating by crossing uve1Δ strains of both mating types under light and dark conditions. We did not find any contribution of this gene in mating, as the uve1Δ strains behaved like wild type for repression of mating by light (Figure S2). Similarly, the phenotype of the uve1Δ mutant in animal studies does not phenocopy that of bwc1Δ or bwc2Δ. A large-scale analysis of virulence has been undertaken in C. neoformans, measuring competitive survival of strains in mouse lungs [38]. Both bwc1Δ and bwc2Δ strains showed reduced proliferation, consistent with their reported role in virulence [17]. In contrast, the uve1Δ mutant had no defect in this virulence assay. We corroborated these results in a wax moth larvae model of virulence (Figure S2). We also examined a possible role of UVE1 under oxidative stress based on the mild phenotype observed in S. pombe [31], but did not find any phenotypic difference in the uve1Δ strains compared to wild type. Thus, we postulate that Bwc1-Bwc2 modulates its function via more than one downstream target (Figure 10A); of which those for virulence and mating remain to be discovered in future studies. We propose a model for the relationship between light-sensing via the WCC and Uve1 function in the mitochondria in C. neoformans (Figure 10B). It is possible that this model applies also to other fungal species for the protection of mitochondrial, nuclear or both genomes under UV stress. The White Collar complex is conserved across the fungal kingdom, with homologs present in the chytrids, Mucoromycotina, Glomeromycotina, Ascomycota and Basidiomycota. However the complex has been lost in some fungal lineages, like its absence from the Saccharomycotina [1], [2], [57]. One important question is whether any WCC downstream targets are conserved. Transcript comparisons by northern blot analysis in N. crassa and P. blakesleeanus, members of the Ascomycota and Mucoromycotina, demonstrate that UVE1 homologs are photo-regulated in at least one species in each of these fungal groups. The UVE1 homolog is also induced by light, as measured by microarray studies, in Aspergillus nidulans and N. crassa [11], [53]. Absence of regulation in bwc1 and madA-madB mutants in N. crassa and P. blakesleeanus further implicate White Collar-dependent regulation of UVE1. This suggests that in fungi that have White Collar, UVE1 is regulated in a light-dependent manner, and this regulation is lost or alternative regulation evolved in fungal species missing White Collar proteins. The presence of LOV domain containing flavin-binding photoreceptor proteins and UVE1 homologs in bacteria, like Bacillus subtilis [25], [58], warrant further examination if through convergent evolution the LOV domain type photoreceptors might be involved in regulation of UVE1 expression even more widely. The repair of photo-damage by Uve1 is conserved in many fungi and important under UV stress, irrespective of the presence of base excision (BER) or nucleotide excision repair (NER) pathways. Repair of DNA damage from UV by Uve1 is faster in comparison to NER [40]; under ancient environments in which ultraviolet levels were higher than today Uve1 could have provided a selective advantage. We estimate from genome sequencing projects that 95% of the fungal genomes encoding WC-1 also encode a copy of UVE1. Many of the exceptions have homologs of photolyase present in their genome, which may play an equivalent role as Uve1, and can also repair mitochondrial DNA [59], [60]. In summary, light triggers a number of physiological and morphological changes in fungi. The advantages of using light as a signal that are conserved have remained unclear although there is increasing evidence for a role in protecting cells from damage. Here, we demonstrate that protection of DNA, including the mitochondrial genome, through photo-regulation of Uve1 provides a benefit that is present in fungi that are able to sense light through the White Collar Complex. Gene knockout cassettes were constructed by fusion of around 1000 bp flanks 5′ and 3′ of the UVE1 gene with nourseothricin acetyltransferase (NAT) coding sequence for strain JEC21 (var. neoformans, serotype D) and neomycin phosphotransferase (NEO) coding sequence for strain KN99α (var. grubii, serotype A). Oligonucleotide primer sequences are listed in Table S1. To make JEC21 uve1Δ, 5′ and 3′ gene flanks were amplified by primer set AISV030/AISV034 and AISV032/AISV035, respectively, using JEC21 genomic DNA. To make KN99α uve1Δ, 5′ and 3′ gene flanks were amplified by primer set ai830/ai831 and ai832/ai833, respectively, using KN99α genomic DNA. The NAT and NEO ORFs were amplified by primer set ai290/ai006. Overlap PCR was performed to obtain 5′-UVE1-NAT-UVE1-3′ and 5′-UVE1-NEO-UVE1-3′ cassettes by mixing equimolar ratio of 5′-UVE1, NAT, UVE1-3′ for JEC21 and 5′-UVE1, NEO, UVE1-3′ for KN99α. Primers used to perform overlap PCR were AISV030/AISV032 and ai830/ai833 for JEC21 and KN99α, respectively. About 2 µg of 5′-UVE1-NAT-UVE1-3′ or 5′-UVE1-NEO-UVE1-3′ cassette was transformed into strains JEC21 and KN99α using biolistic delivery with a PDS 1000/He particle delivery system (Bio-Rad, Hercules, CA) [61]. Gene replacement was confirmed by PCR and Southern blots for the correct integration of the gene cassette. Strains and genotypes are provided in Table S2. For complementation of UVE1 in serotype A, a wild type copy of UVE1 was amplified with primers ALID0001 and ALID0002 and cloned into the pCR2. 1 TOPO plasmid. The insert was excised with BamHI-XhoI and subcloned into the BamHI-SalI site of pPZP-NATcc. The plasmid was transformed into strain AI191 (uve1: : NEO) by biolistics, with positive transformants selected for growth on yeast extract-peptone-dextrose (YPD) +nourseothricin (100 µg/ml) plates. The plasmids used or constructed in this study are listed in Table S3. The UV sensitivities of strains were tested by applying UV stress in an XL-1500 UV cross linker (Spectronics Corporation, Lincoln, NE). Unless otherwise stated, strains were exposed to the laboratory ambient light (400–800 LUX) during experiments. Cultures were prepared for C. neoformans strains KN99α, AI81 (bwc1Δ), JEC21, AI5 (bwc1Δ), C. gattii (R265), S. pombe L972, N. crassa wild-type FGSC 4200, N. crassa (wc-1) FGSC 4398, P. blakesleeanus wild-type NRRL1555, and P. blakesleeanus (madA madB) mutant L51. All strains of each species were plated with equal optical density or numbers of spores in duplicates on 15 cm diameter petri dishes containing YPD, and kept in darkness. For N. crassa only, 50 ml liquid cultures were grown in 50 ml YPD medium. Cultures were grown for 23 h or 47 h depending on the growth kinetics of the species. On completion of 23 h or 47 h, one of the sets was exposed to cool white light (dual Sylvania 4100 K 32W bulbs) of 1600–2600 Lux for 1 h and the other set left in the dark. On completion of the 24 h or 48 h time periods both the light and dark cultures were scraped in the light or under safe red light (GBX LED safelight, Kodak, Rochester, NY). N. crassa cultures were harvested directly from the liquid medium. All cultures were pelleted, frozen using dry ice+ethanol, lyophilized and stored at −80°C. Total RNA was isolated using Trizol reagent (Invitrogen, Grand Island, NY). To address the low transcript abundance of UVE1, total RNA was further purified for polyA mRNA isolation starting with 1 mg total RNA, using the PolyATract Kit (Promega, Madison, WI), except for P. blakesleeanus and C. gattii where 40 µg of total RNA were used. RNA samples were resolved on 1. 4% agarose denaturing formaldehyde gels and blotted on to Zeta Probe membrane (Bio-Rad). Probes for northern analysis were amplified using specific primer sets (Table S4) and radiolabeled with [α-32P] dCTP (PerkinElmer, Waltham, MA) using the RediPrime II labeling kit (Amersham, Pittsburg, PA). The blots were stripped and re-probed with fragments of actin homologs as loading controls. Autoradiograms were scanned, and transcript levels were compared by ImageJ analysis (Dataset S1). The localization of the two isoforms of Uve1 within the cell was assessed by fusions to green fluorescent protein (GFP). C. n. var. neoformans strain JEC21 genomic DNA was used as the template to amplify the UVE1 light (L) isoform using primers AISV001/AISV003 and UVE1 dark (D) isoforms using primers AISV002/AISV003. The histone 3 promoter for Cryptococcus (PH3) and GFP-NAT were amplified from the pPZP-GFP-NATcc plasmid using ai255/AISV005 (overlap primer for L) or ai255/AISV006 (overlap primer for D) for PH3, and AISV004/ai256 for GFP-NAT. The overlap construct was amplified by mixing equimolar ratios of the three amplicons for the light and dark isoforms using primers M13F and M13R. About 2 µg of gel purified UVE1 L and D overlap constructs were transformed into strain AI191 by biolistics. Positives clones were selected by their growth on YPD+100 µg/ml nourseothricin plates and confirmed by PCR, DNA sequencing, western blotting for GFP, and fluorescence signal. Strains AISVCN28 and AISVCN02 were used for localization of the Uve1 light and dark isoforms fused to GFP. Strains were stained with MitoTracker Red CMXRos (Invitrogen) at 3 nM, kept in the dark for 20 min, washed and suspended in phosphate buffered saline (PBS) and used for microscopy. Cells were imaged using Olympus confocal microscopes FLUOVIEW FV10i or FV300. Cultures for C. neoformans strains KN99α (wild type) and AI191 (uve1Δ) were grown overnight in YPD and washed with distilled water. Cells were suspended in phosphate buffered saline to 4×104 cells/ml. For each strain totals of 150 ml cells were distributed in 30 ml aliquots for time points 0 min (after stress), 1 h, 4 h, 6 h and control (no stress). For each aliquot the cells were placed in 15 cm petri dishes and exposed to UV light (50 J/m2) using a UV cross linker. Immediately after the UV stress, cells were transferred to 50 ml tubes and kept on ice. Control and 0 min cells were pelleted and frozen in liquid nitrogen. For 1 h, 4 h and 6 h time points, cells were re-suspended in YPD and incubated at 30°C for these respective times, then centrifuged to pellet and snap frozen. All samples were lyophilized, and DNA was extracted by the CTAB buffer method [62]. The relative DNA damage to the mitochondrial and nuclear genomes were assessed using a PCR assay based on established methods [42]. Concentrations of DNA samples from each treatment were standardized by measuring them by spectrophotometry and making appropriate dilutions. Primers used for amplification of fragments of the mitochondrial genome were AISV87/AISV91 (11 Kb). PCR conditions for long mitochondrial PCR were 94°C 4 min, 23 cycles for 98°C 10 s, 68°C 15 min, and a final extension of 72°C 10 min using Ex Taq (Takara, Kyoto, Japan). For nuclear long amplification (8 kb) primers were AISV85/AISV95. Conditions for long nuclear PCR were 94°C 4 min, 23 cycles for 94°C 20 s, 58°C 20 s, 72°C 6 min and a final extension of 72°C 7 min. Short amplification primers for mitochondrial genome were AISV89/AISV99 and for the nuclear genome AISV85/AISV97 amplifying about 250 bp. PCR conditions for small mitochondrial and nuclear amplicons were 94°C 4 min, 23 cycles for 94°C 20 s, 55°C 20 s, 72°C 1 min, and a 72°C 7 min final extension. All PCR amplicons were resolved on agarose gels, and intensities were quantified using ImageJ software. DNA damage was compared by calculating relative amplification of large PCR fragments of the UV treated samples to that of the respective untreated controls using the method reported in reference [42], and adjusting for differences between nuclear and mitochondrial amplicon sizes. The Galleria mellonella virulence assay followed methods that were previously described [63]. Overnight cultures in YPD medium for strains KN99α, AI191 and AI181 were washed three times with PBS. Cells were suspended in PBS to 2×107 cells/ml. For each strain 11–12 larvae were injected with 5 µl of the cells, as well as the control PBS. Wax moth were incubated at 37°C and survival monitored daily. An S. pombe uve1 knockout strain was constructed to serve for the functional analysis of UVE1 isoforms from C. neoformans. For the construction of the gene knockout cassette, genomic DNA from S. pombe strain L972 was used to amplify around 320 bp 5′-uve1 and 300 bp 3′-uve1 fragments using primer pairs AISV007a/AISV009 and AISV010/AISV011, respectively. The KanMX fragment was amplified using primer set AISV007/AISV008 from plasmid pFA6a-GFP (S65T) -kanMX6. Overlap PCR was performed to generate the gene knockout cassette using primer set AISV007a/AISV011. Around 2 µg of the PCR construct were transformed into S. pombe (strain MM72-4A ura4-D18 h−) by lithium acetate transformation and cells plated on to YPD+100 µg/ml G-418. Gene knockouts were confirmed by PCR and Southern blotting. S. pombe uve1 knockout strain AISVSP1 was selected for C. neoformans UVE1 complementation studies. UVE1 cDNA was reverse transcribed from RNA of C. neoformans strain JEC20 using Superscript III First strand Synthesis System (Invitrogen), as per company instructions (JEC20 is isogenic to JEC21, with a different MAT allele; [64]). The synthesized cDNA was amplified by site directed mutagenesis to abolish an NdeI site inconvenient for subcloning while conserving the encoded amino acid residue, and to introduce NdeI and BamHI restriction sites at the start and end of the UVE1 gene. Primers used for amplification of fragment 1 of the L and D form were AISV014/AISV013 and AISV015/AISV013, respectively. Primers used for amplification of fragment 2 were AISV012/AISV016. Overlap PCR was performed to amplify full L and D genes from fragment 1 and 2, using primers AISV014/AISV016 for UVE1 L form and AISV015/AISV016 for UVE1 D form. The NdeI and BamHI digested cassettes were ligated into the NdeI-BamHI site in the pREP42 vector enabling expression from an nmt promoter [39]. Positive clones were confirmed by sequencing. Plasmids containing UVE1 L and D isoforms were transformed into S. pombe strain AISVSP1 (ura4-D18 uve1: : kanMX) by the lithium acetate method. Empty vector pREP42 was transformed into strain AISVP1 as a control. Positive S. pombe transformants were selected on minimal medium without uracil and were confirmed by PCR. C. neoformans var. neoformans Uve1 (L) C- terminal GFP localization in S. pombe was done by fusion of UVE1 (L) to GFP by overlap PCR. For fragment 1, UVE1 (L) was amplified from pREP42-UVE1 (L) using primers AISV014/AISV003 and the fragment 2, GFP, was derived from pPZP-GFP-NATcc using primers AISV004/AISV066. Overlap PCR joining fragments 1 and 2 was performed by primer set AISV014/AISV066. The overlap PCR product was cloned into pCR 2. 1 TOPO, and transformed by heat shock into E. coli DH5α. Positive clones were selected and sequenced. A plasmid containing the desired Nde1-UVE1-GFP-BamHI overlap was digested with NdeI and BamHI. The Nde1-UVE1-GFP-BamHI digest was ligated with NdeI and BamHI digested pTN157. Plasmid pTN157-UVE1-GFP was transformed into S. pombe strain AISVSP1 (genotype ura4-D18 uve1: : kanMX) by the lithium acetate method. Positive S. pombe transformants were selected on minimal medium without uracil and were confirmed by PCR. Transformants were examined for fluorescence signal and their UV resistant phenotype. Confocal microscopy was performed for strain AISVSP15. For both mitochondrial and nuclear staining, cells were grown in Edinburgh Minimal Medium (EMM). Mitochondrial staining was performed with MitoTracker Red CMXRos (Invitrogen) at a final concentration of 3 nM in water, kept in the dark for 20 min, washed and suspended in PBS and used for microscopy. Hoechst 33342 was used to stain the nucleus. Cells grown in EMM were washed and suspended in Hoechst (1 µg/ml in water) for 10 min. Cells were washed and suspended in PBS, and microscopy was performed. Overnight cultures for strains L972, AISVSP1, AISVSP2, AISVSP3, AISVSP4 and AISVSP15 in YES media were subcultured the following day. Strains in exponential phase were dotted on YES medium in ten-fold serial dilutions. One set of plates was exposed to UV light (120 J/m2) using the UV cross linker, and another plate kept unexposed. Both UV treated and unexposed sets were incubated at 30°C for 2 days. For UV dose response experiments exponentially growing cells for strains at the same optical density were ten-fold serially diluted and equal volumes for all dilutions of the cells were plated on YES media. The control was kept unexposed to UV and others were exposed at UV doses of 60,120 and 180 J/m2. All plates were incubated at 30°C for 3 days, and colony forming unit data analyzed for percentage survival. The UVE1 gene was over-expressed in bwc1 knockout backgrounds to assess if UVE1 can rescue the UV sensitive phenotype of bwc1 mutation. A fusion cassette of the JEC21 GAL7 promoter (PGAL7) and UVE1 gene was made by overlap PCR. The GAL7 promoter was amplified by primer set AISV025/AISV028 and UVE1 was amplified using primer set AISV027/AISV026 on JEC21 genomic DNA. The PGAL7-UVE1 fusion cassette was amplified by mixing GAL7 promoter and UVE1 PCRs in equimolar ratios by primer set AISV025/AISV026. The PGAL7-UVE1 fusion cassette was cloned in a TA vector (pCR 2. 1 TOPO; Invitrogen), and transformed by heat shock in E. coli Top10 cells. Positive clones were selected and sequenced. A plasmid containing PGAL7 -UVE1 was digested with BamHI and XhoI. The fragment was ligated with pPZP-NEO Agrobacterium vector digested with the same enzymes, and transformed into Top10 cells. Positive clones were selected by their growth on kanamycin and verified by PCR and restriction digestion. The pPZP-NEO-PGAL7-UVE1 vector was transformed by electroporation into A. tumefaciens strain EHA105. Selected positive Agrobacterium transformants were co-cultured with C. neoformans strains AI5 and AI81. Positive transformants were selected for growth on YPD+G-418+cefotaxime plates. Transformants were cultured overnight in yeast nitrogen base (YNB) medium +2% galactose or 2% glucose, and 10-fold serial dilutions placed on YPD medium plates. Dotting was performed in duplicate and one set was exposed to UV radiation at 120 J/m2. Both sets were incubated at 30°C for 2 days. JEC21 RNA was reverse transcribed to make cDNA using the Superscript III First strand Synthesis System (Invitrogen). A fragment of BWC2 cDNA was amplified using primers AISV040/AISV041, containing BamHI and EcoRI restriction sites. The amplicon was digested with BamHI and EcoRI, and ligated into the pRSETA vector (Invitrogen) digested with the same enzymes. Top10 cells were transformed with the ligation product. An error free clone was identified by sequencing. The plasmid containing BWC2 was transformed into BL21 (DE3) pLysS cells and selected on ampicillin+chloramphenicol SOB medium plates. Protein induction and expression using 1 mM IPTG was performed as described for the pRSET expression system by Invitrogen. The (Histidine) 6-tagged Bwc2 protein was semi-purified using Pure Proteome Nickel Magnetic beads (Millipore Corporation, Billerica, MA) as per the manufacturer' s instructions. For electrophoretic mobility shift assays (EMSA), a 244 bp (P1) region 5′ of the start codon of UVE1 containing putative Bwc2 binding sites was amplified using primer set AISV019/AISV020. A control nonspecific DNA fragment (NS) of 278 bp was amplified from pRS426 vector using primers ALID1229/ALID1230. About 600 ng of the amplified fragment from UVE1 promoter region and nonspecific probe was radiolabeled with [γ-32P] dATP (PerkinElmer) using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). Labeling conditions were 1X T4 polynucleotide buffer, 5 µl 6000 Ci/mmol γ-32P ATP, 10 units T4 polynucleotide kinase in a 50 µl reaction mixture at 37°C for 30 min. The radiolabeled probes were purified using PCR purification columns (Qiagen, Germantown, MD). The composition of 5X EMSA binding buffer used was 20% w/v glycerol, 5 mM MgCl2,250 mM NaCl, 2. 5 mM EDTA pH 8,10 mM DTT with or without 12 µM ZnSO4 (reference [65] with slight modifications). 20 to 50 µg of purified Bwc2 protein were pre-incubated with 1X EMSA binding buffer for 20 min at room temperature for all reactions. Total reaction mixture of 20 µl consisted of 1X EMSA binding buffer, 20 to 50 µg of purified protein, cold probe (if added), 1 µl of radiolabeled probe and 1X phosphate buffer, followed by 50 min incubation at room temperature. For competition reactions, 600 ng and 900 ng of the cold probes were included in the reaction mixture prior to addition of radiolabeled probes. At the end of 50 min incubation, samples were transferred to ice followed by addition of EMSA loading dye. Samples were loaded on 6% polyacrylamide Tris-borate-EDTA (TBE) gels (Invitrogen), and run at 130 V for 2 h in 1X TBE at 4°C. Autoradiography was performed by exposure to Gene Mate Blue ultra autoradiography films.

Title: The Uve1 Endonuclease Is Regulated by the White Collar Complex to Protect Cryptococcus neoformans from UV Damage Summary: The majority of fungi sense light using the White Collar complex (WCC), a two-protein combination of a photoreceptor and a transcription factor. The WCC regulates circadian rhythms, sexual development, sporulation, metabolism, and virulence. As such, the exposure to light controls properties of fungi that are beneficial and detrimental to people, depending on the species and its interaction with humans. Despite the importance of light on fungal biology, the underlying evolutionary benefit of light-sensing in fungi has remained a mystery. Here we identify a DNA damage repair endonuclease, Uve1, required for UV stress tolerance in the human pathogen Cryptococcus neoformans. UVE1 is a direct target of the WCC in C. neoformans, and UVE1 homologs are also regulated by WCC in two other major lineages of fungi, the Ascomycota and Mucoromycotina. The divergence of the three groups indicates that for about a billion years the same transcription factor complex has regulated a common gene to protect fungal genomes from deleterious effects of light. Curiously, in C. neoformans Uve1 localizes to mitochondria and contributes to mitochondrial DNA repair, implicating its importance in genome repair of this organelle. Thus, light-sensing in fungi exists to protect them against harmful light, and likely all other responses to light relate to or are a secondary consequence of this selective pressure.

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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a method of preventing development of multidrug resistance (hereinafter MDR) and to a method of reversing MDR if one already exists thereby to prevent or correct drug accumulation defect in drug resistant cells. More particularly, the invention relates to the administration of masoprocol along with antineoplastic/cytotoxic drugs to which cells develop multidrug resistance (MDR), e.g. Doxorubicin, Daunorubicin, Amsacrine Mitoxantrone, Dactinomycin, Ellipticine, Etoposide, Teniposide, Chlorambucil, Melphalan, Cyclophosphamide, Nitrosoureas BCNU, CCNU, MeCCNU), Methotrexate, Trimetrexate, 5-FU, Ara-C, Ara-A, Cisplatin, Carboplatin and Taxol. 2. Reported Developments Patients having solid malignant tumors e.g., breast, ovarian, lung, colon and hematological malignant disorders, such as refractory myeloma, often develop MDR.... MDR is associated with the overexpression of an MDR-1 gene that codes for a plasma membrane P-glycoprotein. The expression of the MDR-1 gene is believed to be associated with a decreased cellular accumulation of drug due to an active energy dependent efflux mechanism. The active efflux of drug is believed to be mediated by the P-glycoprotein efflux pump. Attempts have been made to reverse MDR and to correct the drug accumulation defect in drug resistant cells. Among the agents which show promise in vitro are verapamil, quinidine, amioderone, cyclosporin and certain phenothiazines. It appears, however, that in vivo concentrations necessary to reverse MDR cannot be achieved without substantial toxicity to patients. SUMMARY OF THE INVENTION Masoprocol is new MDR modulating agent. We have discovered that the combination of masoprocol known as nordihydroguaiaretic acid (hereinafter sometimes referred to as NDGA) or certain analogues thereof in combination with cytotoxic agents, e.g. Doxorubicin, Daunorubicin, Amsacrine, Mitoxantrone, Dactinomycin, Ellipticine, Etoposide, Teniposide, Chlorambucil, Melphalan, Cyclophosphamide, Nitrosoureas (BCNU, CCNU, MeCCNU), Methotrexate, Trimetrexate, 5-FU, Ara-C, Ara-A, Cisplatin, Carboplatin and Taxol can overcome multidrug resistance of certain diseased cells and has the potential of being effective in the treatment of solid malignant tumors e.g., brain, breast, colon, lung, ovarian cancers and hematological malignant disorders including lymphoma, leukemia (acute nonlymphocytic leukemia, acute myelocytic leukemia), or increase the therapeutic effectiveness of the above-mentioned cytotoxic agents. The naturally occurring meso form of the nordihydroguaiaretic acid [meso- 1,4-bis (3,4-dihydroxyphenyl)-2,3-dimethylbutane] (&#34;NDGA&#34;) was reported as providing a positive result against malignant melanoma. C. R. Smart, et al, Rocky Mountain Medical Journal, November 1970, pp. 39-43. (Unless otherwise indicated, NDGA is used herein to refer to the meso form of nordihydroguaiaretic acid). NDGA is found in the creosote bush, and this plant was used for centuries to brew tea which was the basis for a folk remedy that called for drinking the tea to cure colds, rheumatism and other ailments. However, this remedy has not proven to be successful. A clinical study was conducted by Smart et al, reported in U.S. Pat. No. 4,880,637, in which human cancer patients ingested either a tea made form the creosote bush or doses of pure NDGA. This study indicated that neither NDGA nor the tea were effective anticancer agents and in some cases caused stimulation of tumor cell growth. This confirmed the earlier screening studies of NDGA conducted by Leiter et al of the Cancer Chemotherapy National Service Center of National Cancer Institute which obtained negative results when NDGA was tested against several types of cancer cells. It has been reported in U.S. Pat. No. 4,880,637 that NDGA and its analogues in combination with ionic zinc is effective in the treatment of benign, paramalignant and malignant growth of the skin without detrimental side effects associated with chemotherapy or chemosurgical techniques. NDGA was also reported in U.S. Pat. No. 4,695,590, as having efficacy in retarding senescence or aging in mammals. To our knowledge, NDGA in combination with other cytotoxic/antineoplastic chemotherapeutic drugs is not known, nor have been reported to inhibit multidrug resistance associated with the treatment of solid malignant tumors or hematological disorders in mammals. In the modern clinical oncology practice, it has been established that overexpression of P-glycoprotein (Pgp) in cancer patients is directly related to the increase in the multidrug resistance (MDR) to treatment with presently available chemotherapies. The present inventors have discovered that masoprocol (NDGA) overcomes the Pgp mediated resistance and Pgp antagonism of the cells, thereby increasing the levels of chemotherapeutic drugs which accumulate in the cancer cells. The present invention provides a method and composition for the inhibition and/or reversal of multidrug resistant phenomenon in a patient and thereby treatment of solid malignant tumors and hematological malignancies comprising the administration of NDGA to said patient preferably in a pharmaceutically acceptable vehicle. The general formula comprises of from about 0.1% to about 25% NDGA or an analogue thereof in a pharmaceutically acceptable vehicle for topical application or of from about 1 mg NDGA to about 500 mg of NDGA per kg of body weight for systemic administration. NDGA and its analogues used in the present invention have the following Formula (I): ##STR2## wherein R 1 and R 2 are independently H, lower alkyl or lower acyl; R 3, R 4, R 5, and R 6 are independently H or lower alkyl; R 7 R 8 and R 9 are independently H, hydroxy, lower alkoxy or lower acyloxy; and R 10, R 11, R 12 and R 13 are independently H or lower alkyl. Lower alkyl is intended to generally mean C 1 -C 6 alkyl, and preferably R 3 and R 4 are C 1 -C 3 alkyl. Lower acyl is intended to generally mean C 1 -C 6 acyl, with C 2 -C 6 acyl being preferred. The formula is directed to both the phenolic compounds and the conventional esters and ethers thereof. DETAILED DESCRIPTION OF THE INVENTION NDGA and its analogues (also referred to herein as catecholic butanes) according to the present invention, may be made by the synthetic methods provided in U.S. Pat. Nos. 4,562,298 and 4,954,659 which are incorporated herein by reference in their entirety. Examples of catecholic butanes include: the d-, 1-racemic mixture of d- and 1-, meso-isomers of 1,4-bis(3,4-dihydroxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-dihydroxyphenyl)butane; 1,4-bis(3,4-dimethoxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-diethoxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-dipropoxyphenyl)-2,3-dimethylbutane; 1-(3,4-dihydroxyphenyl)-4-(3&#39;,4&#39;,5&#39;-trihydroxyphenyl)butane; 1,4-bis(3,4-diacetoxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-dipropionyloxyphenyl)2,3-dimethylbutane; 1,4-bis(3,4-dibutyroyloxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-divaleroyloxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-dipivaloyloxyphenyl)-2,3-dimethylbutane; 1,4-bis(3,4-dineopentylcarboxylphenyl)-2,3-dimethylbutane; 1-(3,4-dihydroxyphenyl)-4-phenylbutane and 1-(3,4-dihydroxyphenyl-4-(2,5dihydroxyphenyl)butane. Mixtures of Formula (I) may be used in the instant composition as well. The compositions of the present invention are believed to be effective in the treatment of solid mammalian tumors or hematologic malignancies which can develop multidrug resistance. These solid tumors include cancers of the head and neck, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, rectum, anus, kidney, ureter, bladder, prostate, urethra, penis, testis, gynecological organs, ovarian, breast, endocrine system, skin, central nervous system; sarcomas of the soft tissue and bone; and melanomas of cutaneous and intraocular origin. Itematological malignancies include childhood leukemias and lymphomas, Hodkin&#39;s disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, plasma cell neoplasm and cancers in AIDS. The preferred mammalial species to be subjected to the present invention are humans and domesticated animals. The instant compositions can be applied topically, orally or parentially to the treatment site. When used for topical application, NDGA is usually formulated with a pharmaceutically acceptable carrier. Carrier materials are well known in the pharmaceutical formulation art and include those materials referred to as diluents or vehicles. The carrier may include inorganic or organic materials and should have sufficient viscosity to allow spreading of the composition and provide good adherence to the tissue to which it is topically applied. Examples of such carriers include, without limitation, polyols such as glycerol, propylene glycol, polyethylene glycol, preferably of a molecular weight between about 400 to about 8000, suitable mixtures thereof, vegetable oils, and other materials well known to those skilled in this art. The viscosity of the formulation can be adjusted by methods well known in the art, for example, by the use of a higher molecular weight polyethylene glycol. In addition to NDGA, the formulation can contain pharmacologically-acceptable additives or adjuvants such as antimicrobial agents, e.g. methyl, ethyl, propyl and butyl esters of parahydroxybenzoic acid as well as chlorobutanol, phenol, ascorbic acid, etc. The formulation can also contain thickening or gelling agents, emulsifiers, wetting agents, coloring agents, buffers, stabilizers and preservatives including antioxidants such as butylhydroxyanisole in accordance with the practice of the art. The formulation can also contain penetration enhancers such as dimethylsulfoxide, long-chain alcohols such as nonoxynol, long-chain carboxylic acids, propylene glycol, N-(2-hydroxyethyl)pyrrolidone, 1-dodecyl-azacycloheptan-2-one, and the like. Depending on the method of application and the disease being treated, it may be desirable to use absorption-delaying agents such as aluminum monostearate and gelatin. The compositions of the formulation can be adjusted using components well-known in the formulation art to provide a pharmaceutical formulation which is a gel, cream, ointment, solid, liquid, semi-solid, etc. The particular physical form of the formulation depends on the desired method of treatment and the patient to be treated. For administration by injection, the compositions according to the invention are formulated as solutions or suspensions having a low enough viscosity to be injected. The composition suitable for injectable use must be sterile and fluid to the extent that easy syringe injection exists. It should also be stable under conditions of manufacturer and storage and be preserved against contamination by microorganisms. Preservatives include alcohol, benzoic acid, sorbic acid, and methyl and propyl parabin with and without propylene glycol. Additionally, the pH of the composition must be within a range which does not result in tissue damage, namely, between about 3.0-7.5. The concentration of NDGA in a particular formulation depends on the condition being treated, the method of application, i.e. topical or injection, the rate of delivery of the active ingredient(s) to the treatment site, and the number of applications of the formulation which can be used. Additionally, certain NDGA analogues are more effective in treating particular conditions that are other analogues. The concentration of verapamil in the formulation likewise depends upon the condition being treated and the particular NDGA analogue or combination of analogues being used. The pH of the formulation is important in assuring stability of NDGA or its analogue as well as assuring that the formulation is physiologically acceptable to the patient. Many of the analogues, and particularly nordihydroguaiaretic acid, are susceptible to oxidation, for example, by air. Such oxidation can result in discoloration of the formulation rendering it unacceptable for pharmaceutical use. These catechols are more stable against oxidation at lower pH levels. Therefore, it is preferred that if the formulation is to be exposed to oxidizing conditions the pH to be maintained below about 7 and preferably below about 6 in order to provide maximum stability for the compound against oxidation. However, if the oxidizing conditions can be avoided, for example, by storage of the formulation under an inert atmosphere such as nitrogen, a higher pH can be used. The pH of the formulation may be maintained through the use of toxicologically-acceptable buffers. Such buffers are well-known in the pharmaceutical formulation art, and include hydrochloric acid buffer, acid phthalate buffer, phosphate buffer and citric acid/sodium citrate buffer. The following example will further illustrate the formulations of the present invention. ______________________________________Active Ingredient______________________________________NDGA 10Excipients, Purified water USP 48.25Propylene glycol USP 12.0Polyethylene glycol 400 NF 7.5Stearyl alcohol NF 5.0Light mineral oil NF 5.0Synthetic beeswax 3.5Isostearyl alcohol 3.0Steareth-2 2.75Steareth-21 2.25Xanthum gum NF 0.40Methylparaben, NF 0.16Sodium bisulfite 0.10Propylparaben NF 0.08Citric acid USP 0.01______________________________________ For systemic administration, NDGA or analogues can be given as a suspension in methocel, buffered saline, i.v. as a sterile injectable and orally as a tablet or a capsule. The tests were performed to determine the effect of NDGA on efflux of doxorubicin (hereinafter sometimes referred to as DOX) for doxorubicin sensitive MCF7/0 and doxorubicin resistant MCF7/ADR human breast adenocarcinoma cells in vitro. The cells were exposed to 50 μg/ml NDGA for 24 hrs prior to loading with DOX (doxorubicin) at 37° C. Each cell line was loaded with DOX (5 μg×30 min) and selected samples were treated with verapamil (30 μl of a 1 mM solution) at the end of the loading period and then pelleted and suspended in 37° C. PBS. Flow analysis of cell efflux of DOX was collected over 30 minutes using the time parameter. The results are shown in Table I. TABLE I__________________________________________________________________________THE EFFECT OF NDGA ON STEADY STATE ACCUMULATIONOF DOX IN MCF7/0 AND MCF7/ADR CELLS LINES CHANNEL % DOX % DOX FLUORESCENC INCREASE INCREASE TREATMENT E w/VERAPAMIL w/NDGA__________________________________________________________________________MCF7/0No NDGA DOX 318.7 -- -- DOX + Verapamil 312.8 0.981 --RX NDGA DOX 460.4 -- 44.5 DOX + Verapamil 435.6 0.946 --MCF7/ADRNo NDGA DOX 227.4 -- -- DOX + Verapamil 309.9 36.3 --RX NDGA DOX 285.6 -- 25.6 DOX + Verapamil 365.4 27.9 --__________________________________________________________________________ DOX = Doxorubicin In the table, the loading of DOX in cells (Mean Channel Fluorescence) and the % value represent the increase of DOX measured in the cells after treatment with verapamil or NDGA. Two interesting results are seen with this experiment: 1) the MCF7 cells (sensitive) are loading more DOX after NDGA treatment than controls or verapamil treated cells. This is of interest because verapamil has no effect on sensitive cells. 2) In the resistant MCF7/ADR cells there is an augmentation of DOX retention in both NDGA and NDGA+verapamil. The results indicate NDGA will inhibit the efflux of DOX via the p170 (p-glycoprotein) resistance factor. 3) Treatment of multidrug resistant MCF7/ADR and its parent cell line MCF7/0, human breast adenocarcinoma cells line with 50 μg/ml in clonogenic assay resulted in an increased doxorubicin accumulation of 25.6% and 44.5% respectively. In comparison experiment of same cell lines with verapamil treatment resulted in a 36% increased accumulation in the doxorubicin resistant cells (i.e. MCF7/ADR) and no increase accumulation of doxorubicin concentration in the parent MCF7/0 cells. We conclude that NDGA is a novel agent capable of reversing MDR and potentiation of chemotherapeutic activity of doxorubicin in vitro and its usage as reversing the multidrug resistance in solid malignant tumors and hematological malignancies. Procedure for P-Glycoprotein (PGP) Function Assay of Doxorubicin Effiux is shown hereunder. 1. Verapamil (GFW=491.1) Make a 1 mM solution by dissolving 4.911 mg of Verapamil per 10 mls of distilled water. Store at room temperature. 2. Doxorubicin (GFW=580) Make a solution of doxorubicin to contain 10 μg/ml. Store frozen at 0° C. 3. Rhodamine 123 (GFW=380.0) Make a 1 mM solution by dissolving 3.808 mg per mls of absolute ethanol. Store at -20° C. 4. NDGA (MW=302.36) Make a solution of NDGA to contain 50 μg/ml. Store frozen at 0° C. 5. MCF7/0 (Doxorubicin sensitive) and MCF7/ADR (Doxorubicin resistant), human breast adenocarcinoma cell lines. Procedure 1. Make up 2×10 6 cells per ml in PBS. Expose 1 ml of cells to 50 μg of NDGA for 24 hrs at 37° C. prior to loading with Doxorubicin. 2. Add 1 ml of 10 μg/ml doxorubicin, mix and divide into two aliquots. 3. Add 30 μl of verapamil to one aliquot. 4. Incubate both tubes at 37° C. for 30 minutes. P-glycoprotein function is temperature dependent, so do not place tubes on ice afterwards. 5. Centrifuge the tubes at 37° C. for 5 minutes on a fast spin to pellet cells. Resuspend in 1 ml of buffer. 6. Run immediately on the flow cytometer. Excitation 488 nm, emission 560-600 nm. Collect forward and 90° side scatter and PMT4 linear scale. Collect 10,000-50,000 events and calculate the mean channel fluorescence (MCF) for PMT4. 7. Run both samples as close together as possible, and avoid prolonged exposure to room temperature. Calculations 1. Express results as percent increase in mean doxorubicin fluorescence in the verapamil-containing sample. ##EQU1## Procedural Notes 1. If rhodamine 123 is used in the place of doxorubicin ad fluorescent stain, suspend 2×10 6 viable cells in 2 mls of PBS, add 10 μl of rhodamine to the cells, mix, and divide into two equal aliquots. Add verapamil to one tube and incubate as above. Collect green (PMT2) fluorescence. 2. Keep cells at 37° C. Avoid prolonged exposure to room temperature and do not place cells on ice at any step of the procedure. Table II shows anti-tumor activity of NDGA against various tumor cell lines. TABLE II______________________________________MASOPROCOL - (NDGA) IN VITRO (CYTOTOXICITY)ANTI TUMOR ACTIVITY IC.sub.50 (μM) One Hour *Continuous Pulse Treatment Drug Exposure Doxo- Doxo-Tumor Cell Line Masoprocol rubicin Masoprocol rubicin______________________________________CAK-1 Renal 211 3.8 53.4 0.11DLD-1 Colon 124 3.7 0.08 0.42Lung (NCI-H23) 172 9.3 0.08 0.15Lung (LX- 1) 18 26ChemexLOX-IMVI 38.4 4.8 0.08 0.09MelanomaSNB 312 0.7 0.08 0.03GlioblastomaOVCAR3 258 13.8 35.9 0.7OvarianWI-38 Lung 159 10.0 312 0.6FibroblastsDetroit 551 Skin 62.7 3.2 312 5.5FibroblastsHs 68 Newborn 178 11.6 18 0.12ForeskinFibroblasts______________________________________ *Drug Treatment with observation at 70 hours As various changes might be made in the formulations of the invention herein disclosed, without departing from the spirit and principles of the invention, it is understood that all matter herein described shall be deemed illustrative, and not limiting except as set forth in the appended claims.

Summary: Disclosed are: a method for reversing multidrug resistance in a mammal; and a composition to reverse multidrug resistance comprising: ##STR1## wherein R 1 and R 2 are independently H, lower alkyl or lower acyl; R 3, R 4 and R 5 are independently H or lower alkyl; R 7, R 8 and R 9 are independently H, hydroxy, lower alkoxy or lower acyloxy; and R 10, R 11, R 12 and R 13 are independently H or lower alkyl, in a pharmaceutically acceptable vehicle.

big_patent

en

Summarize: Pictures of the victims of Wednesday's prison bus crash in Texas have been released. The bus skidded off an icy highway near Penwell, Texas, slid down an embankment and collided with a passing freight train, killing eight inmates and two corrections officers, including the bus driver. The Texas Department of Criminal Justice confirmed the 10 deaths in a statement, adding that four prisoners and one corrections officer were also injured. Scroll down for video. Top row, from left, correctional officers Christopher Davis, Eligio Garcia and Jason Self, and inmates, Jesus Reyna and Jeremiah Rodriguez; middle row, from left, inmates Adolfo Ruiz, Michael Stewart, Tyler Townsend, Angel Vasquez and Byron Wilson; bottom row, from left, inmates Kaleb Wise, Terry Johnson, Damien Rodriguez, Hector Rivera and Remigio Pineda. The surviving corrections officer, Jason Self, was in critical condition at a hospital in Lubbock. Inmates Johnson, Damien Rodriguez and Rivera were in critical condition in Odessa, while Pineda was in serious condition. A prison system statement identified the dead as correctional officers Christopher Davis, 53, and Eligio Garcia, 45; and inmates Byron Wilson, 34; Tyler Townsend, 29; Jesus Reyna, 44; Kaleb Wise, 22; Adolfo Ruiz, 32; Michael Sewart, 25; Angel Vasquez, 31; and Jeremiah Rodriguez, 35. The statement did not say which officer was driving the bus. Davis was had more than 17 years of service with the Department of Criminal Justice, and Garcia had nearly 23 years of service. The inmates were serving sentences that ranged from one year for labeling unauthorized recordings to 20 years for drug possession with intent to distribute, according to online prison records. Correctional officer Jason Self, 38, and inmates Terry Johnson, 22, and Damien Rodriguez, 22, were hospitalized in critical condition, the prison system said. Inmates Remigio Pineda, 34, and Hector Rivera, 37, were in serious condition. The bus carrying prisoners and corrections officers fell from an overpass in Penwell, Texas, and crashed onto train tracks below (AP Photo/The Odessa American, Mark Sterkel) Only the driver's seat had a seat belt, he said. Like many buses, the vehicle did not have seat belts on the bench-type seats where the prisoners were seated. The prisoners were handcuffed together in pairs, but did not have leg restraints. Some of them were ejected from the bus after it struck the train, said Trooper Elizabeth Barney of the Texas Department of Public Safety. The overpass on Interstate 20 was slick with ice Wednesday morning when the Texas Department of Criminal Justice bus left the roadway in Penwell, just west of Odessa, according to Ector County Sheriff Mark Donaldson. An earlier accident on the I-20 overpass may have contributed to the prison bus losing control, Donaldson said. 'It's as bad as you can imagine,' Odessa Fire and Rescue Battalion Chief Kavin Tinney told the Odessa American newspaper. 'In 32 years it's as bad as anything I've seen.' Jason Clark, a Department of Criminal Justice spokesman, said the bus was new and had been placed in service only this past summer. It was taking the inmates from the Middleton prison in Abilene to the Sanchez prison in El Paso, which is about 250 miles west of where the accident happened. After the accident around 7:30 a.m., the white bus came to rest on its side, next to the railroad tracks, crumpled with heavy damage to its front and undercarriage. The top of the bus was caved inward. The Union Pacific freight train with four locomotives and 58 cars came to a stop soon after. None of the cars derailed, but two containers at the rear of the train were damaged, said Mark Davis, a railroad spokesman. The containers were carrying hundreds of parcels and packages, many of which were strewn along the tracks. No Union Pacific employees were injured. The freight train containers were carrying hundreds of parcels and packages, many of which were strewn along the tracksin the aftermath of the accident (AP Photo/Odessa American, Mark Sterkel) The train, which was traveling from the Los Angeles area to Marion, Arkansas, remained stopped at the accident site several hours after the accident, Davis said. The National Transportation Safety Board said it was sending a team of inspectors to the scene. Texas Lt. Gov. Dan Patrick issued a statement offering condolences to the families of those killed in the wreck. 'I also pray for a speedy recovery of a third correctional staff member and four offenders who were transported with injuries,' he said. In June, an inmate was killed and several other people were injured when a Department of Criminal Justice van collided with a car in Central Texas. Boxes are strewn out of a UPS train car that was damaged when the bus hit the moving train outside Penwell, Texas, on Wednesday. 10 people were killed in the crash (AP Photo/Odessa American, Mark Sterkel)

Summary: Two corrections officers were killed, and one is in critical condition after bus crashed before collision with a freight train. Eight inmates are dead and four hospitalized following incident outside Penwell, Texas, on Wednesday. Only the driver had a seat belt and the prisoners were handcuffed together in pairs on bench seats. Some of the inmates were ejected from the bus after it struck the train. The bus came to came to rest on its side, crumpled with heavy damage to its front and undercarriage, with the top caved in.

cnn_dailymail

en

Summarize: In an age of streaming media, we're still a culture of digital hoarders. That may partly explain why on Tuesday, Apple introduced an iPad with 128 gigabytes of storage capacity, twice as much as any previous model. But will the average tablet owner need that much space? For casual users, tablets are media-consumption devices. We use them to watch movies and TV shows, listen to music, play games and read books and magazines. All those files take up memory on your internal hard drive. Today's high-resolution photos and videos are increasingly hogging storage, too. It's easy to see how someone with a lot of downloaded files might eventually run out of room on a 64GB tablet. But 128 gigabytes? That's the equivalent of about 100 DVD-quality movies, 30 Blu-ray movies, 30,000 songs or 40,000 photos, according to this graphic by a computer store in the United Kingdom. Everything else about the iPad announced Tuesday is the same as the current fourth-generation models: a 9.7-inch Retina display, a dual-core A6X processor, a FaceTime HD camera, 2 gigabtyes of RAM and an estimated 10 hours of battery life. So the new device, which goes on sale February 5, is all about giving users more storage space. That's why Apple is targeting the 128GB iPad at professional users such as architects, doctors or audio engineers, who often work with large files (and can afford pricey gadgets). "Companies regularly utilizing large amounts of data such as 3D CAD files, X-rays, film edits, music tracks, project blueprints, training videos and service manuals all benefit from having a greater choice of storage options for iPad," Apple said in a news release announcing the new iPad. Some observers say the tablet's cost -- $799 (Wi-Fi only) and $929 (Wi-Fi and cellular), $100 more than the current 64GB model -- isn't worth it in an age when we can store movies, music, photos and documents on cloud-based servers instead of our own machines. "Apple doesn't expect you to buy a 128GB iPad, not unless you're a professional-grade buyer, like an architect or a supervillain," writes Leslie Horn for Gizmodo. "The 128GB iPad is like a $300/head steakhouse dinner. It goes on the corporate account." Other pundits noted that at $929, the top-of-the-line 128GB model costs almost as much as the base-model MacBook Air laptop ($999), which is much easier to type on. "Apple has set its eyes on the dying infrastructure of the PC industry and Microsoft's Windows operating system business," writes Dan Rowinski of ReadWriteWeb. "That is really the only reason that Cupertino would unveil an iPad with 128 GB, a size that challenges many of the 'ultrabooks' that have become popular in the laptop market these days." In that way, the launch of the new iPad seems poised to stave off competition from Microsoft's 128GB Surface Pro tablet, due February 9 in the U.S. and Canada. When it comes to storage capacity, though, these tablets aren't close to being the biggest. That distinction belongs to the Panasonic Toughbook H2, which retails for $3,579 and has a whopping 320GB of internal storage

Summary: Apple introduces iPad with 128 GB of storage capacity. twice as much as previous models. The average user won't need that much internal storage. 128 gigabytes is equivalent of about 100 DVD-quality movies, 30,000 songs or 40,000 photos. Gizmodo: "128 GB iPad is like a $300/head steakhouse dinner. It goes on the corporate account".

cnn_dailymail

en

Summarize: BACKGROUND OF INVENTION The present invention relates to a fish meal manufacturing system. Fish meal is presently manufactured generally in the following manner: Raw fish is fed from a hopper continuously in a limited quantity, and is cooked by steam in a cooker. Then, after separation of almost all boiled liquid the cooked material is pressed for the further separation of pressing water. Fish cake produced by press moisture removal is dried in a dryer, and is ground to produce fish meal. Boiled liquid and pressing water are mixed to produce a fluid (called press water), which is fed at a suitable temperature to a separator. The fluid mixture is separated into fish oil, fish solubles, and wet fish meal. Prior-art processing systems are not of a closed type and the foul gases produced in such systems are discharged into the atmosphere at any point where a solid material must be carried to subsequent processing equipment. Furthermore, not much emphasis is placed upon cleaning-up of foul gases and polluted water. This results in severe environmental problems caused by offensive odors and water pollution in regions where plants under conventional systems are operated. Still further, on existing processing systems, raw fish which can rapidly lose its freshness are continuously fed from a hopper in limited quantities. Such systems are not conducive to obtaining quality products. The quality of the various kinds of products cannot be kept consistantly high from the start to finish of the operation. Raw fish kept for a relatively long time for use at the end of the operation must naturally be less fresh. Thus products of poor quality, which may be even harmful, are quite commonly manufactured in conventional plants. However, this fact of lessened quality is not sufficiently recognized. Generally, a cooker for boiling raw fish is a horizontal type cylindrical device which is slightly slanted and is enclosed with a steam jacket for heating the raw fish in the cooker. In the long cylindrical cooker a given quantity of fish material is pushed and moved therethrough at a constant speed, for example by means of a screw conveyer, which is provided inside the cooker. During this time the fish material is boiled in situ solely with the heat which is conducted through the steam jacket covering the cooker body, without any additional material, such as hot water, being added. The raw fish, upon completion of boiling, is discharged from the cooker so as to be fed to the next processor. Boiling raw fish is a heat processing method in which the raw fish is heated in such a manner that substances constituting the fish body, particularly the fish meat, are contracted and water and fish oil contained therein are extracted. The fish meat is thus converted into a material which is easy to digest and is a good feed for animals. In the case as described above, the fish body is boiled for a given period to extract relatively small quantities of hot water and fish oil. Boiling is continued in the extracted liquids, but the hot water present is insufficient to boil the entire fish body evenly and satisfactorily, and consequently the effect of boiling cannot be produced uniformly. In addition to this disadvantage, the fish body is formed in a layer, which is moved at a fixed speed in the cooker and is not stirred vigorously, as the cooker is not designed for such operation. As a result, the fish body within the core of the cooker cannot be heated as is the portion of the fish body which is held in contact with the inner wall surface of the cooker. In other words, a conventional cooker cannot be employed satisfactorily for evenly boiling the fish body or the fish meat feed material owing to defects in such boiling process and in the design of the cooker. Furthermore, it is noted that such a cooker is not based on the principle of boiling the entire fish body. Rather, emphasis in the prior art is placed on the in situ processing of raw fish with external heat applied to the fish body and on the theory and results of such heating. SUMMARY OF INVENTION The present invention is a result of our development in the technique for treating raw fish with heat, on the basis of a novel theory of heating which is essentially different from conventional concepts and is practically applicable. To overcome such disadvantages as described above, the invention provides a plant system for manufacturing fish products of good quality and high grade. The objects and features of the invention will be clear from what is described below. In accordance with the invention: (1) Raw fish are fed in a batch process system wherein all fish to be processed are fed at the same time into a high-pressure cooker while the fish are still fresh and of uniform quality. Our emphasis is placed upon the quality and consistency of the products, which are obtained from whole fish, fish meat, or by-products in fish processing plants. Precooking raw fish in the high-pressure cooker is conducted quickly and thoroughly under proper conditions of high pressure and high temperature without sacrifice of quality. Thereafter, press moisture is removed from the precooked fish in the same cooker in the absence of high temperature conditions, and press water is discharged from the cooker. The moisture-removed fish meat in the form of a cake is not conveyed to any other device, but is vacuum dried in the same cooker and is removed from the cooker as unfinished or intermediate products. The press water which is discharged from the cooker is further processed for extraction of fish oil, and a stickwater is obtained which is concentrated by means of a vacuum evaporator and is dried by means of a viscous fluid dryer for producing a soluble fish powder. (2) The design of the processing equipment is based on the principle of identical batch processing under identical conditions at the same time. Such processing equipment is operated as a totally closed system, and all processes other than precooking and press moisture removal are carried out under vacuum conditions, due consideration being given to material quality and optimum processing efficiency. (3) From the view point of environmental protection and sanitation, the processing system of this invention is totally enclosed so that the foul-smelling gases present in the steam and the polluted water which result from the processing of raw fish in the system can be thoroughly cleaned with suitable chemical solutions and the use of chemical and physical procesing equipment. Thus, the processing system, in accordance with the invention, is most satisfactory for the purpose of environmental protection and sanitation. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart of the invention. FIG. 2 is a vertical section view of a horizontal cylindrical high-pressure cooker of the invention, omitting the steam manifolds. FIGS. 2A and 2B are sectional views of the steam manifolds. FIG. 3A, 3B, and 3C are views of various main portions of the cooker. FIG. 4 is a schematic longitudinal sectional view of a cyclone steam condenser of the invention. FIG. 5 is a schematic view of gas cleaning equipment of the invention. FIG. 6 is a schematic longitudinal sectional view of a vacuum evaporator of the invention; and FIGS. 7 and 8 are front and side longitudinal sectional view of a viscous fluid dryer. DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to the drawing, the present invention wil be explained in detail. (1) Precooking, Pressing, and Drying of Raw Fish First, raw fish 101 is fed into a horizontal, cylindrical high-pressure cooker 102 as a unit batch of material, so as not to have any deterioration with time. The high-pressure cooker 102 is capable of precooking, pressing and removing moisture from the material and then drying in a single chamber, the structure of which is shown in FIGS. 2 and 3A, 3B, and 3C. Shown in the drawing is outer body or shell 1 of cooker 102 which has two chambers 3 and 3&#39; formed by division with fixed press plates 2, 2 serving as partitions opposed at the center at a given distance, said two chambers 3 and 3&#39; being connected to each other through a vent hole 4. The fixed press plates 2, 2 have slits 5 for passing press water, (FIG. 3B) which are formed on the entire surface of each press plate. Numerals 6, 6 represent jackets which cover the body 1 of the cooker 102, having a thermal or heat exchange medium. 7, 7 are inlets 8, 8 are outlets and 9, 9 are helical passages for the thermal medium. The thermal medium is circulated through a heater not shown, and is employed in a quantitative/qualitative manner so as to leave the outlets 8, 8 at a specific temperture T 2 (°C.) after heat transfer. Temperature T 1 (°C.) at the inlets is controlled in relation to the consumption of fuel for heating the thermal medium. In other words, fuel consumption is controlled for maintaining a constant temperture. The thermal medium is forced to circulate through the heating jackets by means of a pump (not shown). At the bottom of the cooker 102, numerals 10 and 11 represent pipes for discharging press water, which are provided at the center of a chamber 12, and at the outer ends of the chambers 3 and 3&#39; bounded by the fixed press plates 2, 2 and the ends of the cooker. These pipes have automatic control valves 13 for opening and closing. Numeral 14 represents ports for product discharge, which are provided at the inner ends of the chambers 3 and 3&#39; respectively, and likewise have automatic control valves 15. At the upper portion of the cooker 102, numeral 31 represents ports for charging raw fish. Pipes for discharging steam which may be produced in the cooking device 1 are shown at 16, each pipe having an exhaust pump (not shown) and an automatic control valve 17 for opening and closing operation. The pipe 16 has a branch pipe 19, having an automatic control valve 18, which is provided for bleeding the cooker to prevent the danger of a sudden blow of high-pressure steam. In the lower part of the cooker 102, numeral 20 represents jet holes of high-temperature high-pressure steam for precooking operation and 21 represents smaller jet holes of high-pressure steam for breaking and crushing the fish material. As schematically shown in FIGS. 2A and 2B, the steam for these operations is delivered to cooker 102 by means of manifolds 20a and 21a, which have associated valves and the like, known to the art. The fish material, which is wet with press water not yet fully removed as is required, is partly compressed between the press plates and is hardly compressed any more than halfway in the pressing process in the cooker. The smaller jet holes 21 are provided for permitting further pressing of the fish material held between the moving press plate 28 and the fixed plate 2 by breaking and crushing it with high-pressure steam. Numeral 22 represents a rotary driving shaft mounted through the cooker 102. This is a single shaft passing through the two chambers 3 and 3&#39; as indicated in the drawing (FIG. 2). Numerals 23 and 24 represent rotary vane members mounted on the rotary driving shaft 22 at the inner and outer ends of the chambers 3 and 3&#39;. A vane arm (a) (FIG. 3B) has a receiving recess (b) at the end into which a bar-like stirring member (c) is inserted loosely. The stirring member (c), as will be described later, is pushed and moved as the rotary vane members 24 rotate, sliding on the inside wall of the cooker and forced against it by centrifugal force. Such sliding results in positive separation of the material being stirred from the inside wall of the cooker 102. Bosses of rotary vane members 24 extend outside the cooker 1 and have clutch plates 25 at the outer ends. Clutch 26 is mounted integrally on the rotary driving shaft 22 facing the clutch plate 25. When the clutch plates are engaged, the rotary power of the driving shaft 22 is transmitted to the rotary vane members 24, and causes the stirring member (c) to slide against the inside wall of the cooker 102, while the other rotary vane members 23 which are not fixed to the shaft 22 are driven by the moving stirring member (c). Numeral 27 represents square-thread screw portions formed on the driving shaft 22 and 28 represents moving press plates mounted thereon. Each moving press plate 28 has slits 29 formed on the entire surface for discharging press water. Receiving recesses (b) (FIG. 3B) are formed at specific points on the periphery into which the stirring members (c) are inserted loosely. Thus, when the clutches are not engaged, the moving press plate 28 is moved back and forth along the square-thread screw portions 27 as the driving shaft 22 is caused to rotate clockwise and counterclockwise. During engagement of the clutches, the press plate 28 are turned by means of the stirring members (c) without being moved along the driving shaft 22, similar to the rotary vane members 23 which cannot travel along the shaft. In the cooker 102, as shown in the drawing the square-thread screw portions 27 are formed in symmetry as right and lefthand threads, and concomitantly the plates 28 are moved symmetrically in the chambers 3 and 3&#39;. Numeral 30 represents a brake for stopping the rotary vane members 24. Operation of the plant will be explained in the order of processing. (A) Precooking Raw fish are fed through charging holes 31 up to about 60% of full charge and are precooked in a short time under high pressure which is produced by thermal medium supplied through the jackets 6 and by high-temperature high-pressure steam supplied from the jet holes 20, with the stirring members (c) sliding along the inner walls of 1 during engagement of the clutch plates 25 and 26. During this time, pipes 10 and 11 for discharging press water, ports 14 for product discharge, charging holes 31, and pipes 16 and 19 are all closed. In other words, precooking is done under high pressure. The two chambers 3 and 3&#39; are automatically controlled for identical pressure through the vent hole 4 connecting the two chambers. Raw fish is piled evenly on the bottom of the cooker when fed in by operating the stirring members (c) so as to slide in short movements on the inside wall. In this process the moving press plates 28 are kept in their back position, as readily understood. When the pressure controlled within the cooker 1 reaches 3 kg/cm. 2 G, the temperature of all of the material therein becomes 143° C. The precooking of the material is completed under this pressure when this temperature has been reached by continuing the heating for a given period. In this process, almost all of fats and water soluble substances contained in the fish material can be extracted in hot water. The heating period may vary as deemed necessary, but thirty minutes is suitable. One way in which the completion of the precooking step is indicated is by the tenmperature of the thermal medium discharged, i.e., 140° C., which is as high as the temperature when it is introduced into the jackets 6. (B) Pressing and Removing Moisture Pressure in the cooker 102 is reduced after stopping the rotation of the driving shaft 22 and disengaging clutch plate 25 from clutch plate 26. In other words, the control valve 17 in the pipe 16 is opened for discharging steam which is produced in cooker 1 after the opening of the control valve 18 in the pipe 19 for reducing steam pressure. When pressure in the cooker 102 has been reduced, the press plates 28 are moved inward by turning the driving shaft 22 for pressing fully precooked fish therebetween. Press water thus produced is discharged from the pipes 10 and 11 through the slits 29 and 5 to the outside of the processing system. For draining such water it is preferable to install the cooker 102 somewhat at a slant. It is rather difficult to remove moisture from a mass of precooked fish material so that any desired moisture content is evenly distributed therein just by the pressing operation of the inward movement of the press plates 28. This is because the material tends to accumulate to a relatively low height and to become aggregated on the bottom of the cooker. Nevertheless, moisture can be removed evenly and moisture content as desired can be obtained by the method described below. After the pressing operation, as set forth above, stirring by means of the stirring members (c) is started by engagement of the clutch plates 25 and 26, while high-pressure steam for breaking and crushing the material is supplied from the jet holes 21 for making the stirring operation more effective. As a result, the semi-solid material held between the moving press plates 28 in the inward position and the fixed press plates 2 is broken and crushed and is piled up to a greater height; and the volume of the material is increased. The high-pressure steam jets are then cut off, and stirring operation is stopped by disengagement of the clutch plates. The material is thereafter pressed again more tightly. By repeating the above operation, moisture can be removed without difficulty from the material so that any desired moisture content may be evenly maintained therein. For this purpose, high-pressure steam jets are so controlled as to be supplied only at the inner side of the cooking device 1 because no use is made of the steam jets at the outer side. (C) Drying When press moisture removal is finished as desired, the moving press plates 28 are moved back to the original outer position. Then, the material is dried with heat which is conducted from the thermal medium in the jackets 6 under stirring operation with the clutch plates 25 and 26 engaged. During this time the automatic control valves 17 and 18 are kept open. The drying step is carried out under reduced pressure and steam generated in the cooker 102 at this stage is drawn out by vacuum through the pipes 16. (D) Discharge When drying is completed to the degree desired, the stirring operation is stopped, and the ports 14 for product discharge are opened with the automatic control valves 17 and 18 is kept open. The material is pushed and wiped down to the ports 14 by moving the press plates 28 to their innermost positions. Upon completion of precooking operation, the valves 13 are opened, and (referring to the flow chart of FIG. 1) press water is fed to a press water tank 103 by means of a pump. Pressing water produced by further press moisture removal is discharged into the same tank 103. The product (solid portion) delivered from the cooker 102 is made odorless during the process of press moisture removal which is conducted in a vacuum, and is fed to product cooler 104, grinder 105, and product silo 106 as in conventional processes. Treatment of steam discharged under vacuum will be described below. In the system of the invention, all passages of gas and liquid are through pipe members, which serve as closed feed lines. The processing of gas and liquid constituents which are discharged from the cooker 102 will now be described. (2) Steam Producted in the Cooker 102 and Discharged in the Drying Process The steam processing system for such steam is shown as a series of equipment subsequent to the cooker 102 in FIG. 1, comprising two cyclone steam condensers provided in series, a plate condenser, an uncondensable gas cooler, and a water jet ejector which operates to keep the system up to the cooker 102 in a vacuum of about 100 Torr. The steam processing system will now be described in detail. Steam of offensive odor which is discharged from the cooker 102 contains ammonia NH 3, hydrogen sulfide H 2 S, many other kinds of gases, and fish particles which are wet and small in quantity. If such steam is processed in a conventional dry-type cyclone device, some of the dried fish particles will become stuck to the inside wall. Fish particles, thus caught, will deteriorate with atmospheric steam and hydrogen sulfide into a wet viscous substance which can grow thicker and thicker on the inside wall until some malfunction is caused in the operation thereby. In accordance with the invention, such problems are avoided by use of a specially developed cyclone steam condenser. The cyclone steam condenser 107 of this special design is shown in FIG. 4. In the drawing, 31 represents a condenser body, 32 a cooling water jacket provided externally and around the body 31, 33 a condensed water collector of a closed type, 34 a steam inlet, and 35 an outlet for steam discharge. The wall of the condenser body, with which incoming steam is brought into contact, is kept cooled all the time; and part of the steam must touch the inside wall of the condenser 31 during its spiral motion therein. Consequently such steam is cooled and is condensed continuously into water, which flows together on the wall down to the collector 33 and is stored therein. On the other hand, fish particles mixed in the steam, which may also touch the inside wall, are caught on the film of water which is formed thereon and flows down slowly. They are carried down into the collector 33 together with filmy water without adhesion to the wall surface. Thus, the processing system described is trouble-free in operation, which may be otherwise in the prior art dry type cyclone devices, as previously indicated. In accordance with the present invention two condensers of this type are provided in series for more effective operation. Condensed water containing fish particles, which is stored in the collector 33, is mixed with aqueous fish solubles, and this mixture is fed to a cushion tank 123 to be described later. Bad-smelling steam discharged from the condenser 107 is then introduced into a conventional plate condenser 108, where all of such steam is cooled and condensed into water with cooling water by heat exchange on the wall of the condenser. Such condensed water is stored for collection in a closed tank 109 which is installed about 11 meters below the condenser, sufficiently lower to prevent reverse flow by vacuum force. Uncondensable gases consisting of air as a main component, ammonia, hydrogen sulfide, and other gases of foul odor are discharged from the plate condenser 108 into which odorous steam containing such gases are introduced. Processing in the plate condenser 108 is carried out in a vacuum of about 100 Torr, and accordingly the temperature of steam when introduced therein is about 50° C. This means that the temperature of condensed water and uncondensable gases which are discharged from the plate condenser 108 is about 50° C. If such uncondensable gases are fed directly to a water jet ejector 112, the temperature of circulating water employed for gas absorption may be elevated with the result of not only decreasing in the efficiency of the ejector but also producing repugnant odors from such water as the temperature rises. To prevent this occurrence, a fluorinated hydrocarbon gas (Freon) supplied from a freezer 110 is fed to an uncondensable gas cooler 111 so as to reduce the temperature of uncondensable gases to about 15° C., which is a little lower than a normal water temperature before the supply of such gases to the water jet ejector 112. The freezer 110 and the gas cooler 111, which are conventional, are not shown in detail. Condensed water produced here is conveyed to tank 109 for collection and storage. As already described, the processing system down to the water jet ejector 112 is kept in a vacuum of about 100 Torr. Ejector 112 is employed and not a vacuum pump because of higher performance, greater capacity, better reliability and safety in operation. The ejector is also capable of purifying gases. In the drawing, 113 represents a seal tank of circulating water, which is employed by the water jet ejector 112. This tank is conventional and is not shown schematically. Foul-smelling gases which are discharged from the water jet ejector 112 and comprise air as a main component are then introduced into gas cleaning equipment described below. Similarly introduced are the gases which are stored in the seal tanks 113 and 113&#39; and in condensed water collecting tank 106. The gas cleaning equipment employed was developed specially for securing full and positive effects through sufficient contact by mixing the offensive gases with chemical agents. The gas cleaning equipment shown in FIG. 5 consist of three water jet ejectors 114, 115, and 116 provided in series, ion exchanger 117, actived carbon gas absorber 118, and blower 119 for exhausting the gases which are now made odorless and harmless in the cleaning process. In the three water jet ejectors 114, 115, and 116 solutions of 5% H 2 SO 4, 10% NaOH, and 4% NaClO respectively, are employed and circulated in nearly equal amounts. Individual components of the offensive gases are removed by gas-liquid contact with high efficiency when passing through such solutions in the water jet ejectors. After completion of the operation, all water after processing is fed into an industrial water tank. (3) Press Water and Pressing Water Collected from the Cooker 102 led into the Press Water Tank 103 The temperature of such water is controlled properly in the press water tank, as already described, and is fed to an oil-separator self-cleaning unit 121 through a self-cleaning strainer 120, where the water is separated into fish oil, recovered meal, and stickwater. Fish oil is fed to a fish oil tank 122, and is processed into products. Recovered meal is fed to a cushion tank 123, to which is additionally fed a small quantity of fish meat grounds discharged from the self-cleaning strainer 120. Condensed water containing fish particles from the cyclone steam condenser 107, as described above, it also fed into the cushion tank. The aquatic solution in the cushion tank 123 is supplied to a stickwater tank 124, together with stickwater recovered meal which is discharged from the oil-separator self-cleaning unit 121. The self-cleaning strainer 120 and the oil-separator self-cleaning unit 121 are conventional, and are not shown schematically. Stickwater is fed to a vacuum evaporator 125 to be concentrated. The vacuum evaporator 125 is shown schematically in FIG. 6, where 36 represents the body which consists of the lower heating part and the upper part for receiving stickwater, 37 and 38 are, respectively, valves for discharging concentrated liqud and steam generated in the body, and 39 is a valve for charging stickwater to the evaporator. Numeral 40 represents a thermal-medium heating jacket which surrounds the outside of the body 36, 41 a valve for supplying thermal medium, 42 a circulation chamber receiving liquid in the upper part of body 36 and 43 a pump for forced circulation of the liquid through the chamber and the lower part of the body as shown in FIG. 6. Number 44 represents a steam heater which is mounted in the lower part of the body 36, 45 a valve for supplying steam as a heat source, 46 a drain cock, and 47 a pipe for heating hot-water drain. Numeral 48 represents a level gauge for checking the upper level of a full charge of stickwater, and is hung in the upper part of body 31. 49 represents a level gauge for checking the bottom limit of the stickwater to be processed. The operation of vacuum evaporator 125 will now be described. By opening the valve 39, stickwater is charged by means of a pump until the pump is stopped when a given charge of liquid has been indicated by the level gauge 48. During this time the operation of the water jet ejectors is suspended with the valves 38 opened and the valves 45 and 41 closed. When the charging of the liquid is finished, the valve 39 is closed. Then, after the start of operation of all water jet ejectors 112, 112&#39;, 114, 115, 116 connected thereto, concentration starts when the valves 45 and 41 are opened, and the liquid in the lower part of the body is forced to circulate by help of the pump 43. Steam generated in the body of the vacuum evaporator is discharged from the valve 38 by vacuum force, which is produced by a water jet ejector as described above. In other words, such steam is processed in the same manner as in the bad-smelling steam discharged from the condenser 107. The vacuum is generated by the water jet ejector 112&#39; through an uncondensable gas cooler 111&#39;, where florinated hydrocarbon gas supplied from a freezer 110&#39; is circulated. Gases of offensive smells, which are discharged from the water jet ejector 112&#39; are stored in a seal tank 113&#39; and thereafter are fed to the gas cleaning equipment of FIG. 5, previously described. Condensed water produced in a plate condenser 108&#39; and in the uncondensable gas cooler 111&#39; is likewise collected in the tank 109. Upon completion of concentration, the volume of the liquid thus processed is reduced; and consequently, the liquid level goes down to a preset level. When this level has been sensed by the level gauge 49, the valves 45 and 41 are closed, and the pump 43 stops its operation. Then, the water jet ejector 112&#39; for generating vacuum suction stops its operation likewise, and thereafter valve 37 is opened for feeding all concentrated liquid to a tank 126 therefor. At this time, the vacuum evaporator 125 is ready for the next charge of stickwater. The concentrated liqud is then fed to a viscous fluid dryer 127, which was developed for this purpose. The viscous fluid dryer 127 will be described with reference to FIGS. 7 and 8, where 50 represents a closed, horizontal drum, which is driven to rotate and has hollow shafts 52 and 52&#39; mounted on covers 51 and 51&#39; which serve to close both ends of drum 50. The drum thus constructed is supported on bearing members 53 and 53&#39; with a gear 54 mounted on the hollow shaft 52&#39;, which is engaged with a gear 57 mounted on a driving shaft 56 connected to a driving source 55. In the center of the drum 50 is inserted a cylindrical pipe frame 58 extending outside the drum which is supported fixedly on a supporting frame 60 mounted on the base 59 on which the bearing members 53 and 53&#39; are likewise mounted. The cylindrical pipe frame 58, through which a pipe 61 for introducing a fluid to be processed is provided, cannot follow the rotation of the drum 50. The pipe 61 is bent horizontally inside the drum 50 to protrude from cylindrical pipe frame 58, as shown in FIGS. 7 and 8; and a fluid jet portion 62 is formed which has a number of jet holes. Numeral 63 represents a bearing stand mounted on the inside wall of the drum 50 for rotatably receiving the inner end of the cylindrical pipe frame 58, 64 is a scraper fixed at the end of arms 65 which extend from the frame 58, and 66 is a pipe for discharging steam and foul-smelling gases generated in the drum. The pipe 66 is connected at other end to the inlet of cyclone steam condenser 107, as the volume and nature of such gases are found almost identical with those which are discharged from the horizontal, cylindrical cooker 102 previously discussed. Numeral 67 represents a thermal-medium jacket enclosing the drum 50, reference numerals 68 and 69 represent, respectively, a jack and a pin receiver which are provided at both ends on the bottom of the base 59, and 70 represents a product discharge port. With the arrangement of the system of the invention as described heretofore, the fluid to be processed is fed through the pipe 61, and is sprayed through jet holes in the fluid jet portion 62 against the inside wall of the rotating, heated drum 50. As water is evaporated, a thin layer of water 71 is formed on the wall and fish particles contained in the water layer are dried as the heated drum rotates. Dried fish particles are scraped away from the wall by means of the scraper 64, forming flakes 72, which fall onto the bottom of the drum 50. Such flakes are moved back and forth and tumbled up and down, until they are completely dried. Meanwhile, steam and gases produced in the drum are discharged as they are generated to the outside of the processing system under vacuum. As the drying process is conducted in a vacuum, constituents of a fish fluid can be kept fresh and drying can be attained with high efficiency with the simultaneous discharge of foul-smelling gases. In this manner, a mass of flakes 72 produced and held in the drum is made odorless and is dried to the extent required. After the driving motor is stopped at a proper time, and vacuum suction is cut off by closing the valve connected to the cyclone steam condensor 107, the base 59 is inclined by operating a jack 68 so that the dried flakes 72 may be delivered through a product discharge port 70. The mass of flakes thus discharged, which is dry soluble fish material, is used directly as a product of the process after being passed through a cooler and a grinder. In the drawing, a solid material which is discharged from the cooker 102 is fed to the cooler 104 and the grinder 105. However, the soluble fish and material produced in 127 is different in its properties from the solid fish material, produced in cooker 102 and may therefore be processed through a separate cooler and grinder (not shown). After completion of all operations, condensed water collected in the tank 109 is supplied to a separate industrial water tank, to which is added all water stored in the seal tanks provided in connection with the water jet ejectors for vacuum generation and gas cleaning. A mixture of such water in the tank is clean and neutral and may be employed as industrial water. Additional elements of the system shown in FIG. 1 are; thermal medium locater 128 for heating the heat exchange medium, boiler 129 for supplying process steam, water tank 130 to supply water for the boiler, water tank 131 to supply cooling water for the product cooler 104 and plate condenser 108 and 108&#39;, cooling tower 132 for water in tank 131, water tank 134 to supply water to seal tank 113 and 113&#39; and to tank 131, and freezer-cooler 133 to cool the water entering water tank 131. While the invention has been described with reference to treating raw fish, it is to be understood that it can be employed with equally good results to other materials. Included are fish by-products from packing plants and canneries, such as fish entrails, bones, heads and the like; animal slaughter house by-products; krill caught in the Antarcitc waters; cattle dung; and vegetable matter such as grasses, seaweeds and the like.

Summary: A batch process for making fish meal products, which does not pollute the environment, is described. All operations are conducted in equipment not open to the atmosphere and the liquids and gases produced are treated to remove odors. Fish is precooked in a cooker and the steam and vapors are conveyed to scrubbers where the foul-smelling gases are rendered innocuous. The pre-cooked fish meat in the cooker is pressed to removed press liquid which is treated to recover oil, fish meat and stickwater. The pressed fish meat in the cooker is then dried and removed as a product. The stickwater is evaporated under vacuum to recover dry water-soluble fish materials as a product and the overhead vapors are chemically treated to remove odors.

big_patent

en

Summarize: Lotto, SuperEnalotto e 10eLotto di giovedì 19 marzo: la seconda estrazione del Lotto della settimana è terminata. Poco dopo le 20 sono stati comunicati i numeri vincenti del SuperEnalotto e il risultato dell'estrazione del Lotto oggi su tutte le ruote. Il jackpot in palio per chi centra la combinazione vincente è di 35 milioni di euro. Ecco i risultati delle estrazioni del Lotto, SuperEnalotto e 10e Lotto di oggi. Ecco i numeri del Lotto estratti oggi su tutte le ruote: Bari: 32 – 2 – 61 – 56 – 52 Cagliari: 50 – 4 – 71 – 45 – 53 Firenze: 5 – 80 – 21 – 22 – 66 Genova: 68 – 60 – 1 – 76 – 14 Milano: 86 – 89 – 9 – 35 – 27 Napoli: 20 – 3 – 11 – 75 – 78 Palermo: 75 – 50 – 86 – 78 – 13 Roma: 89 – 34 – 76 – 10 – 50 Torino: 61 – 44 – 77 – 2 – 32 Venezia: 38 – 64 – 37 – 70 – 65 Nazionale: 47 – 37 – 34 – 22 – 87 I numeri estratti sulle ruote del Lotto oggi, giovedì 19 marzo, sono appena stati estratti; sul sito di Lottomatica è già possibile verificare l'esito della propria schedina. Le estrazioni del Lotto si svolgono tre volte a settimana e offrono vincite di diverso importo in base a quote fisse. Combinazione vincente SuperEnalotto: 50 – 17 – 28 – 11 – 7 – 29 Numero Jolly: 41 Numero Superstar: 59 Jackpot: 35.000.000€ Non solo Lotto: questa sera sono stati estratti anche i numeri vincenti del SuperEnalotto per il concorso n.34 di giovedì 19 marzo. La sestina vincente del SuperEnalotto vale un montepremi di 35 milioni di euro; dopo l'estrazione si conosceranno anche le vincite e le quote del concorso sul sito di Sisal e su superenalotto.com. I numeri del 10eLotto per l'estrazione serale sono: 2 – 3 – 4 – 5 – 20 – 21 – 32 – 34 – 38 – 44 – 50 – 60 – 61 – 64 – 68 – 71 – 75 – 80 – 86 – 89 Il numero Oro è 32, il Doppio Oro è 32, 2

Summary: Le estrazioni del Lotto oggi, giovedì 19 marzo 2020, e i numeri vincenti del SuperEnalotto per il concorso n.34. Il jackpot vale 35 milioni di euro, a seguire le quote e le vincite. Ecco i risultati delle estrazioni del Lotto, SuperEnalotto e 10e Lotto serale, comunicati dopo le 20 dall'Agenzia delle Dogane e dei Monopoli.

fanpage

it

Summarize: By. Victoria Woollaston. Imagine a home where if you asked your thermostat to change temperature it would, or one that could text your neighbours if an alarm went off while you were on holiday. These scenarios are all now a reality after Google-owned thermostat firm Nest opened up its software for developers to use for the first time. It means that other companies will now be able to take advantage of the smart thermostat’s software - which can be controlled remotely. Nest has opened up its software for third-party developers to use for the first time so that companies will now. be able to take advantage of the smart thermostat's (pictured) software - which can be controlled remotely. Nest Labs was formed in 2011, but earlier this year was purchased by Google for $3.2 billion (£1.8 billion). Co-founders Tony Fadell and Matt Rodgers had both previously worked at Apple, including on the iPod and original iPhone. The company has two central products so far - a smart thermostat that can be controlled via a mobile device, and an alarm system that also monitors carbon monoxide levels. The announcement confirmed the first partners have linked up with Nest to use Google voice recognition and the phrase ‘OK Google’ in order to verbally change temperature on the thermostat. Rodgers, vice president of engineering at Nest, said: ‘The Nest Learning Thermostat and Nest Protect alarm are already helping people save energy, stay comfortable and improve home safety - but that’s only the beginning. The smart LiFx lights (pictured left) were funded by a Kickstarter campaign. They start at $89. (£53) each and an individual light can be customised to be any colour on. the spectrum using an app (right). When connected to the Nest alarm, the smart light bulbs can be used to warn of carbon monoxide leaks, for example. Qualcomm presented its vision for a connected home at Mobile World Congress in February, and showed how almost anything in the home can be controlled by a smartphone or linked together electronically. From smartlocks and security lights to fridges, all the devices in its conceptual dwelling are sold by individual companies, but all of them run AllJoyn software. AllJoyn is an open source project set up by chipmaker Qualcomm in 2011. It is a programme that can be added to smart devices to make them ‘talk’ to other devices running AllJoyn. The company has since partnered with a number of companies that have developed smart household devices to help develop the software further. For example, an August smartlock can be linked to lights that flash if they sense an intruder, using the system. ‘Our goal has always been to bring this kind of thoughtfulness to the rest of your home and life - and that’s what the Nest Developer Programme is all about. ‘To kick off the programme, we’ve worked with iconic brands like Mercedes-Benz and Whirlpool, as well as new industry leaders like Jawbone and LiFx, to build seamless, secure and practical ‘Works with Nest’ experiences for the home.’ The Mercedes-Benz set-up will enable a car to send a message when a driver begins their journey home so the thermostat can start heating or cooling accordingly, in time for their arrival. The Jawbone activity monitor wristband communicates with Nest to set the temperature as a person wakes up and it can do this because the Jawbone UP24 wristband tracks a wearer's sleep. ‘We’re trying to invent experiences you’ll use everyday with products that you’ll use everyday’, said Rodgers. ‘We want to build an experience, not something just for the sake of it.’ The Mercedes-Benz set-up will enable a car (pictured) to send a message when a driver begins their journey home so their thermostat can start heating or cooling accordingly. The Jawbone wristband and sleep monitor (pictured) communicates with Nest to set the temperature as a person wakes up. Nest said that more than 5,000 developers have already registered an interest in working with the software, including light bulb manufacturer LiFx, which has developed a system to turn the internal lighting of your home red should the Nest carbon monoxide monitor detect a leak. Phil Bosua, founder and chief executive of LiFx, said: ‘When we first heard about the Nest Developer Programme, we knew we wanted to be a part of it. ‘Nest brings a whole other dimension to LiFx. Who would have thought by combining Nest products and LiFx products, we could help save lives?’ The launch will further fuel talk of a movement towards the ‘internet of things’ - an ever-growing idea that soon all of the devices we use will be connected together, much like computers when using the web. Technology giant Samsung has confirmed in the past that the company believes that this trend will be the key development of the next decade in the technology sector, with UK president Andy Griffiths saying he believes the future is all about the ‘connected home’. Chipmaker Qualcomm has also unveiled a concept of the connected home where its AllJoyn software connects various smart devices from smartlocks and security lights to the fridge and TV. AllJoyn works in a similar way to Works with Nest, by making it possible for smart gadgets from various manufacturers to 'talk' to each other, however, AllJoyn doesn't need a single product at its centre to function, like Nest does

Summary: Nest has already partnered with Mercedes-Benz, Whirlpool and Jawbone. The program called 'Works with Nest' can be applied to any smart device. Mercedes-Benz set-up gets the car to send a message to the home thermostat so it can start heating or cooling in time for a driver's arrival. Nest said that more than 5,000 developers. have already registered. They include bulb firm LIFX that uses lights to warn of carbon monoxide.

cnn_dailymail

en

Write a title and summarize: Toxin-antitoxin (TA) systems, stress-responsive genetic elements ubiquitous in microbial genomes, are unusually abundant in the major human pathogen Mycobacterium tuberculosis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown. To address these questions, we have taken a comprehensive approach to identify and functionally characterize all the TA systems encoded in the M. tuberculosis genome. Here we show that 88 putative TA system candidates are present in M. tuberculosis, considerably more than previously thought. Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC), but largely absent from other mycobacteria, including close relatives of M. tuberculosis. We found that many of the M. tuberculosis TA systems are located within discernable genomic islands and were thus likely acquired recently via horizontal gene transfer. We discovered a novel TA system located in the core genome that is conserved across the genus, suggesting that it may fulfill a role common to all mycobacteria. By expressing each of the putative TA systems in M. smegmatis, we demonstrate that 30 encode a functional toxin and its cognate antitoxin. We show that the toxins of the largest family of TA systems, VapBC, act by inhibiting translation via mRNA cleavage. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. The expansion and maintenance of TA genes in the MTBC, coupled with the finding that a subset is transcriptionally activated by stress, suggests that TA systems are important for M. tuberculosis pathogenesis. Toxin-antitoxin (TA) systems are ubiquitous in prokaryotic genomes and have been proposed to play a role in several important cellular functions [1]. These systems typically consist of a two-gene operon encoding a toxic protein that targets an essential cellular function and an antitoxin that binds to and inhibits the toxin. Regulation of toxin activity is achieved through differential stability of the stable toxin and the unstable antitoxin [2]. In most cases, the antitoxin also acts as a transcriptional autorepressor of the operon, such that degradation of the antitoxin results in transcriptional induction of the TA genes. Most of what we know about TA systems has come from the pioneering work in E. coli, though their role in bacterial physiology is still controversial. Some of the genome-encoded systems are activated in response to environmental stress, resulting in cell stasis from which these cells can recover under more favorable growth conditions [2], [3]. In contrast, it has also been reported that the MazEF TA system participates in programmed cell death [3]–[6]. Importantly, the HipBA TA system has been implicated in the formation of persister cells, a subpopulation of bacteria that exhibit antibiotic tolerance in an otherwise susceptible population [7], [8] and may thus contribute to the long treatment times required to cure some infections. Because TA systems were discovered as plasmid stability elements, it has also been proposed that genomic TA loci may similarly stabilize or help to ensure the maintenance of genes encoded nearby in the genome [1], [9], [10]. Finally, it has also been postulated that these systems are simply selfish genetic elements that function only to maintain their own existence in a genome [1], [11]. Although these studies have provided a wealth of information regarding the function of TA systems in E. coli, their role in the physiology of other microbes remains largely unexplored. The most common mechanism of TA system toxicity is mediated through mRNA cleavage, resulting in translation inhibition [2], [12], [13]. Two well-characterized TA system families of E. coli, MazEF and RelBE, have been shown to act via this mechanism and cleave specific three-nucleotide sequences [14], [15]. The toxins of the largest family of TA systems in M. tuberculosis, VapBC, contain PIN domains, a motif thought to be associated with ribonuclease function [16] and have been shown to block translation via mRNA cleavage [17]–[19]. Transient activation of these mRNases may allow the bacteria to adapt to stress not only by inhibiting replication, but also by degrading existing transcripts, allowing a rapid change in the metabolic program of the bacteria. Given that a TA system is required for the formation of persisters in E. coli, it has been speculated that the TA systems of M. tuberculosis may govern cell division decisions during infection [10]. In the majority of individuals infected with M. tuberculosis, the bacteria initially grow and then establish a latent, asymptomatic infection that can persist for decades with the potential to reactivate later in life [20], [21]. These persistent bacteria are thought to adopt a slowly or non-replicating state in response to environmental stresses encountered in the host [22], [23], yet the mechanisms by which this non-replicating state is achieved are unknown. Because the majority of current antimicrobials require bacterial growth to exert their killing action, these non-replicating persistent bacteria are thought to comprise an important subpopulation of bacteria that is refractory to antibiotic therapy [24]. A similar antibiotic-tolerant state is elicited by TA system activation in other bacteria [25], suggesting that TA systems may contribute to the long duration of antibiotic therapy required to cure tuberculosis. The most extensively studied culture condition to induce cell stasis in M. tuberculosis is hypoxia [26]–[28]. Gradual oxygen depletion in culture results in significant changes in metabolism and gene expression, leading to a non-replicative persistent (NRP) state [26]. Because bacteria experience an effectively hypoxic environment in vivo as a result of reduced oxygen availability and exposure to nitric oxide (NO), it is thought that hypoxia-induced NRP is similar to the in vivo state [21], [26], [29], [30]. Additionally, these two conditions result in a significant overlap in gene expression as they both induce the dormancy regulon, a set of genes under the control of the transcription factor DosR [26], [31]. Bacilli grown under hypoxia exhibit a tolerance to antimicrobial therapy [26]. Given that hypoxia results in a state of cell stasis and the formation of antibiotic-tolerant persisters, TA systems are prime candidates for mediating this transition both in vitro and in vivo. Recent bioinformatics studies revealed that the M. tuberculosis genome encodes numerous TA system homologs and many PIN domain-containing proteins, far more than any other intracellular pathogen [16], [32]–[34]. Although these studies suggested that there has been a significant expansion of TA systems in M. tuberculosis, these analyses may have missed distantly related homologs and novel families of TA systems and thus the total number of TA systems in M. tuberculosis may be even greater. Additionally, how the M. tuberculosis genome evolved to acquire and maintain these TA systems during its evolution is unclear. To date, there has not been a comprehensive comparative analysis to determine whether the M. tuberculosis TA systems have been selectively maintained in the pathogens of this genus. TA systems are often associated with mobile genetic elements and are thus commonly acquired by horizontal gene transfer [16], [32], yet only three of the M. tuberculosis TA systems have been definitively assigned to a known genomic island [35], [36]. Although the evolutionary history of these genes is uncertain, the vast number of TA systems in M. tuberculosis evokes the question of whether the expansion of TA systems in M. tuberculosis plays an important role in the physiology of the bacteria. A subset of the putative M. tuberculosis TA genes have been partially characterized but our knowledge of the full complement of TA systems thus far is very fragmented [12], [13], [37]–[39]. Therefore, a comprehensive and systematic analysis is needed to provide a foundation on which to investigate the role of this interesting gene family in M. tuberculosis biology. Although recent bioinformatic analyses have expanded the number of putative TA systems encoded in the M. tuberculosis genome [16], [32], [33], the key questions of how many of these genes encode functional TA systems and which of these systems are important in M. tuberculosis biology have not been addressed. Here we report the results of a comprehensive strategy to identify and examine the putative TA systems encoded in the M. tuberculosis genome. Our approach revealed many more putative TA loci than previously appreciated. Expression of each of these systems in M. smegmatis, a fast-growing relative of M. tuberculosis, revealed a subset that encodes bona fide TA systems. Importantly, we identified three novel systems that were not previously recognized and bear no similarity to known TA genes, and thus may represent new families of TA systems. Additionally, by performing comparative genomic analysis across the mycobacterial genus, we made the striking discovery that the vast majority of these systems are conserved only in the MTBC and are absent from mycobacteria outside this complex, including closely-related pathogenic species. The acquisition and expansion of TA systems likely occurred coincident with or after speciation of the MTBC from the last common ancestor, suggesting an important role for these genes in M. tuberculosis evolution. We demonstrate that toxins with homology to RNases inhibit translation and have RNase activity in vitro, while a novel toxin likely functions via a different mechanism. Finally, we show that subsets of these genes are upregulated during hypoxia or macrophage infection, providing strong evidence that these systems are activated during specific stresses likely encountered in the host. To broadly search the M. tuberculosis genome for putative TA systems, we reasoned that a combination of approaches would be more powerful than a single strategy. To this end, we utilized three complementary approaches that took advantage of different characteristics of known TA systems. First, we performed PSI-BLAST searches of the M. tuberculosis genome using the toxin and antitoxin protein sequences from each of eight major TA system families: CcdBA, HigBA, HipBA, MazEF, ParDE, RelBE, VapBC, and Doc/PhD (Table S1). When possible, we used sequences from both distantly-related organisms (Gram-negative) and more closely-related organisms (Gram-positive, high-GC) to search for homologs. Second, we expanded this list to include PIN domain-containing proteins and toxin-antitoxin systems identified in previous analyses [16]. Finally, we used a sequence-independent approach to identify pairs of adjacent genes encoded in the M. tuberculosis genome that bear no homology to known TA systems but share a similar genomic organization [40]. This method, similar to an approach used to identify novel TA systems in E. coli, included constraints on size, orientation, and spacing of putative TA pairs. Candidate genes from all three methods were filtered using four criteria for the genomic organization of TA systems: 1) the putative toxin and antitoxin genes were adjacent to one another, 2) the two putative genes were separated by fewer than 150 bp, likely comprising an operon, 3) neither gene encoded a protein larger than 150 amino acids, and 4) the upstream gene (putative antitoxin) was smaller than the downstream gene (putative toxin). The last criterion was disregarded for cases in which there were small differences in size provided that both the putative toxin and antitoxin had conserved protein domains associated with their proposed function. Additionally, we made an exception in the case of the lone HigBA homolog, as the orientation of the toxin and antitoxin are reversed in this system [32]. In total, we generated a list of 88 putative TA systems in M. tuberculosis (Figure 1A and Table S1). Our list includes 62 gene pairs that were identified by homology. In cases for which the putative toxins and antitoxins belonged to different TA families, the TA system homology was assigned based on the homology of the toxin gene (Table S1). Toxins that contain PIN domains are most closely related to the VapBC family and thus we have classified TA systems with toxins containing these domains as part of the VapBC family (Figure 2, Table S2, and Table S3). We identified an additional 26 putative systems that share no sequence similarity to known TA genes and thus may represent novel systems (Figure 1A and Table S2). Two other groups have incorporated homology-independent methods in addition to traditional homology-dependent searches to aid in the comprehensive identification of TA systems in prokaryotic genomes [33], [41]. Similar to the methods used here, Sevin and Barloy-Hubler utilized a sequence-independent approach that relies on the characteristic size, gene organization and spacing of known TA systems to develop an automated, web-based tool termed RASTA-Bacteria. Makarova, et al. used a novel approach that identifies pairs of genes that are significantly non-uniformly distributed in prokaryotic genomes. Comparison of our results with those of the above studies revealed that our methods identified a large number (23–28) of putative new TA systems, largely of the novel class, not identified in either the Makarova study nor deemed significant by RASTA-Bacteria (Figure S1). To better define when in evolutionary history TA module expansion occurred, we examined both published and unpublished genome sequences spanning the genus Mycobacterium for orthologs and homologs of the known and newly identified TA systems present in M. tuberculosis. We included the draft genomes of several members of the MTBC, including M. canetti, which is the deepest diverging lineage of this group [42], as well as the genomes of M. marinum and M. kansasii, which are the most closely related mycobacterial species that lie outside the MTBC [35], [43]. Finally, completed genomes of rapidly growing environmental mycobacteria and the slow growing pathogens M. leprae, M. ulcerans and M. avium were included. We used reciprocal best BLAST hit and genomic context analyses [44] to identify orthologs and homologs of the toxin portion of 65 of the 84 putative M. tuberculosis TA systems described above. For all top BLAST hits, we then determined whether an associated antitoxin was encoded nearby in the genome. 23 putative toxin genes identified by genomic organization were excluded from this analysis as we found no supporting evidence based on either homology or over-expression experiments (see below) that they encoded functional toxins. Analysis of the MTBC identified orthologs of nearly every TA system in each of the genomes analyzed (Figure 2 and Table S3). All of the TA systems were conserved in M. microti, and only one toxin, Rv2653c, which is encoded on a prophage, was absent in M. bovis and M. africanum. Six toxin genes appeared to be absent in M. canetti, including the prophage-encoded toxin Rv2653c. Analysis of the surrounding genomic sequences, however, revealed that sequences homologous to three of these genes, Rv0624, Rv0627 and Rv1102c, were present in the M. canetti genome but had been disrupted by genomic rearrangements, including the insertion of transposon-like sequences (data not shown). The last two toxins absent from the M. canetti genome, Rv0299 and Rv0301, are encoded on a genomic island in M. tuberculosis that has previously been shown to be absent in several M. canetti strains (Table 1). These results suggest that the vast majority of TA systems were present in the progenitor of the MTBC. The finding that a small number of TA genes have been lost in M. canetti is consistent with its assignment as the deepest branching member of the MTBC lineage [42]. BLASTP analysis identified putative toxins in several mycobacterial genomes outside of the MTBC (Figure 2 and Table S3). In most cases, however, the surrounding genomic regions did not demonstrate conservation of gene content and order with the M. tuberculosis genome, suggesting that these related TA systems were acquired independently in each bacterial species rather than having evolved from a common ancestral acquisition event. Alternatively, because TA systems are often associated with mobile genetic elements, these systems may have been present in a common ancestor and subsequently moved in the genome as each species diverged. Three of the five TA systems (coding and pseudogenes) identified in M. leprae and two of the 12 TA systems found in the M. kansasii genome are in regions with similar gene content and order to that of M. tuberculosis, arguing that they evolved from a common ancestral acquisition event. Intriguingly, the only TA module encoded in all genomes analyzed was the novel TA system Rv0909-Rv0910, identified in the homology-independent search. Strikingly, all but two of the TA systems present in M. tuberculosis are absent in the closely related pathogen M. marinum. Closer analysis of these revealed that the antitoxin gene for one of them, Rv3181c, contains two point mutations resulting a significantly truncated, and likely nonfunctional, protein (data not shown). This lack of conservation of TA systems was surprising given that M. marinum is thought to be the closest genetic relative of M. tuberculosis outside of the MTBC [35], [45]. These findings strongly support the idea that TA gene expansion occurred after the MTBC and M. marinum diverged from their last common ancestor, and suggests that these systems play an important role in the unique biology of the MTBC. In support of this idea and consistent with the known origins of TA systems in other bacteria, we discovered that many of the M. tuberculosis TA systems are encoded in locations in the genome that were previously identified as regions of horizontal gene transfer [35], [46]. By cross referencing the list of TA systems identified here with previously defined genomic islands [35], [46], we discovered that 24 (37%) of these systems are located in these regions (Table 1). It has been proposed that the acquisition of foreign DNA sequences via horizontal gene transfer was a defining event in the speciation of the MTBC [42], [46], [47]. The large number of TA systems that were acquired during this large influx of heterologous DNA, and subsequently maintained, lends support to the idea that these genes are integral to the biology of the MTBC. To begin to understand the functions and biological roles of the numerous TA systems of M. tuberculosis, we first sought to determine the number of putative TA systems that encode functional toxins. We used the inducible acetamidase promoter to conditionally express 78 of the putative toxin genes we identified in M. smegmatis. We were unable to clone and express 10 putative genes, likely due to differences between our strain (Erdman) and the published sequence of H37Rv. Toxicity was assessed by plating 10-fold dilutions of cultures on solid media in the presence or absence of inducer. Genes encoding a toxic protein product, such as Rv0301 and Rv2829c, inhibited growth of cultures on plates with inducer, but did not affect growth of bacteria on plates without inducer (Figure 1B). This method identified a total of 32 genes that resulted in toxicity when expressed, while the remainder of the genes tested did not inhibit growth under inducing or non-inducing conditions (Table S2). Since many genes can be toxic to cells when over-expressed, it was important to show that expression of the cognate antitoxins of these genes could relieve the toxic activity. Therefore, for each gene that was toxic, we then co-expressed the toxin and antitoxin, under the control of the same inducible promoter, to determine if it allowed cell growth in the presence of inducer (Figure 1B). Toxic proteins that were inactivated by their putative antitoxins, as in the case of Rv0300-0301 and Rv2829c-2830c, were then considered functional TA systems. For two of the RelBE homologs, Rv1246c and Rv2866, we were unable to obtain transformants in M. smegmatis. This result is most likely due to low levels of expression from the acetamidase promoter in the absence of inducer. For these toxins, antitoxin activity was assessed by the ability to obtain transformants with the vector containing both the toxin and the antitoxin. In total, we discovered 30 pairs of genes in the M. tuberculosis genome that function as toxin-antitoxin genes in M. smegmatis (Figure 1C and 1D). The majority of the TA systems we identified came from those found by BLAST and PIN-domain containing proteins. Indeed, nearly half of the putative systems identified by these methods are functional TA systems. However, of the genes found by our homology-independent search, a much lower percentage of genes were subsequently found to be functional TA systems. This is not surprising, given that the majority of these genes are not predicted to have protein domains associated with toxin or antitoxin activity. While this method was less efficient, the three novel TA systems identified (Rv0298-0299, Rv0909-0910, and Rv2653c-2654c) are of particular interest. Although Rv0299 appears to be distantly related to MazF, neither Rv0910 nor Rv2653c bear any homology to known TA systems, nor to one another, raising the possibility that they may function by novel mechanisms of toxicity (Figure 1D). Strikingly, Rv0910 was the only toxin present in all genomes analyzed and, in all cases, orthologs of the putative antitoxin Rv0909 were also present nearby (Figure 3A). In contrast to many of the other TA systems identified, this operon is not encoded in a genomic island, and its position in the genome is relatively conserved throughout the genus (Figure 3A and Table 1). These findings suggest that the Rv0909-Rv0910 system may play a conserved role in the physiology of this otherwise diverse group of bacteria. To determine whether this novel, conserved putative TA system acts as a TA system in other mycobacteria, we cloned the Rv0909-0910 orthologs MSMEG_5635-5634 from M. smegmatis. We assessed the ability of MSEMG_5634 to inhibit cell growth as well as the ability of MSEMG_5635 to rescue growth inhibition as described above. Plating cells expressing toxin alone on media containing inducer led to growth inhibition that was ameliorated when the cognate antitoxin was co-expressed with the toxin (Figure 3B). These results show that a second member of this novel TA family functions as a TA pair, supporting the idea that Rv0909-0910 represents the founding member of a new TA family. The VapBC family comprises, by far, the largest family of TA systems in M. tuberculosis. Given that there are a large number of these related genes, we wanted to determine the potential for cross-talk between VapB antitoxins and VapC toxins. Specifically, we wanted to determine if VapB antitoxins can inactivate non-cognate VapC toxins, resulting in the inhibition of toxicity. To address this question we expressed four heterologous VapB and VapC proteins, under the control of separate inducible promoters, and assessed growth in the presence and absence of both inducers. Remarkably, although these are related proteins, our results show that these antitoxins are only able to inhibit their cognate toxins (Figure 4). Although we analyzed only a subset of the VapBC family, this data strongly suggests that VapB antitoxins are highly specific for their associated toxins. Given these results, we conclude that cross-talk is similarly unlikely to occur in vivo. Many toxins of TA systems function as RNases and result in translation inhibition when activated [14], [48]. In particular, PIN domain-containing proteins, including some VapC homologs, have been shown to have RNase function [17]–[19], [49]. We expressed the VapC homolog Rv0301 in M. smegmatis and monitored bulk translation via incorporation of 35S-methionine over a six hour time course. As shown in Figure 5A, Rv0301 expression led to inhibition of translation, an effect that was reversed by co-expression of its antitoxin, Rv0300. The effect on protein synthesis preceded the inhibition of growth caused by this toxin (Figure 5B). Likewise, expression of three other VapC homologs (Rv1561, and Rv2829c, Rv3408) also inhibited translation (Figure 5A). As a control, addition of hygromycin, an antibiotic that targets protein synthesis, inhibited incorporation of 35S-methionine (Figure 5A) and growth (Figure 5B), as early as one hour after addition to the media. The modest difference in the kinetics of translation inhibition between toxin induction and addition of antibiotics is likely due to time required for transcription and synthesis of the toxin. Given the proposed ribonuclease function associated with PIN domains, we reasoned that a likely mechanism for translation inhibition of the toxins tested was RNA cleavage. Indeed, in vitro RNase activity of an M. tuberculosis VapC homolog was recently demonstrated [49]. To test the ability of these proteins to hydrolyze RNA, we performed in vitro RNA cleavage assays with purified VapC proteins and the viral MS2 RNA, a substrate that has been effectively used to detect RNase activity [13]. As shown in Figure 5C, incubation of MS2 RNA with E. coli MazF, or with either of two M. tuberculosis VapC homologs, Rv0301 and Rv1561, resulted in degradation of the RNA, though Rv0301 exhibited less potent RNase activity. The incubation of the toxins MazF and Rv0301 with their cognate antitoxins, MazE and Rv0300, respectively, inhibited this RNase activity (Figure 5D). As controls, we also included MS2 RNA incubated with buffer alone, as well as an MBP-His protein fragment, which both failed to cleave MS2 RNA (Figure 5C). These results show that M. tuberculosis VapC homologs inhibit translation and strongly suggest that these toxins affect translation directly via RNA cleavage. In contrast to our results with the VapC homologs, expression of the novel toxin Rv0910 did not result in inhibition of translation throughout the duration of the assay (Figure 5A). However, expression of this toxin did inhibit cell growth, suggesting that Rv0910 targets a cellular process other than translation (Figure 5B). Additionally, incubation of MS2 RNA with purified Rv0910 yielded intact MS2 RNA, consistent with this idea. The specificity of the translation assay was verified by treating cultures with the antibiotic ciprofloxacin, a DNA replication inhibitor. Although the antibiotic inhibited growth (Figure 5B) and caused DNA damage as measured by recA expression (Figure S2), it did not affect translation over the course of the experiment (Figure 5A). This important specificity control shows that disruption of other macromolecular synthesis pathways does not affect translation during this assay, and is similar to the results obtained with expression of Rv0910. Taken together, these results strongly suggest that Rv0909-0910 represents a novel TA system that inhibits cell growth via a mechanism distinct from the VapBC family. The presence of such a large number of TA systems presents an obvious question: What is the benefit of having so many of these genes in one organism? We postulated that subsets of TA systems important for M. tuberculosis biology may respond to different cellular stresses. To test this hypothesis, we determined if any of the functional TA systems in M. tuberculosis were transcriptionally activated under two conditions encountered during infection. In particular, we examined the response of TA systems under hypoxic conditions in culture and during infection of IFN-γ-stimulated murine bone marrow-derived macrophages. To assess TA activation, we took advantage of the fact that most antitoxins also function as transcriptional autorepressors, and thus degradation of an antitoxin results in increased transcription of its operon. Although this increase in transcription leads to increased protein synthesis of both the toxin and antitoxin, the unstable antitoxin is typically selectively targeted for degradation by a protease, further increasing transcription of the operon, while the more stable toxin interacts with its cellular target [48]. In this manner, we are using the increase in transcription as an indirect read-out of TA system activation via degradation of the antitoxin, thus relieving its transcriptional inhibitory activity. We monitored the expression of each of the 30 functional TA systems by quantitative PCR and obtained detectable signal for 25 TA systems during hypoxia and for 23 systems during macrophage infection (Table S4 and Table S5). Two TA systems, Rv2009-2010 and Rv1955-1956, were induced during hypoxia (Figure 6A). Transcription of hspX and fdxA, two genes belonging to the dormancy regulon that are very highly induced during hypoxia, was also induced, demonstrating that the bacteria experienced a hypoxic environment (Figure 6A). Of the TA systems monitored during macrophage infection, Rv1560-1561 and Rv0549c-0550c were induced. As controls, transcription of hspX and icl were greatly induced, consistent with previous results of the transcriptional response following macrophage infection [50] (Figure 6B). It is interesting that the two TA systems that are activated during hypoxia are not activated during macrophage infection, given the effectively hypoxic environment generated via nitric oxide signaling following IFN-γ stimulation of the macrophages. Indeed, these two conditions both result in induction of the dormancy regulon [50], [51]. However, the TA genes tested here are not part of the DosR dormancy regulon and are likely activated by independent mechanisms. Certainly, it is possible that other TA systems are also activated under these conditions but this was not detectable by the methods used here. In light of these data, it appears that TA systems are regulated independently from one another, and expression of at least four of these TA systems is modulated in response to different environmental stresses. This supports our hypothesis that specific subsets of TA genes are regulated in response to changes in the environment. Here we have shown that of 88 putative M. tuberculosis toxin-antitoxin loci, 30 encode functional TA systems. The numbers of both the putative and functional TA systems are significantly greater than in any other organism studied thus far. Our sequence-based searching of the M. tuberculosis genome, in conjunction with previous bioinformatic approaches, has identified 62 TA systems with homology to known TA systems [16], [32], [33]. Because mycobacterial TA genes may have limited sequence identity with distantly related homologs from other bacteria, there may be additional TA systems that have yet to be discovered. Importantly, incorporation of our sequence-independent method identified an additional 26 loci bearing no homology to known TA genes, allowing us to assemble one of the most comprehensive lists of putative TA systems in M. tuberculosis to date. It is striking that nearly all of the TA systems we identified are well conserved among the MTBC but are largely absent from species outside of this complex, including M. marinum. The paucity of TA systems in M. marinum is particularly notable as the two bacteria are highly related, sharing 3000 orthologs with an average amino acid identity of 85% [35]. Therefore, the massive expansion of TA systems is a distinguishing feature of the MTBC, and the acquisition and maintenance of these genes was likely instrumental for the evolution of M. tuberculosis. We discovered that of the 423 protein-coding genes located within these regions, 48 are TA genes (Table 1). This frequency of TA genes (11%) is significantly higher than that of the entire M. tuberculosis genome (4%), indicating that there is an enrichment of TA loci in these regions. Our results, combined with data from Becq et al. [46] and Stinear et al. [35], lends strong support to the idea that many of the TA systems were recently acquired via horizontal gene transfer after the divergence between M. tuberculosis and M. marinum. It is possible, however, that some of these genes were acquired in a common ancestor of the slow-growing mycobacteria and subsequently lost in a subset of extant species. In support of the latter hypothesis, M. leprae and M. kansasii, bacteria that are thought to be more distantly related to the MTBC, contain TA system orthologs that are clearly absent from M. marinum (Figure 2 and Table S3). Alternatively, the assignment of M. marinum as the closest relative of the MTBC may be incorrect, as supported by whole-genome comparisons that place M. kansasii as the nearest neighbor of the MTBC [43]. Given the evidence that the vast majority of TA systems have no counterparts in mycobacteria outside of the MTBC, the most parsimonious explanation for their expansion is that these genes were acquired after speciation. In addition, further amplification of TA systems may have occurred in the MTBC via gene duplication events. It is curious that so many TA loci are present in the M. tuberculosis genome. One possible reason for this expansion is that genomic TA systems may function to stabilize the M. tuberculosis chromosome, akin to the role of TA systems in stabilizing plasmids [52]. Since toxins are typically more stable than antitoxins, TA systems inhibit post-segregational plasmid loss because cells that do not inherit the episome rapidly deplete the antitoxin protein, leading to toxin activation and inhibition of growth. Indeed, there is recent evidence that chromosomal TA systems may protect adjacent regions of the chromosome from deletion [9]. In support of this notion, many of the TA systems identified here are encoded on genomic islands that include genes important for M. tuberculosis virulence or physiology (Table 1). Alternatively, TA systems may participate more directly in the physiology of M. tuberculosis by functioning as stress response elements, as has been demonstrated in E. coli [2], [3], [48], [53]. There is a growing body of evidence that some TA systems are induced during exposure to adverse environmental conditions, such as exposure to antibiotics, allowing cells to respond and adapt to the assault [54]. Indeed, a screen to identify mutants in M. tuberculosis with altered growth kinetics during transitions in carbon availability revealed numerous TA systems identified in our analysis likely participate in growth rate decisions [55]. In addition to responding to stress directly, the HipBA TA system participates in generating slowly or non-replicating persister cell subpopulations within a larger group of growing bacteria [7]. In this way, TA systems provide resistance to non-favorable environmental conditions as persister cells are better able to survive the onslaughts of severe stress than are replicating bacteria. Given the number and diversity of TA systems in M. tuberculosis, it is possible that some serve to stabilize the genome while others serve to provide stress resistance. Of particular interest are TA systems that participate in the biology of M. tuberculosis, either participating in stress-response or persister formation. Our findings that four TA systems are activated during cellular stress supports the notion that these loci participate directly in M. tuberculosis physiology. For example, both Rv1955-1956 and Rv2009-2010 are induced during the transition to hypoxia (Figure 6A), suggesting they play a role in the adaptation of M. tuberculosis to low oxygen conditions. Curiously, both of the hypoxia-induced TA loci, Rv1955-1956 and Rv2009-2010, are located within the same genomic island [35]. Although these genes are not part of the “dormancy regulon”, notable members of this regulon, dosT and fdxA, are also in the same genomic island. It may be that acquisition of this entire region helped promote M. tuberculosis' ability to respond to a hypoxic environment. In addition to the TA genes upregulated by hypoxia, Rv1560-Rv1561 and Rv0549c-0550c are specifically upregulated during macrophage infection (Figure 6B), in agreement with previous studies suggesting that these genes may be important during infection [56], [57]. Taken together, these data suggest that a subset of TA systems is important for M. tuberculosis in vivo, perhaps as stress-response elements. In an attempt to identify novel classes of TA systems, we incorporated a sequence-independent method in our bioinformatics search. Importantly, this strategy revealed three novel TA systems. One of these, Rv0909-0910, is conserved among the diverse range of the mycobacterial species analyzed (Figure 2 and Figure 3A), suggesting an ancient history and likely fundamental role in mycobacterial physiology. Our results indicate that Rv0910 lacks RNase activity (Figure 5C) and thus probably functions by an alternate mechanism than the majority of M. tuberculosis TA systems. Interestingly, Rv0910 is most similar to the polyketide cyclase group of the START domain superfamily of proteins [58]. Although it is not clear how a polyketide cyclase would function to inhibit cell growth, our results demonstrate for the first time that these two genes have toxin and antitoxin activities. It is interesting to note that mycobacteria encode numerous polyketide synthases and have an enormous capacity for lipid synthesis [35]. Since many TA toxins inhibit macromolecular synthesis (translation, DNA synthesis), it is tempting to speculate that Rv0910 may inhibit lipid biosynthesis. Although our approach yielded a total of 30 functional TA systems, it is likely that there are additional TA systems in the M. tuberculosis genome. Our search for putative systems was biased by using characteristics of known TA systems, including constraints on size and gene organization. Therefore, any TA system with different features would have been excluded from our search. Indeed, two other groups have incorporated homology-independent methods for comprehensive discovery of TA systems in microbial genomes and identify an additional 12–44 putative systems in the M. tuberculosis genome, depending on the level of scoring confidence chosen (Figure S1). We also cannot rule out the possibility that some of the toxins that did not inhibit growth were simply not expressed at sufficiently high levels using our system. Additionally, although M. smegmatis and M. tuberculosis are similar, we cannot discount the possibility that there are some species-specific factors that may be required for the proper function of some of the TA systems tested. Our results differ significantly from two recent reports in which M. tuberculosis TA systems were expressed in E. coli [12], [39]. Of the 78 putative systems we tested in M. smegmatis, only 38 have been evaluated in E. coli. Of the 30 functional TA systems we identified, seven were not functional in E. coli and a further 40 putative systems were not assessed. In contrast, only four systems were uniquely toxic in E. coli. The different results obtained in these studies may be due to the use of a distantly related host, E. coli, rather than the more closely related species, M. smegmatis. For example, specific factors such as transcript GC content, may greatly influence the number of potential targets of an RNase, and these differences are minimized by using another mycobacterial species. Given the huge expansion of VapBC homologs in M. tuberculosis, it seems likely that there are many more toxins that function via RNA cleavage than by other mechanisms. It is perplexing that one genome would have so many genes encoding proteins that perform the same function. One possibility is that most of these TA systems participate solely in genome stability and thus redundant function would not be an issue. However, all four of the stress-responsive TA systems we identified are putative RNAses. Therefore, another possibility is that, although they have the same function, specific TA systems are under different regulatory controls, such that only a subset are activated in response to particular stresses. Having a wide variety of mRNases under diverse regulatory mechanisms would allow the cell to adapt to many different conditions. An alternative explanation is that the different mRNases are actually functionally distinct. Under certain conditions, it may be useful for the cell to express an mRNase with a short, ubiquitous cleavage site that will target most of the existing messages in the cell, and allow for newly transcribed messages to be translated. This provides an efficient way to erase the previous transcriptional profile of the bacterium, allowing the cell to reprogram the proteome and thus rapidly change the metabolic state of the cell during conditions of stress (Figure 7). Alternatively, some TA systems may target only a limited number of messages and, therefore, not inhibit bulk translation. For example, one possible mechanism to tailor the response of the cell upon expression of an mRNase is through the recognition site at which mRNA cleavage occurs. Indeed, the cleavage sites for two M. tuberculosis MazF homologs target pentad sequences, longer than the three-residue recognition site of MazF in E. coli [13]. This may allow the targeting of specific messages, giving rise to more subtle changes in the metabolic state of the cell. It may be the presence of so many mRNases that allows M. tuberculosis to regulate growth, survival and metabolism during a wide range of environmental stresses, including those encountered during infection. This hypothesis is consistent with our findings that the vast majority of TA systems are present only in the virulent mycobacteria of the MTBC. All strains, plasmids and primers used in this study can be found in Table S6. TA system homologs in M. tuberculosis were identified using PSI-BLAST (NCBI) with a cut-off E-value of 10−2. Iterations were repeated until we obtained no new hits below the cut-off E-value. The input sequences for this analysis included the toxins and antitoxins of 8 major TA system families [32]. The origins of the input sequences of each TA system family used for BLAST analysis were as follows: CcdBA (F plasmid from E. coli), MazEF (MazEF from E. coli, PemK from Rhodococcus erythropolis), Doc/Phd (enterobacteria phage P1, Phd from Frankia alni ACN14a), RelBE (RelBE from E. coli, PasBA from plasmid pTCF14 of Acidithiobacillus caldus, YoeB/YefM from E. coli), HipBA (HipBA from E. coli), ParDE (ParDE from plasmid RK2 of E. coli), HigBA (HigBA from plasmid Rts1 of E. coli, HigA of Xylanimonas cellulosilytica DSM 15894), VapBC (VapBC from Frankia sp., StbBC from plasmid pDC3000B of Pseudomonas syringae). Following identification of toxin genes we determined if an adjacent upstream gene smaller than the putative toxin gene was present. Following identification of antitoxin genes we determined if an adjacent downstream gene larger than the putative toxin was present. In both cases, we required a maximum distance of 150 bp between the putative toxin and antitoxin. Homologs for which we were unable to find an adjacent cognate toxin or antitoxin or in which the adjacent gene did not meet our criteria for either size or distance between genes were excluded. In cases where the putative toxin and antitoxin of an adjacent pair were homologous to different TA system families, we assigned the pair based on the homology of the toxin gene. PIN domain-containing proteins were identified as previously described [16]. To find novel TA pairs we searched the M. tuberculosis genome for pairs of genes as previously described [40]. In summary, we identified pairs of open reading frames (ORFs) encoding hypothetical proteins in the M. tuberculosis genome of less than 150 amino acids, were less than 150 bp apart, and in which the upstream ORF was smaller than the downstream ORF. The fully sequenced and annotated genomes were downloaded from the NCBI database. The latest assemblies of unfinished genomes were downloaded from the Sanger Institute website with permission from Dr. Julian Parkhill. or the NCBI database with permission from Dr. Marcel Behr. We used standard BLASTP or TBLASTX to search protein or nucleic acid databases of each genome for homologs of the M. tuberculosis toxin proteins identified in this study. For each toxin homolog or ortholog identified, we determined whether an adjacent putative antitoxin was present. Orthologs were defined as BLAST reciprocal best hits (E-value<10−6) displaying conserved synteny with other orthologs [46]. A detailed description of the phylogenetic analysis can be found in Text S1 and Table S3. All putative M. tuberculosis toxin genes and toxin-antitoxin gene pairs were inserted downstream of the inducible acetamidase promoter in plasmid pHR100. Toxin and antitoxin activity was assessed by growing M. smegmatis carrying the appropriate vector at 37°C on 7H10 solid media with 0. 2% Tween-80,25 µg/ml kanamycin, and 0. 2% acetamide to induce gene expression. Growth was assessed after three days of incubation. Cross-talk between non-cognate VapB and VapC proteins was assessed by co-transforming M. smegmatis with the VapC toxin under the control of the acetamidase promoter and the VapB antitoxins under the control of the tetracycline-inducible promoter in plasmid pUV15tetORm [59]. Toxicity was assessed by patching colonies onto solid media in the absence (7H10 with 0. 2% Tween-80,25 µg/ml kanamycin, 50 µg/ml hygromycin) or presence of both inducers (7H10 with 0. 2% Tween-80,25 µg/ml kanamycin, 50 µg/ml hygromycin, 0. 2% acetamide, 50 ng/ml ATc). M. smegmatis cells carrying the appropriate expression vector were grown to early log phase at 37°C in 7H9 Middlebrook media with 25 µg/ml kanamycin. At OD600 0. 3, acetamide was added to a final concentration of 0. 2% to induce gene expression. Control cultures were treated with either 0. 5 µg/ml ciprofloxacin or 25 µg/ml hygromycin. Samples of 2 ml were harvested by centrifugation at the timepoints indicated and resuspended in 0. 5 ml media containing 5 µCi of 35S-methionine. After one minute of incorporation at 37°C, reactions were stopped by adding 1 ml 40 mM sodium azide and immediately frozen in liquid nitrogen. Proteins were precipitated with 10% trichloroacetic acid and concentrations were determined using Micro BCA Protein Assay Kit (Pierce). Radioactivity incorporated was assessed via a liquid scintillation counter and normalized to protein concentration in each sample. The radioactivity incorporated at t = 0 for each culture was set as 100% translation and all subsequent measurements were compared to this value. Toxin and antitoxin proteins for the RNase assay were expressed in E. coli BL21 (DE3) pLysS cells. The toxins were expressed as N-terminal (His) 6-MBP-TEV tagged fusions while the antitoxins were expressed as N-terminal GST fusions. Protein expression was induced for 3 h at 37°C with 500 µM IPTG. Toxin proteins were purified using Talon metal affinity resin (Clontech). The resin was washed using buffer (50 mM NaPO4,800 mM NaCl, pH 7. 1) containing imidazole at concentrations of 20,40, and 60 mM and eluted using 250 mM imidazole. Antitoxin protein were purified using glutathione resin, washed with 30 column volumes of the buffer indicated above and eluted using 15 mM reduced glutathione. Proteins were subsequently dialyzed in buffer containing 50 mM NaCl and 25 mM Tris-HCl. Toxin proteins were TEV-digested at a ratio of 1∶25 (TEV∶protein) in buffer containing 50 mM NaCl, 2 mM Tris-HCl and 2 mM DTT to remove the tags. 1. 6 µg MS2 RNA (Roche) was incubated for 3 h with 1 µg of each purified protein at 37°C in 10 mM Tris-HCl (pH 7). RNA was purified and samples were heated to 95°C for 5 min and placed on ice for 1 min before loading in a denaturing agarose gel (1% agarose, 6. 5% formaldehyde, 1× MOPS buffer). To assess antitoxin activity, 1 µg of purified toxin protein was incubated with antitoxins Rv0300-GST (5 µg) or MazF-GST (10 µg). RNA from each reaction was electrophoresed in a 2% agarose gel under non-denaturing conditions. NRP was induced essentially as described in [27] with a larger culture volume (830 ml) in 1 liter roller bottles to achieve a headspace ratio of 0. 5. Bacteria were pelleted and lysed at the indicated timepoints by bead beating with 200 µl 0. 1 mm zirconia beads (Biospec) at maximum speed for 90 s in 1 ml Trizol and total RNA was isolated via chloroform extraction and sodium acetate precipitation as previously described. [29] Bacterial RNA was amplified using the MessageAmp II Bacteria Prokaryotic RNA Kit per the manufacturer' s instructions (Ambion). Bone marrow-derived macrophages were isolated from C57BL/6 mice and cultured for 6 d in media containing 30% L-cell supernatant in the presence of antibiotics. Macrophages were stimulated with recombinant mouse IFN-γ at a final concentration of 50 units/ml for 24 h prior to infection. Macrophages were infected using DMEM containing 10% horse serum at a multiplicity of infection of 10, incubated for 2 h, washed and fresh medium was added. At the indicated timepoints, M. tuberculosis RNA from inside macrophages was isolated and amplified as previously described [50]. M. tuberculosis and M. smegmatis from in vitro-grown log-phase cultures were pelleted and lysed by bead beating in Trizol as described above and total RNA was isolated [29] and used for quantitative real-time PCR (qPCR) with the oligonucleotides specified (Table S7). The cDNA used for qPCR was generated with 3 µg of total RNA using the Superscript III First Strand Synthesis for RT-PCR kit (Invitrogen). Standard curves were generated by measuring the concentration of each message in a reference sample of RNA pooled from all indicated conditions and timepoints analyzed for each experiment. All values reported are given as relative expression of each gene compared to 16S RNA (gene/16S).

Title: Comprehensive Functional Analysis of Mycobacterium tuberculosis Toxin-Antitoxin Systems: Implications for Pathogenesis, Stress Responses, and Evolution Summary: Tuberculosis (TB) continues to be a major global health problem, causing 2 million deaths every year. A hallmark of TB pathogenesis is that the bacilli can enter into a slow or non-growing state in response to the host immune system. Because these persistent bacteria are resistant to antibiotic treatment, efforts to eliminate TB from the human population must include therapies to target dormant organisms as they can eventually resume replication to cause active disease. How Mycobacterium tuberculosis, the causative agent of TB, alters its replication dynamics in response to host cues is not understood. Toxin-antitoxin (TA) systems, which may control persistence in other bacteria, are massively expanded in M. tuberculosis, suggesting that they are important for TB pathogenesis. Surprisingly, the vast majority of these numerous TA systems are conserved only in pathogenic mycobacteria, suggesting their acquisition was important in M. tuberculosis evolution. Of the 88 putative TA systems identified, we show that 30 are functional in mycobacteria. A subset of these systems is activated upon exposure to stresses encountered during infection, indicating that specific TA systems are involved in adapting to environmental cues in the host. These genes are promising candidates for the development of novel therapies to target persistent bacteria.

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Summarize: David De Gea has been a rock in Manchester United's shaky defence during a difficult first five months for Louis van Gaal's new-look side, says former Old Trafford legend Peter Schmeichel. De Gea, who signed from Altetico Madrid for £18.9million in 2011, had a difficult start to life with United with many criticising the Spaniard for some shaky early displays between the posts. De Gea, however, has excelled for United in recent seasons and has been consistently excellent behind a questionable defence hit by injuries to key players such as Chris Smalling, Phil Jones and Luke Shaw and without the experienced trio of Nemanja Vidic, Rio Ferdinand and Patrice Evra who all moved on during the summer. David De Gea has been superb for Louis van Gaal's new look Manchester Untied side this season. David De Gea saves a shot from Southampton's Shane Long during his side's recent 2-1 victory at St Mary's. De Gea denies Arsenal forward Alex Oxlade-Chamberlain a shot at goal during United's win at the Emirates. Juan Mata (left) congratulates De Gea after his superb performance against Everton in October. And Schmeichel, who played eight seasons for United, believes De Gea has proven himself to be Van Gaal's most reliable player during the current campaign. 'He's doing really well,' Schmeichel told goal.com. I think he's the only player in the squad who performs consistently every time. Last season he was the only player who consistently brought any kind of quality to Manchester United. 'Last season was good for him. He won the championship in the year before but there was still this doubt about him and I think he washed that away with his performances.' After a disastrous start to the season, including a 5-3 humiliation at Leicester City in September, Van Gaal's has steadied the ship in recent weeks with United winning their last five Premier League games on the trot. Liverpool's Yossi Benayoun shoots past De Gea (left) during his time with Atletico Madrid back in 2010. Recent wins over Crystal Palace, Arsenal, Hull, Stoke City and Southampton have propelled United up to third in the table. 'That's how you win championships. That's how you qualify for the Champions League,' the Dane added. 'Last season, as soon as they came under pressure and went behind, they would lose the game which is very unlike Manchester United. 'To get results and play the way they played against Southampton and win that game, it was hugely important. 'At no point in that game were they impressive but that was the kind of game they would lose last season so that's a very big positive.' United will hoping De Gea's superb form continues as they target a sixth win in a row when they host arch-rivals Liverpool at Old Trafford on Sunday afternoon. Ex-United legend Peter Schmeichel (above) believes De Gea has been the most consistent player at the club

Summary: David De Gea signed from Atletico Madrid for £18.9million in 2011. De Gea received some harsh criticism during his early days at Old Trafford. The Spanish goalkeeper has excelled for United in recent seasons. De Gea has been superb behind an injury-hit United defence this season. Louis van Gaal's men have won their last five Premier League games. De Gea's side are currently third in the league standings. United host Liverpool at Old Trafford on Sunday afternoon.

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Summarize: At least four people are confirmed to have died near Wichita's Mid-Continent Airport after a light aircraft lost an engine during takeoff and plowed into a flight simulator building on Thursday morning. Five injured people were transported to Via-Christi Hospital and four people are currently unaccounted for, including the pilot, said fire Chief Ron Blackwell. By afternoon, one patient was described as being in serious condition, one in fair condition and three in good fair condition, according to a hospital spokeswoman. The roof of the FlightSafety International building - which houses 100 people - burst into flames described as 'horrific' when the twin engine Beechcraft King Air 200 came down shortly after 10am. Scroll down for Video. A small plane lost power after takeoff and crashed into a building Thursday while trying to return to a Kansas airport, killing at least four people. Plumes: Smoke billows from a building at Mid-Continent Airport in Wichita, Kansas on Thursday. Firefighters try to put out a fire at Mid-Continent Airport in Wichita after a small plane crashed into the building killing several people including the pilot. Officials say at least two people are dead after a small plane reported losing engine power and crashed into a building at a Kansas airport. Only the pilot was on the plane, but it wasn't immediately clear how many people were in the building at Wichita Mid-Continent Airport, Fire Chief Ronald D. Blackwell said. Four people remained unaccounted for hours after the crash, but a search was halted at midday after a portion of the building collapsed. Wichita Fire Marshal Brad Crisp assured onlookers the search would resume as soon as the building was deemed stable. 'We understand that this is a very difficult time, especially for folks who have family members who are working out here and they don't know,' Crisp said. 'This is also a very difficult time for first responders.' Huge plumes of smoke that could be seen for miles around the city billowed in the aftermath of the accident. 'I would imagine that there was probably a bit of panic in the early stages of the incident. We are grateful at this point that a large number of people appeared to have gotten out at this point but certainly saddened by the loss of life and anybody who might have gotten hurt,' said Chief Ron Blackwell, Wichita Fire Department according to KSN. Wichita Fire Chief Ronald D. Blackwell says at least four other people were taken to a hospital and five more are unaccounted for after the Thursday morning crash at Mid-Continent Airport. At least four people are confirmed to have died near Wichita's Mid-Continent Airport after a light aircraft lost an engine during takeoff and plowed into a flight simulator building on Thursday. Five injured people were transported to Via-Christi Hospital and four people are currently unaccounted for, including the pilot, said fire Chief Ron Blackwell. Wichita Fire Chief Ron Blackwell speaks with the media at Mid-Continent Airport in Wichita on Thursday. Emergency crews are on the scene at Mid-Continent Airport, and federal officials have confirmed the incident is not believed to be related to terrorism. At least 50 to 60 firefighters from the Wichita Fire Department battled the raging fire. The FAA said that just before the accident the pilot declared an emergency, saying 'we just lost the left engine'. FAA investigators are now at the scene and the National Transportation Safety Board has been notified of the crash. Emergency crews were headed to Wichita's Mid-Continent Airport after huge plumes of black smoke were seen rising into the sky. 'The airport crews arrived first. Seeing smoke and flames and what appears to be a plane striking the building. Firefighters engaged in a horrific fight for several minutes. We have the fire under control. We are in the process of trying to determine if all the employees and visitors who may have been in the building are accounted for,' said Wichita Fire Chief Ron Blackwell to KSN. Brian Youngers, a witness, told Kansas.com he was across the street from the building when the crash happened. 'We heard this 'vroom,''he said. 'It was way too loud, way to close. We were like, 'Holy crap.''The turbo-prop King Air 200, able to carry ten passengers, was being flown by one pilot when it came down on the Kansas airport and fire crews described fighting horrific flames in the aftermath of the crash. Crash: A huge plume of smoke billows out of the flight safety building at Wichita Mid-continental on Thursday. Federal Aviation Administration spokesman Tony Molinaro says a twin-engine Beechcraft King Air reported trouble just after takeoff Thursday morning. Ryan Weatherby said he and several other employees at Yingling Aviation, across the street from the airport, ran outside when they heard the crash and saw rolling black smoke and wreckage. 'It wasn't that loud. It was more like a screech or something,' Weatherby said. Jay Boyle, who works at the airport as a senior field technical adviser, said he saw people standing outside and pointing, then spotted the crash site. 'I could see from a distance the cutout in the side of the building where it looked like a wing had gone through and you could actually see the aircraft landing gear through a hole in the building,' he said. The crash did not appear to be significantly disrupting passenger traffic at the airport as planes could be seen taking off from other runways. Located several mile west of downtown Wichita, a longtime aircraft manufacturing hub, Wichita Mid-Continent is used by private aircraft and served by several airlines and their regional affiliates, including American, Southwest, Delta, United and Allegiant. It saw more than 13,000 departures and about 1.4 million passengers last year, according to the U.S. Department of Transportation. Wichita-area broadcasters posted photos and video of the billowing smoke at the airport shortly after 10 a.m. Thursday. Aircraft: The twin engined Beechcraft Super King Air 200 is capable of carrying up to eight passengers including the pilot

Summary: Twin engined Beechcraft King Air 200 light plane crash landed on take-off at 10am. Came down onto Wichita's Mid-Continent Airport's flight safety building. Four confirmed dead, five seriously injured and four unaccounted for. Fire department has said 100 people were in building when plane crashed. 50 to 60 firefighters from the Wichita Fire Department battled the fire.

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Write a title and summarize: Guantanamo. Reuters/STR Elvire : Est-ce vraiment grâce aux renseignements obtenus à Guantanamo que les Américains sont parvenus à localiser Oussama Ben Laden? Sylvie Kauffmann : En fait, il semble que les renseignements cruciaux qui ont permis d'identifier les contacts de Ben Laden, puis de le localiser au Pakistan ont été recueillis auprès de prisonniers d'Al-Qaida par la CIA, non pas à Guantanamo, mais dans les lieux secrets où ils étaient détenus et interrogés après leur capture. Ces détenus n'ont été transférés à Guantanamo qu'en 2006, où on a continué à les interroger. Guest : Peut-on penser que les méthodes d'interrogatoires musclées utilisées par les Américains ont aidé à l'élimination de Ben Laden? C'est une question qui soulève un débat très intense ces jours-ci aux Etats-Unis. Est-ce grâce à l'utilisation de la torture que ces renseignements cruciaux ont été obtenus? Ce que l'on sait jusqu'ici apporte une réponse nuancée. Les premiers détenus à avoir mentionné l'existence du messager de Ben Laden, c'est-à-dire l'homme grâce auquel les Américains sont finalement arrivés jusqu'à la résidence où se cachait le chef d'Al-Qaida, près d'Islamabad, ne semblent pas avoir été torturés pendant leur interrogatoire. En revanche, Khaled Cheikh Mohamed, l'un des plus importants dirigeants d'Al-Qaida que les Américains considèrent comme le cerveau du 11 septembre 2001, arrêté en 2003 au Pakistan, a été soumis, lui, à la torture du "waterboarding", qui est un simulacre de noyade, 183 fois, de l'aveu même de la CIA. Mais lorsqu'il a été interrogé sur l'existence et l'identité de ce messager, il a induit ses interrogateurs en erreur. Dans l'état des informations dont nous disposons actuellement, on ne peut donc pas affirmer catégoriquement que les renseignements qui ont permis de trouver puis de tuer Ben Laden ont été obtenus grâce aux méthodes d'interrogatoire musclées. Dans les documents de Guantanamo que nous a communiqués WikiLeaks et que nous avons diffusés dans Le Monde et sur LeMonde.fr depuis la semaine dernière, nous avons trouvé trace de ce messager dans l'interrogatoire d'un des détenus considérés comme l'un des plus importants du point de vue des renseignements apportés, Abu Al-Libi, un Libyen de 41 ans arrêté en 2005 au Pakistan. Ce détenu a raconté aux interrogateurs qu'en 2003 il était allé s'installer à Abbottabad, le lieu où a finalement été retrouvé Ben Laden. Il a également mentionné qu'il avait reçu à cette époque-là une lettre de Ben Laden qui lui avait été transmise par ce même messager. Mais on ignore à quelles techniques d'interrogatoire Abu Al-Libi a été soumis. Jarjar : Tous les moyens semblent légitimés aux Etats-Unis pour la guerre contre le terrrorisme : torture des prisonniers (waterboarding), élimination des ennemis (Ben Laden). Quel débat cela suscite-t-il outre-Atlantique? L'administration Bush avait autorisé, dans le cadre de la "guerre mondiale contre le terrorisme", des techniques d'interrogatoire "poussées" pour extraire des renseignements des gens soupçonnés de terrorisme. Cette décision, qui a notamment légalisé le "waterboarding", a été extrêmement controversée à l'étranger, mais aussi aux Etats-Unis. Le président Obama a annulé la plupart de ces dispositions lorsqu'il est arrivé au pouvoir. Mais l'élimination de Ben Laden relance le débat et les anciens de l'équipe Bush refont surface ces jours-ci en tentant de justifier l'usage de la torture. Ils essaient de prouver aujourd'hui que leur politique était la bonne et que l'usage de méthodes d'interrogatoire poussées était justifié. Le débat fait rage, mais le New York Times, par exemple, a pris position aujourd'hui, dans un éditorial très clair : rien ne peut justifier l'usage de la torture. VinX : Obama s'était clairement posé contre Guantanamo et la fermeture de cette prison était l'une de ses promesses de campagne. N'est-il pas dans une position contradictoire, mettant l'opération Ben Laden à son crédit tout en ayant denoncé les méthodes permettant la récuperation d'information? Cela illustre bien le paradoxe américain. Les Américains, et Barack Obama en particulier, sont profondément attachés aux libertés publiques qui sont inscrites dans leur Constitution et que la Cour suprême réaffirme régulièrement. Pourtant, depuis le 11 septembre 2001, ils se sont souvent trouvés en porte-à-faux à ce sujet. Barack Obama a en effet fait campagne sur la promesse de fermer le camp de Guantanamo, mais trois ans plus tard, il y a toujours 172 prisonniers dans ce camp militaire. Il est aussi étonnant, pour des Européens, d'entendre un président d'un pays démocratique dire que Oussama Ben Laden "a eu ce qu'il méritait", alors qu'on peut considérer qu'il a été exécuté sommairement sans autre forme de procès. C'est l'Amérique de l'après-11-septembre. Il faut aussi rappeler que dans plusieurs Etats américains, la peine de mort est toujours en vigueur et pratiquée. BlueRemedy : Existe t-il un cadre juridique au niveau international qui permet d'engager une action contre des terroristes? Le seul ordre juridique mondial qui existe est celui de l'ONU. Mais le terrorisme pose depuis longtemps un défi particulier sur le plan judiciaire, puisque l'on a affaire à des réseaux qui transcendent la compétence des Etats. Manola : Est-ce que tous les pays qui ont des ennemis publics numéro un ne vont pas désormais être fondés à faire des assassinats ciblés? Sur le principe, cette opération pose effectivement la question telle que vous la formulez. Dans la pratique, il faut tenir compte des rapports de force internationaux. Les Etats-Unis sont une superpuissance mondiale et Ben Laden n'était pas un terroriste mineur, mais le chef d'un réseau qui a attaqué de multiples pays à travers le monde. Les Américains savaient donc qu'ils ne risquaient guère une condamnation d'ensemble de la communauté internationale dans cette affaire. Buba : A quoi Guantanamo va-t'-il servir après la mort du leader d'Al-Qaida? Guantanamo est une prison qui abrite donc toujours 172 détenus. Comme le montrent les documents que nous avons obtenus de WikiLeaks, un certain nombre de ces détenus n'ont rien à y faire, mais les Américains ne trouvent pas de pays pour les accueillir. D'autres sont toujours considérés par les militaires américains comme des gens dangereux, susceptibles de reprendre une activité terroriste s'ils sont remis en liberté. Mais ils sortent du droit commun américain, raison pour laquelle ils sont toujours détenus à Guantanamo. C'est un véritable casse-tête juridique, politique et diplomatique dont Barack Obama n'a toujours pas réussi à se sortir. KeyserKev : Comment les Américains apprécient-ils la mort de Oussama Ben Laden : est ce un sentiment de justice qui règne ou de vengeance? Les premiers mots du président Obama en annonçant la mort de Ben Laden sont très révélateurs : "Justice has been done", la justice a été rendue. Cette phrase peut choquer en Europe, en particulier les juristes, mais elle a été très, très bien accueillie aux Etats-Unis. Le souvenir des attentats du 11-septembre et de leurs quelque trois mille victimes est toujours très vivace. Cet événement a marqué les Etats-Unis beaucoup plus profondément qu'on ne le croit en Europe, ce qui explique les réactions de joie que l'on a pu voir à New York et à Washington lundi. Arnaud : Si vous obtenez les photos de la mort d'Oussama Ben Laden de manière détournée, les publieriez vous? Bonne question. Je crois que nous allons voir circuler beaucoup de photos de Ben Laden mort sur Internet et qu'il va être extrêmement difficile de les authentifier. Dès le premier jour, d'ailleurs, la télévision pakistanaise a diffusé une photo d'une tête criblée de balles qui a fait le tour du monde et qui s'est bien sûr révélée ne pas être celle de Ben Laden. Il faut donc être très prudent. Tout dépend de la manière dont ces photos arriveront et de leur source. BlueRemedy : Est-ce important de faire la transparence sur la manière dont les renseignements ont été obtenus? Oui, cela me paraît important. La transparence, dans un événement de cette ampleur, est capitale. Si l'on voit que les autorités américaines cherchent à cacher des détails importants, cela jettera le doute sur la manière dont l'opération a été conduite. Mais par exemple, l'administration Obama a reconnu, après avoir dit dans un premier temps que Ben Laden avait résisté à son arrestation, qu'il n'était pas armé. Ce genre d'information est importante, de même que le fait qu'elle soit communiquée officiellement. La question de la publication de la photo du cadavre est différente. Là, l'argument de la menace que la diffusion de cette photo pourrait poser à la sécurité des troupes américaines peut, me semble-t-il, être entendu. KeyserKev : Comprenez vous les voix qui s'élèvent et qui commence à douter des versions officielles et des informations qui circulent. Comment, dans votre position, vous devez retranscrire cela? Oui, il y aura toujours des gens qui douteront des versions officielles américaines et il y aura toujours des théories conspirationnistes qui se feront jour, sur cette question comme sur d'autres. C'est inévitable. Notre rôle à nous, comme journalistes, est d'enquêter, de vérifier. Certains événements comme l'attaque des Twin Towers ont eu tellement de témoins et ont été tellement documentés qu'ils nous paraissent incontestables. Sur la mort de Ben Laden, l'ensemble des informations communiquées de multiples sources rendent le discours officiel américain très crédible. Même si le corps a été jeté en mer, les Américains ont gardé des éléments physiques qui devraient leur permettre de convaincre, le cas échéant, les sceptiques. Chat modéré par Caroline Monnot

Title: "Mort de Ben Laden:" "Le débat sur les techniques d'interrogatoire est relancé aux Etats-Unis" " Summary: Dans un chat sur LeMonde.fr, Sylvie Kauffmann, directrice de la rédaction du "Monde" explique que les anciens de l'équipe Bush refont surface ces jours-ci en tentant de justifier l'usage de la torture. "Ils essaient de prouver aujourd'hui que leur politique était la bonne et que l'usage de méthodes d'interrogatoire poussées était justifié" indique-t-elle.

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Write a title and summarize: Indian economy At the beginning of the 18th Century, India's share of the world economy was 23%, as large as all of Europe put together. By the time the British departed India, it had dropped to less than 4%. The reason was simple: India was governed for the benefit of Britain. Britain's rise for 200 years was financed by its depredations in India. By the end of the 19th Century, India was Britain's biggest cash-cow, the world's biggest purchaser of British exports and the source of highly paid employment for British civil servants - all at India's own expense. We literally paid for our own oppression. De-industrialisation of India Britain's Industrial Revolution was built on the de-industrialisation of India - the destruction of Indian textiles and their replacement by manufacturing in England, using Indian raw material and exporting the finished products back to India and the rest of the world. The handloom weavers of Bengal had produced and exported some of the world's most desirable fabrics, especially cheap but fine muslins, some light as "woven air". Britain's response was to cut off the thumbs of Bengali weavers, break their looms and impose duties and tariffs on Indian cloth, while flooding India and the world with cheaper fabric from the new satanic steam mills of Britain. Weavers became beggars, manufacturing collapsed; the population of Dhaka, which was once the great centre of muslin production, fell by 90%. So instead of a great exporter of finished products, India became an importer of British ones, while its share of world exports fell from 27% to 2%. 'Clive of India' Colonialists like Robert Clive bought their "rotten boroughs" in England with the proceeds of their loot in India (loot, by the way, was a Hindi word they took into their dictionaries as well as their habits), while publicly marvelling at their own self-restraint in not stealing even more than they did. And the British had the gall to call him "Clive of India", as if he belonged to the country, when all he really did was to ensure that much of the country belonged to him. Bengal famine As Britain ruthlessly exploited India, between 15 and 29 million Indians died tragically unnecessary deaths from starvation. The last large-scale famine to take place in India was under British rule; none has taken place since, since free democracies don't let their people starve to death. Some four million Bengalis died in the Great Bengal Famine of 1943 after Winston Churchill deliberately ordered the diversion of food from starving Indian civilians to well-supplied British soldiers and European stockpiles. "The starvation of anyway underfed Bengalis is less serious than that of sturdy Greeks," he argued. When officers of conscience pointed out in a telegram to the prime minister the scale of the tragedy caused by his decisions, Mr Churchill's only response was to ask peevishly "Why hasn't Gandhi died yet?" Myth of 'enlightened despotism' British imperialism had long justified itself with the pretence that it was enlightened despotism, conducted for the benefit of the governed. Mr Churchill's inhumane conduct in 1943 gave the lie to this myth. But it had been battered for two centuries already: British imperialism had triumphed not just by conquest and deception on a grand scale, but by blowing rebels to bits from the mouths of cannons, massacring unarmed protesters at Jallianwala Bagh and upholding iniquity through institutionalised racism. No Indian in the colonial era was ever allowed to feel British; he was always a subject, never a citizen. Indian railways The construction of the Indian Railways is often pointed to as a benefit of British rule, ignoring the obvious fact that many countries have built railways without having to be colonised to do so. Nor were the railways laid to serve the Indian public. They were intended to help the British get around, and above all to carry Indian raw materials to the ports to be shipped to Britain. The movement of people was incidental except when it served colonial interests; no effort was made to ensure that supply matched demand for mass transport. In fact the Indian Railways were a big British colonial scam. British shareholders made absurd amounts of money by investing in the railways, where the government guaranteed extravagant returns on capital, paid for by Indian taxes. Thanks to British rapacity, a mile of Indian railways cost double that of a mile in Canada and Australia. It was a splendid racket for the British, who made all the profits, controlled the technology and supplied all the equipment, which meant once again that the benefits went out of India. It was a scheme described at the time as "private enterprise at public risk". Private British enterprise, public Indian risk. British aid In recent years, even as the reparations debate has been growing louder, British politicians have in fact been wondering whether countries like India should even receive basic economic aid at the expense of the British taxpayer. To begin with, the aid received is 0.4%, which is less than half of 1% of India's GDP. British aid, which is far from the amounts a reparation debate would throw up, is only a fraction of India's fertiliser subsidy to farmers, which may be an appropriate metaphor for this argument. Britons may see our love of cricket or the English language, or even parliamentary democracy, conjuring up memories of the Raj as in television series like Indian Summers, with Simla, and garden parties, and gentile Indians. For many Indians, however, it is a history of loot, massacres, bloodshed, of the banishing of the last Mughal emperor on a bullock cart to Burma. Indian soldiers in world wars India contributed more soldiers to British forces fighting the First World War than Australia, Canada, New Zealand and South Africa combined. Despite suffering recession, poverty and an influenza epidemic, India's contributions in cash and materiel amount to £8bn ($12bn) in today's money. Two and a half million Indians also fought for British forces in the Second World War, by the end of which £1.25bn of Britain's total £3bn war debt was owed to India, which was merely the tip of the iceberg that was colonial exploitation. It still hasn't been paid. 'Return the Koh-i-Noor diamond' What's important is not the quantum of reparations that Britain should pay, but the principle of atonement. Two hundred years of injustice cannot be compensated for with any specific amount. I, for one, would be happy to accept a symbolic pound a year for the next two hundred years, as a token of apology. And maybe Britain could kindly return the Koh-i-Noor diamond to the country it was taken from!

Title: Viewpoint: Britain must pay reparations to India Summary: At the end of May, the Oxford Union held a debate on the motion "This house believes Britain owes reparations to her former colonies". Speakers included former Conservative MP Sir Richard Ottaway, Indian politician and writer Shashi Tharoor and British historian John Mackenzie. Shashi Tharoor's argument in support of the motion, went viral in India after he tweeted it out from his personal account. The argument has found favour among Indians, where the subject of colonial exploitation remains a sore topic. Here he gives a summary of his views:.

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Write a title and summarize: La Société générale est-elle mêlée aux opérations qui ont permis à la banque d'affaires américaine Goldman Sachs de traverser avec plus de bonheur que d'autres la crise financière? Goldman est dans le collimateur de trois instances américaines : la Commission de contrôle de l'Etat de la Chambre ; une commission d'enquête créée par le Congrès pour élucider les responsabilités dans la débâcle ; et surtout le Sigtarp, qui vérifie l'usage des fonds du plan de sauvetage du secteur financier américain. Elle est soupçonnée d'avoir développé un système lui permettant d'être massivement bénéficiaire - dès lors qu'elle a misé sur l'effondrement des "CDS", des titres de défaut de dette - pour jouir ensuite d'un remboursement rubis sur l'ongle, sur ces mêmes titres, de son assureur AIG. Et ce, pour des pertes dont elle avait elle-même fixé le montant... Une sorte de coup double. Le 16 septembre 2008, l'Etat américain renflouait AIG à hauteur de 85 milliards de dollars. Huit banques - la Société générale, Goldman, Merrill Lynch, la Deutsche Bank, UBS, Calyon, Barclays et Bank of America - se sont vu remettre 67 milliards de dollars par leur assureur AIG. La Société générale et Goldman en ont été, de loin, les principaux bénéficiaires. Jusqu'ici, les élus américains s'interrogeaient sur cette soudaine générosité du Trésor et de la Réserve fédérale (banque centrale) qui, sans négocier des rabais avec ces banques, leur ont offert de récupérer l'intégralité des pertes qu'elles estimaient. Mais le 7 février, le New York Times assure qu'"une partie des 11 milliards de l'argent des contribuables remis à la Société générale (...) a par la suite été transférée chez Goldman, conformément à un accord préalable entre les deux banques". Le lendemain, Goldman réagissait : "Cette assertion est fausse et trompeuse. Goldman Sachs a fourni des financements à de nombreuses contreparties, mais nous n'aurions pas pu savoir si une contrepartie avait contracté une assurance contre le défaut de crédit, sans parler de connaître qui est concerné et pour quel montant." Selon un expert de Wall Street, Goldman explique ne "pas avoir su que la Société générale avait un CDS chez AIG et qu'en conséquence le paiement qu'elle a reçu n'est pas corrélé avec ce fait". Mais la concomitance entre le remboursement des pertes par AIG dès son renflouement et le transfert instantané par la Générale d'une somme X vers Goldman rend l'absence de tout lien entre ces faits peu plausible. Et d'autant plus intriguant. Si Goldman dit vrai, pour quel motif la Société générale lui transfère-t-elle des fonds alors? Soupçons Selon les documents en possession du Sigtarp, Goldman avait vendu à la Générale la moitié des titres que celle-ci a assurés ensuite chez AIG. Goldman connaissait donc la position de la Société générale sur ces titres et leur valeur, qui a servi de base pour calculer les pertes prévisibles. Sollicitée par Le Monde, la Société générale à New York s'est, par écrit, refusée à tout commentaire. A Paris, un proche du dossier réfute la thèse de l'accord secret avec Goldman, et d'une Société générale négociant pour le compte de l'américaine auprès de la Fed. "A aucun moment, il n'y a eu de discussions multilatérales, nous n'avons jamais parlé pour le compte de quelqu'un d'autre, affirme cette source. Les sommes reversées à Goldman ne représentent que quelques pour-cent des 11 milliards. Elles sont liées au débouclage mécanique d'opérations commerciales normales, antérieures à la crise." La Générale aurait cédé à Goldman des CDS et des CDO (obligations adossées à des actifs) assurés par AIG, entre 2002 et 2007, qu'elle a rachetés lors du dénouement des opérations d'AIG. Reste une question : si son versement s'explique facilement, pourquoi ne pas le faire publiquement, au risque d'attiser le soupçon des autorités américaines? Anne Michel, Sylvain Cypel et Sylvain Cypel (à New York) et Anne Michel

Title: Scandale AIG: le rôle de Goldman Sachs et de la Société générale en question Summary: Selon le "New York Times", un accord secret a lié les deux banques lors du sauvetage de AIG. Le 16 septembre 2008, l'Etat américain renflouait l'assureur à hauteur de 85 milliards de dollars. Huit banques, dans la foulée, se sont vu remettre 67 milliards de dollars par l'assureur pour couvrir leurs pertes. Goldman Sachs et la Société générale en ont été les principaux bénéficiaires.

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Write a title and summarize: The spread of the invasive snail Pomacea canaliculata is expanding the rat lungworm disease beyond its native range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on the rat small intestine morphology and physiology. Through a combination of biochemical, histochemical, histopathological, scanning electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate, hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins, as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days. Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of predators of apple snail eggs. The invasive apple snail Pomacea canaliculata (Lamarck, 1822) (Architaenioglossa, Ampullariidae) has become a serious aquatic crop pest in Asia and a vector of the rat lungworm Angiostrongylus cantonensis that causes human eosinophilic meningitis, a potentially fatal disease considered an emerging infectious disease. Unfortunately angiostrongyliasis (rat lungworm disease) continues to be reported in new regions beyond its native range which has been associated with the expansion of this snail [1]; [2]. P. canaliculata is the only freshwater snail listed among the 100 worst invasive species worldwide [3]. Their successful establishment in invaded areas may be related, among other factors, to their high fecundity, and the unusual characteristics of their eggs that increase the risk of the expansion of the disease. Females of P. canaliculata deposit hundreds of bright pink-reddish egg masses, each containing 30–300 eggs [4]; [5]. These egg clutches are remarkable in three respects: they are cemented outside the water, they are brightly colored and have virtually no predators, presumably because they have unusual defenses against predation [5]–[8]. Though filled with a perivitellin fluid (PVF) containing large amounts of carbohydrates and storage proteins (called perivitellins), these toxic eggs have no predators reported in their original South American range and only one in the newly colonized habitats in SE Asia: the fire ant Solenopsis geminata (Fabricius, 1804). The presence of these egg defenses [6]; [8]; [9] would explain the behavior of the snail kite Rostrhamus sociabilis (Vieillot, 1817) and Norway rat Rattus norvegicus (Berkenhout, 1769) that invariably discard the gland that synthesizes the egg defenses when predating on adult female P. canaliculata [10]–[14]. Work in the last two decades identified the perivitellins PcOvo and PcPV2 in the egg defenses against predation [6]; [8]; [13]; [15]–[17]. These are the most abundant perivitellins stored in large quantities in the PVF (57. 0% and 7. 5% of egg total protein for PcOvo and PcPV2, respectively) [15]. Both are resistant to proteolysis reaching the intestine in a biologically active conformation [6]; [8]. PcPV2 is a neurotoxic storage lectin with a strong lethal effect on selected neurons within the spinal cord of mice [9]; [18]. It is a novel combination of a tachylectin-like subunit with a membrane attack complex/perforin (MACPF) -like subunit, not reported in animals before [8]; PcOvo, on the other hand, is a storage carotenoprotein that provides the conspicuously reddish coloration of the clutches which presumably advertises to visual-hunting predators the presence of egg defenses (aposematic warning) [19]. In addition, PcOvo is a proteinase inhibitor limiting the ability of predators to digest egg nutrients. In fact, oral administration of purified PcOvo to rats significantly diminished rat growth rate presumably by a dual mechanism: the inhibition of trypsin activity (antidigestive role) and the resistance of the inhibitor to digestion by gut enzymes (antinutritive) [6]; [20]–[22]. A recent proteomic analysis of P. canaliculata PVF identified a small amount of over 50 other proteins, including two F-type lectins, many proteins involved in innate immunity in other mollusks and some with potential roles against insects and fungi [23]. As the epithelial cells along the digestive tract of animals are fully exposed to food contents, they are possible target sites for defense proteins. In this regard, plants have evolved a wide array of toxic dietary lectins that interact with the membrane glycoproteins of the luminal side of the gut of higher animals having an important role in plant defenses against predation [24]. There are, however, no reports in animals of such a defense mechanism [25]. With the aim to further understand the role of egg defenses of a host of the lungworm disease, in the present work we studied the effect of P. canaliculata PVF on the small intestine of rats. Through a combination of biochemical, histopathological, cell culture and feeding experiments, we provide evidence that oral administration of apple snail PVF adversely affects rat small intestine metabolism and morphology and consequently rat growth rate, presumably by proteins displaying lectin-like activity. This overall effect has not been found in other animals, but it is remarkably similar to that for plant seed lectins on the gastrointestinal tract of rats and other vertebrates. All studies performed with animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals [26] and were approved by the “Comité Institucional de Cuidado y Uso de Animales de Experimentación” of the School of Medicine, UNLP (Assurance No. P08-01-2013). Egg masses of P. canaliculata were collected either from females raised in our laboratory or taken from the wild in streams or ponds near La Plata city, Province of Buenos Aires, Argentina, between November and March of consecutive reproductive seasons. Only egg masses with embryos developed to no more than the morula stage were employed. Embryo development was checked microscopically in each egg mass as described elsewhere [16]. All experiments with rats were performed using male Wistar rats from the Animal Facility of the School of Medicine of the National University of La Plata (UNLP), Argentina. Rats came from a colony started with the strain WKAHlHok (Hokkaido University, Japan). Six-week-old animals weighing 180±2 g at the start of the experiments were housed in cages with 12 h day-night cycle, temperature of 22±1°C and relative humidity of 45–60%. Fertilized eggs were repeatedly rinsed with ice cold 20 mM Tris-HCl, pH 6. 8, containing a protease inhibitor cocktail (Sigma Chemicals, St. Louis) and homogenised in a Potter type homogeniser (Thomas Sci., Swedesvoro, NJ). Ratio of buffer: sample was kept 5∶1 v/w. The crude homogenates were then sonicated for 15 sec and centrifuged sequentially at 10,000×g for 30 min and at 100,000×g for 60 min. The pellet was discarded and the supernatant comprising the egg PVF was equilibrated in 50 mM phosphate buffer pH 7. 4 using a centrifugal filter device of 50 kDa molecular weight cut off (Millipore Corporation, MA) to eliminate potentially interfering compounds. Total protein concentration of the PVF (13. 3 g/L) was measured by the method of Lowry et al. [27]. Cylindrical tissue samples of the small intestine were post fixed in 10% neutral formaldehyde for 24 h at room temperature and then embedded in paraffin wax. Representative 5–7 µm sections were stained with haematoxylin and eosin for histological examination of general morphology. In addition, periodic acid Schiff (PAS) staining was performed to highlight carbohydrate distribution and goblet cells. Fifty properly oriented villi and crypts from duodenum were selected at random from each animal and their length and width measured to calculate mucosal absorptive surface area following the method of Kisielinsky [29] whose results have no significant differences compared with the Harris method, widely used in rats [30]. The method considers a geometric mucosal unit of a cylindrical villous with rounded tip surrounded by cylindrical crypts. It assumes that the whole mucosa is an iteration of this unit, and the surface area can be calculated with mean values of structures that define the mucosal unit: villus length, villus width, and crypt width. Thus, the mucosal-to-serosal amplification ratio M was calculated considering these 3 variables, as follows: Small intestine sections were assayed by immunohistochemistry (IHC) to evaluate cellular proliferation using a primary monoclonal mouse against the proliferating cellular nuclear antigen (PCNA) as a proliferation marker (Dako, Clon PC10). The antibody was diluted in 0. 1% BSA in phosphate buffer and incubated overnight at 4°C. PCNA is a nuclear acid protein which functions as δ DNA polymerase helper. In the presence of PCNA and a replication C factor, δ DNA polymerase starts the synthesis of DNA and the progression of the cellular cycle. Samples were incubated overnight at 4°C as mentioned above, and visualized using the LSAB kit (Dako Cytomation Lab, Carpinteria, USA) detection system which is based on a modified labeled avidin-biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecules. In short, after incubation with the appropriate primary antibody, a sequential 10 min incubation with an anti-mouse biotinylated antibody and peroxidase-labelled streptavidin is performed. Then staining is completed by incubation with 3,3′diaminobenzidine tetrahydrochloride (DAB) and H2O2. Positively stained cells showed a golden, dark-brown color. All sections were counterstained with Maeyer haematoxilyn before analysis. Primary antibody was replaced by normal mouse antiserum in control sections. Small intestine sections were assayed with seven lectins (Table 1) (Lectin Biotinylated BK 1000 Kit, Vector Laboratories Inc., Carpinteria, CA, USA) namely: Con A (Concanavalia ensiformis), DBA (Dolichos biflorus), SBA (Glycine max), PNA (Arachis hypogaea), RCA-I (Ricinus communis-I), UEA-I (Ulex europaeus-I) and WGA (Triticum vulgaris) to reveal possible changes of the glycosylation pattern. In short, paraffin sections were deparaffinized with xylene dehydrated with 100% alcohol twice, 10 min each, and then endogenous peroxidase activity was quenched by incubating 5 min with hydrogen peroxide in methanol 0. 3–3. 0%. They were then hydrated, washed in phosphate-buffered saline, and incubated with biotinylated lectins overnight. Then sections were washed with PBS, followed by 10-min incubation with streptavidin-HRP (streptavidin conjugated to horseradish peroxidase in PBS containing stabilizing protein and anti-microbial agents (Vector Laboratories Inc., USA). Finally the bound lectins were visualized by incubation during 4–10 min with a buffered Tris-HCl solution (0. 05 M, pH = 6. 0) containing 0. 02% 3,3′-diamino-benzidine tetrahydrochloride (DAB) and 0. 05% H2O2 (DAB; Dako, Carpinteria, USA). Positively-stained cells were demonstrated by a dark golden brown coloration. The sections were counterstained with Maeyer haematoxilyn. After 2-hour fixation in 2% (v/v) glutharaldehyde, samples were dehydrated in graded series of ethanol. Then ethanol was replaced by liquid carbon dioxide and samples were dried by critical point in a CP-30 (Balzers). Samples were gold metalized in a JEOL Fine Ion Sputter, JCF-1100. Observations and photomicrographs were obtained with a JEOL JSM 6360 LV SEM (Jeol Technics Ltd., Tokyo, Japan) at the Service of Electron Microscopy, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Argentina. Horse, goat, rabbit and rat erythrocytes were obtained from the animal facilities at the University of La Plata (UNLP). Blood samples were obtained by venous puncture and collected in sterile Elsever' s solution (100 mM glucose, 20 mM NaCl, and 30 mM sodium citrate, pH 7. 2) (Sigma Chemicals, St. Louis). Prior to use, erythrocytes were washed by centrifugation at 1500 g for 10 min in TBS buffer (20 mM Tris, 150 mM NaCl, pH 7. 4). This procedure was repeated several times until the supernatant remained clear. Hemagglutinating activity was assayed in microtiter U plates (Greiner Bio One, Germany) by incubating a two-fold serial dilution of PVF (6 mg/mL) in TBS with 2% erythrocyte suspension in TBS at 37°C for 2 h. Results were expressed as the inverse of the last dilution showing visible hemagglutinating activity by naked eye. Human colorectal adenocarcinoma cells (Caco-2) were cultured in Dulbecco' s modified Eagle' s medium (DMEM) (4. 5 g/liter D-glucose) supplemented with 10% newborn calf serum, penicillin (10 U/mL), streptomycin (10 µg/mL), amino acids and vitamins (Life Technologies-Invitrogen). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Culture medium was replaced every 2 days and subcultured by trypsinization when 95% confluent. Passages 60 through 65 were used for the experiments. Prior to each experiment, the viability of the cells was determined by trypan blue exclusion. Viability of every cell preparation exceeded 90% as determined by counting the stained cells. The cytotoxic effect of the PVF on Caco-2 cells was evaluated using the 3- (4,5-dimethythiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay [31]. Cells were seeded in 200 µL of culture medium on 48-well plates at densities that ensured approximately 90% confluency after 24 h. Once cell cultures reached the desired confluence, 50 µl/well of a serial dilution of PVF (6 mg/mL) in PBS were added and incubated at 37°C for 24 h. Control wells were prepared with 50 µL/well of PBS. After treatments, culture medium was removed and cells were incubated with fresh medium containing 0. 5 g/L of MTT at 37°C for 1 h. Plates were then centrifuged, the supernatant discarded and the cells were washed three times with PBS. Finally the cell monolayers were extracted with 200 µL/well of DMSO and the absorbance of each well recorded at 540 nm with background substraction at 640 nm in a microplate reader Multimode Detector DTX-880 (Beckman Coulter, Inc., CA, USA). Cell viability was expressed as control percentage [31]. %Viability = (OD treated cells/OD control cells) ×100 Data collected from all experiments were analyzed individually by either t test (histology) or ANOVA (bioassays) using Instat v. 3. 05 (Graphpad Software Inc.). Where significant differences between samples occurred, a post-hoc Tukey' s HSD test was performed to identify the differing means. Results were considered significant at the 5% level. GenBank accession numbers for PcOvo subunits: JQ818215, JQ818216 and JQ818217; GenBank accession numbers for PcPV2 subunits: JX155861 and JX155862. During the first 3 days of treatment with PVF, treated rats showed a significantly lower standard growth rate than the control ones (Fig. 1). This effect on growth rate disappeared after the fourth day of treatment and animals began to grow at the same rate as control groups. Daily food ingestion was similar in control and treated rats along the experimental period (results not shown). Oral administration of PVF for 10 days increased the mean intestinal length of the rats though a tendency was already evident after a 4-day treatment (Fig. 2). Within four days of switching the 10-day treated animals to a control diet, the total length of the small intestine returned to control values (Fig. 2). Crypt dimensions and general morphology of intestine were virtually restored to normal. At day 4, samples from control animals showed the characteristic tall, finger-like villi, whereas villi from treated animals showed significantly less height and were wider with some proliferation in the basal zone of the epithelia. In certain areas of the epithelium of treated animals, altered villi with a double, fused or “tongue” shape, displaying a bridge pattern were observed by SEM and light microscopy (Fig. 3). PAS staining was moderate on the glycocalyx of villi and crypt enterocytes while the mucin of goblet cells showed a strong stain in both control and treated samples. Mucose epithelia from treated animals showed an increased number of goblet cells (Fig. 4 A, B). SEM analysis of control and treated animals confirmed the remarkable differences on the length and width of the villi of treated animals (Fig. 3). The increased amount of mucus on the mucosa in the treated animals was also observed with this technique, as well as areas displaying conical, dome-shaped mucosal elevations which seem to connect two villi in a bridge-epithelial pattern (Fig. 3). PCNA labeling showed moderate immunostaining in the basal areas of the epithelium from controls, while a stronger staining was evident on the treated animals. (Fig. 4 C, D arrows). Intestinal epithelial cells were studied using a set of 7 lectins, of which PNA and SBA produced the most remarkable results (Fig. 4). PNA was strongly positive on the supranuclear region of enterocytes of control animals, while in treated animals its binding was observed not only in this region (strong staining) but also in the whole enterocyte (light staining) (Fig. 4, E, F arrows). Besides, SBA lectin binding was strong on the glycocalyx of the apical zone of the enterocytes of treated rats in comparison to the moderate staining in control group, indicating SBA-binding glycans were more expressed on enterocytes exposed to snail egg PVF (Fig. 4 G, H arrows). When the effect of PVF on rat small intestine absorptive surface was quantified on histological sections, a significant decrease of the 4-day treated animals was observed while if the ingestion is continued for 8 days, the absorptive surface reverted to normal (Table 2). When rats were exposed to PcOvo, the small intestine did not show significant changes in absorptive surface for up to 8 days (Table 2) and villi morphology was normal. The egg PVF of P. canaliculata showed hemagglutinating activity against horse red blood cells up to a protein dilution of 0. 15 mg/mL, indicating the presence of active lectins. Moreover, a moderate agglutinating activity against rabbit and rat red blood cells was also observed at 0. 6 mg/mL of PVF protein concentration (Fig. 5). The MTT assay showed that PVF displays cytotoxic activity on Caco-2 cell monolayers in a dose-dependent manner. A very significant reduction of cell viability to only 6. 6±0. 6% in PVF-treated monolayers as compared to control ones was observed at a PVF protein concentration of 0. 6 mg/mL (Fig. 6). The ingestion of apple snail PVF severely affects the gastrointestinal tract rapidly causing a decrease in growth rate. Shortly after feeding a diet containing PVF the rat intestinal morphology undergoes a dramatic change. This included shorter and wider villi and fusion of villi by epithelial bridging, which might be related to the ability of the epithelial cells to stretch in order to cover denuded areas [32]. The observed enlargement of both villous and crypt thickness in treated animals was associated with the presence of hyperplasic crypts and hypertrophic mucosal growth changes. The notable increase in enterocyte proliferation and the presence of immature enterocytes in the crypts suggest increased mitotic activity in treated animals. This in vivo effect was further supported by the analysis of PVF cytotoxicity toward differentiated intestinal cells which indicates the presence of toxins somehow damaging these enterocytes. This damage in turn would induce the proliferative response observed at the crypt. Thus, the ingestion of PVF seems to interfere with gut and systemic metabolism, inducing hyperplasia and hypertrophy of the small intestine and alterations in organ function. Despite this effect on the gut being well established for plant toxic lectins [33] it has not yet been reported for the ingestion of animal proteins. The enterocyte proliferation was also associated with changes of the glycosylation pattern revealed by the differential binding of the plant lectins PNA and SBA. PNA binds to the supranuclear portion of enterocytes where Golgi apparatus is located. It has been reported in humans orally intoxicated with PNA that the perturbation of cell kinetics and the more rapid cell migration and turnover of enterocytes may be reflected as synthesis of incomplete nascent glycoproteins, and expressed by altered PNA binding patterns [34]. This is also a well-known effect caused in rats intoxicated by other plant lectins that, as metabolic signals, can radically alter the pattern of glycosylation of the gut epithelium and thus further amplify their potent physiological effects [33]; [35]. These similarities between the effects of PVF and plant lectins lead us to look for lectin hemagglutinating activity in the PVF, which was found positive for some mammalian erythrocytes. This agrees with the recent identification of two putative lectins in a proteomic study of P. canaliculata PVF [23]. As mentioned before, one of these lectins, PcPV2, is the second most abundant egg protein. A functional study performed after its ingestion by rats showed that PcPV2 has the ability to withstand protease digestion, displaying structural stability within the pH range of the gastrointestinal tract of rats. Moreover, this toxic lectin binds to the glycocalyx of rat enterocytes in vivo and to Caco-2 cells in culture [8]. This interaction is also in agreement with the high cytotoxic effect of the snail PVF on Caco-2 cells observed in this study. These properties are concurrent with those of many plant lectins which are resistant to mammalian gastrointestinal digestion and their toxicity is mainly attributed to the binding to the glycan surface of the small intestine epithelial cells, which leads to interferences with the digestion and anatomical abnormalities [35]–[38]. Besides lectins, PVF also contains the proteinase inhibitor PcOvo. When the effect of a PVF-containing diet on rat growth rate (Fig. 1) is compared with that of a PcOvo-containing diet [6], a larger decrease of rat growth rate was observed with PVF, indicating there are more defensive compounds acting synergistically. In addition, a PVF-supplemented diet, unlike a PcOvo-supplemented one, diminished intestinal absorptive surface. A literature survey reveals no information on animals in this regard but again, a reduction of the absorptive surface area was reported after the administration of diets containing plant lectins to rats, causing malabsorption of nutrients [33]; [39]. As a whole, the decrease on rat growth rate and changes in intestine morphology and absorptive surface caused by PVF ingestion together with the reported ability of PcPV2 toxic lectin to bind intestinal cells were rather similar to the effect observed on rodents fed with diets containing plant lectins strongly suggesting that PVF lectins may be involved in the observed effect of snail toxic eggs on the gut of the rat. If the ingestion of PVF is continued, the rat growth rate becomes indistinguishable from that in control rats indicating an adaptation overcoming the antinutritional effect. Changes in the length of small intestine are often related with the difficulty in digesting the food. Greater length increases the transit time, thus maximizing digestion [40]. The adaptation to the PVF involved a time-dependent increase of the small intestine length, clearly observed after 10-day treatment. Similar effects were also observed in rats 3 days after administering diets containing phytohemagluttinin (PHA) from red kidney beans and other plant lectins [41]; [42]. However, those studies have shown that PHA-treatment of rats resulted in pancreas growth [42]; [43]. No such effect was observed in the current study (results not shown). In addition, the increase in mucous secretion suggests another adaptation allowing the isolation and protection of the intestinal surface from the toxic proteins. The change in length was virtually reverted 4 days after the elimination of the toxins from the diet along with the recovery of the normal tissue morphology. It is worth recalling that the mucosa of the small intestine is lined with epithelium that has the shortest turnover rate of any tissue in the body and in about 3 days' time the entire surface is covered with new cells [44]. Although there is no report of other animal lectins causing this effect, a fast remodeling of intestine by reversible effects on anatomy and morphology are known in rats and pigs administered diets containing plant lectins [41]; [45]. Resting eggs are particularly vulnerable, since they are most attractive to potential parasites and predators and may lack an active defense system (because of their inactive metabolic state). Apple snails seem to have evolved passive defense systems to protect their developing embryos; the preferential accumulation of large quantities of lectins, and protease inhibitors is certainly indicative of that strategy. Moreover, it is believed that the main antinutrients responsible for reducing the nutritional value of many plant seeds are a combination of lectin and trypsin inhibitors [46]. Similarly, in apple snail eggs these two types of proteins may be also the main factors responsible for this effect. This further highlights the previously reported similarities between apple snail egg and plant seed embryo defenses [6]; [8]. In a broader view, the overall effect of P. canaliculata PVF on rats bears many similarities with the effect of plant dietary lectins not only against mammals but also birds, insects and nematodes, preventing these predators from digesting and incorporating nutrients from the tissues consumed [47]–[49]. Unlike plants, P. canaliculata advertises its defenses by a conspicuous coloration of the egg masses. Eggs indeed seem to have a large number of defensive proteins against predation, such as other protease inhibitors, chitinases, glycanases, lectins and antifungal proteins, as the analysis of the PVF proteome revealed [23]. Interestingly, all of these defensive proteins are also present in many plant seeds. It is possible that the combined effect of these defensive perivitellins -some targeting the digestive system while others aiming at other organs- may be an evolutionary adaptation. Although these defenses may not completely protect an egg from consumption, they may very well confer an advantage that increases its fitness helping to explain the virtual absence of egg predators. With more than 80,000 species, gastropods are the second largest class of animals after insects. It is therefore not surprising that a better understanding of gastropod egg biochemical defenses, little studied to date, is unveiling novel strategies not previously recognized among animals. In this regard, this study provides insights on the unique defenses against predators of a snail egg that are advertised by conspicuous coloration, and suggests that the acquisition of this protection may have conferred a survival advantage. This places apple snail eggs in the “winning side” of the predator-prey arms race. In this work we demonstrate that the oral administration of apple snail egg PVF promotes alterations in rat growth rate and small intestine morphophysiology for short periods, whereas prolonged exposure to the toxic PVF induces an adaptation overcoming the antinutritonal effects. This defense has not been reported in animals before, but resembles those well established for plant seeds. The severe effects of PVF on digestive tract adds another line of defense to the previously reported suite of biochemical defenses of apple snail eggs. This study helps to explain the near absence of predators and their successful establishment in invaded areas.

Title: Insights into Embryo Defenses of the Invasive Apple Snail Pomacea canaliculata: Egg Mass Ingestion Affects Rat Intestine Morphology and Growth Summary: Filled with nutritious substances to nourish the embryos, eggs of most animals are often the targets of pathogens and predators. An exception are the eggs of Pomacea canaliculata -known as the apple snail- which have hardly any predators. This freshwater snail is a serious aquatic crop pest in several continents, listed among the 100 worst invasive species. It is the host of a roundworm responsible for the rat lungworm disease causing human eosinophilic meningitis. The spread of this emerging infectious disease has been associated with the expansion of apple snails. They lay eggs above water level in bright pink-reddish masses, presumably a warning coloration. Indeed, eggs have chemical defenses, including neurotoxic and antinutritive proteins. The authors found that the ingestion of egg extracts adversely affects rat small intestine inducing large, reversible changes in the intestinal wall that limits the ability to absorb egg nutrients causing a diminished growth rate. Apple snail eggs are the first animal known to deter predators by this mechanism, but remarkably this defense is rather similar to the toxic effect of plant seeds proteins. These overlapping egg defenses that predators have not managed to overcome yet may partially explain the reproductive success of P. canaliculata.

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Summarize: DONATE USB DRIVES TO HELP US SMUGGLE OUTSIDE INFORMATION INTO NORTH KOREA. Believe it or not, USBs are a significant form of sharing information in North Korea. Many citizens have devices with USB ports. So for many years, North Korean defectors have organized efforts to smuggle outside info into North Korea on USB drives to counter Kim Jong-un’s constant propaganda. But these groups were buying USB drives at cost with limited resources. Flash Drives For Freedom is a campaign that travels the world inspiring people to donate their own USB drives. As a collaboration between the Human Rights Foundation, Forum 280, and USB Memory Direct, Flash Drives for Freedom is significantly increasing the capacities of these North Korean defector groups. In the age of Dropbox and Google Drive, the USB stick has come to seem like a dusty tchotchke that belongs in the drawer with your iPhone 4 cables. But send your chunk of cheap flash memory into North Korea, and it becomes a powerful, even subversive object—one that a new activist project wants use to help chip away at the intellectual control of the hermit kingdom’s fascist government. Late last week, the New York-based Human Rights Foundation and the Silicon Valley nonprofit Forum 280 launched Flash Drives for Freedom, an initiative to collect USB flash drive donations from Americans and then ship those slices of silicon to North Korean defector groups. The Korean activist organizations, starting with the Seoul-based non-profit North Korean Strategy Center, will then fill the drives with Western and South Korean films and TV shows and smuggle them across the border into North Korea, where their contraband contents can break Kim Jong-un’s ban on all foreign media. The glimpse of the outside world that those TV shows and films provide is meant to dispel the ideology and illusions the Kim regime depends on to control its populace: that the outside world is poor, dangerous, hostile, and inferior to North Korea. “You can be involved just by shipping a USB drive to Palo Alto, and we’ll help to get it sent to North Korea,” says HRF’s chief strategy officer Alex Gladstein. “This is a viable technology that works there, that North Koreans have decided is a way to reach people.” Gladstein says that despite Americans’ notion of thumb drives as a near-obsolete technology, they still have the power to pierce North Korea’s internet blackout and give the country’s citizens a radically different view of America, their Southern neighbor and their own impoverished homeland. “Each one has the potential to literally change someone’s life,” he says. PCs remain rare in North Korea, and the internet inaccessible to all but a few elites. But cheap, Chinese-made USB-compatible video players known as notels, and even smartphones, are increasingly common. With those devices in mind, groups like the North Korean Strategy Center, North Korean Intellectual Solidarity, and Fighters for a Free North Korea have all joined a growing movement to smuggle data in the form of USB drives and SD cards into the country. As WIRED detailed in a magazine cover story last March, their tactics range from attaching the contraband to balloons floated over from South Korea to hiding the USB drives in cargo holds of trucks passing over the North border with China. NKSC’s most straightforward tactic has been to arrange handoffs of packages in daring nighttime meetings at the Tumen and Yalu rivers that form North Korea’s northern border. They protect those smugglers by bribing officials on both sides. Using those tactics, NKSC alone now smuggles between 3,000 and 5,000 drives into the country every year, and aims to ramp up to a pace of 2,000 drives a month over the next three years. USB drive donations would help the NKSC as it works to increase its data smuggling, says Sharon Stratton, one of organizers of the group’s USB-smuggling operations. But she admits that the drives themselves are only a fraction of the budget of their missions. For every drive they smuggle in and give to contacts inside North Korea, there’s also the cost of travel, bribes, and even focus groups with defectors to choose the most effective movies and films for opening North Korean minds. (American shows like “Friends” and “Desperate Housewives” are among North Koreans’ favorites, the group has found.) But Stratton argues that the donated drives’ value extends beyond the hardware itself: They also serve as a clever marketing mechanism to get Americans thinking about the plight of intellectually repressed people halfway around the world. And she hopes this could also lead to more awareness of North Korean issues and more donations to their cause. In fact, the group is planning a Indiegogo crowdfunding campaign for later this month. “We’re delighted to be able to get the physical flash drives themselves, since we’re continually sending them in, and it’s costly to keep buying them,” says Stratton. “But we’re really hoping this will develop more awareness in tech communities around what we’re doing…and result in more longterm partnerships with people who can support our information dissemination programs.” When an American donates a USB drive and imagines it in the hands of a North Korean recipient, she argues, it changes their perspective on what might otherwise seem like a very foreign and intractable human rights issue. “It’s always been a challenge to get people to understand why North Koreans’ access to information is important, and this gives us a physical representation…It’s literally a key that will unlock a new world for North Koreans,” she says. “It’s a chance for us to grab hands with the supporter and say ‘this is what we’re going to do with your USB drive, and this is why it’s important.'” Stratton adds that sourcing USB drives from Americans can also win hearts and minds in North Korea. If North Korean recipients find out that someone in the US donated the drives, it could help to counter the Kim regime’s portrayal of Americans as murderous capitalist imperialists. “It’s important that they see ‘people in the outside world that I don’t know are sending me these thumb drives,'” says Stratton. That gesture helps to show “that maybe Americans aren’t the big bad enemy after all.” HRF and Forum280 aren’t the only groups to have hatched the idea of collecting drives from American donors: Stanford’s Korean Students Association has a similar collection project scheduled for next month. And HRF’s Gladstein hints that HRF’s ambitions for USB drive donations also extend beyond NKSC and even North Korea. He points to Cuba, where dissidents use the memory sticks to distribute political materials banned from the press, and where blogger Yoani Sanchez launched an opposition newspaper distributed on thumb drives. In a speech at HRF’s Oslo Freedom Forum in 2014, Sanchez called USB drives a “small technological revolution” for Cuba. “The day there is democratic change in Cuba, we’ll not only have to put up monuments to dissidents and those who opposed the government, we will also have to put up a monument to flash drives,” Sanchez told the Oslo crowd. For now, however, HRF is focusing its campaign on a dictatorship whose digital repression makes even Havana look like Silicon Valley by comparison. “North Korea feels like this monolithic, impenetrable, unknowable black hole,” says NKSC’s Stratton. A project like HRF’s can change that sentiment, she says. “Your tiny, dusty USB drive will be in North Korea. You’ll probably never meet the person who has it, but you can be sure that someone will have it and will be happier for it.” Donate USB drives to HRF’s project at the address here. You can also donate to the North Korean Strategy Center here, and stay tuned for its upcoming Indieogogo campaign. On a cloudy, moonless night somewhere in northeastern China, three men creep through a stand of Japanese Clethra trees. They carry no flashlights, and the sky is so dark that they hear the sound of the rushing Tumen River before they see it: They’ve arrived at the North Korean border. Earlier in the evening at a nearby restaurant, they treated the local Chinese police chief and head of the border patrol to a blowout feast of more than 20 dishes, climaxing with a southern China delicacy—a carp deep-fried and served alive, its mouth and gills still moving. Following an after-meal session of pricey Chunghwa cigarettes and shots of Moutai liquor, the officials made phone calls telling subordinates to abandon their posts for several hours. After dozens of these bribe dinners, they had become routine, practically a tradition among friends; by now the smugglers even had their own key to the rusty bike lock securing the border area’s barbed wire fence. Two hours later the trio’s leader, a middle-aged North Korean defector named Jung Kwang-il, steps into the tall weeds of the riverbank. He pulls out a cheap laser pointer and flashes it across the water. Then he waits for a response: If he sees an X slashed through the air by a laser on the opposite bank, the operation will be called off. Instead, he’s answered with a red circle painted through the darkness. Soon after, a compact man dressed in only a hoodie and boxer shorts wades out of the waist-high water and onto the riverbank where Jung and his companions stand. Jung arranged the meeting earlier in the day using coded language over walkie-talkies. The men embrace and speak softly for a minute about each other’s health, the price of North Korean mushrooms, and Jung’s mother, whom he’d left behind in the North 10 years ago. Then Jung hands the man a tightly wrapped plastic bag containing a trove of precious black-market data: 200 Sandisk USB drives and 300 micro SD cards, each packed with 16 gigabytes of videos like Lucy, Son of God, 22 Jump Street, and entire seasons of South Korean reality television shows, comedies, and soap operas. To bribe the guards on the North Korean side, Jung has included in the bag an HP laptop computer, cigarettes, liquor, and close to $1,000 in cash. The man in the hoodie slings the bag of digital contraband over his shoulder. Then he says good-bye and disappears back into the world’s deepest black hole of information. That smuggling mission was planned and executed last September by the North Korea Strategy Center and its 46-year-old founder, Kang Chol-hwan. Over the past few years, Kang’s organization has become the largest in a movement of political groups who routinely smuggle data into North Korea. NKSC alone annually injects around 3,000 USB drives filled with foreign movies, music, and ebooks. Kang’s goal, as wildly optimistic as it may sound, is nothing less than the overthrow of the North Korean government. He believes that the Kim dynasty’s three-generation stranglehold on the North Korean people—and its draconian restriction on almost any information about the world beyond its borders—will ultimately be broken not by drone strikes or caravans of Humvees but by a gradual, guerrilla invasion of thumb drives filled with bootleg episodes of Friends and Judd Apatow comedies. Kang likens the USB sticks to the red pill from The Matrix: a mind-altering treatment that has the power to shatter a world of illusions. “When North Koreans watch Desperate Housewives, they see that Americans aren’t all war-loving imperialists,” Kang says. “They’re just people having affairs or whatever. They see the leisure, the freedom. They realize that this isn’t the enemy; it’s what they want for themselves. It cancels out everything they’ve been told. And when that happens, it starts a revolution in their mind.” I first meet Kang in a conference room of his office on the ninth floor of a Seoul high-rise. Outside, a bored plainclothes policeman keeps watch, part of a 24/7 security detail provided by the South Korean government after Kang appeared on a top-10 list of North Korean defector assassination targets. Kang answers my questions in a soft voice and maintains a look of calm bemusement. But several NKSC staffers later tell me that his quiet demeanor masks a deep, lifelong anger directed at North Korea’s dictatorship, which held him and his entire family in a prison camp for 10 years of his childhood. (“Compared to some defectors I’ve met, he’s a little more pissed off,” one staffer confides.) Kang Chol-hwan founded the dissident group NKSC, focused on injecting foreign media into North Korea. Here he holds a popular video player known in the country as a notel. Joe Pugliese It doesn’t take a decade in a gulag to see that North Korea needs a revolution. Since the Korean Peninsula split at the end of World War II, seven decades of disastrous financial decisions, isolationist economics, and sociopathic military threats against the rest of the world have turned the country into what Georgetown Asian studies professor and former National Security Council adviser Victor Cha calls simply “the worst place on earth.” Its recent history is a litany of disaster: Despite having a stronger economy and better infrastructure than South Korea in 1945, its GDP is now a fortieth the size of its southern neighbor. Only 16 percent of households have adequate access to food, according to a 2012 study by the World Food Program, stunting growth in 28 percent of the population. In some areas of the country, up to 40 percent of children under 5 are affected. The effects are mental as well as physical. A 2008 study by the National Intelligence Council found that a quarter of North Korean military conscripts are disqualified for cognitive disabilities. The totalitarian government inherited by its 32-year-old leader, Kim Jong-un, punishes any real political resistance with death. And the regime’s most powerful tool for control remains its grip on North Korean minds. The state propaganda system indoctrinates its 25 million citizens from birth, insisting that the Kim family is infallible and that the country enjoys a superior standard of living. In a ranking of 197 countries’ press freedom by research group Freedom House, North Korea places last. It sees any attempt to introduce competing ideas, even the possession of a radio capable of accessing foreign frequencies, as a threat to its power; these infractions are punishable by exile to one of its prison camps, which hold as many as 200,000 people, according to Amnesty International. “The Kim regime needs its ideology,” Cha says. Without it, he argues, North Korea would face the same threats as every dictatorship, such as an internal coup or a popular revolt. “If they get to the point where all they can do is point guns at people, they’ll know their system has failed.” “What I do is what Kim Jong-un fears most,” says Jung, the smuggler. A growing movement of North Korean defector activist groups, including Kang’s NKSC and others, like North Korea Intellectuals Solidarity and Fighters for a Free North Korea, views that reliance on ideological control as a weakness: Outside data is now penetrating North Korea’s borders more than ever before. One group has stashed USB drives in Chinese cargo trucks. Another has passed them over from tourist boats that meet with fishermen mid-river. An NKSC operative showed me a video in which he crawls under a border fence, walks into the Tumen River, and throws two tires to the opposite bank. Each one was filled with South Korean Choco Pies, Chinese cigarettes, and USB sticks loaded with movies like Snowpiercer, The Lives of Others, and Charlie Chaplin’s The Great Dictator. Even The Interview—the Kim Jong-un assassination comedy that the North Korean government tried to keep from being released by using threats, intimidation, and (according to the FBI) a devastating hacking operation against Sony Pictures—has made its way into the country. Chinese traders’ trucks carried 20 copies of the film across the border the day after Christmas, just two days after its online release. “What I do is what Kim Jong-un fears most,” says Jung, the smuggler, who shows me videos and pictures of his missions while seated in the lobby of a hospital in Bucheon, South Korea. Jung, wearing a military-style cap and pajamas, is taking a break from rehabilitation therapy for knee injuries he sustained while being tortured in a North Korean prison 15 years ago. “For every USB drive I send across, there are perhaps 100 North Koreans who begin to question why they live this way. Why they’ve been put in a jar.” Each activist group has its own tactics: Fighters for a Free North Korea loads up 35-foot balloons that float into the country and rain down pamphlets, US dollar bills, and USB drives full of political materials. North Korea Intellectuals Solidarity smuggles in USBs filled with short documentaries about the outside world created by the group’s founder, a former North Korean computer scientist who used to help the government confiscate illicit media. Kang’s NKSC, with its pop cultural offerings, capitalizes on North Korea’s flowering black markets. The group’s smugglers inside the country are motivated by profit as much as politics: A USB stick loaded with contraband films sells for more than a month’s food budget for most middle-class North Korean families. A pack of hundreds represents a small fortune. “In North Korea a USB drive is like gold,” one NKSC smuggler tells me. For Kang, that makes each of those coveted flash drives a self-propelled weapon in a free-market information insurgency. “Right now, perhaps 30 percent of the population in North Korea knows about the outside world,” Kang says. “If you reach 50 percent, that’s enough people to start making demands, to start making changes.” And if that enlightened audience reaches 80 percent? Or 90 percent? Kang leans forward. “Then there’s no way the North Korean government, in its current form, could continue to exist.” A rare photo of a North Korean smuggler, carrying a bag of illegal USB drives on the Chinese side of the Tumen River as he prepares to cross back to his homeland. Activist Jung Kwang-il took the photo in May 2013. Jung Kwang-il Kang Chol-hwan was 9 years old when his grandfather, a high-level government official and ethnic Korean immigrant from Japan, suddenly disappeared. It was the summer of 1977, and within a few weeks, soldiers came for the rest of his family, summarily stating that Kang’s grandfather had been convicted of “high treason” but giving no details. The entire three-generation family would immediately be sent to a reeduca­tion camp. The government confiscated the family’s house and nearly all its possessions, though the soldiers took pity on the tearful Kang and allowed him to carry out an aquarium of his favorite tropical fish. Soon after the family’s arrival at the Yodok concentration camp in the country’s northeastern mountains, the fish floated dead in their tank. The family would spend the next decade in one of Kim Il-sung’s most notorious gulags. Kang’s daily life alternated between school—rote memorization of communist propaganda—and slave labor in the camp’s cornfields, lumberyards, and gold mines. For a time, Kang’s work detail included burying the corpses of prisoners who died daily from starvation or perished in mine cave-ins and dynamite accidents. Children who disobeyed even slightly were beaten. Adult transgressors spent days, or even months, in the sweatbox, a tiny windowless shack in which victims could only crouch on hands and knees. Sometimes prisoners, including Kang, would be required to witness executions. Once he and other inmates were ordered to stone the hanging corpses of would-be escapees. “The skin on the victims’ faces eventually came undone and nothing remained of their clothing but a few bloody shreds,” Kang would later describe it. “I had the strange feeling of being swallowed up in a world where the earth and sky had changed places.” As the years passed, Kang became a resourceful survivor. He learned to eat wild salamanders in a single swallow and catch rats with a lasso he designed out of wire. Their meat sustained him and several family members on the verge of starvation through winters at subzero temperatures. When Kang was 18, the guards announced one day without preamble that his family would be released as a demonstration of leader Kim Il-sung’s generosity. Except Kang’s grandfather—he had been assigned to a different camp, his treason still unexplained. Kang never saw him again. “Kang knows that the more active he is, the closer he gets to his vision, the more his family will suffer.” In his postprison life as a deliveryman in the western county of Pyungsung, Kang harbored few illusions about the corruption of the North Korean regime. But it wasn’t until around three years later that he accessed the information that crystallized his contempt. It came from a pirate radio. A friend gave Kang two radio receivers. Kang paid a bribe to avoid registering one with police, and he learned how to disassemble its case and remove the filament that hardwired it to official regime frequencies. He and his closest confidants would huddle under a blanket—to muffle the sound from eavesdroppers—and listen to Voice of America, Christian stations, and the South’s Korean Broadcasting System. “At first I didn’t believe it,” he says. “Then I started to believe but felt guilty for listening. Eventually, I couldn’t stop.” Under their blanket, they relearned all of North Korea’s history, including the fact that the North, not the South, had started the Korean War. Beginning in 1989, they followed the breakdown of Soviet Eastern Europe and the execution of Romanian dictator Nicolae Ceauşescu, a close friend of Kim Il-sung. They heard the music of Simon and Garfunkel and Michael Jackson, even learning the lyrics and softly singing along. “Listening to the radio gave us the words we needed to express our dissatisfaction,” Kang would later write. “Every program, each new discovery, helped us tear a little freer from the enveloping web of deception.” Soon a contact in the local government warned him: One of his companions had told the police about Kang’s secret radio sessions. He was under surveillance and faced potential arrest and reassignment to a labor camp. Posing as a businessman, he bribed border guards on the Yalu River and escaped to Dalian, China, and finally to Seoul. After his escape Kang wrote a memoir, The Aquariums of Pyongyang, originally published in French in 2000 and a year later translated into English. It was a revelation: the most detailed account yet of life in North Korea’s gulags. Kang was asked to speak around the world, touring Ivy League schools and European conferences. President George W. Bush invited him to visit the White House, where they discussed his homeland’s growing human rights crisis. “It was always just a statistic—hundreds of thousands of people in labor camps,” says Georgetown’s Cha, who advised Bush on North Korea. “But Kang’s book put a name and a face and a story to these abuses.” Back in South Korea, Kang’s story had no such impact. President Kim Dae-jung had won a Nobel Prize for the South’s so-called Sunshine Policy of compromise with the North to reestablish diplomatic ties. Kang’s story was seen as unfashionably antagonistic to the Kim regime and largely ignored. Watching an illegal copy of Titanic as a girl in North Korea made defector activist Yeonmi Park ask questions for the first time about freedom and the outside world. Joe Pugliese Grooming by Casey Geren / ABTP By 2005, Kang had given up hope that South Korea or the rest of the world would act against the North Korean government. Change, he decided, would have to come from within, through the same life-altering education he had received from his illegal radio. He flipped his strategy: Instead of working to tell the world about the horrors of North Korea, he would work to tell North Koreans about the world. That year, a Christian radio station donated 5,000 portable windup radios to Kang’s newly formed organization. Through defector contacts in China, he smuggled them into houses along North Korea’s Tumen River border. “Guards come to these houses to rest and buy cigarettes,” Kang explains. “We would give them these little radios too. So all of these bored kids, during their patrols, could listen to foreign radio broadcasts at night.” With funding from private donors and governments it declines to name, NKSC has since grown to 15 paid staffers, including independent operators along the Chinese border, each with their own contacts in North Korea. Kang hopes to soon expand smuggling operations to 10,000 USB drives a year. He’s also looking at ways the American tech community could advance NKSC’s mission. The group is working with the Wiki­media Foundation to put a North Korean–dialect version of Wikipedia on every flash drive it smuggles over. And in conjunction with the Human Rights Foundation, it’s been talking to Silicon Valley types about building new tools—everything from a small concealable satellite dish to steganographic videogames that hide illegal data. (The activists have considered delivering USBs with miniature drones, but that option remains impractically expensive.) But as his group gains momentum, Kang faces a personal dilemma: Several of his family members remain inside North Korea, including his younger sister, Mi-ho. Despite canvassing his contacts there and filing a special request through the United Nations for information about Mi-ho’s whereabouts, Kang hasn’t been able to find her. She may even have been reimprisoned, says Choi Yoon-cheol, NKSC’s second-most-senior staffer. “Mr. Kang knows that the more active he is, the closer he gets to his vision, the more his family will suffer,” Choi says. “It must be incredibly difficult to know that what you’re doing can hurt the people you love.” When I first ask Kang about his sister, he denies any connection between her safety and his work. Perhaps in an effort to protect her, he argues that the two are now estranged. Besides, he coldly insists, his own family is no longer the issue. “This is a government that doesn’t deserve to survive,” he says. “If someone has to destroy it, I’ll gladly be the one.” Jung works as a data smuggler, coordinating stealthy meetings with contacts along North Korea’s Chinese border. He still suffers from injuries inflicted during torture in one of the country's prisons 15 years ago. Joe Pugliese Yeonmi Park’s family paid around 3,000 North Korean won for a pack of DVDs that contained a bootleg of Titanic. In the early 2000s, she remembers, that was the cost of several pounds of rice in her home city of Hyesan—a significant sacrifice in a starving country. But of all the tween girls who became obsessed with the star-crossed romance of Jack and Rose, Park was one of the very few who saw it as downright revolutionary. “In North Korea they had taught us that you die for the regime. In this movie it was like, whoa, he’s dying for a girl he loves,” she says. “I thought, how can anyone make this and not be killed?” Titanic was hardly Park’s only foreign-­video experience. Her mother had sold DVDs; some of Park’s earliest memories are of waking to the grunts and shouts of her father watching American WWF wrestling. Park loved Cinderella, Snow White, and Pretty Woman. The family would put its tapes and discs in a plastic bag and bury it beneath a potted plant to hide it from the police. But of all those illegal encounters with foreign culture, Titanic was somehow the film that made Park ask herself questions about freedom and the outside world. “It made me feel like something was off with our system,” she says in fluent English, which she perfected by watching the entire run of Friends dozens of times. Park escaped from North Korea in 2007. Now a 21-year-old activist based in Seoul, she’s part of what’s known in Korea as the jangmadang sedae: the black-market generation. During a famine in the North in the mid-1990s, the Kim regime began to tolerate illegal trade because it was the only option to feed a starving population. Since then, black-market commerce has been nearly impossible to stamp out. And some of the hottest commodities—particularly for young people who don’t even remember a North Korea before that underground trade existed—have been foreign music and movies, along with the Chinese-made gadgets to play them. A 2010 study by the US Broadcasting Board of Governors found that 74 percent of North Koreans have access to a TV and 46 percent can access a DVD player. Park says that nearly all of her friends in Hyesan had seen a foreign film or TV show. As a result, her generation is the first to have to square the Kim regime’s propaganda with a keyhole view of the outside world. A group called Liberty in North Korea, which works with young defector refugees, finds that many no longer believe in central tenets of North Korea’s political ideology, such as the country’s superior standard of living or the godlike powers of the Kim family. Even the regime is letting that second illusion slide, admitting that Kim Jong-un has health issues—hardly the norm for heavenly beings. “In North Korea, they taught us that you die for the regime,” says activist Yeonmi Park. Thanks to the flourishing black market, the jangmadang generation’s technology has advanced well beyond radios and DVDs. Despite North Korea’s near-complete lack of Internet access, there are close to 3.5 million PCs in the country and 5 million tablets, according to North Korea Intellectuals Solidarity. But perhaps the most important piece of hardware in North Korea today is what’s known as a notel—a small, portable video player sold for $60 to $100 and capable of handling multiple formats. It has a screen, a rechargeable battery to deal with frequent blackouts, and crucially, USB and SD card ports. In a surprise move in December, the North Korean government legalized the devices, perhaps as part of a bid to modernize its propaganda machine, according to Seoul-based news outlet Daily NK. The result is millions of ready customers for the USB sticks smuggled across the Chinese border. In one of North Korea’s bustling markets, a buyer might quietly ask for something “fun,” meaning foreign, or “from the village below,” referring to South Korea. The seller may lead him or her to a private place, often someone’s home, before turning over the goods. The foreign data is then consumed on a notel among small, discreet groups of mostly young people, friends who enter into an unspoken pact of breaking the law together so that no one can rat out anyone else. The Kim regime has responded by cracking down. In late 2013 the government reportedly executed 80 people across seven cities in a single day, many for trafficking in illegal media. In February last year, the Worker’s Party of Korea held its largest-ever conference of propagandists. Kim Jong-un himself delivered an address calling for the party to “take the initiative in launching operations to make the imperialist moves for ideological and cultural infiltration end in smoke” and to set up “mosquito nets with two or three layers to prevent capitalist ideology, which the enemy is persistently attempting to spread, from infiltrating across our border.” But stamping out illegal media in North Korea has become an intractable problem for the government, according to Sokeel Park, director of research and strategy for Liberty in North Korea. He compares it to the stubborn demand for illegal drugs in the US. “You could call it Kim Jong-un’s War on Information,” he says. “But just like a war on drugs—you can try to slow it down, increase the risks, increase the punishments, put more people in prison. The bribe costs will go up, but it’s still going to happen.” NKSC founder Kang and his family spent 10 years in a North Korean gulag. Joe Pugliese By his third year working for Kim Jong-il’s thought police, Kim Heung-kwang says he could almost sense the presence of illegal data. Going door-to-door with the task force assigned to search out digital contraband in citizens’ homes, he remembers finding forbidden DVDs and players hidden under beds and in books with pages cut away to create hidden compartments. On one occasion he caught a group of video watchers who had, in a panic, hidden together under a blanket in a closet. Early on he found that when he knocked on doors, the guilty watchers would hurriedly hide their DVDs. So he learned to turn off the power to the entire building before making his house calls, trapping discs in their players. “I felt they were watching rotten, capitalist material and ruining the juche mentality,” Kim says, referring to the North Korean communist ideology. The short, bespectacled man, sitting in his austere Seoul office, smiles wearily and crosses his legs with a professorial air. “I felt justified to send these criminals away.” The DVD owners would cry and plead. They’d beg on their knees and pull on the sleeves of his uniform, claiming they had just found the offending media lying in the street. Sometimes he accepted bribes and turned a blind eye. (“You could feel the outside of the envelope between your fingers and tell whether it was a lot of money,” he remembers.) But most of the data criminals he caught, he reported. Many were sentenced to months or years in prison camps. “I show North Koreans what they want to see—what I wanted to see when I was there,” says dissident Kim Heung-kwang. Kim had earned membership in the all-powerful Communist Party through years of work helping to create North Korea’s own computers, including the Paektusan minicomputer, named for the mountain where Kim Jong-il was said to have been born. As a computer science professor at Hamhung University, he had even taught students who would go on to work for North Korea’s cyberwarfare brigade, Unit 121—the group suspected of the Sony breach—in the basics of networking and operation systems. After black markets began to spread, Kim was reassigned in 2000 to a military division that went door-to-door to search for contraband media. “I loved it,” he says. “I had the power to go into homes and take these materials and no one could even question me.” One of the perks of Kim’s position, of course, was nearly infinite access to the media he confiscated. He began to watch the contraband films and TV shows and even loaned out his collection to friends, who rewarded him with gifts like alcohol and meat. In 2002, Kim was given a PC, part of what he describes as a secret aid shipment from South Korea. Its hard drive had been wiped. But using forensic recovery software, Kim was able to reassemble its deleted contents. They included 400 files: films, TV shows, and, most important to his intellectual sensibilities, ebooks. “You can’t imagine how excited I was,” he says. “I had hit a gold mine.” These were what finally transformed Kim’s thinking. He remembers reading a Dale Carnegie self-help book and Alvin Toffler’s The Third Wave. But most influential was a history book about Middle Eastern dictators, including the stories of Saddam Hussein and Muammar Gaddafi, all friends of the Kim regime. “Reading about the crimes happening in these countries, I began to realize that those crimes were happening in my country too,” Kim says. “That was the starting point of the logic shifting in my brain. I began to understand the nature of dictatorship.” Kim’s epiphany came when he read an illegal ebook retrieved from a South Korean hard drive. “That was the starting point of the logic shifting in my brain,” he says. “I began to understand the nature of dictatorship.” Even then, Kim continued busting viewers of the same foreign media he now regularly watched. “I sent a lot of people away, but the karma soon came back to me,” he says. In 2003 he was arrested and taken to a detention center; he’d been ratted out by one of the comrades with whom he’d shared his secret store. He says the police tortured him for a week, forcing him to write hundreds of pages of confession under hot lights and preventing him from sleeping by jabbing his forehead with a needle. When they found that he had only distributed materials to a few friends, he was given a “lenient” sentence: a year at a reeduca­tion farm 40 miles outside Hamhung. “I grew to literally hate the land itself,” he says. “I couldn’t understand why watching a few foreign films should cost me a year of my life.” After the year of drudgery, Kim was released and managed to bribe a border guard to help him escape across the Tumen. He made his way from China to Seoul, where he set up North Korea Intellectuals Solidarity. Kim’s strategy is much like Kang’s with NKSC, using Chinese traders and smuggler contacts. But Kim has only a handful of full-time staffers. Instead of asking his North Korean contacts to wade across the Tumen, he describes throwing a rock tied to the end of a rope across the river. Smugglers on the other side, he says, use it to pull across a plastic-­wrapped bucket of USB drives. (He’s also experimenting with a three-man water balloon slingshot that can catapult contraband hundreds of feet past guards.) Unlike the pop cultural programming proffered by Kang’s group, the content on Kim’s drives includes mostly short educational documentaries created by and starring Kim himself. He explains to North Koreans what democracy is, for instance, or simply shows them what a bookstore or the Internet looks like. “When a North Korean watches an action movie with a chase scene in a grocery store, they want to slow it down to see what’s on the shelves,” he says. “I show them what they want to see—what I wanted to see when I was there.” Kim has also developed what he calls stealth USB drives, designed to avoid detection. To any casual observer, the drive seems empty. But its contents reappear with a simple trigger, the details of which Kim asked that I not publicize. Not even the buyer would necessarily know that the USB contained illegal educational materials, he says. Instead, the files would simply materialize one day, a spontaneous gift Kim hopes will be as life-changing as the hard drive whose wondrous contents he once discovered. Kim denies that his work today is repentance for past sins as a member of Kim Jong-il’s data gestapo. He describes the zealot of those years as almost a different person. But when I ask if he still feels guilt for the lives he wrecked, his polite academic’s smile finally cracks. He massages his temples with one hand. Once, he says, he found a collection of foreign DVDs in the home of a single mother and her two middle-school-aged sons. He could tell by the teen-oriented content that they belonged to the kids. The mother insisted the DVDs were hers, sacrificing herself for her children. Kim says he was inclined to let her go, but a hard-line colleague insisted she be reported, condemning her to a prison camp. “I wanted to forgive her,” Kim says. He pauses. “I still think about that family sometimes.” USB drives filled with foreign content and ready to be taken across the border in a plastic bag, lying on the bank of the Tumen River. On a Friday night in an NKSC conference room, a young North Korean defector who has asked me to call her Yae-un is watching a copy of the teen comedy Superbad. She would later explain to me that she “had never seen a movie on that scale of filthiness before,” and she doesn’t hide her reaction; she spends most of the 113-minute barrage of adolescent sexual angst and dick jokes covering her face with the backs of her hands, as if to cool off her burning cheeks. The movie was supposed to be screened for one of the defector focus groups that NKSC assembles to learn how North Koreans react to different types of media, the better to smuggle in the materials with the most impact. But on this occasion, all the North Koreans but Yae-un are busy or have canceled at the last minute. So, like some kind of Clockwork Orange parody, the focus group has been reduced to one North Korean, watched by me, an NKSC staffer, and volunteers as she reacts to Jonah Hill and Michael Cera trying very hard to get laid. When the movie finishes, Yae-un starts by listing the most astonishing elements from a North Korean perspective: the frank sex talk, constant genitalia references, underage drinking, cops crashing their car, teenage McLovin shooting a gun. All would be seen as indescribably alien, she says. “Even watching it now, I find it vulgar and shocking,” she says. “If I were still in North Korea, it would blow my mind.” So maybe NKSC should skip this one, suggests Rocky Kim, the staffer who organized the screening. “Maybe a documentary would be better?” he asks. Not at all. “I would vote to send it,” Yae-un says without hesitation. “It will blow their minds, but it’s not like they’ll actually explode. They’ll recover.” Predicting North Koreans’ reactions to foreign media isn’t easy. The Interview, for all the furor it elicited from the Kim regime, got an equally negative reaction from North Koreans who saw it on the other side of the border. The smuggler Jung Kwang-il says contacts he spoke to in the country were offended by its low production values and mockery of North Korean culture. “They thought it was poorly made on purpose to mock North Korea, but I explained it was just a bad movie,” he says. “They prefer The Hunger Games.” Other high-profile tactics by the North Korean free-information movement have backfired in their own ways: A balloon launch by Fighters for a Free North Korea in October prompted the North Korean military to fire antiaircraft machine guns over a border village. Some of its balloons, meanwhile, end up stuck in the mountains, blown out to sea, or even back in South Korea. The pamphlets they include, according to some activists, criticize the regime too directly and are dismissed by North Koreans as just another form of propaganda. Joe Pugliese NKSC is more cautious about its content. Ultimately the group decided that Superbad was too risqué for the North; so much for dick jokes defeating dictators. But there’s a question that persisted throughout my conversations with the groups: How does North Korea get from an information revolution to an actual people-in-the-streets-and-toppled-statues revolution? I pose that question to Kang Chol-hwan while we sit in his office one freezing, snowy afternoon, my last day in Korea. He admits there’s not a simple answer, but he offers a few scenarios he considers plausible: The government, for instance, could sense the disconnect between its propaganda and the people’s foreign-media education and launch its own reforms, the kind of gradual opening that took place in Russia and China. Or a disillusioned populace could begin defecting en masse, forcing a border control crisis. Or some spark, like the self-immolation of Tunisian street vendor Mohammed Bouazizi, could coalesce disillusioned North Koreans into their own Arab Spring, a full-scale grassroots uprising. But then Kang surprises me by admitting that all those scenarios are unlikely: The Kim regime is too blind and stubborn to initiate its own reforms, he says, and its totalitarian grip may be too tight for a bottom-up revolution. He puts his highest hopes instead in another scenario: that NKSC’s foreign heresy could penetrate the government and military’s middle ranks and even their elite, eroding the ideology of the Communist Party itself and fracturing Kim Jong-un’s power base from within. A minute later, however, Kang suddenly flips back to his earlier optimism: He predicts that, thanks in part to his information strategy, North Korea’s dictatorship will end within a decade. “They’re already cracking,” he says. “In less than 10 years, I’ll be able to freely go in and out.” That nakedly idealistic statement, beyond its tinge of wishful thinking, seems to reveal something new about how Kang sees his goal. In spite of all his childhood horrors, he wants to transform North Korea not simply into a nation that will let his countrymen go free, but one that will let him back in: He wants to go home again. And whether his smuggling tactics succeed or fail, he’ll continue to send his USB thumb drives into North Korea, like offerings to a mute idol, because it’s the best plan he’s got. “I have no direct power against the North Korean government,” he admits unprompted, his face blank. Outside the window, it’s getting dark and the snow is still falling. A polar vortex has pushed Siberian air southward, bringing winter winds down the Korean Peninsula earlier than most years. And as cold as it is in Seoul, it’s far colder 150 miles north, in the prison camps where Kang spent his childhood and where his sister may still be today. “This is the best way—the only way for me—to open North Korea,” Kang finally says. “Every day until then is a delay to seeing my family again.” Read more about the methods of North Korean data smugglers, and how Silicon Valley is helping the movement. Breaking News Emails Get breaking news alerts and special reports. The news and stories that matter, delivered weekday mornings. SUBSCRIBE A tiny USB memory stick is unlikely to bring down Supreme Leader Kim Jong-un, but thousands upon thousands of them just might help enlighten the North Korean people to the freedoms enjoyed by the outside world, according to human-rights activists. The New York-based Human Rights Foundation and Forum 280, a Silicon Valley nonprofit, have teamed up to launch a program that will collect donated USB flash drives, load them with content ranging from "South Korean soap operas to Hollywood films to Korean-language versions of Wikipedia to interviews with North Korean defectors," and smuggle them into the North for ordinary North Koreans to enjoy. Related: Photos Purportedly Show North Korea's Rocket Debris The goal of the initiative, dubbed "Flash Drives for Freedom," is to turn our dust-collecting electronic has-beens into a cheap conduit of information for some of the nearly 25 million inhabitants living under what the West considers one of the world’s most oppressive regimes. "In the world’s most closed society, flash drives are valuable tools of education and discovery," the program’s website says. "In a society without Internet, with total government censorship, and with no independent media, North Koreans rely on these little pieces of plastic. Filled with films, books, and explainers, they are windows to the outside world."

Summary: Got any old flash drives gathering dust in a drawer? There's a growing movement to get them into the hands of ordinary North Koreans, loaded with Western movies and shows in the hopes of offering a more realistic look at the outside world Pyongyang paints as utterly bleak. Andy Greenberg first did a Wired cover story on the grass-roots efforts in March 2015, and he now follows up on the latest efforts by groups such as the Seoul-based North Korea Strategy Center, the Silicon Valley-based Forum 280, and more. "It's literally a key that will unlock a new world for North Koreans," says Sharon Stratton of North Korea Strategy Center's USB-smuggling operations. "It's important that they see 'people in the outside world [who] I don't know are sending me these thumb drives,' which she says plants the seed"that maybe Americans aren't the big bad enemy after all. "Human Rights Foundation's Alex Gladstein, who helped form Flash Drives for Freedom, tells NBC News:"Obviously, one flash drive is not going to depose Kim Jong Un, but it could change the life of a North Korean. "Gladstein adds that all you have to do is ship your thumb drive to Palo Alto, Calif., and they'll take care of the rest-meaning they'll illegally smuggle the drives (sometimes via cargo trucks, other times attached to balloons) into North Korea, hopefully as many as 2,000 a month, in an effort to pierce the Internet blackout and Kim's ban on all foreign media."North Korea feels like this monolithic, impenetrable, unknowable black hole, "says Stratton. What plays well among North Koreans? Friends and Desperate Housewives seem to be favorites, notes Wired.

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Summarize: “The record is clear that Lynch and Shannon had knowledge that for years Serrano often had several boys, including plaintiff, sleep over at his apartment,” Justice Loren Baily-Schiffman of Kings County Supreme Court wrote in her 2017 order dismissing the church’s motion for summary judgment of the case. “In fact, both Lynch and Shannon testified that they visited Serrano on numerous occasions when young boys were present.” In a deposition, Father Lynch testified that he saw Mr. Serrano kiss an 8- or 9-year-old boy on the mouth and inappropriately embrace the boy. A church secretary, Beatrice Ponnelle, who shared an office with Mr. Serrano, also testified about questionable behavior. She said that although the church had a rule that children were not allowed to be left alone in the office with a staff member, boys as young as 7 or 8 would come into the office to do their homework while sitting on Mr. Serrano’s lap. When she left for the day, he would be the only adult in the office with the boys, Justice Baily-Schiffman wrote. Despite Mr. Serrano’s position as a religious educator — with Mr. Serrano at the church nearly every day between 1997 and 2009, volunteering at the summer camp, and getting keys to the church and rectory — no records were kept regarding him or his employment history at the church, the judge wrote. With the case set for trial, the Diocese of Brooklyn agreed to settle. But in a statement released on Tuesday, the diocese still seemed to minimize its role in allowing the abuse. “The diocese and another defendant have settled these lawsuits brought by the four claimants who were sexually abused by Angelo Serrano at his private apartment many years ago,” the statement said. “Mr. Serrano was a volunteer worker at a local parish; he was not clergy or an employee of the diocese or parish.” The statement added that for three of the claimants, “another defendant” would be contributing “a significant portion of the settlement.” A spokeswoman for the diocese said that the Dorothy Bennett Mercy Center, a local after-school program based next to the church in Clinton Hill, had agreed to pay about one-third of the total settlement. Four men who were sexually abused by a teacher at a Catholic church in Clinton Hill reached a $27.5 million settlement with the Diocese of Brooklyn and a local after-school program on Tuesday. The men were all repeatedly raped as kids by Angelo Serrano, 67, the former director of religion at St. Lucy’s-St. Patrick’s Church, between 2003 and 2009. “These were egregious incidences of sexual abuse,” one of their attorneys, Ben Rabinowitz, told The Post. “And this all happened after the priests were taught to recognize the signs of abuse.” The lawyers say the settlement — which will see the men, now between ages 19 and 21, each receive more than $6.8 million from the diocese and an affiliated after-school program next to the church, according to a New York Times report — is one of the largest made to sexual abuse survivors in the Catholic Church. Serrano was arrested in 2009 for abusing one of the boys, who was then age 10, and pleaded guilty to first-degree sexual conduct charges in 2011. He’s currently serving a 15-year sentence. According to the victims’ suit — which also named two of the church’s pastors, the Rev. Stephen P. Lynch and the Rev. Frank Shannon — Serrano molested boys in the church, at an after-school program and at his nearby apartment, where he often had youngsters sleep over. A church secretary testified that she saw boys as young as 7 or 8 sitting on Serrano’s lap while doing their homework, while both pastors witnessed kids at his home on numerous occasions and — on one occasion — Lynch saw a young boy kiss him on the lips, yet they never kept any records, according to court documents. “The signs were right there in front of their faces,” another attorney, Peter Saghir, told The Post. “One man saw [Serrano] kiss a child on the mouth, and he never reported it. He never asked the child if he was okay.” In a statement, the diocese said it “highly contested its role” in the abuse, claimed it only happened at Serrano’s apartment and noted that he was a volunteer. It said a co-defendant — identified by the Times as the neighboring Dorothy Bennett Mercy Center — is paying a “significant portion” of the payout for three of the men. “The Diocese endeavored to reach this settlement in a way that compensates Mr. Serrano’s victims and respects their privacy. We hope this is another step forward in the healing process for these claimants,” reads the statement. “The Diocese remains committed to ensuring that its parishes, schools and youth programs remain safe and secure for the young people who are entrusted to our care.” Marco Luna, a 35-year-old former neighbor of Serrano’s, said he stopped going to church after the allegations of abuse became public. He said the large settlement was warranted. “That’s a lot of money, but he’s ruined these kids for the rest of their lives. No money in the world can give them what he took from them,” Luna said Tuesday night. “He’s (Serrano) a disgusting individual. What he did to those kids, they didn’t deserve that. He betrayed their parents. Whatever he said to those parents to gain their trust, he did the complete opposite.” Additional reporting by Shari Logan Breaking News Emails Get breaking news alerts and special reports. The news and stories that matter, delivered weekday mornings. By Alex Johnson The New York Diocese of Brooklyn and a co-defendant have reached a $27.5 million settlement with four men who were sexually abused as boys by a lay church education director, the diocese said Tuesday night. Each of the young men will receive $6.875 million from the diocese and an affiliated after-school program under terms of the settlement, said Ben Rubinowitz and Peter Saghir, the attorneys for the young men, who have remained anonymous. In terms of individual payouts, it is the largest settlement on record involving abuse of minors by Roman Catholic Church figures, almost double the amount received by two victims in Rockville Centre, New York, in 2008, according to records maintained by Bishop Accountability, a nonprofit that monitors abuse allegations against Catholic priests and officials. The men, who are now 19 to 21, said in court documents that they were repeatedly raped from 2003 to 2009 by Angelo Serrano, 67, a former director of religious education at St. Lucy's-St. Patrick's Church in Brooklyn. Serrano was arrested in 2009 and pleaded guilty to first-degree sexual conduct. He is serving a 15-year prison sentence, with his earliest possible date of release on July 2022, according to prison records. The plaintiffs alleged in court documents that priests at the church knew or should have known that Serrano was sexually abusing children. One of the priests testified in a deposition that he saw Serrano kiss an 8- or 9-year-old boy on the mouth in the church office but didn't report the conduct. "Unfortunately, we know of other boys who were severely abused by Serrano; however, the statute of limitations prevents their claims from being brought," the young men's attorneys said. In a statement, the diocese confirmed the settlement, saying it hoped the agreement "is another step forward in the healing process for these claimants." Serrano "was not clergy or an employee of the diocese or parish," a spokeswoman said Tuesday night. "The diocese endeavored to reach this settlement in a way that compensates Mr. Serrano's victims and respects their privacy." The settlement was reached less than two weeks after New York Attorney General Barbara Underwood subpoenaed the state's eight dioceses as part of a civil investigation into how the church reviewed allegations of sexual abuse. She cited the bombshell report produced by a Pennsylvania grand jury that concluded that about 300 priests in the state had sexually abused more than 1,000 children over 70 years.

Summary: Four young men who were repeatedly raped by their catechism teacher have received one of the Catholic Church's biggest-ever settlements with abuse victims. The New York Diocese of Brooklyn says the men, who were abused between the ages of 8 and 12 and are now ages 19 to 21, will receive $6.875 million each under the terms of the settlement, NBC News reports. Angelo Serrano, former director of religious education at St. Lucy's-St. Patrick's Church, was arrested in 2009 after one of the boys told his mother about the abuse. The 67-year-old is currently serving a 15-year sentence. Lawyers for the victims say other boys were severely abused, but "the statute of limitations prevents their claims from being brought." After the victims sued, the diocese argued that it shouldn't be held responsible for the abuse because Serrano was officially a volunteer, not an employee, even though he received a stipend from the church, the New York Times reports. A judge, however, found that priests and parish workers ignored signs of abuse, including the fact that Serrano regularly had young boys sleep over at his apartment. In a deposition, Father Stephen Lynch admitted that he had seen Serrano inappropriately embrace an 8- or 9-year-old boy and kiss him on the lips. "These were egregious incidences of sexual abuse," attorney Ben Rabinowitz tells the New York Post. "And this all happened after the priests were taught to recognize the signs of abuse."

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Summarize: The signature of the saint of the poor and neglected, who inspired Pope Francis to choose his name, is nowhere to be seen. Historians agree that he most likely dictated his writings, but certainly his hand touched the papal bulls that in the 1220s registered the pope’s messages to the order. However, these 19 artifacts are the most ancient documents of St. Francis’ life and theological tradition. St. Francis, born the son of a wealthy cloth merchant in Assisi, chose to give up his prosperous, worldly life and live in poverty, preaching peace and respect for all forms of life. “St. Francis was a man, a saint, of the people, who truly stood with those who are the least every day,” Ken Hackett, the United States ambassador to the Vatican, said at a news conference in Rome last week. “We can see Pope Francis exemplified in his trace, as he puts into practice every day his advocacy for the marginalized and the disadvantaged. “This exhibition’s arrival in New York will give Americans the chance to know the history and the spirituality of St. Francis, and the chance to be inspired.” Among the artifacts, the highlight is Manuscript 338, a miscellaneous collection of medieval texts inscribed by at least nine different amanuenses. It contains “Canticle of the Sun,” a praise and thank you to the Lord for such creations as “Brother Fire” and “Sister Water.” “Francis’ hand is not in this poem, not even a line, but there is all of his spirit in it,” said Franco Cardini, professor emeritus of medieval history in the Florence branch of the Scuola Normale Superiore. “It’s unique.” Advertisement Continue reading the main story For this manuscript, the challenge was to restore the original cover, in 13th-century wood covered in goat leather, and then reconstruct the missing fragments, like the spine and the borders, Father Massetti said. Over the past five months, Father Massetti, two other monks and three young restoration experts have cleaned all the manuscripts with a soft paint brush, page by page. Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. In some, the medieval ink had perforated the page; in others it had faded. Some were missing entire figures or miniatures, others the binding cover. The restoration experts have repaired the fissures of the parchment with Japanese vegetable fiber or a bovine membrane. They have consolidated the ink and the colorful paintings through a starch gel. Five of the manuscripts, ranging from the size of a choral book to the pocket format, were unbound, parchment by parchment, and were finally reassembled and stitched back together with a linen thread. The longest restoration work was on Manuscript 328, Father Massetti said, a text by the Franciscan friar Ubertino da Casale, a 14th-century manuscript that interprets the Rule of St. Francis and the poverty of Christ. Artifacts also include a precious fragment, a parchment with the early recounting of the life of St. Francis, commissioned around the time of his canonization in 1228. A more comprehensive recounting of St. Francis’ life and gestures will also be on display in a book written by Father Thomas of Celano in the 1240s. The exhibition was already on display at the Lower House of Parliament this year and has been requested by museums and foundations all over the world, from Russia to Argentina. The friars of the Sacred Convent of St. Francis in Assisi are, however, reluctant to let the founding texts of their order trot the globe and will rather house them in their library after January. Advertisement Continue reading the main story In Assisi, about six million visitors a year, 40 percent of them Americans, visit St. Francis’ basilica, where the saint is buried. Over one and a half million people take a glance at St. Francis’ grave by webcam every month, “the largest virtual pilgrimage in Assisi,” said the Rev. Enzo Fortunato, director of the press office of the Sacred Convent of St. Francis. As Father Massetti prepared to ship the manuscripts across the ocean, he confessed he had had a few agitated months. Restoring the manuscripts was both a professional responsibility and a matter of security for him. He remembers nights in which the alarm for the 19th-century safe of the laboratory would even go off three times, if tremors shook the area. “I know it’s under my responsibility here,” Father Massetti said. “There is an estimated value, but should they go lost it would be an inestimable loss, impossible to pay back.” Founder of the Franciscan Order, born at Assisi in Umbria, in 1181. In 1182, Pietro Bernardone returned from a trip to France to find out his wife had given birth to a son. Far from being excited or apologetic because he'd been gone, Pietro was furious because she'd had his new son baptized Giovanni after John the Baptist. The last thing Pietro wanted in his son was a man of God -- he wanted a man of business, a cloth merchant like he was, and he especially wanted a son who would reflect his infatuation with France. So he renamed his son Francesco -- which is the equivalent of calling him Frenchman. Francis enjoyed a very rich easy life growing up because of his father's wealth and the permissiveness of the times. From the beginning everyone -- and I mean everyone -- loved Francis. He was constantly happy, charming, and a born leader. If he was picky, people excused him. If he was ill, people took care of him. If he was so much of a dreamer he did poorly in school, no one minded. In many ways he was too easy to like for his own good. No one tried to control him or teach him. As he grew up, Francis became the leader of a crowd of young people who spent their nights in wild parties. Thomas of Celano, his biographer who knew him well, said, "In other respects an exquisite youth, he attracted to himself a whole retinue of young people addicted to evil and accustomed to vice." Francis himself said, "I lived in sin" during that time. Francis fulfilled every hope of Pietro's -- even falling in love with France. He loved the songs of France, the romance of France, and especially the free adventurous troubadours of France who wandered through Europe. And despite his dreaming, Francis was also good at business. But Francis wanted more..more than wealth. But not holiness! Francis wanted to be a noble, a knight. Battle was the best place to win the glory and prestige he longed for. He got his first chance when Assisi declared war on their longtime enemy, the nearby town of Perugia. Most of the troops from Assisi were butchered in the fight. Only those wealthy enough to expect to be ransomed were taken prisoner. At last Francis was among the nobility like he always wanted to be...but chained in a harsh, dark dungeon. All accounts say that he never lost his happy manner in that horrible place. Finally, after a year in the dungeon, he was ransomed. Strangely, the experience didn't seem to change him. He gave himself to partying with as much joy and abandon as he had before the battle. The experience didn't change what he wanted from life either: Glory. Finally a call for knights for the Fourth Crusade gave him a chance for his dream. But before he left Francis had to have a suit of armor and a horse -- no problem for the son of a wealthy father. And not just any suit of armor would do but one decorated with gold with a magnificent cloak. Any relief we feel in hearing that Francis gave the cloak to a poor knight will be destroyed by the boasts that Francis left behind that he would return a prince. But Francis never got farther than one day's ride from Assisi. There he had a dream in which God told him he had it all wrong and told him to return home. And return home he did. What must it have been like to return without ever making it to battle -- the boy who wanted nothing more than to be liked was humiliated, laughed at, called a coward by the village and raged at by his father for the money wasted on armor. Francis' conversion did not happen over night. God had waited for him for twenty-five years and now it was Francis' turn to wait. Francis started to spend more time in prayer. He went off to a cave and wept for his sins. Sometimes God's grace overwhelmed him with joy. But life couldn't just stop for God. There was a business to run, customers to wait on. One day while riding through the countryside, Francis, the man who loved beauty, who was so picky about food, who hated deformity, came face to face with a leper. Repelled by the appearance and the smell of the leper, Francis nevertheless jumped down from his horse and kissed the hand of the leper. When his kiss of peace was returned, Francis was filled with joy. As he rode off, he turned around for a last wave, and saw that the leper had disappeared. He always looked upon it as a test from God...that he had passed. His search for conversion led him to the ancient church at San Damiano. While he was praying there, he heard Christ on the crucifix speak to him, "Francis, repair my church." Francis assumed this meant church with a small c -- the crumbling building he was in. Acting again in his impetuous way, he took fabric from his father's shop and sold it to get money to repair the church. His father saw this as an act of theft -- and put together with Francis' cowardice, waste of money, and his growing disinterest in money made Francis seem more like a madman than his son. Pietro dragged Francis before the bishop and in front of the whole town demanded that Francis return the money and renounce all rights as his heir. The bishop was very kind to Francis; he told him to return the money and said God would provide. That was all Francis needed to hear. He not only gave back the money but stripped off all his clothes -- the clothes his father had given him -- until he was wearing only a hair shirt. In front of the crowd that had gathered he said, "Pietro Bernardone is no longer my father. From now on I can say with complete freedom, 'Our Father who art in heaven.'" Wearing nothing but castoff rags, he went off into the freezing woods -- singing. And when robbers beat him later and took his clothes, he climbed out of the ditch and went off singing again. From then on Francis had nothing...and everything. Francis went back to what he considered God's call. He begged for stones and rebuilt the San Damiano church with his own hands, not realizing that it was the Church with a capital C that God wanted repaired. Scandal and avarice were working on the Church from the inside while outside heresies flourished by appealing to those longing for something different or adventurous. Soon Francis started to preach. (He was never a priest, though he was later ordained a deacon under his protest.) Francis was not a reformer; he preached about returning to God and obedience to the Church. Francis must have known about the decay in the Church, but he always showed the Church and its people his utmost respect. When someone told him of a priest living openly with a woman and asked him if that meant the Mass was polluted, Francis went to the priest, knelt before him, and kissed his hands -- because those hands had held God. Slowly companions came to Francis, people who wanted to follow his life of sleeping in the open, begging for garbage to eat...and loving God. With companions, Francis knew he now had to have some kind of direction to this life so he opened the Bible in three places. He read the command to the rich young man to sell all his good and give to the poor, the order to the apostles to take nothing on their journey, and the demand to take up the cross daily. "Here is our rule," Francis said -- as simple, and as seemingly impossible, as that. He was going to do what no one thought possible any more -- live by the Gospel. Francis took these commands so literally that he made one brother run after the thief who stole his hood and offer him his robe! Francis never wanted to found a religious order -- this former knight thought that sounded too military. He thought of what he was doing as expressing God's brotherhood. His companions came from all walks of life, from fields and towns, nobility and common people, universities, the Church, and the merchant class. Francis practiced true equality by showing honor, respect, and love to every person whether they were beggar or pope. Francis' brotherhood included all of God's creation. Much has been written about Francis' love of nature but his relationship was deeper than that. We call someone a lover of nature if they spend their free time in the woods or admire its beauty. But Francis really felt that nature, all God's creations, were part of his brotherhood. The sparrow was as much his brother as the pope. In one famous story, Francis preached to hundreds of birds about being thankful to God for their wonderful clothes, for their independence, and for God's care. The story tells us the birds stood still as he walked among him, only flying off when he said they could leave. Another famous story involves a wolf that had been eating human beings. Francis intervened when the town wanted to kill the wolf and talked the wolf into never killing again. The wolf became a pet of the townspeople who made sure that he always had plenty to eat. Following the Gospel literally, Francis and his companions went out to preach two by two. At first, listeners were understandably hostile to these men in rags trying to talk about God's love. People even ran from them for fear they'd catch this strange madness! And they were right. Because soon these same people noticed that these barefoot beggars wearing sacks seemed filled with constant joy. They celebrated life. And people had to ask themselves: Could one own nothing and be happy? Soon those who had met them with mud and rocks, greeted them with bells and smiles. Francis did not try to abolish poverty, he tried to make it holy. When his friars met someone poorer than they, they would eagerly rip off the sleeve of their habit to give to the person. They worked for all necessities and only begged if they had to. But Francis would not let them accept any money. He told them to treat coins as if they were pebbles in the road. When the bishop showed horror at the friars' hard life, Francis said, "If we had any possessions we should need weapons and laws to defend them." Possessing something was the death of love for Francis. Also, Francis reasoned, what could you do to a man who owns nothing? You can't starve a fasting man, you can't steal from someone who has no money, you can't ruin someone who hates prestige. They were truly free. Francis was a man of action. His simplicity of life extended to ideas and deeds. If there was a simple way, no matter how impossible it seemed, Francis would take it. So when Francis wanted approval for his brotherhood, he went straight to Rome to see Pope Innocent III. You can imagine what the pope thought when this beggar approached him! As a matter of fact he threw Francis out. But when he had a dream that this tiny man in rags held up the tilting Lateran basilica, he quickly called Francis back and gave him permission to preach. Sometimes this direct approach led to mistakes that he corrected with the same spontaneity that he made them. Once he ordered a brother who hesitated to speak because he stuttered to go preach half-naked. When Francis realized how he had hurt someone he loved he ran to town, stopped the brother, took off his own clothes, and preached instead. Francis acted quickly because he acted from the heart; he didn't have time to put on a role. Once he was so sick and exhausted, his companions borrowed a mule for him to ride. When the man who owned the mule recognized Francis he said, "Try to be as virtuous as everyone thinks you are because many have a lot of confidence in you." Francis dropped off the mule and knelt before the man to thank him for his advice. Another example of his directness came when he decided to go to Syria to convert the Moslems while the Fifth Crusade was being fought. In the middle of a battle, Francis decided to do the simplest thing and go straight to the sultan to make peace. When he and his companion were captured, the real miracle was that they weren't killed. Instead Francis was taken to the sultan who was charmed by Francis and his preaching. He told Francis, "I would convert to your religion which is a beautiful one -- but both of us would be murdered." Francis did find persecution and martyrdom of a kind -- not among the Moslems, but among his own brothers. When he returned to Italy, he came back to a brotherhood that had grown to 5000 in ten years. Pressure came from outside to control this great movement, to make them conform to the standards of others. His dream of radical poverty was too harsh, people said. Francis responded, "Lord, didn't I tell you they wouldn't trust you?" He finally gave up authority in his order -- but he probably wasn't too upset about it. Now he was just another brother, like he'd always wanted. Francis' final years were filled with suffering as well as humiliation. Praying to share in Christ's passion he had a vision received the stigmata, the marks of the nails and the lance wound that Christ suffered, in his own body. Years of poverty and wandering had made Francis ill. When he began to go blind, the pope ordered that his eyes be operated on. This meant cauterizing his face with a hot iron. Francis spoke to "Brother Fire": "Brother Fire, the Most High has made you strong and beautiful and useful. Be courteous to me now in this hour, for I have always loved you, and temper your heat so that I can endure it." And Francis reported that Brother Fire had been so kind that he felt nothing at all. How did Francis respond to blindness and suffering? That was when he wrote his beautiful Canticle of the Sun that expresses his brotherhood with creation in praising God. Francis never recovered from this illness. He died on October 4, 1226 at the age of 45. Francis is considered the founder of all Franciscan orders and the patron saint of ecologists and merchants.

Summary: When Francis of Assisi went blind after living a life of poverty, he penned his inspiring "Canticle of the Sun," Catholic Online notes. Now the manuscript that contains that writing, as well as 12 other medieval manuscripts, are heading to the US after a 700-year stay in Italy, the New York Times reports. The artifacts' first stop: the UN, where they'll be on exhibit from Nov. 17-28; they'll then be displayed at Brooklyn Borough Hall through mid-January. "This text may be the foundation of the Italian language, the first text ever known in vernacular," the Rev. Pierangelo Massetti, the monk who led the manuscripts' restoration, tells the Times. The restoration was an arduous task, with at least five others joining Massetti in using a soft paint brush to clean the manuscripts one page at a time Challenges included reconstructing missing parts like the spines, figuring out how to revitalize the faded ink, and fixing the original covers (at least one was made of wood covered in goat leather).Pope Francis chose his name from the patron saint of the poor, and while restorers admit the saint's signature is nowhere to be found in the ancient documents (they believe he dictated most of his writings), they contend his spirit is infused throughout them. While other institutions as far away as Russia and Argentina are eagerly vying to be the next to host the manuscripts, Italian friars at the Sacred Convent of St. Francis in Assisi want them all back after January.

multi_news

en

Summarize: [0001] This application claims priority from U.S. Provisional Application No. 61/347,985 filed May 25, 2010. BACKGROUND OF THE INVENTION [0002] As is generally known, cancer treatment can be both complex and invasive. In some cases, patients need to be subjected to high-dose radiation treatment in order to eradicate and prevent additional cancerous tissue growth. In cases of oral cancer, cancerous tissue growth is typically located on the floor of the mouth, cheek lining, tonsils, pharynx, or the tongue. When radiation treatment is applied in cases of oral cancer, there will always be some unnecessary damage to normal oral tissues that lie between the beam source and the cancer. For example, a patient&#39;s tongue may not be invaded by a cancer of his or her tonsils, but may be damaged as a result of the need to pass radiation energy through the tongue in order to cover the entire tonsillar tumor. Any time normal tissue can be spared by altering the beam path or moving the tissue physically out of the way, the patient benefits. BRIEF DESCRIPTION OF THE DRAWINGS [0003] FIG. 1 illustrates a plurality of elements comprising, when assembled, an intra-oral device in accordance with an embodiment; [0004] FIG. 2 is an exploded perspective view of the assembly of an intra-oral device in accordance with an embodiment; [0005] FIG. 3 is an exploded perspective view of the assembly of an intra-oral device in accordance with an embodiment; [0006] FIG. 4A illustrates in greater detail an upper “horseshoe” coupled with a lower “horseshoe”; and [0007] FIG. 4B is a cross section of a U-shaped member (“horseshoe”) included in the intra-oral device. DETAILED DESCRIPTION OF THE INVENTION [0008] Techniques for protecting oral tissues during radiation treatment are described. The techniques involve an intra-oral device that may reduce unnecessary damage of oral tissues of an oral cancer patient during radiation treatment. The intra-oral device is configured to move the tongue and/or other oral structures out of the way of the radiation beam during treatment, or to hold these structures steady so the beam can be better targeted. In so doing, unnecessary damage to the deflected oral tissues is minimized. In one embodiment, the device is configured to protect the patient&#39;s healthy tongue from the negative effects of radiation. In another embodiment, the device is configured to stabilize a cancerous lesion on a patient&#39;s tongue so that the radiation will have a consistent target volume to treat. [0009] An intra-oral device for protecting oral tissues during radiation treatment comprises several components, assembled into a final product with primarily tongue-deviating and/or tongue-depressing functions. One of the critical elements of the assembly that ensures tongue-deviating or tongue-depressing functions will be called hereinafter a tongue-deviating or tongue-depressing paddle, respectively. The purpose of the deviating paddles is to move the tongue away from the side with the cancer and the purpose of the depressing paddles is to either move the tongue down for a maxillary cancer or to stabilize it in a repeatable position for a mandibular or tongue cancer. The device is configured such that the position of the tongue paddle within the assembly is adjustable so as to allow for customizing to the particular shape of the mouth and tongue of each patient. [0010] Thus, in one embodiment, the tongue paddle (i.e., tongue-depressing paddle) may be disposed horizontally within the assembly comprising an intra-oral device in order to depress or stabilize the patient&#39;s tongue. In another embodiment, the tongue paddle (i.e., tongue-deviating paddle) may be disposed vertically in order to hold the tongue away from the cancerous zone in order to prevent the tongue from being exposed to radiation. If disposed vertically, the tongue paddle may be configured to be deviating to the right in order to move the tongue to the right side of the patient&#39;s mouth. Alternatively, the tongue paddle may be configured to be deviating to the left, in order to move the tongue to the left side of the patient&#39;s mouth. The device may be produced in a few predetermined sizes, for example, small, medium, and large. [0011] FIG. 1 illustrates a plurality of elements comprising, when assembled, an intra-oral device in accordance with one or more embodiments. The elements comprising the intra-oral device include at least the following elements described herein. [0012] A substantially U-shaped upper member (“horseshoe”) 10 is configured to replicate standard maxillary arch forms. The upper member may be sized as small, medium, or large, and possibly other sizes. The upper member defines a slot disposed about an upper side of the upper member and is adapted to receive a fill-in material to customize the fit to the individual patient&#39;s upper teeth. [0013] A substantially U-shaped lower member (“horseshoe”) 12 is configured to replicate standard mandibular arch forms. The lower member may be sized as small, medium, or large, and possibly other sizes. The lower member similarly defines a slot (shown in cross section in FIG. 4A ) disposed about the lower side of the lower member and is adapted to receive a fill-in material to customize the fit to the individual patient&#39;s lower teeth. [0014] The upper and lower members 10 and 12 may be joined to each other at the posterior aspect (the open end of the “U” shape), in one embodiment, by two hinging and/or sliding struts 14 and 16 (assembly shown in FIG. 2 ) that both allow anterior-posterior placement between the upper and lower members to be adjusted, as well as the amount of interincisal opening. These struts may also each have a hole or guide slot 18 or 20 ( FIG. 1 ) running transversely that will allow a tongue deviating paddle or tongue-depressing paddle (described below) to be adjusted in a medial/lateral direction. In these embodiments, guide slots 18 and 20 comprise ball joints with through apertures. The struts 14 and 16 may be adjustable so as to conform to the patient&#39;s jaw and tongue shapes. In one embodiment, the struts may have some version of snap-lock or slide/groove ability to allow some forward/backward motion of the upper and lower members in relation to one another. Posterior rods 22 and 24 may be configured to be inserted through the ball joints 18 and/or 20 and connect to a tongue paddle 32 or 40 as will be described below. [0015] At the anterior aspect of the device, an additional anterior midline strut 26 (shown in place in FIG. 2 ) may be attached to the upper member and the lower member in such a way that it can adjust to differences in the interincisal distance. This strut may act as a housing for a rotating sphere that may have a single hole (guide slot) or a ball joint with a through aperture 28 running through it. The anterior midline strut 26 is configured to support and serve as a placement guide for the anterior/posterior as well as X, Y and Z axis placement of a tongue paddle. A midline rod 30 may be connected to the tongue paddle through the ball joint 28 to provide desired support and guiding for the tongue paddle. [0016] The tongue deviating paddles 32, 40 may be substantially tear-drop shaped with an anterior rod that will fit through the anterior strut&#39;s sphere and protrude extra-orally for control during the fitting and placement stage. The paddles 32, 40 may be convex on the side away from the tongue and concave on the side toward the tongue. On the convex side, they may have an adjustable side rod (e.g., the rod 30 ) that will fit through the appropriate hole in one of the two posterior struts to render strength and support for holding the tongue toward the contralateral side. The tongue-deviating paddles may take different forms and sizes (e.g., small, medium, or large) and be configured to deviate right or deviate left depending on a side of oral tissue that needs to be treated. [0017] Accordingly, a tongue-deviating paddle 32 is a substantially oval-shaped or tear-drop-shaped tongue protection element having an aperture or a ball joint with a through aperture 34 configured to receive at least one of the posterior rods 22 or 24. The tongue-deviating paddle 32 may further include a housing 36 mounted along the narrow side of the “tear” shape of the paddle and configured to receive the midline rod 30. [0018] A tongue-depressing paddle 40 is a substantially oval-shaped tongue protection element having lugs with ball joints 42 and 44 mounted on a convex side of the paddle and configured to receive the posterior rods 22 and 24 and a housing 46 configured to receive the midline rod 30. [0019] Embodiments of the intra-oral device will now be described with reference to FIGS. 2-4. FIG. 2 illustrates an example assembly 200 of an intra-oral device with a tongue-deviating paddle 32. The assembly may utilize the elements described in reference to FIG. 1. However, it should be understood that the elements comprising the assembly are interchangeable and may be replaced by similar elements configured to perform the same functions. The assembly 200 includes two elongate, essentially U-shaped members, a lower member 10 and an upper member 12, coupled by a strut 14 ( 16 ) at each end. [0020] In one embodiment, the member 10 may be further connected to the member 12 by the midline strut 26 as shown in FIG. 2. In another embodiment, the member 10 may be coupled to the member 12 by a joint 417, as shown in FIG. 4(A). Alternatively, the assembly 200 may be manufactured as a uniform element, that is, the member 10 may be directly attached to the member 12. FIG. 4(B) illustrates a cross-section 420 of a U-shaped member 10 ( 12 ). As shown in FIG. 4(B), the U-shaped member 10 ( 12 ) forms a slot (cavity) 424 adapted to be filled with plastic fill-in material. The above structure may act as an intermaxillary scaffold to hold the patient&#39;s upper and lower jaws apart in a repeatable position in three-dimensional space, at a prescribed level of opening, and to support a “tongue paddle” which will displace the tongue in a prescribed direction. [0021] After the U-shaped intermaxillary scaffold is constructed, the tongue paddle may be loosely attached to the scaffold and inserted in the patient&#39;s mouth. Using the anterior rod as a positioning device, rotating the paddle using the housed sphere as a fulcrum point, the tongue may be positioned by the operator in the desired location. Once in this position, the anterior and posterior strut assemblies may be fixed by either addition of light-cured acrylic or by fusing the components with cyanoacrylate or similar bonding agents, locking the tongue paddle in its final, non-movable position. [0022] Referring back to FIG. 2, the assembly 200 further comprises a substantially oval-shaped tongue protection element, e.g., tongue-deviating paddle 32, inserted in the middle of the assembly 200. In one embodiment, the tongue paddle 32 may be disposed within the assembly 200 such that a certain freedom of movement (adjustment) of the tongue paddle 32 within the assembly 200 is ensured. The ball joints included in the struts and on the paddles as described in reference to FIG. 1 may be configured to allow the greatest range of motion of the tongue paddle within the assembly. The ball joints may be secured within respective struts or paddles with some version of adhesive (e.g., cyanoacrylate) once the paddle is fixed in a desired position within the assembly. For example, the tongue-deviating paddle 32 may be coupled through the strut 34 with the rod 22 disposed through the strut 14. The end of the tongue-deviating paddle 32 may be connected with the midline rod 30 disposed through the midline strut 26. The described structure of the assembly allows for a semi-universal movement at the front end of the tongue-deviating paddle when the midline rod 30 is moved. The midline rod may be removed (e.g., cut off or snapped) off once the paddle is secured in a desired position. [0023] As shown in FIG. 2, the tongue-deviating paddle 32 may be disposed substantially vertically relative to the assembly 200. While the tongue-deviating paddle 32 is configured to provide a tongue deviation to the left (relative to the back of a patient&#39;s head), a “right” version of a tongue-deviating paddle may be configured and mounted similarly to that of the “left” version described herein. In one embodiment, the members 10 or 12 may be configured to provide support for the tongue-deviating paddle 32. For example, the tongue-deviating paddle may be attached to the middle section of the member 10 or to the member 12. [0024] An embodiment of the intra-oral device will now be described with reference to FIG. 3, which illustrates an example assembly 300 that includes a tongue-depressing paddle 40. Similar to the tongue deviating paddles 32, the tongue depressing paddles 40 may have an anterior positioning rod 30 that fits through the anterior strut 26 &#39;s ball joint 28. The paddle may be substantially oval-shaped and may be positioned horizontally as shown in FIG. 3. Like the deviating paddles described above, the depressing paddle 40 may be loosely fitted into the intermaxillary scaffold formed by the members 10 and 12, placed intraorally and positioned by the operator using the positioning rod 30. Once in the desired position, the anterior strut assembly formed by elements 26 and 28 may be immobilized, the rod 30 shortened, and two posterior support rods 22 and 24 added for additional strength and secured with adhesive (e.g., cyanoacrylate). [0025] The intra-oral device described herein may be customized for a particular patient. Similarly, a shape of the tongue paddle may be adjusted (generally by material removal, but also by material addition) to replicate the particular shape of a patient&#39;s tongue. In operation, when the customized device is inserted into the patient&#39;s mouth, the tongue paddle will shield the patient&#39;s tongue from the effect of intra-oral cancer radiation treatment, thus protecting the tongue tissues. The materials selected must be capable of withstanding several months of daily high-dose radiation exposure. The intra-oral device may be manufactured of any elastic material which is capable of withstanding radiation (e.g., having low radiation absorption and scatter), such as a plastic material. Accordingly, the device may be manufactured of acrylics, carbon fibers, or any other materials having the required properties. [0026] The fill-in material for the upper and lower U-shaped members described above in reference to FIG. 4(B) may include any plastic material having a molding property. For example, the fill-in material for the U-shaped members may be made of a customizable material such as Triad acrylic, Polyether or Polyvinylsiloxane. When the device is inserted into the patient&#39;s mouth, the patient “molds” the insides of each horseshoe by biting into the fill-in material, which subsequently hardens either autocatalytically or via application of a bright photoactivating light, thus replicating the particular patient&#39;s upper and lower tooth forms. The device may also be customizable such as by addition of light-cured acrylic to add custom devices such as lead-lined lip bumpers, cheek bumpers near metallic crowns, etc. When the device is inserted into the patient&#39;s mouth, a radiation treatment specialist will adjust the tongue paddle using a handle (e.g., midline rod 30 ) by moving the paddle around the struts. When the tongue paddle is adjusted, the handle may be shortened by cutting or snapping off. [0027] While various illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the device disclosed herein.

Summary: An intra-oral device for protecting oral tissues during radiation treatment is disclosed. In an embodiment, the device includes substantially a U-shaped upper member and lower members coupled to the upper member at their first and the second ends, and a substantially oval-shaped protective element disposed between the first ends of the upper and lower members and the second ends of the upper and lower members and movably coupled with one of the upper or lower members so as to provide for a three-dimensional spatial adjustment of the protective element between the first and second ends of the upper and lower members. The upper and lower members are configured to receive upper and lower teeth of a patient and the protective element is configured to engage a tongue of the patient so that the tongue is selectively positioned for treatment and/or diagnostic procedures.

big_patent

en

Summarize: CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/238,922 filed Sep. 1, 2009 and U.S. Provisional Patent Application No. 61/263,610 filed Nov. 23, 2009 the entirety of both of which are hereby incorporated by reference into this application. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to new and useful pesticidal compositions and particularly relates to herbicidal compounds requiring special precautions when being applied to reduce or prevent vapor transfer thereof to plants which are not the target of application of the compounds, and/or for which longer duration pest control is desired. [0004] 2. Description of Related Art [0005] Agricultural chemicals, particularly herbicides, are conventionally used in a wide variety of formulation types, including solid formulations, such as powders, dusts and granules, and liquid formulations, such as solutions, emulsifiable oil concentrates, and suspensions of solids in liquid carriers, or time-release microcapsules dispersed in an aqueous and organic solvent or oil carrier. The choice of which type of selected formulation to be used is generally governed by many considerations, such as the physical and chemical characteristics of the active ingredients, the crop or weed species to which the formulation is to be applied, and whether the application is better made postemergence or preemergence. [0006] Delayed-release formulations are chosen normally to limit the release of the pesticide to regions of the environment other than the location of the pest organism to be controlled. Microencapsulation of the pesticide is one delivery form often selected for providing the desired delayed-release. Applying microencapsulated pesticide has, in some cases, the disadvantage of substantially sacrificing the activity of the pesticide at the proper point of time, e.g., soon after application. Micro-encapsulated formulations have been prepared containing alone or in mixtures trifluralin, alachlor, butachlor, propachlor, triallate, metribuzin, linuron, and pyrethriods (U.S. Pat. No. 4,107,292, U.S. Pat. No. 4,360,376, U.S. Pat. No. 4,280,833, U.S. Pat. No. 4,497,793). The related art also teaches the physical property requirements for successful micro-encapsulation and so microcapsules should be possible for thiobencarb, acetochlor, dimethenamid, terbufos, carbosulfan, cypermethrin, zeta-cypermethrin, permethrin, bifenthrin and selected other pesticides. [0007] Conventionally, the pesticide is, or is dissolved in, an oil and the micro-capsule walls are formed around micro-droplets of this oil suspended in water. Micro-encapsulation can also be carried out with pesticides and other compounds which are soluble or suspendable in water, wherein the micro- capsule walls are formed around micro-droplets of this water suspended in an oil. This technology is generally referred to as reverse-phase micro-encapsulation, as described in U.S. Pat. No. 4,534,783, U.S. Patent No. 6,113,935, U.S. Pat. No. 6,359,031, U.S. Pat. No. 6,534,094, and U.S. Patent Application Publication No. 2009/0053271, all hereby incorporated by reference into this application. [0008] Formulations containing a microencapsulated pesticide have been prepared in which a second pesticide is also present, often present in the suspending liquid medium chosen for the microcapsules and thus able to be released immediately upon application to the crop or other pest-containing location. This combination formulation is especially suited for combinations of an insecticide with a synergist or a herbicide with a safener (U.S. Pat. No. 5,229,122, U.S. Patent Application Publication No. 2005/0255137, DE 2411373, U.S. Pat. No. 5,178,872, U.S. Patent Application Publication No. 2008/0176746). [0009] Thus microencapsulated formulations of selected pesticides are well-known and utilized in agriculture in which the microcapsules contain one or more pesticidally active agents. In these formulations it is known that one can also include pesticidally active insecticides in the suspending medium to provide a high initial “knockdown” activity, or other biologically active agents to pre-condition the pest or target crop so the encapsulated compound will be more active on the pest or more tolerated by the crop (U.S. Pat. No. 5,229,122). Further it is known that microcapsules of two differing pesticidal compounds can be combined in the same suspension, which can be useful if the two compounds are incompatible (U.S. Patent Application Publication No. 2005/0266996, U.S. Patent Application Publication No. 2008/0176746). Compositions containing two or more separately-prepared populations of microcapsules with divergent release rates of the same active ingredient have not been described in the prior art of which applicant is aware. [0010] An excellent selective soil applied herbicide commercially available for controlling many broadleaf and grass weeds, in soybean, cotton, sugarcane, rice, tobacco, oilseed rape, vegetables and other crops has the common name clomazone which chemically is 2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone. Clomazone is an effective herbicide as evidenced by its ability to control, at low application rates in crops, a broad spectrum of grasses and broadleaf weeds that compete with crops. Unfortunately, clomazone has a high vapor pressure and is phytotoxic to some non-targeted crops and naturally occurring plant species when applied to control undesired vegetation. Contact of clomazone with such crops is the result of vapor transfer of the clomazone to sensitive species growing in adjacent areas. Therefore clomazone has been sold for over 20 years now primarily as a micro-encapsulated formulation with significantly reduced risk of vapor transfer to and phytotoxic effects on non-target plant species. The clomazone microcapsules can be prepared as an aqueous suspension or combined with extruded granules (U.S. Pat. No. 5,583,090, U.S. Pat. No. 5,597,780, U.S. Pat. No. 5,783,520, U.S. Pat. No. 6,218,339, U.S. Pat. No. 6,380,133, U.S. Pat. No. 6,440,902, U.S. Pat. No. RE38,675, U.S. Pat. No. 6,797,277). [0011] Growers using clomazone for weed control in rice, especially in the southern United States, have been generally pleased with its control of key weed species. However control of grass weeds by clomazone does not always last until the permanent flood. As a consequence, growers either must incur the cost of a follow-up treatment with post-emergent grass herbicides (herbicide and application costs) or, as some have chosen to do, apply the permitted dose of clomazone in two sequential applications a few weeks apart. This second approach results in sufficient clomazone being present up to the time of the permanent flood to give excellent grass weed control in rice, but growers must bear the additional cost of a second application. [0012] In other crops in which clomazone and other herbicides are used, an increased duration of residual control can also be desired by growers, up to the point of canopy closure. [0013] It is desirable to provide a system for formulating clomazone and other soil-active pesticides so that movement to non-target plants and/or groundwater is significantly reduced and so that the duration of pest control is increased. SUMMARY OF THE INVENTION [0014] The present invention relates generally to the field of pesticidal chemical formulations. The current invention provides a method for extending the duration of pest control for any compound that can be successfully micro-encapsulated by either normal phase or reverse phase micro-encapsulation methods. In the normal phase method, the pesticide is, or is dissolved in, an oil and the micro-capsule walls are formed around micro-droplets of this oil suspended in water. In the reverse phase microencapsulation method, the microencapsulation is carried out with pesticides and other compounds which are soluble or suspendable in water and the micro- capsule walls are formed around micro-droplets of this water suspended in an oil. In particular, the present invention relates to novel compositions of a herbicidal compound, namely clomazone, designed to reduce clomazone&#39;s characteristic volatility, thereby reducing risk of unintended herbicidal activity, and extend its duration of effective weed control, when clomazone is applied and providing growers with more effective weed control. Clomazone chemically is 2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone. The herbicidally active ingredient to which the present invention is concerned will be referred to herein by its common name of clomazone. [0015] The present invention provides adequate suppression of volatility of clomazone (and other compounds) while also providing a longer time of acceptable weed control. It does so by combining micro-encapsulated suspensions of clomazone with micro-encapsulated suspensions of clomazone having a significantly slower release rate. DETAILED DESCRIPTION [0016] The present invention provides a plurality of conditions using effective processes for preparing micro-encapsulated clomazone and other pesticides. In a first condition, the methods and conditions used are similar to those described in U.S. Pat. No. RE38,675, or alternatively in U.S. Pat. No. 5,583,090, hereby incorporated by reference into this application. In a second condition, the conditions are modified so that the walls of the microcapsules are thicker and generally less porous, accordingly the clomazone and other pesticides contained within these microcapsules is released much more slowly than in those prepared under the first condition. Alternatively, faster release microcapsules can be used in the present invention and can have a more rapid release rate than conventional products to give a stronger initial activity. [0017] In the present invention, the ratio of the rates of release from the microcapsules is adjusted so that the slower-released pesticide appears outside the microcapsules at a rate adequate to compensate for metabolism and irreversible soil binding of the previously released pesticide and maintain its effective concentration in the soil. [0018] The microcapsules prepared by the second set of conditions may even appear to be relatively ineffective in normal herbicidal evaluations, as they will have released too little of the pesticide in the time frame for such tests. They will however release the pesticide at a rate sufficient to replace that pesticide which is being lost in the root zone due to microbial metabolism or irreversible soil binding in order to extend the duration of effective weed control. [0019] In one embodiment, a combination of clomazone microcapsules prepared by the first set of conditions and clomazone microcapsules prepared by the second set of conditions is prepared in an appropriate ratio, the mixture provides acceptable control of volatility, acceptable initial activity on emerging weeds and acceptable activity on later-emerging weeds, such as those emerging just before the permanent flood is put into place in rice culture (typically about four weeks) or before canopy closure in other crops. [0020] The present invention, the combination of two sets of microcapsules prepared under differing conditions, can be applied to other soil-active pest control chemicals. In some cases, volatility will not be a concern, but extended duration of control will still be of value. In one embodiment, a composition is prepared which is an aqueous suspension or an extruded granule in which are blended two separate sets of microcapsules of a soil-active pesticide in which the microcapsules have been prepared under two significantly different sets of conditions, to give two significantly different release rates, and then blended in a ratio to give the desired biological activity, and the ratio can vary from about 10:1 to about 1:10. [0021] In one embodiment, the rate of release from the more slowly releasing microcapsules is adjusted to a value that will allow the more slowly released material to replace the pesticide in the soil which has been metabolized or irreversibly bound to the soil. The release rate of the more slowly released pesticide may vary from about ½ to about 1/20 th the release rate of the more rapidly releasing microcapsules, preferably about ⅓ to about ⅙. In one embodiment, the ratio of the faster releasing microcapsules to the slower releasing microcapsules is between about 3:1 and about 1:9, preferably, the ratio of the faster releasing microcapsules to the slower releasing microcapsules is between about 1:1 and about 1:5 and more preferably the ratio of the faster releasing microcapsules to the slower releasing microcapsules is between about 1:1.5 and about 1:3. The average release rate of the slower releasing microcapsules is about two-fold to about six-fold slower than the first set of faster releasing microcapsules. Alternatively, the average release rate of the slower releasing microcapsules is about three-fold to about five-fold slower than the first set of faster releasing microcapsules. [0022] The effect of extended control via a blend of microcapsules, a portion of which have slower release characteristics, can include an ability to reduce the overall amount of chemical applied to the soil, thus reducing environmental contamination. The present invention will also save growers the cost of additional applications of herbicides. Yet another desirable effect may include the ability to use chemicals whose degradation in the soil is so fast that their effectiveness is otherwise too weak for effective and practical pest control. [0023] The capsules can have a shell composed of a polyurea polymer formed around liquid droplets of the pesticide and containing effective dispersants and stabilizers, wherein the shell comprises between about 5% and about 35% by weight of the shell and the microcapsules have a diameter between about one micron and about 100 microns. Suitable pesticides include metolachlor, acetochlor, dimethenamid, triallate, thiobencarb, butachlor, terbufos, carbosulfan, or clomazone. Clomazone is the preferred pesticide. Clomazone can be present between about 1 and about 4 lbs./gallon. Preferably, clomazone is present at between about 2.5 and about 3.5 lbs./gallon. [0024] The present invention includes a method of controlling undesirable plants species (weeds) by applying the compositions to the locus where a crop is to be grown, either to the soil surface or by incorporation into the soil prior to the emergence of the crop. For example, the crop can be rice. The invention can be further illustrated by the following examples thereof, although it will be understood that these examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated. All percentages, ratios, and parts herein, in the Specification, Examples, and Claims, are by weight and are approximations unless otherwise stated. EXAMPLE I [0025] Clomazone is prepared under a first condition as a micro-encapsulated aqueous suspension following procedures described in U.S. Pat. No. RE38,675, hereby incorporated by reference into this application, to give a three pound per gallon concentrate with a release rate of 16% (plus or minus 2%) relative to that of clomazone formulated as a 4 EC in an 18 hour interval as assayed by the method in U.S. Pat. No. No. 5,597,780, hereby incorporated by reference into this application. [0026] Clomazone is also prepared separately under a second condition as a micro-encapsulated aqueous suspension using the same general methods as described in U.S. Pat. No. RE38,675, and using the principles described therein, and in U.S. Pat. No. 5,583,090, to adjust the release rate, such as increased particle size, wall thickness, and solvent content, and in light of the equipment used, to give a three pound per gallon concentrate but with the ratios of components adjusted to give a release rate of 4.0% (plus or minus 0.5%) as assayed by the same method. [0027] The conditions of the microencapsulations thus yield a ratio of release rates equal to 4.0. [0028] These two preparations of a clomazone micro-encapsulated suspension are then blended together at a ratio of 30% of the first, faster-releasing, preparation to 70% of the second, slower releasing, formulation, to give the final blended formulation, a three pound per gallon aqueous concentrate. EXAMPLE II [0029] Clomazone is prepared as a micro-encapsulated aqueous suspension following procedures described in U.S. Pat. No. RE38,675 to give a three pound per gallon concentrate with a release rate of 16% (plus or minus 2.0%) relative to that of clomazone formulated as a 4 EC in an 18 hour interval as assayed by the method in U.S. Pat. No. 5,597,780, hereby incorporated by reference into this application. [0030] Clomazone is also prepared as a micro-encapsulated aqueous suspension using the same general methods as described in U.S. Pat. No. RE38,675, and using the principles described therein, and in U.S. Pat. No. 5,583,090, to adjust the release rate, such as increased particle size, wall thickness, and solvent content, and in light of the equipment used, to give a three pound per gallon concentrate but with the ratios of components adjusted to give a release rate of 3.6% (plus or minus 0.4%) as assayed by the same method. [0031] The conditions of microencapsulation thus yield a ratio of release rates equal to 4.5. [0032] These two preparations of a clomazone micro-encapsulated suspension are then blended together at a ratio of 30% of the first, faster-releasing, preparation to 70% of the second, slower releasing, formulation, to give the final blended formulation, a three pound per gallon aqueous concentrate. EXAMPLE III [0033] Clomazone is prepared as a micro-encapsulated aqueous suspension following procedures described in U.S. Pat. No. RE38,675 to give a three pound per gallon concentrate with a release rate of 16% (plus or minus 2.0%) relative to that of clomazone formulated as a 4 EC in an 18 hour interval as assayed by the method in U.S. Pat. No. 5,597,780, hereby incorporated by reference into this application. [0034] Clomazone is also prepared as a micro-encapsulated aqueous suspension using the same general methods as described in U.S. Pat. No. RE38,675, and using the principles described therein, and in U.S. Pat. No. 5,583,090, to adjust the release rate, such as increased particle size, wall thickness, and solvent content, and in light of the equipment used, to give a three pound per gallon concentrate but with the ratios of components adjusted to give a release rate of 4.6% (plus or minus 0.4%) as assayed by the same method. [0035] The conditions of microencapsulation thus yield a ratio of release rates equal to 3.5. [0036] These two preparations of a clomazone micro-encapsulated suspension are then blended together at a ratio of 30% of the first, faster-releasing, preparation to 70% of the second, slower releasing, formulation, to give the final blended formulation, a three pound per gallon aqueous concentrate. EXAMPLE IV [0037] The preparations of clomazone microcapsules described in the above Example I are blended together at a ratio of 40% of the first, faster-releasing, preparation and 60% of the second, slower releasing, formulation to give final blended formulations of all three pound per gallon aqueous concentrates. EXAMPLE V [0038] The two preparations of clomazone microcapsules described in the above Example II are blended together at a ratio of 40% of the first, faster-releasing, preparation and 60% of the second, slower releasing, formulation to give final blended formulations of all three pound per gallon aqueous concentrates. EXAMPLE VI [0039] The two preparations of clomazone microcapsules described in the above Example III are blended together at a ratio of 40% of the first, faster-releasing, preparation and 60% of the second, slower releasing, formulation to give final blended formulations of all three pound per gallon aqueous concentrates. [0040] It is understood that there may be variations from the specific embodiments described herein without departing from the spirit, scope, or concept of the present invention as defined in the claims. Included in such variations are mixtures in which the encapsulated clomazone is part of a mixture with other herbicides whether or not encapsulated; variations in the release rate of the second slower-releasing microcapsules, variations in the ratios of the two microcapsules preparations. The present invention can as well, be practiced with a variety of other pesticide compounds for which volatility control and/or groundwater contamination control with an extended residual activity is desired. With other pesticides, the rate of release of the active compound from the second set of more slowly releasing microcapsules will need to be adjusted, in light of the soil metabolism and irreversible soil binding characteristics of said pesticide. The more slowly released material will replace that which became ineffective due to metabolism or irreversible soil binding. [0041] It is to be understood that the above-described embodiments are illustrative of only a few of the many possible specific embodiments, which can represent applications of the principles of the invention. The principles of the invention, herein described for normal phase microencapsulation, can also be applied to reverse phase microencapsulation. Numerous and varied other arrangements can be readily devised in accordance with these principles by those skilled in the art without departing from the spirit and scope of the invention.

Summary: The present invention is directed to micro-encapsulated formulations of soil-active pest control and agricultural chemicals such as herbicides, insecticides, nematicides and fungicides (pesticides) that combine a strong initial activity and a longer residual activity in the soil than provided by current micro-encapsulated formulations. These formulations are prepared by combining two separate suspensions of microcapsules, containing one or more pesticides, which were separately prepared under differing conditions and therefore have significantly different chemical properties and field release rates. The present invention, in particular, is useful for the herbicide clomazone, alone or in combination with other herbicides.

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Summarize: The observance of Sunday at Bellomont was chiefly marked by the punctual appearance of the smart omnibus destined to convey the household to the little church at the gates. Whether any one got into the omnibus or not was a matter of secondary importance, since by standing there it not only bore witness to the orthodox intentions of the family, but made Mrs. Trenor feel, when she finally heard it drive away, that she had somehow vicariously made use of it. It was Mrs. Trenor's theory that her daughters actually did go to church every Sunday; but their French governess's convictions calling her to the rival fane, and the fatigues of the week keeping their mother in her room till luncheon, there was seldom any one present to verify the fact. Now and then, in a spasmodic burst of virtue--when the house had been too uproarious over night--Gus Trenor forced his genial bulk into a tight frock-coat and routed his daughters from their slumbers; but habitually, as Lily explained to Mr. Gryce, this parental duty was forgotten till the church bells were ringing across the park, and the omnibus had driven away empty. Lily had hinted to Mr. Gryce that this neglect of religious observances was repugnant to her early traditions, and that during her visits to Bellomont she regularly accompanied Muriel and Hilda to church. This tallied with the assurance, also confidentially imparted, that, never having played bridge before, she had been "dragged into it" on the night of her arrival, and had lost an appalling amount of money in consequence of her ignorance of the game and of the rules of betting. Mr. Gryce was undoubtedly enjoying Bellomont. He liked the ease and glitter of the life, and the lustre conferred on him by being a member of this group of rich and conspicuous people. But he thought it a very materialistic society; there were times when he was frightened by the talk of the men and the looks of the ladies, and he was glad to find that Miss Bart, for all her ease and self-possession, was not at home in so ambiguous an atmosphere. For this reason he had been especially pleased to learn that she would, as usual, attend the young Trenors to church on Sunday morning; and as he paced the gravel sweep before the door, his light overcoat on his arm and his prayer-book in one carefully-gloved hand, he reflected agreeably on the strength of character which kept her true to her early training in surroundings so subversive to religious principles. For a long time Mr. Gryce and the omnibus had the gravel sweep to themselves; but, far from regretting this deplorable indifference on the part of the other guests, he found himself nourishing the hope that Miss Bart might be unaccompanied. The precious minutes were flying, however; the big chestnuts pawed the ground and flecked their impatient sides with foam; the coachman seemed to be slowly petrifying on the box, and the groom on the doorstep; and still the lady did not come. Suddenly, however, there was a sound of voices and a rustle of skirts in the doorway, and Mr. Gryce, restoring his watch to his pocket, turned with a nervous start; but it was only to find himself handing Mrs. Wetherall into the carriage. The Wetheralls always went to church. They belonged to the vast group of human automata who go through life without neglecting to perform a single one of the gestures executed by the surrounding puppets. It is true that the Bellomont puppets did not go to church; but others equally important did--and Mr. and Mrs. Wetherall's circle was so large that God was included in their visiting-list. They appeared, therefore, punctual and resigned, with the air of people bound for a dull "At Home," and after them Hilda and Muriel straggled, yawning and pinning each other's veils and ribbons as they came. They had promised Lily to go to church with her, they declared, and Lily was such a dear old duck that they didn't mind doing it to please her, though they couldn't fancy what had put the idea in her head, and though for their own part they would much rather have played lawn tennis with Jack and Gwen, if she hadn't told them she was coming. The Misses Trenor were followed by Lady Cressida Raith, a weather-beaten person in Liberty silk and ethnological trinkets, who, on seeing the omnibus, expressed her surprise that they were not to walk across the park; but at Mrs. Wetherall's horrified protest that the church was a mile away, her ladyship, after a glance at the height of the other's heels, acquiesced in the necessity of driving, and poor Mr. Gryce found himself rolling off between four ladies for whose spiritual welfare he felt not the least concern. It might have afforded him some consolation could he have known that Miss Bart had really meant to go to church. She had even risen earlier than usual in the execution of her purpose. She had an idea that the sight of her in a grey gown of devotional cut, with her famous lashes drooped above a prayer-book, would put the finishing touch to Mr. Gryce's subjugation, and render inevitable a certain incident which she had resolved should form a part of the walk they were to take together after luncheon. Her intentions in short had never been more definite; but poor Lily, for all the hard glaze of her exterior, was inwardly as malleable as wax. Her faculty for adapting herself, for entering into other people's feelings, if it served her now and then in small contingencies, hampered her in the decisive moments of life. She was like a water-plant in the flux of the tides, and today the whole current of her mood was carrying her toward Lawrence Selden. Why had he come? Was it to see herself or Bertha Dorset? It was the last question which, at that moment, should have engaged her. She might better have contented herself with thinking that he had simply responded to the despairing summons of his hostess, anxious to interpose him between herself and the ill-humour of Mrs. Dorset. But Lily had not rested till she learned from Mrs. Trenor that Selden had come of his own accord. "He didn't even wire me--he just happened to find the trap at the station. Perhaps it's not over with Bertha after all," Mrs. Trenor musingly concluded; and went away to arrange her dinner-cards accordingly. Perhaps it was not, Lily reflected; but it should be soon, unless she had lost her cunning. If Selden had come at Mrs. Dorset's call, it was at her own that he would stay. So much the previous evening had told her. Mrs. Trenor, true to her simple principle of making her married friends happy, had placed Selden and Mrs. Dorset next to each other at dinner; but, in obedience to the time-honoured traditions of the match-maker, she had separated Lily and Mr. Gryce, sending in the former with George Dorset, while Mr. Gryce was coupled with Gwen Van Osburgh. George Dorset's talk did not interfere with the range of his neighbour's thoughts. He was a mournful dyspeptic, intent on finding out the deleterious ingredients of every dish and diverted from this care only by the sound of his wife's voice. On this occasion, however, Mrs. Dorset took no part in the general conversation. She sat talking in low murmurs with Selden, and turning a contemptuous and denuded shoulder toward her host, who, far from resenting his exclusion, plunged into the excesses of the MENU with the joyous irresponsibility of a free man. To Mr. Dorset, however, his wife's attitude was a subject of such evident concern that, when he was not scraping the sauce from his fish, or scooping the moist bread-crumbs from the interior of his roll, he sat straining his thin neck for a glimpse of her between the lights. Mrs. Trenor, as it chanced, had placed the husband and wife on opposite sides of the table, and Lily was therefore able to observe Mrs. Dorset also, and by carrying her glance a few feet farther, to set up a rapid comparison between Lawrence Selden and Mr. Gryce. It was that comparison which was her undoing. Why else had she suddenly grown interested in Selden? She had known him for eight years or more: ever since her return to America he had formed a part of her background. She had always been glad to sit next to him at dinner, had found him more agreeable than most men, and had vaguely wished that he possessed the other qualities needful to fix her attention; but till now she had been too busy with her own affairs to regard him as more than one of the pleasant accessories of life. Miss Bart was a keen reader of her own heart, and she saw that her sudden preoccupation with Selden was due to the fact that his presence shed a new light on her surroundings. Not that he was notably brilliant or exceptional; in his own profession he was surpassed by more than one man who had bored Lily through many a weary dinner. It was rather that he had preserved a certain social detachment, a happy air of viewing the show objectively, of having points of contact outside the great gilt cage in which they were all huddled for the mob to gape at. How alluring the world outside the cage appeared to Lily, as she heard its door clang on her! In reality, as she knew, the door never clanged: it stood always open; but most of the captives were like flies in a bottle, and having once flown in, could never regain their freedom. It was Selden's distinction that he had never forgotten the way out. That was the secret of his way of readjusting her vision. Lily, turning her eyes from him, found herself scanning her little world through his retina: it was as though the pink lamps had been shut off and the dusty daylight let in. She looked down the long table, studying its occupants one by one, from Gus Trenor, with his heavy carnivorous head sunk between his shoulders, as he preyed on a jellied plover, to his wife, at the opposite end of the long bank of orchids, suggestive, with her glaring good-looks, of a jeweller's window lit by electricity. And between the two, what a long stretch of vacuity! How dreary and trivial these people were! Lily reviewed them with a scornful impatience: Carry Fisher, with her shoulders, her eyes, her divorces, her general air of embodying a "spicy paragraph"; young Silverton, who had meant to live on proof-reading and write an epic, and who now lived on his friends and had become critical of truffles; Alice Wetherall, an animated visiting-list, whose most fervid convictions turned on the wording of invitations and the engraving of dinner-cards; Wetherall, with his perpetual nervous nod of acquiescence, his air of agreeing with people before he knew what they were saying; Jack Stepney, with his confident smile and anxious eyes, half way between the sheriff and an heiress; Gwen Van Osburgh, with all the guileless confidence of a young girl who has always been told that there is no one richer than her father. Lily smiled at her classification of her friends. How different they had seemed to her a few hours ago! Then they had symbolized what she was gaining, now they stood for what she was giving up. That very afternoon they had seemed full of brilliant qualities; now she saw that they were merely dull in a loud way. Under the glitter of their opportunities she saw the poverty of their achievement. It was not that she wanted them to be more disinterested; but she would have liked them to be more picturesque. And she had a shamed recollection of the way in which, a few hours since, she had felt the centripetal force of their standards. She closed her eyes an instant, and the vacuous routine of the life she had chosen stretched before her like a long white road without dip or turning: it was true she was to roll over it in a carriage instead of trudging it on foot, but sometimes the pedestrian enjoys the diversion of a short cut which is denied to those on wheels. She was roused by a chuckle which Mr. Dorset seemed to eject from the depths of his lean throat. "I say, do look at her," he exclaimed, turning to Miss Bart with lugubrious merriment--"I beg your pardon, but do just look at my wife making a fool of that poor devil over there! One would really suppose she was gone on him--and it's all the other way round, I assure you." Thus adjured, Lily turned her eyes on the spectacle which was affording Mr. Dorset such legitimate mirth. It certainly appeared, as he said, that Mrs. Dorset was the more active participant in the scene: her neighbour seemed to receive her advances with a temperate zest which did not distract him from his dinner. The sight restored Lily's good humour, and knowing the peculiar disguise which Mr. Dorset's marital fears assumed, she asked gaily: "Aren't you horribly jealous of her?" Dorset greeted the sally with delight. "Oh, abominably--you've just hit it--keeps me awake at night. The doctors tell me that's what has knocked my digestion out--being so infernally jealous of her.--I can't eat a mouthful of this stuff, you know," he added suddenly, pushing back his plate with a clouded countenance; and Lily, unfailingly adaptable, accorded her radiant attention to his prolonged denunciation of other people's cooks, with a supplementary tirade on the toxic qualities of melted butter. It was not often that he found so ready an ear; and, being a man as well as a dyspeptic, it may be that as he poured his grievances into it he was not insensible to its rosy symmetry. At any rate he engaged Lily so long that the sweets were being handed when she caught a phrase on her other side, where Miss Corby, the comic woman of the company, was bantering Jack Stepney on his approaching engagement. Miss Corby's role was jocularity: she always entered the conversation with a handspring. "And of course you'll have Sim Rosedale as best man!" Lily heard her fling out as the climax of her prognostications; and Stepney responded, as if struck: "Jove, that's an idea. What a thumping present I'd get out of him!" SIM ROSEDALE! The name, made more odious by its diminutive, obtruded itself on Lily's thoughts like a leer. It stood for one of the many hated possibilities hovering on the edge of life. If she did not marry Percy Gryce, the day might come when she would have to be civil to such men as Rosedale. IF SHE DID NOT MARRY HIM? But she meant to marry him--she was sure of him and sure of herself. She drew back with a shiver from the pleasant paths in which her thoughts had been straying, and set her feet once more in the middle of the long white road.... When she went upstairs that night she found that the late post had brought her a fresh batch of bills. Mrs. Peniston, who was a conscientious woman, had forwarded them all to Bellomont. Miss Bart, accordingly, rose the next morning with the most earnest conviction that it was her duty to go to church. She tore herself betimes from the lingering enjoyment of her breakfast-tray, rang to have her grey gown laid out, and despatched her maid to borrow a prayer-book from Mrs. Trenor. But her course was too purely reasonable not to contain the germs of rebellion. No sooner were her preparations made than they roused a smothered sense of resistance. A small spark was enough to kindle Lily's imagination, and the sight of the grey dress and the borrowed prayer-book flashed a long light down the years. She would have to go to church with Percy Gryce every Sunday. They would have a front pew in the most expensive church in New York, and his name would figure handsomely in the list of parish charities. In a few years, when he grew stouter, he would be made a warden. Once in the winter the rector would come to dine, and her husband would beg her to go over the list and see that no DIVORCEES were included, except those who had showed signs of penitence by being re-married to the very wealthy. There was nothing especially arduous in this round of religious obligations; but it stood for a fraction of that great bulk of boredom which loomed across her path. And who could consent to be bored on such a morning? Lily had slept well, and her bath had filled her with a pleasant glow, which was becomingly reflected in the clear curve of her cheek. No lines were visible this morning, or else the glass was at a happier angle. And the day was the accomplice of her mood: it was a day for impulse and truancy. The light air seemed full of powdered gold; below the dewy bloom of the lawns the woodlands blushed and smouldered, and the hills across the river swam in molten blue. Every drop of blood in Lily's veins invited her to happiness. The sound of wheels roused her from these musings, and leaning behind her shutters she saw the omnibus take up its freight. She was too late, then--but the fact did not alarm her. A glimpse of Mr. Gryce's crestfallen face even suggested that she had done wisely in absenting herself, since the disappointment he so candidly betrayed would surely whet his appetite for the afternoon walk. That walk she did not mean to miss; one glance at the bills on her writing-table was enough to recall its necessity. But meanwhile she had the morning to herself, and could muse pleasantly on the disposal of its hours. She was familiar enough with the habits of Bellomont to know that she was likely to have a free field till luncheon. She had seen the Wetheralls, the Trenor girls and Lady Cressida packed safely into the omnibus; Judy Trenor was sure to be having her hair shampooed; Carry Fisher had doubtless carried off her host for a drive; Ned Silverton was probably smoking the cigarette of young despair in his bedroom; and Kate Corby was certain to be playing tennis with Jack Stepney and Miss Van Osburgh. Of the ladies, this left only Mrs. Dorset unaccounted for, and Mrs. Dorset never came down till luncheon: her doctors, she averred, had forbidden her to expose herself to the crude air of the morning. To the remaining members of the party Lily gave no special thought; wherever they were, they were not likely to interfere with her plans. These, for the moment, took the shape of assuming a dress somewhat more rustic and summerlike in style than the garment she had first selected, and rustling downstairs, sunshade in hand, with the disengaged air of a lady in quest of exercise. The great hall was empty but for the knot of dogs by the fire, who, taking in at a glance the outdoor aspect of Miss Bart, were upon her at once with lavish offers of companionship. She put aside the ramming paws which conveyed these offers, and assuring the joyous volunteers that she might presently have a use for their company, sauntered on through the empty drawing-room to the library at the end of the house. The library was almost the only surviving portion of the old manor-house of Bellomont: a long spacious room, revealing the traditions of the mother-country in its classically-cased doors, the Dutch tiles of the chimney, and the elaborate hob-grate with its shining brass urns. A few family portraits of lantern-jawed gentlemen in tie-wigs, and ladies with large head-dresses and small bodies, hung between the shelves lined with pleasantly-shabby books: books mostly contemporaneous with the ancestors in question, and to which the subsequent Trenors had made no perceptible additions. The library at Bellomont was in fact never used for reading, though it had a certain popularity as a smoking-room or a quiet retreat for flirtation. It had occurred to Lily, however, that it might on this occasion have been resorted to by the only member of the party in the least likely to put it to its original use. She advanced noiselessly over the dense old rug scattered with easy-chairs, and before she reached the middle of the room she saw that she had not been mistaken. Lawrence Selden was in fact seated at its farther end; but though a book lay on his knee, his attention was not engaged with it, but directed to a lady whose lace-clad figure, as she leaned back in an adjoining chair, detached itself with exaggerated slimness against the dusky leather upholstery. Lily paused as she caught sight of the group; for a moment she seemed about to withdraw, but thinking better of this, she announced her approach by a slight shake of her skirts which made the couple raise their heads, Mrs. Dorset with a look of frank displeasure, and Selden with his usual quiet smile. The sight of his composure had a disturbing effect on Lily; but to be disturbed was in her case to make a more brilliant effort at self-possession. "Dear me, am I late?" she asked, putting a hand in his as he advanced to greet her. "Late for what?" enquired Mrs. Dorset tartly. "Not for luncheon, certainly--but perhaps you had an earlier engagement?" "Yes, I had," said Lily confidingly. "Really? Perhaps I am in the way, then? But Mr. Selden is entirely at your disposal." Mrs. Dorset was pale with temper, and her antagonist felt a certain pleasure in prolonging her distress. "Oh, dear, no--do stay," she said good-humouredly. "I don't in the least want to drive you away." "You're awfully good, dear, but I never interfere with Mr. Selden's engagements." The remark was uttered with a little air of proprietorship not lost on its object, who concealed a faint blush of annoyance by stooping to pick up the book he had dropped at Lily's approach. The latter's eyes widened charmingly and she broke into a light laugh. "But I have no engagement with Mr. Selden! My engagement was to go to church; and I'm afraid the omnibus has started without me. HAS it started, do you know?" She turned to Selden, who replied that he had heard it drive away some time since. "Ah, then I shall have to walk; I promised Hilda and Muriel to go to church with them. It's too late to walk there, you say? Well, I shall have the credit of trying, at any rate--and the advantage of escaping part of the service. I'm not so sorry for myself, after all!" And with a bright nod to the couple on whom she had intruded, Miss Bart strolled through the glass doors and carried her rustling grace down the long perspective of the garden walk. She was taking her way churchward, but at no very quick pace; a fact not lost on one of her observers, who stood in the doorway looking after her with an air of puzzled amusement. The truth is that she was conscious of a somewhat keen shock of disappointment. All her plans for the day had been built on the assumption that it was to see her that Selden had come to Bellomont. She had expected, when she came downstairs, to find him on the watch for her; and she had found him, instead, in a situation which might well denote that he had been on the watch for another lady. Was it possible, after all, that he had come for Bertha Dorset? The latter had acted on the assumption to the extent of appearing at an hour when she never showed herself to ordinary mortals, and Lily, for the moment, saw no way of putting her in the wrong. It did not occur to her that Selden might have been actuated merely by the desire to spend a Sunday out of town: women never learn to dispense with the sentimental motive in their judgments of men. But Lily was not easily disconcerted; competition put her on her mettle, and she reflected that Selden's coming, if it did not declare him to be still in Mrs. Dorset's toils, showed him to be so completely free from them that he was not afraid of her proximity. These thoughts so engaged her that she fell into a gait hardly likely to carry her to church before the sermon, and at length, having passed from the gardens to the wood-path beyond, so far forgot her intention as to sink into a rustic seat at a bend of the walk. The spot was charming, and Lily was not insensible to the charm, or to the fact that her presence enhanced it; but she was not accustomed to taste the joys of solitude except in company, and the combination of a handsome girl and a romantic scene struck her as too good to be wasted. No one, however, appeared to profit by the opportunity; and after a half hour of fruitless waiting she rose and wandered on. She felt a stealing sense of fatigue as she walked; the sparkle had died out of her, and the taste of life was stale on her lips. She hardly knew what she had been seeking, or why the failure to find it had so blotted the light from her sky: she was only aware of a vague sense of failure, of an inner isolation deeper than the loneliness about her. Her footsteps flagged, and she stood gazing listlessly ahead, digging the ferny edge of the path with the tip of her sunshade. As she did so a step sounded behind her, and she saw Selden at her side. "How fast you walk!" he remarked. "I thought I should never catch up with you." She answered gaily: "You must be quite breathless! I've been sitting under that tree for an hour." "Waiting for me, I hope?" he rejoined; and she said with a vague laugh: "Well--waiting to see if you would come." "I seize the distinction, but I don't mind it, since doing the one involved doing the other. But weren't you sure that I should come?" "If I waited long enough--but you see I had only a limited time to give to the experiment." "Why limited? Limited by luncheon?" "No; by my other engagement." "Your engagement to go to church with Muriel and Hilda?" "No; but to come home from church with another person." "Ah, I see; I might have known you were fully provided with alternatives. And is the other person coming home this way?" Lily laughed again. "That's just what I don't know; and to find out, it is my business to get to church before the service is over." "Exactly; and it is my business to prevent your doing so; in which case the other person, piqued by your absence, will form the desperate resolve of driving back in the omnibus." Lily received this with fresh appreciation; his nonsense was like the bubbling of her inner mood. "Is that what you would do in such an emergency?" she enquired. Selden looked at her with solemnity. "I am here to prove to you," he cried, "what I am capable of doing in an emergency!" "Walking a mile in an hour--you must own that the omnibus would be quicker!" "Ah--but will he find you in the end? That's the only test of success." They looked at each other with the same luxury of enjoyment that they had felt in exchanging absurdities over his tea-table; but suddenly Lily's face changed, and she said: "Well, if it is, he has succeeded." Selden, following her glance, perceived a party of people advancing toward them from the farther bend of the path. Lady Cressida had evidently insisted on walking home, and the rest of the church-goers had thought it their duty to accompany her. Lily's companion looked rapidly from one to the other of the two men of the party; Wetherall walking respectfully at Lady Cressida's side with his little sidelong look of nervous attention, and Percy Gryce bringing up the rear with Mrs. Wetherall and the Trenors. "Ah--now I see why you were getting up your Americana!" Selden exclaimed with a note of the freest admiration but the blush with which the sally was received checked whatever amplifications he had meant to give it. That Lily Bart should object to being bantered about her suitors, or even about her means of attracting them, was so new to Selden that he had a momentary flash of surprise, which lit up a number of possibilities; but she rose gallantly to the defence of her confusion, by saying, as its object approached: "That was why I was waiting for you--to thank you for having given me so many points!" "Ah, you can hardly do justice to the subject in such a short time," said Selden, as the Trenor girls caught sight of Miss Bart; and while she signalled a response to their boisterous greeting, he added quickly: "Won't you devote your afternoon to it? You know I must be off tomorrow morning. We'll take a walk, and you can thank me at your leisure."

Summary: Lily attempts to further her designs on Gryce by accompanying the Trenors' daughters to church. She believes that Gryce will see how beautiful she looks while peering through long eyelashes over a hymnal and wearing a modest gray dress, and will fall hopelessly in love with her. In an act of rebellion intended to increase Gryce's longing for her, however, Lily purposely misses the omnibus that takes the group of churchgoers to Sunday services. Instead, she interrupts a private conversation between Selden and Bertha, much to the delight of the former and the consternation of the latter. Lily then sets out on foot for the church, hoping to catch Gryce returning from services. She is met by Selden, who surmises that Lily has designs on Gryce. Gryce does indeed return from church on foot, but as part of a group led by Lady Cressida that also includes the Trenors' daughters. Selden, recognizing that their earlier conversation about Americana was due to Lily's interest in snaring Gryce, offers to further his tutelage at length that afternoon.

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Summarize: <CHAPTER> 6--Thomasin Argues with Her Cousin, and He Writes a Letter Yeobright was at this time at Blooms-End, hoping that Eustacia would return to him. The removal of furniture had been accomplished only that day, though Clym had lived in the old house for more than a week. He had spent the time in working about the premises, sweeping leaves from the garden paths, cutting dead stalks from the flower beds, and nailing up creepers which had been displaced by the autumn winds. He took no particular pleasure in these deeds, but they formed a screen between himself and despair. Moreover, it had become a religion with him to preserve in good condition all that had lapsed from his mother's hands to his own. During these operations he was constantly on the watch for Eustacia. That there should be no mistake about her knowing where to find him he had ordered a notice board to be affixed to the garden gate at Alderworth, signifying in white letters whither he had removed. When a leaf floated to the earth he turned his head, thinking it might be her foot-fall. A bird searching for worms in the mould of the flower-beds sounded like her hand on the latch of the gate; and at dusk, when soft, strange ventriloquisms came from holes in the ground, hollow stalks, curled dead leaves, and other crannies wherein breezes, worms, and insects can work their will, he fancied that they were Eustacia, standing without and breathing wishes of reconciliation. Up to this hour he had persevered in his resolve not to invite her back. At the same time the severity with which he had treated her lulled the sharpness of his regret for his mother, and awoke some of his old solicitude for his mother's supplanter. Harsh feelings produce harsh usage, and this by reaction quenches the sentiments that gave it birth. The more he reflected the more he softened. But to look upon his wife as innocence in distress was impossible, though he could ask himself whether he had given her quite time enough--if he had not come a little too suddenly upon her on that sombre morning. Now that the first flush of his anger had paled he was disinclined to ascribe to her more than an indiscreet friendship with Wildeve, for there had not appeared in her manner the signs of dishonour. And this once admitted, an absolutely dark interpretation of her act towards his mother was no longer forced upon him. On the evening of the fifth November his thoughts of Eustacia were intense. Echoes from those past times when they had exchanged tender words all the day long came like the diffused murmur of a seashore left miles behind. "Surely," he said, "she might have brought herself to communicate with me before now, and confess honestly what Wildeve was to her." Instead of remaining at home that night he determined to go and see Thomasin and her husband. If he found opportunity he would allude to the cause of the separation between Eustacia and himself, keeping silence, however, on the fact that there was a third person in his house when his mother was turned away. If it proved that Wildeve was innocently there he would doubtless openly mention it. If he were there with unjust intentions Wildeve, being a man of quick feeling, might possibly say something to reveal the extent to which Eustacia was compromised. But on reaching his cousin's house he found that only Thomasin was at home, Wildeve being at that time on his way towards the bonfire innocently lit by Charley at Mistover. Thomasin then, as always, was glad to see Clym, and took him to inspect the sleeping baby, carefully screening the candlelight from the infant's eyes with her hand. "Tamsin, have you heard that Eustacia is not with me now?" he said when they had sat down again. "No," said Thomasin, alarmed. "And not that I have left Alderworth?" "No. I never hear tidings from Alderworth unless you bring them. What is the matter?" Clym in a disturbed voice related to her his visit to Susan Nunsuch's boy, the revelation he had made, and what had resulted from his charging Eustacia with having wilfully and heartlessly done the deed. He suppressed all mention of Wildeve's presence with her. "All this, and I not knowing it!" murmured Thomasin in an awestruck tone, "Terrible! What could have made her--O, Eustacia! And when you found it out you went in hot haste to her? Were you too cruel?--or is she really so wicked as she seems?" "Can a man be too cruel to his mother's enemy?" "I can fancy so." "Very well, then--I'll admit that he can. But now what is to be done?" "Make it up again--if a quarrel so deadly can ever be made up. I almost wish you had not told me. But do try to be reconciled. There are ways, after all, if you both wish to." "I don't know that we do both wish to make it up," said Clym. "If she had wished it, would she not have sent to me by this time?" "You seem to wish to, and yet you have not sent to her." "True; but I have been tossed to and fro in doubt if I ought, after such strong provocation. To see me now, Thomasin, gives you no idea of what I have been; of what depths I have descended to in these few last days. O, it was a bitter shame to shut out my mother like that! Can I ever forget it, or even agree to see her again?" "She might not have known that anything serious would come of it, and perhaps she did not mean to keep Aunt out altogether." "She says herself that she did not. But the fact remains that keep her out she did." "Believe her sorry, and send for her." "How if she will not come?" "It will prove her guilty, by showing that it is her habit to nourish enmity. But I do not think that for a moment." "I will do this. I will wait for a day or two longer--not longer than two days certainly; and if she does not send to me in that time I will indeed send to her. I thought to have seen Wildeve here tonight. Is he from home?" Thomasin blushed a little. "No," she said. "He is merely gone out for a walk." "Why didn't he take you with him? The evening is fine. You want fresh air as well as he." "Oh, I don't care for going anywhere; besides, there is baby." "Yes, yes. Well, I have been thinking whether I should not consult your husband about this as well as you," said Clym steadily. "I fancy I would not," she quickly answered. "It can do no good." Her cousin looked her in the face. No doubt Thomasin was ignorant that her husband had any share in the events of that tragic afternoon; but her countenance seemed to signify that she concealed some suspicion or thought of the reputed tender relations between Wildeve and Eustacia in days gone by. Clym, however, could make nothing of it, and he rose to depart, more in doubt than when he came. "You will write to her in a day or two?" said the young woman earnestly. "I do so hope the wretched separation may come to an end." "I will," said Clym; "I don't rejoice in my present state at all." And he left her and climbed over the hill to Blooms-End. Before going to bed he sat down and wrote the following letter:-- MY DEAR EUSTACIA,--I must obey my heart without consulting my reason too closely. Will you come back to me? Do so, and the past shall never be mentioned. I was too severe; but O, Eustacia, the provocation! You don't know, you never will know, what those words of anger cost me which you drew down upon yourself. All that an honest man can promise you I promise now, which is that from me you shall never suffer anything on this score again. After all the vows we have made, Eustacia, I think we had better pass the remainder of our lives in trying to keep them. Come to me, then, even if you reproach me. I have thought of your sufferings that morning on which I parted from you; I know they were genuine, and they are as much as you ought to bear. Our love must still continue. Such hearts as ours would never have been given us but to be concerned with each other. I could not ask you back at first, Eustacia, for I was unable to persuade myself that he who was with you was not there as a lover. But if you will come and explain distracting appearances I do not question that you can show your honesty to me. Why have you not come before? Do you think I will not listen to you? Surely not, when you remember the kisses and vows we exchanged under the summer moon. Return then, and you shall be warmly welcomed. I can no longer think of you to your prejudice--I am but too much absorbed in justifying you.--Your husband as ever, CLYM. "There," he said, as he laid it in his desk, "that's a good thing done. If she does not come before tomorrow night I will send it to her." Meanwhile, at the house he had just left Thomasin sat sighing uneasily. Fidelity to her husband had that evening induced her to conceal all suspicion that Wildeve's interest in Eustacia had not ended with his marriage. But she knew nothing positive; and though Clym was her well-beloved cousin there was one nearer to her still. When, a little later, Wildeve returned from his walk to Mistover, Thomasin said, "Damon, where have you been? I was getting quite frightened, and thought you had fallen into the river. I dislike being in the house by myself." "Frightened?" he said, touching her cheek as if she were some domestic animal. "Why, I thought nothing could frighten you. It is that you are getting proud, I am sure, and don't like living here since we have risen above our business. Well, it is a tedious matter, this getting a new house; but I couldn't have set about it sooner, unless our ten thousand pounds had been a hundred thousand, when we could have afforded to despise caution." "No--I don't mind waiting--I would rather stay here twelve months longer than run any risk with baby. But I don't like your vanishing so in the evenings. There's something on your mind--I know there is, Damon. You go about so gloomily, and look at the heath as if it were somebody's gaol instead of a nice wild place to walk in." He looked towards her with pitying surprise. "What, do you like Egdon Heath?" he said. "I like what I was born near to; I admire its grim old face." "Pooh, my dear. You don't know what you like." "I am sure I do. There's only one thing unpleasant about Egdon." "What's that?" "You never take me with you when you walk there. Why do you wander so much in it yourself if you so dislike it?" The inquiry, though a simple one, was plainly disconcerting, and he sat down before replying. "I don't think you often see me there. Give an instance." "I will," she answered triumphantly. "When you went out this evening I thought that as baby was asleep I would see where you were going to so mysteriously without telling me. So I ran out and followed behind you. You stopped at the place where the road forks, looked round at the bonfires, and then said, 'Damn it, I'll go!' And you went quickly up the left-hand road. Then I stood and watched you." Wildeve frowned, afterwards saying, with a forced smile, "Well, what wonderful discovery did you make?" "There--now you are angry, and we won't talk of this any more." She went across to him, sat on a footstool, and looked up in his face. "Nonsense!" he said, "that's how you always back out. We will go on with it now we have begun. What did you next see? I particularly want to know." "Don't be like that, Damon!" she murmured. "I didn't see anything. You vanished out of sight, and then I looked round at the bonfires and came in." "Perhaps this is not the only time you have dogged my steps. Are you trying to find out something bad about me?" "Not at all! I have never done such a thing before, and I shouldn't have done it now if words had not sometimes been dropped about you." "What DO you mean?" he impatiently asked. "They say--they say you used to go to Alderworth in the evenings, and it puts into my mind what I have heard about--" Wildeve turned angrily and stood up in front of her. "Now," he said, flourishing his hand in the air, "just out with it, madam! I demand to know what remarks you have heard." "Well, I heard that you used to be very fond of Eustacia--nothing more than that, though dropped in a bit-by-bit way. You ought not to be angry!" He observed that her eyes were brimming with tears. "Well," he said, "there is nothing new in that, and of course I don't mean to be rough towards you, so you need not cry. Now, don't let us speak of the subject any more." And no more was said, Thomasin being glad enough of a reason for not mentioning Clym's visit to her that evening, and his story. </CHAPTER>

Summary: Clym has moved from Alderworth into his mother's house in Bloomsend. He waits there, hoping to hear from Eustacia. On the fifth of November, Clym goes to Thomasin's house. She is alone, for her husband has gone for a "walk." Actually he has gone to meet Eustacia. Clym informs Thomasin about the rift between him and his wife and she advises him to make up as soon as possible. Listening to his cousin's advice, he returns home and writes Eustacia a letter asking her to come back to him. Returning from his visit with Eustacia, Wildeve is closely questioned by his wife. He is then surprised when Thomasin reveals that she has followed him and has seen which direction he has taken.

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Summarize: FIELD OF THE INVENTION [0001] This application is a continuation in part of the PCT application PCT/CN02/00665 filed on Sep. 18th, 2002. The present invention relates generally to an automatic cooking method and system, and more particularly to a method and system of recording and simulating a cooking process for a dish by a famous chef. BACKGROUND OF THE INVENTION [0002] Traditionally, cooking is done by chefs one dish at a time. Exceptional dishes sometimes can only be produced by a limited number of famous chefs. Therefore, certain dishes cannot be provided unless a particular chef is invited to a restaurant. For example, Chinese cuisine has many types of cooking styles. For each style there are only limited numbers of chefs who are capable of producing certain exceptional dishes. In the past, the styles and crafts were passed on to the next generation individually, but this method has obvious limitations. It cannot serve a large number of patrons at the same time. The public is not well served by this method. [0003] The objective of the present invention is to provide an automatic cooking method and system that will widely spread the unique techniques of the famous chefs. SUMMARY OF THE INVENTION [0004] In order to achieve the objective of the present invention, the present invention provides an automatic cooking method including the following steps: [0005] 1). Measuring and recording kinds and amounts of main ingredients and seasoning materials prepared by a chef; [0006] 2). Measuring and recording movement tracks of cooking containers and shovels; [0007] 3). Measuring and recording amounts of main ingredients and seasoning materials added by the chef and timing when the main ingredients and seasoning materials are added; [0008] 4). Measuring and recording strength of fire adjusted by the chef and timing when strength of fire is adjusted; [0009] 5). Processing the data recorded by step 1 through step 4 by a computer and providing an operation program of the automatic cooking system that reflects the chef&#39;s cooking process; [0010] 6). Installing the operation program into the automatic cooking system and central control device, enabling the central control device to operate according to the operation program and to complete the cooking process. [0011] Additionally, in order to achieve the objective of the present invention, the automatic cooking system provided by the present invention comprises recording system that records the chef&#39;s cooking process, and mechanical operation system that operates according to recorded cooking process, wherein said recording system comprises: [0012] 1). Equipment for measuring and recording kinds and amounts of main ingredients and seasoning materials prepared by a chef; [0013] 2). Equipment for measuring and recording amounts of main ingredients and seasoning materials added by the chef and timing that main ingredients and seasoning materials are added; [0014] 3). Equipment for measuring and recording movement tracks of cooking containers and shovels; [0015] 4). Equipment for recording and measuring strength of fire adjusted by the chef and timing when main ingredients and seasoning materials are added; [0016] 5). Equipment for processing recorded data, and for providing operation program of the automatic cooking system that reflects the chef&#39;s cooking process. [0017] The mechanical operation system includes: [0018] 1). A controller installed with the operation program that reflects the chef&#39;s cooking process; [0019] 2). Manipulators that is adapted to be connected with the cooking containers and shovels, and complete the cooking tasks according to signals received from the operation program of the controller that reflect the chef&#39;s cooking process; [0020] 3). Fire controlling device that is connected with the stove, and control the strength of fire according to signals received from the operation program of the controller that reflect strength of stove controlled by the chef; [0021] 4). Main ingredient and seasoning material supply devices that is connected with the main ingredient and seasoning material supply equipment, and control the main ingredient and seasoning material supply equipment according to signals received from the operation program of the controller that reflect the kinds and amounts of the main ingredients and seasoning materials added by the chef and timing when the main ingredients and seasoning materials are added. [0022] The present invention records the cooking process of the chef with recording system and provides operation program according to the cooking process, then it operates the mechanical operation system to complete cooking tasks. Thus, delicious dishes by famous chefs will be available to any restaurant or household who purchase the mechanical operation system and operation program. This will not only widely spread the technique for making those exceptional dishes, it will also save manpower. BRIEF DESCRIPTION OF THE DRAWINGS [0023] [0023]FIG. 1 illustrates the recording system for recording the cooking process of a chef; [0024] [0024]FIG. 2 illustrates the mechanical operating system of the automatic cooking system; [0025] [0025]FIG. 3 illustrates the base coordinate system, camera lens coordinate system and cooking container and shovel coordinate systems; [0026] [0026]FIG. 4 illustrates the positions of the specific points in camera lens coordinate system; [0027] [0027]FIG. 5 illustrates the positions of the specific points at the camera receiving membrane; [0028] [0028]FIG. 6 is the calculation of the position and posture of a cooking container based on the coordinates of the specified points at base coordinate system; [0029] [0029]FIG. 7 is the block diagram of calculation of the movement track of the manipulators; [0030] [0030]FIG. 8 illustrates the dynamic model of the manipulators; [0031] [0031]FIG. 9 illustrates the dynamic model of allover components of the mechanical operating system of the automatic cooking system; [0032] [0032]FIG. 10 is the electrical circuit drawing of the recording system that records the demo cooking process of the chef; [0033] [0033]FIG. 11 is the electrical circuit drawing of mechanical operating system of the automatic cooking system; [0034] [0034]FIG. 12 is the manipulator control diagram. [0035] Illustrated below is the detailed description of the embodiment of present invention related to the Figures. DETAILED DESCRIPTION OF THE INVENTION [0036] [0036]FIG. 1 illustrates the cooking process of a chef. As illustrated in FIG. 1, multiple (three were shown) main ingredient containers 8 are used to contain the main ingredients of the dish. Two cameras 1 record the cooking process conducted by the chef, said cameras 1 receive images of the cooking process conducted by the chef and send the signals to a computer 3. Said computer 3 processes the images. The computer 3 measures and calculates the coordinate system positions of each specified points P of cooking container (such as wok) 10 and shovel 11 during the movement in the cooking process (see FIG. 3), and obtain the movement track of cooking container 10 and shovel 11. Multiple (four were shown) seasoning material containers 6 contain seasoning materials such as cooking oil, salt, sauces, and vinegar, etc. An electronic scale 7 is placed under each seasoning material container 6 respectively, the electronic scales 7 measure the amounts of seasoning materials added to the cooking container 10 and transform the data to electronic signals. In the mean time, said electronic scales 7 also record the time when the seasoning materials are added. These data are transformed into numerical signals through first interfaces (such as A/D board, I/O port) 9 A and sent to computer 3 (see FIG. 10). Therefore, the data for the amounts of seasoning materials added and the time when the seasoning materials are added will be stored in computer 3. The rotation sensor set in stove 4 will measure the rotated angle of stove rotating switch 5. Said rotation sensor will record the adjustment of the strength of stove by the chef, and simultaneously record the time when the adjustments are made. A temperature sensor (such as thermocouple thermograph) installed on the cooking container (such as wok or pot) can also measure the temperature of the cooking container to reflect the strength of the fire, the signal for the temperature will also be sent to computer 3. A position sensor (such as infrared transmitter, or touch on switch) 12 can also record the time when the main ingredients are put into the cooking container 10. [0037] [0037]FIG. 2 illustrates the mechanical operating system of automatic cooking system. As shown, the present invention uses one or more manipulators and affiliating devices to construct mechanical operating system. The automatic cooking system comprises multiple, such as three, seasoning material containers 17, the output of which are controlled by a computer program and program controlled funnels 18 placed underneath that can add the seasoning materials for the dish according to operation commends of the program. Each manipulator 21 of the mechanical operating system has at least one mechanical hand 16, and has at least six degrees of freedom (i.e. has at least six joints 20 ). Said mechanical hands 16 can grab and hold multiple, such as three, main ingredient containers 15 that contain main ingredients of the dish and pull out the main ingredients according to the operation commends of the program. The stove switch 19 can control the fire strength of the stove according to operation commends of the program. The central controller 14 comprises exterior storage interface 31 and displayer 32. The exterior memory (such as diskette, CD, etc.) containing operation program is installed into central controller 14 (or the operation program is sent to central controller 14 through network). Once the main ingredients and seasoning materials for the dish are prepared according to the requirement of the operation program, said mechanical operating system will automatically start cooking once the operation program starts. [0038] [0038]FIG. 3 illustrates the setup of base coordinate system Σ b, camera lens coordinate system (Σ cl and Σ cr ) and cookware coordinate system of the recording system. The base coordinate system Σ b of the recording system is correspondence to the base coordinate system Σ b of the mechanical operating system. Through this setup, the intended movement tracks of the cooking container 10 and shovel 11 can be obtained by the operation program. As shown in FIG. 3, two of the camera coordinate systems set at the centers of the lenses of the left and right cameras 1 respectively, and the positions of said two cameras and the positions of base coordinate system are directly related. Said camera coordinate systems are shown as Σ cl, Σ cr (also shown in FIG. 4). Two movement coordinate systems set at the tips of the handles of the cooking container 10 and shovel 11 are presented as Σ g, Σ q. The coordinate system positions of said cooking container 10 and shovel 11 directly relate to the coordinate positions of three specified points P (dots of same color). Calculations based on the images recorded by the cameras provide the relevant positions of the specified points to the camera coordinate systems, and the relevant positions of Σ g, Σ q to base coordinate system Σ b can also be calculated. The details of the calculation are illustrated below. [0039] As shown in FIG. 4, one specific point P is used as an example for the calculation, wherein camera 1 comprises image receiving membrane 33, camera lens 34. The axis center lines cr and cl of left and right cameras 1 are parallel with each other, and in line with said camera lens 34. The centers of lenses O cl and O cr are origins of the camera coordinate system, and the line connect them becomes Y axis of the camera (the Y axis of the left camera is y cl, the Y axis of right camera is y cr ). The axis center lines of two cameras are X axis, wherein the X axis of left and right cameras is x cl, x cr respectively. The plane formed by x cl and y cl of the left camera are the same with the plane formed by x cr and y cr. Therefore, the left and right cameras&#39; center lines are at the same plane. The z cl and z cl axis of the left and right cameras are perpendicular to this plane, which is called Z plane. The plane formed by y cl and z cl axis of the left camera must be the same as the plane formed by y cr and z cr axis of the right camera, that is the left and right camera are in line with each other. The plane formed by x cl and z cl axis of the left camera is parallel to the plane formed by x cr and z cr axis of the right camera, and distance between them is w, which is also the distance between the origins of the coordinate system O cl and O cr. The distances of point P in FIG. 4 to each plane at each moment are its positions at the left and right camera coordinate systems, shown as ( cl x p, cl y p, cl z p, cr x p, cr y p, cr z p ).The projection point from point P to plane Z is P z. Connecting PO cl, PO cr., and P z O cl and P z O cr, the angle between PO cl and P z O cl is called cl θ Py, the angle between PO cr and P z O cr is called cr θ Py, the angle between P z O cl and x cl is called cl θ Pz, the angle between P z O cr and x cr is called cr θ Pz, and the relationship between them can be calculated by the equation below, wherein cl, cr represent the coordinate systems of the left and right cameras: cr x p tan cr θ Pz = cr y p cl x p tan cl θ Pz = cl y p Because cl x p = cr x p, and cl y p + cr y p =w, cr x p tan cr θ Pz + cl x p tan cl θ Pz =w cr x p = cl x p =w (cot cr θ Pz +cot cl θ Pz ) cr y p = cr x p tan cr θ Pz, cl y p = cl x p tan cl θ Pz [0040] [0040] z p cr = z p cl = x p cl  1 sin  θ Pz cl  tan  θ Py cl [0041] [0041]FIG. 5 illustrates how to use the positions of sr P ( sr x p, sr y p ) (the measuring unit of which needs to be transformed from pixel into mm, the transformation ratios are different for different cameras) in the image received by image receiving membrane 33 of the right camera to obtain rotation angles ( cr θ Py, cr θ Pz ). sl, sr represent the image receiving membrane plane coordinate system of the left and right cameras, and l is the distance between image receiving membrane and the center of the lens. [0042] The equation for calculating the rotation angle is shown below: θ Pz cr = - tan - 1  x p sr l  θ Py cr = - tan - 1  y p sr  cos  θ Pz cr l [0043] [0043] cl θ Pz and cl θ Py can also be calculated from sl x p and sl y p of left camera using the same method. [0044] [0044]FIG. 6 illustrates how to use the positions of three specific points ( b g1, b g2, b g3 ) on the cooking container to calculate the related position ( b g ) and posture ( b g ) of the cooking container coordinate system Σ g to the base coordinate system Σ b. The position and posture Matrix of the cooking container ( b g ) can be calculated as shown below: b g1 =[ b x g1, b y g1, b z g1 ] b g2 =[ b x g2, b y g2, b z g2 ] b g3 =[ b x g3, b y g3, b z g3 ] [0045] [0045] T g b = [ R g b p g b 0 T 1 ] b g = g( b P g1, b P g2, b P g3 ) [0046] Wherein b is the base coordinate system. b x g1 represents the distance of point P 1 on the cooking container 10 at base coordinate system Σ b to plane y b -z b. Because camera coordinate system and base coordinate system has fixed relationship, b gn (n=1, 2, 3) may be obtained from ( cr x pg, cr y pg, cr z pg ) and ( cl x pg, cl y pg, cl z pg ) through coordinate transformation. g in the equation is linear algebra function. The function of g is obtained according to the setup of the cooking container coordinate system, and a person with ordinary skill in the art can obtain it according to a well known calculation method, which will be omitted here. The result of the above calculation will be shown at the block diagram of FIG. 7, wherein the calculation process in FIG. 7 is illustrated as follow: The coordinate positions of the three specified points ( sr 1, sr 2, sr 3 and sl 1, sl 2, sl 3 ) on the image receiving membrane plane coordinate systems (x sr −y sr and x sl −y sl ) are obtained using computer image processing, then the coordinate position of each specified point ( cr 1, cr 2, cr 3 and cl 1, cl 2, cl 3 ) in each camera lens coordinate system (x cr −y cr −z cr or x cl −y cl −z cl ) is calculated through coordinate transformation. Further, the coordinate position of each specified point related to base coordinate systems is calculated through coordinate transformation, and the relative position and posture of the cooking device coordinate system Σ g to the base coordinate system Σ b, refereed to as b T g (k), are obtained, where k is the sample cycle number. [0047] As shown in FIG. 7, “other action signals”, such as action signals indicating the action of taking the ingredients or adjusting the strength of the fire, are inserted. Because these actions are decided according to starting points and ending points, and the calculations are relatively simply, and a person of ordinary skill in the art will be able to calculate them easily, the calculation will be omitted here. Each manipulator has its own specific kinematics function, and r is the kinematics function of the right manipulator. r (k) is the rotation angle vector of each joint of the right manipulator (also called object track). The calculation process of the rotation angle vector of the each joint of the left manipulator is the same with the right manipulator, which will be omitted here. The calculation for shovel is the same as for the cooking container, which will also be omitted here. [0048] Because the image processing process for the cooking process recording system is only used to identify some specified marks, ordinary image identification method such as Hough Transform can be used to calculate the position of each mark, which will not be discussed in detail here. Besides, the method of recording the cooking process illustrated above can also be replaced by Direct Teaching method often used in ordinary manipulator (the chef can use his/her arms to direct manipulators in order to directly teach the manipulator the movement for the cooking process), or replaced by Magnetic Field sensor (magnetic sensors can be used to record the positions of the cooking container and shovel in order to record the cooking process). However, the method illustrated here will be able to guarantee accuracy. In practice, these methods can compliment each other. The calculation and manipulator operating program are performed by computer automatically. The image processing and camera recording process may be conducted simultaneously, or the image processing task can be conducted after the images have been recorded. [0049] [0049]FIG. 8 is the structure and kinematics model of the manipulators of the automatic cooking system. As shown in FIG. 8, the structures of these manipulators are similar to the structure of ordinary manipulators for industrial use. θ 0l,θ 2l... θ 5l and θ 0r, θ 1r... θ 5r represent the rotation angles of each of the six elbows of two manipulators. [0050] [0050]FIG. 9 is the kinematics model of all the components of the mechanical operation system of the automatic cooking system. The setups of the coordinate system are as follow: The setups of the coordinate systems of the cooking container and shovel (Σ g, Σ q ) and the base coordinate system Σ b are the same as those of the coordinate systems of the cameras recording chef&#39;s operation. The directions and positions of the coordinate systems of main ingredient containers 15 (Σ v1, Σ v2... Σ vn ) are set that when any of the mechanical hands 16 holds any of the main ingredient containers 15, the coordinate system of this mechanical hand is superimposed upon the coordinate system of the respective main ingredient container 15. The directions and positions of the coordinate systems of the seasoning material containers 17 (Σ f1, Σ f2... Σ fu ) are set that when the cooking container 10 receives the seasoning material from any of the seasoning material containers 17, the coordinate system of the cooking container 10 is superimposed upon the coordinate system of the respective seasoning material container 17. The stove fire switch will be controlled by a computer program controlled motor. [0051] [0051]FIG. 10 is the electrical circuit drawing of the recording system for recording a Chef&#39;s cooking process. As shown in FIGS. 1 and 10, cameras 1 are connected with image processing circuit boards 2. Said cameras 1 will send the images of the cooking process of the Chef through image processing circuit boards 2 to computer 3. The stove rotating switch 5 of the stove having a rotation sensor is connected with the first interface 9 A (such as A/D circuit board). Said stove rotating switch 5 records the signals of the rotating angle (the strength of the fire) chosen by the Chef, and sends the signals through the first interface 9 A to computer 3. The seasoning material containers 6 comprise electronic scales 7, and the changed amounts of the seasoning materials used by the Chef will be recorded by the electronic scales 7. These data will be sent through the first interface 9 A to computer 3. The main ingredient containers 8 comprise ingredient sensors 12 (such as diode infrared transmitters) that are connected with the second interface 9 B. Said ingredient sensors 12 will send signals representing the time when the main ingredients are put into the cooking container through the second interface 9 B to computer 3. A simple program modular can be made according to the above data. According to the result of the above calculation, computer 3 having a ready-made program in addition to program modular will provide operation program imitating chef&#39;s movement when cooking each dish. The program can be adjusted if necessary according to the requirement of each occasion. [0052] [0052]FIG. 11 is the electrical diagram of the mechanical operating system of the automatic cooking system. After the operation program of the automatic cooking system is inserted into computer 35 of the central controller, the system will be started. Computer 35 will send operation program&#39;s control signals for controlling the amounts of seasoning materials through pulse generating board 22 to stepping motor drives 23, and then the stepping motor drives 23 motivate the stepping motors of the program controlled funnel 18 of the seasoning material containers 17, and add the seasoning materials to the cooking container. Computer 35 will send the control signals of the operation program that control the strength of stove fire through pulse generating board 22 to respective stepping motor drive 23. The stepping motor drive 23 will control stepping motor of the program controlled stove rotating switch to control the strength of the fire. The operation program of the computer 35 will control mechanical hands 16 to grab main ingredient containers and pour out main ingredients. The control signals will be sent through pulse generating board 24 to stepping motor drives 25. The stepping motor drives 25 will control mechanical hands 16 to grab and hold main ingredient containers, cooking container, and shovel. The movements of each joint of the manipulators when accomplishing the cooking tasks are controlled by the computer. The computer sends control signals through a third interface 26 (such as A/D circuit board) to drivers 27. The drivers 27 will control the movement of the manipulators through the motors of each joint 20 to accomplish cooking tasks. The angle rotation signals of the manipulators&#39; motor angle rotation transmitters will be amplified through amplifier 29 and send to counter board 28. The feedback signal of the movement of the manipulators will be sent to computer 35, in order to calibrate the movements of the manipulators. The manipulators of this system will be able to imitate the cooking process according the operation program. [0053] [0053]FIG. 12 is the manipulator control diagram, wherein θ r (k) is the target movement track of the right manipulator calculated from operation recording system (the output of FIG. 7), same is for the left manipulator. Said block diagram is the PID control diagram for ordinary controlled manipulators. Once the target track is known, each manipulator can be controlled as shown in FIG. 12. θ ro (k) is the actual rotated angle of each joint of the manipulator, and θ′ ro (k) is the angle velocity of each joint of the manipulator. [0054] As illustrated above, the essence of the present invention is to record and process the images of the movement of the chef when cooking (or real time process). Through calculations of the coordinate positions of three specified points on the cooking container and shovel used by the chef, the movement tracks of the cooking container and shovel when used by the chef to cook certain cuisine can be obtained (although the drawings and detailed description only referred to one cooking container and one shovel, the same method can be used for more than one cooking container and shovel). Then, the cooking program can be produced according to the amounts of main ingredients and seasoning materials added by the chef and the time the main ingredients and seasoning materials are added when cooking, the strength of the stove fire and the movement track of the cooking container and shovel. Furthermore, the manipulators of the mechanical operating system of the present invention will imitate the chef&#39;s cooking movements according the signals of the above program and produce the cuisines similar to what are produced by the chef. In addition, the method and system of the present invention can also be used partially. For example, recording system or mechanical operation system may be used as an independent system. Or, the mechanical operation system may be used as a supplemental device supporting chef&#39;s operation.

Summary: The present invention provides an automatic cooking method and system, wherein the cooking process of a chef is recorded. Then, a program about the cooking process is obtained with information about amounts and kinds of main ingredients and seasoning materials used by the chef, timing of adding main ingredients and seasoning materials and movement tracks of the cooking container and shovel. Thereafter, manipulators of a mechanical operating system of the present invention imitate chef&#39;s cooking process according to commend signal from the program to produce a dish. The present invention uses recording devices to record chef&#39;s cooking process and provide a program, then respective mechanical operating system accomplishes cooking tasks imitating the chef, which provides restaurants and households with dishes by the chef when using the program and mechanical operating system. The present invention not only made exceptional dishes widely available, it can also serve a large number of patrons at the same time.

big_patent

en

Summarize: CROSS REFERENCE TO RELATED APPLICATIONS The present application is a continuation of U.S. Nonprovisional application Ser. No. 11/549,153, filed 13 Oct. 2006 and entitled “Portable Ride-On Bouncing and Spinning Toy,” the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION The present invention relates to a ride-on toy stylized as a friendly character. Such toys are also often styled in a saddle-type configuration including a saddle-type seat. The toy is typically connected to the supporting surface by a connector. The connector can include a motorized member that moves the seat automatically or a biasing member that manually reacts to the movement of the child. Whether the toy and connector are motorized or self-powered, children get excited about and spend endless hours enjoying such ride-on toys. Generally, the connector supports the seat, allowing the seat to move in various directions. Specifically, in addition to an up and down (vertical) riding (bouncing) motion, some connectors of ride-on toys enable rotation or spinning of the seat while the child is sitting on the seat. Although rotation of the seat is desirable after the child has been seated on the toy, the climbing onto or off of a rotating toy may be somewhat difficult. Parents generally encourage children to play independently as early as possible. For a small child, however, the rotation and bouncing of the seat on a conventional ride-on device can make an unsupervised mounting of such toys an unstable and even potentially dangerous undertaking. There is therefore a need to develop a ride-on toy which allows relative rotation between the seat and connector, but which prevents rotation of the seat when the child is mounting the toy and then again allows rotation of the seat after the child has safely mounted the toy. In this way, the child can safely mount the toy and then safely enjoy the freedom of seat rotation and bouncing. SUMMARY OF THE INVENTION Generally, the present specification discloses a children&#39;s ride-on activity toy device. The ride-on toy device includes a seat, a connector and a base. The seat is stylized as a friendly character and includes a saddle/seating area (e.g., a saddle formed on the character&#39;s back). The connector supports the seat above a base, the base contacting and stabilizing the device on a supporting surface in a manner that allows multiple degrees of freedom between the seat and the connector. Specifically, the present invention seat is stylized as an animal character (e.g., a horse, zebra, camel etc.). The back of the animal character may include a seating area stylized a saddle. A connector, in accordance with the present invention, may support the seat above a base (and thus also above the supporting surface) and may include a first connector portion and a second connector portion. The first connector portion being connected to the seat and the second connector portion being connected to the base. A connector in accordance with the present invention may be connected to the seat at a connection portion located on the bottom of the seat. The connector may be in the form of a compressible column and includes an upper column portion or first connector portion that moves telescopically relative to a lower column portion or second connector portion. The upper end of the first connector portion may be connected to the seat and the lower end of the second connector portion may be connected to the base. When a child sits on the seating area of the seat, the force of the child&#39;s weight is transmitted through the first connector portion to a biasing member to compress the biasing member and force the first connector portion toward the second connector portion, thus reducing the overall length of the connector. Furthermore, a child who sits on the seat with their legs touching the ground can adjust the force applied to the biasing member to initiate a bouncing (up and down in the vertical direction) movement with the seat. In order to provide a safe play experience, the present invention includes a safety mechanism that prevents the seat from rotating relative to the base when insufficient force is applied to the biasing member, but allows the seat portion to rotate relative to the base when sufficient compressive force (e.g., the weight of the child) is applied to the seat (and thus, the biasing member). The safety mechanism includes a first series of projections associated with the connector&#39;s first connector portion and a second series of projections that are associated with the connector&#39;s second connector portion. When insufficient compressive force is applied to the biasing member, the biasing member forces the first series of projections toward the second series of projections such that the first and second series of projections are in rotational alignment (i.e., they are interlocked). When the first and second series of projections are in rotational alignment, rotation of the seat, and thus, rotation of the first connector portion, causes the first series projections to engage with the second series of projections to prevent rotation of the seat about a vertical axis. However, when sufficient compressive force (e.g., weight of a child) is applied to the seat and thus to the biasing member, the first series of projections separates from the second series of projections (the first and second series of projections are moved out of rotational alignment). As a result, when a relative rotational force is applied between the seat and the base, the first series of projections rotates freely about a vertical axis relative the second series of projections. In other words, when the seat along with the first connector portion is sufficiently compressed relative the second connector portion, the seat is allowed to rotate freely about a vertical axis relative to the second connector portion and the base. In use, when a child attempts to mount the seat, because the seat is yet unloaded, the biasing member engages the safety mechanism to prevent the seat portion from rotating about a vertical axis relative to the base. However, when the child has mounted the seat, the weight of the child compresses the biasing member to disengage the safety mechanism allowing the seat portion to rotate about a vertical axis relative to the base (as well as bounce up and down on the vertical axis). Along with a seat, the ride-on toy of the present invention may also include a hand grip for stability. A hand grip also helps to allow a child to transfer motion energy to this self-energized toy. In addition, the ride-on toy of the present invention may include an electronic entertainment device with sensors that are added to detect operation (motion energy) of the ride-on toy and trigger sensory stimulating output (e.g., lights, sounds etc.) to increase the entertainment experience of the child. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A illustrates a perspective view of the ride-on activity device in accordance with the present invention. FIG. 1B illustrates a perspective view of the ride-on activity device of the FIG. 1A showing how an electronic entertainment device interconnects with the ride-on activity device. FIG. 1C illustrates an electronic schematic of the electronic entertainment device of FIG. 1B. FIG. 2 illustrates a child (in phantom lines) seated on the ride-on activity device of FIG. 1A with their feet on the base and clutching the handle members of the electronic entertainment device. FIG. 3 illustrates an exploded view of the ride-on activity device of the FIG. 1A showing the seat, the connector, and the base. FIG. 4 illustrates an enlarged perspective view of a connector in accordance with the present invention showing the first (upper) connector portion assembled onto the second (lower) connector portion. FIG. 5 illustrates an enlarged perspective view of the unloaded connector of FIG. 4 (with the cover member of the first connector portion removed to expose the internal workings of the connector). FIG. 6 illustrates an enlarged perspective view of the connector of FIG. 5 with the biasing member and the first connector portion in the loaded position. FIG. 7 illustrates a close-up side view of the connector of FIG. 5 with the side walls of the cover member and flange of the first connector portion removed to expose the connector&#39;s rotational safety feature. FIG. 8 illustrates a close-up perspective view of the loaded connector of FIG. 6 with the side walls of the cover member and flange of the first connector portion removed to expose the connector&#39;s rotational safety feature. FIG. 9 illustrates a child sitting on a ride-on device in accordance with the present invention moving the device in directions indicated by the directional arrows. Like reference numerals have been used to identify like elements throughout this disclosure. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, a ride-on activity device 100 is disclosed. FIG. 1A illustrates a perspective view of the ride-on activity device 100 in accordance with the present invention. The device 100 includes a base 120 for stabilizing the ride-on activity toy on a supporting surface (floor) 101, a seat 102 on which a child sits and a connector 110 for connecting and movably supporting the seat above the supporting surface 101. A child sitting on the seating area 105 of the seat 102 with their feet on the base 120 can bounce up and down (along a vertical axis) relative to supporting surface 101 and spin (about the vertical axis) relative to supporting surface 101. The seat 102 is stylized as a friendly character or other attractive object. Specifically, as illustrated, the toy 100 can be stylized as animal and the seating area 105 can be stylized as a saddle. The base 120 serves as a stabilizer for the device 100 on the supporting surface 101. Thus, the base 120 functions to prevent the device 100 from tipping over. The base 120 also serves as a foot rest for a child using the device 100. The base 120 could be eliminated if the connector 100 is otherwise secured to the supporting surface 101. FIG. 1B illustrates a perspective view of the ride-on activity device 100 of the FIG. 1A showing how an electronic entertainment device 130 interconnects with the ride-on activity device 100. The electronic entertainment device 130 connects to the head portion of the animal character and includes a handle portion 132, 134 and an electronics unit 131. The handle portion includes two handle members 132, 134 that connect to the head of the animal character. The handle members 132, 134 provide handles with which a child can stabilize themselves while the child is bouncing and spinning on the seating area 105. In mounting the electronic entertainment member 130 to the device 100, each handle member 132, 134 includes an end connector 140 A, 140 B which are respectively received in openings 145 A and 145 B ( 145 B not visible in FIG. 1B ) in the head of the animal character. A further support connection is made between the electronic entertainment device 130 and the device 100 as the post 150 of the electronic entertainment device 130 is received in the receptacle 155 in the head of the animal character. The handle members 132, 134 also support the electronics unit 131 therebetween. FIG. 1C illustrates an electronic schematic of the electronics unit 131 of the electronic entertainment device 130 of FIG. 1B. The general operation of the electronics unit 131 is managed by a microprocessor/controller 175 powered when ON/OFF switch 165 is turned to the ON position. The electronics unit 131 further includes a conventional motion switch 170 for triggering sensory output (e.g., sounds, lights, vibration etc.). Other types of switches may be employed that receive external input (e.g., sound, motion, pressed button, etc.) signals from the inputs and transmit those signals to the controller 175 for processing. Upon receipt of activation signals from the various inputs, the controller 175 then triggers a number of colorful LEDs 160 and a speaker 180 to generate sensory output (including music and/or sound effects). Furthermore, the electronic entertainment device 130 includes attractive entertainment characters that are mechanically connected to the electronic electronics unit 131 by resilient members 137 A, 137 B (e.g., springs etc.). In addition, the electronics unit 131 includes a mechanical roller 139 containing a switch for triggering electronic sensory stimulation (e.g., sounds and lights) to encourage a child to spin the roller 139. FIG. 2 illustrates a child 200 (in phantom lines) seated on the ride-on activity device 100 of FIG. 1A with their feet on the base 120 and clutching the handle members 132, 134 of the electronic entertainment device 130. In this position, the child 200 can bend their knees to bounce up and down (along a vertical axis) on the device 100. The connector 110 enables the seat 102 to bounce relative to the base 120 as further described below. FIG. 3 illustrates an exploded view of the ride-on activity device 100 of FIG. 1A showing the seat 102, the connector 110, and the base 120. Specifically, FIG. 3 shows how the connector 110 is positioned between the base 120 and the seat 102. A portion of the connector 110 fits into an opening 305 in the base 120 and is secured to the base 120. The pivotal connection between the connector 110 and the seat 102 will be described below. FIG. 4 illustrates an enlarged perspective view of a connector 110 in accordance with the present invention showing the first (upper) connector portion (generally designated as 420 ) assembled onto the second (lower) connector portion (generally designated as 430 ). First connector portion 420 is separable into a cover member 420 A and a lower ring 420 B. Cover member 420 A and lower ring 420 B are connectable by snapping cover member 420 A onto lower ring 420 B. Cover member 420 A includes projection 420 H, disposed on guide member 420 C. Lower ring 420 B includes a catch member 420 G having an opening for receiving projection 420 H when catch member 420 G is slid onto projection 420 H. Lower ring 420 B also includes a receiver 420 D that is engaged by guide member 420 C to ensure alignment between catch member 420 G and projection 420 H. Also, as the cover member 420 A is snapped onto lower ring 420 B, flange 420 I receives the lower edge (not shown) of the cover member 420 A. Furthermore, FIG. 4 shows reinforcement ribs 420 N and a bias guide 440 extending from an opening in cover member 420 A and also shows securing members 420 E, 420 F for securing the first connector portion 420 to the underside of the seat 102. As mentioned above, the connector 110 securely supports the seat 102 above the base 120 while allowing the seat 102 the freedom to bounce up and down (along a vertical axis) and to rotate relative to the base 120 (about a vertical axis). To this end, the first connector portion 420, moves telescopically up and down relative to second connector portion 430. In other words, as cover member 420 A is compressed downward relative to column post 430 B, cover member 420 A, guide ring 420 J, and the lower ring 420 B slide downward relative to column post 430 B. The relative telescopic movement between the first connector portion 420 and the second connector portion 430 is more clearly illustrated in the figures below. Furthermore, the rotational relationship between the first connector portion 420 and the second connector portion 430 will be discussed below in conjunction with the \rotation safety feature of the device 100. FIG. 5 illustrates an enlarged perspective view of the unloaded connector 110 of FIG. 4 with the cover member 420 A of the first connector portion 420 removed to expose the internal workings of the connector 110. The cover member 420 A is removed to reveal interior portions of the connector 110 including the biasing member 530 that provides the resilience for the vertical bouncing feature of the device 100. FIG. 5 also shows an upper stop 430 A of the column post 430 B that limits the relative compression between the first connector portion 420 and the second connector portion 430 by limiting the overall downward travel of the cover member 420 A. Biasing member opening 550 is disposed in the upper stop 430 A for receiving the biasing member 530. The biasing member 530 rests on a biasing surface (not shown) that is fixed relative to the second connector portion 430. When loaded, the biasing member 530 is compressed between the biasing surface (not shown) and the biasing guide 540. In other words, when the cover member 420 A pushes the bias guide 540 downward, bias guide 540 in turn compresses the biasing member 530 against the biasing surface (not shown). When the compressive force is released, the biasing member 530 exerts a reactive force back against the cover member 420 A to urge the seat 102 back upward. Therefore, the up and down bouncing motion is accomplished by cyclically loading the biasing member 530 and releasing the load as the child bounces up and down on the seat 102. As discussed above, in addition to the up and down bouncing motion, the connection between the connector 110 and the seat 102 allows the seat 102 to rotate about a vertical axis relative to the base 120. However, this rotational connection mechanism of the present invention includes a safety feature that prevents rotation in certain situations when rotation might be inconvenient or unsafe for a child. More specifically, the connector 110 includes a safety mechanism that enables a child to mount and dismount the seat 102 without fear that the rotating seat 102 will cause a potential instability. FIG. 6 illustrates an enlarged perspective view of the loaded connector 110 of FIG. 5 with the biasing member 530 and the first connector portion 420 in the loaded position. In FIG. 6, the bias guide 540 is shown in a lower, more compressed state, than that shown in FIG. 5 to illustrate its configuration under compression by a force F (caused by a child sitting on the seat 102 ). Correspondingly, the lower ring 420 B is shown in a lowered compressed state relative to that shown in FIG. 5. In the compressed configuration of FIG. 6, the inner ring surface 420 K of the lower ring 420 B and the lower stop 430 D can be seen. When the lower ring 420 B is shown in the compressed configuration illustrated in FIG. 6, the ring projections 420 L disposed on the inner ring surface 420 K of the lower ring 420 B are visible and the stop projections 430 M disposed on underside surface the lower stop 430 D are also visible. The rotation safety feature of the device 100 in accordance with the present invention will now be discussed. In a non-compressed state (as illustrated in FIG. 5 ), lower stop 430 D of the second connector portion 430 and ring surface 420 K of the first connector portion 420 remain close to each other such that stop projections 430 M engage with ring projections 420 L to prevent relative rotation between lower ring 420 B and lower stop 430 D. In other words, when an insufficient compressive force F (insufficient to compress the biasing member 530 ) is applied to the connector 110, ring projections 420 L rotatably engage stop projections 430 M to prevent the first connector portion 420 from rotating relative to the second connector portion 430. On the other hand, when the seat 102 is sufficiently loaded (sufficient to compress the biasing member 530 ), it in turn sufficiently loads the first connector portion 420 to cause clearance between ring projections 420 L and stop projections 430 M. Therefore, when sufficient compressive force is present such as illustrated in FIG. 6, lower ring 420 B, cover member 420 A, and thus the seat 102 is freely rotatable relative to second connector portion 430. FIG. 7 illustrates an enlarged cut away view of the connector 110 in an unloaded state as also illustrated in FIG. 5. In the FIG. 7 illustration, flange 420 I is partially removed to more clearly show ring projections 420 L and stop projections 430 M in a rotational alignment which prevents rotation of the first connector portion 420 relative to the second connector portion 430. FIG. 8 illustrates an enlarged perspective view of the connector 110 of the invention in a compressed configuration (as also illustrated in FIG. 6 ) that separates the ring projections 420 L and the stop projections 430 M out of rotational alignment with each other. Again, the separation of ring projections 420 L and stop projections 420 M enable relative rotation between first connector portion 420 and second connector portion 430. FIG. 9 illustrates a ride-on activity device 100 of FIG. 1A in accordance with an embodiment of the present invention showing arrows indicating the direction of a child bouncing and rotating on the device 100. In use, a child 200 approaches the ride-on activity device 100 and attempts to mount the device 100. During mounting, the child 200 benefits from being able to support himself/herself against the seat 102 that does not rotate when urged (e.g., when swinging a leg around the back of the seat 102 ). The device 100 allows the child 200 to mount the seat 102 with maximum support by preventing rotation during mounting. After, the child 200 has mounted the seat 102, the weight of the child will load the bias member 530 and allow the child 200 to bounce up and down on the seat as indicated in FIG. 9 by arrows 910 A, 910 B. In addition, the bias member 530 is chosen such that the weight of the child 200 sufficiently loads the seat 102 and thus the first connector portion 420 to force the connector 110 to the compressed configuration as discussed above (with respect to FIGS. 6 and 8 ). In this compressed configuration, the safety rotation mechanism disengages (causing ring projections 420 L to be separated from stop projections 430 M) to allow the seat 102 to freely rotate as indicated in FIG. 9 by arrow 920. The child 200 will then be able to freely bounce and rotate. When the child 200 is ready to dismount, the child 200 rises from the seat 102 to unload the connector 110. Unloading the device 100 causes the rotation safety mechanism to again engage (causing ring projections 420 L to be in contact with stop projections 430 M) to prevent rotation so that the child 200 can support themselves as they dismount safely. It will be appreciated that the embodiments described above and illustrated in drawings represent only a few of the many ways of implementing the present invention. For example, the relative movement between the seat 102 and the base 120 or supporting surface 101 is due to the connections between the seat 102 and connector&#39;s first connector portion 420, between the connector&#39;s first connector portion 420 and the connector&#39;s second connector portion 430, or the connector&#39;s second connector portion 430 and the base 120. In other words, relative movement between the seat 102 and base 120 can be due to any of the foregoing connections. Specifically, the rotation between the seat 102 and the base 120 may be due to the connection between the second connector portion 430 and the base 120 rather than between the first connector portion 420 and the seat 102. The connection between the seat 102 and the connector 110 can be located anywhere on the seat 102, but is shown on the bottom of the seat 102 in the drawings. The connection between the first connector portion 420 and the second connector portion 430 can be of any type, but is shown as a telescopic connection in the drawings. The connection between the second connector portion 430 and the base 120 can be any type of connection and can be similar to the connection between the first connector portion 420 and the seat 102. The connection between the seat 102 and first connector portion 420 may be in an upper portion of the seat 102 when the connector 110 is an overhead support (not shown in the drawings). Alternatively, the connection between the seat 102 and first connector portion 420 may be in a lower portion of the seat 102 when the connector 110 is a column-type support. The electronics assembly 130 in accordance with the present invention may include any combination of sensors, switches, lights, speakers, animated members, motors, and sensory output generating devices. The microprocessor unit 175 may produce any combination of audio and visual effects including, but not limited to, animation, lights, and sound (music, speech, and sound effects). The output pattern is not limited to that which is discussed herein and includes any pattern of music, lights, and/or sound effects. The electronics assembly 130 may also include additional switches or sensors to provide additional sensory output activation without departing from the scope of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention that come within the scope of the appended claims and their equivalents. For example, it is to be understood that terms such as “left”, “right” “top”, “bottom”, “front”, “rear”, “side”, “height”, “length”, “width”, “upper”, “lower”, “interior”, “exterior”, “inner”, “outer” and the like as may be used herein, merely describe points of reference and do not limit the present invention to any particular orientation or configuration.

Summary: A ride-on activity device is disclosed, wherein the device includes a seat, a base and a connector for movably connecting the seat relative to the base. The connection between the seat and the base allows multiple degrees of freedom such that the seat is capable of bouncing and rotating relative to the base. The connection between the seat and the connector includes a rotation safety mechanism that allows rotation at the connection when the seat is occupied by a user and prevents rotation at the connection when the seat is unoccupied. Furthermore, the connector includes a resilient member that allows the seat to bounce vertically relative to the base.

big_patent

en

Summarize: Ask Alex Song who the best player in the world is and his answer is emphatic: 'Lionel Messi. Oh my God.' On Wednesday night, the East End welcomes the Barcelona superstar with open arms as the Boleyn Ground plays host to a mouth-watering international between Argentina and Croatia. Of course, the likelihood is that Messi won't play the full 90 minutes. But who cares? Even a brief glimpse of the great man on the green grass of Upton Park will be an unforgettable treat for the 17,000 fans expected to be in attendance. Alex Song (left) says Lionel Messi is the best player in the world, and has the hunger to win more and more. Messi practices his shooting as Argentina train at West Ham's Rush Green Stadium. Last week, Messi created history by equalling Raul's all-time European goalscoring record with a brace at Ajax. By doing so, he instantly poured scorn over bizarre comments made by Argentina's national boss Tata Martino. Martino, who lasted just one year in charge at Barcelona, voiced concern that the 27-year-old'may never capture his best form again'. 'I don't know if we'll have the chance to see it happen,' Martino told Spanish sports daily, Marca. 'It's like when people compare Barcelona now to what they were like under Guardiola.' Admittedly, there have been doubts over Messi’s fitness. He’s played every possible minute for Barca in La Liga so far this season. Argentina's players warmed up for their behind-closed-doors game against West Ham's U21 side. And naturally, his form took a dip as he strived to get over the agony of Argentina's World Cup final defeat by Germany. But with his match-winning double in Amsterdam, he proved he is still the menacing Messi of old. He now sits proudly at the top of the queue to eclipse the Champions League feat of Real Madrid legend Raul - and one ahead of his arch-nemesis, Cristiano Ronaldo. The two will again go head-to-head at the beginning of December in their battle to land the coveted Ballon d'Or award. Victory for Messi, who was named the best player at the World Cup last summer despite Argentina's Rio heartache, would surely coronate him as the game's best ever. The Barcelona superstar listens to Argentina coach Gerardo Martino during the training session. But for Song, West Ham's on-loan midfielder from Barcelona, there is no doubt that title already belongs to his mercurial friend. 'Lionel Messi. Oh… I think this guy is fantastic. He’s the best player I have ever seen in my life,' says Song, who spent the past two seasons playing alongside the diminutive genius at the Nou Camp. 'Honestly, I’ve played with a lot of great players in my career. I was very lucky to play with players like Thierry Henry, Robert Pires, Dennis Bergkamp... it was a privilege for me. 'And then I went to Barcelona to play with Andres Iniesta, Xavi… and, of course, Leo Messi. It’s fantastic.' West Ham’s Under 21 side saw first-hand how brilliant the great man is when Argentina played a practice game against them on Tuesday evening at the club’s Rush Green Academy base. Song contests with Messi during Arsenal's 2-1 win over Barcelona in 2011 at the Emirates. So what separates Messi from the rest? His incredible hunger and desire, according to Song. 'For me, Leo is one of the players who is just unbelievable,’ he adds. 'I see young players like Neymar who is the future best player in the world like this guy. 'But the level Messi is at is just unbelievable. Messi is up here [Song stretches his arms far and wide]… and everyone else is below. Let’s just say, it’s a very big gap. A big gap. 'The thing I like about Leo is he is so hungry. He wants to win everything. He’s won everything but still wants to win more. 'He’s a great guy. He’s a winner. The way he manages himself to try and improve himself is a great example to other players and young boys.' Song has been in good form since joining West Ham in the summer from Barcelona on loan. Song's own future is uncertain. He could return to Barcelona next summer but he is happy at West Ham, where he has shone so far this season in Sam Allardyce's side. Perhaps Song could tempt Messi to prolong his short-stay in the East End and join him in January? 'Ha... why not! It would be good.' Indeed it would. Hopefully Wednesday won't mark the last time we ever see the masterful Messi grace English soil

Summary: Alex Song has no doubt who the best player in the world is right now. Former team-mate Lionel Messi is 'unbelievable', according to Song. Song is currently on loan at West Ham from Messi's side Barcelona. Messi is in the East End with Argentina, who are facing Croatia in a friendly at Upton Park on Wednesday night. A behind closed doors game with West Ham U21s featured the likes of Messi, Sergio Aguero, Javier Mascherano and Angel di Maria.

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Summarize: By. Meghan Keneally and Louise Boyle. PUBLISHED:. 07:27 EST, 29 August 2012. |. UPDATED:. 10:37 EST, 29 August 2012. A young woman from a wealthy New York family, who was found dead at the bottom of a Manhattan stairwell, had been due to enter rehab following the collapse of her marriage, sources claimed. Carlisle Brigham, 29, had suffered a. massive wound to her neck, a shattered chin and compound fractures to. her arms - possibly after tripping on her high heels following a night out to celebrate a friend's wedding. Her father James Brigham, the city's former budget chief, was reportedly so concerned about her well-being following the collapse of her year-long marriage that he had flown in from St Louis to take his distraught daughter to a rehab facility. He was believed to have spoken to her after landing at La Guardia airport at 9.30am on Monday and had planned to meet his daughter for lunch. Before their estrangement: Carlisle Brigham and Anthony Champalimaud pose together. Ms Brigham died after what appears to be a tragic accident on the stairwell of the Manhattan apartment she was staying at. Ms Brigham was discovered, dressed all in white, lying in a pool of blood just before 11 am on Monday at the bottom of a treacherously steep stairwell in the Lower East Side. The severity of her injuries made investigators initially believe she had been. murdered. However, the New York City Chief Medical. examiner ruled that Ms Brigham, whose father is now the chairman of a St Louis investment bank, died in a tragic 'freak accident' after suffering blunt trauma to her head and neck. Initial autopsy results were unclear whether alcohol may have played a role in her death. According to the New York Post, the night before her death, Ms Brigham had returned from a wedding in Washington D.C. and gone to Sixth Ward, a bar next door to the apartment where she had been staying. Attending the wedding was difficult for her, her father told the New York Times, coming a week after her first wedding anniversary. Ms Brigham married 34-year-old Anthony Lindley Champalimaud last year in Newport, Rhode Island but the couple were separated three months ago - although hoped to work out their relationship. Talented: Ms Brigham was a skilled model maker at the Natural History Museum in New York. Tragic: Carlisle Brigham (with unidentified friends) died after a fall down stairs in a New York apartment building shortly before she was to meet her father for lunch. She was living in the Lower. East Side with another man at the time of her death, believed. to be old college friend John Madtes, because she had lost the keys. to her own apartment. Though neighbors performed CPR on her. body, she was transported to Beth Israel Medical Center in an ambulance just before her father arrived at the apartment. The young woman was. pronounced dead a short time later. It is believed that a heavy bag and slippery stairs may have contributed to her fatal fall. A spokesman for the medical examiner. said that Ms Brigham did not bleed to death and her injuries were. consistent with a heavy blow to her head and neck. Distraught: The college friend that Ms Brigham was believed to have been staying with on the Lower East Side returns to the building where she died. Late night: Ms Brigham had been at this bar with friends the night before her death after attending a weekend wedding in Washington D.C. One resident of 191 Orchard St said that he attempted to perform CPR on Brigham but she was already dead when he found her. 'Her body was actually quite cold,' said 19-year-old Mizanur Rahman. 'The whole floor was just blood. 'I knew something had gone seriously wrong when I flipped her over. There was blood in nose, there was blood coming out of the mouth and a huge gash on her chin.' Another resident Lippy Khair, 27, said: 'She was wearing all white. White top, white pants. And she had really high heels.' Her father, James Brigham Jr., served. as the New York City budget officer in the late 1970s before moving to. St. Louis and turning to private equity at Warson Capital Partners in. St. Louis. Turmoil: Ms Brigham married her husband Anthony Champalimaud (right) last year but the couple was said to be estranged and he is thought to have been in London on business at the time of her death. Grief: James Brigham, a prominent investment banker, was on the way to meet his daughter for lunch after flying in from St Louis that morning. The former mayor of New York, Ed Koch, for whom James Brigham worked said that her grief-stricken father called him yesterday. 'He was sobbing,' said Koch to the New York Post. 'He told me that she had died and that it was apparently a fall...a freak accident. 'Wonderful young woman. I just was overwhelmed with grief and still am.' Ms Brigham's estranged husband Mr Champalimaud works for a hotel design firm run by his mother and. was in London on business when his wife was found. Mr Champalimaud. is the vice president of YLT Hotels and Properties and has a leading. role in the operations at the five-star Gainsborough Hotel in Bath,. England. Police in front of 191 Orchard Street, investigating the death of Carlisle Brigham, 29, the daughter of a leading investment banker, after she was found with her throat sliced open in the stairwell. The stairwell in Orchard Street in Manhattan's Lower East Side where Carlisle Brigham died. Scene: Ms Brigham was staying at 191 Orchard Street - slippery stairs and a heavy bag may have contributed to her fall. Distraught: The roommate who was living with Ms Brigham in the Lower East Side apartment building said that he got a phone call from the 29-year-old Monday morning and she'sounded distraught' Fatal fall: The 29-year-old's death was described as a 'freak accident' A. law enforcement official told the Journal that Ms Brigham was staying. with a male roommate in an apartment in 191 Orchard Street, and she was. found bleeding on the second floor landing of the building. The. roommate told police that the last time he heard from Ms Brigham was. shortly before her death, when she called him at work and'sounded. distraught'. The Local East Village blog of The New York Times reports that an unnamed resident of the building found her and spoke to police extensively after they arrived on the scene. 'She did not look like she was homeless or a hooker,' the resident, who is also a medic, told The Local East Village reporter. 'She had too many accouterments of the average American young person in her 20s: An iPhone and wallet full of plastic. 'She had compound fractures in her arm and she was cut.' Her death was confirmed by former Mayor Ed Koch, who worked with her father James Brigham Jr. from 1978 to 1981. 'They are wonderful people. She was absolutely extraordinarily beautiful. It just shook me as if I were kicked by a horse. It’s just awful,' Mr Koch said. Ms Brigham worked as an exhibit installation specialist at the American Museum of Natural History from 2007 to April of this year, and graduated from University of Mary Washington. Ms Brigham and Mr Champalimaud married in August 2011 in the chapel at St. George's School, an elite boarding high school in Newport, Rhode Island, from which the groom graduated in 1996

Summary: Carlisle Brigham, 29, 'tripped on her heels' in Lower East Side building. 'Large purse and slippery stairs' may have contributed to fatal fall. Father had arrived in city from St Louis that morning to help daughter who was 'distraught' over collapse of her marriage. Discovered around 11am on Monday dressed all in white and lying in a pool of blood on the floor. Police initially thought she had been murdered but autopsy revealed it was a 'freak accident' Was staying in building with male college friend at time of death.

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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to arts and crafts and health and beauty products, more particularly, to a device for storing swabs and a wetting fluid. 2. The Prior Art Cotton swabs are handy tools for applying fluids to small areas, for example, rubbing alcohol, nail polish remover, or paint. The swabs are stored in one container and the application fluid is stored in a separate covered container. The user removes a swab from its container, removes the cap from the fluid container, wets the swab by dipping it into the fluid, and then replaces the cap. In most cases, the fluid container is substantially deeper than the length of the swab, so the container must be tilted to wet the swab, requiring two hands. Having to manipulate the swab, cap, and fluid container can be awkward and prone to accidents, particularly when putting the cap back on the fluid container while holding a wet swab. There is also the convenience factor of having to deal with two containers which may or may not be stored together. Several solutions to the problem have been posed. For example, in U.S. Pat. No. 3,146,806, the fluid container is fitted with a stopper through which a swab can be pushed for wetting. Although this device substantially reduces the risk of accidental spillage, it does not alleviate the inconvenience of having two separate containers. A different solution is suggested by U.S. Pat. No. 4,747,719. In this patent, the fluid is stored in hollow in the handle of the swab. When wetting is desired, the user pushes the swab onto a pin to prick a hole in the hollow, allowing the fluid to escape and wet the swab. The shortcomings of this device are that it is a one-use-only device that is relatively complicated and expensive to produce. Also, both the swab and the swab container with the pin need to be disposed of after use, no part of the device is reusable. Finally, it is not particularly cost-effective for home use. A third solution is suggested by U.S. Pat. No. 5,378,226. In this patent, the swab is stored in a sealed bag with a smaller burst pouch that holds the fluid. The pouch is burst open while the bag is sealed, and the fluid from the pouch wets the swab within the bag. Then the bag is opened and the swab is removed. Like with the &#39;719 patent above, this is a one-use-only device that is relatively complicated and expensive to produce. The &#39;226 patent does disclose that there may be more than one swab in the bag. They are all wetted at the same time and must either be used or disposed of. Also like the &#39;719 patent, all components of the device need to be disposed of after use, no part of the device is reusable. Finally, this device is not particularly convenient or cost-effective for home use. Thus there continues to be a need for a device to safely and conveniently store swabs and wetting fluid. SUMMARY OF THE INVENTION An object of the present invention is to provide a swab dispenser with an integral fluid reservoir for safely and conveniently storing swabs and a wetting fluid. Another object is to provide a swab dispenser that is cost-effective for home, commercial, industrial use. A further object is to provide a swab dispenser that only requires one hand to remove and wet a swab. The present invention is a swab dispenser adapted for use with a swab that has a relatively straight, rigid handle with an absorbent material attached at an end thereof. The dispenser has a storage bin for swabs and a reservoir for a wetting fluid. The storage bin is an open top compartment that is optionally separated into compartments. The swabs stand generally vertically. The reservoir holds a fluid for wetting the swab, so the walls of the reservoir must be impervious to the fluid. The only opening to the reservoir is an aperture in the ceiling at the low point of a depression in the ceiling. The depression causes the fluid to flow down the depression surface to the aperture. The reservoir floor is concave, with the lowest point directly below the aperture so that the fluid flows to where it is most convenient for wetting the swab. The aperture is covered by a membrane that minimizes evaporation and spillage of the fluid. The membrane has at least two intersecting slits through which the swab is pushed, causing the membrane to deform inwardly and opening a hole for the swab. The membrane is composed of a material that returns the membrane to its original shape when the swab is removed. Preferably, the slits extend across the entire aperture and the membrane. If the slits are shorter, they may tear with repeated use, increasing the size of the opening and allowing more potential evaporation and spillage. Optionally, the swab dispenser of the present invention includes a clear cover for protecting the swabs from contamination, providing some protection against fluid spills, and further retarding evaporation. Other objects of the present invention will become apparent in light of the following drawings and detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS For a fuller understanding of the nature and object of the present invention, reference is made to the accompanying drawings, wherein: FIG. 1 is a top perspective view of the swab dispenser of the present invention; FIG. 2 is a cross-section of the swab dispenser of FIG. 1; and FIG. 3 is an enlarged perspective view of the membrane of FIG. 1 with a swab inserted. DETAILED DESCRIPTION The swab dispenser 10 of the present invention is shown in FIGS. 1 and 2. The basic dispenser 10 has a storage bin 12 for swabs and a reservoir 14 for a wetting fluid. The present invention is intended for use with swabs 20 that have a rigid handle 22 with an absorbent material 24, typically cotton, at one or both ends. The storage bin 12 is preferably an open top compartment where the swabs 20 stand generally vertically. Optionally, the bin 12 is separated into a set of smaller compartments 26 by walls 28. The compartments 26 provides several functions. If the compartments 26 are relatively small, the swabs remain relatively vertical when there are few swabs in the bin 12 to hold each other up. If there are few swabs in the bin 12, the swabs tend to fall over. The walls 28 provide a support to hold the swabs up. More than one compartment 26 also makes it easier to separate different types of swabs so that they do not mingle and makes them easier to locate and remove. The reservoir 14 holds a fluid 36 for wetting the swab 20 prior to use. The fluid 36 depends upon the application and may be, for example, rubbing alcohol, nail polish remover, antiseptic solutions, detergent solutions, plastic model cement, paint, or any kind of fluid that one may wish to apply with a swab. The reservoir 14 must be composed of a material that is impervious to the fluid 36. Alternatively, the inner walls of the reservoir 14 are coated with a material that renders the walls impervious to the fluid 36. The reservoir 14 is nearly fully enclosed, with a ceiling 30, side walls 32, and floor 34. The only opening to the reservoir 14 is an aperture 38 in the ceiling 30 through which the swab 20 is pushed for wetting. The aperture 38 is preferably round, but may have any shape. The aperture 38 is at the low point of a depression 44 in the ceiling 30. The depression 44 is sloped so that most fluid 36 will flow down the depression surface 46 to the aperture 38. The preferred range of angles of slope of the depression surface 46 depends upon the intended application of the present invention 10. The more viscous the fluid 36, the steeper the angle needs to be in order for the fluid 36 to flow down the slope. The aperture 38 is preferably covered by a membrane 40 that retards evaporation and minimizes spillage of the fluid 36. The membrane 40 has at least two intersecting slits 42 through which the swab 20 is pushed. When there are two slits 42, they are preferably at approximately a 90° to each other, forming an X, as in FIG. 1. As a swab 20 is pushed through the slits 42, the membrane 40 deforms inwardly, as in FIG. 3, opening a hole for the swab 42. Preferably, the opening is only large enough to allow the swab 20 to fit through easily. The smaller the opening, the less evaporation and spillage of the fluid 36 they can be. It is also preferred that the slits 42 extend across the entire aperture 38 and the membrane 40. If the aperture 38 is round, the length of the slits 42 is the same as the diameter of the aperture 38 and membrane 40. If the slits 42 are shorter than the membrane diameter, the slits 42 may tear with repeated use, increasing the size of the opening. And because the tearing will be irregular, the edges of the tear will not match, and the opening will no longer close. With this preferred configuration, the membrane 40 will not actually be a unitary component, but will consist of four 90° sections 48 of membrane 40, each attached to the edge of the aperture 38. The present invention does contemplate that the membrane 40 may be larger than the slits 42, provided that the membrane 40 is composed of a material that resists tearing with repeated use. As indicated above, one use of the membrane 40 is to reduce evaporation and spillage. Another possible use is to wipe excess fluid 36 from the swab 20 as it is pulled from the reservoir 14. As a swab 20 is pushed through the slits 42, the membrane sections 48 deform inwardly. As the swab 20 is pulled from the reservoir 14, the membrane sections 48 tend to deform outwardly. As the absorbent material 24 of the swab 20 passes the membrane sections 48, pressure from the membrane sections 48 against the absorbent material 24 squeezes off fluid that would most likely drip off the swab 20 prior to use. The membrane 40 is composed of a material that is resilient so that it deforms inwardly when pushed by the swab 20, and is rigid enough so that it returns to its original state to cover the aperture 38 to retard evaporation when the swab 20 is removed. Preferably, the membrane 40 is composed of a rubber or plastic material. Preferably, the floor 34 of the reservoir 14 is concave, with the lowest point directly below the aperture 38. With a flat floor, as the level of the fluid falls, the user typically needs to tilt the reservoir to wet the swab. The concave floor 34 of the present invention eliminates the need to tilt the reservoir 14 by using gravity to cause the remaining fluid 36 to pool at the lowest point under the aperture 38, where the fluid 36 is easiest to reach. The figures show a circular reservoir 14 in the center of the circular bin 12. This arrangement is merely illustrative. Any arrangement of the bin 12 and reservoir 14 is contemplated by the present invention, as long as they are integrated into a single package. The present invention contemplates that the dispenser 10 may be manufactured and sold with the reservoir 14 already filled with a fluid and/or that the reservoir 14 may be refilled from another container. The reservoir 14 would be refilled through the aperture membrane 40. Optionally, the swab dispenser 10 of the present invention includes a cover 16. The cover 16 fits in a lip 50 on the outer wall of the integral bin/reservoir. The cover 16 provides several advantages. It protects the swabs 20 from contamination, provides some protection against fluid spills if the dispenser 10 should be knocked over or dropped, and further retards evaporation of the fluid 36. Preferably, the cover 16 is clear so that the swabs 20 are visible. Thus it has been shown and described a swab dispenser with an integral fluid reservoir which satisfies the objects set forth above. Since certain changes may be made in the present disclosure without departing from the scope of the present invention, it is intended that all matter described in the foregoing specification and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

Summary: A swab dispenser comprising a bin adapted to store swabs, a fluid reservoir, and optionally, a cover. The bin may be compartmentalized. The reservoir ceiling has a depression in its outer surface and an aperture at the low point of the depression. A membrane covers the aperture. The membrane has at least two intersecting slits that allows a swab to be inserted into the reservoir by temporarily deforming the membrane. Preferably, the slits extend completely across the aperture. Preferably, the floor of the reservoir is concave with the low point directly below the aperture.

big_patent

en