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Summarize: Loic Remy's £8.5 million move to Liverpool dramatically collapsed on Sunday after he failed a medical. The France forward had flown out to Boston earlier this week after Liverpool had met the release clause in his contract and he had agreed personal terms and a long-term contract before beginning his fitness assessment. Given Liverpool, who also completed a £20million deal for Dejan Lovren on Sunday, have been based in unfamiliar surrounds, the medical had taken longer than normal to conduct but when the results came back, Liverpool decided they had no other option but to pull the plug on the move. VIDEO Scroll down to watch Loic Remy in shooting practice during France training. No deal: Liverpool manager Brendan Rodgers, seen here at the end of his team's 1-0 win over Olympiacos in Chicago on Sunday night, described the collapse of an £8.5m move for Remy as 'unfortunate' It's off: Loic Remy was unable to complete his move to Liverpool but the club have not yet explained why. Match winner: Raheem Sterling settled Liverpool's International Champions Cup match with Olympiacos after five minutes. VIDEO Remy deal collapses. Liverpool have declined to comment on the specific reason they have withdrawn from the move but it is understood that the 27-year-old, who has a historical heart condition, is devastated his dream move to Anfield has fallen through. Manager Brendan Rodgers confirmed the deal was off after Liverpool beat Greek champions Olympiacos 1-0 in the International Champions Cup in Chicago. He said: ‘It's very simple we have made a decision as a club not to go ahead with the deal. It's unfortunate for the player. We are disappointed for the player but there’s nothing more to be said and we will move on and look at other targets.’ Having sold Luis Suarez to Barcelona, they must now decide whether they look for another forward as Daniel Sturridge and Rickie Lambert are the only senior strikers. They are in the process of sealing a £10million deal for Divock Origi but the Belgium forward will be loaned back to Lille next year. Liverpool have spent close to £100million this summer yet they have funds available. Manager Brendan Rodgers, however, will not make a decision in haste, preferring instead to consider his options. He may even wait until the winter transfer window to sign a striker. Confirmed: Rodgers was boosted by the completion of Dejan Lovren's £20m move from Southampton. Incoming: Liverpool are also close to wrapping up the signing of Lille and Belgium forward Divock Origi. Rodgers said: ‘There is money to spend, no question, but I won’t spend it for the sake of it. It has to be the right type. If that means I have to wait until January then that’s what I will do. The players at the moment are in great condition, they are working very well and very hard. ‘I have seen the development in some of the young players but if the right ones become available we would like to do something because i still have a lot to do on that front – but if they’re not available, I will wait.’ It is the second blow for Rodgers in the space of 48 hours after he lost Adam Lallana, his £23.6million signing from Southampton, for six week after he damaged his lateral collateral knee ligaments in training. Rather than flying back to England, Lallana – who does not need surgery – will stay with Liverpool’s squad for the remainder of their US tour in order to get to know his new team-mates but there is no disputing his frustration. Still, Rodgers feels it will not impact on Liverpool too severely. Disappointment: Remy has been left devastated after his proposed £8.5m move to Anfield fell through. ‘Last year if someone like Adam had gone down it would have really hurt us,’ Rodgers said. ‘It’s unfortunate for him but thankfully at this stage of the season, where he has got a good level of fitness, which means he can recover quicker. ‘He won’t be too far away after the start of the season, which is great news. This year is vital for us. You’re going to pick up injuries and we need to have the players who can step in and that’s what we are trying to build.’ There was, at least, something positive for Rodgers with the news that his pursuit of Lovren has reached a successful conclusion. The Croatia international has signed a long-term deal and will link up with Liverpool in either New York or Charlotte, their next stops on this American tour. Lovren, who has become the most expensive defender in Liverpool’s history, described the move as a “dream come true” and Rodgers believes he has the type of characteristics that will enable his side to vastly improve on the 51 goals they conceded last season in the Barclays Premier League. The manager believes Lovren can fill the void left in the back four that has been there since Jamie Carragher retired in 2013. Rodgers said: ‘He's right footed so he can play on the right but he prefers to play on the left. It will depend what needs must. The Important thing is that we have got the player and we will use him where we feel he needs to be. He’s a player who can still improve. Meet and greet: Rodgers signs autographs for some of Liverpool's American-based fans as they played Olympiacos in Chicago. Pride and passion: Liverpool fans show their colours during the friendly at Soldier Field in Chicago. ‘I think Dejan is a really commanding central defender. I said since we lost Jamie Carragher we needed that leadership and he's a player who will gives us that. He's strong and can play. I was impressed by him when he was at Lyon and he was a big driving force for Southampton.’ Lovren will join up on Liverpool’s United States tour either in New York or Charlotte after agreeing a five-year contract and Rodgers believes his squad is now taking shape – and he is no longer mourning the sale of Luis Suarez. ‘I’m really pleased with how it's all coming along,’ said Rodgers, who should see Divock Origi become a Liverpool player in the next 24 hours, having agreed a £10million deal with Lille. ‘Our World Cup players have just come back. ‘We have only just been together as a squad over the last week or so. I know the group well and I know those coming in will fit what we're trying to do. When the Southampton game (at Anfield on August 17) comes up, we will be ready for that.' Of the performance against Olympiakos, Rodgers added: ‘I was delighted with the team. As pre-season goes well, we will be getting sharper in our movements and passing but, at this time, we are building up the resistance of players and getting minutes under the belt. ‘In the first half, our passing was crisp and precise. We looked real threat at top end of the field. I was also very pleased with how we defended. We are in good shape and I am delighted.’

Summary: Liverpool have pulled out of proposed £8.5m move for Loic Remy. France international failed a medical on the Reds' US tour. Brendan Rodgers must decide whether to pursue other options. Liverpool beat Olympiacos 1-0 in International Champions Cup match in Chicago on Sunday night. Raheem Sterling settled the contest with an early goal. Luis Suarez was sold for £75m to Barcelona with Rickie Lambert coming in. Adam Lallana has been ruled out for six weeks but will remain in the US.

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Write a title and summarize: Ein Computerspiel oder Videospiel ist ein elektronisches Spiel, das durch Interaktion mit einer Benutzeroberfläche visuelles Feedback auf einem Videobildschirm, meist einem Fernsehgerät oder Computermonitor, generiert. Weitere wissenschaftliche Definitionen des Begriffs werden in der Ludologie, die sich beispielsweise auch mit der Einteilung in verschiedene Genres beschäftigt, gegeben. == Geschichte Die Computerspiele entwickelten sich innerhalb von ca. 50 Jahren von eher technischen Versuchen an Universitäten zu einer der einflussreichsten Freizeitgestaltungsformen des 21. Jahrhunderts. Bereits auf den ersten Computern gab es Versuche, bekannte Spiele, wie etwa das Damespiel, umzusetzen. Als erstes Computerspiel, welches neue Möglichkeiten jenseits altbekannter Spiele bot, wird oft das 1958 von dem Amerikaner William Higinbotham entwickelte Tennis for Two angesehen. Die Entwicklung war stark abhängig vom technischen Fortschritt der Computertechnik. Spielte sie sich anfangs nur "nebenher" auf eigentlich für andere Zwecke vorgesehenen Großrechnern an Universitäten ab, so wurde es in den 1970er Jahren durch die Kombination der inzwischen relativ kostengünstigen einfachen Logikchips mit der existierenden Fernsehtechnologie möglich, Spiele auch auf elektronischen Spielautomaten in der Öffentlichkeit zu spielen. Sehr erfolgreich war zum Beispiel Pong von Nolan Bushnell. Unternehmen wie Atari oder Magnavox brachten das Computerspiel in Form von Videospielkonsolen auch den Heimanwendern nahe. Es entwickelte sich ein rasant wachsender Massenmarkt. Durch die Einführung der Heim- und Personal-Computer (PCs) in den 1980er Jahren entwickelten sich zunächst zwei technisch betrachtet unterschiedliche Arten des Computerspiels: Zum einen das Videospiel (damals "Telespiel"), welches auf speziellen Spielkonsolen fußte und das Computerspiel für Heimcomputer und später zunehmend für PCs. Im Jahr 1983 kam es zu einem Crash auf dem Videospielemarkt, vor allem durch die Überschwemmung des Marktes mit schlechten Videospielen und der wachsenden technischen Überlegenheit der Heimcomputer gegenüber den damaligen Spielkonsolen. In Japan, wo Heimcomputer noch nicht so erfolgreich waren, läutete Nintendo 1983 mit der Konsole Nintendo Entertainment System (kurz: NES) eine neue Ära der Videospiele ein, die etwa zwei Jahre später, 1985, auch Nordamerika und Europa erreichte. Seit Mitte der 1990er Jahre werden die Bereiche für Spielekonsolen und PCs aus Vermarktungsgründen wieder zunehmend zusammengeführt. So bilden einheitliche Speichermedien (wie die CD-ROM oder DVD) und eine kompatible Hardware die Möglichkeit, Spiele für verschiedene Konsolen wie auch für PCs weitgehend parallel und somit kostengünstiger und für einen breiteren Markt zu entwickeln. Computerspiele sind heute eine weit-verbreitete Form der Unterhaltung. Sie zählen zu den produktivsten Bereichen erzählerischer Aktivität in den digitalen Medien. Sie haben den Bereich der Interactive Fiction um sensuelle Eindrücke erweitert und den Benutzern ermöglicht, in Echtzeit zu interagieren. In vielen Ländern hat sich eine eigene Industrie für ihre Entwicklung gebildet, deren Umsätze teilweise die der jeweiligen Filmindustrie übersteigen. == Gesellschaftliche Struktur === Bedeutung Computerspiele prägen heute unsere Kultur. Sie beeinflussen Menschen moderner Gesellschaften ebenso wie andere Massenmedien. Besonders bei Jugendlichen ist zu beobachten, dass sich ihr Alltag durch Computerspiele stark verändert. Die Bedeutung und Akzeptanz eines Computerspiels ist in den einzelnen Industriestaaten sehr unterschiedlich. In manchen Ländern führen Computerspiele gesellschaftlich und kulturell ein Nischendasein, wenn auch nicht zwingend wirtschaftlich. Dagegen hat sich beispielsweise in Südkorea eine bedeutende Kultur rund um Spiel und Spieler gebildet. Computerspiele nehmen dort einen hohen Stellenwert im Alltagsleben ein. Das Computerspiel wird nur zögernd als Kunstform neben Film, Musik, bildender Kunst usw. akzeptiert. Das mag an der kurzen Geschichte und den oft sehr technologiebasierten und auf bloße Unterhaltung fixierten Inhalten liegen, wobei diese zudem bei neuen Titeln sehr oft bloße technisch verbesserte Wiederholungen älterer Titel mit kaum neuen Inhalten sind. Auf der anderen Seite mag auch die abwertend wirkende Bezeichnung Spiel dazu beitragen, die eine Ähnlichkeit zu einem Spielzeug mit bloßem Unterhaltungswert ohne Inhaltsvermittlung vermuten lässt. Es gibt auch Argumente für die Kunstform Computerspiel: Da das Spielen am PC oder an der Konsole interaktiv ist, macht jeder seine eigene "Kunst", indem er seine eigene Spielweise anwendet. Im Internet hat sich im Zusammenhang mit Computerspielen die Let's-Play-Szene entwickelt. Dabei spielt ein sogenannter Let's-Player ein Videospiel und kommentiert das Spielgeschehen. Let's-Player genießen besonders auf YouTube große Beliebt- und Bekanntheit; so ist etwa der zweitmeistabonnierte YouTube-Kanal PewDiePie durch Let's Plays bekannt geworden. === Nutzung Computerspiele werden in allen Altersschichten gespielt. Manche Kinder beginnen bereits im Vorschulalter damit. Im Allgemeinen interessieren sich vor allem männliche Jugendliche und junge Männer für Computerspiele. Der durchschnittliche Computerspieler war 2003 zwischen 18 und 23 Jahren alt. Laut Digitalverband Bitkom spielen im Jahre 2015 rund 44 Prozent der Deutschen Computer- und Videospiele. Dabei gibt es Unterschiede in den einzelnen Altersgruppen: Bei den 14- bis 29-Jährigen liegt der Anteil bei ca. 81 Prozent. In der Altersgruppe zwischen 30 und 49 Jahren sind es etwa 55 Prozent, unter den 50- bis 64-Jährigen ca. 25 Prozent und in der Generation der 65-Jährigen und älteren spielen rund 11 Prozent Computer- oder Videospiele. Die Entertainment Software Association, der Wirtschaftsverband, in dem die meisten Computerspiele Publisher engagiert sind, geht davon aus, dass jeder vierte amerikanische Bürger im Alter von über 50 Jahren regelmäßig am Computer spielt. Weibliche Jugendliche sind Computerspielen nicht abgeneigt, verbringen aber meist weniger Zeit damit. In Deutschland spielten 2007 der Studie "Typologie der Wünsche" nach 38,8 % der Männer und 22,3 % der Frauen Computer- oder Videospiele. 2015 konnte eine repräsentative Umfrage erstmals zeigen, dass in Deutschland der Anteil an Spielern bei Männern und Frauen mit 43 bzw. 42 Prozent in etwa gleich hoch ist. Insbesondere im E-Sport, dem wettbewerbsmäßigen Spielen von Computer- oder Videospielen, gibt es etliche sogenannte "all female", also rein weibliche Clans, die auch ihre eigenen Turniere bestreiten. In der Regel richten Spielkonsolen sich meist an ein jüngeres Publikum und beinhalten deshalb mehr Action. Computerspiele für den PC können durch leistungsfähigere Hardware auch komplexere Simulationen erzeugen und sind daher auch bei Älteren beliebt: Die Hauptkäufergruppe sind nicht Jugendliche, sondern junge Erwachsene, da Jugendliche nicht über das erforderliche Geld verfügen und deswegen kommerzielle Software oft kopieren. Ein ähnliches Problem kennt die Musikindustrie. Eine Nutzung von Computerspielen zum Zweck der Ausbildung ist möglich. Sie entspricht aber nicht der strengen Definition eines Spiels als zweckfrei, so dass man in solchen Fällen meist von Simulationen spricht. Durch die Möglichkeiten der digitalen Medien entsteht aus den Reihen der Spieler eine Bewegung von Menschen, die Computerspiele nicht nur nutzen, sondern diese auch verändern und sogar neue Spiele daraus entwickeln. Sogenannte Mods (Kurzform von Modifikation) sind meist von den Spielern, selten von professionellen Spieleentwicklern, erstellte Veränderungen oder Erweiterungen von Computerspielen. So werden zum Beispiel nach kurzer Zeit schon Fehler oder unerwünschte Beschränkungen in kommerziellen Spielen beseitigt, die Grafik verbessert oder zusätzliche Funktionen eingebaut. Viel bedeutender sind jedoch die Mods, die das ursprüngliche Spiel um neue Erlebnisse erweitern. Die bekannteste Modifikation ist Counter-Strike, ursprünglich als Mehrspieler-Erweiterung zum Spiel Half-Life entstanden. Die Computerspiel-Industrie unterstützt diese Szene zunehmend aktiv, da es eine günstige Möglichkeit darstellt, fertige Spiele zu erweitern und dadurch noch attraktiver zu machen. Computerspielen wird zunehmend auch zum Beruf. Bereits 2008 lebten 500.000 Menschen in Entwicklungsländern vom Computerspielen. === Wirkung ==== Negative Effekte Bei übertriebenem Konsum von Computerspielen und dem damit verbundenen Schlafentzug kann es (wie bei übertriebener Computernutzung allgemein) zu Schlafstörungen, Halluzinationen, Konzentrationsschwächen, Haltungsschäden (hervorgerufen durch Bewegungsmangel), Nervenschäden (Karpaltunnelsyndrom), Augenschäden, Leistungsversagen und Nervosität kommen. Auch das Auftreten von Gaming Sickness (siehe auch Simulator Sickness, Reisekrankheit) ist möglich. In vielen Spielhandbüchern werden außerdem Epilepsiewarnungen ausgesprochen; diese sind in einigen Staaten gesetzlich vorgeschrieben. Eine am 10. November 2005 veröffentlichte Studie der Berliner Charite zeigte, dass etwa jeder zehnte Computerspieler Abhängigkeitskriterien erfüllt, vergleichbar mit denen von anderen Süchtigen wie beispielsweise Alkoholabhängigen. Ein Zusammenhang zwischen Aggressionen und Spielsucht wird in Politik und Medien kontrovers diskutiert. Unabhängig davon scheint wohl auch für Computerspiele derselbe viel zitierte Satz zu gelten, der im Rahmen der Erforschung des Fernsehens entstand: Der Berufsverband Deutscher Psychologinnen und Psychologen (BDP) warnte auf der Spielemesse Gamescom 2016 vor dem Einfluss von gewalthaltigen Spielen ("Killerspielen") auf die Gewaltbereitschaft von Menschen. Bei allen Effekten von Medienkonsum (z. B. Geschicklichkeit, Konzentration) gehe man selbstverständlich davon aus, dass ein Einfluss besteht, jedoch nicht bei "Killerspielen". Hier werde die irrige Meinung verbreitet, dass diese keinen kausalen Einfluss auf die Gewaltbereitschaft hätten. "Genau wie die Produktwerbung im Fernsehen das Kaufverhalten im Supermarkt beeinflusst, wirkt sich das Töten und Verletzen im Rahmen von Killerspielen auf Gedanken, Gefühle und Verhaltensweisen im echten Leben aus. Gewalterfahrungen im realen Leben und in den Medien verstärken sich gegenseitig und führen nicht nur kurzfristig, sondern auch langfristig zu einer positiven Bewertung von Gewalt". Laut einer Expertise der Mediengewaltkommission der Internationalen Gesellschaft für Aggressionsforschung (International Society for Research on Aggression ISRA) gibt es wissenschaftliche Belege für einen Zusammenhang von Amoktaten und ähnlichen Formen extremer Gewalt und "Erfahrung von Gewalt in der virtuellen Realität, sei es durch Killerspiele oder durch Horrorvideos". ==== Positive Effekte Zu den förderlichen Auswirkungen von Videospielen gehört das Training von räumlicher Orientierung, Gedächtnisbildung, strategischem Denken sowie Feinmotorik. Auch die Aufmerksamkeit und Wahrnehmung visueller Details kann verbessert werden. Doch Computerspiele sind nicht nur als reine Freizeitbeschäftigung für die Konsumenten selbst interessant; es gibt inzwischen gezielte Anwendungen durch die Medizin, beispielsweise zur Behandlung von Demenzerkrankungen, Schmerz- oder Schlaganfallpatienten, wobei teilweise speziell entwickelte und teilweise "normale" Spiele erprobt werden. Für die Behandlung einer Schwachsichtigkeit, vornehmlich im Kindesalter, wurde ein Spiel konzipiert, bei dem das seit Langem bekannte Anaglyph-Verfahren für 3D-Stereoskopie zweckentfremdet wird, um statt eines 3D-Eindrucks ein 2D-Bild zu erzeugen, das nur unter Benutzung beider Augen korrekt erkannt werden kann; ein Spielfortschritt ist nicht möglich, wenn nur das dominante Auge benutzt wird. Übliche Therapien konzentrieren sich bislang darauf, das dominante Auge auszuschalten (bspw. durch Augenklappen oder Pflaster), um das schwache Auge ohne das dominante zu trainieren. === Wettbewerbe und Meisterschaften Beim elektronischen Sport (E-Sport) treten Spieler organisiert in Clans im Mehrspielermodus der einzelnen Computerspiele gegeneinander an, um sich sportlich zu messen oder zunehmend auch um finanzielle Interessen zu verfolgen. Wenn hauptsächlich Preisgelder aus den Turnierspielen und Sponsorenverträge angestrebt werden, spricht man vom Progaming. Diese Mannschaften spielen dann auch häufig in Ligen mit. Die wohl bekannteste und größte Liga im deutschen Raum ist die ESL, die Electronic Sports League, bei der die Gewinner Prämien von bis zu 500.000 € gewinnen können. Inzwischen steigern sich aber die Preisgelder enorm, beispielsweise gibt es bei der CPL World Tour ein Preisgeld von 1.000.000 Dollar zu gewinnen. International weitaus prestige- und preisgeldträchtigere Turniere sind der Electronic Sports World Cup oder die World Cyber Games. Neben den Sportligen gibt es mittlerweile Meisterschaften in fast allen Genres der Videospielekultur (Ego-Shooter, Construction Games etc.). == Computerspiele als Industrie === Geschichtliche Entwicklung Während in den frühen 1980er Jahren zur Zeit der Heimcomputer und Videospielkonsolen noch ein einzelner Programmierer nahezu alle Aufgaben der Produktion eines Spiels erledigen konnte, benötigt man heute für kommerzielle Computerspiele aufgrund der gestiegenen Komplexität (wie z. B. durch den technischen Fortschritt oder die höheren Ansprüche an das fertige Produkt im Allgemeinen) Teams aus Spezialisten für die einzelnen Bereiche. === Entwicklerszene Computerspiele/ Videospiele werden von Spieleentwicklern erstellt. Das können zwar auch Einzelpersonen sein, sind jedoch meist sog. Studios (Developer), in denen mindestens ein Game Designer, Produzent, Autor, Grafikdesigner, Programmierer, Level-Designer, Tongestalter, Musiker und Spieltester in Teams an der Entwicklung von Computerspielen zusammenarbeiten. Zu den bekanntesten Entwicklern zählen John Carmack, Sid Meier, Peter Molyneux, Will Wright, Shigeru Miyamoto, Yu Suzuki, Geoff Crammond, Richard Garriott, Hideo Kojima, American McGee, Markus Persson, Chris Sawyer und Warren Spector. Die meisten Teams umfassen zwanzig bis fünfzig Entwickler, es können aber auch über hundert sein. Die durchschnittliche Entwickleranzahl und auch die Entwicklungsdauer sind mit der wachsenden Bedeutung der Industrie und der zunehmend komplexeren Technologie angestiegen. Die Produktion eines modernen, kommerziellen Spiels dauert etwa ein bis drei Jahre und kostet ungefähr eine bis 15 Millionen US-Dollar. Die Produktionskosten werden oftmals von sogenannten Publishern (vergleichbar mit Buchverlagen) getragen, die das fertige Produkt später vertreiben und vermarkten. Besonders in Japan unterscheidet sich die Spieleindustrie recht stark von der in Europa und den USA. Durch die Geschichte der Arcade-Spiele und der immer noch höheren Popularität von Konsolen- und Arcade-Spielen gegenüber PC-Spielen in Japan entwickelten sich dort andere Strukturen der Spielentwicklung. So produzieren viele Entwickler anonym oder unter Pseudonymen. Oft haben die Teams in Japan einen fest zugeordneten Designer (Director genannt) und sind wesentlich größer als bei vergleichbaren Spielen aus anderen Ländern. Da es auch schwieriger ist, ohne Publisher Spiele für Konsolen zu produzieren als beispielsweise für PCs, gibt es kaum unabhängige Produktionen aus Japan. In Europa und den USA haben sich dagegen etliche von Publishern unabhängige Studios gebildet. Vor der Veröffentlichung eines Computerspiels wird es einer Prüfung durch die Unterhaltungssoftware Selbstkontrolle (USK) unterzogen. Diese Prüfung ist keine Pflicht, wird aber bei praktisch jeder Neuveröffentlichung vorgenommen, da das Videospiel sonst nur volljährigen Käufern zugänglich gemacht werden dürfte. Diese Einstufung wird durch einen deutlich sichtbaren Aufdruck auf der Verpackung und dem Datenträger gekennzeichnet. Sollte der Inhalt des Spiels gegen geltendes Recht verstoßen (zum Beispiel bei Kriegsverherrlichung oder der Darstellung von leidenden Menschen in einer die Menschenwürde verletzende Weise), kann das Spiel durch die Bundesprüfstelle für jugendgefährdende Medien (BPjM) indiziert werden. Um das zu verhindern, werden Spiele für den deutschen Markt oft in einer gegenüber der internationalen Version "geschnittenen" Fassung verkauft. Trotz der großen Popularität von Computerspielen ist eine Beschäftigung in dieser Industrie noch immer recht unsicher. Viele Entwicklerstudios entstehen, entwickeln einzelne Spiele und verschwinden schnell wieder vom Markt. Aus diesem Grund ist zu beobachten, dass sich die Entwickler verstärkt in bestimmten geografischen Gebieten ansammeln, um sich schnell wieder benachbarten Studios anzuschließen oder gar neue Teams zu gründen. Nur rund fünf Prozent aller Computerspiele erwirtschaften Profite. Etliche Produktionen werden nicht fertiggestellt und nie veröffentlicht. Deshalb kann es durchaus erfahrene Spieleentwickler geben, deren Arbeiten aber nie der Öffentlichkeit bekannt wurden. Die Spieleentwickler organisieren sich auf internationaler Ebene in der International Game Developers Association (IGDA) und haben sich in Deutschland zum Bundesverband der Entwickler von Computerspielen (G.A.M.E.) zusammengeschlossen. Weitere Verbände zur Interessensvertretung sind die Entertainment Software Association in den Vereinigten Staaten und der Bundesverband Interaktive Unterhaltungssoftware in Deutschland. Die größte Fachmesse ist die E3 Media and Business Summit (ehemals Electronic Entertainment Expo, auch E3), die jährlich in Los Angeles stattfindet. Der Besuch ist Fachbesuchern vorbehalten. In Europa war die Games Convention in Leipzig mit jährlich über 100.000 Besuchern die größte Messe für Computerspiele, seit 2009 wurde diese von der Gamescom auf dem Kölner Messegelände abgelöst. Spieleentwickler präsentieren jedes Jahr auf der Game Developers Conference die neuesten Entwicklungen und tauschen sich über kommende Technologien aus. === Verkaufszahlen und Umsätze in Deutschland Verkaufte Datenträger und Downloads und Umsätze für Computer- und Videospiele in Deutschland: Quelle: BIU Der Markt für Computerspiele in Deutschland ist, nach Aussagen des Branchenverbands G.A.M.E., mit einem Umsatz in Höhe von 2,66 Milliarden im Jahre 2013 der größte in Europa. === Weltweiter Umsatz Die folgende Tabelle stellt die zehn größten Videospielmärkte nach geschätztem Umsatz für das Jahr 2018 dar. == Inhalte Fast alle Computerspiele definieren das Ziel des Spiels durch formalisierte Erfolgskriterien wie eine Punktzählung (Highscore) oder das Erreichen vordefinierter Siegkriterien. Einige Spiele bieten außerdem Spielmodi, in denen kein Ziel definiert wurde und das Spiel beliebig fortgesetzt werden kann oder nur durch einen Misserfolg beendet wird (Endlosspiel). Beispiele dafür sind Lebenssimulationen und Non-Games. === Motive Moderne Computerspiele beschäftigen sich mit sehr unterschiedlichen Inhalten; einige nehmen zudem Bezug auf andere Medien. So werden oft Elemente oder ganze Welten aus bekannten Filmen wie etwa aus Blade Runner, den James-Bond-, Star-Trek- und Star-Wars-Serien übernommen und immer häufiger aus Computerspielen auf andere Medien übertragen - wie etwa die Verfilmungen von Tomb Raider, Resident Evil und Doom. === Kategorien und Genres Obwohl es die unterschiedlichsten Arten von Computerspielen gibt, ist innerhalb der wissenschaftlichen Auseinandersetzung keine klar definierte Kategorisierung möglich. Man unterscheidet zwischen vielen Genres, die auf der einen Seite eher auf semiotischen Schemata basieren (wie etwa Action-Adventures), auf der anderen Seite die Mechaniken und die verwendete Schnittstelle beschreiben (zum Beispiel Ego-Shooter). So gibt es etliche Computerspiele, die mehreren Genres zugeordnet werden können und bei denen deshalb eine Eingliederung schwerfällt. Einige Genres sind sehr bekannt, andere weniger. Zu den bekanntesten Genres zählt seit Mitte der 1990er Jahre der Ego-Shooter oder First-Person-Shooter, bei dem die virtuelle Spielwelt aus der Ich-Perspektive dargestellt wird und der meistens das reaktionsschnelle Abschießen von virtuellen Gegnern zum Inhalt hat (siehe Frag). Weitere bedeutende Genres sind das Adventure, bei dem oft Rätsel in die Geschichte eingefasst sind und die Reaktionsschnelle gegenüber dem Nachdenken in den Hintergrund tritt; Strategiespiele, bei denen es darum geht, eine Basis aufzubauen, Rohstoffe zu sammeln, eine Armee oder Ähnliches aufzustellen und damit strategisch gegen seinen Gegner vorzugehen; Rollenspiele, in denen es vor allem um die spezifische Ausprägung der Fertigkeiten eines virtuellen Charakters ankommt und Jump-'n'-Run-Spiele, in denen sich die Spielfigur laufend und springend fortbewegt und das präzise Springen einen wesentlichen Teil der spielerischen Handlung darstellt. Ein weiteres Genre, das eng mit der Entwicklung von Computern verbunden ist, sind diverse Simulationen, wie Flugsimulationen, die teilweise auch professionell genutzt werden. Dazu zählen auch Wirtschaftssimulationen, in denen ein möglichst hoher Gewinn erwirtschaftet werden muss. In Sportspielen muss durch Geschicklichkeit an der Schnittstelle eine virtuelle Sportsituation gemeistert werden. == Interaktion Der Benutzer interagiert über einen Computer mit anderen Spielern oder künstlichen Spielfiguren durch Eingabe mittels Maus, Tastatur, Gamepad oder zunehmend per Gestensteuerung und erhält in der Regel über einen Bildschirm Reaktionen. Dabei steuert er häufig einen virtuellen Charakter als Stellvertreter durch eine vordefinierte Welt. In dieser kann er sich, je nach Spiel, in unterschiedlichem Maße frei bewegen. Der Spieleentwickler hat zuvor Regeln und Ziele definiert. Diese Regeln muss der Spieler einhalten (siehe auch Cheat), um das Ziel zu erreichen. Ein Qualitätsmerkmal für Computerspiele ist oft die Handlungsfreiheit. Das wechselseitige aufeinander Einwirken des Spielers mit dem Computer im Einzelspielermodus oder über einen Computer mit anderen Spielern im Mehrspielermodus ist grundlegend für das Computerspiel, weshalb man es anders als zum Beispiel das Fernsehen, den Film oder das Buch als interaktives Medium bezeichnen kann. === Einzelspieler Computerspiele werden überwiegend im sogenannten Einzelspieler-Modus gespielt. Dabei wird die Spielsituation nur durch den Spieler selbst und den Computer beeinflusst. Die Handlungen und Reaktionen der Gegner, oft Bots genannt, werden vom Computer berechnet. Das Niveau der künstlichen Intelligenz der Nichtspielercharaktere ist häufig Qualitätskriterium bei Spielen mit Einzelspieler-Modus und mit der Entwicklung der Computertechnik schreitet sie immer weiter fort. Spielstände können in Form von Savegames gespeichert werden, um sie später wieder aufzunehmen oder an andere zu verschicken. === Mehrspieler Viele Computerspiele unterstützen auch den sogenannten Mehrspielermodus, bei dem mehrere menschliche Spieler gegen- oder miteinander (z. B. Koop-Modus) spielen können. Gespielt wird entweder am selben Computer (bei gleichzeitigem Spiel oft mit Hilfe der Split-Screen-Technik oder abwechselnd per Hot-Seat-Modus) oder über vernetzte Geräte: Über das Internet oder ein lokales Netzwerk (in größerem Umfang auch auf LAN-Partys, wo viele Gleichgesinnte ihre Computer miteinander vernetzen). Der Mehrspieler-Modus lässt einen direkten Vergleich der Spielfertigkeiten zu und ermöglicht so das sportliche Messen der Leistungen. Diesen sportlichen Wettkampf mit Computerspielen nennt man E-Sport. Beispiele für solche Spiele sind: League of Legends, Unreal Tournament, Warcraft 3, Counter-Strike und Fortnite. === Onlinespiele mit hoher Spielerzahl (MMO oder MMORPG) Über das Internet ist es möglich, viele Spieler an einem Computerspiel zu beteiligen. Dabei läuft das eigentliche Spiel auf einem Server und jeder Benutzer kann von einem vernetzten Computer aus am Spielgeschehen teilnehmen. Die bedeutendste Form dieser Onlinespiele sind die Massively Multiplayer Online Role-Playing Games, kurz MMORPGs, bei denen mehrere tausend Spieler ein Rollenspiel spielen. Dabei fallen oft neben dem Kaufpreis für das Spiel auch laufende Kosten für die Benutzung der Server an. Diese regelmäßigen Kosten sind eine wichtige Einnahmequelle für die Betreiber solcher Spiele. MMORPGs besitzen, laut einer Studie für den deutschsprachigen Raum, ein gewisses Suchtpotenzial, da der Spieler sein Spieltempo nicht mehr selbst bestimmen kann. Das führt oft zu einem enormen Zeitaufwand für die Entwicklung der virtuellen Spielfigur. Das bisher erfolgreichste MMORPG ist RuneScape, welches 2012 weltweit die 200-Millionen-Account-Grenze überschritt. == Technik Computerspiele werden über Eingabegeräte gesteuert. Der Computer verarbeitet diese Daten und berechnet mithilfe der sogenannten Spiel-Engine Reaktionen, die über Ausgabegeräte ausgegeben werden. === Plattformen Als Plattform bezeichnet man die Hard- und/oder Software, die als Grundlage für das jeweilige Computerspiel dient. Man kann zwischen statischen Plattformen wie extra entwickelten Spielkonsolen wie dem Nintendo Entertainment System oder der PlayStation und generischen Plattformen wie PCs und Mobiltelefonen unterscheiden, die sich mitunter stark verändern. Die erfolgreichste Spielkonsole aller Zeiten gemessen an Verkaufszahlen ist mit Stand 2020 die PlayStation 2 von Sony. Aktuelle Spielkonsolen sind die PlayStation 5 von Sony, die Xbox One S und Xbox One X von Microsoft und die Switch von Nintendo. Daneben existiert ein Markt für tragbare Geräte wie beispielsweise die Nintendo Switch Lite. War früher das mobile Computerspiel ausschließlich die Domäne dieser Handheld-Konsolen, so bieten heute Smartphones zusätzlich zu ihren Kernfunktionen auch eine Spieleunterstützung an. Als Plattform für Computerspiele ist auch der PC beliebt. === Engines Spiel-Engines (englisch Game Engines) sind Programme, die den Spieleentwicklern häufig benutzte Werkzeuge zur Verfügung stellen und als technischer Kern eines Computerspiels verstanden werden können. Sie ermöglichen die Darstellung von 3D-Objekten, Effekten wie Explosionen und Spiegelungen, die Berechnung des physikalischen Verhaltens von Objekten im Spiel, den Zugriff auf Eingabegeräte wie Maus und Tastatur und das Abspielen von Musik. Bei der Produktion eines Computerspiels wird entweder eine neue Game-Engine programmiert - bis Mitte der 1990er war das fast immer der Fall - oder aber eine bereits bestehende lizenziert und evtl. modifiziert genutzt, wodurch die Produktionsdauer verkürzt werden kann. Bekannte kommerzielle Engines sind Unity, die Unreal Engine von Epic Games, die CryEngine des deutschen Entwicklerstudios Crytek und die Source Engine von Valve. Bekannte freie Engines sind die Quake-Engine von id Software und deren Abkömmlinge. Zu den Game-Engines gibt es fast immer auch Editoren - Programme, mit denen man ohne professionelle Programmierkenntnisse eigene Levels erzeugen kann. Diese werden vor allem zur Erweiterung und Modifikation von kommerziellen Spielen, siehe Mods, eingesetzt. === Eingabe Üblicherweise erfolgt die Eingabe per Hand mit der Tastatur und/oder der Maus oder - insbesondere bei Spielkonsolen - dem Gamepad. In den 1980er Jahren waren noch andere Eingabegeräte wie Paddles und Joysticks weiter verbreitet. Spiele mit Sprachsteuerung haben sich auf Grund der Fehleranfälligkeit der Spracherkennung bisher nicht durchgesetzt. Die Füße werden nur selten, vor allem bei Autorennspielen zur Steuerung von Gas und Bremse mit entsprechenden Pedalen genutzt. Außerdem sind noch einige weniger gebräuchliche Geräte wie das PC Dash und der Strategic Commander verwendbar. Es hat verschiedene Versuche gegeben, Spiele zu vermarkten, die auf die Körperbewegung des Spielers reagieren - beispielsweise durch Drucksensoren in Gummimatten oder durch Auswertung eines Kamerabildes. Diese Spiele stellten jedoch lange Zeit ein Nischenprodukt dar. Erst mit der hohen Verbreitung der Wii-Konsole von Nintendo etabliert sich diese Art von Steuerung. Der Controller verfügt über einen Bewegungssensor, der Position und Bewegung im Raum registriert, so kann durch Armbewegungen eine Spielfigur gesteuert werden. === Optische Ausgabe Man kann grob zwischen maschinellem Text im Textmodus, 2D- und 3D-Computergrafik unterscheiden. Es hat sich eine eigene Ästhetik der Computerspiele entwickelt, eine eigene Bildsprache. Die ersten Computerspiele waren einfarbig und geprägt von Text oder Blockgrafik. Mit der Verfügbarkeit immer besserer Grafikprozessoren wurden die Bildwelten immer farbiger und komplexer. Das typische Spieldisplay heute zeigt den Spieler als Avatar im Bild, oder direkt seine eigene Sicht, die First-Person-Ansicht (Egoperspektive) beispielsweise im Ego-Shooter, vergleichbar der subjektiven Kamera im Film. Dazu erscheinen alle möglichen Anzeigen, Punktestände, Meldungen wie Gesundheitszustand oder Missionsziele im Bild (meist in Form eines Head-up-Displays/HUD). Die visuelle Informationsausgabe kann per Monitor, Display oder Fernseher erfolgen und in Verbindung mit einer 3D-Brille kann sogar ein dreidimensionales Erlebnis erzeugt werden. Einige Videospiel-Entwickler benutzen mittlerweile auch die Technologie Virtual Reality um den Spieler noch mehr in ihre Welten einbeziehen zu können. Die Ausgabe erfolgt über ein Headset, meist als Zubehör für entsprechende Plattformen erhältlich. Diese VR-Headsets sind Brillen bestehend aus zwei getrennten nicht-linearen Bildschirmen. Die Kamera-Perspektive in der virtuellen Welt wird durch den Spieler mittels seinen eigenen Kopfbewegungen selbst eingenommen. Häufig wird durch mehrere externe, selten auch eine integrierte Kamera, die Position in der virtuellen Welt bestimmt. === Akustische Ausgabe Akustische Signale, Effekte und gesprochener Text werden in zunehmendem Umfang und immer besser werdender Qualität bei Computerspielen eingesetzt. Von der ehemals überwiegend atmosphärischen Bedeutung haben sie sich zu einer wichtigen Informationsquelle für den Spieler entwickelt (zum Beispiel zur räumlichen Ortung und Orientierung innerhalb des Spiels). Besonders in Mehrspieler-Partien erlangen akustische Informationen durch die Anwendung von Headsets, die eine schnelle und einfache Kommunikation zwischen Teammitgliedern erlauben, eine immer größere Bedeutung. In Deutschland wird die Sprachausgabe importierter Computerspiele immer öfter ähnlich professionell synchronisiert wie bei Kinofilmen. Teilweise wird bei der Lokalisierung auch auf bereits aus anderen Medien bekannte Sprecherstimmen zurückgegriffen. Besondere Bedeutung hat die Musik in Spielen: Anfänglich als reine Untermalung der Spielszene eingeführt, nimmt sie heute eine ähnliche Rolle wie bei Filmen ein: Sie dient der Steigerung der Dramatik und soll das Spielgeschehen szenisch führen. Dabei kommen oft kurze, einprägsame Melodiesätze zur Anwendung, die auch nach häufigerem Anhören nicht langweilig werden. Die Bandbreite bezüglich des Qualitätsanspruchs ist dabei groß: Professionelle Spieleentwickler beschäftigen heute eigene Komponisten, die sich ganz auf die Erstellung der Musik konzentrieren. Diese wird dem Projekt heute einfach als fertige Audiospur in üblichen Datenformaten zugefügt. PC-Spiele bieten dem Anwender bei frei zugänglichen Datenordnern die Möglichkeit, ungeliebte Musikstücke oder Geräusche auszutauschen und dem eigenen Geschmack anzupassen. Das ist nur dann möglich, wenn Standardformate wie Wave, MP3, MIDI oder andere zum Einsatz kommen und das Spiel von Programmiererseite nicht zu einer einzigen ausführbaren Datei zusammengefasst wurde. Bei den ersten Telespielen der 1980er Jahre mussten die Musikentwickler auch über umfangreiches programmiertechnisches Fachwissen verfügen, um ihr Notenmaterial in das Programm integrieren zu können. === Mechanische Ausgabe Neben der optischen und akustischen Ausgabe bietet die mechanische eine weitere Interaktionsmöglichkeit. Die sogenannte Force-Feedback-Technologie ermöglicht die Ausgabe mechanischer Effekte als Reaktion auf Kräfte, die auf die Spielfigur einwirken. Diese Technik wird vor allem in Lenkrädern für Rennsimulationen, Joysticks für Flugsimulationen und in Gamepads sowie bei Maustasten eingesetzt. Wenn beispielsweise der Spieler mit dem Rennwagen gegen ein Hindernis fährt, spürt er am Lenkrad eine Gegenbewegung. == Überschneidung mit anderen Medien, Kunstformen Das Computerspiel zeichnet sich durch wesentliche Unterschiede, aber auch durch wesentliche Gemeinsamkeiten anderen Medien bzw. Kunstformen gegenüber aus. Die drei wesentlichen Elemente eines Computerspiels sind das (bewegte) Bild, die Story und die Interaktivität. Das erste ist sowohl im Film wie auch in der Malerei und der Zeichnung wiederzufinden, das zweite in Film und Literatur, das dritte im experimentellen Theater. Mehr und mehr ist auch die internationale Vernetzbarkeit von Computerspielen eine seiner wesentlichen Eigenschaften. Oft entlehnt das Computerspiel anderen Medien weitere Elemente und entwickelt diese im eigenen Rahmen weiter, etwa die Geschichte, entlehnt vom Drama, dem Film und der Literatur oder die Musik und, wenn auch in einer völlig neuen Art und Weise, das Schauspiel selbst. Ansätze dazu finden sich etwa in Black & White, Deus Ex, World of Warcraft, Die Sims, Dungeon Keeper, Baldur's Gate 2, Fahrenheit, Monkey Island 3 etc. Teilweise können bestimmte Teile eines Videospiels auch komplett einer anderen Kunstform entsprechen. So werden zum Beispiel Cutscenes immer häufiger nach den Methoden aus der Filmtheorie gestaltet. Ein anderer, in den letzten Jahren aufgekommener Trend ist es, parallele Handlungsstränge in Form von geschriebenen Texten als sammelbare Items im Spiel zu "verteilen". Umgekehrt fließen eGames auch in die Literatur ein: In Die drei Sonnen, einem Science-Fiction-Roman des chinesischen Autors Liu Cixi, spielt das Spiel "Threebody" eine Rolle, allerdings sind keine Aktivität oder Interaktivität der Spieler eingebaut, es handelt sich eher um eine parallele Möglichkeit, etwas zu erzählen. In SpielRaum von Alex Acht ist das Designen eines Computerspiels Teil der Handlung, die Interaktionen werden gut beschrieben, mit ihrer Hilfe kann der Kommissar am Ende den Fall lösen. Im Februar 2008 sprach sich Olaf Zimmermann vom Deutschen Kulturrat dafür aus, dass auch Computerspiele-Entwickler als Künstler anzuerkennen wären. Hans-Joachim Otto, Vorsitzender des Ausschusses für Kultur und Medien des Deutschen Bundestages, pflichtete Zimmermann in einem Interview bei und erklärte, dass die Entwicklung von Spielen ein hohes Maß an kreativer und künstlerischer Arbeit erfordere. Bei einer Indizierung durch die BPjM wird der Kunstbegriff oft als nicht so wichtig wie die Jugendgefährdung gewertet. == Kritik === Soziale Auswirkungen Die Auswirkungen von Gewalt in Computerspielen sind Gegenstand kontroverser Diskussionen. Dabei geht es im Wesentlichen darum, wie Gewalt in Spielen eingesetzt und gezeigt wird, deren Auswirkungen auf die Persönlichkeitsentwicklung von computerspielenden Kindern und Jugendlichen, und einen möglichen Zusammenhang zwischen virtueller und realer Gewalt, d. h., ob Gewalt in Computerspielen Menschen mit einer dafür empfänglichen Persönlichkeitsstruktur auch im realen Leben aggressiver und/oder gewaltbereiter macht. Durch diverse Studien, welche zum Teil schon seit Mitte der 1980er Jahre durchgeführt werden, versuchen Forscher zu untersuchen, ob der exzessive Konsum gewalthaltiger Computerspiele Auswirkungen auf die Gewaltbereitschaft der Konsumenten haben kann. Dabei spielen weitere Aspekte hinein, wie zum Beispiel der Rückhalt im sozialen Umfeld und die Beschaffenheit des Umfelds. Jüngste Analysen mittels funktioneller MRT deuten darauf hin, dass die Gehirnaktivität im linken unteren Frontallappen selbst noch nach einer Woche verminderte Reaktion im Stroop-Test auf Gewalt zeigt. Getestet wurde eine Gruppe von 14 Männern und eine gleich große Kontrollgruppe. Ein Mangel der Studie besteht allerdings darin, dass die Kontrollgruppe kein Computerspiel spielte. Es stellt sich die Frage ob bei einer realistischen Kontrollgruppe, die ein gewaltfreies Computerspiel gespielt hätte, nicht ähnliche Ergebnisse wie bei der mit gewalttätigen Computerspielen konfrontierten Gruppe entstanden wären. === Body-Mass-Index (BMI) Aufgrund uneinheitlicher Ergebnisse hinsichtlich des Zusammenhangs zwischen der Intensität des Spielens von Videospielen und des Body-Mass-Index (BMI) wurde in einer Meta-Analyse überprüft, ob sich das Spielen von Videospielen negativ auf den BMI auswirkt und ob das Spielen einen Einfluss auf die Änderung von körperlicher Aktivität bei den Spielern hat. In die Analyse flossen die Ergebnisse von 20 Publikationen ein. Die Ergebnisse ergaben einen kleinen positiven Zusammenhang zwischen nicht-aktiven Videospielen und dem BMI. Dabei wiesen die miteinbezogenen Studien eine signifikante Heterogenität auf. Eine weitere Analyse potenzieller Moderator-Variablen konnte zeigen, dass der Zusammenhang bei Erwachsenen ausgeprägter war. Ein meta-analytisches Strukturgleichungsmodell ergab nur wenige Hinweise auf eine Änderung der körperlichen Aktivität durch die für Videospiele aufgewendete Zeit. Insgesamt konnte durch die Analyse die Annahme eines starken Zusammenhangs zwischen Videospielen und Körpermasse nicht bestätigt werden. === Schulische Leistungen In einer prospektiven Studie zum Einfluss des Spielens von Computer- und Videospielen auf die Schulleistungen konnte gezeigt werden, dass die Intensität des Spielens von Computerspielen eine signifikant schlechtere Schulleistung zwei Jahre später voraussagte. Dieser Effekt blieb auch unter Kontrolle des Einflusses der ursprünglichen Noten und des Denkvermögens signifikant. Zusätzlich zeigte sich, dass die mathematischen Kompetenzen und Lese-Fähigkeiten der Schüler nicht durch die Spielhäufigkeit beeinflusst wurden. Die Autoren schlossen daraus, dass das Computer- und Videospielen zwar zu einem, wenn auch kleinen Verlust an schulischen Erfolgen führt, basale Grundkompetenzen davon jedoch nicht beeinflusst würden. === Spielsucht Von Wissenschaftlern wird auf die Suchtgefahr bei exzessivem Computerspielen hingewiesen. In Computerspielen wird z. B. das Belohnungssystem im Gehirn ständig wieder aktiviert, um den Spieler am Spielen zu halten. In der Praxis müssen in einem Computerspiel oft viele kleine Aufgaben gelöst werden, die im Gegensatz zum realen Leben auch fast immer in sehr kurzer Zeit zur Zufriedenheit des Spielers erledigt werden können. Der Spieler erlebt dann beim Beenden des Spiels einen negativen emotionalen Zustand, den er durch Weiterspielen zu verhindern versucht. In Südkorea kam es 2002 zum ersten bekannt gewordenen Todesfall infolge ununterbrochenen Computerspielens. Ein 24-Jähriger brach nach 86 Stunden ohne Schlaf und Nahrungsaufnahme vor einem Rechner in einem Internetcafe zusammen. Nachdem er sich scheinbar von dem Zusammenbruch erholt hatte, fand ihn wenig später die herbeigerufene Polizei tot auf der Toilette eines PC Bangs. == Zensur und Verbote von Computer- und Videospielen Nach geltendem Recht dürfen Computer- und Videospiele in Deutschland keine Kriegsverherrlichung oder leidende Menschen in einer die Menschenwürde verletzende Weise darstellen. Aus diesen und anderen Gründen werden die deutschen Versionen mancher Spiele zensiert. So schießt der Spieler z. B. bei Ego-Shootern in der zensierten Version auf Außerirdische, während in der Originalversion des Spiels Menschen als Gegner zu sehen sind. Blut wird manchmal grün statt rot dargestellt. International gab und gibt es Verbote auch aus anderen Gründen. So wurde Pokemon Go in Saudi-Arabien (Glücksspiel) und im Iran (Sicherheitsbedenken) verboten. Das Spiel Animal Crossing: New Horizons ist in China verboten, da es in Hong Kong benutzt wurde Proteste zu organisieren. Im Juli 2002 wurde in Griechenland ein Gesetz verabschiedet, dass illegales Glücksspiel stoppen sollte. Stattdessen wurden aber alle elektronischen Spiele verboten und es gab Berichte über Verhaftungen wegen des Spielens von Counter-Strike oder Schach in der Öffentlichkeit. Das Gesetz wurde im September 2002 dahingehend geändert, dass ein geldwerter Vorteil für den Spieler oder eine dritte Partei entscheidend ist. == Literatur

Title: Computerspiel Summary: Ein Computerspiel ist ein Programm, mit dem man am Computer spielt. Eine Person spielt allein, oder mit anderen Spielern. Manchmal sind die Spieler nicht im selben Raum, sondern die Computer sind über das Internet verbunden. Das nennt man dann online spielen. Je besser die Computer wurden, desto ausgefeilter und komplizierter wurden die Spiele. In den Jahren nach 1970 stellte man Automaten auf, an denen man für Geld spielen konnte. In den Jahren nach 1980 gab es mehr und mehr Computer für zuhause. In den Jahren vor 2000 fing es an, dass man gemeinsam über das Internet spielen konnte. Es werden auch heute noch viele Spiele hergestellt, denn damit kann man viel Geld verdienen. Für ein grosses, erfolgreiches Spiel braucht man viele Leute: Manche programmieren, andere zeichnen, wieder andere sorgen für den Ton. Ähnlich viel Aufwand kostet ein Hollywood-Film.

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Summarize: By. Sarah Griffiths. Three premature babies have died and many others seriously injured after being wiped with an antiseptic solution, it has emerged. Now doctors have been urged to be cautious when using chlorhexidine antiseptic on tiny babies after health experts noted a number of cases of chemical burns and other serious side effects in premature infants. The solution has even been linked to the deaths of three babies in the UK, officials said. Doctors have been urged to be careful when using an antiseptic on tiny babies after health officials noted a number of cases of chemical burns in premature infants wiped with the solution. Stock image. Experts at the London-based Medicines. and Healthcare products Regulatory Agency (MHRA) have asked doctors to. use the solution with care after noticing a pattern of burns. among premature babies. A. total of 28 newborns in the UK have suffered ‘serious side effects’ after being wiped down with the antiseptic before a catheter was. inserted. These side effects included chemical burns, which caused skin loss in some cases, or a skin condition called erythema. Chlorhexidine is frequently used to prevent catheter-related blood stream infections among premature babies. The MHRA received 13 reports of ‘serious. side effects’ among children who were treated with a chlorhexidine. solution between 2004 and 2013. Experts at the regulator have also identified a further 16 cases in medical records between 1992 and 2014. Of the 29 cases, four of the babies died including three in the UK and one in the United States. A total of 28 newborns in the UK have suffered ‘serious side effects’ after being wiped down with the antiseptic before a catheter was inserted. Side effects included chemical burns, which caused skin loss in some cases, or a skin condition called erythema. The chemical injuries occurred in premature babies who were born and treated with the solution before 32 weeks of pregnancy. A total of 12 reports of'serious side effects' were reported among children who were treated with a chlorhexidine solution between 2004 and 2013 and 16 cases in medical literature between 1992 and 2014. Of the 29 cases, four of the babies died including three in the UK and one in the United States. Experts have said that the solution itself should be harmless if used correctly and new guidelines have been issued. One baby died in the UK in 2005 and while the cause of death given was kidney failure, chlorhexidine is thought to be a possible contributing factor in the case, an MHRA spokesman said. A further two children died in 2010. The cause of death recorded in the first case was chronic lung disease. and prematurity, and in the second case no cause of death was given. The. baby is thought to have died as a result of complications related to. extreme prematurity. But the spokesman said that in both cases. chlorhexidine is thought to be a possible contributing factor. The case in the U.S. was not linked to the use of the antiseptic. The. MHRA said that the chemical injuries occurred in premature babies who. were born and treated with the solution before 32 weeks of pregnancy. A. drug safety update issued by the regulator gave fresh advice for health. workers on the use of the chemical including, using the minimum amount. of the solution to treat small babies and monitoring them frequently if. they are treated with the antiseptic. Experts at the London-based Medicines and Healthcare products Regulatory Agency (MHRA) have urged doctors to use the solution with care after experts noticed a pattern of burns among premature babies. A chemical diagram for Chlorohexidine is pictured. Any nappies or sheets soaked with the solution should be removed, it added. The MHRA said that European health officials will be reviewing the issue. ‘Hospital use of chlorhexidine has a crucial role in preventing infection in premature infants, which is a leading cause of death in neonatal units and is used safely many thousands of times every year in hospitals,’ said Dr June Raine, director of MHRA’s Vigilance and Risk Management of Medicines Division. ‘Our monthly bulletin, Drug Safety Update, includes information on a small number of historic reports of serious side-effects, including four suspected deaths, in premature infants associated with the use of this product. Two of these were thought to be due to severe complications of prematurity. ‘We have also given guidance to healthcare professionals to use the minimum solution required and to remove any excess bearing in mind the risk in very pre-term infants.’ Dr Martin Ward-Platt, a spokesman for the Royal College of Paediatrics and Child Health, added: ‘This stuff should be harmless, if harm is caused it is likely to be a training or knowledge issue. 'This is avoidable and a harm these babies should not be at risk of. Nobody’s baby should come to harm because of this.’

Summary: Babies have suffered burns and other serious side effects after being disinfected with the chlorhexidine antiseptic. Experts say the solution may have contributed to the deaths of three babies. Twenty eight newborns in the UK have. suffered'serious side effects' after being wiped down with the. antiseptic before a catheter was inserted. Experts at London's Medicines and Healthcare products Regulatory Agency said the burns occurred on babies treated before 32 weeks of pregnancy. New advice has been given to medics and a spokesman for the Royal College of Paediatrics and Child Health said the treatment should be harmless.

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Summarize: BACKGROUND OF THE INVENTION This invention relates to improvements in drawer slides of the type which permit a drawer to be selectively pulled out from opposite sides of a support body such as a desk, file cabinet, service vehicle and the like. Two way or double pull drawers of various types have been known and used for some time and may be classified generally as being two member or three member assemblies. The two member assembly has one member fixed to the frame or support body for the drawer such as a desk or cabinet and a movable member attached to the drawer and to the fixed member. With this arrangement, the drawer cannot be held regidly when the mounting point connecting it to the slide is moved more than one half the length of the drawer and thus there is the inherent disadvantage that only approximately one half of the drawer space is accessible from either side of the support body. Examples of two member slides appear in U.S. Pat. Nos. 2,565,784 and 2,599,865. The three member assembly, as is well known, permits extension of the drawer to nearly one hundred percent of its length and the present invention is directed to improvements in this type of slide assembly. The three member assembly also uses one member fixed to the support body and a movable member attached to the drawer but includes a third member slidably arranged intermediate and operably connected to both of the other two members. Examples of the three member slide assembly are disclosed in U.S. Pat. Nos. 1,736,108 and 2,914,370. Notwithstanding the advantage of the three member slide in permitting substantially one hundred percent extension of the drawer, it has been our observation that malfunction of the slide capability of the intermediate third member is commonly experienced with the result that the drawer frequently cannot be fully retracted. Such malfunctioning is found to exist in various latching and locking arrangements generally found mounted within the support body but exteriorly of the slide assembly itself so as to require considerable adjustment and alignment. With the above observations in mind, one of the important objects of the present invention is to provide an improved and trouble free operating three member two way travel drawer supporting slide assembly. More particularly, it is an object herein to provide a drawer slide assembly of the above class that consists generally of an outer elongated channel section for fixed attachment to a support body, a central channel section slidably mounted within the channel of the outer section and an interior section slidably mounted within the channel of the center section and fixedly attached to a drawer to be moved into and out of a support body together with an improved arrangement of movable stops designed to facilitate and assure the proper movement of the central section relative to the outer and interior sections to permit substantially full extension and trouble free retraction of the drawer relative to respective opposite sides of the support body. Another object herein is to provide a drawer slide assembly as characterized in which all components operable to effect or control the sliding movement of the several sections and particularly the central section are arranged within the confines of the overall assembly and no control elements attached to the support body for such purposes are required. The foregoing objects and such further objects as may appear herein, or be hereinafter pointed out, together with the advantages of this invention will be more fully discussed and developed in the more detailed description of the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a service vehicle having a body portion in which this invention is shown mounted and indicating the two way operation thereof, FIG. 2 is an enlarged foreshortened perspective view of this slide assembly from the interior side shown extended in one direction, FIG. 3 is a side elevational view of this invention from the interior side shown in closed or retracted position, FIG. 4 is a side elevational view of this slide assembly shown extended in one direction, FIG. 5 is an end view taken from the line 5--5 of FIG. 4, FIG. 6 is a sectional view taken on the line 6--6 of FIG. 4, FIG. 7 is a plan view taken from the line 7--7 of FIG. 4, FIG. 8 is a sectional view taken on the line 8--8 of FIG. 4, FIG. 9 is a plan view taken from the line 9--9 of FIG. 4, FIG. 10 is a perspective view of a drawer showing latch means at each end relative to the support body, and FIG. 11 is an enlarged perspective view of the complementary latch components for the support body and the drawer. DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to the drawings, an example of a two way or double pull drawer 10 mounted on our slide assembly designated generally by the numeral 12, is illustrated in FIG. 1 on a service vehicle 14 provided with a suitable compartment 16 in body 18 to support assembly 12 and contain drawer 10. It is not intended that slide assembly 12 be limited to use shown in a service vehicle 14 since it may be utilized in other environments adapted for double pull drawers such as desks, file cabinets and the like as is well known. Slide assembly 12 (FIGS. 2-4) is a three section two way slidable unit which, in general, comprises an outer elongated section 20 adapted to be fixedly attached to one wall of compartment 16 in any suitable manner such as by screws 22 through appropriately placed holes 24, a central or intermediate section 26 which is slidably journalled in section 20 and an inner or interior section 28 slidably journalled in section 26 and adapted to be fixedly secured to one side of drawer 10 in any suitable manner such as by fasteners 30 through appropriately spaced holes 32, it being noted that identical assemblies 12 are mounted relative to opposite sides of compartment 16 and drawer 10 so that a description of only one assembly 12 will suffice for present purposes. No invention is claimed for the three member assembly per se comprising sections 20, 26 and 28 as shown but invention is claimed for the improvement movement control means particularly for the intermediate or central section 26 as will be referred to in detail as this description proceeds. With reference now more particularly to FIGS. 1,5,6, the outer section 20 is a channel bar having the bar member 33 with the respective opposed upper 34 and lower 36 channels. Bar portion 33 is attached to the wall of compartment 16 in body 18 so that channels 34, 36 project into the compartment. Channels 34, 36 are of like formation so, primarily, channel 34 will be described in detail and like parts on channel 36 will be given like numerals primed. Channel 34 extends first perpendicularly from the top edge of bar 33 away from wall 16 to form a flat track surface 38 and then upwardly into a half circle shape to form the curved or grooved track 40 and it will be understood that on the lower channel 36, track 40' extends downwardly from track 38'. The length of channels 34, 36 is slightly less than the length of bar 33 so that bar 33 projects beyond such channels at both ends, indicated at 42 in FIG. 2, and at the upper and lower ends of projection 42 at each end of bar 33, are the respective stops 44, 44' which extend across and define the terminal points of the flat track 38, 38'. The central section 26 is also a channel bar slidably within the outer section 20 as best seen in FIG. 2 and includes the bar member 46 disposed in juxtaposition to bar member 33, and the respective like upper 48 and lower 50 channels so that, primarily, channel 48 will be described in detail and like parts on channel 50 will be given like numerals primed. The top edge of channel 48 is provided with the grooved track 52 with the corresponding track 52' on channel 50 being the bottom edge thereof as seen in FIG. 2 and in juxtaposition to the tracks 52, 52' on the inner sides of channels 48, 50 are the respective grooved tracks 54, 54'. Thus far described and as seen in FIGS. 5,6, the respective tracks 40, 40' on the outer section 20 complement the tracks 52, 52' on the center section 26 to provide a trackway for other components yet to be described. At each end of channels 48, 50 are the respective stops 56, 56' which define the terminal point of the respective tracks 52, 54 and 42', 54'. The construction of channel sections 20, 26 as thus far described are well known in slide assembly devices and it is to such type of device we have made important improvements as follows. At each end portion of channels 48, 50 we have provided respective slots 58, 58' which communicate with the respective tracks 52, 54 and 52', 54' as seen in FIG. 2. In each slot 58, 58', a disk-like dog 60, 60' is pivotally and eccentrically mounted, 61, 61', so that opposed dogs can move towards each other through slots 58, 58' and track 52, 52' and away from each other through slots 58, 58' and track 54, 54' as shown in FIG. 2. It is noted that while dogs 60, 60' are shown as round washer type disks, other shapes such as oval, square, triangular and the like capable of movement as described may be used without any departure from the principles involved. In each of the trackways provided by the complementary tracks 40, 52 and 40', 52', there is disposed the elongated bar stop 62, 62' which is slidable therein by means of the ball bearings 64 disposed in longitudinal spaced relationship in appropriate holes in stops 62, 62', such ball bearings being confined by the curved tracks 40, 52 and 40', 52'. Thus arranged, one longitudinal edge of stop bars 62, 62' is disposed to move in track 38, 38' as seen in FIGS. 5,6 and is a well known arrangement in the conventional construction of the three member slide assembly. The inner section 28 (FIGS.2,8) is also of known construction and includes the bar member 66 for attachment to drawer 10 as described and the respective like upper 68 and lower 70 channels for which channel 68 will be primarily described and like parts on channel 70 will be given like numerals primed. The top edge of channel 68 and corresponding edge of channel 70 are formed with the respective grooved tracks 72, 72' and with section 28 slidably journalled in section 26 as seen in FIG. 2, track 72 complements track 54 on section 26 to form a trackway in which is mounted a ball bearing stop bar 74 similar to bar 62. Likewise, track 72' on section 28 complements track 54' on section 26 to form a trackway for stop bar 74' as shown. Each end of channels 68, 70 are provided with the respective stops 76, 76'. OPERATION The closed or retracted position of slide assembly 12 is seen in FIG. 3 where the position of section 28, attached to drawer 10, positions the dogs 60, 60' through slots 58, 58' generally in the outer rolled tracks 52, 52' of channels 48, 50 of the intermediate section 26. In this position, section 26 cannot be moved in either direction because of the stop engagement capability of dogs 60, 60' with the end of the rolled edges on tracks 40, 40' of section 20. In the opening of drawer 10, being shown in the drawings from left to right, section 28 first moves with the drawer 10 so that the left end of section 28 slides away from the left end of section 26 to no longer support dogs 60, 60' in the position shown in FIG. 3 and such dogs are free to move through slots 58, 58' to the interior portion of section 26. As drawer 10 and section 28 are pulled further out, stop bars 62, 62' will move dogs 60, 60' at the right end of section 26 to th position shown in FIGS. 2,4 and also contact stops 56, 56' on section 26 at the right end thereof whereby section 26 is also moved from left to right. As this occurs, dogs 60, 60' at the left end of section 26 engage the edges of channels 40, 40' and are moved through slots 58, 58' into the interior of section 26 as also seen in FIGS. 2,4. Further movement of the assembly to the right to substantially the full amount of drawer 10 terminates when stop bars 74, 74' engage stops 44, 44' at the right end of section 20. In closing drawer 10, being from right to left as shown, section 28 slides relative to section 26 until the left end of section 28 engages the dogs 60, 60' at the left end of section 26 which, being confined by the rolled channels 40, 40' cannot move at that point through slots 58, 58' and thus serve as temporary stops so that continued pushing inwardly of drawer 10 and section 28 effects the sliding of section 26 from right to left back to closed position and as the left end of section 26 clears the left end of channels 40, 40' on section 20, the continued movement of section 28 pushes the dogs 60, 60' through slots 58, 58' to the position shown in FIG. 3. The opening of drawer 10 to the left is a mirrored action of the sequence just described. In the above arrangement, it is noted particularly that the several dog components are completely contained within the confines of the three member assembly and eliminates the need usually found in other assemblies of this class for additional mountings on the body member in which the drawer is arranged and the inherent problems in correcting and aligning a plurality of separate mechanisms to assure operability particularly of the intermediate section of a three member slide assembly. Reference is now made to FIGS. 10, 11 relative to a simple latch means 78 which we have applied to opposite ends of drawer 10 as best seen in FIG. 10 even though for many purposes in stationary bodies such as desks and cabinets, a latch means such as 78 may not normally be required to prevent any accidental opening of the drawer 10. However, notwithstanding the described control means relative to movement of section 26, it will be appreciated that section 28 attached to drawer 10 is free to move in either direction and since assembly 12 as shown and described has been adapted on a service vehicle 14, latch means 78 prevents any accidental opening of drawer 10 when such vehicle may be in motion. Latch 78 includes a strike plate 80 attached to the outer side of body 18 adjacent compartment 16 and projects slightly into compartment 16 as at 82 but not far enough to interfere with the free movement of drawer 10 out of and into such compartment. Integral with portion 82 is the cam-like edge 84 extending outwardly from body 18. A normally horizontal bar 86 is pivotally secured, 88, intermediate its ends to the end of drawer 10 and its horizontal position is maintained by stop 90 on the drawer in which position, one end of bar 86 rests behind and engages strike plate portion 82 so that drawer 10 cannot be pulled out. A drawer pull or handle 92 on drawer 10 is convenient to the other end of bar 86 so that such bar can be easily pivoted out of engagement with member 82 when the pull is grasped. When drawer 10 is moved to closed position, bar 86 will automatically pivot upon contact with the cam surface 84 and return to latched position. Latch 78 as described is provided on each end of drawer 10 and the respective latches are independent of each other for their intended purpose. The present invention has been described preferably in relation to commonly used three section slide assemblies. However, more than three sections are sometimes used having a plurality of like central sections referred to and the present invention can be adapted to such multiple sections without departing from the principles disclosed. Accordingly, in view of the foregoing, it is thought a full understanding of the construction and operation of this invention will be had and the advantages of the same will be appreciated.

Summary: A two way travel three section drawer slide assembly is provided for cabinets, desks, files, service vehicles and the like to permit a drawer to be selectively extended from opposite sides. The assembly includes an outer channel section for attachment to a support body such as a cabinet wall, a central channel section slidably mounted within the outer section and an interior channel section slidably mounted to the central section and fixedly attached to the drawer to be moved. The present invention particularly provides improved movable stops wholly within the confines of the assembly to facilitate and assure the proper movement of the central section to full extension and full retraction whereby no latching or locking devices exteriorly of the slide assembly are required. Latch members on opposite sides of the drawer and support body prevent accidental outward movement of the drawer. This invention is also usable on drawer assemblies having more than three sections.

big_patent

en

Summarize: IHS dismantled the iPad Air to work out how much each component cost to make. The cheapest 16GB iPad Air costs $274 to make, plus a $6 manufacturing cost, yet sells for $499 in the U.S. Apple launched its iPad Air last month, which is twice as. fast as previous models and 20 per cent thinner - yet a new report has now found it’s. also cheaper to make, meaning Apple's profit margins just increased. Colorado-based analytics firm IHS took the tablet to pieces. and worked out how much each individual component would cost to manufacture. It discovered the cheapest 16GB Wi-Fi-only iPad Air costs. $274 to make, based on preliminary results, yet sells for $499. This is despite the fact the iPad Air is $42 cheaper. to make than the third-generation model. IHS’ Teardown Analysis Service only looked at the price in. dollars. If the figures are converted to UK prices this works out at £170. production costs compared to a retail price of £399. Elsewhere, the 16GB iPad Air with 3G connectivity costs £188 ($304) to make, according to preliminary results, and this represents a 6 per cent drop. in production price of the iPad 3, down from £201 ($325). When the £4 ($6) manufacturing cost of the iPad Air is added in,. the total cost to make the tablets increases to $280 (£174) for the smaller 16GB model and $310 (£192) for the 32GB device. ‘While the iPad Air slims down in size, the profit margins. are getting fatter,’ said Andrew Rassweiler, senior director, cost benchmarking. services for IHS. ‘Although the Air’s new, ultra-thin display and touch screen. are more expensive than for the third-generation iPad, Apple has held the line. on cost by taking advantage of price erosion in other areas. IHS' Teardown Analysis Service checked each of the iPad Air's components, pictured. The firm only looked at the price in dollars, but if the figures are converted to UK prices this works out at £170 production costs compared to a retail price of £399. This works out at a 135 per cent mark-up. ‘Furthermore, the iPad Air leverages the same components and. suppliers that are used in the iPhone 5s and 5c as much as possible.’ The profitability of the iPad Air rises dramatically as the. built-in storage capacity increases. For example, the 32GB model costs Apple only £5.20 ($8.40) more. to produce - but has a retail price that’s £80 more in the UK and $100 higher in the U.S. Apple was criticised for not dropping the prices of its. latest iPhones and iPads in a bid to appeal to emerging markets. This cost breakdown shows that Apple is sticking to its. traditional pricing strategy. This may also be, in part, due to the fact the firm’s. earnings recently fell 9 per cent. It was the third consecutive quarter its earnings dropped. compared with the previous year. US figures. Cost to make (plus $6 overall manufacturing cost): $280. Sales price: $499. In this instance $274 = 100 per cent. To find out the relative percentage for the sales price, $280 is divided by 100 (%) to make 2.8. This is essentially equivalent to one per cent. If the sales price of $499 is divided by 2.8, it works out at 178 per cent, meaning the mark-up is 78 per cent. UK figures. IHS’ Teardown Analysis Service only looked at the price in. dollars, so the figures were directly converted to UK prices for components - the sales price is the price available in the Apple store. Cost to make (plus £4 overall manufacturing cost): £174. Sales price: £399. In this instance £174 = 100 per cent, so £174 is divided by 100 (%) to make 1.74. If the sales price of £399 is divided by 1.74, it works out at 229 per cent, meaning the mark-up is 129 per cent

Summary: Researchers dismantled the iPad Air to work out costs for each component. 16GB model worked out at £174 ($274) to make, yet retails at £399 ($499) This is £26 ($42) less than what Apple paid to make its iPad 3. The 32GB model costs £5.20 ($8.40) more to make than the smaller model, yet retails for £80 extra in the UK and $100 extra in the U.S.

cnn_dailymail

en

Summarize: Loss: Audrie Pott, 15, killed herself last year after she was allegedly sexually assaulted at a party. A teenager who hanged herself after three classmates allegedly sexually assaulted her and circulated photos of the sickening attack desperately pleaded with the boys to delete the images in the days before her death, it has emerged. Audrie Pott, a 15-year-old from Saratoga, California, took her life in September 2012 after sending Facebook messages to the boys who allegedly assaulted her at an alcohol-fueled party. 'I swear to god if u still have those pictures illl killl u [sic],' she wrote to one of her alleged attackers in the days after the party. 'It's gonna get out.' Another male classmate responded: 'lol that s*** gets around haha everyone knows mostly everything hahaah.' Potts was found hanging in the bathroom of her mother's home on September 12, 2012 after the explicit photographs had allegedly led to bullying at school. Her devastated parents blame her death on the horrific sexual attack a week earlier at the hands of three teenage boys who then allegedly tried to cover up their involvement. On the night of the attack, a group of teenagers were at a friend's house drinking heavily. The girl who lived at the house threw the party when her parents left town. Three male classmates allegedly. helped an inebriated Audrie to a bedroom before stripping her naked, drawing over her. body and sexually assaulting her. Scroll down for video. Cruel: Pott, right, was aassaulted after a night of drinking and boys spread photos of the attack. In police interviews, the teenagers admitted. to coloring half of her face black with marker, then. pulling down her bra, taking off her shorts and drawing on her breasts and near her genitals. They allegedly photographed themselves sexually assaulting her and, by the time she started her sophomore year at Saratoga High School two days later, scores of students had seen the images. Rolling Stone magazine has revealed the torment Audrie suffered in the days before her death and has detailed the messages she exchanged with her alleged attackers, whom she had known since middle school. She messaged one boy, vowing to kill him if he still had the images. 'They are deleted and I didn't take them,' he wrote. 'I promise it wasn't me.' Heartbroken: Lawrence Pott and Audrie's stepmother and his second wife Lisa have allegedly sent Facebook messages accepting that Lazarin is Audrie's biological fatehr. Grieving: Audrie's mother, Sheila Pott, found her daughter hanging in the bathroom in September 2012. Audrie Pott wrote a series of desperate Facebook messages before her death that have been excerpted in Rolling Stone. In one exchange, she speaks to one of her alleged attackers:. AUDRIE: i need to talk to u. BOY: What. AUDRIE: one word... marker. BOY: What about marker. AUDRIE: u know what im talking about... i dont remeber anything about that... [A friend] had to tell me everything... i swear to god if u still have those pictures illl killl u. BOY: They are deleted and I didn't take them I promise it wasn't me... And I'm sorry about the marker. AUDRIE: You have no idea what it's like to be a girl. An exchange with another classmate:. CLASSMATE: lol that shit gets around haha everyone knows mostly everything hahaah. AUDRIE: oh my god... i f***ing hate people... Do you know how people view me now?... I now have a reputation I can never get rid of. AUDRIE: My life is over... I ruined my life and I don't even remember how. Source: Rolling Stone magazine. 'I'm sorry about the marker,' he added of the drawings that covered her body. She also messaged another boy who had been at the party to ask if one of the teenagers had photographs of her. 'ur fine,' he responded. 'ill make sure nothing goes around.' She replied: 'It's gonna get out. S*** always does. Especially with the people who were there.' And in an even more concerning exchange, one of her classmates sent her a message reading: 'lol that s*** gets around haha everyone knows mostly everything hahaah.' 'My life is over... I ruined my life and I don't even remember how,' one of her final messages read. 'You have no idea what it's like to be a girl.' When she returned to school, she missed classes to avoid people and had an argument with one of her closest friends, Kathy Atabakhsh, who accused her of becoming a different person. 'She had been, literally, the best person. you could meet – always honest and trustworthy,' Kathy told Rolling Stone. 'And I was so upset that she had. changed. It was hard for her to hear that from a close friend.' Another girl loudly pointed out in class that Audrie had been cutting herself, and the teenager burst out crying. She told friends she had cut herself on a vase. On September 12, 2012, Audrie had texted her mother, Sheila Pott, and pleaded for her to collect her from school - but was silent on the drive home. When they arrived back at the house, Audrie went to her bedroom and her mother went to check on her after about 20 minutes. She saw the bathroom door closed and knocked - to no answer. Desperate: Her parents have claimed that the school failed to help with bullying after they told staff. Becoming increasingly concerned, Mrs Pott frantically worked the lock from the outside and eventually opened the door. But it was too late - she saw her daughter hanging inside the room. She wracked her memory for what could have happened to her daughter. She said she remembered seeing marker on her daughter's chest - which the girl quickly dismissed - after the party, and that her daughter had called her unusually early the night after the party to come and collect her. After the death, Saratoga police agreed with the school to wait until a week later to start an investigation to allow the students time to grieve. But just days later, Audrie's friend Kathy went to talk to school administrators to explain what she knew about the party and the pictures of her friend. When police arrived to interview students, there had already been rumors of an investigation and some students were heard urging others to shut down their Facebook accounts, Rolling Stone reported. Left behind: Audrie is pictured left with her father, step mother and half brother and sisters. Remembered: Photographs show Audrie and her family and an essay she wrote about her happy home life. The Pott family learned of the police investigation when a relatives heard of it from a student and called Audrie's father to urge him not to cremate his daughter's body. When police investigated the three boys, they found that one boy had apparently lost his phone and another claimed his was broken. Due to these setbacks, it took seven months before the boys were charged. But on April 11, they were arrested on charges of misdemeanor sexual battery, felony possession of child pornography and felony sexual penetration. When they were seized, police allegedly found new images of naked teens on their phones and added charges. One boy was even trying to sell the pictures for money, the Rolling Stone reported. Two of the teenagers could receive community service or time in a juvenile detention center, and the third boy may be upgraded to an adult court, where he can expect to face a harsher punishment. Loved: School friends have remembered Audrie by wearing teal - her favorite color - to class. Scene: Saratoga High School, where the teens attended class. Two of the 'attackers' still go to the school. The Pott family has filed a civil suit against the boys and their families and an administrative claim against the school district, alleging that administrators did not respond to bullying against Audrie. The school has said these bullying claims were never discussed. Only one parent of the accused boys returned a call to Rolling Stone and said that the case had been misreported. He passed on their condolences to the Pott family. But he added: 'It was a prank by a few kids, and it's blown out of proportion. Audrie had a lot of other problems in her life, and everybody in Saratoga knows that.'

Summary: Audrie Pott, 15, hanged herself in September 2012 'after images were passed around school showing her naked and being sexually assaulted' Three teenage boys'stripped her naked, drew on her body and attacked her after a night of heavy drinking at a house party' Facebook messages sent that week show her pleading with the 'attackers' to delete photos - while other classmates tell her they're already out. Teenage boys are now awaiting sentencing and her parents are suing their parents and the school district.

cnn_dailymail

en

Summarize: RELATED APPLICATIONS [0001] This application is the U.S. National Stage under 35 USC 371 of PCT Application PCT/IB2015/054026.and claims foreign priority based of Italian patent application BA2014A000036 filed on Jun. 6, 2014. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to a new machine for the production of “cartellate”. In particular, said machine is able to realize accurately all the steps which reproduce the skillful manual production of “cartellate” by means of a specific working procedure. [0004] 2. Brief Description of the Prior Art [0005] As it is known at the state of the art, the “cartellate” are typical cakes coming from Apulia and produced also in the neighbouring regions. The classical preparation process is typically carried out manually and both the recipe and the preparation procedure of the same is handed down from generation to generation. [0006] The classical preparation occurs by making scalloped strips of a thin dough pastry (in the following scalloped strips of dough) obtained with flour, oil and white wine, which is joined and wrapped on oneself to form a sort of choreographic “rose” with recesses and holes, which will be then fried in plenty of oil. The most important part of the preparation of the “cartellata” is in fact the characteristic folding of a scalloped strip of dough of determined length which has to be pinched in more points at regular intervals, by making the dough adhere well so that many little open recesses are obtained on the whole length of the dough strip. Starting then from the end of the pinched strip, the same is rolled on itself so that the “rose” shape is provided. [0007] There exist many variants of “cartellate”, but the typical regional recipe is the one in which they are soaked with warm vincotto or honey, and then covered with cinnamon, icing sugar or almonds. The vincotto is a condiment provided by cooking the must of the grapes from Salento or figs. In other variants there is chocolate instead of vincotto, or simply icing sugar. Once prepared, they are kept in great pans, far from light and indoor. [0008] The manual preparation times of “cartellate” are normally variable according to the result intended to be obtained, but they are definitely long. They are about 4 hours to obtain about 50-60 pieces from one kilogram of dough. In this case, it is a not excellent product, with dough thickness equal to about 1 mm. If one wants to obtain a better quality it occurs to work the dough to 0.5 mm thickness and in this way it is possible to obtain about 100-120 pieces for kilogram of dough. In this case, the times for the product preparation, always related to kilogram of dough, can exceed six hours. [0009] As it can be observed, these are important times which make the preparation of “cartellate” difficult on a greater scale. But, on the other hand, there exist no machines able to reproduce, in each step, the manual preparation procedure of such product. Therefore, there exists the need to automatize the production of “cartellate”, passing from a manual preparation procedure to an industrial “production” procedure of the same. SUMMARY OF THE INVENTION SUMMARY [0010] Therefore, aim of the present invention is to provide a new machine for producing “cartellate” which allows to reproduce optimally each step of the manual procedure with a huge time saving and with optimum quality. [0011] Another aim of the present invention is to provide, by using said machine, a specific working procedure in order to have a final product “as handmade”. [0012] The machine for producing “cartellate” has the features claimed in the independent claim of product. The procedure for working and producing “cartellate”, used thanks to the new machine, has the features claimed in the independent claim of method. [0013] The dependent claims describe other aspects of the machine. BRIEF DESCRIPTION OF THE DRAWINGS [0014] The different embodiments of the invention will be now described as a way of example with reference to the appended drawings, in which: [0015] FIG. 1 shows a scheme of the basic structure of the machine for producing “cartellate” according to an embodiment of the present invention; [0016] FIG. 2 shows a detail of the pinching means and shaping means of the machine for the preparation of “cartellate” of FIG. 1 ; [0017] FIG. 3 shows another detail of the pinching means and cutting means of the machine of FIG. 1 ; [0018] FIG. 4 is a detail of the conveyor means. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0019] As it is shown in FIG. 1, the machine 100 for producing “cartellate” is provided with a support structure 1 and a motorized conveyor belt 3 which advances thanks to the rotation of a couple of pulleys or gears or other similar transmission organs 2. The transmission organs rotate on suitable shafts 2 ′ suitably supported at the ends by support means 2 ′. The machine further comprises other means operating in sequence on the dough strips: conveyor means 5, pinching means 6 suitably moved by respective motors 4, shaping means 7, dough guide means 8, at least a cutting means 9 and a mobile rolling means 10 to roll the dough strips. All these means will be better described in the following, along with the working procedure. [0020] The raw material used for the production of “cartellate” is the dough pastry, or better, a dough strip, scalloped, worked by means yet available on the market. The worked dough strip is positioned on the conveyor belt 3 and a guide means 8, for example a disc, guides the same along a predetermined direction and with an advancement speed adjustable according to the production needs. The machine core consists in the elements which form and pinch the scalloped strip at the same time and which, in the following, cut them to length. In fact firstly two conveyor means 5, for example rollers, as shown in FIG. 4, positioned on both sides of the conveyor belt, lift the edges of the scalloped strip thus simplifying the next step of folding and pinching the dough in more points. The conveyor rollers 5, in practice, realize a first step of strip forming. In the following, the machine carries out the pinching of the dough strip in more points. The pinching is carried out by pinching means 6, for example two counter-rotating discs, each one provided with a plurality of projections 6 ′, and consists in folding the dough strip to half and in carrying out little pinchings, at regular interval, along the whole length of the scalloped strip. In the same step, a shaped matrix 7 ′ of a shaping means 7 penetrates the dough strip inside the lifted and folded edges, synchronously with the pinching discs, thus ending the forming. The shaping means 7 can be made up of a wheel bearing a plurality of angularly equidistant shaped matrices 7 ′. In the following, a cutting means 9, for example a blade integral to the pinching disc, cuts the dough to a predetermined length. Finally, a mobile rolling means 10, for example a mobile disc, rolls the cut and pinched dough strip thus realizing the famous “rose” shape, typical of the “cartellata”. [0021] Specifically, in FIG. 3, going from bottom to the top, it is possible to follow the pinching, forming and cutting steps of the scalloping strip. The pinching means 6, in a preferred configuration, are two pinching discs, moved by motors 4 provided with stabilized variable speed reduction gear, and provided with corresponding shaped projections 6 ′ which realize the narrow section 11 by means of which the strip undergoes the pinching. This is shown in FIG. 3 a. The pinching is carried out at regular intervals each time a projection of a disc is at a projection of the other disc. Between two consecutive pinchings, i.e. between two consecutive couples of projections 6 ′ (see FIG. 3 b ) there forms a section 12, greater than the narrow section 11, inside which a shaped matrix 7 ′ of the shaping means 7 penetrates. As yet said, the shaping means 7 can comprise a wheel bearing a plurality of shaped matrices 7 ′, which penetrate in rotational synchrony the section 12. In this way it is realized the dough strip forming with uniform and constant dimensions. [0022] Advantageously, the number of projections 6 ′ and the number of shaped matrices 7 ′ are coordinated with respect to each other: n the first one, n+ 1 the second one. In this way it is sufficient to move the pinching discs 6, while the wheel of the shaped matrices 7 ′ is driven by the pulses that the projections 6 ′ will give to the corresponding shaped matrices 7 ′, in practice as it occurs in the gear between a driving gear and a driven gear. In FIG. 3 c it is shown a detail of the cutting means 9. Advantageously, said means comprise a blade positioned on one of the pinching discs and in rotation therewith. The blade, in practice, is arranged on the pinching mean instead of a projection 6 ′. At the end of a complete rotation of the disc, in which the above described pinching and shaping steps are alternated, the blade cuts the segment of the pinched and formed strip which makes up the linear development of the “cartellata”. [0023] In the dimensioning of the machine, it is also important to coordinate the advancement speed of the conveyor belt 3 with the rotation speed of the pinching discs 6, so that at each full rotation of the discs, when the strip is cut to length, the same strip is advanced linearly of the useful dimension in order to carry out the rolling and to realized the “rose” shape of the “cartellata”. The values of the advancement speed of the belt and rotation of the pinching discs are such that it is possible to realize about 100 pieces (corresponding to a kilogram of dough) in 15-20 minutes. [0024] According to another aspect, the present invention relates also to a specific working and production method of the “cartellate” by means of the machine 100. The method comprises the flowing steps: positioning the dough scalloped strip on the conveyor belt 3 ; [0000] activating the conveyor belt along a predetermined direction, wherein the dough scalloped strip is kept in position by a guide means 8 ; lifting the side edges of the dough strip by means of conveyor rollers 5 to favour the half folding of the same strip and the next pinching step; pinching and forming the strip carried out by pinching means 6 and shaping means 7 in more points; cutting the strip to a predetermined length carried out by cutting means 9, rolling the strip carried out by a mobile means 10 thus realizing a “rose” shape. [0027] Therefore the method is characterized in that, thanks to the synchronism between the pinching discs and the shaping means, the pinching and shaping steps are carried out simultaneously, alternating with respect to each other during the movement of the dough strip. [0028] In addition to the embodiments of the invention, as above described, it is to be intended that there exist many other variants. Further, it is to be intended that said embodiments are only example and do not limit the object of the invention and its possible application or configurations. On the contrary, even if the above described description allows the experts in the field to carry out the present invention at least according an embodiment thereof, it is to be intended that many variations of the elements above described are possible without departing from the object of the invention, encompassed by the appended claims, literally interpreted and/or according to legal equivalents thereof.

Summary: A machine ( 100 ) for "cartellate" production comprising a support structure ( 1 ), a motorized conveyor belt ( 3 ) for transport of dough strips, on which dough guide means ( 8 ) operate, conveyor means ( 5 ), motorized pinching means ( 6 ), shaping means ( 7 ), cutting means ( 9 ) and movable rolling means ( 10 ); said pinching means ( 6 ) being configured to operate, by rotating around its central axis, the clamping of the dough strips; said pinching means ( 6 ) cooperating with said conveyor means ( 5 ) and with said shaping means ( 7 ) achieving the dough strips forming; said cutting means ( 9 ) being steadily fixed to one of said pinching means ( 6 ).

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Summarize: By. Paul Donnelley. Ex-Emmerdale star Cy Chadwick at Leeds Crown Court where he is facing charges of indecent assault. A former star of the soap Emmerdale star has gone on trial accused of forcing a young boy who looked up to him ‘like a big brother’ to perform a sex act on him. Cy Chadwick, 45, who played Nick Bates for ten years in the programme, is alleged to have indecently assaulted a boy between September 1985 and September 1987, when the schoolboy was eight or nine-years-old. The complainant was sleeping on a camp bed when the alleged abuse took place, a court heard. Now in his 30s, the complainant only reported it to police in May last year, telling police in a video interview that he came forward ‘because it’s always in the news - it’s constantly on my mind’. ‘How did I know he’s not done it to anybody else at the time. I thought he was going to get to be an old man and do it to someone else,’ he said during the interview played to Leeds Crown Court. ‘Something’s got to be done.’ Addressing the jury before the opening of Chadwick’s trial today, Judge Neil Clark told them: ‘The defendant played a part in the soap Emmerdale. It’s important when you hear the evidence, please remember you’re trying the defendant Cy Chadwick – not a character in the show and not the character he played in the show. Ex-Emmerdale star Cy Chadwick leaves Leeds Crown Court earlier today where he is accused of assaulting a small boy. ‘You try the case on what you hear in this room, about this man and not the character he played. You should try him in the same way as anyone else who appears before this court.’ Prosecuting, Geraldine Kelly told the court that Chadwick used to take the schoolboy out on day trips in his car, and giving him gifts, including a record player and records. The complainant, talking in a police interview video which was shown to the court, told: ‘We were friendly. ‘I looked up to him as a big brother to be honest.’ The Fox And Grapes pub in Pudsey, West Yorkshire, was owned by the mother and stepfather of Cy Chadwick and where the abuse is alleged to have happened. Asked what they would usually do together, the complainant said: ‘Just messing around with stuff. Board games, normal stuff. He used to take me out.‘I liked him. I did. ‘He bought me a record player, records, little trinkets which obviously I thought was brilliant. ‘He had the fanciest equipment anyone could buy because he had loads of money, obviously. He had every electrical gadget you could buy. ‘He had records, turntables, computers, fancy TVs.’ The complainant told how on the night in question he had been lying on the camp bed while Chadwick was lying on his bed, when Chadwick pulled back the covers. It is alleged that Chadwick said to the schoolboy: ‘Put it in your mouth, this is what grown-ups do when they’re friends’. The boy allegedly obeyed after which he described feeling ‘sick’ and ‘peculiar’. Afterwards, Chadwick told the boy ‘not to say anything’, and left the room, sleeping elsewhere, the court heard. The complainant said he had not seen Chadwick since. Chadwick, who now works in TV production and lives in Pudsey, West Yorks., watched the evidence silently from the dock. He denies one charge of indecent assault between September 17, 1985 and September 17, 1987. The trial continues. Sorry we are not currently accepting comments on this article

Summary: Cy Chadwick, 45, played Nick Bates. for ten years in the programme. He is alleged to have indecently assaulted a. schoolboy who was. 8 or 9 at the time between 1985 and 1987. Prosecution alleges Chadwick took the boy out on day trips and bought him many gifts including a record player, records and trinkets. Complainant, now in 30s, only made his allegation last year.

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Write a title and summarize: En haut à gauche Aurore Berge, en haut à droite Adrien Quatennens, en bas Julien Dive Thomas Samson/AFP, Jean-Luc Hauser/CC BY-SA 4.0, Thomas Samson/AFP Rencontre sur le renouveau politique en France et sur le rôle du Parlement, avec les députés Aurore Bergé (La République en marche, Yvelines), Adrien Quatennens (La France insoumise, Nord) et Julien Dive (Les Républicains, Aisne) dimanche 24 septembre, de 13 h 30 à 14 h 30, à l’Opéra Bastille. Un débat animé par Alexandre Lemarié. Réservation en ligne. Quelles sont les marges de manœuvre d’un nouveau député à l’Assemblée nationale? Alors que les 314 élus de La République en marche (LRM) disposent d’une majorité écrasante au Palais-Bourbon — et du soutien de poids des 47 députés MoDem — comment un jeune député LRM peut-il se faire entendre? Comment faire face aux critiques de l’inexpérience de ces élus souvent novices, après que de nombreux couacs ont eu lieu à la fin de juillet, et de quelle manière y remédier à la rentrée? Quant aux députés de l’opposition, comment conçoivent-ils leur rôle face aux troupes d’Emmanuel Macron : comme des forces d’opposition devant tout faire pour empêcher l’adoption des projets de loi de l’exécutif, ou comme des forces de proposition susceptibles d’amender les textes? Aurore Bergé, 30 ans, porte-parole du groupe majoritaire ; Adrien Quatennens, 27 ans, député de La France insoumise ; et Julien Dive, 32 ans, qui représente la nouvelle génération de droite, échangeront sur le rôle du Parlement à l’ère Macron et sur ce que peuvent changer les réformes institutionnelles. Ils exposeront ce que signifie pour eux le renouveau en politique. Intervenants : - Aurore Bergé Âgée de 30 ans, Aurore Bergé est l’une des figures montantes de La République en marche (LRM). Porte-parole du groupe majoritaire à l’Assemblée nationale, elle est la voix d’Emmanuel Macron dans les médias, qu’elle a rejoint en février 2017. Élue députée sous les couleurs LRM dans les Yvelines lors des dernières élections législatives, elle se dit « libérale, progressiste, féministe ». Elle a fait ses classes à l’UMP, auprès de Valérie Pécresse, avant de soutenir Nicolas Sarkozy, puis Alain Juppé. Article réservé à nos abonnés Lire aussi Les députés LRM, entre culture d’entreprise et discipline de parti - Adrien Quatennens Âgé de 27 ans, Adrien Quatennens est la révélation du groupe de La France insoumise (LFI) à l’Assemblée nationale. Le jeune protégé de Jean-Luc Mélenchon a fait son entrée au Palais-Bourbon après avoir été élu dans le Nord lors des élections législatives. Ce Lillois d’origine s’est rapidement fait repérer des médias par ses interventions percutantes dans l’hémicycle contre le projet de loi d’habilitation pour réformer le code du travail. Article réservé à nos abonnés Lire aussi Quatre figures montantes du Palais-Bourbon - Julien Dive Âgé de 32 ans, Julien Dive est l’un des représentants de la nouvelle génération de droite. Protégé de Xavier Bertrand, il avait pris la succession de ce dernier à l’Assemblée nationale en étant élu député Les Républicains (LR) de l’Aisne en mars 2016, lors d’une législative partielle, M. Bertrand ayant accédé à la présidence du conseil régional des Hauts-de-France. Il a été réélu dans la même circonscription un an plus tard. Réservation en ligne.

Title: Du renouveau en politique, pour quoi faire Summary: "Le Monde" organise dans le cadre du "Monde Festival" un débat sur le renouveau politique en France et sur le rôle du Parlement, avec les députés Aurore Bergé, Adrien Quatennens et Julien Dive, dimanche 24 septembre, de 13 h 30 à 14 h 30, à l'Opéra Bastille.

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Summarize: By. Nick Enoch. PUBLISHED:. 13:48 EST, 20 December 2013. |. UPDATED:. 13:54 EST, 20 December 2013. A Batman impersonator who was caught on CCTV handing in his friend to the police earlier this year has been spared jail today after being found guilty of burglary. Stan Worby, 40, took Daniel Frayne, 27, to a Bradford police station in February to answer an arrest warrant issued over claims he tried to cash a stolen cheque. Just like with the real Batman, the public wanted to unmask the hero - and father-of-five Worby was eventually identified. But today Worby stood in the dock - minus his costume - to be sentenced for a burglary seven weeks later in which he and Frayne stole almost £800 worth of power tools from a garage. Scroll down for video. Batman impersonator Stan Worby, who was caught on CCTV handing in his friend to the police earlier this year, was spared jail today after being found guilty of burglary. This image of Worby handing over Daniel Frayne to police in Bradford, West Yorks, in February caught the public imagination - but seven weeks later they were both charged with stealing £800 worth of power tools from a garage. During a one-day trial last month, a jury at Bradford Crown Court found Worby guilty of burglary after hearing that just after 4am on April 14, police found him and Frayne in a silver Mondeo car 50 metres away from a garage that had been burgled. Stashed in the boot was £770 worth of power tools including a drill set worth £150, three grinders worth between £50 and £80, a socket set worth £100 and a box of screwdrivers worth £40. Worby claimed he knew nothing about the stolen goods, and that Frayne had piled them into his car while en route to a car boot sale. The jury took just half an hour to convict Worby, and Judge David Hatton QC handed him a six-month jail sentence suspended for 12 months. He was also ordered to carry out 200 hours in unpaid work and pay an £80 victim surcharge. The court heard that Worby had a 'lengthy record' which started at the juvenile courts, and included burglaries, battery, drug offences and motoring offences. He was given a suspended jail term in 2010 for possession with intent to supply cannabis, and last appeared before courts in September 2011 where he was given an absolute discharge for depositing environmental waste. Frayne, left, was previously sentenced to eight months in prison for. burglary, handling stolen goods and breach of a community order. Worby was handed a six-month jail sentence suspended for 12 months. Mitigating, Simon Hustler said that Worby had been employed at the time of the burglary by Kirklees Council but was let go from this job after a local paper printed the story of the burglary on their front page. Judge Hatton told Worby: 'You have committed no offences of burglary or attempted burglary for 13 years. 'That is what prevents you going to prison immediately. 'The offence is so serious that it passes the custody threshold, but I impose a sentence of six months' imprisonment suspended for 12 months.' Frayne was previously sentenced to eight months in prison for burglary, handling stolen goods and breach of a community order

Summary: Stan Worby, 40, was filmed in February handing Daniel Frayne to West Yorks police. Worby and Frayne were charged with burglary seven weeks later. Pair were found by a car stashed with power tools near to where a garage was burgled, in April. Worby given six-month jail sentence, suspended for 12 months; Frayne was previously sentenced to eight months in prison for burglary, handling stolen goods and breach of a community order.

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Summarize: Background ESRA authorizes IES to conduct and support many different types of research and evaluations. Specifically, ESRA contains several key provisions related to the management, core functions, and processes of IES: all research conducted by IES is to use scientifically based research standards that include, where appropriate, making claims of causal relationships only in random assignment experiments; education evaluations conducted by IES are to employ experimental designs using random assignment, when feasible; all research, statistics, and evaluation reports conducted by or supported through IES must be subjected to rigorous peer review before being published or otherwise made available to the public; and the establishment of an advisory board—the NBES—whose duties include (1) advising and consulting with the Director of IES regarding its policies and approving the Director’s overall research priorities, and (2) reviewing and approving procedures for peer review and reviewing the work of IES to ensure the consistency of scientifically valid research. Education administers programs that support education research and technical assistance through grants and contracts involving several research groups—including the RELs and R & D Centers within IES and the Comprehensive Centers within the Office of Elementary and Secondary Education (see fig. 1). The National Center for Education Evaluation and Regional Assistance administers the REL program, a network of 10 regional entities that conduct applied research, develop and disseminate research and products, and conduct technical assistance and other activities to support the needs of state and local education agencies in their region. The Comprehensive Centers Program is a network of 22 technical assistance grantees that help to increase the capacity of state educational agencies (SEA) to assist districts and schools in meeting student achievement goals. Selected Comprehensive Centers focus on specific areas of expertise and produce research-based information and products for use by SEAs. Lastly, ESRA includes specific requirements for the administration of R & D Centers, which are designed to address areas of national need and each of which IES assigns at least one of the broad research topics outlined in the law. R & D Centers are also responsible for the production and dissemination of rigorous evidence and products that provide practical solutions to important education problems in the United States. The National Center for Education Research and the National Center for Special Education Research administer 17 R & D Centers in total. In addition to IES, other entities conduct education-related research and evaluations, and ESRA includes general requirements for the Director of IES to coordinate its research and evaluation work with these entities, both within Education and across the rest of the federal government. Within Education, the Office of Planning, Evaluation, and Policy Development (OPEPD) conducts analyses and program evaluations on behalf of the department. Other federal agencies, such as the National Science Foundation and the National Institute for Child Health and Human Development—part of the National Institutes of Health—also support education-related research and the directors of these agencies serve as nonvoting ex officio members on the NBES. IES Has Supported High-Quality Research, but Lacks Key Processes and Performance Measures in Some Areas In our ongoing work, we found that IES has substantially improved the education research field. In 2007, the Office of Management and Budget (OMB) assessed IES’s research and concluded that since its inception, IES had transformed the quality and rigor of research within Education and increased demand for scientifically based evidence of effectiveness in the education field as a whole. Likewise, many stakeholders we spoke with said IES’s research standards have improved the quality of Education’s research and had a positive influence on education research generally. More specifically, several stakeholders told us that IES products, such as its publications of education statistics reports, were useful for their work. While IES’s research grants and evaluations have resulted in many randomized controlled studies since the agency was established over 10 years ago, IES’s research standards also include guidelines for the implementation of other rigorous research methodologies, and it has recently funded studies using those methodologies, such as regression discontinuity or single-case designs. IES’s support of these multiple types of methodologies allows it to better meet its various stakeholders’ needs. However, IES’s research is sometimes of limited usefulness to policymakers and practitioners. Some stakeholders told us that the research and evaluations supported by IES may not be completed soon enough to inform the decision making of policymakers and practitioners on important questions. For example, officials from one constituency based organization for policymakers said that IES’s evaluation of Education’s Race to the Top and School Improvement Grant Programs will not be released in time to give states the opportunity to implement lessons learned from these studies before these programs’ funding expires. The peer review process may exacerbate timeliness concerns. In order to ensure the high quality of IES’s work, ESRA requires IES-supported research reports to be peer reviewed before being published. In the past year, however, the time it takes to complete this review process has substantially increased, from an average of 117 days in fiscal year 2011 to 175 days in fiscal year 2012. When asked for explanations for the increase in 2012, senior IES officials cited factors such as the complexity of the reports reviewed in that year and the time it took IES to work with its contractors on suitable responses to peer review comments. In accordance with GAO’s internal control standards, program managers should have access to and use operational data to determine whether they are meeting their agencies’ goals for effective and efficient use of resources. However, officials told us that while the peer review office within IES monitors the time taken for its review, IES does not monitor the time its Centers or contractors take to respond to peer review comments, which would allow it to take steps to mitigate delays, such as by holding contractors more accountable. We previously reported on and made several recommendations related to the timeliness and dissemination activities of the What Works Clearinghouse. See GAO, Department of Education: Improved Dissemination and Timely Product Release Would Enhance the Usefulness of the What Works Clearinghouse, GAO-10-644. (Washington, D.C.: July 23, 2010). experiment in Texas and the development of a system to monitor students’ social and emotional learning in a school district in Nevada. Although IES has made recent efforts to increase the relevance of its research, IES does not have a structured process for incorporating feedback from policymakers and practitioners into its research agenda. Though there is no single way for government agencies to conduct research, in prior work we developed a framework to identify key elements that promote a sound federal research program using guidelines from several leading national research organizations. Within that framework, we found that agencies should establish a structured process for developing their research and evaluation priorities that considers key stakeholders’ input. found individual IES Center Commissioners and the Director of IES have at times gathered input from groups of policymakers and practitioners, but that IES did not have an ongoing, structured process for collecting this input. Inconsistent outreach by IES may have contributed to gaps in its research. For example, stakeholders said that there is a shortage of research using varied methodologies that could allow for shorter turnaround times among IES-funded research projects. This framework includes five key elements: (1) agenda setting, (2) selecting research, (3) designing research, (4) conducting research, and (5) disseminating research results. See GAO-11-285. For more information on leading practices for research and evaluation planning, see also National Research Council, Rebuilding the Research Capacity at HUD (Washington, D.C.: National Academies Press, 2008) and American Evaluation Association, An Evaluation Roadmap for a More Effective Government, (September 2010), available at http://www.eval.org/d/do/107. accomplishments. In addition, according to our internal control standards and leading practices on performance management, agencies should establish performance measures for their activities and continually compare actual performance data against these goals. To measure the effectiveness of its research grants, IES counts the number of IES- supported interventions that have been determined to be effective in improving student outcomes in particular areas. However, IES officials told us that its current performance measures, which were developed after the agency was established in 2002, no longer capture the scope of IES’s current research and priorities. Furthermore, in some cases, senior IES officials told us they are not relevant to managing the agency’s operations. For example, one measure relies on the results of a survey of potential users of the What Works Clearinghouse, but IES officials told us they will not conduct this survey because they do not believe it would yield enough useful information to be worth the investment. Moreover, a new performance measure was reported in Education’s fiscal year 2014 budget request for IES that better reflects the results of its recent research. However this measure still does not include certain existing areas of IES research, such as research on the organization and management of schools and education systems. In addition, IES does not publicly report on the performance of the RELs, which constitute one of the agency’s largest investments. As we have reported, without performance measures, agencies may be at risk for failing to achieve their goals. IES officials told us they have begun work on revising their performance measures. Officials told us that they plan to include revised performance measures in Education’s fiscal year 2015 budget request for IES, and that it has begun discussions with OMB to establish these new measures. As of August 2013, IES officials told us they intend for the new performance measures they are developing to include all programs, including the RELs and its new grant programs for researcher-practitioner partnerships. Research and Technical Assistance Groups Take Steps to Disseminate Relevant Research, but IES Has Not Fully Assessed These Efforts Research and technical assistance groups have taken various steps to provide relevant research to the education field. More specifically, to identify topics of relevance, RELs, R & D Centers, and Comprehensive Centers have engaged policymakers and practitioners in planning research and technical assistance activities. For example, beginning in 2012, RELs were required to conduct their work through new or existing partnerships of practitioners, policymakers, and others—called research alliances—which would work together to use data and research to address specific concerns in education, such as improving low-performing schools or college readiness. All three groups use a range of methods to disseminate their research evidence and products, including publications, conferences, and in some cases, technical assistance resources specifically for teachers, such as professional development courses. Despite these efforts, stakeholders—including practitioners and policymakers—have raised concerns about the relevance and dissemination of some of the research and products these groups have produced. For example, a stakeholder group that represents local school districts, as well as two superintendents we spoke with, said they did not find REL research as relevant or as timely as other sources of research information. At the same time, several stakeholders said that the RELs’ new strategy of creating and working through research alliances could help increase the RELs’ focus on research questions of concern to the field. We also heard from stakeholders we spoke with that R & D Center research may have limited relevance to practitioners, because these centers consider the research community the primary audience for their products. As a result, they do not always adapt their research findings in a format that is readily understandable by practitioners, such as by producing non-technical reports and shorter research summaries. In addition to concerns about relevance, REL and R & D Center dissemination efforts are not always reaching policymakers and practitioners. Officials from some intermediaries—such as industry associations—we spoke with said their organizations disseminate research information to policymakers and practitioners. However, some noted that further efforts are needed to leverage intermediary groups to better market REL and R & D Center work to reach IES’s target audiences. IES does not have plans to evaluate the current group of RELs or R & D Centers, nor does it collect sufficient information to manage these groups’ efforts to disseminate relevant research. IES is still in the process of conducting a mandated evaluation of the prior group of RELs—whose contracts ended in 2011—but has no further plans to evaluate the current group of RELs. In addition, as of August 2013, IES had no plans to conduct a formal evaluation of the R & D Centers and there is no requirement to do so. Further, IES does not collect sufficient information about RELs and R & D Centers to manage these groups’ efforts to disseminate relevant research. For example, in 2012, IES, in collaboration with the RELs, developed nearly 50 performance indicators for these groups. IES officials told us that in August 2013 they had prioritized 24 of these indicators on which RELs must now report. However, IES has not established performance targets or goals for these selected measures. Were IES to establish targets or goals, there would be more incentive for the RELs to perform at the agency’s desired level, and IES would be better positioned to determine if RELs are meeting its expectations. Previous GAO work has indicated that agencies successful in measuring performance had performance measures including targets or goals that (1) demonstrate results; (2) are limited to the vital few; (3) cover multiple priorities; and (4) provide useful information for decision making. In addition, IES collects limited information to assess the R & D Centers’ dissemination efforts because IES does not require R & D Centers to report on their specific dissemination strategies or the strategies’ effectiveness. Despite its difficulty, we have previously reported on ways in which agencies can assess research programs with broad dissemination goals. IES Coordinates with Other Federal Agencies, but Education Faces Challenges in Funding Program Evaluations IES coordinates with other federal education research agencies on projects to increase federal agencies’ use of research evidence in guiding funding decisions. For example, IES co-led a joint Education-National Science Foundation working group to develop common evidence guidelines for federally-funded research in education. Several researchers we interviewed during our ongoing work, as well as two federal agencies we spoke with, said these guidelines will benefit the education field. In addition, IES helped develop and incorporate a tiered evidence framework for awarding grants for Education’s Investing in Innovation Fund (i3) grant program. According to an official from the Administration for Children and Families (ACF) at the Department of Health and Human Services, ACF has recently begun awarding its grants using Education’s i3 program model, which uses tiered-evidence standards to award larger grants to applicants with greater evidence of their programs’ effectiveness. In addition to working with other federal agencies, IES coordinates within Education to facilitate collaboration between the REL and Comprehensive Center programs. In our ongoing work, directors of both groups told us coordination has improved with the current group of RELs and Comprehensive Centers, which began in 2012. In addition, some directors said that the RELs’ new structure of research alliances has improved coordination, as some Comprehensive Centers are now members of REL research alliances. This coordination, according to some directors with whom we spoke, has helped to address confusion among some SEA officials about the appropriate role and tasks performed by each. Some directors told us they have conducted joint visits with SEA officials, as well as made efforts to sequence their work for their state clients to better meet their needs. For example, some REL directors said the Comprehensive Center may conduct initial planning work for a project, and the REL would later assist with any aspects of the project requiring data analysis or original research. Within Education, IES and OPEPD are jointly responsible for program and policy evaluation, and together, these offices lead a department-wide planning process to identify evaluation projects to conduct. However, according to Education officials, efforts to prioritize evaluation projects through this annual process are hindered in part because of statutory requirements related to funding and program evaluation, including the fact that amounts designated for evaluation purposes under a program must be used only for evaluating that specific program or its activities. Specifically, officials reported that Education does not have the authority to combine evaluation funds from programs across the Department and use them to evaluate any program. As a result, some evaluations may not occur, and high-priority evaluations may be delayed. In addition, OPEPD and IES officials said that smaller programs often have insufficient funds to conduct an evaluation. We have previously reported that many Education programs, especially smaller programs, have not been evaluated, which can limit the ability of Congress to make informed decisions about which programs to continue, expand, modify, consolidate, or eliminate. Further, the President’s Budget request contained a proposal to increase Education’s flexibility to conduct program evaluations, and bills pending in Congress to reauthorize the Elementary and Secondary Education Act, including that of this committee, would address some of these issues for certain education programs. In conclusion, since its creation more than a decade ago, IES has made significant contributions to strengthening the rigor of the education research field and has considerably elevated the demand for conducting and has promoted the use of scientifically based research in our nation’s education system. However, IES could continue to build on these efforts by improving its ability to release relevant and timely information to policymakers and practitioners. With better management of its product review process to ensure the more timely release of reports and a more structured process for incorporating stakeholders’ needs into its research agenda, IES would position itself to more fully deliver on its mission to be responsive to the needs of its various stakeholders. Similarly, comprehensive performance measures and routine assessment of its grantees’ and contractors’ dissemination strategies would ensure its stewardship of federal investments in these areas. In addition, the ability to prioritize and conduct effective evaluations is critical to helping make the best use of limited resources and to support Congress in making informed decisions about which programs to continue, expand, modify, consolidate, or eliminate. As we complete our ongoing work, we will consider any recommendations needed to address these issues. Chairman Kline, Ranking Member Miller, and Members of the Committee, this concludes my prepared statement. I will be pleased to answer any questions that you may have. GAO Contact and Staff Acknowledgments For future contact regarding this testimony, please contact George A. Scott at (202) 512-7215 or scottg@gao.gov. Key contributors to this testimony were Scott Spicer (Assistant Director), Lucas Alvarez, Deborah Bland, Nora Boretti, David Chrisinger, Elizabeth Curda, Rachel Frisk, Dana Hopings, Jean McSween and James Rebbe. 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: The federal government has a longstanding role in conducting education research and collecting related data. The Education Sciences Reform Act of 2002 established IES as Education's primary research and evaluation arm. With a budget of just under $600 million in fiscal year 2013, IES has a broad mission to provide its research, evaluations, and statistics to a wide variety of stakeholders, including researchers, parents, educators, and the general public. This testimony reports on ongoing GAO work about IES. A full report will be issued later this year. Based on preliminary findings, this testimony will focus on: (1) the extent to which IES supports high-quality research and fulfills its mission, (2) the extent to which selected Education research and technical assistance groups disseminate relevant products to the education field, and (3) IES's coordination within Education and with other federal agencies. For this work, GAO reviewed agency documents and relevant federal legislation, interviewed agency officials and stakeholders, and analyzed information from selected research and technical assistance groups. GAO also compared IES's practices to established guidelines for internal controls and effective program management and performance reporting. The Institute of Education Sciences (IES) supports high-quality research, according to stakeholders, but lacks certain key procedures needed to fulfill other aspects of its mission. Since its inception, IES has substantially improved the quality of education research. However, stakeholders expressed some concerns about IES's ability to produce timely and relevant research that meets their various needs. For example, IES's efforts to respond quickly to its stakeholders are slowed, in part, because the time IES's products have spent in peer review substantially increased this past year, and IES does not monitor some aspects of these timeframes. In addition, IES does not have a structured process for incorporating stakeholder input into its research agenda, which previous GAO work has shown to be key to sound federal research programs. Lastly, IES's performance measures do not fully reflect its current programs, which is not consistent with GAO's leading practices for performance management. IES officials said, however, that they have begun to develop new performance measures for all of their programs. Although the Department of Education's (Education) research and technical assistance groups have taken steps to produce and disseminate relevant research to the field, IES does not always assess these efforts. Some stakeholders raised concerns about the relevance and dissemination of research and products from the Regional Educational Laboratories (REL) and Research and Development Centers (R & D Center). For example, they told us that these groups do not always adapt their products for use by both policymaker and practitioner audiences. Further, IES has not fully assessed REL and R & D Center relevance and dissemination efforts. As a result, IES does not know if these efforts are effective in meeting their mandated goal of providing usable research and information to policymakers and practitioners. GAO's prior work on information dissemination suggests that further assessment could help to inform IES's oversight of the RELs and R & D Centers to improve these groups' dissemination to key audiences. IES works with federal education research agencies to increase the use of research evidence in federal decision-making, but according to officials, has limited ability to prioritize evaluations. IES and the National Science Foundation recently developed guidelines to help improve the quality of evidence resulting from federally-funded education research, which stakeholders said will benefit the education field. Within the department, IES plans evaluations of Education programs in concert with various other offices. However, Education officials said funding and program evaluation requirements prevent the agency from combining evaluation funds across programs, which limits their ability to conduct the evaluation projects they consider most important.

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Write a title and summarize: Neurological involvement occurs throughout the leprosy clinical spectrum and is responsible for the most feared consequences of the disease. Ultrasonography (US) provides objective measurements of nerve thickening and asymmetry. We examined leprosy patients before beginning multi-drug therapy aiming to describe differences in US measurements between classification groups and between patients with and without reactions. Eleven paucibacillary (PB) and 85 multibacillary (MB) patients underwent nerve US. Twenty-seven patients had leprosy reactions (type 1, type 2 and/or acute neuritis) prior to US. The ulnar (at the cubital tunnel–Ut–and proximal to the tunnel–Upt), median (M) and common fibular (CF) nerves were scanned to measure cross-sectional areas (CSAs) in mm2 and to calculate the asymmetry indexes ΔCSA (absolute difference between right and left CSAs) and ΔUtpt (absolute difference between Upt and Ut CSAs). MB patients showed greater (p<0. 05) CSAs than PB at Ut (13. 88±11. 4/9. 53±6. 14) and M (10. 41±5. 4/6. 36±0. 84). ΔCSAs and ΔUtpt were similar between PB and MB. The CSAs, ΔCSAs and ΔUtpt were similar between PB patients with reactions compared to PB patients without reactions. MB patients with reactions showed significantly greater CSAs (Upt, Ut and M), ΔCSAs (Upt and Ut) and ΔUtpt compared to MB patients without reactions. PB and MB showed similar frequencies of abnormal US measurements. Patients with reactions had higher frequency of nerve thickening and similar frequency of asymmetry to those without reactions. This is the first study to investigate differences in nerve involvement among leprosy classification groups using US before treatment. The magnitude of thickening was greater in MB and in patients with reactions. Asymmetry indexes were greater in patients with reactions and did not significantly differ between PB and MB, demonstrating that asymmetry is a characteristic of leprosy neuropathy regardless of its classification. Neurological involvement is present throughout the leprosy clinical spectrum, and nerve impairment is responsible for the most feared consequences of the disease; therefore, some authors advocate that leprosy should be regarded as a chronic neurological condition rather than a skin disease [1–5]. Several authors have postulated that tuberculoid patients have asymmetric nerve thickening, lepromatous patients have symmetric and diffuse involvement, and borderline patients have variable and usually intense nerve enlargement [1,3, 6,7]. Leprosy reactions (acute neuritis, types 1 and 2 reactions) can lead to additional nerve damage due to immune-mediated mechanisms. They may be superimposed on the chronic course of the disease and require immediate treatment [1,3, 5,6]. Neurophysiologic and imaging studies can be used to investigate neurological impairment in leprosy. Although neurophysiology provides detailed information about dysfunction of affected nerves, it does not reveal anatomic changes, such as thickening and fascicular pattern changes [8,9]. High-resolution ultrasonography (US) permits examination of multiple nerve trunks over a long course in a few minutes, and compared with magnetic resonance imaging, US is considered more accessible and reasonably precise [8,10–12]. Furthermore, it is reported that US is more accurate than clinical palpation for assessment of peripheral nerve enlargement [11] and it provides objective measurements of peripheral nerve thickening and asymmetry [13]. To our knowledge, no published studies have investigated differences in US nerve measurements across the leprosy spectrum. The purpose of this study was to investigate peripheral nerve thickening and asymmetry, as evaluated through US measurements, in leprosy patients, examining differences between leprosy types grouped according to the Ridley-Jopling and WHO classifications and to assess the influence of leprosy reactions on US findings. The Ethics Committee of the Clinics Hospital of Ribeirão Preto Medical School approved the study (process n°02663112. 0. 0000. 5440). Written informed consent was obtained from all participants. Some participants were minors and their parents provided written consent on behalf of them. Ninety-six consecutive leprosy patients that were referred to Leprosy Reference Center at the Clinics Hospital of Ribeirão Preto Medical School were included in the study and underwent bilateral high-resolution US of the peripheral nerves before starting World Health Organization (WHO) multi-drug therapy. Leprosy diagnosis was established based on clinical signs and symptoms, skin smears, skin biopsy, and neurophysiologic examination when necessary. Patients were classified according to the Ridley-Jopling [14] classification in five groups: tuberculoid (TT), borderline-tuberculoid (BT), borderline-borderline (BB), borderline-lepromatous (BL), and lepromatous (LL). Patients were also grouped in paucibacillary (PB) and multibacillary (MB) according to the WHO operational classification [15]. Medical charts were reviewed for the identification of any leprosy reactions prior to US evaluation. Leprosy reactions were classified as cutaneous reactions (type 1 or 2) associated or not with acute neuritis. Type 1 cutaneous reactions were defined as presence of erythema and edema of skin lesions. There may be accompanying neuritis and edema of the hands, feet, and face. Type 2 reactions (erythema nodosum leprosum) were defined as presence of tender subcutaneous skin lesions. There may be accompanying neuritis, iritis, arthritis, orchitis, dactylitis, lymphadenopathy, edema, and fever. Neuritis was diagnosed if patients presented acute inflammation of one or more peripheral nerve trunk detected by swelling and/or functional impairment with spontaneous nerve pain and/or nerve tenderness on palpation. To statistical analysis patients were divided according to the presence of any kind of reaction prior to US examination or absence of reactions. Patients with a history of diabetes mellitus, hypothyroidism, human immunodeficiency virus infection, trauma-related peripheral nerve disease or alcoholism were excluded from the study. Musculoskeletal radiologists with previous fellowship training in nerve imaging performed all US sessions using a 12-MHz linear transducer model HDI-11 (Philips Medical Systems, Bothell, Washington, USA). Patients were examined in a seated position with 45° flexed elbows and 90° flexed knees. The ulnar (at the cubital tunnel area–Ut–and proximal to the tunnel–Upt), median (M) and common fibular (CF) nerves were systematically scanned along the transverse and longitudinal axes. Ulnar nerves were scanned from the middle third of the arm to the middle third of the forearm. M nerves were evaluated at the middle and distal thirds of the forearm. CF nerves were evaluated from the distal third of the thigh to the knee at the fibular head. In some cases, it was not possible to examine nerves bilaterally due to amputation, cutaneous ulcers or other cutaneous alterations at the site of examination. Nerve cross-sectional areas (CSAs) were measured by freehand delimitation at the inner borders of the echogenic rims of the nerves. The measurements were performed using the electronic cursor at the time of examination, and the CSAs were assessed at the level of maximum nerve thickening. Ulnar nerve maximum CSAs were measured in two different regions: above the medial epicondyle proximal to the cubital tunnel (Upt) and at the cubital tunnel (Ut). CSA measurements were used to calculate nerve asymmetry as follows: (1) the differential CSA index (ΔCSA), which was calculated as the absolute difference between CSAs for each nerve point from one side to the contralateral side, and (2) the differential Ut-Upt index (ΔUtpt) of the ulnar nerves, which was calculated as the absolute difference between the largest and smallest CSAs of Upt and Ut points of ulnar nerves on the same side. High ΔCSA values reflect nerve asymmetry with the contralateral nerve. High ΔUtpt values reflect non-uniform and focal thickening of the ulnar nerve between the tunnel and pre-tunnel areas. For the classifications of the CSA, ΔCSA and ΔUtpt values as normal or abnormal, we used previously published values obtained from healthy volunteers [13], considering as abnormal values that were greater than the mean plus 2 standard deviations. Additionally, to compare the PB and MB groups, we performed two different analyses: one including all patients and another including only those patients with at least one abnormal measurement for each variable (CSA, ΔCSA and ΔUtpt). With second analysis, we expected to reduce the possible bias due to the existence of different frequencies of abnormalities in PB and MB. Statistical analysis was performed using JMP software version 10. 0 (SAS Institute Inc., Cary, NC). We performed the Wilcoxon test to compare the means of two groups. To analyze differences between the means of three or more groups, we performed the Kruskal-Wallis test. For the comparison of proportions, we used the two-tailed Fisher’s exact test. Probability (p) values less than 0. 05 were considered significant. The average age ± standard deviation of the patients was 45. 9 ± 16 years (age range 16–85 years); 56 (58. 3%) of the patients were men, and 40 (41. 7%) were women. The clinical classifications and incidences of neuritis, type 1 and/or type 2 cutaneous reactions occurring prior to US are presented in Table 1. For clinical data and CSAs values of each patient see supporting information (S1 Table). Table 2 shows the results for CSA, ΔCSA and ΔUtpt of the nerves studied for the five types of leprosy according to the Ridley-Jopling classification. For all nerve points studied, we observed maximum mean values of CSA in LL and BL patients; the percentages of enlarged nerves (abnormal CSAs) were also greater in these two groups, reaching 81. 3% incidence of enlarged ulnar nerves at the cubital tunnel (Ut) in LL patients. Although the mean values of ΔCSAs showed variable results, with maximum values in different types of leprosy, we observed higher incidences of asymmetric nerves (abnormal ΔCSAs) in LL and BL patients. LL patients showed the maximum ΔUtpt mean and the highest percentage of nerve asymmetry considering this measurement. Table 3 shows the CSAs, ΔCSAs and ΔUtpt according to the WHO operational classification for all patients and for the patients with at least one abnormal measurement for each variable. The two analyses yielded similar results, with the MB patients displaying greater CSAs, ΔCSAs, ΔUtpt and frequency of abnormalities at the Upt, Ut and M nerves. On the other hand, at the CF nerve, the PB patients showed similar or slightly greater CSAs and ΔCSAs. Because borderline patients show variable nerve impairment, we compared only the two polar forms, thereby aiming to better characterize and highlight nerve thickening and asymmetry. For this analysis, we selected only TT (n = 8) and LL (n = 8) patients who had at least one abnormal measurement for each variable. Significantly greater CSAs were observed in the LL patients at the Upt (7. 64±1. 91 /14. 9±9. 08 mm2), Ut (9. 53±6. 14 / 15. 56±4. 57 mm2) and M (6. 36±0. 84 / 12. 04±4. 75 mm2) nerves; no significant difference was observed at the CF nerve. Although the ΔCSAs were also greater in LL patients, no statistically significant differences were observed at the Upt (2. 01±2. 12 / 8. 11±8. 96 mm²), Ut (4. 41±5. 25 / 4. 44±2. 23 mm2) and M nerves (0. 86±0. 48 / 1. 94±1. 91 mm2). At the CF nerve, the TT patients showed a non-significantly greater ΔCSA (9. 29±18. 14 / 2. 4±2. 23 mm2). It is important to report that one TT patient had the maximum ΔCSA value observed in the study at this nerve (52. 7mm2). No significant difference in the ΔUtpt was observed between TT and LL (6. 02±6. 34 / 8. 18±5. 93 mm2). The CSAs, ΔCSAs and ΔUtpt were similar between PB patients with previous leprosy reactions (neuritis, type 1 and/or type 2 reactions) compared to PB patients with no reaction. MB patients with reactions showed significantly greater CSAs compared to MB patients without reactions at the Upt (14. 20±11. 09 / 8. 20±5. 12 mm2), Ut (14. 73±15. 73 / 10. 62±6. 13 mm2) and M nerves (10. 88±5. 99 / 8. 40±4. 32 mm2). ΔCSAs were also significantly greater in MB patients with reactions at the Upt (7. 39±11. 07 / 2. 84±5. 59 mm2) and Ut nerves (8. 33±19. 29 / 3. 06±4. 61 mm2). In addition, the ΔUtpt was significantly greater in MB patients with previous leprosy reactions (5. 82±7. 36 / 3. 85±5. 21 mm2). Considering that abnormalities in at least one US measurement reflect neuropathy, we sought to evaluate differences in the frequencies of abnormalities between PB and MB patients and also between patients who did and did not have reactions prior to US. The results of this analysis are presented in Table 4. We found that nerve asymmetry detected on US is characteristic of leprosy, with similar frequencies of abnormal measurements found in PB and MB patients. Furthermore, we observed a tendency toward higher ΔCSA and ΔUtpt values in the latter group. As expected, we also found that thickening (CSA) of the peripheral nerves was more pronounced in MB. Another important finding was that MB patients with previous leprosy reactions (neuritis, type 1 and/or type 2) had greater CSA, ΔCSA and ΔUtpt values than MB patients without reactions; however, among the PB patients there was no significant difference in nerve thickening and asymmetry comparing the groups with and without reactions. This is the first study in which differences in peripheral nerve thickening and asymmetry among patients of different leprosy classification groups were systematically investigated using accurate US measurements prior to specific treatment (WHO multi-drug therapy); the inclusion of patients at different stages of treatment in previous studies [10,11,16] could weaken conclusions about variations in the pattern of nerve involvement. Two studies in which US evaluation was performed before treatment have been reported, but those studies did not address differences between leprosy classification types [8,13]. There is a growing interest in US as a diagnostic tool for peripheral neuropathies. Nerve palpation is subjective and requires expertise [5], and even among trained professionals, the reliability of palpation of superficial peripheral nerves is unsatisfactory, with poor agreement between evaluators [17]. In a study that compared clinical examination and US of peripheral nerves in 20 leprosy patients and 30 healthy volunteers, Jain et al. [11] concluded that clinical examination was subjective and inaccurate, whereas US provided an objective evaluation of nerve damage and could identify more extensive involvement. Another previous study showed that US abnormalities may be present in patients with normal neurophysiological findings [8]. The concept that US should always be performed in addition to neurophysiological studies during the investigation of peripheral neuropathies is currently gaining strength [8,9, 12]. In our study, we found that peripheral nerve involvement, objectively evaluated by US, is common in all types of leprosy. Considering that the presence of one or more abnormal nerve measurements reflects neuropathy, we observed similar frequencies of neuropathy in the PB and MB patients. The frequency of abnormalities was high even among the PB patients (72. 7% of the PB patients had at least one thickened or asymmetric nerve), suggesting that enlargement and asymmetry of the peripheral nerves may be more frequently detected on US, corroborating the findings of Jain et al. [11]. These results support the idea that leprosy is a neurological disease [1–3,5] and reinforce the importance of conducting a detailed neurological exam for all patients. The ulnar nerve was the most commonly involved nerve in MB patients; up to 81. 3% of LL patients showed abnormal CSA values in the cubital tunnel area. Furthermore, thickening of peripheral nerves was also frequent in PB patients, especially at the common fibular nerve. The thickening was clearly more pronounced in MB patients at superior limb nerves, even when we analyzed only the group of patients in which some neuropathy was detected at US: the CSAs were significantly greater in the MB group, and the 95% confidence interval showed no overlap between PB and MB mean values at the ulnar (Upt and Ut) and median nerves. At the common fibular nerve, no difference was found between PB and MB patients; furthermore, the common fibular nerve was the most frequently affected nerve in PB patients, in agreement with observations from clinical studies that suggest that this nerve can be impaired even early in the disease course [7,18]. Clinical expertise and previous studies indicate that PB patients (especially TT) have asymmetric nerve enlargement, whereas MB patients, notably the patients exhibiting the lepromatous polar form of the disease, have symmetric and diffuse involvement [1,6]. Frade et al. [13] have shown that ΔCSA and ΔUtpt possess high specificity for a diagnosis of leprosy when leprosy patients are compared to healthy individuals and that these measurements allow for the objective quantification of peripheral nerve asymmetry. We found that the asymmetry measurements (ΔCSA and ΔUtpt) did not significantly differ between the PB and MB patients; moreover, our data indicate a weak tendency toward greater asymmetry in the latter group. The ΔCSA means were greater in MB at the Ut, Upt, and median nerves, and the highest percentages of abnormal ΔCSA values for all studied nerves were found in the LL and BL patients. ΔUtpt showed the same trend, with greater values in the MB patients and the highest percentage of asymmetry in LL patients. We compared only the two polar forms of the disease (TT and LL), aiming to emphasize the findings. Our results confirmed previous analyses, showing significantly greater values of CSA (Upt, Ut, and median nerves) in LL patients. Although asymmetry measurements did not differ significantly between TT and LL patients, we observed a tendency toward greater asymmetry in LL patients. The results for the common fibular nerve showed an opposite tendency, with greater values of ΔCSA (although without statistical significance) in TT patients; nevertheless, we emphasize that one TT patient had the maximum ΔCSA value at common fibular nerve (52. 7mm2), which could have increased the mean of this group. Leprosy reactions, especially acute neuritis, can lead to severe nerve impairment and require immediate treatment with steroids and other immunosuppressive drugs. Consistent with the results of previous studies, our results indicate that more severe enlargement and asymmetry of nerves occurs in MB patients with previous or active reactions in all evaluated nerves except for the common fibular nerve. The nerve measurements among PB patients with and without reactions did not show significant differences. Despite the fact that all patients included in our study were examined before beginning WHO multi-drug treatment, the majority of patients who had a history of reactions were already receiving anti-reaction treatment (prednisone, thalidomide and/or azathioprine) at the time of the US exam; thus, nerve swelling and asymmetry might have been diminished in these patients. Taken together with previous results, these findings indicate that chronic M. leprae nerve infection and its ability to cause inflammation and fibrosis, as well as the presence of leprosy reactions, are important causes of nerve thickening and asymmetry. Previous studies have addressed the influence of reactions on peripheral nerve imaging findings. Martinoli et al. [10] investigated US and magnetic resonance imaging findings for 23 leprosy patients and concluded that patients with previous or active leprosy reactions had nerve enlargement and fascicular abnormalities. These authors also identified the presence of intraneural color Doppler signal in patients with active reactions. Jain et al. [11] have found that increased blood flow can be present in multiple nerves distant to the affected dermal lesion. In our study, we investigated all patients for the presence of Doppler signal; however, because most of the patients with clinical signs of reactions were already receiving anti-reaction treatment at the time of the US exam, Doppler signals were observed only in a small number of them, and the Doppler results are not reported here. One limitation of our study is the lack of neurophysiological correlation, which could provide useful information concerning nerve function abnormalities. However, our main objective was the evaluation of anatomic alterations, represented by nerve thickening and asymmetry. Two other studies in which ulnar nerve neuropathy was investigated using US and electrophysiology [8,16] found US abnormalities in patients with normal neurophysiological findings. Those studies also demonstrated that leprosy patients can have normal ulnar nerve US findings with significant electrophysiological changes. The authors concluded that leprosy patients can exhibit abnormal nerve anatomy with preserved nerve function and vice versa [8]. Therefore, although we did not perform electrophysiological tests, we consider that our results improve the understanding of anatomic changes in leprosy neuropathy. Despite the fact that the sample size of this study is the largest reported for any study of the use of US for leprosy neuropathy evaluation, the division of the patients into six clinical classification types resulted in the presence of a small number of subjects in each group. Perhaps the numbers of patients included in the groups with the polar forms TT and LL were not large enough to reveal significant differences in asymmetry measurements between the groups. In conclusion, the present study contributes to the understanding of the pattern of peripheral nerve involvement in leprosy patients. The US results reported here have revealed that thickening and asymmetry are common in leprosy patients and that these abnormalities occur at similar frequencies in PB and MB. Moreover, the magnitude of thickening was greater in the MB patients and in those with previous leprosy reactions. Nerve asymmetry did not significantly differ between the PB and MB patients, demonstrating that asymmetry is a characteristic of leprosy neuropathy regardless of its classification.

Title: Asymmetric Nerve Enlargement: A Characteristic of Leprosy Neuropathy Demonstrated by Ultrasonography Summary: Leprosy is an infectious disease that affects the peripheral nerves, leading to nerve thickening, asymmetry and dysfunction; therefore, early detection of leprosy neuropathy is important for preventing deformities and disabilities. We examined peripheral nerve involvement using ultrasonography (US) in 96 leprosy patients prior to treatment, aiming to better understand differences in neuropathy patterns between leprosy classification groups and between patients with and without leprosy reactions. Patients underwent bilateral US of the ulnar (at the cubital tunnel and proximal to the tunnel), median and common fibular nerves to measure thickening and asymmetry. We found that nerve thickening was more severe in patients with a high bacillary load (multibacillary) and in those with previous reactions. Nerve asymmetry measurements were greater in the patients with previous reactions. Asymmetry did not differ significantly between the paucibacillary and multibacillary patients, demonstrating that asymmetry is a characteristic of leprosy neuropathy regardless of its classification.

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Summarize: Gary Player was camped out under the big oak tree behind the clubhouse, signing autographs and posing for pictures, as he did 50 years ago. At the many snack stands scattered around Augusta National, pimento cheese sandwiches in green wrappers were selling for 1962 prices _ a buck fifty. A place stuck in a time warp, just like the men in green jackets who run it. They gathered Wednesday for their annual State of the Masters report and, despite some dying azaleas that will make this year's Masters a bit less colorful, it seems that things have never been better in one of the last bastions of exclusivity in sports. The Cadillacs out front no longer have tail fins the length of belly putters, and the players don't smoke anymore between shots. But the snacks are still cheap, the old-timers keep coming back, and the green jacket for winning remains the most coveted prize in golf. One other thing hasn't changed: Membership in the club is by invitation only, and women, it seems, need not apply. Why that's become an issue again this year is largely a matter of circumstance _ the recent appointment of a woman executive to head IBM, one of the main sponsors of the Masters. As far as anyone knows, Virginia Rometty hasn't asked for her own green jacket, but since the last four CEOs at IBM, all male, were members, she goes to the top of the list by default. Hardly reason to take to the streets, as activist Martha Burk did a decade ago in an ill-fated attempt to open up Augusta National's membership to women. Even the most ardent feminists would be hard-pressed to march on behalf of a millionaire business executive who lives in the rarified air of the privileged elite. Lee Westwood found the whole thing amusing. "What gender issue? I'm a man," the Englishman said. Still, two decades after a black man finally was given a green jacket to wear, the basic issue is one of equality. I'm not going to become a member of Augusta National, and odds are you aren't, either, for reasons that have nothing to do with race or sex. But to automatically exclude half the world's population because it's female just seems so 1962. Not to the men in the green jackets, of course. They bristle when the subject is raised and immediately hide behind the only protective cover they know: It's their club, and they alone will decide who belongs. "As has been the case whenever that question is asked, all issues of membership are now and have been historically subject to the private deliberations of the members," Augusta National chairman Billy Payne said Wednesday. "That statement remains accurate and that remains my statement." Fair enough, I guess. Rich people can be picky when it comes to who they share a tee box with, but in the end it is their club and they can do what they wish. Problem is, when Payne said that he had barely finished talking about Augusta National's role in golf and its responsibilities for helping grow the number of people who play the game. The club wants to get more people playing, he said, especially the girls and boys who are the future of the game. As it stands now, those boys can dream of one day wearing green jackets themselves. The girls can't. That led to an exchange between reporters and Payne that, while testy, bordered on comical. Pressed several times on what he would tell his granddaughters about their chances of joining the club, Payne finally answered: "My conversations with my granddaughters are also personal." OK. What would Payne tell a reporter's daughters? "I don't know your daughters," Payne replied. If IBM's Rometty wanted to make it an issue she certainly could, but so far she and the company, at least publicly, seem satisfied with being one of the three major sponsors of the Masters and leaving the push for change to others. And, as a pressing social issue, equality at Augusta National doesn't exactly rank up there with making sure every child in America grows up able to read and write. Burk herself is watching this one from afar, not about to get burned again. Still, she couldn't help but tweak IBM and the green jackets who caused her so much grief. "I think it's astounding that one of the largest corporations in the world is having their strings pulled by a bunch of old guys in Augusta," she said. That's the way things happen at the Masters. No one dares tell the guardians of Augusta National what to do, or when they should do it. It may be a benevolent dictatorship, but no one doubts it is a dictatorship. By the time Tiger Woods and Rory McIlroy tee off Thursday, the IBM issue likely won't matter much. Attention will turn to the golf itself and the issue will be forgotten for at least another year. Best of all, the pimento cheese sandwiches will still only be a buck fifty. ____ Tim Dahlberg is a national sports columnist for The Associated Press. Write to him at tdahlberg(at)ap.org or follow at http://twitter.com/timdahlberg Bloomberg News William Porter Payne, chairman of Augusta National Golf Club speaks at the green jacket presentation at the 2011 Masters Tournament at Augusta National Golf Club on April 10, 2011 in Augusta, Georgia. Photographer: Jamie Squire/Getty Images Augusta National Golf Club Chairman Billy Payne said he wouldn’t discuss the organization’s lack of female members amid questions about a possible invitation to Virginia “Ginni” Rometty of International Business Machines Corp. (IBM:US) “All issues of membership remain the private deliberations of the membership,” Payne said at his annual news conference on the eve of the Masters Tournament at the club in Augusta, Georgia. “That statement remains accurate.” Augusta National is again under scrutiny for its all-male membership following the appointment of Rometty as chief executive officer of IBM, one of three Masters Tournament sponsors. While Augusta National has no formal policy against female members, it hasn’t admitted one in its eight decades. It has traditionally invited IBM’s chief executive officer for membership. “We don’t talk about our private deliberations,” Payne said. “We especially don’t talk about them when a named candidate is a part of the question.” Edward Barbini, a spokesman for Armonk, New York-based IBM, has previously declined to comment on the issue. Nine years ago, Martha Burk, then head of the National Council of Women’s Organizations, staged a small protest near the front entrance of the club founded in 1933 by golf champion Bobby Jones and Wall Street financier Clifford Roberts. Burk had written the previous June to Hootie Johnson, then Augusta National’s chairman, asking him to to review the club’s membership policy to avoid it “becoming an issue” during the 2003 tournament. Johnson responded that he wouldn’t be “bullied” into allowing female members. “There may well come a day when women will be invited to join our membership,” Johnson said in a media statement in July 2002. “But that timetable will be ours and not at the point of a bayonet.” Sponsor Requests Burk then wrote to sponsors, including IBM, asking them to withdraw their support for the tournament. Augusta National subsequently announced it wouldn’t air commercials during the 2003 event and suspended its sponsorships with IBM, Coca-Cola and Citigroup for the 2003 and 2004 tournaments. Only IBM returned in 2005. As players prepare for tomorrow’s opening round of the Masters, golf’s first major tournament of the year, the issue has become part of the discussion. Players, who are invited to participate, have been reluctant to talk about the club’s membership. “It’s a completely private club and they can do what they want,” Sweden’s Henrik Stenson, the 2009 Players championship winner, said yesterday at the club’s practice range. “I don’t really have anything to say on it.” Previous CEOs While the club doesn’t disclose the names of its members, a 2010 partial list obtained by Bloomberg News and 2004 documents published by the Augusta Chronicle and USA Today show the past four IBM CEOs were members, beginning with John R. Opel, who ran the company from 1981 to 1985 and died last year. John F. Akers, IBM’s chief from 1985 to 1993, and Louis V. Gerstner, who helped turn around IBM as CEO from 1993 to 2002, also were members. The refusal to consider female membership amid Burk’s protest prompted the resignations from the club of then-Treasury Secretary nominee John Snow and former CBS Corp. (CBS:US) Chief Executive Officer Thomas Wyman, who called the policy “pigheaded.” Wyman died about five weeks later. Snow was secretary of the treasury under President George W. Bush from 2003 to 2006, and now is chairman of Cerberus Capital Management LP, an investment firm. IBM, Exxon Mobil Corp. (XOM:US) and AT&T Inc. (T:US) are now the tournament’s only U.S. sponsors. Their agreements enable the companies to entertain executives and clients in private hospitality cabins, tucked into a wooded area along the left side of the 10th hole. More CEO Members Exxon Mobil CEO Rex Tillerson is a member, as is AT&T CEO Randall Stephenson. Ed Whitacre, Stephenson’s predecessor, is a member and in December gave Stephenson his place as a director on the U.S. PGA Tour’s board. Rick Burton, a former chief marketing officer for the U.S. Olympic Committee, said a Masters sponsorship probably costs at least $10 million annually. Augusta didn’t have a black member until 1990, when it extended an invitation to Gannett Co. (GCI:US) television President Ron Townsend, who still belongs. That move followed the PGA of America’s decision to move its annual championship, the season’s final golf major, from Alabama’s Shoal Creek because of that club’s all-white membership. Payne, who succeeded Johnson as chairman of the tournament and the golf club in 2006, has previously said he has “no specific timetable” on possibly ending all-male membership. He repeatedly declined to comment on membership issues at today’s news conference. Atlanta Olympics As the chief organizer of the 1996 Olympics in Atlanta, Payne was known as a progressive leader. His Olympic organizing team included influential women, including Ginger Watkins, Linda Stephenson and Cindy Fowler. Since Payne took over as chairman of Augusta National, the tournament has made changes including a ticketing program for children, the establishment of an Asian amateur championship and the creation of the Masters Tournament Foundation. The charitable foundation invests in organizations to help promote golf globally. Funding comes from private donations, proceeds from the tournament and sales of a 2012 Tiger Woods Masters- related video game. In 2010, Payne publicly criticized the extramarital affairs of Woods, a four-time Masters winner, saying “our hero did not live up to the expectations of a role model that we sought for our children.” Occasional Golfer Unlike her predecessors at IBM, Rometty said she plays golf only occasionally. She and her husband, Mark, prefer scuba diving and split their time between White Plains, New York, and Bonita Springs, Florida. IBM is featured in the tournament’s TV commercials and runs the event’s website, mobile-phone applications and media-center technology. IBM’s official sponsorship began 10 years ago, though it had been involved earlier. The company has run the masters.com website since 1996. To contact the reporter on this story: Mike Buteau in Atlanta at mbuteau@bloomberg.net To contact the editor responsible for this story: Michael Sillup at msillup@bloomberg.net At a Masters press conference Tuesday, England's Lee Westwood was asked about the ongoing "gender issue" at Augusta National Golf Club. Here's how that was reported in the transcript provided by the Masters: Q. – The gender issue thing has come up over here, again? LEE WESTWOOD: What gender issue? I'm a man (Nodding, looking down). There is a gender issue – no women members at Augusta – and the topic came up again Wednesday in club Chairman Billy Payne's annual news conference. As is always the case, Payne wouldn't go there, delivering the standard Augusta response: Membership issues are private. THE MASTERS: Weather could get stormy THE MASTERS: Betting on Tiger Woods not much of a gamble Controversy over no women members at Augusta was fueled a decade ago as a result of a protest campaign led by Martha Burk, who appealed to corporate sponsors to drop their ties with the Masters. The matter is back in the spotlight with the Masters set to begin Thursday. In January, IBM named Virginia ("Ginni") Rometty as its new president and CEO. IBM is a Masters sponsor. The past four CEOs of IBM have been members of Augusta National. At Westwood's press conference, he also was asked whether the matter was anything the players have been chatting about in the locker room. "It's not something I think about," Westwood said.

Summary: The prestigious Augusta National Golf Club, host of the Masters, seems to be sticking to its maddening policy of banning female members. Augusta National Chairman William Porter Payne won't even deign to publicly discuss the issue. Some critics believe it's long past time to extend an invitation to Virginia Rometty, CEO of IBM, one of three sponsors (along with Exxon and AT&T) of the Masters tourney, notes BusinessWeek. "All issues of membership remain the private deliberations of the membership," Payne said at his annual news conference on the eve of the Masters. "We don't talk about our private deliberations." The last four CEOs of IBM-all men-were members. Following the last brouhaha over the policy, John Snow, who served as Treasury secretary under George W. Bush, and CBS CEO Thomas Wyman-who called the ban "pig-headed"-quit the club. Augusta began inviting black members in 1990, but no female black members. "The place is stuck in time warp, just like the men in green jackets who run it," sniffs AP's Tim Dahlberg. Women aren't finding many supporters among the male golf champs participating in the Masters. "What gender issue?" smiled Brit Lee Westwood. "I'm a man."

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Summarize: In this June 2, 2016 photo, Brock Turner, 20, right, makes his way into a Santa Clara Superior Courthouse in California (Dan Honda/Bay Area News Group via AP) Courts records released Friday in the Brock Turner sexual assault case in Santa Clara County, Calif., include numerous exhibits and documents related to the prosecution of the former Stanford University swimmer. [“Did you rage?" In Stanford sexual assault case, court records shed new light] Among them are statements submitted to the court before Turner was sentenced on June 2 to six months in jail and three years of probation for the sexual assault of an unconscious woman in January 2015. At the time Turner was a freshman. Below is a statement from the victim’s sister. The identities of both women were redacted in the court documents. NEW YORK -- Brock Turner, the ex-Stanford swimmer convicted of sexually assaulting an unconscious woman, is allegedly pictured smoking from a pipe in images prosecutors say were extracted from his cell phone. The newly-released images are included as an exhibit attachment to a sentencing memo submitted by Deputy District Attorney Alaleh Kianerci that alleges Turner lied about using drugs in a statement to probation officials. Santa Clara County Superior Court Turner was sentenced to six months in jail and three years of probation for three counts of sexual assault, but can be released in as soon as three months. Santa Clara County Superior Court Judge Aaron Persky's sentence has led to widespread outcry and a campaign to recall him, though some have defended the sentence. Turner has also come under fire for his statement to probation officials, obtained by CBS News, in which he blames his behavior on alcohol and the "college lifestyle." In the statement, he also implies he hadn't had experience with partying and drinking prior to the sex assault. "Coming from a small town in Ohio, I had never really experienced celebrating or partying that involved alcohol," he said in the statement. A probation report also says Turner denied that he had used drugs. Santa Clara County Sheriff's Department/Handout via Reuters In the sentencing memo, however, prosecutors point to cell phone evidence they say proves otherwise. In a search of Turner's cell phone, prosecutors say they found photos of Turner smoking a pipe and a Dec. 27, 2014 video of Turner smoking a bong and drinking out of a liquor bottle immediately after. Several images the prosecutors referenced surfaced Friday as a part of a release of the Turner case file by the County of Santa Clara Superior Court. CBS News had previously obtained Kianerci's sentencing memo. The new images include the photo of Turner smoking a pipe, a close-up photo of a bong, and an image of a person who prosecutors say is Turner's teammate holding a bong. The documents also include a photo of an apparent sexually explicit message sent through the "Group Me" app that investigators saw on Turner's phone the night of his arrest. The memo includes cell phone records of Turner's text messages, but it doesn't include the "Group Me" message because it was sent from a third-party app and may have been deleted, according to the memo. Prosecutors say the texts point to drug use, including a Dec. 18, 2014 message in which Turner asks a friend, "Do you think I could buy some wax so we could do some dabs?" referring to a highly concentrated form of marijuana. Other text messages referenced smoking, buying and sharing "weed" and trying to find a "hook up" to buy acid both while Turner was in high school and at Stanford, the memo says. On Dec. 24, 2014, according to the memo, Turner received a message from a friend that read, "I've got a hankerin for a good acid trip when we get back." Prosecutors say Turner replied: "I'm down for sure." The memo says he responded to a friend bragging about "candyflippin" - referring to taking LSD and MDMA together - by saying, "I gotta [expletive] try that. I've heard it's awesome." The memo says on June 3, 2014 Turner's sister asked him via text, "Did you rage last night?" To which Turner replied, "Yeah kind of. It was hard to find a place to drink. But when we finally did we could only drink for like an hour an a half." Prosecutors say the records show Turner was already an experienced drinker in high school, had routinely smoked marijuana and experimented with hard drugs including LSD, despite his statement. The memo goes on to blast Turner for blaming his "predatory and repulsive" actions on "drinking, peer pressure and college culture." In a letter that has since gone viral, the victim in the case calls on Turner to take responsibility for his actions, saying, "assault is not an accident."

Summary: The sister of Brock Turner's victim says Turner tried to force himself on her the same night he raped her sister. That's one of a number of things revealed in court documents released Friday. The Los Angeles Times reports the victim's sister says Turner "started making out on her cheek" and grabbed her waist unprompted at the frat party in January 2015. She was able to pull away and warn a friend. "The face of the man who assaulted my sister is burned into my memory," the Washington Post quotes her as saying in a statement to the court. She also talks about the guilt she feels for leaving her sister alone at the party. "An entire part of my heart has been permanently broken," she says. "The damage you inflicted is irreversible," she tells Turner, who was sentenced to six months in jail. The newly released documents also include allegations from prosecutors that Turner lied to the court about his alcohol and drug use, CBS News reports. Part of Turner's defense was that he had "never really experienced celebrating or partying that involved alcohol" before coming to Stanford. But the documents include texts, photos, and videos that prosecutors say prove Turner had plenty of experience with alcohol and drugs, including LSD and marijuana, in high school and prior to his assault on the victim. Finally, following the release of statements from Turner's father and grandparents, the new documents include a statement from his mother. In it, she says she cries everyday and can't decorate her new home because of the trauma inflicted on her family, the Fresno Bee reports. She says Turner was just "trying to fit in."

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Summarize: A self-confessed murderer lashed out in the courtroom Monday after he was handed a 50-to-100 year prison sentence for slaying his girlfriend, a mother-of-five, and dumping her body in the garbage. Before learning his fate, Jahleel Hoskins, of Grand Rapids, Michigan, offered a weeping apology to the family of Latrice Maze, whom he killed last March, insisting he loved the woman and didn't meant to take her life. But when Kent County Circuit Court Judge James Redford sentenced him to up to a century behind bars, the handcuffed man snapped, throwing the podium toward the bench and lunging forward before security dragged him from the courtroom. Scroll down for video. Angry: Confessed murderer lashed out in the courtroom Monday after he was handed a 50-to-100 year prison sentence for slaying his girlfriend, a mother-of-five, and dumping her body in the garbage. var p = new anv_pl_def(); p.config = {}; p.config.width = 636; p.config.height = 400; p.loadVideoExpressV3('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|STAGEV3|SPSQA');. But before he even left the premises, Hoskins' family began screaming at the family of the victim, including her mother and father, according to mlive.com. The two families surging towards each other, yelling, and additional court deputies had to tear them apart. Hoskins' devastated mother, who made an emotional statement to the court earlier, yelled at Hoskins cousin to shut up as Redford banged his gavel and demanded order. In almost as dramatic a move, the 26-year-old halted his trial on December 11 - the third day - by pleading guilty to second-degree murder in the middle of testimony. Temper: When Kent County Circuit Court Judge James Redford sentenced him to up to a century behind bars, the handcuffed man snapped, throwing the podium toward the bench and lunging forward before security dragged him from the courtroom. Brawl: The two families surging towards each other, yelling, and additional court deputies had to tear them apart. Victim: Latrice Maze, pictured left and right, was murdered by Hoskins last March. But Assistant Kent County Prosecutor Kellee Koncki urged the judge not to give the killer a lesser sentence because he confessed. Rather, she asked Redford to sentence him to what would result in a lifetime in prison. Maze went missing on March 19. Less than two months later, Grand Rapids police said they believed her body had been incinerated and dumped in a landfill after Hoskins put it in a Dumpster. According to mlive.com, Koncki argued that Hoskins' motive in the slaying was that he wanted to stop her talking to police after he allegedly assaulted the father of two of her children. She said the violent strangulation was premeditated. Apology: Before learning his fate, Hoskins, of Grand Rapids, Michigan, offered a weeping apology to the family of Maze. Lies: He insisted he loved the woman and didn't meant to take her life. Redford also admonished Hoskins for dumping the body, robbing the family of a chance to have a proper funeral for their loved one. Maze' mother, Wanda Rose, spoke before Hoskins was sentenced and recalled the difficult task of trying to explain to her grandchildren about their mother's death. Hoskins said in his piece that he loved Maze and worked hard to support her and her daughter. 'Before this happened, everybody looked at me as a good person,' Hoskins said. 'In the blink of an eye, I'm a monster.' Tragic: Latrice Maze's mother, Wanda Rose, weeps after making statements Monday January 6, 2014

Summary: Confessed murderer Jahleel Hoskins lashed out in the. courtroom Monday after he was handed a 50-to-100 year sentence. for slaying his girlfriend. Latrice Maze, a mother-of-five, was killed last March and dumped in the garbage. When Kent County Judge James Redford handed down the sentence, Hoskins snapped, throwing the. podium and lunging forward. Security had to drag him out of the courtroom. His and the victim's families then surged towards each other, screaming, and additional court deputies had to tear them apart.

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Summarize: DENVER, March 24, 2015 -- Chocolate has many health benefits -- it can potentially lower blood pressure and cholesterol and reduce stroke risk. But just as connoisseurs thought it couldn't get any better, there's this tasty new tidbit: Researchers have found a way to make the treat even more nutritious -- and sweeter. They will describe their research here today at the 249th National Meeting & Exposition of the American Chemical Society (ACS), the world's largest scientific society. The meeting features nearly 11,000 reports on new advances in science and other topics. It is being held here through Thursday. Cocoa undergoes several steps before it takes shape as a candy bar. Workers cut down pods from cocoa trees, then split open the pods to remove the white or purple cocoa beans. They are fermented in banana-lined baskets for a few days and then set out to dry in the sun. Roasting, the next step, brings out the flavor. But some of the healthful polyphenols (antioxidants) are lost during the roasting process, so the researchers wanted to figure out a way to retain as much of the polyphenols and good flavors as possible. "We decided to add a pod-storage step before the beans were even fermented to see whether that would have an effect on the polyphenol content," says Emmanuel Ohene Afoakwa, Ph.D., who is at the University of Ghana. "This is not traditionally done, and this is what makes our research fundamentally different. It's also not known how roasting affects polyphenol content." Afoakwa's team divided 300 pods into four groups that were either not stored at all or stored for three, seven or 10 days before processing. This technique is called "pulp preconditioning." After each storage period passed, fermentation and drying were done as usual. He reports that the seven-day storage resulted in the highest antioxidant activity after roasting. To assess the effects of roasting, the researchers took samples from each of the storage groups and roasted them at the same temperature for different times. The current process is to roast the beans for 10-20 minutes at 248-266 degrees Fahrenheit, he explains. Afoakwa's team adjusted this to 45 minutes at 242 degrees Fahrenheit and discovered that this slower roasting at a lower temperature increased the antioxidant activity compared to beans roasted with the conventional method. In addition, the beans that were stored and then roasted for 45 minutes had more polyphenols and higher antioxidant activity than beans whose pods were not stored prior to fermentation, says Afoakwa. He explains that pulp preconditioning likely allowed the sweet pulp surrounding the beans inside the pod to alter the biochemical and physical constituents of the beans before the fermentation. "This aided the fermentation processes and enhanced antioxidant capacity of the beans, as well as the flavor," he says. He adds that the new technique would be particularly useful for countries in Southeast Asia and Latin America where cocoa beans produce a chocolate with a less intense chocolate flavor and have reduced antioxidant activity. Looking to the future, he says the team will be studying in more detail the effects of roasting on the flavor of freshly picked compared to stored cocoa beans. They will be testing different temperatures and roasting and storing times to determine if even higher amounts of antioxidants can be retained through the process. The researchers acknowledge funding from the Belgium Government under the VLIR TEAM Cocoa Project between Ghent University, Ghent, Belgium, and the University of Ghana, Accra, Ghana. A press conference on this topic will be held Tuesday, March 24, at 11 a.m. Mountain time in the Colorado Convention Center. Reporters may check-in at Room 104 in person, or watch live on YouTube http://bit. ly/ ACSLiveDenver. To ask questions, sign in with a Google account. ### The American Chemical Society is a nonprofit organization chartered by the U.S. Congress. With more than 158,000 members, ACS is the world's largest scientific society and a global leader in providing access to chemistry-related research through its multiple databases, peer-reviewed journals and scientific conferences. Its main offices are in Washington, D.C., and Columbus, Ohio. To automatically receive news releases from the American Chemical Society, contact newsroom@acs.org. Note to journalists: Please report that this research is being presented at a meeting of the American Chemical Society. Follow us: Twitter Facebook Title Roasting effects on phenolic content and free-radical scavenging activities of pulp pre-conditioned and fermented cocoa (Theobroma cacao) beans Abstract Polyphenols are phytochemicals responsible for the astringency, bitterness, green flavours and antioxidant activities in cocoa (Theobroma cacao) beans. These are degraded during fermentation, drying and roasting affecting the antioxidant activity of the beans. However, the extent of degradation of phenolics during roasting remains unknown. This work was aimed at investigating the changes in total polyphenols, anthocyanins, o-diphenols and antioxidant activity (free-radical scavenging activities) during roasting of pulp pre-conditioned and fermented cocoa. A 4×4 full factorial design with the principal experimental factors as pod storage and roasting time were used. Samples were analyzed for total polyphenols, o-diphenols, anthocyanins and antioxidant activity using standard analytical methods. Variable decrease in total polyphenols, o-diphenols and anthocyanins were observed with increase in pod storage and roasting durations. However, variable trends were observed for the % free-radical scavenging activities. The total polyphenols, anthocyanins and o-diphenols in the cocoa beans after 45 minutes roasting decreased from 132.24 to 57.17 mg/g, 6.71 to 1.07 mg/kg and 15.94 to 8.25 mg/g respectively for 0, 3, 7 and 10 days pod storage treatments. The total polyphenols for the fermented, dried and unstored cocoa beans was 132.25 mg/g which reduced to 122.14 mg/g (7.642% degradation), 116.721 mg/g (11.7% degradation) and 92.22 mg/g (30.3% degradation) for pod stored for 3, 7 and 10 days respectively. Increasing roasting time caused continuous decreases in the % free-radical scavenging activity from 89.10% to 74.31% after 45 minutes for the unstored pods. Pulp pre-conditioning by pod storage and roasting duration could be used to reduce the astringency and bitterness caused by polyphenols, o-diphenols and anthocyanins in cocoa beans whilst increasing the antioxidant activity imparted by cocoa. Polyphenols are phytochemicals responsible for the astringency, bitterness, green flavours and antioxidant activities in Theobroma cacao beans. Polyphenols degradation in cocoa beans during roasting is crucial to the flavour outcome and it is influenced by factors such as temperature, time and pod storage. Antioxidants are compounds that help to inhibit oxidation reactions caused by free radicals such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite thereby preventing damage to the cells and tissues. Their mechanisms of action include scavenging reactive oxygen and decreasing localised oxygen concentration thereby reducing molecular oxygen’s oxidation potential, metabolising lipid peroxides to non-radical products and chelating metal ions to prevent generation of free radicals in humans. The study aimed at investigating changes in total polyphenols, anthocyanins, o-diphenols and antioxidant activity (free-radical scavenging activities) after roasting of pulp preconditioned and fermented cocoa beans using standard analytical methods. A 4×4 full factorial design with the principal experimental factors as pod storage time (0, 3, 7 and 10 days) and roasting duration (0, 15, 30 and 45 minutes) at 120oC were used to study the changes in the total polyphenols, anthocyanins, o-diphenols and % free-radical scavenging activities of the cocoa beans. Variable decrease in total polyphenols, odiphenols and anthocyanins were observed with increase in pre-conditioning (pod storage time) and roasting duration. However, variable trends were observed for the % free-radical scavenging activities. The total polyphenols, anthocyanins and o-diphenols in the cocoa beans after 45 minutes roasting decreased in the range 132.24 to 57.17 mg/g, 6.71 to 1.07 mg/kg and 15.94 to 8.25 mg/g respectively at all pod storage treatments. The total polyphenols of the fermented, dried and unstored (freshly harvested) cocoa beans was 132.25 mg/g which reduced to 122.14 mg/g (7.6% degradation), 116.721 mg/g (11.7% degradation) and 92.22 mg/g (30.3% degradation) after storage for 3, 7 and 10 days, respectively. The optimum decrease in the % freeradical scavenging activity was 7 days and above of pods storage. Increasing roasting time caused a continuous decrease in the % free-radical scavenging activity from 89.10% to 74.31% after 45 minutes for beans from the unstored (freshly harvested) pods. However, pod storage caused an increase in the % free radical scavenging activities during roasting. Pulp pre-conditioning (pod storage) and roasting duration could be used to reduce the astringency and bitterness caused by polyphenols, o-diphenols and anthocyanins in cocoa beans as well as increase the antioxidant activity imparted by cocoa. Key words: Cocoa, pod storage, roasting, polyphenols.

Summary: One of your vices could one day be a little more virtuous: Scientists are today announcing that they've figured out how to make chocolate healthier. The findings will be detailed by researchers from Belgium's Ghent University and the University of Ghana at the national meeting of the American Chemical Society in Denver, and center around how antioxidant-rich the sweet is. As a press release explains, it all comes down to tweaking the process. Cocoa beans are removed from pods, fermented in baskets, sun-dried, and then roasted. It's during that last step, the roasting, that polyphenols, which act as antioxidants, are partially lost. In a bid to up the polyphenol content, researchers added a nontraditional step that "makes our research fundamentally different," explains Emmanuel Ohene Afoakwa: pulp preconditioning. That simply means they stored the pods-in the case of their experiments, for zero, three, seven, or 10 days-before removing the beans and beginning the fermentation process. A sweet pulp rests between the pod and the beans, and Afoakwa believes the preconditioning gives the pulp time to affect those beans. Indeed, the researchers found that those stored for a week showed the highest antioxidant activity after roasting-which they also adjusted. Rather than heat the beans for the typical 10 to 20 minutes at 248-266 degrees, they lowered the temp to 242 and upped the roasting time to 45 minutes, and discovered that slower and lower was also best in terms of antioxidant activity. The researchers' abstract notes another benefit: "Pulp preconditioning and roasting duration could be used to reduce the astringency and bitterness," improving chocolate's flavor.

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Write a title and summarize: Diego Armando Maradona Franco (* 30. Oktober 1960 in Lanus, Provinz Buenos Aires; + 25. November 2020 in Tigre, Provinz Buenos Aires) war ein argentinischer Fußballspieler und -trainer. Maradona ist eine der "Legenden des Weltfußballs" und gilt als einer der besten Fußballspieler der Geschichte. Er machte sich zu Beginn seiner Karriere bereits im Alter von 15 Jahren bei den Argentinos Juniors einen Namen, bevor er zu den Boca Juniors wechselte und 1981 argentinischer Meister wurde. Anschließend zog es den "Goldjungen" (El Pibe de Oro) für eine Rekordablösesumme nach Europa zum FC Barcelona. Dort feierte er mit dem Pokalsieg 1983 nur einen wichtigen Titelgewinn. Von Krankheiten und Verletzungen geplagt, musste er den Verein nach nur zwei Jahren wegen zahlreicher Skandale wieder verlassen. Daraufhin schloss er sich erneut für eine Rekordablösesumme der SSC Neapel an. Mit dem Underdog aus Kampanien, der vor seiner Ankunft dem Abstieg nahe war, feierte er zwischen 1984 und 1991 die größten Erfolge seiner Vereinskarriere, darunter 1987 und 1990 die bis heute einzigen Meistertitel der Vereinsgeschichte und den Gewinn des UEFA-Cups 1989. Der Juniorenweltmeister von 1979 führte die argentinische Nationalmannschaft 1986 in Mexiko als Mannschaftskapitän zum Gewinn ihrer zweiten Weltmeisterschaft nach 1978. Dabei erzielte der 25-Jährige beim 2:1-Sieg gegen England im Viertelfinale innerhalb von vier Minuten zwei der berühmtesten Tore der Fußballgeschichte, als er zunächst einen hohen Ball mit der Hand, der "Hand Gottes", regelwidrig ins Tor beförderte und anschließend nach einem Dribbling über etwa 60 Meter das WM-Tor des Jahrhunderts erzielte. Insgesamt nahm Maradona an vier WM-Turnieren (1982, 1986, 1990, 1994) teil und erzielte in 91 Länderspielen 34 Tore. In den 1990er Jahren geriet Maradona wegen Drogenproblemen und Doping in die Schlagzeilen und bekam vom Fußballweltverband FIFA zweimal eine 15-monatige Sperre auferlegt. Nach seiner aktiven Karriere war Maradona als Trainer tätig, hatte aber immer wieder gesundheitliche Schwierigkeiten. Von Oktober 2008 bis Juli 2010 war er Nationaltrainer seines Heimatlandes. == Familie und Kindheit Diego Armando Maradona wurde am 30. Oktober 1960 in Lanus, einer Stadt im Ballungsraum Gran Buenos Aires, geboren. Sein Vater Diego Maradona Senior ("Don Diego" oder "Chitoro"; 1927-2015) war indigener Herkunft, die Mutter Dalma Salvadora Franco ("Dona Tota"; 1930-2011) hatte italienische Vorfahren. Nach vier Töchtern war "Diegito" ihr erster Sohn. Die Eltern stammten ursprünglich aus der Provinz Corrientes und gehörten der armen, sozial benachteiligten Bevölkerungsschicht an, die man in Argentinien abfällig als "Cabecitas negras" bezeichnete. Mit ihren insgesamt acht Kindern bewohnten sie eine ärmliche Behausung ohne Strom oder fließendes Wasser in Villa Fiorito, einer informellen Siedlung am südlichen Rand der Millionenmetropole. Die Lebensbedingungen in dieser Villa Miseria waren durch Kriminalität und eine unzureichende Infrastruktur gekennzeichnet. Während "Don Diego" einer schlecht bezahlten Arbeit in einer Tiermehlfabrik nachging, kümmerte sich die dominante und streng katholische Mutter um den Haushalt und die Kindererziehung. Als Kind war Maradona ein begeisterter Straßenfußballer, der auf den Bolzplätzen seines Viertels für die informelle Jugendmannschaft Estrella Roja spielte. Nachdem er 1969 an einem Auswahltraining des Erstligisten Argentinos Juniors teilgenommen hatte, wurde er umgehend in deren Nachwuchsabteilung aufgenommen und lief fortan für die von Francisco Cornejo trainierten "Los Cebollitas" (Die Zwiebelchen) auf. Maradona spielte regelmäßig für ältere Jahrgänge und durfte als Zwölfjähriger in den Halbzeitpausen der Profispiele zur Unterhaltung des Publikums den Ball jonglieren. Ein Artikel in der Sportzeitung Clarin sowie ein Auftritt in der Fernsehsendung Sabados Circulares festigten seinen Ruf als Ausnahmetalent. Im Umfeld der Argentinos lernte Maradona den drei Jahre älteren Jorge Cyterszpiler kennen und freundete sich mit ihm an. Um dem Talent die beschwerliche Anreise aus Villa Fiorito zu den Trainingseinheiten im Stadtteil La Paternal zu ersparen, ließ Cyterszpiler ihn gelegentlich bei sich übernachten. Die Cebollitas, die zeitweise 136 Spiele in Folge ungeschlagen blieben, entwickelten sich zu einer der erfolgreichsten Jugendmannschaften des Landes, die an Turnieren in Uruguay und Chile teilnahmen und 1974 die nationale Jugendmeisterschaft gewannen. Schon zu dieser Zeit galt der blutjunge Maradona als fußballerische Ausnahmeerscheinung, was ihm den Spitznamen "El Pibe de Oro" (Der Goldjunge) eingebracht hatte. Da sich frühzeitig eine Karriere als Fußballprofi abzeichnete, verließ Maradona die Schule ohne Abschluss. Um ihr größtes Nachwuchstalent an den Verein zu binden, mieteten die Klubverantwortlichen der Argentinos Juniors der Familie 1975 ein Appartement im bürgerlichen Stadtteil Villa del Parque, in unmittelbarer Nähe zum Vereinsgelände. Damit verwirklichten sich die Hoffnungen der Eltern auf einen sozialen Aufstieg, die auf ihrem ältesten Sohn geruht hatten. Maradonas jüngere Brüder Raul (* 1966) und Hugo (* 1969) wurden später ebenfalls Profifußballer. == Vereinskarriere === Argentinos Juniors (1976 bis 1981) Dem Cheftrainer der Profimannschaft, Juan Carlos Montes, blieb das Wunderkind aus der eigenen Nachwuchsabteilung nicht verborgen und er berief Maradona in den Profikader. Am 20. Oktober 1976, zehn Tage vor seinem 16. Geburtstag, debütierte Maradona in der Primera Division für die Argentinos Juniors, als er im Heimspiel gegen Club Atletico Talleres (0:1) zur zweiten Halbzeit eingewechselt wurde. Damit war er zu diesem Zeitpunkt der jüngste Spieler in Argentiniens erster Liga. Der mutige Auftritt überzeugte Montes, ihn trotz seines jugendlichen Alters dauerhaft in der Profimannschaft einzusetzen. Seine ersten beiden Tore gelangen Maradona am 14. November 1976 gegen San Lorenzo Mar del Plata, als er zum 4:2 und 5:2 traf. Im Verlauf der Metropolitano 1977 entwickelte sich "El Pibe" zum entscheidenden Spieler der Argentinos und nur vier Monate nach seinem Debüt folgte die Nominierung für die A-Nationalmannschaft. Innerhalb kürzester Zeit war Maradona zum gefeierten Publikumsliebling avanciert und der rasante Aufstieg zum besten Spieler des Landes nahm seinen Anfang. Die argentinische Öffentlichkeit stürzte sich auf den Lockenkopf mit der Rückennummer 10 und stellte Vergleiche mit dem brasilianischen Ausnahmefußballer Pele an. Maradona spielte insgesamt viereinhalb Jahre für die Argentinos, in denen er 116 Ligatore erzielte und fünfmal in Folge Torschützenkönig wurde (Metropolitano 1978: 22 Tore; Metropolitano 1979: 18 Tore; Nacional 1979: 12 Tore; Metropolitano 1980: 25 Tore und Nacional 1980: 17 Tore). Der nur 1,65 Meter große Dribbelkünstler tauchte auf dem gesamten Spielfeld auf, war Spielmacher und Torjäger zugleich. Sein kompakter Körperbau, seine Finten und überraschenden Tempowechsel stellten die Gegenspieler vor scheinbar unlösbare Probleme. Schon als junger Spieler besaß Maradona die Fähigkeit, durch seine Dynamik und trickreiche Spielweise, ein Spiel alleine zu entscheiden. Die Argentinos Juniors zählten zu den kleineren Vereinen aus Buenos Aires, hatten sich jedoch Dank der individuellen Klasse Maradonas zu einer Mannschaft entwickeln können, die um Platzierungen im oberen Tabellendrittel mitspielte. Nach dem fünften Platz 1978 wurden die Argentinos unter Trainer Miguel Angel Lopez in der Metropolitano 1980 zwar Vizemeister, jedoch reichten die Möglichkeiten des Klubs nicht aus, um Titel zu gewinnen. Obwohl die Argentinos einen Werbevertrag mit der Fluggesellschaft Austral abgeschlossen hatten, die damit erster Trikotsponsor des argentinischen Vereinsfußballs wurde, erlaubte es das enorme Gehalt Maradonas nicht, den Kader durch Spielerkäufe weiter zu verbessern und langfristige finanzielle Stabilität zu garantieren. Auch aus sportlicher Sicht war Maradona, der 1979 und 1980 jeweils die Auszeichnungen als Argentiniens und Südamerikas Fußballer des Jahres erhalten hatte, seinem Klub enteilt, was den Verkauf des Starspielers schließlich unausweichlich machte. Maradona war inzwischen Dollar-Millionär, der sich durch seinen Jugendfreund Jorge Cyterszpiler vertreten ließ, den er 1977 zu seinem Manager gemacht hatte. Cyterszpiler nutzte die stetig wachsende Popularität seines Klienten und schloss lukrative Sponsorendeals mit Puma, Coca-Cola und Agfa ab. Zur besseren Vermarktung gründete er das Unternehmen "Diego Armando Maradona Producciones S.A." und ließ es 1979 in das Unternehmensregister Liechtensteins eintragen. Mit seiner Familie bezog Maradona ein Anwesen im Prominentenviertel Villa Devoto und erwarb in Moreno ein Landhaus mit angeschlossenem Trainingsgelände. === Boca Juniors (1981/82) Nachdem Maradona eine Vielzahl individueller Auszeichnungen erhalten hatte, erreichten ihn Angebote europäischer Spitzenvereine. Doch auf Weisung der Militärjunta untersagte ihm der argentinische Fußballverband AFA einen Transfer ins Ausland. Argentiniens Rekordmeister River Plate war bereit, ihn neben Nationaltorhüter Ubaldo Fillol zum Topverdiener zu machen, allerdings wollte Maradona nicht für den Verein der Oberschicht spielen und lehnte ab. Stattdessen nahm er das Angebot der Boca Juniors an, deren Anhänger traditionell den ärmeren Bevölkerungsschichten des Landes angehören. Da auch sein Vater ein leidenschaftlicher Boca-Fan war und aufgrund der eigenen Herkunft, war der Wechsel für Maradona eine Herzensangelegenheit. Obwohl einer der populärsten und erfolgreichsten Vereine des Landes, mussten die Boca Juniors an ihre finanzielle Schmerzgrenze gehen, um den Transfer realisieren zu können. Die Einigung mit den Argentinos Juniors für die 16-monatige Leihe beinhaltete neben einer Gebühr von vier Millionen US-Dollar den Wechsel von sechs zusätzlichen Kaderspielern. Maradona selbst erhielt ein Handgeld in Höhe von 600.000 Dollar, die größtenteils in Immobilien geleistet wurden und ein Monatsgehalt von 60.000 Dollar. Den ausgehandelten Vertrag unterzeichnete er medienwirksam am 20. Februar 1981 und im anschließenden Freundschaftsspiel bestritt Maradona jeweils eine Halbzeit für seinen alten und neuen Verein. Am 22. Februar 1981 debütierte Maradona für die Boca Juniors und erzielte beim 4:1-Sieg über CA Talleres einen Doppelpack. Ohne Eingewöhnungszeit integrierte sich Maradona in das Mannschaftsgefüge der Xeneizes, die mit Hugo Gatti, Roberto Mouzo, Miguel Brindisi, Vicente Pernia, Marcelo Trobbiani und Oscar Ruggeri weitere Spitzenspieler in ihren Reihen aufwiesen. Anstandslos riss der 20-Jährige das Spiel an sich und wurde zum unangefochtenen Dirigenten der Mannschaft. Allerdings war das Verhältnis des Neuzugangs zu Trainer Silvio Marzolini angespannt, da dieser seinem Star nicht die gewohnte Sonderbehandlung einräumte. In seinem ersten Superclasico gegen den Erzrivalen River Plate vor heimischem Publikum leistete Maradona mit einem Sololauf die Vorarbeit zum Führungstor. Schließlich traf er selbst zum 3:0-Endstand, als er eine Flanke im Strafraum annahm, Nationaltorhüter Fillol umspielte und den auf die Torlinie zurückgeeilten Verteidiger Alberto Tarantini narrte, bevor er den Ball einschob. Durch den Sieg über die verhassten Millonarios erreichte die kultische Verehrung Maradonas neue Höhen und die einflussreichen Barra Bravas erwarteten von ihrer Mannschaft nach fünf Jahren wieder den Gewinn der Meisterschaft. Die Boca Juniors hielten dem Druck stand und entschieden den Titelkampf mit Ferro Carril Oeste am letzten Spieltag der Metropolitano für sich. Nach einem 1:1 gegen Racing Club wurden sie in La Bombonera argentinischer Meister und mit 17 Saisontoren hatte Maradona maßgeblichen Anteil an seinem ersten nationalen Titel. Im anschließenden Torneo Nacional gelangen Maradona elf Tore in zwölf Spielen, jedoch scheiterten die Boca Juniors überraschend im Viertelfinale an CA Velez Sarsfield. Das Rückspiel (1:3-Niederlage) hatte Maradona wegen einer Rotsperre verpasst. 1981 erhielt er zum dritten Mal in Folge die Auszeichnung als Argentiniens Fußballer des Jahres. Die Boca Juniors waren gezwungen, finanziell lukrative Freundschaftsspiele zu absolvieren, da sie ohne die Zusatzeinnahmen die Gehälter ihrer Spieler nicht hätten zahlen können. Die weltweit angesetzten Spiele waren nicht selten mit kraftraubenden Reisen verbunden und führten zu verkürzten Regenerationsphasen. Im Januar 1982 musste die Mannschaft acht Spiele in 21 Tagen bestreiten. Maradonas letzte Partie für Boca absolvierte er am 6. Februar 1982 im Sommerturnier Copa de Oro gegen River Plate. Anschließend reiste er zur Vorbereitung auf die Weltmeisterschaft 1982 zu einem viermonatigen Trainingslager zur Nationalmannschaft ab. === FC Barcelona (1982 bis 1984) Nach der Weltmeisterschaft 1982 wechselte Maradona für die damalige Rekordsumme von 7,3 Millionen US-Dollar zum FC Barcelona nach Spanien. Für den ambitionierten Vereinspräsidenten Josep Lluis Nunez war die Verpflichtung des teuersten Fußballers der Welt eine Frage des Prestiges und sollte wirtschaftliche Potenz gegenüber dem Rivalen Real Madrid demonstrieren. Zur Vorstellung des Spielers kamen 50.000 Zuschauer ins Camp Nou, doch von einigen Höhepunkten abgesehen, konnte Maradona die hochgesteckten Erwartungen nicht erfüllen. Sein Debüt gab er am 3. August 1982 während der Saisonvorbereitung in einem Freundschaftsspiel gegen den SV Meppen. Trainer des FC Barcelona war Udo Lattek, der ausgiebig Kraft und Ausdauer trainieren ließ, was zu anhaltenden Auseinandersetzungen mit Maradona führte, der die körperlich harten Einheiten nicht mochte. Trotz des angespannten Verhältnisses zu seinem Trainer zeigte Maradona ansprechende Leistungen. Durch seine trickreiche Spielweise machte er das katalonische Starensemble unberechenbarer und bildete mit Bernd Schuster ein spielstarkes Duo. Bis Dezember 1982 erzielte er in 13 Ligaspielen sechs Tore und Barca konnte sich erwartungsgemäß in der Spitzengruppe der Primera Division festsetzen. In der 2. Runde des Europapokals der Pokalsieger wurde Roter Stern Belgrad auswärts mit 4:2 bezwungen. Der formstarke Maradona begeisterte das Belgrader Publikum mit einem Tor per Lupfer von der Strafraumgrenze und wurde mit stehenden Ovationen verabschiedet. Im Dezember 1982 erkrankte Maradona jedoch an Hepatitis und musste drei Monate aussetzen. Er zog sich in seine Villa im Vorort Pedralbes zurück und bekämpfte seine Einsamkeit, indem er Freunde aus der argentinischen Heimat nach Barcelona holte. Dieser sogenannte "Maradona-Clan" sollte ihn auch in seiner weiteren Karriere begleiten. In der bürgerlichen Stadt kursierten zu diesem Zeitpunkt erste Gerüchte über sein wildes Privatleben: So soll die Hepatitis-Erkrankung in Wahrheit eine Geschlechtskrankheit gewesen sein. Während er dies zeitlebens bestritt, gab er später zu, in Barcelona erstmals Kokain genommen zu haben. Die lange Rekonvaleszenz bezeichnete er rückblickend als die "unglücklichste Phase meiner Karriere." Nach dem vorzeitigen Ausscheiden aus dem Europapokal wurde Lattek im März 1983 entlassen und durch Cesar Luis Menotti ersetzt, der als Verfechter eines freien, kreativen und offensiven Fußballs galt. Mit der Verpflichtung seines Landsmannes erklärte sich Maradona für genesen, ließ neuen Spaß am Fußball erkennen und wurde mit elf Saisontreffern bester Ligatorschütze seiner Mannschaft. Doch auch mit dem neuen Trainer verpasste Barca am Ende der Saison 1982/83 als Tabellenvierter die Meisterschaft. Durch einen 2:1-Finalsieg über Real Madrid gewann der Verein aber zumindest den Pokal. Auch bei der ersten Austragung des kurzlebigen Ligapokals Ende Juni 1983 setzte sich Barcelona in zwei Finalspielen gegen Real Madrid durch, wobei Maradona in beiden Begegnungen ein Tor erzielte. Insbesondere sein Treffer im Hinspiel, als er alleine auf das gegnerische Tor zusteuerte, den Torhüter umspielte und anschließend noch seinen Gegenspieler Juan Jose ins Leere grätschen ließ, bevor er den Ball einschob, sorgte für Aufsehen. Daraufhin zollten die Zuschauer im Estadio Santiago Bernabeu erstmals einem gegnerischen Spieler Applaus. Nachdem er im Vorjahr krankheitsbedingt nur 20 Ligaspiele hatte absolvieren können, startete Maradona mit viel Ehrgeiz in die Saison 1983/84. Ende September 1983 empfing der FC Barcelona am 4. Spieltag den amtierenden Meister Athletic Bilbao. Das Duell zwischen den Fußball-Ästheten aus Katalonien und den körperbetonten Basken galt zu Beginn der 1980er Jahre als ein Aufeinandertreffen gegensätzlicher Fußballphilosophien. Beim Stand von 2:0 wurde Maradona in der 58. Minute durch den Verteidiger Andoni Goikoetxea mit einem Tackling von hinten brutal gefoult und erlitt die schwerste Verletzung seiner Karriere: Einen Außenbandriss und eine komplizierte Sprunggelenkfraktur, die durch einen operativen Eingriff fixiert werden musste. Unmittelbar nach dem Spiel empörte sich Trainer Menotti über das Foul ("Es muss wohl erst jemand sterben, bis sich etwas ändert") und bezeichnete Goikoetxea als "Anti-Fußballer", während die Presse ihm den Spitznamen "Schlächter von Bilbao" verlieh. Weil der Schiedsrichter keine Absicht erkannt hatte, verurteilte das Sportgericht den baskischen Verteidiger anschließend zu einer Sperre von 18 Spielen, die sich später auf sechs reduzierte. Zur Genesung kehrte Maradona in den Kreis seiner Familie nach Buenos Aires zurück und verpasste auch den Großteil seiner zweiten Saison mit dem FC Barcelona. Unter der Aufsicht des Sportmediziners Ruben Dario Oliva, den er eigens aus Mailand hatte kommen lassen, kämpfte er sich mit Disziplin und Trainingsfleiß wieder heran. Nach 106-tägiger Verletzungspause gab er am 8. Januar 1984 gegen den FC Sevilla sein Comeback und erzielte beim 3:1-Sieg zwei Treffer. Bis zum Saisonende bestritt Maradona insgesamt 16 Ligaspiele und schoss dabei elf Tore. Mit einem Punkt Rückstand verpasste Barca als Tabellendritter erneut die Meisterschaft, erreichte aber wieder das Endspiel der Copa del Rey, das am 5. Mai 1984 ausgerechnet gegen Athletic Bilbao verloren ging (0:1). Nachdem Maradona in der Partie immer wieder hart attackiert und provoziert worden war, sorgte er nach dem Schlusspfiff für einen Eklat, indem er mehrere Gegenspieler und Betreuer mit unkontrollierten Fußtritten attackierte. In Anwesenheit des spanischen Königs Juan Carlos I. auf der Ehrentribüne entwickelte sich zwischen den Mannschaften eine wilde Massenschlägerei. Maradona wurde als einer der Hauptschuldigen identifiziert und musste sich in Begleitung Cyterszpilers beim König in einer Audienz entschuldigen. Das "Wunderkind" aus dem südamerikanischen Elendsviertel wurde mit dem vornehmen Barcelona nie richtig warm und nach den Ereignissen des Pokalfinales war für die entnervte Vereinsführung die Schmerzgrenze erreicht. Bereits in den Monaten zuvor war Maradona aufgrund einiger Eskapaden - Ausflüge in das Nachtleben, Berichte über den Umgang mit Prostituierten oder Handgreiflichkeiten gegen Autogrammsammler - regelmäßig mit Präsident Nunez aneinandergeraten. Der war wegen der zu erwartenden dreimonatigen Sperre und der zunehmend ungemütlichen Presse bereit, Maradona auf die Transferliste zu setzen. Maradona selbst war einem Vereinswechsel nicht abgeneigt. Dies hing mit verlustreichen Geschäften und dem Unvermögen seines Freundes und Managers Cyterszpiler zusammen, in dieser Doppelfunktion Maradonas Ausgaben in den Griff zu bekommen, was dazu führte, dass Maradona trotz eines Jahresgehalts von 1,5 Millionen US-Dollar im Sommer 1984 pleite war. === SSC Neapel (1984 bis 1991) Zur Saison 1984/85 schloss sich Maradona für die Rekordablösesumme von 24 Millionen D-Mark dem italienischen Erstligisten SSC Neapel an. Neapel war bis dato noch nie italienischer Meister geworden und in der Vorsaison nur knapp dem Abstieg entgangen. Bessere Angebote waren jedoch ausgeblieben. Um die enorme Ablöse stemmen zu können, hatte Vereinspräsident Corrado Ferlaino einen Konsortialkredit aufgenommen. Ein Journalist, der bei Maradonas Vorstellung fragte, ob dieser wisse, wer die Camorra sei und welchen Einfluss sie in der süditalienischen Hafenstadt habe, wurde von einem echauffierten Ferlaino des Presseraumes verwiesen. Anschließend wurde Maradona von ca. 75.000 Fans im Stadio San Paolo empfangen. Die Menschen in Neapel verbanden mit Maradona die Hoffnung, endlich den erfolgreicheren Vereinen aus dem wirtschaftlich stärkeren Norditalien wie Juventus Turin, der AC Mailand und Inter Mailand Paroli bieten zu können. Für sie war "Diego" mehr als ein Fußballspieler und Maradona hatte aufgrund seiner Herkunft wenig Mühe, sich mit den Neapolitanern zu identifizieren und deren Zuneigung zu erwidern. Die Serie A war in den achtziger Jahren Anlaufpunkt zahlreicher Weltstars. Maradona benötigte Anlaufzeit, um sich an die italienische Spielweise zu gewöhnen, die von einer robusten Defensive geprägt war, was dazu führte, dass die Liga in jenem Jahrzehnt einen bermerkenswert geringen Toreschnitt aufwies. Zu Beginn der Spielzeit 1984/85 gewann die SSC Neapel nur eines der ersten neun Ligaspiele. Die Ergebnisse verbesserten sich im Saisonverlauf jedoch deutlich. Der Verein schloss die Saison letztlich im gesicherten Mittelfeld ab und Maradona wurde auf Anhieb zu Italiens Fußballer des Jahres gewählt. Anschließend verpflichtete Neapel mit Ottavio Bianchi einen neuen Trainer, der Maradona auf dem Spielfeld mehr Schutz und Freiheiten gewährte, so dass dieser noch mehr Einfluss auf das Angriffsspiel nehmen konnte. Darüber hinaus entwickelten sich Spieler wie Ciro Ferrara, Fernando De Napoli oder Salvatore Bagni an seiner Seite zu wichtigen Leistungsträgern. In der Saison 1985/86 wurde Neapel Tabellendritter und qualifizierte sich für den UEFA-Cup. Maradona wandelte nun auf dem Höhepunkt seiner Karriere. Nachdem er Argentinien im Sommer 1986 zum Gewinn der Weltmeisterschaft verholfen hatte, führte er die SSC Neapel in der Saison 1986/87 als Spielmacher, wichtigster Torschütze und Vorbereiter, Kapitän sowie Integrationsfigur zum ersten Meistertitel der Vereinsgeschichte. Obendrein gewann der Verein am Saisonende auch noch den Pokal. Durch diesen nicht für möglich gehaltenen Erfolg entwickelte sich um Maradona in Neapel ein nie dagewesener Fankult. Die Stadt versank im Ausnahmezustand. Autokorsos, Straßenfeste und Mauergemälde mit Maradonas Konterfei beherrschten die Szenerie. Populärer Gesang der Tifosi wurde Ho visto Maradona! ("Ich sah Maradona!"), eine Art Liebeserklärung an Maradona. Zu Beginn der Saison 1987/88 schlug Maradona ein Angebot der AC Mailand aus und verlängerte seinen ursprünglich bis 1989 laufenden Vertrag in Neapel vorzeitig bis Juni 1993, wobei sein Jahresgehalt auf 5 Millionen US-Dollar angehoben wurde. Im Anschluss deutete vieles auf den erneuten Gewinn der Meisterschaft hin: Neapels Sturmreihe, bestehend aus Maradona, Bruno Giordano und dem neu verpflichteten Careca, erhielt schnell den Spitznamen Ma-Gi-Ca und schoss den Verein von Sieg zu Sieg. Fünf Spieltage vor Saisonende führte Neapel mit vier Punkten Vorsprung souverän die Tabelle an. Im Anschluss holte der Klub allerdings nur noch einen von möglichen zehn Punkten und gab damit die Meisterschaft aus der Hand. Bis heute hält sich das Gerücht, dass die Niederlagenserie zum Saisonende mit der Camorra zusammenhing, die im Falle des erneuten Meistertitels hohe Verluste mit illegalen Sportwetten verbucht hätte. Maradona wurde mit 15 Toren Torschützenkönig. In der Saison 1988/89 wurde Neapel erneut Tabellenzweiter und verlor das Pokalfinale gegen Genua, jedoch schlug der Verein im Europapokal Größen wie Juventus Turin oder Bayern München und gewann gegen den VfB Stuttgart erstmals den UEFA-Cup. Unmittelbar nach diesem Erfolg bat Maradona den Vereinspräsidenten Corrado Ferlaino um die Freigabe. Zum einen hatte er mit Neapel aus sportlicher Sicht bereits die größtmöglichen Erfolge gefeiert. Zum anderen fühlte er sich vom Leben in Neapel und der Zuneigung der Neapolitaner zunehmend erdrückt. Hinzu kamen persönliche Skandale wie die Geburt eines unehelichen Sohnes und Verbindungen zum Giuliano Clan, der Maradona hofierte und vermehrt mit Kokain sowie Prostituierten versorgte. Maradona zufolge hatte ihm Ferlaino in der Vorsaison mündlich zugesichert, dass er den Verein im Falle des Europapokalsieges verlassen dürfe. Nun aber lehnte Ferlaino alle Gesuche ab, darunter ein Angebot von Olympique Marseille. Nach eigener Aussage wurde er damit zu Maradonas Kerkermeister. Maradona antwortete, indem er mit einem Monat Verspätung aus dem Sommerurlaub zurückkehrte und sich ins Nachtleben stürzte. Er feierte nach jedem Wochenendspiel bis mittwochs Kokainexzesse, ehe er donnerstags wieder ins Mannschaftstraining einstieg, um seinen Körper auf das nächste Spiel vorzubereiten. Den Verantwortlichen der SSC Neapel blieb Maradonas Drogenkonsum nicht verborgen. Der Verein half gar, negative Dopingbefunde durch Abgabe falscher Urinproben herbeizuführen. Solange Maradona spielte, hielt Neapels Erfolg an. In der Saison 1989/90 wurde der Verein mit zwei Punkten Vorsprung auf die AC Mailand erneut italienischer Meister. Im Sommer 1990 wurde Maradona durch die Vorkommnisse bei der Weltmeisterschaft in Italien (siehe Abschnitt "WM 1990") zur meistgehassten Person des Landes. Nach dem Turnier kehrte er formschwach und übergewichtig zur SSC Neapel zurück. Seine sechs Tore in der Saison 1990/91 erzielte er alle per Strafstoß. Die Eskapaden abseits des Fußballplatzes nahmen zu, der sportliche Erfolg ab. Im Europapokal der Landesmeister schied Neapel bereits in der 2. Runde aus, in der Liga rückte die Titelverteidigung in weite Ferne. Im Februar 1991 eröffnete die Polizei eine Untersuchung, nachdem bei einer Abhöraktion in Verbindung mit Drogen und Prostituierten mehrfach Maradonas Name gefallen war. Im Zuge dessen wurde er später wegen Besitzes und der Weitergabe von Betäubungsmitteln in Abwesenheit zu 14 Monaten Freiheitsstrafe auf Bewährung verurteilt. Zuvor wurde ihm am 29. März 1991 in einer zwei Wochen zuvor nach einem Heimsieg gegen den AS Bari abgegebenen Dopingprobe die Einnahme von Kokain nachgewiesen. Damit war Maradonas Drogensucht - in Neapel längst ein offenes Geheimnis - zur offiziellen Realität geworden. Am 1. April 1991 verließ er Neapel in Richtung Argentinien. Am 6. April wurde ihm vom italienischen Fußballverband eine 15-monatige Sperre bis zum 30. Juni 1992 auferlegt, die im Anschluss von der FIFA weltweite Wirkung erhielt. Am 26. April 1991 wurde Maradona bei einer Razzia im Stadtteil Caballito mit zwei Freunden wegen Drogenbesitzes verhaftet. Im anschließenden Gerichtsprozess wurde er unter anderem zu einer Entziehungskur verpflichtet. Gleichzeitig engagierte er einen privaten Fitnesstrainer, der ihn für ein Comeback fit machen sollte. Maradonas Vertrag mit der SSC Neapel behielt auch nach Ablauf der Sperre bis zum Sommer 1993 Gültigkeit. Der Spieler zeigte jedoch wenig Interesse an einer Rückkehr zu seinem Verein, da er in Italien mittlerweile als persona non grata galt und mit weiteren Gerichtsprozessen hätte rechnen müssen. Über seinen Manager Marcos Franchi forderte er die vorzeitige Auflösung seines Vertrages, was Klubpräsident Ferlaino ablehnte, da sich Maradona auf diese Weise ablösefrei einem neuen Verein hätte anschließen dürfen. Im August 1992 knüpfte er seine Rückkehr schließlich an zahlreiche Bedingungen und Zugeständnisse, auf die der Verein weitgehend nicht einging. Ende September 1992 wurde schließlich sein Wechsel zum FC Sevilla bekanntgegeben. Nach Maradonas Abschied begann der langsame Niedergang der SSC Neapel, die in finanzielle Schwierigkeiten geriet und mit der Neugründung in der Serie C1 2004 ihren sportlichen Tiefpunkt erreichte. Erst 2012 sollte der Verein mit der Coppa Italia wieder einen nationalen Titel gewinnen. In 259 Pflichtspielen hatte Maradona 115 Tore erzielt. Seine Trikotnummer 10 wird vom Verein seit 2000 nicht mehr vergeben. === FC Sevilla (1992/93) Der Wechsel zum FC Sevilla war von Carlos Bilardo eingefädelt worden. Der ehemalige argentinische Nationaltrainer, mit dem Maradona 1986 Weltmeister geworden war, hatte im Juli 1992 das Amt des Cheftrainers bei den Andalusiern übernommen und die Vereinsführung von der Verpflichtung seines Landsmannes überzeugt. Da Sevilla die von Napoli aufgerufene Ablösesumme nicht zahlen wollte, schaltete sich die FIFA in die festgefahrenen Verhandlungen ein. Maradona hatte den Weltverband in der Vergangenheit zwar öffentlich als "autokratische Organisation" kritisiert, jedoch sorgten sich die Spitzenfunktionäre um seine Teilnahme an der bevorstehenden und kommerziell wichtigen WM 1994 in den Vereinigten Staaten. Daher übernahm FIFA-Generalsekretär Sepp Blatter die Rolle des Vermittlers und erzielte am 21. September 1992 einen für die Fußballöffentlichkeit überraschend schnellen Verhandlungsdurchbruch. Im Ergebnis zahlte der FC Sevilla 7,5 Millionen US-Dollar an Neapel und nahm Maradona unter Vertrag. Um einen Großteil der Transfersumme aufzubringen, verpflichtete sich der Verein zur Austragung zahlreicher Freundschaftsspiele und verkaufte deren TV-Übertragungsrechte an Silvio Berlusconis Mediengruppe Mediaset. Am 4. Oktober 1992 feierte Maradona im Rahmen einer 1:2-Niederlage gegen Athletic Bilbao am fünften Spieltag der Primera Division 1992/93 sein Comeback. Nach eineinhalb Jahren ohne Pflichtspiel lief er seiner Bestform hinterher und war nicht mehr der Spieler, der Mitte der achtziger Jahre den Weltfußball dominiert hatte. Seine athletischen Defizite glich er aus, indem er Spielsituationen vorausahnte, und seine Standards waren immer noch brandgefährlich. Am 19. Dezember 1992 lieferte er im Rahmen eines 2:0-Heimsieges gegen Real Madrid seine wohl beste Vorstellung im Sevilla-Trikot ab. Im Februar 1993 kehrte er sogar in die Nationalmannschaft zurück, für die er letztmals im WM-Finale 1990 aufgelaufen war. Die Reisen zur Nationalmannschaft führten jedoch zu Auseinandersetzungen mit dem Vereinspräsidium. Darüber hinaus sorgte Maradonas Lebensstil auch in Sevilla für zahlreiche Eskapaden. Als er gegen Saisonende an Form verlor und am vorletzten Spieltag gegen Real Burgos vorzeitig ausgewechselt wurde, überwarf er sich in aller Öffentlichkeit mit Trainer Bilardo. Dadurch endete sein zweites Engagement in Spanien bereits nach nur einer Saison. === Newell's Old Boys (1993/94) Im September 1993 wechselte Maradona zurück nach Argentinien zu den Newell's Old Boys. Sein Gastspiel bei dem Verein aus Rosario war von kurzer Dauer. Er bestritt bis Dezember fünf Pflichtspiele. Anschließend führten eine Muskelverletzung und sein angespanntes Verhältnis zu Trainer Jorge Castelli dazu, dass er den Klub bereits Anfang 1994 wieder verließ. Am 2. Februar 1994 schoss er mit einem Luftgewehr auf Journalisten, die seine Villa nahe Lomas de Zamora belagerten, und wurde zu einer 34-monatigen Haftstrafe auf Bewährung verurteilt. === Boca Juniors (1995 bis 1997) Nach Ablauf der 15-monatigen Sperre für die Einnahme verbotener Substanzen bei der WM 1994 feierte Maradona im Oktober 1995 ein Comeback bei den Boca Juniors in der argentinischen Primera Division. Der Verein erreichte in der Apertura 1995 aber nur einen enttäuschenden vierten Platz. Im Anschluss wurde Mauricio Macri zum neuen Klubpräsidenten gewählt und mit Carlos Bilardo ein neuer Trainer verpflichtet. Maradona und Bilardo versöhnten sich und die Boca Juniors spielten in der Clausura 1996 lange um die Meisterschaft mit. In der heißen Phase des Titelrennens verschoss Maradona jedoch gleich fünf Elfmeter in Folge. Boca verlor den Anschluss und wurde nur Fünfter. Am Ende der Saison 1995/96 verließ Maradona den Klub temporär. Im April 1997 unterzeichnete er ein neues Arbeitspapier und am 9. Juli 1997 kehrte er nach elf Monaten gegen Ende der Clausura 1997 auf den Fußballplatz zurück. Zu Beginn der Apertura 1997 trafen die Boca Juniors am 24. August auf die Argentinos Juniors. Nach dem Spiel musste Maradona zur Dopingkontrolle, woraufhin ihm erneut die Einnahme von Kokain nachgewiesen wurde. Maradona ließ das Ergebnis zunächst anfechten und durfte so weiterhin am Spielbetrieb teilnehmen. Am 25. Oktober 1997 bestritt er im Rahmen eines 2:1-Auswärtssieges gegen River Plate sein letztes Profispiel. Als sich die Anzeichen verdichteten, dass die Anfechtung abgelehnt würde, kam Maradona einer weiteren Sperre zuvor und verkündete am 30. Oktober 1997, seinem 37. Geburtstag, sein offizielles Karriereende. == Nationalmannschaftskarriere === Beginn Nur vier Monate nach seinem Debüt in der Primera Division nominierte Nationaltrainer Cesar Luis Menotti den 16-jährigen Maradona für die A-Nationalmannschaft. Am 27. Februar 1977 absolvierte "El Pibe" sein erstes Länderspiel für die Albiceleste, als er im Freundschaftsspiel gegen Ungarn (5:1) in der 62. Minute für Leopoldo Luque eingewechselt wurde. Anschließend versetzte Menotti ihn in die U-20-Auswahl, mit der er im April 1977 an der U-20-Südamerikameisterschaft in Venezuela teilnahm. Dort schieden die enttäuschenden Argentinier sieglos in der Vorrunde aus. Maradona war zweimal eingesetzt worden. Vor der Weltmeisterschaft 1978 gehörte Maradona dem vorläufigen WM-Kader an, wurde jedoch zu seiner großen Enttäuschung nicht für das Turnier berücksichtigt. Aus Sicht der argentinischen Militärdiktatur war die Austragung der WM ein Ereignis von nationaler Bedeutung, die ihre Herrschaft stabilisieren sollte, weshalb Menotti den Jungstar als noch nicht reif genug befunden hatte, um dem Druck standzuhalten. Die argentinische Mannschaft gewann ohne Maradona ihren ersten WM-Titel. In seinem neunten A-Länderspiel, am 2. Juni 1979 beim 3:1-Sieg über Schottland im Glasgower Hampden Park, gelang Maradona sein erstes Tor. === Junioren-WM 1979 Im September 1979 führte Maradona die U-20-Auswahl seines Landes zum Gewinn der Junioren-Weltmeisterschaft. Bei dem in Japan ausgetragenen Turnier schoss er sechs der zwanzig argentinischen Tore und wurde zum besten Spieler gewählt. Anschließend kehrte Maradona in die Seniorenmannschaft der "Albicelestes" zurück und war aus deren Stammformation bald nicht mehr wegzudenken. === WM 1982 Die Erwartungen an El Pibe de Oro waren vor der Weltmeisterschaft 1982 in Spanien enorm hoch. Argentinien ging mit neun Weltmeistern ins Turnier (u. a. Mario Kempes, Daniel Passarella, Osvaldo Ardiles), und mit Maradona wollte man den Titel erfolgreich verteidigen. Sein erstes WM-Spiel bestritt er im Eröffnungsspiel gegen Belgien, in seinem neuen Heimatstadion Camp Nou in Barcelona, das Argentinien jedoch mit 0:1 verlor. Die Belgier hatten Maradona einer harten Manndeckung unterstellt und ihn so weitgehend aus dem Spiel genommen - eine Erfahrung, die der Jungstar im Turnierverlauf noch öfter erleben sollte. Im zweiten Gruppenspiel gegen Ungarn wurde er zwar wieder doppelt bewacht, doch war er in dieser Partie nicht zu halten und traf doppelt beim 4:1-Sieg. In der Zwischenrunde hatte er gegen die erfahrenen italienischen Verteidiger einen schweren Stand, dabei wurde er vom beinharten Claudio Gentile gedeckt, der ihn auch einmal mit einem Faustschlag niederstreckte. Gentile sah dafür lediglich Gelb. Maradona setzte einen Freistoß ans Lattenkreuz und der spätere Turniersieger Italien gewann 2:1. Gegen Brasilien verlor Argentinien klar mit 1:3. Maradona trat dabei seinem Gegenspieler Batista kurz vor Spielende entnervt in den Magen und sah dafür die Rote Karte. === WM 1986 Die Weltmeisterschaft 1986 in Mexiko prägte Maradona wie wohl kein anderer Spieler eine WM zuvor oder danach. Als neuer Kapitän der Nationalmannschaft führte er die argentinische Elf zunächst zum Gruppensieg. Dabei bereitete er beim 3:1-Auftakterfolg gegen Südkorea alle argentinischen Tore vor und traf im zweiten Gruppenspiel gegen Titelverteidiger Italien selbst zum 1:1-Endstand. Im letzten Gruppenspiel gegen Bulgarien (2:0) steuerte er eine weitere Torvorlage bei. In der anschließenden K.-o.-Phase erreichte Maradona Topform. Im Viertelfinale gegen England erzielte er zwei seiner bekanntesten Tore. Beim Führungstor beförderte er den Ball regelwidrig mit seiner Hand über den englischen Torhüter Peter Shilton hinweg ins Netz, wobei er im Anschluss an das Spiel in diesem Zusammenhang von der "Hand Gottes" sprach ("Es war der Kopf Maradonas und die Hand Gottes"). Drei Minuten später traf er nach einem Dribbling über das halbe Spielfeld, bei dem er die gesamte englische Abwehr ausspielte, zum 2:0. Dieser Treffer wurde 2002 von der FIFA zum "WM-Tor des Jahrhunderts" gekürt. Beim 2:0-Halbfinalsieg gegen die Überraschungsmannschaft Belgien schoss Maradona erneut beide Tore. Das Tor zum Endstand, dem erneut ein Dribbling vorausging, landete bei der Wahl zum WM-Tor des Jahrhunderts auf dem vierten Platz. Im Finale gegen Deutschland wurde Maradona teilweise von zwei Spielern manngedeckt (Lothar Matthäus und Karlheinz Förster). Er kam nicht so zur Entfaltung wie im bisherigen Turnier, schaffte aber mehr Räume für seine Mitspieler (v. a. Jorge Valdano und Jorge Burruchaga). Fünf Minuten vor Spielende gab er den entscheidenden Steilpass auf Burruchaga, der zum 3:2-Siegtreffer vollendete. Argentinien war zum zweiten Mal Weltmeister und hatte diesen Titel zum Großteil Maradona zu verdanken (5 Tore und 5 Torvorlagen), der als bester Spieler der WM ausgezeichnet wurde. Zudem erhielt er zum vierten Mal die Auszeichnung zum Fußballer des Jahres in Argentinien und Südamerika. === WM 1990 Bei der WM 1990 in Italien führte Maradona Argentinien wieder als Spielführer ins Turnier. Er war wegen eines geschwollenen Knöchels auf Schmerzmittel angewiesen und spielte eine weitaus weniger dominante Weltmeisterschaft als noch vier Jahre zuvor. Das Eröffnungsspiel gegen Kamerun ging mit 0:1 verloren. Die zwei verbleibenden Gruppenspiele gegen die UdSSR (2:0) und Rumänien (1:1) bestritt Argentinien in Maradonas "Wohnzimmer", dem Stadio San Paolo. Außerhalb von Neapel wurde er in jedem Stadion ausgepfiffen. Argentinien qualifizierte sich letztlich als bester Gruppendritter für das Achtelfinale. Dort traf die Albiceleste auf Brasilien und war weitgehend unterlegen. Ein genialer Moment von Maradona, der in der 80. Minute das Siegtor von Claudio Caniggia vorbereitete, genügte jedoch zum Einzug ins Viertelfinale. Gegen Jugoslawien gewann Argentinien nach 120 torlosen Minuten trotz eines Fehlschusses von Maradona im Elfmeterschießen. Im Halbfinale erwies sich Gastgeber und Turnierfavorit Italien als schwierige Aufgabe. Der Austragungsort Neapel entwickelte sich dabei fast zum Politikum, weil Maradona im Vorfeld des Spiels verkündete, die Stadt gehöre nicht zu Italien, und die Hoffnung zum Ausdruck brachte, die Neapolitaner würden in Anbetracht seiner Beiträge zu den Erfolgen des Heimatklubs in dieser Partie zu Argentinien halten. Argentinien gewann das Spiel erneut im Elfmeterschießen, wobei diesmal auch Maradona vom Elfmeterpunkt traf. Das Finale in Rom war eine Neuauflage des 1986er-Endspiels gegen Deutschland. Als die argentinische Nationalhymne unmittelbar vor der Partie seinetwegen lautstark ausgepfiffen wurde, beschimpfte Maradona die Zuschauer als Hurensöhne. Im Anschluss wurde er erfolgreich von Guido Buchwald manngedeckt und fand zu keiner Zeit ins Spiel. Deutschland wurde mit einem 1:0-Sieg Weltmeister. === WM 1994 Argentinien verpasste ohne Maradona die direkte Qualifikation zur WM 1994 in den USA durch eine 0:5-Niederlage gegen Kolumbien am letzten Spieltag. Für die Entscheidungsspiele gegen Australien im Oktober und November 1993 sah sich Nationaltrainer Alfio Basile gezwungen, den inzwischen 33-Jährigen trotz fehlender Spielpraxis wieder ins Aufgebot zu berufen. Zur Vorbereitung auf seine vierte und wohlwissend letzte WM-Endrunde setzte Maradona auf ein diszipliniertes Trainingsprogramm, das von seinem langjährigen privaten Fitnesstrainer, Fernando Signorini, und Daniel Cerrini ausgearbeitet wurde, mit dem er erstmals für sein Engagement bei Newell's Old Boys zusammengearbeitet hatte. Beim 4:0-Auftaktsieg gegen Griechenland erlebte die Fußballwelt einen sehr motivierten Maradona, der sich in ausgezeichneter körperlicher Verfassung befand und seine bemerkenswerte Leistung mit dem Tor zum 3:0 krönte. Auch im zweiten Vorrundenspiel gegen Nigeria (2:1) bildete er mit Claudio Caniggia ein kongeniales Duo und strafte seine Kritiker Lügen, die ihm eine Wiederholung der Leistung aus dem ersten Spiel nicht zugetraut hatten. In einer nach dem Spiel abgegebenen Urinprobe wurden Maradona jedoch verbotene Substanzen nachgewiesen (u. a. der Appetitzügler Ephedrin). Eine spätere Untersuchung ergab, dass er die Substanzen nicht wissentlich eingenommen hatte; vielmehr waren sie ihm von seinem privaten Fitnesstrainer Cerrini verabreicht worden. Ungeachtet dessen wurde Maradona vom Turnier ausgeschlossen und erhielt zum zweiten Mal in seiner Karriere eine 15-monatige Sperre. Maradonas Nationalmannschaftskarriere war damit beendet. Maradona betrachtete sich als unschuldiges Opfer einer Verschwörung. Er schloss mit dem TV-Sender Canal 13 einen Exklusiv-Vertrag ab, um für eine Gage von 1,5 Millionen Dollar als WM-Kommentator in den USA zu bleiben. Die WM hatte durch den Skandal mit Maradona einen der großen Stars verloren. Argentinien zählte danach nicht mehr zu den Turnierfavoriten und schied im Achtelfinale gegen Rumänien aus. === Copa America Die Copa America, das südamerikanische Äquivalent zur Europameisterschaft, konnte Maradona nie gewinnen. Er nahm an drei Turnieren teil: 1979 schied er mit Argentinien in der Vorrunde aus, 1987 wurde er im eigenen Land Vierter. 1989 erreichte er mit dem dritten Platz die beste Platzierung. 1983 fehlte er verletzungsbedingt. Die Turniersiege in den Jahren 1991 und 1993 verpasste er aufgrund seiner ersten 15-monatigen FIFA-Sperre und einer weiteren Verletzung. == Spielweise Maradona war ein klassischer Spielmacher, zu dessen Stärken Dribblings auf engstem Raum, präzise Pässe, seine Spielintelligenz und seine kreative Spielgestaltung zählten. Er war ein Linksfüßer, der oft auch dann von seinem linken Fuß Gebrauch machte, wenn der Ball eigentlich besser für den rechten Fuß geeignet war. Maradona hatte eine kompakte Physis: Seine starken Beine und der durch seine geringe Größe tiefe Körperschwerpunkt ermöglichten es ihm, beim Laufen mit dem Ball den körperlichen Attacken seiner Gegenspieler standzuhalten. Seine Ballkontrolle war so eng, dass der Ball beim Dribbling zu seinem Körper zu gehören schien. Er war ein technisch versierter Stratege und Teamplayer, konnte sich mühelos mit dem Ball auf engstem Raum bewegen und zog meist eine Vielzahl an Gegenspielern auf sich, woraufhin er entweder mit rasanten Körpertäuschungen zum Dribbling ansetzte oder einem freistehenden Mitspieler eine Torchance ermöglichte. Darüber hinaus war Maradona ein torgefährlicher Freistoß- und Elfmeterschütze. Maradona bewies bemerkenswerte Führungsqualitäten. Als Anführer auf und neben dem Spielfeld war er das Sprachrohr seiner Teams und schreckte auch vor Kritik an Autoritätspersonen nicht zurück. Seine Fähigkeiten als Spieler und seine übermächtige Persönlichkeit wirkten sich dabei positiv auf sein jeweiliges Team aus. Sein Mitspieler bei der WM 1986, Jorge Valdano, erklärte: "Maradona war ein technischer Anführer: Er fand für sämtliche Schwierigkeiten auf dem Spielfeld eine Lösung. An erster Stelle war er dafür verantwortlich, Wunder zu vollbringen. Das gibt der Mannschaft jede Menge Selbstvertrauen. Darüber hinaus war das Ausmaß seiner Berühmtheit so gewaltig, dass er im Namen seiner Teamkollegen den gesamten Druck absorbierte. Als Diegos Mitspieler konnte man vor einem Spiel gut schlafen. Man wusste, dass er Dinge vollbringen konnte, die kein anderer zustande brachte. Und im Unterbewusstsein war uns klar, dass er derjenige sein würde, der im Falle einer Niederlage den Großteil der Verantwortung stemmen müsste. Das war der Einfluss, den er auf das Team ausübte." Maradona war bekannt für seine gewitzte, charismatische Persönlichkeit. Sein Spitzname El Pibe de Oro ("Goldjunge") geht damit einher: Pibe ist die Bezeichnung für einen gewieften und listigen, nonkonformistischen Einzelgänger. Sein kontroverses Tor mit der Hand bei der WM 1986, das er tarnte, indem er zeitgleich eine Kopfbewegung wie bei einem Kopfball ausführte, wird von einigen Kritikern als Verkörperung des Viveza-criolla-Konzepts angesehen, das in der Slumsiedlung vorherrschte, in der Maradona aufwuchs. Einige Publikationen haben Maradonas Charakter mit dem des Artful Dodger verglichen, einer Figur aus Charles Dickens' Roman Oliver Twist. == Karriere als Trainer und Fußballfunktionär === Frühe Trainerkarriere Maradona versuchte sich während seiner Dopingsperre 1994/95 als Trainer der argentinischen Erstligisten Textil Mandiyu und Racing Club, aber ohne Erfolg. Mandiyu spielte unter seiner Regie zwölf Partien und gewann eine, Racing Club gewann zwei von elf. === Argentinischer Nationaltrainer Im Oktober 2008 wurde Maradona Trainer der argentinischen Nationalmannschaft. Obwohl er kaum über Erfahrung als Fußballtrainer verfügte, setzte er sich nach einem Gespräch mit dem argentinischen Verbandspräsidenten Julio Grondona unter anderem gegen Sergio Batista, Diego Simeone, Miguel Angel Russo und Carlos Bianchi durch. Maradonas Vorgänger Carlos Bilardo fungierte als Teammanager. Nach der lange gefährdeten, aber letztlich erfolgreichen Qualifikation zur WM 2010 sorgte er mit Aussagen über seine Kritiker auf einer Pressekonferenz für einen Skandal. Er wurde in der Folge für zwei Monate gesperrt und musste 25.000 Schweizer Franken Strafe zahlen. In der Gruppenphase der Endrunde qualifizierte sich Argentinien ohne Punktverlust für das Achtelfinale und zog nach einem 3:1-Sieg gegen Mexiko ins Viertelfinale ein. Dort unterlag Maradonas Team mit 0:4 gegen Deutschland und schied aus dem Turnier aus. Am 27. Juli 2010 wurde er vom Verband entlassen, nachdem Verhandlungen über die Fortsetzung seiner Arbeit gescheitert waren. Maradona hatte die weitere Zusammenarbeit mit seinen Assistenten zur Bedingung gemacht. === Weitere Stationen Nach seinem Engagement als Nationaltrainer zog es Maradona im Mai 2011 zu Al-Wasl in die UAE Arabian Gulf League. Er unterschrieb einen Zweijahresvertrag bis Sommer 2013. Der Verein schloss die Saison 2011/12 nur als Tabellenachter ab. In der GCC Champions League gelang der Finaleinzug, das Endspiel ging jedoch im Elfmeterschießen gegen Muharraq Club verloren. Daraufhin wurde Maradona im Juli 2012 nach 14 Monaten im Amt vorzeitig entlassen. Im August 2013 schloss sich Maradona dem argentinischen Fünftligisten Deportivo Riestra auf ehrenamtlicher Basis als Mentaltrainer an. Am 7. Mai 2017 unterzeichnete Maradona einen Einjahresvertrag als Cheftrainer des Al-Fujairah SC, einem Zweitligisten der Vereinigten Arabischen Emirate. Nachdem der direkte Aufstieg am Saisonende knapp verpasst worden war, trat er am 27. April 2018 zurück. Knapp zwei Wochen später gelang dem Verein über die Playoffs der Aufstieg in die UAE Arabian Gulf League. Im Juli 2018 wurde Maradona als Vorstandsvorsitzender des weißrussischen Erstligisten Dynamo Brest vorgestellt. Bereits Anfang September trennten sich die Wege wieder. Er blieb jedoch Ehrenvorsitzender des Vereins. Am 6. September 2018 wurde Maradona Trainer des mexikanischen Zweitligisten Dorados de Sinaloa. Dass ausgerechnet der einst kokainsüchtige Maradona in den Bundesstaat Sinaloa übersiedelte, der für das in den Drogenhandel verwickelte Sinaloa-Kartell bekannt ist, wurde zunächst mit Skepsis aufgenommen. Der Verein spielte unter Maradona jedoch eine erfolgreiche Saison 2018/19 und qualifizierte sich sowohl in der Apertura als auch in der Clausura für die Playoffs. Dort unterlag Dorados de Sinaloa jeweils Atletico San Luis im Finale nach Verlängerung. Am 13. Juni 2019 trat Maradona wegen gesundheitlicher Probleme zurück. Seine Zeit in Sinaloa war Thema der im November 2019 veröffentlichten Netflix-Dokuserie Maradona in Mexico. Am 5. September 2019 wurde Maradona Trainer bei Gimnasia y Esgrima La Plata, dem zu diesem Zeitpunkt Tabellenletzten der argentinischen Primera Division. Der Vertrag lief zunächst bis zum Saisonende. Am 19. November 2019 trat er trotz bis dahin guter Ergebnisse aus Solidarität mit Vereinspräsident Gabriel Pellegrino, dessen Bewerbung für eine Wiederwahl an internen Streitigkeiten zu scheitern drohte, zurück. Zwei Tage später revidierte er seine Entscheidung, nachdem es zu einer Einigung gekommen war. Im Dezember 2019 wurde Pellegrino für weitere drei Jahre ins Amt gewählt. Ende April 2020 wurde die Saison wegen der COVID-19-Pandemie in Argentinien auf Geheiß der AFA abgebrochen. Zu diesem Zeitpunkt stand Gimnasia y Esgrima La Plata auf dem 19. von 24 Plätzen. Anfang Juni 2020 verlängerte Maradona seinen Vertrag daraufhin bis zum Ende der Saison 2020/21. == Titel, Auszeichnungen und Ehrungen === Als Vereinsspieler Boca Juniors FC Barcelona SSC Neapel === Individuelle Auszeichnungen * Maradona gewann die von der FIFA initiierte Internetabstimmung unter Fußballfans zum besten Fußballspieler des vergangenen Jahrhunderts; er war für die FIFA wegen seiner zahlreichen Skandale jedoch schwer zu vermarkten, sodass durch eine von der FIFA eingesetzte Jury ein Äquivalent zur Internetabstimmung geschaffen wurde, die der FIFA-konforme Pele gewann; somit wurde der Titel "Fußballer des Jahrhunderts" zweimal vergeben. == Ehe und Nachkommen Maradona heiratete seine langjährige Freundin Claudia Villafane am 7. November 1989 in Buenos Aires. Aus dieser Verbindung stammen zwei Töchter: Dalma Nerea (* 1987) und Giannina Dinorah (* 1989). Die Ehe wurde 2004 geschieden. Seine Tochter Giannina war mit dem argentinischen Fußballer Sergio Agüero liiert. Zu seiner Zeit in Neapel unterhielt Maradona eine Beziehung zu Cristiana Sinagra, aus welcher der Sohn Diego Armando jr. (* 1986) hervorging, der in der italienischen Strandfußball-Nationalmannschaft spielte. 1993 wurde er von einem italienischen Gericht zu Unterhaltszahlungen verpflichtet, nachdem er sich geweigert hatte, eine DNA-Probe abzugeben. Erst im August 2016 bekannte er sich zur Vaterschaft. Darüber hinaus zeugte Maradona eine Tochter (* 1996) mit Valeria Sabalain und einen weiteren Sohn (* 2013) mit Veronica Ojeda. Im März 2019 bekannte sich Maradona zur Vaterschaft von drei weiteren Kindern aus der Zeit seiner Kuraufenthalte in Kuba zu Beginn der 2000er Jahre. == Saisonstatistik Quellen: footballdatabase.eu, national-football-teams.com == Weiterer Werdegang Im Oktober 1985 entließ er Cyterszpiler und ersetzte ihn mit Guillermo Coppola. Coppola ordnete Maradonas Finanzen, erarbeitete sich jedoch einen zweifelhaften Ruf in Bezug auf den Drogenkonsum seines Klienten. Im Oktober 1990 gab er den Posten ab und wurde durch Mario Franchi ersetzt. Im Januar 1995 einigten sich Maradona und Coppola auf eine erneute Zusammenarbeit. Im Oktober 1996 wurde Coppola bei einer Razzia wegen Kokainbesitzes festgenommen. Im Juni 2005 kehrte Maradona nach Neapel zurück, um dem Abschiedsspiel von Ciro Ferrara im Stadio San Paolo beizuwohnen. Anfang August 2005 begann er beim argentinischen Fernsehkanal Canal 13 eine eigene Fernsehshow zu moderieren, La Noche del 10 ("Die Nacht der Nummer 10"). In dieser Show, deren 13 Episoden bis Ende des Jahres im argentinischen Fernsehen liefen, sprach Maradona mit Prominenten und Sportlern aus aller Welt, darunter Fidel Castro, Pele, Roberto Duran und Mike Tyson. === Drogen, Krankheiten und Tod Am 4. Januar 2000 erlitt Maradona während eines Aufenthaltes im uruguayischen Badeort Punta del Este einen schweren Herzinfarkt, der auf eine Überdosis Kokain zurückgeführt wurde. Sein Manager Coppola wurde bei einer anschließenden Befragung durch die Polizei zur Herkunft des Kokains wegen Falschaussagen festgenommen. Nach seiner Genesung unterzog sich Maradona einer sechsmonatigen Entziehungskur auf Kuba, wo er Freundschaft mit Fidel Castro schloss. Am 18. April 2004 wurde Maradona wegen hohen Blutdrucks, Atemnot und einer Lungenentzündung in eine Klinik in Buenos Aires eingeliefert. Nach zwölf Tagen durfte er das Krankenhaus wieder verlassen. Anfang Mai erlitt er jedoch einen Rückfall, der eine weitere Behandlung auf der Intensivstation notwendig machte. Die Krankenhausaufenthalte wurden schließlich auf Maradonas schwere Kokainsucht zurückgeführt, weswegen er Mitte Mai 2004 auf Druck seiner Angehörigen in eine Nervenklinik am Stadtrand von Buenos Aires eingeliefert wurde und ein Rehabilitationsprogramm begann. Am 12. August 2004 traf sich Maradona unter strengen Sicherheitsvorkehrungen mit Staatspräsident Nestor Kirchner (siehe Foto), um eine Ausreisegenehmigung für einen Suchtklinikaufenthalt in der Schweiz zu erhalten. Wegen eines Gerichtsbeschlusses war es Maradona zu dieser Zeit nicht erlaubt, Argentinien zu verlassen. Im Jahr 2005 besserte sich Maradonas Gesundheitszustand nach der Entziehungskur erheblich, insbesondere nachdem er sich in Kolumbien einer Magenverkleinerung unterzogen hatte, um sein chronisches Übergewicht zu reduzieren. Ab 28. März 2007 wurde Maradona wegen starken Alkoholmissbrauchs mit einer toxischen Hepatitis zwei Wochen in einer Klinik in Buenos Aires behandelt. Mitte April erlitt er nur wenige Tage nach seiner Entlassung aus dem Krankenhaus einen Rückfall. Im Anschluss hielt er sich bis zum 7. Mai 2007 zur Behandlung in einer psychiatrischen Klinik auf. Am 8. Mai trat er im argentinischen Fernsehen auf und erklärte, seit zweieinhalb Jahren keine harten Drogen mehr genommen zu haben und fortan auf Alkohol verzichten zu wollen. Am 16. November 2015 unterzog sich Maradona im Krankenhaus von Maracaibo in Venezuela einer zweiten Magen-Bypass-Operation, nachdem sein behandelnder Arzt bei ihm 75 kg Übergewicht diagnostiziert hatte. Anfang November 2020 unterzog er sich einer Kraniotomie, bei der ihm ein Blutgerinnsel entfernt wurde. Er wurde wenige Tage nach der Operation aus dem Krankenhaus entlassen. Am 25. November 2020 verstarb er im Alter von 60 Jahren infolge eines erneuten Herzinfarkts. Daraufhin rief die Regierung Argentiniens eine dreitägige Staatstrauer aus. Gegen mindestens sieben Mitglieder des medizinischen Teams, darunter Maradonas Leibarzt Leopoldo Luque, ermittelt die Staatsanwaltschaft wegen des Verdachts der fahrlässigen Tötung. === Politische Aktivitäten und Positionen Im Jahr 2000 veröffentlichte er seine Autobiografie Yo Soy El Diego, die er unter anderen Fidel Castro und dem kubanischen Volk widmete. Zudem ließ er sich Tattoos von Castro und Che Guevara auf die linke Wade bzw. den rechten Oberarm stechen. Im November 2005 nahm Maradona an einer Protestkundgebung im Umfeld des vierten Amerika-Gipfels teil und skandierte gegen George W. Bush gerichtete anti-US-amerikanische Parolen. Bei der Präsidentschaftswahl in Venezuela 2013 unterstützte Maradona den Sozialisten Nicolas Maduro. Die NZZ sprach ihm ein Faible für autoritäre Staaten zu, dies nach Sympathiekundgebungen für Kuba und den venezolanischen Staatspräsidenten Chavez, der Wahlkampfunterstützung für dessen Nachfolger Maduro oder dem Besuch beim tschetschenischen Präsidenten Ramsan Kadyrow. Maradona selbst bezeichnete sich stets als Peronist. == Filme Der tiefe Eindruck, den Maradona bei einem Auftritt mit Barcelona in Belgrad 1982 hinterließ, als er bei einem 4:2-Auswärtssieg Barcelonas vor 120.000 Zuschauern zwei Tore schoss, führte unter anderem dazu, dass der Regisseur Emir Kusturica 2006 einen Dokumentarfilm über Maradonas Leben mit dem Titel Maradona by Kusturica drehte. Weitere Filme: == Neologismen Aufgrund seiner Qualität als Fußballer war Maradonas Name eine Zeit lang in der Presse Synonym für einen begnadeten Fußballer. So wurden die Techniker Gheorghe Hagi als "Karpaten-Maradona", Andres D'Alessandro als "Mini-Maradona" und Andreas Herzog als Alpen-Maradona bezeichnet. Des Weiteren wird in der argentinischen Presse regelmäßig die Bezeichnung "neuer Maradona" für talentierte Nachwuchsspieler benutzt. So wurden z. B. Javier Saviola, Diego Latorre, Ariel Ortega, Marcelo Gallardo, Juan Roman Riquelme, Pablo Aimar und Lionel Messi als "neue Maradonas" bezeichnet. Lionel Messi wurde zudem nach besonders spektakulären Toren und hochklassigen Spielen als "Messidona" gefeiert.

Title: Diego Maradona Summary: Diego Armando Maradona war ein argentinischer Fussballspieler. Für einige Menschen war er der beste Fussballer, den es je gab. Maradona wurde im Jahr 1960 geboren. Seine Familie lebte in ärmlichen Verhältnissen am südlichen Stadtrand der argentinischen Hauptstadt Buenos Aires. Als er neun Jahre alt war, wurde er beim Spielen mit seiner Strassenmannschaft von einem Talentsucher entdeckt. Er spielte dann in der Kindermannschaft "Die Zwiebelchen". Damals wurde er "Der Goldjunge" genannt. Als zwölfjähriger Balljunge unterhielt er in den Halbzeitpausen die Zuschauer im Stadion mit seinen Dribbelkünsten. Maradona kam als junger Spieler in die Mannschaft Boca Juniors in Buenos Aires. Mit nur 21 Jahren galt er als der neue Superstar im Weltfussball und wurde mit Pele verglichen. Im Jahr 1982 nahm Maradona an seiner ersten Fussball-Weltmeisterschaft teil und wechselte für sehr viel Geld zum spanischen Fussballverein FC Barcelona. Im Jahr 1984 wechselte er für noch mehr Geld zum italienischen Verein SSC Neapel. Der SSC Neapel wurde zum ersten Mal italienischer Meister und die Fans verehrten ihn. Nach Maradonas Zeit wurde das Trikot mit der Nummer 10 nicht mehr vergeben. Er spielte bei vier Weltmeisterschaften mit und wurde mit Argentinien im Jahr 1986 Weltmeister. Gegen England beförderte er den Ball mit der Hand ins Tor. Danach sprach er von der "Hand Gottes": "Es war der Kopf Maradonas und die Hand Gottes." Im gleichen Spiel spielte er die gesamte englische Abwehr aus und schoss das "WM-Tor des Jahrhunderts". Zum Andenken an dieses Tor wurde eine Maradona-Statue am Eingang des Stadions aufgestellt. Am Ende seiner Karriere hatte Maradona Probleme mit seinem Körpergewicht und wurde wegen Drogenkonsums gesperrt. Er versuchte zwar immer wieder, an seine grossen Zeiten anzuknüpfen. Doch es gelang ihm nicht. Anschliessend versuchte er sich als Trainer und wurde sogar argentinischer Nationaltrainer. Weil er zu wenig Erfolg hatte, wurde er aber entlassen. Im Jahr 2020 starb er an einem Herzinfarkt.

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Summarize: Former President Bill Clinton and his wife, former Secretary of State Hillary Clinton, are worth between $10 and $50 million, according to their financial disclosure forms – but taxpayers will still pony up nearly $1 million this year to fund their post-White House lifestyle. Like the three other living former presidents, Jimmy Carter and the two George Bushes, Clinton receives a $201,000 annual pension. But that just scratches the surface of the government benefits he shares with his wife Hillary. The total for 2014 also includes $157,000 for staff salaries and benefits, $414,000 for office space and $9,000 for phone bills. In all, The Washington Post first reported, taxpayers are providing Clinton with $944,000 in post-presidential pay and benefits. SCROLL DOWN FOR VIDEOS. Overkill? Bill Clinton costs taxpayers nearly $1 million every year, five times what his salary was during his presidency. Former Secretary of State Hillary Rodham Clinton is scrambling to redefine her public image, with husband Bill saying Tuesday that focusing on their joint wealth is 'the wrong debate' to have as the 2016 election season approaches. Public art subsidy? Former president George W. Bush got $1.28 million in government payments and benefits this year while he painted portraits. Nice digs: Taxpayers shelled out $414,000 this year for Bill Clinton's office in the Harlem neighborhood of New York City. That tab was $1.28 million in 2002, the year after he left office, according to a Congressional Research Service report cited by the Post and the Washington Free Beacon. Hillary Clinton was lambasted in the press for saying on June 9 that she and Bill were 'dead broke' after their White House tenure. Her rhetorical play at poverty during an ABC News interview included the claim that she and Bill'struggled to, you know, piece together the resources for mortgages for houses, for Chelsea’s education, you know – it was not easy.' The CRS report, issued in April, also noted that government payments and benefits for former President George W. Bush outstripped Clinton's in every full year since Bush left the White House. Bush the younger is costing taxpayers $1.28 million this year, and averages 4 per cent more annual than Clinton. The government's General Services Administration inexplicably budgeted $102,000 for Bush's telephone expenses in 2014, and planned to spend $135,000 more on furniture, computers, office supplies and other miscellany. The former president and first lady both tried to pivot away from the national frenzy over their elite financial status on Tuesday, with Bill Clinton saying that his wife is 'not out of touch' with the middle-class Americans. During an annual Clinton Global Initiative conference in Denver, he told event moderator David Gregory of NBC’s 'Meet the Press' on stage that discussing their millions is 'the wrong debate' to have in the run-up to the 2016 election season. Instead, according to Businessweek, he claimed the public should focus on how to counter the 'demise of the American dream.' He acknowledged that Hillary didn’t 'give the most adept answer' to questions about whether they were as wealthy as Republican politicians who have been tripped up by insinuations of upper-class snobbery. 'You can say, "OK, I gotta clean that up," which she did,' he claimed. But 'it is factually true that we were several million dollars in debt,' Clinton said, and reporters'should put this in some sort of context.' He also claimed that he had 'the lowest net worth of any American president in the twentieth century when I took office, but I still could have been tone deaf. And, you know, now I don’t. And we’ve got a great life and I’m grateful for it.' 'But I still, we go to our local grocery store on the weekend. We talk to people in our town. We know what’s going on. The real issue is, if you’ve been fortunate enough to be successful, are you now out of touch and insensitive to the agonizing struggles other people are facing?' Pivot: On Tuesday in Denver Bill Clinton tried to reposition his wife Hillary, the former secretary of state, describing her as a warrior for poor and middle-class Americans instead of a wealthy, out-of-touch elitist. At the same Denver event, Hillary Clinton repositioned herself as a warrior for the downtrodden, including poor children. Upward mobility'really does take a village,' she said. Mrs. Clinton hasn't yet said whether she will run for president. Her. missteps in recent weeks, along with lackluster sales of her memoir. 'Hard Choices,' have become the first chink in what was thought to be an. unassailable political armor. The Former Presidents Act of 1958 provides one-time residents of the White House with pensions, office expenses, professional staff, coverage for medical care, and lifetime Secret Service protection. Utah Republican Rep. Jason Chaffetz tried to change the arrangement in 2012 with his Presidential Allowance Modernization Act, but it died in committee. The legislation would have capped the government's total spending on any former president to $400,000. And that amount would have been reduced dollar-for-dollar by any money they earned. Ka-ching: Bill Clinton and George W. Bush together collect more than $2 million each year in taxpayer money and benefits -- not including the cost of Secret Service protection -- and will continue that pace until they die. According to the nonpartisan Congressional Research Service, George W. Bush costs taxpayers more in his post-presidency than any other former president. Under that scenario, Clinton would have received nothing this year and last year. His earnings for individual speeches reportedly can top $500,000. 'Nobody wants our former presidents living the remainder of their lives destitute," Chaffetz said at the time, 'but the fact is none of our former presidents are poor.' 'Reports actually indicate that between book tours and speaking fees, these men are making millions of dollars a year. There's little reason why American taxpayers should be subsidizing these former presidents when they're doing fine on their own.' Jimmy Carter, who left office in 1981, spends the least among the four living former presidents. His office space costs just $109,000 per year, and the GSA budgeted $466,000 for his total upkeep in 2014

Summary: Bill Clinton said Tuesday his wife's claim that they were 'dead broke' after his presidency was 'factually true' He and Hillary are not 'out of touch,' he insisted, because they go to the grocery store and 'talk to people in our town' An April Congressional Research Service report found that the federal government has spent $15.9 million on Bill Clinton's retirement since 2001. He got $1.28 million in benefits the year after he left office. Clinton said Tuesday that attacking his wife as a wealthy elite in the run-up to the 2016 election is 'the wrong debate' This year Clinton's costs include a $201,000 pension, $157,000 for staff salaries and benefits, $414,000 for office space and $9,000 for phone bills. Former president George W. Bush spends even more, costing taxpayers $1.28 million this year and averaging 4 per cent more per year than Clinton. The government inexplicably budgeted $102,000 for Bush's telephone expenses in 2014, and planned to spend $135,000 more on furniture, computers, office supplies and other miscellany.

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Summarize: De'Von Pickett had been in town for only two days. The Bronx native - a road manager for rapper Nicki Minaj - was in Philadelphia to start rehearsals for her forthcoming tour, and took the opportunity to meet up with a few friends Tuesday night. By the next morning, he was dead. Pickett, 29, succumbed to stab wounds dealt outside an East Germantown bar in an early-morning attack. On Tuesday evening, Pickett and four friends - some from the tour, some local - had been out drinking, bouncing from Cavanaugh's in University City to Che Bar & Grill, a small, unassuming bar in East Germantown with a pool table and a dance floor. They knew the bartender there, a law enforcement source said. At some point, a former Che employee showed up at the bar, drunk, the source said. She got into a fight with the owner. And when she walked behind the bar and started pouring herself drinks, the source said, the owner kicked everyone out. Pickett and his friends filed out with the rest of the evening crowd. They hadn't been involved in the dispute, the source said. But it was late, past 2 a.m., and the group wanted to make sure their bartender friend made it safely to her car, the source said. Someone called her to make sure she was all right. She'd be out soon, she told them, and so Pickett and his friends waited by her car. That's when, police say, two to three men, at least one carrying a knife, approached. They began pushing and shoving Pickett and his friends, who tried to defuse the situation, the source said. It would be to no avail. In the ensuing attack, Pickett was stabbed to death. A friend of his, a 27-year-old man who also worked for Minaj, was wounded and remained in critical condition at Einstein Medical Center, where Pickett was pronounced dead at 2:45 a.m. Minaj, on Instagram, called the attack "unbelievable." "Another senseless act of violence that took the life of a great guy. So sad," she wrote. The singer Rihanna posted a photo on Instagram showing her smiling next to Pickett - he had worked on Kanye West's 2008 Glow in the Dark tour, which also featured Rihanna. "One of the nicest, coolest, funniest people I've ever met and toured with," she wrote. "Sad to lose a beautiful spirit like yours." On Wednesday, police were still trying to piece together exactly what led to the death of Pickett, nicknamed "Day Day," a longtime roadie who had worked on several high-profile tours, including shows for Britney Spears. It was unclear, the source said, whether the altercation with the former employee inside the bar was connected to the attack outside. Detectives recovered surveillance footage of the incident from nearby businesses. The bar, a two-story brick establishment at 6364 Stenton Ave., anchors a strip of mom-and-pop stores. Residents said the bar, though it has changed owners, had been a neighborhood fixture for nearly 30 years. In that time, neighbors said, they could not recall a similar incident. "It's nothing that's different than any other bar - a little rowdy, but no hostilities, no shootings, nothing," said Barry Kelinson, who owns the clothing store next door to the bar. "It's a nice, quiet, neighborhood bar," said Vanita Cruse, who owns an eyeglass store across the street. A man at the front door who identified himself as the bar's manager declined to comment. Meanwhile, Pickett's friends and colleagues expressed shock and grief at the news. "I am still in shock & don't want to believe this. And it hurts even more knowing how good these two dudes are. How hard these guys work. And how loyal they are to whoever they are down with & each other," the rapper Fabolous wrote on Instagram. Police described Pickett's killer as a man in his 30s, about 5-foot-8 and 160 pounds with a light complexion, thin build, and full beard. He was last seen in dark gold Buick LeSabre. awhelan@philly.com 215-854-2961 @aubreyjwhelan Two members of Nicki Minaj's touring crew were stabbed outside of a bar in Philadelphia on early Wednesday morning (Feb. 18), and one of the team members was killed in the incident, the rapper confirmed on Twitter. Two members of my team were stabbed last night in Philly. One was killed. They had only been there for two days rehearsing for the tour. -- NICKI MINAJ (@NICKIMINAJ) February 18, 2015 Billboard has confirmed with the Philadelphia Police Department that two people were stabbed during an incident in the city last night, with one dead and one in critical condition. The Philadelphia PD would not reveal the names of the victims or any details of the incident, the men in Minaj's Instagram photo, which she posted on Wednesday afternoon, have been confirmed to be De'Von "Day Day" Pickett, who was killed in the incident, and Eric Parker, who is in critical condition. "They had only been there for two days rehearsing for the tour," wrote Minaj on Twitter. She added in an Instagram caption, "These guys flew into Philly for my tour rehearsals 2 days ago and were both stabbed last night. #RIPDAYDAY condolences to your family. Unbelievable. Eric (on the right) is recovering. Another senseless act of violence that took the life of a great guy. So sad." According to an NBC-10 news report, the incident began during an argument inside the Che Bar & Grill in Philadelphia's West Oak Lane neighborhood around 2:30 AM on Wednesday morning. The argument spilled outside, where witnesses say the two victims were stabbed several times. Police are interviewing witnesses to the murder and analyzing surveillance video of the scene. A rep for Minaj did not immediately respond to request for comment. Pickett's position within Minaj's touring crew has yet to be confirmed, although TMZ refers to the Brooklyn resident as a "stage hand." Fabolous also posted about the incident on Instagram, writing that he has worked with both Pickett and Parker and that his "heart is heavy" with the news of their stabbings. "My friend @daydaydoesdis (left) died & my friend Eric Parker is in critical condition in the hospital fighting for his life," he wrote. "I am still in shock & don't want to believe this. And it hurts even more knowing how good these two dudes are. How hard these guys work. And how loyal they are to whoever they are down with & each other. Please send Eric Parker your blessings & a prayer for him to pull thru. And RIP to the fallen soldier Day Day." Minaj's European headlining tour is scheduled to begin in Stockholm on Mar. 16. Additional reporting by Joe Lynch, Billboard. De'Von Pickett, tour manager for music star Nicki Minaj, was stabbed to death overnight in West Oak Lane. He was in town preparing for her upcoming tour. NBC10's Nefertiti Jacquez spoke with Pickett's cousin. (Published Thursday, Feb. 19, 2015) A tour manager who was well-known in the music industry and was working on preparations for rap superstar Nicki Minaj's upcoming tour, died after a bar fight spread into a Philadelphia street. De'Von Pickett, 29, of the Bronx, New York, was stabbed to death outside the Che Bar & Grill along Stenton Avenue in Philadelphia's West Oak Lane neighborhood early Wednesday morning. Pickett was with Eric Reed, 27, and three other friends inside the bar around 2:35 a.m. when they got into an argument with another group of men, according to police. “We believe that both of the victims, as well as the perpetrator, were all inside of this bar and that’s when an argument started,” said Chief Inspector Scott Small. The argument spread outside the bar where one of the men pulled out a knife, police said. Someone stabbed both Pickett and Reed several times. Pickett suffered stab wounds in the torso while Reed suffered stab wounds to his left arm and left side. A private car rushed both men to Albert Einstein Medical Center. Pickett died from his injuries at 2:45 a.m. Reed remained in fair condition late Wednesday. Police later returned to the 6300 block of Stenton Avenue where they found personal items, a cellphone and blood on the sidewalk outside the bar. A Philadelphia Police crime camera and business surveillance cameras possibly captured at least part of the incident, said investigators. Police said the suspect was last seen driving off in a gray Buick LeSabre. He is described as a 30-year-old man standing 5-foot, 8-inches tall and weighing 160 pounds with a full beard. He was last seen wearing a black and gray jacket. Earlier, witnesses told investigators that the dispute possibly had to do with a recently fired employee. Small said that police were working to verify if that was actually the case. Both Pickett and Reed were tour managers who were working on preparations for Minaj's "The Pinkprint Tour," which is set to begin next month in Sweden. Pickett was also the co-founder of BK Nerd & Co. and was well-known in the music industry for his work in branding and public relations. Minaj tweeted that she knew the victims were in Philly to rehearse for her upcoming tour: Two members of my team were stabbed last night in Philly. One was killed. They had only been there for two days rehearsing for the tour. — NICKI MINAJ (@NICKIMINAJ) Feb. 18, 2015 Pickett's cousin, who did not want to be identified, also confirmed with NBC10 that Pickett was in Philly to work on the tour with Philly rapper Meek Mill. "They were all here for her," the cousin said. "I guess they were out celebrating and things went wrong." During his career, Pickett worked with some of the biggest stars in the music industry, including Rihanna, Lady Gaga, Justin Timberlake and Hip-Hop artist, Fabolous, who shared his condolences on his Instagram page. Rihanna also posted photos of her and Pickett on her Instagram page as well. can't believe this is true! one of the nicest, coolest, funniest people I've ever met and toured with! We had so much fun together!!! Sad to lose a beautiful spirit like yours #RIPDayDay A photo posted by badgalriri (@badgalriri) on Feb 18, 2015 at 10:59am PST #RipDayDay you are loved and will be missed. A photo posted by Questlove Gomez (@questlove) on Feb 18, 2015 at 1:37pm PST throwback #RIPDayDay A photo posted by badgalriri (@badgalriri) on Feb 18, 2015 at 11:37am PST Family and friends, including Questlove of the Roots, also shared their condolences on Twitter, using the hashtag #RIPDayDay. Pickett's cousin told NBC10 he came all the way from New York to mourn. "He was a great guy," he said. "He didn't deserve this. He set an example for a lot of guys in this industry and it's sad to see this happen." The City of Philadelphia is offering a $20,000 reward for an arrest and conviction in Pickett's death.

Summary: Two members of Nicki Minaj's touring team were stabbed-one fatally-early yesterday outside a bar in Philly, where they were rehearsing for the tour. Minaj confirmed the news on Twitter, and Billboard confirmed the stabbings with the Philadelphia Police Department, though PPD would not reveal details or names. In her tweet, Minaj noted that the men had only been in the city two days. She also posted a photo of them on Instagram, and they have been confirmed as De'Von "Day Day" Pickett and Eric Parker. Parker survived. Pickett, who was Minaj's 29-year-old tour manager, was out with four friends. Reports via NBC Philadelphia and the Philadelphia Inquirer differ, but an argument broke out inside the Che Bar & Grill around 2:30am. The fight spread into the street, where one man pulled a knife; both Pickett and Parker were stabbed several times. Police are still searching for the suspect. Pickett is well-known in the music industry, and celebrities including Rihanna and Questlove have been mourning him on social media. Minaj's tour is set to begin March 16 in Stockholm.

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Summarize: Il giudice, come ogni altro professionista, In tute queste ipotesi, il giudice può chiedere l'ausilio (può nominare) quello che con un acronimo viene definitivo C.T.U. (consulente tecnico d'ufficio), la cui funzione è quella di fornire al giudice uno strumento (una relazione) che faciliti la decisione. Quanto detto già mette in evidenza un aspetto particolare: lo stretto legame tra il Ctu (le competenze tecniche / professionali del ctu) e la situazione di fatto e la difficoltà del giudice (per mancanza di specifiche conoscenze) con la situazione di fatto (basta pensare ad una causa per plagio sulla base di un testo redatto in lingua non italiana). Questo comporta che rilevano le specifiche competenze professionali del ctu, infatti, per stabilire se vi è stato un errore medico per valutare il danno fisico derivante da un incidente sarà nominato ctu un medico; per valutare la stabilità di una costruzione o le cause di infiltrazioni d'acqua, ed, infine, per analizzare un bilancio di una società sarà nominato un commercialista. Quindi, la peculiare situazione concreta influenza la figura (le caratteristiche professionali) che dovrà avere il soggetto che sarà chiamato ad essere nominato come Ctu. Altro elemento che rileva è dato dal fatto che la consulenza tecnica d'ufficio è uno degli strumenti del giudice per poter giungere alla decisione, ma non è l'unico strumento, in altri termini, la consulenza tecnica d'ufficio, non solleva le parti dagli oneri probatori a loro carico (e, certo, non è un mezzo per sopperire all'eventuale mancato adempimento dell'onere della prova). Infine, l'ultimo elemento è dato dal fatto che il CTU non è il Giudice, in altri termini, il consulente d'ufficio non redige la sentenza e non emette decisioni, ma presta la sua opera per facilitare il compito del giudice e/o per fornire alcune delucidazioni tecniche, all'esisto delle quali, poi, il giudice deciderà quale norma giuridica applicare e quale decisione assumere. Da quanto detto risulta che è molto delicato (per non dire problematico) è il rapporto tra Ctu ed onere della prova. In quanto la nomina di un ctu, per quanto discrezionale del giudice, non può essere concessa o non può essere effettuata quando serve per sopperire all'onere della prova a carico di una delle parti. Questo limite è vero in generale, ma, esisto delle eccezioni, infatti, la consulenza tecnica può costituire essa stessa fonte oggettiva di prova, quando si risolva in uno strumento, oltre che di valutazione tecnica, anche di accertamento di situazioni di fatto rilevabili solo con il ricorso a determinate cognizioni tecniche e percepibili con l'ausilio di specifiche strumentazioni tecniche. Quindi, pur non essendo la consulenza tecnica d'ufficio qualificabile come mezzo di prova in senso proprio e non potendo essere utilizzata per sgravare le parti dai loro oneri probatori, è consentito affidare al consulente non solo l'incarico di valutare i fatti accertati o dati per esistenti (cosiddetta consulenza deducente), ma anche quello di accertare i fatti stessi (cosiddetta consulenza percipiente), quando si tratta di fatti che la parte ha dedotto e posto a fondamento della sua domanda ed il cui accertamento richiede specifiche cognizioni tecniche.

Summary: La Cassazione del 12.2.2015 n. 2761 ha stabilito che la consulenza tecnica d'ufficio (ctu) non è qualificabile come mezzo di prova e non può essere utilizzata per sgravare le parti dall'onere probatorio, però è possibile che la ctu possa costituire prova, quando si risolva in uno strumento di accertamento di situazioni di fatto rilevabili solo con il ricorso a determinate cognizioni tecniche e percepibili con l'ausilio di specifiche strumentazioni tecniche. Inoltre, è consentito affidare al consulente non solo l'incarico di valutare i fatti accertati o dati per esistenti (cosiddetta consulenza deducente), ma anche quello di accertare i fatti stessi (cosiddetta consulenza percipiente), quando si tratta di fatti che la parte ha dedotto e posto a fondamento della sua domanda ed il cui accertamento richiede specifiche cognizioni tecniche.

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Summarize: By. Julian Robinson. A retired footballer has been ordered to pay a woman more than £2,500 in damages - after she stubbed her toe on a 'keep off the grass' sign outside his house. Former goalkeeper Malcolm White, 73, has also been hit with a £25,000 legal bill after he was successfully sued by Emma Grady. Miss Grady, 40, injured her left toe on a homemade wooden sign the pensioner had put on a grass verge outside his home in South Hykeham, Lincolnshire. Malcolm White, left, has been ordered to pay Emma Grady, right, more than £2,500 in damages after she stubbed her toe on a 'keep off the grass' sign outside his house. Emma Jane Grady made a claim against Malcolm White, after she said that she had tripped over a 'Do not park on the grass' sign that he had placed there. The signs can be seen here placed in the grass, circled in red, but they have since been removed. The sign, which measured 4ins by 10in, read ‘Please do not park on the grass’ and was erected outside hisdetached property. Mr White, who played for Lincoln City, Grimbsy Town and Wolverhampton Wanderers, put it there as a favour for villagers who were fed up with parents parking on the grass verge when they collected their children from the local primary school. But he was taken to court after Miss Grady tore the nail off her left big toe on a rusty nail when she stumbled over the sign as she went to collect her daughter from school on November 12, 2012. Miss Grady, who lives half-a-mile away in the neighbouring village of High Hykeham, claimed it took six months for her toe nail to grow back but has been left deformed. A sign on the grass verge in South Hykeham, Lincolnshire said 'Please do not park on the grass' Mr White, who went on to play in America for Los Angeles Wolves, has been ordered to pay Miss Grady £2,541.35 in damages and £24,874.39 in court costs. He denied erecting the sign but his wife Valerie, 72, admitted he did put it up in a telephone conversation with Miss Grady’s solicitor Katherine Trafford. Passing judgement at Lincoln County Court, District Judge Chris Cooper said: 'This case comes down to whether or not Mr and Mrs White or Mrs Trafford is telling the truth. 'It is clear that the claimant suffered sizable injury after the accident. 'I am satisfied that it happened in the way that she said it did and she stubbed her toe on the offending sign. 'I am also satisfied that the signs caused a public nuisance. 'A pedestrian does not have to look at their feet when going about their business.' Mr White has now been forced to pay the cash from his own pocket because his insurance company will not cover his legal bills. In a statement after the hearing, the couple's solicitor Patrick Tedstone said: 'It’s a sad day for Mal and Val. 'They are leaving the village this week after more than 30 very, very happy years, to be closer to family. 'They are very disappointed with the judgement and with the costs, too, but they will respect the judge’s decision. Malcolm White played youth football for his hometown team Wolverhampton Wanderers before signing for Grimsby Town in the late 1950s. He enjoyed a successful trial with the club and stayed with the club for five years making nearly 70 first-team appearances from 1958 to 1963. He went on to play for Walsall, Lincoln City, Bradford City and Halifax Town before going to America to star for Los Angeles Wolves in the late 1960s. Speaking in 2011, he told how, playing for Grimsby, he once saved a penalty from the legendary Brian Clough - who went on to win two consecutive European Cups as manager of Nottingham Forrest. He said: 'When we were in the Second Division, we were competing with the likes of Sunderland, Newcastle, Chelsea, Leeds and Middlesbrough. 'Against Sunderland, I saved a penalty from Brian Clough in front of 46,000 - mind you, he scored a hat-trick in the same game.' 'They wish everyone in the village well.' A friend added: 'They are gutted. It’s a barmy ruling because the sign was put on the verge to stop parents churning it up with their cars. 'The land is publicly owned but was being turned into a car park by inconsiderate parents. I feel very sorry for them. This should never have gone to court.' Miss Grady said: 'I am relieved this is all over. 'I have been through 18 months of hell. My foot will never be the same again. I am satisfied with the result.'

Summary: Malcolm White put sign up next to his home in South Hykeham, Lincolnshire. Wooden sign said 'Please do not park on the grass' as 'favour' to residents. Emma Grady injured her toe on sign as she was picking up child from school. Retired footballer, Mr White, 73, ordered to pay her £2,500 in damages. He has also been hit with a £25,000 legal bill after being sued by Miss Grady.

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Summarize: Article Excerpt A deepening scandal in the futures-trading business has left an elder statesman of the industry hospitalized after a suicide attempt and regulators seeking to find out what happened to about $215 million of customer money allegedly missing from his firm. The firm, Peregrine Financial Group Inc., filed Tuesday evening in Chicago to liquidate under Chapter 7 of the bankruptcy code. Earlier in the day, the Commodity Futures Trading Commission filed a lawsuit in federal court in Chicago accusing Peregrine Financial and its founder, Russell Wasendorf Sr., of fraud, customer-funds violations and making false statements. The CFTC said shortfalls may have... CHICAGO/WASHINGTON/CEDAR FALLS, Iowa The U.S. futures industry reeled on Tuesday as Iowa-based broker PFGBest collapsed after regulators accused it of misappropriating customer funds for more than two years, dealing a new blow to trader trust just months after MF Global's demise. The Commodity Futures Trading Commission (CFTC), which along with industry regulators had given a clean bill of health to dozens of brokers following spot checks in January, alleged that the firm's regulated Peregrine Financial Group (PFG) unit and its owner had defrauded customers and lied to regulators in order to hide a shortfall that now exceeds $200 million. "The whereabouts of the funds is currently unknown," the CFTC said in a complaint against PFG and its founder and chairman, Russell R. Wasendorf Sr., whose suicide attempt on Monday morning outside the firm's Cedar Falls, Iowa, offices appears to have precipitated the crisis. On Tuesday evening, Peregrine filed to liquidate under Chapter 7 of the U.S. bankruptcy code, with between $500 million and $1 billion of assets, between $100 million and $500 million of liabilities, and between 10,000 and 25,000 creditors. The Chapter 7 filing suggests the company is winding down. The funds shortfall represents more than half of PFGBest's client accounts but is modest relative to the estimated $1.6 billion missing from MF Global's accounts. As more details of the scandal became clear, the circumstances began to look more like a Bernard Madoff-style fraud than MF Global CEO Jon Corzine's desperate bid to stay afloat. Wasendorf intercepted confidential regulatory documents that were mailed by the National Futures Association to what the industry group believed was U.S. Bank, PFG's bank, a person close to the situation told Reuters. Instead, they were sending the documents, used to independently verify a broker's bank balances, to a post office box that Wasendorf had set up, the source said, who declined to be identified. The CFTC complaint, which relies on many of the details released on Monday by the NFA, the broker's main regulator, said the bank account that PFG reported was holding $225 million in 1,845 customer accounts actually contained only $5 million. Wasendorf forged signatures and fabricated bank balances on the documents and simply mailed them back to the Chicago-based NFA, the person said. The scheme apparently began to unravel as the NFA shifted to electronic confirmations. The NFA "started getting suspicious. He was resisting this new way of confirming the balance," the source said. Wasendorf only recently signed the authorization, a decision that would quickly have led regulators to uncover the discrepancy. While distinct from MF Global's demise in many ways, news that a second broker has violated sacrosanct segregated customer funds threatens to shatter the fragile confidence in an industry that once prided itself on an unblemished record in protecting client money. "It's déjà vu all over again," said John Roe, co-founder of the Commodity Customer Coalition (CCC), set up in the aftermath of MF Global's collapse last October to help clients recoup their money. Wasendorf, 64, a well-known and mostly well-regarded figure in the industry over a four-decade career as a journalist, trader and executive, was reported to be in a coma, the CFTC said. He was found in his car early on Monday morning in an apparent suicide attempt. A spokesman for University of Iowa Hospitals and Clinics said on Tuesday he was unable to comment on Wasendorf's status due to federal privacy laws. A police report released on Tuesday said he had been found "breathing but incoherent" after running a hose from his exhaust pipe into the car. A suicide note found with him alluded to some kind of discrepancies with accounts at PFG, the report said, confirming what the broker had told its clients a day ago. The bankruptcy filing showed that Russell Wasendorf Jr, the founder's son, had been empowered to act for Wasendorf Sr in the event the latter became incapacitated, under a power of attorney dated July 3. REGULATORY IRE, CUSTOMER GRIEF, EMPLOYEE DISMAY A well-known broker for U.S. foreign exchange and commodity markets for 20 years, PFGBest was among a dozen or so mid-sized, independent brokers that scrambled to reassure customers of the safety of their funds after MF Global's collapse. In February it posted a notice that the firm "reports daily and monthly to regulators concerning customer segregated accounts". A number of former MF Global customers also moved their accounts to PFGBest. "We had personal assurances from Wasendorf Sr. as recently as two weeks ago that they were not like MF Global," said Lauren Nelson, director of communications for Attain Capital, an introducing broker specializing in managed futures in Chicago. "We've been speaking to other (brokers) in the hope we can eventually transfer our accounts over. But the fear is the funds are gone, the regulators have really dropped the ball." But unlike MF Global, which is believed to have misused customer funds in a mad scramble to meet margin calls on proprietary trades in its waning days, PFGBest's abuse seems to have extended back years, according to the complaint. There is no indication yet of how or why the missing money was used. The CFTC case filed in the U.S. District Court for the Northern District for Illinois, Eastern Division, alleges that PFG failed to segregate customer funds, committed fraud by misappropriation and reported false data to regulators. The CFTC complaint says that PFG records showed a balance of $207 million in the 1,845 customer segregated accounts as of February 28, 2010, although the actual bank balance was under $10 million. They said that under $10 million was in the account as of March 30, 2011, when PFG records showed a balance of $218 million. The balance was just $5 million this week, it said. The NFA order said the accounts were held at U.S. Bank. A source familiar with the situation said PFG's balance had been steady at around $5 million for several years. JEFFERIES LIQUIDATES POSITIONS On Tuesday, the firm's clearing broker Jefferies Group Inc, which was responsible for clearing trades through exchanges like those run by CME Group, said it had begun unloading positions held on behalf of PFG's clients after it failed to meet a margin call. It said a "substantial portion" had already been closed and it did not expect to incur losses. As a "non-clearing" futures commission merchant (FCM), PFGBest acted as a middleman for mostly small-scale or retail traders, passing those trades to Jefferies to clear. Because it was not a clearing member of the CME, a not-uncommon arrangement for smaller brokers that do not want or are unable to keep enough capital of their own, the regulatory burden falls to the NFA and the CFTC, letting the CME Group -- which suffered harsh criticism as the front-line regulator of MF Global -- off the hook. PFGBest officials have said nothing beyond a notice to clients on Monday confirming an investigation into "accounting irregularities" and advising customers that they could liquidate open trading positions but would not be able to withdraw cash or initiate any new trades. WASENDORF SHOCK The shock was twofold for many in the tight-knit trading industry, who struggled to reconcile the apparent suicide attempt with the industry veteran known for his hometown philanthropy and passion for peregrine falcons. "I always thought they were straight shooters," said Mark Melin, an author and futures-industry consultant, who worked for PFGBest for about 2-1/2 years. Others expressed less shock. One former employee of the firm said he had grown concerned that Wasendorf did not do more to distance the company from a massive $194 million forex-trading Ponzi scheme run by Trevor Cook in Minnesota, who admitted defrauding more than 700 investors. Cook is serving 25 years in prison. In February, PFGBest, which had acted as Cook's broker, was fined $700,000 by the NFA for failing to notice the scheme. The company was subsequently sued for $48 million by the receiver rounding up the assets from Cook's scheme. "They never admitted they were aware of what was going on, but they didn't deny it either," said the former employee. Others wondered about Wasendorf's decision to move the firm's headquarters from Chicago back to his hometown of Cedar Falls, occupying a 50,000 square-foot, three-story glass headquarters that cost $18 million and was celebrated for its eco-friendly construction and four-star cafeteria. It was a sizeable investment for an industry that has come under enormous strain lately as ultra-low interest rates sap revenue from holding customer funds and electronic trading threatens the role of middleman. (Reporting By David Sheppard, Frank Tang and Jonathan Stempel in New York, Tom Polansek, Ann Saphir and PJ Huffstutter in Chicago, PJ Huffstutter in Cedar Falls, Chuck Abbott and Alexandra Alper in Washington; Editing by Ryan Woo, Jeffrey Benkoe, Jim Marshall and Chris Lewis)

Summary: PFGBest declared Chapter 7 bankruptcy last night, after the Commodity Futures Trading Commission filed a lawsuit accusing it of fraud, lying to regulators, and abusing customer funds. Regulators believe Russell Wasendorf, who tried to kill himself Monday, had been fabricating bank balances and forging signatures on the documents he submitted to the industry's self-regulating body, the National Futures Association, for years, Reuters reports. Now regulators believe at least $215 million in customer funds are missing. The scandal has echoes of that at the larger MF Global-and indeed, regulators first became suspicious when Wasendorf resisted their push to switch to an electronic method of verifying customer account balances in the wake of that scandal, the Wall Street Journal reports. "It's déjà vu all over again," says the cofounder of a group set up to recoup MF Global money for customers.

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Summarize: Prince Harry came to the rescue of a lady who lost her hat after a gust of wind blew it off as he made a speech at RAF Honington in Suffolk this morning. Ever the gentleman, the Prince, 30, rushed to her aid and scooped up the wayward titfer before returning the garment to its delighted owner with a big smile. Harry's moment of gallantry came just after he had made a speech in which he praised the 'professionalism and determination' of the RAF's 26 Squadron. Scroll down for video. There you go: A gallant Prince Harry returns a missing hat to its owner after it was blown off by a gust of wind. Going, going, gone! A gust of wind sends the lady's hat flying as Prince Harry walks away to inspect the troops. Let me help: Harry spots the errant hat and scoops it off the parade ground tarmac, watched by troops and well-wishers. An officer AND a gentleman: A beaming Prince Harry returns the wayward hat to its delighted owner. Praise: Prince Harry praised the RAF's 'determination and professionalism' in the fight against terrorism. Salute: During the visit, he officiated during a ceremonial parade in front of airmen's friends and family. March: RAF Honington serves as the depot for the Royal Air Force's contingent of ground troops. The royal, who is fresh from a visit to Kandahar Airbase in Afghanistan, inspected ground troops before presenting No. 26 Squadron RAF Regiment with a new standard. The regiment is a specialist unit that focuses on thwarting chemical, biological, radiological and nuclear attacks - a role that has put it at the forefront of anti-terrorism efforts. In a speech in front of servicemen and women and their families, he said: 'This role is vital to UK defence and civil response capabilities; is is also highly valued by NATO. 'Alongside 27 Squadron, it is constantly committed at extremely high readiness to support the combined emergency service and military response to a CBRN [chemical, biological, radiological or nuclear] incident in the UK. "Indeed, such is the vital nature of this role that responsibility has been handed over to the remainder of the wing to allow the squadron to be on the parade today.' He added: 'It is clear that every member of the squadron possesses the determination and professionalism to succeed, even in the most arduous of circumstances. Enjoying himself: Prince Harry appeared to enjoy his visit to RAF Honington in Suffolk. Inspection: Harry has held the role of Honorary Air Commandant at RAF Honington since 2008. Important work: The men and women of 26 Squadron are a specialist counter-chemical warfare unit. Atten-shun! The men of the RAF Regiment - who make up the Air Force's ground fighting force - put on a good show for Harry. Courage: Harry praised the troops' determination to succeed even in 'the most arduous of circumstances' Smiles: The visit is Prince Harry's second to an RAF base in the space of less than four days. Work: Harry remains a captain in the Blues and Royals but has taken a step back from combat operations. Dedicated: Instead, the Prince has been concentrating on helping injured servicemen and women. Smart: After the parade - and the hat rescue - Harry and the men of 26 Squadron RAF Regiment posed for a group photo. 'Traits which have ensured the squadron's success since its formation and that are embodied in this new standard and the proud history it represents.' A standard is presented to RAF Regiment squadrons every 25 years and represents hundreds of years of history of service to the monarch and to the country. Royal Air Force Honington is the RAF's centre of Force Protection and is the depot for the Royal Air Force Regiment - the ground-fighting force of the RAF. The regiment protects air bases from attack and is trained in a wide range of RAF protection roles, both at home and abroad. Harry, himself a trained Apache helicopter pilot - albeit in the Army rather than the Royal Air Force - became Honorary Air Commandant of the base at the request of the Queen in 2008. Joking: Prince Harry met Sgt Rick Clements during a visit to Bryan Adam's Wounded: The Legacy of War. Awareness: Mr Adams' photos are intended to capture 'the unique spirit and resolve' of British personnel. Forefront: Harry has spearheaded efforts to help servicemen via charity appearances and the Invictus Games. Nice to meet you: Harry shakes hands with one of the servicemen during the event on Tuesday night. Return to Afghanistan: Harry spent Remembrance Sunday with RAF personnel stationed at Kandahar Airfield. He remains a captain in the Blues and Royals but took a step back from frontline combat operations earlier this year in order to concentrate on the Invictus Games. The Games, which took place in September, saw more than 600 wounded servicemen from more than nine different countries go head-to-head in a range of sports, among them archery and wheelchair rugby. Harry remains a passionate supporter of charities working to rehabilitate injured troops and, on Tuesday night, joined Canadian rocker Bryan Adams at a photo exhibition dedicated to servicemen and women. In typical Harry style, the royal joked and laughed with the veterans, quipping to Sgt Rick Clement who lost his legs in an IED explosion in Afghanistan: 'At least your hair has grown back!' Speaking of Sunday's trip to Kandahar Airfield, he added: 'The visit [to Afghanistan] was great. Fourteen people collapsed, though, during the ceremony, and it was only 12 degrees heat. But they were from the RAF so that's acceptable.' All smiles: Harry looked delighted to be back in the Middle East and shared a joke with the airmen on duty. Clown prince: His trademark high jinks left the men stationed at Kandahar Airfield in fits of laughter. Good work: Harry met some of the pilots charged with flying bombing raids on ISIS jihadis. My turn: He also convinced some of the airmen to let him take a seat in the cockpit of one of the planes. Memories: During the Remembrance service on the base, Harry read a passage from the Bible. Proud: At the end of the visit, the Prince posed for a photo with a group of delighted Tornado pilots

Summary: Harry was at RAF Honington in Bury St Edmunds in Suffolk where he made a speech praising the RAF. Came to the aid of a lady whose hat blew off during his address and returned it with a smile. Prince Harry has been the Honorary Air Commandant of RAF Honington since 2008 at the Queen's request. Praised the 'determination and professionalism' of 26 Squadron which specialises in thwarting chemical attacks. The speech in Suffolk comes days after he made Remembrance Day visit to Kandahar Airfield in Afghanistan.

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Summarize: Jehovah’s Witnesses use 1st Amendment to hide child sex abuse claims If you have any other questions, please contact us at republish@revealnews.org Additionally, we will not provide indemnification if you are located or publishing outside the United States, but you may contact us to obtain a license and indemnification on a case-by-case basis. If you wish to only use portions of the work or create a derivative, you need separate permission and the license and indemnification do not apply. This license only applies to republication of full works. FYI, you can grab HTML code for our stories easily. Click on the “Republish this content” link at the bottom of every story. Please do not alter this code. If we send you a request to remove our content from your website, you must agree to do so immediately. If you plan to republish our content, you must notify us at republish@revealnews.org. 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Do not change or edit our material, except only to reflect changes in time and location. (For example, “yesterday” can be changed to “last week,” and “Portland, Ore.” to “Portland” or “here.”) If you plan to republish our content, please notify us at republish@revealnews.org. Our reporter(s) must be bylined. We prefer the following format: By Emmanuel Martinez, Reveal. Include this language at the beginning of the story: “This story was produced by Reveal from The Center for Investigative Reporting (link organization name to revealnews.org), a nonprofit news organization. Get their investigations emailed to you directly by signing up at revealnews.org/newsletter. Credit and tag Reveal when sharing the story on social. We’re @reveal on Twitter and you can find us on at facebook/ThisIsReveal. You may embed our audio and video content and republish any written story for free under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 license and will indemnify our content as long as you strictly follow these guidelines: Thanks for your interest in republishing this story. As a nonprofit newsroom, we want to share our work freely with as many people as possible. We only ask that you follow a few guidelines. Jehovah’s Witnesses use 1st Amendment to hide child sex abuse claims This story was originally published by Reveal from The Center for Investigative Reporting, a nonprofit news organization based in the San Francisco Bay Area. Learn more at revealnews.org and subscribe to the Reveal podcast, produced with PRX, at revealnews.org/podcast. The leadership of the Jehovah’s Witnesses – one of the world’s most insular religions – for 25 years has instructed its elders to keep cases of child sexual abuse secret from law enforcement and members of their own congregations, according to an examination of thousands of pages of documents in recent cases. The religion’s parent organization, the Watchtower Bible and Tract Society of New York, issued the directives in at least 10 memos dating back to 1989. Although the memos were anonymously written, Watchtower officials have testified that the organization’s Governing Body approved them all. The most recent letter, dated Nov. 6, 2014, instructed elders – the spiritual leaders of local congregations – to form confidential committees to handle potential criminal matters internally. “In some cases, the elders will form a judicial committee to handle the alleged wrongdoing that may also constitute a violation of criminal law (e.g., murder, rape, child abuse, fraud, theft, assault),” the directive stipulates. “Generally, the elders should not delay the judicial committee process, but strict confidentiality must be maintained to avoid unnecessary entanglement with secular authorities who may be conducting a criminal investigation of the matter.” Don't miss the next big story. Brave investigations that change minds, laws and lives. Emailed directly to you. Within the organization, the Watchtower has final say over who is considered a serial child abuser. According to a 2012 Watchtower memo: “Not every individual who has sexually abused a child in the past is considered a ‘predator.’ The (Watchtower), not the local body of elders, determines whether an individual who has sexually abused children in the past will be considered a ‘predator.’ ” The directives are part of a pattern for the organization, which has more than 8 million members worldwide and preaches that Armageddon will soon release the world from Satan’s grip. In the U.S., the Jehovah’s Witnesses operate more than 14,000 congregations with about 1 million members. Internal documents obtained by Reveal show that the Witnesses have systematically instructed elders and other leaders to keep child sexual abuse confidential, while collecting detailed information on congregants who prey on children. Having successfully leveraged the First Amendment as a defense of their right to not serve in the military or salute the American flag, the Jehovah’s Witnesses now are using a similar legal strategy to defend policies that shield serial predators from law enforcement. “ ‘You come to us first. Don’t tell anybody. … You don’t warn parents in the congregation. We’ll decide what happens here.’ That’s their policy.”— Irwin Zalkin, attorney for Jose Lopez, describing the system he believes the Witnesses have created In many ways, the response by the Witnesses in recent child sexual abuse cases has mirrored the actions of another religious group: ultra-Orthodox Jews in New York. There, the community has faced a backlash for asking observant Jews to consult a rabbi instead of going immediately to police with evidence of abuse. With the Witnesses, however, there appears to be far more documentation and a bureaucratic protocol for dealing with allegations of abuse among its members. For Jose Lopez, it took almost three decades to find some semblance of justice after he’d been molested – when he was 7 – by a predator who’d operated within a congregation of Jehovah’s Witnesses in San Diego. When his case against the Witnesses concluded in October, a judge awarded Lopez $13.5 million, a remarkably large sum in an era of frequent payouts in abuse cases. The decision rested in part on the Witnesses’ refusal to hand over documents in the case, prompting the frustrated judge to ban the organization from making a defense. The Lopez case was remarkable for another reason. It forced the Witnesses into a rare admission: Somewhere within the organization, there is a trove of documents with the names and whereabouts of known child sexual abusers in its U.S. congregations. During the trial, a senior official from the Jehovah’s Witnesses headquarters, Richard Ashe, told Lopez’s attorney, Irwin Zalkin, that the organization had collected and electronically scanned internal documents on decades of known abuse cases. Ashe said that the Witnesses keep their child sexual abuse reports in a Microsoft SharePoint database but that it would take years to extract the information because it was mixed up with millions of other documents. “Honestly, Mr. Zalkin, the efforts that we’ve made up to this point is just trying to figure out how on earth we could ever do that in our filing system,” Ashe said. “You’re talking about 14,000 congregations and over 3 million documents that have been scanned and that would have to be searched. … It would take years to do that.” Zalkin called in a software expert who testified that by using simple search terms, the Witnesses could produce the information in less than two months, or maybe two days. At that point, the Watchtower simply refused to provide the database. During the case, Lopez said his mother reported his abuse to the elders in 1986, but they didn’t call police or warn the congregation. Lopez and his mother left the religion soon after. Even as the abuser, Gonzalo Campos, continued to sexually assault children, the elders promoted him within the congregation, first to ministerial servant in 1988, then to elder in 1993, according to a 1995 letter from elders to the Watchtower. In 1994, John and Manuela Dorman learned that Campos had abused their son a decade earlier, court documents show. They called Campos, who confessed and said the elders already were aware of the situation. When Manuela Dorman went to the elders, they told her not to talk about the abuse. They said too much time had passed, and nothing could be done. After a letter from the Dormans reached the Watchtower later that year, the elders confronted Campos. In 1995, he confessed and was disfellowshipped, the Witnesses’ version of excommunication. By then, he had at least seven known victims, according to elders’ letters. But Campos was reinstated to the congregation in 2000, eight months after the elders had sent a letter to the Watchtower explaining that they had managed to keep Campos’ past hidden, court documents show. “The community does not know of all this and there was no publicity about this,” the letter read. “Everything took place in the congregation and because of that he was not prosecuted.” Campos, according to news reports, fled to Mexico. During Lopez’s trial, Ashe testified that the Watchtower instructs elders that child abuse must be kept confidential. “And it directs that that should be kept confidential from prosecuting authorities?” Zalkin asked. “Yes,” Ashe responded. Asked whether the Watchtower’s policy of silence hinders parents’ ability to protect their children from abuse, Ashe told Zalkin, “Not in my view it doesn’t.” Ashe did not respond to a request for comment. In a written statement to Reveal, representatives from the Watchtower said, “We continue to educate parents and provide them with valuable tools to help them educate and protect their children.” But Zalkin, who has been aggressively filing lawsuits against the Witnesses throughout the country, said the pattern is clear. “Keep your mouth shut. Don’t go to law enforcement,” he said, describing the system he believes the Witnesses have created. “ ‘You come to us first. Don’t tell anybody. … You don’t warn parents in the congregation. We’ll decide what happens here.’ That’s their policy.” In the last few years, the Jehovah’s Witnesses have been hit with an increasing barrage of lawsuits claiming the organization has covered up child sexual abuse. A pair of sisters in Vermont filed a suit in September claiming that a member of their congregation molested them when they were as young as 4. When they reported the abuse to the elders in the congregation, they said, they were called liars. The next month in Dallas, five women and one man filed a joint lawsuit alleging that an elder in their congregation sexually abused them while they were all younger than 13. In Oregon in December, two former Witnesses sued the Watchtower and a local congregation, claiming the defendants kept silent after learning that an elder had sexually abused them when they were in grade school. Since 2012, attorneys have filed more than a dozen similar suits against the Watchtower in Connecticut, Florida, New Mexico, Ohio, Oklahoma and other states. The Watchtower’s frequent defense – that such cases violate protections under the free exercise clause of the First Amendment – has led to the dismissal of several lawsuits. Watchtower lawyers argue that judicial questioning of the spiritual beliefs and practices of Jehovah’s Witnesses would trample the organization’s religious freedoms. In a recent court hearing in California, Jehovah’s Witnesses lawyer James McCabe argued that “the religious beliefs of Jehovah’s Witnesses were at play in this case from start to finish.” At the heart of this issue are the Watchtower’s own child abuse policy memos. Each memo has been addressed to “all bodies of elders” and bears the letterhead of either the Watchtower Bible and Tract Society or the Christian Congregation of Jehovah’s Witnesses. Those are the primary corporations used by the organization to administer spiritual guidance, create and disseminate policy, oversee the writing and publication of literature, and manage the organization’s vast real estate holdings. “Often the peace, unity, and spiritual well-being of the congregation are at stake,” a 1989 Watchtower memo reads. “Improper use of the tongue by an elder can result in serious legal problems for the individual, the congregation, and even the Society.” The letter goes on to warn that breaches of confidentiality may lead to costly lawsuits and prosecutions. “Worldly persons are quick to resort to lawsuits if they feel their ‘rights’ have been violated,” according to the document. “Substantial monetary damages could be assessed against the elders or congregation. In some cases where the authorities are involved, certain complications could lead to a fine or imprisonment.” Subsequent memos reinforced the Watchtower’s policies, culminating in a 1997 letter that directed elders to report all known or suspected child sexual abusers – past, present and future – to the organization’s New York headquarters. This memo appears to be the foundation of the database referenced in Jose Lopez’s case. The memo lists 11 questions that must be answered in each case, including the name of the perpetrator, age of the victim and how many times the abuse occurred. Other questions appear to be designed to gauge the perpetrator’s risk of exposure: “How is he viewed in the community and by the authorities? Has he lived down any notoriety in the community? Are members of the congregation aware of what took place?” In the Lopez trial, the Watchtower refused to provide its list of perpetrators, in violation of an order upheld by the California Supreme Court. It also refused to provide the longest-serving member of the Governing Body, Gerrit Lösch, who was subpoenaed. As a result, San Diego Superior Court Judge Joan Lewis disqualified the Watchtower’s defense. “Watchtower’s actions or omissions were ‘reprehensible.’ I think ‘disgraceful’ may be synonymous with ‘reprehensible,’ but I think ‘disgraceful’ doesn’t say enough about it,” Lewis wrote in her decision. “The award of punitive damages against them will hopefully send a message to Watchtower and its managing agents, the governing body of the Jehovah’s Witnesses, that their handling of sex abuse cases within their congregation was absolutely reckless.” Her $13.5 million verdict was based solely on Lopez’s evidence and testimony. “They fight every request for documents, every subpoena,” said attorney Irwin Zalkin. “They’ll take a hit not to produce what they know.” “Not every individual who has sexually abused a child in the past is considered a ‘predator.’ The (Watchtower), not the local body of elders, determines whether an individual who has sexually abused children in the past will be considered a ‘predator.’ ”— 2012 Watchtower memo The Watchtower bases its child abuse policies on Scripture, and the message to elders is clear: Disobey the policy and you disobey God. For emphasis, the authors punctuate many of their policy directives with verses from the Bible. For example, if a child reports to an elder that someone in the congregation has molested him or her, the child must produce another witness to the crime before the elders will investigate the allegation. The so-called two-witness rule, one memo explains, comes from Deuteronomy 19:15: “No single witness should rise up against a man respecting any error or any sin. … At the mouth of two witnesses or at the mouth of three witnesses the matter should stand good.” Although most incidents of child abuse don’t occur in the presence of witnesses, senior official Richard Ashe said in his deposition that elders are bound by the two-witness rule. “You know the truth has the ring of truth to it, but it still has to be established scripturally by the mouth of two witnesses for the congregation to be authorized to take any action,” he said. The only other way elders are allowed to take action is for the perpetrator to confess. But abusers who express repentance often are allowed to remain in the congregation. In either case, elders are not instructed to call police unless required by state law. But state laws are complicated. According to a federal agency, clergy are mandated to report child abuse in 42 states, but laws in 32 of those states, including California, contain a loophole called the clergy-penitent privilege. That exception allows religious leaders to withhold information from authorities when they receive it through a spiritual communication, such as a confession in the Catholic church. Watchtower officials say they instruct their members to obey state laws. Their policy memos direct elders who hear of alleged child abuse to “contact the Society’s Legal Department immediately” to learn whether the laws in their states require them to notify police. Another case from California has exposed the way Jehovah’s Witnesses have hidden abuse from authorities, enabling predators to collect more victims. In 1993 in Fremont, a Jehovah’s Witness named Jonathan Kendrick confessed to two elders that he had sexually abused his 13-year-old stepdaughter as she slept. Kendrick’s wife and her daughter, the victim, were present at the confession. The elders, Michael Clarke and Gary Abrahamson, wrote to the Watchtower for guidance. Two weeks later, a letter from the Watchtower advised the elders that Kendrick’s conduct constituted a “minor uncleanness” and that he could remain a member of the congregation. “However,” the letter said, “it would be appropriate for two elders to meet with him and provide him with strong Scriptural counsel.” The Watchtower determined that Kendrick’s crime didn’t warrant police involvement, disfellowshipping or a warning to the congregation. Because the incident was known outside of the immediate family, it said Kendrick should lose his title of ministerial servant, which meant that he could no longer pass out Watchtower literature at the kingdom hall – the equivalent of a church for Jehovah’s Witnesses – or turn on the microphone at the start of meetings. The elders have testified that they watched Kendrick closely and told him not to be alone with children, but he was allowed to continue preaching the Bible door to door. Although the North Fremont congregation’s elders never reported Kendrick to authorities, he was prosecuted in 1994 after his stepdaughter, during a hospital visit, told police about the abuse, according to a police report. Kendrick pleaded guilty to misdemeanor sexual battery. He was fined $200 and placed on probation. The elders told the congregation that Kendrick had lost his title but, in accordance with Watchtower policy, did not say why. Kendrick and his wife separated in 1997. He moved about 60 miles north to the city of Oakley, where he joined the local congregation and struck up a courtship with a recently widowed Witness named Linda Hood. The courtship caught the attention of the Oakley elders. One of them, Roger Bentley, was charged with getting to know Kendrick on behalf of the congregation. “He told me about an incident with his stepdaughter, but when he told that story, it was accidental touching,” Bentley said. “The version I remember is, he was coming home, it was dark, she was on the couch, he tripped on it and accidentally touched her breast.” Later, in a court deposition, Kendrick acknowledged that he had lied to Bentley. “Whenever I talked to anybody, including Roger, about that incident, I would use the term ‘battery,’ ” he said in the deposition. “I – I dropped the word ‘sexual’ battery. I didn’t want people to think I had been having sex with a child, so I just used the term ‘battery,’ whether it was with Roger or anybody else.” About a year after Kendrick arrived in Oakley, he proposed to Hood. They asked Bentley to perform the wedding ceremony. A letter from North Fremont to the Oakley elders, introducing Kendrick that year – signed by elder Larry Lamerdin – mentioned nothing about Kendrick’s abuse of his stepdaughter. Although the elders had placed restrictions on Kendrick for “loss of temper,” Lamerdin wrote, they had since lifted them. “You will find him to be a fine individual, kind, loving, and appreciates the peace and refreshment of the Christian Brotherhood,” the letter read. “He is a very interesting individual who has taken the lead with some young ones in the congregation and helped them from vearying (sic) off course.” Months later, elder Clarke of North Fremont sent a second report to Oakley. Nowhere did it mention sexual abuse or Kendrick’s conviction. “I can’t overstate how powerful those two letters were to Oakley elders,” former elder Bentley said in an interview. “Where does it say he’s a child abuser? My conclusion was that Jonathan was telling the truth and every document that we saw said that he was telling the truth and that we could go ahead with that wedding.” Still, concerned that Kendrick recently had left a rocky marriage, Bentley urged Hood to call the North Fremont elders and ask them directly whether they believed Kendrick would make a good husband. Hood called Clarke. “Mr. Clarke told me Mr. Kendrick was a good person. Because I was not told any negative information about Jonathan and because that I was in love with him, I went ahead and married him,” Hood wrote in a declaration to the Alameda County Superior Court. The couple wed in Hood’s backyard on New Year’s Day 1999. Clarke would not comment to Reveal for this story. Lamerdin did not return phone messages. Josh Hood, Linda’s eldest son, disliked Kendrick from the beginning. He worried that his mother – still grieving over the death of her first husband a year earlier – had married a man she didn’t really know. One night in 2003, Kendrick broke a glass over the head of Josh’s younger brother in a drunken fit, according to a police report. The adult brothers had had enough and went to their mother’s house one night when no one was home to look for information about Kendrick. When they loaded a recovery software program onto Kendrick’s computer, Josh Hood said in sworn testimony, they found child pornography. “I swear to God, it made my heart almost come out of my chest, ” he said later in an interview, “because he’d been around my daughter for years.” Josh Hood then asked his 8-year-old daughter whether Kendrick had ever abused her, which she confirmed. He called his mother, who then called Kendrick at work. Kendrick said he remembered Linda Hood asking whether it was true. “I don’t think I said much,” he recalled later under oath. “I was cutting my wrists at the time.” Soon after, Josh Hood tracked down Kendrick’s ex-wife, who told him that Kendrick had abused her daughter almost a decade earlier. When Josh Hood learned that the North Fremont elders had withheld that information from his family, he was livid. “They left out that Jonathan wasn’t allowed to be around kids because he had had an incident with his stepdaughter from his previous marriage where he had touched her improperly,” Josh Hood said. “They didn’t want him around their children, so it should have been passed along.” After Kendrick cut his wrists, he spent three days recovering in a hospital. Manuel Iglesias and Jim Dominguez, the Oakley elders charged with investigating Josh Hood’s daughter’s allegations, visited him there. Meanwhile, Josh Hood reported the abuse to police and said he gave them a disk with the child pornography he claimed was copied from Kendrick’s computer. Kendrick never was investigated for the alleged pornography. Although Kendrick confessed to the elders about the abuse, according to court records, neither cooperated with the law enforcement investigation. Sgt. Jeffrey Baldwin of the Contra Costa County Sheriff’s Office said in a deposition that Iglesias did not return his calls. When Baldwin reached Dominguez, the elder told him that his visit with Iglesias and Kendrick was a “penitential visit” and therefore exempt from mandatory reporting laws. “They would not speak to me on the matter; and that they referred me essentially to their attorney,” Baldwin recalled. Reached in person at his home, Dominguez told Reveal: “That was a dark time. I was trying to protect the congregation. I think it’s best for me if I don’t talk now.” Adhering to Watchtower policy, the Oakley elders also did not tell their congregation what Kendrick had done. In 2003, Kendrick pleaded guilty to committing a lewd act on a minor under 14. He spent about eight months in jail and five years on probation, during which time he underwent sex offender treatment. But that was not the end. Years later, a woman claiming to have been another victim of Jonathan Kendrick would come forward, calling into question the testimony of the North Fremont elders and leading to the first U.S. trial in a child abuse case against the Watchtower. Candace Conti was long gone from the Jehovah’s Witnesses in 2009, when she decided to look up Kendrick on the Internet. Years earlier, she said, Kendrick had abused her, but she had never reported the incident. She broke down when she found him on a sex offender registry. “I know it never left me, knowing that he had hurt somebody else,” she said. “Knowing that he went to a different kingdom hall and hurt somebody else, knowing that that person was the same age as me.” Conti was born into the Jehovah’s Witnesses and attended the North Fremont congregation as far back as she can remember. Both of her parents and her grandparents on her mother’s side were Witnesses. She started preaching door to door when she was 5, handing out Watchtower pamphlets with colorful illustrations of paradise on earth. “I just remember my whole opening spiel was, ‘Wouldn’t you love to live in a beautiful place like this?’ ” she said. “You’re bringing them to Jehovah’s organization, you’re saving these people’s lives.” While in grade school, Conti spent 70 hours a month preaching. Kendrick had become close friends with Conti’s father. Although she wasn’t comfortable around Kendrick, Conti said she would end up alone with him during long afternoons of knocking on doors. According to Conti, when she was 9 and 10 years old, Kendrick used the time they were alone to take her to his house and sexually assault her. She says she kept the abuse to herself for years. While there is no dispute that Kendrick molested at least two girls, he emphatically denied to Reveal ever abusing or even being alone with Conti. Kendrick would only say during a brief phone interview: “I did not molest Candace Conti. I have never been questioned by law enforcement involving Candace Conti. I’ve never been charged with a crime involving Candace Conti.” He would not comment on any other cases. Conti’s discovery of Kendrick in the sex offender registry led her back to the Witnesses. She went to elders Michael Clarke and Larry Lamerdin, who had watched her grow up, and told them her story for the first time. Because she didn’t have any witnesses to her abuse, she said, they told her there was nothing they could do. “They got the story from me and they both cried, she said. But they couldn’t come to bat for me. It was out of their hands. They are bound by those rules and regulations that are passed down by the organization.” Clarke and Lamerdin wrote to the Oakley congregation in December 2009 to inform the elders that Kendrick had abused Conti. The letter arrived seven years after Josh Hood learned that Kendrick had abused his daughter. “She claims that there (sic) relationship was inappropriate and her parents and congregation elders should have put a stop to it,” they wrote. “We totally agreed.” “She asked us twice if we were going to report this to the authorities,” they continued. “We told her that if she wishes to make a report, it is her absolute right to do so.” When Conti told them that she didn’t trust the congregation to protect children, the elders wrote, “We shared a number of scriptures with her regarding Jehovah’s love and concern for her.” Frustrated with the response from the Witnesses, Conti sued the Watchtower in 2011. “It was to attack the policies and procedures that were in place, that let a serial molester continue to molest children,” Conti said. “I had this sense of guilt. …What if I had done something to maybe protect this other child?” During the trial, Conti’s attorney Rick Simons zeroed in on the Watchtower’s child abuse policy memos. In his deposition during Conti’s lawsuit, Clarke said elders are required to adhere strictly to the Watchtower’s policies. “Are congregations free to deviate from the practices through which instructions are given through ‘Body of Elder’ letters?” her lawyers asked. “No,” Clarke responded. In an attempt to discover who wrote the memos, Simons also deposed Allen Shuster, a supervisor in the Watchtower’s service department, where the memos were drafted. Shuster testified that he might have contributed to writing some of the memos but that he couldn’t remember for sure. “There would have been a group of elders within the service department that would have reviewed this letter,” he said. “It would have been approved by a committee of the Governing Body.” Simons then asked about the two-witness rule. “These are policies that come from the Governing Body?” Simons asked. “That is an accurate statement, yes,” Shuster said. Before the Conti case, all U.S. child abuse lawsuits against the Watchtower had been either dismissed or settled out of court. Hers was the first to go to trial. The jury found in 2012 that the North Fremont congregation and the Watchtower had been negligent, failing to protect Conti from a known child abuser. She was awarded more than $15 million. In the wake of the Conti verdict, Josh Hood and his daughter filed their own lawsuit against the Watchtower in 2012. Howard Magee, the Hoods’ lawyer, deposed Thomas Jefferson, another Watchtower supervisor in the New York service department. He corroborated Clarke’s testimony in Conti’s case that elders are instructed to report child abuse to the Watchtower’s legal department and expected to follow whatever advice they receive. Jefferson said the Watchtower has files on Kendrick’s abuse history. He was not aware of any Watchtower policy that would prohibit Kendrick from preaching door to door, he said “Child molestation is a confidential matter,” he said. Kendrick, now 61, is still an active member of the Oakley congregation. He and Linda Hood remain together. Linda Hood’s granddaughter’s case was dismissed after the judge affirmed the Watchtower’s First Amendment protection. This month, a California judge in another case ruled against the Watchtower for refusing to provide its database of abusers. In a statement to Reveal punctuated with Bible citations, the Watchtower stated that congregation elders comply with child abuse reporting laws. “The victim and his or her parents have the absolute right to report the matter to the governmental authorities. (Galatians 6:5),” the statement read. “Congregation elders do not shield abusers from the authorities or from the consequences of their actions. (Galatians 6:7).” “We believe that loving and protective parents are the best deterrent to child abuse.” The governing body of the Jehovah’s Witness church received another rebuke this week by a state appeals court for “obstinately” refusing to turn over internal documents about knowledge of church leaders who have been accused of sexually abusing children. The ruling, filed Thursday by the 4th District Court of Appeal, upholds a $4,000-a-day penalty against Watchtower Bible and Tract Society of New York for its failure to comply with a court order in a lawsuit filed by a man who claimed to have been molested in the 1990s. “Here, Watchtower has abused the discovery process. It has zealously advocated its position and lost multiple times. Yet, it cavalierly refuses to acknowledge the consequences of these losses and the validity of the court’s orders requiring it to produce documents…,” the opinion concluded. The fight for these internal documents has been at the center of not only this lawsuit, but a similar one that accuses the same leader of molestation. Church elders knew Gonzalo Campos had molested a boy as early as 1982 but did not remove him from interacting with children, according to evidence revealed in the cases. In one lawsuit filed in San Diego Superior Court in 2012, Jose Lopez said he was 7 when a church elder in a Linda Vista congregation suggested Campos mentor him. Campos molested the boy at Campos’ La Jolla home one day in 1986, according to the lawsuit. When church leaders were told, they said they would handle the situation, the lawsuit says. Campos became more involved with another congregation in La Jolla in 1987. In 1994 or 1995, Campos molested Osbaldo Padron, a church member there, when he was 7 or 8 years old, according to Padron’s 2013 lawsuit. Campos later confessed to abusing at least eight children between 1982 and 1995. He fled to Mexico around 2010, said Irwin Zalkin, the lawyer for both alleged victims. Watchtower has argued that the court’s order to turn over the documents is too burdensome and overbroad, and also that Watchtower does not have access to such records after 2001, but a church corporation does. In both lawsuits, Watchtower has rebuffed court orders to produce documents about current of former leaders accused of molesting children and has heavily redacted the records it has turned over. In the Lopez case, a Superior Court judge found Watchtower to be noncompliant and eventually terminated the organization’s right to be heard in the case. Watchtower appealed, questioning why the judge didn’t use lesser measures to gain compliance, such as monetary sanctions. The appeals court agreed last year, saying the terminating sanction had been too harsh and reversed a $13.5 million judgment that had been imposed. That case is still being litigated. But when the issue came up in the Padron case, and a different Superior Court judge imposed financial sanctions — $4,000 a day for not producing or searching for the ordered documents — Watchtower complained it was unfair. “… We are troubled that Watchtower has taken two inconsistent positions before us,” the three-judge appellate panel said in its Thursday ruling. The court concluded that Watchtower’s conduct was “so egregious” in its handling of the documents that if court orders were not followed, it may be necessary to end the organization’s right to be heard in Padron’s case. Watchtower officials said in a statement Friday that the organization is evaluating its legal options following the ruling. kristina.davis@sduniontribune.com Twitter: @kristinadavis

Summary: The Jehovah's Witnesses have a $2 million tab with the state of California that's growing by $4,000 every day, all because the group won't hand over internal documents that detail alleged child sex abuse in its congregations. Reveal from the Center for Investigative Reporting explains: San Diego man Osbaldo Padron claims he was sexually abused as a child in the 1990s by an adult congregation member and filed suit against the Jehovah's Witnesses for not interceding and continuing to promote the man. The San Diego Union-Tribune reports the church allegedly knew in 1982 that Gonzalo Campos had molested a child; Campos later admitted to abusing at least eight kids between that year and 1995 (he fled to Mexico in 2010). Padron wants internal documents that he believes show a pattern, and the church won't hand them over, in violation of a court order. Hence the $4,000 daily penalty; the order was upheld on appeal last week. Reveal offers details on internal documents it was able to obtain, which reach back three decades and include a 1989 letter sent to all congregations from HQ, known as the Watchtower, noting that elders "must be careful not to divulge information about personal matters to unauthorized personnel." A 1997 letter decried molestation but instructed on how to handle a "former child molester" moving to a new congregation: send a letter about any "needed cautions" in a "Special Blue" envelope to the new congregation's elders, but "this letter should not be read to or discussed with the congregation." The Union-Tribune notes that Watchtower has complained the court order is overly burdensome and broad, and has heavily redacted the documents it has handed over.

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Summarize: Pam Bondi is once again opposing same-sex marriage rights in Florida. On Wednesday, the attorney general filed papers in Broward County Circuit Court saying that Palm Beach resident Heather Brassner and her former partner, Megan Lade, should not be granted a petition for divorce. Brassner and Lade were joined together in a civil union in Vermont back in 2002. In August, Broward County Circuit Court Judge Dale Cohen had become the third Florida judge to rule that the state's ban on same-sex marriage is unconstitutional, as he presided over Brassner's case. See also: South Florida Lesbians Challenge Gay Marriage Law: "We Are Not Second-Class Citizens" Brassner, 41, had been seeking to file for divorce in absentia after the couple split and Lade disappeared. Brassner soon had hired a private investigator try to track down Lade, but those efforts proved unsuccessful. Brassner's main reason for seeking a divorce was security. She fears Lade may resurface and come after her financially, use her name to obtain property, or do any number of things that could be used against her. Basically, Brassner is seeking basic marriage rights afforded to straight divorced couples. Cohen had to declare the state's same-sex marriage ban unconstitutional so Brassner and Lade's civil union could be legally recognized and for the divorce to be official. But on Wednesday, Bondi stepped in and said Cohen should not grant Brassner the petition. A civil union is not the same as a marriage, Bondi's filing says. For this reason, Bondi argues, the ban on same-sex marriage cannot be declared unconstitutional. The deadline to oppose the divorce was Friday. "Attorney General Bondi should not hold our state and our families hostage by calling for the stay to remain in place," Equality Florida CEO Nadine Smith said in a statement. "Let her follow the lead of the 9th Circuit and the U.S. Supreme Court and allow marriages to commence. Every day that Floridians are denied access to the protections only marriage can provide, our families suffer." Brassner, who has since begun dating someone else, told New Times in September that she would keep fighting for the rights of same-sex couples, even if Bondi had appealed Cohen's ruling, as she was expected to do. "I believe in fighting for equality for all," she said. "We are all members of the human race, and my hope is that love and kindness become the rule of the land. We are not second-class citizens, nor is anyone else. I believe that being seen as equal goes hand in hand with human dignity." Bondi has filed several motions in a state appeals court requesting a freeze on appeals by same-sex couples who are challenging Florida's gay marriage ban. With the motions, Bondi wants the U.S. Supreme Court to decide whether states have the right to ban gay marriage. Her contention is that it would be a burden on the state's taxpayers to keep bringing these issues to court. She's also been very vocal about her opposition to same-sex marriage in Florida. In July she argued against six gay and lesbian couples suing the state for the legalization of same-sex marriage. Bondi wrote, "Disrupting Florida's existing marriage laws would impose significant public harm." "Florida's marriage laws have a close, direct and rational relationship to society's legitimate interest in increasing the likelihood that children will be born to and raised by the mothers and fathers who produced them in stable and enduring family units," Bondi added. Send your story tips to the author, Chris Joseph. Follow Chris Joseph on Twitter

Summary: Florida's attorney general isn't going to allow same-sex divorce any sooner than she allows same-sex marriage. In a ruling this week, Pam Bondi opposed granting a divorce petition because doing so would have required recognizing the same-sex civil union and declaring the state's marriage laws unconstitutional, reports the Miami Herald. Some background: Heather Brassner and former partner Megan Lade were granted a civil union in Vermont in 2002, but Brassner says Lade disappeared after cheating on her years ago. The Florida resident can't get a divorce in absentia if the state won't recognize the union, and Vermont can't dissolve the union without an affidavit from Lade, whom a private investigator couldn't track down. Brassner fears that without a divorce, Lade could do things like use her name to obtain property, reports the New Times Broward-Palm Beach. Bondi-who has defended the state's same-sex marriage ban in several lawsuits-argues that a civil union isn't "equivalent to marriage for purposes of Florida's dissolution of marriage laws," so Brassner's case isn't one in which the 2008 ban can be declared unconstitutional. In August, a federal judge struck down the ban, but he issued a stay delaying the effect of his own order.

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Summarize: The only surviving son of fraudster Bernie Madoff has died after a long battle with lymphoma. Andrew Madoff, 48, was surrounded by family as he 'died peacefully' at Sloan Kettering Cancer Center in New York on Wednesday morning, his attorney, Martin Flumenbaum, said in a statement. His death comes four years after his brother Mark killed himself on the second anniversary of their father's arrest for a $65 billion Ponzi scheme. Bernie Madoff is serving an 150-year prison sentence. Andrew Madoff, who leaves behind a fiancée and two daughters, first fought mantle cell lymphoma in 2003 and was diagnosed with the disease again in October 2012. In an interview last year, he blamed the stress of living with his father's arrest for the relapse. Passed: Andrew Madoff, pictured left on the Today show in 2011 and right in February last year, has died of lymphoma. The 48-year-old trader was the last remaining son of fraudster, Bernie Madoff. 'One way to think of this is the scandal and everything that happened killed my brother very quickly,' Andrew told People magazine in April 2013. 'And it's killing me slowly.' As part of his treatment, he had a stem cell transplant in May 2013, followed by chemotherapy. Although he reconciled with his mother, Ruth, shortly before his second diagnosis, he always said that he had no interest in having contact with his father. 'Even on my deathbed, I will never forgive him for what he did,' he told People magazine. 'He's already dead to me.' Before the storm: Andrew Madoff is pictured second right with his father Bernie, brother Mark and mom Ruth. Locked up: Madoff, pictured in January 2009, is serving an 150-year sentence in a North Carolina prison for his $65 billion Ponzi scheme. Andrew Madoff said he would never forgive his father for what he did. Bernie Madoff was arrested on December 11, 2008, following his $65 billion Ponzi scheme, which became the largest financial fraud in history. He pleaded guilty to 11 federal felonies in March 2009. The charges included securities fraud, wire fraud, mail fraud, money laundering, making false statements, perjury, theft from an employee benefit plan and making false filings with the SEC. The scheme was exposed after he told his sons about it and they contacted the FBI. Andrew told People magazine last year that turning his father in was 'at the same time the easiest decision I ever had to make and the hardest'. Andrew, who had been head of equities at his father's New York-based company, always denied knowing anything about the scheme. Family man: Andrew Madoff is pictured with his daughters, Anne and Emily, from his first marriage. Left behind: A photo from 2011 shows Andrew Madoff with his fiancee Catherine Hooper and his mother Ruth. He reconciled with his mother after initially shunning her but said he would never forgive his father. Struggling to deal with the revelations, Mark Madoff, 46, hanged himself in his apartment on the second anniversary of his father's arrest, as his two-year-old son was asleep in the room next door. Speaking on the Today show in 2011, Ruth Madoff revealed that the last time she had seen her son Mark alive was the evening her husband had revealed the scheme. 'I'll never get over that,' she said. 'I blame Bernie. It's the worst thing that can happen,' In January, Bernie Madoff revealed he was battling his own health woes, telling CNBC that he suffered a heart attack last year and had been diagnosed with kidney cancer. Defrauded Madoff investors have long viewed the convicted swindler's sons, wife and other family member suspiciously, arguing it is impossible that they did not know about his lies. No family members were criminally charged. Torn apart: Madoff's other son, Mark, is pictured with his wife Stephanie ahead of his suicide. He committed suicide in December 2010 on the second anniversary of their father's arrest. Scene: A police officers points to the third floor New York City apartment where Mark Madoff's body was found after he hanged himself. He took his life as his two-year-old son slept in the room next door. Mantle cell lymphoma, which most often affects men over the age of 60, is a rare type of non-Hodgkin lymphoma that causes white blood cells to grow and multiply uncontrollably. It is aggressive and often comes back after patients go into remission. Survival rates are generally poor compared to other types of non-Hodgkin lymphomas and most people do not live longer than five years after diagnosis. Initial symptoms can include fever, heavy night sweats and weight loss. Patients may also find a lump on their neck, armpits or groin. Andrew Madoff leaves behind two daughters, Anne and Emily, from his first marriage. He was engaged to Catherine Hooper

Summary: Andrew Madoff, 48, 'died peacefully' in a New York hospital on Wednesday. He fought lymphoma in 2003 and was diagnosed again in October 2012. In an interview last year, he blamed the stress of his father's arrest for his relapse and said he would never forgive him. Bernie Madoff, who has himself been diagnosed with cancer, is serving an 150-year prison sentence in North Carolina for his $65 billion Ponzi scheme. In 2010, his son Mark killed himself on the second anniversary of his arrest.

cnn_dailymail

en

Summarize: BACKGROUND [0001] The present invention relates to shovels and more particularly to foldable shovels which can be disassembled for storage, shipping or length adjustment and which can be easily re-assembled. [0002] Foldable shovels have been in use for a number of years. In general they comprise blades, handles and hand grips which can be disassembled and reassembled from each other for storage, shipping or length adjustment. Known foldable shovels are complex to assemble and disassemble, sometimes require special tools and are expensive and complicated to manufacture and maintain. OBJECTS [0003] The present invention overcomes these drawbacks and has for one of its objects the provision of an improved foldable shovel which may be easily assembled and disassembled. [0004] Another object of the present invention is the provision of an improved foldable shovel which can be easily assembled and disassembled without the use of special tools. [0005] Another object of the present invention is an improved foldable shovel in which the length of the tool may be easily adjusted. [0006] Another object of the present invention is the provision of an improved foldable shovel which is simple to use and inexpensive and simple to manufacture and maintain. [0007] Other and further objects of the invention will be obvious upon an understanding of the illustrative embodiment about to be described, or will be indicated in the appended claims and various advantages not referred to herein will occur to one skilled in the art upon employment of the invention in practice DRAWINGS [0008] A preferred embodiment of the invention has been chosen for purposes of illustration and description and is shown in the accompanying drawings forming a part of the specification wherein: [0009] [0009]FIG. 1 is an exploded perspective view showing the foldable shovel of the present invention. [0010] [0010]FIG. 2 is a top plan view of the shovel. [0011] [0011]FIG. 3 is a side plan view of the shovel. [0012] [0012]FIG. 4 is a sectional view taken above line 4 - 4 of FIG. 3. [0013] [0013]FIG. 4A is an exploded sectional view similar to FIG. 4 showing the position of some of the parts when the shovel is disassembled. [0014] [0014]FIG. 5 is a sectional view taken along line 5 - 5 of FIG. 2. [0015] [0015]FIG. 6 is a sectional view taken along line 6 - 6 of FIG. 3. DESCRIPTION [0016] Referring to the drawings, the foldable shovel 1 of the present invention comprises a blade 2, handle sections 3 and 4 and a hand grip 5. The blade 2 has a stem 6 extending therefrom and the hand grip 5 has a stem 17 extending therefrom. The stem 6 of the blade 2 has an opening 7 therein; section 3 has openings 8 and 9 therein adjacent the ends 10 and 11, respectively; section 4 has openings 12 and 13 therein adjacent the ends 14 and 15, respectively; and stem 17 of the hand grip 5 has an opening 16 therein. The section 3 may have a reduced diameter portion 20 adjacent end 10 so that it may telescope into the end 21 in the blade stem 6. The section 4 may be made entirely of reduced diameter so that it may telescope into the end 11 of the section 3 and the end 22 of the hand grip stem 17. However, it will be understood that the diameters of the various parts may be changed so long as certain parts of the shovel may telescope over and/or into other parts. It will also be seen that the length of the shovel 1 can be shortened or lengthened depending on the number of sections 3 and 4 used and that more or less sections 3 and/or 4 may be used without departing from the invention. [0017] Improved holding means 30 are provided to removably hold the various parts 2, 3, 4 and 5 of the shovel 1 together and to permit easy assembling and disassembling thereof The holding means 30 comprises a resilient lock clip 31 having a pair of opposed legs 32 and 33 which are formed by bending a single strip of resilient material to form opposed legs 32 and 33 into a V having apex 34. The legs 32 - 33 can be pressed together and when released the resiliency of the clip 31 will cause them to spring back to their original position. The clips 31 are mounted within the inner surface 23 of the section 3 reduced portion 20 adjacent end 10 and on the inner surface 24 of the section 4 adjacent ends 14 and 21. The two legs 32 - 33 are sufficiently spread apart and biased outwardly so that they will bear against and be held frictionally within the inner surfaces 23 and 24 of the sections 3 and 4. One leg 32 has a push button 35 which is adapted to be pressed in to compress leg 32 relative to the leg 33 and which is adapted to extend through the openings 7, 8, 9, 12, 13 and 16 to lock the various parts together. The various parts are held in place by the frictional force exerted by the legs 32 - 33 of the clips 31 on the inner surfaces of the sections 3 and 4 and the push buttons 35 inserted into the various openings 7, 8, 9, 12, 13 and 16. If desired, the leg 33 of some or all of the clips 31 may be permanently or removably attached to the inner surface of any one or more of the sections 3 or 4 without departing from the invention. In addition, any of the clips 31 may be completely removed from one shovel or section thereof and used within another shovel or section. [0018] In the shovel 1 shown in the drawings, a clip 31 is positioned in the end 10 of section 3 and has push button 35 extending through its opening 8. The reduced portion 20 of section 3 is then telescopically inserted within the open end 21 of the blade stem 6 and push button 35 is manually depressed so that the legs 32 - 33 of the clip 31 are compressed and the push button 35 slides within and along the inner surface of the blade stem 6 until it reaches the opening 7. At this point, the natural resiliency of the clip 31 snaps the leg 32 outwardly so that the push button 35 moves into the opening 7 to hold the blade 2 and section 3 together. In order to disassemble the two it is merely necessary for the push button 35 to be depressed until it moves out of the opening 7 in the blade stem 6 so that the reduced portion 20 of the section 3 can be telescopically moved out of blade stem 6. [0019] In a similar manner, a clip 31 is positioned within section 4 adjacent its end 14. Push button 35 extends through the opening 12 where the resilient clip 31 is held in place at that point. The push button 35 is pressed down to compress the legs 32 - 33 of the clip 31. Section 4 is then telescopically inserted into section 3 and the push button 35 will slide along the inner surface 23 of section 3 until it reaches the opening 9. At this point, the legs 32 - 33 of the clip 31 snap automatically and push button 35 moves into the opening 9 to hold the two sections 3 and 4 together. When it is desired to disassemble the two sections, it is merely necessary to press the push button 35 down until it is out of the opening 9 after which the section 4 can be telescopically moved out of the section 3. [0020] Likewise, a clip 31 is positioned within the section 4 adjacent the end 15. In this position, the push button 35 extends through the opening 13 and the clip 31 is held in place at this point. The push button 35 of clip 31 is pressed in and the end 15 is telescopically inserted within the stem 17 of the hand grip 5. The push button 35 moves along the inner surface of the hand grip 17 until it reaches the opening 16. At this point, the legs 32 - 33 will spread apart to move the push button 35 into the opening 16. This will hold the hand grip 5 and the section 4 together. When it is desired to remove the hand grip 5 from section 4 it is merely necessary to depress the button 35 until it is out of the opening 16 at which point the section 4 can be telescopically removed from the hand grip stem 17. [0021] It will thus be seen that the present invention provides an improved foldable shovel which may be easily assembled and disassembled, which can be easily assembled and disassembled without the use of special tools, in which the length of the tool may be easily adjusted, and which is simple to use and inexpensive and simple to manufacture and maintain. [0022] As many and varied modifications of the subject matter of this invention will become apparent to those skilled in the art from the detailed description given hereinabove, it will be understood that the present invention is limited only as provided in the claims appended hereto.

Summary: A foldable shovel having a plurality of elements telescopically mounted together. The elements include a blade, a hand grip and an intermediate section with the intermediate section telescopically mounted to the blade and the hand grip. Openings in the blade, section and hand grip and a locking clip for locking the intermediate section to the blade and to the hand grip. The locking clip is a resilient clip having a button removably extending into the openings in order to hold the intermediate section to the blade and to the hand grip.

big_patent

en

Write a title and summarize: SECTION 1. SHORT TITLE; REFERENCES. (a) Short Title.--This Act may be cited as the ``Graduate Opportunities in Higher Education Act of 2003''. (b) References.--Except as otherwise expressly provided, whenever in this Act an amendment or repeal is expressed in terms of an amendment to, or repeal of, a section or other provision, the reference shall be considered to be made to a section or other provision of the Higher Education Act of 1965 (20 U.S.C. 1001 et seq.). SEC. 2. JAVITS FELLOWSHIP PROGRAM. (a) Interruptions of Study.--Section 701(c) (20 U.S.C. 1134(c)) is amended by adding at the end the following new sentence: ``In the case of other exceptional circumstances, such as active duty military service or personal or family member illness, the institution of higher education may also permit the fellowship recipient to interrupt periods of study for the duration of the tour of duty (in the case of military service) or not more than 12 months (in any other case), but without payment of the stipend.''. (b) Allocation of Fellowships.--Section 702(a)(1) (20 U.S.C. 1134a(a)(1)) is amended-- (1) in the first sentence, by inserting ``from diverse geographic regions'' after ``higher education''; and (2) by adding at the end the following new sentence: ``The Secretary shall also assure that at least one representative appointed to the Board represents an institution that is eligible for a grant under title III or V of this Act.''. (c) Stipends.--Section 703 (20 U.S.C. 1134b(a)) is amended-- (1) in subsection (a)-- (A) by striking ``1999-2000'' and inserting ``2004- 2005''; (B) by striking ``shall be set'' and inserting ``may be set''; and (C) by striking ``Foundation graduate fellowships'' and inserting ``Foundation Graduate Research Fellowship Program''; and (2) in subsection (b), by amending paragraph (1)(A) to read as follows: ``(1) In general.--(A) The Secretary shall (in addition to stipends paid to individuals under this subpart) pay to the institution of higher education, for each individual awarded a fellowship under this subpart at such institution, an institutional allowance. Except as provided in subparagraph (B), such allowance shall be, for 2004-2005 and succeeding academic years, the same amount as the institutional payment made for 2003-2004 adjusted for 2004-2005 and annually thereafter in accordance with inflation as determined by the Department of Labor's Consumer Price Index for the previous calendar year.''. (d) Authorization of Appropriations.--Section 705 (20 U.S.C. 1134d) is amended by striking ``fiscal year 1999 and such sums as may be necessary for each of the 4 succeeding fiscal years'' and inserting ``fiscal year 2004 and such sums as may be necessary for each of the 5 succeeding fiscal years''. SEC. 3. GRADUATE ASSISTANCE IN AREAS OF NATIONAL NEED. (a) Designation of Areas of National Need; Priority.--Section 712 (20 U.S.C. 1135a) is amended-- (1) in the last sentence of subsection (b)-- (A) by striking ``and an assessment'' and inserting ``an assessment''; and (B) by inserting before the period at the end the following: ``, and the priority described in subsection (c) of this section''; and (2) by adding at the end the following new subsection: ``(c) Priority.--The Secretary shall establish a priority for grants in order to prepare individuals for the professoriate who will train highly-qualified elementary and secondary school teachers of math, science, and special education, and teachers who provide instruction for limited English proficient individuals. Such grants shall offer program assistance and graduate fellowships for-- ``(1) post-baccalaureate study related to teacher preparation and pedagogy in math and science for students who have completed a master's degree or are pursuing a doctorate of philosophy in math and science; ``(2) post-baccalaureate study related to teacher preparation and pedagogy in special education and English language acquisition and academic proficiency for limited English proficient individuals; and ``(3) support of dissertation research in the fields of math, science, special education, or second language pedagogy and second language acquisition.''. (b) Collaboration Required for Certain Applications.--Section 713(b) (20 U.S.C. 1135b) is amended-- (1) by striking ``and'' at the end of paragraph (9); (2) by redesignating paragraph (10) as paragraph (11); and (3) by inserting after paragraph (9) the following new paragraph: ``(10) in the case of an application for a grant by a department, program, or unit in education or teacher preparation, contain assurances that such department, program, or unit collaborates with departments, programs, or units in all content areas to assure a successful combination of training in both teaching and such content; and''. (c) Stipends.--Section 714(b) (20 U.S.C. 1135c(b)) is amended-- (1) by striking ``1999-2000'' and inserting ``2004-2005''; (2) by striking ``shall be set'' and inserting ``may be set''; and (3) by striking ``Foundation graduate fellowships'' and inserting ``Foundation Graduate Research Fellowship Program''. (d) Additional Assistance.--Section 715(a)(1) (20 U.S.C. 1135d(a)(1)) is amended-- (1) by striking ``1999-2000'' and inserting ``2004-2005''; and (2) by striking ``1998-1999'' and inserting ``2003-2004''. (e) Authorization of Appropriations.--Section 716 (20 U.S.C. 1135e) is amended by striking ``fiscal year 1999 and such sums as may be necessary for each of the 4 succeeding fiscal years'' and inserting ``fiscal year 2004 and such sums as may be necessary for each of the 5 succeeding fiscal years''. (f) Technical Amendments.--Section 714(c) (20 U.S.C. 1135c(c)) is amended-- (1) by striking ``section 716(a)'' and inserting ``section 715(a)''; and (2) by striking ``section 714(b)(2)'' and inserting ``section 713(b)(2)''. SEC. 4. THURGOOD MARSHALL LEGAL EDUCATIONAL OPPORTUNITY PROGRAM. (a) Contract and Grant Purposes.--Section 721(c) (20 U.S.C. 1136(c)) is amended-- (1) by amending paragraph (2) to read as follows: ``(2) to prepare such students for study at accredited law schools and assist them with the development of analytical skills and study methods to enhance their success and promote completion of law school;''; (2) by striking ``and'' at the end of paragraph (4); (3) by striking the period at the end of paragraph (5) and inserting ``; and''; and (4) by adding at the end the following new paragraph: ``(6) to award Thurgood Marshall Fellowships to eligible law school students-- ``(A) who participated in summer institutes authorized by subsection (d) and who are enrolled in an accredited law school; or ``(B) who are eligible law school students who have successfully completed a comparable summer institute program certified by the Council on Legal Educational Opportunity.''. (b) Services Provided.--Section 721(d)(1)(D) (20 U.S.C. 1136(d)(1)(D)) is amended by inserting ``in analytical skills and study methods'' after ``courses''. (c) Authorization of Appropriations.--Section 721(h) (20 U.S.C. 1136(h)) is amended by striking ``1999 and each of the 4 succeeding fiscal years'' and inserting ``2004 and each of the 5 succeeding fiscal years''. (d) General Provisions.--Subsection (e) of section 731 (20 U.S.C. 1137(e)) is repealed. SEC. 5. FUND FOR THE IMPROVEMENT OF POSTSECONDARY EDUCATION. (a) Contract and Grant Purposes.--Section 741(a) (20 U.S.C. 1138(a)) is amended-- (1) by amending paragraph (1) to read as follows: ``(1) the encouragement of the reform and improvement of, and innovation in, postsecondary education and the provision of educational opportunity for all, especially for the non- traditional student populations;''; (2) in paragraph (2), by inserting before the semicolon at the end the following: ``for postsecondary students, especially those that provide academic credit for programs''; (3) by amending paragraph (3) to read as follows: ``(3) the establishment of institutions and programs based on the technology of communications, including delivery by distance education;''; and (4) by amending paragraph (6) to read as follows: ``(6) the introduction of institutional reforms designed to expand individual opportunities for entering and reentering postsecondary institutions and pursuing programs of postsecondary study tailored to individual needs;''. (b) Areas of National Need.--Section 744(c) (20 U.S.C. 1138c(c)) is amended by striking paragraph (4) and inserting the following: ``(4) International cooperation, partnerships, or student exchange among postsecondary educational institutions in the United States and abroad. ``(5) Establishment of academic programs including graduate and undergraduate courses, seminars and lectures, support of research, and development of teaching materials for the purpose of supporting faculty and academic programs that teach traditional American history (including significant constitutional, political, intellectual, economic, diplomatic, and foreign policy trends, issues, and documents; the history, nature, and development of democratic institutions of which American democracy is a part; and significant events and individuals in the history of the United States). ``(6) Support for planning, applied research, training, resource exchanges or technology transfers, the delivery of services, or other activities the purpose of which is to design and implement programs to enable institutions of higher education to work with private and civic organizations to assist communities to meet and address their pressing and severe problems, including economic development, community infrastructure and housing, crime prevention, education, healthcare, self sufficiency, and workforce preparation.''. (c) Authorization of Appropriations.--Section 745 (20 U.S.C. 1138d) is amended by striking ``$30,000,000 for fiscal year 1999 and such sums as may be necessary for each of the 4 succeeding fiscal years'' and inserting ``$40,000,000 for fiscal year 2004 and such sums as may be necessary for each of the 5 succeeding fiscal years''. SEC. 6. URBAN COMMUNITY SERVICE. Part C of title VII (20 U.S.C. 1139 et seq.) is repealed. SEC. 7. DEMONSTRATION PROJECTS TO ENSURE STUDENTS WITH DISABILITIES RECEIVE A QUALITY HIGHER EDUCATION. (a) Serving All Students With Disabilities.--Section 762(a) (20 U.S.C. 1140a(a)) is amended by striking ``students with learning disabilities'' and inserting ``students with disabilities''. (b) Authorized Activities.-- (1) Amendment.--Section 762(b)(2) is amended-- (A) in subparagraph (A), by inserting ``in order to improve retention and completion'' after ``disabilities''; (B) by redesignating subparagraphs (B) and (C) as subparagraphs (C) and (E), respectively; (C) by inserting after subparagraph (A) the following new subparagraph: ``(B) Effective transition practices.--The development of innovative, effective, and efficient teaching methods and strategies to ensure the smooth transition of students with disabilities from high school to postsecondary education.''; and (D) by inserting after subparagraph (C) (as redesignated by subparagraph (B) of this paragraph) the following new subparagraph: ``(D) Distance learning.--The development of innovative, effective, and efficient teaching methods and strategies to provide faculty and administrators with the ability to provide accessible distance education programs or classes that would enhance access of students with disabilities to higher education, including the use of electronic communication for instruction and advisement.''. (2) Conforming amendment.--Section 762(b)(3) is amended by striking ``subparagraphs (A) through (C)'' and inserting ``subparagraphs (A) through (E)''. (c) Applications.--Section 763 (20 U.S.C. 1140b) is amended-- (1) by amending paragraph (1) to read as follows: ``(1) a description of how such institution plans to address the activities allowed under this part;''; (2) by striking ``and'' at the end of paragraph (2); (3) by striking the period at the end of paragraph (3) and inserting ``; and''; and (4) by adding at the end the following new paragraph: ``(4) a description of the extent to which an institution will work to replicate the best practices of institutions of higher education with demonstrated success in serving students with disabilities.''. (d) Authorization of Appropriations.--Section 765 (20 U.S.C. 1140d) is amended by striking ``fiscal year 1999 and such sums as may be necessary for each of the 4 succeeding fiscal years'' and inserting ``fiscal year 2004 and such sums as may be necessary for each of the 5 succeeding fiscal years''. Passed the House of Representatives October 21, 2003. Attest: JEFF TRANDAHL, Clerk.

Title: To amend title VII of the Higher Education Act of 1965 to ensure graduate opportunities in postsecondary education, and for other purposes Summary: Graduate Opportunities in Higher Education Act of 2003 - Amends the Higher Education Act of 1965 (HEA) to revise requirements for Graduate and Postsecondary Improvement Programs (title VII), and to reauthorize appropriations for some of such programs through FY 2009. (Sec. 2) Revises the Jacob K. Javits fellowship program. Directs the Secretary of Education to give grant priority to institutions of higher education (IHEs) for fellowships to students in advanced linguistic studies and courses that prepare teachers to teach students with limited English proficiency. Permits IHEs to allow fellowship recipients an interruption of study due to active duty military service or a personal or family member illness. Revises requirements for allocation of fellowships. Directs the Secretary to ensure that one member of the fellowship board will be from a minority-serving institution. Reauthorizes appropriations through FY 2009. (Sec. 3) Revises the program of graduate assistance in areas of national need. Directs the Secretary to give grant priority to IHEs to prepare mathematics, science and special education faculty who can train highly qualified mathematics, science, or special education teachers for service in elementary and secondary schools. Revises requirements relating to designation of areas of national need, stipends, and additional assistance. Reauthorizes appropriations through FY 2009. (Sec. 4) Revises requirements for the Thurgood Marshall legal educational opportunity program. Revises activities for which the Council on Legal Education Opportunity (CLEO) is to use program contract and grant funds provided by the Secretary to include: (1) assisting students to develop analytical skills and study methods; and (2) awarding such fellowships to eligible law school students who either participated in summer institutes and are enrolled in an accredited law school or have successfully completed a comparable summer institute certified by CLEO. Revises types of program services to provide that undergraduate preparatory courses be in analytical skills and study methods. Reauthorizes appropriations through FY 2009. (Sec. 5) Revises requirements for the Secretary's Fund for the Improvement of Postsecondary Education program contracts and grants. Authorizes consideration of applications for projects relating to: (1) the needs of nontraditional student populations; (2) distance education delivery through communications technology; and (3) expanded opportunities to enter and reenter postsecondary institutions and pursue study programs tailored to individual needs. Includes among special projects international partnerships with postsecondary institutions abroad. Reauthorizes appropriations through FY 2009. Eliminates continuation awards under certain parts of title VII of HEA. (Sec. 6) Eliminates the Urban Community Service program (part C of title VII of HEA). (Sec. 7) Revises requirements for demonstration projects to ensure that students with disabilities receive a quality higher education. Includes among authorized project activities developing innovative, effective, and efficient teaching methods and strategies to: (1) ensure such students' smooth transition from high school to postsecondary education; and (2) enable faculty and administrators to provide accessible distance education programs or classes to enhance such students' access to higher education. Requires project grant applications to describe how the IHE will work to replicate the best practices of IHEs with demonstrated success in serving students with disabilities. Reauthorizes appropriations through FY 2009.

billsum

en

Summarize: The teenaged Texas Tech cheerleader who created a social media firestorm with photos of the African big game she hunted has fired back at her online attackers. In a typo-laden defense that invokes the safari-loving 26th U.S. president Teddy Roosevelt, Kendall Jones, 19, claims that killing rare rhinos and elephants actually helps to save them. 'This is a conservation effort to assure [sic] that they never do become extinct,' the teen from Cleburne, Texas posted to Facebook. 'This is a conservation effort!': 19-year-old Texas Tech cheerleader Kendall Jones is firing back at the critics who say her hunting safaris to Africa are just poaching expeditions disguised as conservations. Unfairly targeted? Kendall Jones, far right, loves Jesus, cheerleading, and hunting African big game. Facebook, of course, is where the controversy began. By Wednesday evening, 150,000 global animal lovers had signed a petition urging CEO Mark Zuckerberg to take down the photos in which Jones smiles proudly over the corpses of her prey she claims to be saving from extinction. '[Roosevelt] was a hunter too, right? He killed the same species that hunters now chase today under a mound of anti-hunting pressure. 'Yet, how can it be possible that someone can love the earth, and take from the Earth in the name of conservation? For some folks, they'll never understand. For the rest of us...we were born that way. God Bless Teddy,' wrote Jones. Jones' Facebook remains active, the photos that started the controversy have vanished. Meanwhile, a Facebook account has now been created as part of Jones' defense called Support Kendall Jones. 'Support this teenage girl who was attacked for posting pictures of her game that she legally harvested while in Africa,' reads the page. Conserving by killing? 19-year-old Texas cheerleader Kendall Jones really likes to kill rare animals in Africa. While she pays for her legal hunts, her critics says she's not the conservationist she claims to be. Big 5: Jones says her first kill was a rare African white rhino, part of her quest to bag the Big 5 African game animals (rhino, elephant, Cape buffalo, leopard and lion) Legal: The young hunter has many critics but also a lot of supporters who say what she's doing is fine, since she pays the governments of African countries to kill the animals. Jones claims photos of dead hippos, elephants, lions and other. beasts on Facebook are a testament to her hunting skills and dedication. to game preservation. But. critics are appalled by the teen and are calling. Kendall sick and depraved for killing the rare animals and boasting. about it online. 'For the sake of all animals,' reads the petition against Jones as it implores. animal lovers to sign, 'especially the animals in the African region... where hunters are going for fun just to kill an animal!' Jones, whose Facebook indicates. she 'is looking to host a TV show in January 2015,' maintains she is. doing what's best for the preserves, where there isn't always space for. even threatened species like elephants or lions. 'Controlling the male lion population. is important within large fenced areas like these,' Jones writes. 'Funds. from a hunt like this goes partially to the government for permits but. also to the farm owner as an incentive to keep and raise lions on their. property.' Hungry: Jones defends her killing of elephants by saying their meat goes on to feed hundreds of thankful village families. Jones's photos show her posing with bagged zebras, hugging a dead leopard, and smiling beside elephants she's killed. One. particular photo, in which she's posing alongside a an extremely. endangered rhinoceros, has her critics especially steaming, but the. Texas Tech cheerleader says it was alive and well. 'The. vet drew blood, took DNA samples, took body and head measurements,. treated a leg injury and administered antibiotics. I felt very lucky to. be part of such a great program and procedure that helps the White Rhino. population through conservation,' she wrote. However, Jones has in her quest to bag the Big 5 African game animals (lion, elephant, Cape buffalo, leopard, and White/Black rhinoceros) shot a white rhino, which number around 20,000. 'The first animal I ever shot was a White Rhino with a.416 Remington!!' the teen writes on her Facebook page. Because of her enthusiasm for killing rare game and what they say is her dubious standing as a conservationist, some critics say Jones should be banned from hunting in Africa completely. family pride: Jones first learned to love the hunt when she started following her father to Africa for his own hunts at age 9. 'Harvest': 'Another harvest for today,' wrote Jones with this photo. 'White springbok, it's 1 of the 4 color shades of this animal! And let me tell you it's one of my favorite kinds of meat so far!' A second petition, this one on change.org and originating from South Africa, is hoping to do just that. 'Kendall Jones is an American born hunter who has entered the continent and has been hunting African wildlife under the facade of conservation,' reads the petition. It continues: 'She has publicly stated that she hopes to have a television hunting show and she is using endangered and helpless African animals as a stepping to further her popularity on social media platforms.' Critics say the 19-year-old is not conservationist and is simply reaping the benefits of her Facebook photos with African animals to help get a television deal. 'This time I got my leopard,' writes Jones of a third safari she took at age 14. 'And also took down a hippo to get 6 of the Dangerous 7'

Summary: Kendall Jones, 19, drew the ire of thousands with her Facebook photos showing her smiling alongside rare African beasts. She fired back Tuesday in a Facebook post invoking 26th American president Teddy Roosevelt. Jones has posted shots of herself posing with dead elephants, hippos and lions among others that she's killed across Africa. Jones claims her kills come after a 'fair chase,' but thousands are demanding that Facebook remove the posts. Jones is a cheerleader at Texas Tech and is gunning for a reality show about her African adventures.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Fee Repeal and Expanded Access Act of 2007''. SEC. 2. RECREATION FEE AUTHORITIES. The Federal Lands Recreation Enhancement Act (16 U.S.C. 6801 et seq.) is amended-- (1) by striking section 801 (16 U.S.C. 6801 note) and inserting the following: ``SEC. 801. SHORT TITLE. ``This Act may be cited as the `Federal Lands Recreation Enhancement Act'.''; (2) by striking sections 802 through 812 (16 U.S.C. 6801 through 6811) and inserting the following: ``SEC. 802. RECREATION FEE AUTHORITY. ``(a) In General.--Beginning January 1, 2008, subject to subsections (c) and (d), the Secretary of the Interior (referred to in this section as the `Secretary') may establish and collect any fee from individuals or groups for-- ``(1) admission to a unit of the National Park System, including a commercial vehicle admission fee for a National Park at a level determined by the Secretary; and ``(2) the use of only the facilities or services described in subsection (b) at Federal recreational land or water under the jurisdiction of the Director of the National Park Service. ``(b) Authorized Facilities and Services.--The facilities and services referred to in subsection (a)(2) are the following: ``(1) Use of developed campgrounds that provide at least a majority of the following: ``(A) Tent or trailer spaces. ``(B) Picnic tables. ``(C) Drinking water. ``(D) Access roads. ``(E) The collection of the fee by an employee or agent of the Federal land management agency. ``(F) Reasonable visitor protection. ``(G) Refuse containers. ``(H) Toilet facilities. ``(I) Simple devices for containing a campfire. ``(2) Use of highly-developed boat launches with specialized facilities or services, such as mechanical or hydraulic boat lifts or facilities, multilane paved ramps, paved parking, restrooms, and other improvements, such as boarding floats, loading ramps, or fish cleaning stations. ``(3) Rental of cabins, boats, stock animals, lookouts, historic structures, group day-use or overnight sites, audio tour devices, portable sanitation devices. ``(4) Use of hookups for electricity, cable, or sewer. ``(5) Use of sanitary dump stations. ``(6) Use of transportation services. ``(7) Use of developed swimming sites that provide at least a majority of the following: ``(A) Bathhouses with showers and flush toilets. ``(B) Refuse containers. ``(C) Picnic areas. ``(D) Paved parking. ``(E) Attendants, including lifeguards. ``(F) Floats encompassing the swimming area. ``(G) Swimming decks. ``(c) Prohibition on Fees for Certain Persons or Places.--The Secretary shall not charge an admission fee under subsection (a) for-- ``(1) a person under 16 years of age; ``(2) an outing conducted for a noncommercial educational purpose by a school or other academic institution; ``(3)(A) the U.S.S. Arizona Memorial; ``(B) the Independence National Historical Park; ``(C) any unit of the National Park System within the District of Columbia; or ``(D) the Arlington House-Robert E. Lee National Memorial; ``(4) the Flight 93 National Memorial; ``(5) an entrance on other route into the Great Smoky Mountains National Park or any part of the Park unless fees are charged for entrance into the Park on main highways and thoroughfares; ``(6) an entrance to a unit of the National Park System containing a deed restriction on charging fees; or ``(7) an area or unit covered under section 203 of the Alaska National Interest Lands Conservation Act (16 U.S.C. 410hh-2), other than the Denali National Park and Preserve. ``(d) Prohibited Sites.--The Secretary shall not charge a fee under subsection (a) for Federal recreational land or water managed by-- ``(1) the Director of the Bureau of Land Management; or ``(2) the Commissioner of Reclamation. ``(e) Requirements.--In establishing fees pursuant to this section, the Secretary shall-- ``(1) establish the minimum practicable number of fees; and ``(2) avoid, to the maximum extent practicable, collection of multiple or layered fees for a variety of activities or programs. ``(f) Analysis.-- ``(1) In general.--Before establishing a fee under subsection (a), the Secretary shall analyze-- ``(A) the benefits and services provided to visitors to National Parks; ``(B) the cumulative effect of the assessment of the fee; ``(C) the direct and indirect cost and benefit to the Federal Government with respect to the fee; ``(D) applicable public policy and management objectives; ``(E) the economic and administrative feasibility of fee collection; and ``(F) such other factors as the Secretary determines to be appropriate. ``(2) Submission to congress.--Not later than the date that is 90 days before the date on which a fee established under subsection (a) is published in the Federal Register, the Secretary shall submit to Congress-- ``(A) the analysis conducted with respect to the fee under paragraph (1); and ``(B) a description of the level of the fee. ``(g) Publication.-- ``(1) In general.--The Secretary shall publish in the Federal Register a notice of-- ``(A) any new fee established pursuant to this section; and ``(B) any change in the amount of such a fee. ``(2) Effective date.--A fee established pursuant to this section, and any modification to such a fee, shall not take effect until the date that is 1 year after the date on which a notification regarding the fee or modification is published in the Federal Register under paragraph (1). ``(h) Administration.-- ``(1) In general.--The Secretary-- ``(A) may waive or discount a fee established pursuant to this section, as the Secretary determines to be appropriate; and ``(B) shall provide information to the public regarding any fee program under this section, including a description of the costs and benefits of the program. ``(2) Administrative costs.--The Secretary may use not more than 15 percent of the total amount of fees collected pursuant to this section for administrative costs of the recreation fee program, including-- ``(A) direct operating or capital costs; ``(B) the costs of fee collection; ``(C) the costs of notification of fee requirements; ``(D) the costs of direct infrastructure; ``(E) fee program management costs; ``(F) the costs of bonding of volunteers; ``(G) start-up costs; and ``(H) the costs of analyzing and reporting on program success and effects. ``(i) Distribution of Receipts.--Of amounts received by the Secretary as a result of a fee collected at a specific area, site, or facility pursuant to this section-- ``(1) not less than 80 percent shall be used at the specific area, site, or facility in accordance with subsection (j); and ``(2) not more than 20 percent shall be used for other activities or facilities of the National Park Service, as the Secretary determines to be appropriate. ``(j) Use of Funds.--Amounts described in subsection (i)(1) may be used at an area, site, or facility for-- ``(1) repair, maintenance, facility enhancement, media services, and infrastructure, including projects relating to visitor enjoyment, visitor access, environmental compliance, and health and safety; ``(2) interpretation, visitor information, visitor service, visitor needs assessments, monitoring, and signs; ``(3) habitat enhancement, resource assessment, preservation, protection, and restoration relating to recreational uses; and ``(4) law enforcement relating to public use and recreation. ``(k) Reports.--On January 1, 2012, and every 3 years thereafter, the Secretary shall submit to Congress a report describing the status of the recreation fee program under this section, including-- ``(1) an evaluation of the program as conducted at each unit of the National Park System; ``(2) a description of projects funded, activities accomplished, and future projects and programs proposed to be conducted using the fees; and ``(3) any recommendations for modifications to the fee system of the Secretary.'' (3) in section 813 (16 U.S.C. 6812), by striking subsections (e) and (f); and (4) by striking section 814 (16 U.S.C. 6813). SEC. 3. REINSTATEMENT OF CERTAIN ADMISSION AND USE FEE AUTHORITIES. (a) Repeal.--Subsections (a), (c), and (d) of section 813 of the Federal Lands Recreation Enhancement Act (16 U.S.C. 6812) are repealed effective December 8, 2004. (b) Applicability.-- (1) Land and water conservation fund act of 1965.-- Subsections (a) through (f), and (g) of section 4 of the Land and Water Conservation Fund Act of 1965 (16 U.S.C. 460l-6a) shall be applied and administered as if section 813(a) of the Federal Lands Recreation Enhancement Act (16 U.S.C. 6812(a)) had not been enacted. (2) Admission permits for refuge units.--Section 201 of the Emergency Wetlands Resources Act of 1986 (16 U.S.C. 3911) shall be applied and administered as if section 813(c) of the Federal Lands Recreation Enhancement Act (16 U.S.C. 6812(c)) had not been enacted. (3) Golden eagle passport.--Section 502 of the National Parks Omnibus Management Act of 1998 (16 U.S.C. 5982) shall be applied and administered as if section 813(d) of the Federal Lands Recreation Enhancement Act (16 U.S.C. 6812(d)) had not been enacted. (4) National park passport program.-- (A) In general.--Title VI of the National Parks Omnibus Management Act of 1998 (16 U.S.C. 5991 et seq.) shall be applied and administered as if section 813(d) of the Federal Lands Recreation Enhancement Act (16 U.S.C. 6812(d)) had not been enacted. (B) Conforming amendment.--Section 603(c) of the National Parks Omnibus Management Act of 1998 (16 U.S.C. 5993(c)) is amended by striking paragraph (2) and inserting the following: ``(2) General use.--Of amounts received by the Secretary as a result of sales of national park passports at a specific area, site, or facility-- ``(A) not less than 50 percent shall remain available for use at the specific area, site, or facility at which the sales occurred; and ``(B) not more than 50 percent shall be used for other activities or facilities of the National Park Service, as the Secretary determines to be appropriate.''. (c) Admission Fees.--Section 4(a) of the Land and Water Conservation Fund Act of 1965 (16 U.S.C. 460l-6a(a)) (as in effect after subsections (a) and (b) take effect) is amended-- (1) in paragraph (1)-- (A) in the first sentence of subparagraph (A)(i), by striking ``$25'' and and inserting ``$65''; and (B) in the second sentence of subparagraph (B), by striking ``$15'' and inserting ``$40''; and (2) in paragraph (2)-- (A) in the fourth sentence, by striking ``$5'' and inserting ``$25''; and (B) in the sixth sentence, by striking ``$3'' and inserting ``$12''.

Title: A bill to repeal certain provisions of the Federal Lands Recreation Enhancement Act Summary: Fee Repeal and Expanded Access Act of 2007 - Rewrites specified provisions of the Federal Lands Recreation Enhancement Act. Provides for the application and administration of certain admission and use fee authorities under the Land and Water Conservation Act of 1965, the Emergency Wetlands Resources Act of 1986, and the National Parks Omnibus Management Act of 1998 as if the Federal Lands Recreation Enhancement Act had not been enacted.

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Write a title and summarize: Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena. In Gram-negative bacteria, envelope machineries connecting the cell interior to the extracellular milieu must span all envelope layers, including the inner membrane, peptidoglycan and outer membrane. Despite these constraints, gram-negative bacteria have evolved sophisticated envelope nano-machines to interact with their environment. Conspicuous examples are bacterial organelles such as flagella, pili, and transport and secretion systems [1], [2]. In general, the structural genes encoding these systems are clustered within large transcriptional units allowing co-regulation of their expression. However, assembly also relies on additional complexity and must involve “just-in time” transcriptional regulations, specific targeting and protein self-assembly properties [3]. This raises the question of the evolutionary processes that led to the emergence of these macromolecular systems [4]. Non-homologous envelope macro-molecular structures mediate motility in bacteria. For example, bacteria swim in extremely viscous environments by means of a rotary flagellum, one of the most sophisticated known biological nano-machines [3]. Bacteria can also crawl across surfaces, for example, polymerization and de-polymerization of pilin fibers from the bacterial cell pole pull the cell forward, a “twitching” motility mechanism which also involves the coordinated assembly of many envelope proteins [5]–[7]. However, gram-negative rod-shaped bacteria are also able to move on surfaces by other means. For example, many bacteria move smoothly along their long axis in the absence of obvious extra-cellular organelles [8]. This gliding motility is associated with unusual flexibility of the cell body and can be found in very diverse bacterial phyla, such as, Bacteroidetes, Cyanobacteria and Deltaproteobacteria [8], [9]. In most species, the mechanism that drives gliding motility remains speculative. For example, in Flavobacterium johnsoniae (Bacteroidetes) gliding motility may be associated with a novel secretion apparatus. However, it is unclear whether this system is involved in assembly of the gliding machinery or constitutes the machinery itself [10]. Finally, gliding may be propelled differently in various species [8]. Despite decades of research, dedicated gliding motility machineries have not been identified unambiguously in any bacterial species, hampering detailed mechanistic studies and asking the question of the emergence of this process in bacteria. In Myxococcus xanthus, a gram negative deltaproteobacterium, surface motility allows the directed aggregation of thousands of cells into mounds that mature into fruiting bodies where the bacteria differentiate into spores [11]. Myxococcus cells can move by twitching motility, but in the absence of pili, the cells are still able to move, unmasking the activity of the gliding engine [11]. Recent cytological work suggested that motility is driven by protein complexes (Focal Adhesion Complexes, FAC) that push against the substratum as they accumulate periodically on the ventral side of the cell [12]–[14]. In a live cell assay, FACs can be observed as bright fluorescent fixed spots in cells expressing a fluorescent gliding motility protein (AglZ-YFP, [12]). The formation of AglZ-YFP foci requires the bacterial MreB-actin cytoskeleton [15] and a FACs-localized proton motive force-driven motor (AglRQS) was recently identified [13]. These observations suggest that AglRQS powers motility in concert with the MreB-cytoskeleton; however, how work from AglRQS is tranduced to the cell surface remains unknown and requires the identification of a motor-associated complex that spans the cell envelope. In the past, 51 genes associated to defects in gliding motility were identified by transposon-based genetic screens, but the functional role of these genes in motility was not established [16], [17]. Recent work by Nan et al. [18] uncovered a new motility complex (AgmU, AglZ, AglT, AgmK, AgmX, AglW and CglB) and suggested that AgmU may be actively transported by PMF-utilizing motors [14]. However, the function of this complex and its direct link with the AglRQS motor remains to be established. In this work, we aimed to identify the motility machinery conclusively. We re-investigated the 51 known M. xanthus gliding genes with the premise that the gliding machinery must have co-evolved with the AglRQS motor. This approach allowed us to identify a novel energy-driven protein complex, which we prove to be the basal gliding machinery. The results reveal the architecture of the gliding machinery and suggest a scenario of its emergence (and evolution) in bacteria. Two independent transposon-based genetic screen studies [16], [17] identified 35 and 23 potential gliding motility genes, respectively (Table S1). Only seven genes overlapped in the two genetic studies, suggesting that the screens are not saturated and thus, the complete set of genes involved in gliding motility has likely not been identified. Nevertheless, these data constituted a good starting point and could indeed contain genes that encode the motility machinery. Irrespective of the exact motility mechanism, a number of cell envelope proteins should be part of the motility machinery. Therefore, we re-visited gene annotations specifically looking for genes encoding predicted membrane proteins, exported proteins and proteins containing motifs mediating protein-protein interaction, such as Tetratricopeptide repeat (TPR) and Coiled-coil domains (Table S1). A total of 28 genes were thus highlighted. A careful survey of these 28 genes revealed that 13 gene hits were in fact clustered into four chromosomal regions of the M. xanthus DK 1622 genome. One region containing three hits, aglW (MXAN_5756, tolB), aglX (MXAN_5753, tolQ) and aglV (MXAN_5754, tolR), encoded together with MXAN_5755 (tolA) and MXAN_5757 (pal), bona fide components of a complete Tol-Pal system. Tol-Pal maintains envelope integrity and supports cell division in all bacteria where it has been studied [19], [20]. Thus, it is unlikely that Tol-Pal constitutes the motility machinery. Consistent with a general envelope function of the Myxococcus Tol-Pal, the aglV (tolR) mutant was also severely impaired in twitching motility [16]. We then focussed our analysis on the remaining three gene clusters (hereafter referred as Gliding1 (G1), Gliding 2 (G2) and Motor 1 (M1), Figure 1A). The G1 cluster contains eight genes, MXAN_4870-62, six of which have been hit by transposons: agmU (MXAN_4870), aglT (MXAN_4869), pglI (MXAN_4867), agmV (MXAN_4864/65, see below), agmK (MXAN_4863) and agmX (MXAN_4862) (Figure 1A). The G2 cluster contains four genes, MXAN_2538-41, two of them inactivated by transposons: agmO (MXAN_2538) and agnA (MXAN_2541). Finally, M1 contains the aglRQS genes themselves (MXAN_6862-60) and two hits by transposon insertions in aglR (MXAN_6862) and aglS (MXAN_6860). So overall, the G1, G2 and M1 clusters involve 15 genes, 10 of which have been previously hit by the transposon screens (Table 1). The M1 cluster encodes the component of a TolQR-like proton conducting motor, which has been characterized elsewhere [13]. The G1 and G2 cluster genes were analysed using public sequences and domain databases. The predicted MXAN_4866 (G1 region) and MXAN_2540 (G2 region) proteins are probably secreted and inserted in the outer membrane because they contain an Autotransporter ß-domain and adopt an OmpA-like fold, respectively (Table 1). AgmO may also be located in the outer-membrane because it carries a typical Outer-membrane Type-II signal sequence. TPR-repeats typically involved in multiprotein assemblies [21] are encoded by four G1 and G2 region genes: agmU, aglT, agmK, and agnA (Table 1). Among them, AgmU, AglT, and AgnA also carry signal peptides, suggesting that they are exported beyond the inner membrane. PglI (G1 region) is a predicted bi-topic transmembrane protein with a cytosolic ForkHead-Associated domain (FHA, [22]) and a periplasmic domain of unknown function. AgmX (G1 region) is also a potential integral membrane protein. MXAN_4868 and MXAN_2539 both carry N-terminal signal peptides but do not contain any conserved functional domains (Table 1). Finally, MXAN_4864 and MXAN_4865 are probably not actual genes and were discarded from this study (see Text S1 for justification) In the rest of this work, we tested if the G1 and G2 genes encode the AglRQS motor-associated gliding machinery. For clarity and to homogenize the nomenclature, we renamed all the G1 and G2 genes glt (gliding transducer, see below), with gltD, E, F, G, H, I and J corresponding respectively to agmU, aglT, MXAN_4868, pglI, MXAN_4866, agmK and agmX (G1) and gltC, A, B and K corfresponding to agnA, MXAN_2540, MXAN_ 2539 and agmO (G2, see below and Text S2 for justification). The G1, G2 and M1 clusters encode a majority of potential envelope proteins and a motor complex (see above). A tempting hypothesis would be therefore that all these components constitute the gliding machinery. We systematically investigated the taxonomic distribution of the 14 genes defining the G1, G2 and M1 clusters in the 1180 complete prokaryotic proteomes available at the beginning of this study (see methods). The 14 genes could be separated in two distinct groups based on taxonomic distribution: A first group (Group A) contained seven genes (gltF, gltH, gltI, gltJ, gltK, gltB and gltA) that were only present (and sometimes in several copies) in Myxococcales (i. e. Sorangium cellulosum, Plesiocystis pacifica, Haliangium ochraceum, Stigmatella aurantiaca, Myxococcus xanthus and the four Anaeromyxobacter sp.) and in Bdellovibrionales (Bdellovibrio bacteriovorus) (Figure 2A). Such restricted taxonomic distribution suggested that these genes appeared only recently during the evolution of the Deltaproteobacteria. By contrast, a second group (Group B) contains seven genes with a much broader taxonomic distribution (Figure 2A). Specifically, blastp and PSI-BLAST queries identified 142 GltD, 2545 GltE, 313 GltG, 83 GltC, 2677 AglR, 2348 AglQ and 2385 AglS homologues. The taxonomic distributions of all these homologues are very different, suggesting that the corresponding genes have undergone different evolutionary histories, which was confirmed by preliminary phylogenetic analyses (not shown). However, in all these phylogenetic trees, the M. xanthus sequences emerge within a monophyletic clade containing homologues from other Deltaproteobacteria but also from a set of unrelated bacteria (i. e. one Betaproteobacteria, several Gammaproteobacteria and one member of Fibrobacteres, Figure S4). This strongly suggests that, although these genes belong to large gene families of distinct evolutionary histories, the M. xanthus gltD, gltE, gltG, gltC, aglR, aglQ and aglS genes and their closest homologues share a similar evolutionary history. The presence of Group B genes (sometime in several copies per genomes) in a few distantly related bacteria (Figure S1) suggests a complex evolutionary history punctuated with horizontal gene transfer (HGT) and gene duplication events (see below). In all non-Deltaproteobacteria and in Geobacter, Group B genes clustered in a single genomic region, possibly an operon, arguing strongly that they encode a single functional unit (i. e. core complex, Figure 2B and Figure S1, Tables S2 and S3). In all these bacteria, the core complex contains an additional gene that has no homologues in Myxococcales and Bdellovibrionales (Figure S1). Remarkably, group B genes (and thus the core complex) group genes from the G1 (gltD, gltE and gltG), G2 (gltC) and M1 (aglQ, R and S) clusters (Figure 2A-2B). This suggests an evolutionary link between the G1, G2 and M1 gene clusters. Strengthening this prediction, homologues of G1 and G2 clusters are grouped on the chromosome of the four Anaeromyxobacter relatives (Figure S1). Then, we proceeded to test the functional relationships between the G1, G2 and M1 genes. In M. xanthus, many of the genes composing the G1 and G2 clusters were previously hit by genetic screens [16], [17]; however, the genes were only partially characterized, and it was not determined how they might be functionally related. More recently, Nan et al. [18] showed that individual deletions of the G1 genes gltD-J impair motility, but their analysis did not test whether these genes are structural. In fact, structural motility components cannot be simply discriminated from regulatory motility components solely based on mutational analysis and colony agar plate assays. First, the absence of motility at colony edges does not necessarily indicate that single cells are completely unable to move: for example, a class of directional mutants (FrzCDc, hyper active Frz-receptor mutants, [23]) forms smooth colony edges, yet, when observed under the microscope, individual cells glide but move back and forth at very high frequencies and thus show no net translocation (hyper-reversing cells, [23]). Thus, motility mutants must also be probed in single cell motility assays. Second, some mutations leading to complete motility defects can be suppressed by second-side mutations, showing that the mutated genes are not structural but regulatory. For example, the motility defect of the aglZ mutant is suppressed when frz, encoding a signal transduction system regulating the directionality of motility, is disrupted [24]. Thus, structural machinery genes must minimally meet the following criteria: (i) gene deletion should result in complete loss of motility in mutants that also lack twitching motility both at colony and single cell scales and (ii), the motility defect should not be suppressed by a frz mutation [24]. Consequently, in this study, we systematically combined the deletions to pilA- or frzE-null mutation (encoding the major pilin sub-unit and the essential FrzE kinase, respectively). It is still possible that regulatory genes may work independently from Frz, but, altogether, the genetic, localization and interaction evidence strongly supports that the Glt proteins are structural (see below). We therefore made markerless in frame deletions in all the G1 and G2 genes (except gltI and gltJ) and showed that the deletions did not create polar effects by quantitative RT-PCR (Table 2 and Table 3). Of note, the expression of gltH was up-regulated 4-5 folds when gltG was deleted, which may point to a regulatory function of GltG (Table 2). On agar plate assays, the gltA-H and gltK mutants retained intact twitching motility but were completely deficient in single cell motility at the colony edges (Figure 3A). gltA-H and gltK pilA double mutants were all completely non-motile both at the colony and single cell levels (Figure 3A and data not shown), showing that the glt genes are specific and essential to gliding motility. In one exception, the gltH pilA mutant showed small scales “jerky” displacements on occasions, but the motility defect was still very severe (Figure 3A and Video S1). In a second step, we observed that gltA-H and gltK frzE double mutants were also completely non-motile in the colony and single cell assays (Figure 3B and data not shown). As a control, we also tested the simultaneous deletion of frzE and aglZ and observed that colony and single motility were both restored, as previously described (Figure 3B, [24]). Interestingly, group swarming appeared enhanced on hard agar plates in all cases, suggesting that the frzE mutation enhanced twitching in those mutants (Figure S2). Nan et al. [18] reported that motility of a gltD mutant allele was restored when a frz mutation was introduced, however this conclusion was based on observation of colony edges. In fact, enhanced twitching motility in the double mutant may have been mis-interpreted for restored gliding motility. To test this, we further introduced a pilA mutation in the double gltD frzE mutant. Motility was completely abolished in the resulting triple mutant. In contrast, the triple aglZ frzE pilA mutant was motile under similar conditions, as expected (Figure 3B, compare middle and right panels). The enhanced twitching in the glt frzE double mutants points to intriguing couplings between gliding and twitching motility, which will need further investigation. In conclusion, the glt genes are genetically separable from aglZ, and may thus encode structural components of the motility machinery. A comparable genetic analysis also suggested that aglR, Q and S are structural [13]. Thus, the genetic results are consistent with a functional link between aglRQS and the glt G1 and G2 group genes. We next aimed to determine the subcellular localization of the suspected Glt protein complex. In absence of specific antibodies to detect all proteins, we only tested some proteins of the G1 cluster: GltD, E, F, G and H, all predicted to localize within the cell envelope (Table 1). We also tested the localization of a functional GltF-mCherry fusion with specific anti-mCherry antibodies. Cell fractionation experiments showed unambiguously that all five proteins localize in the cell envelopes (Figure 4). GltD was also present in the soluble fraction but to minor extents (Figure 4). GltF-mCherry was equally distributed in the soluble and membrane extracts (Figure 4). The GltF-mCherry fusion was functional (Figure S3 and data not shown), however it also seemed to be processed to some extent during the fractionation procedure (Figure 4), thus it cannot be excluded that its presence in the soluble fraction results from improper secretion. We next wanted to discriminate inner- and outer-membrane proteins. Separating the inner membrane from the outer membrane was difficult using standard sucrose density gradients or detergent-based methods (see Methods). We therefore decided to harvest outer-membrane-derived vesicles (see Methods). Extracted vesicles contained PilQ, the pilus Secretin but not PilC, localizing at the inner membrane, confirming that the vesicles were derived from the outer membrane. Only GltH was detected in the vesicle preparation, which is consistent with the presence of an auto-transporter ß-domain in this protein (Figure 4, Table 1). All together these results suggest that GltD-G form an inner membrane localized complex that extends through the periplasm and connect the cell surface via the outer-membrane protein GltH. In a parallel study, we have demonstrated that the M1 cluster (aglRQS) encodes a proton-motive force-driven channel that produces motility traction forces at FACs [13]. The present study suggests that AglRQS and Glt proteins are functionally related, which needed to be proven experimentally. If the Glt proteins interact with the AglZ-AglRQS system, it would be expected that the Glt proteins also localize at FACs. A fluorescent functional GltD-mCherry fusion was already available [18]. We additionally obtained another functional fusion to GltF. In two other studies, GltD-mCherry was found to localize both in fixed clusters [18] and along a dynamic helix-like structure [14]. To rationalize this apparent dual localization pattern, it was proposed that GltD-mCherry molecules traffic along a helix and accumulate at FACs when they become engaged in propulsion [14]. In our hands, the pattern of GltD-, GltF-mCherry fluorescence in live cells was similar: fluorescence was mostly evident around the cell periphery; however, when we collected z-stacks of unprocessed images, fluorescent clusters became clearly apparent when the focal plane was focussed closer to the substratum (Figure 5A, 5B and Video S2). In moving cells, these clusters were fixed and largely co-localized with AglZ-YFP (Figure 5B and 5C). We were not able to resolve a helical pattern of GltD-mCherry around the cell periphery, but observing this structure may require mathematical image-deconvolution processing [14], which would explain this discrepancy. Nevertheless, these results show that GltD and GltF are recruited at FACs, and may be parts of a complex mediating contact between the exterior and the cell interior. GltD-mCherry dynamics are dependent on the PMF [14], suggesting that they result from the activity of a motor, possibly the AglRQS complex (M1). Indeed, in an aglQ mutant, GltD- and GltF-mCherry failed to accumulate at FACs and were only localized around the cell periphery and at the cell poles (Figure 5A. and data not shown). To prove that AglRQS directly fuels trafficking of the Glt proteins, we searched which Glt protein may interact with the motor. By analogy to the Tol/Exb system, the AglR protein would deliver motor work to an output protein through an H+-driven conformational change in its N-terminal transmembrane helix [25]. Thus, the best candidate for direct interaction with AglR is the GltG protein. Indeed, this predicted transmembrane protein has a proline-rich TonB-like motif, typically found in TolA and TonB, the effector transducers in the Tol/Exb systems [20]. Moreover, GltG is the only predicted transmembrane protein that belongs to the core complex together with AglRQS (Figure 2B). We tested a potential interaction between AglR and GltG in a bacterial two-hybrid assay [26] (Figure 6). Highly significant β-galactosidase activity was only obtained when AglR and GltG were expressed together, showing that these proteins interact specifically (Figure 6). Finally, GltD-mCherry cluster localization was also abolished in mutants lacking gltF, G and H, further suggesting that these proteins are parts of one motility complex within the focal adhesion clusters (Figure 5A). All together, the results suggest that the AglRQS-Glt proteins assemble a dynamic envelope spanning motility machinery at the focal adhesion sites. Taken together, the computational and experimental results strongly suggest that the G1, G2 and M1 clusters contain genes encoding the major components of the gliding motility machinery. The most striking result of our in silico analysis is the discovery of a conserved core of genes (Group B) coding for several homologues of the gliding machinery components in non-gliding bacteria. To obtain further insights on the evolutionary mechanisms underlying the emergence of the gliding apparatus, we conducted an in-depth phylogenomic analysis (see Text S3). The phylogenies of the closest homologues of the seven genes defining the conserved core of genes (i. e. Group B) showed similar topologies (Figure S4). However, these analyses were based on a fairly small number of unambiguously aligned positions and as a result most of the nodes of the inferred trees were weakly supported (Bootstrap Values (BV) <90% and Posterior Probabilities (PP) <0. 95, Figure S4). This caveat precluded the precise elucidation of the evolutionary histories of the components. To improve the resolution of the phylogenetic trees, we combined the group B genes gltD, E, G and AglR, Q, S in two distinct supermatrices (See Methods). As expected, the trees based on each supermatrix showed better resolutions than the individual gene trees (compare PP and BV in Figure 6 and Figure S4). Consistent with the single phylogenies (Figure S4), two separate clades (at odds with the species phylogeny) were observed in the resulting phylogenetic trees (PP = 1. 00 and BV = 100%, Figure 7): More precisely, the three Geobacter representatives (Deltaproteobacteria) emerged within the Gammaproteobacteria, whereas F. succinogenes and the other Deltaproteobacteria, belonging to distinct phyla [27], emerged together in the glt and agl phylogenetic trees (Figure 7). Moreover, the relationships among the gammaproteobacterial sequences were mostly incongruent with the species phylogeny (Figure 2 and Figure 7). The discrepancy between the organism and gene trees precluded the clear identification of the precise bacterial lineage where the core complex originated, possibly the Gamma- or Deltaproteobacteria (Figure 8A). Nevertheless, HGT of the core complex is apparent: first, between Gammaproteobacteria and Deltaproteobacteria, then among Gammaproteobacteria and from Deltaproteobacteria to Fibrobacter, and last, from Gammaproteobacteria or Betaproteobacteria to Geobacter (Figure 8A, circles 1 to 4). In contrast, the restricted taxonomic distribution of the Group A genes indicates that they appeared and were recruited more recently during differentiation of the Deltaproteobacteria. An evolutionary scenario may thus be suggested: gltA, B and F likely appeared in the common ancestor of the Myxococcales and Bdellovibrionales, whereas gltI and gltJ (MXAN_4863-62) probably appeared in the ancestor of the Myxococcales, while gltK and H may have been acquired more recently (Figure 8A). The evolutionary history of the genes involved in the gliding machinery is complicated by multiple duplication events, sometime followed by gene losses, which occurred in Myxococcales and Bdellovibrionales but also in Gammaproteobacteria (e. g. two Marinobacter, Teredinibacter turnerae and Saccharophagus degradans) and Fibrobacteres (Figure 8A and Figure S1). As a result, the gene clusters are sometimes present in several copies in the genomes of some species (i. e. the G3, G4, G5 and M2 clusters in M. xanthus, Figure 1B). Interestingly, none of these copies can substitute for the motility functions of the G1, G2 and M1 genes suggesting that duplications were associated with the emergence of novel functions (see below). The data strongly suggest that an ancestral cluster of genes containing GltD, E, G, C and AglR, Q, S (Group B genes) evolved into a motility machinery after sequential recruitment of new components, namely GltF, H, I, J, A B and K (Group A genes). Obviously, ancient genetic linkages were lost during the evolution of the gliding machinery, explaining why it has not been previously identified and precluding the rapid identification of all the motility genes. The Agl/Glt complex is likely the gliding machinery because: (i), individual mutations of all the aglRQS [13] and glt genes resulted in complete motility defects and were not suppressed by a second-site frz mutation. (ii), GltD- and GltF-mCherry fusions showed similar localization patterns and localized to fixed FACs like the AglRQS proteins [13] (iii) GltD localization depended on GltF, G and H and, (iv) AglR interacted with GltG in a bacterial two-hybrid study and the localization of GltD-mCherry depended on AglQ. We thus propose that mechanical work from the motor is transduced to the cell surface by the Glt complex through a specific interaction with GltG. Here, we have identified a minimal motility machinery gene set, and it cannot be excluded that more Glt proteins may emerge as functional and phylogenomic approaches will be continued. For example, interaction studies identified GltD, E, I, J to be part of a complex also containing CglB and AglW [18]. The functional relevance of these interactions still needs to be demonstrated. CglB is an outer membrane motility lipoprotein [28] that harbours a very restricted taxonomy distribution, being present only in Myxococcales genomes (except the four Anaeromyxobacter). CglB may be a surface-exposed components of the complex. However, AglW may not be specifically linked to motility because its genomic localization and its amino acid sequence indicate that it is a bona fide structural component of the Tol/Pal system (TolB). The discovery of the Agl/Glt machinery now provides a new framework to elucidate the gliding motility mechanism (Figure 8B). A low-resolution architecture of the apparatus may be suggested by the genetic/localization/interaction and bioinformatic results (Figure 8B). AglRQS and GltG may constitute an inner membrane platform (this study) linked to the MreB cytoskeleton via proteins such as AglZ and MglA [15] on the cytosolic side and anchored to the substratum by a GltA-K complex in the cell envelope (Figure 8B). Nan et al. [14] proposed that the motility complex does not traverse the peptidoglycan layer but rather deforms it, generating transverse waves propagating down the axis of the cell. While this is plausible, the finding that GltH (and also potentially GltK and GltA based on bioinformatics predictions) is a critical outer-membrane component of the machinery rather argues that the motility complex is continuous through the cell envelope and contacts the cell exterior directly. More work will be required to understand the individual functions of the Glt proteins but the identification of the machinery gene set now opens investigations to understand the motility mechanism as a whole. Elucidating the motility mechanism may be greatly facilitated by functional studies of the core complex (aglR, Q, S and gltD, E, G, C). The conservation and genetic linkage of these genes in gammaproteobacterial genomes suggest that they encode a functional protein complex of unknown function in these bacteria. It is unlikely that the core complex drives motility on its own because gliding is not documented in most bacteria where it is found and our study shows that the corresponding genes are not sufficient to drive motility. Based on this later observation, we propose that additional functional blocks (such as Group A genes) have been added sequentially to the original protein complex to convert it into a motility machinery (Figure 8A). What are these building blocks and how many of them remain to be discovered? A recent study unambiguously showed that Bdellovibrio bacteriovorus is a bona fide glider [29]. While we cannot definitively rule out the independent emergence of gliding motility in this bacterium, we consider it unlikely: the Bdellovibrio genome contains four sets of expanded core complex suggesting that the Bdellovibrionales and Myxococcales gliding apparati are linked evolutionarily. Gliding motility may thus have emerged quite early in the ancestor of the Myxococcales and the Bdellovibrionales. The absence of homologues of GltH, I, J and K, all essential gliding proteins in Myxococcus, in the Bdellovibrio genome suggests that there are species specific requirements for gliding motility (Figure S1). Bdellovibrio cells are unusually small (less than 1 µm in length and 0. 5 in diameter vs > µm in length and 1 µm in diameter for Myxococcus), which could explain some structural differences between gliding apparati. Based on the phylogenetic analysis of the agl components, the genes composing the Bd0828-0838 locus appear more closely related to aglRQS and may therefore constitute the best candidate to encode the Bdellovibrio gliding apparatus (Figure 7B). The Bd0828-0838 cluster also contains many homologues of M. xanthus gliding genes (with the exception of gltH, I, J and K, Figure 7A and Figure S1). Based on the Bdellovibrio example, it is tempting to speculate that any bacterium containing AglR, Q, S, GltD, E, F, G, C, A and B is a potential glider. This is for instance the case of Myxococcus close relatives, Stigmatella aurantiaca and the four Anaeromyxobacter species (Figure S1). Finally, the M. xanthus gliding machinery is not conserved in bacteria belonging in other phyla (e. g. Bacteroidetes or Cyanobacteria), confirming that gliding motility evolved several times independently in Bacteria, as suggested by Jarrell and McBride [8]. The presence of multiple copies of the G1, G2 and M1 clusters in Myxococcales (e. g. G3, G4, G5 and M2, in M. xanthus Figure 1B) likely results from duplication events. These duplications may be linked to the whole genome duplication event that occurred in the ancestor of the Myxococcales [30] and/or resulted from punctual gene-duplications during differentiation of the terminal branch of the Deltaproteobacteria. Duplications provide the raw material for the evolution of new gene functions [31], [32] and, for example, several regulation networks may have emerged this way in Myxococcus [30]. This study shows that the G3, G4, G5 and M2 clusters cannot compensate disruptions in the glt and aglRQS genes, already suggesting that they encode distinct functions. To further test this, we generated polar mutations in all the gltD homologues (MXAN_1922, G4; MXAN_1327, G5 and MXAN_3374, G3) and a deletion in MXAN_3004 (M2), the aglQ homologue [13]. None of these mutations impacted motility at any appreciable level, (Figure S5 and [13]). If the function of the G4, G5 and M2 regions is unknown, the G3 region was recently shown to be critical for sporulation and named nfs (necessary for sporulation, [33]). As expected, our nfsD (MXAN_3374) mutant failed to mature spores (data not shown). The nfsA-H genes are clustered in a single genomic region containing close homologues of G1 and G2 region genes (with the exception of GltI, J and K). Strikingly, the short evolutionary distances separating the nfs and glt genes in individual gene trees and in the glt supermatrix indicate that the nfsA-H genes are in fact the closest homologues of the glt genes (Figure 7 and Figure S4). Thus, the Glt and Nfs systems are a clear example of exquisite machinery specialization: in these cases, the ancestral core machinery has terminally differentiated to drive sporulation or gliding motility. In absence of more mechanistic insights, it is not clear which of the two processes is the most recent but this finding points to unsuspected similarities in these two distinct morphological processes. Comparative molecular analysis of the nsf and glt systems should be powerful to understand how these machineries function and how they can be specialized to enforce sporulation or gliding. In summary, the mechanism of gliding motility has remained mysterious despite three decades of research. A converging array of evidence now shows that motility is not propelled by slime secretion but results from PMF-energized trans-envelope complexes periodically distributed along the cell body (this study and [12], [13], [18]). However, how force is transduced from the AglRQS motor to the Glt proteins through the entire cell envelope and ultimately how that translates into motion, remains to be elucidated. The identification of the components of the gliding machinery now paves the way to address these questions. An immediate goal will be to characterize the motility machinery in an exhaustive manner, which we should be able to resolve combining bioinformatics, genetics and cell biology. In addition, the M. xanthus genome contains several gene clusters deriving from the ancestral core complex, but these copies are not functionally redundant and even specify non-motility related functions (i. e. the sporulation nfs system). Thus, Glt-like systems are remarkably linked to two fundamental processes of the Myxococcus life cycle and their acquisition may thus have been critical to the recent diversification of the Deltaproteobacteria. In the future, comparative analysis in M. xanthus, but also in the Delta and Gammaproteobacteria should be a powerful approach to elucidate the pathways that led to the evolution and diversification of complex bacterial envelope machineries. Primers and plasmids are listed in Tables S4 and S5. See Tables S6 and S7 for strains and their mode of construction. M. xanthus strains were grown at 32°C in CYE rich media as previously described [23]. Plasmids were introduced in M. xanthus by electroporation. Mutants and transformants were obtained by homologous recombination based on a previously reported method. Complementation of gltG and expression of GltF-mCherry were obtained after ectopic integration of the genes of interest at the Mx8-phage attachment site in appropriate deletion backgrounds (Table S6). For phenotypic assays, cells (10 µl), at a concentration of 4×109 cfu ml−1, were spotted on CYE plates containing agar concentrations of 0. 5% or 1. 5%, incubated at 32°C and photographed after 48 h with an Olympus SZ61 binocular or a Nikon Eclipse (model TE2000E) microscope (4x objective). RNA from appropriate strains was extracted using a standard RNA purification kit (Promega). One microgram of total RNA was reverse-transcribed following the recommendations of the iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was then diluted (1/16), and 5 µl were used for the quantitative reverse transcription-PCR (q-RT-PCR) reaction. This step was performed on a Mastercycler ep realplex instrument (Eppendorf), using the SYBR Premix Ex Taq (Perfect Real Time) PCR kit (Takara Bio Group, Japan) according to manufacturer instructions in a final volume of 20 µl. Specific primers used for the reactions are described in Table S4. Melting curves were systematically analyzed to control for the specificity of the PCR reactions. The relative units were calculated from a standard curve plotting four different dilutions (1/80,1/400,1/2,000, and 1/10,000) against the PCR cycle number at which the measured fluorescence intensity reached the threshold (CT), corresponding to ∼10 times the standard deviation and thus significantly above the noise band of the baseline. Western blotting was performed as previously described [34] with 1/1000-1/5000 dilutions of polyclonal α-GltD, α-GltE, α-GltG, α-GltH (all raised for this study) and α-mCherry[13], α-PilC, α-PilQ [35] and α-FrzS [36]. Membrane Fractions and OMVs were purified from exponentially-growing cell cultures. Vegetative cells of M. xanthus were grown in CYE medium to an OD600 nm = 0. 7. For membrane fractions, cells were harvested by centrifugation at 8. 000 rpm for 10 min at RT, resuspended in 50 mM Tris-HCl pH 7. 6 and lysed by sonication. Cell debris were removed by low speed centrifugation (14000 rpm). The supernatants were then centrifuged at 45,000 g for 1 hr at 4°C. The resulting supernatants are enriched in soluble proteins. Pellets containing the crude envelope fractions (Inner and outer membrane) were resuspended in 50 mM Tris-HCl pH 7. 6 and homogenized. The quality of the fractionation procedure was tested with antibodies to FrzS (soluble protein [36]) and PilC (inner membrane protein [35]). Standard procedures to separate the inner membrane from the outer membrane using sucrose density gradients [37] did not successfully separate the two membranes. Detergent-based methods have been used successfully in Myxococcus, however in our case we could not prevent rapid degradation of the Glt proteins during the separation process [35]. OMVs are largely derived from the outer membranes, which was recently confirmed by proteomic analysis of the Myxococcus outer-membranes [38]. Thus, to test which Glt proteins are in the outer membranes we tested their presence in purified vesicules. For OMVs purification, cells and were discarded by centrifugation (8. 000 rpm for 10 min at RT) and the culture supernatant was used for the isolation of vesicles. Culture supernatants (1 L) were passed through a 0. 2 µm vacuum filter (Millipore). The resulting filtrate was centrifuged at 125 000× g for 2 h at 4°C to recover membrane vesicles. The supernatant was carefully removed and the vesicle pellet was resuspended in 50 mM Tris-HCl pH 7. 6 and centrifuged at 180 000× g for 2 h at 4°C to concentrate and wash vesicles. The quality of the purification procedure was tested by electron microscopy (not shown) and antibodies to PilQ (outer membrane protein [35]) and PilC (inner membrane protein [35]). Bacterial two-hybrid experiments, plate and ß-Galactosidase assays were performed as previously described [26] and as recommended by the manufacturer (Euromedex). Time lapse experiments were performed as previously described [39]. Microscopic analysis was performed using an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon, France). The microscope is equipped with “The Perfect Focus System” (PFS) that automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times, in spite of any mechanical or thermal perturbations. Images were recorded with a CoolSNAP HQ 2 (Roper Scientific, Roper Scientific SARL, France) and a 40x/0. 75 DLL “Plan-Apochromat” or a 100x/1. 4 DLL objective. All fluorescence images were acquired with appropriate filters with a minimal exposure time to minimize bleaching and phototoxicity effects. Cell tracking was performed automatically using a previously described macro under the METAMORPH software (Molecular devices), when appropriate, manual measurements were also performed to correct tracking errors with tools built into the software. Typically, the images were equalized, straightened and overlaid under both ImageJ 1. 40 g (National Institute of Health, USA) and METAMORPH. The genetic screens of Youderian et al. ([16]) and Yu and Kaiser ([17]) allowed the identification of 35 and 23 potential gliding motility genes, respectively (Table S1). The function of the corresponding proteins was investigated using sequence similarity based approaches against the non-redundant (nr) database at the National Center for Biotechnology Information (NCBI, http: //www. ncbi. nlm. nih. gov/) and Pfam (release 24. 0) databases ([40]). The presence and location of signal peptide signal cleavage sites and of transmembrane helix were the predicted using the signalP 3. 0 server ([41]) and TMHMM server v. 2. 0 ([42]). Finally, the location and the neighbourhood of each gene in the chromosome of M. xanthus DK 1622 were investigated using the complete genome sequence available at the NCBI ([30]; CP000113). Homologues of each M. xanthus candidate protein were retrieved from a local database containing all complete prokaryotic proteomes available at the NCBI (April 8,2010) using blastp with default parameters [43]. We also include in our analyses the genome sequences of Stigmatella auriantiaca DW4/3-1 and of Plesiocystis pacifica SIR-1 that came out in November 2010 and whose assembly is ongoing, respectively, both genomes being available at the NCBI. Importantly, the distinction between homologous and non-homologous sequences was assessed by visual inspection of each blastp outputs (no arbitrary cut-off on the E-value or score). To ensure the exhaustive sampling of homologues, iterative blastp queries were performed using homologues identified at each step as new seeds. PSI-BLAST queries were also used in order to recover very divergent homologues [43]. The absence of homologue in any complete proteome was systematically verified by tblastn queries against the nucleotide sequence of the corresponding genome. For each candidate protein, the retrieved homologues were gathered in a dataset. The corresponding sequences were aligned using the ClustalW2 program (Default parameters, [44]). Each alignment was visually inspected and manually refined when necessary using the ED program from the MUST package [45]. Regions where the homology between amino acid positions was doubtful were manually removed using NET from the MUST package. Working on complete genomes may introduce major biases due to the taxonomic sampling of available complete genomes. Accordingly, for each candidate protein a second set of datasets based on homologues retrieved from the non-redundant (nr) protein database (the most exhaustive public database) at the NCBI was assembled. The taxonomic distribution and the phylogeny of homologues retrieved from either the nr database or from complete genomes showed similar patterns (data not shown). Thus, our analyses based on complete genomes are representative and reflect the taxonomic distribution of known homologues. Accordingly, only the results based on complete genomes will be presented in the results section. The preliminary phylogenetic analyses of the candidate proteins allowed the identification of closest relatives of M. xanthus sequences. For each protein these homologues were gathered in a second dataset, the sequences were aligned and the resulting alignment was manually refined and cleaned like previously described. For the phylogenetic analyses of some of these datasets, we were removed some divergent sequences that can bias the phylogenetic reconstruction. One approach to improve the resolution of the phylogenetic trees is to combine the genes that share a common evolutionary history in a single large alignment (also called supermatrix), [46]–[48]. Among the seven genes composing the Group B, gltD, E and G homologues are always clustered together in genomes and their individual phylogenies are very similar. Thus, these genes likely share a similar evolutionary history and can be used to build a supermatrix. For similar reasons, we combined the aglR, Q and S alignments in a second supermatrix. In contrast, gltC could neither be included in the glt nor in the agl supermatrix because it does not cluster physically with the corresponding genes in most Deltaproteobacteria. The Glt and Agl supermatrices were manually constructed by combining the cleaned alignments of GltDEG and AglQRS, respectively. When more than one homologue of these genes were present in a given genome, the genes were combined according to their physical linkage on the chromosome. For instance in the case of AglQRS, in M. xanthus the genes were combined as following: (i) MXAN_6860, _6861, _6862 and (ii) MXAN_3005, _3004, _3003. For each individual and concatenated alignment, both Maximum likelihood (ML) and Bayesian phylogenetic trees were computed. ML analyses were run using PHYML version 3. 0 with the Le and Gascuel (LG) model (amino acid frequencies estimated from the dataset) and a gamma distribution (4 discrete categories of sites and an estimated alpha parameter) to take into account evolutionary rate variations across sites [49]. The robustness of each branch was estimated by the non-parametric bootstrap procedure implemented in PhyML (100 replicates of the original dataset with the same parameters). Additional ML analyses were performed using TreeFinder with the same parameters [50]. Bayesian analyses were performed using MrBayes [51] with a mixed model of amino acid substitution including a gamma distribution (4 discrete categories) and an estimated proportion of invariant sites. MrBayes was run with four chains for 1 million generations and trees were sampled every 100 generations. To construct the consensus tree, the first 1500 trees were discarded as “burnin”.

Title: Emergence and Modular Evolution of a Novel Motility Machinery in Bacteria Summary: Motility over solid surfaces (gliding) is an important bacterial mechanism that allows complex social behaviours and pathogenesis. Conflicting models have been suggested to explain this locomotion in the deltaproteobacterium Myxococcus xanthus: propulsion by polymer secretion at the rear of the cells as opposed to energized nano-machines distributed along the cell body. However, in absence of characterized molecular machinery, the exact mechanism of gliding could not be resolved despite several decades of research. In this study, using a combination of experimental and computational approaches, we showed for the first time that the motility machinery is composed of large macromolecular assemblies periodically distributed along the cell envelope. Furthermore, the data suggest that the motility machinery derived from an ancient gene cluster also found in several non-gliding bacterial lineages. Intriguingly, we find that most of the components of the gliding machinery are closely related to a sporulation system, suggesting unsuspected links between these two apparently distinct biological processes. Our findings now pave the way for the first molecular studies of a long mysterious motility mechanism.

lay_plos

en

Summarize: [0001] This application is a Continuation of, and claims priority under 35 U.S.C. §120 to, U.S. patent application Ser. No. 13/443,154, filed Apr. 10, 2012, which is a Divisional of, and claimed priority under 35 U.S.C. §120 to, U.S. patent application Ser. No. 12/494,843, filed Jun. 30, 2009, now U.S. Pat. No. 8,191,553, which claimed priority under 35 U.S.C. §119 to U.S. Provisional application No. 61/076,757, filed 30 Jun. 2008, entitled “Jaw Thrust Device and Method”, the entireties of which are incorporated by reference herein. BACKGROUND [0002] 1. Field of Endeavor [0003] The present invention relates to devices, systems, and processes useful in patient airway maintenance, and more specifically to devices and methods that perform a jaw thrust. [0004] 2. Brief Description of the Related Art [0005] The jaw thrust is a technique used on patients in a supine position to open the patient&#39;s trachea (airway), which has become blocked by the backward movement of the lower jaw (mandible) relative to the rest of the patient&#39;s skull, which in turn can cause the patient&#39;s airway to be blocked. The practitioner typically uses their thumbs to physically push the posterior (back) aspects of the mandible forward and into a position in which the airway is no longer blocked. When the mandible is displaced forward, it pulls the tongue forward and prevents it from blocking (occluding) the entrance to the trachea, helping to ensure a patent (securely unobstructed) airway. [0006] Numerous devices have in the past been proposed for assisting in this procedure, which have been met with limited acceptance. Among the difficulties with prior devices is that many secure the patient&#39;s head to the device and/or to the surface (e.g., an operating table) on which the patient is positioned, which limits the medical practitioner&#39;s ability to perform procedures on the patient&#39;s head and neck. Additionally, many prior devices address only the relative position of the mandible and the associated position of the patient&#39;s tongue, and do not address other portions of the patient&#39;s airway. SUMMARY [0007] According to a first aspect of the invention, a jaw thrust device comprises a frame having a pair of upstanding arms with free ends, two jaw pads and two adjustment mechanisms, each of the adjustment mechanisms mounts a respective one of the jaw pads to a respective one of the free ends, and a neck pad positioned on the frame and between the two jaw pads, the neck pad having first and second ends, the frame holding the neck pad first end such that a portion of the frame and the two neck pad ends together form a triangle shape with the neck pad first end at the triangle apex. [0008] According to another aspect of the present invention, a jaw thrust device comprises a frame having a pair of upstanding arms with free ends two L-shaped jaw pads and two adjustment mechanisms, a jaw pad mounted to each arm free end via one adjustment mechanism, and a neck pad positioned on the frame and between the two jaw pads, the frame holding the neck pad. [0009] According to yet another aspect of the present invention, a method for opening a trachea of a patient comprises hyperextending a neck of a patient, and displacing a mandible of the patient anteriorly. [0010] Still other aspects, features, and attendant advantages of the present invention will become apparent to those skilled in the art from a reading of the following detailed description of embodiments constructed in accordance therewith, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0011] The invention of the present application will now be described in more detail with reference to exemplary embodiments of the apparatus and method, given only by way of example, and with reference to the accompanying drawings, in which: [0012] FIG. 1 illustrates an exemplary use of a device in accordance with the present invention to maintain the patency of a patient&#39;s airway; [0013] FIG. 2 illustrates a perspective view of a first exemplary embodiment of a device in accordance with the present invention; [0014] FIG. 3 illustrates a perspective view of a second exemplary embodiment of a device in accordance with the present invention; [0015] FIG. 4 illustrates a perspective view of a third exemplary embodiment of a device in accordance with the present invention; [0016] FIG. 5 illustrates a top, right, rear perspective view of a fourth exemplary embodiment of a device in accordance with the present invention; [0017] FIG. 6 illustrates a top, left, rear perspective view of the embodiment of FIG. 5 ; [0018] FIG. 7 illustrates a bottom plan view of the embodiment of FIG. 5 ; [0019] FIG. 8 illustrates a top plan view of the embodiment of FIG. 5 ; [0020] FIG. 9 illustrates a top, right, front perspective view of the embodiment of FIG. 5 ; [0021] FIG. 10 illustrates a schematic representation of a bottom view of a fifth exemplary embodiment of a device in accordance with the present invention; and [0022] FIG. 11 illustrates a schematic representation of a side view of the embodiment of right side of the embodiment of FIG. 10. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS [0023] Referring to the drawing figures, like reference numerals designate identical or corresponding elements throughout the several figures. [0024] With reference to FIG. 1, an exemplary device 1 embodying principles of the present invention is illustrated. In FIG. 1, the device 1 is schematically represented. The device 1 can include a base member 2 that can have a generally triangular shape, as see in side profile or sagittal view. The base member 2 can be oriented relative to a support surface S such that an apex 3 of the triangular shaped base member 2 can be positioned at height relative to the support surface S and at least one side 5 of the base member 2 can be inclined relative to the support surface S. When an adult human patient, in a supine position, is positioned on the support surface S with the back of their neck N resting on the base member 2, the angle of the side 5 of the base member 2 and the height of the apex of the base member 2 can cause hyperextension of the patient&#39;s neck N. The amount of hyperextension of the patient&#39;s neck N can be an appropriate amount sufficient to minimize occlusion of the patient&#39;s trachea that may be caused by the patient&#39;s internal anatomy. [0025] In order to maintain patency of the patient&#39;s trachea once the neck N has been properly hyperextended, the device 1 can include a jaw support 4 secured to the base 2. (Only the right side of the jaw support 4 is viewable in FIG. 4 —see FIG. 2, for example, for further illustration of both sides of the exemplary jaw support.) The jaw support 4 can engage and support both sides of the patient&#39;s mandible M at a position relative to the apex 3 and the support surface S. In particular, the jaw support 4 can be oriented relative to the patient such that the jaw support 4 engages the ramus portion Ron each side of the patient&#39;s mandible M. Thus, the jaw support 4 can prevent the patient&#39;s mandible M from slipping backwards when the patient&#39;s neck N is hyperextended, by the cooperation of inclined side 5 with other features of the base 2, as will be described below. [0026] A first exemplary embodiment of the device 1 schematically represented in FIG. 1 is shown in FIG. 2. FIG. 2 illustrates a device 10 that can include a base 12 and a jaw support 14 that can engage and support a patient&#39;s neck and mandible, respectively, as described above. [0027] The base 12 can include a frame 16 and a neck pad 18. The frame 16 can cooperate with the neck pad to hyperextend the patient&#39;s neck an appropriate amount. The jaw support 14 can be connected to the frame 16, as will be described in detail below. The neck pad 18 can be merely placed onto the frame 16 or the neck pad 18 can be positively connected to the frame 16. If the neck pad 18 positively connected to the frame 16, then the neck pad 18 can either be removably connected or permanently connected to the frame 16. [0028] The frame 16 can be configured from a material and with a geometry sufficient to provide an unyielding support of a patent&#39;s neck when the patient is lying supine on a support surface. By way of example, the frame 16 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. [0029] The frame 16 can form at least a portion of the base 12 and can include a polygonal stand 20, a first pad support 22 (underneath the neck pad 18 ) and a second pad support 24. The pad supports 22, 24 can extend from opposite ends of the polygonal stand 20. The pad supports 22, 24 can be configured from a material and with a geometry sufficient to provide an unyielding support of a patent&#39;s neck when the patient is lying supine on a support surface. By way of example, the pad supports 22, 24 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. [0030] The polygonal stand 20 and the pad supports 22, 24 can be integrally formed to define the frame 16 as a single, homogenous component. In this exemplary embodiment, the polygonal stand 20 and the pad supports 22, 24 can be formed by bending the stock into the desired shape. In another exemplary embodiment, the pad supports 22, 24 can be formed as separate components and connected to the frame 20 by any known fastening devices. [0031] The polygonal stand 20 can include a central segment 26, a first lateral segment 28, a second lateral segment 30, a first connector segment 32 and a second connector segment 34. The first lateral segment 28 can extend from one end of the central segment 26 at an obtuse angle. The second lateral segment 30 can extend from the other end of the central segment 26 at an obtuse angle and symmetrically with respect to the first lateral segment 28. The first connector segment 32 can extend from the first lateral segment 28 to the first pad support 22. The second connector segment 34 can extend from the second lateral segment 30 to the second pad support 24. The first and second connector segments 32, 34 can extend at an obtuse angle relative to the respective first and second lateral segments 28, 30, respectively. The first and second connector segments 32, 34 can extend from the first and second pad supports 22, 24 and any angle deemed sufficient to provide an appropriate hyperextension of the patient&#39;s neck. The junction between the first connector segment 32 and the first pad support 22 and the junction between the second connector segment 34 and the second pad support 24 can be arcuate. In another exemplary embodiment, this junction can be angular. [0032] Each of the segments 26, 28, 30, 32, 34 can be integrally formed to define the polygonal stand 20 as a single, homogenous component. Or, each of the segments 26, 28, 30, 32, 34 can be formed as separate components and connected to each other by any known fastening devices to form the polygonal stand 20. [0033] The neck pad 18 can be permanently secured or removably secured to the first and second pad supports 22, 24 in any known manner. If the neck pad 18 is removably connected to the pad supports 22, 24, then the neck pad 18 can be cleaned and reused, or the used neck pad 18 can be disposed and replaced with a new neck pad 18 after each use. The neck pad 18 can span the frame 16 from the first pad support 22 to the second pad support 24. [0034] The neck pad 18 can include a first end 36, a second end 38, a first side 40, a second side 42, a backing 44, and a cushion 46. The cushion 46 can include an engagement surface 48 on a side of the cushion opposite to the backing 44. The backing 44 and the engagement surface 48 can extend from and between the first and second ends 36, 38 and the first and second sides 40, 42. The cushion 46 can be formed as a separate component from the backing 44 and subsequently affixed, permanently or removably, to the backing 44 in any known manner. The backing 44 can have a rigidity sufficient to support the patient&#39;s neck in an appropriate hyperextended position above the support surface without substantial deformation to the backing 44. Any known material providing sufficient rigidity can be used to form the backing 44. The cushion 46 can be formed from any known soft, resilient material used for cushions. One example of the cushion material can be a foam material. [0035] The first and second sides 40, 42 of the neck pad 18 can be positioned adjacent to the respective first and second pad supports 22, 24. The first and second sides 40, 42 can extend substantially parallel to the first and second pad supports 22, 24 and can extend beyond the pad supports 22, 24. [0036] The backing 44 can rest against the pad supports 22, 24 without a positive connection thereto. Or, the backing 44 can be positively secured to the pad supports 22, 24 in any known manner. Any positive connection between the backing 44 and the pad supports 22, 24 can be either a removable connection or a permanent connection. [0037] The engagement surface 48 of the neck pad 18 can be generally concave in its extent from the first side 40 to the second side 42. The engagement surface 48 can have a generally convex curvature along a central portion extending from the first end 36 to the second end 38. This compound curvature of the engagement surface 48 can provide stable support in the posterior, inferior, superior, and lateral directions for the patient&#39;s neck when the patient&#39;s neck is appropriately hyperextended. The backing 44 can mirror the geometry of the engagement surface 48 or the backing 44 can be configured in any suitable geometry. [0038] The first end 36 of the neck pad 18 can be positioned adjacent the junction of the pad supports 22, 24 with their respective connector segments 32, 34. The first end 36 of the neck pad 18 can be spaced from the central segment 26 and the first and second lateral segments 28, 30 of the polygonal stand 20. [0039] The second end 38 of the neck pad 18 can be spaced from the first and second pad supports 22, 24. The second end 38 can curve as it extends from the first side 40 to the second side 42. Alternatively, the second end 38 of the neck pad 18 can be segmented in a manner that corresponds to the central segment 26 and the lateral segments 28, 30 of the polygonal stand 20. [0040] In use, a portion of the second end 38 of the neck pad 18 and a portion of the polygonal stand 20 can be placed on and engage the support surface upon which the patient lies. When placed on the support surface, the engaging portions of the second end 38 and the polygonal stand 20 can define the vertices of a triangular shape, when viewed in profile or sagittal view. The apex of this triangular shape relative to the support surface can lie adjacent the first end 36 of the neck pad 18. The apex of the triangular shape can lie adjacent the junctions of the first and second connector segments 32, 34 with the first and second pad supports 22, 24. [0041] The lengths of the lateral segments 28, 30 and the connector segments 32, 34 of the polygonal stand 20 and the lengths of the sides 40, 42 of the neck pad 18 can be chosen along with the angle defined between the first connector segment 32 and the first pad support 22 and the angle defined between the second connector segment 34 and the second pad support 24 such that the base 12 can elevate the patient&#39;s neck above the support surface an amount to appropriately hyperextend the patient&#39;s neck. Thus, the frame 16 and the neck pad 18 can cooperate to stably support the neck of a supine patient in an appropriate hyperextended position without the need to fix the device 10 to the support surface. [0042] Routinely, a patient can lie on the support surface in a supine position with both shoulders resting against the support surface. When the engagement surface 48 of the neck pad 18 receives the patient&#39;s neck in this supine position, the central segment 26 of the polygonal stand and a central portion of the second end 38 of the neck pad 18 can engage the support surface. In this position, the lateral segments 28, 30 of the polygonal stand 20 can extend away from the support surface. The length of the central segment 26 can be any length sufficient to ensure stable support of the patient&#39;s neck while the central segment 26 engages the support surface without fixing the device to the support surface. [0043] However, it may be advantageous to slightly roll the patient toward one side such that the opposite shoulder is slightly spaced above the supporting surface. The device 10 can also support a patient&#39;s neck in an appropriate hyperextended position when the patient is slightly rolled toward one side while lying on the support surface. The first and second lateral segments 28, 30 can define beveled corners of the frame 16 that can permit rotation of the base 12 in unison with the patient as the patient is rolled slightly toward one side. [0044] In an instance where the patient is rolled slightly on the support surface toward the patient&#39;s left side, the device 10 can be reoriented relative to the support surface in unison with the patient because the device 10 is not fixed to the support surface. When so reoriented, the first lateral segment 28 of the polygonal stand 20 can engage the support surface and the central segment 26 and second lateral segment 30 can be spaced above the support surface. [0045] In an instance where the patient is rolled slightly on the support surface toward the patient&#39;s right side, the device 10 can be reoriented relative to the support surface in unison with the patient because the device 10 is not fixed to the support surface. When so reoriented, the second lateral segment 30 can engage the support surface and the central segment 26 and first lateral segment 26 can be spaced above the support surface. [0046] As with the central segment 26, the length of the lateral segments 28, 30 can be any length sufficient to ensure stable support of the patient&#39;s neck while the appropriate one of the lateral segments 28, 30 engages the support surface without fixing the device 10 to the support surface. Thus, it is not necessary to fix the device 10 to the support surface when the device is in any of the above-mentioned orientations relative to the support surface. However, the device 10 can be removably fixed relative to the support surface in any known manner, as desired. [0047] In addition to providing stable support of the patient&#39;s neck, the concave curvature of the neck pad 18 can accommodate the multiple orientations of the polygonal stand 20 on the support surface. Similarly, the configuration (arcuate or segmented) of the second end 38 of the neck pad can also accommodate the multiple orientations of the polygonal stand 20 on the support surface. [0048] After the patient&#39;s neck has been appropriately hyperextended, the jaw support 14 can be used to position the patient&#39;s jaw relative to the neck such that occlusion of the patient&#39;s trachea by the patient&#39;s internal anatomy can be minimized. The jaw support 14 can include first and second mounting arms 50, 52 and first and second jaw pad assemblies 54, 56 engaging the first and second mounting arms 50, 52, respectively. [0049] The first and second mounting arms 50, 52 can extend from the first and second pad supports 22, 24, respectively, at positions external to the first and second sides 40, 42, respectively, of the neck pad 18. Each of the mounting arms 50, 52 can include a first end connected to the respective pad support 22, 24 and a free end spaced from both the pad supports 22, 24. The first ends of the mounting arms 50, 52 can be connected to the first and second pad supports 22, 24 in any manner known in the art suitable to ensure a substantially rigid relationship therebetween. [0050] By way of example, the first and second mounting arms 50, 52 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. If the first and second mounting arms 50, 52 are fabricated from the same material stock as the frame 16, then the first and second mounting arms 50, 52 can be integrally formed as a single, homogenous component with the frame 16. [0051] The first and second mounting arms 50, 52 can extend away from the engagement surface 48 to a height sufficient to permit an adjustment range for the respective one of the jaw pad assemblies 54, 56 while not impeding placement of either of the lateral segments 28, 30 onto the support surface. Details of the adjustability offered by the mounting arms 50, 52 will be discussed below. [0052] The first and second mounting arm 50, 52 can be arcuate and can be aligned about a common arc. This geometry can promote contact with the patient&#39;s mandible when the neck pad 18 receives the patient&#39;s neck. Simultaneously with promotion of contact with the mandible, this geometry imparts a thrust force onto the patient&#39;s mandible that can push inward (medially) on the mandible. Thus, the jaw support 14 can be self-seating and can resist slippage relative to the mandible. [0053] The curvature of the mounting arms 50, 52 can also be sufficient to allow clearance of the first and second mounting arms 50, 52 with the support surface when either of the lateral segments 28, 30 of the polygonal stand 20 engage the support surface, as discussed above. The curvature of the first and second mounting arms 50, 52 can also be set to ensure sufficient clearance of the patient&#39;s neck as it is moved into and out of contact with the engagement surface 48 of the neck pad 18. [0054] In another exemplary embodiment, the first and second mounting arms 50, 52 can be linear. In another exemplary embodiment, the mounting arms 50, 52 can include an arcuate portion connected to the respective pad support 22, 24 and a linear portion connected to the other end of the arcuate portion. [0055] The second jaw assembly 56 can be substantially identical to or substantially a mirror image of the first jaw assembly 54 and can operate substantially identically to the first jaw assembly 54. Accordingly, the following description of the jaw assemblies 54, 56 will be limited to the second jaw assembly 56. [0056] The second jaw assembly 56 can include a jaw pad 58, an adjustment post 60 and a connector assembly 62. The jaw pad 58 can be secured to one end of the adjustment post 60 in any known manner. The connector assembly 62 can be movably secured to the second mounting arm 52 in either direction indicated by the arrows A. The adjustment post 60 can be movably mounted to the connector assembly 62 in either direction indicated by the arrows B. [0057] The jaw pad 58 can include a deformable core (not shown) and a removable cover. The core can be made from a deformable material and can be secured to the adjustment post 60. The cover can enclose the core in part or in total. The cover can be made from a cloth material and can be removed from around the core for cleaning and reused or the cover can be removed, disposed and replaced with a new cover after each use. [0058] In another exemplary embodiment, the entire jaw pad 58 can be removably mounted to the adjustment post 60. In this exemplary embodiment, the jaw pad 58 can be formed as a single component as compared to a separate core and cover component. This single component jaw pad can be removed, cleaned, and reused. Or, this single component jaw pad can be removed and replaced with a new jaw pad after each use. [0059] The jaw pad 58 can have an engagement surface 64 that can contact the ramus portion of the patient&#39;s mandible when the patient is positioned on the device as described herein. In FIG. 2, the engagement surface 64 is best viewed on the jaw pad 58 associated with the first jaw pad assembly 54. FIG. 2 depicts the engagement surface 64 of the jaw pad 60 as a substantially planar surface. However, the engagement surface 64 of the jaw pad 60 can be configured in any geometry deemed appropriate for stable engagement with the patient&#39;s mandible. [0060] By way of example, the adjustment post 60 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. [0061] The connector assembly 62 can include a housing 63 that can include respective through-holes for the second mounting arm 52 and the adjustment post 60. The connector assembly 62 can slide along the second mounting arm 52 via the respective through-hole and the adjustment post 60 can slide within the respective through-hole of the connector assembly 62. Thus, the first and second jaw pad assemblies 54, 56 can be adjusted to best fit the anatomy of each patient. [0062] The through-hole of the housing 63 that receives the adjustment post 60 can be oriented relative to the base 12 such that the adjustment post extends inwardly and upwardly (supero-medially) over the neck pad 18. The adjustment post 60 can slide along its length within the housing 63 so that the jaw pad 58 can engage and subsequently displace the patient&#39;s mandible forward (anterior) by an amount sufficient to minimize occlusion of the trachea by the patient&#39;s internal anatomy. [0063] Once adjusted to the desired position, the first and second jaw pad assemblies 54, 56 can be fixed in position by the respective connecter assembly 62. By way of example, the connector assembly 62 can include a ratchet assembly (not shown) associated with each of the through-holes. The ratchet assembly can be any known ratchet assembly. [0064] Alternatively, the connector assembly 62 can utilize other fastening devices to secure the second jaw pad assembly 56 relative to the second mounting arm 52 and the adjustment post 60 relative to the connector assembly 62. Examples of these alternate fastening devices can include a set screw, a cam-lever assembly, a ball and detent assembly, etc. In another exemplary embodiment, the through-holes can be dimensioned to provide a friction fit with the mounting arm 52 and the adjustment post 60. [0065] As a independent feature of the jaw pad assemblies 54, 56, the mounting arms 50, 52, adjustment posts 60 and the respective through-holes can have complimentary geometries that can prevent, or at least impede relative rotation between the through holes and the respective one of the mounting arms 50, 52 and the adjustment posts 60. An exemplary geometry can be a square cross-sectional geometry. [0066] Thus, the device 10 can position a patient&#39;s neck in an appropriate hyperextended position while the patient lies on the support surface. The device 10 can also be adjusted for each patient so that the patency of the trachea can be maintained after the neck has been hyperextended by an appropriate amount. Additionally, the device 10 can accommodate multiple orientations of the patient relative to the support surface while providing and maintaining the appropriate hyperextension of the patient&#39;s neck. [0067] FIG. 3 illustrates a second embodiment of the device 1 schematically represent in FIG. 1. In this embodiment, a device 70 can be substantially identical to the device 10 of FIG. 2, except as noted below. Accordingly, substantially identical features of the device 70 are denoted by the same references numerals as used for the device 10 of a FIG. 2. [0068] The device 70 can include a neck pad 72 that can be configured to internally receive the pad supports (not visible—see pad supports 22, 24 of FIG. 2, for example) of the frame 16 and a portion of the mounting arms 50, 52. In particular, the pad supports can extend within the neck pad 72 between the backing 74 and the cushion 76 of the neck pad 72. Thus, the neck pad 72 can be connected to the frame 16. [0069] FIG. 4 illustrates a third embodiment of the device 1 schematically represent in FIG. 1. In this embodiment, a device 80 can be substantially identical to the device 10 of FIG. 2, with any exceptions and/or modifications noted below. Accordingly, substantially identical features of the device 80 are denoted by the same references numerals as used for the device 10 of a FIG. 2. [0070] The device 80 can include a base 82. The base 82 can include a frame 84 and a neck pad 86. The neck pad 86 can include a backing 88 and a cushion 90. The backing 88 and the cushion 90 can include substantially all the features of the backing 44 and the cushion 46 described with reference to the device 10 of FIG. 2. In this exemplary embodiment, the backing 88 of the neck pad 86 can be integrally formed with the frame 84 to define a single, homogenous component. The cushion 90 of the neck pad 86 can be affixed to the backing 88 in any manner described above with reference to FIG. 2. [0071] The frame 82 can include a polygonal stand 92 that can be configured as a solid planar wall. The polygonal stand 92 can include a central edge segment 94, a first lateral edge segment 96, and a second lateral edge segment 98 that lie along the periphery of the polygonal stand 92. The first lateral edge segment 96 can extend from one end of the central edge segment 94 at an obtuse angle. The second lateral edge segment 98 can extend from the other end of the central edge segment 94 at an obtuse angle and symmetrically with respect to the first lateral edge segment 96. The device 80 can be oriented relative to a support surface with any one of the edge segments 94, 96, 98 in contact with the support surface and the remaining edge segments 94, 96, 98 spaced from the support surface in any manner described above with respect the segments 26, 28, 30 of the device 10 of FIG. 2. [0072] The polygonal stand 92 can include an upper edge segment 100, a first side edge segment 102, and a second side edge segment 104. The upper edge 100 can abut the first end 36 of the neck pad 86. The upper edge 100 can be arcuate with a curvature that can conform to the curvature of the first end 36 of the neck pad 86. [0073] The first side edge segment 102 can extend from the first lateral edge segment 96 to the upper edge segment 100. The first side edge 102 can abut the backing 88. The first side edge segment 102 can extend at an obtuse angle relative to the first lateral edge segment 96. The first side edge segment 102 can extend at an acute angle relative to the upper edge segment 100. [0074] The second side edge segment 104 can extend from the second lateral edge segment 98 to the upper edge segment 100. The second side edge segment 104 can abut the backing 88. The second side edge segment 104 can extend at an obtuse angle relative to the second lateral edge segment 98. The second side edge segment 104 can extend at an acute angle relative to the upper edge segment 100. [0075] The edge segments 94, 96, 98, 100, 102, 104 together can define the perimeter of the planar wall of the polygonal stand 92. [0076] The backing 88 can include a groove 107 formed in the surface of the backing 88 that abuts the cushion 90. The groove 107 appears as a convex ridge from the outside of the backing 88, as viewed in the orientation of FIG. 4. The groove 107 can extend from the first side 40 of the neck pad 18 to the second side 42 of the neck pad 90. [0077] The device 80 can include a jaw support 108. The jaw support 108 can include a C-shaped mounting post 109. By way of example, the mounting post 109 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. [0078] The mounting post 109 can be centered in the groove 107 such that first and second free ends 110, 112 can extend beyond the respective ends of the groove 107 and the sides 40, 42 of the neck pad 86. The free ends 110, 112 can extend away from the engagement surface 48 of the cushion 90. [0079] The depth of the groove 107 can be such that the mounting post 109 can lie flush with the surface of the backing 88 that abuts the cushion 90. [0080] In an alternate exemplary embodiment, the mounting post 109 can include two separate post sections. In this alternate exemplary embodiment, the groove 107 can include separate groove sections that receive a respective one of the post sections. These separate groove sections can extend from the respective sides 40, 42 of the neck pad 86 and terminate at respective position on the backing 88 intermediate the sides 40, 42. [0081] A further modification for the groove 107 can include a divider spanning the width of the groove 107 that can divide the groove into two sections. Each of these groove sections can receive a respective one of the post sections just described above. [0082] The jaw support 108 can include first and second jaw pad assemblies 54, 56. The jaw assemblies can be substantially identical in structure and operation to the jaw assemblies 54, 56 described above with reference to the device 10 of FIG. 2. The jaw assemblies 54, 56 can be movably mounted along the respective free ends 110, 112 of the mounting post 109. [0083] The frame 84 can further include a first reinforcing support (not visible in FIG. 4 ) and a second reinforcing support 106. The first reinforcing support can be mirror image of the second reinforcing support 106. Accordingly, further reference is made only to the second reinforcing support 106. [0084] The second reinforcing support 106 can extend from the polygonal stand 92 to the backing 88. The reinforcing support 106 can extend along at least a portion of second side edge segment 104. The second reinforcing support 106 can extend along the bottom surface of the backing 88 and can abut the convex ridge of the groove 107. The second reinforcing support 106 can include a plurality of ribs 114 arranged in a criss-cross pattern. Each of the ribs 114 can span from the bottom surface of the backing 88 to the bottom edge of the reinforcing support 106. The second reinforcing support 106 can provide increased rigidity in the region adjacent the apex of the base 82, where the apex lies adjacent to the junction of the upper edge segment 100 and the first end 36 of the neck pad 86. [0085] The backing 88, the polygonal stand 92, and the reinforcing supports 106 can be formed from any suitable material sufficient to provide an unyielding support of a patent&#39;s neck when the patient is lying supine on a support surface. By way of example, the backing 88, the polygonal stand 92 and the reinforcing supports 106 can be fabricated from ABS. However, these components of the device 80 can be formed separately from dissimilar materials. If these components of the device 80 are formed from the same material, these components of the device 80 can be integrally formed to define a single, homogenous component of the device 80. [0086] FIGS. 5-9 illustrate a fourth embodiment of the device 1 schematically represent in FIG. 1. In this embodiment, a device 120 can be substantially identical to the device 10 of FIG. 2, with any exceptions and/or modifications noted below. Description of the device 120 is provided with specific reference to FIGS. 5 and 6. [0087] The device 120 can include a base 122 and a jaw support 124 that can engage and support a patient&#39;s neck and mandible, respectively, as described above with reference to the device 10 of FIG. 2. [0088] The base 122 can include a frame 126 and a neck pad 128. The jaw support 124 can be connected to the frame 126, as will be described in detail below. The neck pad 128 can be merely placed onto the frame 126 or the neck pad 128 can be positively connected to the frame 126. If the neck pad 128 is positively connected to the frame 126, then the neck pad 128 can either be removably connected or permanently connected to the frame 126. Details of the engagement of the frame 126 by the neck pad 128 will be described below. [0089] The frame 126 can be configured from a material and with a geometry sufficient to provide an unyielding support of a patent&#39;s neck when the patient is lying supine on a support surface. By way of example, the frame 126 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. [0090] The frame 126 can include a polygonal stand 130, a first lateral pad support 132, a second lateral pad support 134 and a transverse pad support 136. The pad supports 132, 134 can extend from opposite ends of the polygonal stand 130. The pad supports 132, 134, 136 can be configured from a material and with geometry sufficient to provide an unyielding support of a patent&#39;s neck when the patient is lying supine on a support surface. By way of example, the pad supports 132, 134, 136 can be fabricated from hollow tubing stock or solid rod stock. This stock can have any cross-section deemed appropriate by one skilled in the art. Examples of materials for the stock can include metals and plastics. In another example, stainless steel can be used for the stock material. Stainless steel is a common material for surgical equipment known to for its ability to withstand repeated sterilizations and it can be readily formed into complex geometric configurations. [0091] The polygonal stand 130 and the pad supports 132, 134, 136 can be integrally formed to define the frame 126 as a single, homogenous component. In this exemplary embodiment, the polygonal stand 130 and the pad supports 132, 134, 136 can be formed by bending the stock into the desired shape. In another exemplary embodiment, the pad supports 132, 134, 136 can be formed as separate components and connected to the frame 126 by any known fastening device. [0092] The polygonal stand 130 can be substantially identical in structure and operation as described above with reference to the polygonal stand 20 of the device 10 of FIG. 2. Accordingly, reference numbers of FIG. 2 are used in FIGS. 5 and 6 to denote the substantially identical structure of these two embodiments. [0093] The neck pad 128 can be permanently secured or removably secured to the any of the pad supports 132, 134, 136 in any manner known in the art. If the neck pad 128 is removably connected to the pad supports 132, 134, 136, then the neck pad 128 can be cleaned and reused, or the used neck pad 128 can be disposed of and replaced with a new neck pad 128 after each use. The neck pad 128 can span the frame 126 from the first pad support 132 to the second pad support 134. [0094] In this exemplary embodiment, the neck pad 128 can be removably connected to each of the lateral pad supports 132, 134. The neck pad 128 can include first and second clips 148, 150. The clips 148, 150 can be configured to resiliently clamp to a respective one of the lateral pad supports 132, 134. [0095] The neck pad 128 can include a first end 152, a second end 154 (see FIG. 9 ), a first side 156, a second side 158, a backing 160, and a cushion. The cushion is omitted from FIGS. 5-9 for clarity and can be configured with structure and operation in manner substantially identical to the cushion 46 of the device 10 of FIG. 1. The backing 160 can extend from and between the first and second ends 152, 154 and the first and second sides 156, 158. The pad supports 132, 134, 136 can extend along the entirety of the respective sides 156, 158 and the second end 154. The backing 160 can have a rigidity sufficient to support the patient&#39;s neck in an appropriate hyperextended position above the support surface without substantial deformation to the backing 160. Any material providing sufficient rigidity can be used to form the backing 160. According to another exemplary embodiment, the backing 160 alternatively, even if it is less preferable, can be made in the form of a taught sling, that is, of a flexible material that is stretched between the sides. [0096] The structural relationship of the neck pad 128 to the frame 126 can be substantially identical to that described above with respect to the neck pad 18 and the frame 16 of the device 10 of FIG. 1. [0097] The jaw support 124 can be substantially identical in structure and operation to the jaw support 14 of the device of FIG. 2. Accordingly, reference numbers of FIG. 2 are used in FIGS. 5 and 6 and structure that can be unique to the jaw support 124 of the device 120 of FIGS. 5-9, as compared to the jaw support 14 of the device 10 of FIG. 1 will be noted in the following description. [0098] The jaw support 124 can include first and second jaw pad assemblies 162, 164. Reference numbers of FIG. 2 are used to denote structure of the jaw pad assemblies 162, 164 that can be substantially identical in structure and operation as the jaw pad assemblies 54, 56 of the device 10 of FIG. 2. [0099] The jaw pad assemblies 162, 164 can include first and second jaw pads 166, 168, respectively. The jaw pads 166, 168 can be connected to a respective one of the adjustment posts 60 in any manner discussed above with respect to the device 10 of FIG. 2. The jaw pads 166, 168 can include respective L-shaped engagement surface 170, 172. The L-shaped engagement surfaces 170, 172 of the jaw pads can provide a thrust to the lower (inferior edge) of the patient&#39;s mandible that can inhibit, and possibly prevent, the patient&#39;s mandible from sliding backwards (posteriorly), while also engaging against the side of the patient&#39;s mandible along the ramus portion, thus inhibiting, and possibly preventing, the jaw pads 166, 168 from sliding medially into the patient&#39;s neck. [0100] FIGS. 10 and 11 schematically illustrate a fifth embodiment of the device 1 schematically represent in FIG. 1. FIGS. 10 and 11 can schematically represent an embodiment of the device 1 substantially identical to the device 80 of FIG. 4. In this embodiment, a device 180 can include a base 182 and a jaw support 184 substantially identical in structure to the base 82 and the jaw mount 14, respectively, of the device 80 of FIG. 4, with at least the following exceptions. [0101] The base 182 can be configured for positive connection to the support surface such that the base 182 can be immobilized relative to the support surface. In this exemplary embodiment, the jaw support 184 is configured to move in either direction along an arcuate path (indicated by the arrows P) relative to the base 182 when the patient is rolled slightly to either side, respectively, while lying supine on the support surface. [0102] The jaw support 182 can include a C-shaped mounting post 186 that can move within an arcuate passage 188 formed though the base 182. The passage 188 can be configured to frictionally engage the mounting post 186 or any known fastening device (not shown) can be used to lock the mounting post 186 in the desired position along the arcuate path P. The passage 188 and the mounting post 186 can be configured with complimentary geometries that can prevent, or at least impede rotation of the mounting post to the left or right, as viewed in FIG. 11. According to yet another exemplary embodiment, similar to that illustrated in FIGS. 10 and 11, the C-shaped mounting post 186 can instead be attached to a platform which is mounted to the base 182. The platform, which includes a triangularly shaped portion which can hyperextend a patient&#39;s neck as described with reference to the other embodiments herein, carrying the post 186 and the other affixed structures, slides relative to the base 182 in the same curved path as the post 186 in the embodiment illustrated in FIGS. 10 and 11. In this manner, the patient can be rotated along the same arcuate path P when resting on the platform, keeping the jaw pads pressing against the remus portion of the patient&#39;s jaw and pressing against the back of the patient&#39;s neck to hyperextend the neck. [0103] As can be readily appreciated from the several illustrations, it can be particularly advantageous when the frame, including the arms, forms at least part of an arc, e.g., a portion of a circle, so that the jaw pads engage the patient&#39;s mandible and simultaneously push medially inward on the patient&#39;s mandible, thus self-seating the jaw pads to the mandible and resisting the jaw pads slipping off the mandible. [0104] The shape of the head rest also can be advantageous, by effectively forming a ‘knee’ over which the patient&#39;s neck rests, which in turn can provide at least two beneficial effects. The ‘knee’ of the head rest can stabilize the patient on the device, because the patient&#39;s head can rest on the side of the ‘knee’ opposite the patient&#39;s shoulders and lower neck, thus inhibiting, and likely preventing, the device and the patient from sliding relative to each other. Stated somewhat differently, the triangle can provide firm support which can resist any downward movement or disengagement of the neck pad from the neck as the jaw pads are actively thrusting the patient&#39;s mandible forward (anteriorly). Additionally, this configuration can hyperextend the patient&#39;s neck, which in turn can further assist in opening the patient&#39;s airway. [0105] Optionally, for all of the embodiments, a strap (not illustrated) can be provided which can extend around the patient&#39;s forehead and around a portion of the device, to assist in holding the device to the patient. [0106] While the invention has been described in detail with reference to exemplary embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. The foregoing description of the preferred embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiments were chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto, and their equivalents. The entirety of each of the aforementioned documents is incorporated by reference herein.

Summary: A pair of pads is held against the remus of a patient&#39;s jaw, to prevent the jaw from slipping back and causing an airway obstruction, while the patient&#39;s neck is hyperextended to also cause the patient&#39;s airway to stay open. A device including the adjustable jaw pads as well as a triangularly shaped portion over which the patient&#39;s neck rests is not required to be attached to the surface on which the patient is lying, and permits the patient to be rolled on either side while still maintaining the patency of the patient&#39;s airway.

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Write a title and summarize: Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4+ T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s. c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4+ T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4+ and CD8+ T cells, persistent expansion of all circulating CD8+ T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4+ T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection. Although HIV-1 establishes a chronic active infection that evolves toward clinical immunodeficiency over a span of several years, accumulating evidence indicates that critical pathogenic events take place during the acute phase of infection, leading to a massive and seemingly irreversible depletion of CD4+ T cells, predominantly of the memory phenotype [1], [2]. A large fraction of the T-cell pool in the body is harbored in the gut-associated lymphoid tissue (GALT) [3], which has been identified as a primary anatomical site for CD4+ T-cell depletion in both HIV-1-infected patients [4]–[6] and SIV-infected nonhuman primates [1], [2], [7], [8]; yet, the loss of CD4+ T cells within the early phase of infection appears to be a systemic phenomenon that involves all secondary lymphoid organs [1], [9], [10]. Taken together, these observations suggest that interventions aimed at preventing or reducing the immunologic damage caused by HIV-1 would be most effective if implemented during the earliest stages of infection, before the pool of memory CD4+ T cells becomes irreversibly compromised. In spite of extensive research over the past three decades, the mechanism of CD4+ T-cell depletion during the course of HIV-1 infection is still debated. Studies in SIV-infected macaques have highlighted the role of direct cytopathic effects of the virus during the course of acute primary infection [1], [9], [10]. However, indirect mechanisms, including bystander apoptosis, may also be important, as suggested by the increased levels of apoptosis detected in blood and lymphoid organs of macaques acutely infected with pathogenic SIV strains [2], [11]–[15], as well as in ex vivo-cultured T cells from individuals with acute HIV-1 infection [16]–[18]. Thus, the use of anti-apoptotic agents during primary HIV-1 infection may have beneficial effects for preserving the integrity of the CD4+ T-cell pool. We previously demonstrated that interleukin-7 (IL-7), a nonredundant cytokine that plays a critical role in the development and homeostasis of the T-lymphoid compartment of the immune system [19]–[21], effectively reduces the levels of spontaneous apoptosis in both CD4+ and CD8+ T cells from HIV-1-infected individuals [22]. In lymphopenic hosts, the levels of endogenous IL-7 increase, causing transient proliferation of naïve and central memory (CM) CD4+ and CD8+ T cells, which eventually leads to the reconstitution of the physiological T-lymphocyte pool [20]–[22]. Owing to these unique biological properties, IL-7 is currently under clinical investigation as an immune-reconstitution agent in various forms of natural and iatrogenic immunodeficiencies, including those associated with AIDS and cancer [23], [24]. Pre-clinical studies in macaques chronically infected with SIV [25]–[28], as well as clinical studies in patients with HIV-1 infection or receiving treatment with immunosuppressive antineoplastic drugs [29]–[32], have documented beneficial effects of short-term courses of IL-7 therapy, resulting in the proliferation and numerical expansion of naïve and CM CD4+ and CD8+ T cells in peripheral blood and secondary lymphoid organs. Whether adjuvant therapy with IL-7 may effectively lead to the long-term reconstitution of the immunologic function remains unclear. Moreover, IL-7 treatment has never been evaluated in acute HIV-1/SIV infection, a phase in which it may still be possible to avert the seemingly irreversible immunologic damage caused by massive viral replication prior to the appearance of virus-specific adaptive immune responses. In the present study, we administered fully glycosylated simian IL-7 to rhesus macaques during the acute phase of infection with a pathogenic SIV strain (mac251). The concomitant use of ART was deliberately avoided in order to exclude its confounding effects on pathogenesis since ART would have suppressed SIV replication, thereby preventing the pathologic depletion of CD4+ T-cells and making it impossible to evaluate the CD4-protective effects of IL-7. Another important goal of our study was to examine the effects of IL-7 on SIV replication since IL-15, a related common-γ-chain cytokine, was recently shown to dramatically increase SIV replication and accelerate disease progression when administered to acutely-infected macaques [33]. Our results demonstrate that treatment with IL-7 during the acute phase of SIV infection is safe and effective in preventing the decline of circulating naïve and memory CD4+ T cells without causing major increases in the levels of SIV replication. None of the 6 rhesus macaques treated with IL-7 exhibited adverse clinical side effects throughout the treatment period. After the first IL-7 injection (day −7 relative to SIV infection), plasma IL-7 levels peaked on day −5 to return to baseline on day 0 (Figure 1A). The two subsequent injections (day 0 and day 7) induced higher peak levels of plasma IL-7 and a greater area under the curve (AUC), resulting in markedly elevated trough levels before each of the following injections. This pattern likely reflects the initial distribution of the cytokine to a high-affinity compartment that was saturated upon subsequent injections. Increased plasma levels of IL-7, albeit considerably lower than in IL-7-treated animals, were also observed in untreated animals starting on day 28 post-infection in parallel with the most pronounced reductions in circulating lymphocyte counts (Figure 1A). No significant correlations were found between plasma levels of IL-7 and various immunological parameters, including circulating CD4+ and CD8+ T-cell counts and expression of the specific chain of the IL-7 receptor (IL-7Rα or CD127) (data not shown). In agreement with previous studies [25]–[32], IL-7 treatment initially caused a significant downmodulation of the IL-7 receptor (CD127) in both naïve and memory T cells (Figure 1B). Comparison of the two groups of animals showed that treatment with IL-7 did not induce significant increases in the levels of SIV plasma viremia at any time during the acute phase of infection and the follow-up period, including the peak of viral replication, the viral set point and the AUC, with the only exception of the earliest time point analyzed (day 4 post-infection; p = 0. 043 for the comparison between the two groups of animals by Wilcoxon rank sum test) (Figure 2A). However, there was a trend towards higher levels of viremia in IL-7-treated animals, particularly on days 35 and 41 post-infection, even though the statistical p values remained far below the threshold for significance (Table S1 in Text S1). Likewise, the two groups of animals showed no significant differences in the levels of SIV p27 antigenemia (Figure 2B and Table S1 in Text S1) and proviral SIV DNA load detected in blood mononuclear cells on days 14 and 77, in the GALT (ileum) on days 14–16, and in axillary lymph nodes on days 25–27 post-infection (Figure 2C and Table S1 in Text S1). All the animals developed SIV-specific IgG antibodies, which became detectable between day 11 and day 21 of infection (data not shown). During the follow-up period, 4 animals (two untreated and two IL-7-treated) developed early signs of progression to AIDS (rapid progressors [RP]) and were euthanized for terminal disease within 5 months of infection. The RP course has been suggested to represent a unique form of SIV disease, distinct from that of conventional progressors (CP) and associated with an unusual pathogenesis characterized by higher levels of SIV viremia, massive SIV replication in mononuclear phagocytic cells rather than in CD4+ T cells (with consequent lack of depletion of CD4+ T cells), and severe SIV-related enteropathy in the absence of opportunistic infections [34]. Additional data on RP animals are presented in Supplementary Data and Figure S1 in Text S1. Since the presence of RP animals could be a confounding factor in our study, all the virological and immunological data were analyzed both with the inclusion and after the exclusion of the 4 RP animals. When the analysis of SIV plasma viremia and antigenemia was restricted to CP animals, the statistical comparisons between IL-7-treated and untreated animals did not show any significant changes from those obtained with the inclusion of all the animals (Figure S2A in Text S1). While all the animals in the untreated group experienced a marked and sustained decline of circulating CD4+ T lymphocytes starting at the time of peak SIV replication (day 14 post-infection), IL-7-treated animals showed no decline of CD4+ T-cell counts over the entire treatment period, with even a significant increase, relative to baseline, on day 41 (Figure 3A). When the two groups of animals were compared, IL-7-treated macaques had significantly higher absolute numbers of peripheral CD4+ T cells at several time points, including day 14 post-infection (statistical data not shown); similar results were obtained by comparison of the changes in CD4+ T-cell counts from baseline in IL-7-treated versus untreated animals (Figure S3 in Text S1). To better characterize the effects of IL-7 on CD4+ T cells, the naïve, memory and effector subpopulations were analyzed separately. In the absence of IL-7 treatment, SIV infection caused significant decreases in the absolute numbers of naïve and memory CD4+ T cells, compared to pre-infection levels, throughout the acute phase of the infection (Figure 3A). In contrast, IL-7 caused an initial increase in memory CD4+ T cells (day 7) and subsequently prevented the decline of both naïve and memory CD4+ T cells throughout the treatment period; effector CD4+ T cells were increased at several time points (Figure 3A). However, the protective effects of IL-7 were not sustained after treatment interruption with both naïve and memory CD4+ T cells becoming significantly decreased, compared to baseline values, on day 62 post-infection, 4 weeks after the last injection of IL-7 (Figure 3A). When the absolute numbers of circulating naïve, memory and effector CD4+ T cells in the two groups of animals were compared, significant differences were detected at several time points (statistical data not shown); similar results were obtained by comparison of changes from baseline in IL-7-treated versus untreated animals (Figure S3 in Text S1). The statistical differences between treated and untreated animals remained significant when the analysis of total, naïve, memory and effector CD4+ T cells was restricted to CP animals, after exclusion of the 4 RP animals (Figure S2B in Text S1). A detailed subset analysis of the memory CD4+ T-cell population was performed at selected time points. While in untreated animals all memory CD4+ T-cell subsets (CM, transitional memory [TM] and effector memory [EM]) dramatically declined during the acute phase of infection, none of these subsets was quantitatively reduced in animals receiving IL-7 (Figure 3B). The protective effects of IL-7 on memory CD4+ T cells were not sustained after treatment interruption as shown by the significant decline of all three subpopulations, compared to baseline, on day 62 post-infection (Figure 3B). IL-7-treated animals experienced sustained increases in all subsets of circulating CD8+ T cells throughout the acute phase of infection, whereas a transient decline of naïve and memory CD8+ T cells was observed in untreated animals (Figure 3A). When the two groups of animals were compared, IL-7-treated monkeys showed higher absolute numbers of peripheral CD8+ T cells at several time points (statistical data not shown); similar results were obtained by comparison of changes from baseline between IL-7-treated and untreated animals (Figure S3 in Text S1). Subset analysis of memory CD8+ T cells revealed no major changes in untreated animals, whereas IL-7 induced significant increases in all memory CD8+ T-cell subsets (Figure 3B). However, on day 62 post-infection, CM CD8+ T cells returned to baseline values, while TM and EM CD8+ T cells were significantly decreased in both IL-7-treated and untreated animals (Figure 3A and 3B). The statistical differences between treated and untreated animals remained significant when the analysis of total, naïve, memory and effector CD8+ T cells was restricted to CP animals, after exclusion of the 4 RP animals (Figure S2B in Text S1). Longitudinal analysis of the levels of proliferation in freshly isolated peripheral blood T cells revealed that IL-7 treatment induced only a transient increase in the proportion of Ki67-expressing CD4+ T cells during the first week of treatment (day −5 and −3 prior to SIV infection), which returned to baseline levels at the time of SIV inoculation (day 0); remarkably, there was no further increase in proliferation after any of the subsequent IL-7 injections (Figure 4A). In contrast, CD8+ T cells showed elevated Ki67 expression both before SIV inoculation and at two time points (day 4 and 11) after infection, even though the proportion of cycling cells returned to baseline thereafter (Figure 4A). Analysis of apoptosis in circulating T cells by Annexin-V binding did not show significant elevations, compared to baseline, in either untreated or IL-7-treated animals throughout the acute phase of SIV infection (Figure 4B), suggesting that the peripheral blood compartment is largely spared from in vivo apoptosis during primary infection; however, IL-7-treated animals exhibited significant and sustained increases in the intracellular levels of the anti-apoptotic protein Bcl-2 in both CD4+ and CD8+ T cells during the first three weeks of treatment (Figure 4C). Representative histograms illustrating Ki67 expression, Annexin-V binding and Bcl-2 expression in CD4+ and CD8+ T cells from one untreated (#749) and one IL-7-treated (#746) animals are shown in Figure S4 in Text S1. Altogether, these results suggested that numerical expansion due to proliferation was not a major factor contributing to the lack of decline of circulating CD4+ T cells seen in IL-7-treated animals while a decreased sensitivity to apoptosis was likely involved. In contrast, both proliferation and apoptosis reduction may have contributed to the sustained numerical increases documented in circulating CD8+ T cells. Next, we studied the effects of IL-7 in peripheral lymphoid tissues. Lymph node biopsies were collected from treated and untreated macaques on days 25–27 post-infection. The relative proportions of total, naïve, memory and effector CD4+ and CD8+ T cells in lymph nodes were not significantly different between IL-7-treated and untreated macaques, and the level of Ki67 expression was very low (<1%) in both groups of animals (data not shown). Analysis of T-cell apoptosis by Annexin-V binding and intracellular Bcl-2 expression did not show significant differences between the two groups of animals despite a trend toward reduced Annexin-V binding in memory CD4+ and CD8+ T cells and effector CD8+ T cells from IL-7-treated macaques (Figure 5A, upper panel); however, these differences became statistically significant when the analysis was restricted to CP animals, after exclusion of the 4 RP animals (Figure 5B, upper panel). In accordance with the Annexin-V data, lymph node CD4+ T cells from IL-7-treated macaques showed higher levels of intracellular Bcl-2 when RP animals were excluded from the analysis (data not shown). Terminal ileum biopsies were obtained from all animals on days 14–16 post-infection. The yield of CD3+ T cells from these biopsies was highly variable (range = 1. 2–15. 0% of the total cell populations), underscoring the inherent difficulties in obtaining mucosal specimens with comparable representation of the GALT via retrograde ileoscopy. Regardless of this limitation, no significant differences were detected in the relative proportions of total, naïve, memory and effector CD4+ T cells, as well as in the CD4/CD8 ratio in the intestinal tissues of IL-7-treated vs. untreated macaques (data not shown); likewise, the proportion of Annexin-V-positive CD4+ T cells was similar in the two groups (Figure 5A, lower panel). In contrast, the proportion of Annexin-V-positive CD8+ T cells was lower in IL-7-treated animals (p = 0. 041), primarily due to a reduction of apoptosis among memory CD8+ T cells (p = 0. 048) (Figure 5A, lower panel), associated with a lower proportion of apoptosis-prone naïve CD8+ T cells (p = 0. 024) (data not shown). The difference in the proportion of Annexin-V-positive CD8+ T cells remained significant when RP animals were excluded from the analysis (Figure 5B, lower panel). SIV-specific T-cell responses were evaluated at multiple time points during and after the IL-7 treatment period by measuring the intracellular production of IFN-γ, IL-2 and MIP-1β in CD4+ and CD8+ T cells stimulated with peptide pools derived from the SIV Tat and Gag proteins. SIV-specific T-cell responses were detected at multiple time points in all animals, while we were unable to document the presence of SIV-neutralizing antibodies in serum at any time during acute primary infection in both untreated and IL-7-treated macaques (data not shown). The total number of Tat-specific CD8+ T cells at the first time point analyzed (day 21 post-infection) was significantly higher in IL-7-treated than in untreated animals (p = 0. 017), whereas the difference in Tat-specific CD4+ T-cell responses was close to but did not reach statistical significance (p = 0. 051) (Figure 6). Overall, IL-7-treated animals displayed higher numbers of Gag-responding CD4+ and CD8+ T cells at several time points, but the differences did not reach statistical significance (Figure 6). Qualitative analysis of SIV-specific T-cell responses revealed that initially most Tat-specific (Figure 7A) and Gag-specific (Figure S5 in Text S1) CD4+ and CD8+ T cells were monofunctional in both groups of animals, producing a single cytokine (single-producer, SP). When SP cells were analyzed separately from double- and triple-producer cells (DP and TP), the difference between IL-7-treated and untreated animals on day 21 was significant for both CD4+ and CD8+ Tat-specific T cells (p = 0. 030 and 0. 017, respectively; Figure 7B). The quality of the T-cell responses evolved over time, with both Tat-specific (Figure 7B) and Gag-specific (Figure S5 in Text S1) T cells acquiring some degree of polyfunctionality over time. This phenomenon was more prominent among IL-7-treated animals, as indicated by a significant difference in the proportion of the various functional subpopulations of Tat-specific CD8+ T cells on day 62 post-infection (Figure 7C). While the progressive refinement of multi-drug ART protocols has led to extraordinary advances in the treatment of chronic HIV-1 infection, there is still uncertainty as to whether a complete reconstitution of the immunologic function can be achieved even after years of sustained virologic suppression [35]. The often incomplete immunologic reconstitution in patients treated with ART, which is linked to an increased incidence of adverse clinical events [36], has prompted consideration of adjuvant therapies, including treatment with cytokines of the common-γ-chain family, such as IL-2, IL-7 and IL-15, which are known to induce T-cell expansions. However, the ability of such treatments to regenerate fully competent naïve and memory T-cell pools remains questionable as multiple lines of evidence indicate that the initial damage caused by HIV-1 during acute primary infection is irreversible [1], [2], [10], marking a critical event in the pathogenesis of HIV-1 disease and precluding the success of any restoration attempts enacted during the chronic phase. These considerations underscore the need to aim adjuvant treatment strategies toward immunologic preservation rather than reconstitution and, therefore, to implement such strategies at the earliest possible time after the diagnosis of HIV-1 infection. In this study, we used a macaque model to demonstrate that treatment with IL-7, the principal T-cell homeostatic cytokine, can prevent the dramatic decline of circulating naïve and memory CD4+ T cells that occurs during acute primary SIV infection. Although in the clinical setting IL-7 would presumably be associated with ART, as it was done in phase-1 studies in patients with chronic HIV-1 infection [29], [30], we deliberately avoided the use of virus-suppressive drugs in order to allow for an unbiased evaluation of the effects of IL-7 on CD4+ T-cell depletion, which is the hallmark of HIV-1/SIV-induced pathogenesis during primary infection. In fact, ART by itself would have prevented the loss of CD4+ T cells, limiting the scope of our study. The fact that in acutely-infected macaques treatment with IL-7 was able to protect naïve and memory CD4+ T cells even in the absence of ART suggests that the combination of IL-7 with virus-suppressive drugs would be even more effective in maintaining the integrity of the CD4+ T-cell pool during one of the most critical phases in the pathogenesis of HIV-1 disease. To elucidate the mechanisms responsible for the CD4-protective effects of IL-7, we examined the kinetics of CD4+ T-cell proliferation and apoptosis, as well as the induction of SIV-specific cellular immune responses during the course of IL-7 treatment. Some levels of proliferation, albeit low, were documented following the first injection of IL-7, presumably leading to the observed initial expansion of the memory CD4+ T-cell subset. However, in agreement with data of IL-7 treatment in chronically SIV-infected macaques [28], no further proliferation was seen after any subsequent IL-7 injection, suggesting that repeated administrations of fully glycosylated IL-7 at weekly intervals may in fact induce tachyphylaxis, at least concerning the proliferative effects of the cytokine. Conversely and in agreement with our previous ex vivo findings [22], several observations pointed to a reduction of apoptosis as one of the mechanisms responsible for CD4+ T-cell preservation in our IL-7-treated macaques. First, while the levels of apoptosis in peripheral blood T cells did not change significantly during primary SIV infection in either treated or untreated animals, preventing the evaluation of the anti-apoptotic effects of IL-7 in this compartment, there was a sustained increase in the expression of the anti-apoptotic protein Bcl-2, a marker of increased resistance to apoptosis, in circulating CD4+ and CD8+ T cells from IL-7-treated animals. Second, analysis of secondary lymphoid tissues (lymph nodes and GALT) demonstrated a reduction of apoptosis in selected CD4+ and CD8+ T-cell populations in IL-7-treated macaques, even though these tissues were only sampled at a single time point during the acute phase due to the inherent difficulties in obtaining multiple tissues from the same animal within a short period of time. This limitation reduced our chances to document the full spectrum of anti-apoptotic effects of IL-7. Of note, analysis of the GALT at the time of peak SIV replication demonstrated that IL-7 reduced the proportion of Annexin-V-binding CD8+ T cells, particularly of the memory phenotype, but not CD4+ T cells. An important caveat that must be considered in this setting is the inconsistent quality of ileum specimens obtained by retrograde ileoscopy, which have a high probability of sampling error due to a markedly variable yield of lymphoid cells. Regardless, the fact that we did not observe even a trend toward reduced apoptosis among GALT CD4+ T cells may reflect the fact that IL-7 is inactive against the direct cytopathic effect of the virus, which has been identified as the principal mechanism responsible for CD4+ T-cell depletion in the gut during the acute phase of the infection, whereas the role of apoptosis in this context remains controversial [1], [2]. Although a more extensive investigation is warranted to confirm this hypothesis, our data suggest that IL-7 treatment prevented the decline of circulating CD4+ T cells during the acute phase of SIV infection by inducing an initial expansion of these cells combined with their subsequent preservation via sustained pro-survival effects. Since IL-7 seemed to effectively preserve the pool of CD4+ T cells, which are the main cellular targets for SIV, one could have anticipated considerable increases in the levels of virus replication in acutely infected macaques treated with IL-7. However, despite the absence of concomitant ART, we did not observe dramatic increases in the levels of SIV replication during both the treatment phase and the follow up period, with the only exception of the earliest time point analyzed (day 4 post-infection) in which the levels of SIV plasma viremia were significantly higher in IL-7-treated than in untreated macaques. A trend toward higher levels of SIV plasma viremia was seen at certain time points, particularly day 35 and day 41 post-infection, but it did not reach statistical significance even after exclusion from the analysis of the 4 animals with rapid disease progression. We cannot rule out that this lack of statistical significance was due to the low number of animals included in the study (n = 6 per group), even though the p values remained far below the threshold for significance. Nevertheless, it is undisputable that, if an enhancing effect of IL-7 on SIV replication did occur, this effect was far less dramatic than that documented for a related common-γ-chain cytokine, IL-15, in a similar experimental setting [33]. Indeed, IL-15 treatment of macaques during the acute phase of SIV infection was shown to induce a ∼3 log increase in viral load, most likely due to increased CD4+ T-cell proliferation and activation, and to accelerate the disease progression [33]. Various hypotheses can be formulated to explain these striking differences between the outcomes of IL-7 and IL-15 treatment. First, the lack of strong induction of CD4+ T-cell proliferation by IL-7, unlike IL-15, which was detectable only transiently and only after the first IL-7 injection; of note, this transient proliferative effect of IL-7 could have been responsible for the significant increase in SIV plasma viremia at the earliest time point tested (day 4 post-infection). Second, the induction of early and vigorous SIV Tat-specific CD4+ and CD8+ T-cell responses in IL-7-treated animals, as compared to untreated animals, associated with the proliferation, expansion and activation of all CD8+ T-cell subpopulations. Since Tat is a regulatory protein expressed during the early phase of the viral life cycle, Tat-specific T-cell responses presumably were able to halt the infection before completion of a full replicative cycle, thereby preventing the spread of infectious virus to other target cells. Of note, a pronounced numerical increase was detected in EM CD8+ T cells, a functionally competent subset that was recently associated with effective vaccine-elicited protection in macaques [37]. Thus, it is plausible that the increased availability of target CD4+ T cells in IL-7-treated animals was counterbalanced by the effectiveness of virus-specific cell-mediated immune responses resulting in the observed minimal increases in SIV replication levels. An interesting correlate of our findings was provided by a recent study in mice acutely infected with LCMV, in which early treatment with IL-7 augmented the number and functionality of specific antiviral effector T cells, thereby reducing organ pathology and promoting viral clearance [38]. Conversely, it is also possible that the earlier and stronger Tat-specific CD4+ and CD8+ T-cell responses observed in IL-7-treated animals resulted from a more robust antigenic stimulation due to the higher amount of virus detected in these animals at day 4 post-infection; indeed, strong CD8+ T-cell responses were also detected in IL-15-treated animals, although they were incapable of controlling viral replication [33]. Thus, the different mechanisms of action of IL-7 and IL-15 on CD4+ T cells, with IL-15 inducing high levels of activation and proliferation mainly in memory CD4+ T cells, the preferential target of the virus, and IL-7 stimulating the proliferation and renewal of naïve T cells too, thereby contributing to the preservation and replenishment of the peripheral T-cell pool, may account for the divergent effects of treatment with these two cytokines. Although a more extensive investigation is warranted in the perspective of a potential clinical use of IL-7 in acute HIV-1 infection, it is important to emphasize that in a therapeutic regimen IL-7 would always be administered in combination with ART, thereby minimizing its possible enhancing effects on viral replication. Our results may have implications for devising new treatment strategies for acute HIV-1 infection, whose clinical management remains a challenge. A critical hurdle is the inherent difficulty in identifying and treating patients at the earliest possible stage in order to reduce the massive HIV-1 replication that occurs before the development of virus-specific adaptive immune responses and its deleterious effects on the immune system. The extent of viral replication during the acute phase of SIV infection in macaques has indeed been identified as a critical determinant of the natural history of the disease [39]. Although early ART treatment was reported to be beneficial on the induction and maintenance of HIV-specific cellular immune responses [39]–[41], additional studies in patients [10], [42] and macaques [43] have shown limited effects of ART alone on T-cell preservation in the intestinal lamina propria, underscoring the importance of devising effective adjuvant therapies. Indeed, even if ART is promptly initiated during primary infection, its effects may not be sufficient for fully preventing the immunologic damage caused by HIV-1, particularly in the gastrointestinal tract, due to dishomogeneous drug biodistribution or inactivation by P-glycoproteins within the intestinal mucosa. Moreover, complete suppression of viral replication could take several weeks, and indirect mechanisms of cell destruction, such as bystander apoptosis, may remain active for some time after the virus has ceased to replicate. Our data provide a scientific basis for the clinical evaluation of IL-7, in combination with ART, for the treatment of acute primary HIV-1 infection. The animals were housed and fed according to regulations established in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. The animal experiments performed in this study were approved by the NIH Animal Care and Use Committee (ACUC). Twelve colony-bred juvenile Rhesus macaques, housed at Bioqual Inc. (Rockville, MD), were divided in two groups, according to HLA haplotype and baseline CD4+ T-cell counts (Table S2 in Text S1): untreated controls (group 1) and IL-7-treated (group 2). Blood samples were obtained three times during the first week of IL-7 treatment, then twice weekly for the entire treatment period and then once per week; terminal ileum biopsies were obtained on day 14–16 post-infection; lymph node biopsies were obtained on day 25–27 post-SIV infection. Since one animal in the control group (H744) was lost on day 14 post-infection during intestinal biopsy, all the data from day 14 onwards refer to a total of 11 animals. Recombinant glycosylated simian IL-7 (Cytheris, France) was employed in this study since it has a considerably longer half-life and greater stability (M. Morre et al., unpublished results) than the non-glycosylated form that had been employed in several previous studies [27], [28]. IL-7 was administered subcutaneously to the 6 monkeys included in group 2 at a concentration of 50 µg/kg of body weight once per week for a total of 7 consecutive administrations. To allow for the achievement of steady-state plasma levels of the cytokine prior to SIV infection, treatment was initiated 1 week before SIV inoculation (day −7). On day 0, all 12 monkeys were inoculated intravenously with 100 macaque infectious doses of the pathogenic strain SIVmac251, kindly provided by Dr. R. C. Desrosiers. Multiple clinical, immunological and virological parameters were monitored throughout the acute phase of infection, as well as for a follow-up period of 6 months post-infection. The concentration of IL-7 in serial plasma samples was measured using a high-sensitivity commercial ELISA (Quantikine HS, R&D Systems, Minneapolis, MN). Peripheral blood was collected under sterile conditions in vacutainer tubes with EDTA as anticoagulant and complete blood cell count with differential was performed by a commercial laboratory (Antech Diagnostics, Rockville, MD). Plasma was separated by spinning whole blood at 500×g for 20 min at 4°C without brake and stored at −80°C. PBMC were isolated by gradient centrifugation using Lymphocyte Separation Medium (LSM; MP Biomedicals). Blood was diluted in Phosphate Buffered Saline (PBS), stratified over LSM and centrifuged at 500×g for 25 min at 4°C without brake. The mononuclear cell ring was collected, and the PBMC were washed twice with PBS, counted and used for cytofluorimetric analyses. The following monoclonal antibodies (mAbs) were used for surface staining: CD28-FITC (clone CD28. 2), CD4-PerCpCy5. 5 (clone L200), CD95-APC or -PE (clone DX2), CD8-PECy7 (clone SK1), CD3-APCCy7 (clone SP34-2), all from BD Biosciences. The naïve (CD28+CD95−), memory (CD28+CD95+) and effector (CD28−CD95+) T-cell subsets were identified as previously described [43]. At selected time points, a more detailed characterization of the memory T-cell compartment was performed by using a combination of the following mAbs: CD45RA-FITC (clone 5H9), CD28-PECy7, CCR7-APC (clone 3D12), CD3-V450, CD4-APCH7 (all from BD Biosciences), and CD8-eFLOUR 605NC (clone RPA-T8; eBioScience). As illustrated in Figure S6 in Text S1, central memory T cells (TCM) were defined as CD45RA− CCR7+ CD28+, transitional memory T cells (TTM) as CD45RA− CCR7− CD28+, and effector memory T cells (TEM) as CD45RA− CCR7− CD28−. Additional mAbs were used to measure the expression of other cell-surface markers, including the IL-7 receptor-α/CD127-PE (clone hIL-7R-M21), CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5) and the activation markers HLA-DR-PE (clone L243/G46-6) and CD25-PE (clone M-A251) (all from BD Biosciences). Stained cells were analyzed by flow cytometry using a BD FACSCanto collecting a minimum of 100,000 events per sample. Flow data were analyzed using the Flowjo software (Tree Star) or FCS Express (DeNovo Software). Apoptosis was assessed by measuring the levels of Annexin-V binding, using Annexin V-APC conjugated (BD Biosciences), after surface staining, as previously reported [22]. Bcl-2 expression levels and cellular proliferation were assessed as follows: after surface staining, the cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and incubated with mAbs anti-Bcl-2-PE (clone Bcl-2/100) and anti-Ki67-PE (clone B56) (BD Biosciences). Annexin-V binding and Bcl-2 expression (Mean Fluorescence Intensity, MFI) were evaluated on various CD4+ and CD8+ T-cell subpopulation by multicolor flow cytometry using a BD FACSCanto, collecting a minimum of 100,000 events per sample, and flow data were analyzed with the Flowjo software (Tree Star) or FCS Express (DeNovo Software) applying a progressive gating strategy. Naïve memory and effector T-cell subsets were identified using a combination of anti-CD95 and anti-CD28 antibodies [44] on CD3+CD4+ and CD3+CD8+ gated cells, as described above for surface staining experiments. Plasma SIV RNA levels were measured using a quantitative real-time RT-PCR assay. Viral RNA was purified from 280 µl of cell-free plasma using the QIAamp Viral RNA kit (Qiagen, USA), and stored at −80°C. The number of SIV RNA genome equivalents was determined using a single-tube real-time RT-RCR assay based on the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, USA), under the following reaction conditions: 10 min at 45°C (RT), 10 min at 95°C and 40 cycles of 15 sec at 95°C and 45 sec at 60°C (PCR). Primers (300 nm) and probe (200 nm) were specifically designed within the SIV gag gene to amplify a fragment of 91 bp. Forward primer: 5′-GCAGAGGAGGAAATTACCCAGT-3′; reverse primer: 5′-ATTTTACCCAGGCATTTAATGTTC-3′ (used for the RT phase); TaqMan MGB probe, FAM-labelled: 5′-ACAAATAGGTGGTAACTATG-3′. The copy number was determined by interpolation on a standard curve of a DNA plasmid carrying a fragment of the SIV gag gene containing the RT-PCR amplicon (serial 10-fold dilutions from 100 copies/reaction to 106 copies/reaction). Forward cloning primer: 5′-GCAGAGACACCTAGTGATGGAAAC-3′; reverse cloning primer: 5′-TCTCCCACACAATTTAACATCTG-3′. The SIV proviral DNA load was measured by real-time PCR using the same primers and probe as in the plasma viremia assay. Normalization for cell number was performed by quantification of a non-polymorphic single-copy gene, CCR5. Additional details about the method have been reported [45]. SIV p27Gag antigenemia was measured by using the SIV Core Antigen Assay (Coulter Corporation, Miami, USA) according to the manufacturer' s instructions. All samples were initially tested undiluted and retested at 1∶10 dilution if necessary. Plates were read using an end-point protocol with a microplate spectrophotometer (Bio-Rad Instruments). Anti-SIV monkey IgGs were quantified using a home-made ELISA. Briefly, plates were coated overnight at 4°C with 1 µg/ml of total SIV protein extract, obtained by lysing with 1% Triton x-100 for 1 hour at room temperature the SIV isolate SIVmac251/SupT1-CCR5 CL30 (from the AIDS & Cancer Virus Program, NCI, NIH, Frederick). The following day the plates were washed and blocked for 1 hour. Samples were then added to each well, initially undiluted and re-tested diluted 1∶5 or 1∶20, whenever needed. Plates were then incubated with biotinylated goat anti-human IgG (Sigma-Aldrich, #B1140) and HRP-conjugated streptavidin (R&D Systems), and read at a wavelength of 450 nm with a reference set at 570 nm on a microplate spectrophotometer (Bio-Rad Instruments). The presence of neutralizing anti-SIV antibodies in the sera of infected untreated and IL-7-treated animals was tested using an envelope-mediated fusion assay with PM1 cells chronically infected with SIVmac251 as effector cells and Hela (TZM-bl) cells expressing human CD4 and CCR5 as target cells. Ileum biopsies were performed by retrograde ileoscopy on day 14–16 post-infection. At least 6–8 punch biopsies were obtained from the terminal ileum of each animal, immediately placed in cold RPMI medium and then processed within 3 hours of excision. During the procedure, one animal (H744) suffered an intestinal perforation and was lost. Ileum biopsies were digested in Iscove' s media supplemented with 2 mg/ml Type II collagenase (Sigma-Aldrich) and 1 U/ml DNase I (Sigma-Aldrich) for 30 min at 37°C. After digestion, the samples were passed through a 70 µm strainer, and the suspended cells were washed twice with RPMI media supplemented with 10% heat-inactivated FBS. Lymph node excisional biopsies were performed on day 25–27 post-infection on axillary lymph nodes from all animals. Lymph nodes were cut longitudinally in two halves, one of which was stored at −80°C. The remaining half was finely minced using sterile scalpels and mechanically smashed to release lymphoid cells into the media. Cells were then washed, passed through a 70 µm strainer and stained for surface and intracellular markers as described above. SIV-specific CD4+ and CD8+ T-cell responses were analyzed by measuring intracellular cytokine production after antigen stimulation. Frozen samples of PBMC were thawed in RPMI 10% FBS and rested at 37°C for at least 5 hrs. Cells were then plated at 5×105 cells/well in a 96-well round-bottom plate in RPMI 10% FBS in the presence of purified anti-CD28 and anti-CD49d mAbs both at a final concentration of 1 ng/ml. Two pools of SIV-gag peptides and one pool of SIV-tat peptides (from NIBSC, EVA Centre for AIDS reagents) at a final concentration of 1 µg/ml (each peptide) and Staphylococcus aureus Enterotoxin B (SEB, Sigma) as positive control at a final concentration of 2 µg/ml were added to the samples in a total volume of 200 µl. A negative control with no stimulation was included. After 1 hr incubation at 37°C, 5% CO2,1 µl of Brefeldin A (BD GolgiPlug, BD Biosciences) was added to each well, and the plates were incubated for an additional 11 hrs. At the end of the incubation period the cells were transferred to 96-well V-bottom plates and washed twice with PBS before surface intracellular staining. Cells were stained first with LIVE/DEAD Fixable Dead Cell Stain reagent (Invitrogen by Life Technologies) at a concentration of 1 µl/106 cells for 20 min at 4°C and then with the mAbs to lineage antigens (CD3-V450 and CD4-APCH7, BD Biosciences), before fixation and permeabilization with BD Cytofix/Cytoperm Buffer (BD Biosciences). After washing with BD PermWash Buffer (BD Biosciences), the cells were incubated for 20 min at 4°C with anti-MIP-1β-PE (clone D21-1351), anti-IFN-γ-PECy7 (clone P2G10) and anti-IL-2-APC (clone MQ1-17H12; all from BD Biosciences), washed again and analyzed on a BD FACSCanto instrument. T-cell responses were analyzed using Flowjo (Tree Star) and Spice softwares. Statistical analysis was conducted using the softwares SAS (version 9. 1 for Windows), S-Plus (version 6. 2 for Windows), StatView (version 5. 0. 1 for Macintosh) and GraphPad Prism (version 4. 0b for Macintosh). Paired Student' s t-tests were used for the comparison between different time points within the same animal group (untreated or IL-7-treated). Non-parametric Wilcoxon rank sum tests were used to analyze differences between IL-7-treated and untreated animals. To compare untreated and IL-7-treated animals with respect to changes from baseline to multiple time points simultaneously the O' Brien test was used (for a more detailed description see Supporting Information online).

Title: Treatment with IL-7 Prevents the Decline of Circulating CD4+ T Cells during the Acute Phase of SIV Infection in Rhesus Macaques Summary: The development of highly effective cocktails of antiretroviral drugs has had a major impact on the survival and quality of life of individuals with HIV-1 infection. Yet, current protocols often fail to fully restore the immunologic function, a limitation that has prompted the clinical evaluation of immune-reconstitution agents, such as IL-7, as adjuvant therapies. To date, however, IL-7 has been tested exclusively in patients with chronic HIV-1 infection, while it appears that the immune system is irreparably damaged during acute primary infection, within the first few weeks after encountering the virus. We used a macaque model to show that treatment with IL-7 has beneficial effects if implemented during the acute phase of infection with SIV, the simian AIDS virus. Early administration of IL-7 was safe and effectively protected CD4+ T cells, the primary target cells for the virus, from the marked decline that typically occurs during acute SIV infection. Furthermore, IL-7 boosted the development of antiviral immune responses. Thus, IL-7 might be an effective adjuvant therapy in acute HIV-1 infection, which can protect the pool of CD4+ T cells before it is irreversibly compromised by the action of the virus.

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Summarize: Gannett offered to pay $815 million including debt to buy Tribune Publishing, which emerged from bankruptcy less than two years ago. WSJ's Lukas Alpert explains on Lunch Break with Tanya Rivero. Photo: AP Gannett Co. on Monday went public with its proposal to acquire Tribune Publishing Co. in a deal valued at about $400 million that would combine titles like USA Today, the Los Angeles Times and Chicago Tribune, as the struggling newspaper industry increasingly consolidates. Gannett is offering $12.25 in cash for each share of Tribune, a 63% premium to the stock’s closing price Friday. Including the assumption of Tribune’s debt, Gannett said the deal has a total value of $815 million.... A Los Angeles Times newspaper vending box is shown in front of the Times building in Los Angeles, California December 8, 2008. Fred Prouser/Reuters Gannett has proposed to buy Tribune Publishing for $12.25 a share in cash, or $815 million. In a statement Monday, the publisher of USA Today took public an offer it made April 12. Tribune Publishing owns several newspapers including the Los Angeles Times and the Chicago Tribune. Gannett chairman John Jeffry Louis explained why the company wanted Tribune: A combination with Tribune would rapidly advance Gannett's strategy to grow the USA Today network, the largest local to national network of journalists in the country, to include more local markets and new platforms, which we believe will benefit readers and result in significant and sustained value creation for Gannett stockholders. The consolidation of both newspaper companies would also be aimed at cutting costs at a time of uncertainty for the industry. Gannett said it expected annual synergies of about $50 million. Its offer represents a 63% premium to Tribune's closing share price on Friday of $7.52. Tribune shares surged by as much as 60% in premarket trading. Gannett's release Monday also included a letter from CEO Robert Dickey to Tribune CEO Justin Dearborn. Here's the full text: Dear Mr. Dearborn: We are disappointed by the response we received from you in your letter of April 22, 2016 regarding our proposal to acquire all of the outstanding shares of Tribune Publishing Company ("Tribune") for an all-cash purchase price of $12.25 per share, and Tribune's continued refusal to begin constructive discussions with us. We believe our proposal, which we first made in my letter to your Board dated April 12, 2016 and reiterated in several phone discussions with Michael Ferro and you since, is highly compelling for Tribune's stockholders and represents substantial value and immediate liquidity for them. I want to remind you that Gannett's $12.25 per share offer price represents a 63% premium to Friday's closing stock price of Tribune, a 58% premium to the volume weighted average trading price over the past 90 days, and a multiple of 5.6x (including estimated pension and post-retirement benefits payable) your 2016 EBITDA estimate based on consensus research. The $12.25 per share offer price also represents a significant premium to the $8.50 share price at which Tribune recently issued common shares. With our capability to commit to a deal without financing contingencies, we believe that Gannett is uniquely positioned to offer this level of premium to your stockholders and to quickly evaluate and finalize this transaction, allowing your stockholders to receive immediate and certain value. As expressed previously, we believe the financial and strategic logic of a combination of our two companies is clear. The challenges for our industry in the digital age continue. Tribune has itself faced numerous challenges and leadership changes over the last few years. We believe Gannett is uniquely willing and able to propel Tribune into the position of strength that will allow its beloved and historic publications and other assets to survive and thrive in this challenging environment. By combining, we would create a company with the financial stability and flexibility equipped to preserve journalistic integrity, high standards and excellence for years to come. We would be able to both empower our journalists and facilitate the creation of exceptional content while delivering stockholder value. Given the opportunity to benefit from the significant premium and near-term liquidity, we are confident that Tribune's stockholders will embrace our offer. As we have indicated previously, we would prefer to negotiate a transaction with Tribune, but we have determined that making your stockholders aware of our all-cash proposal is necessary, given Tribune's attempts to delay constructive engagement. This matter is of the highest priority to us, and we continue to be ready to dedicate significant resources to completing due diligence and negotiating a transaction on an expedited basis. We have been working closely with our financial advisors at Methuselah Advisors and our legal advisors at Skadden, Arps, Slate, Meagher & Flom LLP and have completed an extensive analysis of the proposed transaction based on publicly available information. As well, we are confident that the regulatory approvals necessary to consummate the proposed transaction will be obtained. This proposal, which is unanimously supported by our Board, is a non-binding expression of our current views, which remains, among other things, subject to satisfactory completion of due diligence, the negotiation, execution and delivery of a mutually satisfactory definitive merger agreement, approval of the definitive agreement by your and our Boards of Directors, approval of the transaction by your stockholders, and receipt of customary regulatory approvals. Given the substantial value represented by our offer and the other compelling benefits of a combination of Gannett and Tribune, we are confident that Tribune's non-management stockholders will support our proposal. Continuing to refuse to engage in a dialogue with us will only serve to delay the ability of your stockholders to receive the value represented by our all-cash offer. We therefore are prepared to consider all alternatives to complete this transaction. In the meantime, we remain eager to meet with you and your team as soon as possible to progress the transaction. CLOSE USA TODAY's Roger Yu breaks down the offer on the offer on the table for Gannett to purchase Tribune Publishing Company. USA TODAY 10/8/2015, McLean, VA. Gannett headquarters building in McLean, Va. (Photo: Tim Loehrke, USAT) Gannett Co. (GCI), which owns USA TODAY and more than 100 other media properties across the country, said Monday it offered to buy Tribune Publishing (TPUB) for about $815 million, its second big expansion move since spinning off from its former parent less than a year ago. In a letter to Justin Dearborn, CEO of Tribune, which owns the Los Angeles Times, Chicago Tribune and nine other dailies, Gannett CEO Robert Dickey reiterated Monday a private April 12 offer to pay $12.25 per share, a 63% premium to Tribune’s closing stock price last Friday. Gannett’s deal includes assuming $390 million of Tribune’s debt outstanding as of Dec. 31, 2015. The offer price is about 5.6 times Tribune’s estimated 2016 earnings before interest, taxes and other items (EBITDA). Gannett estimates about $50 million a year in “synergies” savings. Gannett owns USA TODAY plus 107 local news organizations including the Detroit Free Press, Cincinnati Enquirer, Des Moines Register, the Milwaukee Journal Sentinel and Arizona Republic. “We believe Tribune shares the new Gannett’s unwavering commitment to journalistic excellence and delivering superior content on all platforms,” Dickey said in a statement Monday. “In this respect, the proposed combination of Gannett and Tribune would bring together two highly complementary organizations with a shared goal of providing trusted, premium content for the readers and communities we serve.” Dickey said in an interview that since the original offer, he has had several phone calls with Tribune’s non-executive chairman, Michael Ferro, and Dearborn. But the letter says Tribune has refused to begin formal negotiations, prompting Dickey to reveal the bid publicly. “What we’re hoping for is to sit down with Tribune’s board and work out a transaction. We’re confident that, with cooperation between the companies, we can complete due diligence in a very timely fashion and execute an agreement,” Dickey said. Shares of Tribune closed Friday at $7.52, up 2.6%, and shot up 53% Monday to close at $11.50. Gannett stock was up 6.5% to close at $16.79. "The board is now engaged, with the assistance of its advisors, in a thorough review," Tribune Publishing said Monday in a statement. "The board is committed to acting in the best interests of shareholders and will respond to Gannett as quickly as feasible." In August 2014, Tribune Publishing was spun off from its former parent, Tribune Co., which was looking to focus on broadcasting and eliminate its exposure to the declining print advertising market. Tribune Co. then renamed itself Tribune Media Co. "Since the beginning of 2016, Tribune Publishing has been undertaking a transformation and has made significant organizational changes," Tribune Publishing said. "With a focused strategy, unmatched collection of award-winning content and brands, and the right leadership team in place, Tribune Publishing is well-positioned to create value for shareholders." The Los Angeles Times building in downtown Los Angeles is seen on Oct. 5. (Photo: Richard Vogel, AP) The bid, unanimously supported by Gannett’s board, comes less than a month after the McLean, Va.-based company completed its $280 million acquisition of Journal Media Group, which includes the Milwaukee Journal Sentinel and The (Memphis) Commercial Appeal. Gannett spun off from its former parent in June of last year, retaining the publishing business but not its broadcast assets. At the time of the spin-off, Dickey made it clear that his strategy amid the turbulent print advertising market involved consolidating more media properties to strengthen its position on local reporting and local marketing and advertising. Its 108 newspapers and their affiliated digital properties now comprise the newly created USA TODAY NETWORK, a nationwide news organization that taps into the combined resources of its 3,800 journalists to report on major national as well as local issues, and that focuses heavily on investigative and watchdog reporting. If Gannett were to complete the deal, it would expand the NETWORK in strategic markets by owning dominant newspapers in major metro areas, such as the Los Angeles Times, The Baltimore Sun, Hartford Courant, Chicago Tribune and Orlando Sentinel. Tribune “fills a number of geographical gaps for us,” Dickey said. “We think bringing their publications to the USA TODAY NETWORK strengthens the overall NETWORK.” While it owns some of the most august brands in journalism, Tribune has undergone some turbulent changes in recent months. Beset by falling print ad sales, the Chicago-based company reported a net loss of $2.8 million in 2015, swinging from a profit of $42.3 million a year earlier. In February, Ferro, who made his fortunes in technology and health care, paid $44.4 million to buy a 16.6% stake in Tribune to become its largest single shareholder. Soon after the acquisition, Ferro removed Tribune’s CEO, Jack Griffin, and replaced him with a longtime associate, Dearborn. “We believe Gannett is uniquely willing and able to propel Tribune into the position of strength that will allow its beloved and historic publications and other assets to survive and thrive in this challenging environment,” Dickey wrote in his letter to Dearborn. “Given the opportunity to benefit from the significant premium and near-term liquidity, we are confident that Tribune’s stockholders will embrace our offer.” Gannett, which was virtually debt free when it spun off from TEGNA, plans to finance the deal using its existing $500 million line of credit and tapping the debt market for additional funds required. That will leave the company’s debt-to-EBITDA ratio “at about 1 to 1.5,” Dickey said. That is “considerably below industry standards,” he said. While he will seek savings in duplicative functions, Dickey said the goal isn’t to cut back on editorial resources. “We have tremendous respect for the employees of Tribune Publishing. Our goal is to write great journalism at every location. And you can’t do that without having proper resources. I’m committed to investigative, public service journalism. We will always be efficient at our job. I focus on areas that don't impact journalism,” he said. “Tribune Publishing has little history operating as an independent public company and may be unable to reach earnings targets,” wrote Lance Vitanza, an analyst at CRT Capital, in an investor note Monday. “Amid the turmoil, we find Gannett's interest intriguing…The bid doesn't appear to be out-of-line with recent public and private market multiples for newspaper companies.” Follow Roger Yu on Twitter: @RogerYu_ Read or Share this story: http://usat.ly/1StX87b

Summary: On April 12, USA Today owner Gannett made a private offer to Tribune Publishing: We want you, for roughly $815 million, or $12.25 per share. USA Today's own report on the offer notes that at $12.25 a share, Gannett is offering a 63% premium over Friday's closing stock price. The Wall Street Journal notes the deal is valued at $400 million, as Gannett would be acquiring Tribune's debt. Gannett CEO Robert Dickey says that after making the offer he communicated with Tribune CEO Justin Dearborn and Chair Michael Ferro via phone. But after what Dickey frames as "Tribune's continued refusal to begin constructive discussions with us," Gannett went public with the proposal. "What we're hoping for is to sit down with Tribune's board and work out a transaction," says Dickey. What that transaction would mean: Joining USA Today and 107 local outlets (the Detroit Free Press, Milwaukee Journal Sentinel, Arizona Republic, etc.), would be 11 dailies, chief among them the Los Angeles Times, Chicago Tribune, and Baltimore Sun. Tribune "fills a number of geographical gaps for us," Dickey says. Gannett thinks it could shave $50 million in costs due to overlaps, though Dickey says editorial wouldn't be slashed. The Journal notes that Gannett claims 12% of America's daily newspaper circulation; Tribune Publishing, 5%.

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Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention This invention is directed to surgical masks worn by surgeons, dentists and the like and which covers the nose, mouth, chin and portions of the cheeks and more particularly to such a mask that does not interfere with the breathing of the mask wearer. 2. Description of the Prior Art One type of prior art mask is molded of a rubber composition in the general shape of a cup. Strengthening ribs are formed across the central portion of the mask and an elasticized band is attached to the mask at diametrically located positions adjacent the mask periphery and may be placed about the wearer&#39;s head to hold the mask in the desired position. A deformable metal band is place across the outer surface of the mask at a location which will overlie the nose of the wearer. The metal band is generally deformed to fit the nose just above the flare of the nostrils to prevent inhalation of unfiltered airborne particles. However, the forces applied by the band tend to close the nostrils making breathing more difficult. Another type of mask widely used is one made of paper and having a generally rectangular shape in its initial condition. The mask is made of a two-ply blank, fan-folded with the fold lines extending in the mask&#39;s long dimension. A border is formed along the four edges of the mask to prevent the mask being over extended by the user. The user pulls the top and bottom edges in opposite directions to extend the mask from the wearer&#39;s nose bridge to below his chin. A cord is joined to the mask along the two ends of each of the two short dimensions and each cord looped over a wearer&#39;s ear to hold the mask in place. A deformable metal band is mounted between the two plies within the top edge border. The band is also deformed about the nose above the flare of the nostrils. The manner in which such metal band is deformed may also make breathing more difficult as described above. SUMMARY OF THE INVENTION The instant invention overcomes the difficulties noted above with respect to the prior art surgical masks and provides a structure and teaching which can be applied to such prior art masks, and to new masks, to overcome the problems noted with respect to them. The structure constitutes an easier breathing device which can be added to existing masks or built into masks initially. On the interior surface of the mask in the portion that will overlie the nose, there is placed a pliable, central support member in the shape of an elongated rectangle with two parallel, spaced apart surfaces. On each surface is placed a pressure sensitive adhesive. One surface is applied to the mask interior surface, while the second is attached to the exterior of the wearer&#39;s nose after the protective release layer has been removed. Between the interior surface of the mask and the one surface of the central support member that engages the interior surface of the mask is placed a support means. The support means is shorter and narrower than the central support member so that the support means is adhered to the central support member and the support means and the central support member are coupled to the interior surface of said mask by the adhesive portion beyond that covered by the support means. The support means is made of a flexible, resilient material, such as a plastic strip, which is caused to take on an arch-type configuration when the central support member is attached to the wearer&#39;s nose. The elastic memory of the plastic strip will endeavor to restore it to its initial flat condition. By selecting the material of the support means and its dimensions, the restoration forces applied by the support member will substantially equal the forces applied to the wearer&#39;s nose by the central support member so as to cancel them out and thus not interfere with the wearer&#39;s breathing. It is an object of this invention to provide a novel facial surgical mask with an easier breathing device. It is an object of this invention to provide a novel facial surgical mask which does not interfere with the breathing of the wearer of the mask. It is an object of this invention to provide a mechanism which can be used with presently available facial surgical masks to improve the breathing of the wearer of the mask. It is a further object of this invention to provide a facial surgical mask with a mechanism according to the teachings of this invention which adheres to the nose of the wearer of such mask. It is yet another object of this invention to provide a facial surgical mask with a mechanism according to the teachings of this invention which reduces the possibility of the mask interfering with the breathing of the wearer. It is still another object of this to provide a facial surgical mask with a mechanism according to the teachings of this invention which adheres to the nose of the wearer of such mask and which comprises a support means to counter the closing effects on the nostrils of the wearer&#39;s nose so that there is no interference with the breathing of the wearer of the mask. Other objects and features of the invention will be pointed out in the following description and claims and illustrated in the accompanying drawings, which disclose, by way of example, the principles of the invention, and the best mode which is presently contemplated for carrying them out. BRIEF DESCRIPTION OF THE DRAWINGS In the drawings, in which similar elements are given similar reference characters: FIG. 1 is a front elevational view of a molded, cone-type facial surgical mask presently available in the field. FIG. 2 is a rear elevational view of the surgical mask of FIG. 1 FIG. 3 is a fragmentary, front elevational view of an alternative fastening means which can be used with the facial mask of FIGS. 1 and 2. FIG. 4 is a side elevational view, partly in section, of the mask of FIG. 2 taken along the line 4--4. FIG. 5 is a front elevational view of a rectangular-type facial surgical mask presently available in the field. FIG. 6 is a rear elevational view of the surgical mask of FIG. 5. FIG. 7 is a rear elevational view of a surgical mask similar to that shown in FIG. 6 and showing other construction details found on different available rectangular type masks. FIG. 8 is a rear elevational view of a surgical mask as shown in FIG. 2 and incorporating an easier breathing device constructed in accordance with the concepts of the invention. FIG. 9 is a top plan view of the component layers which make up an easier breathing device. FIG. 10 is a front elevational view of an easier breathing device with the release layer removed to make certain details visible. FIG. 11 is a fragmentary top view, taken along the inclined surface of a nose to which the easier breathing device mask is applied. FIG. 12 is a fragmentary, simplified, side elevational view of an easier breathing device applied to a wearer&#39;s nose and with most components removed to permit a better understanding of the operation of the easier breathing device. FIG. 13 is a rear elevational view of a rectangular-type facial mask similar to that shown in FIG. 6 and incorporating an easier breathing device constructed in accordance with the concepts of the invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS Turning now to FIGS. 1, 2 and 4, there is shown a cone-type facial surgical mask 20 of the type sold by 3M Company under its trademark ASEPTEX and designated 1800+Single Use Fluid Resistant Molded Surgical Mask. The mask is molded from a material which contains natural latex. It could also be fabricated from paper, cardboard or composite materials and coated with latex or other rubber materials, if desired. The body 22 has a generally cup-shaped configuration with a generally oval-shaped periphery 24. The desired position of the mask 20 is to overlay most of the wearer&#39;s nose, mouth, chin and part of the cheeks. The periphery 24 is somewhat flattened at 26 to better accept the wearer&#39;s nose. The front surface 28 of body 22 is formed with a plurality of raised strengthening ribs 30 which extend substantially across the width of body 22. Between adjacent ribs 30 are concave recesses 32. The rear surface 34 of body 22 is the reverse. Raised broad, convex ribs 36 extend between narrow recesses 38. At various points along the periphery 24, the body 22 is flared to better match the user&#39;s face. The depth of the cup, as shown by arrow D in FIG. 4, is chosen such that the rear surface 34 of body 22 does not contact the user&#39;s nose. The mask 20 is held in its desired position on the wearer&#39;s face by an elastic strip 40 anchored at its ends to body 22 by metal clips 42 or the like. The length of strip 40 is too short to go about the wearer&#39;s head in its at-rest position and is stretched in order to pass over the head and then allowed to relax to securely grip the wearer&#39;s head. If desired, the single, elastic strip 40 can be replaced with two strings 44, 46, as shown in FIG. 3, which can be tied at the back of the wearer&#39;s head. A malleable metal band 50 of aluminum or another suitable material is placed adjacent the flattened portion 26 of periphery 24 of body 22. Once mask 20 is comfortably in the desired position on the wearer&#39;s face, the wearer can distort the band 50 to conform to the wearer&#39;s nose and form a tight seal around the nose to prevent inhalation of unfiltered airborne particles. However, the tight seal about the nose to prevent entry into the mask of unfiltered airborne particles often has the effect of closing or substantially reducing the size of the nasal passages thus making it very hard for the wearer to breathe. Thus, the wearer is forced to make a choice or compromise. Turning now to FIGS. 5, 6 and 7, there is shown a rectangular-type facial surgical mask 60 of a type presently available in the field. A version of such mask is sold by Astra USA, Inc. under the trademarks ASTRA PRO™ MAGIC ARCH® POSITIVE FACIAL LOCK® Mask. A multi-ply body 62, made of soft paper is joined adjacent its top edges 64, bottom edges 66 and side edges 68, 70. The mask could also be made of cloth or composite materials, if desired. The plies can be joined by using pressure, heat or a combination thereof and may employ a heat or chemically activated adhesive. Bottom edge 66 is continuous since it represents the folding of the web to provide two plies which make up the body 62. A zone 72, adjacent bottom edge 66, is used to further seal the plies. The plies are joined along side edges 68 and 70 by applying a sealing device in the zones 74, 76, respectively. Top edge 64 is sealed using two spaced apart, parallel sealing lines 78, 80 which provides a pocket 82 therebetween to receive a malleable metal strip 84 as is shown in phantom line in FIG. 6. The body 62 is fan-folded flat having fold edges 86, 88 and 90 visible on front face 63 of body 62. When the mask 60 is subjected to forces operating in two opposite directions each such force applied to one of the top edge 64 or bottom edge 66, the central part of mask 60 within the zones 72, 74, 76 and 80, the mask body 62 expands so that it can fit the wearer&#39;s face from below the bridge of the nose to under the chin. The width of body 62 is such that the side edges 68, 70 cover portions of the cheeks of the wearer. The sealed edges of the body 62 do not expand but act as a pivot for the expansion of the main body 62 portion between such sealed edges. A loop 92 is joined at its ends 94, 96 to mask body 62 adjacent edge 70 and fits over one ear of the wearer. A similar loop 92 is joined at its ends 94, 96 to mask body 62 adjacent edge 68 and fits over the wearer&#39;s other ear to hold mask 60 in the desired position over the wearer&#39;s face. On the back face 65 of body 62 the fold edges 98, 100 and 102 are visible. FIG. 7 shows some modifications of the mask 60 shown in FIG. 6. The loop 92 end 96 can be positioned closer end 94 along side zone 74 of mask 60&#39;. The end 96 of the loop 92 in zone 76 can also be moved closer to end 94. Also, plastic strips 104, 106 (shown in phantom line) can be inserted between the plies. By choice of the plastic employed and the dimensions of such strips 104, 106 and due to the fact that the ends of strips 104, 106 are confined by the seal zones 74, 76, strip 104 will form an arch over the lower portion of the wearer&#39;s nose while strip 106 will form an arch below the nose, whereby direct contact is prevented between back face 65 of body 62 and the nose of a wearer which could block the nostrils and impede or prevent breathing. To prevent access to this area of the mask and the inhalation of unfiltered airborne particles, in addition to the previously described metal strip 84, there is a metal strip 108 (shown in phantom line) in the zone 72, which strip 108 is parallel to side edges 68, 70. This strip 108, made of a metal similar to that used for strip 84, is deformed by the wearer just behind the jaw bone to get a tight seal of the mask 60&#39; to the face of the wearer. The deformation of strip 84 of mask 60&#39; will have the same effect on breathing as described above. Referring now to FIGS. 8 to 13, the details of easier breathing devices and their use with presently available facial surgical masks is shown. FIG. 8 shows a cone-type facial surgical mask 20 with the easier breathing device 118 installed to the rear surface 34 of body 22 adjacent the somewhat flattened portion 26 that fits over the nose of the wearer. The metal strip 50 on the front surface 28 may be removed or left in place without distortion to fit the wearer&#39;s nose. An easier breathing device 118, constructed in accordance with the concepts of the invention is shown in FIG. 9. An attachment means 120 has a central support means or base layer 122 of the desired length, width and thickness, which may be made of foam or other materials which are solid but pliable and can be deformed as needed without applying undue force. A closed cell foam strip was found to work well. On a first surface 124 of central support means 122 is placed a further means or a second layer 126 of a pressure sensitive adhesive which will adhere to the face of a wearer of the mask 20. This layer 126 of adhesive is covered by a release layer 128 which is removed just before the mask 20 is applied to the wearer&#39;s face. On the second surface 130 of the central support means or base layer 122 is a first layer 132 of an adhesive material. The adhesive material of first layer 132 may be a heat, light, UV-light or chemically activated or cured adhesive, or as shown, may be a pressure-sensitive layer upon which is placed support means or counter-balance strip 134. As shown in FIG. 10, the support means or counter-balance strip 134 is shorter and narrower than the first adhesive layer 132 of attachment means 120 and thus first adhesive layer 132 extends beyond the perimeter of counter-balance strip 134. A release layer 136 is placed over the first adhesive layer 132 and counter-balance strip 134 to prevent contact with the adhesive layer 132 until it is desired to mount the easier breathing device 118 on the inner surface 34 of mask 20. FIG. 11 shows the easier breathing device 118 installed to a wearer&#39;s nose N. The view is from the top but taken along the top ridge of the nose N itself. As shown by FIG. 12 the device 118 is generally perpendicular to the top ridge of the nose N. By removing the release layer 136 (not shown in FIG. 11) the first adhesive layer 132 beyond the periphery of support means 134 is exposed for attachment to the rear surface 34 of mask body 22. The device 118 is positioned with respect to mask 20 so that mask 20 occupies the desired position whereby the mask 20 covers the wearer&#39;s nose, mouth, chin and portions of both cheeks and attachment means 120 and support means 134 are positioned with respect to the top of the flare of the nostrils of the nose N of the wearer as shown in FIG. 12. The manner of operation of the easier breathing device 118 is now set forth with respect to FIG. 12 which shows the nose N of a wearer of the facial surgical mask 20 and only the attachment means 120 and support means 134. The pliable central support means 122 of attachment means 120 be formed so that it conforms to the outer contour of the nose N and is then attached to the skin of the nose N by means of second adhesive layer 126. The combined weight of device 118 and mask 20 presses upon the outside of the nose N which tends to restrict or may even fully close the nasal passages interfering with or preventing breathing. The counter-balance strip or support means 134 is initially a flat strip and has an elastic memory which tends to return the strip 134 to its initial condition providing the modulus of elasticity of the strip has not been exceeded and the strip permanently deformed. The material from which strip 134 is fabricated, and its length, width and thickness are so chosen that the forces produced by strip 134 in trying to go from the arch shape shown in FIG. 11 to its initial flat shape equal the forces applied to the nose N by mask 20 and device 118. The forces produced by strip 134 are in the opposite direction to those applied by mask 20 and device 118 and thus cancel such forces. Accordingly, it is as if no forces are applied to the nostrils and there is no interference with the normal breathing of the mask wearer. FIG. 13 shows the placement of an easier breathing device 118 on a rectangular-type facial surgical mask 60. The top edge of device 118 is aligned with top edge 64 of the mask 60 and centered with respect to side edges 68 and 70. The metal strip 84 may be removed or allowed to deform as the mask 60 is applied providing it is not made to closely conform to the nose. The installation and use of device 118 will be substantially the same as described above with respect to FIGS. 9 to 12. While there have been shown and described and pointed out the fundamental novel features of the invention as applied to the preferred embodiments, as are presently contemplated for carrying them out, it will be understood that various omissions and substitutions and changes of the form and details of the devices illustrated and in their operation may be made by those skilled in the art, without departing from the spirit of the invention.

Summary: A facial surgical mask of the type worn over the nose, mouth, chin and portions of the wearer&#39;s cheeks and held in place by one or more bands extending about the back of the head or about the ears. A first band on the inside surface of the mask attaches to the wearer&#39;s nose while a second band counterbalances the first to minimize the forces applied to the wearer&#39;s nose.

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Summarize: By. Alexandra Klausner. The brother of Sochi Olympic skeleton silver medalist Noelle Pikus-Pace along with his three children were finally found today after going missing while on an Idaho camping trip near the Gospel lakes over the weekend. Jared and his children were found walking near mountains about an hour away from their campsite. They were unharmed other than bravely sporting some poison ivy and mosquito bites. Noelle Pikus-Pace posted an update on her Facebook just 15 minutes ago saying that Saturday morning after camping for the night, her brother and his children aged five, seven, and nine went fishing a couple of miles from their campsite and got lost after a fellow camper misdirected them. A miracle: Noelle Pikus-Pace's(right) brother, Jared Pikus (left), and three of his children were found. yesterday walking along this road in Idaho. Spotted: This is the road in Idaho hours from Jared Pikus Pace's campsite where he was found walking with his three kids. Off the map: Noelle Pikus-Pace posted this map of her brother's approximate location near the Gospel Lake in Idaho where she believes he went missing. 'They kept going thinking it would be around the bend. Night came so he used the raft that they had with them and slept beneath it. They drank water from a snow stream and he said he bribed the kids to keep moving forward with a bag of jolly ranchers that he had in his backpack. The terrain was very rocky and steep. He said the kids were amazing,' wrote Noelle. Rescue crews first started looking for Jared Pikus-Pace and his children on Sunday morning after they didn't return home on Saturday afternoon, reported NBC. Pikus and his family were last seen by camper on Saturday in a non-hikeable area of the campground. Jared has a wife at home along with three more children, including a very young baby. Noelle called upon others to help find her brother and his family on her Facebook account today. Her first post read, 'PLEASE say a prayer! My brother, Jared, and 3 of his kids went camping Friday and were supposed to come home yesterday morning. Search and rescue found their vehicle but can't find them! They are in northern Idaho. His wife is at home with their other 3 kids including a very new baby. Please say a prayer for them and those looking for them!' Later in the day Pikus took to her social media accounts again and even posted a map of her brother and his family's approximate location. Found: Sochi Olympic skeleton silver medalist Noelle Pikus-Pace's brother, Jared Pikus (top left), was rescued with his three children today after going missing on a camping trip in Idaho. Swift as silver: Noelle Pikus-Pace won the silver Sochi Olympic medal for skeleton, a sport in which a person rides a sled face down at very high speeds. Her prayers were answered: Noelle asked friends and family to pray for her missing brother who was found with his three children today out of harm's way. 'Search and rescue are still looking for my brother and his three little kids. This is where they are. They have helicopters and teams searching. They found their SUV and campsite. Still no sign of them. He was supposed to be back yesterday afternoon. Please keep them in your prayers! I'll keep you updated,' wrote Noelle Pikus-Pace. Noelle feared for her brother's life because she heard that the terrain around the Gospel lakes is very rough. 'They said the terrain is pretty rough in the area. He went fishing with the kids at one of the Gospel lakes but they didn’t take a boat. They have life jackets and usually fish from the lake side, Pikus Pace told NBC. 'My husband, kids and I just spent a few days with them and were making our way home (stopping along the way). We were in Burley, about 6 hours south of their home and are now driving up there,' said Noelle. Noelle asked friends and family to prayer for her brother and his family and thankfully her prayers were answered and her brother and his family are out of harm's way

Summary: Jared Pikus-Pace, the brother of Sochi Olympic skeleton silver medalist Noelle Pikus-Pace, was found today. Jared along with three of his six children were found walking on a road after they got lost while hiking near the Gospel Lake on Saturday. Jared and his three children aged five,seven, and nine are alive and in good health. Jared had to bribe his children to keep walking with a bag of jolly ranchers and they drank water from a snow stream. Jared has a wife at home and is a father of six children including a very young baby.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Women's Health Research Act of 1993''. SEC. 2. ESTABLISHMENT OF OFFICE OF RESEARCH ON WOMEN'S HEALTH. (a) In General.--Title IV of the Public Health Service Act, as amended by section 2 of Public Law 101-613, is amended-- (1) by redesignating section 486 as section 485A; (2) by redesignating parts F through H as parts G through I, respectively; and (3) by inserting after part E the following new part: ``Part F--Research on Women's Health ``SEC. 486. OFFICE OF RESEARCH ON WOMEN'S HEALTH. ``(a) Establishment.--There is established within the Office of the Director of NIH an office to be known as the Office of Research on Women's Health (in this part referred to as the `Office'). The Office shall be headed by a director, who shall be appointed by the Director of NIH. ``(b) Purpose.--The Director of the Office shall-- ``(1) identify projects of research on women's health that should be conducted or supported by the national research institutes; ``(2) identify multidisciplinary research relating to research on women's health that should be so conducted or supported; ``(3) carry out paragraphs (1) and (2) with respect to the aging process in women, with priority given to menopause; ``(4) promote coordination and collaboration among entities conducting research identified under any of paragraphs (1) through (3); ``(5) encourage the conduct of such research by entities receiving funds from the national research institutes; ``(6) recommend an agenda for conducting and supporting such research; ``(7) promote the sufficient allocation of the resources of the national research institutes for conducting and supporting such research; ``(8) ensure that women are appropriately represented as subjects in projects of clinical research conducted or supported by the national research institutes; and ``(9) prepare the report required in section 486B. ``(c) Coordinating Committee.-- ``(1) In carrying out subsection (b), the Director of the Office shall establish a committee to be known as the Coordinating Committee on Research on Women's Health (hereafter in this subsection referred to as the `Coordinating Committee'). ``(2) The Coordinating Committee shall be composed of the Directors of the national research institutes (or the designees of the Directors). ``(3) The Director of the Office shall serve as the chair of the Coordinating Committee. ``(4) With respect to research on women's health, the Coordinating Committee shall assist the Director of the Office in-- ``(A) identifying the need for such research, and making an estimate each fiscal year of the funds needed to adequately support the research; ``(B) identifying needs regarding the coordination of research activities, including intramural and extramural multidisciplinary activities; ``(C) supporting the development of methodologies to determine the circumstances in which obtaining data specific to women (including data relating to the age of women and the membership of women in ethnic or racial groups) is an appropriate function of clinical trials of treatments and therapies; ``(D) supporting the development and expansion of clinical trials of treatments and therapies for which obtaining such data has been determined to be an appropriate function; and ``(E) encouraging the national research institutes to conduct and support such research, including such clinical trials. ``(d) Advisory Committee.-- ``(1) In carrying out subsection (b), the Director of the Office shall establish an advisory committee to be known as the Advisory Committee on Research on Women's Health (hereafter in this subsection referred to as the `Advisory Committee'). ``(2) The Advisory Committee shall be composed of no fewer than 12, and not more than 18 individuals, who are not officers or employees of the Federal Government. The Director of the Office shall make appointments to the Advisory Committee from among physicians, practitioners, scientists, and other health professionals, whose clinical practice, research specialization, or professional expertise includes a significant focus on research on women's health. A majority of the members of the Advisory Committee shall be women. ``(3) The Director of the Office shall serve as the chair of the Advisory Committee. ``(4) The Advisory Committee shall-- ``(A) advise the Director of the Office on appropriate research activities to be undertaken by the national research institutes with respect to-- ``(i) research on women's health; ``(ii) research on gender differences in clinical drug trials, including responses to pharmacological drugs; ``(iii) research on gender differences in disease etiology, course, and treatment; ``(iv) research on obstetrical and gynecological health conditions, diseases, and treatments; and ``(v) research on women's health conditions which require a multidisciplinary approach; ``(B) report to the Director of the Office on such research; ``(C) provide recommendations to such Director regarding activities of the Office (including recommendations on the development of the methodologies described in subsection (c)(4)(C) and recommendations on priorities in carrying out research described in subparagraph (A)); and ``(D) assist in monitoring compliance with section 486(b)(8) regarding the inclusion of women in clinical research. ``(5)(A) The Advisory Committee shall prepare a biennial report describing the activities of the Committee, including findings made by the Committee regarding-- ``(i) compliance with section 486(b)(8); ``(ii) the extent of expenditures made for research on women's health by the agencies of the National Institutes of Health; and ``(iii) the level of funding needed for such research. ``(B) The report required in subparagraph (A) shall be submitted to the Director of NIH for inclusion in the report required in section 403. ``(e) Representation of Women Among Researchers.--The Secretary, acting through the Assistant Secretary for Personnel and in collaboration with the Director of the Office, shall determine the extent to which women are represented among senior physicians and scientists of the national research institutes and among physicians and scientists conducting research with funds provided by such institutes, and as appropriate, carry out activities to increase the extent of such representation. ``(f) Definitions.--For purposes of this part: ``(1) The term `women's health conditions', with respect to women of all age, ethnic, and racial groups, means all diseases, disorders, and conditions (including with respect to mental health)-- ``(A) unique to, more serious, or more prevalent in women; ``(B) for which the factors of medical risk or types of medical intervention are different for women, or for which it is unknown whether such factors or types are different for women; or ``(C) with respect to which there has been insufficient clinical research involving women as subjects or insufficient clinical data on women. ``(2) The term `research on women's health' means research on women's health conditions, including research on preventing such conditions. ``SEC. 486A. NATIONAL DATA SYSTEM AND CLEARINGHOUSE ON RESEARCH ON WOMEN'S HEALTH. ``(a) Data System.-- ``(1) The Director of NIH, in consultation with the Director of the Office, shall establish a data system for the collection, storage, analysis, retrieval, and dissemination of information regarding research on women's health that is conducted or supported by the national research institutes. Information from the data system shall be available through information systems available to health care professionals and providers, researchers, and members of the public. ``(2) The data system established under paragraph (1) shall include a registry of clinical trials of experimental treatments that have been developed for research on women's health. Such registry shall include information on subject eligibility criteria, sex, age, ethnicity or race, and the location of the trial site or sites. Principal investigators of such clinical trials shall provide this information to the registry within 30 days after it is available. Once a trial has been completed, the principal investigator shall provide the registry with information pertaining to the results, including potential toxicities or adverse effects associated with the experimental treatment or treatments evaluated. ``(b) Clearinghouse.--The Director of NIH, in consultation with the Director of the Office and with the National Library of Medicine, shall establish, maintain, and operate a program to provide information on research and prevention activities of the national research institutes that relate to research on women's health. ``SEC. 486B. BIENNIAL REPORT. ``(a) In General.--With respect to research on women's health, the Director of the Office shall, not later than February 1, 1994, and biennially thereafter, prepare a report-- ``(1) describing and evaluating the progress made during the preceding 2 fiscal years in research and treatment conducted or supported by the National Institutes of Health; ``(2) describing and analyzing the professional status of women physicians and scientists of such Institutes, including the identification of problems and barriers regarding advancements; ``(3) summarizing and analyzing expenditures made by the agencies of such Institutes (and by such Office) during the preceding 2 fiscal years; and ``(4) making such recommendations for legislative and administrative initiatives as the Director of the Office determines to be appropriate. ``(b) Inclusion in Biennial Report of Director of NIH.--The Director of the Office shall submit each report prepared under subsection (a) to the Director of NIH for inclusion in the report submitted to the President and the Congress under section 403. ``SEC. 486C. AUTHORIZATION OF APPROPRIATIONS. ``For the purpose of carrying out this part, there are authorized to be appropriated $25,000,000 for fiscal year 1994, and such sums as may be necessary for each of the fiscal years 1995 and 1996.''. (b) Requirement of Sufficient Allocation of Resources of Institutes.--Section 402(b) of the Public Health Service Act (42 U.S.C. 282(b)) is amended-- (1) in paragraph (10), by striking ``and'' after the semicolon at the end; (2) in paragraph (11), by striking the period at the end and inserting ``; and''; and (3) by inserting after paragraph (11) the following new paragraph: ``(12) after consultation with the Director of the Office of Research on Women's Health, shall ensure that resources of the National Institutes of Health are sufficiently allocated for projects of research on women's health that are identified under section 486(b).''. SEC. 3. OBSTETRICS AND GYNECOLOGY PROGRAM OF NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT. Subpart 7 of part C of title IV of the Public Health Service Act (42 U.S.C. 285g et seq.) is amended by adding at the end the following section: ``program regarding obstetrics and gynecology ``Sec. 452A. The Director of the Institute shall establish and maintain within the Institute an intramural laboratory and clinical research program in obstetrics and gynecology.''.

Title: Women's Health Research Act of 1993 Summary: Women's Health Research Act of 1993 - Amends the Public Health Service Act to establish within the Office of the Director of the National Institutes of Health (NIH) the Office of Research on Women's Health (Office). Establishes in the Office the Coordinating Committee on Research on Women's Health and the Advisory Committee on Research on Women's Health. Directs the Secretary to: (1) determine the extent to which women are represented among senior physicians and scientists of the national research institutes and among physicians and scientists conducting research with funds provided by such institutes; and (2) carry out activities, as appropriate, to increase the extent of such representation. Requires the NIH Director to establish a data system for the collection, analysis, and dissemination of information regarding research on women's health conducted or supported by the national research institutes, including a registry of clinical trials of experimental treatments. Requires the NIH Director to establish and operate a program to provide information on research and prevention activities of the the national research institutes that relate to research on women's health. Authorizes appropriations. Directs the Secretary of Health and Human Services to ensure that NIH resources are sufficiently allocated for projects of research on women's health.

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Summarize: Background The Congress generally provides budget authority to an agency for use during a specific period, referred to as the period of availability. During this period of availability, the agency may incur new obligations, for example, those for goods and services, and charge them against the appropriation. At the end of the period of availability, the appropriation expires, meaning that it may not be used to incur new obligations. Prior to fiscal year 1991, an appropriation account maintained its fiscal year identity for 2 years after its period of availability expired. After the 2- year period, the remaining obligated balance and unobligated balance were transferred and merged into “M” accounts and merged surplus authority, respectively, with the obligated and unobligated balances of previously expired appropriation accounts available for the same general purpose. The M accounts and merged surplus authority were available to pay and adjust valid obligations incurred prior to expiration. However, because M accounts and merged surplus authority had no fiscal year identity, upward adjustments were not limited to the balances from any particular appropriation. From 1956, when the Congress established the M accounts and merged surplus authority, through December 1990, federal agencies’ reported that M accounts and merged surplus authority had grown to over $70 billion, with DOD reported to have about $50 billion, over 70 percent, of this total. In addition, the use of the merged surplus authority to fund upward adjustments to M accounts increased dramatically. For example, DOD’s use of large amounts from the merged surplus authority to cover upward adjustments to M accounts and other expired surplus accounts increased each year over a 5-year period from about $57 million in fiscal year 1985 to $560 million in fiscal year 1989. DOD’s use of large amounts from the merged surplus authority to cover upward adjustments to obligations prompted the Congress to pass legislation in 1990 to strengthen its oversight and control over expired appropriations. Among other things, the 1990 law eliminated the merged surplus authority and M accounts. Instead, under the 1990 law, an expired appropriation account remains available for 5 years, during which it may be used for recording, adjusting, and making disbursements to liquidate obligations that were properly chargeable to the account. At the end of the 5-year period, the appropriation account closes, and any remaining obligated and unobligated balances are canceled. The closed appropriation account may not be used for obligation or expenditure for any purpose. After an account closes, obligations and adjustments to obligations that would have been properly chargeable to the account before closing may be charged to currently available appropriations subject to certain limitations. Even in those cases in which current appropriations must be used to make payments after appropriation accounts close, the closed appropriation accounts are still subject to the provisions of the Antideficiency Act. Among other things, the act prohibits officers or employees of the government from making or authorizing obligations or expenditures that exceed the amounts available in appropriations or funds. Therefore, agencies must continue to maintain current and accurate records of disbursements attributable to the canceled appropriations to ensure that payments do not exceed the originally appropriated amounts. If a violation of the Antideficiency Act occurs, section 1351 of title 31 requires the head of the agency to immediately report to the President and the Congress all relevant facts and a statement of actions taken. Also, under section 1349 of title 31, the officer or employee who violates the act is subject to administrative discipline. The Office of Management and Budget (OMB) issues guidelines for budget execution for executive branch agencies in OMB Circular A-34. Section 40 of that circular sets forth the requirements for reporting Antideficiency Act violations. Section 40.6 requires that an agency head transmit a report to the President, through OMB, providing information about the violation, including the appropriation involved, the date and amount of the violation, the identity of the person(s) responsible for the violation, a statement of administrative discipline taken, a statement regarding the sufficiency of the agency’s fund control regulations, and a statement of any actions taken to prevent similar future violations. Section 40.6 also requires the agency head to send an identical report to the Speaker of the House of Representatives and the President of the Senate. DOD implements the provisions of the Antideficiency Act and OMB’s Circular A-34 guidance in Volume 14 of its Financial Management Regulation. Chapters 4 through 7 of that volume contain detailed instructions on investigating and reporting on possible Antideficiency Act violations. Chapter 9 contains instructions on discipline for violations. Contract Reconciliation As stated previously, because of the need to keep accurate records, agencies may, in limited circumstances, adjust their records pertaining to closed appropriation accounts. For example, if an agency determines that the balances reflected in the records of a closed account are erroneous because of reporting and clerical errors, it may adjust those records to correct the errors. An agency may also adjust its records if it discovers that a disbursement actually made before an appropriation account closed and properly chargeable to an obligation incurred during the appropriation’s period of availability was either not recorded at all or was charged to the wrong appropriation. Neither of these types of adjustments constitutes charging obligations against or disbursing funds from the closed appropriation accounts. They represent corrections of the accounting records. Since the appropriations, in effect, no longer exist, these adjustments affect only the agency’s records. They have no effect on the availability or use of obligated or unobligated balances formerly contained in those appropriation accounts. According to DFAS headquarters officials, DFAS Columbus makes about 99 percent of DOD’s annual closed appropriation account adjustments. DFAS Columbus relies on the Mechanization of Contract Administration Services (MOCAS) system to process DOD contract payment transactions and CRS for performing and processing reconciliation transactions. MOCAS and CRS transactions are recorded in various DFAS accounting systems used for the military services and other DOD organizations that maintain the official accounting records, including the status of canceled funds. During fiscal years 1997 through 2000, DFAS Columbus’ records showed that it made about $10 billion of adjustments affecting closed appropriation accounts. DFAS Columbus conducts two types of contract reconciliations—limited and full scope. Limited scope reconciliations are usually performed to resolve problems that require immediate action, such as when there are insufficient funds on the cited accounting classification reference number (ACRN) to pay a contractor’s invoice. These reconciliations generally result in making only those adjustments necessary to resolve the problems that are preventing payment of the invoice. A full scope reconciliation is generally performed if a limited scope reconciliation failed to adequately resolve a problem or a contract is being prepared for final close-out due to contract completion. Depending on the size of the contracts, these type of reconciliations can result in large numbers of adjustments since they involve identifying and correcting all errors made over the life of the contract. DFAS Columbus uses CRS to help identify discrepancies between a contract’s hard copy documentation and information recorded in MOCAS. To complete this process, reconciliation staff re-enters into CRS all hard copy documents pertaining to the contract. These include documentation for obligations, invoices, disbursements, shipments, and modifications. After all the hard copy information has been entered into CRS, it reconstructs the payments in accordance with the payment instructions input by the reconciliation staff. For example, if the contract payment terms specified that certain payments were to be applied to specific ACRNs in the contract and the reconciliation staff input these data into CRS, it would apply the payments accordingly. However, if the staff failed to instruct CRS to apply the payments as specified in the contract, CRS’ default program would redistribute the payments on an “oldest funds first” basis. Either way, after redistributing the payments, CRS generates accounting transactions to adjust MOCAS’ contract obligation and payment history so that it will agree with CRS. Before the adjustments to the closed appropriation account records can be made, however, reconciliation staffs are required to request approval from the appropriate fund holder. Once DFAS Columbus has provided the appropriate fund holder with the request for approval, the fund holder has 45 days to accept or reject the proposed adjustments. DOD procedures provide that if DFAS Columbus does not receive a response to the request for approval within 45 days of notification, the adjustment will be considered approved and processed accordingly. $615 Million of Adjustments Were Illegal or Otherwise Improper Our review of $2.2 billion of the fiscal year 2000 closed appropriation account adjustments found that $615 million (28 percent) were either illegal ($146 million) or otherwise improper ($469 million). These adjustments should not have been made because the initial disbursements (1) occurred after the appropriation being charged had already canceled, (2) occurred before the appropriation being charged was enacted, or (3) were charged to the correct appropriation in the first place and no adjustment was necessary. Also included in the $615 million were $105 million of adjustments that were not sufficiently documented to establish that they were proper. These were considered to be improper because agencies must be able to provide documentation to show that adjustments are legal and that they changed an incorrect charge to a correct one. Table 1 provides additional details on the $615 million of adjustments that should not have been made. See appendix II for a detailed list of illegal or otherwise improper adjustments. Appropriation Already Canceled When Disbursement Was Made The 1990 account closing law specifically provides that closed appropriation accounts are not available for expenditures. We found that about $108 million of the adjustments resulted in charging appropriation accounts that had closed before the disbursements were made. These adjustments produced the same result as if DOD had made expenditures from and charged closed appropriation accounts at the time the disbursements were made. Therefore, these adjustments violated the 1990 account closing law. Following are several examples in which the adjustments had the effect of spending canceled funds from the closed accounts. In December 1999, DFAS Columbus recorded an adjustment that changed $79 million of disbursements from charges against fiscal years 1993 through 1995 research and development appropriations to charges against a fiscal year 1992 research and development appropriation. According to documentation in the contract files, the adjustment was to correct previous disbursing errors by redistributing the payments in accordance with the payment terms specified in the contract. The payment terms of the contract specified that payments should be made using “oldest funds first.” Under this instruction, payments should be charged to the oldest appropriation cited on the contract until the obligated balance has been exhausted for that appropriation. Subsequent payments are then charged to the next oldest available appropriation, and so on, until all the funds are used up or the contract is complete. Making the adjustment that charged the $79 million of disbursements to the closed fiscal year 1992 research and development account used up the unspent balances in that appropriation account and freed up funds on still open 1993 through 1995 research and development appropriation accounts for other disbursement charges. We found that charging the $79 million of disbursements to the fiscal year 1992 research and development appropriation was illegal because the disbursements were made in February 1999—4 months after the fiscal year 1992 research and development appropriation account had closed on September 30, 1998. DFAS Columbus officials agreed that the adjustment violated the 1990 law and should not have been made. According to the officials, the adjustment was made by CRS, which lacked the necessary controls to prevent this from occurring. (CRS weaknesses are discussed in more detail later in this report.) After we pointed out this illegal adjustment, DFAS Columbus personnel responsible for resolving this problem told us that they would reverse the transaction and begin a review of the contract to determine what they will need to do to properly record the $79 million of disbursements, including using current-year appropriations if necessary. In another case, DOD was not able to pay an $832,907 invoice properly chargeable to a fiscal year 1993 appropriation account because sufficient funds were not available in the cited ACRN to pay the invoice. This prompted a reconciliation of the contract to determine why sufficient funds were not available to pay the invoice. Among other things, the reconciliation identified $721,037 of overpayments made from August 1991 through February 1992 that were charged to a fiscal year 1989 aircraft procurement appropriation—an appropriation account that was closed at the time of the reconciliation. To recover this overpayment, DFAS Columbus offset the amount against a current invoice. On September 28, 2000, DFAS Columbus paid the contractor $111,870, which DFAS Columbus calculated as the net amount owed to the contractor after deducting the $721,037 of overpayments from the $832,907 invoice amount. The 1990 account closing law requires that collections related to closed appropriation accounts be deposited in the Treasury as miscellaneous receipts. However, we found that instead of forwarding the $721,037 of overpayments, which it collected through payment offset, to Treasury, DFAS Columbus processed adjustments to move the overpayments to a fiscal year 1993 ACRN on the contract that was still open and available for new disbursements. It then used the overpayments to offset the amount owed to the contractor. In discussing this adjustment with DFAS Columbus officials, they told us that it is standard practice to offset collections against invoices when making payments, even if closed accounts are involved. However, they agreed that, regarding collections of disbursements made from accounts that closed before collection, the proper procedure would be to forward those collections to the Treasury and use other sources of funding to pay the invoice. Therefore, in this case, the proper procedure would have been to (1) record the collection of the overpayments as a deposit to Treasury’s miscellaneous receipts and (2) use available appropriations to cover the $721,037 needed to replace the funds returned to Treasury. While DFAS Columbus officials could not tell us why they had not followed proper procedures in this case, they told us they would review this transaction to determine how to correct the error. Appropriation Not Yet Enacted When Disbursement Was Made Under 31 U.S.C. 1502 (a), an appropriation may be used to pay only those expenses properly incurred during the appropriation’s period of availability. However, we found that over $38 million of the closed appropriation account adjustments resulted in charging disbursements to appropriation accounts that had not yet been enacted at the time the disbursements were actually made. For example, in January 2000, a total of $21 million of disbursements charged to fiscal years 1989 and 1990 appropriations were changed to charges against fiscal years 1998 and 1999 missile procurement appropriations. Since the actual disbursements were for expenses that were incurred before the fiscal years 1998 and 1999 appropriations were enacted, charging disbursements to these two appropriations violated 31 U.S.C. 1502 (a). Further, included in the $21 million were $9.9 million in overpayments, which the contractor identified as a return of funds that were paid from the fiscal years 1988 through 1990 appropriations. However, these appropriations had been canceled before the overpayments were returned. As discussed earlier in this report, the 1990 law requires that the collection of canceled funds be deposited into the Treasury as miscellaneous receipts. However, we found that instead of forwarding the overpayments to the Treasury, DFAS Columbus redistributed the $9.9 million to current and expired appropriations that were funding the still-open contract. In discussing these errors with responsible DOD officials, they agreed that the $21 million adjustment and the $9.9 million redistribution were incorrect and should not have been made. According to the officials, they plan to reverse the adjustments and determine what actions are required to correct the accounting records, including returning the $9.9 million to the Treasury. No Adjustment Was Necessary Closed account adjustments totaling $364 million were not necessary because the initial payments had been charged to the correct appropriations and should not have been adjusted. DOD made these adjustments during contract reconciliations to try to correct errors in recording disbursements made under the contracts. Generally, these reconciliations were initiated if DOD could not pay invoices because other disbursements had been erroneously recorded against the wrong appropriations funding contracts. For example, in November 1999, DFAS Columbus received an invoice from a contractor for $685,000. DFAS Columbus could not pay the contractor because there were not sufficient funds available in the cited ACRN to pay the invoice. As a result, DFAS Columbus reconciled the fiscal year 1988 contract, which resulted in over $590 million of adjustments affecting closed appropriation accounts. Our review of these found that $210 million of the adjustments should not have been made because the actual disbursements—some of which were made over 10 years earlier—were recorded correctly. As a result of this process to free up sufficient funds to pay the $685,000 invoice, DFAS Columbus made unnecessary adjustments affecting the closed accounts. Thus, the reconciliation resulted in at least $210 million of accounting errors that did not exist before the reconciliation took place. Insufficient Documentation In order to adjust its records, an agency must have sufficient documentation to show that the adjustment is legal and changed an incorrect charge to a correct one. However, neither DOD nor we could find sufficient documentation in DOD’s accounting and contract records to support about $105 million of closed appropriation account adjustments. For example, in June 2000, DFAS Columbus made an adjustment that changed over $2.4 million of disbursements from charges against a fiscal year 1993 appropriation that had not yet canceled to a fiscal year 1992 appropriation that had canceled. According to the contract files, the adjustment was to correct a previous disbursing error. However, in reviewing the contract files for this adjustment, neither DOD nor we could identify the original invoice or other supporting documentation to show which appropriation should have been charged for the goods or service. We considered these types of unsupported adjustments improper because DOD must be able to provide documentation to show that the adjustments are legal and that they changed incorrect charges to correct ones. DOD is researching these transactions further to determine if additional documentation can be located to support the adjustments. Contract Reconciliation Process Lacks Certain Fundamental Controls The contract reconciliation process lacks the controls necessary to ensure that adjustments to closed appropriation accounts are proper. Our review disclosed that CRS routinely processed billions of dollars of closed appropriation account adjustments without regard to the requirements of the 1990 account closing law, which prohibits making disbursements or obligations from closed accounts. Further compounding this shortfall was the lack of oversight on how contract modifications were written and processed, which, when combined with the deficiencies in CRS, changed the payment terms of some contracts to free up current and expired funds. As noted earlier, these deficiencies contributed to at least $615 million in illegal or otherwise improper closed account adjustments during fiscal year 2000. Contract Reconciliation System Weaknesses CRS does not contain a control to determine if an appropriation was available at the time the disbursement was made. Specifically, CRS does not compare the actual disbursement date with the appropriation involved in the adjustment to ensure that the adjustment does not result in charging disbursements (1) back to an appropriation that had canceled before the actual disbursement was made or (2) forward to an appropriation that had not yet been enacted at the time the actual disbursement was made. Furthermore, unless reconciliation staff input specific payment terms into CRS, it will redistribute disbursement charges on an “oldest funds first” basis. As a result, it will change disbursements charged to current and expired appropriation accounts to charges against older appropriation accounts, even if they are closed, to use up any unspent balances in the closed appropriation accounts. This is a violation of the 1990 act. DOD officials responsible for contract reconciliations told us that they have been using CRS to perform reconciliations since 1995 and that redistribution of disbursement charges to the closed appropriation accounts was a routine practice. They also acknowledged that, in 1996, they identified the CRS control weakness that allowed them to make adjustments that charged disbursements to closed accounts. However, they could not tell us why they had not taken action to correct the problem—a problem (1) they estimated would have cost $24,460 to fix in 1996 and (2) that resulted in illegal and otherwise improper adjustments. After we identified the problem during our review and brought it to their attention, DOD officials implemented the control in May 2001. In regard to the other needed control that would prevent making adjustments that charge disbursements against appropriation accounts that had not yet been enacted when the disbursement was actually made, DOD officials told us that they were not aware of this problem until we brought it to their attention during our audit. They told us they were reviewing the process to determine how to include this control in CRS and planned to have it implemented by September 2001. In an effort to address some of the control weaknesses we identified, DFAS Columbus required all reconciliation staff to attend a 3-day refresher course on the adjustment process. According to DFAS Columbus officials, all of the 235 DFAS Columbus employees involved in performing contract reconciliations had completed this course as of March 2001. DFAS Columbus also issued interim guidance in January 2001 in response to our audit findings on the weaknesses in CRS. The implementing memorandum noted that the current process using CRS was flawed because it did not require that disbursements be compared with dates relevant to the use of appropriations before the adjustments were processed. It specifically noted that the current process allowed for the processing of an adjustment even if the date of the disbursement occurred after the date the funds canceled. To address these problems, the interim guidance provides detailed steps for staff to follow to ensure that the person preparing the adjustments performs the manual comparison of dates and that documentation is prepared to support the comparison process. DFAS Columbus’ efforts are a step in the right direction. However, addressing adjustments that involve moving disbursement charges forward to appropriations that were not enacted at the time the disbursement occurred could enhance the interim guidance. DFAS Columbus officials agreed that the interim guidance should also address this issue and were in the process of determining what needs to be done. They did not provide us an estimated date for completing this guidance update. Contract Modifications or Other Instructions Led to Improper Adjustments During our review of transactions, we noticed that DFAS Columbus personnel were relying on contract modifications or other contracting officers’ informal instructions to justify adjustments that changed disbursements from charges against current or expired appropriations to charges against closed appropriation accounts. The modifications or informal guidance either instructed that payments be made using “oldest funds first” or specifically instructed that disbursements be moved from one ACRN to another. According to DFAS Columbus officials, when contracting officers modify contracts or issue informal instructions to make adjustments that charge disbursements against older appropriations, DFAS Columbus will usually comply with the modifications without regard to the 1990 closed account law. We found several instances in which DFAS Columbus followed either a contract modification or informal instructions and made adjustments that resulted in improperly charging closed appropriation accounts. We met with DOD accounting and acquisition officials to discuss the use of contract modifications to change payment terms to move disbursements back to closed appropriation accounts. According to the officials, there was no DOD policy that prohibited changing the payment terms of the contract. In fact, DFAS Columbus personnel responsible for reconciliations told us that when they receive a contract modification from a contracting officer to change payment terms, they take it for granted that the contracting officer wants DFAS Columbus to apply the modification retroactively on an “oldest funds first” basis in order to use up the unspent canceled funds. Air Force contracting officials also acknowledged that they intended for DFAS Columbus to apply the modification retroactively. They told us that the use of modifications was intended to redistribute the disbursements to the unspent canceled appropriations in order to avoid having to request current-year funds to replace the canceled appropriations. After pointing out this practice to DOD accounting and acquisition managers, they agreed that contract modifications should not be written with the purpose of using up the unspent balances in closed appropriation accounts. They told us that they would review this issue and would revise their policy to prevent this type of contract modification. Factors Contributing to the Need to Correct Disbursement Errors The $1.6 billion of closed account adjustments to which we do not take exception are part of a bigger problem facing DOD. While these adjustments were adequately documented corrections of prior errors, the fact that this large amount of transactions required adjustment in the first place is a result of a long-standing and well documented problem DOD has with correctly accounting for and recording obligations and the corresponding disbursements. For example, for fiscal year 1999, DFAS data showed that almost $1 of every $3 in contract payment transactions was for adjustments to previously recorded payments—$51 billion of adjustments out of $157 billion in transactions. Over the years we have issued numerous reports discussing DOD’s financial management problems, and we have designated DOD financial management as a high-risk area since 1995. In July 2000, we testified that DOD’s inadequate process and control problems contribute to billions of dollars in improper payments. For example, we noted that for fiscal years 1994 through 1999, DFAS records showed that defense contractors returned over $5.3 billion of overpayments and erroneous payments due to contract administration actions and payment-processing errors. In 1997, we reported several key factors contributed significantly to problems in DOD’s payment process that, for the most part, still exist today. Among these is the “long line of accounting” DOD uses to allocate payment information among numerous accounting categories. The following discussion from our 1997 report describes this complex and convoluted process, which, we found, also contributed to DOD’s $2.7 billion closed account adjustments for fiscal year 2000. DOD uses what it refers to as a “long line of accounting” to accumulate appropriations, budget, and management information for contract payments. This long line of accounting can contain over 50 alphabetical and numerical symbols that identify such things as the military service, appropriation, beginning and ending fiscal years, and appropriation suballotment. The buying activities (generally military services that are responsible for administering these funding segments) assign a two- character ACRN to each accounting line containing unique information. DFAS Columbus allocates the payments to the ACRNS. Compounding the problem is that the type, quantity, and format of information vary among the services since there is not standardization of account transactions. Figure 1 is a sample of an accounting line. Contracts can be assigned anywhere from 1 to over 1,000 ACRNs. A contract with numerous ACRNs involves extensive data entry, increasing the chance for errors, and manual payment processing. When buying activities assign numerous ACRNs to a complex contract, payment allocations to the ACRNs can be time-consuming. For example, as we noted in our 1997 report, a single payment on a contract with many ACRNs took DFAS Columbus 6 to 8 hours to process. The contractor, required to bill by ACRN, took 487 pages to assign $2.1 million in costs and fees to 267 ACRNs. Ten of the ACRNs cited by the contractor had insufficient obligation balances to cover the payment, according to DFAS Columbus’ records. The remaining 257 ACRNs corresponded to eight annual appropriations covering from 1 to 5 fiscal years and included Army, Air Force, and general defense funds. Of the 257 transactions processed, 38 were for less than $10, and some involved debits or credits for pennies. Unresolved discrepancies, such as insufficient funds on some ACRNs, had persisted for about 3 years. Our 1997 report pointed out that even a simple purchase could cause extensive and costly rework if assigned numerous ACRNs. We noted that a $1,209 Navy contract for children’s toys, candy, and holiday decorations for a child care center was written with most line items (e.g., bubble gum, tootsie rolls, and balloons) assigned separate ACRNs. A separate requisition number was generated for each item ordered, and a separate ACRN was assigned for each requisition. In total, the contract was assigned 46 ACRNs to account for contract obligations against a single appropriation. To record this payment against the one appropriation, DFAS Columbus had to manually allocate the payment to all 46 ACRNs. In addition, the contract was modified three times—twice to correct funding data and once to delete (deobligate) the funding on the contract for out-of-stock items. The modification deleting funding did not cite all the affected ACRNs. DFAS Columbus made errors in both entering and allocating payment data, compounding errors made in the modification. Consequently, DFAS Columbus allocated payment for the toy jewelry line item to fruit chew, jump rope, and jack set ACRNs—all of which should have been deleted by modification. Contract delivery was completed in March 1995, but payment was delayed until October 1995. DFAS Columbus officials acknowledged that this payment consumed an excessive amount of time and effort when compared to the time to process a payment charged to only one ACRN. A single ACRN would also have significantly reduced the amount of data entered into the system and the opportunities for errors. Our 1997 report also noted that sometimes contracts do not require contractors to provide the accounting detail on the invoices necessary to allocate the payments. In these instances, DFAS Columbus prorates payments among ACRNs. How the payments are prorated may have little relationship to which activities received what goods and services, which may cause funds to be initially paid from the wrong appropriations. These errors will require correction at some later date. We found that this problem still exists. DFAS Columbus officials agreed that they are still experiencing many of the same problems today that we identified in our 1997 report. They told us that when contracts do not include detailed payment terms, they would generally pay a contractor’s invoice by prorating the payment across all the applicable ACRNs on the contract. When this happens, sufficient funds may not be available at some later date because unspent balances in the older appropriations cancel. This can lead to contract reconciliations that redistribute the payments using some other form of payment allocation. Conclusion Because DOD had not established the requisite systems, controls, and managerial attention required to properly account for its disbursements consistent with the 1990 account closing law, DOD made at least $615 million of illegal or otherwise improper adjustments during fiscal year 2000 alone. DOD was aware of the limitations the account closing law placed on the availability of canceled appropriations and that the law was enacted because of previous abuses by DOD’s use of old appropriations. The department also knew that a major system used to control its use of appropriations allowed for disbursements to be charged in a way that was inconsistent with that law. However, it did nothing to fix the system, although it estimated the cost to do so to be minimal. The $615 million of adjustments we identified in this report as illegal or otherwise improper must be immediately reversed. Furthermore, at a minimum, DOD will need to effect changes to its systems, policies, procedures, and the overall weak control environment that fostered these practices and served to perpetuate this problem. Top management must clearly demonstrate its commitment to adhering to the account closing law and eliminating the abuses of appropriations law. In the short term, this will require that DOD immediately fix the system, contract modification problems, and inadequate policies and procedures identified in this report. In the long term, DOD will need to resolve its overall financial management problems, including the lack of leadership and accountability that have been the subject of numerous reports and recommended corrective actions over the years. Recommendations for Executive Action We recommend that the Secretary of Defense direct the Under Secretary of Defense (Comptroller) to direct the Director of the Defense Finance and Accounting Service to immediately reverse adjustments to closed accounts identified in this report as illegal or otherwise improper; determine the correct accounting for these adjustments after they have ensure that the requisite controls are properly included and operating effectively in CRS so that it will prohibit charging disbursements against appropriation accounts that (1) are closed or (2) have not yet been enacted at the time the disbursements are actually made; revise current policies and procedures pertaining to closed account adjustments to include specific detailed guidance to require that future adjustments to closed appropriation accounts satisfy the criteria discussed in this report; and establish a monitoring program for future adjustments to closed appropriation accounts and make clear to managers that they will be held accountable if abuses are identified. To the extent that DFAS Columbus is unable to make correcting adjustments because insufficient balances remain in the correct accounts, we also recommend that the Secretary of Defense direct the Under Secretary of Defense (Comptroller) to investigate and report on these adjustments as required by the Antideficiency Act, 31 U.S.C. 1351, and implementing guidance. We also recommend that the Secretary of Defense direct the Under Secretary of Defense for Acquisition, Technology, and Logistics to issue a policy that prohibits the writing of contract modifications to change the payment terms of a contract if the change would result in illegal or otherwise improper adjustments, as defined in this report, affecting closed appropriation accounts. We are sending copies of this report to interested congressional committees. We are also sending copies of this report to the Secretary of Defense; the Principal Deputy Under Secretary of Defense for Acquisition, Technology, and Logistics; the Secretaries of the Army, Navy, and Air Force; the Director of the Defense Finance and Accounting Service; and the Director of the Office of Management and Budget. We will make copies available to others upon request. If you have any questions regarding this report, please contact me at (202) 512-9505. Key contributors to this report are listed in appendix III. Appendix I: Scope and Methodology To meet our first objective of assessing the adequacy of DOD procedures for adjusting closed appropriation accounts, we reviewed applicable laws, regulations, administrative guidelines, policies, and procedures. These included title 31 U.S.C. “Money and Finance,” Chapter 13, “Appropriations,” and Chapter 15, “Appropriation Accounting;” OMB Circular A-34; Volume 3, Chapter 10 of DOD’s Financial Management Regulation, “Accounting Requirements for Expired and Canceled Accounts;” and DFAS Columbus’ Responsible Contract Reconciliation Agency Guide, which defines the responsible contract reconciliation agent’s missions and responsibilities in conducting reconciliations in accordance with Office of the Secretary of Defense policy and DFAS procedures. We also reviewed DFAS Columbus’ Desk Procedure 808, “Coding of Adjustments,” that DFAS Columbus employees are to follow when using CRS to perform contract reconciliations and to correct errors. This review included the process used by DFAS Columbus to code, report, and record obligation, disbursement, and appropriation adjustments to closed accounts. We met with DFAS Columbus officials responsible for performing contract reconciliations to discuss and obtain an understanding of the process for reconciling closed account adjustments, including the roles and responsibilities of the military services in the overall process to make and approve adjustments to closed appropriation accounts. We also met with responsible DFAS Columbus officials that had responsibility for CRS to obtain an understanding of how the system processed and reported on closed account adjustments. To meet our second objective to determine if closed appropriation account adjustments complied with the 1990 account closing law, we selected 268 such adjustments valued at over $2.2 billion from a population of 4,470 adjustments valued at over $2.7 billion. The 268 adjustments were selected based on their large dollar value—generally at least $1.7 million each. The 268 adjustments were limited to closed appropriation account adjustments made during fiscal year 2000 as recorded in the DFAS Columbus CRS database. We selected the adjustments from fiscal year 2000 data because they were the most complete fiscal year data available at the time. The reviewed adjustments represented 81 percent of the closed appropriation account adjustment dollars recorded in CRS during fiscal year 2000. To assess if DFAS Columbus had adequate documentation to support the propriety of the adjustments, we analyzed financial information from DFAS Columbus’ records and reports, including contracts, contract modifications, shipping notices, invoices, payment vouchers, and schedules of adjustments. We identified and met with the DFAS Columbus officials knowledgeable about each adjustment and obtained their views on the results of our analysis. We also identified the responsible DFAS or military service locations that maintained the official account records and obtained documentation to show how each adjustment was recorded in the accounting records. We compared these documents with the DFAS Columbus adjustments and resolved any differences. We performed our work primarily at the Defense Finance and Accounting Service Center in Columbus, Ohio. We also obtained documentation from the following DFAS locations that were responsible for maintaining official accounting records: Cleveland, and Dayton, Ohio; Limestone, Maine; Omaha, Nebraska; San Bernardino, California; and St Louis, Missouri. Our review was conducted from May 2000 through March 2001 in accordance with U.S. generally accepted government auditing standards, except that we did not validate the accuracy of the number of closed account adjustments and their related dollar values in the CRS database, which was provided to us by DFAS Columbus. On May 25, 2001, we requested comments on a draft of this report from the Secretary of Defense or his designee, but none had been provided at the time we finalized our report on July 17, 2001. Appendix II: Illegal or Otherwise Improper Adjustments Appendix III: GAO Contact and Staff Acknowledgments GAO Contact Acknowledgments In addition to the contact named above, Bertram J. Berlin, Dennis B. Fauber, Jeffrey A. Jacobson, Mary Jo Lewnard, Keith E. McDaniel, Michael S. Peacock, and Harold P. Santarelli made key contributions to this report. Ordering Information The first copy of each GAO report is free. Additional copies of reports are $2 each. A check or money order should be made out to the Superintendent of Documents. VISA and MasterCard credit cards are also accepted. Orders for 100 or more copies to be mailed to a single address are discounted 25 percent. Orders by mail: U.S. General Accounting Office P.O. 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Summary: This report reviews the Department of Defense's (DOD) handling of appropriated funds from expired appropriation accounts. In 1990, Congress changed the law governing the use of appropriation accounts because it concluded that controls over them were not working. Without adequate controls, Congress was concerned that agencies could disburse money in amounts and for purposes that it had not approved. GAO found that DOD improperly charged appropriation accounts after they were closed. GAO also found that DOD did not establish the requisite systems, controls, and managerial attention required to properly account for its disbursements consistent with the 1990 account closing law. As a result, DOD made at least $615 million of illegal or otherwise improper adjustments during fiscal year 2000 alone. DOD was aware of the limitations the account closing law placed on the availability of canceled appropriations and that the law was enacted because of previous abuses by DOD. DOD also knew that a major system used to control its use of appropriations allowed for disbursements to be charged in a way that was inconsistent with the law. However, DOD did nothing to fix the system, even though the cost to do so was minimal. The $615 million of adjustments GAO identified as illegal or otherwise improper must be immediately reversed. Also, at a minimum, DOD will need to change its systems, policies, and procedures, along with the weak control environment that fostered these practices, and served to perpetuate the problem. GAO summarized this report in testimony before Congress (GAO-01-994T).

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Summarize: Il codice civile permette al possessore Se situazioni che possono essere tutelate con le azioni possessorie sono infinite e vanno dalla chiusura di un passaggio con un cancello, fino all'apposizione di fioriere per impedire l'ingresso di auto in un cortile, anche le immissioni possono generare una tutela possessoria (es. condizionatori d'aria rumorosi). Naturalmente il primo problema concreto è valutare se si è in presenza di una molestia o di un vero e proprio spoglio del bene. Come ogni azione legale, anche le azioni possessorie vanno indirizzate contro il c.d. legittimato passivo (di solito colui che ha messo in opera la molestia o la turbativa del possesso), ma come in ogni campo del diritto potrebbe non essere semplice individuare il legittimato passivo, basta pensare all'ipotesi in cui il possesso del bene non è di un unica persona, ma si è in presenza di situazioni di compossesso. Per rendere più chiara la situazione basta pensare al caso in cui uno dei proprietari di un appartamento (in comunione) cambi le chiavi dell'appartamento e impedisca agli altri comproprietari di accedere all'immobile oppure si può fare l'esempio di un lastrico solare in condominio (1117 c.c.) sul quale uno condomino apponga un condizionatore d'aria e i relativi tubi. Se si può affermare che le azioni possessorie sono applicabili anche ai beni sui quali è esercitato il compossesso, in presenza di un compossesso occorre anche coordinare le azioni possessorie con l'art. 1102 c.c., ora, risulta chiaro che i due esempi sopra fatti sono agli antipodi, in quanto uno (cambio delle chiavi) rientra nella tutela possessoria, l'altro (apposizione di un condizionatore sul lastrico comune) può rientrare nell'uso del bene comune ex art. 1102 c.c. ammesso e non contestabile tramite le azioni possessorie (in altre parole, in presenza di compossesso, le azioni possessorie trovano un limite nell'art. 1102 c.c.). Sempre in tema di legittimazione si potrebbe pensare all'ipotesi in cui il medesimo soggetto (tizio) loca un bene immobile (da caio) e stipula un contratto di comodato per alcuni beni mobili (con sempronio) per arredare la casa locata (da caio), in questa situazione l'azione di restituzione dei beni mobili in comodato va esercitata contro il proprietario dell'immobile dove sono allocati i beni mobili o contro colui che ha stipulato il contratto di comodato? Oppure si potrebbe pensare all'ipotesi in cui il soggetto che ha posto in essere la molestia perde la titolarità del bene (es. vende il fondo servente sul quale è stato apposto un cancello che impedisce il passaggio al fondo dominate) e, di conseguenza, occorre anche valutare se (e come) l'eventuale provvedimento di reintegra nel possesso possa essere "opponibile" al nuovo titolare del bene.

Summary: La Cassazione del 13.4.2015 n.7365 ha stabilito che in tema di azioni possessorie, quando la successione nel possesso (1169 cc) si verifica dopo la proposizione della domanda di reintegrazione (1170 cc) o di manutenzione (1168 cc), nei confronti dell'autore dello spoglio opera l'art. 111 cpc senza che abbia rilievo la trascrizione prevista da detta norma, poiché la domanda di reintegrazione o di manutenzione non va trascritta ex art. 2653 n. 1 cc. Per cui la sentenza pronunciata contro il dante causa è titolo eseguibile anche nei confronti dell'acquirente.

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Summarize: How To Make A Mom Friend in 20 Simple Steps: 1. Visit a park at a high traffic time (between 10AM-Noon or 4-6PM) 2. Select your MARK. Choose a woman with a ch...ild around your child's age. For compatibility's sake, make sure her grooming/hygiene level matches yours (if you look like a swamp dwelling yoga instructor who has never heard of shampoo, find another mom who looks like a swamp dwelling yoga instructor who has never heard of shampoo). 3. Discreetly observe your mark's interactions with her child. Does she look a little bored, like she'd rather by in bed with a caramel macchiato scrolling Instagram? Good she's normal. 4. Convince your child to play in the vicinity of your new best friend. 5. Creepily encourage your child to share/play with her child whether or not they want you. If your child resists, whisper "Stop being selfish, all you do is take, Mommy needs this," in their ear. Promise them chocolate if you have to. It's go time. 6. Using your best preschool teacher voice, say "Hello" to your mark's child so that the mom notices you. Then to your child say, "Hey (your kid's name), isn't (her kid's name) nice/fun/beautiful/gifted?" 7. Smile at your new best friend. (If she has already picked up her child and moved away from you with an alarmed look on your face abandon the mission entirely and go home before the police arrive). 8. If she smiles back, make a funny remark about the weather or life. Don't get too heavy right away. Things like, "I'm desperately lonely" or "I crave human touch" or "What's your opinion on blood oaths?" would all fall into the "Too Much" category. 9. Tell the mark you first name. "Mommy" is not your first name. If you can't remember your government name, check your driver's license. It should be right on there. Ask your mark her name. 10. If your mark hasn't abruptly left yet, ask her if she lives in the area. Use a relaxed, disinterested voice so she doesn't think you're a burglar. 11. Talk to your mark about life. Be truthful but avoid controversial subjects such as politics, religion, or forward-facing carseats. 12. Make sure your chid is still at the park with you. 13. Ask your mark about her life. While she's talking, maintain eye contact. If she fidgets, don't ask her if she has to potty. That's only for children. 14. At some point during the conversation, look at your child and your mark's child and say, "Wow, they play so well together" even if your kid has her kid in a headlock. Just say it. 15. When one of you is getting ready to leave, admit you've already forgotten her name and have her say it back to you. Repeat it three times to help burn it into your brain. Then say, "Hey, we're both legally responsible for minors, would you like to be legally responsible for our minors together sometime in my home if you don't think I'm a murderer or in yours unless I get a bad vibe upon entry?" 16. If she says yes, exchange phone numbers. Let her know that you only text and that under no circumstances should she ever attempt to call you, ever. Use a threatening tone so she gets it. 17. Go home and get lost in your life. 18. Wonder why you don't have friends. 19. Remember the park interaction but figure your mark probably forgot about you or hates you by now, who knows. 20. Two weeks later, visit the park. Begin again from Step 1. My first novel Confessions of a Domestic Failure about a first-time mom who feels like motherhood has personally slapped her in the face comes out in four days! It's fiction but based on my experiences trying not to ruin my children. I hope you guys like it. :) Preordering it so it arrives sooner than later is easy and fun: Amazon print: https://www.amazon.com/Confessions-Domestic-Fa…/…/0778330680 · Amazon Kindle: https://www.amazon.com/Confessions-Domestic-Fa…/…/B01N3JKY1D · iBooks: https://geo.itunes.apple.com/…/confessions-do…/id1173523939… · Nook: http://www.barnesandnoble.com/w/confessions-of-…/1125091896… · B&N Print: http://www.barnesandnoble.com/w/confessions-of-…/1125091896… · Kobo: https://www.kobo.com/…/eb…/confessions-of-a-domestic-failure · Google: https://play.google.com/…/Bunmi_Laditan_Confessions_of_a_Do… share tweet pin email While followers of Honest Toddler may spend their days chuckling at the comical musings of the pretend toddler voiced by author Bunmi Laditan, it's Laditan's own social media accounts that have recently gotten parents laughing. One of Laditan's recent posts shows the chicken nuggets and french fries she served to her own toddler for dinner. Lest anyone place judgement on the nutritional value of the meal, Laditan cleared a few things up in the viral post. "The chicken nuggets are organic, free-range, rescue chickens who communicated to me in chicken language that they wanted to die to nourish my children. I coated them in gluten-free almond meal and probiotics before baking them on a tray lightly sprayed with coconut oil and colostrum," she wrote. "The box was a craft I did with my children this afternoon while I was being mindful with them in our television-free living room. Those aren't grease stains on the box, they are tears of joy from my toddler. He's so thankful for every meal." In another post, Laditan shared a photo of leftover-meatballs-turned-tacos, which she explained caused some confusion for her three children, ages 9, 6 and 2. "My kid asked me why there was no shredded cheese, guacamole, or anything else and I explained that the theme for tonight is prison and that these are prison tacos so stay in school," Laditan wrote in the post. Other posts to the account show Laditan's attempts to do yoga amidst the mess of children's toys, the fruit she added to her wine in hopes of a more nutritious diet and tri-colored pasta she served her kids, telling them it was "Skittles pasta" and to "taste the rainbow." Laditan, who lives with her family in Quebec, Canada, says that she considers her writing to be a love letter to other moms. "I struggle with insecurities about mothering all the time — if I'm doing enough, being too tough, being too weak, feeding he right foods," Laditan told TODAY Parents. "It's such a hard gig, being a mom. I just want people to read my work to feel understood and like they're enough. I want them to laugh. I guess deep down, that's what I want for myself. My posts help me find community with other moms." Bunmi Laditan "It's such a hard gig, being a mom," said Laditan. "I just want people who read my work to feel understood and like they're enough." While Laditan pokes fun at varying parenting styles in her posts, the author of "The Honest Toddler: A Child's Guide to Parenting" and "Toddlers are A**Holes: It's Not Your Fault" says she considers herself to be a fairly "crunchy" mom. "I had one home birth. I breastfed — probably not long enough, but I did," said Laditan. "(The posts) are more about making fun of my own insecurities that always swirl around about how I can do things better. I think moms enjoy them because they help relieve some of the internal pressure we carry around all the time." Bunmi Laditan talks about giving up something very important for the sake of her son For parents, bedtime is sacred. Our children’s bedtime, that is. It’s the time of day where we can finally do the things we want to do without tiny hands and voices beckoning us. That’s why a recent viral post from a mom who’s been there is striking a chord with parents. Because we’ve all been there. Bunmi Laditan, the hilarious mom behind Honest Toddler, wrote about a recent night where she had to set aside her hopes and dreams of sweet silence to be with her young son. She talks of that amazing feeling when a parent finally has the kids asleep and they can do whatever they want. And then, the cry from one of the bedrooms. We all know that cry. She writes, “Night time is my time. While the days are for work, cleaning, and errands, once the last child breathes heavily and steadily in their bed, I come alive in a new way. Silence descends upon my home and I’m free to do whatever I’d like.” It is a precious time, especially when your kids are very small. And when it’s intruded upon, the urge to quash whatever your child is asking for is strong. But as Laditan shares, sometimes, you just need to be there. She explains that he cried out an hour after having gone to sleep and after listening for a moment, she knew he wasn’t going back to bed on his own. After going into his room and trying all the usual “tricks” to help him fall back asleep, she surrendered to it, despite her desire to do otherwise. “Nothing worked and I felt that familiar frustration rising. I didn’t want to be here, in his room, battling with the most difficult version of him. I wanted to lie down, read, watch Netflix, or eat something I shouldn’t. I deserved it. I only had an hour or so left before I’d fall prey to the sleep that’s always behind my eyes. And what if he wakes the others? The only thing worse than one awake child past their bedtime is three in the same predicament.” Her concerns are highly relatable — who among us hasn’t been there? After a tough evening of meeting your child’s every need, all you want is to sink into the couch with a glass or wine or bowl of ice cream and zone out on some mindless television. My husband and I often call that cry from the bedroom after we’ve settled in for adult time “the final insult.” Like, everything else we did all day and night for our kids wasn’t enough? They have to cut into our beloved evening hours too? Well, yes. Sometimes they do. As Laditan says, her first instinct was to protect her time to herself but quickly realized it wasn’t going to be so simple this time. So she let it happen. She was there for her son when he needed her. “I relaxed into the thin rug on the wood floor and surrendered, not to him or his needs, but to what the moment needed of me. I needed to be there and I knew it. There was no escaping this, no convincing, bribing, or threatening my way out of it. The parenting books and experienced grandmas might say different, but I could feel in my bones where I needed to be: here.” Losing out on evening alone time is a huge deal to a parent, no question, but these are the sacrifices we make. There are times where our children’s needs supersede ours and yes, it sucks. But if we are honest, we wouldn’t have it any other way. As Laditan says, our kids can teach us some important lessons, and the one she learned that night? “Sometimes you have to sit.” These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites.

Summary: If you find yourself wishing you'd had kids in a simpler time, like a few decades ago, meet Bunmi Laditan, whose satiric Facebook post about vitamin shopping has gone viral. "I own two good bras but I'm ready to spend upwards of $100 on children's vitamins," Laditan writes in describing her 45-minute chore of comparing vitamins, supplements, probiotics, and who knows what else. "Do you know what vitamins I had growing up? NONE. DAYLIGHT WAS MY VITAMIN," she writes. "Occasionally, once a year tops, my mom would get us those chalky (Flintstones) vitamins that looked liked kidney stones but we'd only have to eat them for a few days before she lost interest in our health." But that just won't do today. She goes on to gripe about the fact that, although she basically lived on Tampico ( "that's orange juice for poor people") and turned out fine, nowadays, "you don't really love your kids if you're not a paranoid mess about their physical well being and willing to spend a small fortune on dye-free toothpaste made in the woods that tastes like (elderberry) and privilege." Laditan is the author of several books, including one titled, Toddlers Are A--holes, and is the voice behind "the Honest Toddler." She's been praised before for her posts "blowing the lid off dinnertime desperation," per Today, and "reminding us of the sacrifices we make as parents," per ScaryMommy. Indeed, "nothing about modern parenting is simple and it irritates me," sums up Laditan.

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Summarize: By. Alex Greig. Getting high is going high-end in Colorado, where the state's 'ganja-preneurs' are cashing in on white-collar cannabis enthusiasts desire to partake in a refined environment. Now that Colorado citizens can buy marijuana for recreational purposes legally, catered pot parties are the new wine and cheese mixers. Event planner Jane West of Edible Events threw her first weed-friendly event at downtown Denver's high-end Space Gallery. Ganja-preneur: Jane West dreamed up Edible Events to create parties where people can consume cannabis along with quality food and drink in the company of like-minded weed-enthusiasts. Pairing cannabis with canapes is a winning formula for West, whose guests forked out $125 per ticket for the sold-out event where the wine and food were provided but the weed was BYO. West, 37, dreamed up the event soon after Colorado became the first U.S. state to legalize the sale of cannabis for recreational use. She quickly realized that there was a market for a more discerning demographic to consume pot in a social environment. 'When I looked around Denver there was nothing that really fitted my demographic, that I felt comfortable with,' she told the UK Telegraph. The inaugural party for Edible Events was themed End of Prohibition, and photographed by cannabis education group Kind Reviews. Recreational: Cannabis is becoming more popular with professional women, who love the lack of hangover and the relaxation it provides. High brow: Surrounded by artworks, guests chat, eat and consume cannabis at West first party, themed End of Prohibition. Munchies: Edible Events is fully catered with foods your average stoner can only dream about. Among her guests are sisters Candy Nuss, 59 and CynDee Williams, 62, dressed smartly, their silvery hair perfectly coiffed. Successful real estate broker Wendy Bruner, 67, also enjoyed the event, telling the Telegraph that she never liked to smoke and hates the taste of alcohol but loves the high cannabis gives her when she consumes it in the form of cakes and granola bars. 'It blows my mind how many people our age are doing it. My brain's always racing and it mellows me out. It just takes all the worry out of things. I think people will realise this is not a stepping stone and you're not going to be a cocaine freak in three months,' she said. Edible Events' soirees, to be held monthly hereafter, 'combine visually stunning venues with decadent food and libations to create a unique, unforgettable evening affair' and showcase the best Denver has to offer in 'the culinary, cannabis and art scenes,' according to the company's Facebook profile. 'I decided to have events that I would like to attend. It's really about normalising cannabis, making using it as ordinary as ordering a glass of wine. This is for people who would go to an art gallery opening, or a four course dinner, but also like cannabis,' West told the Telegraph. Pot party: Guests brought their own vaporizers, cannabis-infused baked goods or joints to the event. All aboard: The luxury bus where guests could smoke instead of huddling in a corner of the parking lot as in days of old. High times: Colorado is blazing trail after becoming the first state to legalize the sale of marijuana for recreational use. Her guests brought their own cannabis in the form most pleasing to them - edible, vaporiser, joint - and in turn were provided with a delicious menu of weed-friendly foods such as chicken parmesan lollipops in sofrito and brie, mango and poblano chilli quesadillas. The food is specifically designed with the weed-consumer's mouth in mind, moist with sauces, cheese and cream and full of more-ish flavors. 'Munchies for foodies,' West calls it. It's a far cry from the stereotype of the dim stoner slacker munching down a bag of Cheeto's and watching Beavis and Butthead. Guests who preferred to consume their weed the traditional way - rolled and smoked - were provided with a luxury bus bedecked with peacock feathers where they could smoke to their hearts' content. The company's goal, reads the Edible Events Facebook page is to'maximize your cannabis experience and stimulate your heightened awareness of taste, smell and sight.' Tunes: A dj spins some tracks to enhance the mellow vibe. Wrapped up: The burgeoning cannabis economy is creating new business opportunities for entrepreneurial Coloradans. The conversation flowed at the inaugural Edible Events cannabis party. 'I wanted to create an event where consuming cannabis is just the same as consuming alcohol, so it's really just normalizing it,' she said. 'I don’t want to use the word "pot" or "weed" or "smoke" or "joint,"' West told The New York Times. 'If we redefine it as consuming cannabis, then maybe people will be more open to that. There are only so many hoodie-wearing stoners in town. This needs to be opened up to other demographics.'

Summary: The first Edible Events party was held in Denver Saturday, catering to the city's well-heeled cannabis enthusiasts. The party was held at a high end art gallery in downtown Denver. Guests paid $125 per ticket for the fully catered event which was BYOC (bring your own cannabis) Event planner Jane West says she wants consuming weed to become just as acceptable as drinking wine. Guests brought their own pot-infused foods, vaporizers or joints to the event. Joint smokers were provided with a luxury bus outside the non-smoking gallery. The event will be held monthly for guests keen to create a new, high-end pot scene.

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Summarize: If it was up to Kanye West, we'd all be strutting around in flesh-coloured body stockings next season. As ridiculous as it may sound, that may well be the case as the musician-come-designer has received an abundance of praise for his range for Adidas from fashion editors, authorities, and influencers. The collection made its way down the runway on the first day of New York Fashion Week yesterday, comprising of full body stockings, see-through skin-coloured crop tops and visible nude pants. It's certainly a new take on underwear as outerwear - one certainly not for the faint hearted. Scroll down for video. Deni Kirkova, 24, from London, stepped out on a London High Street to find out what the reaction from onlookers would be. She wore a High Street makeshift version of the types of outfits seen on the Kanye West X Adidas catwalk, right. The Kanye West Adidas Fall 2015 collection received an abundance of praise from fashion elite - but do the public agree it's a hot look? To win praise for his fashion is certainly a step forward for Kanye - now Femail are testing whether his clothes will be as popular with the public. Writer Deni Kirkova, 24, from London, trialled the full flesh-coloured body stocking look, stepping out on a London High Street to find out what the reaction from onlookers would be. She paired an American Apparel bralet (£12.50), crop top (£10), leggings (£14) and - of course - a £6 pair of pants, from Marks & Spencer, worn over the top, all in a beige flesh tone, topping off the look with a Kim K style parka jacket from Topshop. Speaking about her outfit, and whether she thought it worked, Deni said: 'The problem is visible pants. It's quite funny to walk around dressed like this now, and it's a bit of a laugh, but I wouldn't dare go out like this seriously. 'A nude crop top and leggings, though, could work. You'd have to tone this down a notch to take it from catwalk to High Street. 'I'm quite surprised that people on the street generally liked my outfit to be honest - some fashion know-it-alls praised how bold it was, noting the Kanye influence, and said that the jacket and lipstick went well with the all-nude look. 'Some though, of course, said that the pants were too much.' Deni paired an American Apparel bralet, crop top, leggings and pair of M&S pants worn over the top, all in a beige flesh tone, with a parka. Deni's look was inspired by Adidas Originals x Kanye West Yeezy collection seen in New York on Thursday. The designer's wife Kim Kardashian, 34, turned up wearing an ensemble from the range. She liked the look so much, she even posted an Instagram photo of herself, adding: 'My look for the Yeezy show!!!!!! Yeezy head to toe!!!!!' Critics similarly seemed to enjoy Kanye's latest fashion offerings with Vogue saying: 'Kanye West's New collection with Adidas lives up to the hype.' He had the fashion bible's editor-in-chief, Anna Wintour, and top designer Alexander Wang sitting front row. Kanye's wife Kim Kardashian showed up to the event wearing a brown body stocking with a grey crop top and a camo jacket. Everyone from builders to couples and fashionistas to old ladies seemed to like the bold look, much to Deni's surprise. Everyone from builders to couples and fashionistas to old ladies seemed to like the bold look, much to Deni's surprise. The problem for Deni was visible pants. 'It's quite funny to walk around dressed like this now, but I wouldn't dare go out like this seriously' A review posted on the fashion magazine's website said: 'Hems were raw and frayed, tops billowy, bottoms either skintight to slide into newly unveiled suede stiletto boots or cinched and ready to be tucked into aforementioned Boosts. 'There were immaculate flak vests and officer sweaters that looked like they’ve actually taken shrapnel, and everything was said to be unisex... On display, in rigid lines of expressionless youth, it was at the very least satisfyingly instigative for a line of sportswear.' Refinery 29 noted that Kanye's collection probably would sell well saying: 'It was a lineup of oversized jackets, sweatshirts, sweaters, and leggings, interspersed with Vanessa Beecroft-esque nude-colored underwear. 'A camo parka that Kim Kardashian also wore was a highlight, and we thought the oversized backpack was a clever accessory that'll probably make it into a healthy stack of editorials.' Kanye claims that he wanted the collection to be made up of'solutions based clothing,' saying, 'I don't want the clothes to be the life, I want the clothes to help the life.' It is also thought that it will have a relatively low price point compared to other designer clothes meaning the pieces can be snapped up by his fans. Now that the fully nude spray-on look has won approval from fashion darlings and the man on the street, will you be trying it out? Deni says maybe a nude crop top and leggings could work - without the pants. You'd have to tone this down a notch. The show is the first time Kanye has received praise for his attempts at fashion design. Kanye's collection featured baggy coats which sat over the body-stocking. Models stood in an army-like formation wearing their outfits designed by the rapper

Summary: Kanye launched his debut collection of nude body suits and crop tops for Adidas at New York Fashion Week. Wife Kim Kardashian arrived wearing the daring designs and critics have praised the collection of sports-wear. Femail takes the style from catwalk to High Street to see if a real woman can pull off underwear as outerwear look. Sent writer Deni Kirkova to test the trend and captured the public's reaction on camera (it may surprise you!)

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Summarize: Roseanne Barr I'm Done with the U.S.... I'm Moving to Israel Roseanne Barr Says She's Moving to Israel Roseanne Barr is throwing in the towel and leaving the U.S. Barr appeared on Rabbi Shmuley's podcast and said she's headed for Israel... first she says for a few months to study, but then she talks about a full-on move -- "I have an opportunity to go to Israel for a few months and study with my favorite teachers over there, and that's where I'm going to go and probably move somewhere there and study with my favorite teachers." Barr says she made a "fatal mistake" -- apologizing for her racist comment about former Obama aide Valerie Jarrett. She says when the left gets hold of an apology from someone on the other side of the political spectrum they don't let it go and eventually destroy their adversary. Barr, who is a deeply religious Jew, goes on to say, "I have some mental health issues and depression and stuff. I got to stay in the middle or I'll go dark and I don't want to go dark again." John Goodman just said Roseanne's gonna be killed off at the beginning of the reboot, "The Conners." As for moving to Israel, Barr says, "I have saved a few pennies and I'm so lucky I can go." (CNN) Roseanne Barr says she plans to be abroad when "The Conners" premieres without her this fall. Barr told her longtime friend, Rabbi Shmuley Boteach, during the latest installment of his podcast, that she won't be tuning into the "Roseanne" spinoff. Instead, she'll be halfway around the world. "I have an opportunity to go to Israel for a few months and study with my favorite teachers over there," Barr said. "And that's where I'm going to go and probably move somewhere there and study with my favorite teachers. I have saved a few pennies and I'm so lucky I can go. It's my great joy and privilege to be a Jewish woman." Barr was fired by ABC after she went on a Twitter tirade in which she made racist comments about Valerie Jarrett, a former aide to President Barack Obama. Barr also made offensive comments about Chelsea Clinton and George Soros. Read More

Summary: Roseanne Barr may be killed off in the reboot of her ABC sitcom, and the controversial comedian says she won't be tuning in. Instead, TMZ reports she's decided to be in Israel for the fall premiere of the newly re-named The Conners. Barr, whose eponymous series was canceled following her infamous tweet about Obama aide Valerie Jarrett, made the announcement on celebrity rabbi Shmuley Boteach's podcast. "I have an opportunity to go to Israel for a few months and study with my favorite teachers over there," Barr said. And it may even be permanent. Per CNN, Barr said she would "probably move somewhere there.... I have saved a few pennies and I'm so lucky I can go. It's my great joy and privilege to be a Jewish woman." Barr, who ABC has announced will have absolutely no creative or financial ties to The Conners when it debuts, also addressed her firing in the interview. The vocal supporter of President Trump said she made a "fatal mistake" when she decided to apologize for the tweet in which she compared Jarrett, who is black, with a character from Planet of the Apes. "Once you apologize to them they never forgive, they just try to beat you down until you don't exist," Barr said in reference to the political left. "That's how they do things. They don't accept apologies." The Conners airs its first episode Oct. 16.

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Write a title and summarize: Le livre, au-delà de sa lecture, peut être aussi un bel objet auquel le lecteur s'attache par un rapport quasi charnel, qui n'a rien de commun avec ce que peut susciter l'enveloppe plastique translucide d'un CD. BENOIT PAILLEY POUR "LE MONDE" Miracle : deux livres qui traitent d'art italien et qui ne sont pas encore des monographies de Raphaël ou Titien. Mieux même : deux livres qui se donnent des sujets originaux et sur lesquels il n'existait jusqu'ici rien d'équivalent. Ils ont encore un dernier point commun, tout aussi remarquable : s'intéresser à des formes de création d'ordinaire tenues pour subalternes et simplement décoratives. Ainsi, en étudiant les stucs, Alessandra Zamperini rend à ce matériau - un enduit à base de chaux qui se modèle et se colore à volonté - et à ses spécialistes leur dignité. Comme il se doit, elle rappelle que blasons, animaux, anges, guirlandes de fruits et de fleurs ont des fonctions bien au-delà de l'ornement et doivent s'interpréter comment des éléments d'un langage plastique qui répond à l'architecture qu'il revêt et à la peinture qu'il environne. SENS DU RYTHME Mais, en forçant le lecteur à regarder attentivement, elle démontre aussi l'étonnante maîtrise et l'inventivité de tous ceux qui, tout en respectant ces programmes symboliques, en profitaient pour rivaliser avec les sculpteurs de l'Antiquité ou tenter l'impossible. Quand Giuseppe Ferrari, vers 1760, déploie un quadrillage souple, une sorte de filet, dans le palais Loredan à Venise, il fait preuve d'un sens du rythme et de la géométrie non moins remarquable que son aisance technique. Les cheminées en gueules de monstre de Bartolemeo Ridolfi à Vicence ou les bucranes et crocodiles ailés de Marcantonio Barbaro sont aussi fort réussis. Cet art qui n'admet pas le vide et ne s'arrête que quand la loi de la gravité l'y contraint touche au délire durant l'âge baroque. Il en va de même des Sacri Monti, spécialité essentiellement alpine. On appelle ainsi des chapelles étagées sur une pente, chacune occupée par des groupes sculptés et peints décrivant une scène de la Passion du Christ. Des grilles les fermaient et les pèlerins ne pouvaient les voir que selon certains angles, déterminés par les scénographes de ces annonciations, flagellations ou crucifixions. Elles ont pour acteurs des statues animées de mouvements excessifs, environnées d'animaux et de draperies aux couleurs souvent intenses.

Title: "Extensions du domaine du baroque italien." "Sacri Monti, Incandescence baroque en Italie du Nord" ", de Valère Novarina et Marc Bayard, et" "Les Stucs. Chefs-d'oeuvre méconnus de l'histoire de l'art" ", d'A Summary: Deux ouvrages originaux, "Sacri Monti, Incandescence baroque en Italie du Nord", de Valère Novarina et Marc Bayard, et "Les Stucs. Chefs-d'oeuvre méconnus de l'histoire de l'art", d'Alessandra Zamperini permettent d'approfondir la connaissance d'une riche période de l'art de la Péninsule.

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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to co-pending provisional patent application No. 60/911,830 filed Apr. 13, 2007. The entire disclosure of that provisional application is incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to orthopedic devices and in particular to cervical support devices. BACKGROUND OF THE INVENTION [0003] A wide variety of work and recreational activities require participants to look up for extended periods of time. For example, automobile mechanics working under a car that has been raised on a lift have to look up to work on the car. The person belaying for a rock climber must continuously look up at the climber during the entire time that the climber is ascending or descending. Since looking up requires tilting the head backwards, these activities can place strain on a participant&#39;s cervical and upper thoracic spine areas. Prolonged arching of the neck can lead to muscle strain and neck pain. Supports for the back of the head or cervical area should help to relieve the strain and fatigue and potentially avoid related cumulative stress injuries. [0004] A wide variety of cervical support devices are known. Many of the devices are intended for medical or therapeutic purposes. These devices are typically constructed of rigid or soft materials such as plastic or foam and fabric. The device typically rests on the shoulders, supports the neck and holds the head-and-neck in a normal eyes (or face)-forward anatomical position. These devices are not well suited for relatively unrestricted physical activities and typically prevent the user from looking upwards. Other devices are intended to support the neck and head when the wearer is in a sitting or back-lying position. These devices are intended to provide support and proper alignment of the head-and-neck during rest or sleep. One such device is illustrated in U.S. Pat. No. 5,738,640. This device includes an upper cervical horizontal cushion, and an attached upper thoracic spine vertical cushion with straps to hold the device against the upper portion of the spine. It is intended to be used in a chair or bed with the wearer&#39;s head-and-neck resting against the chair or bed pillow. Again, this device is not intended to be used to provide neck support during physical activities. Because of the device&#39;s top-of-shoulder-straps attachment site onto the cervical (top or horizontal) component of this device&#39;s cushions, there is no effective forward pull and consequently that device would not successfully support the head-and-neck during upward-looking activities. In fact, the cervical horizontal cushion would effectively fall away from the back of the head-and-neck. Therefore, a head-and-neck support is desired that provides support for the back of the head-and-neck and is suitable for use during physical activities, especially during activities that require sustained extension of the cervical spine, i.e., looking-up activities. SUMMARY OF THE INVENTION [0005] The present invention is directed to a head-and-neck support collar that can be worn during physical activities and that provides support for the base of the skull and the posterior cervical region of the spine, i.e., the neck. Suitable activities for the use of this device include, but are not limited to, belaying in rock climbing, bird watching, astronomy, painting, dry-walling, seam-sealing overhead, continuously looking up from a wheelchair, car mechanics standing and working under cars overhead and electricians working overhead. The support collar includes an elongated neck cushion attached to a harness that includes a main strap. When the neck cushion is placed behind the neck, extending slightly upwards to the base of the skull, the main strap extends from either end of the neck cushion over the shoulders and behind the back. The strap only contacts the cushion at the ends, eliminating any other points of contact or back pieces that restrict movement. This end-of-cushion attachment of straps also provides for a forward pull on the cushion, thus allowing adequate support when the head-and-neck are in a tilted-back position. [0006] In one embodiment, a webbed strap is sewn on either end of the cushion. Each short strap is threaded into a tensioning connector that allows for tightening and loosening of the cushion. Each tensioning connector is attached to one end of the main strap. From the tensioning connector, the webbing of the main strap continues over the shoulder, under the corresponding arm and across the back. The main strap proceeds from either tensioning connector using the same routing. Between the two tensioning connectors located at the ends of the main strap is a buckle. The buckle can be a snap connection and can include an additional tensioner. [0007] In addition to the main strap, the harness can include a horizontal sternum or chest strap. The chest strap is constructed from a similar webbed belt material as the short straps and the main strap. The chest strap is connected to the main strap in two places between the ends of the main strap. In one embodiment, the chest strap includes a stair buckle. One end of the horizontal chest strap is laced through a male component of a snap buckle-tensioner, and the other end of the horizontal chest strap is laced through the female end of the snap buckle and is sewn back on itself, holding the female end of the snap buckle in place. The chest strap tensions the main strap and provides a point through which the harness and neck cushion can be put on or taken off. [0008] The head-and-neck cushion supports the head-and-neck during cervical extension activities, i.e., head tilted-back activities. The cushion can be U-shaped to conform to the shape of the neck. In one embodiment, the cushion has a split-cylindrical shape with a generally flat side extending between the two ends and a generally rounded side opposite the flat side. In one embodiment, the rounded side is intended to be the surface making contact with the back of the neck and the base of the skull. The cushion is secured to the user by the harness. Having the attachment points on the ends of the cushion uniformly and conformingly draws the cushion around the neck when the tensioners or length adjusters in the harness are tightened. [0009] In one embodiment, the harness holds the neck cushion in place and provides adjustability or tensioning. In another embodiment, the harness includes a quick-release mechanism that completely separates the harness from the neck cushion. In this tear-away safety harness embodiment, the collar can be tom-away in an emergency situation where a person would need to be quickly released from the setup. In one embodiment, the straps extending from the ends of the neck cushion are the first part of a two-part fastener, for example a hook-and-loop type fastener. In addition, the corresponding second parts of the two-part fastener are attached to either end of the main strap. Loops can also be provided near either end of the main strap to serve as handles to be used in the activating the quick release mechanism. [0010] In accordance with one exemplary embodiment, the present invention is directed to a head-and-neck support collar that includes an elongated cushion having two opposing ends and a harness. In one embodiment, the cushion has a generally flat face running between the opposing ends and a generally rounded face opposite the flat face. The collar also includes two first parts of a two-part quick release mechanism. Each one of the two first parts is attached to and extends from one of the opposing ends of the elongated cushion. The harness includes a main strap having two ends and two second parts of the two-part quick release mechanism. Suitable materials for the main strap include polymer webbing. Each one of the two second parts is attached to one of the two main strap ends such that the elongated cushion and the main strap form a closed shape when each of the two first parts of the quick release mechanism is connected to one of the second parts of the quick release mechanism. This forms two separate quick release mechanisms, one each adjacent one of the opposing ends of the elongated cushion. The elongated cushion is completely disengaged from the harness when the first parts are released from the corresponding second parts of the quick release mechanism. [0011] Preferably, the quick release mechanism is a hook and loop fastener. Suitable hook and loop type fasteners are available under the tradename Velcro® from Velcro Industries B.V. of Middlewich—Cheshire, UK. In this embodiment, the two first parts are each a loop portion of the hook and loop fastener, and the second parts are each a hook portion of the hook and loop fastener. In one embodiment, the harness includes a loop disposed at each end of the main strap. These loops provide a handle to engage the quick release mechanism. In one embodiment, the harness includes at least one length adjustment mechanism disposed between the two ends of the main strap. In one embodiment, the harness includes a buckle disposed between the two ends of the main strap. The buckle is separate from and independent of the two quick release mechanisms. The harness can also include a chest strap attached to the main strap at two distinct locations. The chest strap includes a buckle and a length adjustment mechanism and divides the closed shape of the cushion and strap into two closed shapes. [0012] In one embodiment, the first parts of the two-part quick release mechanism each include a first length of a double-sided hook and loop fastener tape, and the second parts of the two-part quick release mechanism each include a second length of the double-sided hook and loop fastener tape. A portion of the second length of each second part is attached to a corresponding main strap end. In one embodiment, each second part is formed into a loop extending from the corresponding end of the main strap and having the hook portion of the hook and loop fastener tape on an exterior circumference. In one embodiment, each of the first lengths of the hook and loop fastener tape are equal to one half the circumference of the loops formed in the second parts. Each second length of the hook and loop fastener tape can include a portion between the end of the main strap and the loop having a length equal to one half the circumference of the loop. [0013] The present invention is also directed to head-and-neck support collar having an elongated cushion having two opposing ends and a harness. The collar includes two first lengths of a double-sided hook and loop fastener tape. Each one of the two first lengths is attached to and extends from one of the opposing ends of the elongated cushion. The harness includes a main strap having two ends and two second lengths of the double-sided hook and loop fastener tape. Each one of the two second lengths is attached to one of the two main strap ends and includes a loop extending from a corresponding end of the main strap. The elongated cushion and main strap form a closed shape when each of the two first lengths is connected to one of the second lengths to form two separate quick release mechanisms. In addition, the elongated cushion is completely disengaged from the harness when the first lengths are released from the corresponding second lengths of the quick release mechanism. In one embodiment, each loop has a hook portion of the hook and loop fastener tape on an exterior circumference. In one embodiment, each of the first lengths is equal to one half the circumferences of the loops formed in the second length. In addition, each second length of the hook and loop fastener tape includes a portion between the end of the main strap and the loop having a length equal to one half the circumference of the loop. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIG. 1A is a front view of one embodiment of the head-and-neck support collar of the present invention; [0015] FIG. 1B is a front view of another embodiment of the head-and-neck support collar with a quick-release mechanism; [0016] FIG. 1C is a front view of head-and-neck support collar with a quick-release mechanism with the neck cushion removed from the harness; [0017] FIG. 2A is a front view of one embodiment of the head-and-neck support collar of the present invention attached to a user; [0018] FIG. 2B is a side view of the embodiment of FIG. 2A attached to a user; [0019] FIG. 2C is a back view of the embodiment of FIG. 2A attached to a user; [0020] FIG. 3A is a front view of another embodiment of the head-and-neck support collar and quick-release mechanism of the present invention attached to a user; [0021] FIG. 3B is a side view of the embodiment of FIG. 3A attached to a user; [0022] FIG. 3C is a back view of the embodiment of FIG. 3A attached to a user; [0023] FIG. 4 is a top view of an embodiment of a neck cushion to be used in the head-and-neck support collar of the present invention; [0024] FIG. 5 is a view through line 5 - 5 of FIG. 4 ; [0025] FIG. 6 is an illustration of an embodiment of a second part of a two part quick-release mechanism for use in the present invention; and [0026] FIG. 7 is the illustration of FIG. 6, with the loop portion of the second part secured. DETAILED DESCRIPTION [0027] Referring initially to FIG. 1A, an exemplary embodiment of a head-and-neck support collar 10 in accordance with the present invention is illustrated. The head-and-neck support collar includes an elongated cushion 12 having two opposing ends 15. In one embodiment, the elongated cushion has a curved or U-shape that generally conforms to the contours of the neck of the user. Referring to FIGS. 4 and 5, in another embodiment, the elongated cushion 402 has a generally cylindrical or half-cylindrical shape having, for example, a flat surface 502 running between the two opposing ends 404 and a generally round or semi-circular surface 508 opposite the flat surface. The round surface is intended to make contact with the back of the neck and the base of the skull. In one embodiment, the elongated cushion is constructed of one or more cushioning materials 506 surrounded by a fabric cover. Suitable materials for the elongated cushion include foam rubber and polyester or other cushioning materials. Any suitable fabric can be used including natural and synthetic fabrics as well as fabric that provides for the wicking of moisture away from the skin. Alternatively, the cushion can be one piece molded foam rubber with a protective outer coating. Other suitable cushions include air bladders. In one embodiment, an additional removable cover (not shown) is placed over the cushion member. [0028] Returning to FIG. 1A, in one embodiment, a short length of a webbing strap 17 is attached to each end of the cushion 12. Suitable webbing straps include synthetic or polymer webbings such as nylon webbing. This webbing, as well as all other strapping used herein can have a length of from about 0.5″ up to about 1″ and preferably about 0.75″. The webbing strap is secured to the ends of the neck cushion using adhesives or stitching. In another embodiment, a first part of a two-part quick release mechanism is attached to each one of the ends of the elongated neck cushion. Any suitable two-part quick release mechanism can be used. Referring to FIG. 1C, in one embodiment, two first parts 40 of a two-part quick release mechanism are provided, and each one of the two first parts is attached to and extends from one of the opposing ends 15 of the elongated cushion. A preferred quick release mechanism is a two-part hook and loop type fastener. For example, synthetic webbing can be attached to the ends of the elongated cushion and the desired portion of the hook and loop fastener can be attached to the synthetic webbing, for example, using stitching. Alternatively, a tape containing the hook, the loop or both the hook and loop portion of the hook and loop fastener is attached to both of the opposing ends of the elongated cushion. In one embodiment, the first parts of the two-part quick release mechanism is the loop portion of the hook and loop fastener. Referring to FIG. 4, in one embodiment, the first parts 440 are double-sided hook and loop fastener tape having a loop portion 406 side and an opposing hook portion 408 side. [0029] Returning to FIG. 1A, in one embodiment of the neck and head support collar 10, the collar includes a harness 19 that includes a main strap 21 having two ends 18. Suitable main straps include synthetic or polymer webbings such as nylon webbing. This webbing, as well as all other strapping used herein can have a width of from about 0.5″ up to about 1″ and preferably about 0.75″. In this embodiment, tension adjusting clips 14 are provided at each of the two ends, and each tension adjusting clip accepts one of the two webbing straps 17 attached to the elongated cushion. A buckle 13 is included in the main strap between the two ends, and an optional expandable back strap 20 can also be provided between the two ends of the main strap. In one embodiment, the harness 19 includes a chest strap 23 attached to the main strap at two distinct locations. The chest strap 23 includes a buckle 16 and a length adjustment mechanism 25 and divides the closed shape of the cushion and strap into two closed shapes. [0030] Referring to FIGS. 1B and 1C, in another embodiment the neck and head support collar 100 includes a harness 119 having a main strap 121 that includes two second parts 24 of the two-part quick release mechanism. Each one of the two second parts is attached to one of the two main strap ends 118. The elongated cushion 12 and main strap 121 form a closed shape ( FIG. 1B ) when each of the two first parts 40 of the quick release mechanism is connected to one of the second parts of the quick release mechanism 40, forming two separate quick release mechanisms. The elongated cushion 12 completely disengages from the harness 119 when the first parts 40 are released from the corresponding second parts 24 of the quick release mechanism ( FIG. 1C ). In one embodiment, the harness 119 includes a chest strap 23 attached to the main strap at two distinct locations. The chest strap 23 includes a buckle 16 and a length adjustment mechanism 25 and divides the closed shape of the cushion and strap into two closed shapes. [0031] A preferred quick release mechanism is a two-part hook and loop type fastener, and in one embodiment, the second parts are the hook portion of the hook and loop type fastener. For example, the main strap 121 is constructed from synthetic webbing, and the desired portion of the hook and loop fastener can be attached to the synthetic webbing at the ends 118, for example, using stitching. Alternatively, a tape containing the hook, the loop or both the hook and loop portion of the hook and loop fastener is attached to both ends of the main strap. In one embodiment, each second part of the two-part quick release mechanism is the hook portion of the hook and loop fastener. In one embodiment, the second parts 24 are double-sided hook and loop fastener tape having a hook portion 42 side and an opposing loop portion side 44. A buckle 113 and a tension adjustment clip 114, i.e., a length adjustment mechanism, can be included in the main strap between the two ends, and an optional expandable back strap 20 can also be provided between the two ends of the main strap. The buckle is separate from and independent of the two quick release mechanisms. [0032] In order to provide activation of the quick release mechanism, a handle or grip is provided for the user to grasp and to activate the quick release mechanism. This handle can take the form of a loop, for example, a loop of the main strap or a loop in the second part of the quick-release mechanism. As illustrated, the harness further includes a loop 22 disposed at each end of the main strap, each loop providing a handle to engage the quick release mechanism. In one embodiment, the first parts of the two-part quick release mechanism are a first length 46, 446 of a double-sided hook and loop fastener tape. This first length is the length of tape extending past the end of the neck cushion. Each first part may include additional lengths that are used for attaching the first parts to the ends of the cushion. The second parts of the two-part quick release mechanism are a second length of the double-sided hook and loop fastener tape. A portion of the second length of each second part is used for attachment to a corresponding main strap end. In one embodiment, each second part is formed into a loop 22 extending from the corresponding end of the main strap and having the hook portion of the hook and loop fastener tape on an exterior circumference 42. In one embodiment, each of the first lengths of the hook and loop fastener tape are equal to one half the circumference of the loops formed in the second parts. This length can be from about 4″ to about 5″. [0033] Referring to FIGS. 6 and 7, an embodiment of the second length of the second part 624 with the loop 622 is illustrated. In this embodiment, a second length of the hook and loop fastener tape includes a portion 602 of the tape between the end 618 of the main strap 621 and the loop 622 having a length equal to one half the circumference of the loop. This length is from about 4″ to about 5″. This length is also made of the double-side hook and loop fastener tape and allows the loop, i.e., the exterior circumference of the loop, to be secured against that portion of the tape as shown in FIG. 7. [0034] FIGS. 2A, 2 B and 2 C illustrate a first embodiment of the head-and-neck support collar of the present invention attached to a user 26. FIGS. 3A, 3 B and 3 C illustrate a second embodiment of the head-and-neck support collar with the quick release mechanism of the present invention attached to a user 26. The neck cushion is placed behind the neck of the user, and the main strap is routed over the shoulders, under the arms and behind the back of the user. The length can be adjusted as desired, and this adjustment, since it is pulling forward on the ends of the neck cushion, conforms the cushion to the back of the user&#39;s neck, extending partially to each side of the user&#39;s cervical region, i.e., neck. There is no connection between the main strap and the neck cushion behind the user, and no pads or straps run vertically up the spine between the shoulder blades. This provides a greater degree of unobstructed movement to both the head and arms. The quick release mechanism is located in front of the user on the upper chest. This placement makes it easy for the user to access and to activate the quick release mechanism. [0035] While it is apparent that the illustrative embodiments of the invention disclosed herein fulfill the objectives of the present invention, it is appreciated that numerous modifications and other embodiments may be devised by those skilled in the art. Additionally, feature(s) and/or element(s) from any embodiment may be used singly or in combination with other embodiment(s) and steps or elements from methods in accordance with the present invention can be executed or performed in any suitable order. Therefore, it will be understood that the appended claims are intended to cover all such modifications and embodiments, which would come within the spirit and scope of the present invention.

Summary: A head-and-neck support collar is provided that supports the cervical region of the spine and the base of the skull when the head is in a tilted-back position. The collar is designed to reduce head-and-neck stress, strain and pain during physical activities that require the wearer to look upwards, especially for extended periods of time. The collar includes an elongated neck cushion that is positioned behind the neck of the user. A harness is attached to either end of the elongated neck cushion and is routed over the shoulders, under the arms and across the back. A two part quick release mechanism is provided between the harness and each end of the cushion to facilitate quick and complete release of the cushion from the harness. This quick release mechanism is preferably a hook and loop type fastener.

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Summarize: Shi'te Muslim Iran is so alarmed by Sunni insurgent gains in Iraq that it may be willing to cooperate with Washington in helping Baghdad fight back, a senior Iranian official told Reuters today. The idea is being discussed internally among the Islamic Republic's leadership, the senior Iranian official told Reuters, speaking on condition of anonymity. The official had no word on whether the idea had been raised with any other party. Officials say Iran will send its neighbor advisers and weaponry, although probably not troops, to help its ally and Iraq's Prime Minister Nuri al-Maliki check what Tehran sees as a profound threat to regional stability, officials and analysts say. Scroll down for video. Iranian President Hassan Rouhani, pictured here on Monday in Turkey with Turkish President Abdulah Gul, promised 'constructive engagement' with the world when he was elected. Tehran is open to the possibility of working with the United States to support Baghdad, a senior Iranian official said today. Islamist militants have captured swathes of territory including the country's second biggest city Mosul. Tehran is open to the possibility of working with the United States to support Baghdad, the senior official said. 'We can work with Americans to end the insurgency in the Middle East,' the official said, referring to events in Iraq. 'We are very influential in Iraq, Syria and many other countries.' For many years, Iran has been aggrieved by what it sees as U.S. efforts to marginalize it. Tehran wants to be recognized as a significant player in regional security. Sabre-rattling: An Islamic militant issues a call to arms, saying: 'Declare Allah the Greatest! Allah is the Greatest!' in a video released by ISIS. Trail of destruction: Militants of Islamic State of Iraq and the Levant damage a patrol car of Iraq army in the city of Mosul. Relations between Iran and Washington have improved modestly since the 2013 election of President Hassan Rouhani, who promised 'constructive engagement' with the world. And while Tehran and the United States pursue talks to resolve the Islamic state's decade-old nuclear standoff with the West, they also acknowledge some common threats, including the rise of al Qaeda-style militancy across the Middle East. On Thursday, President Barack Obama said the United States was not ruling out air strikes to help Baghdad fight the insurgents, in what would be the first U.S. armed intervention in Iraq since the end of the U.S.-led war. Rouhani on Thursday strongly condemned what he called violent acts by insurgent groups in the Middle East. 'Today, in our region, unfortunately, we are witnessing violence, killing, terror and displacement," Rouhani said. 'Iran will not tolerate the terror and violence... we will fight against terrorism, factionalism and violence.' Asked on Thursday about Iranian comments, U.S. State Department spokeswoman Jen Psaki said: 'Clearly, we've encouraged them in many cases to play a constructive role. But I don't have any other readouts or views from our end to portray here today.' Men pose with automatic rifles and a stationary machine gun, with the ISIS flag propped up behind them. An extremist group linked to the jihadists has claimed responsibility for the alleged kidnappings of three teens in Israel. No resistance: The masked ISIS fighters waved the black flag of the Islamic State and flashed the 'V' sign while some shouted 'towards Baghdad' Warlike: The Kurdish Peshmerga armed forces, pictured yesterday in Kirkuk, Iraq, could defeat ISIS, but are in no mood to. Fearing Iraq's war could spill into Iran, Foreign Minister Mohammad Javad Zarif has urged the international community to back Maliki's administration 'in its fight against terrorism.' Brigadier-General Mohammad Hejazi said Iran was ready to supply Iraq with'military equipment or consultations,' the Tasnim news agency reported. 'I do not think the deployment of Iranian troops would be necessary,' he was quoted as adding. The senior Iranian official said Iran was extremely worried about the advance of ISIL, also a major force in the war against Iran's close ally Syrian President Bashar al-Assad, carving out a swathe of Syria territory along the Iraqi border. 'The danger of extremist Sunni terrorist in Iraq and the region is increasing... There have been several high-ranking security meetings since yesterday in Tehran,' the official said. 'We are on alert and we also follow the developments in Iraq very closely.'

Summary: Sunni insurgents have already taken the Iraqi cities Tikrit and Mosul and are now on the march to Baghdad. Senior Iranian official said the Islamic Republican was also open to working with the United States to keep Baghdad secure. Asked about talks with Iran, U.S. State Department spokeswoman Jen Psaki. said on Thursday, 'Clearly, we've encouraged them in many cases to play a. constructive role' 'But I don't have any other readouts or views from our. end to portray here today,' she said. Iran is Shi'te and the insurgents Sunni. Iraq is ruled by a Shi'te elite.

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Summarize: WASHINGTON (AP) — Food companies and restaurants could soon face government pressure to make their foods less salty — a long-awaited federal effort to try to prevent thousands of deaths each year from heart disease and stroke. FILE - This May 30, 2014, file photo shows Food and Drug Administration Commissioner Dr. Margaret Hamburg in Washington. Food companies and restaurants could soon face government pressure to make their... (Associated Press) FILE - This Feb. 7, 2012 file photo shows a shopper walking down the canned soup aisle at a grocery store in Cincinnati. Food companies and restaurants could soon face government pressure to make their... (Associated Press) The Food and Drug Administration is preparing voluntary guidelines asking the food industry to lower sodium levels, FDA Commissioner Margaret Hamburg told The Associated Press. Hamburg said in a recent interview that the sodium is "of huge interest and concern" to the agency. "We believe we can make a big impact working with the industry to bring sodium levels down, because the current level of consumption really is higher than it should be for health," Hamburg said. It's still unclear when FDA will release the guidelines, despite its 2013 goal to have them completed this year. Hamburg said she hoped the agency would be able to publicly discuss the issue "relatively soon." On Tuesday, FDA spokeswoman Erica Jefferson said there is no set timeline for their release. The food industry has already made some reductions, and has prepared for government action since a 2010 Institute of Medicine report said companies had not made enough progress on making foods less salty. The IOM advised the government to establish maximum sodium levels for different foods, though the FDA said then — and maintains now — that it favors a voluntary route. Americans eat about 1½ teaspoons of salt daily, about a third more than the government recommends for good health and enough to increase the risk of high blood pressure, strokes and other problems. Most of that sodium is hidden inside common processed foods and restaurant meals. In addition to flavor, companies use sodium to increase shelf life, prevent the growth of bacteria, or improve texture and appearance. That makes it more difficult to remove from some products, Hamburg noted. Once the guidelines are issued, Americans won't notice an immediate taste difference in higher-sodium foods like pizza, pasta, bread and soups. The idea would be to encourage gradual change so consumers' taste buds can adjust, and to give the companies time to develop lower-sodium foods. "I think one of the things we are very mindful of is that we need to have a realistic timeline," Hamburg said. Health groups would prefer mandatory standards, but say voluntary guidelines are a good first step. Still, Michael Jacobson of the Center for Science in the Public Interest says he is concerned companies may hesitate, worried that their competitors won't lower sodium in their products. If that happens, "then FDA should start a process of mandatory limits," Jacobson says. That's what companies are worried about. Though the limits would be voluntary, the FDA is at heart a regulatory agency, and the guidelines would be interpreted as a stern warning. Brian Kennedy of the Grocery Manufacturers Association, which represents the country's biggest food companies, says the group is concerned about the FDA setting targets and any guidelines should be based on a "rigorous assessment of all available scientific evidence." The food industry has pointed to a separate 2013 IOM report that said there is no good evidence that eating sodium at very low levels — below the 2,300 milligrams a day that the government recommends — offers benefits. The government recommends that those older than 50, African-Americans and people with high blood pressure, diabetes or chronic kidney disease eat 1,500 milligrams a day. The American Heart Association recommends that everyone eat no more than 1,500 milligrams a day. Lanie Friedman of ConAgra Foods, one of the companies that would be subject to the voluntary guidelines, says the newer IOM report is a "paradigm change" and more research is needed. But those pushing for sodium limits say it's pointless to debate how low the recommendations should go — Americans are still eating around 3,400 milligrams a day. Many food companies and retailers already have pushed to reduce salt. Wal-Mart pledged to reduce sodium in many items by 25 percent by next year, and food giant ConAgra Foods says it made a 20 percent reduction. Subway restaurants said it has made a 30 percent reduction restaurant-wide. The companies say that in some cases, just removing added salt or switching ingredients does the trick. Potassium chloride can also substitute for common salt (sodium chloride), though too much can cause a metallic taste. Levels of sodium in food can vary widely. According to the Centers for Disease Control and Prevention, sodium in a slice of white bread ranges from 80 milligrams to 230 milligrams. Three ounces of turkey deli meat can have 450 milligrams to 1,050 milligrams. Those ranges give health advocates hope. "Those differences say to me that the companies that make the highest-sodium products could certainly reduce levels to the same as the companies that make the lower-sodium products," Jacobson says. Still, the guidelines could be a hard sell. In recent years, congressional Republicans have fought the Obama administration over efforts to require calorie labels on menus and make school lunches healthier. When the administration attempted to create voluntary guidelines for advertising junk food for children, the industry balked and Republicans in Congress fought the idea, prompting the administration to put them aside. Other members of Congress are pushing the agency to act. "As the clock ticks, America's blood pressure, along with health costs due to chronic disease, continues to rise," says Sen. Tom Harkin, chairman of the Senate committee that oversees the FDA. ___ Find Mary Clare Jalonick on Twitter at http://twitter.com/MCJalonick Food and restaurant companies are under increasing pressure to make products healthier, but sometimes they don't want customers to know when they have cut the salt or fat. Companies have employed the tactic, which some executives call "stealth health," in tweaking products including Hamburger Helper, Oreo cookies and McDonald's french...

Summary: In the minds of customers, healthy food often means less-tasty food. At the same time, however, many clamor for healthier options. All this puts restaurants and food makers in a bind-and the solution, the Wall Street Journal reports, is "stealth health." This refers to companies secretly improving the healthiness of their offerings without telling the customers, at least not until they've already gotten used to the altered version. "When you tell people something's healthy, they think it doesn't taste good," says an exec at Boston Market. In the fourth quarter of last year, that restaurant chain reduced sodium in several products; it finally told customers about it in February. Kraft didn't tell anyone when it cut trans fats from Oreos in 2006. And General Mills kept quiet about sodium cuts over six years in Hamburger Helper, using ingredients such as garlic to maintain flavor. "It takes multiple months, if not years, to get the right equation between taste and health," says a company health officer. But it all depends on the product and its audience: Fans of Progresso Soups, also made by General Mills, want to eat more healthily-so when sodium was cut in the soups, the company made sure customers knew. Like it or not, we may be seeing lower salt levels in our food soon: The FDA is readying new sodium guidelines, the AP reports.

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Summarize: Image copyright Reuters Image caption The freed students being helped into a truck by paramilitary force in Bamenda Schoolchildren kidnapped from a boarding school in Cameroon's North-West region have met the regional governor after being freed. The 78 boys and girls and three others were seized early on Sunday in the region's capital, Bamenda. A driver was also released, but the school's principal and a teacher are still being held. The government has accused separatists in the English-speaking region of being behind the kidnapping. The Anglophone separatists have denied they were involved. The secessionist movement took up arms last year to demand independence for the North-West and South-West regions - the two English-speaking regions in a country where French is the most widely spoken official language. The kidnapped students, aged between 11-17 years old, were "frightened and traumatised but in good shape", Rev Fonki Samuel, Presbyterian Church Moderator in Cameroon, told the BBC Focus on Africa programme. He said that they were being given food and being checked by the doctors before being reunited with their parents. One of the hostages, 17-year-old, Alain, said the kidnappers forced them to run after seizing them. He told Reuters news agency that they were not mistreated and the captors gave them food. "They gave us kontchap [a mix of corn and beans] to eat... It was not enough but they still gave us some. They also gave us water," he said. Rev Samuel said the Bamenda's Presbyterian Secondary School - where the students were seized - had been closed. How were the children freed? According to the Presbyterian Church of Cameroon, the students were abandoned in one of their buildings in the town of Bafut, about 24km (15 miles) from Bamenda. "The release was done peacefully... by unidentified gunmen. They [students] were brought into the church premises," Rev Samuel said. "The first information we got from them [kidnappers] is their call and they were telling us they intended to release the children yesterday [Tuesday] morning... but unfortunately it rained so heavily that could not happen. "So [on] the evening of yesterday, surprisingly and by God's grace, the children were brought back to us." Image caption The North-West and South-West regions are Cameroon's two English-speaking regions Rev Samuel told the BBC that 78 students, not 79 as earlier reported, had been released. He also revealed that Sunday's kidnapping was the second such case at the school in less than a week. In the earlier 31 October incident, 11 boys were taken and then released. It is unclear who the kidnappers were but the church paid a ransom of $4,000 (£3,000) to secure their release, he said. The army had been deployed to try and find the children taken on Sunday. Who was behind the kidnap? Rev Samuel told the BBC he was not concerned about who was behind the kidnapping, only "overwhelmed and happy" that the schoolchildren had been freed. He said, "armed groups, gangsters and thieves" could be taking advantage of the insecurity in the region to kidnap people, and blame it on the government and separatists. Cameroon's authorities have blamed the kidnap on Anglophone separatist militias - who have called for schools in English-speaking regions to be closed. They want to create an independent state called Ambazonia. There have been a spate of kidnappings in the Anglophone regions at other schools but Sunday's incident involved the largest number abducted at one time, the AP reports. It said that the separatists had set fire to at least 100 schools and taken them over as training grounds. On Tuesday, anxious parents gathered at the Presbyterian Secondary School to try to get information about their children. Image copyright Reuters Image caption Parents gathered at the Presbyterian Secondary School in Bamenda after getting news their children had been kidnapped A video of the hostages released on Monday showed one of the captives saying they had been seized by "Amba Boys" - the widely-used term to describe the separatist rebels. An Anglophone group, the Ambazonia International Policy Commission (AIPC), has however denied that the separatists were behind the kidnapping, noting that the person recording the video appears to have a poor grasp of Pidgin-English, the language spoken widely in the Anglophone area of the country. One of the kidnappers was also apparently heard speaking French. What is happening in English-speaking parts of Cameroon? English-speakers in Cameroon have long complained that they face discrimination from Cameroon's Francophone majority. They say that they are excluded from top civil service jobs and that government documents are often only published in French, even though English is also an official language. Cameroon - still divided along colonial lines Image copyright Alamy Image caption Africa's borders were "carved up" up by colonial powers Colonised by Germany in 1884 British and French troops force Germans to leave in 1916 Cameroon is split three years later - 80% goes to the French and 20% to the British French-run Cameroon becomes independent in 1960 Following a referendum, the (British) Southern Cameroons join Cameroon, while Northern Cameroons join English-speaking Nigeria Read more: Cameroon timeline (CNN) Two children who were among dozens kidnapped by gunmen from their boarding school in Cameroon Monday are still missing, the school's moderator told CNN, updating an earlier statement that all 78 students had been freed. Rev. Fonki Samuel Forba, the moderator of the Presbyterian Church in Cameroon, told CNN that authorities realized the two children were missing after the other children underwent interviews and medical checks. "When we handed the children to their parents this evening, we learned two students are still with the kidnappers, and the principal and one teacher," Forba said. On Tuesday, Forba said all 78 children had been freed. Forba said the kidnappers had asked the students to tell if them if their parents had high-ranking government positions. "These two raised their hands. So they kept them," he said. The group of 42 girls and 36 boys was seized early Monday by gunmen, along with their principal, a teacher and a driver, from the Presbyterian Secondary School in Bamenda, in the northwest of the central African nation. One other girl managed to escape from the kidnappers. Read More

Summary: All 78 students kidnapped by anti-government separatists in Cameroon Sunday have been freed, officials now say, but the ordeal isn't over for everyone. The BBC reports that the children taken from Presbyterian Secondary School in Bamenda are now being questioned by authorities before they get to go home to their families. It hasn't been definitively established who abducted the 42 girls and 36 boys, per CNN, with the government and the so-called "Amba Boys"-an English-speaking minority movement opposed to President Paul Biya-both accusing each other. Still reportedly held by the kidnappers: a teacher and the school's principal, though a driver has also been said to have been released.

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Summarize: No matter how high his international stature rises, Chinese dissident artist Ai Weiwei never seems to take himself too seriously. That's my takeaway, anyway, from his parody of the South Korean pop hit "Gangnam Style." Well, it's sort of a parody. Ai and his team donned some pink clothes and jumped around to the song's audio, splicing in some shots from the video (with its star, Psy, always cut out). At one point, the artist starts waving a set of handcuffs, which he later uses to attach himself to another man in the video. I can't claim to understand the significance of this, if there is any. Chinese police arrested Ai for almost three months in 2011 and later placed him under house arrest. Does anyone have any insight to this, or is it just Ai having a bit of fun? Update: Answer here. Ai showed his more serious side to the New Yorker's Evan Osnos, who just posted video of his interview with the artist. A poignant moment comes about eight minutes in, when Osnos asks Ai, "Have you thought about how you will explain your life to [your son]?" Ai: I had a very funny notion during my detention, even after or now: I want my son to grow slower. I don't want him to be mature too soon, to understand what I'm doing. Osnos: Because it's impossible to explain it? Ai: It doesn't really make sense to me. Of course I don't want to teach my children a sense of suffering, it's not necessary. It doesn't have to be this way. UN Secretary General Ban Ki-moon has been taking dancing lessons from South Korean rapper Psy. Mr Ban, who is also South Korean, joked that he felt overshadowed by the Gangnam Style singer, whose video has clocked up more than half a billion views on YouTube. "I'm a bit jealous. Until two days ago someone told me I am the most famous Korean in the world. Now I have to relinquish. I have no regrets," he said. The two lavished praise on each other at the UN's headquarters in New York, with Mr Ban risking a few of Psy's trademark horse-riding dance moves from Gangnam Style. "So now you have first and second famous Korean in the same building," Psy told reporters, adding that he had nothing but praise for Mr Ban. "For all the Koreans he is the guy, you know, in everyone's heart in Korea, the best among the best," said the 34-year-old. "To be here and he knows me, even the thing that he knows me is so touching right now, and he's saying he saw my video. He counted my video views. "This is much more better feeling than when I did No 2 on Billboard (US music chart)." Psy does his much-copied horse-riding dance (YG Entertainment) Although Psy has been a hit in his home country for years, it took Gangnam Style to propel him to worldwide stardom, getting to No 1 in the UK Singles Chart. He has appeared on numerous television programmes in the US, including The Ellen DeGeneres Show, Saturday Night Live and The X Factor, and his moves have been performed by the likes of Nelly Furtado and Britney Spears. The official video accompanying Gangnam Style has prompted numerous parodies, including a group of US lifeguards and some pupils from Eton. Mr Ban's meeting with Psy, whose real name is Park Jae-sang, was a break from the conflict and wars the UN leader usually deals with. Mr Ban's spokesman said the Secretary General thinks it is important to engage different parts of society. Forget Dancing With the Stars. You want to see an A-lister bust some really impressive moves, look no further than Hugh Jackman's sexy, smooth "Gangnam Style" lesson. The action star took a break from filming The Wolverine to dance with Psy, the South Korean pop star who sparked the dance craze. It's not clear why Psy hit the movie set. Maybe they are negotiating a dance montage for the heavy action flick to lighten things up a bit. (Stranger things have happened!) Whatever the reason, the Twitter pics they each posted of the visit are priceless. Hugh and Psy are both wearing those crazy sharp Wolverine talons while gettin' jiggy with it. "Had a great time," tweeted the singer. Hugh was definitely psyched too. "Slicing gangnam style!!!! Great to meet @psy_oppa who visited set yesterday," he later tweeted along with the following pic. How cool is that? Hugh is such a good sport. Do you still love "Gangnam Style" or are you over it? Images via Twitter & Twitter

Summary: South Korean rapper PSY has taught his insanely popular "Gangnam Style" dance to Britney Spears and Ellen DeGeneres and even Wolverine, aka Hugh Jackman-but his latest student may be his most impressive yet. UN Secretary General Ban Ki-moon did the "horse dance" alongside PSY at UN headquarters in New York yesterday, Sky News reports. Ban also joked that he's jealous of his fellow South Korean. "Until two days ago someone told me I am the most famous Korean in the world," he said. "Now I have to relinquish." And PSY has another high-profile fan: Chinese dissident artist Ai Weiwei. Ai has released what the Washington Post calls an "odd" parody of the "Gangnam Style" video, which basically features a pink-shirted Ai dancing around to the song as scenes from PSY's video are spliced in. "At one point, the artist starts waving a set of handcuffs, which he later uses to attach himself to another man in the video. I can't claim to understand the significance of this, if there is any," writes Max Fisher. "Chinese police arrested Ai for almost three months in 2011 and later placed him under house arrest. Does anyone have any insight to this, or is it just Ai having a bit of fun?"

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Write a title and summarize: Microscopy, being relatively easy to perform at low cost, is the universal diagnostic method for detection of most globally important parasitic infections. As quality control is hard to maintain, misdiagnosis is common, which affects both estimates of parasite burdens and patient care. Novel techniques for high-resolution imaging and image transfer over data networks may offer solutions to these problems through provision of education, quality assurance and diagnostics. Imaging can be done directly on image sensor chips, a technique possible to exploit commercially for the development of inexpensive “mini-microscopes”. Images can be transferred for analysis both visually and by computer vision both at point-of-care and at remote locations. Here we describe imaging of helminth eggs using mini-microscopes constructed from webcams and mobile phone cameras. The results show that an inexpensive webcam, stripped off its optics to allow direct application of the test sample on the exposed surface of the sensor, yields images of Schistosoma haematobium eggs, which can be identified visually. Using a highly specific image pattern recognition algorithm, 4 out of 5 eggs observed visually could be identified. As proof of concept we show that an inexpensive imaging device, such as a webcam, may be easily modified into a microscope, for the detection of helminth eggs based on on-chip imaging. Furthermore, algorithms for helminth egg detection by machine vision can be generated for automated diagnostics. The results can be exploited for constructing simple imaging devices for low-cost diagnostics of urogenital schistosomiasis and other neglected tropical infectious diseases. Microbiological diagnostics at the basic levels of the health care system has to meet the challenges of harsh environmental conditions, inadequately trained personnel and difficulties to maintain routines for quality assurance. There is a widespread failure to understand that diagnosis is essential to the prevention and treatment of disease [1] A major problem is that methods developed in well-equipped laboratories are difficult to maintain due to lack of resources [2], [3]. The methodological requirements differ depending on the purpose, diagnostics, epidemiology, effect of intervention etc. [4], [5]. Effective control measures put the focus on diagnostics, which needs to be adapted to the stage of control. The number of countries reporting on schistosomiasis treatment increased from 17 in 2008 to 21 in 2009 but the number of people treated for schistosomiasis in 2009 is only 8. 2% of the estimated number of people infected [6]. Even more disturbing is the calculation that fewer than 5% of the infected population is receiving antischistosomal treatment and the conclusion that we may be facing “one of the first great failures of the global health decade” that began in 2000 [7]. The focus is increasingly on diagnostics of chronic multiple parasitic infections affecting populations in poor rural areas [8], [9]. There is a definite shift from morbidity control to transmission control after long-term treatment campaigns [10], [11]. This underlines the need for monitoring tools. With decreasing transmission rates, factors related to chronic infection - not parasite load - become important. Diagnostics of urogenital schistosomiasis based on the presence of blood in urine has been successfully used as a low-cost method in a high prevalence situation [12], but such indirect methods become less useful in low prevalence and low intensity situations [13]. With decreasing endemicity the requirement for high specificity becomes more and more important – and increasingly difficult to maintain. Flawed information becomes a growing problem, which may have an unexpected impact on the fundamentals of a control effort. A large-scale commitment to eliminate major neglected infectious diseases by the end of the decade, the “London Declaration on Neglected Tropical Diseases” [14] will require reliable tools for diagnostics and monitoring. It is obvious that recent developments are going to have an impact on our possibilities to perform diagnostics even under difficult conditions in poor endemic regions. These developments affect information transfer and analysis but also novel tools for obtaining basic information. Digital web-based microscopy over the Internet offers the possibility not only for education and quality assurance [15], a central computer may also serve as a diagnostic unit as mobile imaging devices such as mobile phones can bring microscopy in contact with diagnostics at a distance. The interpretation of an image can be performed by an expert or even by crowd-sourcing diagnostics to non-experts [16] located essentially anywhere. It is the basis for much of telemedicine and used in radiology, cardiology etc. The use of a mobile phone to transfer a microscope image as described by Frean [17], has an unexploited potential - as shown e. g. by Zimic [18] - considering the proliferation of mobile networks and the possibility of integrating various types of health care related information [19]. Novel tools are emerging for producing high magnification images. By placing a small ball lens over the mobile phone camera lens [20] or by placing objects in close proximity to the surface of a sensor chip, “on-chip imaging” [21]. These methods have the potential to revolutionize diagnostic imaging, which today can be achieved only using a microscope. A current limitation of the methods is that either the sharp image area is very small, as in case a ball lens is used, or the resolution is limited as in case of on-chip imaging. On-chip imaging using rather elaborate computational holographic techniques such as diffraction analysis and super-resolution by the pixel shift technique [22], [23], [24] has shown that high-resolution imaging can be performed. In this study we wanted to test if the basic technique of imaging an object directly on the surface of an image sensor chip of a webcam or a mobile phone camera - which is so simple that it has been presented as a hobby project (http: //makeprojects. com/Project/Lensless-Microscope/220/1) - can be used for the diagnostics of helminth infections. As “proof-of-principle”, analysis of webcam images was done using computer algorithms for the identification of Schistosoma haematobium eggs in the urine. We show that it is possible to use pattern recognition to “duplicate the abilities of human vision by electronically perceiving and understanding an image approach to image analysis” [25] in the diagnostics of urogenital schistosomiasis. Patient urine and stool materials containing excreted helminth eggs were handled in accordance with the Swedish “Biobanks in Medical Care Act” (2002: 297) and the “New Biobanks Act” (Swedish government Report, SOU 2010: 81) stating that “…. samples may be collected, stored and used for certain purposes (including research and cross-border exchanges of samples and data), with respect for the individual integrity and privacy. ” (http: //www. hsern. eu/index. php/news/show/sw-swedish-government-published-a-report-sou-2010-81-entitled-a-new-biobank-act) Samples were not collected specifically for this study. All human samples obtained under oral consent and anonymized were from an already-existing sample collection for education and quality assurance (“Panel för Cystor & Maskägg” at SMI). Anonymized stool samples provided by Jürg Utzinger at the Swiss Tropical and Public Health Institute, Basel, Switzerland were collected for collaborative evaluation of diagnostics and quality assurance and approved by the ethics committees of Basel (EKBB, 377/09) and Côte d' Ivoire (reference no. 1993 MSHP/CNER; date 2010-05-10). Schistosoma mansoni eggs were obtained from mice kept according to national guidelines (Swedish Board of Agriculture SJVFS 2012: 26). Mice were experimentally infected to provide materials for diagnostics of human infections. The protocol was evaluated and approved by the regional ethical committee Stockholm North, Dpt. 1, Stockholm District Court (reference no. N527/11; date 2011-01-26). Experiments reported here were performed on a urine sediment obtained by pooling urines from individuals shown to excrete S. haematobium eggs. The formalin fixed sediment was stored at +4°C. For on-chip experiments, aliquots of the sediment were diluted in saline to give a concentration of about 250 eggs per ml. The concentration corresponds to a 10-fold concentration of 250 eggs in 10 ml of urine allowed to sediment and then re-suspended into 1 ml. The concentration of more than 50 eggs per 10 ml is considered to reflect an infection of high intensity [26], [27]. The S. haematobium sample was obtained from the diagnostic parasitology laboratory of the Swedish institute for communicable disease control (SMI), Solna, Sweden. Samples containing intestinal helminth eggs (S. mansoni, Trichuris trichiura and Diphyllobothrium latum) and Strongyloides larvae were from standard formalin or SAF-fixed (fixative containing sodium acetate, acetic acid and formalin) human stool samples. Pooled isolated S. mansoni eggs used for some on-chip experiments were obtained from experimentally infected mice as previously described [28], [29]. Images of helminth eggs were captured for reference purposes using established techniques: For part of the samples we used a microscope (Leica DMRB, Leica, Leitz) equipped with a digital camera (AxioCam; Carl Zeiss; Oberkochen, Germany) and using image capture. Imaging software (Openlab, Improvision; Coventry, United Kingdom) on a desktop computer (Apple Macintosh G4 with McOS 9; Cupertino, CA) was used for image capture. Specimens containing S. haematobium eggs and Strongyloides larvae were digitized for web-based virtual microscopy as described before [15]. Some samples were also digitized with an automated whole slide scanner (Pannoramic P250,3DHistech Ltd, Budapest, Hungary), using a 20×objective (numerical aperture 0. 8) equipped with a three-CCD (charge-coupled device) digital camera (CIS 3CCD, 2 megapixel, CIS Corporation, Tokyo, Japan). The pixel resolution was 0. 22 µm. The images were compressed with a conservative compression ratio of 1∶5 to a wavelet file format (Enhanced Compressed Wavelet, ECW, ER Mapper, Erdas Inc, Atlanta, Georgia) and made available for web-based virtual microscopy [15]. To enable fixation of liquid samples for the whole-slide imaging, specimens of helminth eggs were mounted and immobilized under coverslips on microscope slides with a drop of glycerin-gelatin (Sigma-Aldrich product GG1 aqueous slide mounting medium) at 55–60°C. Samples were scanned not only in the x and y planes, but also in different focal planes in order to generate z stacks to enable focusing in the web-based viewer. On-chip imaging was performed essentially by placing the specimen in contact with an image sensor, which was then illuminated to produce a shadow of objects present in the specimen. The CMOS (Complementary Metal Oxide Semiconductor) sensor chip of an imaging device was made available for imaging experiments by removing the optics (see supporting information S1 and S2). The main results reported here were obtained with the exposed sensor of a low cost webcam (Live! Cam Sync; (VFO520,640×480 pixel, Creative Technology Ltd. Singapore, sold by Clas Ohlson Co; Insjön, Sweden as Webbkamera, product 38-3612 for 99,00 SEK (i. e. approximately 11€) with a calculated pixel size of 3. 658 µm, in which the sensor was covered with a protective glass at a level allowing direct contact with microscope slides and resolution slide (see below). In reality, the sensor area is smaller than the cover glass and thus pixel size is smaller if calculated based on images obtained (see results). We could not test the effect of bringing objects closer to the surface of this particular actual sensor as we were unable to remove the protective covering glass without causing damage to the sensor surface. Depending on the physical appearance of the exposed sensor, additional modified imaging devices were used in experiments related to specific issues, such as the effect of image sensor pixel size on image resolution and construction of a chamber on top of the image sensor for the analysis of fluid samples. The image sensors were surrounded by protruding components of the camera circuit board, which prevented positioning of a flat microscope glass slide directly on top of the sensor chip surface. In such cases a drop of the sample was placed directly in contact with the surface of the sensor. This was done after protecting the components of the circuit board from exposure to fluid using silicone or acrylate polymer. Such on-chip experiments were performed with the exposed sensor of another webcam (“Venus” USB 2. 0 PC Camera, Vimicro Corporation; Beijing, China, sold by Clas Ohlson Co; Insjön, Sweden as Webbkamera, product 38-4068). Some experiments were performed using the exposed 8 megapixel (3624×2448 pixels; pixel size 1. 75 µm) sensor of mobile phone after removal of the thick protective glass and replacing it with a piece of coverslip with a thickness of 0. 1 mm (Sony Ericsson C905, Sony, Japan). Superior resolution was obtained using the exposed sensor of a mobile phone (Nokia E71,3. 2 megapixel; 2048×1536 pixels, pixel size 1. 75 µm, Nokia, Finland) camera. For the experiments, replacement camera modules (n = 60) were acquired for the mobile phone (E71 Camera w/Flex Ribbon; eBay, Unclemartin; China). The sensor surface was hard to access and it was difficult to protect the surrounding circuit board components from damage caused by fluid samples. As the image sensor of this particular camera module was not protected by a cover glass it was easily damaged by the drying urine sample, which became attached to the microlens polymers on the sensor surface. Thus a new camera had to be installed for each experiment. In some modifications, a chamber consisting of a plastic test tube was fitted above the image sensor: A rectangular hole corresponding to the size of the sensor was cut in the lid of the tube and fixed to the circuit board with silicone as described above. A sedimentation chamber was obtained by attaching a test-tube to the lid. Sedimentation was allowed to take place by inverting the test tube to allow particles to sediment onto the sensor surface. The test tube with supernatant was then removed and replaced with a light source. In all on-chip imaging experiments the resolution of images depended on the intensity, the size of the light source and the degree of collimation of light hitting the sensor. Near collimated light was obtained placing a small LED light at a distance of about 20 cm from the sensor. The distance from the light source could be decreased to about 25 mm using a plano-convex lens (radius curvature 12. 7 mm, diameter 25. 4 mm BK 7 KPX043, Newport Corporation; Irvine, CA, United States of America). As an alternative to LED light, we used indirect daylight from a 1 mm plastic monofilament core 2. 2 mm diameter fibre optic cable (HARTING; Sibiu, România, purchased from Elfa Distrelec AB; Solna, Sweden). Resolution was measured using an optical resolution slide (NBS USAF 1951 Test chart – R70 TN8 6HA. Pyser-SGI; Fircroft Way, Edenbridge, United Kingdom). The square glass slide was cut to a width of 25 mm in order to fit in close approximation to the exposed sensor chip of the webcam Live! Cam Sync. Calibration beads of similar size as helminth eggs were polydisperse glass particle standards (refractive Index 1. 51–1. 52) for image analysis calibration with a range of 50–350 µm (WhitehouseScientific Co. ; Waverton, Chester, CH3 7PB, United Kingdom, http: //www. whitehousescientific. com/) Image-capture and transfer was done using the camera imaging software provided by the manufacturer. Still images 640×480 pixels (VGA resolution) of S. haematobium eggs were used to create algorithms for computer vision (see supporting information S3 and below). The purpose was to identify, by computer vision, S. haematobium eggs in on-chip images of urine sediment obtained with the modified lensless webcam (Live! Cam Sync). Image analysis algorithms were used to detect S. haematobium eggs in images. Images with eggs detected by the observer were classified as positive samples. (see Supporting Information S4, Algorithms for the detection of S. haematobium eggs by computer vision). To develop the detection method (Algorithm 1), 243 images were used for training a parametric model. Images were preprocessed to normalize brightness differences and to enhance contrast [30]. The preprocessed images were thresholded based on grey values [31]. On the thresholded images, regions of interest (ROIs) were generated based on morphological methods [32]: the images were morphologically opened in order to remove small structures. Too large and too small blobs (binary large objects, [31]) were eliminated. The ROIs were classified into positive (eggs) and negative (no eggs) based on the area, shape and contrast of regions in the original image. As shape features, eccentricity and major and minor axis were used. Also, pairs of blobs within a certain distance from each other were combined to one egg hypothesis. The parameters for the grey value threshold and for the features to classify the ROIs were derived from 660 manually labeled eggs in 243 training images. A second set of 545 labeled eggs in 119 images with was then used for testing the detection method. The initial results of image analysis based on the Algorithm 1 described above gave results (see below) which warranted further image analysis studies using a more advanced algorithm (Algorithm 2) where images are processed by a sequence of classifiers each stage rejecting false positive samples passed through the previous stages. (See S3) The Haar-feature based cascade classifier [33] with 45 stages containing a total of 454 weak classifiers was trained using 500 cropped egg images. 400 of these were the confirmed detections from the first classification method. The training set was extended by 100 images, which were generated by applying small distortions of randomly selected cropped images. Ten thousand negative ROIs were obtained from images of urine sediment with no eggs present. Due to the limited number of samples, the classifier was tested using seventy-five synthetically generated images where egg images were rotated and added to background. The size variation of detections was limited between ±15% of the expected egg size. (S. Varjo and J. Hannuksela: A Mobile Imaging System for Medical Diagnostics, Proc. Advanced Concepts for Intelligent Vision Systems (ACIVS 2013), Poznan, Poland, due to appear in volume 8192 of the Lecture Notes in Computer Science series.) Sensitivity (recall/completeness) was calculated as the percentage of true positive (TP) divided by true positives and false negatives (FN). Positive predictive value (precision/correctness) was calculated as the percentage of true positives divided by true positives and false positives (FP). S. haematobium eggs could be recognized in on-chip images obtained using a simple modified webcam (Figure 1; Supporting information S1, S2 and S3). Samples on microscope slides or in liquid form could be placed directly or pipetted on the exposed sensor (Figure 1A) after protecting surrounding components with acrylic resin or silicone (Figure 1B). A chamber could easily be fitted on top of the sensor, e. g., using a pierced test tube lid (Figure 1C) and the inverted test tube could function as a sedimentation chamber. After removing the test tube, it could be replaced with an appropriate light source (see below). After trimming the edges of the glass supported USAF 1951 resolution chart glass slide to 2 cm width with a glass cutter it could be positioned onto the exposed webcam image sensor. The sensors of the other devices tested were inaccessible to the resolution test slide due to components of the circuit board protruding above the sensor level. The maximum resolution of on-chip images seen on this webcam was about 40 lines per mm (group number 5, element 3 or 4 (Figure 2A). The length of schistosome eggs equaled roughly that of the line length of the first element of group 4 in the USAF 1951 resolution chart, which is 0. 15625 mm. The sample field of view dimensions correspond to the dimensions of the sensor size, which is 2. 341×1. 756 mm, i. e. approximately 4. 11 mm2. As the sensor has 640×480 pixels, the calculated pixel size is 3. 658 µm. Figure 2B shows on-chip image of 50–350 µm calibration beads. The images of S. haematobium eggs obtained by on-chip imaging (Figure 2C and supportive information S3) were of low resolution as compared to microscope images. For reference, see scanned slide “Schistosoma haematobium”, in the virtual webmicroscope (fimm. webmicroscope. net/Research/Momic/helmintex). However, it was possible to use such images obtained by on-chip imaging for the development of a computer algorithm (see below). It was necessary to adjust the amount of light directed towards the sensor. In fact adjusting the amount of light was necessary for obtaining an image. When a LED light source at a distance of about 10 cm from the sensor chip surface was used together with a pin-hole aperture of 2 mm diameter, sufficient light was obtained. For the parameter tuning of the computer vision classifier, 660 eggs in the 243 training images were annotated as certain eggs (egg identified without doubt; n = 564) uncertain eggs (may be an egg; n = 96) or negative (not an egg) (Figure 2D). Five hundred sixty four were labeled “positive” and 96 were labeled “uncertain”. After training, the approach was tested on a second image set consisting of 119 test images, which were taken from a new sample at a later date. A total of 545 parasite eggs were manually labeled; 414 certain and 131 uncertain eggs and the object co-ordinates in the image used as reference in the evaluation of the computer vision algorithm (Figure 3 and 4). When the performance of the algorithm (Algorithm 1) in detecting individual eggs (detection at the object level) was calculated, we obtained a sensitivity (recall/completeness) of 26% and positive predictive value (precision/correctness) of 91%. Using the more complex cascade classifier involving 45 stages (Algorithm 2), Using the more complex cascade classifier, the sensitivity was 71% and positive predictive value 79% calculated from detections in 75 test image pairs, each image pair containing an image with a single egg and its companion image from which the egg had been replaced by background. (Sensitivity = TP/ (TP+FN) = 53/ (53+22) = 0. 7066→70,7%), positive predictive value = TP/ (TP+FP) = 53/ (53+14) = 0. 7910→79. 1%.) (TP = true positive; FP = false positive; FN = false negative; TN = true negative). The calculated high specificity was based on the large number of true negative images used for generating the cascade classifier (with 10000 true negative samples the specificity - TN/ (FP+TN) - approached 100%. When the performance of Algorithms 1 and 2 was compared using the same set of 75 images the difference in precision and recall was evident, but less pronounced (see supporting information S4 and S5). On-chip imaging experiments performed using stool samples containing various helminth eggs showed that eggs from helminths of different species could be distinguished from each other. Morphological features such as the lateral spine of S. mansoni eggs could be identified with certainty in some eggs (Figure 5A). The shape of S. mansoni eggs was clearly distinct from that of, e. g., T. trichiura eggs. (Figure 5C), and Strongyloides larvae were visible both in still images (Figure 5E) and in video recordings (see supporting information S6). On-chip images obtained with the webcam and Ericsson mobile phone sensors had a relatively poor resolution in comparison with images obtained by conventional microscopy (Figure 5B). However, despite technical problems due to the inaccessibility of the exposed image sensor (see above), we were able to obtain the highest resolution on-chip images with one of the mobile phone cameras in which the sensor surface was not covered by protective glass (Nokia E71; Figure 6). This setup minimized the distance between the sample and the sensor surface. On-chip imaging provides further diagnostic possibilities based on motion detection. On-chip video recordings of stool samples showed that moving Strongyloides larvae in fluid specimens (Figure 5E) may be identified (see supporting information S6). In the present study we show that helminth eggs placed directly on the image sensor of inexpensive imaging devices can be visualized in sufficient detail to be identified on standard displays. (se presentation; supporting information S7) If a mobile phone image sensor is used, the image can be directly sent for analysis at a distance. In this study we had access only to pooled samples and no attempt was made to establish the sensitivity of egg detection on a sample level in comparison to the standard reference method, which is expressed as egg counts per 10 ml of urine [17]. Image analysis could be performed either locally or centrally. Centralized diagnostics after image transfer may be performed based not only on visual inspection, but a central computer may also perform diagnostics more or less independently based on a computer vision algorithm. Image analysis may be performed by a remote computer [15] accessible through a network of servers [34] in much the same way as pattern recognition is applied for diverse purposes such as identification of immunoelectrophoretic patterns [35] and automated facial recognition for checking the passport at border controls [36]. Field studies are necessary in order to establish the sensitivity and specificity of both visual image interpretation and computer vision. The methods need to be assessed with ordinary microscopy as reference. Visual identification of helminth eggs present in on-chip images obtained with the simple webcam was successful, but the high specificity of image analysis was linked to a sensitivity, which needs to be improved before the method can be established in real-world situations. The performance of the computer algorithms in comparison to visual identification suggested, that we need to consider variables such as variation in egg size and shape, presence of inflammatory cells, casts, bacteria etc. in the background. To identify S. haematobium eggs in urine, we developed two computer algorithms. The original algorithm based on morphological features, was capable of identifying eggs correctly (precision/positive predictive value/true positive rate) only in 26% of on-chip images with a specificity of 90%. The precision of the algorithm was 71%. The second algorithm, based on cascading classifiers had a specificity approaching 100% and an improved sensitivity of 79% as compared to visual identification. In the present study we compared computer vision to visual image interpretation (as gold standard) and generated computer algorithms with high specificity for the evaluation of algorithm sensitivities (recall rates, which depend on the rate of false negatives) and precision (which is affected by the number of false positives). The computer algorithms generated both false positive and false negative results (about one out of four eggs), which reflect the superiority of visual interpretation of images. For a real-world test the 79% sensitivity should probably be improved to above 90%. On chip imaging as a potential field assay for automated diagnostics depends on two parameters, first, the capacity of imaging objects (helminth eggs) with sufficient resolution to permit identification. Second, diagnostics depends on correct identification of objects in the test sample. The image quality can be determined in terms of resolution – lines per mm -as in the present study. Clearly resolution depends on several parameters in addition to pixel size of the sensor; distance from the sample to the sensor surface and a well-controlled illumination are critical for the acquisition of on-chip images suitable for computer vision. A higher resolution will improve the accuracy of image analysis. There is a theoretical limit to the resolution, which can be achieved, set by pixel size as stated by the Nyquist-Shannon sampling theorem, stating that the maximum achievable resolution is twice the sampling frequency. Our experimental setup using the inexpensive webcam, Live! Cam Sync, allowed us to detect objects with a resolution of 12. 41 µm. The pixel size of the webcam image sensor was calculated to be 3. 7 µm and the observed resolution therefore somewhat poorer than the theoretical resolution limit of 7. 4 µm. In practice the resolution is less than the theoretical limit depending on the distance of an object to the sensor surface and due to imperfect collimation of light. The observed image resolution was much better with the Nokia E71 3. 2 megapixel image sensor (2048×1536 pixels, pixel size 1. 75 µm) without a protective cover glass. The 3. 5 µm theoretical resolution-limit reflects the small pixel size. Further improvements of on-chip mini-microscopes are therefore within reach, since recently introduced camera sensors have a pixel size close to 1 µm. We envision that an on-chip imaging device can be incorporated as an add-on to mobile phones capable of image transfer. On-chip imaging can benefit from the proliferation of mobile phones and the expanding data communication networks may provide the necessary infrastructure for functioning communication with a central server. Thus on-chip imaging may become an integrated part of telemedicine platform based on image capture, -transfer, -analysis and feedback. To meet the Millennium Development Goals complex political and social re-thinking is needed at different levels [2], [37]. Health care is not isolated from the social and economic life of humans and we need to understand in detail how novel tools can be integrated in point-of-care diagnostics in much the same way as novel tools for microeconomics have revolutionized the life of “the bottom billion” [38], [39]. Limitations posed by current microscopy-based diagnostics - and national surveillance systems depending upon them- need to be resolved. Especially among poor populations of the world, microscopy needs to be adequate [40]. However complex these issues are, there is a widespread opinion that telemedicine will play an increasingly important role in managing health care in affluent and resource-poor societies alike, and tele-medical solutions will without doubt contribute to democratization of the relationship between patient-physician and family [41], [42]. Numerous compact and cost-effective optical imaging platforms, “mini-microscopes” have been developed in recent years to improve access to effective and affordable healthcare [23]. In a recent study it was shown that soil transmitted helminths can be detected in images obtained with a mobile phone [43] equipped with a small ball lens, a technique shown to generate high magnification high resolution images [21]. Like other described “mini-microscopes” on-chip imaging as described in the present paper does not require any new procedures since microscopy is the ‘gold’ standard for identification of parasitic infections. Standardized methods exist for sample collection, handling and preparation [44], [45]. The on-chip image quality is subject to diffraction artifacts caused by the absence of optical components. Proposed solutions to this problem include computational reconstruction methods (partially coherent in-line holography approach) [reviewed in 23] to obtain microscope-like images. However, in the case of imaging helminth eggs, diffraction artifacts or distortions do not seem to undermine visual identification, as seen in e. g. Fig. 6A. One big advantage of the on-chip method described here is the large field of view - a simple webcam sensor has an area of over 10 mm2, e. g. more than 6 times the visual field of a conventional microscope using a 20×objective and more than 10 times the field of view using a typical ball lens [23]. On-chip microscopy, even without a computer algorithm, involves examining fewer visual fields. It can alter the tiresome routine microscopy for finding and correctly identifying parasites present in low numbers - one of the major reasons for perceived low status of the method and its failure [46], [47]. Our results suggest that automated diagnostics of helminth infections for field use using a simple imaging device and appropriate algorithms are within reach. A decisive advantage of a mini-microscope such as the one we describe, may prove to be the potential of providing diagnostic support by computer vision at a distance. Furthermore, our results suggest that diagnostics based image analysis has a potential to compete with laborious conventional microscopy e. g. by providing automated motion recognition for the detection of live nematode larvae.

Title: On-Chip Imaging of Schistosoma haematobium Eggs in Urine for Diagnosis by Computer Vision Summary: There is a need to develop diagnostic methods for parasitic infections specifically designed for use in resource-deficient situations. Worm infections are common in many poor countries and even if repeated treatment can be arranged at low cost, diagnostics and identification of treatment failures demand resources not easily available. With the proliferation of mobile phones, data transfer networks and digital microscopy applications the stage is set for alternatives to conventional microscopy in endemic areas. Our aim was to show, as proof of concept, that it is possible to achieve point-of-care diagnostics by an inexpensive mini-microscope for direct visualization on a display and remote diagnostics by computer vision. The results show that parasitic worm eggs can be recognized by on-chip imaging using a webcam stripped off the optics. Images of eggs from the blood fluke S. haematobium present in urine of an infected patient could be interpreted visually and by computer vision. The method offers both an inexpensive alternative to conventional microscopy and diagnostic assistance by computer vision.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Veterans' Hospice Care Services Act of 1995''. SEC. 2. PROGRAMS FOR FURNISHING HOSPICE CARE TO VETERANS. (a) Establishment of Programs.--Chapter 17 of title 38, United States Code, is amended by adding at the end the following: ``subchapter vii--hospice care pilot program; hospice care services ``Sec. 1761. Definitions ``For the purposes of this subchapter-- ``(1) The term `terminally ill veteran' means any veteran-- ``(A) who is (i) entitled to receive hospital care in a medical facility of the Department under section 1710(a)(1) of this title, (ii) eligible for hospital or nursing home care in such a facility and receiving such care, (iii) receiving care in a State home facility for which care the Secretary is paying per diem under section 1741 of this title, or (iv) transferred to a non-Department nursing home for nursing home care under section 1720 of this title and receiving such care; and ``(B) who has a medical prognosis (as certified by a Department physician) of a life expectancy of six months or less. ``(2) The term `hospice care services' means-- ``(A) the care, items, and services referred to in subparagraphs (A) through (H) of section 1861(dd)(1) of the Social Security Act (42 U.S.C. 1395x(dd)(1)); and ``(B) personal care services. ``(3) The term `hospice program' means any program that satisfies the requirements of section 1861(dd)(2) of the Social Security Act (42 U.S.C. 1395x(dd)(2)). ``(4) The term `medical facility of the Department' means a facility referred to in section 1701(4)(A) of this title. ``(5) The term `non-Department facility' means a facility (other than a medical facility of the Department) at which care to terminally ill veterans is furnished, regardless of whether such care is furnished pursuant to a contract, agreement, or other arrangement referred to in section 1762(b)(1)(D) of this title. ``(6) The term `personal care services' means any care or service furnished to a person that is necessary to maintain a person's health and safety within the home or nursing home of the person, including care or services related to dressing and personal hygiene, feeding and nutrition, and environmental support. ``Sec. 1762. Hospice care: pilot program requirements ``(a)(1) During the period beginning on October 1, 1995, and ending on December 31, 2000, the Secretary shall conduct a pilot program in order-- ``(A) to assess the desirability of furnishing hospice care services to terminally ill veterans; and ``(B) to determine the most effective and efficient means of furnishing such services to such veterans. ``(2) The Secretary shall conduct the pilot program in accordance with this section. ``(b)(1) Under the pilot program, the Secretary shall-- ``(A) designate not less than 15 nor more than 30 medical facilities of the Department at or through which to conduct hospice care services demonstration projects; ``(B) designate the means by which hospice care services shall be provided to terminally ill veterans under each demonstration project pursuant to subsection (c); ``(C) allocate such personnel and other resources of the Department as the Secretary considers necessary to ensure that services are provided to terminally ill veterans by the designated means under each demonstration project; and ``(D) enter into any contract, agreement, or other arrangement that the Secretary considers necessary to ensure the provision of such services by the designated means under each such project. ``(2) In carrying out the responsibilities referred to in paragraph (1) the Secretary shall take into account the need to provide for and conduct the demonstration projects so as to provide the Secretary with such information as is necessary for the Secretary to evaluate and assess the furnishing of hospice care services to terminally ill veterans by a variety of means and in a variety of circumstances. ``(3) In carrying out the requirement described in paragraph (2), the Secretary shall, to the maximum extent feasible, ensure that-- ``(A) the medical facilities of the Department selected to conduct demonstration projects under the pilot program include facilities located in urban areas of the United States and rural areas of the United States; ``(B) the full range of affiliations between medical facilities of the Department and medical schools is represented by the facilities selected to conduct demonstration projects under the pilot program, including no affiliation, minimal affiliation, and extensive affiliation; ``(C) such facilities vary in the number of beds that they operate and maintain; and ``(D) the demonstration projects are located or conducted in accordance with any other criteria or standards that the Secretary considers relevant or necessary to furnish and to evaluate and assess fully the furnishing of hospice care services to terminally ill veterans. ``(c)(1) Subject to paragraph (2), hospice care to terminally ill veterans shall be furnished under a demonstration project by one or more of the following means designated by the Secretary: ``(A) By the personnel of a medical facility of the Department providing hospice care services pursuant to a hospice program established by the Secretary at that facility. ``(B) By a hospice program providing hospice care services under a contract with that program and pursuant to which contract any necessary inpatient services are provided at a medical facility of the Department. ``(C) By a hospice program providing hospice care services under a contract with that program and pursuant to which contract any necessary inpatient services are provided at a non-Department medical facility. ``(2)(A) The Secretary shall provide that-- ``(i) care is furnished by the means described in paragraph (1)(A) at not less than five medical facilities of the Department; and ``(ii) care is furnished by the means described in subparagraphs (B) and (C) of paragraph (1) in connection with not less than five such facilities for each such means. ``(B) The Secretary shall provide in any contract under subparagraph (B) or (C) of paragraph (1) that inpatient care may be provided to terminally ill veterans at a medical facility other than that designated in the contract if the provision of such care at such other facility is necessary under the circumstances. ``(d)(1) Except as provided in paragraph (2), the amount paid to a hospice program for care furnished pursuant to subparagraph (B) or (C) of subsection (c)(1) may not exceed the amount that would be paid to that program for such care under section 1814(i) of the Social Security Act (42 U.S.C. 1395f(i)) if such care were hospice care for which payment would be made under part A of title XVIII of such Act. ``(2) The Secretary may pay an amount in excess of the amount referred to in paragraph (1) (or furnish services whose value, together with any payment by the Secretary, exceeds such amount) to a hospice program for furnishing care to a terminally ill veteran pursuant to subparagraph (B) or (C) of subsection (c)(1) if the Secretary determines, on a case-by-case basis, that-- ``(A) the furnishing of such care to the veteran is necessary and appropriate; and ``(B) the amount that would be paid to that program under section 1814(i) of the Social Security Act would not compensate the program for the cost of furnishing such care. ``Sec. 1763. Care for terminally ill veterans ``(a) During the period referred to in section 1762(a)(1) of this title, the Secretary shall designate not less than 10 medical facilities of the Department at which hospital care is being furnished to terminally ill veterans in order to furnish the care referred to in subsection (b)(1). ``(b)(1) Palliative care to terminally ill veterans shall be furnished at the facilities referred to in subsection (a) by one of the following means designated by the Secretary: ``(A) By personnel of the Department providing one or more hospice care services to such veterans at or through medical facilities of the Department. ``(B) By personnel of the Department monitoring the furnishing of one or more of such services to such veterans at or through non-Department facilities. ``(2) The Secretary shall furnish care by the means referred to in each of subparagraphs (A) and (B) of paragraph (1) at not less than five medical facilities designated under subsection (a). ``Sec. 1764. Information relating to hospice care services ``The Secretary shall ensure to the extent practicable that terminally ill veterans who have been informed of their medical prognosis receive information relating to the eligibility, if any, of such veterans for hospice care and services under title XVIII of the Social Security Act (42 U.S.C. 1395 et seq.). ``Sec. 1765. Evaluation and reports ``(a) Not later than September 30, 1996, and on an annual basis thereafter until October 1, 2001, the Secretary shall submit a written report to the Committees on Veterans' Affairs of the Senate and House of Representatives relating to the conduct of the pilot program under section 1762 of this title and the furnishing of hospice care services under section 1763 of this title. Each report shall include the following information: ``(1) The location of the sites of the demonstration projects provided for under the pilot program. ``(2) The location of the medical facilities of the Department at or through which hospice care services are being furnished under section 1763 of this title. ``(3) The means by which care to terminally ill veterans is being furnished under each such project and at or through each such facility. ``(4) The number of veterans being furnished such care under each such project and at or through each such facility. ``(5) An assessment by the Secretary of any difficulties in furnishing such care and the actions taken to resolve such difficulties. ``(b) Not later than August 1, 1999, the Secretary shall submit to the committees referred to in subsection (a) a report containing an evaluation and assessment by the Under Secretary for Health of the hospice care pilot program under section 1762 of this title and the furnishing of hospice care services under section 1763 of this title. The report shall contain such information (and shall be presented in such form) as will enable the committees to evaluate fully the desirability of furnishing hospice care services to terminally ill veterans. ``(c) The report under subsection (b) shall include the following: ``(1) A description and summary of the pilot program. ``(2) With respect to each demonstration project conducted under the pilot program-- ``(A) a description and summary of the project; ``(B) a description of the facility conducting the demonstration project and a discussion of how such facility was selected in accordance with the criteria set out in, or prescribed by the Secretary pursuant to, subparagraphs (A) through (D) of section 1762(b)(3) of this title; ``(C) the means by which hospice care services care are being furnished to terminally ill veterans under the demonstration project; ``(D) the personnel used to furnish such services under the demonstration project; ``(E) a detailed factual analysis with respect to the furnishing of such services, including (i) the number of veterans being furnished such services, (ii) the number, if any, of inpatient admissions for each veteran being furnished such services and the length of stay for each such admission, (iii) the number, if any, of outpatient visits for each such veteran, and (iv) the number, if any, of home-care visits provided to each such veteran; ``(F) the direct costs, if any, incurred by terminally ill veterans, the members of the families of such veterans, and other individuals in close relationships with such veterans in connection with the participation of veterans in the demonstration project; ``(G) the costs incurred by the Department in conducting the demonstration project, including an analysis of the costs, if any, of the demonstration project that are attributable to (i) furnishing such services in facilities of the Department, (ii) furnishing such services in non-Department facilities, and (iii) administering the furnishing of such services; and ``(H) the unreimbursed costs, if any, incurred by any other entity in furnishing services to terminally ill veterans under the project pursuant to section 1762(c)(1)(C) of this title. ``(3) An analysis of the level of the following persons' satisfaction with the services furnished to terminally ill veterans under each demonstration project: ``(A) Terminally ill veterans who receive such services, members of the families of such veterans, and other individuals in close relationships with such veterans. ``(B) Personnel of the Department responsible for furnishing such services under the project. ``(C) Personnel of non-Department facilities responsible for furnishing such services under the project. ``(4) A description and summary of the means of furnishing hospice care services at or through each medical facility of the Department designated under section 1763(a)(1) of this title. ``(5) With respect to each such means, the information referred to in paragraphs (2) and (3). ``(6) A comparative analysis by the Under Secretary for Health of the services furnished to terminally ill veterans under the various demonstration projects referred to in section 1762 of this title and at or through the designated facilities referred to in section 1763 of this title, with an emphasis in such analysis on a comparison relating to-- ``(A) the management of pain and health symptoms of terminally ill veterans by such projects and facilities; ``(B) the number of inpatient admissions of such veterans and the length of inpatient stays for such admissions under such projects and facilities; ``(C) the number and type of medical procedures employed with respect to such veterans by such projects and facilities; and ``(D) the effectiveness of such projects and facilities in providing care to such veterans at the homes of such veterans or in nursing homes. ``(7) An assessment by the Under Secretary for Health of the desirability of furnishing hospice care services by various means to terminally ill veterans, including an assessment by the Director of the optimal means of furnishing such services to such veterans. ``(8) Any recommendations for additional legislation regarding the furnishing of care to terminally ill veterans that the Secretary considers appropriate.''. (b) Clerical Amendment.--The table of sections at the beginning of such chapter is amended by adding at the end the following: ``subchapter vii--hospice care pilot program; hospice care services ``1761. Definitions. ``1762. Hospice care: pilot program requirements. ``1763. Care for terminally ill veterans. ``1764. Information relating to hospice care services. ``1765. Evaluation and reports.''. (c) Authority To Carry Out Other Hospice Care Programs.--The amendments made by subsection (a) may not be construed as terminating the authority of the Secretary of Veterans Affairs to provide hospice care services to terminally ill veterans under any program in addition to the programs required under the provisions added by such amendments. (d) Authorization of Appropriations.--Funds are authorized to be appropriated for the Department of Veterans Affairs for the purposes of carrying out the evaluation of the hospice care pilot programs under section 1765 of title 38, United States Code (as added by subsection (a)), as follows: (1) For fiscal year 1996, $1,200,000. (2) For fiscal year 1997, $2,500,000. (3) For fiscal year 1998, $2,200,000. (4) For fiscal year 1999, $100,000.

Title: Veterans' Hospice Care Services Act of 1995 Summary: Veterans' Hospice Care Services Act of 1995 - Directs the Secretary of Veterans Affairs to conduct a pilot program to: (1) assess the feasibility and desirability of furnishing hospice care to terminally ill veterans; and (2) determine the most efficient and effective means of providing such care. Directs the Secretary to designate 15 to 30 Department of Veterans Affairs medical facilities for hospice care demonstration projects. Allows such hospice care to be provided by Department medical facilities and personnel or by contract with a non-Department medical facility. Limits the amount paid for such care to amounts paid for hospice care programs under title XVIII (Medicare) of the Social Security Act. Directs the Secretary, during the pilot program period of October 1, 1995, through December 31, 2000, to designate no fewer than ten Department medical facilities at which hospice care is being provided to furnish palliative care to such veterans. Provides for: (1) informing terminally ill veterans of their eligibility for hospice and palliative care; and (2) hospice program evaluation, assessment, and congressional reports by the Secretary and the Under Secretary for Health of the hospice care pilot program. Authorizes appropriations for FY 1996 through 1999.

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Summarize: RELATED APPLICATIONS This application is a national phase entry of International Application No. PCT/EP2010/006840, filed Nov. 10, 2010 published in German, which claims the benefit of German Patent Application No. 10 2009 052 396.0 filed Nov. 10, 2009, the disclosure of which is hereby incorporated herein by reference. FIELD OF THE INVENTION The invention relates to a lounger and to a drive unit for a lounger. TECHNOLOGICAL BACKGROUND In large and small gardens throughout the world, on boats and ships, seating and lounge furniture are commonly used and are offered in different variations. For example, seating furniture designed for garden use and consisting of plastics netting and water-repellent cushioning exist, which seating furniture is barely distinguishable from common seating furniture for indoor use. Besides that, there also exist relatively classic sun loungers, which comprise a large backrest and a foot part whose angle may be changed in steps, such that sitting or lying in several positions may be provided. The simplest models are comprised of a wooden frame with several frame parts which are movably mounted to each other, such that for example one frame part may be inserted or attached in recesses of another frame part for adapting the sitting or lying angle. Similarly, also sun loungers are known which are made of a frame of hollow profiles of aluminium or the like, and which have armrests which may be locked in different positions on a base structure and which, depending on the locking position, define an angle of the backrest. In such furniture, the movement of the foot part may often be coupled to the movement of the back part. The described known furniture for use in the garden have a number of technical features that enhance the comfort of the sitting or lying person, however they also have several distinct disadvantages. For example, if a person is situated on a classic sun lounger described above and if this person likes to change the position, from a sitting to a lying position or vice versa, he often has to get up from the sun lounger for disengaging the locking of the back part from a position behind the backrest and re-establishing the locking at another position, or the person has at least to lean forward to remove the weight from backrest, such that with the help of the arm rests or the like, an adjusting of the sitting or lying angle may take place. This may be particularly disadvantageous, if the person under good weather conditions decides to sleep on the sun lounger, at the same time the standing up or the shifting of weight for adjusting the sitting or lying angle reduces the tiredness, such that falling asleep may be delayed or prevented, or the person has to sleep in a position uncomfortable for the person. Also for senior persons it may be exhausting to get up from a lying position into a sitting position without shifting the weight onto the backrest, or laboriously to adjust the angle of the back element. In the state of the art, lying furniture is known which particularly enhance the comfort for senior persons, for example in hospitals or for domestic care, however, this furniture is adapted exclusively for the use in dry rooms and is not suited for use in a garden. Furthermore, it would be relatively inconvenient to have to provide a power supply in the garden for operating such known lying furniture during good and dry weather in the garden. SUMMARY OF THE INVENTION Thus, there may be a need for a lounger which provides a lying and seating opportunity which is as comfortable as possible, wherein adjusting of the sitting or lying position is feasible as simple as possible, without the necessity of getting up or of load removal of a back element, or the like. The term of the lounger is not to be construed as limiting, but can at the same time encompass lying furniture for the use on ships or boats. At the same time, there may be a need for a lounger which may be operated as comfortable as possible, which however may be robust against external influences and which may be portable. According to a first aspect of the invention, the lounger according to the invention comprises a base frame, a lounger frame, and a drive unit. In the following, a base frame denotes a support or another frame-like structure which defines the outer shape of the lounger. Accordingly, a substantially rectangular-shaped structure may be used as a base frame, which structure may be adapted for being placed on a floor and for receiving a lounger frame which allows seating or lying on the lounger according to the invention. Basically, the lounger frame may be constructed using several lounger frame elements which may be movably connected with each other, and which for example should allow sitting and lying positions of a person situated thereon. In the simplest case, the lounger frame may be designed in the form of an adjustable slatted frame or the like, and for example comprise a foot part and a back part. The drive unit may be understood as a compact unit which may be adapted for providing for an energy supply for an automatic adjustment of the lounger elements. Therein, the invention is not confined to a certain functional principle, it may be quite possible to employ several different functional principles for providing a sufficient energy supply. According to an embodiment the invention may be seen in designing the construction of base frame, lounger frame, and drive unit in such a way that an operation may be possible independent from an external electric power supply and such that a weather resistance is ensured. This implies on the one hand, that the base frame may be produced with a weatherproof material. For example, this may be a thermoplastic material, a duromer material, a fiber composite material, a weatherproof metallic material such as for example aluminium, anodized aluminium, steel with a corresponding galvanization, stainless steel, or the like, wherein also pressure-impregnated wood or other weatherproof materials may be considered. The lounger frame may also be comprised of one of the materials mentioned above, wherein several different materials may be used for both frames at the same time. The several lounger frame elements may preferably be connected with each other by way of hinge joints, wherein the hinge joints may be produced of plastics, metal, a combination of plastics and metal, and the like. In the conception of the material composition, a point should be made of choosing plastics which may particularly be UV-proof. As may be often seen in particular in the state of the art, plastics garden furniture may become brittle and fragile after a period of time in which they are exposed to solar radiation, and after which they may not be used any more. A particularly good UV-resistance may be achieved by polymethyl methacrylate, polycarbonate and polyvinylidene fluoride. The lounger frame may preferably be designed as a kind of slatted frame, wherein the different lounger frame elements may be movable by way of one or several drive devices. For example, a lounger frame element may be provided in the form of a foot element, which may be moved in parallel to the base frame by a drive device. This may, for example, be realized by a kind of parallelogram guide, in which two levers which are mounted in parallel to each other at the base frame, guide the foot part. The foot part may be moved in parallel to the base frame by pivoting one of these levers by way of a drive device or by deflecting a hinge point at the foot part. However, the invention may not be limited to actually pivot single elements of the lounger elements, thus the parallel guiding of the foot part to the base frame should not be construed as a limitation. In fact, any kind of movable or deformable lounger frame which may be realized with one or several areas, for example with so-called multi-zone slatted frames or the like, and which enhance the comfort of a user, may be possible. A drive device for moving the lounger frame or individual lounger frame elements may according to the invention be designed waterproof and weatherproof. This may for example imply that the drive device may completely be encapsulated, for example by a rubber bellows. The functionality of such a drive device may be that two ends of the drive device move towards or away from each other, and by way of this contraction or expansion, two defined hinge points of the lounger frame move away or towards each other. In this way, in the example mentioned above, a rodding of the foot part may be extended by the parallelogram guide and at the extended end of the foot part, a drive device may be fastened, which drive device may be mounted with its other end at a fixed point of the base frame. A movement of the foot part may be forced by the expansion or contraction. A complete enclosure of the drive device exclusively results in a protection for a possible spindle drive or any other linear drive against external effects such as rain, snow, dust, sand, or dirt. The connection of a drive device to the drive unit may be realized by a cable which at the connection to the drive device may be grouted, welded, or connected in any other way to the rubber bellows. The drive unit may be understood as a compact, closed unit which provides the drive devices with sufficient electricity. With a combination of an accumulator, a solar cell, a charging device, a voltage transformer, and a control unit, a possibility may be suggested for providing a sufficient energy supply to one or several drive devices independently of an electric power supply, such that the lounger according to the invention may be placed at any place within a garden, without being dependent on a socket outlet and cables. By combining the features mentioned above, a lounger is suggested which may be particularly robust against environmental influences and at the same time may provide a maximum in convenience for a user. A lounger frame may meet different needs of users in that different heights and angles of lounger frame elements may be adjusted. An encapsulation of the drive device for the lounger frame and an electric power supply independent from a grid serve for enhancing the comfort, while at the same time rainproof and weatherproof materials may be used. According to an advantageous embodiment of the lounger according to the invention, the lounger frame comprises several lounger frame areas which may be adjusted independently from each other by way of one or several drive devices. The adapting may comprise adjusting of a height, an angle and a distance, furthermore it may comprise an adjustment of a hardness, or the like. Thus, the comfort of a user may be adjusted depending on the number of areas very individually. According to a further similarly advantageous embodiment of the lounger according to the invention, the lounger frame may be adapted for receiving a lounger overlay, such that the comfort of the user may be chosen depending on his liking in that an individual lounger overlay may be used. According to a similarly advantageous embodiment of the lounger according to the invention, the lounger overlay may be realized as a mattress, which for example may be produced of a foam, and which depending the number, position and size of the lounger frame elements, may be adapted for not obstructing a bending of the lounger frame. According to a particularly preferred embodiment, the lounger overlay may be designed such that the lateral surfaces and the upper side may be manufactured from a water-repellent material, which should be as UV-proof as possible. The upper side and the lateral surfaces of the lounger overlay may comprise more than one material, wherein an upper material for example should comprise particularly optical and haptic qualities, which a user of seating and lying furniture for the garden may expect, while a layer positioned below the upper layer may be primarily used for repelling water. The lower side of the lounger overlay should preferably be designed from a material which may be water-repellent and at the same time may be breathable. If the mattress is manufactured from a foamed material, a particularly good compression movement may be allowed, at the same time, sweat or condensed water which entered through small cracks, seams, or the like may escape from the lounger overlay, thus enhancing its durability. It is also preferable, that the lounger overlay is manufactured in form of a self-inflating mattress, which in its shape may also be adapted to the individual areas of the lounger frame, such that a movement of the lounger frame may not be obstructed. For this purpose, the lounger overlay should all-around be designed from a water-repellent material, which may be designed airtight and at the same time breathable at least at the lower side. If a relatively soft foam core is chosen for the self-inflating function, it may be appropriate to design the entire lounger overlay completely airtight and waterproof. The entry of air into the lounger overlay may be made possible by a corresponding manually closable ventilation opening. It might also be preferable to design a lounger overlay in form of an inflatable mattress which comprises a ventilation connection and a vent connection which may be connected to a compressor, which compressor may for example be arranged at the drive unit. In this way, a user may be able to remove the lounger overlay during winter months, and to store it space-savingly in the house. On the other hand, in this way at the same time the possibility may be provided to adjust the hardness of the lounger overlay according to the liking of a user, such that in cooperation with several lounger frame elements and lounger frame areas, a particularly good adaptability to the needs of a user may be realized. At the same time, it may be particularly preferable to provide a receiving compartment within the base frame which may be used for accommodating the drive unit. Thus, it may not be absolutely necessary to provide a separate container or the like beside or behind the inventive lounger, in which the drive unit is to be placed. At the same time, it may also be preferable to provide one end of the inventive lounger with two wheels which may be used for moving the lounger in the garden. For example, one end of the lounger may comprise only feet, while the opposing end comprises two wheels. By way of lifting the end with the feet, a simple moving of the inventive lounger may be possible. According to a preferred embodiment of the lounger according to the invention, the drive unit may be designed as a separate component which may provide an electric power supply for enhancing the functionality of several different components. In addition to an accumulator, which may be charged manually by way of an integrated charging electronics, a solar cell may be arranged at an upper side of the drive unit, which solar cell in particular during good weather, that is, when a use of the lounger is to be expected, makes possible a recharging or a maintaining of the charge of the accumulator, or a providing of electric energy. Also for this purpose, a dedicated charging electronics may be necessary, which may also be combined with the charging electronics which effects a charging of the accumulator with grid electricity. At the same time, a separate independent drive unit may be adapted for forming a connection with a connection device of the lounger according to the invention. Preferably, an individual connection device or a combined connection device may be necessary for each drive device, and thus particularly for each lounger frame area to be adjusted, which connection device makes possible a connecting of several electrical circuits between the drive unit and several drive devices. In this way, it may be ensured that a controlling of the lounger according to the invention may be carried out by the drive unit itself, as for example by way of a remote control which may be coupled to the drive unit wirelessly or wire-based. By providing an electrical circuit between the drive unit and the drive device, a drive device may be put into operation, such that a corresponding lounger frame element may be moved. Accordingly, it would be possible to remove the drive unit together with the electronics and the accumulator from the lounger according to the invention, and to store it in the house, despite the weatherproof design. This may be particularly advantageous for the electronics and the accumulator, whose functionality otherwise may be compromised at low temperatures. At the same time, the removability of the drive unit serves as protection against contamination, particularly during autumn or winter weather. The connecting device may be realized in various ways. If, for example, the drive unit is arranged in the receiving compartment described above in the base frame of the lounger according to the invention, a conventional electrical plug connection may be completely sufficient, as the plug connection in the receiving compartment or the like is relatively well protected within the base frame and below the lying surface, and which should not get wet, also during heavy rain. On the other hand, an inductive electrical connection between the drive unit and the individual drive devices may be created, so that it may be only necessary to provide the drive unit with induction coils, preferably with core elements, which correspond to induction coils with core elements at the lounger according to the invention, and in this way transmit electricity. In this way, the drive unit as well as the connection device directly at the lounger according to the invention may be encapsulated. Furthermore, it may be particularly advantageous to provide the drive unit with additional interfaces, such that for example a charging of the accumulator may be possible, without prior opening or disassembling of the drive unit. As an example, the power connection socket is mentioned here, which may be made weatherproof by way of the sealed screw top. Therein, the screw top may be manufactured from a metallic material, which does not become brittle due to external environmental influences, wherein at the inner surface of the screw top, a sealing ring may be arranged, which sealing ring makes possible a particularly good sealing of the power connection plug. Similarly, it may be conceivable, to provide further interfaces which may allow a user to operate electronic devices, particularly during good weather. As an example, a low voltage connection is mentioned here, which may, for example be designed as an USB-connection for modern portable electronic devices. This connection may also be encapsulated by way of a screw top or the like, wherein the screw top may be removed by a user when required, to make possible a use of the USB connection. In this way, mobile phones, MP 3 players, or the like may be used over a considerably prolonged time period. For a better usability of the lounger according to the invention on board of ships or boats, the frame should be produced of a material which may be extremely durable, also when constantly being subjected to moisture and salted water. This material may a metallic material, as for example steel or aluminium, which may be provided with a saltwater proof priming and/or finishing. At the same time, also various kinds of wood may be suitable for a use on board of ships, which preferably are treated with such primings and/or finishings. Basically, it may be appropriate to use quite robust kinds of wood, which additionally may be provided with a clear, shiny, robust, and saltwater-resistant finishing layer for all of the considered climate regions. For the use on ships, it may be advantageous to adapt the choice of material, and thus the weight of the lounger to the size and kind of ship. When used on bigger cruise ships, relatively heavy loungers which comprise a steel frame or a solid wood frame may be used, thus preventing an easy slipping on deck. This may be further enhanced by providing a rubber coating or another slip-proof coating at areas of the lounger which contact the floor of the ship. On board of smaller boats, which for example have clearer weight limitations, lighter frame constructions may be appropriate, for example using hollow profiles from aluminium. Additionally, it may be appropriate to enhance the weather protection of the drive unit and of the drive device for use on board of ships in such a way that all joint patches of a housing of the drive unit may be packed and sealed, and in that radial sealings of the drive device are adjusted for use in a surrounding comprising saltwater. Additionally, it may be advantageous to produce moveable elements of a drive device from aluminium or stainless steel, and/or to coat the moveable elements completely with a bellows construction, for minimizing the risk of rusting. In a particularly advantageous embodiment of the lounger according to the invention, the drive unit which provides energy may be adapted for providing energy to several drive devices of several loungers. This implies that, for example on deck of a cruise ship, large numbers of loungers according to the invention may be connected to a common drive unit, which provides all of the loungers coupled to it with energy with the help of available energy sources, such as an onboard power grid, solar energy, an accumulator, or the like. This system of loungers accordingly comprises a common drive unit with several drive devices of several loungers coupled to the drive unit. For example, a drive unit may provide energy to 10-50 loungers. The need mentioned above may be also met by the already described drive unit. At the same time, a use of a drive unit according to the invention for driving of an electrical lounger may also cover the need. BRIEF DESCRIPTION OF THE DRAWINGS Further features, advantages, and application possibilities of the present invention may be derived from the following description of embodiments and from the figures. Therein, the described and depicted features form the subject-matter of the invention separately and in any combination, independently from their combination in the individual claims or their back references. Furthermore, in the figures same reference numbers are used for same or similar objects. FIG. 1 shows a view of a first embodiment of a lounger according to the invention. FIG. 2 shows a view of further embodiment of a lounger according to the invention. FIG. 3 shows a view of a drive unit according to the invention for use in a lounger according to the invention. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS In FIG. 1, an embodiment of a lounger 2 according to the invention is shown exemplarily, the lounger 2 exemplarily comprises a base frame 4, a lounger frame 6, and a drive unit 8, which may be arranged in a receiving compartment 10 below the base frame 4. The lounger frame 6 may exemplarily be divided into five different areas 12 - 20 on which a lounger overlay 22 is arranged. Exemplarily, a foot part formed by the lowest lounger frame area 12 may be lifted in parallel to the base frame 4, wherein the parallel guidance may be provided by a lever 24 which is arranged in parallel to the lounger surface area 14. The movement of the foot part may be realized by a drive device 26 which moves the area 14 of the lounger frame at a point 36, and which alternatively may also deflect the lever 24. The invention is not to be limited thereto. Furthermore, a deflection direction of the drive device 26 should not be construed as a limitation of the invention. Rather, the shown example makes clear, that a lounger 2 may be provided which may absolutely flexibly be adapted to individual user needs for enhancing the comfort of the user. Furthermore, it may be possible to move a lounger frame area 18 or 20 by way of a further drive device 34, which engages the lounger frame element 18 at a point 28. This point 28 is chosen absolutely arbitrarily in the shown presentation, the invention is not to be limited thereto. In the shown embodiment, a weather protection for the drive devices 26 and 34 may be provided in that the drive device 34 is arranged in a receiving compartment 10 and is only in contact with the surroundings by way of a slot 30. This slot may be furthermore protected by a blind-like device which encloses the drive device 34 on both sides, and which may be movable within the slot 30. At the same time, also the receiving compartment 10 may be designed shorter, wherein it may extend up to the dashed line 32, such that the drive device 34 would have to be mounted at another point, and would then be completely exposed to the surroundings. Here, it may be appropriate to put a rubber bellows or the like at the drive device 34, which rubber bellows provides for a complete encapsulation of the drive device 34. Analogously, the same may be possible for the drive device 26. Within the receiving compartment 10, in other words within the drive unit 8, exemplarily an accumulator 38 may be arranged, which accumulator is connected to an electronics unit 40. This electronics unit may be adapted for converting inputs of a remote control 42 into a current flow from an accumulator 38 or from another current source to the driving devices 26 and 34. At the same time, the electronics unit 14 may be adapted for making possible a charging or a charge maintaining of the accumulator 38. In the shown case, the lounger overlay 22 may also be designed with five areas 44 - 52, which may be adapted to the lounger frame areas 12 - 20. In this way, an easy movability may be provided. For example, the lounger overlay 22 may be provided as an integrally formed component which may be pivoted easily due to recesses or cut-outs in the bending positions. The lounger overlay 22 may be any kind of lounger overlay. Conventional mattress structures which for example may be formed of a foamed material may be possible. At the same time, also a self-inflating mattress may be possible which for example comprises an airtight casing 54, and which may be supplied with pressurized air by way of a pressurized air connection. It may be possible to attach a pressurized air line 58 to the pressurized air connection 56, and to connect the pressurized air line to the compressor 60 within the drive unit 8. In this way, a user may be able, for example by operating a remote control 42 or the like not only to adjust the location or position of the individual lounger frame elements 12 - 20, but also to adjust the fill level of the lounger overlay 22, and in this way to obtain a further degree of freedom for adjusting the personal convenience. Basically, it may be possible to design the lounger 2 according to the invention also transportable, which in the shown embodiments may be achieved by way of a pair of wheels 62, which for example may be arranged below a foot end of the lounger. At an opposing end, the lounger 2 according to the invention rests for example on two feet 64, and further comprises a handle 66 which serves for lifting the lounger 2 according to the invention at one side, such that the lounger 2 may be moved on a ground by way of the wheels 62. In the shown embodiment of FIG. 1, the drive unit 8, which coordinates the current supply and the connection to a control element, for example in the form of a remote control 42, may be arranged in a receiving compartment 10 below the base frame 4. It may however also be possible to conceive or to provide a drive unit which is designed as a completely separate component, and which may be mounted to a lounger according to the invention manually. This is shown in FIG. 2, where a drive unit 68 is arranged at a base 70, and which supplies sufficient electric current to the lounger 2 according to the invention. The drive unit 68 is shown in more detail in FIG. 3. The drive unit 68 may for example comprise a base 70 and a top 72, wherein an accumulator 38, an electronics unit 40, and a compressor 60 may be arranged at the base 70. At a lateral surface 74, for example a series of plug sockets 76 may be arranged, which provide manifold connection possibilities. For example, a grid connection 78 may be provided, at which a cable enables the operation of the lounger 2 according to the invention from a public power grid. Equally, this grid connection 78 may also serve for charging the accumulator 38, wherein the electronics unit 40 also functions as a charging electronics. In this way, a fast operation with a strongly discharged accumulator 38 may be made possible, for example after a longer operation pause of the lounger 2 according to the invention. Furthermore, a connection 80, for example for small electronic devices which may be used at the lounger 2 according to the invention, may be also provided. Exemplarily, this connection 80 for small devices is shown as an USB connection, as nowadays lot of mobile electronic devices may be supplied with electricity by way of such a connection. Here, the electronics unit 40 functions as a voltage converter, which provides a constant voltage of 5 Volt to this connection 80, and at the same time limits the current to 0.5 Ampere or to another, predefined common maximal value. As the lounger 2 according to the invention may mainly be used during good weather, it may be appropriate to arrange one or several solar cells 82 at the top 72, which solar cells are also to be connected to the electronics unit 40. In this way, it may be ensured that the lounger 2 according to the invention may always be ready for use, as the accumulator 38 may also be charged by the operation of the solar cells 82. In such a structure of the drive unit 68, the electronics unit 40 functions as a voltage converter which converts the voltage provided by the solar cells 82 into a charging voltage. The electronics unit 40 should furthermore be adapted for maintaining the accumulator 38, that is to say, it is to be made sure that the accumulator 38 may neither be overcharged nor be discharged too deeply. The provided plug sockets 78 and 80, or all further possible connection sockets may be arranged in a recess of the housing for protection against weather influences or against saltwater, which recess of the housing may be provided with a thread which respectively allows a screwed connection with a screw cap 84. At this point it should be noted, that the shown embodiments in FIGS. 1-3 only serve for illustration purposes, the invention is in no way confined to the shown embodiments. This applies in particular to the dividing of the lounger frame into five lounger frame areas, also more or less areas may be possible. Substantially, in the design of such loungers, at least a one pivotable backrest and preferably also an adjustable foot part are to be provided. The configuration of the drive unit according to the invention is also not limited to being arranged in a receiving compartment below the foot end, or to being placed on a base beside the foot part. In fact, there may be no limits to the creative freedom of the person skilled in the art, however, at the time of the application it appeared that the shown positions and arrangements may be quite advantageous. Furthermore, the invention is not limited to using certain kinds of drive devices. All drive devices may be possible which are capable of performing a linear and/or rotational movement. Besides spindle drives with electro-motors, pneumatic and hydraulic drives may be used, wherein a pneumatic drive in the form of a pneumatic cylinder may be coupled to the compressor for an air mattress. In this way, a very variable design of the drive may be possible, as within the lounger according to the invention, only pressurized air has to be transferred. Additionally, it should be noted, that “comprising” does not exclude other elements or steps, and that “a” or “an” does not exclude a plurality. Furthermore, it should be noted that features described with reference to one of the embodiments above may be used also in combination with other features of other embodiments described above. Reference signs in the claims should not be construed as limitations. REFERENCE SIGNS 2 lounger 4 base frame 6 lounger frame 8 drive unit 10 receiving compartment 12 area of the lounger frame 14 area of the lounger frame 16 area of the lounger frame 18 area of the lounger frame 20 area of the lounger frame 22 lounger overlay 24 lever 26 drive device 28 point 30 slot 32 dashed line 34 drive device 36 point 38 accumulator 40 electronics unit 42 remote control 44 area of the lounger overlay 46 area of the lounger overlay 48 area of the lounger overlay 50 area of the lounger overlay 52 area of the lounger overlay 54 airtight casing 56 pressurized air connection 58 pressurized air line 60 compressor 62 wheel 64 foot 66 handle 68 drive unit 70 base 72 top 74 lateral surface 76 plug socket 78 grid connection 80 connection for small devices 82 solar cell 84 screw cap

Summary: A lounger includes at least a weatherproof base frame, a weatherproof lounger frame with at least two movably mounted lounger frame elements, and at least one weatherproof drive unit for supplying at least one drive device with electric energy. The drive unit preferably includes a combination of an accumulator, a solar cell, and a grid connection, and contributes in connection with a weatherproof design of the frames to portable and grid-power-independent lounger with an automatic adjusting functionality.

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en

Summarize: Background Health centers are private, nonprofit community-based organizations or, less commonly, public organizations such as public health department clinics. Section 330 of the Public Health Service Act, which authorizes the Health Center Program, requires health centers to provide a comprehensive set of primary health care services, including enabling services—such as language translation and transportation—that facilitate access to health care. Among other things, health centers are also required to have a sliding fee scale based on a patient’s ability to pay and to be governed by a community board of which at least 51 percent of the members are patients of the health center. Health Center Patients, Revenue, and Grants In 2010, nearly 93 percent of all health center patients had incomes at or below 200 percent of the federal poverty level, and nearly 72 percent had incomes at or below 100 percent.insured by Medicaid, and nearly 38 percent were uninsured. See figure 1 for more information on insurance coverage of health center patients. HRSA Revised 2011 Award Process, and Most New Access Point Grants Went to Health Centers Serving Special Populations HRSA revised its New Access Point award process for fiscal year 2011 to increase the emphasis on need and on the designated special populations. As a result of these changes and HRSA’s receiving less fiscal year 2011 funding than it had anticipated, a high proportion of grants went to health centers serving the designated special populations. HRSA Increased Weight Given to Need and Emphasized Special Populations in 2011 Award Process HRSA revised the New Access Point grant application and award process for fiscal year 2011. According to HRSA officials, it did so to better target resources to communities with high need because it found that the previous process did not adequately factor need into the application score, and because past applicants and grantees expressed concerns about this issue. One step HRSA took was to increase the need score from 10 to 30 points, out of a maximum of 100 base points. (See table 2.) Twenty of the 30 points are available for applicant responses provided on an attached form that documents barriers to access to care and various health indicators in the proposed service area, and the remaining 10 points are available based on the applicant’s narrative describing health care need. HRSA also revised its award process in fiscal year 2011 to award extra points, which HRSA calls priority points, over the maximum 100 base points to applicants seeking to serve the three designated special populations and sparsely populated areas. HRSA did this to help it continue to meet the Public Health Service Act requirements regarding these populations.applicants seeking to serve high-poverty areas to further increase the emphasis on need in the award process. This was the first time HRSA In addition, HRSA decided to award extra points to awarded such extra points, and the application guidance described how the points would be awarded, providing transparency for this aspect of the process. (See table 3.) HRSA officials applied the extra points to applicants’ base scores out of 100; these adjusted scores were used to update and finalize the rank order list of all applicants. HRSA awarded from 5 to 10 extra points for serving one or more of the designated special populations, 5 extra points for serving a sparsely populated area, and up to 5 extra points for serving a high-poverty area. Health Centers and Other Providers Reported Collaboration and Little or No Competition, but Rural Areas Have Greater Potential for Competition Health centers in the communities we studied collaborate with other providers in their service area. Health centers and other providers said they generally did not compete for patients, but we found greater potential for competition in rural areas. Health Centers in Communities We Studied Collaborate with Other Providers Health centers in the communities we studied collaborate with other providers to meet the health care needs of patients in the health center’s service area. Officials we interviewed from each of the health centers described at least one collaborative relationship with another provider— such as local hospitals and specialty care providers—to provide access to services not available through the health center. For example, officials from one of the health centers told us they collaborate with specialists such as a pediatric cardiologist, podiatrist, and ophthalmologist. In addition, officials from several hospitals said that collaborating with health centers is important because the centers help reduce the non-urgent use of hospital emergency departments. However, in some of the rural communities we studied we also found that the relationship between the health center and a nearby hospital was strained. For example, officials from a hospital in one community we studied told us that the health center did not always send the medical records of admitted patients in a timely way. HRSA has encouraged health centers to collaborate with other providers in their service area. HRSA issued a Program Assistance Letter in fiscal year 2011 that provides guidance to health centers on collaborating and establishing contractual agreements with other providers to maximize resources and efficiencies in their service area. For example, the letter includes a list of suggested resources to help health centers maximize collaboration with other safety net providers, such as the UDS Mapper tool, PCAs, and HRSA project officers, who are responsible for overseeing health centers and providing technical assistance. For fiscal year 2011, HRSA added a collaboration component to the New Access Point application scoring to encourage collaboration by health centers. The fiscal year 2008 and 2009 applicants had been required to include a written description of existing collaborative relationships with other providers and had also been encouraged to submit letters of support, but these were not scored separately. However, in fiscal year 2011, applicants could receive up to 10 points for submitting letters of support—from other providers or community organizations—and a written description of their existing and proposed efforts to establish collaborative relationships with other providers in their proposed service area. During the period included in our study, letters of support were written by, among others, neighboring health centers, local hospitals, private physicians, local government agencies, and PCAs. The letters of support generally included similar types of information—such as a description of the specific health care needs of the community and support for the applicant’s efforts to care for underserved patients—regardless of the type of organization expressing support. A few letters included information about specific support the writer had provided or planned to provide to a health center, such as pediatric or obstetrical care to health center patients. PCAs often work with applicants and grantees to help them develop collaborative relationships. Officials from several PCAs told us they used applicants’ requests to the PCA for a letter of support as an opportunity to assist them in developing relationships with other providers. For example, officials from one PCA told us that for the fiscal year 2011 New Access Point award cycle, they hosted over 20 town hall meetings in applicants’ communities to facilitate community involvement, collaboration, and understanding of the Health Center Program. Several PCAs told us they also work with potential applicants to determine whether it would be better for them to combine efforts with an existing health center grantee or to establish a new health center. Officials from one PCA explained that it may be better for a new organization to become a satellite site of an existing organization because existing organizations already have the resources and infrastructure in place to operate a health center. Officials we interviewed identified various factors that contribute to successful collaboration between health centers and other providers. Officials from hospitals, other providers, and community groups said that leadership commitment to collaboration, community participation in developing a new health center, and other providers’ understanding of the role of health centers are important factors that contribute to successful collaboration. For example, officials from one hospital and a community group in the same area noted improved collaboration as a result of a new director coming to a health center. They told us that the previous director was difficult to collaborate with and did not acknowledge the abilities of other primary care providers to serve the safety net population. These officials also said that the current relationship is much more collaborative and that the health center and hospital share a board member and a physician. In addition, officials from one PCA told us that a former state government official had, over many years, discouraged hospitals from collaborating on efforts to establish new health centers in their communities, warning the hospitals that they could lose patients to the health centers. Regarding the importance of community participation, officials from one hospital said that the hospital led the effort to develop the health center in its community, because previously physicians voluntarily provided services for low-income patients two evenings a week, and that effort was unsustainable. Health Centers and Other Providers Generally Did Not Encounter Significant Competition for Patients, but Rural Areas Have Greater Potential for Competition In the communities we studied, health centers and other providers in their service area generally do not compete for patients. HRSA and PCA officials told us that health centers typically serve patients not treated elsewhere, such as uninsured and Medicaid patients. Nationwide, 37.5 percent of health center patients are uninsured, and for the eight health centers we studied, the rate of uninsured patients averaged 30.4 percent. Similarly, Medicaid patients make up 38.5 percent of health center patients nationwide, and 35 percent in the health centers we studied. Officials from most of the PCAs we spoke with said health centers and other providers generally do not compete for uninsured patients; some also noted that other providers rarely provide care for uninsured patients. Similarly, officials from one health center told us that Medicaid patients in their area had difficulty finding other providers that would accept them. We have previously reported on the difficulties certain Medicaid patients, such as children, face in finding providers who are willing to serve them. HRSA’s service area overlap policy is designed to help the agency avoid awarding grants for new delivery sites in areas where other safety net providers are already serving the population’s need, and this may reduce competition between health centers and other safety net providers. The agency did not award grants to two applicants in fiscal years 2008 and 2009—one in each year—because awarding grants to these applicants would have resulted in overlap with existing providers that had the capacity to meet the needs of the area. HRSA officials told us that they did not find any significant service area overlap during the fiscal year 2011 award cycle. They also said that since the agency increased its emphasis on collaboration—and applicants have increased their outreach in their communities—it has received fewer complaints from other safety net providers about service area overlap than it received in prior years. See GAO, Community Health Centers: Adapting to Changing Health Care Environment Key to Continued Success, GAO/HEHS-00-39 (Washington, D.C.: Mar. 10, 2000). care if health centers did not offer these services, because some dentists are unwilling to serve Medicaid patients. Insurance coverage for patients served by the health centers we studied includes coverage by Medicaid, CHIP, Medicare, other public insurance (such as state insurance programs), and private insurance. receive grant funding for construction or renovations, which gives them a competitive advantage. They said the health centers might be better able to attract insured patients because of the improved facilities or might be able to attract staff because these grant funds free up resources that can be used for higher salaries. PCA and health center officials we interviewed more frequently raised concerns about the potential for competition between health centers and rural health clinics, in part because there are more similarities in the services they provide to patients in rural communities. For example, several PCA officials told us that while there is no competition between health centers and rural health clinics for serving uninsured patients, they do compete for patients with insurance, including Medicaid and Medicare. Although patients in rural areas often face access barriers because of a shortage of providers, HRSA officials said the addition of a health center to an area can increase competition for insured patients when such patients seek treatment from a health center that is more conveniently located than other providers. HRSA officials also told us that they may award grants in rural areas where there are other providers if those providers do not fully meet the needs of the safety net population. For example, existing providers may not offer a sliding fee scale or be willing to serve uninsured people. Conclusions Health centers funded by HRSA’s Health Center Program are a critical component of the nation’s health care safety net, and New Access Point grants provide the agency with an important means for increasing access to health care for vulnerable populations—those who may have difficulty obtaining needed health care services because of financial or other limitations. To better target resources to communities with a high need for health center services, in fiscal year 2011 HRSA increased the weight of the criterion assessing the need for services in the New Access Point grant application. Certain populations—migrant and seasonal farmworkers, homeless people, and residents of public housing—are particularly vulnerable and often have specific health and access problems. In its 1996 consolidation of the Health Center Program, Congress began requiring that specific proportions of the program’s funds be used to serve these populations. Over the years, HRSA has taken various actions to ensure it was meeting the required funding proportions. During its fiscal year 2011 New Access Point award process, HRSA for the first time gave extra points to applicants serving these designated special populations. Congress also requires HRSA to give special consideration to organizations serving sparsely populated areas, and in fiscal year 2011 HRSA also gave extra points to applicants serving sparsely populated and high-poverty areas. HRSA’s approach of assigning these extra points—and its description in its application guidance of how the points would be awarded—increased the transparency of the grant award process compared to previous years. However, because the extra points were not sufficient to ensure that HRSA met its statutorily required funding proportion for migrant health centers, HRSA also moved applicants serving this population ahead of other applicants to ensure the required proportion was met, a step that was not specifically described in the application guidance. Although HRSA had used such an approach before, the effect in fiscal year 2011 was magnified by the combined effect of the reduction in program funding and HRSA’s need to increase the share of funding awarded to the designated special populations as a result of not applying the proportions when awarding grants with Recovery Act funds in fiscal year 2009. HRSA has periodically needed to take actions to meet its statutory obligations and may face such a situation in the future. Evaluating the effectiveness and transparency of the New Access Point grant award process it used most recently could help HRSA identify lessons learned and possible improvements that it could apply to future funding cycles to ensure the most effective use of limited Health Center Program resources. Recommendation for Executive Action To ensure that in the future HRSA can effectively target limited Health Center Program resources through a transparent grant award process, the Secretary of HHS should direct the Administrator of HRSA to evaluate the fiscal year 2011 New Access Point grant award process to identify lessons learned and potential improvements for future funding cycles, including consideration of (1) the effect of the change in the need score on targeting grants to communities with demonstrated need, (2) the effect of actions taken to target grants to applicants proposing to serve the designated special populations and sparsely populated and high-poverty areas, and (3) the transparency of the process to applicants, Congress, and the public. The Secretary should also direct the Administrator of HRSA to complete the evaluation before the next New Access Point funding opportunity is announced, make the results of the evaluation publicly available, and incorporate any improvements identified into the award process for that funding opportunity. Agency Comments We provided a draft of this report to HHS for review, and HHS provided written and oral comments. (HHS’s written comments are printed in app. III.) HHS agreed with our findings and recommendation. In its general comments, HHS restated and provided additional information on our discussion of the Health Center Program and the New Access Point grant process. HHS said that the increased score for need and use of extra points improved the agency’s awarding of New Access Point grants in fiscal year 2011 by targeting resources to higher need communities and populations while still ensuring that organizations with sound health center service delivery plans were funded. HHS also noted that increased emphasis on collaboration contributed to health centers and other area providers maximizing available resources while enhancing the service delivery system to better address the community’s primary health care needs. HHS said that these factors support HRSA’s goal to expand the current safety net on a national basis by creating new delivery sites in areas not currently served by federally funded health centers. Regarding the GAO recommendation, HHS said HRSA is taking steps to evaluate the effects of the change in the need score and other actions taken to target grants, including for special populations. According to HHS, HRSA plans to use the findings from its evaluation to improve the New Access Point application guidance and will make its findings available to the public. In its oral comments, HHS suggested that the title of the draft report did not fully reflect the contents of the report, which provides a detailed discussion of the changes HRSA made to its fiscal year 2011 New Access Point grant award process, including increased weight given to need. We revised the report title to reflect this. HHS also provided technical comments, and we incorporated information from its general and technical comments as appropriate. As agreed with your offices, unless you publicly announce the contents of this report earlier, we plan no further distribution until 30 days from the report date. At that time, we will send copies to the Secretary of HHS and the Administrator of HRSA. The report also will be available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at (202) 512-7114 or draperd@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made major contributions to this report are listed in appendix IV. Appendix I: New Access Point Grant Awards, Fiscal Years 2008-2011, and Total Health Center Grantees, 2010 Table 5 shows the distribution of New Access Point grants awarded to applicants in each state and territory in fiscal years 2008 through 2011. It also shows the number of grantees in each state and territory and the percentage of total grantees in each state and territory in 2010. Appendix II: Ratio of Health Center Grantees to Population Living in Poverty, by State, 2010 We calculated the ratio of total health center grantees to the population living in poverty for every state, a measure of the availability of care for the medically underserved. We then ranked them in order from highest to lowest ratio. (See table 6.) Appendix III: Comments from the Department of Health and Human Services Appendix IV: GAO Contact and Staff Acknowledgments GAO Contact Acknowledgments In addition to the contact named above, Helene F. Toiv, Assistant Director; Giselle Hicks; Coy J. Nesbitt; Roseanne Price; Julie T. Stewart; E. Jane Whipple; Jennifer Whitworth; and Monique Williams made key contributions to this report. Related GAO Products Health Center Program: Improved Oversight Needed to Ensure Grantee Compliance with Requirements. GAO-12-546. Washington, D.C.: May 29, 2012. Hospital Emergency Departments: Health Center Strategies That May Help Reduce Their Use. GAO-11-643T. Washington, D.C.: May 11, 2011. Hospital Emergency Departments: Health Center Strategies That May Help Reduce Their Use. GAO-11-414R. Washington, D.C.: April 11, 2011. Health Resources and Services Administration: Many Underserved Areas Lack a Health Center Site, and Data Are Needed on Service Provision at Sites. GAO-09-667T. Washington, D.C.: April 30, 2009. Health Resources and Services Administration: Many Underserved Areas Lack a Health Center Site, and the Health Center Program Needs More Oversight. GAO-08-723. Washington, D.C.: August 8, 2008. Health Centers: Competition for Grants and Efforts to Measure Performance Have Increased. GAO-05-645. Washington, D.C.: July 13, 2005. Community Health Centers: Adapting to Changing Health Care Environment Key to Continued Success. GAO/HEHS-00-39. Washington, D.C.: March 10, 2000.

Summary: Health centers funded in part by grants from HRSA’s Health Center Program, under Section 330 of the Public Health Service Act, provide comprehensive primary care services for the medically underserved, including many poor, uninsured, and Medicaid patients. Legislation enacted in 2009 and 2010 provided additional funding that could significantly expand health center capacity over the next several years. GAO was asked to review HRSA’s process for awarding grants for new delivery sites and possible effects of health centers, such as competition, on other providers. This report examines (1) the actions HRSA has recently taken to target its grants for new delivery sites to health centers in communities with demonstrated need and the outcome of HRSA’s award process in recent years, and (2) the extent to which HRSA-funded health centers collaborate and compete with other health care providers in their service area. GAO focused its work on NAP grants, HRSA’s primary means of establishing new health centers and delivery sites, during fiscal years 2008 through 2011. GAO analyzed HRSA documents and interviewed HRSA officials, and interviewed officials from 11 health centers and providers and officials in their service areas. The Department of Health and Human Services’ (HHS) Health Resources and Services Administration (HRSA) revised its New Access Point (NAP) competitive award process in fiscal year 2011 to increase the emphasis on the need for services in the applicant’s proposed service area, and on the three special populations—migrant and seasonal farmworkers, homeless people, and residents of public housing—designated by the Public Health Service Act. The act requires that certain proportions of Health Center Program funding go to health centers serving the special populations. To increase the emphasis on need, HRSA increased the weight given to need in the application review process. To target health centers serving special populations, HRSA gave extra points in the application process to applicants proposing to serve them. When this was insufficient to meet the required proportions, HRSA moved some applicants ahead of others in the award rank order list, a method it had used in the past. The effect of HRSA’s actions on the award outcome was magnified in fiscal year 2011 because (1) HRSA received less program funding than it had anticipated, and (2) it needed to increase the share of grants going to health centers serving the special populations because HRSA had not applied the statutory proportions when it used American Recovery and Reinvestment Act funding to award grants in fiscal year 2009. As a result, HRSA awarded 67 NAP grants in fiscal year 2011, 57 of which went to applicants proposing to serve at least one special population; 13 of the 57 received grants by being moved ahead of other applicants with equal or higher review scores. HRSA announced the extra points in application guidance, but not the potential moving of some applicants ahead of others. As HRSA has periodically needed to take actions to meet its statutory obligations and may need to do so again, evaluating the effectiveness and transparency of its most recent New Access Point grant award process could help it identify lessons and possible improvements for the future. Health centers in the communities GAO studied collaborate with other providers and generally do not compete with them for patients, but GAO found greater potential for competition in rural areas. Health center officials described collaborative relationships with other providers that give patients access to services not available through the health center. Health centers and other providers told GAO they generally do not compete for patients; health centers typically serve patients not treated elsewhere, such as uninsured and Medicaid patients. However, because the health center grant covers, on average, about 20 percent of a center’s budget, other funding must also be secured, such as by serving insured patients, for the center to be financially sustainable. This can result in competition with other providers in its service area. During the award process, HRSA takes steps to reduce competition by identifying nearby safety net providers and assessing whether the level of unmet need in the area warrants a grant for a new health center or delivery site. Greater potential for competition exists in rural areas because patients there are more likely to be insured and rural health clinics and certain hospitals might seek to serve some of the same patients as health centers, although they may not offer all of the services required of health centers.

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Summarize: Mr. Sherlock Holmes, who was usually very late in the mornings, save upon those not infrequent occasions when he was up all night, was seated at the breakfast table. I stood upon the hearth-rug and picked up the stick which our visitor had left behind him the night before. It was a fine, thick piece of wood, bulbous-headed, of the sort which is known as a "Penang lawyer." Just under the head was a broad silver band nearly an inch across. "To James Mortimer, M.R.C.S., from his friends of the C.C.H.," was engraved upon it, with the date "1884." It was just such a stick as the old-fashioned family practitioner used to carry--dignified, solid, and reassuring. "Well, Watson, what do you make of it?" Holmes was sitting with his back to me, and I had given him no sign of my occupation. "How did you know what I was doing? I believe you have eyes in the back of your head." "I have, at least, a well-polished, silver-plated coffee-pot in front of me," said he. "But, tell me, Watson, what do you make of our visitor's stick? Since we have been so unfortunate as to miss him and have no notion of his errand, this accidental souvenir becomes of importance. Let me hear you reconstruct the man by an examination of it." "I think," said I, following as far as I could the methods of my companion, "that Dr. Mortimer is a successful, elderly medical man, well-esteemed since those who know him give him this mark of their appreciation." "Good!" said Holmes. "Excellent!" "I think also that the probability is in favour of his being a country practitioner who does a great deal of his visiting on foot." "Why so?" "Because this stick, though originally a very handsome one has been so knocked about that I can hardly imagine a town practitioner carrying it. The thick-iron ferrule is worn down, so it is evident that he has done a great amount of walking with it." "Perfectly sound!" said Holmes. "And then again, there is the 'friends of the C.C.H.' I should guess that to be the Something Hunt, the local hunt to whose members he has possibly given some surgical assistance, and which has made him a small presentation in return." "Really, Watson, you excel yourself," said Holmes, pushing back his chair and lighting a cigarette. "I am bound to say that in all the accounts which you have been so good as to give of my own small achievements you have habitually underrated your own abilities. It may be that you are not yourself luminous, but you are a conductor of light. Some people without possessing genius have a remarkable power of stimulating it. I confess, my dear fellow, that I am very much in your debt." He had never said as much before, and I must admit that his words gave me keen pleasure, for I had often been piqued by his indifference to my admiration and to the attempts which I had made to give publicity to his methods. I was proud, too, to think that I had so far mastered his system as to apply it in a way which earned his approval. He now took the stick from my hands and examined it for a few minutes with his naked eyes. Then with an expression of interest he laid down his cigarette, and carrying the cane to the window, he looked over it again with a convex lens. "Interesting, though elementary," said he as he returned to his favourite corner of the settee. "There are certainly one or two indications upon the stick. It gives us the basis for several deductions." "Has anything escaped me?" I asked with some self-importance. "I trust that there is nothing of consequence which I have overlooked?" "I am afraid, my dear Watson, that most of your conclusions were erroneous. When I said that you stimulated me I meant, to be frank, that in noting your fallacies I was occasionally guided towards the truth. Not that you are entirely wrong in this instance. The man is certainly a country practitioner. And he walks a good deal." "Then I was right." "To that extent." "But that was all." "No, no, my dear Watson, not all--by no means all. I would suggest, for example, that a presentation to a doctor is more likely to come from a hospital than from a hunt, and that when the initials 'C.C.' are placed before that hospital the words 'Charing Cross' very naturally suggest themselves." "You may be right." "The probability lies in that direction. And if we take this as a working hypothesis we have a fresh basis from which to start our construction of this unknown visitor." "Well, then, supposing that 'C.C.H.' does stand for 'Charing Cross Hospital,' what further inferences may we draw?" "Do none suggest themselves? You know my methods. Apply them!" "I can only think of the obvious conclusion that the man has practised in town before going to the country." "I think that we might venture a little farther than this. Look at it in this light. On what occasion would it be most probable that such a presentation would be made? When would his friends unite to give him a pledge of their good will? Obviously at the moment when Dr. Mortimer withdrew from the service of the hospital in order to start a practice for himself. We know there has been a presentation. We believe there has been a change from a town hospital to a country practice. Is it, then, stretching our inference too far to say that the presentation was on the occasion of the change?" "It certainly seems probable." "Now, you will observe that he could not have been on the staff of the hospital, since only a man well-established in a London practice could hold such a position, and such a one would not drift into the country. What was he, then? If he was in the hospital and yet not on the staff he could only have been a house-surgeon or a house-physician--little more than a senior student. And he left five years ago--the date is on the stick. So your grave, middle-aged family practitioner vanishes into thin air, my dear Watson, and there emerges a young fellow under thirty, amiable, unambitious, absent-minded, and the possessor of a favourite dog, which I should describe roughly as being larger than a terrier and smaller than a mastiff." I laughed incredulously as Sherlock Holmes leaned back in his settee and blew little wavering rings of smoke up to the ceiling. "As to the latter part, I have no means of checking you," said I, "but at least it is not difficult to find out a few particulars about the man's age and professional career." From my small medical shelf I took down the Medical Directory and turned up the name. There were several Mortimers, but only one who could be our visitor. I read his record aloud. "Mortimer, James, M.R.C.S., 1882, Grimpen, Dartmoor, Devon. House-surgeon, from 1882 to 1884, at Charing Cross Hospital. Winner of the Jackson prize for Comparative Pathology, with essay entitled 'Is Disease a Reversion?' Corresponding member of the Swedish Pathological Society. Author of 'Some Freaks of Atavism' (Lancet 1882). 'Do We Progress?' (Journal of Psychology, March, 1883). Medical Officer for the parishes of Grimpen, Thorsley, and High Barrow." "No mention of that local hunt, Watson," said Holmes with a mischievous smile, "but a country doctor, as you very astutely observed. I think that I am fairly justified in my inferences. As to the adjectives, I said, if I remember right, amiable, unambitious, and absent-minded. It is my experience that it is only an amiable man in this world who receives testimonials, only an unambitious one who abandons a London career for the country, and only an absent-minded one who leaves his stick and not his visiting-card after waiting an hour in your room." "And the dog?" "Has been in the habit of carrying this stick behind his master. Being a heavy stick the dog has held it tightly by the middle, and the marks of his teeth are very plainly visible. The dog's jaw, as shown in the space between these marks, is too broad in my opinion for a terrier and not broad enough for a mastiff. It may have been--yes, by Jove, it is a curly-haired spaniel." He had risen and paced the room as he spoke. Now he halted in the recess of the window. There was such a ring of conviction in his voice that I glanced up in surprise. "My dear fellow, how can you possibly be so sure of that?" "For the very simple reason that I see the dog himself on our very door-step, and there is the ring of its owner. Don't move, I beg you, Watson. He is a professional brother of yours, and your presence may be of assistance to me. Now is the dramatic moment of fate, Watson, when you hear a step upon the stair which is walking into your life, and you know not whether for good or ill. What does Dr. James Mortimer, the man of science, ask of Sherlock Holmes, the specialist in crime? Come in!" The appearance of our visitor was a surprise to me, since I had expected a typical country practitioner. He was a very tall, thin man, with a long nose like a beak, which jutted out between two keen, gray eyes, set closely together and sparkling brightly from behind a pair of gold-rimmed glasses. He was clad in a professional but rather slovenly fashion, for his frock-coat was dingy and his trousers frayed. Though young, his long back was already bowed, and he walked with a forward thrust of his head and a general air of peering benevolence. As he entered his eyes fell upon the stick in Holmes's hand, and he ran towards it with an exclamation of joy. "I am so very glad," said he. "I was not sure whether I had left it here or in the Shipping Office. I would not lose that stick for the world." "A presentation, I see," said Holmes. "Yes, sir." "From Charing Cross Hospital?" "From one or two friends there on the occasion of my marriage." "Dear, dear, that's bad!" said Holmes, shaking his head. Dr. Mortimer blinked through his glasses in mild astonishment. "Why was it bad?" "Only that you have disarranged our little deductions. Your marriage, you say?" "Yes, sir. I married, and so left the hospital, and with it all hopes of a consulting practice. It was necessary to make a home of my own." "Come, come, we are not so far wrong, after all," said Holmes. "And now, Dr. James Mortimer--" "Mister, sir, Mister--a humble M.R.C.S." "And a man of precise mind, evidently." "A dabbler in science, Mr. Holmes, a picker up of shells on the shores of the great unknown ocean. I presume that it is Mr. Sherlock Holmes whom I am addressing and not--" "No, this is my friend Dr. Watson." "Glad to meet you, sir. I have heard your name mentioned in connection with that of your friend. You interest me very much, Mr. Holmes. I had hardly expected so dolichocephalic a skull or such well-marked supra-orbital development. Would you have any objection to my running my finger along your parietal fissure? A cast of your skull, sir, until the original is available, would be an ornament to any anthropological museum. It is not my intention to be fulsome, but I confess that I covet your skull." Sherlock Holmes waved our strange visitor into a chair. "You are an enthusiast in your line of thought, I perceive, sir, as I am in mine," said he. "I observe from your forefinger that you make your own cigarettes. Have no hesitation in lighting one." The man drew out paper and tobacco and twirled the one up in the other with surprising dexterity. He had long, quivering fingers as agile and restless as the antennae of an insect. Holmes was silent, but his little darting glances showed me the interest which he took in our curious companion. "I presume, sir," said he at last, "that it was not merely for the purpose of examining my skull that you have done me the honour to call here last night and again today?" "No, sir, no; though I am happy to have had the opportunity of doing that as well. I came to you, Mr. Holmes, because I recognized that I am myself an unpractical man and because I am suddenly confronted with a most serious and extraordinary problem. Recognizing, as I do, that you are the second highest expert in Europe--" "Indeed, sir! May I inquire who has the honour to be the first?" asked Holmes with some asperity. "To the man of precisely scientific mind the work of Monsieur Bertillon must always appeal strongly." "Then had you not better consult him?" "I said, sir, to the precisely scientific mind. But as a practical man of affairs it is acknowledged that you stand alone. I trust, sir, that I have not inadvertently--" "Just a little," said Holmes. "I think, Dr. Mortimer, you would do wisely if without more ado you would kindly tell me plainly what the exact nature of the problem is in which you demand my assistance."

Summary: An unknown visitor has come by the house that Sherlock Holmes and John Watson share, but they weren't home to meet him. Watson inspects a walking stick that the visitor mistakenly left behind. Watson notices that it's made of nice wood and it has a band of silver under the handle dedicated "To James Mortimer, M.R.C.S., from his friends of the C.C.H.," dated 1884. Watson guesses that the stick belongs to an older country doctor, and that it was a present from the local hunting organization. Holmes breaks the news to Watson: he's mostly wrong. But his dumb ideas have helped Holmes to get the right idea. Yeah, James Mortimer is a doctor, and he does live in the countryside. But the "H" in "C.C.H." probably means hospital rather than hunt. Holmes concludes that Mortimer must be a young man who did his medical residency at the Charing Cross Hospital before moving out to the countryside to start his own practice. Also, Holmes guesses from tooth marks on the stick that Dr. Mortimer owns a smallish dog. According to Holmes' records, there is a Dr. James Mortimer living in Dartmoor, in a town called Grimpen. Just then, Dr. Mortimer appears at their door, and it's all as Holmes says. He's young, he has a smallish dog, he left Charing Cross Hospital some time ago to set up his practice in the countryside. Dr. Mortimer is here because he has a most extraordinary problem.

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Summarize: CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of copending application Ser. No. 760,182, filed Jan. 17, 1977, now abandoned. BACKGROUND OF THE INVENTION The present invention relates generally to the dental alloys which are used for filling teeth from which decayed portions have been removed. More particularly, the invention relates to a corrosion resistant dental alloy which permits adjusting the handling characteristics of amalgams made with such alloy. The prior art emphasized the development of alloys which are corrosion resistant. While typical dental alloys are principally composed of silver and tin, they usually contain small amounts of copper and zinc. A typical alloy of the prior art would contain at least 65 wgt.% silver, about 1-2 wgt.% zinc, and about 2-4 wgt.% copper, with the remainder being tin. Such alloys are not completely resistant to corrosion. It has been found that increasing the copper content of such alloys provides increased strength and also avoids the formation of what is known in the art as the gamma-two phase, a tin and mercury phase which has low resistance to corrosion and thus may lead to early deterioration of fillings. Typical of such high copper alloys are those disclosed in U.S. Pat. No. 3,871,876 and U.S. Pat. No. 3,997,328. Such dental alloy compositions increase the copper content from the typical 2-4 wgt.% to the range of 8-27 wgt.% in the first mentioned patent, and in the latter patent, from 20-40 wgt.%. While such alloys have improved corrosion resistance, another important characteristic of dental alloys has been neglected heretofore. The success of a dentist in filling a dental cavity is related to the handling characteristics of the alloy after it is amalgamated with mercury. For example, the high copper alloy disclosed in U.S. Pat. No. 3,871,876, is typically produced by air atomization from the molten state which results in a spherical or spheriodal form for the finished alloy. It is characteristic of alloys having a spherical shape that they feel relatively soft to the dentist and appear to require delicate handling. They are sometimes difficult to pack into a dental cavity since they have a tendency to be formed up the wall of the cavity if too much pressure is exerted or an instrument is used which has a small bearing area. Consequently, many dentists find that such spherical material is not well-adapted to their individual technique. As a result, they may be unable to take advantage of the corrosion resistance inherent with spherical alloys having a high copper content. One method of improving handling characteristics of conventional dental alloys is disclosed and claimed in U.S. Pat. No. 3,997,327. In that invention a major portion of spherical particles is combined with a minor portion of microcut irregular particles, or flakes. Typical dental alloys in the prior art generally have been of the flake type, which inherently requires a higher pressure in order to pack it into a dental cavity than is characteristic of the spherical particles. By combining spherical particles with flake particles having the same or a similar composition, it is possible to improve the handling characteristics of the resulting mixture. Such a combination, having a conventionally low copper content, has less resistance to corrosion than the higher copper content alloys previously discussed. Another method of improving handling characteristics applied to a corrosion resistant alloy is disclosed and claimed in a copending application filed concurrently with the present application in the name of the inventors hereof and assigned to a common assignee, entitled &#34;Corrosion-Resistant Dental Alloy Having Improved Handling Characteristics&#34; (hereinafter &#34;Improved Alloy&#34;). The alloy of U.S. Pat. No. 3,871,876 is produced as particles having a unique randomly-shaped microcrystalline morphology which is characterized by having a higher than average silver and copper content at the surface of the particles than in the interior of the particles and a surface area about 20-30% greater than that of spherical particles and about 20-30% less than that of flake-like particles. The unique form of the particles provides handling characteristics similar to those of flake-like particles, while retaining the corrosion resistance characteristic of the spherical form. Although the unique particles of Improved Alloy are advantageous in providing excellent handling characteristics, fine adjustment of the handling characteristics is not readily made with it. The present invention has as its objective providing adjustment of handling characteristics of corrosion-resistant dental alloys. SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a corrosion-resistant dental alloy mixture for use as a filling for dental cavities after amalgamation with mercury, comprising a substantially uniform mixture of a first corrosion-resistant alloy comprising a mixture of silver, tin and copper, the first alloy being in the form of spherical particles having a mean particle size of from about 20 to 26.5 microns, and further having a particle size distribution such that substantially all of the particles fall within a particle size range of from about 1 to 75 microns. The second corrosion-resistant alloy is included and comprises a mixture of silver, tin and copper, the second alloy having a mean particle size of from about 20 to 26.5 microns, and further having a particle size distribution such that substantially all of the particles fall within a particle size range of from about 1 to 75 microns. These particles have a surface area ranging from about 0.23 to 0.26 m 2 /gm. A third alloy is also included and comprises a mixture of silver, tin and copper, the third alloy comprising flake-like particles having a mean particle size of between about 25 and 30 microns, and further having a particle size distribution such that substantially all of these particles fall within a particle size range of from about 1 to 75 microns. These particles have a surface area of about 0.33 m 2 /gm, and comprise not more than about 25% by weight of the dental alloy mixture. In accordance with one aspect of the invention, the first and said second alloys comprise from about 47 to 70 percent by weight of silver, from about 20 to 32 percent by weight of tin, and from about 7 to 27 percent by weight of copper and the third alloy comprises from about 55 to 75 percent by weight of silver, from about 20 to 40 percent by weight of tin, and up to about 10 percent by weight of copper. The first and second alloys need not be identical to each other, so long as they fall within the above range. One aspect of the invention provides that corrosion-resistant dental alloy comprises a mixture of from about 25 to 60 percent by weight of the first alloy, from about 25 to 60 percent by weight of the second alloy, and from about 10 or 15 to 25 percent by weight of the third alloy. The invention also provides for a dental amalgam comprising a combination of the above corrosion-resistant dental alloy with mercury, preferably one comprising about 1 part by weight of mercury for each part by weight of corrosion-resistant dental alloy. The first, second and third particles are each sized such that at least about 90% by weight thereof fall within a particle size range of from about 10 to 52 microns, in accordance with another aspect of the invention. Generally, the dental alloy of the invention combines corrosion resistance, in that it has a relatively high copper content, and good handling qualities. The alloy is composed of a mixture of three forms of particles. The compositions of two of the forms of particles correspond generally to that of the spherical material disclosed in United States patent 3,871,876, being within the range of about 47% to 70% by weight silver, 20% to 32% by weight tin, and 7% to 27% by weight copper. As is true of the material of the 3,871,871 patent, the two forms of particles may have a higher than average silver and copper content at the surface of the particles, but while one particle is spherical, the second particle has the unique form of Improved Alloy. The third type of particle has the relatively low copper and high silver content typical of the prior art. In a preferred embodiment, the third type of particles will have a composition of about 55% to 75% by weight silver, 20% to 40% by weight tin, 0% to 10% by weight copper and 0% to 2% by weight zinc. By combining suitable proportions of spherical particles, randomly-shaped microcrystalline particles, and flake-like particles, the handling characteristics of amalgams prepared from such mixtures can be adjusted to suit the requirements of the individual user. Although any suitable proportions of the first and second types of particles may be employed, the third type of particle is limited to a maximum of 25% by weight of the alloy mixture in order to retain the corrosion resistance provided by the first and second types of particles. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the spherical particles of the prior art corresponding to U.S. Pat. No. 3,871,876. FIG. 2 shows the randomly-shaped microcrystalline particles of Improved Alloy. FIG. 3 shows the flake-like particles of the prior art. FIG. 4 shows a mixture of the particles of FIGS. 1-3 according to the invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS A dentist in packing an amalgam prepared from a dental alloy and mercury into a dental cavity considers two factors to be of particular importance. First, what may be termed &#34;condensation&#34; relates to the resistance of the alloy to being packed into the cavity by the dentist using typical instruments. It will be clear that an amalgam must have sufficient plasticity when under pressure to enable it to flow into and completely fill all portions of the cavity, thereby preventing the formation of open spaces in the finished filling which could weaken it or permit further decay to the tooth structure. At the same time, the amalgam must not be so fluid as to flow out from beneath the dental instruments during condensation of the amalgam and move up the wall of the cavity. In such situations, a nonuniform degree of packing necessarily results, with poor adaptation to the cavity and increased porosity which weakens the filling and may result in further decay. Thus, one important handling characteristic of an amalgam is its ability to be pressed into a dental cavity to fill all the small openings under the desired condensation pressure, while not being so soft that the dentist cannot adequately compact the amalgam. This condensation pressure may be approximated by an empirical test which will be hereinafter described and which is useful in connection with the present invention. The second handling characteristic of importance to the dentist is the ability of an amalgam to be carved or shaped in order to finish the exterior surface of the compacted filling. An amalgam also must be of a desired plasticity in order to be satisfactorily carved or shaped. An amalgam may be satisfactorily carved or shaped. An amalgam may be satisfactorily packed into a dental cavity but be difficult to smooth and shape when the packing process is completed. On the other hand, an amalgam which is easy to carve and shape may be difficult to pack properly into a dental cavity. Another empirical test to be described hereinafter may be related to the carving characteristic of the amalgams derived from various dental alloys. As described in U.S. Pat. No. 3,253,783 and elsewhere, the gas atomization technique may be used to produce spherical or spheroidal particles from molten dental alloys. Particles are screened after cooling to provide a powdered alloy having aprticles in the size range of about 1 micron to about 65 microns. Larger and smaller particles are separated and recycled to be remelted and recast. Spherical particles such as are illustrated in FIG. 1 have an average surface to volume ratio of about 0.21 m 2 /gm as measured by the usual BET apparatus. The randomly-shaped microcrystalline particles of Improved Alloy typically have a BET surface area of about 0.23-0.26 m 2 /gm. By way of contrast, the flake-like particles commonly used heretofore have a BET surface area of about 0.33 m 2 /gm. It should be noted that the surface area is related in part to the particle size, thus the values given herein relate to a particle size distribution suitable for dental alloys and as specifically reported hereinafter for the alloy of the invention. It should be further noted that the surface area measured by the BET apparatus is much larger than the geometric exterior surface of the particles. For example, a perfect sphere would have a surface area only about 10% of that measured for the generally spherical particles of FIG. 1. The additional 90% of the measured surface is evidently due to surface roughness and porosity. Since this additional surface seems less likely to have a large effect on the handling properties of amalgams than the geometric surface, the geometric surface of the particles should be compared rather than the BET surface. However, the geometric surface of the alloy particles has not been measured although it may be approximated by substracting about 90% of the BET value for comparison purposes. In the specification and claims, all surface measurements are those as measured by the known BET apparatus. Amalgams are produced by mixing mercury with dental alloys of the invention. At the completion of the amalgamation process, the amalgam is condensed into a tooth cavity by a dentist and then the filling is carved or shaped until the amalgam has become so hard that it cannot be worked. This period is typically about six minutes. The dentist packs or condenses the amalgam into the tooth cavity while the amalgam is still soft enough to do so. The pressure required is quite important to the dentist as has been previously discussed and to characterize dental alloys of the invention we have chosen to designate the resistance of the amalgam one minute after amalgamation is complete as the condensation factor. A lower value indicates that an amalgam is stiffer and requires more pressure to pack or condense it into a tooth cavity than an amalgam having a higher numerical value. The test used to obtain values reported herein for condensation factors may be described as follows. A pellet of dental alloy is mixed with the recommended amount of mercury in an amalgamator for the manufacturer&#39;s recommended time. A commercially available Wig-L-Bug Model 5AR manufactured by Crescent Corporation was used in the tests reported herein, although other amalgamators would be acceptable. After the amalgamation is complete, the amalgam is immediately placed on a flat glass plate and covered by another such glass plate and pressed to a one millimeter thickness, as determined by one millimeter spacers placed between the plates. The top plate is removed and measurements are made of the resistance of the flattened amalgam disc during the hardening period. For the measurements reported herein an Instron testing unit model 1101 produced by Instron Corporation was employed. A constant load of five pounds was placed on a two millimeter steel ball in contact with the amalgam. The depth of the indentation made by the ball when the load was applied for fifteen seconds is used as a measure of the resistance of the amalgam. Tests were made at one minute intervals for a period of five minutes, or until no further change in the resistance was measured. The period of time during which measurements were made approximates the time which a dentist uses to fill a tooth cavity and to carve the filling. Test results obtained with prior art dental alloys in spherical and flake form are compared with the dental alloy of the invention in the examples below. The carvability factor relates to the ability of a hardening dental amalgam to be carved and shaped by dental instruments after it has been compacted. It will be apparent that after the compaction or condensation period (about 2 minutes) the dentist will have a limited time in which to shape or carve the hardening amalgam. A variant of the test previously described is sued to obtain a carving factor. The two millimeter ball loaded by a five pound weight is replaced with a one pound Gilmore needle having a one milli-meter point. The Gilmore needle is normally used for measuring setting rates of cements and plastic materials and has been described in an article by Peyton and Craig in Restorative Dental Materials, 4th ed., 1971. It has been found that the lighter loaded Gilmore needle will fail to penetrate an amalgam after it is sufficiently hardened. The time between the end of the amalgamation process and the failure of the Gilmore needle to penetrate the hardening amalgam may be used as an index of the carvability of the amalgam. EXAMPLE 1 A dental alloy is prepared by mixing individual metal powders to provide an overall composition of 58 wt.% Ag, 29 wt.% Sn, 13 wt.% Cu. The powdered mixture is melted and processed in an air atomization apparatus modified to minimize the formation of spherical particles by contacting the molten droplets during the cooling process, thereby producing the randomly-shaped microcrystalline particles of Improved Alloy. The particles formed have a surface area of 0.24 m 2 /gm. They are sieved to produce a powdered alloy as shown in FIG. 2 and having particles sized within the range of about 1 to 45 microns. The powdered alloy is then pelleted and mixed with sufficient mercury to form an amalgam having an alloy to mercury ratio of 1:1. The amalgam is measured for its resistance to condensation pressure according to the test hereinbefore described. EXAMPLE 2 A dental alloy is prepared by mixing individual metal powders to provide an overall composition of 58 wt.% Ag, 29 wt.% Sn, 13 wt.% Cu. The powdered mixture is melted and processed in an air atomization apparatus according to U.S. Pat. No. 3,871,876 to produce spherical particles and shown in FIG. 1. The particles have a surface area of 0.21 m 2 /gm. The particles are sieved to provide particles within the size range of from about 1 to 40 microns and a mean particle size of from about 20 to 26.5 microns. The powdered alloy is then pelleted and mixed with sufficient mercury to form an amalgam having an alloy to mercury ratio of 1 to 1. The amalgam is subjected to the condensation factor test described hereinbefore. EXAMPLE 3 A dental alloy is prepared by mixing individual metal particles to provide an overall composition of 68 wt.% Ag, 27 wt.% Sn, 5 wt.% Cu. The mixture is melted and cast in a bar, from which it is cut into flake-like particles as shown in FIG. 3 according to the usual technique of the prior art. The particles have a surface area of 0.33 m 2 /gm and are within the size range of about 1 to 50 microns. The powdered alloy is then pelleted and mixed with sufficient mercury to form an amalgam having an alloy to mercury ratio of 1.2 to 1. The amalgam is subjected to the condensation factor test described hereinbefore. EXAMPLE 4 A dental alloy is prepared by mixing 25% by weight of the particles of Example 1 with 60% by weight of the particles of Example 2 and with 15% by weight of the particles of Example 3. The mixed particles have a surface area of about 0.24 m 2 /gm and are within the size range of 1 micron to 45 microns. The powdered alloy is then pelleted and mixed with sufficient mercury to form an amalgam having an alloy to mercury ratio of 1 to 1. The amalgam is subjected to the condensation factor test described hereinbefore. The mixed particles of Example 4 and FIG. 4 provide amalgams having handling properties intermediate amalgams made with the spherical particles of Example 2 and the flakelike particles of Example 3. The condensation factors expressed as millimeters indentation after one minute from completion of the amalgamation process are 10.75, 18, 14.0, and 13.0 for the alloys of Examples 1-4 respectively. The spherical particles of Example 2 and FIG. 1 produce an amalgam which is soft when freshly mixed with mercury. As previously indicated, dentists often find amalgams made with spherical particles to be delicate to handle and difficult to condense properly. The randomly-shaped particles (FIG. 2 and Example 1) have a unique morphology which provides handling characteristics similar to those of the flake-like particles of the prior art during the condensation of the amalgam into a tooth cavity. The mixture of spherical particles with randomly-shaped particles and flake-like particles (FIG. 4 and Example 4) provide intermediate handling characteristics which will be experienced by the dentist as a moderately soft amalgam which requires less pressure for proper condensation into a cavity. The combination of Example 4 is only one possible mixture. Clearly, other mixtures could be made to suit the individual requirements of the user. Another satisfactory mixture combines 60% by weight of the particles of Example 1 with 25% by weight of the particles of Example 2 and 15% by weight of the particles of Example 3. The surface area of such a mixture is about 0.25 m 2 /gm and the size distribution is within the range of about 1 micron to about 45 microns. An amalgam made of such a mixture will be generally firmer than the mixture of Example 4 and its condensation factor after one minute would be about 12.5 millimeters. The particles of Examples 1 and 2, having a relatively high copper content overall and a higher than average silver and copper content at the surface than in the interior of the particles are corrosion resistant and may be combined in any proportions found desirable to adjust the handling characteristics of amalgams made from alloys of the invention. The relatively high silver and low copper content of the flake-like particles of Example 3 are not so resistant to corrosion. Consequently, the flake-like particles may be included in an alloy according to the invention as desired to adjust handling characteristics of amalgams made therewith, but limited to a maximum of 25% by weight of the alloy in order to retain the corrosion resistance of the other two particles. The flake-like particles typically will have a composition of about 55% to 75% by weight silver, 20% to 40% by weight tin, 0% to 10% by weight copper and 0% to 2% by weight zinc. The carving period (typically 2 to 5 minutes after amalgamation) represents the time period when the dentist shapes the compacted filling to suit the patient&#39;s bite. After a certain period the amalgam becomes unduly hard and can no longer be worked with the usual dental instruments. After about one hour a typical amalgam has reached substantial strength and can withstand the pressure of normal use. Another test may be used to discriminate between amalgams made from alloy particles of various shapes. Measurements of the three particles in the preceding examples made by substituting a Gilmore needle for the two millimeter ball as previously described, give the following results. TABLE I______________________________________ Carving FactorParticle Type Time, minutes - Penetration Ceased______________________________________Spherical (Ex. 2) 4.15Microcrystalline (Ex. 1) 3.15Flake-like (Ex. 3) 2.15Mixed particles (Ex. 4) 3.50______________________________________ The above results indicate that spherical particles can be carved with less force and for a longer time than the randomly-shaped microcrystalline particles, which have the same advantage over flake-like particles. The mixture of particles, as would be expected, can be carved with less force for a longer period than the amalgams made with the flake-like particles of Improved Alloy (Example 1) but the mixture is firmer and hardens quicker than amalgams made with spherical particles. Mixing particles according to the invention provides a means by which the handling characteristics of dental amalgams may be adjusted to suit the requirements of the individual user. At the same time, when the flake-like particles are limited to a maximum of 25 weight percent, the resulting mixtures are found to have satisfactory corrosion resistance. Alloy particles are sieved to provide a typical particle size distribution as follows: ______________________________________ Microns Wt.%______________________________________ 52-75 0.2 to 1.4 44-52 1.4 to 12.2 38-44 1.6 to 8.9 30-38 20.9 to 24.6 20-30 26.1 to 35.7 10-20 24.0 to 35.4 1-10 3.6 to 7.2______________________________________ The mean particle size is typically 20 to 26.5 microns. Although some variation about the above typical size distribution may be made to adjust the handling characteristics, an amalgam prepared with particles having a significantly different size distribution from that given above will have handling characteristics differing from those reported herein. In general, the smaller the average particle size, the firmer the amalgam will be and the shorter the working time. As previously discussed, the surface area of the alloy particles of the invention having the size distribution as given above will be found to have a BET surface area between those of the component particles, namely from about 0.24 m 2 /gm to about 0.27 m 2 /gm. With other size distributions, the surface to volume ratio may be wider. Particles may be used directly to form amalgams, especially if employed in pre-mixed dental capsules. Often the particles are pelletized for use in dispensers designed to provide the desired amount of mercury needed to amalgamate with the pelleted alloy. The pelletizing process has been found to alter the handling properties of the resulting amalgam, generally providing a dry and less plastic amalgam than if the powdered alloy were used directly. It has been found that by heat treating the pellets in a vacuum or under an inert atmosphere (e.g., argon, nitrogen) for a suitable time, the mechanical properties and useful working time of the alloy can be returned to their original and more desirable values. Typically a vacuum of about ten microns (0.01 mm Hg absolute pressure) has been found to be acceptable, the determining factor being the need to avoid oxidation of the metals with the consequent degradation of physical properties and corrosion resistance. The heat treatment is carried out typically between 100° and 700° F. (37.8° to 370° C.) as required until the handling characteristics of an amalgam made from the pellets matches those of the unpelleted powder, as measured by the condensation and carving factors. Generally, at least about 90% of the particles of the alloy of the invention will fall within the size range of from about 10 to 52 microns. Particles of a size greater than 52 microns should comprise not more than about 1.4% by weight of the alloy particles. With particles larger than about 52 microns, such oversized particles could pose difficulties in filling small apertures in a tooth. The lower limit of particle size is determined by the fact that with very small particle sizes the desired effect provided by the defined specific shape of the particles of the invention is lost. Further, very fine particle sizes of the alloy use up a proportionately greater amount of mercury in the amalgam and tend to increase the proportion of mercury beyond the desired limit. Obviously, particle range sizes expressed herein are maximum ranges; the actual particle size range of specific embodiments of the invention may fall within a narrower range encompassed by the broadly stated ranges. The foregoing discussion of the preferred embodiments of the invention is not intended to limit the scope of the invention, which is defined by the claims which follow.

Summary: A corrosion-resistant dental alloy is disclosed which has improved handling characteristics during the filling of a dental cavity. The alloy is a substantially uniform blend of three types of particles having the same chemical components, but differing in morphology and, optionally, in proportions of components. One type of particle is spherical or spheroidal in form. The second type of particle is a randomly-shaped microcrystalline form. The third type of particle is a flake-like particle. Handling characteristics of an amalgam prepared from such three particles can be adjusted to suit the requirements of the user by varying the relative proportions of the three types of particles while still retaining the corrosion resistance of the particles.

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Summarize: A DC inner city school teaches rugby to a school for the deaf and the results are loud and clear. A DC inner city school teaches rugby to a school for the deaf and the results are loud and clear. Aaron Dworkin brings classical music to the inner city, and opens up a world of possibilities. Can it get any more American than steak, onions and cheese whiz on a homemade bun? Bringing wounded vets fly-fishing to heal their mind and soul. A Holocaust survivor, she was one of five children that survived from her town. He talks about seeing the last two 9/11 survivors being pulled from the wreckage. Some call air guitar part perf. art, pro wrestling and rock’n’roll. Whatever it is, it sure is fun. This lovable mascot of the Philadelphia Phillies brings imagination to life. She talks about coming over from Italy as a child and the chances America has given her. An Iraq War veteran, James talks about surviving a suicide attack and serving our country. This July 4th patriotic pig out is rooted in love of country. Her parents left Albania so she could grow up with the freedom to live out her “American Dream.” She’s following in grandfather’s footsteps, paying it forward, and living up to her good name. He took his son to his first day of Kindergarten in NYC on 9/11. A firefighter who helped with the rescue efforts at the WTC after the 9/11 attacks. She raised nine children and talks about overcoming adversity and following your dreams. The father of entertainer Beyonce, he talks of the importance of reaching for your dreams. A 1st grade teacher talks about teaching the Pledge of Allegiance to her students. An Iraqi veteran, he talks about the price of freedom. Like her mother, she teaches her children that in America, anything is possible. Inspired by wounded soldiers, he’s in med school training to become a doctor in the U.S. Army. She shares her ancestors’ sacrifices and struggles for the freedoms we enjoy in America. As devoted parents, they provided a safe and loving environment for over 40 foster babies. They adpoted two children from Russia and share how the experience changed their lives. He talks about his journey from homelessness to financial freedom. Julie Smith & Joyce Hogan have served our nation through their husbands’ military service. He shares his admiration for his grandfather and the importance of unity in America. Chairman and CEO of GE from 1981-2001, he talks about following your dreams. He compares football to unity and talks about the importance of America coming together. He looks back and remembers what NYC was like on the day they buried JFK. Vietnam Vet who talks about how service changed his life and his feeling for this country. He emigrated from Turkey to go to college and follow his dreams. The couple talks about raising their kids and the freedoms we cherish in this country. A Vietnam Vet, Anthony talks about surviving a vicious attack, and the costs of freedom. She’s a 3rd generation American who talks of fulfilling the dreams of our founding fathers. Married for 52 years, they talk about growing up and raising a family in a small town. A Cuban American who talks about how her father risked his life to bring his family here. She remembers the Oklahoma City bombing and how Americans stand together. A teacher who credits her ancestors for giving us the freedoms we share in this country. Oleg Haskel He’s a Russian immigrant who talks about coming to America and achieving his dreams. Andria Mellon She talks about her ancestors’ hard work and sacrifice emigrating here from Greece. George Padavil He emigrated from India and talks about the opportunities America has given him. Well this is an interesting twist – actor/musician LL Cool J, who is one of the celebrities who will be part of Sarah Palin’s Thursday show (and is in the promo), isn’t happy about the ‘misrepresentation.’ He is reacting on Twitter – but is he ‘misrepresenting’ the circumstances as well? Do me a favor. Start this video now – it will provide the soundtrack to the rest of the post: Ok, we’re good? Now let’s get started. So Palin’s special on Thursday at 10pmET is called Real American Stories (here’s a preview), and although it is the premiere episode, the concept of “Real American Stories” is not new for Fox News. It began as a website back in July 2008 – a website, it is important to note, that is owned by Fox News. Well the website is still there, as is the interview LL Cool J did for it (watch the video here). But here’s what Ladies Love Cool James had to say on Twitter last night: Fox lifted an old interview I gave in 2008 to someone else & are misrepresenting to the public in order to promote Sarah Palins Show. WOW So here’s where he’s right, and wrong: • “Fox lifted an old interview” – Yes, it appears they did. The LL (and Jack Welch) segments were never billed as actual Palin interviews, just that they were to be featured in the show. It sounds like Palin will just be fronting these pieces, as opposed to the Toby Keith interview, which sounds more like a sit-down. • “…I gave in 2008 to someone else” – No, it looks like he did indeed give the interview to Fox News. Or, at the very least, to a Fox News-run website. • “…& are misrepresenting to the public” – It has been a little unclear through the promo what exactly Palin’s role will be. It sounds like it won’t be a pure interview show, although the Los Angeles Times reports she “interviewed people in studio for some of the segments.” Clearly, though, not LL. • “…in order to promote Sarah Palins Show” – Honestly, I can’t imagine how many FNC viewers come over because LL Cool J will be featured. Toby Keith, sure, but LL? If you were planning on watching the special before, you likely still would have if LL Cool J wasn’t involved. No offense, LL. • “…WOW.” – Yeah, wow is right. This special will likely garner huge ratings, and this extra publicity isn’t necessarily a bad thing. But it does make it a little surreal. Rap battle! > Update: A Fox News spokesperson responds: Real American Stories features uplifting tales about overcoming adversity and we believe Mr. Smith’s interview fit that criteria. However, as it appears that Mr. Smith does not want to be associated with a program that could serve as an inspiration to others, we are cutting his interview from the special and wish him the best with his fledgling acting career. Love it…apparently not NCIS: Los Angeles fans. —– » Follow Steve Krakauer on Twitter Have a tip we should know? tips@mediaite.com

Summary: LL Cool J is in the unlikeliest of "rap battles" with Sarah Palin over his appearance on her new Fox show. The rapper apparently gave an interview to Real American Stories, a website owned by Fox News, in 2008. That interview will now be featured on the premiere episode of the show of the same name, hosted by Palin, on Thursday-and Ladies Love Cool James is none too pleased about it. "Fox lifted an old interview I gave in 2008 to someone else & are misrepresenting to the public in order to promote Sarah Palins Show. WOW," he tweeted. Though some of his complaints are valid, is he really promoting the show? "I can't imagine how many FNC viewers come over because LL Cool J will be featured. Toby Keith, sure, but LL?" writes Steve Krakauer on Mediaite. "If you were planning on watching the special before, you likely still would have if LL Cool J wasn't involved. No offense, LL."

multi_news

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Summarize: Patients are increasingly turning to the internet to self-diagnose their health problems as they struggle to get an appointment with a GP. The number of visitors to NHS Choices, the Government’s health website, jumped by 40 per cent compared to last year and nearly 160 per cent since 2012, data released today reveals. And two thirds of visits were to pages about the diagnosis and treatment of conditions. The most popular topics were pregnancy and baby health, viewed 29.5million times so far this year. File image. The most popular topics were pregnancy and baby health, viewed 29.5million times so far this year. Other common searches were about mental health, stomach ache, chicken pox and back pain, according to the Health and Social Care Information Centre, which runs the website. Campaigners said patients are turning to the internet because it is increasingly difficult to get a GP appointment or to see the same GP to get consistent advice about a chronic condition. Roger Goss, of Patient Concern, said the situation was ‘terrible’. Earlier this year, the Royal College of GPs warned of a ‘crisis’ as surgeries struggle to cope with a growing and ageing population. Campaigners said patients are turning to the internet because it is increasingly difficult to get a GP appointment or to see the same GP to get consistent advice about a chronic condition. Above, file image. It also said patients face a ‘postcode lottery’ – in some areas there are just 22 full-time equivalent GPs for every 100,000 patients. Dr Maureen Baker, the college’s chairman, said: ‘There is no harm in using reputable websites, such as NHS Choices, but people should be cautious about trying to diagnose themselves. ‘GPs are the most trusted professionals in the NHS and it is important that we remain the first port of call for patients.’ The NHS Choices site has been viewed one billion times since its launch in 2007

Summary: Patients are turning to NHS Choices to self-diagnose their health problems. Two thirds of visits were to pages about diagnosis and treatment. The most popular topics searched for were pregnancy and baby health. Other common ones were about mental health, stomach ache, chicken pox. Broken nails, blocked noses and hair loss are prompting hundreds of calls to the NHS's 111 urgent care helpline. Other minor problems such as sunburn, stubbed toes and even an itch mean calls to the number have doubled in a year, with almost one million made every month.

cnn_dailymail

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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. patent application Ser. No. 11/225,645 filed on Sep. 13, 2005, which is a continuation-in-part application of U.S. patent application Ser. No. 10/930,044 filed on Aug. 30, 2004 and issued Nov. 27, 2012 as U.S. Pat. No. 8,317,871, which is a continuation-in-part application of U.S. patent application Ser. No. 10/205,386 filed Jul. 25, 2002 and issued Aug. 31, 2004 as U.S. Pat. No. 6,783,549, which claims the benefit of U.S. Provisional Application No. 60/308,340, filed on Jul. 27, 2001. The disclosure of the above applications is incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates generally to an apparatus and method for shoulder arthroplasty and, more particularly, to a humeral component and other associated surgical components and instruments for use in shoulder arthroplasty. [0004] 2. Discussion of the Related Art [0005] A natural shoulder joint may undergo degenerative changes due to a variety of etiologies. When these degenerative changes become so far advanced and irreversible, it may ultimately become necessary to replace a natural shoulder joint with a prosthetic shoulder joint. When implantation of such a shoulder joint prosthesis becomes necessary, the natural head portion of the humerus is resected and a cavity is created in the intramedullary canal of the host humerus for accepting a humeral component. The humeral component includes a head portion used to replace the natural head of the humerus. Once the humeral component has been implanted, the humeral cavity positioned at the scapula socket may also be resurfaced and shaped to accept a glenoid component. The humeral component generally includes an articulating surface which engages and articulates with the socket portion of the glenoid component. [0006] It is generally known in the art to provide a shoulder joint prosthesis having a humeral component, as discussed above. However, current prior art humeral components along with the associated surgical components and instruments utilized during shoulder arthroplasty suffer from many disadvantages. [0007] For example, since the humeral component is subject to various types of loading by the glenoid component, the humeral component must offer a stable and secure articulating surface. To achieve this, some humeral components provide a post or stem attached to a lateral surface of the prosthetic humeral head. These humeral components are generally a single piece system with a single stem, which is inserted and cemented into a hole bored deeply into the intramedullary cavity. However, such existing humeral components also exhibit several disadvantages. For example, these types of stemmed humeral components utilize a large stem to stabilize and secure the humeral component to the humerus. Such humeral components increase the amount of bone tissue removed, while also increasing the labor and complexity of the shoulder arthroplasty. Other stemmed humeral components may offer a larger diameter stem. However, the larger diameter stem also requires excess bone tissue to be removed which may not be practical in some patients. [0008] Other prior art humeral components, such as that disclosed in WO 01/67988 A2 sets out a stemless humeral component or head that provides an integral cruciform shape that includes two planar intersecting fins. While this type of humeral component addresses the amount of bone tissue removed, this type of system provides little versatility or adjustments to a surgeon performing the shoulder arthroplasty. Moreover, this type of system does not provide additional enhanced fixation other than the planar intersecting fins. [0009] Additionally, most prior art humeral components only rely on the stem to secure the humeral component into the intramedullary canal, via a cement mantle or bone attachment. The stem may also include grooves or holes, which act as an anchor, once the stem is cemented within the intramedullary canal. The medial surface of most humeral components are thus generally overlooked to enhance cement fixation and are therefore generally smooth. Although some humeral components may include a few longitudinal grooves and others may include both grooves and depressions on the medial surface, such surface enhancements only utilize or texture a portion of the medial surface, thereby not advantageously using the entire medial surface. [0010] What is needed then is a modular humeral component and associated surgical components for use in shoulder arthroplasty which do not suffer from the above-mentioned disadvantages. This in turn, will provide a humeral component which is stable and secure, reduces the overall amount of bone tissue required to be removed, increases a surgeon&#39;s available components utilizing a single sized post, reduces the overall surgical time and complexity, increases overall medial surface area, enhances and increases post strength without increasing overall post diameter, provides a fully enhanced or textured medial surface for enhanced cement fixation or bone fixation and increased overall stability, provides for a uniform cement mantle, and provides increased tensile and shear strength. It is, therefore, an object of the present invention to provide such a humeral component and associated surgical components for use in shoulder arthroplasty. SUMMARY OF THE INVENTION [0011] In accordance with the teachings of the present invention, an apparatus and method for shoulder arthroplasty is disclosed. The apparatus and method employs a modular humeral component and other associated surgical components for use in the shoulder arthroplasty. In this regard, the modular humeral component is adapted to be implanted into a humerus and engaged by a glenoid portion of a scapular component. [0012] In one preferred embodiment, a modular humeral component is used for shoulder arthroplasty such that the humeral component is adapted to be implanted into a humerus and engage a glenoid component. The humeral component includes a head member having a first articulating surface and a second fixation surface, which is opposite to the first articulating surface. The first articulating surface is adapted to engage the articulating surface of the glenoid component and the second fixation surface is adapted to engage a fixation component. The fixation component has a first surface adapted to be secured to the head member and a second surface that is generally opposite the first surface. The second surface includes a fixation member adapted to be secured to the humerus. [0013] Use of the present invention provides an apparatus and method for shoulder arthroplasty, and specifically, a modular humeral component and associated surgical components for use in shoulder arthroplasty. As a result, the aforementioned disadvantages associated with the currently available humeral components and associated surgical components for shoulder arthroplasty have been substantially reduced or eliminated. BRIEF DESCRIPTION OF THE DRAWINGS [0014] Still other advantages of the present invention will become apparent to those skilled in the art after reading the following specification and by reference to the drawings in which: [0015] FIG. 1 is a perspective view of the humeral component according to the teachings of the preferred embodiment of the invention shown implanted in a skeletal structure; [0016] FIGS. 2 a - 2 c are views of the fixation member of humeral component of FIG. 1 ; [0017] FIGS. 3 a - 9 are alternate embodiments for the fixation member of the humeral component of the present invention; [0018] FIGS. 10-12 represent alternate peg configurations for the fixation member of the humeral component of the present invention; [0019] FIGS. 13 and 14 represent alternate texturing, which is usable in the humeral components of the present invention; [0020] FIGS. 15 and 16 represent cross-sectional views of implanted humeral components of the present invention; [0021] FIGS. 17 through 22 depict an alternate embodiment of the present invention having an insert member disposed between the head and the base member; [0022] FIGS. 23 a - 23 e depict another alternate embodiment of the present invention having flanges disposed on a shelfless base member; [0023] FIGS. 24-26 b illustrate a method for preparing the humerus implantation of the humeral component using associated surgical components according to the teachings of the preferred embodiment of the present invention; [0024] FIGS. 27-28 further illustrate methods for implanting the humeral components into the prepared humerus according to the teachings of the preferred embodiment of the present invention; [0025] FIGS. 29 a - c represent side and perspective views of an alternate fixation member having a through stem fixation bore; [0026] FIGS. 30 a - c represent side and perspective views of an alternate fixation member having a male Morse taper with through stem fixation bore; [0027] FIGS. 31 a - c represent side and perspective views of an alternate fixation member having a female Morse taper with through stem fixation bore; [0028] FIGS. 32 a - c represent side and perspective views of an alternate fixation member [0029] FIG. 33 represents a perspective view of an alternate fixation member; [0030] FIG. 34 represents a cross-sectional view of the fixation member shown in FIG. 33 ; [0031] FIG. 35 represents a cross-sectional view of an alternate fixation member; [0032] FIGS. 36 a and 36 b represent an alternate fixation component having a fixation member; [0033] FIG. 37 represents the coupling of a bone engagement member to a fixation shelf; [0034] FIG. 38 represents a fixation member having a concave shelf member; [0035] FIG. 39 a - 39 b represents the intention of a fixation member; [0036] FIG. 40 represents a prosthetic according to another embodiment to the invention; [0037] FIGS. 41 and 42 are side views of the prosthetic shown in FIG. 40 ; [0038] FIG. 43 represents a perspective view of the implantation of the coupling member shown in FIG. 40 ; and [0039] FIGS. 44 and 45 represent side views of the implantation of a humeral prosthetic head into the coupling member shown in FIGS. 43 and 42. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0040] The following description of the preferred embodiments concerning an apparatus and method for shoulder arthroplasty is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. [0041] FIG. 1 depicts the components used in the shoulder arthroplasty of the current invention. As shown, the modular humeral component 31 has a base member 32 and a head member 33. The base member 32 has a fixation peg 34, which is used to attach the humeral component to the resected portion 36 of the humerus 38. If a total shoulder arthroplasty is performed, a glenoid component 40 is first implanted into the scapula 42 using techniques well known in the art. The glenoid component 40 is preferably of the type disclosed in U.S. Pat. No. 5,800,551, which is hereby incorporated by reference, or other suitable conventional glenoid components. The humeral component 31 is designed to allow rotational and transitional movement of the head member 33 with respect to the glenoid component 40. [0042] FIGS. 2 a - 2 c depict the base member 32 of one embodiment of the current invention. The base member 32 is defined by a shelf member 44, which may have a plurality of through holes 46. The shelf member 44 can have at least one mating member 48 for engaging the head member 33 of the humeral component 31. It is preferred that the mating member 48 be a defined Morse taper or other suitable attaching mechanism. In addition to the mating member 48, each base member 32 has a fixation peg 34 disposed on the lower lateral surface 50 of shelf member 44. The fixation peg 34 is generally perpendicular to the shelf member for its entire length of the fixation peg 34. As depicted in FIGS. 2 a - 3, the shelf member 44 of the base member 32 can define flat lower lateral surface 50 and flat generally parallel upper surfaces 51. FIG. 3 a shows the second embodiment of the current invention, which has a cavity 52 defined in the shelf member 44. The cavity 52 is preferably the female side of a Morse taper, which would engage a male Morse taper on the head member 33. [0043] FIG. 3 b shows the head member 33 which mates with the base member 32 of FIG. 3 a. It should be noted that a surgical kit of the components would contain numerous head members 33, each having a varied radius of curvature, diameter, and height to allow a surgeon to optimize joint movement. Additionally, a surgical kit would contain the instruments needed for implantation (shown later). [0044] The head member 33 and base member 32 must be made of bio-compatible materials such as, without limitation, titanium, titanium alloys, surgical alloys, stainless steels, bio-compatible ceramics, and cobalt alloys. Optionally, the base member 32 can additionally be made of materials such as biocompatible ceramics and resorbable and non-resorbable polymers and other anticipated bio-compatible metallic or polymeric materials. Should the base member 32 be made of non-metallic components, a fastener would be needed to couple the head 33 to the base 32. [0045] As shown in FIGS. 4-7, the shelf members 44 need not to be planar. FIGS. 4 and 5 show the base member 32 and having an interior concave surface 56 and a convex medial surface 58. Base members, as disclosed in FIGS. 4 and 5 would be used in situations where maximum bone removal in the humerus 38 is required. In each situation, the head member 33 would have a convex medial surface 59 for engaging the concave surface 56. It is envisioned as with all of the embodiments that the base members 32 and head member 33 can be coupled using the mating member 48, i.e., Morse taper. The use of the convex-concave interface provides a coupling interface which is self centering under a multitude of loading conditions. The interface reduces the occurrence of micro-motions which can disrupt the normal functioning of the joint prosthesis as well as lead to premature component failures. Any loads applied to the articulating surface of the head member 33, are transferred as a perpendicular force into the base member 32 of the modular humeral component 31 through the non-planar shelf member 44. [0046] FIGS. 6 and 7 define base members 32 having the shelf member 60 having a convex outer surface 62. Additionally shown is a concave inner surface 64 for mating with a resected head 36 of the humerus 38. The base members as depicted in FIGS. 6 and 7 can be used when minimal bone removal is possible and will generally encapsulate the hemispheric shape cut into the humerus 38 as described later. As with the base members as shown in FIGS. 4 and 5, any loads applied to the articulating surface of the head member 33, are transferred as a perpendicular force into the base member 32 of the modular humeral component 31. [0047] FIGS. 8 and 9 disclose alternate embodiments of the base member 32 for the humeral component 31. FIG. 8 depicts the base member 32 having a convex outer surface 66 and a flat lateral surface 68. The base members as depicted in FIGS. 2 a - 3 and 8 can be utilized when a moderate amount of bone material must be removed from the resected head 36 of the humerus 38. FIG. 9 depicts the base member 32 having a flat upper surface 51 and a convex lateral surface 58. This base member would readily utilize the head member 33 as used with the base member as depicted in FIG. 2 a. It is envisioned that either base member can have a defined male or female mating member 48 in the form of a Morse taper. [0048] FIGS. 10-12 depict possible configurations for the fixation peg 34. FIG. 10 shows the fixation peg 34 defining a plurality of flutes 70 therein. As can be seen, the modular system does not need a shelf member 44. Without the shelf member 44, the base can have either a male or female Morse taper. FIGS. 11 and 12 depict the fixation peg 34 being at a tapered prism with the base of the prism coupled to the lower lateral surface 50 of the shelf member 44. [0049] FIGS. 13 and 14 depict possible surface treatments for the lower lateral surface 50 of the shelf member 44 and fixation peg 34. All of the possible fixation pegs 34 can have a porous coated region 72, which will assist in the fixation of the component to the humerus 38. Additionally, all of the lower lateral surfaces 50 of the shelf member 44 can define a waffle pattern 74 to assist in the incorporation of bone cement. Each fixation peg 34 extends from the lower lateral surface 50 to define or fill in a coupling region 75 having a diameter of about 0.50 inches. Each coupling region 75 also includes a sidewall 77 formed with and from the lower lateral surface 50. The coupling region 75 provides a smooth flat surface for which the fixation peg 34 extends out, and reduces or eliminates any stress risers about each fixation peg 34, which could be caused by positioning the lower lateral surface 50 immediately adjacent the fixation peg 34. [0050] The fixation peg 34 includes a first end 79, which is inserted into or engages a cavity or hole formed within a cavity in the humerus and a second end 81, which extends from or is integral with the shelf member 44. The first end 79 is semi-spherical and the second end has a 0.25 inch radius about the circumference of the second end 81 of the peg, which blends into a flat or smooth portion of the coupling region 75 to decrease the overall sheer stress of the fixation peg 34. Optionally, should the fixation peg be non-metallic, embedded within the first end of each fixation peg 34 is a tantalum ball 83. The tantalum ball 83 enables the humeral component 31 to be easily identified in an x-ray. [0051] FIGS. 15 and 16 depict cross-sectional views of various embodiments of the current invention implanted into resected head 36 of humerus 38. As depicted in FIG. 16, when a large amount of bone mass must be removed during the arthroplasty, the base member 32 as depicted in FIG. 9 can be used. As is shown, the base member 32 is fixed to the humerus 38 using a plurality of screws 85. The lateral surface 59 of the head member 33 defines a cavity 52 for receiving the mating member 48 or Morse taper post. [0052] As with the base member depicted in FIG. 15, the base member 32 is held to the humerus 38 by use of screws 85 disposed through the holes 46. FIG. 16 discloses the use of the base member 32 as depicted in FIG. 3 a which is similarly held in place by use of fixation screws 85 to the humerus 38. [0053] FIGS. 17 through 18 show an alternate embodiment of the humeral component 109. Base member 110 is shown having a modified Morse taper cavity 116. The humeral component 109 further has a head portion 112 with a male Morse taper portion 121. Disposed between the head portion 112 and the base member 110 is a coupling member 114. Coupling member 114 has an outer surface 118 which acts as the male portion of a Morse taper to bond with the cavity 116 of the base member 110. Coupling member 114 further defines a female portion 120 of a Morse taper which corresponds to the male portion 121 of the Morse taper of the head portion 112. The coupling portion 114 functions to move the center of curvature of the head portion 112 a fixed distance 123 from the center line of the base member 110. This functions to effectively change the centering location of the head portion 112 with respect to the humerus 138, thus allowing the surgeon more flexibility. [0054] FIG. 17 a shows the alternate humeral component 109 in its assembled configuration. FIG. 18 shows an exploded view of the alternate humeral component 109, coupling member 114, and base member 110. Rotation of coupling member 114 allows for translation of the head portion 112 on the base member 110. FIGS. 19 through 19 b depict a head portion 112 having a female Morse taper cavity 116 which engages a male Morse taper 115 on alternate coupling member 114. FIG. 19 a depicts an assembled view of the alternate humeral component 109. [0055] FIGS. 21 through 21 b depict an alternate embodiment of the humeral component 122. Shown is the base member 124 which has a modified female cavity defining a Morse taper 116. The head portion 126 has a coupling male Morse taper 132 disposed on the medial surface of the head component 126. Disposed between the head portion 126 and the base portion 124 is the coupling member 128. The coupling member 128 defines an outer surface 118 which functions as the male portion of the Morse taper and couples to the female portion 116 of the base member 124. The coupling member 128 further defines an interior cavity 130 which functions as a female Morse taper for the male Morse taper 132 of the head 126. The interior cavity 130 of the coupling member has an offset angle 134, which functions to rotate the center of curvature of the head portion 126 with respect to the base member 124. Similarly, shown in FIG. 21 b is a coupling member 114 having a male Morse taper 115 being angled. [0056] FIGS. 20 and 22 show the alternate humeral components 122 inserted into a resected humerus. As with the other humeral components, the base member is fixed to the head of the humerus using fasteners. [0057] FIGS. 23 a - 23 e depict another alternate embodiment of the present invention. Shown is a shelfless base member 232 which is formed by a fixation peg 234. Each fixation peg 234 has three evenly spaced triangular fins 236 disposed thereon. The triangular fins 236 have an edge 239 which is co-planar to a top surface 238 of fixation peg 234. Incorporated into a top surface 238 of the fixation peg 234 is a fixing mechanism 240. [0058] FIGS. 23 a and 23 b disclose fixing mechanism 240 in the form of a female Morse taper as the fixation which functions to couple the head 30 onto the base member 232 (see FIG. 23 d ). As can be seen FIG. 23 e, the top surface 238 alternately can have a fixing mechanism 240 in the form of a male Morse taper disposed thereon. It is envisioned that a head member 30 being used in this embodiment can have a lower surface 244 which has a porous coat, plasma spray, grit blast, or smooth surface to facilitate the coupling to the bone. [0059] When the base member 240 is coupled to head member 30, there is a defined gap between the lower surface 244 of the head 30 and the upper surface 238 of the base member 232. After implantation, the lower surface 244 of head member 30 rests upon the resected bone, not the top surface 238 of the base member 232. [0060] The method for implanting the humeral component 31, along with associate surgical components utilized will now be described with reference to FIGS. 24 a - 28. The head of the humerus 38 is resected using a saw, chisel then planed flat or with a concavity. With the resected head 36 of the humerus 38 exposed, an alignment or guide hole 90 is first drilled substantially through the center of resected head 36 of the humerus 38 using a quick release drill bit 96 and driver 98. Optionally, the resected head 36 of humerus 38 can be resected to provide a flat surface during the drilling of pilot hole 90. [0061] Once the guide hole 90 is drilled, the resected head 36 of humerus 38 is optionally reamed using a concave spherical reamer shaft 102 with the driver 98. The concave reamer 102 includes a guide pin 104 and a roughened spherical surface 106 substantially corresponding to the spherical shape of the lower medial surface of the shelf member 44 of base member 32. An optional convex reamer surface 108 permits rasping or drilling of tight humeral cavities (see FIGS. 26 a and 26 b ). Upon rotating the surface of the reamer, the bone of the resected head 36 of the humerus 38 is prepared to mate or conform to the shape of the lower lateral surface 50 of the shelf member 44 of the base member 32. As depicted in FIGS. 25 a and 26 b, the reamer 102 can have a convex shape or alternatively a flat shape, which reams a concave shape into the resected head 36 of the humerus 38. Determining which reamer is used is a function of the preoperative degenerative changes in the humerus 38. [0062] With reference to FIGS. 27-28, which depict the insertion of the humeral components 30, once the surface of the resected head 36 of the humerus 38 has been resected, the base member 32 is inserted into the guide hole 90. It is envisioned that fixation peg 34 of the base member 32 can be forced into the guide hole 90 to displace the bone material around the intramedullary canal. Optionally, the guide hole 90 can also be reamed to a larger interior diameter to accept the base member 32 without displacement of the bone material by the fixation peg 34. [0063] Once the base member 32 has been inserted into the guide hole 90, the optional screws 85 are disposed through the holes 46 to couple the base member 32 to the humerus 38. At this point, a surgeon may use any number of test head portions and/or adapter portions to determine the proper size needed to mate with the glenoid component. Once a proper head member 33 size has been determined, the final head member 33 can be fixed to the shelf member 44 of the base member 32. [0064] FIGS. 26 and 27 show the use of the base member 32 as depicted in FIG. 3. As can be seen, the base member of FIG. 3 is utilized when a minimal amount of bone is required to be removed. [0065] FIGS. 29 a - c represent side and perspective views of an alternate fixation member 246 defining a through member fixation bore 248. The shelf member 250 can have at least one mating member 252 for engaging the head member of the humeral component. The mating member 252 can be a defined female Morse taper 254 or other suitable attaching mechanism. In addition to the mating member 252, each fixation member 246 has a fixation peg 256 disposed on the lower lateral surface 258 of shelf member 250. The fixation peg 256 may be perpendicular to the shelf member for the entire length of the fixation peg 256. As depicted in FIGS. 29 a - c, the shelf member 250 of the fixation member 246 can define a convex spherical lower lateral surface 258 and concave spherical upper lateral surface 260. FIG. 29 a shows hidden lines representing the through member fixation bore 248. As shown, the female Morse taper 254 is optionally co-linear or concentric with both the through member fixation bore 248 and the fixation peg 256. Adjacent to the bottom 262 of the female Morse taper is a defined conical portion 264 which is configured to accept a head of a bone coupling screw. [0066] FIGS. 30 a - c represent side and perspective views of an alternate fixation member 266 having a mating member 268 in the form of a male Morse taper with a through stem fixation bore 270 for engaging the head member of the humeral component. In addition to the mating member 268, the fixation member 266 has a fixation peg 272 disposed on the lower lateral surface 274 of shelf member 276. The shelf member 276 can define convex spherical lower lateral surface 258 and concave spherical upper lateral surface 260. FIG. 30 a shows hidden lines representing the through member fixation bore 270. As shown, the mating member 268, in the form of a Morse taper, is optionally co-linear with both the through member fixation bore 270 and the fixation peg 272. Disposed through the top 278 of the fixation member 268 is a defined conical portion 280 which is configured to accept a tapered head of a bone coupling screw. [0067] FIGS. 31 a - c and 32 a - c represent side and perspective views of an alternate fixation members 282 a and 282 b. The members 282 a and 282 b have a shelf member 280 with mating features 284 a and 284 b for engaging the head member of the humeral component. The shelf member 280 of the fixation members 282 a and 282 b define a flat lower lateral surface 286 and convex spherical upper lateral surface 288. FIGS. 31 a and 32 a show hidden lines representing the through member fixation bore 290. The mating features 284 a and 284 b in the form of a Morse tapers are optionally co-linear with both the through member fixation bore 290 and a fixation peg 292. Disposed through the top 278 of the mating features 284 a and 248 b and fully though the fixation members 282 a and 282 b is a defined fixation bore 248 having a conical screw head supporting portion 298 which is configured to accept a tapered head of a bone coupling screw. This bone coupling screw can be positioned into or extended through the resected bone. [0068] The fixation members depicted in FIGS. 29 a through 32 c are generally implanted as described above. Generally, a portion of the head of the joint is resected using a rasp or other appropriate cutting tool. A bore is then formed in the resected head. The fixation member is then fixed to the prepared surface. A fastener such as a bone screw then disposed through the fixation bore 290 to fasten the fixation member to the resected bone. At this point, a articulating head (shown in FIG. 1 or 15 through 22 ) is coupled to the fixation member. While specific fixation members are show in FIGS. 29-32 c, the through bore feature is equally applicable to the fixation members shown in FIG. 1-14. [0069] The modular nature of the humeral component 31 of the present invention allow a set of various types of both replacement base members 32 and head members 33 to be formed. In using such a set, a surgeon can interoperably choose the appropriate base member depending on the patient&#39;s particular degenerative condition. Additionally, the surgeon can then choose from a set of head members 33, which both have the proper articulating surface radius and a proper coupling to the base member 32. [0070] FIGS. 33 and 34 represents a perspective and cross sectional views of an alternate fixation member 300. The fixation member 300 is formed of two components, a generally circular shelf member 302, and a bone engaging member 304. As described below, after an articulating surface is resected, an appropriate shelf member 302 is positioned against the resected surface. The bone engagement member 304 is then coupled to and through the shelf member 302 into the resected bone. An articulating head (not shown) is coupled to the bone fixation member to form a prosthetic with an articulating surface. [0071] The bone engaging member 304 has a head engagement portion 306 and a threaded bone engagement portion 308. The bone engagement member 304 additionally has a shelf member engagement region 310 and drive feature 312. The shelf engagement region 310 functions to distribute and translate forces from the articulating head into the resected bone. It is envisioned that the shelf engagement region 310 can be an annular engagement flange 318 or textured cylindrical interface surface. The drive feature 312 can be an aperture having at least one defined flat drive surface, such as a hex aperture. The head engagement portion 306 can be a defined male 314 or female 316 Morse taper. [0072] The shelf member 302 can be generally flat or curved. As described in previous embodiments, the shelf member 302 has upper and lower surfaces 320 and 322 which can be flat, concave or convex. The shelf members can additionally define a plurality of through apertures 324 that are configured to accept bone engaging screws to prevent rotation of the shelf member 302 during the insertion of the bone engaging member. [0073] FIG. 34 shows the annular engagement flange 318 is positioned an upper surface 320 of the annular shelf member 302. The shelf member 302 defines an aperture which annularly supports the bone engaging member 304. It is envisioned that the male Morse taper 316 can be used to couple the fixation member to an articulating head (see FIGS. 15-21 ). [0074] FIG. 35 represents a cross-sectional view of an alternate fixation member 302. Shown is the bone engaging member 304 having a head engagement portion 306 which is generally located below the shelf member 302. The head engagement portion 306 is shown as a female Morse taper 316. The annular engagement flange 318 is disposed within an annular coupling groove 326 which is formed about the aperture 328 of the shelf member 302. Defined in the bottom of the Female Morse taper 316 is the drive mechanism 312. It is envisioned that the annular coupling groove 326 can take the form of a tapered countersink. [0075] FIGS. 36 a and 36 b represent an alternate fixation component having a bone fixation member 304. The upper surface 320 of the shelf member 302 is show being concave as previously described. The shelf member has a annular coupling groove 326 which mates with the annular flange 318 of the bone engaging member 304. [0076] FIG. 37 represents a cross-sectional view of an alternate fixation member. Shown is the bone engaging member 304 having a head engagement portion 306 which is generally located below the shelf member 302. The head engagement portion 306 is shown as a female Morse taper 316. The annular engagement flange 318 is disposed on the upper surface 320 about the aperture 328 of the shelf member 302 Defined in the bottom of the Female Morse taper 316 is the drive mechanism 312. [0077] FIG. 38 shows the bone engagement member 304 with annular engagement flange 318. The annular engagement flange 318 is positioned on the convex upper surface 320 of the annular shelf member 302. The shelf member 302 defines an annular engagement groove adjacent the aperture which annularly supports the male Morse taper of the bone engaging member 304. [0078] FIG. 39 a - 39 b represents the implantation of a fixation member. Once the surface of the resected head 36 of the humerus 38 has been resected, the shelf member 302 positioned adjacent to the resected head 36. The bone engagement member 304 is inserted into the guide hole 90. It is envisioned that the bone engagement member 302 can be forced into the guide hole 90 to displace the bone material around the intramedullary canal. [0079] Once the bone engagement member 302 has been inserted into the guide hole 90, the bone engagement member 302 is rotated until the annular engagement flange 318 engages the upper surface 320 of the annular shelf member 302. Optional screws 85 are disposed through the holes 324 to couple the base member 302 to the humerus 38. At this point, a surgeon may use any number of test head portions and/or adapter portions to determine the proper size needed to mate with the glenoid component. Once a proper head member size has been determined, the final head member 330 can be fixed to the head engagement portion 306 of the bone engaging member 302. [0080] FIG. 40 represents a humeral implant 400 according to another embodiment. Shown is a coupling member 402 and a humeral head 403. The coupling member 402 has a generally cylindrical body portion 404 with an exterior coupling surface 406. The coupling surface 406 can be tapered or can be cylindrical. The exterior coupling surface 406 further has at least one bone engaging thread 407 which is configured to couple the cylindrical body portion to a hole defined within the receptive humerous as previously described. Appended to the proximal end 408 of the body portion 404 is a pair of linearly aligned coupling flanges 410 and 412, which define a pair of fastening member coupling apertures 414. The body portion 404 defines a female Morse taper 416 which is used to couple the head 403 to the coupling member 402. The coupling member 402 is configured to allow the fixation of the head without relying on the head seating on the resection for stability. [0081] The head portion 403 is shown with an axially centered fixation stem 418. It is envisioned the stem 418 can take the form of an offset male Morse taper as is shown in FIGS. 17-22. Additionally, the head has an exterior articulation surface 405 which is configured to articulate with a natural or prosthetic glenoid (not shown). [0082] FIGS. 41 and 42 represent side views of the prosthetic shown in FIG. 40. The coupling flanges 410 and 412 have a coupling surface 420 which is configured to be mated to a resected humerus 422. As shown in FIGS. 44 and 45, the resected humerus 422 can be prepared as previously described to have a flat 424 or convex surface 426. The body 404 is inserted into a hole within the resected humerus 422. In this regard, the body 404 is rotated in a hole formed in the resected humerus to engage the threads with the humeral head and neck bone. [0083] A pair of fasteners 434 are positioned through the coupling apertures 414 in the coupling flanges 410 and 412. As seen in FIGS. 43 and 45, a coupling surface 420 of the flanges 410 and 412 can have a predefined concave coupling surface. Additionally, the concave surface can be formed by a physician bending the flanges 410 and 412 in the operating room prior to the insertion of the coupling fasteners 434 into the apertures 414. [0084] After the coupling member 402 is inserted into the humerus, a trialing head 60 ′ is optionally used to determine the proper size and orientation of the humeral head 403. The humeral head 403 is then coupled to the coupling member as previously described. [0085] The description of the invention is merely exemplary embodiments in the present invention. One skilled in the art would readily recognize from such discussion and from accompanying drawings and claims that various changes, modifications, variations may be made therein without departing from the spirit and scope of the invention.

Summary: A two piece humeral component for use in joint arthroplasty which is adapted to be implanted into a joint and engaged by a socket component of the joint. The joint component includes a body having a first articulating surface and a second medial surface opposite the first articulating surface. The first articulating surface is adapted to be engaged by the socket and the second medial surface is adapted to be secured to mounting portion. The mounting portion has a first surface and a second medial surface. The first surface is adapted to be fixably engaged to the second mounting portion of the humeral component. The second medial surface is adapted to be secured to the humerus. A peg which has a first end adapted to engage a cavity found in the humerus is disposed on the mounting portion&#39;s second medial surface.

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Summarize: ACT IV. SCENE I. _The moated grange at ST LUKE'S._ _Enter MARIANA and a BOY._ _BOY sings._ Take, O, take those lips away, That so sweetly were forsworn; And those eyes, the break of day, Lights that do mislead the morn: But my kisses bring again, bring again; 5 Seals of love, but sealed in vain, sealed in vain. _Mari._ Break off thy song, and haste thee quick away: Here comes a man of comfort, whose advice Hath often still'd my brawling discontent. [_Exit Boy._ _Enter DUKE disguised as before._ I cry you mercy, sir; and well could wish 10 You had not found me here so musical: Let me excuse me, and believe me so, My mirth it much displeased, but pleased my woe. _Duke._ 'Tis good; though music oft hath such a charm To make bad good, and good provoke to harm. 15 I pray you, tell me, hath any body inquired for me here to-day? much upon this time have I promised here to meet. _Mari._ You have not been inquired after: I have sat here all day. _Enter ISABELLA._ _Duke._ I do constantly believe you. The time is come 20 even now. I shall crave your forbearance a little: may be I will call upon you anon, for some advantage to yourself. _Mari._ I am always bound to you. [_Exit._ _Duke._ Very well met, and well come. What is the news from this good Deputy? 25 _Isab._ He hath a garden circummured with brick, Whose western side is with a vineyard back'd; And to that vineyard is a planched gate, That makes his opening with this bigger key: This other doth command a little door 30 Which from the vineyard to the garden leads; There have I made my promise Upon the heavy middle of the night To call upon him. _Duke._ But shall you on your knowledge find this way? 35 _Isab._ I have ta'en a due and wary note upon't: With whispering and most guilty diligence, In action all of precept, he did show me The way twice o'er. _Duke._ Are there no other tokens Between you 'greed concerning her observance? 40 _Isab._ No, none, but only a repair i' the dark; And that I have possess'd him my most stay Can be but brief; for I have made him know I have a servant comes with me along, That stays upon me, whose persuasion is 45 I come about my brother. _Duke._ 'Tis well borne up. I have not yet made known to Mariana A word of this. What, ho! within! come forth! _Re-enter MARIANA._ I pray you, be acquainted with this maid; She comes to do you good. _Isab._ I do desire the like. 50 _Duke._ Do you persuade yourself that I respect you? _Mari._ Good friar, I know you do, and have found it. _Duke._ Take, then, this your companion by the hand, Who hath a story ready for your ear. I shall attend your leisure: but make haste; 55 The vaporous night approaches. _Mari._ Will't please you walk aside? [_Exeunt Mariana and Isabella._ _Duke._ O place and greatness, millions of false eyes Are stuck upon thee! volumes of report Run with these false and most contrarious quests 60 Upon thy doings! thousand escapes of wit Make thee the father of their idle dreams, And rack thee in their fancies! _Re-enter MARIANA and ISABELLA._ Welcome, how agreed? _Isab._ She'll take the enterprise upon her, father, If you advise it. _Duke._ It is not my consent, 65 But my entreaty too. _Isab._ Little have you to say When you depart from him, but, soft and low, 'Remember now my brother.' _Mari._ Fear me not. _Duke._ Nor, gentle daughter, fear you not at all. He is your husband on a pre-contract: 70 To bring you thus together, 'tis no sin, Sith that the justice of your title to him Doth flourish the deceit. Come, let us go: Our corn's to reap, for yet our tithe's to sow. [_Exeunt._ NOTES: IV, 1. SCENE I. Enter M.] Ff. M. discovered sitting. Steevens. 5, 6: F4 omits the refrain in l. 6. Rowe omits it in both lines. 6: _but_] _though_ Fletcher's version. 13: _it_] _is_ Warburton. 17: _meet_] _meet one_ Hanmer. 19: Enter I.] Transferred by Singer to line 23. 24: SCENE II. Pope. _well come_] Ff. _welcome_ Warburton. 32, 33, 34: _There have I made my promise Upon the heavy middle of the night To call upon him._] S. Walker conj. _There have I made my promise, upon the Heavy middle of the night to call upon him._ Ff. _There on the heavy middle of the night Have I my promise made to call upon him._ Pope. _There have I made my promise to call on him Upon the heavy middle of the night._ Capell. _There have I made my promise in the heavy Middle...._ Singer. _There have I made my promise on the heavy Middle...._ Dyce. Delius and Staunton read with Ff. but print as prose. 38: _action all of precept_] _precept of all action_ Johnson conj. 49: SCENE III. Pope. 52: _have_] _I have_ Pope. 58-63: _O place... fancies_] These lines to precede III. 2. 178. Warburton conj. 60: _these_] _their_ Hanmer. _base_ Collier MS. _quests_] _quest_ F1. 61: _escapes_] _'scapes_ Pope. 62: _their idle dreams_] Pope. _their idle dreame_ Ff. _an idle dream_ Rowe. 63: _Welcome, how agreed?_] _Well! agreed?_ Hanmer. SCENE IV. Pope. 65: _It is_] _'Tis_ Pope. 74: _tithe's_] _Tithes_ F1 F2 F3. _Tythes_ F4. _tilth's_ Hanmer (Warburton). _Our... sow_] _Our tythe's to reap, for yet our corn's to sow_ Capell conj. MS. SCENE II. _A room in the prison._ _Enter PROVOST and POMPEY._ _Prov._ Come hither, sirrah. Can you cut off a man's head? _Pom._ If the man be a bachelor, sir, I can; but if he be a married man, he's his wife's head, and I can never cut off a woman's head. _Prov._ Come, sir, leave me your snatches, and yield me 5 a direct answer. To-morrow morning are to die Claudio and Barnardine. Here is in our prison a common executioner, who in his office lacks a helper: if you will take it on you to assist him, it shall redeem you from your gyves; if not, you shall have your full time of imprisonment, and 10 your deliverance with an unpitied whipping, for you have been a notorious bawd. _Pom._ Sir, I have been an unlawful bawd time out of mind; but yet I will be content to be a lawful hangman. I would be glad to receive some instruction from my fellow 15 partner. _Prov._ What, ho! Abhorson! Where's Abhorson, there? _Enter ABHORSON._ _Abhor._ Do you call, sir? _Prov._ Sirrah, here's a fellow will help you to-morrow in your execution. If you think it meet, compound with 20 him by the year, and let him abide here with you; if not, use him for the present, and dismiss him. He cannot plead his estimation with you; he hath been a bawd. _Abhor._ A bawd, sir? fie upon him! he will discredit our mystery. 25 _Prov._ Go to, sir; you weigh equally; a feather will turn the scale. [_Exit._ _Pom._ Pray, sir, by your good favour,--for surely, sir, a good favour you have, but that you have a hanging look,-- do you call, sir, your occupation a mystery? 30 _Abhor._ Ay, sir; a mystery. _Pom._ Painting, sir, I have heard say, is a mystery; and your whores, sir, being members of my occupation, using painting, do prove my occupation a mystery: but what mystery there should be in hanging, if I should be 35 hanged, I cannot imagine. _Abhor._ Sir, it is a mystery. _Pom._ Proof? _Abhor._ Every true man's apparel fits your thief: if it be too little for your thief, your true man thinks it big 40 enough; if it be too big for your thief, your thief thinks it little enough: so every true man's apparel fits your thief. _Re-enter PROVOST._ _Prov._ Are you agreed? _Pom._ Sir, I will serve him; for I do find your hangman is a more penitent trade than your bawd; he doth 45 oftener ask forgiveness. _Prov._ You, sirrah, provide your block and your axe to-morrow four o'clock. _Abhor._ Come on, bawd; I will instruct thee in my trade; follow. 50 _Pom._ I do desire to learn, sir: and I hope, if you have occasion to use me for your own turn, you shall find me yare; for, truly, sir, for your kindness I owe you a good turn. _Prov._ Call hither Barnardine and Claudio: [_Exeunt Pompey and Abhorson._ 55 The one has my pity; not a jot the other, Being a murderer, though he were my brother. _Enter CLAUDIO._ Look, here's the warrant, Claudio, for thy death: 'Tis now dead midnight, and by eight to-morrow Thou must be made immortal. Where's Barnardine? 60 _Claud._ As fast lock'd up in sleep as guiltless labour When it lies starkly in the traveller's bones: He will not wake. _Prov._ Who can do good on him? Well, go, prepare yourself. [_Knocking within._] But, hark, what noise?-- Heaven give your spirits comfort! [_Exit Clandio._] By and by.-- 65 I hope it is some pardon or reprieve For the most gentle Claudio. _Enter DUKE disguised as before._ Welcome, father. _Duke._ The best and wholesomest spirits of the night Envelop you, good Provost! Who call'd here of late? _Prov._ None, since the curfew rung. 70 _Duke._ Not Isabel? _Prov._ No. _Duke._ They will, then, ere't be long. _Prov._ What comfort is for Claudio? _Duke._ There's some in hope. _Prov._ It is a bitter Deputy. _Duke._ Not so, not so; his life is parallel'd 75 Even with the stroke and line of his great justice: He doth with holy abstinence subdue That in himself which he spurs on his power To qualify in others: were he meal'd with that Which he corrects, then were he tyrannous; 80 But this being so, he's just. [_Knocking within._ Now are they come. [_Exit Provost._ This is a gentle provost: seldom when The steeled gaoler is the friend of men. [_Knocking within._ How now! what noise? That spirit's possessed with haste That wounds the unsisting postern with these strokes. 85 _Re-enter PROVOST._ _Prov._ There he must stay until the officer Arise to let him in: he is call'd up. _Duke._ Have you no countermand for Claudio yet, But he must die to-morrow? _Prov._ None, sir, none. _Duke._ As near the dawning, provost, as it is, 90 You shall hear more ere morning. _Prov._ Happily You something know; yet I believe there comes No countermand; no such example have we: Besides, upon the very siege of justice Lord Angelo hath to the public ear 95 Profess'd the contrary. _Enter a MESSENGER._ This is his lordship's man. _Duke._ And here comes Claudio's pardon. _Mes._ [_Giving a paper_] My lord hath sent you this note; and by me this further charge, that you swerve not from the smallest article of it, neither in time, matter, or other circumstance. 100 Good morrow; for, as I take it, it is almost day. _Prov._ I shall obey him. [_Exit Messenger._ _Duke._ [_Aside_] This is his pardon, purchased by such sin For which the pardoner himself is in. Hence hath offence his quick celerity, 105 When it is borne in high authority: When vice makes mercy, mercy's so extended, That for the fault's love is the offender friended. Now, sir, what news? _Prov._ I told you. Lord Angelo, belike thinking me remiss 110 in mine office, awakens me with this unwonted putting-on; methinks strangely, for he hath not used it before. _Duke._ Pray you, let's hear. [Transcriber's Note: In order to preserve the marked line breaks without losing readability, each line of the quoted message has been split into two equal halves.] _Prov._ [_Reads_] Whatsoever you may hear to the contrary, let Claudio be executed by four of the clock; and in the afternoon Barnardine: for my 115 better satisfaction, let me have Claudio's head sent me by five. Let this be duly performed; with a thought that more depends on it than we must yet deliver. Thus fail not to do your office, as you will answer it at your peril. What say you to this, sir? 120 _Duke._ What is that Barnardine who is to be executed in the afternoon? _Prov._ A Bohemian born, but here nursed up and bred; one that is a prisoner nine years old. _Duke._ How came it that the absent Duke had not 125 either delivered him to his liberty or executed him? I have heard it was ever his manner to do so. _Prov._ His friends still wrought reprieves for him: and, indeed, his fact, till now in the government of Lord Angclo, came not to an undoubtful proof. 130 _Duke._ It is now apparent? _Prov._ Most manifest, and not denied by himself. _Duke._ Hath he borne himself penitently in prison? how seems he to be touched? _Prov._ A man that apprehends death no more dreadfully 135 but as a drunken sleep; careless, reckless, and fearless of what's past, present, or to come; insensible of mortality, and desperately mortal. _Duke._ He wants advice. _Prov._ He will hear none: he hath evermore had the 140 liberty of the prison; give him leave to escape hence, he would not: drunk many times a day, if not many days entirely drunk. We have very oft awaked him, as if to carry him to execution, and showed him a seeming warrant for it: it hath not moved him at all. 145 _Duke._ More of him anon. There is written in your brow, provost, honesty and constancy: if I read it not truly, my ancient skill beguiles me; but, in the boldness of my cunning, I will lay my self in hazard. Claudio, whom here you have warrant to execute, is no greater forfeit to the 150 law than Angelo who hath sentenced him. To make you understand this in a manifested effect, I crave but four days' respite; for the which you are to do me both a present and a dangerous courtesy. _Prov._ Pray, sir, in what? 155 _Duke._ In the delaying death. _Prov._ Alack, how may I do it, having the hour limited, and an express command, under penalty, to deliver his head in the view of Angelo? I may make my case as Claudio's, to cross this in the smallest. 160 _Duke._ By the vow of mine order I warrant you, if my instructions may be your guide. Let this Barnardine be this morning executed, and his head borne to Angelo. _Prov._ Angelo hath seen them both, and will discover the favour. 165 _Duke._ O, death's a great disguiser; and you may add to it. Shave the head, and tie the beard; and say it was the desire of the penitent to be so bared before his death: you know the course is common. If any thing fall to you upon this, more than thanks and good fortune, by the Saint 170 whom I profess, I will plead against it with my life. _Prov._ Pardon me, good father; it is against my oath. _Duke._ Were you sworn to the Duke, or to the Deputy? _Prov._ To him, and to his substitutes. _Duke._ You will think you have made no offence, if the 175 Duke avouch the justice of your dealing? _Prov._ But what likelihood is in that? _Duke._ Not a resemblance, but a certainty. Yet since I see you fearful, that neither my coat, integrity, nor persuasion can with ease attempt you, I will go further than I 180 meant, to pluck all fears out of you. Look you, sir, here is the hand and seal of the Duke: you know the character, I doubt not; and the signet is not strange to you. _Prov._ I know them both. _Duke._ The contents of this is the return of the Duke: 185 you shall anon over-read it at your pleasure; where you shall find, within these two days he will be here. This is a thing that Angelo knows not; for he this very day receives letters of strange tenour; perchance of the Duke's death; perchance entering into some monastery; but, by 190 chance, nothing of what is writ. Look, the unfolding star calls up the shepherd. Put not yourself into amazement how these things should be: all difficulties are but easy when they are known. Call your executioner, and off with Barnardine's head: I will give him a present shrift and 195 advise him for a better place. Yet you are amazed; but this shall absolutely resolve you. Come away; it is almost clear dawn. [_Exeunt._ NOTES: IV, 2. SCENE II.] SCENE V. Pope. 2-4: Printed as verse in Ff. 37-42: Abhor. _Sir,.......thief_] Abhor. ***Clown.*** _Sir, it is a mystery._ Abhor. _Proof.--_ Clown. _Every... thief_ (42) Hanmer. Pom. _Proof... thief_ (42) Lloyd conj. 39-42: _Every......thief_] Capell. Abh. _Every....thief_ (39). Clo. _If it be... thief_ (41) Ff. Abh. _Every... thief, Clown: if it be......thief_ (42) Theobald. 45: _your_] _you_ F2. 53: _yare_] Theobald. _y'are_ Ff. _yours_ Rowe. 56: _The one_] _Th' one_ Ff. _One_ Hamner. 58: SCENE VI. Pope. 63: _He will not wake_] F1 F2. _He will not awake_ F3 F4. _He'll not awake_ Pope. 64: _yourself_] _yourself_ [Ex. Claudio.] Theobald. 65: _comfort!_ [Exit Claudio.] _By and by.--_] Capell. _comfort: by and by,_ Ff. 70: _None_] F1. _Now_ F2 F3 F4. 71: _They_] _She_ Hawkins conj. _There_ Collier MS. 85: _unsisting_] F1 F2 F3. _insisting_ F4. _unresisting_ Rowe. _unresting_ Hanmer. _unshifting_ Capell. _unlist'ning_ Steevens conj. _resisting_ Collier conj. _unlisting_ Mason conj. _unfeeling_ Johnson conj. _unwisting_ Singer. 86:....Provost]....Provost, speaking to one at the door, after which he comes forward. Capell. 91: _Happily_] _Happely_ F1 F2. _Happily_ F3 F4. See note (XVIII). 96: SCENE VII. Pope. _lordship's_] Pope. _lords_ Ff. om. Capell. 96, 97: _This... man._ Duke. _And... pardon_] Knight (Tyrwhitt conj.). Duke. _This... man._ Pro. _And... pardon_ Ff. 98-101: Printed as verse in Ff. 113: _you_] om. F4. 114: Prov. [Reads] Rowe. The letter. Ff. 117: _duly_] _truly_ Capell (a misprint). 131: _It is_] Ff. _Is it_ Pope. 136: _reckless_] Theobald. _wreaklesse_ F1 F2 F3. _wreakless_ F4. _rechless_ Pope. 138: _desperately mortal_] _mortally desperate_ Hanmer. 161-165: Printed as verse in Ff. Rowe. 167: _tie_] F1 F4. _tye_ F2 F3. _tire_ Theobald conj. _dye_ Simpson conj. 168: _bared_] Malone. _bar'de_ F1 F2 F3. _barb'd_ F4. 179: _persuasion_] Ff. _my persuasion_ Rowe. 188: _that_] F1 F2 F3. _which_ F4. 191: _writ_] _here writ_ Hanmer. SCENE III. _Another room in the same._ _Enter POMPEY._ _Pom._ I am as well acquainted here as I was in our house of profession: one would think it were Mistress Overdone's own house, for here be many of her old customers. First, here's young Master Rash; he's in for a commodity of brown paper and old ginger, nine-score and seventeen 5 pounds; of which he made five marks, ready money: marry, then ginger was not much in request, for the old women were all dead. Then is there here one Master Caper, at the suit of Master Three-pile the mercer, for some four suits of peach-coloured satin, which now peaches him a 10 beggar. Then have we here young Dizy, and young Master Deep-vow, and Master Copper-spur, and Master Starve-lackey the rapier and dagger man, and young Drop-heir that killed lusty Pudding, and Master Forthlight the tilter, and brave Master Shooty the great traveller, and 15 wild Half-can that stabbed Pots, and, I think, forty more; all great doers in our trade, and are now 'for the Lord's sake.' _Enter ABHORSON._ _Abhor._ Sirrah, bring Barnardine hither. _Pom._ Master Barnardine! you must rise and be hanged, 20 Master Barnardine! _Abhor._ What, ho, Barnardine! _Bar._ [_Within_] A pox o' your throats! Who makes that noise there? What are you? _Pom._ Your friends, sir; the hangman. You must be 25 so good, sir, to rise and be put to death. _Bar._ [_Within_] Away, you rogue, away! I am sleepy. _Abhor._ Tell him he must awake, and that quickly too. _Pom._ Pray, Master Barnardine, awake till you are executed, and sleep afterwards. 30 _Abhor._ Go in to him, and fetch him out. _Pom._ He is coming, sir, he is coming; I hear his straw rustle. _Abhor._ Is the axe upon the block, sirrah? _Pom._ Very ready, sir. 35 _Enter BARNARDINE._ _Bar._ How now, Abhorson? what's the news with you? _Abhor._ Truly, sir, I would desire you to clap into your prayers; for, look you, the warrant's come. _Bar._ You rogue, I have been drinking all night; I am not fitted for 't. 40 _Pom._ O, the better, sir; for he that drinks all night, and is hanged betimes in the morning, may sleep the sounder all the next day. _Abhor._ Look you, sir; here comes your ghostly father: do we jest now, think you? 45 _Enter DUKE disguised as before._ _Duke._ Sir, induced by my charity, and hearing how hastily you are to depart, I am come to advise you, comfort you and pray with you. _Bar._ Friar, not I: I have been drinking hard all night, and I will have more time to prepare me, or they shall beat 50 out my brains with billets: I will not consent to die this day, that's certain. _Duke._ O, sir, you must: and therefore I beseech you Look forward on the journey you shall go. _Bar._ I swear I will not die to-day for any man's persuasion. 55 _Duke._ But hear you. _Bar._ Not a word: if you have any thing to say to me, come to my ward; for thence will not I to-day. [_Exit._ _Duke._ Unfit to live or die: O gravel heart! 60 After him, fellows; bring him to the block. [_Exeunt Abhorson and Pompey._ _Re-enter PROVOST._ _Prov._ Now, sir, how do you find the prisoner? _Duke._ A creature unprepared, unmeet for death; And to transport him in the mind he is Were damnable. _Prov._ Here in the prison, father, 65 There died this morning of a cruel fever One Ragozine, a most notorious pirate, A man of Claudio's years; his beard and head Just of his colour. What if we do omit This reprobate till he were well inclined; 70 And satisfy the Deputy with the visage Of Ragozine, more like to Claudio? _Duke._ O, 'tis an accident that heaven provides! Dispatch it presently; the hour draws on Prefix'd by Angelo: see this be done, 75 And sent according to command; whiles I Persuade this rude wretch willingly to die. _Prov._ This shall be done, good father, presently. But Barnardine must die this afternoon: And how shall we continue Claudio, 80 To save me from the danger that might come If he were known alive? _Duke._ Let this be done. Put them in secret holds, both Barnardine and Claudio: Ere twice the sun hath made his journal greeting To the under generation, you shall find 85 Your safety manifested. _Prov._ I am your free dependant. _Duke._ Quick, dispatch, and send the head to Angelo. [_Exit Provost._ Now will I write letters to Angelo,-- The provost, he shall bear them,--whose contents 90 Shall witness to him I am near at home, And that, by great injunctions, I am bound To enter publicly: him I'll desire To meet me at the consecrated fount, A league below the city; and from thence, 95 By cold gradation and well-balanced form, We shall proceed with Angelo. _Re-enter PROVOST._ _Prov._ Here is the head; I'll carry it myself. _Duke._ Convenient is it. Make a swift return; For I would commune with you of such things 100 That want no ear but yours. _Prov._ I'll make all speed. [_Exit._ _Isab._ [_Within_] Peace, ho, be here! _Duke._ The tongue of Isabel. She's come to know If yet her brother's pardon be come hither: But I will keep her ignorant of her good, 105 To make her heavenly comforts of despair, When it is least expected. _Enter ISABELLA._ _Isab._ Ho, by your leave! _Duke._ Good morning to you, fair and gracious daughter. _Isab._ The better, given me by so holy a man. Hath yet the Deputy sent my brother's pardon? 110 _Duke._ He hath released him, Isabel, from the world: His head is off, and sent to Angelo. _Isab._ Nay, but it is not so. _Duke._ It is no other: show your wisdom, daughter, In your close patience. 115 _Isab._ O, I will to him and pluck out his eyes! _Duke._ You shall not be admitted to his sight. _Isab._ Unhappy Claudio! wretched Isabel! Injurious world! most damned Angelo! _Duke._ This nor hurts him nor profits you a jot; 120 Forbear it therefore; give your cause to heaven. Mark what I say, which you shall find By every syllable a faithful verity: The Duke comes home to-morrow;--nay, dry your eyes; One of our covent, and his confessor, 125 Gives me this instance: already he hath carried Notice to Escalus and Angelo; Who do prepare to meet him at the gates, There to give up their power. If you can, pace your wisdom In that good path that I would wish it go; 130 And you shall have your bosom on this wretch, Grace of the Duke, revenges to your heart, And general honour. _Isab._ I am directed by you. _Duke._ This letter, then, to Friar Peter give; 'Tis that he sent me of the Duke's return: 135 Say, by this token, I desire his company At Mariana's house to-night. Her cause and yours I'll perfect him withal; and he shall bring you Before the Duke; and to the head of Angelo Accuse him home and home. For my poor self, 140 I am combined by a sacred vow, And shall be absent. Wend you with this letter: Command these fretting waters from your eyes With a light heart; trust not my holy order, If I pervert your course.--Who's here? 145 _Enter LUCIO._ _Lucio._ Good even. Friar, where's the provost? _Duke._ Not within, sir. _Lucio._ O pretty Isabella, I am pale at mine heart to see thine eyes so red: thou must be patient. I am fain to dine and sup with water and bran; I dare not for my 150 head fill my belly; one fruitful meal would set me to't. But they say the Duke will be here to-morrow. By my troth, Isabel, I loved thy brother: if the old fantastical Duke of dark corners had been at home, he had lived. [_Exit Isabella._ _Duke._ Sir, the Duke is marvellous little beholding to 155 your reports; but the best is, he lives not in them. _Lucio._ Friar, thou knowest not the Duke so well as I do: he's a better woodman than thou takest him for. _Duke._ Well, you'll answer this one day. Fare ye well. _Lucio._ Nay, tarry; I'll go along with thee: I can tell 160 thee pretty tales of the Duke. _Duke._ You have told me too many of him already, sir, if they be true; if not true, none were enough. _Lucio._ I was once before him for getting a wench with child. 165 _Duke._ Did you such a thing? _Lucio._ Yes, marry, did I: but I was fain to forswear it; they would else have married me to the rotten medlar. _Duke._ Sir, your company is fairer than honest. Rest you well. 170 _Lucio._ By my troth, I'll go with thee to the lane's end: if bawdy talk offend you, we'll have very little of it. Nay, friar, I am a kind of burr; I shall stick. [_Exeunt._ NOTES: IV, 3. SCENE III.] SCENE VIII. Pope. 5: _paper_] _pepper_ Rowe. 11: _Dizy_] F2 F3 F4. _Dizie_ F1. _Dizzy_ Pope. _Dicey_ Steevens conj. 14: _Forthlight_] Ff. _Forthright_ Warburton. 15: _Shooty_] F2 F3 F4. _Shootie_ F1. _Shooter_ Warburton. _Shoo-tye_ Capell. 17: _are_] _cry_ Anon. conj. See note (XIX). _now_] _now in_ Pope. 25: _friends_] F1 F2. _friend_ F3 F4. 32: _his_] _the_ Pope. 49: _I_] om. F4. [Transcriber's Note: The text does not specify which occurrence of "I" is meant. The speech begins "Not I: I have..."] 57: _hear_] _heave_ F2. 59: SCENE IX. Pope. 60: _gravel heart_] _grovelling beast_ Collier MS. 61: Given by Hanmer to _Prov._ 69: _his_] F1. om. F2 F3 F4. _do_] om. Pope. 76: _whiles_] _while_ Pope. 83: _both Barnardine and Claudio_] _Claudio and Barnardine_ Hanmer. See note (XX). 85: _the under_] Hanmer. _yond_ Ff. _yonder_ Pope. 86: _manifested_] _manifest_ Hanmer. 88: _Quick_] _Quick, then,_ Capell. 96: _well-_] Rowe. _weale-_ F1 F2 F3. _weal_ F4. 102: SCENE X. Pope. 103: _She's come_] _She comes_ Pope. 106: _comforts_] _comfort_ Hanmer. 107: _Ho,_] om. Pope. 113, 114, 115: Ff make two lines ending at _other... patience._ Text as proposed by Spedding. 114, 115: _show... patience_] _In your close patience, daughter, shew your wisdom_ Capell. 114: _your wisdom_] _wisdom_ Pope. 115: _close_] _closest_ Pope. 119: _Injurious_] _perjurious_ Collier MS. 120: _nor hurts_] _not hurts_ F4. _hurts not_ Rowe. 122: _say_] _say to you_ Collier MS. _find_] _surely find_ Pope. 124: _nay_] om. Pope. 125: _covent_] Ff. _convent_ Rowe. 126: _instance_] _news_ Pope. 129: _If you can, pace_] Rowe. _If you can pace_ Ff. _Pace_ Pope. S. Walker thinks a line is lost after 131. 129, 130: _If you can pace... wish it, go,_ Edd. conj. 137: _to-night_] om. Pope. 141: _combined_] _confined_ Johnson conj. (withdrawn). 145: _Who's_] _whose_ F1. 146: SCENE XI. Pope. 154: [Exit ISABELLA] Theobald. om. Ff. 155: _beholding_] Ff. _beholden_ Rowe. 163: _not true_] _not_ Rowe. 172: _it_] om. F2. SCENE IV. _A room in ANGELO'S house._ _Enter ANGELO and ESCALUS._ _Escal._ Every letter he hath writ hath disvouched other. _Ang._ In most uneven and distracted manner. His actions show much like to madness: pray heaven his wisdom be not tainted! And why meet him at the gates, and redeliver our authorities there? 5 _Escal._ I guess not. _Ang._ And why should we proclaim it in an hour before his entering, that if any crave redress of injustice, they should exhibit their petitions in the street? _Escal._ He shows his reason for that: to have a dispatch 10 of complaints, and to deliver us from devices hereafter, which shall then have no power to stand against us. _Ang._ Well, I beseech you, let it be proclaimed betimes i' the morn; I'll call you at your house: give notice to such men of sort and suit as are to meet him. 15 _Escal._ I shall, sir. Fare you well. _Ang._ Good night. [_Exit Escalus._ This deed unshapes me quite, makes me unpregnant, And dull to all proceedings. A deflower'd maid! And by an eminent body that enforced 20 The law against it! But that her tender shame Will not proclaim against her maiden loss, How might she tongue me! Yet reason dares her no; For my authority bears of a credent bulk, That no particular scandal once can touch 25 But it confounds the breather. He should have lived, Save that his riotous youth, with dangerous sense, Might in the times to come have ta'en revenge, By so receiving a dishonour'd life With ransom of such shame. Would yet he had lived! 30 Alack, when once our grace we have forgot, Nothing goes right: we would, and we would not. [_Exit._ NOTES: IV, 4. SCENE IV.] SCENE XII. Pope. A room... house.] Capell. The palace. Rowe. 2, sqq.: Angelo's speeches in this scene Collier prints as verse. 5: _redeliver_] Capell. _re-liver_] F1. _deliver_ F2 F3 F4. 13: A colon is put after _proclaim'd_ by Capell, who prints lines 13-16 as verse. 19: _And_] om. Hanmer. 23: _dares her no;_] Ff. _dares her:_ Pope. _dares her: no,_ Hanmer. _dares her No_ Warburton. _dares her? no:_ Capell. _dares her note_ Theobald conj. _dares her not_ Steevens conj. _dares her on_ Grant White (Becket conj.). _reason... no_] _treason dares her?--No_ Jackson conj. 24: _bears of a credent bulk_] F1 F2 F3. _bears off a credent bulk_ F4. _bears off all credence_ Pope. _bears a credent bulk_ Theobald. _bears such a credent bulk_ Collier MS. _here's of a credent bulk_ Singer. _bears so credent bulk_ Dyce. _bears up a credent bulk_ Grant White. SCENE V. _Fields without the town._ _Enter DUKE in his own habit, and FRIAR PETER._ _Duke._ These letters at fit time deliver me: [_Giving letters._ The provost knows our purpose and our plot. The matter being afoot, keep your instruction, And hold you ever to our special drift; Though sometimes you do blench from this to that, 5 As cause doth minister. Go call at Flavius' house, And tell him where I stay: give the like notice To Valentius, Rowland, and to Crassus, And bid them bring the trumpets to the gate; But send me Flavius first. _Fri. P._ It shall be speeded well. [_Exit._ 10 _Enter VARRIUS._ _Duke._ I thank thee, Varrius; thou hast made good haste: Come, we will walk. There's other of our friends Will greet us here anon, my gentle Varrius. [_Exeunt._ NOTES: IV, 5. SCENE V.] SCENE XIII. Pope. FRIAR PETER] See note (XXI). 6: _Go_] om. Hanmer. _Flavius'_] Rowe. _Flavio's_ Ff. 8: _To Valentius_] _To Valencius_ Ff. _Unto Valentius_ Pope. _To Valentinus_ Capell. SCENE VI. _Street near the city-gate._ _Enter ISABELLA and MARIANA._ _Isab._ To speak so indirectly I am loath: I would say the truth; but to accuse him so, That is your part: yet I am advised to do it; He says, to veil full purpose. _Mari._ Be ruled by him. _Isab._ Besides, he tells me that, if peradventure 5 He speak against me on the adverse side, I should not think it strange; for 'tis a physic That's bitter to sweet end. _Mari._ I would Friar Peter-- _Isab._ O, peace! the friar is come. _Enter FRIAR PETER._ _Fri. P._ Come, I have found you out a stand most fit, 10 Where you may have such vantage on the Duke, He shall not pass you. Twice have the trumpets sounded; The generous and gravest citizens Have hent the gates, and very near upon The Duke is entering: therefore, hence, away! [_Exeunt._ 15 NOTES: IV, 6. SCENE VI.] SCENE XIV. Pope. 2: _I would_] _I'd_ Pope. 3: _I am_] _I'm_ Pope. 4: _to veil full_] Malone. _to vaile full_ F1 F2 F3. _to vail full_ F4. _t' availful_ Theobald. _to 'vailful_ Hanmer.

Summary: The Duke finds Mariana, and exchanges a few cursory words with her. Isabella enters as Mariana leaves, to tell the Duke that she has agreed to Angelo's plan, and describes the place of meeting. Isabella said that she told Angelo she could only stay briefly, and that she would be bringing a servant with her, which means she can bring Mariana without suspicion. Isabella has a word with Mariana, and Mariana agrees to go with the plan, provided the "friar" agrees, which he does. The Duke still has to assure her that she is doing no sin, because she is only fulfilling the contract she had with Angelo some time ago.

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Summarize: The money was far too much even for a fee in a fairy-tale, and in the absence of Mrs. Beale, who, though the hour was now late, had not yet returned to the Regent's Park, Susan Ash, in the hall, as loud as Maisie was low and as bold as she was bland, produced, on the exhibition offered under the dim vigil of the lamp that made the place a contrast to the child's recent scene of light, the half-crown that an unsophisticated cabman could pronounce to be the least he would take. It was apparently long before Mrs. Beale would arrive, and in the interval Maisie had been induced by the prompt Susan not only to go to bed like a darling dear, but, in still richer expression of that character, to devote to the repayment of obligations general as well as particular one of the sovereigns in the ordered array that, on the dressing-table upstairs, was naturally not less dazzling to a lone orphan of a housemaid than to the subject of the manoeuvres of a quartette. This subject went to sleep with her property gathered into a knotted handkerchief, the largest that could be produced and lodged under her pillow; but the explanations that on the morrow were inevitably more complete with Mrs. Beale than they had been with her humble friend found their climax in a surrender also more becomingly free. There were explanations indeed that Mrs. Beale had to give as well as to ask, and the most striking of these was to the effect that it was dreadful for a little girl to take money from a woman who was simply the vilest of their sex. The sovereigns were examined with some attention, the result of which, however, was to make the author of that statement desire to know what, if one really went into the matter, they could be called but the wages of sin. Her companion went into it merely so far as the question of what then they were to do with them; on which Mrs. Beale, who had by this time put them into her pocket, replied with dignity and with her hand on the place: "We're to send them back on the spot!" Susan, the child soon afterwards learnt, had been invited to contribute to this act of restitution her one appropriated coin; but a closer clutch of the treasure showed in her private assurance to Maisie that there was a limit to the way she could be "done." Maisie had been open with Mrs. Beale about the whole of last night's transaction; but she now found herself on the part of their indignant inferior a recipient of remarks that were so many ringing tokens of that lady's own suppressions. One of these bore upon the extraordinary hour--it was three in the morning if she really wanted to know--at which Mrs. Beale had re-entered the house; another, in accents as to which Maisie's criticism was still intensely tacit, characterised her appeal as such a "gime," such a "shime," as one had never had to put up with; a third treated with some vigour the question of the enormous sums due belowstairs, in every department, for gratuitous labour and wasted zeal. Our young lady's consciousness was indeed mainly filled for several days with the apprehension created by the too slow subsidence of her attendant's sense of wrong. These days would become terrific like the Revolutions she had learnt by heart in Histories if an outbreak in the kitchen should crown them; and to promote that prospect she had through Susan's eyes more than one glimpse of the way in which Revolutions are prepared. To listen to Susan was to gather that the spark applied to the inflammables and already causing them to crackle would prove to have been the circumstance of one's being called a horrid low thief for refusing to part with one's own. The redeeming point of this tension was, on the fifth day, that it actually appeared to have had to do with a breathless perception in our heroine's breast that scarcely more as the centre of Sir Claude's than as that of Susan's energies she had soon after breakfast been conveyed from London to Folkestone and established at a lovely hotel. These agents, before her wondering eyes, had combined to carry through the adventure and to give it the air of having owed its success to the fact that Mrs. Beale had, as Susan said, but just stepped out. When Sir Claude, watch in hand, had met this fact with the exclamation "Then pack Miss Farange and come off with us!" there had ensued on the stairs a series of gymnastics of a nature to bring Miss Farange's heart into Miss Farange's mouth. She sat with Sir Claude in a four-wheeler while he still held his watch; held it longer than any doctor who had ever felt her pulse; long enough to give her a vision of something like the ecstasy of neglecting such an opportunity to show impatience. The ecstasy had begun in the schoolroom and over the Berceuse, quite in the manner of the same foretaste on the day, a little while back, when Susan had panted up and she herself, after the hint about the duchess, had sailed down; for what harm then had there been in drops and disappointments if she could still have, even only a moment, the sensation of such a name "brought up"? It had remained with her that her father had foretold her she would some day be in the street, but it clearly wouldn't be this day, and she felt justified of the preference betrayed to that parent as soon as her visitor had set Susan in motion and laid his hand, while she waited with him, kindly on her own. This was what the Captain, in Kensington Gardens, had done; her present situation reminded her a little of that one and renewed the dim wonder of the fashion after which, from the first, such pats and pulls had struck her as the steps and signs of other people's business and even a little as the wriggle or the overflow of their difficulties. What had failed her and what had frightened her on the night of the Exhibition lost themselves at present alike in the impression that any "surprise" now about to burst from Sir Claude would be too big to burst all at once. Any awe that might have sprung from his air of leaving out her stepmother was corrected by the force of a general rule, the odd truth that if Mrs. Beale now never came nor went without making her think of him, it was never, to balance that, the main mark of his own renewed reality to appear to be a reference to Mrs. Beale. To be with Sir Claude was to think of Sir Claude, and that law governed Maisie's mind until, through a sudden lurch of the cab, which had at last taken in Susan and ever so many bundles and almost reached Charing Cross, it popped again somehow into her dizzy head the long-lost image of Mrs. Wix. It was singular, but from this time she understood and she followed, followed with the sense of an ample filling-out of any void created by symptoms of avoidance and of flight. Her ecstasy was a thing that had yet more of a face than of a back to turn, a pair of eyes still directed to Mrs. Wix even after the slight surprise of their not finding her, as the journey expanded, either at the London station or at the Folkestone hotel. It took few hours to make the child feel that if she was in neither of these places she was at least everywhere else. Maisie had known all along a great deal, but never so much as she was to know from this moment on and as she learned in particular during the couple of days that she was to hang in the air, as it were, over the sea which represented in breezy blueness and with a summer charm a crossing of more spaces than the Channel. It was granted her at this time to arrive at divinations so ample that I shall have no room for the goal if I attempt to trace the stages; as to which therefore I must be content to say that the fullest expression we may give to Sir Claude's conduct is a poor and pale copy of the picture it presented to his young friend. Abruptly, that morning, he had yielded to the action of the idea pumped into him for weeks by Mrs. Wix on lines of approach that she had been capable of the extraordinary art of preserving from entanglement in the fine network of his relations with Mrs. Beale. The breath of her sincerity, blowing without a break, had puffed him up to the flight by which, in the degree I have indicated, Maisie too was carried off her feet. This consisted neither in more nor in less than the brave stroke of his getting off from Mrs. Beale as well as from his wife--of making with the child straight for some such foreign land as would give a support to Mrs. Wix's dream that she might still see his errors renounced and his delinquencies redeemed. It would all be a sacrifice--under eyes that would miss no faintest shade--to what even the strange frequenters of her ladyship's earlier period used to call the real good of the little unfortunate. Maisie's head held a suspicion of much that, during the last long interval, had confusedly, but quite candidly, come and gone in his own; a glimpse, almost awe-stricken in its gratitude, of the miracle her old governess had wrought. That functionary could not in this connexion have been more impressive, even at second-hand, if she had been a prophetess with an open scroll or some ardent abbess speaking with the lips of the Church. She had clung day by day to their plastic associate, plying him with her deep, narrow passion, doing her simple utmost to convert him, and so working on him that he had at last really embraced his fine chance. That the chance was not delusive was sufficiently guaranteed by the completeness with which he could finally figure it out that, in case of his taking action, neither Ida nor Beale, whose book, on each side, it would only too well suit, would make any sort of row. It sounds, no doubt, too penetrating, but it was not all as an effect of Sir Claude's betrayals that Maisie was able to piece together the beauty of the special influence through which, for such stretches of time, he had refined upon propriety by keeping, so far as possible, his sentimental interests distinct. She had ever of course in her mind fewer names than conceptions, but it was only with this drawback that she now made out her companion's absences to have had for their ground that he was the lover of her stepmother and that the lover of her stepmother could scarce logically pretend to a superior right to look after her. Maisie had by this time embraced the implication of a kind of natural divergence between lovers and little girls. It was just this indeed that could throw light on the probable contents of the pencilled note deposited on the hall-table in the Regent's Park and which would greet Mrs. Beale on her return. Maisie freely figured it as provisionally jocular in tone, even though to herself on this occasion Sir Claude turned a graver face than he had shown in any crisis but that of putting her into the cab when she had been horrid to him after her parting with the Captain. He might really be embarrassed, but he would be sure, to her view, to have muffled in some bravado of pleasantry the disturbance produced at her father's by the removal of a valued servant. Not that there wasn't a great deal too that wouldn't be in the note--a great deal for which a more comfortable place was Maisie's light little brain, where it hummed away hour after hour and caused the first outlook at Folkestone to swim in a softness of colour and sound. It became clear in this medium that her stepfather had really now only to take into account his entanglement with Mrs. Beale. Wasn't he at last disentangled from every one and every thing else? The obstacle to the rupture pressed upon him by Mrs. Wix in the interest of his virtue would be simply that he was in love, or rather, to put it more precisely, that Mrs. Beale had left him no doubt of the degree in which SHE was. She was so much so as to have succeeded in making him accept for a time her infatuated grasp of him and even to some extent the idea of what they yet might do together with a little diplomacy and a good deal of patience. I may not even answer for it that Maisie was not aware of how, in this, Mrs. Beale failed to share his all but insurmountable distaste for their allowing their little charge to breathe the air of their gross irregularity--his contention, in a word, that they should either cease to be irregular or cease to be parental. Their little charge, for herself, had long ago adopted the view that even Mrs. Wix had at one time not thought prohibitively coarse--the view that she was after all, AS a little charge, morally at home in atmospheres it would be appalling to analyse. If Mrs. Wix, however, ultimately appalled, had now set her heart on strong measures, Maisie, as I have intimated, could also work round both to the reasons for them and to the quite other reasons for that lady's not, as yet at least, appearing in them at first-hand. Oh decidedly I shall never get you to believe the number of things she saw and the number of secrets she discovered! Why in the world, for instance, couldn't Sir Claude have kept it from her--except on the hypothesis of his not caring to--that, when you came to look at it and so far as it was a question of vested interests, he had quite as much right in her as her stepmother, not to say a right that Mrs. Beale was in no position to dispute? He failed at all events of any such successful ambiguity as could keep her, when once they began to look across at France, from regarding even what was least explained as most in the spirit of their old happy times, their rambles and expeditions in the easier better days of their first acquaintance. Never before had she had so the sense of giving him a lead for the sort of treatment of what was between them that would best carry it off, or of his being grateful to her for meeting him so much in the right place. She met him literally at the very point where Mrs. Beale was most to be reckoned with, the point of the jealousy that was sharp in that lady and of the need of their keeping it as long as possible obscure to her that poor Mrs. Wix had still a hand. Yes, she met him too in the truth of the matter that, as her stepmother had had no one else to be jealous of, she had made up for so gross a privation by directing the sentiment to a moral influence. Sir Claude appeared absolutely to convey in a wink that a moral influence capable of pulling a string was after all a moral influence exposed to the scratching out of its eyes; and that, this being the case, there was somebody they couldn't afford to leave unprotected before they should see a little better what Mrs. Beale was likely to do. Maisie, true enough, had not to put it into words to rejoin, in the coffee-room, at luncheon: "What CAN she do but come to you if papa does take a step that will amount to legal desertion?" Neither had he then, in answer, to articulate anything but the jollity of their having found a table at a window from which, as they partook of cold beef and apollinaris--for he hinted they would have to save lots of money--they could let their eyes hover tenderly on the far-off white cliffs that so often had signalled to the embarrassed English a promise of safety. Maisie stared at them as if she might really make out after a little a queer dear figure perched on them--a figure as to which she had already the subtle sense that, wherever perched, it would be the very oddest yet seen in France. But it was at least as exciting to feel where Mrs. Wix wasn't as it would have been to know where she was, and if she wasn't yet at Boulogne this only thickened the plot. If she was not to be seen that day, however, the evening was marked by an apparition before which, none the less, overstrained suspense folded on the spot its wings. Adjusting her respirations and attaching, under dropped lashes, all her thoughts to a smartness of frock and frill for which she could reflect that she had not appealed in vain to a loyalty in Susan Ash triumphant over the nice things their feverish flight had left behind, Maisie spent on a bench in the garden of the hotel the half-hour before dinner, that mysterious ceremony of the _table d'hote_ for which she had prepared with a punctuality of flutter. Sir Claude, beside her, was occupied with a cigarette and the afternoon papers; and though the hotel was full the garden shewed the particular void that ensues upon the sound of the dressing-bell. She had almost had time to weary of the human scene; her own humanity at any rate, in the shape of a smutch on her scanty skirt, had held her so long that as soon as she raised her eyes they rested on a high fair drapery by which smutches were put to shame and which had glided toward her over the grass without her noting its rustle. She followed up its stiff sheen--up and up from the ground, where it had stopped--till at the end of a considerable journey her impression felt the shock of the fixed face which, surmounting it, seemed to offer the climax of the dressed condition. "Why mamma!" she cried the next instant--cried in a tone that, as she sprang to her feet, brought Sir Claude to his own beside her and gave her ladyship, a few yards off, the advantage of their momentary confusion. Poor Maisie's was immense; her mother's drop had the effect of one of the iron shutters that, in evening walks with Susan Ash, she had seen suddenly, at the touch of a spring, rattle down over shining shop-fronts. The light of foreign travel was darkened at a stroke; she had a horrible sense that they were caught; and for the first time of her life in Ida's presence she so far translated an impulse into an invidious act as to clutch straight at the hand of her responsible confederate. It didn't help her that he appeared at first equally hushed with horror; a minute during which, in the empty garden, with its long shadows on the lawn, its blue sea over the hedge and its startled peace in the air, both her elders remained as stiff as tall tumblers filled to the brim and held straight for fear of a spill. At last, in a tone that enriched the whole surprise by its unexpected softness, her mother said to Sir Claude: "Do you mind at all my speaking to her?" "Oh no; DO you?" His reply was so long in coming that Maisie was the first to find the right note. He laughed as he seemed to take it from her, and she felt a sufficient concession in his manner of addressing their visitor. "How in the world did you know we were here?" His wife, at this, came the rest of the way and sat down on the bench with a hand laid on her daughter, whom she gracefully drew to her and in whom, at her touch, the fear just kindled gave a second jump, but now in quite another direction. Sir Claude, on the further side, resumed his seat and his newspapers, so that the three grouped themselves like a family party; his connexion, in the oddest way in the world, almost cynically and in a flash acknowledged, and the mother patting the child into conformities unspeakable. Maisie could already feel how little it was Sir Claude and she who were caught. She had the positive sense of their catching their relative, catching her in the act of getting rid of her burden with a finality that showed her as unprecedentedly relaxed. Oh yes, the fear had dropped, and she had never been so irrevocably parted with as in the pressure of possession now supremely exerted by Ida's long-gloved and much-bangled arm. "I went to the Regent's Park"--this was presently her ladyship's answer to Sir Claude. "Do you mean to-day?" "This morning, just after your own call there. That's how I found you out; that's what has brought me." Sir Claude considered and Maisie waited. "Whom then did you see?" Ida gave a sound of indulgent mockery. "I like your scare. I know your game. I didn't see the person I risked seeing, but I had been ready to take my chance of her." She addressed herself to Maisie; she had encircled her more closely. "I asked for YOU, my dear, but I saw no one but a dirty parlourmaid. She was red in the face with the great things that, as she told me, had just happened in the absence of her mistress; and she luckily had the sense to have made out the place to which Sir Claude had come to take you. If he hadn't given a false scent I should find you here: that was the supposition on which I've proceeded." Ida had never been so explicit about proceeding or supposing, and Maisie, drinking this in, noted too how Sir Claude shared her fine impression of it. "I wanted to see you," his wife continued, "and now you can judge of the trouble I've taken. I had everything to do in town to-day, but I managed to get off." Maisie and her companion, for a moment, did justice to this achievement; but Maisie was the first to express it. "I'm glad you wanted to see me, mamma." Then after a concentration more deep and with a plunge more brave: "A little more and you'd have been too late." It stuck in her throat, but she brought it out: "We're going to France." Ida was magnificent; Ida kissed her on the forehead. "That's just what I thought likely; it made me decide to run down. I fancied that in spite of your scramble you'd wait to cross, and it added to the reason I have for seeing you." Maisie wondered intensely what the reason could be, but she knew ever so much better than to ask. She was slightly surprised indeed to perceive that Sir Claude didn't, and to hear him immediately enquire: "What in the name of goodness can you have to say to her?" His tone was not exactly rude, but it was impatient enough to make his wife's response a fresh specimen of the new softness. "That, my dear man, is all my own business." "Do you mean," Sir Claude asked, "that you wish me to leave you with her?" "Yes, if you'll be so good; that's the extraordinary request I take the liberty of making." Her ladyship had dropped to a mildness of irony by which, for a moment, poor Maisie was mystified and charmed, puzzled with a glimpse of something that in all the years had at intervals peeped out. Ida smiled at Sir Claude with the strange air she had on such occasions of defying an interlocutor to keep it up as long; her huge eyes, her red lips, the intense marks in her face formed an _eclairage_ as distinct and public as a lamp set in a window. The child seemed quite to see in it the very beacon that had lighted her path; she suddenly found herself reflecting that it was no wonder the gentlemen were guided. This must have been the way mamma had first looked at Sir Claude; it brought back the lustre of the time they had outlived. It must have been the way she looked also at Mr. Perriam and Lord Eric; above all it contributed in Maisie's mind to a completer view of that satisfied state of the Captain. Our young lady grasped this idea with a quick lifting of the heart; there was a stillness during which her mother flooded her with a wealth of support to the Captain's striking tribute. This stillness remained long enough unbroken to represent that Sir Claude too might but be gasping again under the spell originally strong for him; so that Maisie quite hoped he would at least say something to show a recognition of how charming she could be. What he presently said was: "Are you putting up for the night?" His wife cast grandly about. "Not here--I've come from Dover." Over Maisie's head, at this, they still faced each other. "You spend the night there?" "Yes, I brought some things. I went to the hotel and hastily arranged; then I caught the train that whisked me on here. You see what a day I've had of it." The statement may surprise, but these were really as obliging if not as lucid words as, into her daughter's ears at least, Ida's lips had ever dropped; and there was a quick desire in the daughter that for the hour at any rate they should duly be welcomed as a ground of intercourse. Certainly mamma had a charm which, when turned on, became a large explanation; and the only danger now in an impulse to applaud it would be that of appearing to signalise its rarity. Maisie, however, risked the peril in the geniality of an admission that Ida had indeed had a rush; and she invited Sir Claude to expose himself by agreeing with her that the rush had been even worse than theirs. He appeared to meet this appeal by saying with detachment enough: "You go back there to-night?" "Oh yes--there are plenty of trains." Again Sir Claude hesitated; it would have been hard to say if the child, between them, more connected or divided them. Then he brought out quietly: "It will be late for you to knock about. I'll see you over." "You needn't trouble, thank you. I think you won't deny that I can help myself and that it isn't the first time in my dreadful life that I've somehow managed it." Save for this allusion to her dreadful life they talked there, Maisie noted, as if they were only rather superficial friends; a special effect that she had often wondered at before in the midst of what she supposed to be intimacies. This effect was augmented by the almost casual manner in which her ladyship went on: "I dare say I shall go abroad." "From Dover do you mean, straight?" "How straight I can't say. I'm excessively ill." This for a minute struck Maisie as but a part of the conversation; at the end of which time she became aware that it ought to strike her--though it apparently didn't strike Sir Claude--as a part of something graver. It helped her to twist nearer. "Ill, mamma--really ill?" She regretted her "really" as soon as she had spoken it; but there couldn't be a better proof of her mother's present polish than that Ida showed no gleam of a temper to take it up. She had taken up at other times much tinier things. She only pressed Maisie's head against her bosom and said: "Shockingly, my dear. I must go to that new place." "What new place?" Sir Claude enquired. Ida thought, but couldn't recall it. "Oh 'Chose,' don't you know? --where every one goes. I want some proper treatment. It's all I've ever asked for on earth. But that's not what I came to say." Sir Claude, in silence, folded one by one his newspapers; then he rose and stood whacking the palm of his hand with the bundle. "You'll stop and dine with us?" "Dear no--I can't dine at this sort of hour. I ordered dinner at Dover." Her ladyship's tone in this one instance showed a certain superiority to those conditions in which her daughter had artlessly found Folkestone a paradise. It was yet not so crushing as to nip in the bud the eagerness with which the latter broke out: "But won't you at least have a cup of tea?" Ida kissed her again on the brow. "Thanks, love. I had tea before coming." She raised her eyes to Sir Claude. "She IS sweet!" He made no more answer than if he didn't agree; but Maisie was at ease about that and was still taken up with the joy of this happier pitch of their talk, which put more and more of a meaning into the Captain's version of her ladyship and literally kindled a conjecture that such an admirer might, over there at the other place, be waiting for her to dine. Was the same conjecture in Sir Claude's mind? He partly puzzled her, if it had risen there, by the slight perversity with which he returned to a question that his wife evidently thought she had disposed of. He whacked his hand again with his paper. "I had really much better take you." "And leave Maisie here alone?" Mamma so clearly didn't want it that Maisie leaped at the vision of a Captain who had seen her on from Dover and who, while he waited to take her back, would be hovering just at the same distance at which, in Kensington Gardens, the companion of his walk had herself hovered. Of course, however, instead of breathing any such guess she let Sir Claude reply; all the more that his reply could contribute so much to her own present grandeur. "She won't be alone when she has a maid in attendance." Maisie had never before had so much of a retinue, and she waited also to enjoy the action of it on her ladyship. "You mean the woman you brought from town?" Ida considered. "The person at the house spoke of her in a way that scarcely made her out company for my child." Her tone was that her child had never wanted, in her hands, for prodigious company. But she as distinctly continued to decline Sir Claude's. "Don't be an old goose," she said charmingly. "Let us alone." In front of them on the grass he looked graver than Maisie at all now thought the occasion warranted. "I don't see why you can't say it before me." His wife smoothed one of her daughter's curls. "Say what, dear?" "Why what you came to say." At this Maisie at last interposed: she appealed to Sir Claude. "Do let her say it to me." He looked hard for a moment at his little friend. "How do you know what she may say?" "She must risk it," Ida remarked. "I only want to protect you," he continued to the child. "You want to protect yourself--that's what you mean," his wife replied. "Don't be afraid. I won't touch you." "She won't touch you--she WON'T!" Maisie declared. She felt by this time that she could really answer for it, and something of the emotion with which she had listened to the Captain came back to her. It made her so happy and so secure that she could positively patronise mamma. She did so in the Captain's very language. "She's good, she's good!" she proclaimed. "Oh Lord!"--Sir Claude, at this, let himself go. He appeared to have emitted some sound of derision that was smothered, to Maisie's ears, by her being again embraced by his wife. Ida released her and held her off a little, looking at her with a very queer face. Then the child became aware that their companion had left them and that from the face in question a confirmatory remark had proceeded. "I AM good, love," said her ladyship.

Summary: The cab fare that the Countess gave Maisie turns out to be way too generous. Back at Mrs. Beale's, a debate about what to do with the extra money ensues. Susan Ash, the maid who sometimes looks after Maisie, pockets some. Mrs. Beale insists on giving all of the extra money back. Sir Claude takes Maisie and Susan Ash to Folkestone, a city on England's southeast coast, on the English Channel. Here, Maisie is able to put a lot of the puzzle pieces together. She is getting smarter by the day. Sir Claude has taken her to Folkestone to go along with Mrs. Wix's plan, Maisie thinks. It seems like Mrs. Wix's urging of Sir Claude to do the right thing has finally made a difference. Maisie is sad that Mrs. Wix herself hasn't yet appeared, but she expects to see her again any day. All of a sudden, Maisie's mother, Ida, appears out of nowhere. Ida tells Sir Claude that she wants to talk to Maisie alone.

booksum

en

Summarize: Crystal Palace chairman Steve Parish came under fire from Liverpool supporters on Sunday night after making a comment on Twitter about their celebrations following the FA Cup win. Making reference to his own club’s progress since he took over at Selhurst Park in 2010, Parish tweeted: ‘When we took over we just avoided going to the third tier. Yesterday #LFC beat us. They are celebrating like they won the league #progress’ However, several Liverpool fans on the social media site took offence and accused Parish of being bitter and classless, forcing the Palace owner to defend his statement, insisting he was simply paying tribute to how far the Eagles have come in the past five years. Crystal Palace chairman Steve Parish (left) came under fire from Liverpool supporters on Sunday night. Parish tweeted this about Liverpool's celebrations following the FA Cup win over Palace. Parish was forced to defend his statement to Liverpool fans on Twitter. Parish insists he was simply paying tribute to how far the Eagles have come in the past five years. Fraizer Campbell gave Palace the lead at Selhurst Park on Saturday before goals from Daniel Sturridge and Adam Lallana saw Liverpool come from behind to win 2-1. It means Liverpool qualify for the FA Cup quarter-finals and will be in the draw on Monday night. Palace will switch their focus to the Premier League as they look to avoid being drawn into a relegation battle with the London club sitting 13th - five points above the bottom three. Parish made a comment on Twitter about Liverpool's celebrations following the FA Cup win. Several Liverpool fans on the social media site took offence and accused Parish of being bitter and classless

Summary: Crystal Palace chairman Steve Parish was forced to respond to Liverpool fans after making a comment on Twitter about their celebrations. 'When we took over we just avoided going to the third tier. Yesterday #LFC beat us. They are celebrating like they won the league #progress,' he wrote. Parish insists he was simply paying tribute to how far Palace have come. Liverpool came from behind to beat Palace 2-1 in the FA Cup on Saturday.

cnn_dailymail

en

Summarize: BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention is generally related to the nucleic acid fluid from the kind of chlorella (the chlorella hot water to extract “ Chlorella Extracts; C.E.; C.G. F.”) Making use of the quality of the solute change in which is produced from leaching action, extraction of the essence in the alga contain the nucleic acid is designated as achievement purpose from operation of freezing and thawing. [0003] 2. Description of the Prior Art [0004] Chlorella ( Chlorella sp.) It is a kind of unicellular alga plant in fresh water, it was discovered by the Dutch microbiologist “M W Bei Yonick” in 1890, the size is similar to the red corpuscle of the mankind, it can be observed through the microscope, generally being called “green blood corpuscle”, it is the unicellular plant which does not have maneuverability. According to the classification in botany, the chlorella is spherical monad plankton from the fresh water; mainly it is globose or is elliptical shape. Photosynthesis efficiency of the alga is several dozen faster or more than the other plant, also the chlorophyll which contained more abundant than the other plant. Simultaneously, the natural chlorella having the special separation structure which the other photosynthesis living things does not have, the new born natural alga absorbs nutrition and light energy from underwater and to grow, which it matures to blast cell from 20 to 24 hours and furthermore it will separates to four new cells, such a quick speed propagation, is because where the abundant special growth stimulus hormone is included. [0005] The alga includes 55% or more quantity of the vegetable protein, it is something rejoices by the vegetarians. In the alga it included nutrition component such as the chlorophyll and the vegetable fiber, vitamin A, B, C, D and E, the nicotinic acid, folic acid, calcium, iron, magnesium, and various mineral substance and amino acid etc. Furthermore as for the chlorella it will offer the vegetarian whom easily to lack the vitamin B group and, especially vitamin B12; The vitamin B12 it is the important substance which maintains the health of the red corpuscle and nervous system, it is difficult to find in the general vegetables and fruits, but in 5 grams of green algae it contains 4 milligrams of vitamin B12, in which, green algae nucleic acid included the higher quantity than the other food, an attached table as follows. [0000] TABLE 1 The quantity of nucleic acid is included of various foods Food name Green Sea Bonito Green Salmon Blue algae cucumber Sardine paragraph Salmon Yeast onion Egg extract alga Content 13000 3605 539 907 289 1399 78 86 10600 4600 nucleic DNA&amp;RNA RNA DNA acid mg/100 g [0006] The alga is a kind of alkaline food, long term in take it, can adjust one&#39;s physique, many nutrition component which are included in it can helps adjustment of physiological function, maintains digestion performance, promotion of health and beauty care, strength physical ability, health maintenance and longevity prolongation of life, furthermore may take as nourishment for pre-natal, post-natal or after illness, at present time many countries are doing research of the chlorella, to mass produce it and develop into the products to sale. [0007] In the chlorella includes alga essence ( Chlorella Extracts C.E.; C.G. F.), is also known as the alga growth stimulation factor, it is the essence of the alga. In every 100 grams of alga usually contains only 4˜5 grams of alga concentrated essence fluid. And in the alga concentrated essence fluid includes the Nucleic Acid, Nucleotide, Folic Acid, Niacin, Lysine, Alanine, Glycine, Praline, Glutamic Acid, Aspartic Acid, Punting Ten, Small Molecular Protein, the water soluble Vitamin and Mineral Substance, is a similar material and ingredient with the animal placenta element. Therefore, it is also named as plant placenta element; and it may stimulate the alga to grow faster. [0008] Generally during the process of concentration and extraction of alga essence, this concentration procedural is a key method to increase the application scope and the commercial value of the alga. [0009] The present technology of concentration procedural is to heat up the alga directly, and evaporate part of moisture to achieve the goal of concentration, and it may be used with the related auxiliary depressurize equipment to increase the efficiency of this part of process. [0010] Due to the long heating process of alga essence fluid, the destruction of the complicated nutrition ingredient is unavoidable. There is unpredictable reaction between various ingredients during the process. Furthermore the long heating process reduces the luster and flavor of the alga concentration fluid. The fact that to solve the fault of the above-mentioned traditional alga essence fluid concentration method and to offer the concentrated method to extracting the nucleic acid include within alga is immediate urgent matter. SUMMARY OF THE INVENTION [0011] According to the background of this application, an alga essence nucleic acid fluid concentration method is developed and disclosed. [0012] The main purpose of this invention is to solve the fault from above mentioned traditional alga essence fluid concentration method. It is to offer the concentration method to extract its content of nucleic acid from alga, by applying the leaching function will create the characteristic of solute transformation phenomena, the refrigeration method will achieve it purpose of extract the nucleic acid ingredient from the concentrated alga essence fluid. This invention produces the alga nucleic acid fluid with it color and it luster leaning green with the pure natural flavor, and sweet taste. [0013] In order to achieve above goal, by applying the method with this invention, from chlorella to extract nucleic acid, include following; First, the formation of the chlorella mixture fluid, which made possible by at least intermix with the raw materials of chlorella and to infiltration with the mixed liquid to form the chlorella mixture fluid. Next, process of extraction is proceed, to heats the chlorella mixed liquid evenly at approximately from between 90 degree centigrade up to 120 degrees, and to maintain it for a period of time, then cooling the heated chlorella mixed fluid, it forms the chlorella extracted fluid. Continuously, the dregs and the suspended matter in the chlorella extract are removed and only the chlorophyll is left to form the prototype product of alga essence nucleic acid. Lastly, at least once the leaching process is done, depending on this leaching action to freezes and at the same time nature melting the prototype product of the alga essence nucleic acid fluid, and to complete the concentrated alga essence nucleic acid fluid. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIG. 1 is a flow chart of the alga essence nucleic acid fluid concentrated production method according to the first embodiment of the present invention; [0015] FIG. 2 is a flow chart of the alga essence nucleic acid fluid concentrated production method according to the second embodiment of the present invention; [0016] FIG. 3 is a flow chart of the alga essence nucleic acid fluid concentrated production method according to the third embodiment of the present invention; [0017] FIG. 4 is a operating procedure of the alga essence nucleic acid fluid concentrated production method according to the present invention; and [0018] FIG. 5 is during the concentration stages where collected the leaching solution at the time of each stage the related figure of the solution include it quantity and the time according to the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS [0019] What is probed into the invention is an alga essence nucleic acid fluid concentration method. Detail descriptions of the structure and elements will be provided as followed in order to make the invention thoroughly understood. The application of the invention is not confined to specific details familiar to those who are skilled in the art. On the other hand, the common structures and elements that are known to everyone are not described in details to avoid unnecessary limits of the invention. Some preferred embodiments of the present invention will now be described in greater detail as followed. However, it should be recognized that the present invention can be practiced in a wide range of other embodiments besides those explicitly described, that is, this invention can also be applied extensively to other embodiments, and the scope of the present invention is expressly not limited except as specified in the accompanying claims. [0020] The invention is to provide an alga essence nucleic acid fluid concentration method, to eliminate traditional direct heating method, but to extract the concentration by carry out the freezes, and defrost operation, this method may further achieve the simplification and maintain establishing of the production equipment, and then reduces the production cost, the concentrates extract of the chlorella essence nucleic acid fluid by this invention, may maintain its sweet flavor, and to improve the luster by join the tradition facture to obtained the concentrated chlorella essence. [0021] The alga nucleic acid fluid referred in this invention is a kind of chlorella extracted from hot water concentration, the ingredient of this extracts are the protein, the polysaccharide, the Vitamin and the mineral substance etc the aqueous solution, long-term edible has the function of adjust one&#39;s physique, and to improve health. Moreover, in the chlorella nucleic acid fluid&#39;s special nutrition ingredient the alga essence accelerate growth factor (CGF) has many biological activity function, therefore the alga hot water extraction receives special attention, therefore, to judge the chlorella nucleic acid fluid quality is fit or unfit, usually rests on its nature quality (luster) and takes it CGF quantity (density). [0022] To determinate its content of chlorella CGF and its legal expression system act according to Taiwan Commodity Inspection Bureau the CNS4202 and N5134 stipulation method. Naturally raised alga its CGF content majorities are situated between 1.3˜2.5; it will be different along with the different kind of plants and growthraise condition. The alga nucleic acid fluid&#39;s CGF content by the nature most density indicated the absorbance quality under light wave length 260 nm, for example OD200 or OD400. (OD: Optical Density). [0023] As shown in FIG. 1, a first embodiment of the present application discloses an alga essence nucleic acid fluid concentrated production method 100, comprising: forming a Chlorella mixed fluid 110, performing a extraction process 120, removing the dregs and the suspended matter and keeping the Chlorophyll in the Chlorella extract 130, and performing a leaching process 140. Depending on above description, forming a Chlorella mixed fluid 110, wherein the Chlorella mixed fluid is mixed and dampened by at least one of Chlorella raw materials and a mixed fluid. As for mixed liquid it can be water, secondary water, deionizer water or all nonpoisonous solvents etc. The above-mentioned the OD (Optical Density) value of the Chlorella raw materials is between 1.5 and 2.5. The Best, the OD (Optical Density) value of the Chlorella raw materials is approximately 2.3. [0024] Continuously, an extraction process 120 is performed to heat the Chlorella mixed fluid evenly with first temperature and maintaining fixed time, and the heated Chlorella mixed fluid is to cooling down with the second temperature to form a Chlorella extract fluid. The above-mentioned first temperature is between 80 degree centigrade and 120 degree centigrade. As for being better, the above-mentioned first temperature is between 80 degrees centigrade to 95 degree centigrade. The above-mentioned chlorella mixed fluid is heated evenly at the first temperature, particular time is maintained, the particular time is between 30˜40 minutes. The above-mentioned second temperature is between 20 degree centigrade and 30 degree centigrade. [0025] After the completing the extraction process 120, removing the dregs and the suspended matter and keeping the Chlorophyll in the Chlorella extract 130 to form an alga essence nucleic acid prototype product. Finally, a leaching process 140 is performed, the alga essence nucleic acid prototype product is frozen and then thawed naturally by leaching action to form a concentrated alga essence nucleic acid fluid. The above-mentioned concentrated alga essence nucleic acid fluid is more than or equal to 400. To repeat it at least 3 times as for relatively better ones with the above-mentioned leaching process 140. [0026] As shown in FIG. 2, a second embodiment of the present application discloses an alga essence nucleic acid fluid concentrated production method 200, comprising: forming a Chlorella mixed fluid 210, performing a extraction process 220, removing the dregs and the suspended matter and keeping the Chlorophyll in the Chlorella extract 230, performing a frozen process 240, and performing a thawing process 250. Depending on above description, forming a Chlorella mixed fluid 210, wherein the Chlorella mixed fluid is mixed and dampened by at least one of Chlorella raw materials and a mixed fluid. Then, an extraction process 220 is performed to heat the Chlorella mixed fluid evenly with first temperature and maintaining fixed time, and the heated Chlorella mixed fluid is to cooling down with the second temperature to form a Chlorella extract fluid. Next, removing the dregs and the suspended matter and keeping the Chlorophyll in the Chlorella extract 230 to form an alga essence nucleic acid prototype product. Next, a frozen process 240 is performed, the alga essence nucleic acid prototype product is frozen below freezing point of zero centigrade. Finally, a thawing process 250 is performed, the frozen alga essence nucleic acid fluid prototype product is naturally thawed approximately between 24 centigrade degree to 28 degrees to form said concentrated alga essence nucleic acid fluid. The above-mentioned chlorella raw materials OD value, first temperature, maintain a particular time at first temperature, second temperature, essence nucleic acid prototype product OD value and concentrated alga essence nucleic acid fluid OD value are same condition with the first embodiment of the present application. [0027] As shown in FIG. 3, a third embodiment of the present application discloses an alga essence nucleic acid fluid concentrated production method 300, comprising: forming a Chlorella mixed fluid 310, performing a extraction process 320, removing the dregs and the suspended matter and keeping the Chlorophyll in the Chlorella extract 330, performing a first frozen process 340 A, performing a first thawing process 350 A, performing a second frozen process 340 B, and performing a second thawing process 350 B. Depending on above description, forming a Chlorella mixed fluid 310, wherein the Chlorella mixed fluid is mixed and dampened by at least one of Chlorella raw materials and a mixed fluid. Then, an extraction process 320 is performed to heat the Chlorella mixed fluid evenly with first temperature and maintaining fixed time, and the heated Chlorella mixed fluid is to cooling down with the second temperature to form a Chlorella extract fluid. Next, removing the dregs and the suspended matter and keeping the Chlorophyll in the Chlorella extract 330 to form an alga essence nucleic acid prototype product. Next, a first frozen process 340 A is performed, the alga essence nucleic acid prototype product is frozen below freezing point of zero centigrade. Next, a first thawing process 350 A is performed, the frozen alga essence nucleic acid fluid prototype product is naturally thawed approximately between 24 centigrade degree to 28 degrees. Next, a second thawing process 340 B is performed, the defrost alga essence nucleic acid fluid prototype product to freeze it again below the freezing point of the centigrade. Finally, a second thawing process 350 B is performed, the frozen alga essence nucleic acid fluid prototype product is naturally thawed approximately between 24 centigrade degree to 28 degrees to form the concentrated alga essence nucleic acid fluid. The above-mentioned chlorella raw materials OD value, first temperature, maintain a particular time at first temperature, second temperature, essence nucleic acid prototype product OD value and concentrated alga essence nucleic acid fluid OD value are same condition with the first embodiment of the present application. The above-mentioned frozen process ( 340 A; 340 B) and with thawing process ( 350 A; 350 B) in between it can proceed to repeat several time. EXAMPLE 1 [0028] With regular chlorella powder raw material ( Chlorella sp. ) and adds on 1:8˜10 time (w/w) water to infiltration mix with the alga raw material powder, to evenly add heating process until reach to 90˜100° C., and maintains 50˜60 minutes then carries on to cool downit to the normal temperature (room temperature), will obtain the mixture and after the separation of the solid material (wreckage of algae body), and after the steps of extracting suspension to keep the chlorophyll etc, it clarifies the amber leaning green liquid, namely the chlorella extracted liquid include the alga essence accelerate growth factor (CGF), I.e. the alga essence of nucleic acid fluid has not concentrated is the initial prototypeend product (the usual OD value situated between 130˜170), placeput this has not yet concentrated alga essence nucleic acid fluid of the prototype initial end product into a regular vessel, store it in the refrigerator to freeze it, this frozen prototypeinitial end product of un-concentrated alga essence nucleic acid fluid, take it out from the refrigerator and causes it to dissolve under the room temperature, because of the dissolved state continues to carry on (coexistent and mix with a solid condition) has the leaching function, After gathering this dissolution, the liquid state part (to be called leaching fluid or drop filtrate liquates), namely the concentrated alga essence of nucleic acid fluid; the un-dissolved portion content extremely few solute; may get rid of it or to redo it, to repeat the above step. [0029] So-called “the leaching function” is refers to the ice in the initial dissolve liquid state portion, the solute content is much higher than average content in the ice solute; the ice dissolves the water is the function to cause the chemical composition to have the migration phenomenon in the ice. [0030] As show in FIG. 4, to operate the above steps repeatedly altogether by 3 times, at the prototype product of alga essence nucleic acid fluid it OD value is 170, it may be concentrates the OD value over above 400 of the green alga nucleic acid fluid. [0031] In which, takes the raw material of chlorella to target for OD value 1.5, after depends on above step to manufacture, obtains the OD130 alga essence nucleic acid fluid at the prototype initial end product, after carry out the first freeze, defrosting, and the collection procedure, to obtains the density is above OD170; if this raw material chlorella OD value target is 2.3, after depend on above manufacture step, obtains the initial prototypeend product of the alga essence nucleic acid fluid at OD170, after carries on again to freeze it, defrosting, withand the collection procedure, the final product density of the OD value is above 230, repeatedly operates the above freezing, the dissolvinged and collection steps reaches to 3 times, the alga essence nucleic acid fluid may result in the OD value is above 400. [0032] The concentration method apply to the production line operation has the extremely good flexibility, and ease to operate, the prototype initial end product of this alga essence nucleic acid fluid and place itat put in a regular 25˜35 liter barrel, place it in the refrigerator to freezecause it freeze, and take out this frozen prototype initial end product of alga essence nucleic acid fluid from the refrigerator and inverts it, causes it to dissolve naturally under the room temperature, continues to carry on with the dissolved state to forms the liquid-solid coexistent condition, to produce the leaching function, and then to collecting this leaching fluid (or calls drop filtrate), namely alga essence nucleic acid fluid from this invention. [0033] As show in FIG. 5, when carries on the concentration step again with this invention, is also works as the leaching function that to produce, continually dissolves in the process, during various stages the collection of leaching fluid (or called drop filtrate) is the solute content and the time relational graph. EXAMPLE 2 [0034] The Food Analysis Center of Japan in Aug. 6, 2007 (Heisei 19) analyzed assay with result report based on the concentrated alga essence nucleic acid fluid report send in for exam arranged by the applicant, as show in below: [0000] TABLE 1 Analytical Investigation Result Detection Assay Items Result limit Note Method Moisture 97.4 g/100 g Normal pressure heating drying method Protein 1.6 g/100 g 1 Kjeldahl method Lipid 0.1 g/100 g Acid resolution Under Ash 0.4 g/100 g Directly ashing method Carbohydrate 0.6 g/100 g 2 Energy 9 kcal/100 g 3 Note 1. Nitrogen, protein conversion factor: 6.25 Note 2. Formula: 100 − (Moisture + Protein + Lipid + Ash) Note 3. Nutrition indicatory standard [Heisei 15(2003) Public Welfare Ministry of Labor Public Notice No. 176] About energy conversion factor: Protein, 4; Lipid, 9; Carbohydrate, 4 [0000] TABLE 2 Analytical Investigation Result Detection Assay Items Result limit Note Method Moisture 97.4 g/100 g Normal pressure heating drying method Protein 1.6 g/100 g 1 Kjeldahl method Lipid 0.1 g/100 g Acid resolution Under Ash 0.4 g/100 g Directly ashing method Sugar 0.4 g/100 g 2 Dietary Fiber 0.2 g/100 g Enzyme - Gravimetric method Energy 8 kcal/100 g 3 Note 1. Nitrogen, protein conversion factor: 6.25 Note 2. Nutrition indicatory standard [Heisei 15(2003) Public Welfare Ministry of Labor Public Notice No. 176] About Formula: 100 − (Moisture + Protein + Lipid + Dietary Fiber) Note 3. Nutrition indicatory standard [Heisei 15(2003) Public Welfare Ministry of Labor Public Notice No. 176] About energy conversion factor: Protein, 4; Lipid, 9; Suger, 4; Dietary fiber, 2 [0000] TABLE 3 Analytical Investigation Result Assay Items Result Detection limit Note Method Sodium 2.7 mg/100 g Atom extinction brightness method ICP Iron 0.44 mg/100 g luminous analysis Calcium 9.9 mg/100 g ICP luminous analysis Kalium 117 mg/100 g Atom extinction brightness method ICP Magnesium 20.5 mg/100 g luminous analysis Zinc 0.14 mg/100 g ICP luminous analysis Selenium Not detect 5 μg/100 g Fluorescent brightness method Entire chrome Not detect 0.05 mg/100 g ICP luminous analysis Vitamin A(Retinol equivalence) 3 μg/100 g 1 α-Carotin 8 μg/100 g High speed liquid chromatograph method β-Carotin 27 μg/100 g High speed liquid chromatograph method Thiamine (Vitamin B 1 ) 0.03 mg/100 g 2 High speed liquid chromatograph method Ribo Flavin (Vitamin B 2 ) 0.42 mg/100 g High speed liquid chromatograph method VitaminB6 0.13 mg/100 g 3 Bio assay method VitaminB 12 1.1 μg/100 g 4 Bio assay method Ascorbic acid(Total Vitamin C) Not detect 1 mg/100 g 5 High speed liquid chromatograph method Vitamin D Not detect 0.1 μg/100 g High speed liquid chromatograph method Vitamin D (International unit) — Vitamin E Not detect High speed liquid chromatograph method Folic acid 82 μg/100 g 0.1 mg/100 g 6 Bio assay method —: Because it does not detect, it does not calculate. Induction embody it measured Note 1. α-Carotin 24 μg and β-Carotin 12 μg, respectively take the retinol equivalence of 1 μg Note 2. As a thiamine salt Note 3. Fungus strain used: Saccharomyces cerevisiae ( S. uvarum ) ATCC 9080 Note 4. Fungus strain used: Lactobacillus delbrueckii subsp. Lactis ( L. leichmannii ) ATCC 7830 Note 5. Apply hydrazine and measured after changes into induction embody. Note 6. Fungus strain used: Lactobacillus rhamnosus ( L. casei ) ATCC 7469 [0000] TABLE 4 Analytical Investigation Result Assay Items Result Detection limit Note Method Punting ten 0.19 mg/100 g 1 Bio assay method Biotin 5.6 μg/100 g 1 Bio assay method Inositol 5 mg/100 g 2 Bio assay method Niacin 3.02 mg/100 g 3 Niacin(Nicotine suitable quantity) 2.85 mg/100 g 1 Bio assay method Toriputohuan 10 mg/100 g High speed liquid chromatograph method Choline Not detect 0.03 g/100 g 4 Dextro glucose 0.22 g/100 g 5 High speed liquid chromatograph method Arabinose Not detect 0.02 g/100 g 5 High speed liquid chromatograph method Xylose Not detect 0.02 g/100 g 5 High speed liquid chromatograph method Rhamnose 0.04 g/100 g 5 High speed liquid chromatograph method Mannose Not detect 0.02 g/100 g 5 High speed liquid chromatograph method Galactose 0.05 g/100 g 5 High speed liquid chromatograph method Lysine 66 mg/100 g Amino acid automatic analysis Alanine 112 mg/100 g Amino acid automatic analysis Glycine 69 mg/100 g Amino acid automatic analysis Proline 57 mg/100 g Amino acid automatic analysis Glutamic acid 169 mg/100 g Amino acid automatic analysis Ceric 45 mg/100 g Amino acid automatic analysis Aspartic acid 99 mg/100 g Amino acid automatic analysis Note 1. Fungus strain used: Lactobacillus plantarum ATCC 8014 Note 2. Fungus strain used: Saccharomyces cerevisiae ( S. uvarum ) ATCC 9080 Note 3. Niacin(Nicotine suitable quantity) and 1/60 of total quantity was made by niacin equivalent Note 4. Based on salt settling method. The room temperature mixes the approximately 1 hour later Note 5. Acid adding water to disassembled it and then measured. Hydrolysis condition: 72% of Sulfuric acid, after agitating it for one hour at room temperature, 4% sulfuric acid, put in autoclave (121° C.), one hour [0000] TABLE 5 Analytical Investigation Result Detection Assay Items Result limit Note Method Total 3.0 mg/100 g Extinction brightness Chlorophyll method (Visible) Chlorophyll a 2.7 mg/100 g Chlorophyll b 0.3 mg/100 g Cobalt Not detect 0.05 ppm Atom extinction brightness method Germanium Not detect 1 ppm ICP luminous analysis [0035] Other modifications and variations are possibly developed in light of the above demonstrations. It is therefore to be understood that within the scope of the appended claims the present invention can be practiced otherwise than as specifically described herein. Although specific embodiments have been illustrated and described herein, it is obvious to those skilled in the art that many modifications of the present invention may be made without departing from what is intended to be limited solely by the appended claims.

Summary: The present invention discloses an alga essence nucleic acid fluid concentration method, to apply the leaching function principle with the operation of freezes defreeze to achieve the goal of extract nucleic acid from the concentrated chlorella. Because of above operation steps with this invention, it will obtain better density of alga nucleic acid fluid. This invention is also to prepare above mentioned particular method of concentrate the alga nucleic acid and produce the alga nucleic acid fluid. The alga nucleic acid fluid may maintain it sweet flavor, and the alga nucleic acid fluid assumes the amber leaning to green color, it is sweet flavor and purely natural taste.

big_patent

en

Summarize: FIELD OF THE INVENTION The invention concerns a wrapping implement for the enveloping of a cylindrical bale with a run of enveloping material. BACKGROUND OF THE INVENTION DE 196 54 982 A1 discloses a wrapping arrangement for a small bale of grass or straw that can be attached to a vehicle and wraps small bales, having either a circular or a rectangular cross section, with foil. For this purpose, the small bales are lined up in the form of a queue, aligned with each other with their end faces, but may also be wrapped individually. It is not apparent how the small bales are brought into this position and it seems that these small bales are of a small diameter. DE 40 21 307 A1 reveals a large baler with a comparable wrapping arrangement for slab-shaped bales that are conducted to the wrapping arrangement in the same way and wrapped by it. Another embodiment of a large round baler combined with a wrapping arrangement is revealed by U.S. Pat No. 5,822,967 A1, in which indeed two rolls with wrapping material rotate about a vertical axis and the cylindrical bale can be rotated about a horizontal axis during the wrapping process. An arm to take up the wrapping arrangement extends considerably beyond the large round baler, even in a non-operating position, which is not conducive to permitting good maneuverability. Furthermore, the rotating arms with the rolls must be brought to the side in a non-operating position during each loading and unloading process of the large round baler. EP 1 210 861 A2 is a large round baler with a wrapping arrangement attached at the rear that can be removed for a non-operating condition. During operation, however, the combination provides a great length of the entire vehicle train. EP 1 050 207 A2 discloses a bale wrapping arrangement, in the form of a trailer, that can be coupled behind an agricultural tractor, and raises cylindrical bales lying on the ground by means of a fork to a wrapping table. SUMMARY OF THE INVENTION The problem underlying the invention is seen in the fact that the combination of a known large round baler with a wrapping arrangement that is also known always leads to an extended vehicle train. According to the present invention, there is provided a novel bale wrapping implement for being mounted to large round baler. An object of the invention is to provide a bale wrapping implement which is designed such that an unduly long combined implement does not result when it is mounted to the rear of a large round baler. More specifically, there is provided a bale wrapping implement, wherein the guidance ring for supporting the wrapping material supply roll or rolls is oriented in a near vertical plane. In this way, the wrapping implement can be held relatively short in the extent of its length and is appropriate for its operation as an attachment to a large round baler, as well as a free-standing implement, that is either equipped with a loading arrangement or is loaded by means of a front loader or the like. The pulley or the roll can be configured as smooth, with conveyer bridges or configured in some other manner in order to attain the rotating movement of the cylindrical bale or to increase it. The run of enveloping material is predominantly a stretch foil that is appropriate for improving the silage process. It is sufficient if either the pulley or the roll is driven in order to bring the cylindrical bale into rotation where the rotational speed is preferably variable. The pulley and the roll may be arranged together on the carrier; however, the roll can be attached independently of the carrier, for example, in the guidance ring or provided in a conveyor associated with the wrapping implement. If the carrier can be repositioned relative to the guidance ring, it is possible to bring the cylindrical bale into various positions relative to it. This also makes it possible to deposit the cylindrical bale on the ground or even to lift it up. The wrapping implement can be positioned in a stationary location on the ground in order to remain in a non-operating position or to be operated. In the non-operating position, the carrier can also be used for retaining. If operation is to be performed with the guidance ring set down, or the guidance ring is to be stabilized, it is useful to provide another support. The support may selectively be adjustable in length, in a known manner, and/or it may be repositioned by outside force. The use of a buttress on the carrier provides the advantage that the cylindrical bale does not fall off the carrier during a repositioning of the carrier, for example, for the unloading of the cylindrical bale, which otherwise could possibly occur if the spacing between the pulley and the roll should change. A circulating movement of the wrapping material carrier along a circular path can be generated by the guidance ring if it is provided with a rotating ring that can be rotated by a motor in a slotted guidance housing. The wrapping material carrier conventionally is a spool, supported in bearings, free to rotate, on which hundreds or thousands of meters of stretch foil or the like are wrapped. For the guidance ring, one or more wrapping material carriers may be provided. The slot in the guidance housing may be provided on a radially inner, as well as a radially outer, side of the guidance housing, that is, also on a forward or a rear end face. By the same token, an Omega-shaped or Hat-shaped profile could be used, the essential thing is only that the ring is guided and can accommodate at least one wrapping material carrier. On the other hand, the guidance housing can also be enclosed by a wrapping material carrier that then moves as on a rail or along a pipe, rod or the like. The movement can be produced by means of a rope pull, a chain or by means of a friction drive or a gear motor. The velocity of the wrapping material carrier may be constant as well as variable, and may conform, for example, to the number of wrapping material carriers used. In order for the cylindrical bale to be held, on the one hand, by a carrier, and on the other hand, be enclosed, trouble free, by enveloping material, the carrier is equipped with a frame or configured as a frame, that leaves an interior space through which the wrapping material carrier can be moved during the wrapping operation. Accordingly, the components of the frame, such as transverse and lengthwise struts, are located outside the path of movement of the wrapping material carriers. The frame may always be configured in that way or it may be arranged in that way only for the wrapping operation. The frame is not necessarily a closed structure; a fork-shaped or other configuration can also be considered, as long as it guarantees that the cylindrical bale is carried safely. If the guidance ring is to be transportable, and if necessary, can be attached to another implement, this can be accomplished basically as a permanent attachment or be difficult to remove. However, assembly time can be saved and flexibility can be increased if the guidance ring or quick connection devices are provided, such as hooks, couplings, pin connections, etc. Elements that are advantageous and reliable include, for example, elements that are used for the attachment of tools to front loaders or three-point implement hitches. Analogously, the wrapping implement could also be attached to a front loader and be easily transported. Similarly, the connection at the outlet of a large round baler or another type of baler is possible that produces, for example, slab-shaped bales. The cylindrical bale or basically any bale can be deposited on various surfaces, for example, its circumferential surface or one of its end faces. The deposit on an end face has the advantage that the cylindrical bale does not roll away when deposited on a slope. Such a deposit can be attained if the carrier is attached to the guidance ring at its circumferential direction so as to be repositioned, and thereby changes the inclination of the longitudinal center line of the cylindrical bale during the deposit process, until it is tilted onto its end face. In an alternative way, the bale can be deposited on its end face if the carrier can be adjusted vertically relative to the guidance ring, in particular pivoted. Particularly with cylindrical bales with a small diameter, an ejection sleeve provides the assurance that the cylindrical bale does not remain in the interval between the pulley and the roll when the carrier is moved for the deposit of the cylindrical bale. This ejection sleeve is brought into contact with the cylindrical bale as soon as the cylindrical bale is to leave its wrapping position. This can be performed at that time and to that degree that the carrier is repositioned, that is, it depends on this. It can also be performed by means of a pusher or the like that can act upon the cylindrical bale under the control of outside force. An actuating arrangement may be flexible if it is supported, for example, by a gas spring or a mechanical spring or by a pressurized medium such as oil or air escapes while being throttled by valves, etc. This flexibility has the advantage that the impact of an oncoming cylindrical bale is damped and that either a free-standing wrapping implement remains standing safely or that an attached wrapping implement does not transmit excessive forces to the connections. If the rotational speed of the pulley or the roll can be varied, either manually or automatically, the number and the overlap of the layers on the cylindrical bale can be varied or maintained at a constant value in case of a failure of a wrapping material carrier. Difficult conditions at the beginning or the end of the wrapping process can also be considered. The repositioning of the location of the pulley makes it possible to position the bale that is to be wrapped correctly on the carrier, as well as to assure that it lies on the carrier at the correct angle and thereby, safely. The repositioning is performed preferably by means of stepper motors and automatically, but can be performed in a simple version by means of a perforated rail or perforated plate or by means of a clamping arrangement. BRIEF DESCRIPTION OF THE DRAWINGS The drawing is several embodiments of the invention that shall be described in greater detail in the following. FIG. 1 is a schematic left side view of a combination of a large round baler and a wrapping implement according to a first embodiment with a conveyor. FIG. 2 is a rear view of the wrapping implement of the combination according FIG. 1. FIG. 3 is a schematic left side view of the large round baler without the wrapping implement. FIG. 4 is a schematic left side view of the wrapping implement detached from the large round baler. FIG. 5 is the combination according to FIG. 1 during the transfer of a cylindrical bale from the large round baler to the wrapping implement. FIG. 6 is the combination according to FIG. 1 during the wrapping of the cylindrical bale in the wrapping implement. FIG. 7 is the combination according to FIG. 1 during the ejection of a cylindrical bale from the wrapping implement. FIG. 8 is the large round baler of the combination according to FIG. 1 with the conveyor and without the wrapping implement during the ejection of a cylindrical bale. FIG. 9 is a schematic left side view of a combination of a large round baler with a wrapping implement according to a second embodiment with a conveyor. FIG. 10 is the combination according to FIG. 9 in a transfer condition of the conveyor. FIG. 11 is the combination according to FIG. 9 during a wrapping operation. FIG. 12 is the combination according to FIG. 9 during the unloading of a cylindrical bale. FIG. 13 is a schematic left side view of a combination of a large round baler with a wrapping implement according to a third embodiment with a conveyor and a pivoted carrier. FIG. 14 is a rear view of the wrapping implement according to FIG. 13. FIG. 15 is a plan view of the wrapping implement of the combination according to FIG. 13. FIG. 16 is a side view of the combination according to FIG. 13 during the delivery of a cylindrical bale. FIG. 17 is a rear view of the wrapping implement according to FIG. 13 during the delivery of the cylindrical bale. FIG. 18 is a schematic side view of a combination of a large round baler with a wrapping implement according to a fourth embodiment with a conveyor and a pivoted carrier. FIG. 19 is a rear view of the wrapping implement of the combination according to FIG. 18 during the delivery of the cylindrical bale. DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to FIG. 1, there is shown a combination 10 of a large round baler 12 and a wrapping implement 14. The large round baler 10 is preferably configured according to U.S. patent application Ser. No. 10/281,475, filed 25 Oct. 2002, whose disclosure is hereby incorporated herein. Basically the large round baler 10 may, however, be of any conventional configuration with a fixed or a variable baling chamber and may be applied in agriculture as well as in industrial operation. For this purpose, the large round baler 10 is provided with a frame 16, a chassis 18, baling elements 20, a baling chamber 22, a supply arrangement 24, and a conveyor 26. The frame 16 is formed by a welded and/or bolted assembly that ends at the front in a towbar 28, is connected at the bottom with the chassis 18, and is provided at the rear with upper and lower connecting points 30, 32. The frame 16 includes side walls 34 that are not described in any further detail. The side walls 34 form opposite sides of a baling chamber 22. In the region of the upper connecting point 30 on each side of the frame 16, there is a hook element 76 that opens upwardly and is pivotally mounted for being repositioned by an actuating arrangement 78. The chassis 18 can be configured in a tandem axle configuration with two axles 36, as shown, or it may be provided with only a single sprung or unsprung axle 36 that carries a wheel on each side. The chassis 18 is located underneath the baling chamber 22 and is offset to the rear of it. In the embodiment shown, the baling elements 20 are configured as endless, flexible tensioning means, for example, as belts, bands, bar chains, etc., and generally surround, and thereby define, the circumference of the baling chamber 22. The baling elements 20 are conducted over rolls 38, whose position is in part rigid and in part movable. The use of rolls 38 that are movable in their attachment results in a baling chamber 22 of variable size. The movable rolls 38 are engaged by pivoted arms, in a way not shown, and repositioned as a function of operating condition. In the forward region of the baling chamber 22 facing the supply arrangement 24, the baling elements 20 leave an inlet 40, through which crop to be baled can be conveyed into the baling chamber 22. In the rear lower region of the baling chamber 22, an outlet 42 is formed when the corresponding rolls 38 are raised with the baling elements 20 and thereby open the baling chamber 22. The baling chamber 22 is variable in its size, since the baling elements 20 are flexible. Instead of that, a baling chamber 22 could be formed whose size is invariable and that is surrounded, for example, by rolls or bands on axes that are unchangeable in their location. The baling chamber 22 is oriented in such a way that a cylindrical bale 44 contained in it rotates about a horizontal, central, cylindrical axis that extends transverse to the direction of operation. In this embodiment, the supply arrangement 24 contains a take-up device 46 in the form of a so-called pick-up that is followed by a conveying arrangement 48, if necessary configured as a cutting arrangement, in order to grasp crop to be baled that is lying on the ground and convey it through the inlet 40 into the baling chamber 22, where it is formed into a cylindrical bale 44. As is illustrated in FIG. 5, the conveyor 26 is provided with a frame 50, a conveying table 52, a guide arrangement 54, a first actuating arrangement 56, and a second actuating arrangement 58. The assignment of the conveyor 26 consists of taking up a cylindrical bale 44 delivered by the baling chamber 22 and to either convey it to the wrapping arrangement 14 or to deposit it on the ground. The frame 50 is configured as a two-piece component and includes a slide 60, with rolls 62, that is connected in a bearing 64 with a pivoting frame 66 so as to pivot vertically. The slide 60 with its rolls 62 is engaged in the guide arrangement 54 and can be moved inclined to the ground in the direction of operation. The rolls 62 are attached to the slide 60 in such a way that they are still retained in the guide arrangement 54 in each end position of the slide 60. In a simplified configuration, guide shoes or the like may also be provided in place of the rolls 62. The conveying table 52 contains at least two rolls 68 that are spaced from each other at a distance of less than the diameter of a completed cylindrical bale 44 and are mounted, free to rotate, on the pivoting frame 66. In the preferred embodiment, an endless, flexible band 70 is slung over the rolls 68 that bridges the spacing between the rolls 68 and sags to a small amount. Fundamentally, such a band 70 is not required, and in its place, further rolls could be provided in case that the spacing is then to be bridged. An axis of rotation 72 of the roll 68 facing the wrapping implement 14 is used simultaneously as a bearing 64. On the side of the pivoting frame 66 facing away from the wrapping implement 14, a further roll 74 is provided alongside the roll 68 that is remote from the axis of rotation 72. The roll 74 may be provided with a smaller diameter than the remaining rolls 68 and is offset relative to a plane through the axes of rotation of the rolls 68 so that a trough shape results in the shape of the conveying table 52. The conveying table 52 can occupy a lower end position inclined forward and downward (see FIG. 5 in dashed lines), in which the cylindrical bale 44 is accepted from the baling chamber 22, and an upper end position inclined upward and to the rear (see FIG. 5 in solid lines), in which it can deliver the cylindrical bale 44 into the wrapping implement 14. The rolls 68 may be configured as free to rotate or may be driven. In the preferred embodiment, the rear roll 38 is driven, for example, by means of a hydraulic motor. In the simplest case, the guidance arrangement 54 consists of two U-shaped rails extending parallel to the longitudinal center plane of the large round baler 12 that can be bolted to the frame 16 of the large round baler 12 and that are open toward its longitudinal center plane. The guidance arrangement 54 extends at a small inclination of, for example, 30° from the ground upward and to the rear, and extends above the rear axle 36. The interior of the guidance arrangement 54 is appropriate to take up the rolls 38, free to rotate. The guidance arrangement 54 is configured in such a way that it still safely guides the slide 60 even in the operating condition according to FIG. 8. For this purpose, one-piece rails could be used, that for example, can be tilted about a horizontal bearing by means of a further actuating arrangement, not shown, if this should be necessary. In the preferred embodiment, the rails are configured, in particular, as can be seen in FIG. 8. According to that, the rails are two-piece components and contain a joint 116 above the rear axle 36 that connects both sections with each other so as to pivot vertically. If necessary, a section developing during the pivoting can be bridged by means of a curved piece. In this way it is possible that the slide 60 again slides downward after the cylindrical bale 44 is raised over the rear axle 36 and deposits the cylindrical bale 44 gently on the ground. The first actuating arrangement 56 is configured as a double-acting hydraulic motor having its cylinder end pivotally connected to the frame 16 of the large round baler 12 and having its rod end pivotally connected to the slide 60. The orientation of the first actuating arrangement 56 is selected in such a way that its change in length produces a repositioning of the slide 60 in the guidance arrangement 54. The first actuating arrangement 56 is controlled by means of electromagnetically controlled valves preferably from an on-board computer or manually. The stroke of the first actuating arrangement 56 is sufficient to move the slide 60 in both directions over the entire length of the guidance arrangement 54. The second actuating arrangement 58 is also configured as a hydraulic motor, however, it is single-acting. The second actuating arrangement 58 has its head end pivotally to the slide 60 and has its rod end pivotally connected to the pivoting frame 66 of the conveying table 52. The second actuating arrangement 58 is used to pivot the pivoting frame 66 out of its lower end position, if necessary with the cylindrical bale 44 lying on it, into its upper end position, and to lower it under control on the basis of the force of gravity. The second actuating arrangement 58 is also controlled electrically and preferably by means of an on-board computer. Fundamentally, the two actuating arrangements 56, 58 could also be operated mechanically, electrically or pneumatically. At this state of the description, it will be appreciated that the conveying table 52 could perform without the attached wrapping implement 14. According to FIG. 8, only the large round baler 12 is provided, which holds the conveying table 52 at the outlet side of the baling chamber 22. As soon as the baling elements 20 and the rolls 38 carrying them are raised in order to open the baling chamber 22, the cylindrical bale 44 can leave it and fall onto the conveying table 52, which can also be seen in FIG. 5. The conveying table 52 is then operated to move the bale 44 to the rear over the axle 36 and slides downward on the section of the guidance arrangement 54 that is tilted downward, after crossing one-half of the path, in order to deposit the cylindrical bale 44 on the ground. In the preferred embodiments, the wrapping implement 14 is configured as an independent unit, that can be connected to the large round baler 12 in a quick assembly method without the use of tools. The wrapping implement 14 can also be configured in such a way, which is not shown, that it can be attached to a front loader or a rear loader, a telehandler, a three-point implement hitch or the like in order to be easily removable. As can best be seen in FIGS. 2 and 4, the wrapping implement 14 includes a guidance ring 80, a carrier 82, at least one wrapping material carrier 84, a third actuating arrangement 86, and a support 88. The guidance ring 80 has a circular cross section that is almost completely closed, but remains open only for a slot, not shown or described in any further detail, in a guidance housing 108. In the installed condition of the wrapping implement 14, the guidance ring 80 assumes an upright position of nearly 90° to the ground and is carried by the frame 16 of the large round baler 12. In place of a nearly closed cross section, an Omega profile or the like could be used. In the upper one-fourth of the guidance ring 80, a retainer 90 is provided at the guidance housing 108 that can be grasped by the hook element 76 at the frame 16 and that is configured, for example, as a pin or the like. The guidance ring 80 or the guidance housing 108 is provided with a diameter that permits the cylindrical bale 44 to pass without any change in its orientation, so that is can be deposited on the carrier 82. In the guidance housing 108, a ring 110 is located that is preferably closed and that can rotate in the guidance housing 108. The ring 110 is moved, in particular, by means of a hydraulically driven motor 112 that makes contact with the ring 110 through an opening in the guidance housing 108. The drive of the motor 112 is performed either by a positive locking connection, for example, a rack, pinion or through a friction locking connection, for example, friction wheel, and transmitted to the ring 110. In this embodiment, the motor 112 is located and protected in the lower region of the guidance housing 108 and is brought into motion by means of electromagnetically controlled valves, where the control signals again may originate from an on-board computer. Indeed, the motor 112, or several like motors, may be attached to other locations of the guidance housing 108, which depends on the spacial possibilities and environmental conditions (incidence of dirt, mechanical effects). The movement of the ring 110 within the guidance housing 108 can proceed with little friction if guide rolls, not shown, or the like support and guide the ring 110. The ring 110 may consist of metal as well as a plastic. Nearly at the lower end of the guidance housing 108, coupling elements 114 are provided at its front side for engagement with the lower connecting points 32 on the baler frame 16. The carrier 82 includes a frame 92, a roll 94, and a side panel 96. The carrier 82 is required to carry the cylindrical bale 44 during the wrapping process and to rotate it about its cylinder axis or to engage it, free to rotate. After the wrapping process, the carrier 82 is required to deposit the wrapped cylindrical bale 44 on the ground. The carrier 82 is located at the lower one-fourth or one-fifth of the guidance ring 80. As can be seen in FIG. 2 the frame 92 is provided with a transverse strut 98 and lengthwise struts 100 in a horizontal position. The transverse strut 98 is located at such a distance from the guidance ring 80 that the wrapping material carrier 84 can be moved through the interior space. The transverse strut 98 has a length approximately equal to the diameter of the guidance ring 80. The lengthwise struts 100 connect the transverse strut 98 with the guidance ring 80. For this purpose, the lengthwise struts 100 extend in the direction of operation and engage the guidance ring 80 in a bearing 102 so as to pivot vertically. For this purpose, the lengthwise struts 100 are bent at right angles, if necessary, from the forward end region towards the guidance housing 108 or pillow blocks are applied to the latter that bridge the sideways distance between the guidance housing 108 and the lengthwise struts 100. Within the space enclosed by the frame 92 in the plan view and connected to the rear side of the guidance housing 108, an ejection sleeve 118 is located. This ejection sleeve 118 consists of a small rigid frame that projects beyond the lengthwise struts 100 and projects beyond the enclosed space when the carrier 82 is tilted downward (see FIGS. 4 and 7 ). This ejection sleeve 118 prevents a small cylindrical bale 44 from resting on the rear roll 94 even when the carrier 82 has been tilted downward. Rather, upon the lowering of the carrier 82, its center is moved over the roll 94 so that it rolls down from the carrier 82. The roll 94 is applied to the transverse strut 98, free to rotate in pillow blocks, and extends horizontally transverse to the direction of operation of the combination 10. Hence, it is in contact with the circumferential surface of the cylindrical bale 44 that was taken up. The roll 94 is approximately as wide as the cylindrical bale 44 and is driven in case that the roll 38 is not driven. The panel 96 is configured as a bow, a sheet metal shoulder or the like, and is located on the opposite side of the frame 92 from the roll 94. The panel 96 is formed or attached in such a way that it is supported on the rear pulley 68, on its axis of rotation or the like, during corresponding positions of the conveying table 52 and the carrier 82, as this is shown in FIG. 5. For this purpose, the panel 96 need not extend over the entire width of the pulley 68. The requirement of the panel 96 is to retain the cylindrical bale 44 on the carrier 82 even after the conveying table 52 is again tilted forward and downward. The position of the panel 96 with the rear roll 68 and the pulley 94 are selected in such a way that they position the cylindrical bale 44 during the wrapping process centrally to the path of movement of the wrapping material carrier 84. As can be seen in FIG. 4, the wrapping material carrier 84 includes a spindle 104 and a wrapping material roll 106. In addition, and not shown, on the wrapping material carrier 84, there is also a pre-loading arrangement, a cutting arrangement, and/or a contact pressure arrangement as these are known in themselves. The spindle 104 is fastened in the axial direction on the ring 110, extends through the slot in the guidance housing 108, and carries the wrapping material roll 106 in its outer region, free to rotate and secured axially. The wrapping material roll 106 consists of a considerable length of wrapping material wrapped onto the roll, consisting of plastic, that is commercially available. A third actuating arrangement 86 is also configured as a hydraulic cylinder, has its head end pivotally coupled to the guidance housing 108, and has its rod end pivotally coupled to the underside of the carrier 82. As are the other actuating arrangements, the third actuating arrangement 86 is preferably controlled electromagnetically by means of an on-board computer. By means of the third actuating arrangement 86, the carrier 82 can be brought into an intermediate position in which it extends generally vertically to the plane of the principal extent of the guidance housing 108, as shown in FIG. 1, and can be brought into a raised position, wherein it is displaced approximately 30° upward from its intermediate position, as shown in FIG. 5. The carrier 82 can also be moved to a lowered position, wherein the carrier 82 is used as a support for the guidance ring 80 when the latter is detached from the baler 12. The third actuating arrangement 86 may be equipped or connected with a pressure accumulator so that it can deflect under pressure in a direction, particularly a downward direction. The support 88 is attached in the lower half of the guidance housing 108. It may consist of two individual legs or struts or it may consist of a U-shaped bow that folds away from the plane of the guidance housing 108 to the front and can be locked by means not shown. As can be seen, in particular from FIG. 4, the result is that the wrapping implement 14 in the unassembled condition rests in front on the support 88 and at the rear on the carrier 82 or the lengthwise struts 98 and/or transverse struts 100. On the basis of the foregoing description, the first embodiment operates as follows, where in addition it should be noted that in every case, the cylindrical bale 44 is wrapped or enclosed in net before its release from the baling chamber 22, in order to maintain its compact and cylindrical shape. The combination 10 and the wrapping implement 14 could operate without this wrapping or enveloping process but the quality of the bale would be worse. The large round baler 12 can be driven individually or in the combination 10 with the wrapping implement 14. If it is operated individually, only the conveyor 26 is attached that can take up the cylindrical bale 44 and deposit it on the ground, as was described above. When the large round baler 12 and the wrapping implement 14 are connected with each other, the conveyor 26 is not used to deposit the cylindrical bale 44 on the ground, but is used for its transfer to the wrapping implement 14. According to FIG. 5, the slide 60 is located in its forward, lower acceptance position for the acceptance of the finished cylindrical bale 44 from the baling chamber 22. As soon as the cylindrical bale 44 reaches the conveying table 52, it is in contact with the forward, further roll 74 and cannot roll down off the conveyor 26 when this is conducted by means of the first actuating arrangement 56 at an angle to the rear within the guidance arrangement 54. As soon as the slide 60 has reached its rear and upper end position, the second actuating arrangement 58 is extended so that the slide 60 tilts upward at the front about the bearing 64 and the cylindrical bale 44 lies upon the carrier 82. When it is lying on the carrier 82, the latter is pivoted upward at the rear through approximately 30°, and the panel 96 lies on the rear roll 68 or in its region so that no troublesome gap develops between the conveying table 52 and the carrier 82. In this condition, the load carrying surfaces of the conveying table 52 and the carrier 82 extend in the shape of a throat to each other, so that the cylindrical bale 44 is grasped securely and cannot roll down. If the third actuating arrangement 86 interacts with a pressure accumulator, which is not necessarily required, it deflects slightly under the impact of the cylindrical bale 44, and reduces its impact somewhat. Then the cylindrical bale 44 rests on the rear roll 68 of the conveying table 52 and the roll 94, where its center of gravity is located between the two. Following this, the carrier 82 is lowered into its position perpendicular to the guidance housing 108, in which it continues to rest on the roll 68 and the roll 94, as is shown in FIG. 6. In the position according to FIG. 6, the roll 68 or the pulley 94 is driven so that the cylindrical bale 44 will rotate about its central cylindrical axis, extending horizontally, transverse to the direction of operation of the combination 10. Following this, the ring 110 in the guidance housing 108, is brought into rotation by means of the motor or motors 112, so that the wrapping material carriers 84 are moved along a vertical track about the cylindrical bale 44 and apply foil to it in a known manner. Since this is known in itself, the application, pressing, and cutting of the foil will not be further explained. It can be seen that the large round baler 12 in this position can already be operated to form a new cylindrical bale 44, although the present cylindrical bale 44 is still being wrapped. During the wrapping process, the wrapping material carriers 84 also move through the interior space enclosed by the frame 92. When the wrapping process has been completed and the foil has also been separated, the third actuating arrangement 86 is retracted and thereby the carrier 82 is lowered. During the lowering of the carrier 82, it retracts behind the rear edge of the ejection sleeve 118, so that the cylindrical bale 44 receives an additional impulse to the rear and rolls over the pulley 94 out of the region of the carrier 82. Now the wrapping and depositing process is completed and a further cylindrical bale 44 can be accepted and wrapped. The further description concerns the second embodiment which generally corresponds to the first embodiment; differences exist in the repositioning of the slide 60, the conveying table 52, and in the position of the carrier 82 during the take-up of the cylindrical bale 44. Thus, referring to FIG. 9, it can be seen that in place of two actuating arrangements 56 and 58, only a single actuating arrangement 58 ′ is used that is connected in joints and configured as a hydraulic motor in the same way as the second actuating arrangement 58 according to the first embodiment. However, the actuating arrangement 58 ′ is provided with a considerably longer stroke and is preferably double-acting. Furthermore, a retainer 120 is attached to the slide 60, in particular, at least on one side. Finally, a locking arrangement 122 is provided on the guidance housing 108. The locking arrangement contains at least one hook 124 and a linkage 126. The same number of hooks 124 as of retainers 120 are provided, and the hook or the hooks 124 are arranged and configured in such a way that in each case they are able to overlap a retainer 124 in a positive lock and retain it. Each hook 124 can pivot vertically about a bearing 128 and is provided with a hook nose 130 extending from the bearing 128 to the front and a lever 132 extending to the rear. Each hook 124 is attached to the guidance housing 108 preferably on its outside, so as to pivot vertically, in particular, in such a way that it can grasp the retainer 120 with the hook nose 130, when the wrapping implement 14 is attached to the large round baler 12 and the slide 60 is moved to the rear up to the stop at the guidance housing 108. Although the drawing is a hook nose 130 that is open upwards, a hook-nose opening downward could also be used. At least one linkage 126 is provided that is configured as a compression spring in this embodiment and that is arranged between the lever 132 and an extension of the frame 92 projecting forward beyond the bearing 102. Instead of being configured as a compression spring, the linkage could also be configured as a rod, a rope pull, an extension spring, a rocker arm or the like. The essential thing here is that a relationship be established between the position of the hook 124 and of the carrier 82. It is possible that two hooks 124 are provided, but that are controlled together, so that only one linkage 126 is required. In contrast to the illustration of FIG. 5 for the first embodiment, the carrier 82 is not raised when taking up a cylindrical bale 44, but remains in its generally horizontal position. However, the operation of the actuating arrangement 86 as a shock absorber is preferably maintained. The operation of this second embodiment is described as follows. As soon as the cylindrical bale 44 lies upon the conveying table 52, the actuating arrangement 58 ′ is extended and the slide 60 moves in the guidance arrangement 54 to the rear and upward so that the cylindrical bale 44 is conveyed to the carrier 82. As soon as the slide 60 has reached its rear end position, the hook 124 grasps the retainer 120 and holds it rigidly in this position. The actuating arrangement 58 ′ is extended further and the conveying table 52 pivots upward about the bearing 64, in the clockwise direction as seen in FIG. 10, in order to deposit the cylindrical bale 44 on the carrier 82, until it rests on the pulley 94. Following this, the actuating arrangement 58 ′ is either drained of its pressure, so that it is lowered, or pressure is applied to it and it retracts. As a result, the conveying table 52 is also lowered, and except for the rear roll 68, is released from the circumferential surface of the cylindrical bale 44. In the condition that now obtains and is shown in FIG. 11, the wrapping process can be performed as in the case of the first embodiment. As soon as the wrapping process is ended and the carrier 82 is tilted to the rear and downward, as is shown in FIG. 12, in order to deposit the cylindrical bale 44 on the ground, the extension of the carrier 82 presses on the linkage 126, this in turn on the lever 132, and thereby the hook nose 130 is lifted from the stop. The release of this positive lock now makes it possible for the actuating arrangement 58 ′ to be retracted completely and to move the slide 60 with the conveying table 52 into its accepting position underneath the baling chamber 22, as shown in FIG. 12. A further embodiment is illustrated in FIGS. 13 through 17, which generally follows the first embodiment, but is provided with another carrier 82 ′. In contrast to the carrier 82 described so far, the carrier 82 ′ now used is provided with only one lengthwise strut 100. For example, the strut 100 provided on the left side in the previous embodiments is missing. The carrier 82 ′ is the approximate shape of a fork, which however, is repositioned by means of the actuating arrangement 86 in the same way as in the first or the second embodiment. However, the bearing 102 and the actuating arrangement 86 are not provided on the guidance housing 108, but on a sliding stand 134. The sliding stand 134 is curved corresponding to the guidance housing 108 and engages on its outer circumferential surface in a positive lock along a curve of approximately 90°. For example, an Omega or a hat profile and rolls could be provided that are used to accommodate the sliding stand 134 on the guidance housing 108. The sliding stand 134 is moved about the central axis of the guidance housing 108 by means of a motor 136, which engages in a sprocket 138 on the circumferential surface of the housing 108. Instead, a rope pull, a hydraulic cylinder, an electric motor or the like could be used, that cause at least an upward movement, while the downward movement is performed by the force of gravity alone. While the wrapping operation with this carrier 82 ′ corresponds to that of the other embodiments, this embodiment is provided with another and possibly additional deposit possibility, while the previous possibilities using the actuating arrangement 86 remain in effect. A further advantage of this carrier 82 ′ consists of the fact that it can deposit the cylindrical bale 44 on its end face, as it is shown in FIGS. 16 and 17. This type of deposit is particularly advantageous on a slope, since there the cylindrical bale 44 cannot roll away. As can be seen, in particular, from FIG. 17, the sliding stand 134 can be pivoted about the horizontal lengthwise axis of the wrapping implement 14 so far until the roll 94 and a buttress 140 located opposite them assume an inclination of approximately 45° to the ground, the buttress taking up a load only when the roll 68 of the conveying table 52 is not located in its rear end position. The buttress 140 may be a sheet metal plate, a roll, a strut or the like. In this position, the cylindrical bale 44 will tilt over the lower edge of the roll 94 and the buttress 140 and land on its end face and remain lying there. Depending on the configuration and the size of the cylindrical bale 44, the tilting may occur earlier or later, with the timing in any case depending on when the center of gravity comes to lie to the side of the lower edge of the roll 94. As soon as the cylindrical bale 44 has been deposited and the combination 10 has been operated past it, the carrier 82 ′ can be pivoted again into its original horizontal position, in which it can accept a new cylindrical bale 44. The fourth embodiment, as shown in FIGS. 18 and 19, corresponds generally to the third embodiment. However, the deposit of the cylindrical bale 44 on its end face is performed in a different manner. The configuration of the large round baler 12 and the conveyor 26 corresponds to that of the previous embodiments. In contrast to the third embodiment, the carrier 82 is not fastened to a sliding stand 134, but can be pivoted about a horizontal axis 142 extending generally in the direction of operation on which an arm 144 is supported in bearings, free to pivot. The axis 142 engages, on the one hand, the guidance housing 108, and on the other hand, the arm 144, rigidly or movably. The axis 142 is configured as a steel journal or the like and is preferably oriented with a slight inclination to the direction of operation, particularly to the side. The axis 142 or the shaft extends to the rear past the guidance housing 108 in order to take up the arm 144, and extends to the front in order to be connected with a lever arm 146. The arm 144 is rigidly connected with the lengthwise strut 100 and extends in a plane oriented parallel to the transverse strut 98. The arm 144 extends very close to the guidance housing 108 and the buttress 140. Between the arm 144 and the lengthwise strut 100, the third actuating arrangement 86 extends, which brings the carrier 82 into its take-up and delivery position, as already described above. The lever arm 146 extends radially from the axis 142 and is connected at its end with a motor 136 ′. In the embodiment shown, the lever arm 146 extends in front of the guidance housing 108 and can be repositioned between an approximately 3 o&#39;clock and an approximately 5 o&#39;clock position, as considered when viewing the wrapping implement 14 from the rear. The motor 136 ′ is configured as a single-acting or as a double-acting hydraulic cylinder, that is connected at its other end to the guidance housing 108, free to pivot. The motor 136 ′ is again repositioned automatically, that is, extended or retracted, preferably by means of an on-board computer using electromagnetic valves, not shown. On the basis of the foregoing description, the fourth embodiment operates as follows. The formation, the wrapping, the delivery and transport of the cylindrical bale 44 to the carrier 82, and the enveloping of it with foil is performed in the manner described previously. In this position, the motor 136 ′ is extended and the carrier 82 with its roll 94 extend in a horizontal plane. As soon as the wrapping process is completed and the cylindrical bale 44 can be deposited on the ground, the motor 136 ′ is retracted and the carrier 82 tilts about or with the axis 142 in the counterclockwise direction as seen in FIG. 19, until it is inclined approximately 45° to the ground. In this position, the cylindrical bale 44 falls onto its end face. If the cylindrical bale 44 is to be deposited on its circumferential surface, the motor 136 ′ remains rigid and the actuating arrangement 86 is operated so that the cylindrical bale 44 reaches the ground in its usual manner. Having described the preferred embodiment, it will become apparent that various modifications can be made without departing from the scope of the invention as defined in the accompanying claims.

Summary: A wrapping implement is mounted to a rear location of a large round baler with connections permitting its quick attachment to, and detachment from, the baler. The wrapping implement includes a guidance ring that is essentially oriented vertically for guiding at least one wrapping material carrier about its periphery. The baler is equipped with a bale conveyor that receives a bale discharged from the baling chamber and transfers the bale to a bale carrier mounted to the guidance ring. The carrier supports and effects or permits the rotation of the supported cylindrical bale, where during the wrapping process the axis of rotation of the cylindrical bale extends perpendicular to the central axis of the circle of movement of the wrapping material carrier.

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Summarize: The new Terry Gilliam film, “The Imaginarium of Dr. Parnassus,” has one of those roll-up, roll-up titles that appeal to Gilliam, who is not so much a movie director as a carnival barker with a bent for motion pictures. The last example was “The Adventures of Baron Munchausen,” and some of its tropes return here: the team of travelling players, and the elderly fabulist who tries to dictate the proceedings. In this case, our aging hero, Dr. Parnassus (Christopher Plummer), is perched inside a mobile theatre, of antiquated design, which is trundled through modern London; part of the movie’s charm is its brazen anachronism—a sense that the past can seep through time and burst into the here and now. (And the now is pretty wretched: rusting wastelands beside the Thames.) Also in attendance are Tom Waits, as a smiling Beelzebub in a bowler hat; a dwarf, sadly compulsory in any work that flirts with the surreal; and, as Parnassus’s daughter, the British model Lily Cole, whose unearthly heart of a face is like a special effect. Above all, we get four types of Tony, the gadabout who consorts with Parnassus’s troupe. He is played by Johnny Depp, Colin Farrell, Jude Law, and Heath Ledger, the first three deputizing for the last, who died before the film’s completion. It should not be mistaken for his finest hour; we see his larking, but not the undertow of frailty that tugged at “Brokeback Mountain,” and his principal duty here, like everyone else’s, is to go with the flow of phantasmagoria. Thus, people are invited onto the Doctor’s stage and sent through a rickety mirror, into a world where anything goes—a candy-colored landscape out of “Mary Poppins,” an oily river that curls up into a snake’s head, you name it. Are these embodiments of each person’s fancy, or did Parnassus himself cook them up? I have no idea, any more than I can decide whether C.G.I. was the best or the worst thing that could have happened to Terry Gilliam. His gifts of invention were already so fecund, and so prolix, that this newfound ability to construct anything that drifts into his mind’s eye—as opposed to the ramshackle, hand-drawn delight of his earlier animation—spells both enchantment and chaos. He can follow any train of thought, so he does, and it’s no surprise when the trains run out of steam. No such mania infects “The Young Victoria,” Jean-Marc Vallée’s account of the monarch’s early years, which veers in the other direction. From the start, it feels handsome, steady, and stuck; the ties that bind the historical bio-pic are no looser than those which constrain a royal personage, and the frustration to which Victoria would later admit (“I had led a very unhappy life as a child—had no scope for my very violent feelings of affection”) is legible in the face of Emily Blunt, who takes the title role. She is attended by a host of British actors—Paul Bettany, though too young, makes a winning and cynical Lord Melbourne, and Jim Broadbent, resembling a Gillray cartoon, detonates the stolid tale with a loud, and well-attested, explosion of rage at a banquet—but the movie, like the age that it depicts, stands or falls on the woman at its core. Blunt strikes me as the real deal: languid but biting, like Jeanne Moreau, yet able to command a scene while somehow appearing to shift to one side (as Moreau would never do) and observe with a skeptic’s smile. The little puff of relief that Victoria gives after addressing the Privy Council on the morning of her accession is, despite her duties, the exhalation of a free spirit, and one prays that films more liberating than this, and more likely to feed Blunt’s appetites, will come her way. The Colin Firth of “A Single Man,” likewise, rises above the atmosphere that encircles him. He plays George, who teaches literature at a college in Los Angeles, in 1962, while mourning the loss of his lover, Jim (Matthew Goode), in a car accident. When George walks through the snow toward the crash site, not only are his pants perfectly creased but even the corpse preserves a certain je ne sais quoi. True, this is a dream sequence, but then the whole movie, with its glazed and polished air, could be a dream. We know, for instance, how English professors dress: some trim-suited and clerical, others avuncular in tweed, many too deep in Dryden to be bothered with their outward crust, and one or two no better than compost heaps. Not this lot. They look as spry and as spotless as an advertising spread in L’Uomo Vogue. Who’s in charge here, for heaven’s sake—a fashion designer? Well, yes, for “A Single Man” is the first feature to be directed by Tom Ford. The hero of the story, adapted from the novel by Christopher Isherwood, explains himself in voice-overs: “Just get through the goddamned day: bit melodramatic, perhaps, but then again, my heart has been broken. Feel as if I’m drowning, sinking, can’t breathe,” he says. Such wordiness seems unjust to Firth, who is perfectly capable of showing any congestion of spirit by body language alone. The film is slowed by its own beauty, but it is salvaged by two majestic scenes. In one, George learns of Jim’s death in a phone call from a relative, during which his voice (this being 1962) must betray nothing, leaving his face (on which Ford is smart enough to keep the camera) to do all the work; in another, George goes around for a long evening with his friend Charley (Julianne Moore), who likes to start boozing as she puts on her face in the morning. Two characters trying and failing to drown their hopes and regrets, and two strong actors refusing to be tight-laced by a director’s exercise in style: here is a mood piece looking for a fight. ♦ Cert PG, 105mins, 2/5 Co-produced by none other than the Duchess of York, here's yet another queasy entry into the ever-growing Queensploitation genre. Like a Franklin Mint wallplate of Her Majesty advertised in the Sunday supplements, it's a glossy, insipid and toe-curlingly reverential affair. Following the similarly deferential The Duchess, the clock's turned back to the 1800s for a look at the life and times of Queen Victoria. Charting the monarch's days from childhood to her 1819 marriage to Germany's Prince Alfred, it's down to Emily Blunt to slip into the Royal corset and wrestle with the age-old - and depressingly familiar - battle between duty and love. Complicating things further are her meddling mum (Miranda Richardson) and scheming suitor Sir John Conroy (Mark Strong). Should our heroine go for the inappropriate Teutonic prince or deny her heart for the good of the Union? Who cares? A film this concerned with powder-wigged toffs inspires little but apathy and a deep longing for a Republic of Britain. IF YOU LIKED... The Other Boleyn Girl, Becoming Jane... YOU'LL LIKE THIS. Watch my video review of The Young Victoria in the video below: Over the centuries, Queen Victoria has become as much a fixed image as a flesh-and-blood historical figure. Stern, stout, and unyielding, she’s been transformed into a stand-in for everything repressive and staid in 19th-century Britain. Though at heart a perfectly polite costume drama, The Young Victoria gets a minor charge simply by portraying Victoria in the years leading up to and after her coronation at the age of 18. Winningly played by the anything-but-stern Emily Blunt, Victoria is presented as a bright, vivacious, willful, and sometimes rash monarch-in-the-making who took the reins away from those who expected to hold them for her. The film around her performance is never so commanding, but as a colorful, only mildly juiced-up history lesson, it’s effective enough. Director Jean-Marc Vallée bathes the frames in the details of its luxurious surroundings, and the screenplay, by Gosford Park writer Julian Fellowes, does a passable job of laying out all the factions jockeying for power around Blunt’s Victoria. The film lays on its politics-as-chess-game metaphor a little thick, however, and its refusal to leave the corridors of power to see the impact of its developments on the country at large makes it feel stuffy after a while. Maybe that’s why—in spite of nice work by Miranda Richardson as the Victoria’s mother, and Paul Bettany as Lord Melbourne, whose mentorship carries a hint of self-interest—The Young Victoria is at its liveliest when focusing on the romance between its heroine and her cousin Albert Of Saxe-Coburg And Gotha (Rupert Friend), which—here at least—begins as an attempt at power consolidation and blooms into true love. Blunt and Friend generate an impressive amount of sexual tension via courtly letters and chaperoned conversations. Even under constricted circumstances, and in the middle of constricted movies, hearts can’t hide their passions. Movie Review In one portrait of the young Queen Victoria, the 18-year-old who ascended the British throne in 1837 and gave the Victorians their name, she sits in her coronation robes staring straight ahead with a somewhat glazed expression. Her arms are white and fashionably plump, her dress cream and splashed with gold. Her coronation robe, a massive ermine-and-red-velvet cloak, spills off her bared shoulders and onto the ground, creating a luxurious puddle. She looks a bit bored — heavy hangs the crown, or at least those opulent threads. This isn’t our familiar image of Victoria, the globular old lady shrouded in widow’s weeds and vehement melancholia, and wearing a dour look that suggests that she was never amused, not now, not ever. That woman, who became known as the Widow of Windsor after the death of her husband, Prince Albert, in 1861, is nowhere to be seen in “The Young Victoria,” a frivolously entertaining film, with Emily Blunt, about the young monarch. Ms. Blunt, who as Meryl Streep’s viperous assistant in “The Devil Wears Prada” slyly upstaged Anne Hathaway, doesn’t have Victoria’s padding or bearing, which is probably why she was cast in the role. No one wants to watch the lives and loves of the rich and dowdy. That at least seems to be the prevailing filmmaker wisdom, which is why what we usually get in movies about royal personages from dusty and distant worlds are sumptuous frocks, soaring music, a dash of intrigue and thunderous nonsense. “Young Victoria” — the latest in a long line of fictional entertainments about the dramatic, if more often than not melodramatic, ups and downs of female British monarchs — fits the bill nicely. There’s something about a queen that inspires filmmakers (audiences too), who enjoy pulling the curtain away from these most private lives and taking peeks under the royal robes. Because this queen spent so much of her life draped in black and gloom, her younger self would seem particularly ripe material. She is and she isn’t, at least here. Directed with some snap by Jean-Marc Vallée from a screenplay by Julian Fellowes, “The Young Victoria” opens with some turgid on-screen text about her birth in 1819: “A child is born.” No, not Jesus, but little Victoria, heir to the throne and the only daughter of the Duchess of Kent (Miranda Richardson) and the unseen, soon-to-be-dead Duke of Kent, the fourth son of George III. But while she was born with a royal spoon in her mouth, little Victoria, like so many rich girls before and since, proves deeply unhappy. “What little girl does not dream of growing up as a princess,” Victoria asks early in dolorous voice-over, before lowering the boom: “Even a palace can be a prison.” A chill fills that palace’s rooms, a coldness largely emanating from the duchess and her close adviser, Sir John Conroy (Mark Strong), who keep Victoria under lock and key to control her and the power she will assume. Isolated from other children, with only servants and a spaniel for company, Victoria amuses herself as best she can. Ms. Blunt tries her hardest to interest us as well, but there is limited entertainment in watching even an appealing actress wander forlornly, talking to herself and the dog. Victoria, it seems, wasn’t all that interesting at this stage in her life (or so it appears here), which might explain why Mr. Vallée tweaks his material to the point of exaggeration, sometimes to the point of parody, as with Mr. Strong’s virtual mustache-twirling. Happily, the story of young Victoria is also the tale of her relationship with her young consort, Albert (Rupert Friend, pretty and pink-cheeked), her first cousin. Born on the Continent, in part of what would become Germany, Albert enters Victoria’s life obliquely, first through the machinations of their uncle, King Leopold of Belgium (Thomas Kretschmann), and then during tentative visits. There’s a political dimension to Leopold’s matchmaking, but those details are immaterial to the romance that develops and deepens. Despite the filmmakers’ efforts to persuade us that “The Young Victoria” is a serious work, and despite some tense moments and gunfire, the movie’s pleasures are as light as its story. No matter. Albert may never rip Victoria’s bodice, but he does eventually loosen it, to her delight and ours. “The Young Victoria” is rated PG (Parental guidance suggested). Flirting and nuzzling. THE YOUNG VICTORIA Opens on Friday in Manhattan. Directed by Jean-Marc Vallée; written by Julian Fellowes; director of photography, Hagen Bogdanski; edited by Jill Bilcock and Matt Garner; music by Ilan Eshkeri; production designer, Patrice Vermette; produced by Graham King, Martin Scorsese, Tim Headington and Sarah Ferguson; released by Apparition. Running time: 1 hour 44 minutes. WITH: Emily Blunt (Queen Victoria), Rupert Friend (Prince Albert), Paul Bettany (Lord Melbourne), Miranda Richardson (Duchess of Kent), Jim Broadbent (King William), Thomas Kretschmann (King Leopold), Mark Strong (Sir John Conroy), Jesper Christensen (Baron Stockmar) and Harriet Walter (Queen Adelaide).

Summary: Whether they find the film delightful costumed fluff or a dreary museum piece, most critics agree Emily Blunt is lovely as The Young Victoria. Some takes: "Winningly played" by Blunt, Keith Phipps writes for the Onion AV Club, Victoria is "a bright, vivacious, willful, and sometimes rash monarch-in-the-making," a welcome antidote to the familiar dour characterizations of the monarch. The film has its ups and downs, says Manohla Dargis of the New York Times. "There is limited entertainment in watching even an appealing actress wander forlornly, talking to herself and the dog," but the romance is a delight. "Albert may never rip Victoria's bodice, but he does eventually loosen it, to her delight and ours." Yuck, writes David Edwards in the Daily Mirror. "Yet another queasy entry into the ever-growing Queensploitation genre" about the "depressingly familiar battle between duty and love." "The movie, like the age that it depicts, stands or falls on the woman at its core," and Blunt strikes New Yorker critic Anthony Lane as "the real deal." For her sake, he writes, "one prays that films more liberating than this, and more likely to feed Blunt's appetites, will come her way."

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Summarize: Background VA comprises three major components: the Veterans Benefits Administration (VBA), the Veterans Health Administration (VHA), and the National Cemetery Administration (NCA). VA’s mission is summed up in its mission statement, a quotation from Abraham Lincoln: “to care for him who shall have borne the battle and for his widow and his orphan.” VA carries out this mission by providing benefits and other services to veterans and dependents. The department’s vision is to be a more customer-focused organization, functioning as “One VA.” This vision stemmed from the recognition that veterans think of VA as a single entity, but often encountered a confusing, bureaucratic maze of uncoordinated programs that put them through repetitive and frustrating administrative procedures and delays. The “One VA” vision is to create versatile new ways for veterans to obtain services and information by streamlining interactions with customers and integrating IT resources to enable VA employees to help customers more quickly and effectively. This vision will require modifying or replacing separate information systems with integrated systems using common standards to share information across VA programs and with external partner organizations, such as the Department of Defense. Accordingly, effective management of its IT programs is vital to VA’s successful achievement of its vision and mission. Table 1 shows a breakdown of VA’s approximately $2.1 billion IT budget request for fiscal year 2006. Of the total, VHA accounted for approximately $1.8 billion, VBA approximately $150 million, and the National Cemetery Administration (NCA) approximately $11 million. The remaining $84 million was allocated to the department level. CIO Plays Major Role in IT Management The Congress has long recognized that IT has the potential to enable federal agencies to accomplish their missions more quickly, effectively, and economically. However, fully exploiting this potential presents challenges to agencies. Despite substantial IT investments, the federal government’s management of information resources has produced mixed results. One of the ways in which the Congress has addressed this issue was to establish the CIO position; an agency’s CIO serves as the focal point for information and technology management within an agency. Legislative Evolution of Agency CIO Role For more than 20 years, federal law has structured the management of IT and information-related activities under the umbrella of information resources management (IRM). The IRM approach was first enacted into law in the Paperwork Reduction Act of 1980. The intention of the Congress was to provide for a coordinated approach to managing federal agencies’ information resources, addressing the entire information life cycle, from collection through disposition, with the ultimate goal of improving the efficiency and effectiveness of government while reducing the “paperwork burden” on the public. The 1980 Paperwork Reduction Act centralized governmentwide IRM responsibilities in the Office of Management and Budget (OMB), giving OMB specific policy-setting and oversight duties regarding individual IRM areas, such as records management, privacy, and the acquisition and use of IT. Agencies were given responsibility for carrying out their IRM activities in an efficient, effective, and economical manner in compliance with OMB policies and guidelines. The law also required that each agency head designate a senior official, reporting directly to the agency head, to carry out the agency’s responsibilities under the law. In 1996, the Clinger-Cohen Act established the position of agency CIO by giving this title to the “senior IRM official” mentioned in the Paperwork Reduction Act and specifying additional responsibilities for this position. Among these responsibilities, the Clinger-Cohen act required that the CIOs in the 24 major departments and agencies have IRM as their “primary duty.” The view of the Congress as reflected in current law is thus that CIOs should play a key leadership role in ensuring that agencies manage their information functions in a coordinated and integrated fashion in order to improve the efficiency and effectiveness of government programs and operations. CIO Responsibilities and Reporting Relationships Besides their statutory responsibilities, CIOs have other responsibilities that can contribute significantly to the successful implementation of information systems and processes. In July 2004, we interviewed 27 CIOs at major agencies on their roles, responsibilities, and challenges, among other things. For this report, we identified major areas of CIO responsibilities that were either statutory requirements or critical to effective information and technology management. Altogether, we identified the 13 areas shown in table 2. According to our report, CIOs were generally responsible for the key information and technology management areas shown in the table, although not all CIOs were completely responsible for all areas. For example: ● All the CIOs were responsible for five areas (capital planning and investment management, enterprise architecture, information security, IT/IRM strategic planning, and IT workforce planning). ● More than half had responsibility for six additional areas (systems acquisition, major e-government initiatives, information collection/paperwork reduction, records management, information dissemination, and privacy). ● Fewer than half were responsible for two areas (information disclosure and statistics). It was common for CIOs to share responsibility for certain functions, and in some cases responsibilities were assigned to other offices. For example, systems acquisition responsibility could be shared among the CIO and other officials, such as a procurement executive or program executive; disclosure could be assigned to general counsel and public affairs, while statistical policy could be assigned to offices that deal with the agency’s data analysis. Nevertheless, even for areas of responsibility that were not assigned to CIOs, agency CIOs generally reported that they contributed to the successful execution of the agency’s overall responsibilities in that area. In carrying out their responsibilities, CIOs generally reported to their agency heads. The Paperwork Reduction Act—as well as our guidance—generally calls for CIOs to report to their agency heads, forging relationships that ensure high visibility and support for far- reaching information management initiatives. For 19 of the agencies in our review, the CIOs stated that they had this reporting relationship. In the other 8 agencies, the CIOs stated that they reported instead to another senior official, such as a deputy secretary, under secretary, or assistant secretary. In addition, 8 of the 19 CIOs who said they had a direct reporting relationship with the agency head noted that they also reported to another senior executive, usually the deputy secretary or under secretary for management, on an operational basis. According to members of our Executive Council on Information Management and Technology, what is most critical is for the CIO to report to a top level official. Tenure and Backgrounds of CIOs Federal CIOs often remained in their positions for less than the length of time that some experts consider necessary for them to be effective and implement changes. At the major departments and agencies included in our review, the median time in the position of permanent CIOs whose time in office had been completed was about 23 months. For career CIOs, the median was 32 months; the median for political appointees was 19 months. To the question of how long a CIO needed to stay in office to be effective, the most common response of the CIOs (and former agency IT executives whom we consulted) was 3 to 5 years. Between February 10, l996, and March 1, 2004, only about 35 percent of the permanent CIOs who had completed their time in office reportedly had stayed in office for a minimum of 3 years. The gap between actual time in office and the time needed to be effective is consistent with the view of many agency CIOs that the turnover rate was high, and that this rate was influenced by the political environment, the pay differentials between the public and private sectors, and the challenges that CIOs face. In contrast, the CIOs interviewed for our report were generally helped in carrying out their responsibilities by the background and experience they brought to the job. The background of the CIOs varied in that they had previously worked in the government, the private sector, or academia, and they had a mix of technical and management experience. However, virtually all had work experience or educational backgrounds in IT or IT-related fields; 12 agency CIOs had previously served in a CIO or deputy CIO capacity. Moreover, most of the them had business knowledge related to their agencies because they had previously worked at the agency or had worked in an area related to the agency’s mission. Success Factors and Challenges of CIOs To allow CIOs to serve effectively in the key leadership role envisioned by the Congress, federal agencies must use the full potential of CIOs as information and technology management leaders and active participants in the development of the agency’s strategic plans and policies. The CIOs, in turn, must meet the challenges of building credible organizations and developing and organizing information and technology management capabilities to meet mission needs. In February 2001, we issued guidance on the effective use of CIOs, which describes the following three factors as key contributors to CIO success: ● Supportive senior executives embrace the central role of technology in accomplishing mission objectives and include the CIO as a full participant in senior executive decision making. ● Effective CIOs have legitimate and influential roles in leading top managers to apply IT to business problems and needs. Placement of the position at an executive management level in the organization is important, but in addition, effective CIOs earn credibility and produce results by establishing effective working relationships with business unit heads. ● Successful CIOs structure their organizations in ways that reflect a clear understanding of business and mission needs. Along with knowledge of business processes, market trends, internal legacy structures, and available IT skills, this understanding is necessary to ensure that the CIO’s office is aligned to best serve agency needs. The CIO study that we reported on in July 2004 also provides information on the major challenges that federal CIOs face in fulfilling their duties. In particular, CIOs view IT governance processes, funding, and human capital as critical to their success, as indicated by two challenges that were cited by over 80 percent of the CIOs: implementing effective information technology management and obtaining sufficient and relevant resources. ● Effective IT management. Leading organizations execute their information technology management responsibilities reliably and efficiently. A little over 80 percent of the CIOs reported that they faced one or more challenges related to implementing effective IT management practices at their agencies. This is not surprising given that, as we have previously reported, the government has not always successfully executed the IT management areas that were most frequently cited as challenges by the CIOs—information security, enterprise architecture, investment management, and e-gov. ● Sufficient and relevant resources. One key element in ensuring an agency’s information and technology success is having adequate resources. Virtually all agency CIOs cited resources, both in dollars and staff, as major challenges. The funding issues cited generally concerned the development and implementation of agency IT budgets and whether certain IT projects, programs, or operations were being adequately funded. We have previously reported that the way agency initiatives are originated can create funding challenges that are not found in the private sector. For example, certain information systems may be mandated or legislated, so the agency does not have the flexibility to decide whether to pursue them. Additionally, there is a great deal of uncertainty about the funding levels that may be available from year to year. The government also faces long-standing and widely recognized challenges in maintaining a high-quality IT workforce. In 1994 and 2001, we reported on the importance that leading organizations placed on making sure they had the right mix of skills in their IT workforce. About 70 percent of the agency CIOs reported on a number of substantial IT human capital challenges, including, in some cases, the need for additional staff. Other challenges included recruiting, retention, training and development, and succession planning. In addition, two other commonly cited challenges were communicating and collaborating (both internally and externally) and managing change. ● Communicating and collaborating. Our prior work has shown the importance of communication and collaboration, both within an agency and with its external partners. For example, one of the critical success factors we identified in our guide focuses on the CIO’s ability to establish his or her organization as a central player in the enterprise. Ten agency CIOs reported that communication and collaboration were challenges. Examples of internal communication and collaboration challenges included (1) cultivating, nurturing, and maintaining partnerships and alliances while producing results in the best interest of the enterprise and (2) establishing supporting governance structures that ensure two- way communication with the agency head and effective communication with the business part of the organization and component entities. Other CIOs cited activities associated with communicating and collaborating with outside entities as challenges, including sharing information with partners and influencing the Congress and OMB. ● Managing change. Top leadership involvement and clear lines of accountability for making management improvements are critical to overcoming an organization’s natural resistance to change, marshaling the resources needed to improve management, and building and maintaining organizationwide commitment to new ways of doing business. Some CIOs reported challenges associated with implementing both changes originating from their own initiative and changes from outside forces. Implementing major IT changes can involve not only technical risks but also nontechnical risks, such as those associated with people and the organization’s culture. Six CIOs cited dealing with the government’s culture and bureaucracy as challenges to implementing change. Former agency IT executives also cited the need for cultural changes as a major challenge facing CIOs. Accordingly, in order to effectively implement change, it is important that CIOs build understanding, commitment, and support among those who will be affected by the change. Effectively tackling these reported challenges can improve the likelihood of a CIO’s success. Until these challenges are overcome, federal agencies are unlikely to optimize their use of information and technology, which can affect an organization’s ability to effectively and efficiently implement its programs and missions. Roles and Responsibilities of the CIO Position at VA Have Evolved over Time Since enactment of the Clinger-Cohen Act in 1996, the roles and responsibilities of VA’s Chief Information Officer have evolved. From lacking a CIO entirely, the department has taken steps to address the challenges posed by its multiple widespread components and its decentralized information technology and services. In June 1998, VA assigned CIO responsibility to a top manager. However, we reported in July 1998 that the person holding the CIO position at VA had multiple additional major responsibilities, as this person also served as Assistant Secretary for Management, Chief Financial Officer, and Deputy Assistant Secretary for Budget. According to the act, the CIO’s primary responsibility should be information and technology management. Noting that VA’s structure was decentralized, its IT budget was large, and its CIO faced serious information and technology management issues, we recommended that the Secretary appoint a CIO with full-time responsibilities for IRM. Concurring with the recommendation, VA established the position of Assistant Secretary for Information and Technology to serve as its CIO. As of May 2000, however, the position of Assistant Secretary for Information and Technology was vacant, and as we reported at the time, it had been unfilled since its creation in 1998. The Secretary then created and filled the position of Principal Deputy Assistant Secretary for Information and Technology, designating that person as VA’s acting CIO until an Assistant Secretary could be appointed. The Secretary also realigned IRM functions within VA under this position, which reported directly to the Secretary. As we reported, the Principal Deputy Assistant Secretary was involved in IT planning issues across the department. In addition to advising the Secretary on IT issues, he served as chair of the department’s CIO Council and as a member of the department’s Capital Investment Board, and he worked with the CIOs in VBA and VHA (at the time, NCA had no CIO). According to this official, one of his priorities was to ensure that IT activities in VBA and VHA were in concert with VA’s departmentwide efforts. In August 2001, VA filled the CIO position. In March 2002, we testified that this hiring was one of the important strides that the Secretary of Veterans Affairs had made to improve the department’s IT leadership and management, along with making a commitment to reform the department’s use of IT. On June 29, 2003, the CIO retired after a tenure of almost 2 years (about the median length of tenure for federal CIOs, as discussed above); the current CIO was confirmed in January 2004. Figure 1 is a time line showing the history of the CIO position at VA since the passage of the Clinger-Cohen Act. VA Proposed to Realign its IT Organization in Response to IT Management Challenges Our prior work highlighted some of the challenges that the CIO faced as a result of the way the department was organized to carry out its IT mission. Among these challenges was that information systems and services were highly decentralized, and the VA administrations and staff offices controlled a majority of the department’s IT budget. For example, in VA’s information technology budget for fiscal year 2002 of approximately $1.25 billion, VHA controlled about $1.02 billion (over 80 percent), whereas the department level controlled about $60.2 million (less than 5 percent). In addition, we noted that there was neither direct nor indirect reporting to VA’s cyber security officer—the department’s senior security official—thus raising questions about this person’s ability to enforce compliance with security policies and procedures and ensure accountability for actions taken throughout the department. The more than 600 information security officers in VA’s three administrations and its many medical facilities throughout the country were responsible for ensuring the department’s information security, although they reported only to their facility’s director or to the chief information officer of their administration. Given the large annual funding base and decentralized management structure, we testified that it was crucial for the departmental CIO to ensure that well-established and integrated processes for leading, managing, and controlling investments are commonplace and followed throughout the department. This is consistent with the finding in our CIO review that implementation of IT management practices was a challenge; over half of federal CIOs identified IT investment management specifically. Recognizing weaknesses in accountability for the department’s IT resources and the need to reorganize IT management and financing, the Secretary announced a realignment of the department’s IT operations in a memorandum dated August 2002. According to the memorandum, the realignment would centralize IT functions, programs, workforce personnel, and funding into the office of the department-level CIO. In particular, several significant changes were described: ● The CIOs in each of the three administrations—VHA, VBA, and NCA—were to be designated deputy CIOs and were to report directly to the department-level CIO. Previously, these officials served as component-level CIOs who reported only to their respective administrations’ under secretaries. ● All administration-level cyber security functions were to be consolidated under the department’s cyber security office, and all monies earmarked by VA for these functions were to be placed under the authority of the cyber security officer. Information security officers previously assigned to VHA’s 21 veterans integrated service networks would report directly to the cyber security officer, thus extending the responsibilities of the cyber security office to the field. ● Beginning in fiscal year 2003, the department-level CIO would assume executive authority over VA’s IT funding. In September 2002, we testified that in pursuing these reforms, the Secretary demonstrated the significance of establishing an effective management structure for building credibility in the way IT is used, and took a significant step toward achieving a “One VA” vision. The Secretary’s initiative was also a bold and innovative step by the department—one that has been undertaken by few other federal agencies. For example, of 17 agencies contacted in 2002, 8 reported having component-level CIOs, none of which reported to the department-level CIO. Only one agency with component-level CIOs reported that its department-level CIO had authority over all IT funding. We also noted that the CIO’s success in managing IT operations under the realignment would hinge on effective collaboration with business counterparts to guide IT solutions that meet mission needs, and we pointed out the importance of the three key contributors to CIO success described in our 2001 guidance (discussed earlier). Although we have not reviewed the current status of this proposed realignment or VA’s current organizational structure, it remains our view that the proposed realignment held promise for building a more solid foundation for investing in and improving the department’s accountability over IT resources. Specifically, under the realignment the CIO would assume budget authority over all IT funding, including authority to veto proposals submitted from subdepartment levels. This could have a significant effect on VA’s accountability for how components are spending money. To sum up, the CIO plays a vital role in ensuring that VA’s funds are well spent and in managing information technology to serve our nation’s veterans. In our view, the realignment of VA’s IT organization proposed in 2002 held promise for improving accountability and enabling the department to accomplish its mission. The additional oversight afforded the CIO could have a significant impact on the department’s ability to more effectively account for and manage its proposed $2.1 billion in planned IT spending. Mr. Chairman, this concludes my statement. I would be pleased to respond to any questions that you or other members of this Committee may have at this time. Contacts and Acknowledgements For information about this testimony, please contact Linda D. Koontz, Director, Information Management Issues, at (202) 512-6240 or at koontzl@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this statement. Individuals making key contributions to this testimony include Barbara Collier, Lester Diamond, Barbara Oliver, and Eric Trout. 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 carrying out VA's mission of serving the nation's veterans and their dependents, the agency relies extensively on information technology (IT), for which it is requesting about $2.1 billion in fiscal year 2006. VA's vision is to integrate its IT resources and streamline interactions with customers, so that it can provide services and information to veterans more quickly and effectively. Fully exploiting the potential of IT to improve performance is a challenging goal for VA, as it is throughout government. The Clinger-Cohen Act of 1996 addressed this challenge by, among other things, establishing the position of chief information officer (CIO) to serve as the focal point for information and technology management within departments and agencies. The Committee requested that GAO discuss the role of CIOs in the federal government, as well as provide a historical perspective on the roles and responsibilities of VA's CIO. In developing this testimony, GAO relied on its previous work at VA as well as on the CIO role across government, including a 2004 review of CIOs at major departments and agencies. CIOs play a critical role in managing information and technology within federal agencies. According to GAO's 2004 review, CIOs generally held wide responsibilities and reported to their agency heads or other top level managers. In general, CIOs reported that they were responsible for key information and technology management areas; for example, all the CIOs were responsible for five key areas (capital planning and investment management, enterprise architecture, information security, strategic planning for information technology and information resource management, and information technology workforce planning). However, in carrying out their responsibilities, the tenure of federal CIOs was often less than the length of time that some experts consider necessary for them to be effective and implement changes: the median tenure was about 2 years, and the most common response regarding time required to be effective was 3 to 5 years. In contrast, CIOs were generally helped in carrying out their responsibilities by the background and experience they brought to the job: most had background in information technology (IT) or related fields, and many also had business knowledge related to their agencies. Other factors that help CIOs meet their responsibilities include (1) being supported by senior executives who recognize the importance to their missions of IT and an effective CIO; (2) playing an influential role in applying IT to business needs; and (3) being able to structure their organizations appropriately. At the same time, CIOs cited several challenges, of which the two most frequently mentioned were implementing effective IT management and obtaining sufficient and relevant resources. Over time, the CIO position at VA, as well as information and technology management as a whole, has received increased attention at the department. After several years with CIOs whose primary duty was not information and technology management or who were serving in an acting capacity, the department appointed a full-time permanent CIO in August 2001. In 2002, the department proposed further strengthening the position and centralizing IT management, recognizing that aspects of its computing environment were particularly challenging and required substantial management attention. In particular, the department's information systems and services were highly decentralized, and a large proportion of the department's IT budget was controlled by the VA's administrations and staff offices. To address these challenges, the Secretary issued a memo in 2002 announcing that IT functions, programs, and funding would be centralized under the department-level CIO. This realignment held promise for improving accountability and enabling the department to accomplish its mission. The additional oversight afforded the CIO could have a significant impact on the department's ability to more effectively account for and manage its IT spending.

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Summarize: By. Frank Coletta for Daily Mail Australia. The Prime Minister says Australian extremists linked with terror organisations 'hate us' and will try to return 'accustomed to kill'. Tony Abbott has explained that, so far, no Australian troops have been committed to the crisis in northern Iraq but has not ruled out the possibility. 'Australia cannot leave the Iraqi people to face this horror, this pure evil, alone,' Mr Abbott said. Scroll down for video. The Prime Minister has not ruled out Australian ground forces being deployed in northern Iraq, suggesting if the call was made for military action by the US, that would be assessed against certain criteria. The US President Barack Obama has authorised air strikes against Islamist militants in Iraq, if they are deemed necessary to stop the advance of extremists in northern Iraq. 'So far, Australian aircraft have participated in humanitarian air drops to people trapped on Mount Sinjar and just yesterday to the besieged inhabitants of the town of Amerli.' The next stage will include the air-lift of of supplies 'including military equipment' to the Kurdish military. 'This involvement has been at the request of the Obama administration,' Mr Abbott confirmed. 'So far we have met requests for humanitarian relief and for logistical support. 'There has been no request for military action itself. Should such a request come it would be considered against these criteria. 'Is there a clear and achievable overall objective? Is there a clear and proportionate role for Australian forces? Have all the risks been property assessed and is there an overall humanitarian objective in accordance with Australia's national interests?.' Mr Abbott went on to say that 'Australia has no intention to commit combat troops on the ground but we are not inclined to stand by in the face of preventable genocide either'. 'Australia is not a country that goes looking for trouble but Australia is prepared to do what we can in the wider world. This conflict is reaching out to us. 'At least 60 Australians are fighting with terrorist groups across Iraq and Syria. They are supported by about 100 more and we know, or should assume that many of them will seek to return to Australia. 'They will return accustomed to kill. 'The Australians and their supporters who have joined terrorist groups in the Middle East are a serious and growing threat to our security. 'They have come to hate us no less than they hate their victims in Iraq and Syria.'

Summary: There has been no call from the United States, as yet, for Australia to involve itself in military action in northern Iraq. If that request is made for ground forces, an assessment against a list of objectives and criteria would be made. Australian involvement has, so far, been limited to humanitarian air drops but will provide'military equipment' in the next air-lift. He has called the extremist group Islamic State of Iraq and Levant (ISIL) 'a death cult' Australian extremists 'hate us' and will return 'accustomed to kill' PM says 60 Australians are fighting with terrorist groups and 'are supported by at least 100 more'

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Summarize: By. Sophie Jane Evans. Jailed: Michael Webster, 49, from Streatham Hill, London, was today jailed for stealing £75,000 from his clients to prop up his diamond business in Ghana. One of Britain’s top black lawyers was today jailed for stealing £75,000 from his clients to prop up his diamond business. Michael Webster, 49, took tens of thousands of pounds from the client account of his firm Webster Dixon LLP, which made history as first company founded by black lawyers in the City of London. He then funnelled the cash into the account of a business associate who hoped to make money on diamonds in Ghana. When. the deal failed to go through, he tried to take out a loan to cover. the loss, but was arrested after his bookkeeper spotted discrepancies in his accounts. Today Webster, a former chairman of the Black Solicitors Network, has been jailed for eight months after pleading guilty to one count of fraud by abuse of position at London's Old Bailey. The court heard that the lawyer had made four withdrawals totalling £75,605.57 from the client account at Webster Dixon LLP between December 3, 2012, and February 1, 2013. Leee Ingham, prosecuting, said the law firm had been in financial difficulty at the time. He told the court: ‘What Webster was really doing was speculating with someone else’s money. The firm was suffering financially and that is the primary motivation.’ He added that the diamond deal had, in fact, turned out to be an advance fee fraud operated by criminals in Ghana. Webster, a former lawyer at Lehman Brothers International, set up his own firm in 1998 after four years as a partner at Conway & Co. Solicitors. He was named in the Lawyer Magazine ‘Hot 100’ in 2004 and described as ‘one of an influential new generation’ of lawyers. In July that year, he was elected as a. Council Member of the Law Society and the following year, he joined the. board of the City of London Law Society. Shamed: Webster (above) took tens of thousands of pounds from the client account of his firm Webster Dixon LLP. He then funnelled the cash into the account of an associate who hoped to make money on diamonds. In November 2007, Webster was elected chairman of the Black Solicitors Network, the largest organisation for minority lawyers in Europe. He also spent four years as secretary for the Society of Black Lawyers. Tyrone Smith, defending, told the court that Webster's good character and references from dozens of fellow lawyers, including a former President of the Law Society, meant he should be spared jail. He said: ‘This man has been broken by his conduct. He will never be able to. practise in the job he loves, his standing amongst his friends is. indelibly stained by his actions. Firm: Webster Dixon LLP (above) made history as first company founded by black lawyers in the City of London. He has brought the good name of his family into disrepute. There is not a day that goes by that he doesn’t consider it.’ He added that the lawyer had always. intended for the money to be returned to the client account once the diamond. deal had been successful. ‘The firm stood to receive significant. fees for the success of the deal,’ he said. ‘This is not a case where. there is a simple taking of clients’ money to enrich the defendant.’ But Judge Nic Madge told Webster that his behaviour had been so serious that he had no option but to send him to prison. He told me: 'You abused a position of trust not just as a solicitor but as a compliance officer for finance and administration in the firm. ‘I accept the motivation for the fraud was financial pressure. This is a personal tragedy for you and your family. ‘You have let yourself down, you have let your family down. You have let your professional partners and your clients down. You have thrown away what you have worked so very hard to achieve.’ Webster, from Streatham Hill, south London, was originally accused of plundering a total £104,000, but the prosecution later admitted the true figure was £75,605.57. He has already paid back £27,000 and has promised to repay the rest of the money. He also faces being struck off by the Solicitors Regulation Authority

Summary: Michael Webster stole £75,000 from clients of his firm Webster Dixon LLP. Funnelled the cash into associate's account to prop up diamond business. But deal turned out to be advance fee fraud operated by foreign criminals. Webster was today jailed for eight months for fraud by abuse of position. Lawyer, of Streatham Hill, is ex-chairman of the Black Solicitors network. Webster Dixon LLP was the first firm founded by black lawyers in London.

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Summarize: Have an extra million dollars lying around and an interest in vintage tech? It's your lucky day. An old-school Apple-1 — aka the first Apple computer — is now up for auction. Apple fans know that this doesn't happen often, so if you're a serious tech collector with money to burn, you better grab your wallet. Any Apple-1 that hits the auction block is sure to bring in some serious coin, but this one may top the others. According to the Charitybuzz listing, the model now up for grabs is the "most unique, and quite possibly the first, Apple-1 ever created." The "Celebration" Apple-1 on the auction block "is extremely rare not only because of the scarcity of Apple-1 computers, but according to Steve Wozniak, co-founder of Apple Computer, no known PCB boards of this type were ever sold to the public," the listing reads. "At this time, this is the only known Apple-1 to show the signs of starting out as a blank original-run board and not part of the two known production runs, so this board appears to be unique from all other known Apple-1 boards." The coveted model is currently sitting at $270,000 with six days left to go in the auction, which will benefit the The Leukemia & Lymphoma Society of Arizona. That's actually quite a deal for this model, considering it's worth an estimated $1 million, according to Charitybuzz. The highest bidder will walk away with the Apple-1, copyright dated 1976, plus a whole lot of other goodies: 4K byte RAM expansion memory (for a total of 8K), a cassette interface daughter board, BASIC program cassette tape, Star Trek and blackjack program cassette tape, all the original manuals and fliers, a sales receipt from previous owner to current owner, and a notarized condition summary report by Apple expert and historian Corey Cohen vouching for its authenticity. Cohen says this model has the potential to be powered up with a little work, though he advises against doing so. A rare Apple 1 computer, possibly even a prototype, is up for auction on Charitybuzz; bids have already topped $270,000 with six days remaining. The computer differs from many of the production units in ways experts say make it likely the unit is one of the earliest of the Apple machines. “It’s the rookie card, for lack of a better word,” Apple expert Corey Cohen told Recode. The Apple 1 up for auction contains original manuals, a cassette tape with BASIC programming language and other accessories and documentation. Cohen said most of the early machines were hand finished by either Dan Kottke or Steve Jobs, and there’s a good chance this one was put together by Jobs. “Dan doesn’t remember assembling this one,” Cohen said, saying he talked to the early Apple employee about the machine up for auction. The BASIC cassette, which Kottke did create, contains the words “Good Luck” written in his handwriting. Cohen said the original owner of the unit is not known. The present owner bought it back in 2000 for $18,000, then the going rate for such machines. While it’s hard to say how much the machine will fetch now, other Apple 1s have garnered as much as $900,000. A portion of the proceeds from this auction are set to benefit the Leukemia & Lymphoma Society of Arizona. Enlarge Image Photo by CharityBuzz If you are a fan of Apple, want to own a piece of tech history and have oodles of cash to burn, you'll be happy to learn a very rare Apple 1 computer has just found its way on to the auction block. Apple's first ever computer, the Apple 1, was originally released in 1976 for the ominous price of $666.66 (equivalent to around $2,770 in today's money). About 200 Apple 1s were made. Of those, 175 were sold and only around 70 are accounted for today. The auction is happening right now on Fundraising site Charitybuzz, with the highest bid standing at $500,000 -- half the site's estimated value of $1 million. While this number may seem nuts, a similar Apple 1 was auctioned off for $900,000 in late 2014. The early Apple 1s were actually handmade by Jobs and Wozniak, which is what makes this particular model on auction special. Called the "Celebration" Apple 1, this computer is rare, according to Wozniak, because no known printed circuit boards of this type had ever been sold to the public. That makes this particular Apple 1 most likely a prototype or a pre-production unit of some sort. In addition to the machine, the winner of the auction will get a cassette interface board for running programs, a BASIC program cassette tape and two games: Star Trek and Blackjack on cassette tape. The winner will also get all the manuals, schematics, a sales receipt from the previous owner and a notarised condition report by Apple historian Corey Cohen that vouches for its authenticity.

Summary: An early Apple computer is up for auction, and it's a safe bet the winning bidder will be shelling out north of $1 million. The reason? The Apple-1 being sold at Charitybuzz appears to be a rare prototype likely hand-finished by Steve Jobs himself four decades ago, reports Recode. In fact, it is "quite possibly the first Apple-1 ever created," says the site. With three days left, the top bid was $505,000 on Monday morning, but given the lineage of this one-and the fact that the last Apple-1 up for auction fetched $900,000-it won't be a big surprise if the million-dollar barrier is broken, notes CNET. The current owner picked it up for $18,000 in 2000. "It's the rookie card," Apple expert Corey Cohen tells Recode. He says these early models, built by Steve Wozniak, were finished by Jobs or Dan Kottke, and Kottke doesn't recall working on this particular one. What's more, the auction site says the computer has a circuit board that appears to be unique to all other Apple-1s. The board didn't end up in production, further suggesting this is a prototype. The winning bidder gets a variety of add-ons, including a BASIC program cassette and the games Star Trek and Blackjack, also on cassette. Cohen, however, advises against trying to power up the computer, notes PC Mag. Some proceeds will go to the Leukemia & Lymphoma Society of Arizona.

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Write a title and summarize: SECTION 1. SHORT TITLE; FINDINGS. (a) Short Title.--This Act may be cited as the ``Christopher Bryski Student Loan Protection Act'' or ``Christopher's Law''. (b) Findings.--Congress finds the following: (1) No requirement exists for private educational lenders' promissory notes to include a clear and conspicuous description of the responsibilities of a borrower and cosigner in the event the borrower or cosigner becomes disabled, incapacitated, or dies. (2) An estimated 1,700,000 people sustain a traumatic brain injury each year, with older adolescents aged 15 to 19 years old more likely to sustain a traumatic brain injury than other age groups. (3) It has been estimated that the annual incidence of spinal cord injury, not including those who die at the scene of an accident, is approximately 40 cases per 1,000,000 people in the United States or approximately 12,000 new cases each year. These injuries can lead to permanent disability or loss of movement and can prohibit the victim from engaging in any substantial gainful activity. (4) In the 2007-2008 academic year, 13 percent of students attending a 4-year public institution of higher education, and 26.2 percent of students attending a 4-year private institution of higher education, borrowed monies from private educational lenders. (5) According to Sallie Mae, in 2009, the percentage of cosigned private education loans increased from 66 percent to 84 percent of all private education loans. SEC. 2. ADDITIONAL STUDENT LOAN PROTECTIONS. (a) In General.--Section 140 of the Truth in Lending Act (15 U.S.C. 1650) is amended by adding at the end the following: ``(g) Additional Protections Relating to Death or Disability of Borrower or Cosigner of a Private Education Loan.-- ``(1) Clear and conspicuous description of cosigner's obligation.--In the case of any private educational lender who extends a private education loan for which any cosigner is jointly liable, the lender shall clearly and conspicuously describe, in writing, the cosigner's obligations with respect to the loan, including the effect the death, disability, or inability to engage in any substantial gainful activity of the borrower or any cosigner would have on any such obligation, in language that the Bureau determines would give a reasonable person a reasonable understanding of the obligation being assumed by becoming a cosigner for the loan. ``(2) Model form.--The Bureau shall publish a model form under section 105 for describing a cosigner's obligation for purposes of paragraph (1). ``(3) Definition of death, disability, or inability to engage in any substantial gainful activity.--For the purposes of this subsection with respect to a borrower or cosigner, the term `death, disability, or inability to engage in any substantial gainful activity'-- ``(A) means any condition described in section 437(a) of the Higher Education Act of 1965 (20 U.S.C. 1087(a)); and ``(B) shall be interpreted by the Bureau in such a manner as to conform with the regulations prescribed by the Secretary of Education under section 437(a) of such Act (20 U.S.C. 1087(a)) to the fullest extent practicable, including safeguards to prevent fraud and abuse.''. (b) Definitions.--Section 140(a) of the Truth in Lending Act (15 U.S.C. 1650(a)) is amended-- (1) by redesignating paragraphs (1) through (8) as paragraphs (2) through (9), respectively; and (2) by inserting before paragraph (2) (as redesignated by paragraph (1)) the following: ``(1) the term `cosigner'-- ``(A) means any individual who is liable for the obligation of another without compensation, regardless of how designated in the contract or instrument; ``(B) includes any person whose signature is requested as condition to grant credit or to forbear on collection; and ``(C) does not include a spouse of an individual referred to in subparagraph (A) whose signature is needed to perfect the security interest in the loan;''. SEC. 3. FEDERAL STUDENT LOANS. Section 485(l)(2) of the Higher Education Act of 1965 (20 U.S.C. 1092(l)(2)) is amended by adding at the end the following: ``(L) Information on the conditions required to discharge the loan due to the death, disability, or inability to engage in any substantial gainful activity of the borrower in accordance with section 437(a), and an explanation that, in the case of a private education loan made through a private educational lender (as such terms are defined in section 140 of the Truth in Lending Act (15 U.S.C. 1650)), the borrower, the borrower's estate, and any cosigner of such a private education loan may be obligated to repay the full amount of the loan, regardless of the death or disability of the borrower or any other condition described in section 437(a).''.

Title: A bill to amend the Truth in Lending Act and the Higher Education Act of 1965 to require additional disclosures and protections for students and cosigners with respect to student loans, and for other purposes Summary: Christopher Bryski Student Loan Protection Act or Christopher's Law - Amends the Truth in Lending Act to require lenders of private educational loans for which cosigners are held jointly liable to describe clearly and conspicuously, in writing, the cosigners' obligations regarding such loans, including the effect a borrower's or cosigner's death, disability, or inability to engage in any substantial gainful activity would have on such obligations. Directs the Consumer Financial Protection Bureau (CFPB) to publish a model form for describing those obligations. Amends the Higher Education Act of 1965 to require institutions of higher education to provide borrowers of federal educational loans information at their entrance counseling on the effect their death, disability, or inability to engage in any substantial gainful activity would have on their federal and private educational loans.

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Summarize: I sat one evening in my laboratory; the sun had set, and the moon was just rising from the sea; I had not sufficient light for my employment, and I remained idle, in a pause of consideration of whether I should leave my labour for the night, or hasten its conclusion by an unremitting attention to it. As I sat, a train of reflection occurred to me, which led me to consider the effects of what I was now doing. Three years before I was engaged in the same manner, and had created a fiend whose unparalleled barbarity had desolated my heart, and filled it for ever with the bitterest remorse. I was now about to form another being, of whose dispositions I was alike ignorant; she might become ten thousand times more malignant than her mate, and delight, for its own sake, in murder and wretchedness. He had sworn to quit the neighbourhood of man, and hide himself in deserts; but she had not; and she, who in all probability was to become a thinking and reasoning animal, might refuse to comply with a compact made before her creation. They might even hate each other; the creature who already lived loathed his own deformity, and might he not conceive a greater abhorence for it when it came before his eyes in the female form? She also might turn with disgust from him to the superior beauty of man; she might quit him, and he be again alone, exasperated by the fresh provocation of being deserted by one of his own species. Even if they were to leave Europe, and inhabit the deserts of the new world, yet one of the first results of those sympathies for which the daemon thirsted would be children, and a race of devils would be propagated upon the earth, who might make the very existence of the species of man a condition precarious and full of terror. Had I a right, for my own benefit, to inflict this curse upon everlasting generations? I had before been moved by the sophisms of the being I had created; I had been struck senseless by his fiendish threats: but now, for the first time, the wickedness of my promise burst upon me; I shuddered to think that future ages might curse me as their pest, whose selfishness had not hesitated to buy its own peace at the price perhaps of the existence of the whole human race. I trembled, and my heart failed within me; when, on looking up, I saw, by the light of the moon, the daemon at the casement. A ghastly grin wrinkled his lips as he gazed on me, where I sat fulfilling the task which he had allotted to me. Yes, he had followed me in my travels; he had loitered in forests, hid himself in caves, or taken refuge in wide and desert heaths; and he now came to mark my progress, and claim the fulfilment of my promise. As I looked on him, his countenance expressed the utmost extent of malice and treachery. I thought with a sensation of madness on my promise of creating another like to him, and, trembling with passion, tore to pieces the thing on which I was engaged. The wretch saw me destroy the creature on whose future existence he depended for happiness, and, with a howl of devilish despair and revenge, withdrew. I left the room, and, locking the door, made a solemn vow in my own heart never to resume my labours; and then, with trembling steps, I sought my own apartment. I was alone; none were near me to dissipate the gloom, and relieve me from the sickening oppression of the most terrible reveries. Several hours past, and I remained near my window gazing on the sea; it was almost motionless, for the winds were hushed, and all nature reposed under the eye of the quiet moon. A few fishing vessels alone specked the water, and now and then the gentle breeze wafted the sound of voices, as the fishermen called to one another. I felt the silence, although I was hardly conscious of its extreme profundity until my ear was suddenly arrested by the paddling of oars near the shore, and a person landed close to my house. In a few minutes after, I heard the creaking of my door, as if some one endeavoured to open it softly. I trembled from head to foot; I felt a presentiment of who it was, and wished to rouse one of the peasants who dwelt in a cottage not far from mine; but I was overcome by the sensation of helplessness, so often felt in frightful dreams, when you in vain endeavour to fly from an impending danger, and was rooted to the spot. Presently I heard the sound of footsteps along the passage; the door opened, and the wretch whom I dreaded appeared. Shutting the door, he approached me, and said, in a smothered voice-- "You have destroyed the work which you began; what is it that you intend? Do you dare to break your promise? I have endured toil and misery: I left Switzerland with you; I crept along the shores of the Rhine, among its willow islands, and over the summits of its hills. I have dwelt many months in the heaths of England, and among the deserts of Scotland. I have endured incalculable fatigue, and cold, and hunger; do you dare destroy my hopes?" "Begone! I do break my promise; never will I create another like yourself, equal in deformity and wickedness." "Slave, I before reasoned with you, but you have proved yourself unworthy of my condescension. Remember that I have power; you believe yourself miserable, but I can make you so wretched that the light of day will be hateful to you. You are my creator, but I am your master;--obey!" "The hour of my weakness is past, and the period of your power is arrived. Your threats cannot move me to do an act of wickedness; but they confirm me in a resolution of not creating you a companion in vice. Shall I, in cool blood, set loose upon the earth a daemon, whose delight is in death and wretchedness. Begone! I am firm, and your words will only exasperate my rage." The monster saw my determination in my face, and gnashed his teeth in the impotence of anger. "Shall each man," cried he, "find a wife for his bosom, and each beast have his mate, and I be alone? I had feelings of affection, and they were requited by detestation and scorn. Man, you may hate; but beware! Your hours will pass in dread and misery, and soon the bolt will fall which must ravish from you your happiness for ever. Are you to be happy, while I grovel in the intensity of my wretchedness? You can blast my other passions; but revenge remains--revenge, henceforth dearer than light or food! I may die; but first you, my tyrant and tormentor, shall curse the sun that gazes on your misery. Beware; for I am fearless, and therefore powerful. I will watch with the wiliness of a snake, that I may sting with its venom. Man, you shall repent of the injuries you inflict." "Devil, cease; and do not poison the air with these sounds of malice. I have declared my resolution to you, and I am no coward to bend beneath words. Leave me; I am inexorable." "It is well. I go; but remember, I shall be with you on your wedding-night." I started forward, and exclaimed, "Villain! before you sign my death-warrant, be sure that you are yourself safe." I would have seized him; but he eluded me, and quitted the house with precipitation: in a few moments I saw him in his boat, which shot across the waters with an arrowy swiftness, and was soon lost amidst the waves. All was again silent; but his words rung in my ears. I burned with rage to pursue the murderer of my peace, and precipitate him into the ocean. I walked up and down my room hastily and perturbed, while my imagination conjured up a thousand images to torment and sting me. Why had I not followed him, and closed with him in mortal strife? But I had suffered him to depart, and he had directed his course towards the main land. I shuddered to think who might be the next victim sacrificed to his insatiate revenge. And then I thought again of his words--"_I will be with you on your wedding-night._" That then was the period fixed for the fulfilment of my destiny. In that hour I should die, and at once satisfy and extinguish his malice. The prospect did not move me to fear; yet when I thought of my beloved Elizabeth,--of her tears and endless sorrow, when she should find her lover so barbarously snatched from her,--tears, the first I had shed for many months, streamed from my eyes, and I resolved not to fall before my enemy without a bitter struggle. The night passed away, and the sun rose from the ocean; my feelings became calmer, if it may be called calmness, when the violence of rage sinks into the depths of despair. I left the house, the horrid scene of the last night's contention, and walked on the beach of the sea, which I almost regarded as an insuperable barrier between me and my fellow-creatures; nay, a wish that such should prove the fact stole across me. I desired that I might pass my life on that barren rock, wearily it is true, but uninterrupted by any sudden shock of misery. If I returned, it was to be sacrificed, or to see those whom I most loved die under the grasp of a daemon whom I had myself created. I walked about the isle like a restless spectre, separated from all it loved, and miserable in the separation. When it became noon, and the sun rose higher, I lay down on the grass, and was overpowered by a deep sleep. I had been awake the whole of the preceding night, my nerves were agitated, and my eyes inflamed by watching and misery. The sleep into which I now sunk refreshed me; and when I awoke, I again felt as if I belonged to a race of human beings like myself, and I began to reflect upon what had passed with greater composure; yet still the words of the fiend rung in my ears like a death-knell, they appeared like a dream, yet distinct and oppressive as a reality. The sun had far descended, and I still sat on the shore, satisfying my appetite, which had become ravenous, with an oaten cake, when I saw a fishing-boat land close to me, and one of the men brought me a packet; it contained letters from Geneva, and one from Clerval, entreating me to join him. He said that nearly a year had elapsed since we had quitted Switzerland, and France was yet unvisited. He entreated me, therefore, to leave my solitary isle, and meet him at Perth, in a week from that time, when we might arrange the plan of our future proceedings. This letter in a degree recalled me to life, and I determined to quit my island at the expiration of two days. Yet, before I departed, there was a task to perform, on which I shuddered to reflect: I must pack my chemical instruments; and for that purpose I must enter the room which had been the scene of my odious work, and I must handle those utensils, the sight of which was sickening to me. The next morning, at day-break, I summoned sufficient courage, and unlocked the door of my laboratory. The remains of the half-finished creature, whom I had destroyed, lay scattered on the floor, and I almost felt as if I had mangled the living flesh of a human being. I paused to collect myself, and then entered the chamber. With trembling hand I conveyed the instruments out of the room; but I reflected that I ought not to leave the relics of my work to excite the horror and suspicion of the peasants, and I accordingly put them into a basket, with a great quantity of stones, and laying them up, determined to throw them into the sea that very night; and in the mean time I sat upon the beach, employed in cleaning and arranging my chemical apparatus. Nothing could be more complete than the alteration that had taken place in my feelings since the night of the appearance of the daemon. I had before regarded my promise with a gloomy despair, as a thing that, with whatever consequences, must be fulfilled; but I now felt as if a film had been taken from before my eyes, and that I, for the first time, saw clearly. The idea of renewing my labours did not for one instant occur to me; the threat I had heard weighed on my thoughts, but I did not reflect that a voluntary act of mine could avert it. I had resolved in my own mind, that to create another like the fiend I had first made would be an act of the basest and most atrocious selfishness; and I banished from my mind every thought that could lead to a different conclusion. Between two and three in the morning the moon rose; and I then, putting my basket aboard a little skiff, sailed out about four miles from the shore. The scene was perfectly solitary: a few boats were returning towards land, but I sailed away from them. I felt as if I was about the commission of a dreadful crime, and avoided with shuddering anxiety any encounter with my fellow-creatures. At one time the moon, which had before been clear, was suddenly overspread by a thick cloud, and I took advantage of the moment of darkness, and cast my basket into the sea; I listened to the gurgling sound as it sunk, and then sailed away from the spot. The sky became clouded; but the air was pure, although chilled by the north-east breeze that was then rising. But it refreshed me, and filled me with such agreeable sensations, that I resolved to prolong my stay on the water, and fixing the rudder in a direct position, stretched myself at the bottom of the boat. Clouds hid the moon, every thing was obscure, and I heard only the sound of the boat, as its keel cut through the waves; the murmur lulled me, and in a short time I slept soundly. I do not know how long I remained in this situation, but when I awoke I found that the sun had already mounted considerably. The wind was high, and the waves continually threatened the safety of my little skiff. I found that the wind was north-east, and must have driven me far from the coast from which I had embarked. I endeavoured to change my course, but quickly found that if I again made the attempt the boat would be instantly filled with water. Thus situated, my only resource was to drive before the wind. I confess that I felt a few sensations of terror. I had no compass with me, and was so little acquainted with the geography of this part of the world that the sun was of little benefit to me. I might be driven into the wide Atlantic, and feel all the tortures of starvation, or be swallowed up in the immeasurable waters that roared and buffeted around me. I had already been out many hours, and felt the torment of a burning thirst, a prelude to my other sufferings. I looked on the heavens, which were covered by clouds that flew before the wind only to be replaced by others: I looked upon the sea, it was to be my grave. "Fiend," I exclaimed, "your task is already fulfilled!" I thought of Elizabeth, of my father, and of Clerval; and sunk into a reverie, so despairing and frightful, that even now, when the scene is on the point of closing before me for ever, I shudder to reflect on it. Some hours passed thus; but by degrees, as the sun declined towards the horizon, the wind died away into a gentle breeze, and the sea became free from breakers. But these gave place to a heavy swell; I felt sick, and hardly able to hold the rudder, when suddenly I saw a line of high land towards the south. Almost spent, as I was, by fatigue, and the dreadful suspense I endured for several hours, this sudden certainty of life rushed like a flood of warm joy to my heart, and tears gushed from my eyes. How mutable are our feelings, and how strange is that clinging love we have of life even in the excess of misery! I constructed another sail with a part of my dress, and eagerly steered my course towards the land. It had a wild and rocky appearance; but as I approached nearer, I easily perceived the traces of cultivation. I saw vessels near the shore, and found myself suddenly transported back to the neighbourhood of civilized man. I eagerly traced the windings of the land, and hailed a steeple which I at length saw issuing from behind a small promontory. As I was in a state of extreme debility, I resolved to sail directly towards the town as a place where I could most easily procure nourishment. Fortunately I had money with me. As I turned the promontory, I perceived a small neat town and a good harbour, which I entered, my heart bounding with joy at my unexpected escape. As I was occupied in fixing the boat and arranging the sails, several people crowded towards the spot. They seemed very much surprised at my appearance; but, instead of offering me any assistance, whispered together with gestures that at any other time might have produced in me a slight sensation of alarm. As it was, I merely remarked that they spoke English; and I therefore addressed them in that language: "My good friends," said I, "will you be so kind as to tell me the name of this town, and inform me where I am?" "You will know that soon enough," replied a man with a gruff voice. "May be you are come to a place that will not prove much to your taste; but you will not be consulted as to your quarters, I promise you." I was exceedingly surprised on receiving so rude an answer from a stranger; and I was also disconcerted on perceiving the frowning and angry countenances of his companions. "Why do you answer me so roughly?" I replied: "surely it is not the custom of Englishmen to receive strangers so inhospitably." "I do not know," said the man, "what the custom of the English may be; but it is the custom of the Irish to hate villains." While this strange dialogue continued, I perceived the crowd rapidly increase. Their faces expressed a mixture of curiosity and anger, which annoyed, and in some degree alarmed me. I inquired the way to the inn; but no one replied. I then moved forward, and a murmuring sound arose from the crowd as they followed and surrounded me; when an ill-looking man approaching, tapped me on the shoulder, and said, "Come, Sir, you must follow me to Mr. Kirwin's, to give an account of yourself." "Who is Mr. Kirwin? Why am I to give an account of myself? Is not this a free country?" "Aye, Sir, free enough for honest folks. Mr. Kirwin is a magistrate; and you are to give an account of the death of a gentleman who was found murdered here last night." This answer startled me; but I presently recovered myself. I was innocent; that could easily be proved: accordingly I followed my conductor in silence, and was led to one of the best houses in the town. I was ready to sink from fatigue and hunger; but, being surrounded by a crowd, I thought it politic to rouse all my strength, that no physical debility might be construed into apprehension or conscious guilt. Little did I then expect the calamity that was in a few moments to overwhelm me, and extinguish in horror and despair all fear of ignominy or death. I must pause here; for it requires all my fortitude to recall the memory of the frightful events which I am about to relate, in proper detail, to my recollection.

Summary: At last Victor sets about creating the second monster. As he suspected, the monster had trailed Victor and Henry's tour through Europe into England. He is anxious to see his mate and he approaches Victor's workshop on the remote island in the Orkneys. With mixed feelings of guilt and temper Victor destroys the half-finished creature in front of the monster, saying that he will not continue. The monster says, Do you dare to break your promise? I have endured toil and misery '''. I have dwelt many months in the heaths of England and among the deserts of Scotland. I have endured incalculable fatigue and cold and hunger. Do you dare destroy my hopes? "Victor responds, Begone! I do break my promise; never will I create another like yourself, equal in deformity and wickedness." The monster disappears into the night and Victor worries who the next victim of the creature's evil will be next. Victor prepares to return home, but he must first destroy his laboratory, and dispose of the body parts he has accumulated. He sets out in a boat in order to dispose of the body parts, but a storm pushes him out to sea and eventually he is cast ashore in Ireland and he is unceremoniously taken to the local magistrate, Mr. Kirwin, accused of murder.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Federal Consent Decree Fairness Act''. SEC. 2. FINDINGS. Congress finds that: (1) Consent decrees are for remedying violations of requirements of Federal law, and they should not be used to advance any policy extraneous to that purpose. (2) Consent decrees are also for protecting the party or class facing injury and should not be expanded to apply to parties not involved in the litigation. (3) In structuring consent decrees, courts should take into account the interests of State and local governments in managing their own affairs. (4) Consent decrees should be structured and administered to give due deference to the policy judgments of State and local officials, and their successors, as to how to obey the law. (5) Whenever possible, courts should not impose consent decrees that require technically complex and evolving policy choices, especially in the absence of judicially discoverable and manageable standards. (6) Consent decrees should not be unlimited, but should contain an explicit and realistic strategy for ending court supervision. SEC. 3. LIMITATION ON CONSENT DECREES. (a) In General.--Chapter 111 of title 28, United States Code, is amended by adding at the end the following: ``Sec. 1660. Consent decrees ``(a) Definitions.--In this section, the term `consent decree'-- ``(1) means any order imposing injunctive or other prospective relief against a State or local government, or a State or local official sued, entered by a court of the United States that is based in whole or part upon the consent or acquiescence of the parties; and ``(2) does not include-- ``(A) private settlements agreements; ``(B) any order arising from an action filed against a government official that is unrelated to his or her official duties; ``(C) any order entered by a court of the United States to implement a plan to end segregation of students or faculty on the basis of race, color, or national origin in elementary schools, secondary schools, or institutions of higher education; and ``(D) any order entered in any action-- ``(i) filed by the United States or any agency of the United States, except for reporting requirements provided under section 4 of the Federal Consent Decree Fairness Act; or ``(ii) in which 1 State is an adverse party to another State. ``(b) Limitation on Duration.-- ``(1) In general.--A State or local government, or a State or local official who was a party to the consent decree (or the successor to that individual) may file a motion under this section with the court that entered a consent decree to modify or terminate the consent decree upon the earlier of-- ``(A) 4 years after a consent decree is originally entered by a court of the United States, regardless if the consent decree has been modified or reentered during that period; or ``(B) in the case of a civil action in which-- ``(i) a State or an elected State official is a party, the expiration of the term of office of the highest elected State official who was a party to the consent decree; ``(ii) a local government or elected local government official is a party, the expiration of the term of office of the highest elected local government official who was a party to the consent decree; or ``(iii) the consent to the decree was authorized by an appointed State or local official, upon the expiration of the term of office of the elected official who appointed that State or local official, or the highest elected official in that State or local government; or ``(C) the date otherwise provided by law. ``(2) Burden of proof.-- ``(A) In general.--With respect to any motion filed under paragraph (1), the burden of proof shall be on the party who originally filed the civil action to demonstrate that the denial of the motion to modify or terminate a consent decree or any part of a consent decree is necessary to prevent the violation of a requirement of Federal law that-- ``(i) was actionable by such party; and ``(ii) was addressed in the original consent decree. ``(B) Failure to meet burden of proof.--If a party fails to meet the burden of proof described in subparagraph (A), the court shall terminate the consent decree. ``(C) Satisfaction of burden of proof.--If a party meets the burden of proof described in subparagraph (A), the court shall ensure that any remaining provisions of the consent decree represent the least restrictive means by which to prevent such a violation. ``(3) Ruling on motion.-- ``(A) In general.--The court shall rule expeditiously on a motion filed under this subsection. ``(B) Scheduling order.--Not later than 30 days after the filing of a motion under this subsection, the court shall enter a scheduling order that-- ``(i) limits the time of the parties to-- ``(I) file motions; and ``(II) complete any required discovery; and ``(ii) sets the date or dates of any hearings determined necessary. ``(C) Stay of injunctive or prospective relief.--In addition to any other orders authorized by law, the court may stay the injunctive or prospective relief set forth in the consent decree in an action under this subsection if a party opposing the motion to modify or terminate the consent decree seeks any continuance or delay that prevents the court from entering a final ruling on the motion within 180 days of the filing of the motion. ``(c) Other Federal Court Remedies.--The provisions of this section shall not be interpreted to prohibit a Federal court from entering a new order for injunctive or prospective relief to the extent that it is otherwise authorized by Federal law. ``(d) Available State Court Remedies.--The provisions of this section shall not prohibit the parties from seeking appropriate relief under State law.''. (b) Technical and Conforming Amendment.--The table of sections for chapter 111 of title 28, United States Code, is amended by adding at the end the following: ``1660. Consent decrees.''. SEC. 4. DEPARTMENT OF JUSTICE REPORT. (a) In General.--Not later than October 1 of each year, the Attorney General shall submit a report to Congress on all consent decrees in which the United States is a party where the consent decrees were entered 4 or more years prior to the date of the report. (b) Content of Reports.--The report required under subsection (a) shall include-- (1) copies of any consent decrees described in subsection (a); and (2) a written statement by the Attorney General or other agency head explaining-- (A) why each consent decree listed in the report requires continued court supervision; and (B) any efforts the United States had made to limit the scope or duration of the consent decree. (c) Preparation of Report.-- (1) In general.--Federal agencies other than the Department of Justice shall provide the information required in this section to the Attorney General not later than September 1 of each year. (2) Input.--In preparing the report required under subsection (a), the Attorney General or other agency head shall solicit, and include in the report, statements relating to each consent decree from State and local officials who-- (A) support continued court supervision; and (B) oppose continued court supervision. (d) Electronic Submission.--Copies of consent decrees required by subsection (b)(1)(B) may be submitted in electronic format. SEC. 5. GENERAL PRINCIPLES. (a) No Effect on Other Laws Relating to Modifying or Vacating Consent Decrees.--Nothing in the amendments made by section 3 shall be construed to preempt or modify any other provision of law providing for the modification or vacating of a consent decree. (b) Further Proceedings Not Required.--Nothing in the amendments made by section 3 shall be construed to affect or require further judicial proceedings relating to prior adjudications of liability or class certifications. SEC. 6. EFFECTIVE DATE. The amendments made by this Act shall take effect on the date of enactment of this Act and apply to all consent decrees regardless of-- (1) the date on which the order of a consent decree is entered; or (2) whether any relief has been obtained under a consent decree before the date of enactment of this Act.

Title: A bill to amend chapter 111 of title 28, United States Code, to limit the duration of Federal consent decrees to which State and local governments are a party, and for other purposes Summary: Federal Consent Decree Fairness Act - Amends the federal judicial code to authorize any state or local government or related official (or successor) to file a motion to modify or terminate a federal consent decree upon the earlier of: (1) four years after the consent decree is originally entered; or (2) in the case of a civil action in which a state or state official, or a local government or local government official, is a party, the expiration of the term of office of the highest state or local government official who was a party to the consent decree; or (3) the date otherwise provided by law. Places the burden of proof with respect to such motions on the party originally filing the action to demonstrate that the denial of the motion to modify or terminate a consent decree (or any part of it) is necessary to prevent the violation of a federal requirement that: (1) was actionable by such party; and (2) was addressed in the original consent decree. Requires a court, within 30 days after the filing of a motion, to enter a scheduling order that: (1) limits the time of the parties to file motions and complete discovery; and (2) sets the date or dates of any necessary hearings. Authorizes a court to stay the injunctive or prospective relief set forth in the consent decree if a party opposing the motion to modify or terminate it seeks any continuance or delay that prevents the court from entering a final ruling on the motion within 180 days of its filing. Requires the Attorney General to report annually to Congress on all consent decrees in which the United States is a party that were entered four or more years before the date of the report.

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Summarize: Phil Ivey pauses during a hand at the final table at the 2009 World Series of Poker at the Penn & Teller Theater at the Rio Hotel in Las Vegas, Nevada, on Nov. 7, 2009. Phil Ivey pauses during a hand at the final table at the 2009 World Series of Poker at... Read More Phil Ivey pauses during a hand at the final table at the 2009 World Series of Poker at the Penn & Teller Theater at the Rio Hotel in Las Vegas, Nevada, on Nov. 7, 2009. Close Professional poker player Phil Ivey’s use of edge sorting to win 7.7 million pounds ($12.4 million) at a form of Baccarat was tantamount to cheating, a London judge ruled today. Judge John Mitting, who said Ivey was an honest witness, ruled against the 38-year-old gambler’s bid to recoup money a Genting Bhd. (GENT) casino had withheld. While he might not have realized it was cheating, Mitting said Ivey and a companion influenced a croupier to deal the cards in certain ways. Ivey, a 10-time winner of the World Series of Poker tournament, won the money playing Punto Banco at Genting’s Crockfords casino in London. He argued that edge sorting was a legitimate tactic to gain an advantage over the casino. “He gave himself an advantage which the game precludes,” Mitting said today following a week-long trial. “This is in my view cheating.” Genting is Southeast Asia’s largest casino operator with a market capitalization of about $35 billion, according to data compiled by Bloomberg. Last year it bought a Las Vegas site, once home to the Stardust resort, for $350 million. “We attach the greatest importance to our exemplary reputation for fair, honest and professional conduct and today’s ruling vindicates the steps we have taken in this matter,” Crockfords said in an e-mailed statement after the verdict. 2012 Game Both sides agreed at trial that Ivey was in the casino in August 2012 and that he won the money. “The issue is whether it amounted to cheating,” Christopher Pymont, Genting’s lawyer, said in documents filed at London’s High Court. Edge sorting is a way a card player can gain an advantage by working out the value of a card by spotting flaws or particular patterns on the back of some cards. Ivey and a companion unfairly influenced a croupier to move and deal the cards in certain ways without her knowing what she was doing, the judge said. Ivey cheated “by using the croupier as his innocent agent or tool,” Mitting said. Ivey has career earnings of more than $21 million from live tournaments alone, according to his website. The judge described him as one of the “world’s finest poker players.” Ivey describes himself as an “advantage player,” someone who is highly skilled at trying to tip the odds in his favor. “It is not in my nature to cheat,” Ivey said through a spokesman after today’s ruling. “I believe what we did was nothing more than exploit Crockford’s failures. Clearly the judge did not agree.” Judge Mitting turned down Ivey’s permission to appeal his verdict. To contact the reporter on this story: Jeremy Hodges in London at jhodges17@bloomberg.net To contact the editors responsible for this story: Anthony Aarons at aaarons@bloomberg.net Peter Chapman Top poker player Phil Ivey has lost his high court case against the owners of Crockfords Club in London over his £7.7m ($12.5m) winnings. The 38-year-old American sued over a version of baccarat known as Punto Banco that he played at the Mayfair casino over two days in August 2012. After four sessions, Ivey was told the money would be wired to him and he left for the US, but it never arrived, although his £1m stake was returned. Genting Casinos UK, which owns more than 40 casinos in the UK including Crockfords, said the technique of “edge-sorting” Ivey used – which aims to provide the customer with an element of “first card advantage” – was not a legitimate strategy and the casino had no liability to pay him. Its lawyers told Mr Justice Mitting in London that Ivey’s conduct defeated the essential premise of the game of baccarat so there was no gaming contract, or constituted cheating. A spokesman for Crockfords said later: “Crockfords is pleased with the judgment of the high court today, supporting its defence of a claim by Ivey. “It is our policy not to discuss our clients’ affairs in public and we very much regret that proceedings were brought against us. We attach the greatest importance to our exemplary reputation for fair, honest and professional conduct and today’s ruling vindicates the steps we have taken in this matter.” Speaking through a spokesman, Ivey said: “ I am obviously disappointed with this judge’s decision. As I said in court, it is not my nature to cheat and I would never do anything to risk my reputation. “I am pleased that the judge acknowledged in court that I was a truthful witness. “ I believe that what we did was a legitimate strategy and we did nothing more than exploit Crockfords’ failures to take proper steps to protect themselves against a player of my ability. “Clearly today the judge did not agree.” Lawyers for Ivey were refused permission to appeal although they can renew their application to the court of appeal directly. Ivey’s counsel, Richard Spearman QC, told the court that edge-sorting involved nothing more than using information that was available to any player simply from viewing the backs of the cards that the casino chose to use and making requests of the casino – which it could accept or refuse – as to the manner in which play was conducted. The casino’s counsel, Christopher Pymont QC, said that Ivey, who lives in Las Vegas and is described on the World Series of Poker website as “arguably the best poker player in the world”, was not a well-known advantage player at the time of his visit but was, in their eyes, an old VIP customer and they trusted him accordingly. It argued that edge-sorting was not a widely known or practised way of playing baccarat in the UK. Ivey, who was accompanied at Crockfords by another professional gambler, Cheung Yin Sun, who introduced him to edge-sorting, said cheating was anathema to advantage players like him. “We observe the unwritten doctrine: how do I find a legal way to beat the house? Any method that could amount to cheating would breach the doctrine and cause you to be ostracised by your fellow players – we are all very careful to stay the right side of the line and we discuss advantage play strategies at length.” He said he was very angry when he heard the casino would not be paying out his winnings: “I was upset as I had played an honest game and won fairly. “My integrity is infinitely more important to me than a big win, which is why I have brought these proceedings to demonstrate that I have been unjustly treated.” In his ruling, the judge said that the case turned on whether there was cheating: “If Mr Ivey cheated, he is not entitled to recover his winnings. If he did not, he is. “What Mr Ivey and Ms Sun did was to persuade the croupier to turn some of the cards in the dealing shoe to permit them to know that they were or were very likely to be sevens, eights or nines, and in circumstances where she did not realise she had done so – and, if she had, would have immediately stopped play. “The fact that Mr Ivey was genuinely convinced that he did not cheat and that the practice commanded considerable support from others was not determinative of the question of whether it amounted to cheating. “Mr Ivey had gained himself an advantage and did so by using a croupier as his innocent agent or tool. “It was not simply taking advantage of error on her part or an anomaly practised by the casino, for which he was not responsible. “He was doing it in circumstances where he knew that she and her superiors did not know the consequences of what she had done at his instigation. The judge concluded: “This is, in my view, cheating for the purpose of civil law.” Dismissing the case, with costs, he said it was immaterial that the casino could have protected itself by simple measures.

Summary: A London judge ruled yesterday that a professional poker player used a croupier "as his innocent agent or tool" to cheat a London casino out of $12.4 million, Bloomberg reports. Phil Ivey-described on the World Series of Poker website as "arguably the best poker player in the world" and winner of 10 WSoP tourneys-and a companion influenced the croupier to "move and deal the cards in certain ways without her knowing what she was doing" during a baccarat game at Genting's Crockfords casino in August 2012, according to Judge John Mitting. Mitting said the technique Ivey used during the game of punto banco "gave [Ivey] an advantage which the game precludes," while Ivey insists it was a perfectly legitimate way to "exploit Crockford's failures." Ivey never got the money wired to him after he returned to America, the Guardian reports, so he sued for it. Edge-sorting, the tactic in question, involves carefully studying the backs of cards for any unique markings or flaws to gain an advantage in the game. Ivey reportedly asked for a specific brand of cards to be used, then had his female companion ask the croupier to turn the cards a certain way, "effectively sorting the deck to make the design flaws stand out," ESPN reports. An upset Ivey insists he's no cheater and that he won fair and square. "We observe the unwritten doctrine: How do I find a legal way to beat the house?" he says. "Any method that could amount to cheating would breach the doctrine and cause you to be ostracized by your fellow players-we are all very careful to stay [on] the right side of the line, and we discuss advantage play strategies at length." An Atlantic City casino has also accused Ivey of using the same trick.

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Write a title and summarize: Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine. Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus infecting most of the human population [1,2]. In immunocompetent individuals infection is usually asymptomatic, yet results in the establishment of latency. However, HCMV is the leading cause of congenitally related diseases causing severe and often irreparable birth defects [3–5]. HCMV is also the most common opportunistic infection causing morbidity in immunocompromised patients [6–8]. Due to the high economic and health burden, development of an HCMV vaccine has key public health priority [9,10]. Vaccination of adolescents, or ideally of all children at young age, would be the most effective strategy to reduce the incidence of congenital CMV infection [11]. The immune correlates preventing transmission of CMV across the placenta are not completely defined yet; however, it seems likely that both humoral and cellular immunity are contributing to protection. Vaccination of immunocompromised transplant recipients would be more challenging, because of lower ability to mount immune responses and also due to safety concerns; nonetheless, at least in solid organ transplant patients, inducing or boosting immunity before transplantation would be feasible and promising. However, in both settings, clinical trials with subunit vaccines were only partially efficacious in preventing infection [12,13]. Humoral and cellular immunity can more effectively be achieved by application of a live vaccine. Indeed several preclinical studies in animal models, including in non-human primates, revealed a robust capacity of attenuated CMVs to elicit a potent memory T cell response [14–20]. With the exception of the well-established Oka vaccine that provides excellent protection against varicella-zoster virus, no attenuated vaccine against other herpesviruses has been approved. There are several difficulties that hamper the development of an effective live HCMV vaccine. The ability of HCMV to reactivate and to re-infect seropositive individuals indicates that immunity resulting from primary infection cannot completely prevent subsequent infections [21–25]. Another challenge is to accomplish an adequate balance between safety and immunogenicity. For instance, a live HCMV vaccine based on the Towne strain could not prevent infection of renal transplant recipients, but lowered severity of CMV disease [26,27], suggesting that this vaccine strain was over-attenuated. Thus, there is a need to rationally engineer an HCMV vaccine that induces comparable or ideally better immunity than natural infection and at the same time presents an excellent safety profile. One approach is the generation of chimeras between the Towne and Toledo strains and these are currently investigated in clinical trials [28,29]. Numerous viral encoded immunoevasins prevent the development of full-blown CMV-specific immunity. Accordingly, genetically modified animal CMVs lacking immunoevasins exhibited outstanding vaccine properties in mouse and guinea pig models [30,31]. Recently, we found that a mouse CMV (MCMV) expressing a host ligand for the activating receptor NKG2D induces an efficient CD8+ T cell response despite being profoundly attenuated [15,32]. Moreover, challenge infection experiments indicated that the protection obtained after immunization is superior to the one seen after natural infection [15,32]. It is highly desirable to test the suitability of similar approaches for a recombinant HCMV vaccine. However, the strategy successfully established for MCMV cannot simply be translated to HCMV. Divergence between rodents and primates started ~90 million years ago and resulted in some differences of the immune system of mouse and man [33]. Similarly, co-evolution with the respective hosts led to even larger dissimilarity of viral virulence factors [34,35]. In this study, we report on the construction and evaluation of a recombinant HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor. We found that cells infected with the ULBP2-expressing strain increased the cytolytic activity of NK cells in an NKG2D-dependent manner, thereby preventing viral spread and containing the viral infection to few cells. Still, the strain exhibited a robust capacity to activate HCMV-specific T cells. Interestingly, the mechanisms steering T cell activation turned out to be different for the viral strains. Altogether, our data imply that the chosen strategy allows attenuation of an HCMV strain, while retaining its ability to stimulate innate NK cell and adaptive T cell immunity. To generate an HCMV strain for testing a novel vaccination approach that aims at activation of NK cells as well as T cells we used the bacterial artificial chromosome (BAC) -cloned strain TB40/E (herein referred as TB40) [36] that displays several features useful for vaccine development. In contrast to most other HCMV isolates, after extended passages in fibroblasts TB40 retains a complex of the glycoproteins gH/gL/UL128-UL131A (called pentameric complex) on the envelope of its virion important for broad cell tropism and produces a high rate of cell free virus, ultimately facilitating large scale production under GMP conditions [36]. TB40 lacks the immunoevasins US2, US3 and US6 [36] involved in MHC class I downregulation, which is expected to improve antigen presentation and priming of the CD8+ T cell response [37]. Note that US11 –another MHC class I immunoevasin–is retained. In order to attenuate the virus (named ULBP2-TB40) and to render it sensitive to NK cell control we inserted the gene for the host NKG2D ligand ULBP2 in the viral genome and replaced UL16, which normally interferes with expression of NKG2D ligands in infected cells [38–40] (Fig 1A). The other NK cell evasion genes present in the parental strain [36] were retained. Insertion of the ULBP2 open reading frame (ORF) had no influence on the viral growth kinetics in vitro (S1 Fig). In human foreskin fibroblasts (HFF) infected with ULBP2-TB40, strong ULBP2 surface expression was observed, whereas in cells infected with the parental strain TB40 no ULBP2 could be detected (Fig 1B). Expression of ULBP2 in HCMV infected cells led to substantially increased lysis by NK cells (for all donors tested) when compared to cells infected with TB40 (Fig 1C and 1D). Blocking of the NKG2D receptor with a specific antibody indicated that for NK cells of all donors analyzed the observed gain of cytotoxicity against ULBP2-TB40 infected cells was mediated via an NKG2D-dependent mechanism (Fig 1D). To determine the effect of the improved NK cell response on viral spread, we compared the ability of primary human NK cells to control the transmission of the viruses from infected to non-infected neighboring cells by applying a focus expansion assay [41]. NK cells could limit the spread of the TB40 virus as reported previously [41]. However, spread of the ULBP2 expressing strain was markedly more inhibited leading to less infected cells per focus of infection (Fig 1E) as well as a lower number of infectious foci in cell cultures (S2 Fig). Blocking of the NKG2D receptor with antibodies reduced the NK cell control of ULBP2-TB40 to the level observed for TB40 (Fig 1F), confirming that the observed additional gain of control is mediated by an NKG2D-dependent mechanism. Taken together, viral expression of ULBP2 leads to increased NK cell-mediated cytotoxicity that limits viral spread. Mice reconstituted with human hematopoietic stem cells can serve as models for infection with several human-specific viruses [42,43] and the use of such a model has also been reported for HCMV [44]. Thus, to assess whether the ULBP2-TB40 strain can induce an immune response in vivo we performed infection experiments in humanized mice. For a pilot experiment NOD/SCID/IL2Rcγc mice expressing human HLA-A02: 01 molecules and engrafted with fetal liver-derived human CD34+CD38- hematopoietic stem cells (humanized NSG-A2 mice) were injected with HCMV-infected fibroblasts as described [44]. Although viral DNA could be detected in spleens of infected animals, we did not find hints for the generation of HCMV-specific CD8+ T cells, probably due to only low numbers of human myeloid cells present in these mice [45]. Thus, for subsequent experiments we injected humanized mice with uninfected or TB40 or ULBP2-TB40 infected DC (Fig 2A). Human CD3+ T cells present in periarteriolar lymphoid sheaths (PALS) were found in spleens at day 18 p. i., when the humanized NSG-A2 mice were sacrificed and organs examined, irrespectively on whether mice were injected with uninfected or infected DC (Fig 2B). The frequency of human CD8+ and CD4+ T cells, as well as of CD19+ B cells and CD56+ NK cells in spleens was in a similar range for the groups receiving infected DC (Fig 2C). In livers slightly higher frequencies of NK cells were detected in the group receiving ULBP2-TB40 infected DC than in the group receiving TB40-infected DC (although the differences were not significant) (S3A Fig). Immunophenotyping of the NK cells in spleen revealed that mice injected with ULBP2-TB40 infected cells had a higher percentage of CD57+ NK cells when compared to mice receiving the parental virus TB40 (S3B Fig). Moreover, in some mice injected with ULBP2-TB40 DC IFNγ-expressing NK cells were found (S3C Fig). The CD57 marker is expressed on mature NK cells [46,47] and the detection of IFNγ producing NK cells suggests that they were functional. CMV-specific antibodies appear particularly important for prevention of congenital infection [48]. We therefore examined the ability of the ULBP2-TB40 strain to mount a humoral immune response. Although NSG humanized mice are not ideal for analyzing antibody responses [49,50], CMV-specific IgM antibodies were detected at comparable levels in the serum of both groups following immunization with either strain (Fig 2D). We concluded that the ULBP2-TB40 strain elicits CMV-specific antibodies with similar efficiency as the TB40 strain. Finally, we investigated whether CMV-specific T cells were primed in the humanized mice. When examined at day 14 p. i., CD8+ T cells specific for the HCMV epitopes NLVPMVATV and VLEETSVML (derived from the viral proteins pp65 and IE1) were found in blood of several animals (S4 Fig). Moreover, HCMV-specific CD8+ T cells were also detected in spleens at day 18 p. i. (Fig 2E and S5A Fig). The percentage of IE1-specific CD8+ T cells was comparable between both infected groups (Fig 2E), and this applied also to the absolute numbers of IE1-specific CD8+ T cells (S5A Fig). IFNγ positive CD8+ T cells could be detected at low level in few animals (S5B Fig) following stimulation with a specific peptide, confirming priming of IE1-specific CD8+ T cells. The majority of the CD8+ T cells displayed a phenotype of terminally differentiated effector memory (TEMRA) cells (S5C Fig), independently of the treatment applied. To investigate to which extent the two strains can establish infection in the humanized animals, viral loads were measured in several organs on day 18 p. i. using quantitative PCR (qPCR). Viral DNA was found only in spleens of infected animals, and not in the other organs analyzed. In spleen of most of the animals infected with the TB40 virus viral DNA was detected (Fig 2F), whereas in the animals of the ULBP2-TB40 infected group the viral DNA loads were lower, although the difference in the viral loads was not statistically significant. Taken together, we concluded that the ULBP2-TB40 virus is able to prime HCMV-specific CD8+T cells and induce CMV-specific antibodies in humanized mice to a similar extent as the TB40 strain despite low viral loads. Encouraged by the observation that the ULBP2-TB40 strain could prime CD8 T cells in the humanized mice, we further investigated the activation of CMV-specific T cells by DC infected with this strain. It is well known that HCMV inhibits maturation of infected DC [51–53], and since DC are central for stimulation of T cells, it was important to know how the ULBP2-TB40 virus influences this process. In particular it was hard to predict whether expression of ULBP2 in HCMV infected DC increases the CD8+ T cell response by engaging the costimulatory NKG2D receptor [54,55], or whether it would perhaps lead to T cell exhaustion [56]. To test the ability of TB40 and ULBP2-TB40 infected DC to expand HCMV-specific CD8+ T cells, primary CD8+ T cells isolated from HLA-A02: 01 HCMV-seropositive healthy donors were co-cultured with infected autologous monocyte-derived DC or for control with uninfected DC (Fig 3). Mature DC loaded with the HLA-A02: 01 specific immunodominant peptide NLVPMVATV derived from the pp65 protein were used as a positive control (S6 Fig). DC infected with either of the two viruses induced a comparable percentage of pp65-specific CD8+ T cells (Fig 3A). When tested against pp65 peptide-loaded DC, the pp65-specific CD8+ T cells had comparable cytokine production and degranulation capacity (Fig 3B). The simultaneous measurement of IFNγ, TNFα and degranulation allowed us to determine the multifunctionality of the pp65-specific CD8+ T cells, revealing that there was a trend toward higher multifunctionality of the T cells that were expanded with ULBP2-TB40 infected DC (Fig 3C), although the differences were not statistically significant. Expansion with ULBP2-TB40 infected DC resulted in a lower percentage of CD8+ T cells with central memory phenotype than expansion with pp65-peptide loaded DC (S7A Fig). Both viruses induced similar percentages of effector memory and central memory CD8+ T cells and of antigen-specific CD8+ T cells (S7A and S7B Fig). The frequency of exhausted PD-1+ CD8+ T cells after expansion with DC infected with either virus was in the same range (10 to 30% of total CD8+ T cells) (S7C and S7D Fig). Overall, we concluded that ULBP2-TB40-infected DC induced a comparable percentage and quality of HCMV-specific CD8+ T cells as TB40-infected DC. NK cells have the ability to modulate the outcome of T cell responses against CMV [57,58]. They can influence activation of CMV-specific T cells in a positive manner by providing an appropriate microenvironment and by lysis of infected cells, thereby increasing the amount of available antigens [59,60]. In contrast, NK cells can potentially also limit the activation of T cells by eliminating CMV infected DC [61,62]. Since expression of the ULBP2 molecule by ULBP2-TB40 led to strong NK cell-mediated lysis of infected cells (compare Fig 1), it was important to analyze the effect of NK cells on the induction of T cell responses. To this end, we set up autologous stimulation assays using peripheral blood mononuclear cells (PBMC). PBMC isolated from HCMV-seropositive donors were co-cultured with autologous monocyte-derived DC either uninfected or infected with the respective viruses or–for control–matured and loaded with pp65 peptides. The infection rate of the DC was comparable for both viruses (S8A Fig). Activation of NK cells as assessed by intracellular cytokine staining for proinflammatory cytokines (IFNγ+, TNFα+) and degranulation (CD107a+) was in a similar range for cultures containing either ULBP2-TB40 or TB40-infected DC (Fig 4A). Likewise, the percentage of CD8+ T cells producing IFNγ+ and degranulating (CD107a+) was comparable upon co-cultivation with TB40 or ULBP2-TB40 infected DC (Fig 4B). Similar results were obtained for CD4+ T cells–ULBP2 expression did not impair the ability of CD4+ T cells to produce proinflammatory cytokines (IFNγ and TNFα) in response to infected DC (S8B and S8C Fig). In summary, PBMC co-cultures with ULBP2-TB40 infected DC activated CD8+ and CD4+ T cells as well as NK cells to the same extent as TB40-infected DC and most importantly, ULBP2 expression in the infected DC did not impair the stimulation of the immune effector cells. Previous studies suggested that ULBP2 can co-stimulate CD8+ T cells via the NKG2D receptor [54]. In order to understand how ULBP2-TB40 infected DC activate CD8+ T cells, we first analyzed the maturation phenotype of the DC. Maturation of HCMV-infected DC in terms of CD80, CD86 and HLA DR expression was found to be impaired–as described by others [51–53]–to the same extent for both viruses (S9 Fig). Interestingly, HLA class I downregulation was more pronounced in ULBP2-TB40 infected DC (Fig 5A and 5B), which became particularly obvious when the surface expression level of HLA-A02: 01 molecules was analyzed (Fig 5A and 5B, bottom panels). To attribute this phenotype to the individual genetic alterations introduced into the ULBP2-TB40 strain, we performed infection experiments with different viruses. In particular, we included the original TB40/E strain (referred to as TB40/E WT), harboring a complete complement of the MHC immunoevasins (US2, US3, US6, US11), and the strain dUL16 TB40 that lacks ORF UL16 as well as ORFs US1 to US6. When analyzed 1 and 2 days post infection (p. i.) the strongest HLA-I downregulation was observed for TB40/E WT infected fibroblast (Fig 5C), substantially more pronounced than in TB40-infected cells, reflecting the effect of the US2 to US6 genes on HLA-I surface expression. Remarkably, HLA-I downregulation in ULBP2-TB40 infected cells was as strong as in cells infected with the TB40E WT strain (Fig 5C, compare second panel and bottom panel), indicating an additional effect of ULBP2 expression (Fig 5C, bottom panel on the right). The dUL16 TB40 virus induced an intermediate phenotype (Fig 5C, second last panel), most likely due to upregulation of ULBP2 as a consequence of missing UL16 (Fig 5C, second last panel on the right). The degree of HLA-I downregulation in dUL16 TB40 and ULBP2-TB40 infected cells correlated with ULBP2 expression on the surface of these cells (Fig 5C). Intriguingly, plotting of HLA class I and ULBP2 surface expression for ULBP2-TB40 infected cells indicates that there is an inverse relationship of the expression of these molecules (Fig 5D). Taken together, these results show that expression of the NKG2D ligand ULBP2 increases MHC class I down-regulation in HCMV-infected cells. The observed lower surface expression of HLA-I molecules in ULBP2-TB40 infected cells was expected to impair antigen presentation, leading to less CD8+ T cell activation than upon stimulation with TB40-infected cells. However, this was in contrast to the results presented in Figs 3 and 4. To investigate this puzzle, we decided to examine in more detail the role of ULBP2 in T cell activation. To this end, we took advantage of a CD8+ T cell clone [63] specific for the peptide NLVPMVATV of the HCMV antigen pp65 (pp65-CTL) and co-cultured it with ULBP2-TB40 or TB40 infected DC. We noticed that a comparable percentage of the pp65-CTL became activated as assessed by IFNγ and TNFα intracellular cytokine staining, independent of which virus was used for the infection of DC (Fig 6A). In addition, comparable results were observed for the degranulation capacity (CD107a) of the pp65-CTL (Fig 6A). Data were confirmed with DC derived from three different donors. Next, we analyzed the expression of NKG2D on the surface of the pp65-CTL. Whereas the majority of the pp65-CTL expressed NKG2D when co-cultured with uninfected or TB40 infected DC, NKG2D surface expression was strongly diminished in the presence of ULBP2-TB40 infected DC (Fig 6B). The finding was confirmed with pp65-specific CD8+ T cells obtained by expansion with pp65-peptide loaded DC (S10A and S10B Fig). We conclude that although the outcome in terms of T cell activation is similar for DC infected with either strain, the underlying activating mechanism is different for T cells co-cultivated with ULBP2-TB40 infected DC and involves the NKG2D receptor as indicated by its strong, almost complete downregulation. In this report we propose a novel vaccination strategy that can be applied to the development of an HCMV vaccine. The ULBP2 ligand for the NKG2D receptor that is present on both NK and CD8+ T cells was expressed by an HCMV strain with the intention to stimulate innate as well as adaptive immunity. We investigated the properties of the resulting recombinant HCMV strain by in vitro assays using ex vivo-derived primary human immune cells and provide a first in vivo assessment in a humanized mouse model. An important goal in development of a live vaccine is attenuation. To this end, we endowed the HCMV strain with the ULBP2 gene, encoding a ligand for the activating NKG2D receptor of NK cells, and deleted the viral gene UL16, which normally counteracts the surface exposure of ULBP2 as well as of several other NKG2D ligands [38,40]. This secured that there is no interference with ULBP2 expression and, even in the unlikely case that the ULBP2 gene is accidentally inactivated, the HCMV strain will remain susceptible to NK cell control. Although NK cells are well known for their contribution to contain acute HCMV infection, these innate immune cells cannot deploy their full efficacy because a series of NK cell evasion functions encoded by wild-type HCMV strains dampen their activity [64]. UL16 it is one of the important immunoevasins, which confers NK cell resistance of HCMV infected cells [39,40]. By breaking NK cell evasion, cells infected with the ULBP2-expressing HCMV strain were expected to become highly vulnerable to recognition and elimination by NK cells. This was exactly what we observed when we performed cytotoxicity assays (Fig 1C and 1D). Consequently, spread of the ULBP2-expressing viral strain was strongly limited in the presence of NK cells (Fig 1E and 1F). These data suggest that the attenuation of the ULBP2-expressing HCMV strain will not depend on pre-existing immunity against HCMV, but on the presence of NK cells. Indeed, we detected human NK cells in the humanized mice which could contribute to the low viral load observed in animals receiving the ULBP2-expressing viral strain. This is supported by the fact that in a humanized mouse model for Epstein–Barr virus (EBV) human NK cells were capable to control the infection [65]. One difference between the EBV and HCMV infection models is that B cells in humanized mice support productive EBV infection, whereas important target cells of productive HCMV infection such as human hepatocytes, endothelial and epithelial cells are missing [66] and thus, primarily a latent HCMV infection is established at low level in few myeloid cells present in these animals [44]. Such limitations hamper the investigation of HCMV-specific immune responses in humanized NSG-A2 mice [67]. We found it nevertheless interesting that there was a trend towards a higher frequency of mature CD57+ NK cells in humanized mice receiving the ULBP2-TB40 strain when compared to the group receiving TB40. Emergence of a subset of CD57+ NK cells occurs in humans during primary HCMV infection [46]. Whether the observation for the CD57+ NK cells points to a stronger expansion in mice injected with the ULBP2-TB40 strain warrants further investigation. Similarly encouraging was the detection of CMV-specific IgM antibodies and a comparable frequency of B cells and CD4+ T cells as well as CMV-specific CD8+ T cells in mice receiving the ULBP2-TB40 and TB40 strains. Humoral immunity is one of the important factors in preventing congenital CMV infection and thus it is essential that a CMV vaccine can also provoke a strong antibody response. NSG mice transplanted with HSC are not ideal for analyzing humoral immunity. In several publications impaired antibody responses were reported [49], and only low levels of IgM were seen and no IgG [50] as in our study. Despite this constraint, our results indicate that ULBP2 expression by the ULBP2-TB40 strain does not interfere with the ability to elicit an IgM response. Induction of CMV-specific IgG antibodies must be analyzed in the future in novel humanized models that support antibody isotype switching [68–70]. We assess the observed HCMV-specific T and B cell responses as very promising results, particularly in view of the low load of ULBP2-TB40 genomes in the humanized mice. However, our study can only provide a first assessment of the vaccination approach with the ULBP2-TB40 strain, and further investigation of the protective capacity of the immune response has to await the establishment of improved humanized mouse models for HCMV infection. As recently pointed out by Crawford et al. (2015) [67], BLT (bone marrow/liver/thymus) humanized mice [71,72] may allow to reproduce acute HCMV infection and analysis of the specific immune responses much better. Although attenuation is a central objective concerning safety of a live vaccine, too much attenuation could impair immunogenicity. Rapid control of the vaccine strain may for instance limit the production of viral antigens–below a level necessary for priming of the adaptive T cell response. To circumvent this problem, we chose an HCMV strain that lacked several of the MHC immunoevasins. Such strains modulate antigen presentation to a lesser extent than HCMV isolates expressing the full complement of immunoevasins [73,74]. In view of these considerations, we value the comparable activation and proliferation of HCMV-specific T cells observed upon co-cultivation with ULBP2-TB40 and TB40 infected DCs (Figs 3 and 4C) as a positive result. As a matter of fact, we expected a stronger CD8+ T cell response, because there was a second reason to express ULBP2 by the HCMV strain. It has been reported that NKG2D - besides being an activating receptor on NK cells—is also a co-stimulatory receptor expressed on CD8+ T cells [54,75]. Indeed, when we co-cultured HCMV-specific T cells with ULBP2-TB40 infected DC, strong downregulation of NKG2D was observed (Figs 6B and S10B Fig). This did not occur when the T cells were co-cultured with uninfected or TB40-infected DC. We concluded therefore that reduced NKG2D surface expression reflects triggering of the receptor in the T cells. Regulation of NKG2D expression has been described as a physiologic mechanism [76] and therefore NKG2D downregulation in our assays should not be misinterpreted as a negative effect as previously suggested [77–79]. Along the same line, we observed that the HCMV-specific T cells that we obtained in the expansion assays expressed high levels of NKG2D (S11 Fig), suggesting that initial NKG2D downregulation is transient and rather indicates co-stimulation and activation of the T cells. The seemingly neutral outcome of ULBP2 expression with respect to the frequency of activated CD8+ T cells (Figs 3 and 4) can best be explained by masking of NKG2D-mediated costimulation, an effect imposed by the lower MHC-I surface level that came as unexpected consequence of ULBP2 expression (Fig 5). It is long known that under conditions of low MHC-I expression and limiting amounts of antigenic peptides, extensive activation of T cells cannot occur [80]; in particular, when it comes to priming of naïve CD8+ T cells because they have a higher activation threshold than memory or effector CD8+ T cells. Our data imply that precisely in this setting when only low peptide concentrations are present, costimulation via NKG2D can secure the induction of specific T cells. This view is supported by the results of our previous mouse studies, which indicated that immunization with MCMV strains expressing the mouse NKG2D ligand RAE-1γ provided stronger protective immunity than infection with wild-type MCMV [15,32]. It will be of interest to understand how NKG2D ligands influence MHC-I surface expression. Interestingly, a previous publication reported that expression of the murine NKG2D ligand RAE-1ε in RMA cells decreases MHC class I expression as well [81], suggesting that this is a more general phenomenon applying to different NKG2D ligands. Unraveling the exact underlying mechanism is beyond the scope of this work and must be the subject of further studies. An excellent safety profile would increase the acceptance of a potential HCMV vaccine and will be an important goal, particularly when vaccination of immunocompromised patients is considered. Further attenuation can probably be achieved by deletion of additional genes for the numerous viral immunomodulatory functions, although it has to be examined that this does not go at the expense of reduced vaccination efficacy. Another option to increase safety is the application of replication- and spread-deficient vaccine strains. Studies in the mouse model revealed that such strains preserve the ability to stimulate CMV-specific T cell immunity [82,83], and in a guinea pig model of congenital CMV infection partial protection of pups was observed following immunization with a non-infectious guinea pig CMV BAC DNA vaccine [84] or with a non-replication-competent virus [85]. We have recently pioneered the development of conditional-replicating CMV strains [86], and this technique has already successfully been used to generate a conditionally replicating HCMV vaccine candidate, which is currently in phase 1 testing (ClinicalTrials. gov number, NCT01986010). The combination of the superior immunization approach presented here in this study with the safety features of a replication-deficient strain could lead to an HCMV vaccine that may even be safely applicable to immunocompromised patients. Additional preclinical studies are therefore warranted to further analyze the protective efficacy as well as the safety of such vaccination strategies against HCMV, particularly when improved humanized mouse models become available, ultimately paving the way for clinical trials. Buffy coat samples were obtained from the Institute for Transfusion Medicine at Hannover Medical School from voluntary healthy blood donors with known HCMV serological status. All materials and data were analyzed anonymously. All studies were performed in accordance with guidelines on human cell research and the approval of the Hannover Medical School Ethics Review Board. All animal research protocols were approved by the authorized Ethics Committee for Biomedical Research of the Clinical Hospital Rijeka and of the University of Rijeka (Cl. 003-08/12-01/40; No. 2170-24-01-12-03) in accordance with the guidance of the European Parliament (Directive 2010/63/EU) and Croatian Federal Law about animal protection (Official Gazette, 135/2006 and 47/2011). PBMC were purified from buffy coat samples obtained from healthy blood donors using standard Biocoll (Biochrom) density gradient centrifugation according to a published protocol (Miltenyi Biotec). Cells were cryopreserved in fetal bovine serum (FBS) with 10% DMSO. Before NK cell isolation or prior to setting-up co-cultures, PBMC were thawed and cultured overnight with 500 IU/ml interleukin-2 (IL-2) (ImmunoTools) in X-VIVO 15 medium (Lonza) supplemented with 10% FBS and 5% human serum (inactivated at 56°C; Sigma Aldrich). Individual cell subsets (CD56+ NK cells, CD8+ T cells and CD14+ monocytes) were isolated using human cell isolation kits (Miltenyi Biotec). NK cells were negatively selected, while CD8+ T cells and monocytes were positively selected. Purity of selection was analyzed by flow cytometry and was more than 94%. The pp65-specific T cell clone (CMV-CTL; kindly provided by L. Hambach) was thawed 2 days before setting up assays in MEM-alpha medium (Lonza) supplemented with 120 IU/ml of IL-2 and 10% human serum. The CMV-CTL was tested for the absence of mycoplasma and its specificity was determined by intracellular cytokine staining and proliferation assays using pp65-peptide loaded cells as positive control. Human monocyte-derived dendritic cells (DC) were generated using a standard protocol [87]. Briefly, isolated monocytes were cultured for 5 days in X-VIVO 15 medium supplemented with recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) (50 ng/ml each; ImmunoTools). After 5 days, DC were used for infection or were matured for 24 h by supplementing the medium with maturation cocktail: recombinant human TNF-α (200 IU/ml), IL-1β (5 ng/ml), IL-6 (10 ng/ml), Prostaglandin E2 (1 μg/ml) (ImmunoTools). Two hours before establishing co-cultures with PBMC or CD8+ T cells, mature DC were loaded with a peptide pool spanning the entire CMV pp65 antigen (20 μg/ml; JPT Peptide Technologies). Primary human foreskin fibroblasts (HFF) were generated following standard methods as described [88] and were maintained in DMEM containing 10% FBS, penicillin (100 units/ml), streptomycin sulfate (100 μg/ml), and 2 mM L-glutamine. For the generation of the recombinant ULBP2-TB40 virus the ULBP2 ORF was amplified using primers 5´-GTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGAGAGCTCCACCATGGCAGCAGCCGCCGCTACC-3´ and 5´-CCCGGATCCCTCTCCTCAGATGCCAGGGAGGATGAAG-3´ and an ULBP2 cDNA clone (Open Biosystems; Genbank accession number: BC034689), and was cloned (via KpnI and BamHI) between the MCMV major immediate-early promoter sequence (corresponding to nucleotides 182849 to 183094 of the MCMV Smith Strain (Genbank accession number NC004065. 1) ) and a kanamycin resistance (KanR) cassette flanked by FRT sites. The whole insert was amplified with primers 5´-GACACCGGGCTCCATGCTGACGTAGGTACCGACTGGGGTCAAAAGCCTTTAAACGGTACTTTCCCATAGC-3´ and 5´-CTTATAGCAGCGTGAACGTTGCACGTGGCCTTTGCGGTTATCCGTTCAGGAACACTTAACGGCTGA-3´, and inserted into the BAC-cloned genome of the HCMV strain TB40/E [36] by red-α, -β, -γ-mediated recombineering as described previously [89] replacing ORF UL16. The KanR cassette was excised by FLP recombinase. Correct insertion was verified by restriction analysis (BglII and NotI) and sequencing (with primers 5´-GGCGATGCGGTATCGCGCACA-3´ and 5´-GACACCTGTTCGTCCAGAATC-3´). The mutant lacking ORF UL16 that was derived from the BAC-cloned TB40/E strain was described previously [41]. All virus stocks were produced by propagation on HFF. Briefly, supernatant was harvested at day 6 p. i., and virus was pelleted by ultracentrifugation (100,000 × g, 1 h) and finally resuspended in X-VIVO 15 medium. Viral titers were determined using standard plaque assay on HFF. DC and fibroblasts were infected at an MOI of 3 and 1, respectively, followed by centrifugal enhancement (800 × g, 30 min). HFF were infected with TB40 or ULBP2-TB40 as described above. One day p. i. the infection rate of the HFF was determined by flow cytometry following intranuclear staining with an Alexa Fluor 488-conjugated CMV IE1/2 antibody (Merck Millipore). Cells were cryopreserved and thawed at the day of establishing the cytotoxicity assay. Prior establishing co-cultures, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; eBioscience) and co-cultured with freshly isolated human NK cells at effector to target ratios of 16: 1,8: 1 and 4: 1 in X-VIVO 15 medium supplemented with 500 IU/ml IL-2. After 4 h incubation at 37°C, specific lysis was determined by flow cytometry as measured by determination of dead cells using the Fixable Viability Kit (L/D) (BioLegend). To evaluate the spontaneous death of cells, samples containing target cells only were used. Cytolytic activity of NK cells expressed as percentage of specific lysis was determined by the following calculation: (% CFSE+ L/D+ cell specific lysis—% CFSE+ L/D+ cell spontaneous lysis) / (100 - % CFSE+ L/D+ cell spontaneous lysis) × 100. Focus expansion assays were established as previously described [41]. Briefly, infected HFF (10 cells/well; 3 d p. i.) were co-cultured with uninfected HFF (2 × 104 cells/well). NK cells were added at the ratio 1: 0. 25 (HFF: NK cells). After 3 days, the cultures were fixed with 80% acetone, and stained with the IE1/2 antibody. Infected cells per focus of infection and number of infectious foci were counted using fluorescence microscopy. Each sample was performed in quadruplicates. Blocking of NKG2D of human NK cells in vitro was performed using the NKG2D-specific antibody clone 1D11 and isotype control mouse IgG1 (BioLegend) (both at 10 μg/ml). In addition, human Fc Block (25 μg/ml; BD Biosciences) was added. For expansion of CMV-specific T cells, CD8+ T cells (105 cells/well) isolated from HLA-A*02: 01 HCMV-seropositive healthy donors were co-cultured with autologous monocyte-derived DC (uninfected, infected with respective viruses or mature peptide-loaded) and with γ-irradiated autologous CD14- feeder cells (2–3 × 105 cells/well). Co-cultures were set up in 96-well plates at the ratio 1: 10 (DC: T cell) in X-VIVO 15 medium supplemented with 5% human serum and IL-2 (500 IU/ml), IL-7 and IL-15 (10 ng/ml each; ImmunoTools). Every 2 days fresh medium containing cytokines was replenished. After 7 days, expanded cells were transferred to 48-well plates, and freshly prepared DC and feeder cells were added. After 2 weeks, the percentage of pp65-specific CD8+ T cells was determined by flow cytometry following staining with the PE-conjugated tetramer HLA-A*02. 01-NLVPMVATV (MoBiTec). For intracellular cytokine staining, the expanded T cells, PBMC or the pp65-specific CTL clone were stimulated in 96-well V-bottom plates for 6 h with DC loaded with the pp65 peptide pool or the NLVPMVATV peptide or with uninfected/infected DC for PBMC and the pp65-specific T cell clone (at a ratio 1: 10 [DC: T cells]). Protein transport inhibitor cocktail and a PE-Cy7-conjugated antibody against CD107a (clone H4A3; Biolegend) were added at the beginning of the assay. For surface staining we used PerCp-Cy5. 5-conjugated anti-human CD3 (clone OKT3; eBioscience), PE-Texas Red-conjugated anti-human CD8 (clone 3B5; Thermo Fisher Scientific) and in case of expanded T cells additionally the PE-conjugated tetramer HLA-A*02: 01-NLVPMVATV. After fixation with 3% paraformaldehyde for 5 min at RT and permeabilization with 0. 1% saponin for 20 min at RT, FITC-conjugated (for expanded T cells) or PE-conjugated anti-human IFNγ (clone 4S. B3; eBioscience) and APC-conjugated anti-human TNFα Abs (clone Mab11; eBioscience) were used for staining. One million of splenocytes isolated from humanized mice were incubated with 1 μg of the peptide 316-VLEETSVML-324 (HLA-A2 restricted immunodominant epitope from the CMV IE1 antigen) for 5 h at 37°C, with brefeldin A (eBioscience) added for the last 4 h of stimulation. For evaluation of IFNγ production by NK cells, 5 × 106 splenocytes were resuspended in 400 μl of RPMI medium supplemented with 10% FCS and IL2 (500 U/ml) and cultured for 5 h at 37°C in the presence of brefeldin A. Humanization of mice was performed at the Helmholtz Center for Infection Research (Braunschweig, Germany). Briefly, fetal liver mononuclear cells were isolated over Ficoll-density gradients, and CD38- CD34+ cells were enriched using a MACS selection kit (Miltenyi Biotec). Newborn (3–5 days old) NOD. Cg-Prkdcscid Il2rgtm1WjlTg (HLA-A2. 1) 1Enge/SzJ mice expressing human HLA-A*02: 01 (NSG-A2) were irradiated with 0. 7 Gy and 4–6 h after irradiation injected with 1 × 105 CD34+ cells intrahepatically. 3 months post transfer engraftment of human cells was determined in blood. Humanization status ranged from 70–90% of human leukocytes (hCD45+ cells). Animals were randomly assigned to experimental groups with the humanization status being equal among the experimental groups. Mice were injected intraperitoneally with uninfected or TB40 or ULBP2-TB40 infected DC (3 × 105 cells/mouse) generated from monocytes of HLA-A*02: 01 positive HCMV seronegative donors. Mice were daily treated with subcutaneous injections of recombinant human granulocyte colony-stimulating factor (G-CSF) (3. 6 μg/mouse s. c. ; Filgrastim HEXAL). Mice were boosted after 7 days with freshly prepared, uninfected or infected DC (3 × 105 cells/mouse). Analysis of CMV-specific IgM antibodies in serum of infected animals was performed using anti-CMV IgM Human in vitro ELISA kit (Abcam) according to the manufacturer’s instructions. Briefly, a 96-well plate was precoated with CMV antigens to bind cognate antibodies. Control and test samples were added to the wells in duplicates. Serum was isolated from blood using density gradient centrifugation applying the standard PBMC isolation protocol (Miltenyi Biotec) and stored at -20°C. The following controls were included: CMV IgM positive control, CMV IgM negative control and CMV IgM cut-off calibrator. Following washing, a horseradish peroxidase (HRP) labelled anti-human IgM conjugate was added to the wells and after incubation as according to the instructions of the manufacturer, the reaction was stopped by adding an acidic stop solution. Absorbance at 450 nm was measured immediately using a TriStar Version 1. 04 microplate reader (BertholdTech). Sections from formalin fixed, paraffin-embedded spleens were stained with hematoxylin and eosin. Labelling of tissue sections was performed by an anti-human CD3 (F7. 2. 38) Ab (Dako) followed by biotinylated goat anti-mouse IgG (BD Pharmingen), streptavidin covalently coupled to horseradish peroxidase conjugate (Roche Applied Science) and DAB chromogene (Dako). Slides were examined with an Olympus BX51 microscope, images were acquired by an Olympus digital camera (DP71) and analyzed using Cell^B software. DNA was isolated from spleen, liver, bone-marrow, blood and salivary glands using the ReliaPrep Blood gDNA Miniprep Kit (Promega) according to the manufacturer’s protocol. Presence of HCMV DNA was determined by 7500 Fast Real-Time PCR system (Applied Biosystems) using a predefined two step PCR: 1 cycle at 95°C for 15 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min using Power SYBR Green PCR master mix and TaqMan Universal PCR mix (Applied Biosystems). To detect viral DNA primers specific for gB (gB_Fw 5’-AGG TCT TCA AGG AAC TCA GCA AGA and gB_Rv 5’-CGG CAA TCG GTT TGT TGT AAA) and a TaqMan gB probe with TAMRA quencher (6FAM—AAC CCG TCA GCC ATT CTC TCG GC—TAMRA) was used as previously described [90]. For the standardization curve we used the gB and IE1-TOPO TA pCR2. 1 plasmid (kindly provided by S. Boppana; University of Alabama, Birmingham, AL). For flow cytometry analysis the following antibodies were used: anti-human Alexa Fluor 700-conjugated CD80 (L307. 4; BD Biosciences), Brilliant Violet 510 conjugated CD86 (IT2. 2; BioLegend), PE-conjugated CD209 (5H10; eBioscience), PE-Cy7-conjugated CD14 (61D3; eBioscience), PE-CF594-conjugated HLA-DR (G46-6; BD Biosciences), APC- -conjugated CD3 (OKT3; eBioscience), Alexa Fluor780-conjugated CD8 (SK1; conjugated HLA-ABC (W6/32; eBioscience), BV510-conjugated CD1a (HI149; BD Biosciences), APC-conjugated HLA-A2 (BB7. 2; eBioscience), PE-conjugated ULBP2/5/6 (165903; R&D Systems), APC eBioscience), PE-Cy7–conjugated CD19 (HIB19; eBioscience), PE-conjugated CD56 (CMSSB; eBioscience), APC-conjugated PD-1 (MIH4; eBioscience), FITC-conjugated CD57 (TB01; eBiosceince), FITC-CD45RA (HI100; eBioscience), PE-Cy7-CCR7 (3D12; eBioscience), PE-Cy7-CD62L (DREG-56; eBioscience) and FITC-CD45RO (UCHL1; eBioscience). As isotype controls we used PE‑conjugated mouse IgG2a (R&D Systems) and APC-conjugated mouse IgG2b (eBioscience). For dead cells exclusion Zombie NIR Fixable Viability Kit (BioLegend) was used. Intranuclear staining with an Alexa Fluor 488-conjugated IE1/2-specific antibody (Merck Millipore) after PFA fixation and permeabilization with 75% ice-cold ethanol was used to detect infected cells. For evaluation of the phenotype of CD8+ T cell we used anti-human APC-conjugated NKG2D (1D11; eBioscience). CMV-specific CD8+ T cells from blood and spleen of humanized mice were analyzed on days 14 and 18 p. i, respectively, using the PE-conjugated pp65-specific tetramer HLA-A*02. 01-NLVPMVATV and the APC-conjugated IE1-specific tetramer HLA-A*02: 01- VLEETSVML synthesized by the NIH tetramer core facility. Flow cytometry was performed using FACSVerse, FACSAria or LSRII (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). Statistical analyses were performed with GraphPad Prism5 software and statistical tests used are indicated in the figure legends. No statistical method was used to predetermine sample size, and data analysis was not blinded. Exact sample sizes and number of independent experiments performed are indicated in each figure legend. No samples were excluded. All error bars represent SEM or range as noted in the figure legends. Differences were considered to be statistically significant for P values <0. 05.

Title: Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand Summary: Human cytomegalovirus (CMV) is a major cause of morbidity and mortality in congenitally infected newborns and immunocompromised individuals, indicating an utmost need for a vaccine to protect these vulnerable groups. Recent experimental studies in animal models, including non-human primates, have shown that attenuated CMVs trigger a potent immune response and are attractive vaccine candidates. However, an effective CMV vaccine is still not available. Here, we demonstrate that rational engineering of a live attenuated human CMV vaccine candidate is feasible. We equipped a CMV strain with an immunostimulatory molecule that is a ligand for an activating receptor present on both Natural Killer cells and CD8+ T cells. Moreover, we deleted several immunoevasins involved in downregulation of MHC class I molecules and of a ligand for Natural Killer cells in order to elicit stronger immune responses. In vitro assays using human immune cells and a first assessment in a humanized mouse model in vivo suggest that the generated CMV strain is attenuated and has the ability to induce a virus-specific immune response. Our study proposes this novel approach for the development of a rationally engineered CMV vaccine.

lay_plos

en

Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a combined package and/or storage container and a device utilized in the cooking of the food product in a manner which automatically adds predetermined additives such as spices in preselected amounts to the food product during the cooking process wherein the package and contained food products may be taken directly from a storage facility, such as a freezer, and placed in a cooking utensil for heating and cooking thereof. 2. Description of the Prior Art Container and packages used for the storage and subsequent cooking of food products are of course well known in the art. Such devices which allow prolonged storage as well as facilitate cooking of the product are particularly popular in today&#39;s modern rapid paced society. The sophistication of prior art devices of this type even provide for the addition or regulation of flavoring to the food products being cooked as well as providing the substantially concurrent cooking of different portions of the same food product or meal. The following U.S. Patents are representative of relatively modern advances in the prior art relating to the above set forth subject matter and include the patents U.S. Pat. Nos. 3,881,094 to Rogers et al; 4,133,896 Standing et al; 4,143,165 Daswick; 4,154,860 Daswick; 4,172,903 Daswick; and 4,299,851 Lowe. Of the above, the Daswick patents show the adding of condiment to the food product during the cooking process and the heating of one product serving to aid in the heating of an adjacently packaged product through the passage of heated or pressurized gases. Also, the patent to Lowe shows a flavoring dispenser which may be added to cooking food. It is readily apparent therefore that the prior art structures directed to both storage and subsequent cooking as well as the additions of condiments, flavorings, juices, gravies, etc. which are added to cooking products in order to increase the overall flavoring or desirability thereof are well known. However, the prior art still appears to be absent any type of selective seasoning in terms of adding one or all of a variety of additives, including spices, flavorings, juices, gravies, etc. to a food product, during the cooking thereof. Such additives or flavorings could be added in preselected and regulated amounts during the entire cooking process so as to expose the food product to the desired additives for a prolonged period. Also, this could be accomplished automatically without need of constant care or adjustment by the user of the device. In addition to the above, a preferred device, container or package should also be specifically structured to serve as a storage container and/or ultimate retail packaging of the subject food product and additives and therefore be relatively condensed in size and shape while at the same time providing an attractive and more importantly functional product to accomplish the intended result. SUMMARY OF THE INVENTION A container assembly of the present invention includes a base generally formed of a rigid material structured to at least partially support the subject assembly, in a manner to be described in greater detail hereinafter, and a depending pouch itself formed of a flexible or more specifically, expandable material which could include, but is not limited to, a metal foil or wire material. The depending pouch holds the primary food product in communicating relation with a hollow interior of the base. Such hollow interior is primarily defined by a chamber having an open mouth designed to cooperate with a closure structure such that cooking liquid, such as water, may be added to the interior of the chamber. By means of a first path of fluid flow, the cooking liquid may pass into the interior of the pouch and effectively &#34;baste&#34; the food product on the interior thereof. Another important feature of the present invention is the provision of a compartment means formed within the base wherein the compartment means are specifically structured to house a plurality of additives therein. Such additives may be in the form of spices, condiments, gravies, etc. In order to preselect the amount of additives to be added to the food product, the compartment means is divided into a plurality of compartments wherein each compartment is divided into a plurality of segments. The segments are structured so as to communicate with the interior of the chamber and the cooking liquid therein by a second path of fluid flow. Seal means are provided to selectively open or remain closed certain ones of a plurality of apertures which define the second path of fluid flow such that all or a fraction of the additives, such as spices, may be allowed to flow onto the contained food product within the pouch during the cooking process. A third path of fluid flow is temporarily closed by the specific structure of the pouch or other blocking means but may be opened upon a build-up of fluid pressure within the pouch due to a cooking of the food product and the heating of the cooking liquid to vapor. Such a build-up of pressure releases any type of blocking closure defining a third path of fluid flow and the steam or pressurized vapor passes through and along the third path of fluid flow into a rear opening of each of the segments of the compartments such that the additives or spices contained therein are effectively fed through the apertures defining the second path of fluid flow into the chamber and again back onto the primary food product contained within the pouch. It is readily seen therefore, that a continuing closed path of travel is effectively defined by the cooking liquid and contained additives dripping or passing slowly onto the primary food product; a heating of the cooking liquid and a rising of the juices and vapors from the food product containing at least a partially pressurized atmosphere which then allows the cooking vapors to pass along a third path of fluid flow into the compartment segments with the additives, wherein the additives are then forced into the centrally disposed chamber and the condensed gases or vapors flow along therewith and subsequently again onto the food product within the pouch. Accordingly, it is apparent that the structure of the present invention allows for a continuous basting and &#34;supply&#34; of both cooking liquid and additives, in preselected amounts, during the entire cooking process wherein the liquid, in the form of pressurized vapor, etc., is &#34;recycled&#34; and the food product is constantly exposed to both the additives and the cooking liquid resulting in a moist, flavorful product, when the cooking process is completed. The invention accordingly comprises the features of construction, combination of elements, and arrangement of parts which will be exemplified in the construction hereinafter set forth, and the scope of the invention will be indicated in the claims. BRIEF DESCRIPTION OF THE DRAWINGS For a fuller understanding of the nature of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings in which: FIG. 1 is an isometric view of the container assembly of the present invention shown in a state capable of storage of a food product. FIG. 2 is a sectional view showing the interior structural features and location of the food product therein. FIG. 3 is a sectional view along line 3--3 of FIG. 2 showing additional structural features on the interior of the container assembly. FIG. 4 is a sectional view along line 4--4 of FIG. 2. FIG. 5 is an end view along line 5--5 of FIG. 2. FIG. 6 is a side view of another embodiment of the container assembly. FIG. 7 is a sectional view along line 7--7 of FIG. 6 showing another embodiment of a closure assembly for the embodiment of the container assembly of FIG. 6. FIG. 8 is a sectional view of another embodiment of the structure of the container assembly. FIG. 9 is a sectional view along line 9--9 of FIG. 8. Like reference numerals refer to like parts throughout the several views of the drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT As shown in FIG. 1, the container assembly of the present invention is generally indicated as 10 and includes a base 12 generally made of rigid material of sufficient strength to support the assembly by an uppermost flange 14, generally about the periphery thereof as at 14&#39;, supported on the upper surrounding peripheral rim of a cooking container or pot 16 as shown in FIG. 2. Also, an extending support 15 is secured to the uppermost flange 14 and may extend outwardly therefrom so as to accommodate the support of the base 12 on the peripheral rim of pots or kettles of a larger size than that represented in FIG. 2. It should further be noted that the outward extension 15 may be a part of the flange 14 or otherwise structured to be collapsible relative thereto to facilitate storage and packaging of the base 12 prior to use. In addition, the container assembly includes a pouch 18 formed from a flexible or more specifically an expandable material which is water resistant, such as foil or the like, wherein the pouch 18 may be disposed on the interior of the cooking vessel 16 and subjected to water or like liquid contained therein 20 for the direct heating of the pouch 18 and the food product and/or contents 22 contained therein. The base 12 is structured to have at least a partial hollow interior portion defined by a centrally disposed chamber 24. The chamber has an open mouth 26 disposed at one end thereof wherein the open mouth 26 is capable of being closed or covered by a closure means 28. The chamber 24 is further defined by a chamber receptacle 25 protruding upwardly from the outer flange or exposed portion 14. This outer protrusion extends the dimension or capacity of the chamber 24 and in addition, provides an external threaded surface 27 to which the closure 28 may be connected as by an internally threaded depending cylindrical wall 29. The closure means 28 has a knob or handle member 30 protruding outwardly from an exposed portion thereof and further a vent means or vent aperture 32 is provided to allow escape of a build-up of fluid pressure within the chamber 24 as will be explained in greater detail hereinafter. It should be apparent that removal of the cover means 28 from its threaded connection 27 at side wall 29 with the upward extension of the chamber vessel 25 allows for access to the interior of the chamber 24 and the placement of a cooking liquid, such as water, juices, gravies, etc., as at 33 therein. The cooking liquid 33 is allowed to drip or pass slowly onto the food product 22 through one or more apertures 36 defining a first path of fluid flow between the interior chamber 24 and the interior of the pouch 18 and of course food product 22. The apertures 36 allow the cooking fluid 33 to pass in a regulated fashion as by drops 33&#39; and collect within the pouch in surrounding or engaging relation to the food product 22. The regulation of flow of the cooking liquid 33 may be accomplished by varying the relative positions between apertures 36&#39; (see FIGS. 5 and 8) formed in the floor 39 of the base which supports a bottom 38 of the vessel 25. In that vessel 25 can be rotated relative to floor 39, the positions of apertures 36 and 36&#39; may vary and of course be reduced to allow only a minimal amount of liquid to drip down as at 33&#39; onto the food product 22. See directional arrows 37 in FIG. 3 showing relative rotation of the bottom 38 of the chamber 25 relative to the floor 29. The base of the assembly further includes an under portion 15 extending downwardly from the upper flange portion 14 in fixed or integral relation to the floor 39 of the base which is supporting the bottom 38 of the chamber 25. Further, the bottom portion 15 extends continuously around and at least partially defines an additional hollow interior portion of the base including a plurality of collecting chambers 40 disposed on the outside of but in communication with a plurality of compartments 42, 44, 46, 48, 50 52, 54 and 56. As shown in FIGS. 2 and 3, each of the compartments are divided into a plurality of compartment segments including an uppermost segment 60, a first and second middle segment 62 and 64 respectively, and a bottom-most segment 66. Each of the compartments 42 through 56 is specifically structured to hold a specific additive such as a spice, flavor, condiment, etc. therein. In addition, since each of the compartments are divided into compartment segments, the spice or additive in each compartment may be equally divided into equal portions wherein each compartment segment contains an equal portion. This permits regulation of the amount of additive to be delivered or forced into the central chamber 44 for mixing with the cooking liquid 33 and eventual delivery and/or basting of the food product 22. With reference to FIG. 4, a plurality of seal means 70 are provided in covering relation to each of a plurality of apertures 72 disposed in the cylindrical wall 74 of the base wherein such cylindrical wall 74 is disposed in surrounding relation with the cylindrical side wall 76 of chamber 25. In addition, it is readily apparent that each of the apertures 72 (FIG. 4) may be arranged in mating, communicating relation with similarly disposed apertures 75 in the cylindrical side wall 76. Accordingly, operation of the device is as follows: Liquid 33 is added to the central chamber 24 after removal of closure 28. This liquid passes down and effectively bastes the food product 22 within pouch 18 as represented as 33&#39;. Passage of the cooking liquid 33 onto the food product 22 is regualated to a slow drip flow by orienting floor 39 and bottom 38 of the base and of the vessel 25 respectively so that only a minimal amount of liquid 33&#39; passes therethrough. These apertures 36 and 36&#39; accordingly define a path of first fluid flow. A path of second fluid flow is defined by the aligned apertures 72 and 75 respectively formed in the cylindrical wall 74 of the base disposed in surrounding relation to the cylindrical side wall 76 of the vessel 25 (see FIG. 3). Through these apertures 72, 75 defining the path of second fluid flow, the additives or spices, etc. pass therethrough as indicated by directional arrows shown leading into the central chamber 24. As set forth above and with reference to FIG. 4, removal of the sealing element 70 determines how much of the spice or additive is allowed to flow into the central chamber 24 and cooking liquid 33. Removal of all the sealing elements 70 would allow the full quantity of additive to be supplied to the chamber 24 contained in each of the compartment segments 60, 62, 64, and 66. However, removal of, for instance, the lowermost sealing structure 70&#39; (FIG. 4) will enable only the contained quantity to pass through the respective apertures 72 and 75 since only compartment segment 66 is in communication with the interior chamber 24. A third path of fluid flow is defined by apertures 80 formed in the depending bottom walls 15 of the base and communicating apertures 82 formed in communicating relation with the various compartment segments 60 through 66. It is readily apparent therefore that aperture 80 allows the vapors or gases to pass into the collection chamber 40 and into the various compartment segments 60 through 66 depending upon which of the seal structures 70 and 70&#39; are removed. Passing of the vapors into the various compartment segments will serve to feed or force out the additives in the respective compartment segments into the central chamber 24 so as to mix with the cooking liquid 33 and pass onto the food product 22. Initially, the apertures 80 are sealed by correspondingly positioned peripheral portions of foil 18 as at 18&#39;. However, the build-up of vapor or fluid pressure within the interior of the pouch 18 forces away this peripheral portion 18&#39; and leaves open the various apertures 80 leading to this respective collection chamber 40. It can be readily seen in FIG. 2 that once the heating liquid 20 is placed on an appropriate burner or heating element 90, the liquid will heat up causing the pouch 18 to expand and fluid pressure, through vapors, to build-up on the interior thereof. The various apertures of the first, second, and third fluid flow paths are open thereby setting the entire container into process and causing an automatic and continuous basting or placement of the additives onto the food product 22. In FIGS. 6 and 7, a varying embodiment of the present invention is provided wherein the closure element 28&#39; extends over the entire outwardly extending flange 14 and is threadedly engaged at upstanding flange segments 15&#39; as shown. In addition, the vent 32 is provided in the uppermost wall portion of the closure means 28&#39; for the venting of excess pressure therefrom. With reference to FIGS. 8 and 9, the structure and use or operation of the assembly is the same as the embodiment of FIGS. 2 and 3 with the exception that the cylindrical side wall 74 of the base surrounding the cylindrical side wall 75 of container 25 has an upward extension as at 74&#39; which is externally threaded as at 27 for mating threaded engagement with the side wall 29 of the closure 28. This differs from the embodiment of FIG. 2 in that the upward extension or extremity of the cylindrical side wall 75 of container 25 is not in threaded engagement but may be removed totally from its position as shown in FIG. 8. Also, upward movement or travel of the container 25 is prevented due to the closure 28 being threadedly secured to the upward extension 74&#39; as shown in FIG. 8. It is therefore to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which as a matter of language, might be said to fall therebetween. Now that the invention has been described,

Summary: A container assembly used for the storage of food products plus additives such as spices, etc. therefor, wherein the additives are stored in segregated relation to one another and the food product. The container assembly is designed for the storage of the food product such as by freezing and also for the cooking of the food product in a manner which facilitates the automatic and selective mixing of the additives with the food product wherein the food product and additives may be stored for prolonged periods prior to cooking and user of the device need not come into direct contact with the food product until it is ready for consumption.

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Summarize: E-cigarettes should be banned indoors over fears that they can be as toxic to bystanders as normal cigarettes, the World Health Organisation has said. Despite releasing vapour instead of smoke, the devices still pollute the air with harmful chemicals, health experts warned. Many smokers use e-cigarettes as a way to quit, as they deliver the nicotine hit but without the carcinogens associated with breathing in smoke. There are no laws currently banning their use inside. But a report by the WHO questioned the safety of e-cigarettes, officially known as electronic nicotine delivery systems (ENDS). "The fact that ENDS exhaled aerosol contains on average lower levels of toxicants than the emissions from combusted tobacco does not mean that these levels are acceptable to involuntarily exposed bystanders,” said the report. "In fact, exhaled aerosol is likely to increase, above background levels, the risk of disease to bystanders, especially in the case of some ENDS that produce toxicant levels in the range of that produced by some cigarettes." Some early studies have shown that e-cigarettes can be more effective than over-the-counter products like gum or nicotine patches. But the report recommends preventing manufacturers from marketing e-cigs as "smoking cessation aids" until they provide robust scientific evidence to back the claim. "Although anecdotal reports indicate that an undetermined proportion of ENDS users have quit smoking using these products, their efficacy has not been systematically evaluated yet,” the report added. "Only a few studies have examined whether the use of ENDS is an effective method for quitting tobacco smoking." The WHO also advised banning sales to under-18s and said vending machines should be removed "in almost all locations.” The report was welcomed by health officials. Professor John Ashton of the Faculty of Public Health said: “Most adult smokers start smoking before the age of 18. That's why many public health experts are concerned that the advertising of electronic cigarettes could make it seem normal again to think smoking is glamorous, when it is anything but. “We also don’t know enough yet about the harms and side effects of electronic cigarettes, and it will take years before we can be sure what they are.” However anti-smoking campaigners said that e-cigarettes were ‘considerably less-harmful’ than tobacco and warned against restricting their sale and use. Hazel Cheeseman, director of policy and research at Action on Smoking and Health, said there was "no evidence of any harm to bystanders from use of these devices" and said regulation needed to be proportionate. She said: "Smoking kills 100,000 people in the UK alone. Smokers who switch to using electronic cigarettes in whole or in part are likely to substantially reduce their health risks. "Although we cannot be sure that electronic cigarettes are completely safe, as the WHO acknowledges, they are considerably less harmful than smoking tobacco and research suggests that they are already helping smokers to quit.” The ingredients in e-cigarettes vary but they generally include nicotine and chemicals to vaporise the nicotine such as propylene glycol. Previous studies have shown that inhaling nicotine, even without tobacco smoke, may contribute to heart disease. Some e-cigarettes have also been found to give off formaldehyde, a known carcinogen, at higher levels than normal cigarettes. Silicate particles, a cause of lung disease, are also present in some e-cigarette vapour. A study published last year by the Bavarian Health and Food Safety Authority found that vaping worsened indoor air quality by increasing the concentration of nicotine, particulates and aluminium. The Department of Health is not considering banning e-cigarettes indoors but said it was planning to prohibit the sale to under 18s. Electronic cigarettes are currently regulated as consumer products in the UK. But by 2016 they will be regulated by the Medicines and Healthcare Products Regulatory Agency. “More and more people are using e-cigarettes and we want to make sure they are properly regulated so we can be sure of their safety,” said a Department of Health spokesman. GENEVA/LONDON The World Health Organization (WHO) called for stiff regulation of electronic cigarettes as well as bans on indoor use, advertising and sales to minors, in the latest bid to control the booming new market. In a long-awaited report that will be debated by member states at a meeting in October in Moscow, the United Nations health agency on Tuesday also voiced concern about the concentration of the $3 billion market in the hands of big tobacco companies. "In a nutshell, the WHO report shows that e-cigarettes and similar devices pose threats to public health," Douglas Bettcher, director of the agency's department on non-communicable diseases, told a news briefing in Geneva. The uptake of e-cigarettes, which use battery-powered cartridges to produce a nicotine-laced vapour, has rocketed in the past two years, but there is fierce debate about the risks. Because they are so new, there is a lack of long-term scientific evidence to support their safety, and some fear they could lead to nicotine addiction and tobacco smoking. "We must emphasise that the onus of responsibility for showing safety, for answering many of these questions, must be on the companies and the industries owning them," Bettcher said. "The reports finds, at this point in time anyway, that there is insufficient evidence to conclude that e-cigarettes help users to quit smoking or not. The jury is still out," he said. The European Union has already agreed to requirements around advertising and packaging to ensure the safety and quality of e-cigarettes. The U.S. Food and Drug Administration has proposed banning sales to anyone under 18 but no curbs on advertising. Activists welcomed the WHO recommendations. "As Big Tobacco corners the e-cigarette market, it is using e-cigarettes as a global PR scheme to gloss over its tarnished image, positioning itself as a'solution' to the problem it drives. In reality, the e-cigarette industry is taking advantage of the regulatory vacuum to employ the Big Tobacco playbook to hook a new generation on its products," said John Stewart of the U.S.-based group Corporate Accountability International. REGULATORY OPTIONS The WHO launched a public health campaign against tobacco a decade ago. The WHO Framework Convention on Tobacco Control, which entered into force in 2005, has been ratified by 179 states, although not the United States. There are 466 brands of e-cigarettes, and the industry represents "an evolving frontier filled with promise and threat for tobacco control", the WHO said in the report. It urged a range of regulatory options including banning vending machines in most locations and preventing e-cigarette makers from making health claims, such as that they help people quit smoking, until there is hard evidence. Smokers should use a combination of already approved treatments for kicking the habit, it said. While e-cigarettes are likely to be less toxic than conventional ones, the WHO dismissed the idea that e-cigarettes merely produced "water vapour", arguing they exposed bystanders and non-smokers to nicotine and other toxic substances. Dr. Armando Peruga, of the WHO's Tobacco Free Initiative, said the contents of e-cigarettes vary but that the aerosol expelled by their users contains nicotine, which is known to alter brain development, and other toxins. "There are brands for example that contain formaldehyde, which is a cancer-causing element, at the same level as some cigarettes," Peruga told reporters. "Depending on the brand, some studies have found that they contain heavy metals, for example cadmium which is completely a cancer-causing agent," Peruga said. Others have been found to contain nickel or acrolein, a respiratory irritant, he said. Their use also posed a threat to adolescents and the foetuses of pregnant women, the WHO said. BACON TO BUBBLE GUM One concern is that e-cigarettes may tempt children, and the report called for a ban on flavours until there was proof they did not attract adolescents. E-cigarettes can be customised with flavours ranging from bacon to bubble gum. Scientists are divided on the risks and potential benefits of e-cigarettes. One group of researchers warned the WHO in May not to classify them as tobacco products, arguing that doing so would jeopardise an opportunity to slash disease and deaths caused by smoking. Opposing experts argued a month later that the WHO should hold firm to its plan for strict regulations. Major tobacco companies including Imperial Tobacco (IMT.L), Altria Group (MO.N), Philip Morris International (PM.N) and British American Tobacco (BATS.L) are increasingly launching their own e-cigarette brands as sales of conventional products stall in Western markets. Two major national producers, China Tobacco and Indian Tobacco Company, have recently become producers, Bettcher said. A Wells Fargo analyst report in July projected that U.S. sales of e-cigarettes would outpace conventional ones by 2020. A BAT spokesman said overly restrictive regulations could prevent smokers from being aware of a less risky alternative to smoking, and "this can only be bad thing for public health". (Additional reporting by Martinne Geller in London; Editing by Angus MacSwan, David Clarke and Jane Baird)

Summary: More support for those who think it's too early to jump on the e-cigarette bandwagon: The vapor-producing devices may still pose a threat to users' and bystanders' health, says WHO, which suggests stronger regulations on the relatively new industry in a report released today, reports Reuters. The health organization also asks for a ban on puffing away on the battery-driven units indoors, as well as on advertising and flavored e-cigs that could lure underage users. Although e-cigs "are likely less toxic than conventional ones," writes Stephanie Nebehay at Reuters, WHO researchers say that nicotine and other chemicals emitted by e-cigs are still a health hazard, especially for teens and pregnant women. Those chemicals can include formaldehyde, aluminum, and silicate particles, reports the Telegraph. The WHO report is lobbying against e-cig vending machines and says manufacturers shouldn't be able to tout their products as "smoking cessation aids" until more research is completed to back that claim up. The main debate right now seems to be between those who think that e-cigs can help cut down on tobacco-related deaths and those who argue that using e-cigs could lead to the real thing for youngsters-especially with flavors such as bacon, bubble gum, and even Thin Mint. "Many public health experts are concerned that the advertising of electronic cigarettes could make it seem normal again to think smoking is glamorous," a health official tells the Telegraph.

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Write a title and summarize: Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted. Solid tumors are aberrant tissues where stromal cell types co-develop with and influence cancer cells [1]. Significant epigenetic alterations and gene expression changes occur in stromal cells as tumors progress, and the stromal changes are as strikingly different as those observed in the cancer epithelial compartments [2]–[5]. Many of these stromal cell changes are elicited by factors secreted by cancer cells, such as vascular endothelial growth factor (VEGF; MIM: 192240), which helps recruit and induce proliferation of endothelial cells [6]. Cancer cells also secrete factors that alter surrounding fibroblasts, such as transforming growth factor (TGF) -β (MIM: 190180) which induces fibroblasts to differentiate into myofibroblasts and secrete collagen, thereby contributing to the abundant extracellular matrix often observed in epithelial tumors [7]. In addition to TGF-β factors secreted by cancer cells that influence stromal fibroblasts include platelet derived growth factor (PDGF; MIM: 173430), interleukin (IL) -6 (MIM: 147620), IL1-α (MIM: 147760), and WNT1 inducible signaling pathway protein (WISP) -1 and -2 (MIM: 603398 and 603399) [8], [9]. Tumor associated fibroblasts have been shown to promote cancer cell proliferation, angiogenesis, extracellular matrix (ECM) remodeling, inflammation, invasion and metastasis [10], [11]. Several fibroblast-secreted or membrane-bound factors that mediate these effects have been identified, including CXCL12 (MIM: 600835), hepatocyte growth factor (HGF; MIM: 142409), matrix metalloproteinase MMP14 (MIM: 600754), osteopontin (MIM: 166490), TGF-β, and CCL2 (MIM: 158105) [12]–[17]. Some basic underlying processes involved in regulating the interactions between the epithelial cancer cells and the stromal fibroblasts have been identified. For example, several fibroblast-secreted factors are inflammatory cytokines whose expression is driven by NF-kappaB-dependent transcription in a process similar to the senescent secretory phenotype observed in aging fibroblasts [18], [19]. Additionally, a study of how fibroblasts co-evolve with tumor cells determined that fibroblasts gradually implement two signaling loops, involving TGF-β and CXCL12, which act together through both autocrine and cross-signaling mechanisms [20]. What remains unclear is whether the different factors involved in cancer cell-fibroblast interactions reflects a requirement of a multitude of fibroblast factors acting in parallel to promote tumorigenicity, or whether it reflects the diversity of the approaches and systems used to identity important interactions. Here, we designed a study to explore how the entire repertoire of fibroblast-secreted factors that are induced by human cancer cells function as a whole and compared the factors. This systems-level study was designed to be complimentary to approaches that focus on single genes or single processes. Our study indicates that the majority of induced fibroblast-secreted factors play a role in promoting tumorigenicity and that they do so through diverse effects on the tumor tissue. We adapted previously used systems of human cancer cells and fibroblasts [21]–[23], by using fibroblast lines that were amenable for the use of stable RNA interference (shRNA) so the relevance of candidate mediators of tumor-stromal interactions could be tested. We determined using co-injection assays that two human fibroblast lines previously shown to promote tumorigenicity in other systems (HFFF2 and HFF1) were able to promote tumorigenicity of two basal breast cancer subtype cell lines, MDA-MB-231 and Cal51, whereas two other human fibroblast lines (Wi-38 and CCD1112Sk) were not (Figure S1). We used the two tumor-supportive fibroblast lines (HFFF2 and HFF1) as models for patient-derived breast carcinoma associated fibroblasts and the two non-supportive fibroblasts as models for normal breast tissue derived fibroblasts. Using fibroblasts cell lines allowed shRNA transfection, selection and subsequent validation of gene silencing, whereas patient-derived breast fibroblasts cannot be passaged in culture long enough for such manipulations (data not shown; Ahmet Acar, personal communication). To determine at which point the host murine fibroblasts replaced the co-injected human fibroblasts, we tagged the human fibroblasts with green fluorescent protein (GFP) so that we could visualize how long they survived ensconced within developing tumors. We found that after three weeks, the number of co-injected GFP-tagged human fibroblasts comprised roughly 20% of the tumor stroma as judged by co-localization of GFP and the fibroblast stromal marker alpha-smooth muscle actin (α-SMA) (Figure S2), but the relative contribution steadily declined from week 3 to week 8, although the GFP-fibroblasts never completely disappear. The level of α-SMA positive cells within tumor stroma persisted during this same time period (Figure S2). We performed transcriptional profiling and pathway analysis to determine if exposure of our model tumor-supportive fibroblasts to breast cancer cells in co-culture resembled expression changes seen in patient-derived breast carcinoma fibroblasts. We compared expression profiles of HFF1 and HFFF2 tumor-supportive fibroblasts co-cultured with either Cal51 or MDA-MB-231 (four combinations total) to those of Wi-38 and CCD1112Sk non-supportive fibroblasts co-cultured with either Cal51 or MDA-MB-231 (another four combinations). We chose to work with basal subtype breast cancer cell lines due to the greater need to develop new treatments for this poor prognosis subtype. We then compared the pathways selectively enriched in tumor-supportive fibroblasts co-cultured with Cal51 and MDA-MB-231 to pathways enriched by comparing cultures of patient-derived breast carcinoma fibroblasts to their normal counterparts. Strikingly, the top four pathways identified by gene-set enrichment analysis (GSEA) that are activated by exposure of tumor-promoting human fibroblasts to breast cancer cells are also amongst the top ten pathways activated in cultures of patient-derived breast carcinoma fibroblasts relative to normal fibroblasts (Figure 1). These pathways are ECM-receptor interaction, focal adhesion, integrin signaling and integrin cell-surface interactions; interrelated pathways that have been shown to be involved in the activation of cancer associated fibroblasts [21], [24], [25]. Additionally, most of the other top ten activated pathways in both systems were activated in the other system at lower ranking but still significant levels. These pathways included cytokine cytokine-receptor interactions, PDGF signaling, and Rho GTPase signaling; which likewise have shown to be involved in activation of cancer associated fibroblasts [7], [13], [26] (Figure 1). On the whole, the overlap in all significantly activated pathways is 44% (Table S1). This affirmed that this system of tumor-promoting human fibroblasts resembled in vitro cultures of patient-derived breast cancer fibroblasts. We next wanted to test whether this system also reflected changes observed in vivo in human primary breast cancer stroma. To address this, we used gene-set enrichment analysis to determine the pathways that were activated in microdissected human breast stroma relative to normal breast stroma [3]. Despite the fact that human breast stroma contains many cell types in addition to fibroblasts, four of the eight significantly activated pathways in human breast stroma were also significantly activated by exposure of tumor-promoting fibroblasts to breast cancer cells, including cytokine/cytokine-receptor interactions and JAK-STAT signaling (Figure 1). Additionally, we developed a gene signature based on stimulation of tumor-promoting fibroblasts by breast cancer cells. Based on both clustering and principal component analysis, this gene signature was able to correctly predict whether microdissected human breast stroma was derived from cancerous or normal breast tissue for 98 of 99 samples (Figure 1). These comparative genomic analyses establish the close correspondence of our system of interaction of breast cancer cells with tumor-promoting fibroblasts to stroma and stromal fibroblasts isolated from primary human breast cancer. We used genome-wide analysis to determine the full repertoire of secreted factors induced by breast cancer cells in our tumor-promoting fibroblasts. As the first step, we compared genes induced in tumor-promoting fibroblast lines to genes induced in fibroblast lines incapable of promoting tumorigenicity. We found 320 genes that were more than 2-fold greater induced in tumor-promoting fibroblasts (Table S2) and within this group were 62 genes encoding secreted proteins. Of these, 42 were also significantly upregulated (p<0. 05) in stromal cells isolated from primary breast cancer relative to normal breast stroma cells [3], [4], [27]. This group contained cytokines (15), extracellular matrix proteins (7), proteases (6), and growth factors and hormones (6) (Table 1). Cytokines as a class were significantly enriched in our screen relative to their representation in the set of all secreted proteins (36% vs. 12%, p = 5. 6e-6). This enrichment is consistent with the prior reports that cytokines play key roles in the tumor-supportive function of tumor-associated fibroblasts [19]. In contrast, the other functional classes of secreted proteins were not significantly enriched. The cytokines upregulated in tumor-supportive fibroblasts included CC- and CXC-chemokines (CCL-2, -5, -7, -8, -20, and CXCL-5, -10; MIM: 158105,187011,158106,602283,600324 and 147310 respectively), pro-inflammatory interleukins (IL-1α, -1β, -8, -11, -24; MIM: 147760,147720,146930,147681 and 604136 respectively), colony stimulating factor (G-CSF; MIM: 138970), interleukin receptor antagonist (IL1RN; MIM: 147679), and TNF superfamily member TNSF15 (MIM: 604052) (Table 1). Systematic literature searches revealed that 16 of the 42 secreted proteins had one or more publication (s) implicating them in the tumor-supportive function of fibroblasts while 26 did not (Table 1). The genes not previously implicated as mediators of the tumor-supportive function of fibroblasts included the majority (8/15) of cytokines, one-half (3/6) of the proteases, and the majority (4/6) of the growth factors and hormones. Examples include CCL8, encoding a chemokine not previously linked to cancer that is involved in homing of memory T lymphocytes to inflamed skin [28]; pregnancy-associated plasma protein-a (PPAPA; MIM: 176385), a protease that degrades IGF-binding proteins and acts as a positive modulator of local IGF signaling in skin repair [29]; and EGFL6 (MIM: 300239), encoding an epidermal growth factor (EGF) repeat protein expressed in osteoblastic-like cells and capable of inducing migration of endothelial cells [30]. Some of the fibroblast-secreted candidate tumor-supportive factors fall outside of the known classes of proteins involved in tumor-stromal interactions, including ISG15 (MIM: 147571), an interferon-inducible, ubiquitin-like protein whose secretion plays a critical role in mediating an effective immune response to mycobacteria [31]; and complement component C3 (MIM: 120700), which in addition to its role in the complement cascade helps mobilize hematopoietic stem/progenitor cells to wounds [32]. In order to characterize the effects of fibroblasts on tumor progression, we co-injected tumor-promoting fibroblasts and examined their effects on both tumor cells and associated non-tumor cells within the tumor microenvironment. Consistent with a faster tumor growth rate, cancer cells in the co-injected tumors exhibited a two-fold higher proliferative rate based on Ki-67 labeling (Figure 2). To visualize and quantify the presence of three of the most important features of the tumor microenvironment - inflammation, vascularization and fibroblast activation - we performed immunohistochemical analysis at week six after co-injection. We used antibodies to the 7/4-antigen, which is highly expressed in neutrophils and inflammatory monocytes [33], the CD31 endothelial antigen, which visualizes blood vessels [34], and α-SMA, which is expressed by activated fibroblasts [35]. Strikingly, the presence of inflammatory cells, degree of vascularization, and number of activated fibroblasts were all 3 to 4 fold higher in the co-injected tumors (Figure 2), indicating a profound influence of tumor-supportive fibroblasts on the composition of the tumor microenvironment. We decided to analyze a mix of previously implicated and novel candidate pro-tumorigenic fibroblast-secreted factors. Additionally, we wanted to test whether seemingly redundant factors were indeed functionally redundant. We focused on cytokines, growth factors, and hormones, and chose CCL2 and CCL7 (both previously validated as functionally important fibroblast secreted factors [17], [36]); the related factor CCL8 which, like CCL7, binds CCR1 (MIM: 601159); amphiregulin (AREG; MIM: 104640), which has been implicated in tumor stromal-interactions but as a ligand produced by cancer cells acting on fibroblasts [37]; and stanniocalcin1 (STC1; MIM: 601185), which has been shown to act as a cancer cell autonomous factor, but not as a stromally produced factor [38]. For each of these five genes, we confirmed by quantitative RT-PCR that there was a significant induction in the co-cultured tumor supportive fibroblasts relative to the co-cultured neutral fibroblasts (Figure S3). CCL2, CCL7, and CCL8 are structurally related chemokines that share a common function of recruitment of monocytes to areas of injury and inflammation [39]. Based on their structural similarity and overlapping functions in inflammation, we wanted to determine if they had redundant roles in the tumor supportive function of co-injected fibroblasts. Remarkably, shRNAs directed against each of these three cytokines suppressed tumorigenicity in the co-injection assay, with the strongest effects observed when silencing CCL2 (53%) or CCL7 (66%) compared to weaker effects exerted by silencing of CCL8 (25%) (two validated shRNAs per gene, Figure 3). Interestingly, despite their related structure, silencing of each of the three cytokines had a distinct impact on the tumors: silencing of CCL2 suppressed recruitment of innate immune cells and angiogenesis (Figure 4) almost to the same levels as in tumors without co-injected fibroblasts. In contrast, silencing of CCL8 suppressed only the recruitment of innate immune cells, while silencing of CCL7 reduced tumor cell proliferation almost to the levels observed in tumors growing in the absence of co-injected fibroblasts (Figure 4 and Figure S4). Our data therefore showed that these related chemokines had non-redundant roles in mediating fibroblast-supportive functions. We next tested whether silencing of AREG in fibroblasts affected tumor supportive function. Silencing of AREG had a pronounced effect, with an average reduction in tumor size at six weeks of 55% to 65% (Figure 5). In contrast to the chemokines, silencing AREG in fibroblasts had no effect on the number of blood vessels or innate immune cells (Figure 5). However, immunohistochemical analysis of the percentage of cells within the tumor that expressed α-SMA, a marker of mesenchymal-derived cells such as activated fibroblasts, revealed a 78% reduction. This level was comparable to the low levels of α-SMA positive cells in tumors formed in the absence of co-injected fibroblasts (Figure 5). Although α-SMA is expressed by activated pericytes, a cell type that surrounds endothelial cells of blood vessels, we did not observe a difference in number of α-SMA cells associated with blood vessels (Figure 5). This suggested that secretion of amphiregulin by the co-injected human fibroblasts plays a major role in establishing a tumor microenvironment that is enriched for activated fibroblasts, and since the co-injected human fibroblasts are almost entirely replaced by mouse fibroblasts at the point of excision (6 weeks), this effect must be propagated through recruited mouse fibroblasts. Consistent with this, we found that amphiregulin increased proliferation of mouse fibroblasts, as well as human fibroblasts (Figure 5). We also found that amphiregulin was a chemoattractant for mouse fibroblasts in migration and invasion assays, suggesting a mechanism of recruitment into the tumor, and that amphiregulin directly activated fibroblasts as judged by induction of α-SMA expression (Figure 6). In addition to the pronounced reduction of activated fibroblasts when AREG was silenced, we also noted changes in activation of the amphiregulin receptor EGFR (MIM: 131550) on tumor cells. Compared to tumor cells co-injected with control tumor-supportive fibroblasts, tumor cells when co-injected with AREG-silenced fibroblasts showed a <2-fold reduction of activated, phospho-EGFR as measured by immunohistochemistry (Figures 6). This was accompanied by a minor, barely significant (p = 0. 06), negative effect on tumor cell proliferative rate in vivo, with no direct effect of amphiregulin on tumor cell proliferation in vitro (Figure 5 and Figure S5). In contrast, we found a significant increase in necrosis in tumors after silencing of fibroblast-secreted amphiregulin (Figure 6). Notably, amphiregulin significantly protected the breast cancer cells from cell death induced by detachment from their normal extracellular matrix (anoikis, Figure 6). Together, these results suggest that fibroblast-secreted amphiregulin has potent effects on tumor progression, with autocrine effects leading to activation of fibroblasts and paracrine effects protecting cancer cells from cell death. Co-culturing also lead to changes in gene expression in the breast cancer cells, which upregulated a shared receptor for CCL2 and CCL7, the chemokine receptor CCR1 upon co-culturing with tumor-supportive fibroblasts (Figure 7). We therefore tested whether CCR1 expression by cancer cells was critical for some of the tumor supportive functions of fibroblasts by stably expressing shRNAs targeting CCR1 in Cal51 breast cancer cell line (knockdown efficiency was quantified by both qRT-PCR and immunoblotting, Figure S6). Silencing of CCR1 had no effect on tumor growth when cancer cells were injected alone. However, in the context of co-injection with fibroblasts, silencing of cancer cell CCR1 resulted in a 3-fold reduction in tumor size, almost eliminating the effect of the fibroblasts (Figure 7 and Figure S6). We further observed a 40% reduction in the proliferative index of cancer cells and a 2-fold reduction in recruitment of neutrophils and inflammatory monocytes to the tumor, but only minor effects on the number of blood vessels and mesenchymal cells (Figure 7 and Figure S6). Thus, expression of CCR1 by cancer cells plays a critical role in enabling fibroblasts to exert tumor supportive function, through increased tumor cell proliferation and potentially indirectly through recruitment of leukocytes. We next asked whether blocking two interactions between fibroblasts and cancer cells was more effective than blocking either one alone. We therefore asked whether simultaneously silencing fibroblast-secreted amphiregulin and cancer cell expressed CCR1 was more efficacious than blocking either alone. We found that tumor growth was significantly more reduced when both pathways were targeted (Figure 7). A number of studies have implicated specific cancer-cell secreted factors in the activation of neighboring fibroblasts, including TGF-β [40] and IL-1β [19]. We wanted to determine whether the repertoire of tumor-promoting fibroblast secreted factors could be induced by single specific inducers, or whether multiple pathways were acting in parallel. To test this, we used quantitative RT-PCR to measure gene expression changes in tumor-promoting fibroblasts of the five factors (CCL-2, -7, -8, AREG, and STC1) that we had determined all had functional relevance, along with two others factors (NRG1; MIM: 142445 and WISP1) that also were induced in the system. Surprisingly, TGF-β, did not induce expression of any of the seven factors (Table 2), despite its ability to induce activation of fibroblasts to myofibroblasts, which resembles many aspects of carcinoma-associated fibroblasts [10]. AREG also did not induce expression of any of the seven factors, despite its key role in stimulating mammary fibroblasts during normal mammary development [37]. IL-1β, a potent activator of NF-κB signaling, produced an upregulation in chemokines CCL-2, -7, -8 similar to that seen by co-culture with breast cancer cells (Table 2). However, IL-1β did not induce expression of WISP1, STC1, AREG, or NRG1. Interestingly, a combination of IL-1β and AREG lead to significant upregulation of WISP1 in addition to stimulation of CCL-2, -7, -8. In contrast, none of the fibroblast factors were induced when TGF-β and IL-1β were combined (Table 2). Based on these results, it seems likely that the ability of breast cancer cells to induce the full spectrum of pro-tumorigenic fibroblast-secreted proteins involves a multitude of interacting factors, including some not previously identified. We also found that co-culture of tumor-promoting fibroblasts with a normal, non-malignant breast epithelial cell line, MCF10A, was able to induce fibroblast expression of two out of the seven factors induced by breast cancer cells, namely AREG and WISP2 (Table 2). For these two factors, it would appear that breast epithelial cells per se, regardless of tumorigenic properties, elicit the same response in fibroblasts. This result is consistent with the key role that stromal AREG plays in normal mammary development [37]. Numerous studies have used the co-injection assay to identify the factors produced by fibroblasts that are responsible for promoting tumorigenicity. These studies have identified single factors that when inhibited strongly suppress the ability of activated fibroblasts to promote tumorigenicity, and they include scatter factor, SDF-1, MMP14, NF-κB, osteopontin, TGF-β, and CCL2 [12]–[17]. However, these studies did not address whether the single factors were unique in their capacity of promoting fibroblast-supported tumorigenicity, nor did they compare different factors. Here, we used comparative genomics to identify 42 candidate mediators of fibroblast-promoted tumorigenicity and we tested the functional impact of a set of five of these factors. Surprisingly, we found that four of the five tested factors promoted tumorigenicity, three of them strongly (CCL7, CCL2, and AREG) and one of them weakly (CCL8) (Table 3). Although the fifth factor STC1 significantly affected tumorigenicity by the area under the tumor growth curve test (Table 3), it failed to show significant effects using t-tests at any single time points (data not shown). Since we only tested five of the 42 fibroblast-secreted-factors that were induced by breast cancer cells, it seems highly likely that an even greater number of fibroblast-secreted factors play a role in promoting tumorigenicity. Thus, our study indicates that even in a single system there are a large number of secreted factors involved in the ability of fibroblasts to promote carcinomas, rather than a single important mediator. Intriguingly, our results also indicate widely diverse mechanisms for fibroblast-secreted factors in the promotion of tumorigenicity. The strong effects of fibroblast CCL7 appeared to be caused by a significant effect on cancer cell proliferation (Table 3), which was unique among the factors tested. We also found that reducing cancer cell expression of the CCL7 receptor, CCR1, also reduced fibroblast-induced proliferation. In contrast, the strong tumor promoting effects of fibroblast CCL2 was associated with different effects on the tumor microenvironment, as we found significant decreases in both angiogenesis and recruitment of innate immune cell upon silencing of CCL2. CCL8 had weaker effects on tumor growth than the other tested chemokines, which likely reflects it inability to affect either tumor proliferation or vascularity (Table 3). Fibroblast AREG also had very strong effects on tumor growth, and influenced both the total number of activated fibroblasts in the tumors and the survival of the cancer cells, a combination not observed with any other tested factor. Interestingly, secretion of amphiregulin by fibroblasts appeared to potentially act as a chemoattractant to recruit new fibroblasts and induce their proliferation and activation, resulting in a tumor microenvironment that is enriched in activated fibroblasts. Thus, in a single system of carcinoma cells and tumor-supportive fibroblasts, several factors play key roles. Our study also sheds light on how cancer cells modify the stromal cells to enable them to promote tumorigenicity. We tested several cancer cell-secreted factors, previously reported to influence stromal cells, but none of them were able to induce the full panel of verified tumor-promoting fibroblast factors as well as the cancer cells themselves. Even in one simple system, it appears that cancer cells act on fibroblasts through multiple factors, resulting in the secretion of another complex set of factors that influence cancer cells and other components of the tumor microenvironment. One of the key findings of our study was that inhibiting multiple interactions between cancer cells and fibroblasts is more efficacious than blocking individual pathways. This finding is not completely unexpected in light of the complexity of the tumor microenvironment and in fact previous reports have suggested this as a possibility [20], [41]. Nevertheless, this result highlight that the different interactions are non-redundant and act in parallel, but it also suggests that effective anti-stromal fibroblast therapeutic strategies can be achieved by taking a multi-targeted approach. Future research towards this end will need to employ models that closely resemble the type of tumors to be targeted in human patients. This presents many challenges, including the ability to selectively target endogenous fibroblasts in tumor tissues, along with the ability to monitor the in vivo response of tumor-associated fibroblasts. Despite these challenges, our study shows that there are several potential combinatorial targets for future fibroblast-targeted therapeutic approaches. All genomic data for this study, including expression analysis of both fibroblasts and breast cancer cells, have been deposited in the Gene Expression Omnibus (GEO) repository (GSE41678). http: //www. ncbi. nlm. nih. gov/geo/query/acc. cgi? token=bnepxkssyoamgdu&acc=GSE41678 Breast cancer cell lines and human fibroblast strains were obtained from ATCC (Manassas, VA), DSMZ (Braunschweig, Germany) or Sigma (St. Louis, MO) and grown under standard tissue culture conditions in growth medium recommended by the supplier. For dual-color co-culture experiments, breast cancer cells stably expressing Discosoma sp. red fluorescent protein (DsRed) and human fibroblasts stably expressing Zoanthus sp. green fluorescent protein (ZsGreen) were generated using pRetroX-IRES-DsRedExpress vector and pRetroX-IRES-ZsGreen1 vector (Clontech, CA) respectively via retroviral transduction (detailed description in Supplemental Experimental Procedures). None of the experiments utilized multiple retroviral transfections. For the co-culture experiments, we transfected the original fibroblasts with a fluorescent marker in order to facilitate cell separation before transcriptome analysis (the breast cancer cells were transfected with a different fluorescent marker). For the shRNA experiments, we transfected the original fibroblasts with validated shRNA constructs from the Broad library. Co-culture of fluorescently-tagged breast cancer cells and fibroblasts was initiated by plating 1. 5 million fibroblasts into 10 cm dishes, and after 18 hours 1 million breast cancer cells were added and then incubated for six days. Monocultures were performed in parallel for the same duration. Following co-culture or mono-culture, cells were trypsinized and resuspended in FACS sorting buffer (PBS+1% FBS) for separation into DsRed+ and ZsGreen+ populations using an ARIA II flow cytometer and analyzed using FACS DiVA software (Becton Dickenson, CA). Total RNA was isolated using RNeasy kit (Qiagen, Netherlands) and hybridized to Gene 1. 0 ST arrays (Affymetrix, CA). Data was extracted, background corrected, normalized, and converted from probe values to gene values using the AROMA R package (www. aroma-project. org). All studies utilizing human xenograft experiments were approved by and in accordance with Cold Spring Harbor Laboratory' s Institutional Animal Care and Use Committee. Five to six week old female nude mice (NCR nu/nu; Charles River Inc., Wilmington, MA) were irradiated at 400 cGy 24–36 hours prior to injections. One million breast cancer cells were trypsinized, resuspended with or without 1. 5 million fibroblasts in 100 µl DMEM and injected subcutaneously into both flanks of irradiated, nude mice. Growth was followed over time by taking caliper measurements at indicated time points. Tumor volume was measured as 0. 52×length×width2. Tumors were excised six-eight weeks post injections or when one of the measurements reached 2 cm. Immunostaining procedure is described in detail in Supplementary Experimental Procedures. The primary antibodies used for immunostaining are as follows: α-SMA (1∶2000; # 1A4; Sigma-Aldrich), CD31 (1∶100; ab28364; Abcam), antigen 7/4 (1∶400, CL8993AP, Cedarlane), Ki-67 (1∶2000; MIB5; Dako), pEGFR (1∶100; 1138-1; Epitomics) and GFP (1∶1000; ab290; Abcam). Immunostained slides were quantified by counting (for 7/4, Ki-67 and CD31), by percentage of stained area (for α –SMA and pEGFR) using Image J software (NIH, Bethesda, MD). Additional methods are found in the file Text S1.

Title: System-Wide Analysis Reveals a Complex Network of Tumor-Fibroblast Interactions Involved in Tumorigenicity Summary: There is increasing interest in developing methods to treat cancer by targeting non-cancer cells that play supportive roles in the tumor microenvironment. One type of non-cancer cell that has received considerable attention along these lines is cancer-associated fibroblasts, which can promote tumor formation and tumor growth. There have been several studies showing that inhibition of individual fibroblast genes or proteins dramatically reduces the tumor supportive function of fibroblasts. From the perspective of developing a therapeutic strategy, what remains unclear is whether the several different important factors discovered to date reflect the requirement of a multitude of fibroblast factors to promote tumorigenicity, or whether it reflects the diversity of the epithelial cancer cells and fibroblasts used in these different studies. Here, we addressed this question directly using a single system of fibroblasts and breast cancer epithelial cells. Importantly, we found that a multitude of fibroblast factors are indeed required to promote tumorigenicity, and that they have different effects on the tumor microenvironment. Furthermore, we found that inhibiting multiple fibroblast-secreted factors is more efficacious than blocking individual factors. These results suggest that fibroblasts and cancer cells act through multiple parallel pathways and that effective anti-stromal therapeutic strategies will need to be multi-targeted.

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Summarize: VII. AMY'S VALLEY OF HUMILIATION. [Illustration: The Cyclops] "That boy is a perfect Cyclops, isn't he?" said Amy, one day, as Laurie clattered by on horseback, with a flourish of his whip as he passed. "How dare you say so, when he's got both his eyes? and very handsome ones they are, too," cried Jo, who resented any slighting remarks about her friend. "I didn't say anything about his eyes, and I don't see why you need fire up when I admire his riding." "Oh, my goodness! that little goose means a centaur, and she called him a Cyclops," exclaimed Jo, with a burst of laughter. "You needn't be so rude; it's only a 'lapse of lingy,' as Mr. Davis says," retorted Amy, finishing Jo with her Latin. "I just wish I had a little of the money Laurie spends on that horse," she added, as if to herself, yet hoping her sisters would hear. "Why?" asked Meg kindly, for Jo had gone off in another laugh at Amy's second blunder. "I need it so much; I'm dreadfully in debt, and it won't be my turn to have the rag-money for a month." "In debt, Amy? What do you mean?" and Meg looked sober. "Why, I owe at least a dozen pickled limes, and I can't pay them, you know, till I have money, for Marmee forbade my having anything charged at the shop." "Tell me all about it. Are limes the fashion now? It used to be pricking bits of rubber to make balls;" and Meg tried to keep her countenance, Amy looked so grave and important. "Why, you see, the girls are always buying them, and unless you want to be thought mean, you must do it, too. It's nothing but limes now, for every one is sucking them in their desks in school-time, and trading them off for pencils, bead-rings, paper dolls, or something else, at recess. If one girl likes another, she gives her a lime; if she's mad with her, she eats one before her face, and don't offer even a suck. They treat by turns; and I've had ever so many, but haven't returned them; and I ought, for they are debts of honor, you know." "How much will pay them off, and restore your credit?" asked Meg, taking out her purse. "A quarter would more than do it, and leave a few cents over for a treat for you. Don't you like limes?" "Not much; you may have my share. Here's the money. Make it last as long as you can, for it isn't very plenty, you know." "Oh, thank you! It must be so nice to have pocket-money! I'll have a grand feast, for I haven't tasted a lime this week. I felt delicate about taking any, as I couldn't return them, and I'm actually suffering for one." Next day Amy was rather late at school; but could not resist the temptation of displaying, with pardonable pride, a moist brown-paper parcel, before she consigned it to the inmost recesses of her desk. During the next few minutes the rumor that Amy March had got twenty-four delicious limes (she ate one on the way), and was going to treat, circulated through her "set," and the attentions of her friends became quite overwhelming. Katy Brown invited her to her next party on the spot; Mary Kingsley insisted on lending her her watch till recess; and Jenny Snow, a satirical young lady, who had basely twitted Amy upon her limeless state, promptly buried the hatchet, and offered to furnish answers to certain appalling sums. But Amy had not forgotten Miss Snow's cutting remarks about "some persons whose noses were not too flat to smell other people's limes, and stuck-up people, who were not too proud to ask for them;" and she instantly crushed "that Snow girl's" hopes by the withering telegram, "You needn't be so polite all of a sudden, for you won't get any." A distinguished personage happened to visit the school that morning, and Amy's beautifully drawn maps received praise, which honor to her foe rankled in the soul of Miss Snow, and caused Miss March to assume the airs of a studious young peacock. But, alas, alas! pride goes before a fall, and the revengeful Snow turned the tables with disastrous success. No sooner had the guest paid the usual stale compliments, and bowed himself out, than Jenny, under pretence of asking an important question, informed Mr. Davis, the teacher, that Amy March had pickled limes in her desk. Now Mr. Davis had declared limes a contraband article, and solemnly vowed to publicly ferrule the first person who was found breaking the law. This much-enduring man had succeeded in banishing chewing-gum after a long and stormy war, had made a bonfire of the confiscated novels and newspapers, had suppressed a private post-office, had forbidden distortions of the face, nicknames, and caricatures, and done all that one man could do to keep half a hundred rebellious girls in order. Boys are trying enough to human patience, goodness knows! but girls are infinitely more so, especially to nervous gentlemen, with tyrannical tempers, and no more talent for teaching than Dr. Blimber. Mr. Davis knew any quantity of Greek, Latin, Algebra, and ologies of all sorts, so he was called a fine teacher; and manners, morals, feelings, and examples were not considered of any particular importance. It was a most unfortunate moment for denouncing Amy, and Jenny knew it. Mr. Davis had evidently taken his coffee too strong that morning; there was an east wind, which always affected his neuralgia; and his pupils had not done him the credit which he felt he deserved: therefore, to use the expressive, if not elegant, language of a school-girl, "he was as nervous as a witch and as cross as a bear." The word "limes" was like fire to powder; his yellow face flushed, and he rapped on his desk with an energy which made Jenny skip to her seat with unusual rapidity. "Young ladies, attention, if you please!" At the stern order the buzz ceased, and fifty pairs of blue, black, gray, and brown eyes were obediently fixed upon his awful countenance. "Miss March, come to the desk." Amy rose to comply with outward composure, but a secret fear oppressed her, for the limes weighed upon her conscience. "Bring with you the limes you have in your desk," was the unexpected command which arrested her before she got out of her seat. "Don't take all," whispered her neighbor, a young lady of great presence of mind. Amy hastily shook out half a dozen, and laid the rest down before Mr. Davis, feeling that any man possessing a human heart would relent when that delicious perfume met his nose. Unfortunately, Mr. Davis particularly detested the odor of the fashionable pickle, and disgust added to his wrath. "Is that all?" "Not quite," stammered Amy. "Bring the rest immediately." With a despairing glance at her set, she obeyed. "You are sure there are no more?" "I never lie, sir." "So I see. Now take these disgusting things two by two, and throw them out of the window." There was a simultaneous sigh, which created quite a little gust, as the last hope fled, and the treat was ravished from their longing lips. Scarlet with shame and anger, Amy went to and fro six dreadful times; and as each doomed couple--looking oh! so plump and juicy--fell from her reluctant hands, a shout from the street completed the anguish of the girls, for it told them that their feast was being exulted over by the little Irish children, who were their sworn foes. This--this was too much; all flashed indignant or appealing glances at the inexorable Davis, and one passionate lime-lover burst into tears. As Amy returned from her last trip, Mr. Davis gave a portentous "Hem!" and said, in his most impressive manner,-- "Young ladies, you remember what I said to you a week ago. I am sorry this has happened, but I never allow my rules to be infringed, and I _never_ break my word. Miss March, hold out your hand." Amy started, and put both hands behind her, turning on him an imploring look which pleaded for her better than the words she could not utter. She was rather a favorite with "old Davis," as, of course, he was called, and it's my private belief that he _would_ have broken his word if the indignation of one irrepressible young lady had not found vent in a hiss. That hiss, faint as it was, irritated the irascible gentleman, and sealed the culprit's fate. "Your hand, Miss March!" was the only answer her mute appeal received; and, too proud to cry or beseech, Amy set her teeth, threw back her head defiantly, and bore without flinching several tingling blows on her little palm. They were neither many nor heavy, but that made no difference to her. For the first time in her life she had been struck; and the disgrace, in her eyes, was as deep as if he had knocked her down. [Illustration: Amy bore without flinching several tingling blows] "You will now stand on the platform till recess," said Mr. Davis, resolved to do the thing thoroughly, since he had begun. That was dreadful. It would have been bad enough to go to her seat, and see the pitying faces of her friends, or the satisfied ones of her few enemies; but to face the whole school, with that shame fresh upon her, seemed impossible, and for a second she felt as if she could only drop down where she stood, and break her heart with crying. A bitter sense of wrong, and the thought of Jenny Snow, helped her to bear it; and, taking the ignominious place, she fixed her eyes on the stove-funnel above what now seemed a sea of faces, and stood there, so motionless and white that the girls found it very hard to study, with that pathetic figure before them. During the fifteen minutes that followed, the proud and sensitive little girl suffered a shame and pain which she never forgot. To others it might seem a ludicrous or trivial affair, but to her it was a hard experience; for during the twelve years of her life she had been governed by love alone, and a blow of that sort had never touched her before. The smart of her hand and the ache of her heart were forgotten in the sting of the thought,-- "I shall have to tell at home, and they will be so disappointed in me!" The fifteen minutes seemed an hour; but they came to an end at last, and the word "Recess!" had never seemed so welcome to her before. "You can go, Miss March," said Mr. Davis, looking, as he felt, uncomfortable. He did not soon forget the reproachful glance Amy gave him, as she went, without a word to any one, straight into the ante-room, snatched her things, and left the place "forever," as she passionately declared to herself. She was in a sad state when she got home; and when the older girls arrived, some time later, an indignation meeting was held at once. Mrs. March did not say much, but looked disturbed, and comforted her afflicted little daughter in her tenderest manner. Meg bathed the insulted hand with glycerine and tears; Beth felt that even her beloved kittens would fail as a balm for griefs like this; Jo wrathfully proposed that Mr. Davis be arrested without delay; and Hannah shook her fist at the "villain," and pounded potatoes for dinner as if she had him under her pestle. No notice was taken of Amy's flight, except by her mates; but the sharp-eyed demoiselles discovered that Mr. Davis was quite benignant in the afternoon, also unusually nervous. Just before school closed, Jo appeared, wearing a grim expression, as she stalked up to the desk, and delivered a letter from her mother; then collected Amy's property, and departed, carefully scraping the mud from her boots on the door-mat, as if she shook the dust of the place off her feet. "Yes, you can have a vacation from school, but I want you to study a little every day, with Beth," said Mrs. March, that evening. "I don't approve of corporal punishment, especially for girls. I dislike Mr. Davis's manner of teaching, and don't think the girls you associate with are doing you any good, so I shall ask your father's advice before I send you anywhere else." "That's good! I wish all the girls would leave, and spoil his old school. It's perfectly maddening to think of those lovely limes," sighed Amy, with the air of a martyr. "I am not sorry you lost them, for you broke the rules, and deserved some punishment for disobedience," was the severe reply, which rather disappointed the young lady, who expected nothing but sympathy. "Do you mean you are glad I was disgraced before the whole school?" cried Amy. "I should not have chosen that way of mending a fault," replied her mother; "but I'm not sure that it won't do you more good than a milder method. You are getting to be rather conceited, my dear, and it is quite time you set about correcting it. You have a good many little gifts and virtues, but there is no need of parading them, for conceit spoils the finest genius. There is not much danger that real talent or goodness will be overlooked long; even if it is, the consciousness of possessing and using it well should satisfy one, and the great charm of all power is modesty." "So it is!" cried Laurie, who was playing chess in a corner with Jo. "I knew a girl, once, who had a really remarkable talent for music, and she didn't know it; never guessed what sweet little things she composed when she was alone, and wouldn't have believed it if any one had told her." "I wish I'd known that nice girl; maybe she would have helped me, I'm so stupid," said Beth, who stood beside him, listening eagerly. "You do know her, and she helps you better than any one else could," answered Laurie, looking at her with such mischievous meaning in his merry black eyes, that Beth suddenly turned very red, and hid her face in the sofa-cushion, quite overcome by such an unexpected discovery. [Illustration: You do know her] Jo let Laurie win the game, to pay for that praise of her Beth, who could not be prevailed upon to play for them after her compliment. So Laurie did his best, and sung delightfully, being in a particularly lively humor, for to the Marches he seldom showed the moody side of his character. When he was gone, Amy, who had been pensive all the evening, said suddenly, as if busy over some new idea,-- "Is Laurie an accomplished boy?" "Yes; he has had an excellent education, and has much talent; he will make a fine man, if not spoilt by petting," replied her mother. "And he isn't conceited, is he?" asked Amy. "Not in the least; that is why he is so charming, and we all like him so much." "I see; it's nice to have accomplishments, and be elegant; but not to show off, or get perked up," said Amy thoughtfully. "These things are always seen and felt in a person's manner and conversation, if modestly used; but it is not necessary to display them," said Mrs. March. "Any more than it's proper to wear all your bonnets and gowns and ribbons at once, that folks may know you've got them," added Jo; and the lecture ended in a laugh. [Illustration: Girls, where are you going?]

Summary: Amy's Valley of Humiliation Amy sighs for money, wishing she could buy pickled limes to treat her friends at school. Meg gives her a quarter, and Amy brings the limes to school. One unkind girl reports the limes to Mr. Davis, the teacher. Mr. Davis makes Amy throw the limes into the snow, then strikes her palm and makes her stand in front of the class until lunch. For Amy, the experience is deeply humiliating, since Amy's parents had never hit her. At recess, Amy takes her possessions and goes straight home. Marmee withdraws Amy from the school, but she lectures Amy on breaking the rules, and encourages her to be more modest. Amy, upon reflection, realizes that Laurie is accomplished, but not conceited, so people enjoy his natural charm.

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Summarize: Just 13 games in to the Sky Bet Championship season and we have already had five different league leaders. The latest of those, Derby County, ascended to the top of the table with a 1-0 win at Blackpool on Tuesday night. It was their fifth victory in seven games. Perhaps more poignantly, Derby are unbeaten in 10 games. Yes, four of those results have been draws, some not pretty, but they only serve to demonstrate the resiliance which makes the Rams arguably the most likely of the five table-toppers so far this season to be there come May 2. Chris Martin steps up to slot home Derby's winner at Bloomfield Road on Tuesday night. Johnny Russell (right) leads the celebrations after Martin scored the only goal of the game. Steve McClaren consoles defender Richard Keogh after Wembley's play-off final defeat by QPR. The quartet of Watford, Norwich, Nottingham Forest and to a lesser extent Bournemouth have the attacking flair to challenge Derby for automatic promotion, but may all lack the consistency to keep in touch with Steve McClaren's relentless Rams. Since the former England manager took the helm at the iPro Stadium just over a year ago, Derby have maintained a handy knack of churning out points, only losing consecutive matches twice and never going more than four games without a win. In fact, were McClaren in charge for more than 36 games last season, then surely Derby would have joined Midlands rivals Leicester in the automatic promotion places, rather than being consigned to that agonising defeat by QPR at Wembley (which the quality of their football simply did not deserve). It seems almost incredible to think of the furious fallout from Nigel Clough's sacking in late September last year given McClaren's popularity in Derby. Clough's foundation work has to be appreciated; much of McClaren's side was inherited from the now-Sheffield United boss, but the 'Wally with the Brolly' has truly rebuilt his reputation on these shores. European football aficionados will point out that he did that when winning the Dutch league with the unfashionable Twente in 2010, but it was very much out of sight, out of mind with regards to McClaren for the English football fan. Given his success in Holland, adopting the same fluid 4-3-3 system he uses at Derby, McClaren was expected to bring verve and flair to the iPro, which he has, but it has been his ability to get the best out of seasoned Football League pros that has impressed and surprised most in the last year. While neither started on Tuesday night, defender Jake Buxton and midfielder John Eustace have been key men for Derby in the last year, McClaren getting them to fit in to a modern, European system despite their reputations as uncompromising journeymen. Derby's two key men over the last year, striker Chris Martin and midfielder Craig Bryson, were both underperforming under Clough, but now look like two of the best players outside the Premier League, and more then capable of making the step up. Chris Martin has been a revelation since McClaren took over and has scored 10 goals already this season. McClaren has continued to put faith in youth as Clough did before him, with academy-product midfielders Jeff Hendrick and Will Hughes flourishing in the first-team and Liverpool winger Jordon Ibe excelling on loan, just as fellow Red Andre Wisdom did the season before. All in all, it has been a brilliant year or so for Derby under McClaren, with 34 wins coming from 56 games; the Rams losing just 10 games. The only blot on the copybook being that last minute Bobby Zamora strike at Wembley. The last two champions in the second tier, Leicester and Cardiff, got their noses out in front early and never looked back. It's taken Derby 13 games to get to the top - and McClaren 13 months - but they may just be there for good. Good week for… SHREWSBURY. The last four days could not have gone any better for Micky Mellon’s side. Two wins, six points, six goals scored, zero conceded. Pre-season promotion favourites in League Two, Shrewsbury started strongly but a run of just one win in six games had seen them slip some way off the automatic places. Tuesday night’s 5-0 thrashing of pace-setters Bury bodes well though; it’s now three wins on the spin for Mellon’s men, who are back up to fifth and just four points off Wycombe at the top of the table. Jordan Clark is mobbed by his team-mates after scoring Shrewsbury's fifth goal on Tuesday night. Bad week for… COVENTRY. Coventry’s performances have plummeted this season along with their attendances. On their return to the Ricoh Arena in September the Sky Blues drew in a crowd of 27,306, beat Gillingham 1-0 and moved up to eighth place in League One. On Saturday, just 11,888 were in attendance as Steven Pressley’s side lost to unbeaten league leaders Bristol City, a result which was compounded by Tuesday night’s 4-1 thrashing at Oldham. Pressley's side are now just one point off the bottom of the table in 20th place. Talent scout: Michael Petrasso (Notts County) QPR have all but abandoned their academy in recent years as Tony Fernandes has ploughed tens of millions of pounds into signing players, but young Canadian winger Petrasso may be hard to ignore if his career continues on its upward trajectory. The 19-year-old, just 5ft 6in, scored twice as Notts County moved up to fifth place in League One with an impressive 3-2 win at Barnsley on Tuesday night. Petrasso, playing just his second game for Shaun Derry’s side, also looked good on loan at Coventry last season. Michael Petrasso brings the ball down before scoring Notts County's winner at Barnsley

Summary: Derby top of the Championship after beating Blackpool 1-0 on Tuesday. Steve McClaren took over at Derby 13 months ago with club 14th. Lost play-off final in May to Bobby Zamora's last-minute winner for QPR. Shrewsbury have won two games in four days without conceding. Coventry just one point off bottom of League One table after two defeats. Notts County's on-loan QPR winger Michael Petrasso impressing.

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Write a title and summarize: DNA interstrand crosslinks (ICLs) are toxic lesions that block the progression of replication and transcription. CtIP is a conserved DNA repair protein that facilitates DNA end resection in the double-strand break (DSB) repair pathway. Here we show that CtIP plays a critical role during initiation of ICL processing in replicating human cells that is distinct from its role in DSB repair. CtIP depletion sensitizes human cells to ICL inducing agents and significantly impairs the accumulation of DNA damage response proteins RPA, ATR, FANCD2, γH2AX, and phosphorylated ATM at sites of laser generated ICLs. In contrast, the appearance of γH2AX and phosphorylated ATM at sites of laser generated double strand breaks (DSBs) is CtIP-independent. We present a model in which CtIP functions early in ICL repair in a BRCA1– and FANCM–dependent manner prior to generation of DSB repair intermediates. Cellular DNA can be chemically modified and damaged when exposed to environmental agents, metabolic byproducts, or chemotherapeutic agents. The most toxic of these lesions is the interstrand crosslink (ICL), a covalent bridge formed between complementary strands of DNA. If not repaired, ICLs prevent DNA strand separation resulting in a block to replication and transcription. ICL generating agents are commonly used in the treatment of cancer. Sensitivity to crosslinking agents is a defining characteristic of Fanconi Anemia (FA), a rare hereditary syndrome characterized by an increased risk in cancer development and hematopoetic abnormalities frequently resulting in bone marrow failure [1]. Elucidation of the cellular pathways that repair ICLs is highly relevant to understanding carcinogenesis, development of novel therapies to treat FA patients, and to the development of better targeted chemotherapeutic drugs. Sensitivity assays suggest that eukaryotic cells have evolved multiple complex systems to repair ICLs that involve the intersection of several different repair pathways (reviewed in [2], [3]). However, the specific mechanism by which ICLs are detected and repair is initiatedremains unknown. A major ICL repair pathway in higher eukaryotes functions during S-phase and is thought to be replication dependent [4]–[7]. ICLs can also be repaired in a replication independent manner [8]–[10]. Current models of replication mediated ICL repair, suggest that repair is initiated when a fork stalls due to encountering an ICL [6], [11]. FANCM/FAAP24 then binds to the ICL stalled fork [12]–[17]. Next, single stranded DNA (ssDNA) is generated and bound by RPA [11], [15], [18] and the DNA damage response kinase ATR/ATRIP localizes to the damaged chromatin through binding to RPA [19]. Localization of ATR/ATRIP to damaged DNA is essential for activation of the S-phase checkpoint and ICL repair [20], [21]. The ability of ICLs to activate the checkpoint is dependent on the FA core complex (FANCA/B/C/E/F/G/M) [10], but not FANCI-FAND2 [18]. The generation of ssDNA at stalled replication forks is thought to be critical for ATR activation. However, the factors required to generate ssDNA under circumstances in which the ICL poses a structural barrier to helicase uncoupling from the DNA polymerase at the replication fork are not known [22]. It has been shown ssDNA arises at an ICL stalled fork in Xenopus extracts due to resection of the lagging strand [11]. In addition this ssDNA is competent for Rad51 loading prior to generation of a DSB ICL repair intermediate [23]. The FANCI-FANCD2 complex is phosphorylated by activated ATR in response to ICL stalled replication forks [20], [24], [25]. This phosphorylation facilitates FANCI-FANCD2 monoubiquitination by the FA core complex [24], [25]. Monoubiquitination is essential for localization of the FANCI-FANCD2 complex to damaged chromatin where it directs downstream repair steps [18], [26]–[28]. The FANCI-FANCD2 complex is required for the initial ICL incision step in replication competent Xenopus extracts [18]. Several candidate nucleases have been identified that may function to excise the ICL. These nucleases include XPF/ERCC1, MUS81/EME1, their regulator SLX4 (also known as the Fanconi Anemia gene FANCP) [29]–[37], SNM1A [37], [38] and SNM1B [39]. Whether they act redundantly or in concert is not clear, however they are all characterized by showing higher sensitivity to crosslinking agents compared to other forms of damage. MUS81/EME1 and XPF/ERCC1 have been proposed to function both early [40], [41]and late in the ICL repair process [29]–[32], [42], [43]. The nuclease FAN1 also confers resistance to ICL inducing agents and has been shown to interact physically with the monoubiquitinated form of the FANCD2 nuclease [44]–[46]. Although a large number of nucleases are implicated in ICL repair, it remains unclear at which specific step these proteins exert their functions. Following ICL incision, translesion synthesis by an error prone polymerase repairs the strand opposite the incised ICL [11], [47]–[50]. Excision of the ICL and polymerase extension up to the excised region results in the generation of double stranded ends resembling a double strand break (DSB). The DSB is repaired by homologous recombination as evidenced by the extreme sensitivity of homologous recombination impaired cell lines to ICL inducing agents [51]–[53]. We hypothesized that CtIP might play an important and unique role in replication dependent ICL repair for the following reasons. First, studies of the CtIP homologs in A. thaliana (AtGR1) and S. cerevisiae (SAE2) have revealed a conserved role for CtIP in ICL repair due to their ability to confer resistance to the ICL inducing agent mitomycin C (MMC) [54], [55]. Second, CtIP expression is enhanced in S through G2 phase [56], [57] when ICL repair primarily occurs. Third, CtIP plays a conserved role in coordinating the recognition and resection of double strand breaks with the MRE11-RAD50-NBS1 (MRN) complex [58]–[63] and may act to facilitate the generation of ssDNA ends following ICL incision. To investigate the mechanism of ICL repair in live mammalian cells we developed a system that takes advantage of the chemical and physical characteristics of the cross-linking agent, 8-methoxy psoralen (8-MOP). 8-MOP intercalates into DNA and forms DNA ICLs upon exposure to long wavelength UVA light (365 nm) [64]. In this system we used laser directed 2-photon activation of 8-MOP via infrared light (730 nm) to generate ICLs in individual human S-phase nuclei. This approach restricts damage to precisely defined subnuclear volumes allowing one to monitor the spatiotemporal dynamics of DNA repair factors after ICL induction. This system was used to determine what role CtIP plays in ICL repair. We show that CtIP plays an important and unexpectedly early role in ICL repair in S phase human cells. In addition to the known role of CtIP and MRE11 in DSB end resection [59], [60], [62], the data presented here demonstrates that CtIP plays an additional role in ICL repair prior to generation of DSB repair intermediates. The data show that CtIP acts prior to RPA loading and localization of ATR and FANCD2 to ICL containing DNA. CtIP is required for the accumulation of known DSB repair factors phospho-histone H2AX (γH2AX), and ATM autophosphorylated on serine-1981 (ATM-pS1981) at ICLs, but not at DSBs. The data support a model in which CtIP is recruited to ICLs in a FANCM dependent manner and is required to initiate ICL processing of stalled forks prior to ICL incision. To determine if CtIP plays a role in ICL repair, we assayed CtIP depleted human cells for sensitivity to the ICL inducing agents MMC or 8-MOP plus whole cell UVA irradiation (365 nm). The products of activated 8-MOP on DNA are predominantly ICLs, but monoadducts are also formed [64], [65]. The psoralen angelicin, which is structurally related to 8-MOPs, forms monoadducts upon UVA exposure and was used to control for cellular sensitivity to DNA monoadducts [66]. CtIP was depleted from Human Embryonic Kidney Cells (HEK293) by transient transfection of two previously described independent small interfering RNAs (siRNAs) [59]. Luciferase siRNA was used as a nontargeting control. CtIP RNA levels were monitored by RT-qPCR and were found to be reduced on average by 70%. Sensitivity to ICL and monoadduct inducing compounds was determined by assessing the fraction of surviving cells after 8 days. As shown in Figure 1A, CtIP depleted cells were sensitive to the ICL inducing agents 8-MOP+UVA and to MMC. CtIP depleted cells were not sensitive to angelicin plus UVA. CtIP depleted cells were only slightly sensitive to IR induced DSBs (Figure S1). This sensitivity profile is similar to that reported for A. thaliana AtGR1/AtCom1 mutated cells [55], and suggests that CtIP plays an important and conserved role in ICL repair. To define the role of CtIP in ICL repair, we established a system that enables the examination of the spatial and temporal recruitment and retention of repair proteins at region specific ICLs. An analogous approach has been used to generate study ICL repair using a UV laser plus light activated psoralen [9], [67]. Our approach uses a near infrared femtosecond laser to produce 730 nm light which activates 8-methoxypsoralen (8-MOP) by two-photon activation (effective wavelength of 365 nm at the focal point). This enabled us to define precisely a subnuclear volume in which DNA damage was created while avoiding damage outside the focal points [68]. This method allowed specific activation of 8-MOP with doses of light that caused no detectable damage in psoralen free control cells (Figure 1B). To define the optimal laser power, we first identified the lowest laser power that gave a robust γH2AX signal in cells treated with 8-MOP, and no observable γH2AX by laser alone (Figure 1B). Cells were subject to thymidine block and release to enrich for replicating cells [69]. One hour after thymidine release the indicated drug was added, and cells were microirradiated with 730 nm light using a femtosecond laser. Following microirradiation, cells were maintained in culture for 2 hours allowing time for replication forks to encounter the damage prior to fixation (Figure S2). Angelicin was used as a control for the amount of γH2AX at the laser tracks that resulted from the generation and/or processing of monoadducts. To verify that the γH2AX along laser tracks was replication dependent a control experiment was performed in which cells were held in thymidine during drug treatment and laser microirradiation. γH2AX staining signal along microirradiated tracks was quantified in individual cells using Image J (See Materials and Methods). The proportion of cells with positive γH2AX staining along microirradiated tracks are summarized in Figure 1C. The number of cells scored positive for γH2AX signal along laser tracks was highly increased in S-phase cells microirradiated in the presence of 8-MOP (83%) relative to microirradiated drug free cells (0%) (n = 29 and 25 cells respectively). Blocking replication in 8-MOP microirradiated cells reduced the number of γH2AX positive cells almost to background levels (4. 5%) (n = 21). Angelicin treated microirradiated cells did not yield bright γH2AX signal (0%) (n = 25) (Figure 1B, 1C), however we do not exclude the possibility that adduct formation by angelicin may be less efficient than that of 8-MOP. Thus we conclude that the γH2AX signal detected in 8-MOP microirradiated cells is primarily due to the replication dependent processing of ICLs. Previously published reports have shown that CtIP plays a role in G1/S progression in mouse fibroblasts (MEFs, NIH 3T3) [57], [70]. We wanted to examine whether CtIP depletion affected cell cycle and S-phase progression in HeLa which could have an effect on the efficiency of ICL repair. Cell cycle analysis was performed on CtIP depleted and control HeLa cells. No significant difference in cell cycle distribution was observed between control cells and 2 independent CtIP siRNAs in 3 independent experiments (Figure 2A, 2B). Figure 2C shows that both CtIP siRNAs effectively depleted CtIP in the samples analyzed for cell cycle distribution. Quantification of the nucleotide analog bromodeoxyuridine (BrdU) incorporation in individual siRNA transfected cells was determined to compare the number of S phase cells and the relative rates of replication associated nucleotide incorporation under the same conditions used in laser experiments. ICL detection and initiation of repair in S phase occurs primarily when a replication fork encounters and stalls at an ICL. Therefore it was important to verify that fork progression, as determined by BrdU incorporation, is not affected in CtIP depleted cells relative to control cells. As judged by quantification of pixel intensities in individual cells there was no significant difference in replication rate between control and CtIP depleted cells Figure 2D. In addition, under the conditions used for laser irradiation experiments >95% of the cells were in S phase. The lack of an effect on cell cycle distribution or BrdU incorporation in CtIP depleted HeLa cells suggests that the decrease in γH2AX generation at ICLs in CtIP depleted cells is due to a defect in ICL processing as opposed to a cell cycle progression defect. To determine when CtIP acts in the ICL repair process, known markers of the DNA damage response and repair process were monitored in CtIP depleted HeLa cells. The first marker examined was γH2AX. To compare the function of CtIP in ICL repair to its known role at directly generated DSBs [59], [62], parallel experiments were conducted in which DSBS were generated directly by microirradiation with 532 nm laser light (64). Laser doses for 730 nm and 532 nm light were predetermined to use the minimal doses that give readily detectable γH2AX signals 2 hour post microirradiation. (Figure S2). γH2AX phosphorylation was found to be strongly reduced in CtIP depleted S-phase HeLa cells along ICL containing microirradiated tracks (Figure 3A, top panel). In contrast, CtIP depletion had no detectable effect on H2AX phosphorylation at DSBs induced directly by laser irradiation (Figure 3A, bottom panel). The percentage of cells containing bright γH2AX signal, as determined by quantification of pixel intensity in individual cells along ICL containing laser tracks, was reduced significantly, over 4 fold, in cells depleted with 2 independent siRNAS targeting CtIP (13% and 19% respectively) relative to control cells (76%), (n = 48,29 and 66 cells respectively; P-values = 0. 003 (CtIP siRNA_1) and 0. 009 (CtIP siRNA_2) (Figure 3B). In contrast, the percent cells with bright γH2AX along DSB containing laser tracks was not reduced in CtIP depleted cells (CtIP siRNA_1 82%, CtIP siRNA_2 94%) relative to control cells (85%), (n = 19,19 and 26 cells respectively; P-value > = 0. 5). The differential effects of CtIP depletion on H2AX phosphorylation at ICLs and DSBs suggests that CtIP plays a role in ICL repair that can be distinguished from its role in DSB repair. To examine the function of CtIP relative to MUS81 and XPF, two nucleases that have been suggested to act early in ICL repair [40], [71], the effects of MUS81 and XPF depletion on γH2AX accumulation at ICLs was examined. Depletion of either XPF or MUS81 did not have a measurable effect on γH2AX accumulation along ICL containing laser tracks (Figure S3). This indicates that Mus81 and XPF function downstream of γH2AX generation in ICL repair. ATM becomes activated in an MRN dependent manner and phosphorylates H2AX at DSBs early in DSB repair [72]–[74], while ATR contributes to ATM activation and H2AX phosphorylation as a result of replication stress [75]–[77]. ATM has also been found to function in the FA pathway [24]. To test if CtIP is required for ATM phosphorylation at ICLs, and thus functions upstream of DSB generation, we examined the effects of CtIP depletion on phosphorylation at serine-1981, a characterized autophosphorylation site. [72]. Consistent with the premise that DSBs are formed as an intermediate during ICL repair, ATM-pS1981 signal was readily detectable in control cells treated with 8-MOP and irradiated to form ICLs. In contrast, ATM-pS1981 signal along ICL containing laser tracks was reduced significantly in CtIP depleted S-phase HeLa cells (38%) relative to control cells (69%), (n = 32 and 27 cells respectively; P-value = 0. 05) (Figure 4A, upper panel). By contrast, CtIP depletion did not significantly reduce the number of cells with bright ATM-pS1981 along DSBs (86%) relative to control cells (79%) (Figure 4A, lower panel), (n = 33 and 29 cells respectively; P-value > = 0. 1) (Figure 4B). Biochemical analysis has demonstrated that the FANCI-FANCD2 complex is required for the incision of interstrand crosslinks in replication competent Xenopus extracts [18]. We therefore examined whether FANCD2 was properly localized to laser activated ICLs in CtIP depleted cells. FANCD2 accumulation was found to be reduced along ICL containing microirradiated tracks in CtIP depleted cells (Figure 5A). The number of cells containing bright FANCD2 signal was reduced significantly by 2. 7 fold in CtIP depleted cells (33%) relative to the control cells (88%) (n = 24 and 26 respectively; P-value = 0. 026) (Figure 5B). These results demonstrate that CtIP acts upstream of FANCD2 localization to chromatin and point to a role for CtIP in ICL repair prior to ICL incision and unhooking. ATR phosphorylates the FANCI-FANCD2 complex which is required for its monoubiquitination and localization to ICLs [20]. RPA coated ssDNA is required for ATR activation [19] and CtIP/MRN is required for resection at double strand breaks [59], [62]. We hypothesized that CtIP might facilitate resection of DNA ends present at an ICL stalled replication fork. Therefore we examined RPA2 accumulation at ICL containing microirradiated tracks in CtIP depleted cells compared to control cells. RPA2 accumulation at laser tracks was found to be strongly reduced in CtIP depleted S-phase HeLa (Figure 6A). The percent cells containing bright RPA2 tracks was reduced 2. 7 fold in CtIP depleted cells (27%) relative to control cells (73%) (n = 29 and 41 cells respectively, P value = 0. 005) (Figure 6B). CtIP is targeted for phosphorylation at Thr847 by cyclin dependent kinase (CDK) [63]. Phosphorylation at this site is required to promote resection at DSBs, but is not required for CtIP recruitment to DSBs [63], [78]. We tested whether a Thr-847 to Ala CtIP mutation would also affect resection at ICLs. Endogenous CtIP was depleted by siRNA and replaced by either wildtype GFP-CtIP or GFP-CtIPT847A in U2OS cells. HeLa were found to be sensitive to increased levels of CtIP, therefore we used U2OS cells for experiments involving ectopic expression of CtIP. U2OS cells were shown to react similarly to CtIP depletion compared to HeLa cells (Figure S4). S-phase cells were irradiated to form ICLs and stained for RPA2. GFP-CtIPT847A accumulated at ICL containing laser tracks comparable to wildtype levels, however RPA2 accumulation was impaired relative to cells expressing wildtype GFP-CtIP (Figure 6C). The percent cells containing bright RPA2 tracks was reduced 7. 9 fold in cells expressing the CtIP phosphorylation mutant (8%) relative to control cells (63%) (n = 20 and 20 cells respectively, P value = 0. 03) (Figure 6D). We next examined whether reduced RPA accumulation at ICLs in CtIP depleted cells was associated with reduced ATR recruitment. The percent cells containing bright ATR signal along ICL containing laser tracks was reduced over 7 fold in CtIP depleted cells (8%) relative to control cells (58%) (n = 24 and 34 cells respectively; P value = 0. 003) (Figure 6E, 6F). The requirement of CtIP for both RPA and ATR localization to ICL containing chromatin suggests that CtIP acts early in ICL repair prior to ssDNA generation and ICL incision. BRCA1 ubiquitinates CtIP and is required for its localization to DSBs [79], [80]. In order to further characterize the requirements for CtIP recruitment to ICLs we examined whether BRCA1 was required for GFP-CtIP accumulation at ICLs. Cells were either BRCA1 depleted using two previously described independent siRNAs [81], [82] or treated with control siRNA. Endogenous CtIP was depleted by siRNA and CtIP expression reconstituted with silencing resistant GFP-CtIP. GFP-CtIP accumulation at ICL containing laser tracks was examined in S phase siRNA treated cells. CtIP accumulation at ICLs was found to be dependent on the presence of BRCA1 (Figure 7). In 3 independent experiments all cells (>25) treated with BRCA1 siRNA that lacked visible BRCA1 expression as verified by immunofluorescence staining and imaging, did not contain visible GFP-CtIP along the micro-irradiated region. In contrast, all control siRNA treated cells (>25) that had robust BRCA1 expression levels also contained GFP-CtIP at the ICL containing laser tracks. These results demonstrate that BRCA1 is required for CtIP recruitment to ICLs. FANCM has been shown to be required for RPA loading at ICLs as well as for activating the S-phase checkpoint [16], [83]. Data presented in Figure 6 indicates that CtIP is required for both RPA and ATR accumulation at laser activated ICLs in replicating cells. In order to determine when CtIP acts relative to FANCM we examined whether FANCM is required for CtIP localization to ICLs. U2OS cells stably expressing GFP-CtIP in which endogenous CtIP is depleted were treated with two independent FANCM siRNA s or control siRNA. Depletion was confirmed by immunoblot (Figure 8A). Cells were microirradiated to form ICLs and CtIP accumulation was examined. The percent of cells containing GFP-CtIP along ICL containing laser tracks was significantly reduced in FANCM depleted cells (22% FANCM siRNA_1,0% FANCM siRNA_2) relative to control cells (67%) (n = 26,11 and 30 cells respectively; P value = 0. 0002) (Figure 8B, 8C). Next we examined whether FANCM was required for GFP-CtIP recruitment to DSBs. Cells treated with FANCM siRNA_2 or control siRNA were irradiated with 532 nm light to form DSBs and GFP-CtIP localization was monitored. In contrast to what was observed at ICLs, FANCM depletion did not have a significant effect on the percent of cells containing GFP-CtIP at DSB containing laser tracks (75% FANCM siRNA_2) relative to control cells (76%) (n = 20 and 17 cells respectively) (Figure 8D, 8E). This data suggests that FANCM acts upstream of CtIP and is required for its localization to ICL but not DSB containing chromatin. The cellular response to ICLs involves the intersection of the DNA repair and replication checkpoint pathways. The FANCM/FAAP24 complex binds to, remodels and stabilizes stalled replication forks at an early step in ICL repair [13], [67]. FANCM/FAAP24 is also required for ATR mediated checkpoint activation in response to ICL stalled replication forks [15], [83]. In addition, FANCM deficient cells have decreased levels of FANCD2 monoubiquitination and chromatin bound FANCD2 [17], [84], [85]. We have shown that FANCM is required for CtIP localization to ICLs in replicating cells. CtIP in turn is required for proper accumulation of RPA, ATR and FANCD2 at ICLs. This places CtIP within the initiating steps of ICL repair, downstream of FANCM and upstream of RPA (Figure 8F). We propose the following model. First, FANCM/FAAP24 remodels ICL stalled replication forks to enable CtIP access. BRCA1 interacts with and ubiquitinates CtIP enabling it to bind to the damaged chromatin. Next, CtIP, presumably in concert with MRN, supports initiation of resection of the lagging strands of the previously active replication fork [59], [62], [63]. This resection activity is dependent on CDK phosphorylation site T847 on CtIP. ssDNA-RPA provides a platform for ATR-ATRIP binding. Once localized to the stalled fork activated ATR promotes S phase checkpoint activation and phosphorylates the FANCI-FANCD2 complex. The phosphorylated FANCI-FANCD2 complex is then monoubiquitinated, localized to damaged chromatin to facilitate ICL incision and generation of a DSB repair intermediate. This proposed order of events is consistent with that described for Xenopus extracts in which replication dependent ICL repair initiates at an ICL stalled fork, followed by generation of ssDNA, activation of ATR as measured by Chk1 phosphorylation, and excision of the crosslink in a FANCI-FANCD2 dependent manner [11], [18]. Cell cycle distribution and rate of BrdU incorporation are unaffected in CtIP depleted HeLa cells compared to control cells. This suggests that ICL processing defects observed in CtIP depleted cells are due to a deficiency in initiation of repair as opposed to an inhibition of cell cycle progression. The difference observed in cell cycle distribution of our CtIP depleted cells compared to those observed in mouse fibroblasts [70] are likely due to a difference in cell type as well as to a difference in the timing and method used to deplete CtIP. A function for CtIP prior to ICL incision, and upstream of a repair intermediate that contains double stranded ends, is supported by the observation that CtIP depleted cells show a striking reduction in the accumulation of the DSB markers ATM-pS1981 and γH2AX at laser generated ICL tracks. In contrast, the accumulation of ATM-pS1981 and γH2AX at DSBs produced by direct laser irradiation are not affected by CtIP depletion. The effect of CtIP depletion on γH2AX at DSBs is in agreement with previously published observations in CtIP depleted U2OS cells [59]. This indicates that initiation of DSB repair, is not grossly affected by CtIP depletion. These results do not preclude an additional downstream role for CtIP where it may act together with MRN in a manner analogous to its function at directly generated DSBs to facilitate the processing of the double stranded DNA ends produced following ICL incision. In summary, our data supports a model in which CtIP functions early in replication associated ICL repair downstream of FANCM and is required for the accumulation of RPA, ATR, FANCD2, ATM-pS1981, and γH2AX at ICLs. Thus, CtIP plays a critical role in initiating the DNA damage response at ICL stalled replication forks, prior to the generation of a double stranded DNA repair intermediate. It will be of interest to see how CtIP and MRN are regulated at ICL stalled forks and whether this response is different from that observed at other stalled forks in which helicase uncoupling contributes to checkpoint activation. Robolase III (RLIII) is a multi-modality laser ablation system that is based on a femtosecond pulsed Ti: Sapphire laser (Mai Tai, Spectraphysics, Newport Corp., Mountain view, CA) and a motorized inverted microscope (Axiovert 200 M, Zeiss) that utilizes a custom Labview-based software package developed specifically for the Robolase series of microscope systems [86]. In this study, the laser wavelength was set to 730 nm. The effective wavelength at the focal point is 365 nm at which psoralen is activated by a 2-photon mechanism [87]. The pulse duration was estimated at 200 fs and the repetition rate was 76 MHz. The laser was focused by a Zeiss 63×/1. 4 plan-apochromat PH3 oil objective with measured transmission of 67% at 730 nm using the double-objective method to determine transmission [88]. The power used in this study was 2 mW before the objective which yielded a peak irradiance of 2. 8×1010 W/cm2 at the focal point. For each cell, a 10 µm by 636 nm region of interest (ROI) was chosen inside the nucleus using real-time phase contrast imaging. An image of the designated ROI was recorded as reference. Laser exposure over each 10 µm long ROI was performed four times over each ROI with a total energy of 2. 4 mJ. H2AX phosphorylation was used as a marker to determine the minimum power that provided a signal in the presence of 8-MOP. Double strand breaks were generated with the Robolase II (RLII) system that uses a frequency doubled 532 nm12 ps pulsed Nd: YVO4 laser [62], [86]. The ROI for each cell was 10 µm by 464 nm. The laser power used in this study was 5 mW in the focal spot for an irradiance of 2. 3×109 W/cm2 and the total energy deposited along the laser track inside each cell was 3. 2 mJ. For experiments in which DSBs were generated cells were laser microirradiated on the same dish as cells treated to form ICLs and fixed at 2 hours 10 minutes post laser. Asynchronous or rapidly proliferating HeLa and HEK293 cells were maintained in Dulbecco' s modified Eagle' s medium (Invitrogen, Carlsbad, CA) supplemented with 10% bovine calf serum L-glutamine, and sodium pyruvate) at 37°C and 5% CO2. Small interfering double stranded RNAs (siRNAs) were introduced into HeLa cells by transfecting cells in a 6 well dish with 50 pmol siRNA, and 5 µL Dharmafect 1 (Dharmacon, Lafayette, CO) per reaction. The following synthetic siRNAs used were obtained from Dharmacon, siLuciferase (control) and CtIP (siCtIP_1 and siCtIP_2) [59], FANCM_1 (GCAAAGUAGCCUAAAGAAAUU), FANCM_2 [83], siMUS81 (D-016043) and siRNA pool containing 4 siRNAs targeting XPF (siERCC4 D-019946-01.) The following siRNAs were obtained from Integrated DNA Technologies, BRCA1_1 (AATGCCAAAGTAGCTAATGTAUU) [82] and BRCA1_3 (AAGGAACCUGUCUCC ACA AAG UU) [81]. HEK293 cells were transfected with the indicated siRNA using Dharmafect 1, sixteen hours later the cells were washed with sterile PBS and fresh media was added. After an eight hour recovery the cells were trypsinized and seeded at 104 per well in a six well plate. After 12 hours 10 µg/ml 8-MOP or angelicin were added to the cells. After 30 minutes incubation the cells were then exposed to the indicated amount of UVA radiation at approximately 360 nm using a Stratalinker (Stratagene, La Jolla, CA). Alternatively, cells were treated with indicated amount of MMC for 2 hours. Post treatment the cells were washed with sterile PBS, fresh media added and replaced every three days. On day 8 the cells were trypsinized and viable cells were counted using a CASY cell counter (Scharfe systems). A retroviral expression vector containing EGFP-CtIP (pBabepuro) was infected into U2OS cells for 48 hrs, followed by puromycin selection for 2–3 days to generate stable cell lines. For transient expression endogenous CtIP was first depleted by transfecting CtIP siRNA_1 into cells. Cells were transfected 24 hours later with silencing resistant GFP-CtIP using Effectene [59] (Qiagen). Experiments were performed 24 hours post GFP-CtIP transfection. GFP-CtIPT847A was generated using Quikchange Site Directed Mutagenesis Kit (Agilent Technologies). Total RNA was isolated from control or CtIP siRNA transfected cells 48 hours post transfection using the RNeasy Kit (Qiagen). Target RNA was amplified using the Bio-rad iScript One Step RT-PCR kit with SYBR green kit (Bio-rad, Hercules, CA) on a Bio-rad real time quantitative PCR machine. The One step kit was used according to manufacturer' s directions with the exception of 20 µL reactions, 2 ng total RNA per reaction, and 2 µL of each primer (5 uM stock). The following primers were used, housekeeping gene PBGD was amplified as a control (PBGDF- TCC AAG CGG AGC CAT GTC TG, PBGDR- AGAATC TTG TCC CCT GTG GTG GA) CtIP (CTIPF- AAG AGG AGG AAT TGT CTA CTG C, CTIPR- AGA ATC TTG TCC CCT GTG GTG GA). Reactions were run on Chromo-4 qPCR I system (MJ Research, Waltham, MA). CtIP RNA depletion was verified to be between 60 and 70% by RT-QPCR for CtIP siRNA 1 and CtIP siRNA 2 in all experiments relative to control depleted cells. HeLa cells were transfected with siRNA as described above and seeded onto coverslips in 12-well plates. Thirty-six hours later, 2 mM thymidine was added to cultures for 16 hours. Cells were released from the thymidine block by washing 2 times with fresh media containing 100 uM bromodeoxyuridine (BrdU). Cells were incubated in BrdU containing media for 20 minutes to label S phase nuclei.. Cultures were fixed with 3. 7% formaldehyde, stained with mouse monoclonal anti-BrdU antibody coupled to FITC (eBioscience, San Diego, CA), and counterstained with DAPI. ImageJ software was used to determine the total number of nuclei, BrdU-positive nuclei, and relative levels of fluorescence in individual cells. siRNA and control cells were prepared for flow cytometry analysis 48 hours post transfection. Cells were treated with trypsin/EDTA and washed twice in PBS before fixing them in 70% ethanol overnight at −20°C. Cells were then washed twice in PBS and incubated in staining solution (20 µg/ml propidium iodide, 20 µg/ml RNase A, 0. 1% Triton X-100 in PBS) for 30 min at 37°C. Data were acquired on a BD FACS Calibur flow cytometer. Percentages of G1, S, and G2 phase cells were determined from cell cycle profiles by using the Watson pragmatic algorithm of the cell cycle platform within FlowJo software (Tree Star Inc., Ashland, OR) with the “remove doublets” and “remove debris” options enabled. P values were determined using an unpaired two-tailed t test. Cells for laser microirradiation were cultured on gridded glass bottom dishes (Mattek, Ashland, MA) for 16 hours in 2 mM thymidine. Cells were washed twice with warm media to remove thymidine one hour prior to microirradiation and fresh media was added. Twenty minutes prior to laser microirradiation drugs were added to the cells; 10 µg/ml 8-MOP (MP Biomedicals, Solon, Ohio), or an equivalent molar amount of angelicin (8. 6 µg/ml Sigma-Aldrich, St. Louis, MO). Cells were allowed to recover for 2 hours post micoirradiation prior to fixation in all experiments except where noted. Soluble proteins were extracted in cytoskeletal buffer on ice for 5 minutes (10 mM Pipes pH6. 8,100 mM NaCl, 300 mM sucrose, 3 mM MgCl2,1 mM EDTA and 0. 5% TritonX-100) [89], followed by fixation in 3. 7% formaldehyde phosphate buffered saline (PBS) at room temperature for 10 minutes, and permeabilized with 0. 2% Triton-X. Cells were then stained with primary antibody in 3% BSA/PBS. The following primary antibodies were used in immunofluorescent staining: 1∶100 rabbit anti-RAD51 1∶100 (Abcam, Cambridge, MA), 1∶1000 mouse anti-γH2AX (Upstate Biotechnology, Waltham, MA), 1∶250 Rabbit anti-NBS1 NB100-143 (Novus Biologicals, Littleton, CO), 1∶200 Mouse anti ATM-pS1981 clone 10H11. E12 (Chemicon, Billerica, MA), 1∶200 Rabbit anti-FANCD2 NB100-182 (Novus Biologicals, Littleton, CO), 1∶50 Mouse Anti-RPA2 (Calbiochem, Gibbstown, NJ), 1∶100 Mouse anti-BrdU (ebioscience, San Diego, CA), 1∶500 Rabbit anti-RAD51 H92 sc-8349 (Santa Cruz Biotechnology, Santa Cruz, CA), 1∶1000 Mouse anti-MUS81 ab14387 (Abcam), 1∶500 XPF Ab17798 (Abcam), 1∶500 Rabbit anti-BRCA1 PA1-14072 (Thermo Fisher) 1∶1000 Rabbit anti-Ku86 SC9034 (Santa Cruz Biotechnology, Santa Cruz, CA). The following secondary antibodies were used at a 1∶5000 dilution in 3%BSA/PBS 1∶5000 Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR) and 1∶5000 Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Nuclei were visualized by staining with 1 µg/ml Dapi (Invitrogen). Glass coverslips were mounted with Vectashield (Vectorlabs, Burlingham, CA). Samples were visualized and images acquired using a 63× objective on a Leica DM IRE2 microscope equipped with a Hamamatsu C4742-95 digital charge-coupled-device camera. Images of cells were analyzed using Image J software (NIH, Bethesda, MD). The fluorescence along the microirradiated track was quantified in each individual cell as follows. Images were thresholded and converted into binary images. The same threshold was applied to all images from a single experiment. The number of pixels above threshold in 3 oval ROIs (150×50 pixels) were acquired per cell: one measurement for the area containing the laser track, and two measurements to determine the level of background signal in the nuclear region outside the laser track. The average number of background pixels was subtracted from the number of pixels measured along the microirradiated track for each individual cell. This resulted in a corrected pixel number for the intensity of a fluorescent signal along a laser track. For each positive control the average pixel number along the laser track was determined similarly. The average value from the positive control group per experiment was used to define the cut-off for experimental signals designated “bright” (corrected pixel number >50% of average positive control) or “low” (corrected pixel number <50% of average positive control). Each cell in the control and experimental siRNA group was then assigned a low signal or bright signal accordingly. The number of cells that fell into the “bright” and “low” categories were determined for each experiment. Cells which did not contain pan-nuclear γH2AX or RPA2 foci were considered to be outside of S-phase and were excluded from analysis. For analysis of GFP-CtIP expressing cells, cells were scored as positive if there was any detectable GFP-CtIP along laser track. Statistical analysis was performed using Microsoft Excel. Differences between control and experimental groups were considered statistically significant when the P-value, determined by a two-tailed unpaired Student' s t-test, was ≤0. 05. HeLa cells were lysed (10 mM Hepes pH 7. 9,10 mM KCl, 1. 5 mM MgCl2,0. 34 M sucrose, 10% glycerol, 1 mM DTT, 0. 1% triton, and protease inhibitors). Nuclear fraction was harvested by centrifugation at 1,399×g for 5 minutes. Pellet was resuspended in loading buffer, boiled for 5 mintues and put through 25 gauge syringe. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose. Immunoblots were probed with indicated primary antibody, anti-FANCM (1∶1000) ab35620 (Abcam) and anti-Ku86 (1∶1000) (Santa Cruz). R. Baer provided a mouse monoclonal antibody to CtIP [90]. Membranes were washed, incubated with HRP-linked secondary antibodies (Invitrogen), and detected by chemiluminescence (Pierce, Fisher Scientific). For Mus81 and XPF blots cells were lysed by boiling in Laemmli buffer (4% SDS, 10% glycerol, 62. 5 mM Tris pH6. 8,10% β-mercaptoethanol), and sonicated on ice. Immunoblots were probed with indicated primary antibody, anti-MUS81 (1∶1000) (Abcam) and anti-XPF (1∶1000) (Abcam), and anti-Ku86 (1∶1000) (Santa Cruz).

Title: CtIP Is Required to Initiate Replication-Dependent Interstrand Crosslink Repair Summary: One of the most lethal forms of DNA damage is the interstrand crosslink (ICL). An ICL is a chemical bridge between two nucleotides on complementary strands of DNA. An unrepaired ICL is toxic because it poses an unsurpassable block to DNA replication and transcription. Certain forms of cancer treatment exploit the toxicity of ICL generating agents to target rapidly dividing cells. Sensitivity to crosslinking agents is a defining characteristic of Fanconi Anemia (FA), a hereditary syndrome characterized by an increased risk in cancer development and hematopoietic abnormalities frequently resulting in bone marrow failure. The mechanism underlying ICL repair is important to human health; however, the sequence of molecular events governing ICL repair is poorly understood. Here we describe how the repair protein CtIP functions to initiate ICL repair in replicating cells in a manner distinct from its previously described role in other forms of DNA repair.

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Summarize: Dr. Milton Wolf has a problem with a member of his family: He says his second cousin, Barack Obama, may just be “the worst president in history.” Now the doctor is on a mission to win a seat in the U.S. Senate and undo some of the damage caused by his kin. His goal? Repeal Obama’s signature legislation, Obamacare, and replace it with a system based on conservative principles. Wolf, 42, is a diagnostic radiologist who runs to the right of incumbent Republican Sen. Pat Roberts in the Kansas GOP primary. He admits he didn’t know he was related to Obama until 2008 and didn’t meet him until they were health-care policy adversaries in 2010. Wolf said being related to a president would be a great experience if Obama weren’t so far left in his ideology. “Of course, who wouldn’t be honored to have a president in your family and sit on the front row of history? We’re related,” he said. “Of course, I remind people you cannot choose your family. Barack Obama is the worst president in our lifetimes, if not in our history. He has been a disaster. It’s nothing personal, but his policies have been disastrous in America. “It’s mostly because he either doesn’t understand or has forgotten what America is all about. The American idea itself is about individual liberty, limited government and free-market values,” he said. “When we have embraced those, we have become the most prosperous and powerful nation in history. And when we abandon those, we suffer. We have suffered under Barack Obama.” Listen to the WND/Radio America interview with Dr. Milton Wolf below: Wolf is making no secret he would be a fierce opponent of President Obama in Washington. The home page of his campaign website reads, “Want to drive Barack Obama crazy? Send his very own fearless conservative cousin – ‘the next Ted Cruz’ – to the United States Senate!” Wolf says he touts himself as the next Ted Cruz because he believes the freshman Texas senator is approaching his office the right way while Sen. Roberts is not. “We need more senators like Ted Cruz, like Mike Lee, like Rand Paul. They stand by the Constitution fearlessly, unapologetically. They don’t need an election year conversion because they’re the real deal,” Wolf said. “Pat Roberts, in an amazing election-year conversion, is following the leadership of Ted Cruz, who’s only been there for one year,” he said. “That’s because Ted Cruz understands something that Pat Roberts has never quite figured out – that a United States senator should have something more powerful than just a vote. He should have a voice, and he should use that voice. He should stand up and fight for our Constitution and for the American idea itself. Wolf continued, “Instead, what we have in our establishment Republicans are these go-along-to-get-along Republicans. Pat Roberts voted for Barack Obama’s $600 million tax increase just a year ago. He’s voted to raise our debt ceiling 11 times. And Pat Roberts voted to put Kathleen Sebelius in charge of Obamacare. That’s not conservative. It’s not good for Kansas, and it’s not good for America.” Defenders of Roberts counter Wolf’s arguments by asserting Roberts spent many years of his Senate tenure in key intelligence committee positions that were not conducive to bold public statements. They also note he was one of only 18 Republicans to oppose the spending bill that ended last year’s partial government shutdown and that he was the first U.S. senator to publicly call for the resignation of Sebelius. “He only bothered to say Kathleen Sebelius should resign three days after I announced my candidacy. The Kansas City Star reported on it and said, ‘If you think those two facts are unrelated, you probably think the Kansas Jayhawks are going to win the national championship this year in football,'” said Wolf, who added that voters need to take a close look at Roberts’ voting record throughout his Senate tenure and not just leading up to elections. “He claims to be in the top five conservatives in the Senate. That’s according to Heritage Action. What he doesn’t want you to know is his lifetime score from Heritage Action, which is a 67. Before I came along, in 2012 Pat Roberts had a 65 from Heritage Action. Before I came along, in 2012 Pat Roberts had a 55 from Club for Growth and a 54 from FreedomWorks. That is not a conservative. That is somebody who is going along to get along, and that’s been the problem with our Republican Party,” he said. So what qualifies Wolf for the Senate, and what would his priorities be if elected? “I confess I don’t have the Washington experience Pat Roberts has. I’ve never voted to raise your taxes,” he said. “I’ve never spent trillions of dollars that aren’t mine, and I’ve never paid $800 for a toilet seat. But what I have done is this: I have met payroll. I have balanced budgets. I have run a company and, far more importantly, I know every day what it’s like to have patients come to me and put their lives in my hands and ask me to make the humbling, sometimes gut-wrenching decisions that are the difference between life and death. That’s the kind of humility I think Washington lacks.” His top legislative priority is PatientCare, his plan to repeal Obamacare and replace it with a system based on conservative principles. “We need to fully repeal Obamacare, and we need to replace it with patient-centered, free-market health-care reform that’s being described as, by far, the best alternative to Obamacare,” he said. Wolf is not only running against a three-term incumbent, but the state’s other senator, Jerry Moran, runs the National Republican Senatorial Committee, or NRSC. That’s the group tasked with re-electing Republican senators and recruiting candidates to run for Democrat-held seats. Wolf said Kansas is in no danger of falling to the Democrats, so the NRSC should stay out of the primary. He says if it doesn’t, it will show the group is not about electing conservatives but simply protecting incumbents. A Kansas radiologist running for Senate posted grisly X-rays of patients who had been shot on his Facebook page and engaged in joking online banter about the images. Milton Wolf, challenging Sen. Pat Roberts in a Republican primary, acknowledged Sunday that he posted “insensitive” comments online, which he described as “mistakes.” Text Size - + reset He was responding to a devastating story in the Topeka Capital-Journal, which reported that he wrote that a patient could not complain about the awkward way his head was positioned for an X-ray, published on Facebook, because he was dead. He said a man decapitated by gunfire resembled an alien in the movie “Terminator.” And he posted another image of a man who had been shot in the temple. Wolf accused Roberts of “character assassination” in a 424-word statement. “To those I have offended, I am truly sorry and I ask for your forgiveness,” he said. Accompanying the newspaper’s story is an eight-minute video of a testy interview with the candidate. Wolf notes that he has authored medical textbooks and argues that it is okay to post images as long as names are redacted, something the paper quotes medical ethics professionals disputing. The reporter asks, “Do you still post images of dead people on the Internet?” “I’m not going to play these kind of gotcha games with you,” Wolf responded. Wolf has the backing of conservative outside groups such as the Senate Conservatives Fund and the Madison Project. The National Republican Senatorial Committee, which supports incumbents, quickly attacked what it described as “freakish behavior.” Officials faulted the outside groups, who often antagonize the establishment, for failing to properly vet primary challengers before offering endorsements. “Wolf is now embroiled in serious ethical and legal questions and challenges, effectively destroying any small hope that he had for a serious campaign,” said NRSC spokesman Brad Dayspring. The tea party challenger, who often notes that he is a distant cousin of President Barack Obama, said he removed the old posts years ago. “Senator Pat Roberts wants to attack me as a doctor rather than giving Kansans a reason to vote for him,” Wolf said. “It’s sad.” CORRECTION: An earlier version of this story incorrectly stated that FreedomWorks has endorsed Wolf. OVERLAND PARK — U.S. Senate candidate Milton Wolf posted a collection of gruesome X-ray images of gunshot fatalities and medical injuries to his Facebook page and participated in online commentary layered with macabre jokes and descriptions of carnage. Wolf, a Johnson County radiologist anchoring a campaign for the Republican nomination with calls for federal heath care reform, said in an interview the medical images were legally uploaded to public social media sites and other online venues for educational purposes. They also served, he said, to demonstrate evil lurking in the world. However, Wolf and others viewing these Facebook postings relentlessly poked fun at the dead or wounded. The gunshot victim, Wolf joked online, wasn't going to complain about the awkward positioning of his head for an X-ray. In a separate Facebook comment, Wolf wrote that an X-ray of a man decapitated by gunfire resembled a wounded alien in a "Terminator" film and that the image offered evidence people "find beauty in different things." Wolf declined in an interview with The Topeka Capital-Journal to clearly answer questions about whether he continued to place images of deceased people on the Internet. He asked to keep copies of the Facebook posts shown to him, but when denied, he walked away. "I'm not going to play these kinds of gotcha games," he said. An array of professionals involved in medical ethics who viewed the images or were provided a description of the materials made public by Wolf condemned his airing of the information outside confines of a doctor-to-doctor consultation or for the purpose of formal medical research or textbook instruction. "The dignity and privacy of the individual should be protected," said John Carney, president of the Center for Practical Bioethics in Kansas City, Mo. "It doesn't sound like they're being protected if they're, obviously, on Facebook." Carney said the summary of Wolf's postings provided to him would be widely viewed as "beyond alarming for a professional in the field of medicine." Truman Medical Centers in Kansas City, Mo., where Wolf said he obtained the decapitation X-ray, said in response to an inquiry Friday it wouldn’t have granted Wolf permission to use images of a shooting victim in this manner. Officials at Shawnee Mission Medical Center, linked to X-rays on the Internet depicting a person embedded with shotgun pellets and marked as property of TheWolfFile.com, responded to inquiries Friday by revealing Wolf had pledged to request removal of the X-rays from a California political website. The Facebook site operated by Wolf appears to have been disabled prior to launching the campaign to oust U.S. Sen. Pat Roberts, R-Kan. It has been replaced by a Facebook site devoted to Wolf's senatorial campaign. Leroy Towns, spokesman for Roberts, said revelations about Wolf's interaction on Facebook raised questions about the doctor's legal and professional responsibilities to maintain privacy of patient medical information. The disclosures also raise questions about Wolf's viability as a candidate, he said. "For any doctor to make patient records public and then use the records for public discussion and entertainment is just unthinkable," Towns said. "Allegations of such lack of judgment demand extensive scrutiny and investigation." On Saturday, Wolf released a statement alleging Roberts was launching a character attack against him. "The attack will not only target me," Wolf said, "but will, through its implications, cast a wider net to vilify all doctors." Wolf, who frequently mentions in campaign stops that he is a distant cousin to Democratic President Barack Obama, has been endorsed by groups aligned with tea party organizers. He authored a book, "First, Do No Harm: The President's Cousin Explains Why His Hippocratic Oath Requires Him to Oppose ObamaCare." He graduated from The University of Kansas School of Medicine, has been a licensed doctor in Kansas since 2004, and is employed at the Shawnee Mission division of Alliance Radiology. The Kansas Board of Healing Arts hasn't reported Wolf to have been the subject of sanctions related to private hospital privileges or through disciplinary action of the state agency. In the interview Thursday in Johnson County, Wolf said he received permission from patients when required before making use of records or images. He claimed usage, including Facebook posts, that didn't reveal an individual's identity didn’t require prior authorization. "It's an educational thing for people," Wolf said. "I take my charge of being a doctor very seriously." On Jan. 25, 2010, Wolf uploaded to Facebook a high-resolution rendering of a deceased man shot in the temple. Wolf also posted an X-ray depicting that victim's fractured skull and a cluster of bullet fragments lodged throughout the brain. Wolf launched a Facebook chat about the 3D image by explaining it was taken from a postmortem examination. A Facebook friend, Melissa Ring-Pessen, responded that she performed the scan on Jan. 22, 2010, and was admonished for improperly positioning the man's head. "Seriously?" she wrote. "Sheesh Melissa," Wolf replied, "it's not like the patient was going to complain." In a Facebook discussion of an image of the person decapitated by gunfire, Wolf shared that the X-ray was among cherished artifacts from time spent working as a medical resident at Truman Medical Centers. The graphic image shows a skull broken apart like a smashed pumpkin. Chunks of skull remain attached by tissue with vertebra exposed at the neck. "One of my all-time favorites," Wolf posted to the Facebook picture. "From my residency days there was a pretty active 'knife and gun club' at Truman Medical Center. What kind of gun blows somebody's head completely off? I've got to get one of those." Facebook friend Angie Rosini responded, "Ya know, the cool thing is, is that this dude walked away from this unscathed." Wolf: "It reminds (me) of the scene from 'Terminator 2' when they shoot the liquid metal terminator guy in the face at close range and it kind of splits him open temporarily almost like a flower blooming. We all find beauty in different things." Shane Kovac, spokesman for Truman Medical Centers, said patient information could only be released from the medical facility with written consent of a patient. "We can say that no one within Truman Medical Centers would have or could have given permission for a physician or anyone associated with the hospital to post an image, diagnostic or otherwise, to a personal social media page that includes patient information," he said. Mallory Laur, marketing specialist at Shawnee Mission Medical Center, said the facility responded when informed X-rays associated with Shawnee Mission Medical Center of a gunshot victim were visible on the website, "Left Coast Rebel – Freedom, Abundance, Responsibility," in conjunction with an interview of Wolf. She suggested the X-rays on the political site weren’t a violation of federal medical privacy law. "De-identified health care images are often used for education and other purposes," Laur said. "While not a HIPAA violation, Dr. Wolf is in contact with the website on which this image was posted to have it removed." However, Wolf said in the interview there was merit to exposing the general population to the end result of violent acts. "These are real consequences," he said. "You know, there are real consequences to some of the evil out there in the world and some of the tragedies that are out there in the world. Do you think there would be so many of these tragedies if people saw the consequences of them?" While discussing the Facebook posts, he also made reference to life and death pressures placed upon some medical professionals. "It's difficult as a health care professional to have to deal with those every single day," Wolf said. Jerry Slaughter, executive director of the Kansas Medical Society, said it wasn’t the place of physicians, nurses and others providers at any level to reveal on Facebook or other public sites the result of criminal violence or accidental injury suffered by patients. He said an assertion Facebook constituted legitimate educational outreach didn't ring true. "If it's patient information, identifiable in any way, it's inappropriate," Slaughter said. "Absent any legitimate educational purpose or context, this is not ethical behavior." Barbara Bollier, a Kansas House Republican and retired doctor who taught in a bioethics master’s program at Kansas City University of Medicine and Biosciences, said apparent transgressions by Wolf could be reported to the Kansas Board of Healing Arts as a potential violation of professional conduct by the radiologist. "I am surprised," she said. "I've never heard of another physician doing this."

Summary: A Kansas candidate for US Senate is acknowledging a "mistake" following reports he posted grisly X-rays on Facebook and joked about them. The now-removed images allegedly posted by radiologist Milton Wolf featured gun victims, one of whom suffered decapitation, the Topeka Capital-Journal reports. "One of my all-time favorites," he commented, per the newspaper. "What kind of gun blows somebody's head completely off? I've got to get one of those." He likened it to a Terminator scene, saying people "find beauty in different things." Wolf reportedly noted of another gunshot victim that the patient isn't "going to complain" about his head's position in the image. The president of a Kansas City bioethics center called the reports "beyond alarming for a professional in the field of medicine." Meanwhile, a spokesman for Sen. Pat Roberts, whom Wolf is battling for the Republican nomination, said that "for any doctor to make patient records public and then use the records for public discussion and entertainment is just unthinkable." Wolf has apologized for his comments, Politico notes, but the pro-incumbent National Republican Senatorial Committee calls his behavior "freakish," saying it is "effectively destroying any small hope that he had for a serious campaign." On another note, Wolf frequently says he's a distant cousin of President Obama, whom he's called possibly "the worst president in history," WND reports.

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Summarize: The Family Leader, an Iowa-based political group with the stated mission to promote Christian conservative social values, has been asking Republican candidates to sign a pledge titled “The Marriage Vow – A Declaration of Dependence upon Marriage and Family.” Rep. Michele Bachmann recently signed the pledge, and now some are in uproar that she may also have agreed to ban pornography. The actual pledge that she signed isn’t terribly surprising for a social conservative like Bachmann; it contains many platitudes like “marriage is between man and a woman.” Oh, and there are parts to the pledge that calls homosexuality a “choice,” which is then likened to bigamy, adultery and polyandry. Writing for the Des Moines Register, William Petroski reports: Presidential candidates who sign the pledge must agree to personal fidelity to his or her spouse, the appointment of “faithful constitutionalists” as judges, opposition to any redefinition of marriage, and prompt reform of uneconomic and anti-marriage aspects of welfare policy, tax policy and divorce law. The Marriage Vow also outlines support for the legal advocacy for the federal Defense of Marriage Act, humane efforts to protect women and children, rejection of Sharia Islam, safeguards for all married and unmarried U.S. military service members, and commitment to downsizing government and the burden upon American families. In addition, candidates are asked to recognize that “robust childrearing and reproduction is beneficial to U.S. demographic, economic, strategic and actuarial health and security.” Alice Stewart, a Bachmann aide, said the congresswoman had no qualms about signing the Family Leader’s pledge. “She has been married for over 30 years and has a strong marriage and faith.” ThinkProgress has noted that, in their esteem at least, the pledge also include a stipulation that bans pornography: PORNOGRAPHY SHOULD BE BANNED: Vow 9 stipulates that the candidate must “support human protection of women and the innocent fruit of conjugal intimacy” and protect them from “seduction into promiscuity and all forms of pornography…and other types of coercion or stolen innocence.” But the actual vow signed by Bachmann doesn’t explicitly call for a ban on pornography, per se. Read the vow portion below (click here for the.pdf file) The Candidate Vow: Therefore, in any elected or appointed capacity by which I may have the honor of serving our fellow citizens in these United States, I the undersigned do hereby solemnly vow* to honor and to cherish, to defend and to uphold, the Institution of Marriage as only between one man and one woman. I vow* to do so through my: –Personal fidelity to my spouse. –Respect for the marital bonds of others. –Official fidelity to the U.S. Constitution, supporting the elevation of none but faithful constitutionalists as judges or justices. –Vigorous opposition to any redefinition of the Institution of Marriage – faithful monogamy between one man and one woman – through statutory-, bureaucratic-, or court-imposed recognition of intimate unions which are bigamous, polygamous, polyandrous, same-sex, etc. –Recognition of the overwhelming statistical evidence that married people enjoy better health, better sex, longer lives, greater financial stability, and that children raised by a mother and a father together experience better learning, less addiction, less legal trouble, and less extramarital pregnancy. –Support for prompt reform of uneconomic, anti-marriage aspects of welfare policy, tax policy, and marital/divorce law, and extended “second chance” or “cooling-off” periods for those seeking a “quickie divorce.” –Earnest, bona fide legal advocacy for the Defense of Marriage Act (DOMA) at the federal and state levels. –Steadfast embrace of a federal Marriage Amendment to the U.S. Constitution which protects the definition of marriage as between one man and one woman in all of the United States. –Humane protection of women and the innocent fruit of conjugal intimacy – our next generation of American children – from human trafficking, sexual slavery, seduction into promiscuity, and all forms of pornography and prostitution, infanticide, abortion and other types of coercion or stolen innocence. –Support for the enactment of safeguards for all married and unmarried U.S. Military and National Guard personnel, especially our combat troops, from inappropriate same-gender or opposite-gender sexual harassment, adultery or intrusively intimate commingling among attracteds (restrooms, showers, barracks, tents, etc.); plus prompt termination of military policymakers who would expose American wives and daughters to rape or sexual harassment, torture, enslavement or sexual leveraging by the enemy in forward combat roles. –Rejection of Sharia Islam and all other anti-woman, anti-human rights forms of totalitarian control.1 –Recognition that robust childbearing and reproduction is beneficial to U.S. demographic, economic, strategic and actuarial health and security. –Commitment to downsizing government and the enormous burden upon American families of the USA‟s $14.3 trillion public debt, its $77 trillion in unfunded liabilities, its $1.5 trillion federal deficit, and its $3.5 trillion federal budget. –Fierce defense of the First Amendment‟s rights of Religious Liberty and Freedom of Speech22, especially against the intolerance of any who would undermine law-abiding American citizens and institutions of faith and conscience for their adherence to, and defense of, faithful heterosexual monogamy. There is plenty in this vow with which one can take issue, but to simply distill it down to Bachmann pledging to outlaw porn (as some have done) seems to be missing a much larger point. Still, there is a lot to digest in this pledge… which will likely be accomplished below by the Mediaite commenter community, with their trademark tolerance and broadness of sensibility. Have a tip we should know? tips@mediaite.com LGBT Iowa Group Asks Republican Candidates To Agree That Homosexuality Is A Choice, Pornography Should Be Banned This morning, Iowa’s FAMiLY Leader unveiled “The Marriage Vow: A Declaration of Dependence upon MARRIAGE and FAMILY,” a 14 bullet pledge presidential candidates will have to sign to secure any “future endorsement” from the organization and its influential leader, Bob Vander Plaats. Throughout the early primary process, Vander Plaats — a three-time unsuccessful gubernatorial candidate who helped propel Gov. Mike Huckabee (R) to victory in the state in 2008 and ran a successful campaign to recall three justices who overturned Iowa’s anti same-sex marriage law — has sought to position himself as a gatekeeper to the conservative voters who participate in the Iowa caucuses. Leader has hosted a presidential forum with almost all of the GOP’s 2012 contenders and Plaats has traveled to Washington, DC to present himself as a national Tea Party leader. Today’s pledge seeks to solidify the organization’s reputation as the state’s conservative moral authority, and it reiterates many of the group’s extreme social views and anti-gay rhetoric. If any candidate signs on to Leader’s vows, they will be agreeing to the following radical constructs: — HOMOSEXUALITY IS A CHOICE: The preface to the pledge reads, “Social protections…have been evaporating as we have collectively ‘debased the currency’ of marriage…in complete absence of empirical proof, that non-heterosexual inclination are genetically determined, irresistible and akin to innate traits like race, gender and eye color; as well as anti-scientific bias which holds, against all empirical evidence, that homosexual behavior in particular, and sexual promiscuity in general, optimizes individual or public health.” Footnote 8 reiterates this notion. — HOMOSEXUALITY IS LIKE POLYGAMY, ADULTERY, POLYANDRY: Vow 4 requires the candidate to pledge “Vigorous opposition to any redefinition of the Institution of Marriage…through statutory, bureaucratic, or court-imposed recognition of intimate unions which are bigamous, polygamous, polyandrous, same-sex.” — HOMOSEXUALITY IS A PUBLIC HEALTH RISK: Footnote 4 claims that homosexuality causes shorter life expectancy and a higher probability of a long list of sexually transmitted diseases. The Leader has previously compared same-sex marriage to second-hand smoking. — SEX IS BETTER AFTER MARRIAGE: Vow 5 requires the candidate to support the notion that “married people enjoy better health, better sex.” — PORNOGRAPHY SHOULD BE BANNED: Vow 9 stipulates that the candidate must “support human protection of women and the innocent fruit of conjugal intimacy” and protect them from “seduction into promiscuity and all forms of pornography…and other types of coercion or stolen innocence.” — REJECT SHARIA ISLAM: Vow 11 requires the candidate to reject Sharia law. Despite Vander Plaats’ best efforts to woo the GOP establishment, many of the Republicans who spoke at the FAMiLY Leader’s presidential forum have already dismissed its extremest anti-gay beliefs. Rick Santorum, Tim Pawlenty and Michele Bachmann, have all told ThinkProgress that they do not believe that homosexuality is akin to second hand smoking. Next week, the group will host former Speaker of the House Newt Gingrich, who — given his past admissions of infidelity — may not be able to commit to a pledge that requires “personal fidelity to my spouse” as its first vow. Republicans have until Aug. 1 to sign the pledge.

Summary: Michele Bachmann became the first GOP presidential hopeful to sign an extremely socially conservative pledge that might sorta call for a ban on porn. The pledge, which is being put forth by an Iowa-based group called The Family Leader, is ostensibly primarily about marriage; it's called "The Marriage Vow-a Declaration of Dependence upon Marriage and Family." But some, like ThinkProgress, have been criticizing it for also including a clause condemning porn. The clause in question is confusing, Mediaite observes. It insists candidates support the "humane protection of women and the innocent fruit of conjugal intimacy"-meaning children- "from human trafficking, sexual slavery, seduction into promiscuity, and all forms of pornography." That's not actually calling for a "porn ban," as some are reporting, but does lump porn together with sex slavery. Other eyebrow-raising things the pledge calls for include: "Rejection of Sharia Islam" (Though a later clause calls for a "fierce defense" of "religious liberty") "Recognition that robust childbearing and reproduction is beneficial" to the US Support for a "cooling-off" period for anyone wanting a divorce "Vigorous opposition" to any recognition of "unions which are bigamous, polygamous, polyandrous, same-sex, etc."

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Summarize: Background Mercury enters the environment through natural and man-made sources, including volcanoes, chemical manufacturing, and coal combustion, and poses ecological threats when it enters water bodies, where small aquatic organisms convert it into its highly toxic form—methylmercury. This form of mercury may then migrate up the food chain as predator species consume the smaller organisms. Through a process known as bio-accumulation, predator species may develop high mercury concentrations in their tissue as they take in more mercury than they can metabolize or excrete. Fish contaminated with methylmercury may pose health threats to those that rely on fish as part of their diet. According to EPA, mercury harms fetuses and can cause neurological disorders in children, including poor performance on behavioral tests, such as those measuring attention, motor and language skills, and visual-spatial abilities (such as drawing). In addition, populations that consume larger amounts of fish than the general population—including subsistence fishers, as well as certain Native Americans and Southeast Asian Americans—may face higher risk of exposure to contaminated fish, according to EPA. The Food and Drug Administration (FDA) and EPA recommend that expectant mothers, young children, and those nursing children avoid eating swordfish, king mackerel, shark, and tilefish and limit consumption of other potentially contaminated fish, such as tuna. These agencies also recommend checking local advisories for recreationally caught freshwater and saltwater fish. According to EPA, 45 states issued mercury advisories in 2003 (the most recent data available). Because mercury released to the atmosphere can circulate for long periods of time and be transported thousands of miles before it gets deposited, it is difficult to link mercury accumulation in the food chain with sources of mercury emissions. EPA estimates that about half of the mercury deposited in the United States is emitted by sources within this country. In 1999, the most recent year for which data were available, EPA estimated that man-made sources within the United States emitted about 115 tons of mercury. Of these emissions, the agency estimates that about 48 tons, 42 percent of the total, came from coal-fired power plants. While power plants are not required to limit their mercury emissions, EPA estimates that the plants currently capture about 27 tons of mercury each year, primarily through the use of controls for other pollutants, such as those used to control nitrogen oxides, particles, and sulfur dioxide. EPA estimates that power plants would otherwise emit about 75 tons of mercury per year. The Clean Air Act (CAA) Amendments of 1990 required EPA to study the environmental and health effects of hazardous air pollutants from coal-fired power plants and determine whether it was “appropriate and necessary” to regulate these pollutants. In 2000, EPA determined that mercury was a hazardous air pollutant and that it was appropriate and necessary to regulate mercury using the technology-based option. Under this section of the act, the emissions limit had to be at least as strict as the average emissions of the facilities with the best-controlled emissions. Because power plants did not already use controls specifically intended to control mercury, EPA analyzed the effectiveness of controls for other pollutants that capture mercury as a side benefit. This effort culminated in EPA’s January 2004 proposal for a technology-based option that would reduce mercury emissions from a current level of 48 tons per year to a projected 34 tons per year (a 29 percent reduction) by 2008. At the same time, however, EPA proposed an alternate policy option that would limit mercury emissions in two phases: to 15 tons in 2018 (a 69 percent reduction from current levels), preceded by an as-yet-unspecified interim cap starting in 2010. The alternate policy option, which would rely on a cap-and-trade system similar to that currently used to control emissions that cause acid rain, differs from the technology-based option in that it would not require each facility to meet emission standards based on control technology. Instead, EPA would set a nationwide “cap” for mercury emissions from coal-fired power plants and then distribute tradable emissions allowances that represent a certain amount of the total cap. At the end of each year, each power plant would have to hold sufficient allowances for the mercury it emitted that year. Plants that reduced their emissions below the levels represented by their allowances could sell their extra allowances to other plants. In addition to its proposed mercury rule, EPA has proposed another rule for power plants, the Clean Air Interstate Rule, which is intended to reduce emissions of nitrogen oxides and sulfur dioxide beginning in 2010. EPA expects that this proposed rule would result in the installation of pollution controls that capture mercury as a side benefit, and thereby decrease mercury emissions to 34 tons per year by 2010, the same level of reduction as the technology-based option. Under the cap-and-trade option, EPA has indicated that it may establish a mercury cap for 2010 equal to the control level expected through the interstate rule. EPA postponed its decision on finalizing the interstate rule until March 2005 while the agency awaits congressional action on pending legislation, known as the Clear Skies Act, that would establish emissions caps and an allowance system similar to those in the interstate rule and the cap-and-trade mercury control option. EPA has stated a preference for achieving reductions of mercury, nitrogen oxides, and sulfur dioxide simultaneously through legislation rather than regulations. Responsibility for analyzing the economic impacts—including costs to industry and expected public health effects—of air pollution control policies rests with EPA’s Office of Air and Radiation. EPA provided documentation of its economic analysis for the proposed mercury rule in three primary documents, some of which refer readers to additional documentation on the agency’s Web site or in the public rule-making docket. According to EPA, the agency did not have time to assemble its economic assessment of the proposed rule in a single document prior to issuing the proposed rule. To assist in estimating costs that air quality regulations will impose on the power industry, EPA uses the Integrated Planning Model (IPM), which estimates how power plants would respond to various environmental policies. The assumptions underlying this model, such as those regarding fuel costs, the costs of pollution controls, and future electricity demand, can affect the modeling results, according to EPA officials responsible for the modeling. EPA’s Economic Analysis Is of Limited Use for Informing Decision Makers about the Economic Trade-offs of Different Policy Options We identified four major shortcomings in the economic analysis underlying EPA’s proposed mercury rule that limit its usefulness for informing decision makers and the public about the economic trade-offs of the two options. First, EPA did not consistently analyze each of its two mercury policy options or provide estimates of the total costs and benefits of the two options, making it difficult to ascertain which policy option would provide the greatest net benefits. Second, EPA did not document some of its analysis or provide consistent information on the anticipated economic effects of different mercury control levels under the two options. Third, the agency did not estimate the economic benefits directly related to decreased mercury emissions. Finally, the agency did not analyze some of the key uncertainties underlying its cost-and-benefit estimates. EPA Did Not Consistently Analyze Each Policy Option or Provide a Complete Accounting of Costs and Benefits EPA’s estimates of the costs and benefits of its two proposed policy options are not comparable because the agency used inconsistent approaches in analyzing the two options. As shown in table 1, EPA analyzed the technology-based option alone, while it analyzed the cap-and-trade option in combination with the interstate rule. In analyzing the technology-based option by itself, EPA estimated the rule would cost about $2 billion annually, and achieve benefits of $15 billion or more annually, yielding net benefits (benefits minus costs) of $13 billion or more annually. In contrast, EPA analyzed the effects of the cap-and-trade option in combination with the proposed interstate rule by combining the costs and benefits of the two proposed rules without separately identifying and documenting those associated with the cap-and-trade option alone. This analysis found that the two proposed rules together would impose costs of $3 billion to $5 billion or more annually, while generating annual benefits of $58 billion to $73 billion or more and annual net benefits of $55 billion to $68 billion or more. Because the estimates for the two options are not comparable, however, it is not clear which option would provide the greatest net benefits. This is particularly important in light of EPA’s decision to delay finalization of the interstate rule. EPA officials responsible for the rule acknowledged the lack of comparability with its analysis of the two proposed options. These officials said the agency analyzed the cap-and-trade option alongside the interstate rule because it viewed these two proposed policies as complementary. They also said it would have been useful to analyze the technology-based option alongside the interstate rule, but the agency did not do so because of time constraints. Nonetheless, it is important for EPA to consistently analyze each policy option and provide decision makers with comparable estimates of net economic benefits. The comparability of EPA’s analysis is further limited because the agency did not provide consistent information on the total costs and benefits of the two options over their entire implementation periods. Specifically, EPA provided cost-and-benefit estimates for 2010, rather than estimates of the total costs and benefits over the entire implementation period. This is important because the economic impact of the policy options could vary from year to year and because the two options have different implementation timelines. For example, under the proposed cap-and-trade option, a second level of mercury reductions would take effect in 2018, which would likely generate additional costs and benefits at that time. Thus, the estimates EPA provided for 2010 did not fully account for the expected costs and benefits over the implementation period for this option. In contrast, EPA officials said that its estimate of the technology-based option in 2010 reflects the full implementation cost because its analysis assumes that power plants would achieve compliance with the technology-based option by that date. However, without estimates of the total value of benefits and costs of each option over the entire implementation period, it is difficult to ascertain which option would generate the greatest net benefits. EPA Did Not Document Some of Its Analysis Supporting the Policy Options or Provide Consistent Information on the Economic Impacts of Different Control Levels The economic analysis underlying the proposed mercury rule does not consistently reflect OMB’s guidance to agencies in terms of adhering to the principles of full disclosure and transparency when analyzing the economic effects of regulations. Specifically, we identified two primary cases where EPA’s analysis does not adhere to these principles, further limiting the usefulness of the agency’s analysis in decision making and diminishing the transparency of the analysis to the public. First, while EPA provides substantial information on its analysis of the technology-based option in the documents supporting its economic analysis of the proposed rule, the agency does not do so for the cap-and-trade option. For the technology-based option, EPA provides documents that describe its findings. In contrast, the agency provides only a summary of its findings for the cap-and-trade option in the rule’s preamble and refers to its findings as “rough estimates” that are based on consideration of available analysis of the interstate rule, the technology-based option, and the proposed Clear Skies legislation. EPA does not describe specifically how the agency used this analysis of other proposed rules and legislation to estimate the costs and benefits of the cap-and-trade option, and it does not identify the key analytical assumptions underlying its cost-and-benefit estimates. This lack of documentation and transparency leaves decision makers and the public with limited information on EPA’s analysis of the cap-and-trade option. Second, EPA officials responsible for the economic analysis told us that they analyzed two variations of the proposed technology-based option with more stringent mercury limits than the option included in the proposal, but the agency did not include this analysis in the documents supporting its economic analysis or in the public rule-making docket. This is inconsistent with EPA’s analysis of the cap-and-trade option, in which it provided a range of costs and benefits associated with different levels of stringency. This omission is also at odds with OMB guidance directing agencies to conduct their economic analysis in accordance with the principles of full disclosure and transparency. With respect to the analysis of the technology-based scenarios that the agency did not make publicly available, EPA officials said the additional modeling showed that the more stringent scenarios were not as cost-effective as the proposed technology-based option. However, EPA did not estimate the benefits of these two scenarios, thereby precluding a comparison of the net economic benefits under the proposed mercury policy options. As a result, it is unclear whether the reduction levels and implementation timelines under either proposed option represent the regulatory scenario that would provide the greatest net benefits. In January 2005, EPA officials responsible for the mercury rule said the agency does not have an obligation to analyze and document every control scenario. We recognize that OMB guidance gives agencies latitude in determining the number of regulatory alternatives to consider and that agencies must balance the thoroughness of their analysis with the practical limits of their ability to carry out analysis. Nonetheless, providing information on the costs and benefits of even a limited range of control scenarios under both proposed options would help decision makers and the public in assessing how different levels of stringency would affect overall estimates of costs and benefits. In December 2004, EPA solicited public comment on additional economic analyses the agency received from commenters on the January 2004 proposed rule, including some that relied on models, assumptions, and levels of stringency that were different from the scenarios EPA analyzed. EPA Did Not Estimate the Human Health Benefits of Mercury Reductions Although EPA’s analysis states that a mercury regulation would generate a variety of benefits, the agency did not estimate in monetary terms all of the benefits expected from reducing mercury emissions. Most notably, EPA did not quantify the human health benefits of decreased exposure to mercury, such as reduced incidence of developmental delays, learning disabilities, and neurological disorders. Instead, EPA estimated only some of the health benefits it anticipates would occur from decreased exposure to fine particles and discussed other impacts qualitatively. Because the two options in the proposed rule differed significantly in both the amount of mercury emission reductions and the time frames in which these reductions would occur, the lack of estimates of the mercury-specific benefits of each policy option represents a significant limitation of EPA’s economic analysis. That is, to the extent that each proposed option would yield measurable mercury-specific health benefits, EPA’s analysis may underestimate the total expected benefits of both options. Moreover, because the options may yield different mercury-related health benefits, the lack of estimates of these benefits makes it difficult to weigh the relative merits of the two proposed options. According to EPA, its analysis did not estimate key mercury-related health benefits because of technical, time, and resource limitations. Specifically, agency officials responsible for the analysis said the agency did not have a method for determining the extent to which mercury reductions from power plants would translate into decreased incidence of mercury-related health problems. According to EPA, estimating these benefits involves a number of complex chemical, physical, and biological processes, as well as a wide variety of human behaviors, such as fish consumption practices. Although EPA did not estimate the expected human health and other benefits of decreased exposure to mercury emissions in the analysis supporting the proposed rule, the agency did list the various human health and other benefits it expects would stem from a mercury rule. Importantly, in December 2004, the agency announced that it was revising its benefit estimates and solicited public comment on a proposed method for estimating mercury-specific benefits. According to EPA, this method would focus on (1) quantifying projected emissions from coal-fired power plants relative to other sources, (2) modeling the dispersion and deposition of mercury, (3) modeling the link between changes in mercury deposition and changes in the methylmercury concentrations in fish, (4) assessing the methylmercury exposure from consuming fish, and (5) assessing how reductions in methylmercury exposure affect human health. According to EPA officials responsible for analyzing the proposed rule’s effects, the agency will consider public comments on this approach and revise its analysis before finalizing a rule. In January 2005, EPA officials responsible for the analysis agreed that providing monetary estimates of mercury-specific benefits would enhance their analysis, and said that the agency might have sufficient information to estimate some, but not all, of the expected human health benefits of reducing mercury emissions. EPA Did Not Assess Key Analytical Uncertainties That Could Affect Its Cost-and-Benefit Estimates OMB guidance under Executive Order 12866 stipulates that agencies should analyze and present information on uncertainties with their cost-and-benefit estimates. According to EPA officials responsible for the economic analysis, the agency’s cost model is generally sensitive to assumptions about future electricity demand and fuel prices, as well as the availability, cost, and performance of pollution controls. Because these assumptions involve long-term projections, they also involve a substantial amount of uncertainty. EPA conducted a limited uncertainty analysis of natural gas prices and electricity demand growth on the cost estimates by examining the impact of alternative projections and concluded that its cost estimates were not particularly sensitive to changes in these variables. However, EPA did not assess how the distribution of estimated benefits and costs would differ given changes in its assumptions about the availability, cost, and performance of mercury control technologies, even though the agency believes that these assumptions could affect its economic modeling. Furthermore, EPA’s December 2004 notice for additional public comment on the mercury proposal highlighted the uncertainty surrounding the ability of its computer model to estimate mercury control costs, primarily because of the power industry’s limited experience with implementing mercury controls. This notice solicited public comment on, among other things, the assumptions in its economic modeling related to the cost, availability, and performance of mercury control technologies. According to senior EPA officials responsible for analyzing the mercury proposal, changes in these assumptions could have a sizable impact on the agency’s cost-and-benefit estimates. This acknowledgment of key uncertainties in its economic modeling highlights the need to determine how they could affect the overall cost-and-benefit estimates for each proposed option. In addition, EPA did not analyze the key uncertainties surrounding its benefit estimates. For example, EPA used economic data from its earlier assessment of the proposed Clear Skies legislation to approximate the impact of emissions reductions that would be expected under the mercury rule. According to EPA, the agency used this approach—referred to as a “benefits-transfer approach”—because time and resource constraints prevented it from performing new research to measure the value of health impacts under a mercury rule. OMB’s September 2003 guidance, which applies to economically significant final rules issued after January 1, 2005, states that although such an approach can provide a quick and low-cost means of obtaining monetary values, the method may be characterized by uncertainty and potential biases of unknown magnitude and should be treated as a last-resort option. Furthermore, EPA’s economic analysis states that the benefits analysis has many sources of uncertainty, including those associated with emissions data, air quality modeling, and the effect of emissions on human health. The agency did not, however, formally assess the impact of these uncertainties. In January 2005, EPA officials responsible for the proposed mercury rule acknowledged this limited analysis of key uncertainties and said that the agency plans to conduct a more formal assessment of these uncertainties prior to issuing a final rule, as directed by OMB’s September 2003 guidance. This guidance directs agencies to assess the sources of uncertainty in their regulatory analyses and the way in which cost-and-benefit estimates may be affected under plausible assumptions. Furthermore, in cases where the annual economic effects total $1 billion or more, the guidance states that agencies should provide a formal quantitative assessment of the key uncertainties about costs and benefits. Conclusions Because EPA estimates that regulating mercury emissions would have significant economic impacts totaling billions of dollars per year, it is important for the agency to have a credible basis for selecting a policy that will maximize the return on this investment. However, EPA’s initial economic analysis of the two policies it is considering has a number of shortcomings. Specifically, because EPA did not analyze and document the economic effects of each policy option by itself—as well as in combination with the interstate rule—over their varying full implementation periods, the results cannot be meaningfully compared. In addition, EPA did not document the analysis supporting the cap-and-trade option or provide consistent information on the economic impacts of different mercury control levels for the two options, limiting the transparency and usefulness of the analysis. Further, without monetary estimates of the human health benefits of mercury emissions reductions—a primary purpose of a mercury regulation—over the full implementation period of each option or, at a minimum, a qualitative comparison of these benefits, EPA’s analysis does not provide decision makers with a strong basis for comparing the net benefits under each option. Finally, because EPA did not analyze some of the key analytical uncertainties that could affect its estimates of net benefits, the agency could enhance its economic analysis by further evaluating these uncertainties and how they could affect its overall findings. Unless EPA conducts and documents further economic analysis, decision makers and the public may lack assurance that the agency has evaluated the economic trade-offs of each option and taken the appropriate steps to identify which mercury control option would provide the greatest net benefits. Recommendations for Executive Action To improve the usefulness of the agency’s economic analysis for informing decision makers and the public, and to help ensure consistency with OMB guidance for economic analysis, we recommend that, as the agency revises its economic analysis prior to selecting a mercury control option, the EPA Administrator take the following four actions: Analyze and fully document the economic effects of each policy option by itself, as well as in combination with the interstate rule, over their full implementation periods. Ensure that the agency documents its analysis supporting the final rule and consistently analyzes the effect that different levels of mercury control would have on cost-and-benefit estimates under each policy option. Include monetary estimates, where possible, of the human health benefits of reductions in mercury emissions from power plants or, at a minimum, provide qualitative information on how these benefits are likely to compare under the two options over a consistent time frame, reflecting full implementation of both options. Further analyze uncertainties surrounding estimates of costs and benefits, as directed by OMB guidance, and evaluate how these uncertainties could affect overall estimates of the rule’s impacts. Agency Comments We provided EPA with a draft of this report for review and comment. In commenting on the draft report, the Assistant Administrator for Air and Radiation said that, prior to issuing a final mercury regulation by March 15, 2005, EPA will conduct additional analysis that will largely address the findings and recommendations identified in our report. EPA’s letter is included as appendix II. As agreed with your offices, unless you publicly announce the contents of this letter earlier, we plan no further distribution until 7 days from the report date. At that time, we will send copies of the report to the EPA Administrator and other interested parties. We will also make copies available to others upon request. In addition, the report will be available at no charge on the GAO Web site at http://www.gao.gov. If you have any questions about this report, please contact me at (202) 512-3841 or stephensonj@gao.gov. Key contributors to this report are listed in appendix III. Objectives, Scope, and Methodology Congressional requesters asked us to assess the usefulness of the economic analysis underlying EPA’s proposed mercury rule for decision making. To respond to this objective, we, among other things, reviewed EPA’s analysis of the proposed rule’s economic effects using standard economic principles, OMB guidance, Executive Order 12866, and the Unfunded Mandates Reform Act of 1995. We also discussed the analysis with senior officials within EPA’s Office of Air and Radiation responsible for developing the proposed rule and analyzing its economic effects. In doing this work, we did not independently estimate the costs or benefits of the mercury control options, evaluate EPA’s process for developing the options, or assess legal issues surrounding the extent to which the options comply with the provisions of the Clean Air Act or its amendments. We took several steps to assess the validity and reliability of computer data underlying EPA’s estimates of economic impacts discussed in our findings, including reviewing the documentation and assumptions underlying EPA’s economic model and assessing the agency’s process for ensuring that the model data are sufficient, competent, and relevant. We also discussed these assumptions and procedures with agency officials responsible for the modeling data. (For the background section of this report, we obtained data on mercury emissions. Because they are used for background purposes only, we did not assess their reliability.) We assessed compliance with internal controls related to the availability of timely, relevant, and reliable information. Our concerns about EPA data and analysis are discussed in the body of this report. We performed our work between May 2004 and February 2005 in accordance with generally accepted government auditing standards. Comments from the Environmental Protection Agency GAO Contacts and Staff Acknowledgments GAO Contacts Staff Acknowledgments In addition to the individuals named above, Tim Guinane and Michael Hix made key contributions to this report. Kate Cardamone, Jessica Fast, Cynthia Norris, Judy Pagano, Janice Poling, and Amy Webbink also made important contributions.

Summary: Mercury is a toxic element that can cause neurological disorders in children. In January 2004, the Environmental Protection Agency (EPA) proposed two options for limiting mercury from power plants, and plans to finalize a rule in March 2005. The first would require each plant to meet emissions standards reflecting the application of control technology (the technology-based option), while the second would enable plants to either reduce emissions or buy excess credits from other plants (the cap-and-trade option). EPA received over 680,000 written comments on the proposal. EPA is directed by statute and executive order to analyze the costs and benefits of proposed rules, and the agency summarized its analysis underlying the two options in the proposal. In this context, GAO was asked to assess the usefulness of EPA's economic analysis for decision making. In doing so, GAO neither independently estimated the options' costs and benefits nor evaluated the process for developing the options or their consistency with the Clean Air Act, as amended. GAO identified four major shortcomings in the economic analysis underlying EPA's proposed mercury control options that limit its usefulness for informing decision makers about the economic trade-offs of the different policy options. First, while Office of Management and Budget (OMB) guidance directs agencies to identify a policy that produces the greatest net benefits, EPA's analysis is of limited use in doing so because the agency did not consistently analyze the options or provide an estimate of the total costs and benefits of each option. For example, EPA analyzed the effects of the technology-based option by itself, but analyzed the effects of the cap-and-trade option alongside those of another proposed rule affecting power plants, the Clean Air Interstate Rule (the interstate rule), without separately identifying the effects of the cap-and-trade option. As a result, EPA's estimates are not comparable and are of limited use for assessing economic trade-offs. EPA officials said they analyzed the cap-and-trade option alongside the interstate rule because the agency views the two proposed rules as complementary. Nonetheless, to provide comparable estimates, EPA would have to analyze each option alone and in combination with the interstate rule. Second, EPA did not document some of its analysis or provide information on how changes in the proposed level of mercury control would affect the cost-and-benefit estimates for the technology-based option, as it did for the cap-and-trade option. Third, EPA did not estimate the value of the health benefits directly related to decreased mercury emissions and instead estimated only some secondary benefits, such as decreased exposure to harmful fine particles. However, EPA has asked for comments on a methodology to estimate the benefits directly related to mercury. Fourth, EPA did not analyze some of the key uncertainties underlying its cost-and-benefit estimates.

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Summarize: A lawsuit filed Tuesday accuses Prisoner Transportation Services of negligence, intentional infliction of emotional distress and violating the 14th Amendment for the conditions detainees are held in the “mobile jails” in the back of these vans. (Weatherford Daily News/AP) Edward Kovari’s 18-day journey caged in the back of a private prisoner transport van began at a gas station in Winchester, Va., where the waiter had recently moved from Houston. As Kovari left the station's convenience store, a Virginia police officer checking out-of-state license plates informed him that his 2005 Pontiac sedan had been reported stolen in Houston. The report turned out to be false, and the resulting charges were ultimately dismissed — but not before Kovari, now 39, was arrested, arraigned on a fugitive warrant and extradited to Houston in September 2016 in what his attorneys describe in a federal lawsuit filed Tuesday as a tortuous ordeal. The lawsuit filed in U.S. district court in western Virginia against three companies — Brevard Extraditions, which conducts business as U.S. Prisoner Transport; Prisoner Transportation Services of America; and its parent company, Prisoner Transportation Services — illustrates the risks posed by the increasing privatization of prisoner extradition, Kovari's lawyers said. [Private-prison giant, resurgent in Trump era, gathers at president’s resort] The financial incentive to pick up as many detainees as possible — with few stops for rest, water, food and bathroom breaks — led to unsanitary and unsafe conditions of confinement, in violation of the 14th Amendment, the lawsuit alleges. It also accuses the companies and their corrections officers of negligence and intentional infliction of emotional distress for denying Kovari needed medical attention. “The realization that this could happen to potentially anyone is frightening,” said Jia Cobb, one of Kovari’s attorneys. “No one should be treated like this, no matter what they are accused of, even if they are convicted of a crime. It’s against the law.” Prisoner Transportation Services, the nation's largest for-profit extradition company, did not immediately respond to a request for comment. Joel Brasfield, the company's president, had denied prior allegations of wrongdoing. The pattern of abuse and neglect during prisoner transport by for-profit companies has been previously documented by the Marshall Project, but Kovari’s experience has not been reported. Robert Downs, then chief operating officer of Prisoner Transportation Services, had told the Marshall Project in response to its investigation that guards were instructed to contact local officials when a serious medical emergency arises. “Unless it’s life or death, we can’t open the cage on the vehicle,” Downs said. “We don’t know if they’re setting us up for something.” Shackled in chains so tight that they left marks on his body, Kovari was crammed in the back of a van with other detainees and deprived of adequate food and water, according to the lawsuit. Kovari also was denied his daily prescription medication for hypertension and medical care as his blood pressure spiked during the circuitous journey in three vans through seven states over 18 days. It normally takes 20 hours to drive the 1,350 miles between Winchester and Houston. [These GOP lawmakers say it’s okay for imprisoned immigrants to work for a $1 a day] At some points, as many as 15 people were crammed into the cargo van — exceeding capacity so that Kovari had to lie on the floor with other detainees's feet resting on his stomach, the lawsuit said. Without regular bathroom stops, Kovari spent the duration of the journey sitting in human waste and filth, both his own and that of other detainees locked in the dark, sweltering cage, according to the lawsuit. Instead of bathroom breaks, drivers demanded detainees relieve themselves in empty bottles or on themselves, according to the lawsuit. At least one person defecated on the floor of the van and another detainee vomited, but the driver did not stop to clean up the mess, the lawsuit alleged. Water was rationed, and detainees were occasionally fed fast food. Kovari’s attorney said his client suffered from heartburn and an upset stomach throughout the journey. Without access to his medication, Kovari began to feel sick after three days on the road. He developed a headache, started to see spots, felt clammy, disoriented and nauseated, the lawsuit said. He pounded on the steel wall to try to get the attention of the corrections officers for medical attention, but repeated requests to be taken to a hospital were ignored, the lawsuit said. Instead, the officers told him to “shut up or we’re going to taze you,” the lawsuit alleged. To maximize profits, the companies schedule drivers to pick up as many detainees as requested, without regard for where they ultimately will be dropped off, the lawsuit said. It is common for detainees such as Kovari to be locked in the back of the “mobile jails” for weeks at a time, the lawsuit said. Occasionally, local jails will house the detainees for a night. Otherwise, detainees, tightly shackled, are locked in steel cages and sit shoulder to shoulder along two metal benches without seat belts or other safety restraints. Kovari’s head would hit the steel wall in front and behind him whenever the van abruptly swerved. The lawsuit alleged that Kovari was unable to move, walk or stand for up to 12 hours at a time. His legs often went numb, and he was in constant excruciating pain, the lawsuit said. He went days without sleeping. At one point along the way, the van had a flat tire. Despite the extreme heat, detainees were kept inside the van during the three-hour wait for assistance. The lawsuit alleges trips like Kovari's can be life-threatening for people with medical conditions because basic medical needs are not met. Prisoner Transportation Services employees are not trained on how to treat detainees with medical conditions, “routinely refuse to provide necessary prescription medications to the individuals who need them, and have even instructed their drivers to ignore individuals’ requests for medical assistance, so as not to put their transports behind schedule,” the lawsuit said. By the time Kovari arrived in Houston, wet with sweat and vomit, he was unable to walk. His blood pressure, which was measured at the time of his intake at the Harris County Sheriff's Office, was higher than 200, well above normal, his attorney said. Kovari’s experience was the result of the companies’ customs, policies and practices, the lawsuit alleges. States and municipalities have increasingly outsourced prisoner transport to private companies that say they can provide the service for less money. At least five people have died on Prisoner Transportation Services vans from alleged medical neglect, according to the Marshall Project, which investigated the industry in a 2016 report published in the New York Times. Two dozen others have been killed or seriously injured in more than 50 crashes involving private extradition vehicles since 2000, the report said. The Justice Department was already investigating Prisoner Transportation Services for other abuse allegations before Kovari's arrest, according to the Marshall Project. Nevertheless, Kovari's attorney said, the company still subjected him to “overcrowded, unsanitary and unsafe conditions of confinement.” Our general interest e-newsletter keeps you up to date on a wide variety of health topics. Blood pressure chart: What your reading means By Mayo Clinic Staff This blood pressure chart can help you figure out if your blood pressure is at a healthy level or if you'll need to take some steps to improve your numbers. Your total blood pressure reading is determined by measuring your systolic and diastolic blood pressures. Systolic blood pressure, the top number, measures the force your heart exerts on the walls of your arteries each time it beats. Diastolic blood pressure, the bottom number, measures the force your heart exerts on the walls of your arteries in between beats. Blood pressure readings fall into four general categories, ranging from normal to stage 2 high blood pressure (hypertension). The level of your blood pressure determines what kind of treatment you may need. To get an accurate blood pressure measurement, your doctor should evaluate your readings based on the average of two or more blood pressure readings at three or more office visits. Here's a look at the four blood pressure categories and what they mean for you. If your systolic and diastolic readings fall into two different categories, your correct blood pressure category is the higher category. For example, if your blood pressure reading is 125/85 millimeters of mercury (mm Hg), you have stage 1 hypertension. Top number (systolic) in mm Hg And/or Bottom number (diastolic) in mm Hg Your category* What to do† *Ranges may be lower for children and teenagers. Talk to your child's doctor if you're concerned your child has high blood pressure. †These recommendations address high blood pressure as a single health condition. If you also have heart disease, diabetes, chronic kidney disease or certain other conditions, you may need to treat your blood pressure more aggressively. Below 120 and Below 80 Normal blood pressure Maintain or adopt a healthy lifestyle. 120-129 and Below 80 Elevated blood pressure Maintain or adopt a healthy lifestyle. 130-139 or 80-89 Stage 1 high blood pressure (hypertension) Maintain or adopt a healthy lifestyle. Talk to your doctor about taking one or more medications. 140 or higher or 90 or higher Stage 2 high blood pressure (hypertension) Maintain or adopt a healthy lifestyle. Talk to your doctor about taking more than one medication. If you are an adult with a 10 percent or higher risk of developing cardiovascular disease in the next 10 years, or if you have chronic kidney disease, diabetes or coronary artery disease, your treatment goal is less than 130/80 mm Hg. If you're a healthy adult age 65 or older, your treatment goal is also less than 130/80 mm Hg. If your blood pressure is normal, maintaining or adopting a healthy lifestyle can prevent or delay the onset of high blood pressure or other health problems. If your blood pressure isn't normal, a healthy lifestyle — oftentimes along with medication — can help bring it under control and reduce your risk of life-threatening complications. Graphic by Bill Marsh Additional research by Dartunorro Clark and Nick Tucker In July 2012, Steven Galack, the former owner of a home remodeling business, was living in Florida when he was arrested on an out-of-state warrant for failing to pay child support. Galack, 46, had come to the end of a long downward spiral, overcoming a painkiller addiction only to struggle with crippling anxiety. Now, he was to be driven more than a thousand miles to Butler County, Ohio, where his ex-wife and three children lived, to face a judge. Like dozens of states and countless localities, Butler County outsources the long-distance transport of suspects and fugitives. Galack was loaded into a van run by Prisoner Transportation Services of America, the nation’s largest for-profit extradition company. Crammed around him were 10 other people, both men and women, all handcuffed and shackled at the waist and ankles. They sat tightly packed on seats inside a cage, with no way to lie down to sleep. The air conditioning faltered amid 90-degree heat. Galack soon grew delusional, keeping everyone awake with a barrage of chatter and odd behavior. On the third day, the van stopped in Georgia, and one of two guards onboard gave a directive to the prisoners. “Only body shots,” one prisoner said she heard the guard say. The others began to stomp on Galack, two prisoners said. The guards said later in depositions that they had first noticed Galack’s slumped, bloodied body more than 70 miles later, in Tennessee. A homicide investigation lasted less than a day, and the van continued on its journey. The cause of death was later found to be undetermined. “This is someone’s brother, father, and it’s like nobody even cared,” said Galack’s ex-wife, Kristin Galack. Every year, tens of thousands of fugitives and suspects — many of whom have not been convicted of a crime — are entrusted to a handful of small private companies that specialize in state and local extraditions. A Marshall Project review of thousands of court documents, federal records and local news articles and interviews with more than 50 current or former guards and executives reveals a pattern of prisoner abuse and neglect in an industry that operates with almost no oversight. Since 2012, at least four people, including Galack, have died on private extradition vans, all of them run by the Tennessee-based Prisoner Transportation Services. In one case, a Mississippi man complained of pain for a day and a half before dying from an ulcer. In another, a Kentucky woman suffered a fatal withdrawal from anti-anxiety medication. And in another, guards mocked a prisoner’s pain before he, too, died from a perforated ulcer. Robert Downs, the chief operating officer of PTS, declined to comment on the deaths. He said guards were instructed to contact local officials when a serious medical emergency arises. “Unless it’s life or death, we can’t open the cage on the vehicle,” Downs said. “We don’t know if they’re setting us up for something.” This concern was echoed by guards at several companies, who said prisoners often feigned illnesses and injuries. Training for guards, many of whom are military veterans, is often limited to a tutorial on handcuffs and pepper spray and a review of policies and paperwork, leaving them unprepared for the hazards of driving a van full of prisoners. At least 60 prisoners have escaped from private extradition vehicles since 2000, including one who later stabbed a police officer and another who was accused of sexual assault on a minor and is still missing. The companies are usually paid per prisoner per mile, giving them incentive to pack the vans and take as few breaks as possible. Crashes have killed a dozen prisoners and guards. Operating primarily across the South and Midwest, guards travel up to weeks at a time along circuitous routes, typically picking up and dropping off prisoners in 15-passenger vans or sometimes minivans retrofitted with interior caging and darkened windows. These vans do not have prisoner beds, toilets or medical services. Violent felons are mixed with first-time suspects. A plexiglass divider is usually the only thing separating women from men. At least 14 women have alleged in criminal or civil court since 2000 that they were sexually assaulted by guards while being transported by these companies. “Just stay in jail. It’s better,” said Lauren Sierra, 21, who said she was repeatedly sexually assaulted by a guard in 2014 while being transported by U.S. Corrections, a rapidly growing company registered in North Carolina. Sierra, who is suing the company, was taken into custody after she faced charges, later dropped, that she used someone else’s Bed, Bath & Beyond gift card. Dustin Baldwin, the executive director of U.S. Corrections, declined to comment beyond saying that the accusations had not been proved. Because the vans cross state lines, accountability falls into a gray zone. Jurisdictions that hire the companies often disavow responsibility for prisoners not under their direct custody, and federal regulators have largely ignored the industry. “It’s like the airport shuttle from hell,” said Zachary Raines, a former PTS guard. Strained Jails and Budgets At a time when a swollen United States prison and jail population has strained law enforcement budgets, transport companies offer a significantly cheaper alternative to traditional extradition, in which local deputies are sent miles out of state for one person. “Some agencies take huge advantage of the taxpayers’ money by sending deputies ‘on vacation’ to extradite an inmate,” said Baldwin of U.S. Corrections, and pay them “a considerable amount of overtime” for doing so. They also have to cover fuel costs or plane tickets and, often, hotel rooms. Private vans can save considerably by picking up and dropping off other prisoners along the way, charging 75 cents to $1.50 a mile per prisoner. Corrections departments in 26 states, law enforcement in cities such as Chicago, Atlanta and Las Vegas, and local agencies nationwide use extradition companies. Although about two dozen private prisoner transport companies have registered with the Department of Transportation, only seven have state-level extradition contracts, with PTS having the most by far. But maintaining tight profit margins depends on relentlessly shaving time and costs on the road, industry veterans said. “You route the prisoner like a package, but miss a single deadline, and you lose money,” said Kent Bradford, a former director of operations for TransCor America, a subsidiary of Corrections Corporation of America, the largest private prison company in the United States. TransCor stopped performing extraditions in 2008 because of liability and cost concerns, but still moves prisoners between CCA locations. Guards — who earn about $150 to $250 per 24-hour shift, and who rotate driving duty — are generally paid only while they are on the road. Because they often have to pay out-of-pocket for a hotel room, most said they rarely chose to stop. Bunking overnight also requires finding a jail willing to offer beds and showers to prisoners, which is difficult because jails do not always want to house unknown prisoners from other jurisdictions. “I’d have an exhaust fan installed in the hall to get that smell out,” said David Osborne, who runs the Daviess County Detention Center in Kentucky, which used to be a PTS hub for transferring and housing prisoners en route. To keep up with demand, vans drive across as many as a dozen states on a single trip. “The bosses would be on the phone, saying, ‘What, you can’t do it? You can’t push it, you can’t make it to the next jail?’” said Fernando Colon, who worked as a guard for two years, first for a company that is now defunct and then for U.S. Corrections. On most trips, every meal for days is a fast-food sandwich. Water is rationed and bathroom stops limited. Prisoners who cannot wait often urinate in bottles or on themselves, and sometimes defecate on the floor of the van, according to guards and lawsuits. “People were screaming, complaining, passing out. I threw up,” said Roberta Blake, 37, who spent two weeks in 2014 being transported by PTS from California to Alabama, including a week in a stifling van. Lacking both privacy and sanitary napkins, she had to use a cup in front of the male guards and prisoners when she began menstruating. After another prisoner ripped off her shirt, she spent the rest of the trip in a sports bra. Blake, whose account was confirmed by two other prisoners in the van, had been arrested on a warrant issued after she failed to return a rental car on time. Medical Skills Not Required For some prisoners, the ride ends in serious injury, or even death. Michael Dykes, who has diabetes, had both of his legs amputated after three days in an Inmate Services Corporation van in July 2012. Dykes said he had already been in declining health when he got into the van after spending nearly three weeks in a South Carolina jail with poor medical care. But once in transport to Missouri, his condition worsened, he said. Black sores on his toes were exacerbated by pressure from ankle shackles, a lawsuit alleges, and his repeated requests for medical care were ignored. His insulin, which must be kept cold, was stored on the dashboard in the sun, Dykes said. Randy Cagle Jr., the president of the Arkansas-based Inmate Services Corporation, denied the accusations. “We always follow protocol and get medical information when we pick an inmate up,” he wrote in an email. “I am confident that we will be vindicated.” Cagle said in a brief phone interview that some prisoners lied or sued frivolously. “You are not going to get through this business without hurting people’s feelings,” he said. “You just have to remember to treat people fair.” When suspects are arrested on a warrant, they often spend considerable time in a local jail before being picked up for extradition. About a dozen guards from several transport companies said jails provided substandard medical care and little information about prisoners’ health status or prescribed medications, which the guards are expected to dispense en route. Guards are not required by law to have any medical experience other than training in cardiopulmonary resuscitation. “They did an hourlong class on their policies, taught us to put on handcuffs, gave us our uniforms and put us on the road. And then we’re expected to deal with this stuff,” said Kenneth Adams, one of two guards aboard a PTS van in which Denise Isaacs, 54, died in Miami in 2014. Like Galack, Isaacs began experiencing bizarre symptoms while on board: muttering, drooling and gasping. When she was unable to climb back into the van after a stop, the guards phoned PTS headquarters. But their supervisors said to keep going, Adams told investigators with the Miami-Dade Police Department. Opening Statement Sign up for our daily newsletter covering the best in criminal justice news. “I would have taken her to the hospital,” the other guard, Kirk Westbrooks, said in an interview with The Marshall Project. “I wanted to.” Isaacs, who had been arrested on charges of violating probation on a theft conviction, died a few hours later in a Taco Bell parking lot. An autopsy later found that she had been experiencing delirium tremens caused by withdrawal from diazepam, an anti-anxiety medication that PTS staff members said they were never informed she was taking. The Miami-Dade police closed the investigation after determining that the death was from natural causes. In January of this year, PTS guards transporting William Culpepper Jr., 36, from Kentucky to Mississippi told officials at a stop at a company jail hub in Missouri that they believed he was faking stomach pains, according to a sheriff’s report. Culpepper, who was wanted for a parole violation, died minutes later from what the coroner handling his case called a “perfectly treatable” perforated ulcer. It was the second time in two years that a PTS prisoner had died from a perforated ulcer. In 2014, William Weintraub, 47, a former physics professor charged with threatening a South Carolina newspaper over an article he disputed, was found blue and covered in urine in the back of a PTS van when it reached Georgia. Investigators there determined that PTS guards had mocked Weintraub’s complaints of severe stomach pain. The investigation was closed. Attempts at Reform Kyle Bell was no ordinary prisoner. In 1993, he molested and murdered his 11-year-old North Dakota neighbor, Jeanna North. Six years later, he escaped from a private transport van. His absence was not noticed for nine hours, and guards did not notify the police for another two hours. The escape warranted a segment on “America’s Most Wanted.” After the episode, Byron Dorgan, then a Democratic United States senator from North Dakota, introduced a measure to impose controls on the industry. “My colleagues and I were all shocked that a guy and his wife with an S.U.V. could start a business to haul violent offenders around with no requirements,” Dorgan said. The law, commonly known as Jeanna’s Act, passed in 2000. Jeanna’s Act mandates that extradition companies must notify local law enforcement immediately after an escape, dress violent prisoners in brightly colored clothing and maintain a ratio of one guard for every six prisoners. It also sets broad standards for training and background checks of guards, and for treatment of prisoners. But the federal law is almost never enforced. The Justice Department could identify just one instance: In 2011, a suspect accused of child molestation escaped from an unlocked van in North Dakota, a few hours from where Jeanna had been murdered. Local farmers cleared a cornfield to flush him out. The company, Extradition Transport of America, was fined $80,000 and went out of business. “Well, it’s regulated by the Department of Justice, but I’ve never seen anybody come out to actually check on us,” said Downs, the chief operating officer of PTS. “We’re just supposed to follow the guidelines.” Extradition companies are not required to report escapes to federal regulators, and there is no centralized tracking. But a review of dozens of local news accounts shows that since Jeanna’s Act was passed, at least 56 prisoners were reported to have escaped from for-profit extradition vehicles. At least 16 were reported to have committed new crimes while on the run. By comparison, the prison systems of California, Florida and Texas — which together transport more than 800,000 inmates every year, most of them in-state — have each had just one prisoner escape from transport vehicles over the same period. “We thought we’d closed the door on this,” Dorgan said in reference to the widespread use of small extradition companies and the escapes that have occurred. While the Department of Transportation has no role in responding to escapes or prisoner mistreatment, it is responsible for monitoring vehicle and driver safety, including whether guards get enough downtime away from the wheel, under the same regulations that govern all passenger carriers. A Marshall Project review of Department of Transportation records shows that the agency’s monitoring is infrequent, and companies are typically given advance notice of an audit. Between 2000 and 2015, records indicate, the department issued fines 20 times, most below $10,000. While PTS has been registered with the department since at least 2005, the agency did not audit the company until 2009, records show. U.S. Corrections, which was founded in 2014, was audited for the first time in March. Because passenger carriers are not required to specify to the Transportation Department what kinds of people they move around, a department spokesman said he could not comment on specifics about the prisoner transport industry. Local news reports and court records show that there have been more than 50 crashes involving private extradition vehicles since 2000. In almost every instance, the prisoners were shackled but not wearing seatbelts, leaving them unable to brace themselves. In addition to the dozen deaths, a dozen prisoners have suffered injuries to their necks, skulls or spines, according to lawsuits, hospital reports and accident reports obtained from state and local agencies. Fatigue seems to have played a role in many of the accidents. Of 26 accidents for which a time could be determined, 14 occurred between midnight and 6 a.m. Downs, who took over operations at PTS after it merged last year with one of its biggest competitors, the Florida-based U.S. Prisoner Transport, said he had taken steps to make the company safer. The company had already installed sleeper berths for guards in its vans. Downs said its agents were now required to stay in a company-paid hotel room every 36 hours, although he said that was not always possible because of scheduling pressures. The company also has three full-size buses and has bought four larger shuttle buses, all with bathrooms on board, in addition to its fleet of nearly 30 vans. Guards are monitored by GPS, and their pay has been increased, Downs said. “It’s a tough industry,” he said. “The profit margins aren’t as good as you would think they are.” He declined to answer a list of written questions about specific occurrences in the company’s vans. Security Transport Services, which is based in Topeka, Kan., and has been in the business since 1990, says it puts all prisoners in seatbelts and requires agents to stay in a hotel every night. A Kansas sheriff said the company had also partly reimbursed his department for the cost of a manhunt after a 2012 escape, which is required by law in cases of negligence but rarely happens, according to a survey of law enforcement officials in jurisdictions where escapes occurred. But the company charges about 30 percent more than its competitors, said Tom Rork, its vice president. Security Transport Services has contracts with three state corrections departments, compared with nearly 20 held by PTS, and it recently lost its Pennsylvania contract to U.S. Corrections. PTS says in federal filings that it has “contracts or relationships” with about 800 agencies. It is also poised to acquire U.S. Corrections, one of its main competitors, next month, according to a filing with the national Surface Transportation Board. Answers Are Elusive After Galack’s death, his brother, Robert, made repeated calls to the Tennessee authorities, trying to determine what had happened. “I mean, he was fully in shackles and ended up dead?” he said. It was hard to find answers. Only one prisoner in the van, Chelsie Hogsett, told investigators that Galack had been beaten. Another, Joseph Allen, did not confirm the account until a later civil suit. The Tennessee Bureau of Investigation decided within eight hours of arriving at the scene that if a crime had occurred, it had happened in Georgia. It sent the van on its way. The Georgia Bureau of Investigation declined to follow up, records show. Galack in van Butler County, Ohio Galack’s intended destination Not in van OHIO 1. Galack is picked up at the Palm Beach County Jail on the morning of July 30, 2012. Two other prisoners picked up shortly after him say he is acting normally. 2. But overnight, as the van travels north, Galack keeps the other prisoners awake, calling out for medicine and complaining of pain. 3. Increasingly delusional, Galack keeps the other prisoners awake for a second night. 4. Between Athens and Dalton, Ga., the driver pulls over and allegedly encourages the other prisoners to beat Galack. 5. Galack is found dead in Madisonville, Tenn. Lexington KENTUCKY Owensboro START Nashville 5 END Madisonville TENNESSEE Dalton 4 Athens 3 Atlanta ALABAMA GEORGIA Dothan FLORIDA Gainesville 2 Ocala Orlando West Palm Beach 1 Fort Myers Fort Lauderdale 100 MILES Galack in van Butler County, Ohio Galack’s intended destination OHIO Not in van 1. Galack is picked up at the Palm Beach County Jail on the morning of July 30, 2012. Two other prisoners picked up shortly after him say he is acting normally. 2. But overnight, as the van travels north, Galack keeps the other prisoners awake, calling out for medicine and complaining of pain. 3. Increasingly delusional, Galack keeps the other prisoners awake for a second night. 4. Between Athens and Dalton, Ga., the driver pulls over and allegedly encourages the other prisoners to beat Galack. 5. Galack is found dead in Madisonville, Tenn. Lexington KENTUCKY Owensboro START Nashville 5 END Madisonville TENNESSEE Dalton 4 Athens 3 Atlanta ALABAMA GEORGIA Dothan FLORIDA Gainesville 2 Ocala Orlando West Palm Beach 1 Fort Myers Fort Lauderdale 100 MILES Galack in van Not in van 1. Galack is picked up at the Palm Beach County Jail on the morning of July 30, 2012. Two other prisoners picked up shortly after him say he is acting normally. 2. But overnight, as the van travels north, Galack keeps the other prisoners awake, calling out for medicine and complaining of pain. 3. Increasingly delusional, Galack keeps the other prisoners awake for a second night. 4. Between Athens and Dalton, Ga., the driver pulls over and allegedly encourages the other prisoners to beat Galack. 5. Galack is found dead in Madisonville, Tenn. Butler County, Ohio Galack’s intended destination OHIO KENTUCKY START END 5 TENNESSEE First stop 4 3 ALABAMA GEORGIA FLORIDA 2 1 100 MILES The medical examiner noted Galack’s injuries — a broken rib, bruises on his head, torso, arms and legs, a broken tooth and cuts around his nose and eyes — but did not believe they had led to his death. The investigation was determined to be “as thorough as the circumstances warranted,” said Josh DeVine, a spokesman for the Tennessee Bureau of Investigation. Anthony Dwyer, the chief deputy of the sheriff’s office in Butler County, Ohio, said he had been told only that a prisoner had died en route, not that a beating might have been involved. “It wasn’t really our responsibility,” he said. He said he monitored PTS’s performance by speaking to prisoners when they arrive. Darnell Ball, one of the guards in the van that transported Galack, declined to comment, citing a confidentiality agreement. The other, Leroy Creese, did not respond to two attempts to contact him at an address believed to be his home. A PTS official said in a deposition taken in a civil lawsuit that Galack had sustained the injuries in a fall in the van. This spring, Galack’s family won a confidential settlement against PTS. But Galack’s son, Jordan, found it paltry consolation. Now 20, he had talked to his father every day on the phone and lost 30 pounds after his father’s death. Kristin Galack said she had never had any idea what her ex-husband would face when he was arrested. “Steve and the other people on these vans, they’ve made mistakes,” she said. “But that doesn’t mean he couldn’t come back from it. People do.” Three months after Galack was found in the back of the van, PTS sent Butler County a bill for $1,061 — the cost of the 752 miles he was transported before dying. McLEAN, Va. (AP) — Edward Kovari's 18-day ordeal began Sept. 12, 2016, when some guys in a van showed up to take him from a jail in Virginia to Texas, where he was wanted on charges that he had stolen a car. But the trip from Winchester, Virginia, to Houston took more than two weeks in a crowded van where inmates had to urinate in bottles and take turns sleeping on the van's floor, according to a federal lawsuit filed Tuesday by Kovari. The private company that contracted with the jail to transport Kovari, 39, kept him shackled in the back of that van for 18 days as it wound through the country picking up inmates in an effort at cost efficiency. The charge on which Kovari was brought to Texas was later dismissed. The company that transferred him, Nashville-based Prisoner Transportation Services, bills itself as the nation's largest prisoner extradition company. It did not respond Tuesday to messages seeking comment. The lawsuit, filed by civil rights lawyers Jia Cobb, Glenn Schlactus and Orly May, alleges Prisoner Transportation Services prioritizes "transporting as many detainees, with as few stops for rest or care, as possible over their obligation to safely transport those in their custody." In Kovari's case, that meant 18 days, most of which he spent in shackles. On only two or three nights did the van actually stop at a jail where prisoners were allowed to get out and spend the night in a bed. Inside the van, no provisions were made for restroom stops, and inmates urinated into bottles which spilled and sloshed on the floor of the van. In one case, a prisoner defecated on the van floor, and in another instance, a prisoner vomited. The lawsuit alleges that nothing was done to clean the mess. "He spent the duration of the transport sitting in human waste and filth," the lawyers wrote. The van was poorly ventilated with no functioning air conditioning, according to the lawsuit. At one point, according to the suit, the van pulled over on the highway after a flat tire and inmates were left in the back of the van. When police responded to the breakdown, one officer threatened the guards with arrest if they did not let the inmates out for fresh air and an opportunity to relieve themselves. The van was so crowded — at times with up to 15 passengers — that inmates including Kovari took turns lying on the floor with other inmate's feet on top for the opportunity to sleep. Kovari was also not permitted to take his medication and asked to be taken to the hospital on a daily basis, according to the lawsuit. But the drivers responded that that any stop at the hospital would require all the other inmates to wait in the van for as long as Kovari was hospitalized. As a result, according to the suit, the other inmates threatened Kovari if he insisted on a hospital stop. When Kovari finally made it to the Harris County Jail, his systolic blood pressure — the so-called top number — exceeded 200 and he was admitted to the jail infirmary, where his condition did not stabilize for two days. Kovari's lawyers declined to make him available for an interview. Alex Friedmann, managing editor of Prison Legal News, a publication focusing on prisoner rights and geared toward inmates, said stories like Kovari's are not uncommon. He said the private prisoner transport industry has been plagued with problems for more than a dozen years, with little effort toward reform. The only federal law governing the issue is designed to protect the public from inmate escapes as opposed to securing inmate safety. "Dropping people off in a timely manner is not the priority," he said. The inmates who are most often subjected to these transports are, like Kovari, pretrial detainees who are presumed innocent, Friedmann said.

Summary: It's bad enough to be detained for a crime you didn't commit; it's worse when that detention leads to nearly three weeks in the back of a van, deprived of a bathroom. The Washington Post reports a Virginia man is suing for just that, saying he was arrested in September 2016 in Winchester, Va., with a cop telling him the car he was driving had been reported stolen in Houston. He was then extradited to Texas in an 18-day trip in a private prison transport van. Edward Kovari's suit-filed against Brevard Extraditions, Prisoner Transportation Services of America, and its parent company, Prisoner Transportation Services-says he and the 15 or so others crammed in the van received small water rations and occasional fast food, that he was denied his hypertension meds, and that, due to no bathroom breaks, he spent much of the seven-state trip sitting in his own and others' waste. An editor for a prisoner legal publication tells the AP conditions like these are common in the private prison transport arena, as the goal is to pick up as many prisoners around the nation as possible for "cost efficiency." (The Marshall Project has previously investigated this "deadly world," including PTS' role.) When Kovari finally arrived in Houston, he couldn't walk and his blood pressure had spiked to above 200 (a systolic number above 130 is considered high). "The realization that this could happen to potentially anyone is frightening," one of the attorneys for Kovari, now 39, tells the Post. The charges against Kovari were eventually dismissed. His suit alleges negligence, intentional infliction of emotional distress, and a violation of his 14th Amendment rights.

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Write a title and summarize: Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2 (RTR313EEE) or L2 (IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei. Over 170 human papillomavirus (HPV) types and numerous PV types from vertebrate animals constitute the family of Papillomaviridae that contains 39 distinct genera [1]. PVs infect skin, oral and anogenital epithelia, where they either persist asymptomatically or cause neoplasia with a range of malignancies. For example, the HPV types 16 and 18 account for more than half of the cervical carcinoma cases worldwide [2,3]. Like most DNA viruses, PVs need to deliver their genome to the host cell nucleus to initiate viral transcription and replication. For this, most intranuclearly replicating viruses use the nuclear import machinery of the host cell (reviewed in [4]). As an exception, HPVs depend on mitosis for nuclear entry during which the viral DNA (vDNA) gains access to the nuclear space upon nuclear envelope breakdown (NEBD) [5,6]. In line with the restriction of PV entry into dividing cells, PVs enter the basal stem cells or transit-amplifying cells in squamous epithelia during initial infection [7,8]. PVs are non-enveloped DNA viruses with an icosahedral (T = 7d) capsid of about 55 nm in diameter. The capsid is formed by 72 pentamers of the major capsid protein L1, and contains up to 72 copies of the minor capsid protein L2 [9,10]. The encapsidated, circular, double-stranded DNA genome of about 8-kb is chromatinized with cellular histones [11]. Since the HPV life cycle and thus progeny virus production require epithelial differentiation [12], so-called pseudoviruses (PsVs) are typically used as a surrogate to study initial infection [13]. PsVs are composed of L1/L2 capsids and a pseudogenome, e. g. a plasmid encoding a reporter gene that indicates successful entry upon expression. Upon initial binding of the HPV16 particle to heparan sulphate proteoglycans at the cell surface or in the extracellular matrix, a series of extracellular conformational changes are triggered in the viral capsid [14–20]. These structural alterations appear to prime the virus capsid for engagement of an elusive secondary receptor that triggers endocytosis (discussed in [21]). HPV16,18 and 31 are endocytosed by a clathrin-, caveolin-, lipid raft-, and dynamin-independent pathway that depends on actin dynamics [22,23]. The virus traffics through endosomal compartments, where the major capsid protein L1 dissociates from a subviral complex formed by L2 and vDNA (L2/vDNA) [22,24–26]. This subviral complex is routed to the trans-Golgi network (TGN) via retrograde pathways, to eventually reach the nucleus, where it localizes to PML nuclear bodies (PML-NBs) [24,27,28]. Further, the subviral complex requires mitotic NEBD to gain access to the nuclear space [5]. However, how the subviral complex penetrates limiting membranes and is recruited to mitotic chromatin, and how it is retained in the nucleus following mitosis is not understood mechanistically. The PV minor capsid protein L2 is a multifunctional protein that exerts crucial roles during virus entry as well as during virus assembly (reviewed in [29]). The L2 protein sequence can be divided into N-terminal, central, and C-terminal domains according to their functions. For assembly of progeny virions, newly synthesized L2 is imported into and retained within the nucleus via its N- and C-terminal nuclear localization signals (NLSs) and a central arginine-rich nuclear retention signal (NRS), respectively [30,31]. In the nucleus, L2 likely packages vDNA into assembling capsids through its N- and C-terminal DNA-binding sites, and through interaction of its C-terminal domain with L1 in the nucleus [32–35]. During PV entry, L2 is important for a number of steps that guide the vDNA to the site of replication. Exposure of the L2 N-terminus to the capsid surface through interaction with cyclophilins and cleavage by extracellular furin are required for infectious cell entry to potentially facilitate the interaction with the elusive secondary receptor [14,19,21]. Furthermore, L2 is implicated in vesicular trafficking since domains in the central part of L2 interact with the cytosolic regulators of endosomal recycling SNX17 and SNX27 [36,37]. L2 is also required for retrograde trafficking from early endosomes to the TGN through direct interaction of the L2 C-terminal part with the retromer [38]. L2 would need to be cytosolically exposed to interact with these cellular sorting factors. It remains unknown exactly when and how L2 spans across or penetrates the membranes of vesicular compartments to interact with the cytosolic cellular interaction partners. The N-terminal transmembrane (TM) domain and C-terminal membrane-destabilizing peptide have been implicated in translocation across membranes originally presumed to occur within endosomal compartments [39,40]. Recently, it has been shown that L2 is capable of using the N-terminal TM domain to span across intracellular membranes during post-entry subcellular trafficking [41]. The orientation of L2 within membranes appears to be consistent with a type I topology. In this model, some or all of the L2 protein C-terminal of the TM domain would be cytosolic, whereas a small N-terminal portion would be luminal. This would place the SNX and retromer binding sites of L2 within the cytosol, explaining how L2 could recruit these cytosolic trafficking factors to facilitate endosomal and retrograde sorting of the vDNA. It has been proposed that the vDNA remains within a vesicular compartment prior to and after passage through the TGN, where the vesicular vDNA would be tethered to mitotic chromosomes until the nascent daughter nuclei would have been formed [42]. In this model, it has been suggested that L2 commandeers microtubule and spindle transport systems to achieve localization to mitotic chromosomes. As the C-terminus of L2 is also capable of binding to the microtubular motor protein dynein, vesicular or free L2/vDNA may utilize microtubules to traverse the cytoplasm to facilitate TGN localization and achieve localization at the microtubule organizing center prior to nuclear entry [43,44]. Upon entry into the nucleus, the C-terminal PML-NB localization domain and the central SUMO interacting motif of L2 may target L2/vDNA to PML-NBs, where L2 could facilitate the early transcription and replication events [28,45,46]. Given that L2 binds vDNA to escort it into the nucleus, and because L2 and vDNA associate with chromosomes during mitosis, we previously proposed that L2 tethers vDNA to mitotic chromosomes upon NEBD to facilitate the nuclear delivery of the PV genome during cell division [5,27,33]. In this study, we used quantitative fluorescence microscopy to identify the required and/or sufficient domains in L2 that mediate chromosomal association. When N-and/or C-terminally truncated HPV16 L2 proteins were tested for their association with mitotic chromosomes, a conserved, central region of 147 amino acids (residues 188–334) in HPV16 L2 conferred binding to chromosomes. Site-directed mutagenesis conserved residues within this newly defined region of 147 amino acids identified several chromosome-binding deficient L2 mutants. Importantly, PsVs containing chromosome-binding deficient L2 mutants were unable to deliver the vDNA to the nucleus. Interestingly, these mutants were also unable to exit vesicular compartments as determined by a novel L2-BirA translocation assay described in detail in [47]. Thus, our overall data indicated a nuclear entry strategy common to various tested PVs, in which a central conserved region in L2 mediated tethering of the vDNA to mitotic chromosomes thereby directing viral genomes to the nascent nuclei during cell division. Further, we propose that the ability to bind mitotic chromatin is functionally linked to translocation of the L2/vDNA complex across a post-Golgi limiting membrane. Our previous work using infection of HPV16 PsVs containing EdU-labeled vDNA (EdU-HPV16) showed that incoming vDNA associates increasingly with condensed, mitotic chromosomes until metaphase, whereupon the degree of association remained constant [5]. As a reference for the metaphase localization of EdU-labeled DNA in EdU-HPV16 infected HeLa and HaCaT cells, please refer to Fig 1A. Live-cell imaging of L2-EGFP-expressing cells showed that HPV16 L2-EGFP localizes dynamically within the cell during cell division [5]. L2 is nuclear during interphase, disperses throughout the cell upon NEBD, and associates with chromosomes in metaphase. Thus, we proposed that L2 tethers the viral genome to mitotic chromosomes upon NEBD. While live-cell imaging provides an excellent dynamic resolution of events, its primary caveat is a low spatial resolution. For a higher spatial resolution, the localization of HPV16 L2-EGFP was examined in transfected HeLa cells using laser scanning confocal microscopy (Fig 1C). After nuclear localization in interphase and prophase (Fig 1C, images i, ii), HPV16 L2-EGFP first associated with chromosomes during prometaphase (Fig 1C, image iii), and remained associated throughout metaphase, anaphase and telophase (Fig 1C, images v-vii). L2-EGFP association also occurred in HaCaT keratinocytes, or when L2 was tagged with a short HA epitope instead of EGFP suggesting that recruitment to mitotic chromatin is cell-type- and EGFP tag-independent (S1A and S1B Fig). Similar to the vDNA, L2 from incoming HPV16 PsV localized to metaphase chromosomes indicating that the L2-EGFP association with mitotic chromosomes in transfected cells phenocopies the recruitment during infection (Fig 1B). The chromosomal recruitment of L2 during a specific stage of mitosis suggests that L2 may interact with a cellular protein that itself is specifically recruited to prometaphase chromosomes, rather than a direct L2 engagement of histones or cellular DNA. Based on the low spatially resolved live-cell imaging data, we had previously concluded that HPV16 L2 is recruited to mitotic chromosomes during metaphase [5]. However, the high-resolution microscopy data clearly indicates that recruitment of L2 occurs specifically during prometaphase, and does not require establishment of the metaphase plate. Furthermore, the accompanying paper by Calton et al. demonstrates that during HPV16 PsV infection of synchronized cells, EdU-labeled L2/vDNA complex begins to associate with mitotic chromosomes specifically during prometaphase, and essentially all of the incoming L2/vDNA is chromosomally associated by metaphase, confirming our previous observations [5,47]. Importantly, this suggests that transfected L2-EGFP associates with mitotic chromosomes using the same mechanisms as incoming vDNA-associated L2 during PsV infection. To verify recruitment of L2 to prometaphase chromosomes, L2-EGFP expressing cell populations were enriched in prometaphase using nocodazole. Nocodazole is a microtubule-depolymerizing drug that prevents the formation of the mitotic spindle, and therefore congression of mitotic chromosomes to the metaphase plate [48]. Thus, HeLa cells that stably expressed the histone marker H2B-mCherry (HeLa H2B-mCherry) were transfected with an L2-EGFP expression plasmid, and subsequently treated overnight with nocodazole prior to fixation and microscopy (S2A Fig). While only a small fraction of cells was in mitosis in the absence of nocodazole (S2B Fig, left, arrowheads), cells were efficiently enriched in prometaphase in the presence of the drug (S2B Fig, right). In these prometaphase-arrested cells, L2-EGFP associated with the chromosomes, whereas EGFP alone failed to accumulate on chromatin (S2C and S2D Fig). These results further validate that L2 is recruited to mitotic chromatin during prometaphase. Moreover, enriching cells in prometaphase allows for a steady-state analysis of mitotic chromosomal association of L2-EGFP in statistically significant numbers of cells. L2 is multifunctional, facilitating several steps of the viral life cycle in concert with certain host factors: extracellular structural modifications of the capsid prior to infectious cell entry (furin cleavage, cyclophilin B binding domain, annexin A2 binding [19,25,49]), vesicular trafficking and endosome escape (cyclophilin B, sorting nexin 17, sorting nexin 27, retromer, syntaxin 18 binding domains, TM domain, membrane-destabilizing peptide, NRS [25,26,37–40,50–53]), cytosolic transport (dynein, beta-actin [43,44,54]), and particle assembly (NLSs, nuclear export signal, DNA-binding, L1-binding, PML nuclear body-localization, SUMO-interaction domain [28,30,31,33,45]). Thereby, most of the known functional domains cluster towards the termini of the PV L2 protein, and only a few are located in the centre (Fig 2A, [29]). To identify the potential chromosome-binding site (s) in HPV16 L2, chromosomal association of L2-EGFP truncation mutants was quantified in prometaphase-arrested cells. As an unbiased and quantitative measure, the chromosomal association index (CAI) was developed. Instead of using a threshold-based algorithm like for example colocalization analysis, the CAI uses non-arbitrary intensity based measurements. It is defined as mean fluorescence intensity ratio of chromosome-associated over cytoplasmic L2-EGFP. To correct for artificial background, these values were normalized to control EGFP-expressing cells. First, HPV16 L2 was sequentially truncated from the N-terminus (Fig 2B). L2 (13–473) -EGFP and L2 (141–473) -EGFP, lacking all the N-terminal functional domains, visually associated with mitotic chromosomes similar to full-length L2-EGFP (Fig 2C, S3A Fig). The CAI analysis of transfected cells revealed that chromosomal association of L2 (13–473) -EGFP and L2 (141–473) -EGFP was decreased by a small but significant amount (Fig 2D), which suggests that N-terminal residues up to amino acid (aa) 140 may partially stabilize chromosomal association. Remarkably, chromosomal association is completely abolished upon deletion of further N-terminal residues with unknown function: L2 (221–473) -EGFP and L2 (356–473) -EGFP were excluded from the chromosomes exhibiting CAIs similar to the non-associating EGFP (Fig 2C and 2D). Thus, residues in between aa 141 and 220 are crucial for mitotic chromosome binding, whereas the functionally described N-terminal domains are dispensable. Next, L2 C-terminal deletions were tested (Fig 2E). L2 (1–443) -EGFP lacking the C-terminal DNA-binding domain, NLS, nuclear export signal, dynein-interacting domain, and membrane-destabilizing peptide [30,31,33,40,43], and L2 (1–350) -EGFP additionally lacking the PML localization, L1 and retromer interaction domains [28,32,34], clearly associated with mitotic chromosomes (Fig 2F and 2G; S3B Fig), suggesting that the C-terminal functional domains are dispensable for chromatin association. Interestingly, sequential C-terminal truncations up to aa 334 as in L2 (1–443) -EGFP, L2 (1–350) -EGFP, and L2 (1–334) -EGFP resulted in increased association, reflected by median CAIs up to 60% higher than that of full-length L2-EGFP (Fig 2F and 2G, S3B Fig). Thus, the C-terminal functional domains are partially inhibitory for chromosomal association. This may indicate that L2 interactions with, for example, retromer or dynein, need to be reversed for efficient chromosomal association during initial infection and subcellular trafficking of L2/vDNA. Notably, chromosomal association was completely lost in truncations lacking 155 or more amino acids from the C-terminus such as in L2 (1–318) -EGFP, L2 (1–220) -EGFP and L2 (1–140) -EGFP (Fig 2F and 2G, S3B Fig), suggesting a crucial role for aa 319 to 334 in mitotic chromosomal association of L2. In summary, the C-terminal functional domains are dispensable for chromosomal association similar to the N-terminal domains. To further exclude that the N- and C-terminal domains compensate each other for mitotic chromosome recruitment, bilaterally truncated L2-EGFP mutants were tested (Fig 3A). Since, for example, L2 (141–355) -EGFP was fully capable to bind to mitotic chromosomes, it is unlikely that compensation occurs (Fig 3B and 3C, S3C Fig). The minimal peptide that binds to chromosomes was L2 (188–334) -EGFP (Fig 3B and 3C). Further truncation of this central L2 fragment from either end abrogated chromosomal association, as evidenced by L2 (188–318) -EGFP and L2 (221–334) -EGFP (Fig 3B and 3C). The requirement for residues directly upstream of aa 221, and residues between aa 319 and 334 correlated with the abolished chromosome-binding of L2 (221–473) -EGFP and L2 (1–318) -EGFP (Figs 2,3 and S3). Since the central region of 147 amino acids (aa 188 to 334) is necessary and sufficient to mediate association of HPV16 L2 with mitotic chromosomes, we designate it the chromosome-binding region (CBR). To assess the functional conservation of L2-chromosome interactions among PVs of different genera, L2 association assays were carried out for HPV18 (another mucosal high-risk alpha-PV), HPV5 (cutaneous low-risk beta-PV), BPV1 (bovine delta-PV), and MnPV (Mastomys natalensis iota-PV). The L2 sequences of HPV16 and HPV18 are evolutionary most closely related, followed by HPV5, MnPV and BPV1 (Fig 4A). L2-EGFP of all tested PVs associated with prometaphase chromosomes, albeit to varying extents (Fig 4B and 4C). L2-EGFP of HPV5, BPV1 and MnPV were distributed evenly over the mitotic chromosomes similar to HPV16 (Fig 4B). In contrast, HPV18 L2-EGFP localized to the peripheral rim of chromosomes (Fig 4B), indicating that HPV18 L2 may be recruited in a different mode or to a different extent. Quantitatively, HPV18 L2-EGFP associated considerably less with chromosomes than HPV16 L2-EGFP (Fig 4B and 4C). In contrast, L2-EGFP of HPV5, and the animal PVs had substantially higher CAIs than HPV16 (Fig 4B and 4C). Hence, certain L2s may engage a common cellular recruiting factor with different affinities, or engage different cellular factors for recruitment to mitotic chromatin. Overall, the tested PV L2s were all recruited to mitotic chromatin suggesting a similar tethering strategy. A comparison of the CBR of the analyzed PV types revealed that it contains two parts, a variable N-terminal region from aa 188 to 301 that contains eight conserved residues (aa 251 to 258), and a highly conserved C-terminal region from aa 302 to 334, overlapping with the previously defined NRS (Fig 5A). The different degrees and patterns of association are likely the result of the variable regions within the CBR. To further elucidate the role of the CBR, we focused on conserved residues within L2 assuming that conservation likely correlates with functional importance. Directly upstream of, or within the highly conserved NRS, the CBR contains residues of several previously described mutations (Fig 5B). Upstream of the NRS, the SUMO-interacting motif IVAL286 is required for efficient HPV infection and important for intranuclear targeting of the incoming vDNA to PML-NBs [45]. Glutamic acid substitutions of critical arginine residues within the NRS, as e. g. RR297 and RTR313, reduce the fraction of exogenously expressed L2 in the nucleus despite the presence of both NLSs [31]. Further alanine-substitution studies of residues RR302/5 revealed that the NRS is essential for infection [26,42]. Interestingly, these studies from the Sapp laboratory variably attribute the function to different entry steps, i. e. traversal of the TGN or nuclear import [26,42]. To directly test whether these residues would be crucial for recruitment of L2 to mitotic chromosomes, respective mutations were introduced into HPV16 L2-EGFP by site-directed mutagenesis, and tested for chromosomal association (Fig 5B). Of those mutants, only L2 (RR297EE) -EGFP associated with mitotic chromosomes although to a lesser extent compared to wild-type L2-EGFP (Fig 5C and 5D). In contrast, L2 (IVAL286AAAA) -EGFP, L2 (RR302/5AA) -EGFP, and L2 (RTR313EEE) -EGFP are unable to bind mitotic chromatin (Fig 5C and 5D). For ease of experimentation, HeLa H2B-mCherry cells were used for most of our assays. For confirmation, association was also tested for L2 (IVAL286AAAA) -EGFP and L2 (RTR313EEE) -EGFP in HaCaT keratinocytes. Similar to HeLa cells, association was abrogated for these mutants in HaCaT cells (S1B and S1C Fig). Thus, L2 residues IVAL286, RR302/5, and RTR313 are involved in L2 association with mitotic chromosomes, and likely play a role in viral genome tethering to mitotic chromatin for nuclear entry. To verify the role of these mutations under more physiological conditions, HPV16 PsVs harbouring L2 (RTR313EEE) and L2 (IVAL286AAAA) were tested in infection studies. As a structural component of the virus capsid, L2 is involved in virion assembly as well as virus entry (reviewed in [29]). Since virions containing L2 (IVAL286AAAA) are known to be assembly-competent [45], we tested whether mutant HPV16 PsVs harbouring L2 (RTR313EEE) would also assemble properly. Morphologic analysis of HPV16-L2 (RTR313EEE) PsVs by negative stain EM revealed no detectable differences between wild-type and mutant HPV16 particles (S4A Fig). In addition, wildtype and mutant L2 proteins, and the EGFP-reporter pseudogenome were incorporated into virus particles with comparable efficiency as shown by densitometric analysis of Coomassie-stained SDS-PAGE gels of PsVs (S4B Fig). Thus, the mutant HPV16-PsVs assemble without considerable defects. Next, we assessed whether incorporation of mutant L2 into virions would affect infectious entry. For this, HeLa or HaCaT cells were infected with HPV16, HPV16-L2 (RTR313EEE), and HPV16-L2 (IVAL286AAAA) at low and high multiplicities. In line with published results [45], incorporation of L2 (IVAL286AAAA) severely reduced infectivity of the PsVs in both HeLa and HaCaT cells (Fig 6A), however, some infectivity of this mutant was retained at the higher MOI (Fig 6A). HPV16-L2 (RTR313EEE) PsVs were non-infectious in both cell types (Fig 6A and 6B). While both motifs are important for infectivity, they differ in their ability to perturb virus entry and possibly virus tethering. Interestingly, chromosomal association was similarly abrogated for both mutants, suggesting that this assay is extremely sensitive. Next, the subcellular localization of vDNA upon infection was analyzed to test whether mutant PsVs failed to deliver vDNA to the nucleus. Intensity-based colocalization analysis showed that at 20 hours post infection (h p. i.) EdU-HPV16 had successfully delivered 60%±10% and 40±12% of vDNA to HeLa and HaCaT nuclei, respectively (Fig 6B, 6C, 6E and 6F). In contrast, intranuclear localization of vDNA was reduced or absent, when cells were infected with EdU-HPV16-L2 (IVAL286AAAA) or EdU-HPV16-L2 (RTR313EEE) PsVs, respectively (Fig 6B, 6C, 6E and 6F). Consistent with a defect in viral genome tethering, both mutants exhibited defects to direct vDNA to nuclei of infected host cells. Instead, the vDNA of mutant virions accumulated in perinuclear clusters, reminiscent of TGN localization. Similar to the infection studies, the L2 (RTR313EEE) mutant PsV exhibited stronger defects than L2 (IVAL286AAAA). To verify these results, the localization of L2 was analyzed by antibody staining. Similar to the vDNA, L2 failed to localize to host cell nuclei after HPV16-L2 (RTR313EEE) infection (Fig 7A and 7B). The perinuclear accumulation of vDNA suggested that the mutant L2 proteins were able to direct pseudogenomes to the TGN. Accordingly, vDNA of EdU-HPV16 and EdU-HPV16-L2 (RTR313EEE) were detectable in the TGN (Fig 6B and 6D). In fact, the mutation caused increased accumulation of vDNA at the TGN (Fig 6B and 6D), similar to the “Golgi retention” phenotype previously observed with EdU-labeled RR302/5AA mutant PsV [26]. As a control, cells were treated with aphidicolin, which arrests cells in interphase, and inhibits infection by blocking nuclear entry of wild-type HPV16 [5,6]. As expected, vDNA of EdU-HPV16 was prevented from entering the nucleus under these conditions and accumulated in the TGN (Fig 6B and 6D). Thus, the mutants were able to traffic vDNA to the TGN but failed to deliver vDNA to the nucleus, instead resulting in vDNA accumulation at the TGN, presumably due to a defect in tethering to mitotic chromosomes for nuclear import. In support, association of vDNA with metaphase chromosomes of mitotic HeLa and HaCaT cells was substantially impaired for EdU-HPV16-L2 (IVAL286AAAA) and virtually absent for EdU-HPV16-L2 (RTR313EEE) (Fig 8A and 8B). Similarly, L2 (RTR313EEE) did not localize to metaphase chromosomes after infection of HeLa cells, whereas wild-type L2 accumulated on mitotic chromatin (Fig 7C and 7D). The Golgi retention phenotype and defective nuclear entry shared between the defective CBR mutants L2 (IVAL286AAAA), L2 (RTR313AAA), and L2 (RR302/5AA) (the latter reported in [26]) prompted us to determine whether these mutants could translocate across limiting membranes during infection. The accompanying report by Calton and colleagues [47] describes a novel system for measuring L2 membrane penetration. In brief, the assay utilizes PsVs that encapsidate L2, which is C-terminally fused to the BirA biotin ligase from E. coli. BirA catalyzes the covalent addition of free biotin to a specific 15-residue biotin acceptor peptide (BAP) substrate [55]. L2-BirA PsVs are used to infect a HaCaT cell clone that stably expresses a cytosolic GFP-BAP fusion [47]. Only upon translocation of the C-terminus of L2-BirA across limiting membranes can the BirA moiety encounter and biotinylate the GFP-BAP substrate. Thus, the degree of biotinylation of GFP-BAP is a proxy for the efficiency of translocation of the L2 C-terminus across the limiting membrane of vesicular compartments [47]. Using this system, Calton et al. observed that translocation is dependent upon the cell cycle, with a strict requirement for entry into mitosis. Furthermore the timing of translocation in synchronized cells was coincident with or just subsequent to mitotic onset [47]. Using the L2-BirA system, Calton et al. show that the L2 (RR302/5AA) mutant, which is defective for chromatin binding (Fig 5), was impaired for translocation of the L2 C-terminus across the limiting membrane. Thus, we tested whether L2 (RTR313EEE) and L2 (IVAL286AAAA) would also be unable to penetrate. HaCaT GFP-BAP cells were infected with HPV16 L2-BirA PsVs containing L2 (RTR313EEE), L2 (IVAL286AAAA), or, as control, L2 (RR302/5AA). In comparison to the wild-type particles, the mutant L2-BirA PsVs were noninfectious and unable biotinylate GFP-BAP, indicating that the L2 region important for binding mitotic chromosomes is also required for translocation of the L2 C-terminus into the cytoplasm (Fig 8C and 8D). Overall, this study further validates the model of PV nuclear entry, in which L2 tethers the viral genome to mitotic chromosomes upon NEBD in order to direct it to the nascent nuclei during cell division. Moreover, a central region in the HPV16 minor capsid protein L2 from aa 188 to 334 was identified as sufficient and required for association of L2 with mitotic chromosomes. In particular, we characterized the chromosome-binding deficient mutant L2 (RTR313EEE) that rendered mutant PsVs non-infectious. HPV16-L2 (RTR313EEE) PsVs were unable to deliver the vDNA to the nucleus and were impaired for C-terminal translocation across the membranes of the vesicular compartment. Taken together, these results supported a new model in which L2 chromatin binding and translocation are interdependent processes. Finally, we provide evidence that Papillomaviridae across different genera share a common L2-mediated nuclear entry strategy by tethering vDNA to mitotic chromosomes. Our recent work demonstrated that nuclear delivery of HPV16 genomes requires NEBD during mitosis, and it provided first evidence for the existence of a tethering mechanism of the viral genome to mitotic chromatin by the minor capsid protein L2 [5]. In this study, we demonstrated that a conserved, central CBR encompassing amino acid residues 188 to 334 of HPV16 L2 mediates recruitment to mitotic chromosomes specifically during prometaphase. By tethering vDNA to mitotic chromosomes, L2 facilitates nuclear delivery of incoming vDNA and infection of both daughter cells. Our data also show that conserved residues within the CBR are also critical for membrane penetration of the L2 C-terminus, suggesting that these two processes are functionally linked. To ensure inclusion into the nascent daughter nuclei after cell division, the vDNA and L2 interact with host mitotic chromatin. This interaction is consistent with L2 acting as a viral tethering factor. A tethering function implies that one part of the protein holds on to vDNA, whereas another part interacts with host chromatin [56]. In PV L2, both the N- and C-terminal polybasic regions of L2 are capable of binding to DNA in vitro [33,57,58] and these linear DNA-binding domains may bind the vDNA during assembly of the virions [33,57–59]. Prior work with purified full length and truncated BPV1 L2 suggests that in particular the C-terminal domain mediates direct binding to DNA [58]. Furthermore, for incoming virions, the N-terminal DNA-binding domain of L2 is cleaved off by furin to mediate localization of L2/vDNA to the TGN [19] [24]. Thus, the vDNA is likely attached to the L2 C-terminal DNA-binding domain upon virus entry and it is unlikely that any of these terminal DNA-binding domains would be available for host chromatin binding. Moreover, our results demonstrate that mitotic chromosomal association of HPV16 L2 is independent of both N-terminal and C-terminal DNA-binding domains, and that the central CBR in HPV16 L2 was sufficient and necessary for chromosomal association. Hence, the use of two different sites in L2 would clearly allow simultaneous binding to mitotic chromatin and the vDNA, the prerequisite for a tethering factor. Two distant sites within the CBR, L2 (189–220) and L2 (319–334), are alone insufficient but indispensable for chromosomal association. Both sites may be necessary to form an interface that binds host mitotic chromosomes upon folding. Since L2 is for the most part, predicted to be an intrinsically disordered protein (DisEMBL, [60]), it is likely that these sites act as linear motifs independent of protein secondary and tertiary structures. In support, computational analysis predicted that both sites contain disordered loops with high mobility, which may fold upon binding to the corresponding cellular partner. Alternatively, L2 may function as a scaffold, in which each of these sites interacts with a different cellular chromosomal target, thereby stabilizing the association with chromatin. However, a simple stabilization appears less likely, as deletion of both L2 sites abrogates mitotic chromatin recruitment. Nevertheless, the potential for multivalent interactions to tether vDNA to chromatin is not without precedent: EBNA1 and LANA1 of Epstein Barr virus and Kaposi’s sarcoma associated herpesvirus, respectively, both use two N- and C-terminal chromosome-binding sites to tether the viral episome to chromosomes for maintenance (reviewed in [56]). The N-terminal chromosome-binding domain of LANA1 interacts with histones H2A-H2B at the nucleosomal surface and modulates the C-terminal interaction with chromosomally associated methyl-CpG-binding protein 2 [61,62]. Interestingly, the C-terminal part (aa 302–334) of the central CBR of HPV16 L2 is highly conserved among PVs, and HPV16 L2 residues 312 to 322 exhibit high conservation with residues 6 to 16 of the N-terminal chromosome-binding domain of LANA1 [63]. While this might point to a putative interaction of HPV16 L2 with histones, such an interaction appears unlikely, as exogenously expressed L2 is recruited specifically and dynamically to mitotic chromosomes and does not interact with interphase and prophase chromatin. The prometaphase-dependent chromosomal association also argues against use of the L2 terminal DNA-binding domains to attach to host chromosomes, as these interactions are non-sequence specific and would be expected to occur throughout the cell cycle rather than at a specific stage of mitosis. In order to engage a cellular factor for tethering during mitosis, the L2 CBR has to be cytosolic. Despite the growing knowledge of molecular players involved in PV intracellular trafficking, it still remains controversial when and how L2/vDNA penetrates and egresses from membrane-bound organelles. L2 contains an N-terminal TM domain and a C-terminal membrane-destabilizing peptide that are essential for PV infection [39,40]. Precisely how these domains insert into the membrane remains unknown, but previous work suggests that L2 may adopt a type-I transmembrane topology during infection, whereby residues upstream of the TM domain are luminal and residues immediately downstream are cytosolic [41]. It should be noted, however, that the topology of the extreme C-terminus was never determined in this prior work. The cytosolic exposure of residues downstream of the TM domain, including the central CBR, likely occurs within endosomal compartments because this portion of L2 contains motifs that are involved in endosomal sorting and trafficking of L2/vDNA to the TGN through interaction with cytosolic SNX17, SNX27, and the retromer [37,38,50]. Interestingly, the accompanying report from the Campos group provides indirect evidence that the extreme C-terminus of L2 together with the vDNA remains luminal within endosomes [47]. This may point to a topology with two membrane-spanning domains, a previously described N-terminally located domain, and a yet elusive C-terminally located domain. Thus, both termini of L2 would be luminal, and the CBR would be cytosolic in this model. The previously described C-terminal membrane destabilizing peptide could be a candidate for this additional membrane spanning domain, but this region does not have the biochemical characteristics of a TM domain [40]. Alternatively, it has been proposed that multiple L2 molecules may oligomerize within the membrane to form a pore, because the N-terminal TM domain of L2 can self-associate within biological membranes [39]. A pore-like structure built from the N-terminal TM domain might allow passage of the L2 C-terminus across the membrane without the assistance of another TM domain. Clearly, further work is necessary to determine the unique topology of L2 as it exists in this membrane-spanning state. In any case, all of these models would suggest that the L2 CBR is accessible for interaction with cytosolic cellular interaction partners. Further, the CBR is located in between the SNX17 binding site at residues 254–257 and the retromer binding sites near the C-terminus. That both of these regions become exposed to the cytosol to engage their respective sorting molecules during infection strongly suggests that the CBR does as well. Point mutational analyses of the highly conserved NRS, which is located within the identified CBR, further strengthened the potential role of this region in delivering vDNA to the nucleus by tethering the viral genome to mitotic chromosomes. The L2 (RTR313EEE) mutation of mostly highly conserved residues has previously been reported to impair nuclear retention of exogenously expressed L2 [31]. Here, we show that this mutation abolished binding of L2 to mitotic chromatin, and rendered the assembly-competent HPV16 particles non-infectious. HPV16-L2 (RTR313EEE) PsVs trafficked to the TGN but failed to reach the nucleus. Moreover, the mutant L2 was unable to tether vDNA to chromosomes during mitosis, which could explain the defect in nuclear entry. Our analysis of the previously described L2 (RR302/5AA) mutation of conserved residues in the NRS in our chromosomal association assay revealed that this mutant was also unable to bind to mitotic chromosomes. Similar to our findings with HPV16-L2 (RTR313EEE) PsVs, recent reports indicate that HPV16-L2 (RR302/5AA) PsVs traffic to the TGN but fail to reach the nucleus [26,42]. Interestingly, the L2 (RTR313EEE) and the L2 (RR302/5AA) mutations blocked translocation of the L2 C-terminus across the limiting membrane as well as L2 association with mitotic chromatin (Figs 5 and 8; [47]). Together these data strongly suggest that translocation and tethering to mitotic chromosomes are functionally linked. In support of this hypothesis, L2 translocation coincides with mitosis, and is inhibited if the cell cycle is arrested prior to M-phase [47]. Moreover, since the L2 (RR302/5AA) and L2 (RTR313EEE) mutations impair both the membrane translocation of the L2 C-terminus and the recruitment to mitotic chromatin[47], the interaction with a cellular tethering factor may additionally act as a translocation factor, functionally coupling L2 chromatin binding to membrane translocation of the L2/vDNA. If this is the case, the L2 (RR302/5AA) and L2 (RTR313EEE) mutations are likely located within the interface for an interaction with a cellular partner. In addition to the mutations within the NRS, mutation of the less conserved SUMO interacting motif (IVAL286AAA) of HPV16 L2 adjacent to the NRS impaired mitotic chromatin association and membrane penetration. Interestingly, this mutation was only partially defective for nuclear import in infection studies in line with previous work [45], which may suggest that the conserved C-terminal part of the NRS contains the more crucial part of the interface for an interaction with a cellular partner. Alternatively, the mutations may have varying structural relevance by affecting the folding dynamics upon engagement of cellular targets at different sites of L2. It has been suggested that L2 (RR302/5AA) mutation may disrupt interaction of L2 with factors mediating plus end-directed transport along microtubules towards condensed chromosomes [42]. Our data show that recruitment of PV L2 to mitotic chromosomes can occur independently of microtubules, because L2 associates with prometaphase chromosomes in the presence of the microtubule-depolymerizing drug nocodazole. While this data does not exclude the possibility that kinesin-mediated transport occurs and contributes to the efficiency of chromosomal association during infection, L2 recruitment to mitotic chromatin is clearly efficient in the absence of kinesin-mediated microtubule transport. Our work strongly indicates a common mechanism among PVs in which L2 tethers the vDNA to mitotic chromosomes for nuclear entry during cell division. For the first time, our results demonstrate that L2 proteins from three different host species (human, bovine, rodent) and four PV genera (alpha, beta, delta, iota) are recruited to mitotic chromosomes. Interestingly, differences in the pattern and extent of association between the different PV L2 proteins may indicate either differential affinities to the same cellular interaction partners or the engagement of different tethering factors. It is tempting to speculate about the latter, as this feature would not be unprecedented even among PVs. The PV E2 protein mediates genome maintenance during persistent PV infection in dividing cells by tethering the episomal genome to host chromatin (reviewed in [64]). While the N-terminus of the E2 protein of BPV1 interacts with bromodomain-containing protein 4 (Brd4) on mitotic chromosomes, HPV8 E2 binds pericentromeric regions, independently of Brd4, via its hinge region and C-terminal domain [65,66]. If L2 proteins of different PVs would interact with different cellular factors, one would expect that the interaction would be primarily defined with less conserved residues of the CBR, as found in the N-terminal portion. If all PV L2 proteins would employ the same cellular chromosomal factors, the conserved residues of the L2 CBR may be responsible for the principle interaction, whereas the less conserved flanking regions may be important to modulate the affinities of interaction. Here, the less conserved L2 residues 189–220 or the less conserved SUMO-interacting motif IVAL286 may be important. The fact that infectivity and nuclear import were less affected for L2 IVAL286AAAA than RTR313EEE virions support an accessory rather than essential role for the SUMO-interacting motif. Unfortunately, we are precluded from further experimental validation of these hypotheses as long as the cellular interaction partners of L2 for chromosomal association remain elusive. With this study, we propose a unified model for all PVs, where membrane translocation and nuclear entry of the L2/vDNA complex occur simultaneously in dividing cells by tethering of vDNA to mitotic chromosomes through L2 upon NEBD. This complex would remain associated with chromosomes throughout mitosis, and is thus directed into the nascent nuclei during cell division. L2 and the vDNA are recruited to prometaphase chromosomes through the interaction of a dedicated L2 CBR (residues 188–334 for HPV16 L2) with a mitosis-specific chromosomal cellular protein. The affinity of L2 to a common cellular protein may be PV type dependent, or different PV may engage different cellular targets. After mitosis, the subviral complex would have to be released from the chromosomes either upon dissociation of the cellular tethering factor from chromosomes, or upon high affinity binding to another nuclear factor that would direct the viral genome, for example, to PML-NBs [45,46,67]. In line with the latter possibility of regulated interactions, mitotic chromosomal association of HPV16 L2-EGFP was increased in the absence of, for example, the C-terminal PML-NB localization domain (Fig 2). The identification of the CBR of L2 for tethering of vDNA to mitotic chromatin now allows a more targeted search for a cellular recruiting factor. Moreover, the observed differences between PV types in their binding pattern to mitotic chromatin prompt future studies on the functional significance of these differences and on how they occur. Importantly, our understanding of the dynamic interactions of the L2/vDNA complex has been inferred from steady state images of incoming virions and of live cell imaging from exogenously expressed L2. However, to fully understand the mechanistic dynamics of these processes, live cell imaging will be required. Since live cell imaging of this subviral complex is currently technically impossible, future efforts will aim at developing methods that allow dynamic analysis of incoming L2/vDNA. HeLa cells were from ATCC. HeLa H2B-mCherry cells were a gift of D. Gerlich, Vienna [68]. Cells were maintained in DMEM (Sigma Aldrich) supplemented with 10% fetal calf serum (Merck Millipore). Medium was supplemented with 500 mg/ml G418 for HeLa H2B-mCherry cells. HaCaT NES-GFP-BAP cells were generated from HaCaT cells [69] and cultured as described in the accompanying report [47]. HPV16 L2 deletion mutants were constructed either by inverse PCR (L2 (13–473), L2 (1–220), L2 (1–350), L2 (1–140), L2 (356–473) ) or conventional cloning. For the inverse PCR, deletion was achieved by using primer pairs flanking the deleted region for outward PCR amplification from the HPV16 L2-EGFP plasmid (EF1alpha promoter-driven; generated by Chris Buck, provided by Martin Müller). The amplified products were digested with DpnI at 37°C for 1 h to remove the parental template before 5’-ends were phosphorylated and then self-ligated either at RT for 4 h or at 16°C overnight. In the conventional cloning approach, other HPV16 L2 deletion mutants were constructed by replacing the L2 gene in the HPV16 L2-EGFP plasmid between NotI and NheI sites with the coding sequence for the respective amino acid region. The insert was prepared in two steps: First, a DNA fragment was amplified from the HPV16 L2-EGFP plasmid with a forward Kozak consensus sequence-containing primer and a reverse NheI site-containing primer annealing to the desired N-terminal and C-terminal amino acid positions, respectively. Second, the obtained product was extended at the 5’-end by a NotI site-containing primer in a PCR with the previous reverse primer before digestion by NotI and NheI. The coding sequence for L2 of HPV5 was amplified from p5sheLL [70] and cloned into NotI/NheI-cleaved HPV16 L2-EGFP as above. To construct HPV16 L2 (269–334) -EGFP, the DNA fragment comprising the Kozak consensus sequence, the coding region for amino acids 269–334 and additional linker residues SGG was released by NotI/NheI digestion from a plasmid containing the synthetically produced insert (Eurofins). The DNA fragment was cloned into NotI/NheI-cleaved HPV16 L2-EGFP plasmid as described above. To clone the coding sequence for L2 of HPV18 and MnPV into NotI/NheI-cleaved HPV16 L2-EGFP plasmid, internal NheI and NotI restriction sites in the respective L2 DNA sequences were mutated in the template plasmids HPV18L2h [71] and pJET-MnPVL2hum (provided by Frank Rösl, DKFZ) by site-directed mutagenesis before conventional cloning was performed as described above. To generate BPV1 L2-EGFP, the L2 gene of BPV1 was amplified from pSheLL [72] with forward and reverse primers carrying KpnI and XmaI sites, respectively, and cloned into pEGFP-N3 (CMV promoter-driven) between KpnI and XmaI sites. All point mutations were introduced with mutagenic primers by using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions. The HPV16 L2-EGFP plasmid was used as template for generating the point mutants L2 (IVAL286AAAA) -EGFP, L2 (RR297EE) -EGFP, L2 (RR302/5AA) -EGFP and L2 (RTR313EEE) -EGFP. p16SheLL [70] was used as template for introducing the point mutations encoding L2 (RTR313EEE) and L2 (IVAL286AAAA). The sequences of primers that were employed in PCRs are listed in S1–S3 Tables. Sequences of obtained expression constructs were verified by Sanger sequencing. Preparations of HPV16 PsVs containing GFP reporter plasmid or 5-ethynyl-2’-deoxyuridine (EdU) -labeled- DNA were performed as previously described using the plasmids p16SheLL and pCIneo-GFP [73,74]. HPV16-L2 (RTR313EEE) and HPV16-L2 (IVAL286AAAA) PsVs were prepared like wild-type HPV16 PsVs but using p16SheLL mutated in the L2 gene. L2-BirA particles encapsidating a luciferase expression plasmid were generated by calcium phosphate transfection and CsCl purification as described in accompanying manuscript [47]. 5x104 HeLa or HaCaT cells were plated in 12-well plates one day prior to infection with HPV16 to result in about 20% infection 48 h p. i. and with comparable amounts of HPV16-L2 (RTR313EEE) or HPV16-L2 (IVAL286AAAA). Additionally, high virus amounts were used to be able to detect any possible residual infectivity for the mutant virus. The inoculum was exchanged at 2 h p. i. with fresh medium. Cells were fixed 48 h p. i. in 4% PFA, and infectivity was assessed by flow cytometry (BD FACSCalibur) for GFP expression. To analyze vDNA of incoming HPV16 PsVs, 3x104 HeLa Kyoto cells or HaCaT cells were seeded on glass cover slips one day prior to infection with either EdU-HPV16, EdU-HPV16-L2 (RTR313EEE), or L2 (IVAL286AAAA) PsVs. To determine trafficking of PsVs to the TGN under conditions, where nuclear entry is blocked by interphase arrest, cells were pretreated after adhesion with 15 μM aphidicolin (Sigma) for 16 h prior to infection, and infected in the presence of the inhibitor. Cells were fixed 20 h p. i. in 4% PFA, and EdU incorporated into the vDNA was detected using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen). After the EdU-Click-iT reaction, nuclei were visualized by staining with Hoechst 33258. Confocal image slices were acquired at the Zeiss LSM 780 confocal microscope with a 63x objective. Intensity-based colocalization analysis using the ImarisColoc module (Imaris, Bitplane). For the colocalization study of vDNA with the TGN cells were immunostained with an antibody against the TGN marker p230 (BD Biosciences #611280,1: 1000 dilution), and a z interval of 0. 8 μm was chosen for z-stacks covering the height of the interphase nucleus. Intensity-based colocalization analysis using the ImarisColoc module was performed to analyze localization of vDNA to the nucleus or TGN within three medial slices per cell. 10 to 20 cells were analyzed per condition in each experiment. For studying colocalization of vDNA with mitotic chromosomes (in metaphase and anaphase cells) a z interval of 0. 6 μm was chosen for z-stacks covering the height of the chromosome bulk. Colocalization of the vDNA with mitotic chromosomes was analyzed within the entire z-stack using ImarisColoc. 10 to 20 cells were analyzed in each experiment. To analyze the L2 of incoming HPV16 PsVs, 3x104 HeLa cells or HaCaT cells were seeded on glass cover slips one day prior to infection with either HPV16 or HPV16-L2 (RTR313EEE) PsVs. Cells were fixed 20 h p. i. in 4% PFA, and L2 protein was detected using the L2 antibody K1L2 [75]. After the antibody staining, nuclei were visualized by staining with Hoechst 33258. Confocal image slices were acquired at the Zeiss LSM 780 confocal microscope with a 63x objective. Intensity-based colocalization analysis using the ImarisColoc module (Imaris, Bitplane). For studying colocalization of L2 with mitotic chromosomes (in metaphase and anaphase cells) an interval of 0. 6 μm was chosen for z-stacks covering the height of the chromosome bulk. Colocalization of the vDNA with mitotic chromosomes was analyzed within the entire z-stack using ImarisColoc. 10 to 20 cells were analyzed in each experiment. 1-5x106 purified HPV16 or HPV16-L2 (RTR313EEE) PsVs in PBS/0. 8 M NaCl were absorbed for 1 min on formvar-coated, carbon-sputtered grids. Particles were contrasted for 4 min with 1% phosphotungstic acid, pH 7. 2, and then for 10 s with 1% uranylacetate/ddH2O. Directly after drying, samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands). Photographs of selected areas were documented with Olympus Veleta 4k CCD camera. Similar amounts of purified HPV16 and HPV16-L2 (RTR313EEE) PsVs were subjected to electrophoretic separation in a 12% SDS-polyacrylamide gel and Coomassie-staining. The relative amounts of L1, L2, and cellular histones derived from the encapsidated chromatinized reporter plasmid in virions were determined by densitometric analysis. To investigate the subcellular localization of HPV16 L2 in cells during the different mitotic stages, 5x104 HeLa H2B-mCherry cells were plated on glass cover slips in 12-well plates, and transfected with a HPV16 L2-EGFP expression plasmid the next day using Lipofectamine2000 (Invitrogen). 9 h post transfection, cells were incubated with 2 mM thymidine for a single thymidine block for 15 h. When cells reached the first peak of mitosis 9–10 h after washing out the thymidine, they were fixed for 20 min in 4% PFA. Single slice images of transfected cells were acquired at the spinning disc microscope (Zeiss Axio Observer Z1, equipped with a Yokogawa CSU22 spinning disc module; Visitron Systems GmbH) with a 63x objective. To analyze the chromosomal association of PV L2-EGFP in prometaphase cells, 5000 HeLa H2B-mCherry cells or 7000 HaCaT cells were plated per well on optical 96-well plates one day prior to transfection with PV L2-EGFP constructs or EGFP expression plasmid as control. 24 h after transfection, cells were treated with 100 ng/ml nocodazole for 16 h in order to enrich cells in prometaphase. Then, cells were fixed for 30 min by carefully adding 12% PFA to the medium in the wells to yield a final concentration of 4%. The actin cytoskeleton was stained using Alexa Fluor 647 Phalloidin in PHEM Triton buffer (60 mM PIPES [piperazine-N, N′-bis (2-ethanesulfonic acid) ], 10 mM EGTA, 2 mM MgCl2,25 mM HEPES, pH 6. 9,0. 1% Triton X-100). Single slice images were acquired at the spinning disc microscope with a 40x objective and analyzed with the cell image analysis software CellProfiler (version 2. 1. 1.). The chromosome and respective cellular area were segmented based on the H2B-mCherry (HeLa H2B-mCherry) or RedDot2 (HaCaT) and actin signal, respectively. The cytoplasmic area was determined by creating an inverted mask of the chromosome within the plasma membrane border. Due to differences in expression levels between the constructs, cells were classified based on their total EGFP intensities. Only cells with expression levels at which full-length HPV16 L2-EGFP showed association with mitotic chromosomes were quantified. The degree of chromosomal association was assessed by chromosomal association index (CAI), i. e. the ratio of mean EGFP fluorescence intensity (intensity per area) of chromosomally associated L2 over cytoplasmic L2 normalized by subtracting the median ratio for EGFP. If not otherwise stated, at least 50 cells per construct were analyzed this way from one to two experiments. The alignment of the L2 amino acid sequences of HPV16,18,5, BPV1 and MnPV by Clustal Omega (Sievers, 2011) was used to determine the evolutionary relationships with the approximate Likelihood-Ratio Test in PhyML 3. 0 [76]. The phylogram was visualized with TreeDyn [77]. The branch length represents the amount of genetic changes in substitutions per site. The L2 amino acid sequences of HPV16,18,5, BPV1 and MnPV were analyzed for amino acid conservation by homology-extended multiple sequence alignment strategy in PRALINE [78]. The output scores from 0 for the least conserved alignment position, up to 10 for the most conserved alignment position were binned into five new color-coded categories: no (0–1, white), low (2–3, blue), intermediate low (4–5, green), intermediate high (6–7, orange), and high (8–10, red) conservation. As described in [47], 50,000 HaCaT NES-GFP-BAP cells were seeded in 24 wells plates one day prior to infection. To assay infectivity, the cells were infected with L2-BirA PsV at 2 x 108 viral genomes/well. At 24 h p. i., the cells were washed with PBS and lysed in reporter lysis buffer from Promega (E3971). Luciferase activity was measured on a Beckman Coulter DTX-800 multimode plate reader using luciferase assay reagent from Promega (E4550) according to the manufacturer’s instructions. Luciferase signal was normalized to GAPDH signal from western blots of infected cell lysates. To assay translocation, the cells were infected with 150ng L1/well L2-BirA virus. At 24 h p. i., the cells were treated with pH 10. 7 PBS for 2. 5 minutes and then washed 2x with pH 7. 2 PBS to remove non-internalized PsV. The cells were then lysed in 1X RIPA buffer (50 mM Tris-HCl pH 8. 0,150 mM NaCl, 1% NP40,0. 5% sodium deoxycholate, 0. 1% SDS) supplemented with 1x reducing SDS-PAGE loading buffer, 1X protease inhibitor cocktail (Sigma P1860), and 1mM PMSF. The samples were then boiled for 5 minutes. After boiling, the samples were then resolved using SDS-PAGE and transferred onto a 0. 45mm nitrocellulose membrane. The membrane was then cut in half at the 50kD marker. The upper half of the gel was blocked with 5% nonfat milk in TBST and stained with anti-L2 K4 at 1: 5000. The lower half of the gel blocked in 100% Odyssey blocking buffer (Licor 927–40000) and stained with neutravidin DyLight 800 (Pierce 22853). The lower half of the blot was then reprobed sequentially with anti-GFP (Clontech 6323770) at 1: 5000 and Goat anti-rabbit DyLight 680 (Pierce 35568) in 50% Odyssey blocker buffer/TBST. Blots were imaged using the Licor Odyssey Infrared Imaging System.

Title: A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry Summary: Papillomaviruses can cause carcinogenic malignancies such as cervical cancer. Like most DNA viruses, papillomaviruses must deliver their genome to the cell nucleus during initial infection, where it is expressed and replicated. However, papillomaviruses make use of unconventional mechanisms for genome delivery. They reside on the cell surface for protracted, hour-long times, before they are taken up by a novel endocytic mechanism. Moreover, they are delivered to the trans-Golgi-network by non-canonical endosomal trafficking prior to nuclear delivery. For entry into the nucleus, papillomaviruses access the nuclear space after nuclear envelope breakdown during mitosis unlike most other intranuclear viruses. The detailed mechanism how the viral genome is directed to nascent nuclei during mitosis remains elusive. Our previous work suggested that the minor capsid protein L2 may tether the incoming viral genome to mitotic chromosomes to direct it to the nascent nuclei. This work identifies a conserved central region in L2 protein to be necessary and sufficient for tethering. Moreover, it demonstrates that this mechanism is conserved across different papillomavirus genera. Importantly, this report also provides evidence that the processes of nuclear import by tethering and membrane penetration are mechanistically linked.

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Summarize: FIELD OF THE INVENTION [0001] The present invention relates to hair pigmentation in man. BACKGROUND ART [0002] The human hair follicle is a complex organ wherein there are interactions of epithelial (for example, different lines of keratinocytes, endothelium), mesenchymal (for example dermal papilla fibroblasts, connective tissue sheath fibroblasts), neuroectodermal (nerves, melanocytes) cellular populations and transitory migrating cells (immune cells, mastocytes). [0003] The growth and pigmentation of hair fibers are affected by several intrinsic factors comprising changes depending on hair cycle, body distribution, age and genre differences, variable hormone sensitivity, genetic defects and age-related changes. The study of hair growth is also complicated by the effects of extrinsic variables that comprise climate and seasons, polluting substances, toxins and exposure to chemicals. The differences found between the pigmentation regulation in the epidermis and in hair follicles reflect the division into compartments of the mammal skin pigmentation system. [0004] The melanocytes of epidermis, of the hair follicle bulb and of the sheath of the hair follicle outer root are very different from each other, despite the fact that the skin pigmentation in mammals must be understood as an open system. The major differences are those of the nature of their respective melanocyte-keratinocyte functional units. The melamine unit of the hair bulb is found in the proximal anagen bulb, which is an immunologically distinct region of skin and overall, is formed by one melanocyte every 5 keratinocytes in the hair bulb and of one melanocyte for each keratinocyte in the basal layer of the hair bulb matrix. On the contrary, each epidermal melanocyte is associated to 36 vital keratinocytes in the immunocompetent epidermal melamine unit. [0005] However, the most obvious difference between these two melanocyte skin populations and with considerable implications for the regulation of the hair pigmentation, is the observation that the hair bulb melanocyte activity is subject to a cyclic control and that melanogenesis is strictly associated to the hair growth cycle. On the other hand, the skin melanogenesis appears to be continuous. SUMMARY OF THE INVENTION [0006] It has now surprisingly been found, and this is the object of the present invention, that the spermidine compound, that is N-(3-aminopropyl)butan-1,4-diamine, as such or in the form of a pharmaceutically acceptable derivative such as a salt, is provided with a melanogenesis activity towards hair and can therefore be effectively used for promoting their pigmentation, in particular the shaft pigmentation. [0007] Such activity allows configuring the use of the active compound in man as a natural pigmentation agent free from negative side effects, for example typical of hair dies. [0008] The object of the invention is also a pharmaceutical or cosmetic or dietetic composition suitable to promote such pigmentation effect and therefore containing spermidine as active principle, as such or in the form of a pharmaceutically acceptable derivative such as a salt, for either topical or oral administration. DETAILED DESCRIPTION OF THE INVENTION [0009] A preferred salt according to the invention is spermidine trichlorohydrate, namely N-(3-aminopropyl)butan-1,4-diamine.3HCl. A composition of the invention preferably comprises spermidine trichlorohydrate in a solution formulated for topical use. Suitable forms for topical use are, for example, a lotion, a conditioner, a shampoo, a mask. [0010] A different composition of the invention preferably comprises spermidine trichlorohydrate in administration unit formulated for oral use. Suitable forms for oral use for example are a tablet or a capsule, either coated or not, or a granulate to disperse in water or other liquid. [0011] Spermidine, as such or in the form of a pharmaceutically acceptable derivative, as a salt, is contained in a composition of the invention according to an amount preferably comprised within the following ranges: 10 −7 to 1 g/100 ml, corresponding to 0.004 to 4·10 4 μM 10 −5 to 1 g/100 ml, corresponding to 0.4 to 4·10 4 μM 10 −4 to 2.4·10 −2 g/100 ml, corresponding to 4 to 9·10 2 μM. [0015] Further preferred concentration ranges are as follows: 10 −6 to 10 −1 g/100 ml 10 −5 to 10 −2 g/100 ml 10 −4 to 10 −3 g/100 ml 10 −7 to 10 −6 g/100 ml 10 −6 to 10 −5 g/100 ml 10 −5 to 10 −4 g/100 ml 10 −4 to 10 −3 g/100 ml 10 −3 to 10 −2 g/100 ml 10 −2 to 10 −1 g/100 ml 10 −1 to 1 g/100 ml [0026] The following formulation examples illustrate the invention, but are not intended to be limiting in any manner. The component amounts are expressed in grams or milligrams and in the case of examples 1 to 4, by concentration ranges. Example 1 [0027] [0000] Shampoo Composition for 100 ml solution Component (INCI nomenclature) Magnesium laureth sulfate 2-10 g Sodium lauroyl sarcosinate 2-10 g Disodium laureth sulfosuccinate 0.5-5 g PEG-200 hydrogenated glyceryl palmate 0.5-5 g Cocamide MIPA 0.5-5 g Glycol distearate 0.5-5 g Glycerin 0.5-5 g Laureth-7 0.1-3 g PEG-7 glyceryl cocoate 0.1-3 g Lauryl methyl gluceth-10 hydroxypropyldimonium 0.1-3 g chloride Polyquaternium-10 0.1-3 g Potassium undecilenoyl wheat protein 0.1-3 g Panthenol 0.1-3 g Tetrasodium EDTA 0.1-3 g Spermidine trihydrochloride 10 −7 -1 g Preservative q.s. pH corrector (to a final pH of 5.0-5.5) q.s. Parfum q.s. Aqua q.s. to 100 ml Example 2 [0028] [0000] Hair mask Composition for 100 ml solution Component (INCI) Glycerin 1-10 g Ammonium acrylolyl-dimethyltaurate/vp copolymer 1-10 g Cyclopentasiloxane 1-10 g Silicone quaternium-15 0.1-3 g Tocopheryl acetate 0.1-3 g Dimethicone 0.1-3 g Sericin 0.1-3 g Methylparaben 0.05-0.1 g C11-15 pareth-5 0.05-0.1 g C11-15 pareth-9 0.05-0.1 g Trideceth-12 0.05-0.1 g Decyl glucoside 0.01-0.5 g Panthenyl ethyl ether 0.01-0.5 g Disodium EDTA 0.01-0.5 g Ethyl hexyl methoxycinnamate 0.01-0.5 g Lactic acid q.s. Preservative q.s. Spermidine trihydrochloride 10 −7 -1 g Parfum q.s. g Aqua q.s. to 100 ml Example 3 [0029] [0000] Hair conditioner Composition for 100 ml solution Component (INCI) Cetearyl alcohol 1-10 g Glyceryl stearate 1-10 g Dimethicone 1-10 g C12-13 alkyl lactate 0.5-5 g Cetrimonium chloride 0.5-5 g PEG-100 stearate 0.5-5 g Cyclopentasiloxane 0.5-5 g Hydroxyethylcellulose 0.1-3 g Dimethiconol 0.1-3 g Panthenol 0.1-3 g Bis-isobutyl Peg/Ppg-20/35/amodimethicone copolymer 0.05-2 g Phytantriol 0.05-2 g Cetyl ethylhexanoate 0.05-2 g Butylene glycol 0.05-2 g Disodium edta 0.05-2 g Polysorbate 80 0.05-2 g Sericin 0.05-2 g Spermidine trihydrochloride 10 −7 -1 g Preservative q.s. g Parfum q.s. g Aqua q.s. to 100 ml Example 4 [0030] [0000] Hair lotion Composition for 100 ml solution Component (INCI) Alcohol 10-20 g PEG-40 Hydrogenated Castor Oil 0.2-2 g Disodium EDTA 0.01-0.5 g Parfum q.s. Spermidine trihydrochloride 10 −7 -1 g Aqua q.s. to 100 ml Example 5 [0031] [0000] Hard gelatine capsules Composition for a single capsule Component Lactose Monohydrate 85 mg Corn starch 25 mg Talc 5 mg Spermidine trihydrochloride 1.25 mg Magnesium stearate 1.5 mg Hard gelatine capsule shell N. 4 1 n. Example 6 [0032] [0000] Soft gelatine capsules Composition for a single capsule Component Soy oil 263.07 mg Gelatine 129.7 mg Glycerine 36.5 mg Sorbitol 70% 24.2 mg Water 23.274 mg Yellow wax 22 mg Fatty acids mono-diglycerides 20 mg Soy lecithin 10 mg Titanium Dioxide 0.686 mg Spermidine trihydrochloride 0.25 mg Example 7 [0033] [0000] Tablet Composition for a single tablet Component Microcrystalline cellulose 168.97 mg Lactose 150 mg Methylcellulose 45 mg Mono- and diglycerides of fatty acids 9 mg Colloidal silicon dioxide 8 mg Magnesium stearate 2 mg Spermidine trihydrochloride 1.0 mg Example 8 [0034] [0000] Slow-release coated tablet Composition for a single tablet Component Microcrystalline cellulose 105 mg Calcium Phosphate bibasic dihydrate 85 mg Hydroxypropylmethylcellulose K100 45 mg Sepifilm TM LP 770 white 15 mg Magnesium stearate 8 mg Hydroxypropylmethylcellulose 6 mg Colloidal silicon dioxide 3.5 mg Spermidine trihydrochloride 0.55 mg Example 9 [0035] [0000] Effervescent granulate in sachet for making an improptu solution Composition for a single sachet Component Mannitol 960 mg Tartaric acid 530 mg Anhydrous sodium bicarbonate 280 mg Flavor 130 mg Polyvinyl pyrrolidone 45 mg Trometamol 32 mg Aspartame 20 mg Spermidine trihydrochloride 1.25 mg Anhydrous colloidal silicon 2 mg [0036] Below is the description of an experimental study relating to the activity in the use of spermidine according to the present invention. Activity Study Tissue Samples [0037] The skin of normal human scalp was taken from a woman who had undergone a routine face lifting surgery after receiving the informed consent. All the experiments were carried out according to the Helsinki principles, with the ethical committee&#39;s approval. [0000] Skin Organ Culture with Complete Thickness [0038] The tissues subject to biopsy with 3-4 mm cylindrical scalpel were cultured at 37° C. for 6 days in Williams E medium (Biochrom, Cambridge, U.K.), integrated with 100 IU ml −1 penicillin, 10 μg ml −1 streptomycin (Gibco, Karlsruhe, Germany), 10 μg ml −1 insulin (Sigma, Taukfirchen, Germany), 10 ng ml −1 hydrocortisone (Sigma) and 2 mmol L −1 (L-glutamine (Invitrogen, Paisley, U.K.). [0039] Spermidine trichlorohydrate, or the vehicle as a reference substance, was then administered at a concentration of 0.1 μM, once at each medium change (i.e., every 48 hours). Micro-Dissection of Hair Follicles and Organ Culture [0040] The hair follicles (HF) in anagen VI phase with normal pigmentation (gray/white hair follicles were excluded from the study) were micro-dissected from normal human scalp skin and subject to organ culture based on the Philpott model. Spermidine or the vehicle were administered once at each medium change (i.e., every 48 hours). LDH Measurement [0041] The LDH activity in the supernatant served as a cytotoxicity indicator and was measured every day according to the manufacturer&#39;s instructions (Cytotoxicity Detection Kit; Roche, Mannheim, Germany). The sample absorbance was measured at 490 nm using an ELISA plate reader. Hair Shaft Elongation [0042] The measurements of the hair follicle shaft length were taken every second day on the single hair follicles using a Zeiss inverted binocular microscope with an ocular measurement reticle. Determination of the Hair Follicle Cycle Stage [0043] The determination of the hair follicle cycle stage was carried out based on the morphological criteria defined before, and the percentage of hair follicles in anagen phase and in early, intermediate or late catagen phase was determined. Hair Pigmentation [0044] The Masson-Fontana staining was carried out for the histochemical display of melanin on frozen sections. Melanin was stained in the form of brown granules and the pigmentation level was determined through the quantitative Masson-Fontana technique (Ito N., Ito T., Kromminga A., Bettermann A., Takigawa M., Kees F., Straub R. H., and Paus R. (2005): Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol. FASEB J 19, 1332-4). [0045] This method is a particularly sensitive and reliable indicator of melanin synthesis variations, as proven by enzymatic activity assays and standard tyrosinase expression (Kauser S., Slominski A., Wei E. T., and Tobin D. J. (2006): Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides. FASEB J 20, 882-95). [0046] The staining intensity was analyzed in a defined reference region of the hair follicle pigmentation unit using the ImageJ software (National Institute of Health). Proliferation and Apoptosis Measurement [0047] In order to evaluate the apoptotic cells in co-location with a proliferation marker Ki-67, the TUNEL (terminal dUTP nick-end labeling) dual staining method with Ki-67 was used. The cryostat sections were fixed in paraformaldehyde and ethanol-acetic acid (2:1) and marked with a digoxigenin-deoxy-UTP (kit for the identification of apoptosis in situ with ApopTag fluorescein; Intergen, Purchase, N.Y.) in the presence of terminal deoxynucleotidyl transferase, followed by incubation with a murine anti-Ki-67 antiserum (1:20 in PBS overnight at 4° C.; Dako, Glostrup, Denmark). TUNEL-positive cells were displayed by a conjugate isothiocyanate fluorescein anti digoxigenin antibody (kit ApopTag), whereas Ki-67 was detected by a goat anti-mouse antibody marked with rhodamine (Jackson ImmunoResearch, West Grove, Pa.). Negative controls were carried out omitting the terminal deoxynucleotidyl transferase and the Ki-67 antibody. The counter-staining was carried out with 4′,6-diamidino-2-phenylindole (DAPI) (Roche Molecular Biochemicals GmbH, Mannheim, Germany). The quantitative histomorphometric assessment was carried out; Ki-67, TUNEL or DAPI-positive cells were counted in a reference region defined beforehand of the hair follicle and skin matrix and the percentage of positive Ki-67/TUNEL cells was determined. Quantitative Immunohistochemistry of K15 [0048] The tyramide signal amplification method described before was used for examining the expression of keratin K15 (Kloepper et al., 2008). In brief, cryosections fixed with acetone were washed three times for 5 minutes using the TNT (tris-HCL NaCl Tween) buffer (0.1 mol/l Tris-HCl, pH 7.5; containing 0.15 mol/l NaCl and 0.05% Tween 20). Radish peroxidase was then blocked through wash with 3% H 2 O 2 in an isotonic phosphate buffer (PBS) for 15 minutes. Preincubation was carried out with the incubation of avidin and biotin for 15 minutes and with 5% normal goat serum in TNT for 30 minutes with intermediate washing steps. Murine anti-human K15 (clone LHK15, Chemicon, Billerica, USA) were diluted in TNT and incubated overnight at 4° C., followed by a secondary goat anti-mouse biotinylate antibody (1:200 in TNT) for 45 minutes at room temperature. Radish streptavidin-peroxidase was then administered (kit TSA; Perkin-Elmer, Boston, Mass., USA) (1:100 in TNT) for 30 minutes at room temperature. The reaction was amplified with a FITC-tyramide amplification agent at room temperature for 5 minutes (1:50 in an amplification diluent supplied with the kit). The intensity of this immuno-staining was quantified by the ImageJ (National Institutes of Health) software. The staining intensity of reference regions defined in hair follicles was measured and compared between the control groups treated with vehicle only and the groups treated with spermidine. Statistical Analysis [0049] The statistical analysis was carried out using a bilateral Student t-test for unpaired samples. Results [0050] The figures of the annexed drawings show the results of the experimental study described. BRIEF DESCRIPTION OF THE FIGURES [0051] FIG. 1 shows a diagram relating to the pigmentation intensity in hair follicles as measured and compared between the control group treated with vehicle only and the group treated with spermidine 3HCl in a concentration of 0.1 μM. [0052] FIG. 2 shows the corresponding images taken from the hystochemical display of melanin through Masson-Fontana staining. [0053] The increase of melanin is clear from both figures in the case of treatment with spermidine, therefore a significant melanogenesis activity towards hair treated with such compound compared to the reference vehicle.

Summary: The invention relates to the use of spermidine or a pharmaceutically acceptable derivative thereof as the active principle in a pharmaceutical, cosmetic or dietetic composition. The composition is used for promoting pigmentation of the hair, particularly the shaft of the hair. The invention also relates to the composition which promotes this pigmentation effect, the composition containing spermidine or a derivative thereof (such as a salt) as an active principle and is intended for topical or oral administration.

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Summarize: It's the apartment no one knew about, and the one that millions of Harry Potter fans around the world would love to see. Renowned actor Daniel Radcliffe has put his exclusive Melbourne apartment on the market after using the property as a home base in Australia for almost ten years. 98B St Georges Road in Toorak was purchased for $1.8 million by Radcliffe's parents Alan Radcliffe, a casting agent, and BBC casting director Marcia Gresham, and is expected to fetch around $2.4 million. Scroll down for video. Harry Potter actor Daniel Radcliffe has put his penthouse apartment in Toorak, Melbourne on the market. The three-bedroom apartment on Georges Street was bought for %1.8 million. The 25-year-old Englishman had the property bought for him by his parents in 2006 for the filming of Rod Harvy's December Boys, in which the then-16-year-old actor played an Australian orphan. Radcliffe shot to fame at 11 years old when he was cast as the eponymous Harry Potter in the Warner Brothers film franchise, and is reported to have property in New York and London, as well as Melbourne. The three-bedroom property was transferred to the Kill Your Darlings actor in 2007 when he turned 18, and was made use of by family and friends when Radcliffe was occupied with projects overseas, reported Domain. Marshall White's Marcus Chiminello told Daily Mail Australia that despite loving the privacy the apartment offered, Radcliffe has found himself visiting Australia less frequently than in previous years and had decided to put the much-loved apartment up for sale. 'He's based himself full-time in New York and has sadly decided to move on,' said Mr Chiminello,. The property was bought in 2006 by Radcliffe's parents, who transferred ownership to him when he turned 18. The apartment is one of only two in the apartment complex in the exclusive Toorak residence. Radcliffe shot to fame at 11 years old when he was cast as the eponymous Harry Potter in the Warner Brothers film franchise. The estate agent said that Radcliffe was a big fan of the large living area, which entailed almost half of the apartment, which made the residence an ideal place for entertaining friends and family. Mr Chiminello said that the real estate agency had already had significant interest from Harry Potter enthusiasts, and said that he was intrigued to see whether that would play a role in the sale. 'I'm interested to see how many people come to the open house inspection dressed as Harry Potter,' Mr Chiminello said. 'Some people have said that they want to come down purely for the Harry factor, and I'd be intrigued to see if someone buys it purely for that reason.' The north facing St Georges Road apartment is spectacular in it's own right, winning the Victorian Apartment Project of the Year and nestled on one of Toorak's most exclusive streets. Estate agent Marcus Chiminello said that Radcliffe had made himself a permanent home in New York. Radcliffe reportedly loved the large living areas which were used for entertaining family and friends. The open plan living and dining area is panelled with timber floors. The suburb has the highest average property values in Melbourne, and is home to other well-known Australians including media personality Eddie McGuire, former Australian Prime Minister Malcolm Fraser, and and footballer Nathan Buckley. The Toorak residence is one of only two penthouse-style apartments in the development, and is elevated to provide exposure to sunlight and offer privacy. The apartment occupies the entire top level, and overlooks Melbourne's CBD and the Yarra River, located just five kilometres away in Melbourne's most expensive suburb. Developed by prestige builder Stonehenge, the residence is accessed by a private lift, and features timber floors, floor-to-ceiling windows, and opens onto a rooftop terrace. The 25-year-old had the property bought for him by his parents in 2006 for the filming of The December Boys. Mr Chiminello said that the agency had already had significant interest from Harry Potter enthusiasts. The estate agent said that he was intrigued to see whether a fan would purchase the apartment. The kitchen is fully equipped with Miele appliances and also boasts a butler's pantry and laundry, and a walk-in pantry to aid entertaining. The apartment houses three bedrooms, and the main features a built-in wardrobe, a walk-in wardrobe, and a grand ensuite. Radcliffe's parents reportedly bought the apartment after seeing photographs of it online, out of a love for Australia. 'Someone my dad went to drama school with lives here [Melbourne]. We just got on with the place really, really well and so we just love it here,' Radcliffe told The Herald Sun in 2007. The apartment houses three bedrooms, and the main features a built-in wardrobe, a walk-in wardrobe, and a grand ensuite. The apartment occupies the entire top level, and overlooks Melbourne's CBD and the Yarra River

Summary: Daniel Radcliffe has put his penthouse apartment in Melbourne up for sale. The house was bought in 2006 by his parents for $1.8 million. The three-bedroom apartment is expected to fetch $2.4 million when it sells. The property boasts CBD views, and an enormous entertaining area. The selling agent expects fans may come dressed up to the inspection. The estate agency has had significant interest from Harry Potter fans. Radcliffe has made New York his home base, and owns three properties.

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Write a title and summarize: The postembryonic brain exhibits experience-dependent development, in which sensory experience guides normal brain growth. This neuroplasticity is thought to occur primarily through structural and functional changes in pre-existing neurons. Whether neurogenesis also mediates the effects of experience on brain growth is unclear. Here, we characterized the importance of motor experience on postembryonic neurogenesis in larval zebrafish. We found that movement maintains an expanded pool of forebrain neural precursors by promoting progenitor self-renewal over the production of neurons. Physical cues associated with swimming (bodily movement) increase neurogenesis and these cues appear to be conveyed by dorsal root ganglia (DRG) in the zebrafish body: DRG-deficient larvae exhibit attenuated neurogenic responses to movement and targeted photoactivation of DRG in immobilized larvae expands the pallial pool of proliferative cells. Our results demonstrate the importance of movement in neurogenic brain growth and reveal a fundamental sensorimotor association that may couple early motor and brain development. During postembryonic development, the brain begins processing sensory information from the environment for the first time and continues to grow, exhibiting elevated levels of neuroplasticity compared to later stages of life. The combination of these factors makes postembryonic brain development highly susceptible to sensory experience (Knudsen, 2004). This susceptibility to experience is evident in the ‘critical’ and ‘sensitive’ periods early in life, in which sensory experiences drive permanent or near permanent changes in brain structure and function, respectively (Knudsen, 2004). Historically, neuroplastic changes associated with early sensory experience were thought to be restricted to structural and functional changes in pre-existing neurons, such as visual experience-dependent synaptic remodeling in thalamocortical projections associated with the development of ocular dominance (Coleman et al., 2010). However, neurogenesis persists postembryonically throughout the brain, either ceasing or curtailing in adolescence or adulthood (Lindsey and Tropepe, 2006). Some neuronal populations appear to be uniquely generated during postembryonic development (Wei et al., 2011), and this process may be regulated by sensory experience (He et al., 2014). Thus, neurogenesis may also mediate the effects of early experience on brain development. Outside of postembryonic development, one of the best-characterized models of experience-dependent regulation of neurogenesis is the increase in cell proliferation in the subgranular zone (SGZ) of the dentate gyrus in the adult mammalian hippocampus (HP) following periods of aerobic running exercise (van Praag et al., 1999a). Since its initial discovery, studies have gone on to link exercise-induced neurogenesis to improvements in cognition, such as spatial learning (van Praag et al., 1999b) and cognitive flexibility (Anacker and Hen, 2017). Studies have also incorporated exercise as a therapeutic intervention to combat neuropsychiatric disorders associated with impaired neurogenesis (Vakhrusheva et al., 2016; Kandola et al., 2016). However, whether physical activity affects forebrain neurogenesis during postembryonic development, when animals first gain control of their movements while exhibiting elevated levels of neurogenesis throughout the brain compared to adulthood, remains unexplored. Furthermore, such a relationship between movement and forebrain growth early in development may help explain the positive correlation between physical activity and cognitive function reported in human children (Tomporowski et al., 2008; Best, 2010). Here, we sought to investigate the relationship between movement and neurogenesis in larval zebrafish during a developmental period in which they first begin to exhibit voluntary movements (Buss and Drapeau, 2001), have brains and peripheral nervous systems sufficiently developed to process sensory input, and continue to exhibit elevated rates of neurogenesis in many brain divisions compared to adulthood (Lindsey and Tropepe, 2006; Feliciano et al., 2015). In addition to the well-documented advantages of larval zebrafish as models for genetic and pharmacological tractability, here we also use them for our ability to control the sensory experiences of larvae, enabling the isolation of different sensory cues associated with movement to identify the nature of sensory feedback driving neurogenic change. We first developed paradigms to both reduce and increase swimming behaviour in larvae and sample for changes in neurogenesis in the forebrain. We then sought to isolate the different sensory cues associated with movement and identify which cue drives a neurogenic response in the forebrain. Finally, we tested whether dorsal root ganglia (DRG), sensory neurons that convey mechanical sensations from the zebrafish trunk, are required to mediate movement-dependent neurogenesis. We did this by both pharmacologically generating larvae deficient in DRG along the trunk and by stimulating the DRG via photoactivation of ankyrin-containing transient receptor potential channels (TRPA1b) in completely immobilized larvae. Altogether, we present a novel and robust relationship between movement and postembryonic forebrain neurogenesis, demonstrating that neural feedback associated with physical movement may provide a simple mechanism through which motor and brain development become coupled early in life. We first established a paradigm through which we could control the amount of swimming exhibited by zebrafish larvae noninvasively. We used movement restraint, in which larvae were confined to a smaller portion of 6-well plates by a mesh cylinder (Figure 1A) and tested if such restraint would reduce swimming from 3 to 9 days post fertilization (dpf). Movement restraint significantly reduced the hourly distance swam by 6 and 8 dpf in both non-repeated (Figure 1B; Treatment x Age Interaction: 1F2,80 = 14. 08, p<0. 01) and repeated (Figure 1C; Video 1; Treatment x Age Interaction: 2F2,34 = 14. 16, p<0. 01) experimental designs compared to unrestrained controls. Furthermore, movement restraint also prevented the increase in the proportion of fast swims (>10 mm/s) first exhibited by control larvae on 6 dpf (Figure 1D–E; Treatment x Age Interaction: 3F2,68 = 14. 90, p<0. 01). Because of the possibility that chronic movement restraint may impair larval development, we sampled body length of control and restrained larvae throughout the restraint period. We found that movement restraint did not affect larval body length by 6 dpf, but reduced body length by 9 dpf (Figure 1—figure supplement 1A; 4F2,111 = 13. 10, p<0. 01). To test if this reduction in body length affected motor ability, we repeated our movement restraint paradigm and, on 8 dpf, we moved restraint larvae into control wells to record unrestrained swimming behaviour. Prior movement restraint did not impair either hourly distance swam (Figure 1—figure supplement 1B; 5t20 = 0. 98, p=0. 34) or proportion of fast swims (Figure 1—figure supplement 1C, 6t20 = 0. 16, p=0. 87) in unrestrained conditions. Collectively, we found that our restraint paradigm reduced motor experience in larvae by 6 dpf without impairing swimming ability. To test for changes in neurogenesis in the restrained larval zebrafish brain, we sampled the proportion of PCNA+ cells in the pallium, subpallium, olfactory bulb, and optic tectum of 6 dpf larval zebrafish. Movement restraint significantly reduced the proportion of proliferative (PCNA+) cells in the forebrain of zebrafish by 6 dpf (Figure 2A–C; 7t9 = 4. 07, p<0. 01) sampled across consecutive coronal sections (Figure 2—figure supplement 1), without affecting forebrain size (Table 2). This difference was attributed to a reduction in the proportion of PCNA+ cells in both the subpallium (Figure 2—figure supplement 2A; 8t5 = 3. 77, p=0. 01) and pallium (Figure 2—figure supplement 2B; 9t5 = 7. 36, p<0. 01). Conversely, movement restraint did not affect the size (Table 2) or proportion of PCNA+ cells in the olfactory bulb (Figure 2—figure supplement 2C; 10t9 = 0. 53, p=0. 61) or optic tectum (Figure 2—figure supplement 2D; 11t8 = 0. 87, p=0. 41) by 6 dpf. Movement restraint reduced forebrain size by 9 dpf (Table 2); however, after correcting for forebrain size, chronic restraint still significantly reduced the proportion of PCNA+ cells in the forebrain by 9 dpf compared to controls (Figure 2D–F; 12U = 0, p<0. 01). Movement restraint also reduced the proportion of tbr2+ cells, a protein marker of intermediate progenitors and newly generated neurons (Englund et al., 2005), in the pallium by 9 dpf (Figure 2G–I; 13t16 = 3. 37, p<0. 01) without affecting the proportion of pallial GFAP+ radial neural stem cells in Tg (GFAP: gfp) embryos (Figure 2J–L; 14t12 = 0. 35, p=0. 73). Thus, movement restraint reduced the size of the pool of proliferative cells, presumably neural progenitors, in the forebrain specifically, without affecting the size of the resident radial stem cell population. We then asked how movement restraint results in a reduced forebrain proliferative cell population. We reasoned that a reduction in this cell population might occur when either proliferative cells generate more differentiated cells at the expense of self-renewal, reducing the size of the proliferative population over successive divisions, or by apoptosis in the proliferative population. To sample cell differentiation in these forebrain populations, we exposed larvae to 5 mM 5-Ethynyl-2' -deoxyuridine (EdU), a synthetic thymidine analog that is incorporated in dividing cells, for 24 hr starting on 5 dpf, then sampled the proportion of EdU+ cells that also express Elavl3 protein, a marker of cells with a differentiated neuronal fate (Lindsey et al., 2012). If movement restraint biased the production of differentiating cells over progenitor self-renewal, we would predict that restrained larvae would exhibit more EdU+ cells that co-expressed Elavl3. Movement restraint significantly increased the proportion of newly generated cells that co-label with Elavl3 in both the pallium (Figure 2M; 15t6 = 6. 02, p<0. 01) and subpallium (Figure 2N–T; 16t6 = 3. 43, p=0. 01) without affecting the absolute number of EdU+ cells produced in the pallium (Figure 2—figure supplement 2E; 17t6 = 0. 35, p=0. 74) or subpallium (Figure 2—figure supplement 2F; 18t7 = 0. 73, p=0. 49). Conversely, movement restraint did not affect the number of cells expressing the apoptotic marker activated caspase-3 (Casp3) in the forebrain (Figure 2—figure supplement 2G; 19t7 = 1. 06, p=0. 32) by 6 dpf. By 9 dpf, however, movement restraint significantly increased the number of activated Casp3+ cells in the forebrain (Figure 2—figure supplement 2H; 20t13 = 3. 56, p<0. 01). This increase in apoptosis at 9 dpf was specific to the pallium (Figure 2—figure supplement 2I; 21U = 1, p<0. 01) and not found in the subpallium (Figure 2—figure supplement 2J; 22t13 = 1. 76, p=0. 10). Despite this increase in pallial apoptosis, cell death rates remained low by 9 dpf and Casp3+ cells were not observed along the midline or dorsal surface of the brain, where the neurogenic niche lies. Together, these findings suggest that movement restraint biased newly generated cells to differentiate into neurons, ultimately at the expense of self-renewal. Because physical restraint may restrict more than just movement (i. e., reducing sensory input in a smaller space), we tested whether increasing movement could also impact forebrain cell proliferation. We raised larvae in groups (n = 15–20) housed in transparent plastic canals against different strengths of water current. Control larvae experienced no displacing current (Figure 3A; water dripping in and out, current did not displace larvae) and ‘exercised’ larvae experienced a strong current (Figure 3B; water flow strong enough to displace larvae) from 3 to 9 dpf following a daily schedule (Figure 3C). In the current condition, larvae would have to swim to counteract the flow of water and maintain their position in the canal, akin to forced exercise paradigms in rodents (Leasure and Jones, 2008). On 9 dpf, larvae reared against a strong current exhibited a greater proportion of PCNA+ cells in the pallium (Figure 3D; 25t15 = 2. 80, p=0. 01), but not the subpallium (Figure 3E; 26t19 = 1. 22, p=0. 24). On 9 dpf, the size of both brain regions was not affected by rearing treatment (Table 2). When we sampled pallial proliferation in larvae earlier, at 6 dpf, we again found an increase in the proportion of PCNA+ cells in the pallium in larvae reared against a strong current (Figure 3—figure supplement 1A; 23t7 = 2. 76, p=0. 03), while the subpallium was unaffected (Figure 3—figure supplement 1B; 24t6 = 1. 27, p=0. 25). Again, the size of both brain regions was not affected by rearing in a strong current on 6 dpf (Table 2). To test if increased movement affected cell differentiation as in our restraint paradigm, we exposed larvae to EdU in a petri dish overnight for 13 hr from 8 to 9 dpf prior to being returned to their swimming canals for a final 5 hr of current-rearing. Rearing larvae against a current reduced the number of newly generated (EdU+) cells that also expressed Elavl3 compared to controls (Figure 3F; 27t11 = 2. 39, p=0. 04), consistent with increased movement maintaining an expanded proliferative cell population over the generation of differentiated neurons. Rearing larvae against a current from 3 to 9 dpf did not affect body length (Figure 3G, 28U = 102, p=0. 5081), suggesting these effects on pallial neurogenesis are not a product of overall growth. Together with our movement restraint data, the increase in pallial cell proliferation following exercise suggests that motor experience regulates forebrain neurogenesis specifically in the pallium, similar to the neurogenic effect of exercise in the mammalian SGZ. Upon establishing a link between motor experience and pallial neurogenesis, we asked if we could identify the modality of sensory feedback associated with movement driving cell proliferation. To isolate visual and physical components of movement, we restrained larvae entirely in agarose from 3 to 6 dpf, preventing locomotion. We then re-introduced visual stimulation associated with movement (optic flow) by exposing immobilized larvae to computer-generated visual gratings to simulate visual motion. Physical input associated with movement was re-introduced to immobilized larvae by cutting the tail of larvae free from agarose embedding, enabling swimming tail movement without bodily displacement in the environment. Control larvae were provided both visual stimulation (gratings) and tail movement (Figure 4A), whereas treatment groups experienced only either tail movement (Figure 4B) or visual stimulation (Figure 4C). Blocking tail movement (complete immobilization) significantly reduced the proportion of PCNA+ cells in the pallium by 6 dpf (Figure 4D; 29F2,14 = 7. 89, p<0. 01). Conversely, removing just visual stimulation had no impact on the number of PCNA+ cells in the pallium. Neither removing visual stimulation nor tail movement affected the proportion of PCNA+ cells in the subpallium (Figure 4E; 30F2,14 = 2. 42, p=0. 13). Because immobilization could impair brain growth globally, we also sampled the number of Hoechst+ cells per section as a proxy for absolute forebrain size. Immobilization did not reduce the total number of cells in the pallium or subpallium (Table 2), instead affecting the PCNA+ cell population specifically. These results suggest that physical input associated with locomotion, specifically tail movement during swimming, drives changes in pallial neuroproliferation. One source of neural feedback that could detect physical movement is the lateral line, a system of hair cells distributed along the teleost body that detects changes in water flow (Dijkgraaf, 1963). We treated 3 dpf larvae with 30 µM copper sulfate for 30 min, an ototoxin that ablates lateral line hair cells and impedes subsequent regeneration of these cells over the following days (Mackenzie and Raible, 2012). We confirmed hair cell ablation by the complete absence of beta-acetylated tubulin (AcTub) expression in hair cell cuppulae following treatment with copper sulfate (Figure 5A). If the lateral line is involved in mediating movement-dependent neurogenesis, then removal of this feedback should affect PCNA+ cell populations in the pallium. However, copper sulfate treatment did not affect the proportion of PCNA+ cells in the 6 dpf larvae pallium (Figure 5B; 31t12 = 0. 51, p=0. 62) when all larvae were reared in unrestrained wells. Intact lateral line signaling does not appear to mediate movement-dependent changes in pallial neurogenesis. In vertebrates, DRG collect sensory feedback from the body and communicate these signals via ascending pathways to the CNS in the spinal cord (Vandewauw et al., 2013). Accordingly, DRG represent another system of neural feedback that could convey physical cues associated with movement. We tested whether blocking DRG development in the trunk would reduce PCNA+ cell populations in the pallium associated with swimming. We blocked development of DRG along the larval trunk by treating embryos with the ErbB receptor antagonist AG1478 in a limited window from 8 to 30 hpf followed by a wash-out period of almost 2 days (Honjo et al., 2008). By 3 dpf, we confirmed that earlier AG1478 treatment reduced DRG development in Tg (isl2b: mgfp) transgenic embryos (Figure 6Ai–iii). However, AG1478 treatment affected neither the pallium size (Table 2) nor proportion of pallial PCNA+ cells on 3 dpf, prior to any motor treatments (Figure 6—figure supplement 1A; 32t7 = 0. 04, p=0. 97). Thus, we divided 3 dpf AG1478- and DMSO-treated larvae into control and movement restraint conditions and sampled PCNA+ cell populations as above. By 6 dpf, prior AG1478 treatment did not affect swimming compared to DMSO-treated controls (Figure 6—figure supplement 1B; 33F3,17 = 16. 16, p<0. 01). Whereas DMSO-treated larvae exhibited a movement-dependent change in the proportion of pallial PCNA+ cells, prior AG1478 treatment blocked this effect (Figure 6—figure supplement 1C; 34F3,77 = 4. 17, p<0. 01). Prior AG1478 treatment did not affect 6 dpf pallium size between unrestrained larvae (Table 2). Furthermore, when unrestrained larvae were exposed to EdU from 5 to 6 dpf as in our restraint paradigm, prior AG1478 treatment increased the number of newly generated (EdU+) cells that also express Elavl3 (Figure 6—figure supplement 1D; 35t8 = 2. 53, p=0. 04) compared to DMSO-treated controls, suggesting early AG1478 treatment affects pallial neurogenesis similarly to chronic restraint, albeit at a reduced magnitude. To resolve differences in the proportion of PCNA+ cells in the pallium of AG1478- and DMSO-treated larvae, we repeated this experiment and extended control and movement restraint conditions until 9 dpf as above. Prior AG1478 treatment also did not affect swimming on 8 dpf (Figure 6B; 36F3,20 = 27. 59, p<0. 01). By 9 dpf, AG1478-treated larvae exhibited movement-dependent differences in the proportion of pallial PCNA+ cells, however, the magnitude of this effect was significantly attenuated compared to DMSO-treated controls (Figure 6C–G; 37F3,23 = 26. 68, p<0. 01). Prior AG1478 treatment did not affect 9 dpf pallium size between unrestrained larvae (Table 2). Together, these results suggest that neural feedback from DRG mediates, at least in part, movement-dependent forebrain neuroproliferation. If DRGs mediate neural feedback during movement to stimulate pallial cell proliferation, then direct stimulation of DRGs independent of movement should also drive pallial neurogenesis. We used an optopharmacological approach to activate DRGs by exposing larvae to a combination of light and Optovin, a small molecule that enables photoactivation of TRPA1 receptors (Kokel et al., 2013). TRPA1 receptors are found in DRG (Vandewauw et al., 2013), trigeminal neurons and Rohon-Beard cells in larval zebrafish (Prober et al., 2008). In zebrafish, Optovin acts specifically on the TRPA1b paralog, which is exclusively expressed in sensory ganglia up to 5 dpf (Prober et al., 2008). To repeatedly photoactivate DRGs using Optovin, we exposed unrestrained 5 dpf larvae isolated in a 24-well plate to either Optovin or DMSO and adjusted light exposures and intermittent darkness to achieve repeatable behavioural activation. Unrestrained larval zebrafish incubated in Optovin exhibited intense, sporadic bouts of movement during light exposure, presumably as spinal reflexes in response to intense DRG activation (Kokel et al., 2013). 5 dpf larvae exhibited repeatable, photo-activated motor responses to 2 s of exposure to light every 5 min, whereas larvae treated with DMSO exhibited no such responses (Figure 7A–B, Figure 7—figure supplement 1; Treatment x Timebin Interaction: 38F2,44 = 6. 36, p<0. 01). Therefore, we used 2 s of light stimulation every 5 min as a paradigm to regularly stimulate DRG in immobilized larvae. We immobilized 3 dpf larvae in agarose individually in 24-well plates. On 5 dpf, larvae were incubated with either DMSO or Optovin and exposed to either darkness or a 5 hr session of light presentations as above. Twelve hours following the end of this session, light exposures significantly increased the proportion of PCNA+ cells in the pallium of larvae exposed to Optovin (Figure 7C; Drug x Light Treatment interaction; 39F1,32 = 4. 47, p=0. 04), whereas light treatments had no effect on the proportion of PCNA+ cells in DMSO-incubated larvae. Furthermore, 6 dpf larvae deficient in trunk DRGs (using a transient 8–30 hpf treatment with AG1478 as above) did not exhibit this optovin-and-light-dependent increase in the proportion of PCNA+ cells in the pallium compared to controls (8–30 hpf DMSO treatment; Figure 7D; 40t13 = 2. 33, p=0. 04). Thus, DRG activation appears sufficient to increase pallial neurogenesis in the zebrafish larvae in the absence of physical movement. We found that movement plays a critical role in determining the number of neural progenitors in the zebrafish forebrain during postembryonic development. Previous work has focused on coupling increased physical activity via aerobic exercise with increases in cell proliferation in the adult mammalian SGZ (Fabel and Kempermann, 2008). Here, we found that physical activity also modulates forebrain cell proliferation postembryonically in the larval zebrafish pallium. Whereas we found that increased physical activity in fish led to an increase in pallial cell proliferation, we also report a negative neurogenic response when movement is reduced via restraint or immobilization. In the most extreme case, restricting larval movement resulted in the near absence of a proliferative population in the pallium by 9 dpf, even though these larvae were fully capable of swimming normally thereafter. Furthermore, we found that these changes in progenitor populations had subsequent impacts on neurogenic brain growth: restrained larvae, who exhibit reduced pallial cell proliferation by 6 dpf, develop smaller forebrains by 9 dpf due to a combination of reduced neurogenesis and, to a lesser extent, pallial cell apoptosis. The mechanisms through which the neurogenic niche is affected by exercise in the adult rodent hippocampus include proposed changes in cell fate, cell cycling, and apoptosis in neural precursors (Overall et al., 2016). Here, we found that, postembryonically, movement appears to maintain proliferative cell populations in the zebrafish pallium primarily by promoting self-renewal in neural progenitor cell populations whereas restraining movement promoted their premature differentiation. Because control and restrained larvae produced the same number of cells in the forebrain from 5 to 6 dpf, movement-dependent regulation of postembryonic forebrain cell proliferation appears to occur predominantly through regulating self-renewal and the production of differentiated cells over factors that might affect the absolute number of cells produced, such as cell cycle length. Within the neurogenic niche, movement-dependent maintenance of the progenitor pool may involve the Shh signaling pathway, which expands progenitor populations via symmetric cell division (Lai et al., 2003; Machold et al., 2003; Yang et al., 2015). Collectively, our findings demonstrate the importance of movement in maintaining a source of new neurons to support forebrain growth postembryonically and present zebrafish as a novel model in which movement modulates early brain development over the course of a few days. In addition to characterizing the relationship between movement and postembryonic neurogenesis in the forebrain, we also sought to identify the nature of the feedback signal associated with movement that drives this neurogenic change. We found that physical cues associated with movement send ascending neural feedback to the brain via DRGs to drive changes in neurogenesis. Specifically, larvae deficient in DRGs along their trunk exhibited an attenuated neurogenic responses to swimming compared to controls. However, we still found significant modulation of pallial cell proliferation on 9 dpf in DRG-deficient larvae. This continued modulation of neurogenesis in the older larvae may be attributed to the nature of our treatment, which blocks the development of DRGs along the trunk, but does not affect DRG populations that are derived from neural crest cells in the head that may also signal movement (Honjo et al., 2008). Other proposed mechanisms of movement-dependent neurogenesis, such as the circulation of growth factors proposed to mediate exercise-dependent adult neurogenesis (Cotman et al., 2007), and other mechanosensory cell populations, such as Rohon-Beard cells, may also play a role in driving motor experience-dependent neurogenic brain development. A previous study has demonstrated that treating zebrafish embryos with AG1478 can impair proliferation during embryogenesis in the zebrafish optic tectum (Sato et al., 2015). In that study, changes in tectal neurogenesis were observed using a near 8-fold increase in AG1478 concentration (compared to that used here) and neurogenesis was found to resume normally within hours following drug washout. Recognizing the possibility of a lasting effect of early AG1478 treatment, we sampled forebrain neurogenesis in 3 dpf larvae treated earlier with AG1478 or DMSO and found no effect of AG1478 treatment on pallial neurogenesis prior to movement or Optovin manipulations. We also found that earlier AG1478 treatment had no effect of pallium size in restrained or unrestrained control larvae by 6 dpf. In conjunction with our original movement and Optovin experiments, which do not include AG1478 manipulations, our results suggest that motor experience-dependent neurogenesis is mediated, in part, via peripheral neural feedback and is likely not attributed to early AG1478 treatment. However, future work would benefit by contrasting the results obtained here with larvae in which DRG development is blocked using alternative means. Furthermore, the ErbB signaling inhibitor used in our studies may have non-neural effects on skin or heart development. Although it is unlikely that these could be a factor in determining transmission of movement information to the brain to alter forebrain neurogenesis, this could be examined in the future. Using completely immobilized larvae, we were able to stimulate pallial cell proliferation by stimulating DRG along with other cells. Furthermore, this increase in pallial cell proliferation due to stimulation was not observed in larvae deficient in trunk DRGs. In conjunction with our studies reducing specific DRG populations along the trunk, our results suggest that neural feedback associated with movement is sensed predominantly by DRG and that DRG activation is sufficient to expand a progenitor pool in the forebrain. This previously undocumented role for DRG in conveying physical cues associated with movement to expand pools of forebrain progenitors may in turn provide a larger source of neurons and support more neurogenic brain growth in the most active animals. We found that physical movement of the body was the most important component of movement driving pallial neurogenesis. Accordingly, we propose that movement triggers mechanosensory input detected by DRGs that are then sent to the brain. In zebrafish larvae, mechanosensory input is most likely to come from one of three sources. The first possibility is the lateral line, which detects changes in water flow and vibration in the environment (Dijkgraaf, 1963). Here, ototoxic ablation of this system had no impact on pallial proliferation, suggesting it does not play a role in maintaining pallial progenitor populations. Second, Rohon-Beard (RB) cells, an early-developing population of spinal neurons, transmit mechanosensory signals (Faucherre et al., 2013) and contain TRPA1b receptors (Prober et al., 2008) that can be activated by Optovin stimulation (Kokel et al., 2013). Originally, RB cells were thought to die off entirely by 4 dpf (Reyes et al., 2004), but subsequent work using transgenic markers suggests they may persist up to 1–2 weeks post-fertilization (Kucenas et al., 2006; Palanca et al., 2013). Our studies showed that early AG1478 treatment, which specifically affected DRG development with resident RB cell populations remaining unaffected, blocked the Optovin-induced increase in proportional PCNA+ cells in the pallium following light presentations, suggesting DRG and not RB are responsible for the increase in pallial neurogenesis following Optovin and light treatment. However, our data indicate that RBs may also contribute to motor experience-dependent pallial cell proliferation, particularly in DRG-deficient larvae by 9 dpf. Finally, DRGs can transmit mechanosensory information from the trunk owing to the array of sensory channels they contain including TRPA1b (Kokel et al., 2013). Because both the removal and activation of DRGs produced neurogenic consequences in the pallium, we propose that these sensory neurons are the primary mediators of movement-dependent postembryonic neurogenesis. TRPA1 channels exhibit deep evolutionary conservation across vertebrates (Christensen and Corey, 2007). Zebrafish have two orthologs of the TRPA1 channel: only TRPA1b, however, likely processes external signals (Prober et al., 2008) and is activated by Optovin treatment (Kokel et al., 2013). Whereas TRPA1 function has been implicated in touch stimuli in DRG of mice (Kwan et al., 2006; Brierley et al., 2011) and sensory neurons in C. elegans (Kindt et al., 2007), this channel is also associated with transducing chemosensory and nociceptive input (Prober et al., 2008). Here, we found evidence suggesting a novel function for TRPA1 in transducing physical cues associated with bodily movement during locomotion. Our results demonstrate a robust connection between motor and brain development during postembryonic development. Motor development in most vertebrates begins early in the postembryonic period, including both viviparous species, such as with fetal motor development in humans (de Vries et al., 1982), and oviparous species, such as the larvae studied here. Therefore, if conserved across taxa, this close relationship between movement and neurogenesis may couple early motor and brain development. Furthermore, this relationship could help explain correlations between early physical and mental development, such as the long-observed comorbidity of physical and mental impairments (Barnett et al., 2012) and correlation between sedentary lifestyle and depression (Anton et al., 2006), which has been previously associated with impaired neurogenesis (Jacobs et al., 2000), in children. All zebrafish used in this study were of an AB genetic background. Larval strains used in this study include: Tg (dlx5/6: gfp) (generously provided by Dr. Marc Ekker, University of Ottawa) and Tg (GFAP: gfp) (generously provided by Dr. Pierre Drapeau, Université de Montreal) in motor restraint and visual vs. physical movement cue experiments; Tg (βactin: gfp) (generously provided by Dr. Ashley Bruce, University of Toronto) for copper sulfate treatments; and Tg (isl2b: gfp) (generously provided by the late Dr. Chi-Bin Chien, University of Utah) larvae for all AG1478 and Optovin treatment experiments. All adult zebrafish crossings included 2–3 male and female fish. Larvae were collected on the day of fertilization in system water and moved to a dark incubator held at 28°C. On 1 dpf, larval water was bleached for 30 s, rinsed four times with fresh system water, and larvae were dechorionated using forceps, before being returned to the incubator. From 3 dpf onward (dpf), larvae were housed in a facility room held under a 14/10 light/dark cycle at 28°C (Lights on at 08: 00/Lights off at 22: 00; light intensity = 300 lux). Larvae housed individually in well plates had half of their system water replaced twice daily (at 08: 00 and 14: 00). From 5 dpf onward, larvae were also fed size 0 zebrafish food (Gemma Micro; Skretting, Tooele, Utah) twice daily immediately following water changes. Zebralab (ViewPoint, Montreal, Canada) recordings were made in a separate testing room with similar environmental conditions as the fish facility. After recordings, all larvae were returned to the housing facility. In all experiments aside from those involving movement tracking (see below), larvae were randomly assigned to experimental conditions from the same clutch prior to experimental manipulations. Minimum sample sizes were selected to mirror those in preliminary experiments and our initial findings reported here, in which we found that restraint influenced the number of proliferative cells in the forebrain. Following all experimental procedures, larvae were sacrificed using an overdose of tricaine prior to tissue collection and fixation (see below). All animal experiments were performed with the approval of the University of Toronto Animal Care Committee in accordance with the guidelines from the Canadian Council for Animal Care (CCAC). To restrict movement, we reared isolated 3 dpf larvae in wells of either unmodified 6-well plastic plates (well diameter = 3. 5 cm) or modified 6-well plates in which larvae were confined to a central portion of each well within a cylinder of plastic mesh (Figure 1A; cylinder diameter = 1 cm). This mesh barrier was selected to both allow water flow in and out of the confined region and the rest of the well and prevent the larvae from escaping. To track swimming of larvae throughout experiments, we used the Zebralab automated tracking system. Larvae were recorded on 4,6, and 8 dpf. On each recording day, one tray of larvae was moved into the Zebrabox (ViewPoint; held at 800 lux light intensity) recording apparatus by 08: 30. Following a 30 min habituation period, swimming was recorded for 4 hr before larvae were moved into fresh system water in a new tray and returned into the Zebrabox recording apparatus for a 30 min habituation and then an additional 4 hr of swimming before being returned to the facility room (at 17: 00). On 6 and 8 dpf, larvae were also fed prior to habituation in the morning (at 08: 00) and between recording sessions (at 13: 00). Because only single trays of larvae were recorded in a session, restraint and control groups were reared as cohorts offset by 1 day and, accordingly, were recorded on alternating days. In all movement tracking experiments, we used 2 tanks of mixed-sex adult zebrafish (each containing 3 males and 3 females) from the same genetic background to generate each cohort. To control for genetic variation between families, parentage was reversed and balanced between treatment groups in subsequent cohorts. For example, the tank of adult zebrafish crossed to generate the control group in our first cohort was crossed to generate the restraint group in our second cohort. Tracking experiments were repeated to achieve the sample sizes reported here and included at least two cohorts. Zebralab tracking thresholds were set up as follows: Inactive/Small Swim Threshold = 5 mm/s; Small/Large Swim Threshold = 10 mm/s. To prevent physical movement from 3 to 6 dpf, we embedded larvae in 1. 2% agarose dissolved in system water at 12: 00 on 3 dpf. We moved larvae into plastic wells in a droplet of system water, anesthetized larvae using tricaine (4 g/L; Sigma-Aldrich, Oakville, Canada), and introduced a drop of 1. 2% agarose to mix with the system water. Once set, additional warmed agarose was added around the embedded larvae to secure the embedded larvae in the well. For treatments requiring free tail movement, newly embedded larvae were submerged in system water and a scalpel was used to cut a block of agarose free from below the larvae’s neck to beyond the base of the tail. To simulate optic flow, we generated a visual grating stimulus using PsychoPy (Peirce, 2008) in which a black-and-white striped gradient moves along a randomly selected axis (between 0–360°) for 30 s, remains stationary for 30 s, and then begins again along a new, randomly selected axis. Gratings were displayed on a Dell P2212Hb computer monitor mounted horizontally, with plastic trays containing immobilized larvae sitting on top of the screen. In pilot experiments, grating bandwidth and speed were adjusted such that they would drive 6 dpf free-swimming larvae in a petri dish placed on the screen to swim along the grating axis. Gratings were presented to immobilized larvae starting on 3 dpf from 15: 00-19: 00, on 4–5 dpf for two 4 hr sessions (08: 00-12: 00 and 15: 00-19: 00), and on 6 dpf for just the morning session. On 3 dpf, larvae were exposed to either system water or 30 µM copper sulfate (Sigma-Aldrich) added to the swimming media for 30 min starting at 09: 00. Following copper sulfate exposure, both groups of larvae were rinsed three times with fresh system water. A subset of each treatment group was kept in a petri dish for an additional 30 min before sacrifice and tissue collection to validate ototoxicity of the copper treatment. The rest of the larvae were all isolated in control 6-well plates for the remainder of the experiment. We dechorionated Tg (isl2b: mgfp) embryos by 6 hpf and treated them with either 4 µM 4- (3-chloroanilino) −6,7-dimethoxyquinazoline (AG1478), an ErbB receptor antagonist (Sigma-Aldrich) dissolved in 0. 4% DMSO in system water or 0. 4% DMSO in system water from 8 to 30 hpf (Honjo et al., 2008). At 30 hpf, larvae were rinsed three times with fresh system water and kept in an incubator until 3 dpf. On 3 dpf, AG1478 treatment was confirmed using fluorescence microscopy to count trunk DRG, which express GFP in Tg (isl2b: mgfp), in treated transgenic larvae. We excluded any larvae treated with AG1478 that exhibited more than 4 DRGs in the trunk. These larvae were removed from all experiments to ensure experimental manipulations only included larvae with most or all of trunk DRGs missing. We incubated larvae in Optovin (Hit2Lead, San Diego, California), a photo-activated small molecule that activates TRPA1b receptors found in zebrafish sensory neurons (Kokel et al., 2013). To establish a paradigm involving repeated photoactivation of TRPA1b, we incubated 5 dpf unrestrained larvae housed individually in 24 well plates in either 10 µM Optovin (in 0. 1% DMSO in system water) or just 0. 1% DMSO in system water for 2 hr in the Zebrabox (Viewpoint) tracking apparatus with the lights off. Following incubation, larvae were exposed to 2 s of white light (800 lux) alternating with 5 min of dark and movement was recorded using the automated tracking parameters outlined above. After validating the utility of Optovin in unrestrained larvae, we incubated half of the larvae in 24-well plates containing 5 dpf larvae immobilized in agarose from 3 dpf (as above) in either Optovin or a DMSO vehicle. One tray of larvae was kept in the Zebrabox apparatus while the other was kept in complete darkness on the counter adjacent to the Zebrabox, beneath an opaque black plastic cover. From 15: 00-20: 00, larvae in the Zebrabox were exposed to 2 s of light (800 lux) alternating with 5 min of darkness. At 20: 00, all larvae trays were rinsed using fresh system water three times and moved into the dark incubator overnight. For coronal section immunohistochemistry, larvae were sectioned using a freezing cryostat (20 μm sections), thaw-mounted on Superfrost Plus slides (Sigma-Aldrich), and dried for 3 hr in the dark at room temperature. Tissue was rehydrated in 0. 2% Tween20 in phosphate-buffered saline (PBT) for 30 min at room temperature. At this point, tissue that was labeled for Elavl3 production was refixed with 4% paraformaldehyde for 20 min at room temperature and exposed to 50 mM Tris (pH = 8. 0) for 60 min at 75–80°C before being rinsed with PBS three times and PBT once. All tissue was washed with PBT three times and blocked with 2% Normal Goat Serum (NGS) in PBT for at least 2 hr at room temperature. Tissue was incubated with the primary antibody in 2% NGS in PBT at 4°C overnight. Primary antibodies used in this study included mouse anti-Human Neuronal Protein HuC/HuD, also called ELAV like neuron-specific RNA binding protein three in zebrafish (Elavl3; Life Technologies, Waltham, Massachusetts, 1: 400), PCNA (Invitrogen, Carlsbad, California, 1: 500), rabbit anti-activated caspase 3, a marker of apoptosis (Cell Signaling Technology, Danvers, Massachusetts, 1: 500), rabbit anti-GFP (Alexa-488 conjugated, Life Technologies, 1: 1000), and tbr2 (Abcam, Cambridge, United Kingdom, 1: 500). On the next day, tissue was rinsed three times with PBT before being incubated in a secondary antibody in 2% NGS in PBT for 1–2 hr at room temperature. Secondary antibodies used included Cy3-conjugated Goat Anti-Mouse IgG (Jackson ImmunoResearch, West Grove, Pennsylvania, 1: 500), Cy3-conjugated Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 1: 500), and Cy2-conjugated Goad Anti-Rabbit IgG (Jackson ImmunoResearch, 111-225-144,1: 500). Tissue was rinsed with PBT three times. To visualize EdU, a Click-iT EdU reaction was performed as per the instructions included in the kit using the Alexa 647 azide (Invitrogen). Next, tissue was rinsed with PBT, counterstained with Hoechst for 10 min at room temperature, rinsed four times with PBS and coverslipped using 90% glycerol in PBS. Coverslips were sealed with clear nail polish and stored at 4°C until imaging. Images were captured with a Leica SP8 confocal microscope using a 40x objective as image stacks throughout the focus of sections compared as z-stacks with a z sampling distance of 1 μm. The treatment identity of larvae was masked prior to image analysis, which was all performed using IMARIS (Bitplane, Belfast, United Kingdom). For whole mount immunohistochemistry larvae were fixed in 4% paraformaldehyde for 2 hr at room temperature, rinsed in PBS, exposed to acetone for 7 min at −20°C, rinsed again with PBS, and a mixture of 1% bovine serum albumin, 1% DMSO, and 0. 1% TritonX-100 (PBDT) before being blocked in 10% NGS in PBDT for 1 hr. Following blocking, larvae were incubated overnight in mouse anti-alpha acetylated tubulin (Abcam, Cat No: 6-11B-1; 1: 500). The second day of immunohistochemistry was completed as above, except exposure to the secondary antibody was extended to 5 hr at room temperature and whole mount larvae were kept in PBS at the end of staining, not coverslipped or sectioned. Images were captured with a Leica SP8 confocal microscope using a 40x objective as image stacks throughout the focus of sections compared as z-stacks with a z sampling distance of 1 μm. Image analyses performed using IMARIS (Bitplane, Belfast, United Kingdom). PCNA +cell sampling was performed using landmarks summarized in Figure 2—figure supplement 1. Olfactory bulb sampling was only possible in single, 20 µm sections and both hemispheres were sampled together. Both pallial and subpallial PCNA+, Hoechst+, tbr2+, and GFAP+ cell sampling were performed across both hemispheres in three consecutive, 20 µm sections for all histology involving 6 and 9 dpf larvae. PCNA +cell sampling in 3 dpf used only two consecutive coronal sections, as the telencephalon was not sufficiently grown to span 3 consecutive sections. Optic tectum sampling on 6 dpf was performed on the first coronal section posterior to the tectal neuropil, sampled in and averaged across both hemispheres in each brain. We define a biological replicate as an individual larvae derived from a mixed clutch borne of at least two male and two female adult zebrafish. Whereas we did not perform any technical replicates of our experiments, which we define as a complete repetition of a single experiment, we instead replicated our main findings by either repeating them in later, more elaborate experiments (for example, control vs. restraint paradigms in both initial experiments and AG1478 experiments) or repeating experiments over multiple time courses (for example, identifying the effects of exercise on forebrain neuroproliferation by both 6 and 9 dpf). All statistical test results are preceded by a superscript numeral enabling reference to each test in our calculations of statistical power summarized in Table 1. In experiments involving two groups, treatment groups were compared using Student’s t-test or, when parametric assumptions were not met, Mann-Whitney U tests. In experiments involving three or more groups, treatment groups were compared using either one-way ANOVA or two-way ANOVA (with a within-groups variable for repeated data). All post hoc comparisons were made using Tukey’s test with a correction for multiple comparisons. When neural precursor counts (PCNA+, tbr2+, and GFAP+ cells) were reanalyzed using the absolute number of cells/section (instead of corrected for the number of Hoechst+/cells), similar results were obtained as present here.

Title: Movement maintains forebrain neurogenesis via peripheral neural feedback in larval zebrafish Summary: Sensory experiences early in life help the brain to grow and develop. For example, raising animals in complete darkness stops the visual areas of their brain from forming properly. Previous studies have shown that sensory input helps to strengthen the connections between already existing brain cells, but it is unclear if it affects the actual creation of new brain cells. Conditions that reduce the mobility of young children, such as muscular disease, are often accompanied by learning difficulties. This suggests that physical movement may be important for healthy brain development. Scientists have previously found a link between exercise and an increased production of new brain cells in adults. However, such a link has not been established earlier in life, when the brain is developing the most. To address this, Hall and Tropepe studied how movement affects the brain development in zebrafish larvae, at an age when many of their brain cells are created. Restraining the larvae decreased their physical movement, while making them swim against a current increased their movement. Hall et al. looked at how this affected the larvae's number of so called progenitor cells - the cells from which brain cells are created. When the larvae moved less, the number of progenitor cells decreased. But when they moved more frequently, the amount of progenitor cells increased. The experiments also showed that some sensory cells, which detect sensations associated with movement of the body during swimming, are linked to brain cell production. Blocking the development of these sensory cells prevented the rise in progenitor cells seen with increased movement in the larvae. However, activating these sensory cells in immobilised larvae increased the number of progenitor cells, even though the larvae could not move. These findings suggest that measures to increase physical movement in young children could be used to help the brain develop normally.

lay_elife

en

Summarize: TECHNICAL AREA The present invention relates to a medicament delivery device and in particular a medicament delivery device utilizing multi-chamber medicament containers that require mixing before drug delivery to a patient. BACKGROUND OF INVENTION It is becoming more and more common to use multi-chamber medicament containers in medicament delivery devices such as injectors. The reason for this is that the medicament can be stored for much longer time periods without being degraded in comparison with medicament dissolved in some liquid. Thus the medicament and the liquid are kept in different compartments in the medicament container and are mixed just before use by moving a dividing wall or stopper such that the compartments can communicate with each other. However, the multi-chamber medicament containers entail more handling steps before a dose of medicament can be injected in that the plunger rod of the injector has to move the stopper or stoppers of the medicament container in order to initiate the mixing. Another feature of many medicament delivery devices and in particular injectors is the attachment of a medicament delivery member, in particular an injection needle to a medicament container and then how to avoid unintentional needle sticks. Document WO2010/000559 discloses a medicament delivery device utilizing a multi-chamber medicament container where the mixing is obtained by rotating a medicament container holder, positioned in the proximal housing part, into a distal housing part whereby the stopper of the medicament container is moved against a plunger rod. In the initial position the proximal end of the medicament container is protruding beyond the proximal housing part, where the latter also acts as a needle shield, such that a medicament delivery device can be attached to the medicament container holder. When the mixing has been performed, the medicament delivery member is drawn into the proximal housing/needle shield. The needle shield is now used for actuating the device in that when a penetration is performed, the needle shield is pushed in the distal direction, whereby it triggers an auto-injection sequence. Thus the needle shield assembly extends almost to the distal end of the device in order to be able to actuate the injection. However, for some devices it is neither necessary nor desirable to have so many components and functions since they tend to make the devices more complex and more expensive. Also the holding of the device during mixing may be performed with one hand rather close to the proximal end of the device instead of the distal end of the device. There is thus room for development of medicament delivery devices. BRIEF DESCRIPTION OF INVENTION The aim of the present invention is to provide a medicament delivery device that can handle multi-chamber medicament containers in a simple and intuitive but yet safe way. According to a main aspect of the invention it is characterised by a medicament delivery device according to the features of the independent patent claim. Preferable embodiments of the invention form the subject of the dependent patent claims. According to a main aspect of the invention it is characterised by a medicament delivery device comprising a body which comprises a housing and a medicament container holder accommodating a multi-chamber medicament container, wherein said housing and said medicament container holder are interactively connected to and movable relative each other; the medicament container holder comprising attachment means for attaching a delivery member; a mixing guard mechanism interactively connected to the body for driving the medicament container holder within the housing and thereby achieving a reconstitution wherein the mixing guard mechanism comprises a mixing cylinder unit and a tubular medicament delivery member guard operably connected to the medicament container holder, such that when the mixing cylinder unit is operated, the medicament container holder is moved in relation to the medicament delivery member guard between a pre-mix position in which said attachment means protrudes through a proximal end of said medicament delivery member guard for allowing the attachment of a delivery member and a mixed position in which said attachment means and said attached delivery member is positioned within the medicament delivery member guard, wherein said mixing guard mechanism further comprises a guard lock and release member operably connected to said guard and to said medicament container holder, such that said guard lock and release member is configured to lock said guard when the container holder is in the pre-mix position and to release said guard when the container holder is moved to the mixed position. According to another aspect of the invention movement in the distal direction of the guard member, when the device is pressed against a dose delivery site, causes a turning of a rotator member comprised in said mixing cylinder unit. According to further aspect of the invention said turning is caused by said rotator member comprising at least one groove inclined in relation to the longitudinal direction of the device, cooperating with at least one protrusion arranged on the guard during distal movement of said guard. According to yet another aspect of the invention said mixing guard mechanism further comprises a resilient member operably arranged to said guard for urging said guard in the proximal direction. According to yet a further aspect of the invention the rotator member comprises a first guard locking means interactively connected to the rotator member such that proximal movement of said guard, after removal from a medicament delivery site, causes a locking of said guard in a proximal position covering said medicament delivery member. According to another aspect of the invention said mixing guard mechanism comprises first connection means, interactively connecting said housing and said medicament container holder for allowing the medicament container holder to be displaced within the housing. According to a further aspect of the invention said first connection means comprises cooperating threads on said housing and said medicament container holder. According to yet another aspect of the invention said first guard locking means comprises at least one proximally directed resilient tongue arranged to said rotator member and at least one protrusion arranged to said guard such that when said guard is proximally displaced, said at least one protrusion passes said at least one tongue, thereby preventing subsequent distal displacement of said guard. According to yet a further aspect of the invention said medicament container holder and said guard are slidably connected but rotationally locked to each other by second connection means. According to another aspect of the invention said mixing cylinder is arranged to said guard such that they are slidably connected but rotationally locked to each other by third connection means. According to a still further aspect of the invention said mixing cylinder and a connector sleeve are fixedly connected to each other by fourth connection means. According to yet another aspect of the invention said connector sleeve and said housing are connected to each other by fifth connection means which prevent a longitudinal movement in relation to each other but allow rotation in relation to each other. According to a further aspect of the invention said rotator member and said connector sleeve are connected to each other by sixth connection means which prevent a longitudinal movement in relation to each other but allow partial rotation in relation to each other, and wherein said connector sleeve and said rotator member are coaxially arranged within said mixing cylinder. According to yet a further aspect of the invention said rotator member and said mixing cylinder are one-direction-rotatably connected to each other by seventh connection means. According to another aspect of the invention it further comprises drive means arranged within said housing and adapted to drive a stopper positioned within said multi-chamber container. According to yet another aspect of the invention said drive means comprises a plunger rod and a force member operatively acting on said plunger rod for urging it in the proximal direction. According to a further aspect of the invention it further comprises a holding means for holding said drive means in a pre-tensioned state. According to yet a further aspect of the invention it comprises activation means capable of acting on said holding means for releasing the drive means from the pre-tensioned state According to another aspect of the invention the device is an injection device. There are several advantages with the present invention. The use of a tubular medicament delivery member guard operatively connected to the medicament container holder and the mixing means provides the possibility of attaching a medicament delivery member to the device and then during the mixing operation cover the medicament delivery member by activating the tubular guard member. By using a rotator member, several functions of the device are obtained with few components and provides the interaction of functions. Further the mixing guard mechanism enables a pushing of the tubular guard in the distal direction when the device is pressed against a dose delivery site. This movement of the tubular guard causes components of the mixing guard mechanism to be moved and thus prepared for a locking of the tubular guard member when the device is removed from the dose delivery site after completed dose delivery, wherein the tubular guard is pushed in the proximal direction by the spring force, thereby surrounding the medicament delivery member in a locked state, whereby the medicament delivery member cannot be tampered with. These and other aspects of, and advantages with, the present invention will become apparent from the following detailed description of the invention and from the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS In the following detailed description of the invention, reference will be made to the accompanying drawings, of which FIG. 1 is a perspective view of a medicament delivery device according to the present invention, FIG. 2 is a cross-sectional view in perspective of the device of FIG. 1, FIG. 3 is an exploded view of the device of FIG. 1, FIG. 4 is a detailed exploded view of a medicament delivery member guard and a proximal housing part, FIG. 5 is a detailed exploded view of key components of the present invention, FIG. 6 is the same view as FIG. 4 turned 180°, FIG. 7 is a detailed cross-sectional view in of a proximal part of the device of FIG. 1, FIG. 8 is a detailed view of a component comprised in the present invention, and FIGS. 9-13 are cross-sectional views in perspective, partly in detail, displaying different functional states. DETAILED DESCRIPTION OF THE INVENTION In the present application, when the term “distal part/end” is used, this refers to the part/end of the delivery device, or the parts/ends of the members thereof, which is/are located the furthest away from the medicament delivery site. Correspondingly, when the term “proximal part/end” is used, this refers to the part/end of the delivery device, or the parts/ends of the members thereof, which, is/are located closest to the medicament delivery site. The medicament delivery device shown in the drawings comprises a body, which in turn comprises a generally elongated housing 10 and a generally tubular medicament container holder 12, FIG. 3. The proximal end of the medicament container holder comprises a neck portion 14 onto which a medicament delivery member 16 may be releasably attached. The interior of the medicament container holder is arranged to house a multi-chamber medicament container 18, having a proximal end fitting into the neck portion 14 of the medicament container holder 12. The interior of the medicament container 12 contains a number of movable stoppers 20, 22, which stoppers form a number of chambers 24, 26 containing medicament in powder form and a diluent. A drive mechanism is further arranged in the housing, comprising an elongated plunger rod 28 having a proximal end in contact with the stopper 20 at the distal end of the medicament container 12. The plunger rod 28 is operatively connected to a drive force member 30 of the drive mechanism operatively acting on the plunger rod 28 for urging it in the proximal direction. The device further comprises a holding member 32 capable of holding the drive force member 30 in a pre-tensioned state as well as an actuation member 34 capable of acting on said holding member 32 for releasing the drive force member 30 from the pre-tensioned state, FIG. 2. The actuation member 34 comprises an actuation button 36 extending through a distal end of the housing. The device further comprises a mixing guard mechanism arranged for mixing the content of the chambers of the multi-chamber medicament container. Thereby the housing 10 and the medicament container 12 are interactively connected to each other by first connection means for allowing the medicament container holder to be displaced within the housing. The first connection means comprises threads 38 on the outer surface of the medicament container holder 12, which threads 38 cooperate with corresponding threads 40, FIG. 2, on the inner surface of the housing 10. The mixing means further comprises longitudinally extending grooves 42, FIG. 3, on the outer surface of the medicament container holder 12, which grooves 42 cooperate with corresponding ribs 44, FIG. 5, on an inner surface of a generally tubular medicament delivery member guard 46, forming second connection means. Further a guard member lock and release member is arranged wherein the outer surface of the medicament container holder 12 at its proximal end is arranged with two oppositely positioned outwardly directed protrusions 49, FIG. 4. These protrusions 49 are arranged to fit into longitudinal slits or grooves 48 arranged in the medicament delivery member guard 46. The medicament delivery member guard 46 is further arranged with a circumferential ledge 47 around the opening at the proximal end. The ledge is arranged to abut a proximal end surface 51 of the medicament container holder surrounding the neck portion 14, thereby preventing movement of the medicament delivery member guard 46. Further the medicament delivery member guard 46 is arranged slidable inside a generally tubular mixing cylinder 50 but rotationally locked, via longitudinal grooves 52 on the outer surface of the medicament delivery member guard 46 cooperating with inwardly protruding ledges 54 on the proximal end of the mixing cylinder 50, FIG. 6, forming third connection means. The medicament delivery member guard 46 is urged in the proximal direction in relation to the mixing cylinder 50 by a spring member 56, FIG. 7, acting between a distal end surface of the medicament delivery member guard 46 and a proximal end surface of the housing 10. Further the mixing cylinder 50 is rotatably attached to the proximal end of the housing 10 via a connector sleeve 58 by a fourth connection means such that it fits into said mixing cylinder 50 and rotationally locked by ribs 57 on the outer surface of the connector sleeve 58 fitting into grooves 59 on the inner surface of the mixing cylinder 50, FIGS. 5, 6. The connector sleeve 58 is attached to the mixing cylinder by outwardly flexible tongues 60 fitting into recesses 62 on the inner surface of the mixing cylinder 50, FIGS. 5 and 6. The connector sleeve 58 is further arranged with a circumferential, inwardly extending ledge 64 in contact with a circumferential ledge 66 of the outer surface of the proximal end of the housing 10, forming a fifth connection means. Further a generally cylindrical rotator 68 is arranged rotatable inside said mixing cylinder 50 in its proximal end and held in place by the connector sleeve 58 with a sixth connection means. The proximal end surface of the rotator 68 is arranged with flexible tongues 70 extending along the circumference, which are provided with proximally directed wedge-shaped ledges 72, FIG. 8. The ledges 72 cooperate with wedge-shaped ratchets 74 on a distally directed surface of a circumferential ledge 76 of the mixing cylinder 50, such that the rotator 68 can only rotate in one direction in relation to the mixing cylinder 50, forming seventh connection means. The rotator 68 is further arranged with proximally directed, and inwardly flexing, tongues 78 having proximally directed end surfaces 79, FIG. 8, which tongues 78 are arranged in grooves 80 on the inner surface of the rotator 68, which grooves 80 extend in the longitudinal direction of the device, forming eighth connection means. Further the longitudinal grooves 80 are cooperating with inclined grooves 82 that meet at the distal end of the longitudinal grooves 80, the function of which will be described below. Protrusions 84 are further arranged on the outer surface of the medicament delivery member guard 46, which protrusions 84 are arranged to cooperate with the grooves 80, 82 of the rotator 68, as will be described below. The device is intended to function as follows. When the device is delivered to a user, the proximal end of the medicament container holder 12 protrudes through the proximal end of the medicament delivery member guard 46, FIG. 2. This enables a medicament delivery member 14 to be attached to the proximal end of the medicament container holder 12. In this position the proximal end surface 51 of the medicament container holder 12 is in contact with the circumferential ledge 47 of the medicament delivery member guard 46, whereby the latter is locked against distal movement, FIG. 9. After a medicament delivery member 16 has been attached, the next step is to perform the mixing. This is performed by rotating the mixing cylinder 50 in relation to the housing 10. This causes the medicament container holder 12 to be moved in the distal direction because of the threads 38 of the medicament container holder 12 cooperating with the threads 40 of the housing. The distal movement of the medicament container holder causes the medicament delivery member 14 to be moved into the medicament delivery member guard 46, FIG. 10. The protrusion 49 on the container holder 12 will slide in the groove 48 of the medicament delivery member guard 46 until the container holder and thus the proximal end of the medicament delivery member has reached a certain distance in relation to the proximal end of the guard 46, FIG. 11, which also releases the guard such that it is movable in the longitudinal direction against the force of the spring member 56. In this position the protrusions 49 will come in contact with the distal end of the grooves 48 whereby the guard 46 will also be moved in the distal direction until the mixing sequence has been completed. The next step is to press the proximal end of the device against the medicament delivery site, such an injection site. The pressing of the device causes the medicament delivery member guard 46 to be pushed in the distal direction into the device and against the force of the medicament delivery member guard spring drive member 56, thereby exposing the medicament delivery member 16. The distance that the container holder 12 and its medicament delivery member 16 have been previously moved distally in relation to the guard, i.e. the length of the groove 48, correspond to a desired and set injection depth when the medicament delivery member is an injection needle. The distal movement of the medicament delivery member guard 46 causes its protrusions 84 to slide along the side surfaces of the inclined grooves 82 whereby the rotator 68 is turned. When the device is fully pressed against the delivery site, the medicament delivery mechanism is activated and a dose of medicament is delivered through the medicament delivery member 16, FIG. 12. Thus, when the actuation button 36 is pressed, the lock member releases the drive member for delivering a dose. However, instead of an automatic dose delivery function, there could instead be a purely manual dose delivery function such that the actuation button acts directly on the plunger rod, whereby the latter is pushed in the proximal direction when the actuation button is pressed. Also, the device may be provided with some kind of actuation locking and release means which acts to keep the actuation mechanism locked until a mixing operation is completed, in order to avoid unintentional activation until the device is ready for use. When a dose of medicament has been delivered, the device may be withdrawn from the site. This causes the medicament delivery member guard 46 to be again pushed in the proximal direction, whereby its protrusions 84 slide along the longitudinally directed grooves 80 of the rotator 68 until the protrusions 82 slide over and pass the tongues 78, after which the medicament delivery member guard 46 is covering the medicament delivery member. This will lock the medicament delivery member guard 46 against being pushed again in the distal direction in that its protrusions 84 will come in contact with the proximal end surfaces 79 of the tongues 78, which, in the case when the medicament delivery member is an injection needle, prevents unintentional needle sticks, FIG. 13. The device may now be discarded. It is to be understood that the embodiment described above and shown in the drawings is to be regarded only as a non-limiting example of the invention and that it may be modified in many ways within the scope of the patent claims.

Summary: A medicament delivery device includes a housing arranged to receive a medicament container, a drive unit arranged to act on a stopper in the medicament container, which drive unit includes a torsion drive spring. A dose setting member is operably connected to the drive unit such that a manual turning of the dose setting member causes a tensioning of the torsion drive spring. The device also includes an activation mechanism having an activation button protruding through a distal end of the device. The activation mechanism is operably connected to the drive unit such that actuation of the activation mechanism causes a release of the drive unit, with a set dose of medicament delivered.

big_patent

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Write a title and summarize: Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p) ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p) ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues. Families of small P-loop GTPases are universally employed as molecular switches in all domains of life [1]. In E. coli there are ∼20 known GTPases, and most of them are involved in cellular processes related to ribosome functions. Among them, ObgE (Obg in E. coli, also known as CgtA or YhbZ) belongs to the highly conserved Obg GTPase family (spo0B-associated GTP-binding protein). Obg proteins are essential for cell growth in all tested bacterial species ([2] and references therein). The ObgE homologs are also widely present in eukaryotic organelles, including both chloroplasts and mitochondria. Despite extensive genetic studies of Obg proteins in several bacterial species, the exact molecular role of this protein family is still unclear. On the cellular level, it has been implicated in a variety of regulatory events in different species, including cell cycle control, DNA replication, stress response, sporulation, morphological development, and ribosome assembly (reviewed in [2], [3]). The pleiotropic phenotypes of Obg proteins indicate that this protein family might participate in certain essential processes that are central to various cellular functions. In line with this view, ObgE and its homolog in Vibrio cholera have been implicated in the (p) ppGpp-mediated stringent response [4]–[6]. The ppGpp (guanosine tetraphosphate) and pppGpp (guanosine pentaphosphate), collectively called as (p) ppGpp, are important secondary messengers in bacteria and plants, regulating many cellular activities in response to various environmental stresses (reviewed in [7], [8]) by targeting transcription factors, GTPases, as well as proteins in nucleotide and lipid metabolism [9]. The first evidence implicating Obg in the (p) ppGpp pathway was from the crystal structure of the Bacillus subtilis Obg protein, in which a ppGpp molecule was found in the active site of its GTPase domain (GD) [10]. Later, it was shown that ObgE binds to ppGpp with a physiological affinity in vitro [4], and perturbation of ObgE function by different non-lethal mutations affects the total level of (p) ppGpp or the relative ratio of pppGpp/ppGpp during different growth phases [4], [5]. Furthermore, ObgE and its V. cholera homolog were found to co-purify and interact with SpoT [5], [6], [11], the cellular enzyme responsible for hydrolyzing (p) ppGpp to GTP or GDP [12], postulating a possible role of ObgE on (p) ppGpp degradation by SpoT. These observations have established a link between Obg proteins and the regulation of (p) ppGpp, and suggested that many phenotypes associated with mutant Obg proteins might originate from impaired temporal control of the cellular (p) ppGpp level. In the meantime, converging biochemical evidences show that Obg proteins also physically interact with the ribosome or ribosomal subunits; the observations include bacteria Vibrio harveyi, E. coli, Caulobacter crescentus, Salmonella typhimurium, B. ubtilis, Chlamydia abortus, and Mycobacterium tuberculosis (reviewed in [3]). It was also revealed that mutations in ObgE lead to cellular defects in the 50S subunit maturation [13], [14]. Moreover, Obg proteins from E. coli and S. typhimurium were shown to have physical [15] or genetic interaction [16] with 23S rRNA modification enzymes. On the basis of these data, a primary role of ObgE in the 50S subunit maturation was proposed [13], [14]. Given the absolute requirement of ribosome function for all cellular activities, this provides an alternative explanation of pleiotropic phenotypes of Obg proteins in multiple disparate cellular events. In the present work, we demonstrate that ppGpp enhances the binding of ObgE to the 50S subunit and promotes dissociation of the 70S ribosome into subunits. Interestingly, we find that ObgE plays an important role as a 50S based anti-association factor, which inhibits the formation of 70S ribosomes from the naked subunits as well as from an mRNA programmed 30S-preinitiation complex (30S-preIC). The inhibition in subunit association also leads to a slower dipeptide formation when ObgE is bound to the 50S subunit. Importantly, a C-terminus deleted construct of ObgE (ObgE-NG), containing the N-terminal domain (NTD) and the central GD, is sufficient for its anti-association activity, suggesting that the NTDs constitute the main activity center of ObgE. Next, we solved the cryo-EM structure of the 50S subunit bound with ObgE. Structural data reveal that the NTD of ObgE is a structural mimic of the A-site tRNA, which exhibits specific interactions with the ribosomal peptidyl-transferase center. Together with previously published data, our results suggest that ObgE might be an important player in the (p) ppGpp regulatory circuit, regulating the assembly of the 50S subunit, and blocking the subunit association and further downstream events in protein translation in response to signal of the nutrient availability. Previous studies showed that endogenous ObgE co-fractionates mainly with the 50S fraction [5], [11], [13], [14], In addition, GTP or GMPPNP was proven to stimulate the binding of ObgE to the 50S subunit [13] or to the 23S rRNA [14]. However, it is not clear whether (p) ppGpp could also modulate the binding of ObgE to the 50S subunit. To test this possibility, we performed co-sedimentation assay to examine the binding of ObgE to the 50S subunit in the presence of different nucleotides. Although ObgE in the apo state showed weak binding to the 50S subunit, addition of guanine nucleotides significantly enhanced its binding to the 50S subunit (Figure 1A). Especially, ppGpp increased the occupancy of ObgE on the 50S subunit by over 5-fold, compared to the apo state. In addition, similar to other ribosome-interacting GTPases, ObgE showed a higher affinity to the 50S subunit in the presence of GTP or GMPPNP than GDP (Figure 1A). These observations indicate that ppGpp binding, similar to GDP and GTP, indeed influences the interaction between ObgE and the 50S subunit. The marked effect of ppGpp, as well as the subtle difference with other nucleotides, on the 50S binding ability of ObgE, suggests that ObgE might sense changes in the nucleotide pool during different growth phases and adjust its behavior accordingly. Previously, it was shown that overexpression of the plasmid-encoded ObgE leads to the co-fractionation of the protein with the 70S, as well as polysome fractions [5]. We then sought to examine whether ObgE could bind to the 70S ribosome in vitro. However, unexpectedly, ObgE was found incompatible with the 70S ribosome when added in excess, and the latter was disassembled into subunits (Figure 1B and 1C). Upon subunit dissociation, ObgE remained associated with the separated 50S subunits (Figure 1D). Importantly, this dissociation of 70S ribosomes did not seem to require energy input from GTP-hydrolysis, because both GDP and ppGpp also enabled this 70S-spliting activity (Figure 1C). As illustrated in Figure 1B, the 70S dissociation was dependent on ObgE concentration. Further comparison of the extent of 70S disassembly in the presence of different nucleotides indicates that the 70S dissociation activity of ObgE is well correlated with its 50S binding ability (Figure 1A). As expected from the results presented in Figure 1A, the maximal dissociation was observed when ObgE was saturated with ppGpp (Figure 1C). Obg proteins from different species contain very diverse C-terminal domains (CTDs), connected to the GDs by long flexible linkers [3], [17]. We therefore tested whether the CTD plays any role for the dissociation of 70S ribosomes. As shown in Figure S1, deletion of the CTD does not impair the binding of ObgE-NG to the 50S subunit (Figure S1A), and ObgE-NG is sufficient to promote dissociation of 70S ribosomes in a way similar to full-length ObgE; the maximal dissociation is seen in the presence of ppGpp (Figure S1B and S1C). Altogether, these data indicate that ObgE binds to the 50S subunit preferentially over the 70S ribosome, and the association is likely mediated by its conserved NTD. Most likely, by binding to the 50S subunit ObgE prevents the re-association of the 30S subunit thereby shifting the 70S dissociation equilibrium toward free subunits in the steady-state. We have studied the effect of ObgE in ribosomal subunit association using naked 30S subunits or an mRNA programmed 30S-preinitiation complex (30S-preIC) containing fMet-tRNAfMet in the P-site. The kinetics of subunit association was followed by monitoring the increase in light scattering after rapid mixing of the 30S (or 30S-preIC) and 50S subunits in a stopped-flow instrument. In both cases, the rates of subunit association, in the absence of ObgE, matched closely with previously published results [18], [19]. ObgE, as expected from its preferential binding to the 50S subunit, showed a strong inhibition on the subunit association both for naked 30S (Figure 2A) and 30S-preIC (Figure 2B). The rates of the subunit association [ (kobs 30S = 4±1 s−1) and (kobs 30S-preIC = 20±0. 2 s−1) ] decreased gradually with increasing concentration of ObgE (Figure 2C). Subsequently, the mean time for subunit association, estimated as the reciprocal of the observed rate (1/kobs), increased linearly with ObgE concentration (inset in Figure 2C), thereby suggesting that ObgE competitively inhibits the 30S subunit for binding to the 50S subunit. By comparing the concentration of ObgE required for half-maximal inhibition (Figure 2C), it is evident that the ObgE-mediated inhibition is stronger in the 30S-preIC association than the naked subunit association. For the 30S-preIC association the Ki is around 0. 3 µM, while the same for naked 30S association is 2 µΜ. Highly consistent with full-length ObgE, the C-terminal deleted ObgE-NG also showed inhibition of subunit association in an extent comparable to ObgE (Figure 2A and 2B). For ObgE-NG the estimated Ki values for inhibition of 30S-preIC and naked 30S association are 1 and 3. 7 µΜ, respectively (Figure 2D). The inhibition was more profound in the presence of guanine nucleotides with ObgE, both GTP and ppGpp led to higher inhibition of subunit association (Figure 2E), as expected from the higher affinity of ObgE to the 50S subunit in the presence of these guanine nucleotides (Figure 1A). Thus, our results demonstrate that ObgE blocks subunit association and hence translation initiation by binding to the 50S subunit, and the NTD of ObgE is likely the main activity center. We have further tested the effect of ObgE in dipeptide synthesis starting from 30S-preIC or 70S-initiation complex (70S-IC). The formation of fMet-Leu (ML) dipeptide starting from 30S-preIC required about 200 msec (kobs dipep 30S-preIC = 4. 7±0. 2 s−1), which involved subunit association followed by peptide bond formation. In the presence of ObgE with 50S containing elongation mix (see Materials and Methods for details), the rate of dipeptide formation slowed down about four times (Figure 2F, kobs dipep 30S-preIC obgE = 1. 2±0. 05 s−1). However, ObgE did not show any effect on the rate of dipeptide formation when 70S-IC was previously associated and ObgE was added with the ternary complex (kobs dipep 70S-IC = 33±2 s−1 and kobs dipep 70S-IC ObgE = 30. 3±1 s−1) (Figure 2F, inset). Thus, our results suggest that the ObgE has no effect on peptide bond formation. The defect seen in dipeptide formation starting from 30S-preIC was due to its anti-association activity. When ObgE is bound to the 50S subunit it blocks association and consequently the downstream steps in protein synthesis get inhibited. Following these observations, we tested the effect of ObgE on translation in a multiple turn-over reaction. As expected, ObgE inhibits the in vitro translation of a reporter gene in a dose-dependent manner (Figure S2). Consistent with the in vitro data, overexpression of ObgE in E. coli cells leads to a slower growth (Figure S3A and S3B) and a substantial increase of free 50S fractions in the ribosome profile (Figure S3C and S3D). Thus, both our in vitro and in vivo results suggest that ObgE acts as an anti-association factor. The role of IF3 as an anti-association factor is well-known [20], which binds primarily to the 30S subunit and prevents premature association of the ribosomal subunits. We show that ObgE demonstrates a similar anti-association activity by binding to the 50S subunit. We next determined the cryo-EM structure of the 50S subunit bound with ObgE·GMPPNP. The cryo-EM density map was solved at a nominal resolution of 5. 5 Å. As shown in Figure 3, ObgE binds to the intersubunit face of the 50S subunit, at a position commonly used for the docking of translational GTPases. Specifically, the NTD of ObgE protrudes into the peptidyl-transfer center (PTC), and its GD is situated between the bL12 (adopted after a newly proposed ribosomal protein naming system described in [21]) stalk base and the sarcin-ricin loop (SRL) of the 23S rRNA (Figure 3A and 3B). However, we did not find extra densities that could be attributed to the CTD of ObgE, indicating that this domain is highly flexible. Structural superimpositions of ObgE with four translational GTPases (IF2, EF-Tu, EF-G, and RF3) on the 50S subunit all report a large steric clash (Figure S4), indicating that the binding of ObgE is incompatible with these translation factors on the 50S or 70S ribosomes. Both the 50S subunit and ObgE undergo conformational changes upon the complex formation. Compared with crystal structures of Obg proteins [10], [17], a large scale rotation between the NTD and GD (Figure S5) is necessary to assume the 50S bound conformation. On the 50S subunit side, significant conformational changes are seen on uL1 stalk, bL12 stalk, helix 38, helix 34, helix 58, as well as helix 69 of the 23S rRNA (Figure 3C), all localized in the intersubunit face. These changes are well correlated with the local resolution map of the 50S·ObgE complex (Figure 3D). Very interestingly, upon binding to ObgE, helix 69 is seen to have a massive movement by about 19 Å (Figure 3C and 3F), which, if mapped on the 70S ribosome structure, would directly disrupt a strong intersubunit bridge (B2a). The B2a is essential for intersubunit association to form the 70S ribosome [22], and ribosome recycling factor (RRF) employs exactly the same mechanism to break the B2a during the ribosome recycling [23]. This large movement of helix 69, as well as the observed conformational changes on bridges B1a (helix 38) (Figure 3C and 3E) and B4 (helix 34) (Figure 3C and 3G), perfectly explains the anti-association activity of ObgE. Another intriguing conformational change takes place on the NTD of uL11. Upon the binding of ObgE, the uL11-NTD becomes “invisible” in the cryo-EM density map (Figure S6). The flexibility of the uL11-NTD probably results from the interaction between the ObgE-GD and the bL12 stalk base (H43-44). The ObgE-NTD is composed of an eight-stranded β-barrel base and a unique glycine-rich protrusion containing six left-handed helices of the poly-Pro type II conformation (Figure S5) [10], [17]. At the tip of the protrusion, connecting the six helices are three loops (Figure S5B). These loops are conserved in both sequence and length among Obg family proteins (Figure S7). The binding environment of the ObgE-NTD on the 50S subunit is exclusively in rRNA helices (Figure 4), including helix 89, helix 90, helix 91, helix 93, and the A-loop (helix 92). Consistent with our structural model, most of the conserved lysine and arginine residues in the ObgE-NTD are located at the rRNA interface (Figures 4C–4E and S7). Specifically, the tip of the NTD shows tight polar interaction with the PTC (Figure 4C and 4D). At the opposite end, a short helical insertion from the β-barrel base is also seen to interact with the junction between helix 89 and helix 91 (Figure 4E). To be more specific, several highly conserved basic residues from the three intervening loops, including R24, R25, K27, and K31 from loop 1; R76, K81, and R82 from loop 2; and R136 and R139 from loop 3, are within 4 Å distance from a number of the PTC residues, such as U2493, G2494, U2504, A2602, C2573, U2555, C2558, and C2507 (Figure 4). To confirm these structural observations, we introduced mutations to a few selected arginine or lysine residues on the three loops and tested the binding of ObgE mutants to the 50S subunit (Figure 5). As a result, all of the mutations impaired the binding, and especially, loop 1 (K27EK31E) and loop 3 (R136GR139G) mutants exhibited almost abolished binding activity. The sequence (Figure S7) and mutational data (Figure 5) indicate that the polar interactions between the ObgE-NTD and the PTC are highly likely very specific and conserved across species, which suggests that the recognition of the PTC is a universal function for the NTDs of all Obg proteins. Interestingly, the loop regions of the ObgE-NTD occupy the space that accommodates the acceptor arm of the A-site tRNA (Figure 6A). A comparison with the crystal structure of the 70S·RF2 complex [24] indicates that the tip of the ObgE-NTD overlaps exactly with the GGQ-motif containing domain of RF2 (Figure 6B). A close inspection at the PTC suggests that the two factors employ a very similar way to interact with this functional center (Figure 6C–6H). Specifically, when the P-site tRNA is superimposed with the 50S·ObgE structure, residues I29–K31 from loop 1 of ObgE are capable of forming interaction with CCA-end of the P-site tRNA, and K31 is inserted between A76 of the P-site tRNA and A2451 of the 23S rRNA (Figure 6E), displaying an astonishing resemblance to the GGQ-motif of RF2 (Figure 6D). Besides the possible interaction with the P-site tRNA, the ObgE-NTD also interacts with the A-loop of the 23S rRNA, in a strikingly similar way as RF2 does (Figure 6G and 6H). The structural similarity between the acceptor arm of a tRNA and the NTD of ObgE indicates that like many translation factors, ObgE also adopts a tRNA mimicry strategy to interact with the ribosome. The GDs of classical translational GTPases, such as IF2 [25], EF-G [26], EF-Tu [27], and RF3 [28], [29], all show only limited contact with the bL12 stalk base (containing uL11, H43, and H44) on the ribosome (Figure 7). Unlike these, the ObgE-GD itself interacts directly with the bL12 stalk base, and bridges the gap between SRL and bL12 stalk base (Figures 7A and S6). Other than that, compared with translational GTPases, the ObgE-GD is distinctively orientated on the 50S subunit, which places the Switch regions and the nucleotide binding pocket of the GD rather distant from the conserved A2662 of the SRL (Figure 7A and 7B). This unprecedented placement of the ObgE-GD on the 50S subunit immediately raises the question whether the 50S subunit serves as the GTPase-activating protein (GAP) for ObgE, as does for classical translational GTPases. Therefore, we performed a GTPase activity assay on ObgE in the presence or absence of purified 50S subunits. As expected, the basal GTPase level of ObgE is relatively low, similar to previous measurements [4], [11], [16]. The hydrolysis rate in the absence of the 50S subunit is ∼0. 0268 min−1; with a sufficient time ObgE is capable of converting all GTP to GDP (Figure 7G). In the presence of increasing amounts of 50S subunits, the phosphate production is accelerated accordingly (Figure 7H). When supplied with equal amount of 50S subunits, the hydrolysis rate is stimulated by about 120-fold, being about 3. 15 min−1. This moderate stimulation by the 50S subunit is in sharp contrast to translational GTPases, e. g., the GTP hydrolysis on EF-Tu and EF-G is enhanced by the 70S ribosome by over seven orders of magnitude [30]. More importantly, the binding and subsequent GTP hydrolysis of translational GTPases are regulated by the dynamic bL12 stalk on the ribosome in very distinct ways [19], [31]–[33]. It remains to be tested whether the bL12 stalk has any role in activating the GTPase of ObgE. Nevertheless, it is clear that ObgE represents a novel class of ribosome-interacting GTPases, whose GTPase activation mechanism should be different from those of classical translational GTPases. In the present work, we reveal that the interaction between the PTC and the evolutionarily conserved NTD of ObgE is highly specific (Figures 4 and 5). This suggests that a primary molecular role of ObgE is directly related to the ribosome, which is consistent with the proposed role of ObgE in the ribosome assembly [13], [14]. The assembly function of ObgE started with a genetic study showing that overexpression of ObgE could suppress the slow growth and ribosome profile defect of the ΔrrmJ strain [16]. RrmJ is a 23S rRNA methyl-transferase (U2552 of the A-loop) required for late-stage 50S subunit assembly [34]. Later it was also shown that Obg homolog from S. typhimurium physically interacts with another 23S rRNA modification enzyme RluD (pseudouridine synthatase for ψ1911, ψ1915, and ψ1917 of helix 69) in vitro [15]. Consistently, pull-down experiment indicates ObgE also co-localizes with factors required for the 50S assembly, such as CsdA and DnaK [14]. Further analysis of the pre-50S subunits accumulated in an ObgE mutant (G80ED85N) strain [13] revealed several 50S maturation defects, including reduced binding of several 50S proteins (e. g., bL33, bL34, and uL16), impaired 23S rRNA processing, and prolonged association of RluC (pseudouridine synthase for ψ955, ψ2504, and ψ2580) and RrmJ with the pre-50S subunits. From our structural data, the binding position of ObgE is exactly next to the modification sites of RrmJ, RluD, and RluC (Figure S8). The release of ObgE from the 50S subunit, therefore, might mark the finish point of the 50S assembly, considering that ObgE might act very late in the assembly pathway [13]. In this sense, ObgE could be a checkpoint protein during the late-stage assembly, which monitors the modification status of these critical residues and local conformation of the PTC. The correctly modified and assembled 50S subunit then signals the GTP-hydrolysis and subsequent release of ObgE (Figure 8A). Escape from this quality control mechanism results in hypo-modified 50S subunits into the translation pool, leading to a profound impact on translation. For examples, lack of methylation at U2552 increases translation accuracy at the expense of efficiency [35], and deletion of rluD gene in E. coli results in a defect in translation termination, with an increased rate of stop codon read-throughs [36], [37]. Therefore, the reported diverse phenotypes, associated with various ObgE mutants, might be at least partially originated from subtle changes on cellular translatome profile, related to specific proteins in different cellular events. The stronger interaction of ObgE with the 50S subunit in the presence of ppGpp (Figure 1), therefore, highly likely reflects another level of regulation on the ribosome assembly by (p) ppGpp during stringent response, which is to delay the 50S assembly by a prolonged association of ObgE·ppGpp with the pre-50S subunits, in addition to the well-known direct role of the (p) ppGpp on rRNA transcription [7]. When GTP is plenty in the mid-log phase, ObgE functions primarily as a 50S assembly factor to facilitate the 50S subunit maturation. In contrast, when the cells are challenged by nutrient limitation or enter stationary phase, the intracellular level of ppGpp sharply rises, and ObgE is dominantly modulated by ppGpp. As an effector, ObgE·ppGpp over-stays on the 50S subunits, and consequently, downregulates the subunit production (Figure 8B). In the process of eukaryotic ribosome assembly, many 60S and 40S maturation factors also possess anti-association activity, and some of them have functional implications in translation initiation (reviewed in [38], [39]). One such example is a 60S subunit assembly factor eIF6 (Tif6 in yeast), which binds to the subunit interface of the pre-60S particles beside the SRL [40], [41] and blocks the subunit association and downstream initiation events. Mammalian eIF6 has also been shown to have a profound role in translation control (reviewed in [42]). Interestingly, in yeast the release of Tif6 from the pre-60S particles by an assembly GTPase Efl1 is triggered by the maturation state of the P-site on the 60S subunit [43]. Taken together, it is apparent that there are common quality control mechanisms in the assembly of bacterial and eukaryotic ribosomes, which make use of the maturation states of functional centers (such as PTC) on the subunits as structural checkpoints. Our biochemical results demonstrate that ObgE possesses anti-association activity in addition to its role in the 50S subunit maturation. By binding to the 50S subunit ObgE prevents association of the naked 30S subunit as well as the properly programmed 30S-preIC to the 50S subunit. Our structural analysis shows that ObgE obstructs the binding of the 30S subunit by inducing significant conformational changes at several intersubunit bridging contacts on the 50S subunit, including B1a, B2a, and B4 (Figure 3). The anti-association activity of ObgE can have deep implications in protein synthesis and bacterial physiology. Under normal growth conditions, the binding of ObgE to pre-50S subunits prevents defective subunits from being engaged in translation, thereby minimizing the chance of inefficient and faulty protein synthesis. However, under stress conditions, when ppGpp level increases in the cell, ppGpp bound ObgE not only delays the maturation of the 50S subunit, but also sequesters a large number of mature 50S subunits from taking part into translation, thereby lowering the number of active 70S ribosomes and thus, regulating the rate of total protein synthesis in the cell. Given the universal distribution of Obg proteins and (p) ppGpp system in bacteria and eukaryotic organelles [44], the action of ObgE might represent a conserved regulatory mechanism on translation in response to fluctuations in cellular energy level caused by nutrient availability. It must be noted that in bacteria both (p) ppGpp synthetase RelA and hydrolase SpoT are associated with the ribosome, and especially, the (p) ppGpp production by RelA is well documented to be strictly dependent on deacylated A-site tRNA on the 70S ribosome [45], [46]. Our structural data show that the NTD of ObgE exactly mimics the CCA-end of the A-site tRNA. Furthermore, ObgE and SpoT were shown to co-fractionate with the pre-50S fractions [5], [11]. All these pieces of information seem to suggest that ObgE might also have additional functional interplay with RelA and SpoT. Whether or not ObgE could directly act as a regulator of the (p) ppGpp pathway remains to be investigated. A handful of genetic studies have also implicated Obg proteins in other cellular processes, such as DNA replication, chromosome segregation, and other stress response pathways (reviewed in [2], [3]). Interestingly, many of these pleiotropic phenotypes associated with Obg dysfunction appear to be species-specific, and could be attributed to the high sequence diversity within the CTDs of Obg proteins (Figure S7). For example, dysfunction of ObgE in E. coli causes cellular defects in DNA replication and chromosome segregation [47]–[50]. Intriguingly, these mutations with defects in DNA synthesis are primarily located to the CTD and GD of ObgE. For another example, Obg proteins in B. subtilis and M. tuberculosis were demonstrated to be involved in σB-controlled general stress response [51], [52], and again, the CTD of the B. subtilis Obg is required for the binding to the anti-σB factor RsbW [53]. However, it must be stressed that many reported functions of Obg proteins are not independent of the ribosome or (p) ppGpp-mediated pathways. The DNA replication is known to be regulated by (p) ppGpp [54], and regulators of the σB-dependent general stress response in B. subtilis also appear to be ribosome-associated [55], [56]. Our structural data suggest that the binding of ObgE induces a conformational change on the uL11-NTD, resulting in the displacement of the uL11-NTD from its normal position (Figure S6). This is highly consistent with roles of uL11 as key regulators in both the stringent response in E. coli [46] and the σB-dependent general stress response in B. subtilis [57]. In addition, we show that the conserved function of the ObgE-NTD is to interact with the 50S subunit, in a similar way as the A-site tRNA does, and the CTD is not required for its 50S binding and anti-association activity. Therefore, the species-specific functions of Obg proteins suggest that Obg proteins might act as a specialized translation factor, partnering, through their CTDs, with distinct players in different growth control pathways to regulate ribosome assembly and protein synthesis at given energy status [3]. The gene for ObgE was amplified from E. coli DH5α genomic DNA using PCR with the following two primers: 5-GCCATATGATGAAGTTTGTTGATGAA-3 and 5-GCGGATCCTTAACGCTTGTAAATGAA-3. The 1. 17 Kb PCR products were digested by NdeI and BamHI (New England Biolabs) and ligated into the pET28a vector (Novagen). A CTD deleted construct of ObgE, including coding sequence for residues 1–340 (ObgE-NG), was similarly constructed. For site-directed mutations, pET28a-obgE was used as PCR templates and the following primers were designed: ObgE-K27EK31E, 5-CGCCGCGAAGAGTATATTCCGGAAGGCGGC-3, and 5-GCCGCCTTCCGGAATATACTCTTCGCGGCG-3; ObgE-R76GR82G, 5-GCAAGCGGCGACTGTACCGGTAAGGGCGGTAAA-3, and 5-TTTACCGCCCTTACCGGTACAGTCGCCGCTTGC-3; ObgE-R136GR139G, 5-TCCGTTAACGGTACACCGGGGCAGAAAACC-3, and 5-GGTTTTCTGCCCCGGTGTACCGTTAACGGA-3 (mutated bases were underlined). The PCR products were digested with DpnI (New England Biolabs) to remove the template. The mutant of ObgE-K27EK31E/R76GR82G/R136GR139G was generated similarly using double-mutant plasmids as templates. E. coli BL21 (DE3) cells transformed with wild type pET28a-obgE were grown in 1. 0 liter LB medium at 37°C to OD600 of approximately 0. 6 to 0. 8. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30°C for 5 h. Cells were collected at 5,000 rpm in a JLA 10. 500 rotor (Beckman Coulter) for 10 min and resuspended in 40 ml lysis buffer (20 mM Tris-HCl [pH = 7. 5], 500 mM NaCl and 50 mM imidazole). Cells were lysed by sonication, and clarified lysates were obtained by centrifugation at 15,000 rpm in a JA 25. 50 (Beckman Coulter) for 30 min. Lysates were loaded onto a Ni-NTA column (GE Healthcare), washed with 20 ml lysis buffer, and eluted with 10 ml elution buffer (20 mM Tris-HCl [pH = 7. 5], 500 mM NaCl and 500 mM imidazole). Protein fractions were desalted through a desalting column (HiPrep 26/10 Desalting, GE Healthcare) with desalting buffer (20 mM Tris-HCl [pH = 7. 5], 150 mM NaCl), then subjected to a RESOURCE Q column (1 ml, GE Healthcare), and eluted with a 20 ml linear gradient of NaCl from 150 to 1,000 mM. Mutant variants of ObgE (point mutations and CTD truncation) were similarly expressed and purified. Purified proteins were finally concentrated to 10 mg/ml on a 6 ml spin filter (Satorius Stedim Biotech). E. coli 70S ribosomes were purified as described previously [58]. Purified 70S ribosomes were further centrifuged through a 10%–40% sucrose gradient with 2 mM MgCl2 to obtain separated 30S and 50S subunits. 50S fractions were pooled and with buffer changed to Binding buffer (20 mM Tris-HCl [pH = 7. 5], 100 mM NH4Cl, 10 mM MgCl2). E. coli cells of the BL21 strain, with or without the pET28a-obgE were grown at 37°C to OD600 of ∼0. 5. The cell cultures were diluted to a series of concentrations (10−3,10−4,10−5,10−6, and 10−7). 2 µl of each dilution was dropped on LB plate (1 mM IPTG) and incubated at 37°C for 10 h. E. coli cells of BL21 strain with or without pET28a- obgE were grown at 37°C to OD600 of ∼0. 5, and 1 mM IPTG was added to both cultures. After 5 h incubation at 30°C, cells were lysed by sonication, and clarified at 15,000 rpm in a JA 25. 50 (Beckman Coulter) for 30 min. Equal amounts of cell extracts were loaded gently onto the top of a12 ml 10% to 40% sucrose gradient in Binding buffer, and the gradients were centrifuged in a SW41 rotor (Beckman Coulter) for 3. 5 h at 39,000 rpm and 4°C. Gradients were analyzed using a Teledyne Isco fractionation system (Teledyne Isco). Each reaction contained a mixture of equal amounts of purified 50S subunits and His-ObgE (∼30 pmole, final concentration 1 µM) in the absence or presence of 0. 5 mM GTP, GMPPNP, GDP (Sigma-Aldrich), or ppGpp (TriLink BioTechnologies). After incubation in Binding buffer for 10 min at 37°C, samples were gently loaded onto the top of 150 µl 33% sucrose cushion in Binding buffer and centrifuged at 330,000 g at 4°C for 4 h in a TLA120. 1 rotor (Beckman Coulter). The supernatants were rapidly removed and the pellets were resuspended with 20 µl of Binding buffer. 10 µl of resolved pellets were loaded onto a 12% SDS-PAGE and the presence of His-ObgE was examined by Western blot analysis using a mouse anti-His antibody as the primary antibody, and a goat anti-mouse IgG (coupled to horseradish peroxidase) as the secondary antibody. Each reaction contained a fixed amount of 70S ribosomes (90 pmole, final concentration 1 µM), with varying amounts of ObgE or ObgE-NG, in the absence or presence of 2 mM GTP, GMPPNP, GDP, or ppGpp. The mixtures were incubated at 37°C for 10 min, loaded onto the top of a 10%–40% sucrose gradient in Binding buffer, and centrifuged in an SW41 rotor (Beckman Coulter) for 3. 5 h at 39,000 rpm and 4°C. Gradients were analyzed using a Teledyne Isco fractionation system (Teledyne Isco). In Figure 1D, fractions were treated with 20% trichloroacetic acid at 4°C overnight. The pellets were isolated by centrifugation, resolved by SDS-PAGE, and then examined by Western blot analysis. >All translation components were from E. coli and purified as previously described [18], [19]. Two reaction mixes were prepared in HEPES polymix buffer (pH 7. 5) [18]. Mix A contained either only 30S subunit (0. 5 µM) (referred to as “naked subunit”) or a 30S-preinitiation complex (30S-preIC) containing 30S (0. 5 µM), 2 µM of each of XR7 mRNA encoding MLL, fMet-tRNAfMet initiation factors 1 and 2 (IF1 and IF2), and GTP (100 µM). Mix B contained 50S subunit (0. 5 µM) alone, with ObgE or ObgE-NG in varying concentrations (0. 5–20 µM). After 5 min incubation at 37°C, equal volumes of mix A and B were mixed rapidly in a stopped flow instrument equipped with fluorescence detector set at 37°C. The extent of 70S formation was monitored by following the increase in Rayleigh light scattering at 425 nm and the observed rates (kobs) were derived by fitting the data using subunit association model described in [59]. The rates of subunit association were plotted as a function of final concentration of ObgE (or ObgE-NG). The Ki values were estimated from the midpoint of the curves fitted with hyperbolic equation. To check the effect of guanine nucleotides, naked subunit association was followed in the absence or presence GTP and ppGpp (100 µM) in the 50S mix containing ObgE (2. 5 µM). The formation of fMet-Leu (ML) dipeptide was followed starting either from 30S-preIC or 70S-initiation complex (70S-IC). The initiation complexes were prepared by incubating 30S subunit or 70S ribosome (1 µM) with XR7 mRNA encoding MLL (1 µM), f[3H]Met-tRNAfMet (2 µM), IF1 (1 µM), and IF2 (2 µM) at 37°C for 10 min. In parallel, an elongation mix was prepared containing leu-tRNA synthetase (0. 5 µM), tRNALeu (10 µM), leucine (0. 2 mM), EF-Tu (10 µM), and EF-Ts (5 µM). For dipeptide formation starting from 30S-preIC, 50S (1 µM) was added in the elongation mix. Both mixes were prepared in HEPES polymix buffer and contained GTP (1 mM), ATP (1 mM), phosphoenol pyruvate (10 µM), pyruvate kinase (50 µg/ml), and myokinase (2 µg/ml). For specific reactions ObgE (10 µM) was incubated with the elongation mix at 37°C for 10 min. Equal volumes of the initiation and the elongation mixes were rapidly mixed in a quench-flow instrument (RQF-3; KinTek Corp.). After definite time intervals the reactions were quenched, precipitated, and the peptides were analyzed by RP-HPLC as described in [18]. The PURExpress in vitro protein synthesis kit (New England Biolabs, E6800S) was used for analyzing the effect of ObgE on translation. Transcription and translation components from the kit were mixed with purified ObgE, in a ribosome to ObgE ratio of 1∶1,1∶2,1∶5,1∶10, or 1∶20. Reactions were started by adding a plasmid DNA template of dihydrofolate reductase (DHFR), and carried out at 37°C for 1 or 2 h and terminated on ice. The reaction mixtures were separated by 12% SDS-PAGE to examine the production of DHFR. 7-methyl-6-thioguanosine (MESG) assay [60] was used to determine the GTPase activity of ObgE by measuring the absorbance increase at 360 nm. In this system, the inorganic phosphate (Pi) release during GTP hydrolysis by ObgE was quantified by a coupled enzyme reaction, in which a purine nucleoside phosphorylase (PNPase) and its chromogenic substrate MESG were used. The 1. 6 ml reaction system contained 100 mM MOPS (pH = 7. 0), 100 mM NaCl, 10 mM MgCl2,100 µM MESG, 0. 1 mg/ml PNPase, and 7. 5 µM GTP. Reactions were initiated by adding ObgE to a final concentration of 3. 5 µM and carried out at 25°C. The time courses of absorbance change were monitored using a Pharmacia Ultraspec III spectrometer and the Pi release was estimated with the molar extinction coefficient 11,200 M−1cm−1 for a phosphate-dependent reaction at 360 nm and pH 7. 0. To determine whether the 50S subunit could simulate the GTP hydrolysis by ObgE, increasing amounts of the 50S subunits (0. 02 µM, 0. 1 µM, 0. 69 µM, 1 µM) were mixed with 1 µM ObgE. The initial rates of reactions were calculated from the linear parts of the progress curves obtained. Nonenzymatic GTP hydrolysis was corrected by measuring the control reaction in the absence of ObgE. To reconstitute the 50S·ObgE·GMPPNP complex, purified 50S subunits (50 nM) were incubated with ObgE in a ratio of 1∶80 in the presence of 2 mM GMPPNP for 10 min at 37°C. Aliquots (5 µl) of samples were applied to carbon-coated Quantifoil 2/2 grids (Quantifoil Micro Tools GmbH), and cryo-grids were prepared as previously described [58]. The specimen was examined on an FEI Titan Krios at 300 kV at liquid-nitrogen temperature. Images were recorded on an FEI eagle CCD camera (4K × 4K) under low dose conditions (∼20 e−/Å2), using a nominal 59,000× magnification (effective pixel size of 1. 502 Å). The data collection was performed with the software AutoEMation [61]. Micrographs were processed following standard reference projection matching procedures using SPIDER [62] with some modifications. Particles were first picked using a method based on a locally normalized cross-correlation function [63] with a 256×256 window size, subjected to correspondence analysis and then manually verified [64]. Due to the sub-stoichiometric binding of ObgE, all particles (223,274 in number) were first classified in two groups, according to the presence or absence of ObgE on the 50S subunit using a modified supervised classification method (Figure S9) [65]. The resulting 188,814 ObgE-containing particles were further applied to another round of 3D classification using RELION [66]. The particles were finally split into four groups in 30 iterations using a final angle sampling of 1. 8 degree. One of the four groups, which displays the highest ObgE occupancy, with a total particle number of 102,814, was used for final refinement (Figure S10). The refinement was performed using RELION [66] with the final sampling angle of 0. 1 degree. The final resolution was reported by gold-standard FSC calculations [67], as 5. 5 Å according to FSC 0. 143 criterion (Figure S11). Amplitude correction using the B-factor sharpening approach [68] was applied to the final volume. Local resolution map was calculated using the blocres program of the Bsoft package [69]. The atomic model of the E. coli ObgE was built with MODELLER [70], using the B. subtilis and Thermus thermophiles Obg crystal structures (PDB IDs 1LNZ and 1UDX) [10], [17] as templates. A crystal structure of the 50S subunit (PDB ID 3OFC) [71], NTD and GD of the ObgE model were first manually docked as rigid bodies into the cryo-EM density map. The flexible fitting was performed using the molecular dynamics flexible fitting approach [72]. PyMol [73] and Chimera [74] were used for graphic visualization and figure preparation. Cryo-EM map of the 50S·ObgE complex has been deposited in the EMDataBank (EMD-2605). The atomic model has been deposited in the Protein Data Bank (4CSU).

Title: Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase Summary: GTPases commonly act as molecular switches in biological systems. By oscillating between two conformational states, depending on the type of guanine nucleotide bound (GTP or GDP), GTPases are essential regulators of many aspects of cell biology. Additional levels of regulation can be acquired through the synthesis of other guanine nucleotide derivatives that target GTPases; for instance, when nutrients are limited, bacterial cells produce guanine tetraphosphate/pentaphosphate- (p) ppGpp-as part of the "stringent response" to adjust the balance between growth and survival. ObgE is a GTPase with many reported cellular functions that include ribosome biogenesis, but none of its functions is understood at the molecular level. Here we characterize, both biochemically and structurally, the binding of ObgE to its cellular partner, the 50S ribosomal subunit. Our results show that ObgE is an anti-association factor, which binds to the 50S subunit to block the formation of the 70S ribosome, thereby inhibiting the initiation of translation. Furthermore, the binding and anti-association activities of ObgE are regulated by guanine nucleotides, as well as by (p) ppGpp. We thus propose that ObgE is a checkpoint protein in the assembly of the 50S subunit, which senses the cellular energy stress via levels of (p) ppGpp and links ribosome assembly to other global growth control pathways.

lay_plos

en

Summarize: FIELD OF THE INVENTION This device pertains to the measurement of electrical effects in living tissue near high voltage discharges. It specifically measures the rate and duration of charge-limited breakdown in gas near the contact boundary of an electrically stressed skin-dielectric interface. BACKGROUND OF THE INVENTION &#34;Kirlian photography&#34; or the photographic recording of &#34;coronal&#34; images is an area of questionable research carried on for more than 50 years mostly by persons with little education or training in the disciplines of engineering and physical science. Recently, data collected in India with a Kirlian apparatus, by Ramesh Chouhan has suggested a relationship between percent transmission figures as measured by densitometer readings and the presence and progress of a variety of carcinomas. His unique discovery, is that this correlation appears when the percent transmission variable is measured as a function of time after the skin surface is washed. Thus a new dimension has been added to a procedure previously of doubtful value. The equipment used to collect this data, however, was unwieldy and not suited to research into the physical processes behind the time variance. There is also a need to make the recording method more accurate and repeatable; a need to make the equipment less expensive and more transportable to make it more reliable and safer to operate; and, further, to make an instrument that will operate in the normal ambient illumination conditions of a clinical setting. Traditionally, special meanings have been ascribed, and marginal scientific theories put forth, to explain the shapes and even the colors that are present in Kirlian images. And, it has been claimed that these images are a visible manifestation of &#34;auras&#34; that are visible only to certain specially-gifted people. This invention begins working from the assumption that, (given present science and technology), no useful information will be found in the spatial gradations of density in Kirlian film images. It is also assumed, that information extracted from the images recorded by Ramesh Chouhan, using the SINGLE POINT percent transmission method, can be collected equally well by measurement of total illumination from the discharge surrounding a defined AREA of skin. Further, it is also assumed, for a given patient at least, that total charge transferred in breakdown-mediated events is proportional to the integrated value of the associated illumination. The scientifically accurate view is taken that the illumination producing discharge process occurs as follows: 1) a dielectric plate is polarized by an adjacent polarizing conductor at high electrical potential, 2) the resultant induced surface charge on the far side of the plate will in turn create a potential in that vicinity which is higher than it would otherwise be in a free space condition (no dielectric), 3) skin or some other conductor, where it contacts the dielectric, will supply mobile charge (because it&#39;s a conductor), to neutralize the induced surface charge, 4) where the dielectric is NOT contacted, a high potential difference will exist between the skin and the surface of the dielectric, 5) as the potential of the polarizing plate increases, the potential difference between dielectric and skin will increase to the point where electrical breakdown of the intervening gas will occur and charge will be transferred between the skin and dielectric surface, 6) but, because the surface of the dielectric is not a conductor, this breakdown process does not occur all at once, but small area by small area of dielectric is neutralized via breakdown. When the polarizer plate&#39;s polarity is reversed, a similar process occurs, but the charge transfer direction is reversed. Polarizer voltage rises to just beyond the breakdown level, so it must be emphasized that the continued breakdown process is dependent upon alternation of the high voltage polarity; multiple high voltage pulses of the same polarity will have little effect after the first breakdown mediated transfer. Once charge has moved, there won&#39;t be quite enough potential difference between the skin and any location on the dielectric to start a breakdown in the reverse direction when polarizer voltage drops to zero. This sequence of limited tiny charge transfers, which is qualitatively different than an arc discharge in lightening, for example, makes the process safe from a medical standpoint. This patent describes the construction of devices that apply basic science and that exploit the stated assumptions. The devices described are new and significant departures from the existing state of the art, such as it is. SUMMARY OF THE INVENTION The principle objects of the present invention are to measure as cheaply as possible, the electrical behavior of conductive tissue used as an electrode in a charge-limited electrical breakdown process, and to make the process as repeatable as possible. A thumb will be used for purposes of illustration as an example of skin. There are provided, two methods of measuring charge transfer rate. One collects illumination from the entire area of discharge around a thumb and directs it to an inexpensive large area photocell or solar cell. Imaging or non-imaging optics may be used to collect the light. Whatever method is used, there must be no significant change in total illumination delivered to the photocell for small changes in thumb position. The other method detects the high frequency variations in current that arise when thousands of small surface charge regions are individually neutralized at a high rate. This high frequency current flows in the lead connecting the polarizer supply and the polarizer plate. A properly designed transformer can not only isolate the high voltages on this lead from the low level detection circuitry; it can also block low frequency signals associated with the displacement current that flows through the area of thumb which contacts the dielectric. And, it can transform the high frequency source impedance to better match the high frequency signal detector. And, there are provided, methods to increase reliability and repeatability. Reliability is greatly increased by the use of a non-imaging light pipe to conduct illumination from the dielectric plate to the photocell. Intimate contact at all points along the optical path exclude dust and the chemically reactive ozone which tend to occlude optical surfaces. Repeatability is improved by locating the thumb on a reference platform and mounting the entire dielectric plate assembly on a linear movable track with a constant force mechanism. This sets the contact force between thumb and dielectric to a repeatable value. Repeatability is also improved by a baseline technique that subtracts background illumination from measured illumination. The high frequency current measuring method described, eliminates the effect of normal ambient illumination altogether. Lastly, a method is provided to simulate the old Kirlian technique of single point percent transmission measurements on film, for research purposes. BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, there are shown in the accompanying drawings forms which are presently preferred; it being understood that the invention is not intended to be limited to the precise arrangements and instrumentalities shown. FIG. 1 shows the relationship between the essential components of a device that uses the illumination measurement method. It shows a sectional view of the elements in the optical path which are solids of revolution. Exceptions are, a human thumb 36, and an optional pinhole mask 22. Other components are indicated schematically; and FIG. 2 likewise, illustrates inter-relationships between the essential components of a device that employs the high frequency current detection method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Refer first, to FIG. 1, in the following description of construction details for a device based upon the optical method. The disc-shaped indium tin oxide (ITO) coated glass plate 10 conductive transparent coating is surrounded by an insulating ring 8 that prevents unwanted light and current generating corona discharge at the sharp edges of the ITO conductive coating. The upper surface of the glass dielectric plate 10 is coating free, and should be sealed to the insulating ring 8 with a high voltage elastomeric O-ring or some adhesive material such as &#34;acetoxy&#34; type silicone rubber adhesive. Differential expansion and contraction of the insulating ring and glass plate make this necessary unless the ring 8 is glass, which is difficult to fabricate. The lower surface of the glass plate 10 has a conductive transparent ITO coating shown in exaggerated form as element 12 in FIG. 1. Electrical connection to this coating must be extremely intimate and extend completely around the periphery of the lower side of the glass plate 10. If contact with the ITO coating is not intimate enough, small sparks will appear, and slowly vaporize the adjacent coating, and eventually create an ITO-free moat around the entire center area (and an inoperable device). Conductive cement such as &#34;nickel print&#34; can be recommended as a reliable, low-resistance contact material. This connection should be passed to a flexible exterior lead 24, and all exposed conductor surfaces and transitions should be thoroughly protected with a high voltage insulating material. Again, acetoxy silicone rubber is highly recommended where moisture can be expelled. A pinhole-aperture mask 22 may be inserted between the light pipe 28, and ITO coating 12 to simulate percent transmission readings of film, but in conjunction with this, it will be necessary to substitute a photomultiplier tube plus support circuitry for the semiconductor photocell 14. The thickness of the mask 22 is exaggerated. If there is no mask, the edges of the light pipe 28 should be sealed to the adjacent insulating ring 8 to permanently exclude dust and ozone. The semiconductor photocell 14 should be placed directly against the face of the small end of the light pipe 28, and the edges sealed with an O-ring or with a small line of elastomeric adhesive. The guard ring 6 reduces the level of induced surface charge appearing at the small end of the light pipe 28 where it is in contact with the photocell 14. The guard ring 6 should, optimally, be a vacuum deposited aluminum coating that does not interfere with TIR on the surface of the light pipe 28. But, a grounded disk of ITO coated glass will also do a good job of reducing electrostatically induced currents in the face of the semiconductor photocell 14, and is much less expensive in small volume. The above described optical unit is driven through lead 24 by the alternating high voltage polarizer supply 16. The high voltage drive is most easily generated with a step-up transformer of the TV flyback or automobile ignition variety. The important thing is that the frequency of the alternating voltage be high enough that significant current will flow into the parasitic capacitance of the human body, but low enough so that breakdown has time to occur at a reasonable drive voltage. This places frequency nominally between 20 KHz and 50 KHz, and the peak voltage between 10 KV and 20 KV with a 1/16&#34; glass plate 10. The exact waveform of the alternating high voltage drive is unimportant as long as its frequency components are in the proper range. The ringing behavior of the setup transformer will be fixed by its construction, and provides a convenient method of generating a high voltage that alternates, so that the charge transfer process may be reversed. The voltage level however, may be varied conveniently by generating the low voltage primary winding drive signal in a general purpose microprocessor. Within a range of pulse widths less than half the ringing period, a range of peak high voltages may be chosen. This is because the peak current flowing in the high voltage primary is a linear function of the time that a drive voltage is applied, for resultant output voltages in the design range of the transformer. The general purpose microprocessor can also control the period of time between pulses. In the preferred embodiment, this microprocessor communicates with a terminal or host computer for convenient control by an operator. Methods for doing this are well known to any person skilled in the art of constructing computer interfaces, microprocessor assemblies, and electronics at the time of this filing. The photocell should be connected to the signal processing electronics 18 via a flexible pair 26, which optionally may be shielded. Every time the polarizer supply 16 generates a series of alternating pulses, the photocell 14 detects illumination generated by breakdown events near the dielectric surface 38. The processing electronics 18, should contain an integrator to integrate the illuminating signal over time. Each time a series of ringing pulses are generated, an analog-to-digital converter digitizes the photocell integrator output. At this point, the integrator should be reset so that it is ready for the next integration period. Again, as with the polarizer, these processes can be easily controlled using microprocessor technology that is practiced by many who are skilled in the art. The microprocessor should contain instructions that supply an integrated light reading when the high voltage is not applied so that the effect of ambient light leaking into the light-tight case that encloses the above described device may be subtracted from integrator readings taken with the high voltage on. This reduces the error caused by variable ambient light levels. An extraneous variable in the discharge is the force between the thumb 36 and the dielectric plate 10. This should be held constant. A reference platform 34 should be located above the movable surface of the glass plate 10, so that when the thumb is in place, the optical assembly will be approximately at the center of its travel. The sliding frame 30, which might have a bar, riding in a set of rollers 32, thus creating a linear translation bearing assembly, should be connected to a negator spring assembly 31 or other constant force device of similar function. Refer to, FIG. 2, in the description that follows of the construction of a device based upon the high frequency current measurement method. This second device is essentially the same in all respects as the optical device, excepting the method of measuring breakdown-mediated charge transfer, and excepting that the polarizer electrode 12 needn&#39;t be ITO. It should, however, at least be in intimate contact with the dielectric plate 10. Some type of dielectric cement ought to be used with a metal plate; or, conductive cement might be used alone. The flexible lead 24 does not go directly to the polarizer supply 16. Instead, the primary of an isolation transformer 50 is placed in series. This can be accomplished by simply passing the flexible lead 24 through the center of a high frequency ferrite toroid, and providing very ample insulation where the lead 24 passes near the ferrite to suppress local corona discharge. The secondary should consist of several turns of litz wire, the number of turns chosen to match the input voltage needs of the high frequency rectifier 52 circuit. The signal so generated will contain frequency components in the nominal radio frequency range, 1 MHz to 10 MHz. The rectifier 52 should produce a signal of amplitude similar to the photocell 14 described in the previous method. A higher signal to noise ratio will be obtained if parasitic inductance can be kept to a minimum. A further increase will be noted if a small, low-inductance, capacitive bypass 54 to ground is added to the polarizer supply 16 output. This is especially true if the polarizer design relies upon a step-up transformer. In this case though, the capacitor should not be so large as to lower the ringing frequency of the transformer below about 20 KHz. From this point on, the function of the integrating and processing electronics 18, are essentially identical to those described for the optical device. The one exception is that there is now little need for a reference measurement, since normal ambient light will have little effect. Caution should be taken though, to exclude UV. UV will abnormally lower all observed breakdown thresholds where it is able to penetrate. The high voltage polarizer supply ground should be taken to building ground.

Summary: Breakdown-mediated charge transfer is made to occur at the surface of a dielectric and its rate is limited by a defined dielectric polarization level and frequency for safe use in a clinical setting. Measurement is accomplished directly by an optical method and indirectly by a high frequency current detection method. The optical method is made reliable through a sealed construction technique. The quality of diagnostic readings supplied by both methods are made more reliable by a repeatable means of force application.

big_patent

en

Summarize: A single mother on benefits has admitted spending £3,000 of taxpayers' cash on a dream round-the-world trip to far flung destinations with her 10-month-old baby daughter. Kay Bird, 28, from Gainsborough in Lincolnshire, is unemployed and lives comfortably with her middle-class parents. Her mother Jill, 54, and stepfather lawyer Bob, 61, pay for all her rent, bills and all other outgoings. Unemployed single mother Kay Bird, who has admitted spending £3,000 of taxpayers' cash on a round-the-world trip to far flung destinations with her 10-month-old baby Chloe. She is pictured with her daughter in Dubai. Miss Bird and her young daughter splash around in the Indian Ocean during the trip. The young mother receives more than £8,500 in various benefits. But she still receives more than £8,500 a year in child benefit, income support and tax credits as it is considered that she has a low income. And now she has admitted that she has spent £3,000 of her welfare benefits on a five-week-long trip with her daughter Chloe, where she visited places such as Australia, Bali and Dubai. Miss Bird says she could work but chooses not to so she can take care of her young child. She said: 'No, I don’t need the money as such and I didn't need to go travelling either but I wanted to so I did. 'If someone’s offering you free money and telling you to take it, you’d have to be a fool not to – that’s all I did. 'Once it’s in my bank account it’s up to me how I use it and I decided to spend it on taking the trip of a lifetime. Miss Bird, pictured with her daughter Chloe, says she doesn't need the benefit money as her parents pay for all of her rent and other outgoings but would be a 'fool' not to take 'free money' The round-the-world trip began last month when Miss Bird jetted off to Sydney, right, with Chloe via London. She also visited destinations such as Bali and Sri Lanka, left. 'I don’t feel guilty and I don’t regret it. It started off just as a ­holiday to Athens, then things started to fall into place. 'Each time some more money landed in my account, I booked something. 'I started booking flights and accommodation in Europe in October and was booking something with every payment until a few days before I went.' Miss Bird set off on her round-the-world trip last month when she flew to Sydney via London on January 10. She also visited Athens, Istanbul, Dubai, Colombo in Sri Lanka, Kuala Lumpur, Jakarta, Bali and Darwin before returning home via Amsterdam. In total, she spent four months worth of her benefits cash on the trip, paying for 13 flights, travel visas, accommodation and spending money. Her benefits continued to be paid into her bank account while she was away and she returned to the UK just before the five-week travel limit imposed on people claiming Jobseekers' Allowance. It is reported that the 28-year-old spent four months worth of her benefits cash on the trip, paying for 13 flights, travel visas, accommodation and spending money for her and daughter Chloe, right. She added: 'I went to the job centre and told them I wanted to go travelling and they told me there was a five-week limit. I came home just within those five weeks so my benefits didn't get cut off.' Miss Bird had been living in Rome after studying psychology at La Sapienza university in the Italian capital when she became pregnant. After she found out she was expecting a baby, she returned to the UK to live with her parents and visited her local Jobcentre Plus office. Soon after she was claiming £90 a week income support, £90 a month child benefit and £230 a month in tax credits. She said: 'I told them I wanted to register back in the country and they told me I was already eligible for Jobseekers’ Allowance. 'Then a couple of weeks later they said I could switch to income support which meant I didn’t even have to apply for jobs. 'Then I was told I could get tax credits, too. I was really shocked at how generous it was but I wasn’t going to turn it down.' However, she does not receive any money from her daughter's father, a professional rugby player she met in Rome but now lives in Australia, saying she asked him not to pay maintenance because she doesn't need it. Now she says she is planning her next luxury trip for herself and daughter which will be to New Zealand. Among the places that Miss Bird visited were Athens in Greece, left, and Kuala Lumpur in Malaysia, right. But Miss Bird says her way of life could help change the perception of single mothers on benefits. She explained: 'I’m not your regular single mum on benefits who spends it all in McDonald’s and never leaves the town they were born in. 'I’m changing the image of what it is to be a benefits mum and proving that if you do it the right way, you can have ­anything you want. 'They've chosen to eat takeaways and buy the latest gadgets. I chose a different route. 'OK, I don’t have any outgoings so I have been lucky to spend my benefits on this amazing experience but I think single mums get more than enough. 'Of course people are negative and many people get very jealous. 'But I had only been out of Europe once before I went on benefits and now I’ve had the chance to see some incredible things from tropical beaches to the ­skyscrapers of Dubai. 'I never would have been able to afford it without benefits.'

Summary: Single mother Kay Bird, 28, is on benefits and lives with her parents. Receives over £8,500 a year in child benefit, income support and tax credit. Her parents pay all of her bills including all of her rent and other outgoings. Has admitted spending £3,000 of taxpayers' cash on a round-the-world trip. Visited destinations such as Australia, Bali and Dubai with her daughter. Says she doesn't need benefits but would be a 'fool' not to take 'free money'

cnn_dailymail

en

Summarize: By. Sara Malm. and Harriet Arkell. Pictured: Convicted paedophile David Reid, from Belfast, was living in the Algarve, Portugal at the time of the disappearance of Madeleine McCann. A convicted British paedophile was. living in the same area on the Portuguese coast at the time of Madeleine. McCann's disappearance seven years ago, it has been revealed. David Reid moved to Carvoeiro on the Algarve in 2004, after he was released from prison for sexually abusing young girls. He. was still living in the area in 2007, when three-year-old Maddie was. snatched from her family's holiday apartment while her parents were. having dinner nearby. The. same year Reid arrived on the Algarve, several British families on. holiday suffered break-ins and five young girls were abused in their. beds. Chillingly, just. months before Maddie McCann's disappearance, one of his victims had. warned that Reid remained a threat to young girls and would offend. again. 'It's part of his being. He'll never change,' the victim said, according to The Mirror. 'Get your kids and your family away from him, Never out temptation in front of him. He just won't be able to resist it. Reid,. from Northern Ireland, was living just 30 miles from Praia da Luz, the. holiday resort where the McCann family were staying, in 2007. He. was convicted and jailed for gross indecency and indecent assault in. Belfast in 1995, but was able to move freely and unmonitored in Portugal. as the sex offenders' register was not set up at the time of his. release. According to locals. in Carvoeiro, Reid died last year aged 61 or 62, but his family in. Britain has not been informed of his death, The Mirror reports. The. police investigation into the disappearance of Madeleine McCann took a. step forward last week when detective announced that they. are looking for a prolific paedophile who sexually abused five girls at. holiday homes in the Algarve before the British toddler went missing. The. man, described as tanned, dark-haired and speaking English with an. accent, is suspected of breaking into holiday properties where British. families were staying and sexually abusing five white British girls aged. between seven and ten,. Still missing: Three-year-old Madeleine McCann disappeared from a Portuguese resort in 2007, close to where Northern Irish paedophile David Reid, pictured playing in The Roundup Bar in Carvoeiro in November 2006, lived. Hideout: David Reid's apartment on the cliffs in Carvoeiro, just 30 miles from Praia da Luz where the McCanns holidayed in 2007. Reid, who died last year, moved from Belfast to Portugal after he was released from prison where he had served time for sexually assaulting young girls and a boy. The sex attacks took place between 2004-2006, shortly before Madeleine vanished in 2007, and are among a series of 12 break-ins to holiday homes in the Algarve that police believe were committed by the same man. In six of the break-ins, the man sat. on or got into bed with young girls. On one occasion, he abused two. young girls in the same villa. Two. of the attacks were in the resort of Praia da Luz, where Madeleine was. staying in a holiday apartment with her family when she was taken. There were also four in Carvoeiro, where Reid was living, and six in the Vale da Parra, Praia da Gale district. Most of the attacks took place in low season, police said. They. had previously been discounted by Portuguese investigators because they. are spread over a wide geographical area and there were no apparent. attempts at abduction. Nine. of the 12 incidents were reported to Portuguese police at the time they. happened, and details of three of those became known to British. investigators only after they made televised appeals last autumn. Witnesses. describe the man as having dark, tanned skin with short dark unkempt. hair. He spoke in English with a foreign accent, his voice was described. as slow, or possibly slurred. He was sometimes bare chested, some describe him as having a pot belly, and three victims said that he had a noticeable odour. New clue: Detectives on the Madeleine case say the man they are looking for wore a distinctive burgundy top. Still looking: Madeleine's parents, Kate and Gerry McCann, pictured with an image of how she might look now, were having dinner with friends when 'Maddie' disappeared in Praia da Luz. On. two occasions in Vale de Parra and Praia da Gale he was wearing a. distinctive burgundy long sleeve top, on one of those occasions it was. described as having a white circle on the back. DCI. Redwood said: 'This is an offender who has got a very, very unhealthy. interest in young, white, female children whom he is attacking whilst. they are on holiday in their beds.' Police said the suspect may have been. in the villa or looking round the villa for some time before committing. the offences or being disturbed either by a parent, or the child waking. up, and said he remained calm, even when disturbed. On two occasions, they said, the noise of a bin collection lorry could be heard nearby. DCI. Redwood said today that tracing the man is one of his priority lines. of inquiry. He said: 'We. need to establish the identity of this man - these offences are very. serious and no-one has been charged in connection with them.' He. said some of the offences were previously known about, but three of. them, including what is believed to be the first in the series of 12. break-ins, were new reports to police and came about as a result of. their appeal for more information last October. Resort: Madeleine went missing from this holiday apartment at the Ocean Club in Praia da Luz in May 2007. Mr. Redwood's team currently have 38 people classed as 'persons of. interest' to the inquiry, and are also sifting through details of 530. known sex offenders whose whereabouts they cannot account for.Of those 59 are classed as high priority, and some of those are British. British. investigators have so far sent three international letters of request. to Portuguese authorities over the investigation, covering 41 priority. areas for the team, involving 287 separate requests. Deputy. Assistant Commissioner Martin Hewitt showed his anger at the slow pace. of the legal process, saying: 'That's causing us frustration because we. know what we want to do and we are ready to go with that. But the. process is the process.' Another 30 letters have been written to other European countries, but the force would not reveal where. Madeleine, who was then nearly four, disappeared from her family's holiday apartment in Praia da Luz in the Algarve on May 3 2007 as her parents dined at a nearby restaurant with friends. British detectives launched a fresh investigation into the youngster's disappearance in July last year - two years into a review of the case - and made renewed appeals on television in the UK, the Netherlands and Germany. After shelving their inquiry into Madeleine's disappearance in 2008, Portuguese authorities said last October that a review had uncovered enough new information to justify reopening it

Summary: Convicted paedophile David Reid lived in the area at the time of abduction. He moved to Algarve resort in 2004 after being freed from jail in Britain. After this, a man broke into holiday homes and abused five British girls. A tanned, dark-haired man suspected of child sex assaults from 2004-2006. Madeleine McCann, aged three, was taken from Praia da Luz in May 2007. Police say paedophile is suspected of committing 12 break-ins in Algarve. Four were in Carvoeiro, six in the Vale da Parra, and two in Praia da Luz.

cnn_dailymail

en

Write a title and summarize: Soil-transmitted helminth (STH) infections remain highly endemic across the Philippines, and are believed to be important contributors to delayed cognitive development of school-aged children. Identification of communities where children are at risk of functional illiteracy is important for the attainment of Sustainable Development Goals target for literacy. We aimed to quantify the associations between the spatial variation of STH infections and functional literacy indicators adjusting for other important contributors, and identify priority areas in the Philippines in need of interventions. We used data from 11,313 school-aged children on functional literacy indicators collected in 2008. Nested fixed-effects multinomial regression models were built to determine associations between STH endemicity and geographical distribution of functional literacy, adjusting for demographics, household level variables, and the prevalence of malaria. Bayesian multinomial geostatistical models were built to geographically predict the prevalence of each level of functional literacy. The number of school-aged children belonging to each of the functional literacy indicator classes was forecast for 2017. We estimated 4. 20% of functional illiteracy burden among school-aged children in Mindanao might be averted by preventing T. trichiura infections. Areas predicted with the highest prevalence of functional illiteracy were observed in localised areas of the eastern region of the Visayas, and the south-eastern portion of Mindanao. The study demonstrates significant geographical variation in burden of functional illiteracy in school-aged children associated with STH infections suggesting that targeted helminth control could potentially promote the development of cognitive function of school-aged children in the Philippines. The benefits of a spatially targeted strategy should be tested by future studies. Functional literacy is one of the targets of the Sustainable Development Goals (SDG) of the United Nations, launched in September 2015 [1]. Functional literacy is a key indicator of cognitive function, especially information processing and comprehension, and it has been used to measure cognitive function in school-aged children [2]. According to the latest United Nations Educational Scientific and Cultural Organization report on literacy, there are globally 114 million illiterate adolescents and youths [3]. Despite widespread acknowledgement of this problem, between 2000 and 2015, global literacy rates were estimated to have improved by just 4%. Of particular relevance to the current work, progress in addressing national literacy rates in the Philippines has been slow [4]. Debate has recently intensified regarding the role of soil-transmitted helminths (STH), Ascaris lumbricoides, Trichuris trichiura, and hookworms (Ancylostoma duodenale, A. ceylanicum and Necator americanus) in childhood cognitive function, and by extension functional literacy [5]. To date, relatively few studies have investigated this link and evidence remains inconclusive, partly because differences in study designs and the use of cognitive development measurement tools makes it difficult to compare results of studies [6]. STH infections are among the most common infections in school-aged populations, and are particularly common in impoverished communities where the provision of water, sanitation, and hygiene education is limited [7]. STH infections are estimated to incur 4. 98 million years lived with disability, related to anaemia, chronic nutritional imbalances, stunting, and cognitive and motor developmental delay [8]. Possible mechanisms for the effect of STH on cognitive function in children may include the interaction between the host immune system and the species of STH, the direct effect of metabolites excreted during infection, anaemia, or inflammation, and through secondary effects such as malnutrition and micronutrient deficiencies [9]. A recent experimental study demonstrated that T. trichiura infection may influence cognitive function of animals, although a definitive prospective human study has not been performed [10]. STH infections remain highly endemic across the Philippines among both primary and secondary school children [11,12]. While previous studies in the Philippines indicated an effect of STH infection on children’s cognitive development, the contribution of STH infections on the overall functional illiteracy burden in the Philippines is unknown [13–15]. Processes that result in reduced functional literacy are complex and multifactorial. Compromised nutritional and family contextual factors such as poverty, unemployment, low maternal education, and household education stimuli have been linked consistently with cognitive function and educational performance of school-aged children [16]. In developing countries such as the Philippines, cognitive dysfunction could be caused by malnutrition in complex combinations with other factors including deprivation of environmental and emotional stimulation, biological factors, and infections such as pneumonia, meningitis, STH, and malaria [17]. Predictive risk maps, together with geospatial modelling, are emerging as important decision tools for public health policy makers [18]. However, to date there have been no studies looking at geographical variation in functional literacy and the associations with its key determinants. In this study, we aimed to quantify the associations between the spatial variation of STH infections and functional literacy in school-aged children in the Philippines, adjusting for probable confounders. In doing so, we also aimed to develop the first prediction maps of each functional literacy indicator in order to quantify the number of school-aged children at risk of reduced functional literacy in the Philippines. Ethical clearance for this analytical study was provided by the University of Queensland School of Medicine Low Risk Ethical Review Committee (clearance number 2014-SOMILRE-0100). We obtained population level data on functional literacy indicators of 11,313 school-aged children (aged 10–19 years) collected in 2008 during the nationwide Functional Literacy, Education and Mass Media Survey (FLEMMS). In brief, a total of 1,506 barangays (the smallest administrative unit in the Philippines: average diameter of 11 km) were included, 851 located in Luzon, 254 in the Visayas, and 401 in Mindanao (A map of 2008 FLEMMS survey locations is shown in S1 Fig [19]). All data analysed were anonymised. Functional literacy levels were stratified into four classifications – 1) those who cannot read, write, compute and comprehend were classified as functional illiterate; 2) those who can only read and write (with understanding a simple message) were considered as low functional literate; 3) those who can read, write and compute were considered as moderate functional literate; and 4) those who can read, write, compute and comprehend (with a significantly higher level of literacy that includes not only reading and writing skills but also numerical and comprehension skills) were considered as functional literate. Further detailed information on the FLEMMS can be found in S1 Text. A wide array of plausible contributing factors were obtained from the FLEMMS individual questionnaires and FLEMMS household questionnaires, including individual level sociodemographic indicators (e. g. age, sex, education attainment level, marital status, and employment status), and household level factors such as socioeconomic status (SES), access to water, sanitation, and hygiene (WASH), and household education stimuli. Information regarding head of households such as adult functional literacy was measured for a total of 10,339 heads of households. We obtained data of spatial predicted values of Plasmodium infections from the Malaria Atlas Project (S2 Fig) [20]. We used predictive maps of STH infection prevalence generated from spatial analysis of the data collected during the National Schistosomiasis Survey in the Philippines in 2005 to 2007 (S3 and S4 Figs) [12,21]. Further information on each indicator is detailed in S2 Text. A total of 11,313 school-aged children and 10,339 heads of household with complete information were included in the analysis. The highest prevalence of functional illiteracy in school-aged children was observed in the Visayas, followed by Mindanao and Luzon (7. 5%, 6. 9%, and 3%, respectively; Table 5). The observed prevalence of functional illiteracy of heads of households was also higher in the Visayas and Mindanao compared to Luzon (15%, 15%, and 6%, respectively; S2 Table). Full descriptive results of the dataset are presented in S3 and S4 Tables, and S5 Fig [19]. While the relative importance of determinants of functional literacy varied between regions, some findings were consistent across all regions (Table 6). We found that highest education attainment, low socioeconomic status (SES) and adult functional illiteracy rates are major contributors to functional illiteracy across Luzon, the Visayas and Mindanao. Population attributable fraction (PAF) due to highest education attainment: 83. 30% ([ratios of relative risks (RRR) ], 9. 69), 85. 49% ([RRR], 10. 69), and 81. 96% ([RRR], 8. 33), respectively; PAF due to low SES: 24. 36% ([RRR], 1. 97), 29. 45% ([RRR], 1. 98), and 53. 22% ([RRR], 3. 28), respectively; PAF due to adult functional illiteracy: 6. 61% ([RRR], 2. 16), 18. 57% ([RRR], 2. 53), and 26. 71% ([RRR], 3. 43), respectively. Our results indicated that the estimated risk of functional illiteracy attributable to poor sanitation facilities in the Visayas is 13. 21% ([RRR], 1. 86). Our results indicated that the estimated risk of functional illiteracy attributable to P. vivax infection, T. trichiura monoinfection, and moderate/high infection intensity class for T. trichiura in Mindanao were 0. 53% ([RRR], 1. 26), 4. 20% ([RRR], 1. 40), and 3. 96% ([RRR], 1. 82), respectively (Table 7). In Luzon and Mindanao, being female was negatively and significantly associated with the prevalence of functional illiteracy compared to being male (Table 6). Full results of the multinomial logistic regression models are presented in S3 Text. Across all regions, we observed reduced propensity of clustering and larger cluster sizes after adjusting for the effect of covariates (Table 8 and S6 Fig). Our predictive maps indicate significant spatial heterogeneity in the prevalence of each level of functional literacy within each region of the Philippines (Fig 1 and S7 Fig). Our predictive maps also demonstrate that the prevalence of functional illiteracy ranges between 1 and 3%, with the highest rates predicted in localized areas of the eastern region of the Visayas (Eastern Samar), and the centre (Davao Del Norte province) and the southwestern tip of Mindanao (Davao Occidental province) (up to 10. 5%) (Fig 1). The models showed reasonable discriminative ability for functional illiteracy (area under the curve [AUC] = 0. 75 for Luzon, 0. 75 for the Visayas, and 0. 71 for Mindanao), low functional literacy (AUC = 0. 70 for Luzon, and 0. 74 for the Visayas), and moderate functional literacy (AUC = 0. 74 for Luzon, 0. 72 for the Visayas and 0. 82 for Mindanao) (Table 9). For 2017, it was estimated that Luzon had the highest estimated number of school-aged individuals with functional illiteracy (estimated total 2,185), followed by Mindanao (estimated total 1,550) and the Visayas (estimated total 1,212) (Table 10). The highest number of school-aged individuals with functional illiteracy (Fig 2), low functional literacy (Fig 3), and moderate functional literacy (S8 Fig) was circumscribed to areas around metropolitan Manila, with some municipalities exceeding 63 people per square kilometre. In the Visayas (Fig 2) and Mindanao (Figs 2 and 4), the predicted number of school-aged individuals with functional illiteracy was widely distributed in a number of provinces. The highest numbers of school-aged individuals with moderate (S8 Fig) or low functional literacy (Fig 3) were predominantly localised in the western region of the Visayas (some municipalities with more than 24 persons per square kilometre). In Mindanao, the highest numbers of school-aged individuals with moderate (S8 and S9 Figs) or low functional literacy (Figs 3 and 5) were localised in the central and southern parts of Mindanao with some municipalities exceeding 50 people per square kilometre. Names of provinces and municipalities with the poorest functional literacy indicators based on our analyses are provided in Table 11. Overall, our findings suggest significant spatial heterogeneity in the prevalence of functional literacy indicators within each region of the Philippines, reflecting variability in the determinants and the need for location-specific interventions. Our findings are consistent with previous studies, indicating that multiple factors exert a negative impact on functional literacy in school-aged children. Evidence suggests a possible link between low socioeconomic status and cognitive function, including children’s levels of language and literacy skills [17]. This relationship is mediated by different mechanisms such as parenting behaviour and household linguistic stimulation [17]. The observed association could also be explained by the effect of malnutrition in the poorest areas of the Philippines [27]. We found that the determinants of functional literacy are region-specific. For example, in Luzon and Mindanao, our results indicate that female school-aged children are at less risk of functional illiteracy compared to males, suggesting that girls may be less exposed to factors that affect their cognitive development compared to boys. The gender difference identified in our study could also be partly explained by integrated helminth control programs, which provide more attention to female adolescents [28], and the fact that boys are often involved with agricultural activities in resource-limited areas, limiting their participation in schooling or academic learning as agriculture in the Philippines has been dominated by males [29]. In contrast, our results for the Visayas showed that higher prevalence of functional illiteracy was associated with households with poor sanitation facilities. School-aged children who live in socioeconomically deprived environments face multiple challenges such as non-utilisation of sanitary facilities, open defecation, and limited education services. Previous studies have demonstrated that open defecation, a practice highly prevalent in the Visayas [11], is associated with high prevalence of STH infections [30]. Deficient sanitation promotes not only the transmission of STH infections, but also water-borne infections and diarrhoea. It also increases the associated risks of malabsorption, malnutrition, and iron-deficiency anaemia, which could aggravate cognitive dysfunction [31]. Our results also showed that the utilisation of unprotected water sources such as wells and lakes as main sources of drinking water at home is negatively associated with the prevalence of functional illiteracy in the Visayas. Our findings may be confounded by the fact that unprotected drinking water sources are more likely to be present in agricultural communities where access to food and nutritional security are assured through local food production [32]. Further investigation is needed to examine the factors mediating the relation between access to water sources and the prevalence of functional illiteracy identified in this study. The risk of STH-associated morbidity depends on the intensity of STH infection and the species of STH [33]. This study demonstrated significant geographical variation in the burden of functional illiteracy in school-aged children, which could possibly be explained by T. trichiura infection. Indeed, our findings suggest the risk of functional illiteracy among school-aged children in Mindanao might be reduced by 4. 20% by preventing T. trichiura infection. These results could be explained by the pathophysiological impact of T. trichiura infection, including chronic diarrhoea, malnutrition, and iron-deficiency anaemia, all of which are associated with impaired cognitive function [34]. Furthermore, a recent experimental study demonstrated that T. trichiura contributes to pathological changes in the hippocampus and amygdala [10]. Existing preventive chemotherapy shows low to moderate efficacy against T. trichiura in high endemic countries [35]. This finding suggests that new solutions such as alternative treatment (e. g. oxantel pamoate) are needed to eliminate STH-associated morbidity. Our results show that large areas in the Philippines still lag in meeting functional literacy targets. Our estimates indicated that for 2017, Luzon had the highest estimated number of school-aged individuals with low levels of functional literacy. However, when these estimates were adjusted by the geographical variation in population density we found that areas in Mindanao had the highest density of school-aged individuals with functional illiteracy. Geographically targeted functional literacy interventions should thus be prioritised in at-risk areas identified in each region. These interventions should consider the region-specific determinants highlighted above. For example, in Luzon, the gender difference in the prevalence of reduced functional literacy identified in our study suggests that the current integrated helminth control program, which recently expanded its target age group from 1 to 12 years old to individuals 1 to 18 years old [28], should provide more attention to male adolescents. In the Visayas, given the high attributable risk of functional illiteracy from poor sanitation facilities, health educational programs promoting appropriate hygiene and sanitation practice such as educational videos (e. g. The Magic Glasses), which have proven efficacy in China [36], and are currently being tested in the Philippines are recommended. In Mindanao, given the perceived risk of functional illiteracy that could be associated with T. trichiura infections, and the chemotherapeutic failure for this particular parasite, educational and health promotion programs to this region should be considered. This study has some limitations that need consideration. Our data are from the 2008 Functional Literacy, Education and Mass Media Survey (FLEMMS) and may not accurately reflect the current situation. That said, these data constitute the best available and most contemporaneous dataset available and represents a cross section of the school-age population in the Philippines. Additionally, the rate of functional literacy has not seen much improvement in the last three FLEMMS (83. 8% in 1994,84. 1% in 2003, and 86. 4% in 2008) suggesting that data from the 2008 survey were unlikely to differ notably from the current situation. The prediction figures for 2017 may have overestimated the numbers of children currently at risk of functional illiteracy because this model assumes a constant increase in population and it does not account for changes in the prevalence of risk factors of functional illiteracy and STH infection such as poverty and WASH, with the rapid urbanisation that is happening in some parts of the Philippines [37]. However, according to the Philippines Statistics Authority (PSA), while the Philippines has shown some progress achieving the SDGs for literacy, poverty, and water sanitation, the levels have fluctuated between each survey period, showing no distinct trend, however there remain some parts of the Philippines, such as Autonomous Region in Muslim Mindanao (ARMM), that constantly record above average rates of poverty and less access to safe drinking water and sanitation facilities [38,39]. Presently, there are no data available on functional literacy in the Philippines for 2017 to validate our prediction results, therefore further investigation is required. In addition, our predictive prevalence for P. falciparum and P. vivax are likely to represent underestimates, as malaria data for the targeted age group of our study were not available. However, given the low endemicity level of both species of malaria across the Philippines, the effect of malaria was subtle. Small-scale factors such as nutrition are also known to be important determinants of cognitive dysfunction [6]. Unfortunately, data on these factors were not available. In addition, our estimated PAF could be biased in the presence of unaccounted confounding factors. In conclusion, our findings support the need for spatially targeted strategies that can lead to a reduction in the transmission of STH infections and other determinants of functional illiteracy in school-aged children in the Philippines. In the context of the current work, this is particularly relevant in order for the Philippines to achieve the SDG target for functional literacy by 2030.

Title: Functional illiteracy burden in soil-transmitted helminth (STH) endemic regions of the Philippines: An ecological study and geographical prediction for 2017 Summary: While previous studies in the Philippines indicated an association between STH infection and cognitive development measured by memory and school performance, the contribution of STH infections on the overall functional illiteracy burden in the Philippines is unknown. This study presents the first use of geographical risk models of functional literacy adjusted for a wide array of probable confounders to uncover the associations with STH infections. This study also explores how the application of spatial epidemiology in mapping functional illiteracy provides an evaluation-planning tool for the design and implementation of STH-associated morbidity control intervention strategies, estimating the number of school-aged children with functional illiteracy associated with STH infections, and the number of interventions needed in the Philippines.

lay_plos

en

Summarize: Roxane, The duenna, Cyrano. ROXANE: We are going to Clomire's house. (She points to the door opposite): Alcandre and Lysimon are to discourse! THE DUENNA (putting her little finger in her ear): Yes! But my little finger tells me we shall miss them. CYRANO: 'Twere a pity to miss such apes! (They have come to Clomire's door.) THE DUENNA: Oh, see! The knocker is muffled up! (Speaking to the knocker): So they have gagged that metal tongue of yours, little noisy one, lest it should disturb the fine orators! (She lifts it carefully and knocks with precaution.) ROXANE (seeing that the door opens): Let us enter! (On the threshold, to Cyrano): If Christian comes, as I feel sure he will, bid him wait for me! CYRANO (quickly, as she is going in): Listen! (She turns): What mean you to question him on, as is your wont, to-night? ROXANE: Oh-- CYRANO (eagerly): Well, say. ROXANE: But you will be mute? CYRANO: Mute as a fish. ROXANE: I shall not question him at all, but say: Give rein to your fancy! Prepare not your speeches,--but speak the thoughts as they come! Speak to me of love, and speak splendidly! CYRANO (smiling): Very good! ROXANE: But secret!... CYRANO: Secret. ROXANE: Not a word! (She enters and shuts the door.) CYRANO (when the door is shut, bowing to her): A thousand thanks! (The door opens again, and Roxane puts her head out.) ROXANE: Lest he prepare himself! CYRANO: The devil!--no, no! BOTH TOGETHER: Secret. (The door shuts.) CYRANO (calling): Christian!

Summary: Roxane and the Duenna prepare to go to the talk on love at the house opposite. She tells Cyrano that if Christian comes to visit her, Cyrano should ask him to wait. She reveals that she plans to ask Christian to improvise around the subject of love, but asks Cyrano not to pass this on to Christian, as she believes he would practice a speech beforehand. Roxane and the Duenna leave. Cyrano calls Christian, who has been waiting nearby.

booksum

en

Summarize: CROSS REFERENCE TO RELATED APPLICATION The present application is a Continuation Application of U.S. patent application Ser. No. 11/048,233 filed on Feb. 1, 2005, which claims priority to German Utility Model Application Serial No. 20 2004 001 504.8, filed Feb. 2, 2004, the entire content of these applications are expressly incorporated herein by reference thereto. FIELD OF THE INVENTION The present invention relates to a device for manipulating bone and, in particular, a device for clamping bone fixation elements, which are attached to bone, for manipulating fragments of a fractured bone. BACKGROUND OF THE INVENTION Bone fractures, especially fractures of the proximal femoral shaft, have proven difficult to manipulate in preparation for internal fixation. For example, when proximal shaft fractures of the femur occur, the distal end of the proximal fragment rotates anterior (flexion) and lateral (abduction) creating difficulty in accessing the piriformis fossa and/or the desired entry point for intramedullary nailing or in performing other methods of internal fixation. Several devices for aligning fractured bones are described in the prior art. For example, WO 02/096294 discloses a device for aligning bones. The device includes an elongated shaft which connects a handle, located at a proximal end of the device, to a bone grappling claw, located at a distal end of the device. The shaft and handle define a channel which may receive a compression rod. The claw is positioned around the bone and the rod is moved in the channel towards the claw thereby positioning the bone between the rod and the claw. The positioning of the claw around the bone may necessitate the detachment of the soft tissue surrounding the bone. There remains a need for a device which can be used to manipulate a bone without the need to detach soft tissue surrounding the bone. SUMMARY OF THE INVENTION The device of the present invention may include a hollow body defining a cavity, a longitudinal axis, a front end, a rear end, a clamping body which may be moved axially within the cavity, and a clamping device which may be operably associated with the clamping body. The clamping device may be connected to the clamping body by a connecting member (e.g., a rod or bar) and may be used to move the clamping body axially within the cavity and along the longitudinal axis of the hollow body. A free end of at least one bone fixation element (e.g., Kirschner wires, bone pins, screws) may be inserted into the cavity of the hollow body, the other end of the at least one bone fixation element may be inserted into bone. The cavity may have a wall and may comprise a conical segment and a cylindrical segment. A first end of the conical segment may be positioned proximate the front end of the cavity and a second end of the conical segment may be positioned within the hollow body. The cylindrical segment may be positioned adjacent the second end of the conical segment. The conical segment may taper from the first end to the second end such that a first dimension at the first end of the conical segment may be greater than a second dimension at the second end of the conical segment. The second dimension may be the same as the diameter of the cylindrical segment. In another embodiment, the cavity may comprise only a conical segment. The clamping device may include a screw connection having a sleeve which may be rotated to move the clamping body axially within the cavity. Additionally, the clamping device may also include a mechanism having a lever for locking the clamping body in a fixed position in the cavity of the hollow body. Using the clamping device, an operator may move the clamping body out of the cavity so that the fixation element(s) may be inserted between an external surface of the clamping body and the wall of the cavity. The clamping device may also be used to move the clamping body into the cavity to wedge the fixation element(s) between the external surface of the clamping body and the wall of the cavity. In one embodiment, the lever may have a loosened or unlocked position and a tightened or locked position. The sleeve of the screw connection may include external threads which may engage internal threads formed in a borehole in the hollow body. With the lever in the loosened position, the sleeve may be rotated in a first direction, drawing the sleeve into the hollow body thereby causing the clamping body to move out of the cavity of the hollow body so that a free end of a fixation element, which may have another end positioned in a bone fragment, may be inserted into the cavity between the clamping body and the cavity wall. The sleeve may then be rotated in a second direction, drawing the sleeve out of the hollow body and causing the clamping body to move into the cavity of the hollow body. The clamping body may include grooves for receiving the fixation element(s). As the clamping body moves further into the cavity, the fixation element(s) may be wedged between the clamping body and the wall of the cavity. Thereafter, the lever may be moved to the tightened position, thereby locking the clamping body within the cavity and fixing the fixation element(s) with respect to the device of the present invention. An operator may use the device to manipulate the fixation element(s)s and, thus, manipulate bone. Some advantages achieved by the present invention include: only small incisions in the body may be necessary and detachment of the soft tissue surrounding the bones may be unnecessary; fixation elements may be attached to the bone such that the fixation elements may not penetrate into the medullary space or pass through the bone and, therefore, a medullary pin may be introduced into the medullary space of the bone without removing the device of the present invention, and the fixation elements may be disposed in the bone so that a bone plate may be placed on the surface of the bone without removing the device of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS The present invention may be better understood by reference to the following drawings, wherein like reference numerals represent like elements. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present invention should not be limited to the embodiments shown. FIG. 1 is a cross-sectional view of an embodiment of the device of the present invention; and FIG. 2 is another cross-sectional view of an embodiment of the device of the present invention engaging fixation elements inserted into bone. DETAILED DESCRIPTION As shown in FIG. 1, the device 10 of the present invention includes a hollow body 1 having a longitudinal axis 2, a clamping body 6 which may be moved axially and fixed in a cavity 5 of the hollow body 1, and a clamping device 16 which may be disposed at a rear end 3 of the hollow body 1. It should, however, be understood that those of ordinary skill in the art will recognize many modifications and substitutions which may be made to various elements of the present invention. The clamping body 6 may be moved between a first position, for example, as shown in FIG. 1, and a second position, for example, as shown in FIG. 2. In the first position, there may be a first distance A between the clamping body 6 and the wall 7 of the cavity 5. The first distance A may sized and configured for introducing one or more bone fixation elements (e.g., Kirschner wires 8 ) into a front end 4 of the hollow body 1, past the clamping body 6 and into the cavity 5 between the clamping body 6 and the wall 7 of the cavity 5. Distance A may be between about 3.0 mm and about 6.0 mm. In the second position, there may be a second distance “a” between the clamping body 6 and the wall 7 of the cavity 5. Preferably, distance “a” is less than distance A so that a fixation element may be wedged between the clamping body 6 and the wall 7 of the cavity 5. Distance “a” may be between about 1.0 mm and about 3.0 mm. In one preferred embodiment, the ratio of distance “a” to distance A (a:A) may be between about 0.1 and about 0.9. As shown in FIG. 1, the cavity 5 may include two separate segments. For example, the cavity 5 may include a conical segment 12 at the front end 4 of the hollow body 1 and a hollow cylindrical segment 11 which may adjoin the conical segment 12. Alternatively, the cavity 5 may include only a single conical segment 12. It will be appreciated, however, by one skilled in the art that other shapes may be used to form the cavity 5. The cylindrical segment 11 and the conical segment 12 may have axes which may be coaxial with each other and with the longitudinal axis 2. Moreover, the conical segment 12 may have a first end proximate the front end 4 of the hollow body 1 and a second end located within the hollow body 1 and proximate the cylindrical segment 11. The first end of the conical segment 12 may have a first dimension and the second end of the conical segment 12 may have a second dimension. The conical segment 12 may expand towards the front end 4 of the hollow body 1 such that the first dimension may be greater than the second dimension. The angle α of the conical segment 12 may be between about 5° and about 35″ and the wall 7 of the conical segment 12 of the cavity 5 may extend along at least a longitudinal section X of the cavity 5. It will be appreciated by one skilled in the art that the minimum internal diameter D of the conical segment 12 may correspond to the internal diameter of the hollow cylindrical segment 11. The clamping body 6 may move within the cavity 5 and may have a maximum external diameter d, which may be larger than the minimum internal diameter D of the conical segment 12. The clamping body 6 may be positioned within the hollow body 1 so that the clamping body 6 may be centered in the device 10 and may be used to clamp fixation elements simultaneously. Fixation elements of different sizes may be used with the device 10. For example, Kirschner wires 8 having diameters ranging, for example, from about 1.0 mm to about 3.5 mm, may be used with the device 10. To clamp Kirschner wires 8 in the device 10, the clamping body 6 may be moved towards the rear end 3 of the hollow body 1 such that the Kirschner wires 8 may be clamped in the gap between the wall 7 of the conical segment 12 and an external surface 9 of the clamping body 6. By moving the clamping body 6 towards the rear end 3 of the hollow body 1, the gap between the wall 7 of the conical segment 12 and the external surface 9 of the clamping body 6 may be decreased. Moreover, the external surface 9 of the clamping body 6 may include grooves 13, which may be distributed uniformly on the periphery of the surface 9 and extend on meridians. Such a configuration may enable fixation elements, such as Kirschner wires 8, to be captured positively in the grooves 13 and guided laterally. In this way, an increased torque may be transferred about the longitudinal axis 2. To simplify the introduction of the fixation elements into the gap between the wall 7 of the cavity 5 and the external surface 9 of the clamping body 6, the clamping body 6 may be spherically convex in shape on an axial section 14, which may be directed towards the rear end 3 of the hollow body 1, and conical in shape on an axial section 15, which may be directed towards the front end 4 of the hollow body 1. This configuration may produce small contact zone(s) between the fixation elements and the clamping body 6 and may result in larger clamping forces being exerted on the fixation elements. A clamping device 16 may be used to move the clamping body 6 with respect to the hollow body 1 and to axially fix the clamping body 6 during the clamping of the fixation elements. The clamping device 16 may be connected to a connecting member, for example, rod 21 which, in turn, may be operably attached to the clamping body 6 so that movement of the clamping device 16 may result in corresponding movement of the clamping body 6. The clamping body 6 may be attached to the front end 22 of the rod 21. The rod 21 may be guided in a central borehole 18 passing coaxially through the hollow body 1. The clamping body 6 may be connected with the rod 21 such that the clamping body 6 may be moved axially along the longitudinal axis 2 and may be centered within the cavity 5. This may enable fixation elements of different diameters to be clamped between the clamping body 6 and the wall 7 of the cavity 5. In one embodiment, the device 10 may incorporate a mechanism 19 or a screw connection 20 to move the clamping body 6 with respect to the hollow body 1. In other embodiments, the device 10 may incorporate both the mechanism 19 and the screw connection 20 to move the clamping body 6 within the cavity 5. The mechanism 19 may be located at the rear end 23 of the rod 21 and the screw connection 20 may be located at the rear end 3 of the hollow body 1. The mechanism 19 may include a clamping lever 25 with a contact surface 26. The contact surface 26 may be, for example, cylindrical in shape and may be eccentric with respect to an axis of rotation 24. The clamping lever 25 may swivel about the axis of rotation 24, which may be orthogonal to the longitudinal axis 2. For example, the clamping lever 25 may be rotated between a first position, as shown in FIG. 1, and a second position, as shown in FIG. 2. Moreover, the construction of the mechanism 19 may enable an operator to assert a large clamping force on a fixation element. The mechanism 19 may also function as a safeguard preventing the clamping device 16 from loosening unintentionally and, thereby, releasing the fixation elements. The screw connection 20 may have an axis which may be coaxial with the longitudinal axis 2. Rotating the screw connection 20 may cause the clamping body 6 to move towards the rear end 3 of the hollow body 1 in an axial direction along the longitudinal axis 2. The screw connection 20 may be rotated until the external surface 9 of the clamping body 6 contacts the fixation elements and causes the fixation elements to be pressed against the wall 7 of the cavity 5. Rotating the screw connection 20 in the opposite direction may cause the clamping body 6 to move towards the front end 4 of the hollow body 1. The axially adjustable screw connection 20 may include a sleeve 30 having an external threaded portion 28 for engaging an internal threaded portion 27 formed in an enlarged portion of the borehole 18 at the rear end 3 of the hollow body 1. The external threaded portion 28 may engage the internal threaded portion 27 such that when the sleeve 30 is rotated in a first direction, the clamping body 6 may move out of the cavity 5 (away from the rear end 3 ), and when the sleeve is rotated in a second direction, the clamping body 6 may move into the cavity 5 (towards the rear end 3 ). The rod 21 may pass through the sleeve 30 and may be dimensioned to extend between the clamping body 6 and the clamping lever 25 such that the contact surface 26 of the clamping lever 25 may rest on the outer end surface 31 of the sleeve 30. In use, a first end of a pair of fixation elements (e.g., Kirschner wires 8 ) may be inserted (e.g., drilled, hammered, etc.) by a surgeon into a bone 17. Alternatively, as appreciated by one skilled in the art, any number of fixation elements may be inserted into bone 17. In an embodiment wherein the device 10 includes both a mechanism 19 and a screw connection 20, the clamp lever 25 may be rotated into the position shown in FIG. 1 (i.e., the loosened or unlocked position). The clamping body 6 may be moved axially out of the cavity 5 to a certain extent by rotating the sleeve 30 in the first direction until the clamping body 6 is in a first position. In the first position, a second end of the fixation element(s) (i.e., the end of the fixation element(s) which has not been inserted into bone) may be introduced into the gap between the clamping body 6 and the wall 7 of the cavity 5. Thereafter, the sleeve 30 may be rotated in the second direction until the clamping body 6 is moved into the second position so that the external surface 9 of the clamping body 6 contacts the second end of the fixation element(s) and causes the fixation element(s) to be pressed against the wall 7 of the cavity 5. The lever 25 may then be rotated into the position shown in FIG. 2 (i.e., the tightened or locked position) to tension the lever 25 and assert an additional upward force on the clamping body 6, which may move the clamping body 6 further into the cavity 5. In this way, the fixation element(s) may be fixed firmly in the gap between the clamping body 6 and the wall 7 of the cavity 5. While the foregoing description and drawings represent the preferred embodiments of the present invention, it will be understood that various additions, modifications and substitutions may be made therein without departing from the spirit and scope of the present invention as defined in the accompanying claims. In particular, it will be clear to those skilled in the art that the present invention may be embodied in other specific forms, structures, arrangements, proportions, and with other elements, materials, and components, without departing from the spirit or essential characteristics thereof. One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, proportions, materials, and components and otherwise, used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not limited to the foregoing description.

Summary: A device for clamping fixation elements, which have been inserted into bone, for manipulating fragments of a fractured bone. The device incorporating a hollow body with a cavity having a wall, a clamping body moveable within the cavity between a first position and a second position, and a clamping device operably connected to the clamping body for moving the clamping body between the first position and the second position. The device being sized and configured so that when the clamping body is in the first position a free end of the fixation element(s) may be positioned between the clamping body and the wall of the cavity. Once the fixation element(s) is properly positioned within the cavity, the clamping device may be used to move the clamping body into the cavity, thereby wedging the fixation element(s) between the clamping body and the wall of the cavity. A bone fragment may then be manipulated using the device.

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Summarize: CROSS-REFERENCE(S) [0001] Not Applicable. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not Applicable. cl REFERENCE TO A “MICROFICHE APPENDIX” [0003] Not Applicable. DESCRIPTION OF RELATED ART INCLUDING INFORMATION DISCLOSED UNDER 37 CFR 1.97AND 37 CFR 1.98 BACKGROUND [0004] U.S. Pat. No. 3,865,110 discloses a disposable diaper with a removably attached bag. In use, one end of the bag is detached from the diaper, the soiled diaper is folded, and the bag is inverted over to enclose the soiled diaper. [0005] U.S. Pat. No. 3,877,432 discloses a disposable diaper with associated disposal bag formed of two sheets of attached fluid-impervious back sheet. The used diaper is enclosed in the disposal bag by turning the back sheet inside out. [0006] U.S. Pat. No. 4,430,087 discloses a disposable diaper with a folded bag attached at one end between the absorbent pad and backing. The bag is inverted over the soiled diaper. The bag containing the soiled diaper is sealed with an adhesive tab before disposal. [0007] U.S. Pat. No. 4,923,455 discloses a disposable diaper with an integral envelope into which the soiled diaper is placed. The envelope is sealed with a resealable adhesive. [0008] U.S. Pat. No. 4,964,859 discloses a disposable diaper with a folded integral changing pad. The pad has a drawstring which is used to close the pad as a bag about the soiled diaper. [0009] U.S. Pat. No. 4,493,713 discloses disposable diaper with a detachable bag. The soiled diaper is placed in the bag which is sealed with a fastening strip. [0010] U.S. Pat. No. 5,071,414 discloses a disposable diaper with an extra layer to the backing layer which forms a pocket at one end. In use, the soiled diaper is rolled up to the end with the pocket, ears on the sides of the diaper are extended over the roll and secured by adhesive strips, and the pocket is inverted over the rolled diaper, enclosing it. [0011] U.S. Pat. No. 5,304,158 discloses a disposable diaper with an attached pouch containing a changing pad. The soiled diaper is wrapped in the pad which is placed in the pouch. [0012] U.S. Pat. No. 6,454,748 discloses a diaper with a pocket. The pocket contains baby-changing related objects. The pocket has adhesive or other closure devices on the interior surface of the external layer for closing the pocket after the soiled diaper is rolled up and turned into the pocket. [0013] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings. BRIEF SUMMARY [0014] Embodiments include disposable diapers comprised of a fluid impervious backing sheet, fluid absorbent material, and a bag for enclosing the diaper for disposal, wherein the improvement comprises attachment of the bag to the impervious backing sheet with the bag opening adjacent to one end of the diaper, the bag capable of inversion with enclosure of the diaper, and a two element interactive closure on the outside of the uninverted bag, [0015] Embodiments include a removable fabric cover panel which covers the attached bag. In embodiments the fabric cover, bag, and fluid impervious sheet is decorated with designs and indicia which may be personalized. In embodiments the bag is scented to mask offensive odors. In embodiments the bag contains a hand sanitizer or wipe for use in hand cleaning. [0016] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tool and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements. [0017] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions. BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIG. 1 is a plan view of the back of a first embodiment diaper. [0019] FIG. 2 is a cross section view of a first embodiment diaper taken at arrow 2 of FIG. 1. [0020] FIG. 3 is a plan view of the back of a second embodiment diaper. [0021] FIG. 4 is a cross section view of a second embodiment diaper taken at arrow 3 of FIG. 3. [0022] FIG. 5 is a cross sectional view of a closed bag with projecting ridge closure elements. [0023] FIG. 6 is a cross sectional view of a closed bag with projecting ridge and slider closure elements. [0024] FIG. 7 is a cross sectional view of a closed bag with male and female closure elements. [0025] FIG. 8 is a cross sectional view of a closed bag with hook and loop closure elements. [0026] FIG. 9 is a cross sectional view of a closed bag with adhesive closure elements. [0027] FIG. 10 is a cross sectional view of a closed bag with self adhesive closure elements. [0028] FIG. 11 is a flow chart of the process of bagging a first embodiment diaper. [0029] FIG. 12 is a flow chart of the process of bagging a second embodiment diaper. DETAILED DESCRIPTION [0030] FIG. 1 is a plan view of the back of a first embodiment diaper 100. A fluid impermeable backing sheet 110 has a top end 104 and a bottom end 102. A left tab 106 and right tab 108 are adhesive coated tabs which are used to fasten the top end 104 to the bottom end 102 when the diaper is in use. A graphic 111 comprising a design or personalized indicia may appear on the backing sheet 110. A bag 120 is folded and attached near the top end 104 of the backing sheet 110. The bag opening 130 is near the top end 104. Visible in FIG. 1 are the front panel 122 of the bag and the back panel 126 of the bag. The front panel closure element 124 is visible in FIG. 1. A graphic 111 comprising a design or personalized indicia may appear on the front panel 124. The backing sheet 110 is overlaid by a cover 118 depicted by diagonal lines in FIG. 1. A U-shaped weakened area 114 of the cover 118, such as cuts in a dashed line, allows tearing of the cover from the bag when it is desired to bag the diaper. A graphic 112 comprising a design or personalized indicia may appear on the cover 118. [0031] FIG. 2 is a cross section view of a first embodiment diaper 100 taken at arrow 2 of FIG. 1. Visible in FIG. 2 is a fluid impermeable sheet 110 with the diaper top 104 and bottom 102. Absorbent material 119 which absorbs body wastes is attached to the sheet 110. The bag 120 is comprised of outer panel 122 and inner panel 126 which are joined at the bag bottom 129 and an opening 130 at the top of the bag. Outer panel 122 has an exterior side 121 and an interior side 123. Inner panel 126 has an exterior side 125 and an interior side 127. An outer panel connector element 124 is located at the exterior side 121 of the outer panel 122. An inner panel connector element 131 is located at the exterior side 125 of the inner panel 126. When the bag 120 is inverted the outer panel connector element 124 interacts with the inner panel connector element 131 and secures the inverted bag in a closed condition. The inner panel 126 is connected and secured to the fluid impermeable sheet 110 at connection site 103 which secures the bag 120 to the sheet 110 near the diaper top 104. In the first embodiment diaper 100 the bag 120 is folded at least once. The fluid impermeable sheet 110 and the bag 120 is enclosed by a cover 118. [0032] FIG. 3 is a plan view of the back of a second embodiment diaper 200. A fluid impermeable backing sheet 210 has a top end 204 and a bottom end 202. A left tab 206 and right tab 208 are adhesive coated tabs which are used to fasten the top end 204 to the bottom end 202 when the diaper is in use. A bag 220 is comprised of a bag front panel 222 and the fluid impermeable backing sheet 210 which plays the role of the back bag panel of the first embodiment diaper. The front panel 222 is connected to the backing sheet 210 at the left side 232, right side 234, and bottom 229. The bag opening 230 is near the top end 204. The front panel closure element 224 is visible in FIG. 3. A graphic 212 comprising a design or personalized indicia may appear on the front panel 222. The front panel 222 is overlaid by a cover 228, depicted by diagonal lines in FIG. 3. A weakened area 214 of the cover material, such as cuts in a dashed line, allows tearing of the cover and allows access to the opening 230 of the bag when it is desired to bag the diaper. A graphic 227 comprising a design or personalized indicia may appear on the cover 228. [0033] FIG. 4 is a cross section view of the second embodiment diaper 200 taken at arrows 3 of FIG. 3. Visible in FIG. 4 is a fluid impermeable sheet 210 (also bag inner panel) with the diaper top 204 and bottom 202. Absorbent material 219 which absorbs body wastes is attached to the exterior side 212 of the fluid impermeable sheet 210. The bag 220 is comprised of outer panel 222 and inner panel 210 (also fluid impermeable sheet) which are joined at the bag bottom 229 and an opening 330 at the top of the bag. In the second embodiment the fluid impermeable sheet 210 is also the bag inner panel 210. Outer panel 222 has an exterior side 221 and an interior side 223. Inner panel or fluid impermeable sheet 210 has an exterior side 212 and an interior side 211. An outer panel connector element 224 is located at the exterior side 221 of the outer panel 222. An inner panel connector element 231 is located at the exterior side 212 of the inner panel or fluid impermeable sheet 210. When the bag 220 is inverted the outer panel connector element 224 interacts with the inner panel connector element 231 and secures the inverted bag in a closed condition. In the second embodiment diaper 200 the bag 220 is unfolded and extends approximately from the top 204 to the bottom 202 of the diaper. The fluid impermeable sheet 210 and the bag 220 is enclosed by a cover 218. [0034] Any diaper having a fluid-impermeable backing sheet can be used with this invention. Suitable diapers include HUGGIES. HUGGIES is a trademark for diapers owned by Kimberly-Clark, Dallas Tex. Suitable diapers also include PAMPERS. PAMPERS is a trademark for diapers owned by The Procter &amp; Gamble Company, Cincinnati, Ohio. [0035] Any suitable fabric material capable of concealing the bag yet allowing easy removal may be used for the cover. Suitable materials include woven or unwoven plastic, gauze, paper, and cotton fabric. [0036] Any suitable two element closure system which provides for secure closure of an inverted bag containing a soiled diaper may be used. The following embodiments illustrate a variety of suitable closures. These elements are illustrated with the bag elements of the first embodiment, but they can be used with any embodiment. [0037] FIG. 5 is a cross section view of an embodiment with an inverted bag closed with parallel projecting ridge closure. Visible in FIG. 5 is the outer panel 122 of the bag 120 with the interior side 123 and exterior side 121. Also visible is the inner panel 126 with the interior side 127 and exterior side 125. Two or more parallel projecting ridges 140 are spaced apart and arrayed near the bag top 130 attached to the exterior side 121 of the outer panel 120 and the exterior side 125 of the inner panel 126. Each ridge 140 is comprised of an elongated web 142 and an expanded head 144. In use the ridges are pressed together and the expanded heads 144 interact and hold the closure elements in a closed position. Suitable bags include ZIPLOC brand bags. ZIPLOC is a trademark for bags owned by S.C. Johnson &amp; Son, Inc., Racine, Wis. [0038] FIG. 6 is a cross sectional view of an embodiment in which the closed lo 0 inverted bag with parallel projecting ridge closure elements are urged together by a slider 148 which is manually drawn along the fasteners from one end to another. The other elements of FIG. 6 are the same as in FIG. 5. The parallel projecting ridge closure elements are attached about the circumference of the outside of the uninverted bag with the slider 148 at one intersection between the front and back panels of the bag. Suitable bags include HEFTY bags with ONEZIP sliders, also termed EASY GRIP sliders. HEFTY, ONEZIP, and EASY GRIP are trademarks owned by Pactiv Corporation, Lake Forest, Ill. for sliders sold as a component part of multi-purpose household bags, plastic bags for multipurpose household use, namely, closeable storage and freezer bags, and a ribbed slider sold as a component of plastic bags for food storage and general purpose use, respectively. [0039] FIG. 7 is an embodiment in which the bag is closed using male and female element fasteners which interact to close the opening of the bag. Visible in FIG. 7 is the outer panel 122 of the bag 120 with the interior side 123 and exterior side 121. Also visible is the inner panel 126 with the interior side 127 and exterior side 125. The male closure element is an elongated ridge 170 comprising a web 172 and an expanded head 174. The female closure element 178 is comprised of an elongated groove 176 with adjoining lips 173 and 175. The fastener elements are attached about the circumference of the outside of the uninverted bag at the top 130 of the bag. The inverted bag is closed by pressing together the male 170 and female 178 elements of the fasteners. In the closed orientation, the expanded head 174 is retained in the groove 176 by the lips 173 and 175. [0040] FIG. 8 is an embodiment in which the bag is closed using hook and loop fasteners elements attached to the circumference of the outside of the uninverted bag. Visible in FIG. 8 is the outer panel 122 of the bag 120 with the interior side 123 and exterior side 121. Also visible is the inner panel 126 with the interior side 127 and exterior side 125. The loop element 180 of the fastener is comprised of a attachment strip 182 to which a multiplicity of loops 183 is connected. The hook element 186 is comprised of an attachment strip 188 to which a multiplicity of loops 187 is connected. The fastener elements are attached about the circumference of the outside of the uninverted bag at the top of the bag 130. Suitable fasteners include VELCRO hook and loop fasteners. VELCRO is a trademark for hook and loop fasteners owned by Velcro Industries B.V., a Dutch Company, and may be obtained from Velcro USA Inc., Manchester, N.H. [0041] FIG. 9 is another embodiment in which a strip of pressure sensitive adhesive is applied to both the front and rear panels of the uninverted bag. Visible in FIG. 9 is the outer panel 122 of the bag 120 with the interior side 123 and exterior side 121. Also visible is the inner panel 126 with the interior side 127 and exterior side 125. A strip of pressure sensitive adhesive 190 is spread on the exterior side 121 of the outer panel 122 and another strip of pressure sensitive adhesive 192 is spread on the exterior side 125 of the inner panel 126 near the top 130 of the bag 120. These adhesives use polymers, such as acrylics, rubbers, and polyurethanes, together with plasticizers and tackifying resins to form a permanently tacky or sticky adhesive. A separate release strip of paper or plastic covers the adhesive strips and protects the strips until the release strip is removed before the bag is inverted. The strips of pressure sensitive adhesive on the front and rear panels of the inverted bag is then pressed together, thereby sealing the bag. Suitable materials include pressure sensitive adhesives obtainable from Adchem Manufacturing, Riverhead, N.Y. [0042] FIG. 10 is an embodiment in which the bag is manufactured of a plastic film having one surface which is adhesive when pressed against a similar surface. Visible in FIG. 10 is the outer panel 122 of the bag 120 with the interior side 123 and exterior side 121. Also visible is the inner panel 126 with the interior side 127 and exterior side 125. The self adhesive surface is on the exterior side 123 of the outer panel 122 and on the exterior side 121 of the inner panel 127 of the uninverted bag near the top 130 of the bag 120. One element of the closure of this embodiment is the exterior surface of the outer panel of the bag and the other element is the exterior side of the inner panel of the bag. When inverted over the soiled diaper, the self adhesive surfaces about the circumference of the inverted bag are manually pressed together, thereby sealing the soiled diaper into the bag for disposal. Any suitable strong film material having at lease one surface self adhesive can be used in this embodiment diaper. Suitable materials include films obtainable from The Glad Products Company, Oakland Calif. using the trademarks PRESS&#39;N SEAL, PRESS&#39;N SEAL FREEZER, and GRIPTEX. PRESS&#39;N SEAL and PRESS&#39;N SEAL FREEZER are trademarks for plastic wrap; plastic bags owned by The Glad Products Company, Oakland Calif. GRIPTEX is a trademark for general purpose plastic bags and plastic wrap owned by Procter &amp; Gamble Company, Cincinnati, Ohio and licensed to The Glad Products Company, Oakland, Calif. [0043] Although the closure elements illustrated in FIGS. 5-10 are illustrated in connection with the first embodiment diaper, the closure elements may be used with the second and any other embodiment. [0044] The interior surfaces of the bag of embodiments are scented to eliminate or suppress unpleasant odors associated with soiled diapers. [0045] The bags of embodiments contain moistened tissues for use in sanitizing and cleaning associated with the changing of a diaper. The used wipes are placed in the inverted bag for disposal after use. [0046] Diapers of embodiments may be used by infants, babies, toddlers, or adults. [0047] The material of the bag in embodiments is opaque thereby concealing the contents when the bag contains a soiled diaper. [0048] FIG. 11 is a flow chart of the process of bagging a first embodiment diaper. The first step 150 is to remove the bag cover by tearing at the weakened areas. The next step is unfolding the bag 152. The user inserts one hand into the bag 154, with the palm toward the back side of the diaper. The user then grasps the diaper 156 with the inserted hand and, optionally, folds the diaper by closing the fingers. The user the inverts the bag over the diaper 158 with the user&#39;s other hand. The user then seals the bag 160 using the closure elements of the now inverted bag. [0049] FIG. 12 is a flow chart of the process of bagging a second embodiment diaper. The first step 250 is to open the bag cover at the weakened area. The user inserts one hand into the bag 252, with the palm toward the back side of the diaper. The user then grasps the diaper 254 with the inserted hand and, optionally, folds the diaper by closing the fingers. The user the inverts the bag over the diaper 256 with the user&#39;s other hand. The user then seals the bag 258 using the closure elements of the now inverted bag. [0050] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Summary: An exemplary embodiment providing one or more improvements includes a fluid-impermeable bag attached to or integral with a disposable diaper, the bag having closure elements on the outside of the front and back panels of the uninverted bag. After use, the bag is inverted over the soiled diaper and the closure elements interact, thereby closing and sealing the soiled diaper inside the bag. In some embodiments the bag is covered by a removable fabric element, such as a gauze panel, which may be decorated.

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Summarize: By. Associated Press. A southwest Ohio woman accused with her husband of abandoning the adopted nine-year-old son they raised since infancy pleaded guilty Monday to a lesser charge and received a suspended jail sentence, a prosecutor said. Lisa Cox and husband Cleveland Cox, of Butler County's Liberty Township, had been charged with nonsupport of dependents. The misdemeanor charge alleging that they recklessly abandoned or failed to provide adequate support for the boy could have resulted in sentences of six months in jail and a $1,000 fine for each of them. Lisa Cox, 52, and her husband Cleveland, 49, of Liberty Township, arrive for a hearing at Butler County Common Pleas Court at the Government Services Center in Hamilton, Ohio, on Wednesday. Lisa Cox pleaded guilty to the lesser misdemeanor of attempted nonsupport of dependents and received a suspended sentence of 90 days in jail, Butler County Prosecutor Michael Gmoser said. The charge against Cleveland Cox was dropped because jeopardizing the father's employment 'would not have been in the best interest of justice or society,' Gmoser said. The couple's attorney, Anthony VanNoy, did not immediately return calls to his office Monday. Authorities alleged the couple left the boy with children services in October after saying he displayed aggressive behavior and earlier threatened the family with a knife. Documents filed by the prosecutor say the parents didn't tell the boy when they left him on October 24 that he wouldn't be returning home. The boy was left with a bag containing some clothes and a handwritten letter from Lisa Cox in which she said that she loved him and would never forget him. Well-to-do: The couple live in this lovely neighborhood where the median income is around $100,000. They had raised their son since he was three months old. VanNoy said last week after a pre-trial hearing that the boy has gotten some much-needed help and the parents have been in counseling with him. VanNoy said the family's goal had always been to get help for the boy. Adolfo Olivas, an attorney appointed by. the court to protect the boy's interests, has said the emotionally hurt. and confused child is now receiving help that the parents should have. gotten for him. Gmoser said the mother's sentence was suspended conditioned upon her good behavior and continued cooperation with the county's children services toward reunification with the boy. Not a money problem: The Coxes live in this home valued at more than $330,000 home in Liberty Township. Threatening? Neighbors said the Coxes are good parents and people. One of them described the 9-year-old adopted boy as a 'bad seed.' The couple claims the boy has threatened them with a knife. Gmoser said he was encouraged by Lisa Cox taking responsibility and by the reunification efforts. 'I feel justice was served,' the prosecutor said in a telephone interview Monday. 'There was no justification for abandonment, but there were mitigating circumstances with respect to the frustration that the family was having toward the issues they were facing on a daily basis.' The prosecutor said he hoped the case would stand as a message and a deterrent to parents who seek to abandon their children in a similar fashion and would focus attention on issues raised by the case. People within the adoption community say they worry about emotional trauma to the child. They say giving up a child after so much time is rare and undermines the stability and commitment that adopted children need. The median annual income in the region where the parents live is more than $100,000, and the median home value is more than $280,000, according to census data

Summary: Lisa Cox of Ohio pleaded was given a suspended jail sentence. Cox, along with her husband, was charged last November with misdemeanor counts of nonsupport and pleaded guilty to a lesser charge. Charges against husband Cleveland Cox were dropped as they jeopardized his employment. The couple tried to return their son to child services after he become violent and threatened to stab them. Their lawyer said that the couple are visiting the boy and hope to be reunited with him. All three are receiving counseling.

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Summarize: BERLIN (AP) — A comet-chasing space probe that has been in hibernation for almost three years has woken up and sent its first signal back to Earth. FILE - In this Dec. 10, 2013 file picture a European Space Agency, ESA, employee sits in the control room for the Rosetta mission at the ESA in Darmstadt, Germany. Scientists at the European Space Agency... (Associated Press) FILE - This image provided by the European Space Agency ESA shows an artist’s impression of the Rosetta orbiter deploying the Philae lander to comet 67P/Churyumov–Gerasimenko. The image is not to scale;... (Associated Press) FILE - This undated image provided by the European Space Agency ESA shows an artist's impression of the Philae lander. Scientists at the European Space Agency are expecting their comet-chasing probe Rosetta... (Associated Press) FILE - In this 2013 file photo provided by the European Space Agency, ESA, employees work in the control room of ESA in Darmstadt, Germany. Scientists at the European Space Agency are expecting their... (Associated Press) The European Space Agency received the all-clear message "Hello World!" from its Rosetta spacecraft some 800 million kilometers (500 million miles) away shortly after 7 p.m. (1800 GMT; 1 p.m. EST). Rosetta was put into hibernation in 2011 to conserve energy for its long journey to meet with comet 67P/Churyumov-Gerasimenko. If all goes as planned the probe will rendezvous with the comet in the coming months and drop a lander onto its icy surface in November. (Reuters) - After a 10-year journey, Europe’s Rosetta spacecraft is due to end its hibernation on Monday and prepare for an unprecedented mission to orbit a comet and dispatch a lander to the surface. Rosetta’s on-board alarm clock is due to go off at 5 a.m. EST (1000 GMT), but it will take the spacecraft about seven hours to warm up its star tracking navigation gear, fire up rocket thrusters to slow its spin, turn on its transmitter and beam a message back to Earth, the European Space Agency said in a status report posted on its website. The probe, presently located about 500 million miles (about 800 million km) from Earth and just shy of Jupiter’s orbit, is so far away that its radio transmissions, traveling at the speed of light, will take 45 minutes to reach listening stations in California and Australia. Ground control teams hope to have confirmation of Rosetta’s resuscitation by 1:30 p.m. EST (1830 GMT), the European Space Agency said. The spacecraft, which carries a 220-pound (100 kg) lander called Philae, has been hibernating for most of the past three years to save power. It is due to reach a 2.4-mile diameter comet called 67P/Churyumov-Gerasimenko in August. Unlike previous comet probes, Rosetta won’t just sail by. The spacecraft is designed to put itself into orbit around 67P for more than a year of close-up studies. Comets are believed to be the pristine leftover remains from the formation of the solar system some 4.6 billion years ago. Scientists hope the mission will provide more clues about how the solar system came into existence, much like the Rosetta Stone, for which the spacecraft is named, provided a blueprint for deciphering ancient Egyptian hieroglyphs. “Rosetta should become a key element for our understanding of the history of the solar system,” Stephan Ulamec, a Rosetta project manager, said in an interview with Reuters last month. One of Rosetta’s first tasks will be to scout for a suitable landing location for its piggyback-riding Philae probe. Scientists are particularly keen to conduct organic chemistry experiments on samples drilled out from inside the comet’s body. “It would be really interesting to find out whether the organic chemistry that is relevant for life is there on comets,” Ulamec said. Engineers who designed the lander did not know what type of terrain they would find on the comet’s surface. It is outfitted with twin harpoons laced with tethers that will be fired into the comet’s surface to anchor Philae and keep it from bouncing back into space after touchdown. Europe spent about 1 billion euros ($1.35 billion) on the mission, which is due to run at least until the end of 2015. (This story was refiled to fix typo in paragraph 5) Media playback is unsupported on your device Media caption Engineers will now finesse Rosetta's trajectory for rendezvous with a comet Rosetta, Europe's decade-long quest to put a robotic lander on a comet, has reached a key milestone. The probe, which has spent the past two-and-half-years moving through space in a deep sleep, was expected to rouse itself at 10:00 GMT, ready to send a signal to Earth. Receipt of this "I'm awake" message will confirm the great endeavour is still on course. Rosetta is due to rendezvous with Comet 67P/Churyumov-Gerasimenko in August. The despatch and landing of the small robot currently piggybacking the probe is set for November. The reactivation of Rosetta is occurring some 800 million km from Earth, out near the orbit of the planet Jupiter. Controllers at the European Space Agency's (Esa) operations centre here in Darmstadt, Germany, do not know precisely when Monday's all-important contact will be made, but they anticipate their consoles lighting up sometime between 17:30 and 18:30 GMT. "It will be transmitting just the 'carrier signal', so at that point there's no data coming down from the spacecraft," explained Andrea Accomazzo, Rosetta's spacecraft operations manager. "We just receive a firm frequency. In theory, it would be like a continuous beep if you were to convert it into sound. "We will see it on a screen that is basically a spectrum analyser. Although, we will have no information from the spacecraft, we will know just from that transmission that it must have done everything it had to do automatically and is in a safe status; and that everything that happens next is in our hands," he told BBC News. Rosetta must work through a sequence of pre-programmed activities before sending the signal. None of these activities has a fixed time length. They include warming the spacecraft's navigation instruments, and finding Earth on the sky so that the probe's main communications antenna can be pointed in the right direction for the call home. Media playback is unsupported on your device Media caption Andrea Accomazzo, Rosetta's Space Operations: "Milestone" in the project First contact is likely to come through the US space agency's 70m Goldstone radio dish in California. If the signal does not arrive as expected, controllers will hold off any intervention until Tuesday morning. The Darmstadt team can send commands to Rosetta that would forcibly bring it out of its slumber, but the preference is to give the probe sufficient time to complete the necessary tasks automatically. Rosetta was put into hibernation in June 2011 because its trajectory through the Solar System was about to take it so far from the Sun that its solar panels would produce minimal power. The decision was therefore taken to put the spacecraft in a deep sleep. Image copyright AIRBUS Image caption Europe's Rosetta spacecraft was launched from Earth in 2004 Launched back in 2004, the probe has taken a rather circuitous route out to its comet target. This has involved making a number of flybys of the inner planets, using their gravity to pick up sufficient speed for the eventual comet encounter. It has already delivered some fascinating science, particularly from the close passes it made to two asteroids - the rocks Steins, in 2008, and Lutetia, in 2010. Once controllers have a full assessment of the health of Rosetta, they will initiate a series of burns on its thrusters to close the gap to 67P. Currently at a separation of nine million km, this will be reduced to a mere 10km by mid-September. The landing of the three-legged robot called Philae in November is sure to be a nail-biting event. "I know that Nasa colleagues talked about'seven minutes of terror' for the landing of the Curiosity rover on Mars; it'll be more like four hours of terror between the separation of Philae and its 'docking' on the comet," said Esa director general Jean-Jacques Dordain. The intention is for Rosetta to follow the comet as it moves closer towards the Sun, monitoring the changes that take place on the body. Philae will report changes that occur at the surface. Comets - giant "dirty snowballs", as some have called them - are believed to contain materials that have remained largely unchanged since the formation of the Solar System 4.6bn years ago, and Rosetta data should therefore help researchers understand better how our local space environment has evolved over time. "Rosetta is a unique mission - unique technologically, unique scientifically, and unique philosophically because comets may be at the origin of who we are," Mr Dordain told BBC News. Image copyright Airbus Image caption Philae robot's task in November: No mission has ever attempted to make a soft landing on a comet before Jonathan.Amos-INTERNET@bbc.co.uk and follow me on Twitter: @BBCAmos

Summary: After snoozing since 2011, the comet-chasing Rosetta probe awoke today, sending the message "Hello World!" to the European Space Agency just after 1pm EST, according to the AP. The European probe, launched a decade ago, has been in a long hibernation to save power. Now, it's ready for its unprecedented mission to orbit a comet and send a lander to its surface, the BBC reports. The message indicates that the probe has finished its wake-up routine, including warming its navigation instruments and locating Earth. Rosetta, currently around 500 million miles from Earth near Jupiter's orbit, is due to rendezvous with the comet R67P/Churyumov-Gerasimenko in August. Scientists believe analysis of the comet-believed to be a leftover from the formation of our solar system-will provide vital clues about how the solar system came to be. "It would be really interesting to find out whether the organic chemistry that is relevant for life is there on comets," a Rosetta project manager tells Reuters.

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Write a title and summarize: Yeast biofilms are complex multicellular structures, in which the cells are well protected against drugs and other treatments and thus highly resistant to antifungal therapies. Colony biofilms represent an ideal system for studying molecular mechanisms and regulations involved in development and internal organization of biofilm structure as well as those that are involved in fungal domestication. We have identified here antagonistic functional interactions between transcriptional regulators Cyc8p and Tup1p that modulate the life-style of natural S. cerevisiae strains between biofilm and domesticated mode. Herein, strains with different levels of Cyc8p and Tup1p regulators were constructed, analyzed for processes involved in colony biofilm development and used in the identification of modes of regulation of Flo11p, a key adhesin in biofilm formation. Our data show that Tup1p and Cyc8p regulate biofilm formation in the opposite manner, being positive and negative regulators of colony complexity, cell-cell interaction and adhesion to surfaces. Notably, in-depth analysis of regulation of expression of Flo11p adhesin revealed that Cyc8p itself is the key repressor of FLO11 expression, whereas Tup1p counteracts Cyc8p’s repressive function and, in addition, counters Flo11p degradation by an extracellular protease. Interestingly, the opposing actions of Tup1p and Cyc8p concern processes crucial to the biofilm mode of yeast multicellularity, whereas other multicellular processes such as cell flocculation are co-repressed by both regulators. This study provides insight into the mechanisms regulating complexity of the biofilm lifestyle of yeast grown on semisolid surfaces. In nature, microorganisms preferentially live within multicellular communities such as different types of biofilms and colonies [1–5]. Yeast biofilms are complex uniquely organized structures, in which cells are protected from hostile environments, including antifungals, host immune systems, and starvation. Active multidrug resistance transporters and protective extracellular matrix that are produced by subpopulations of differentiated cells in colony biofilms formed by wild Saccharomyces cerevisiae strains [6], contribute to this protection. Despite the identification of various genes and processes (including chromosome reorganization) involved in formation of colony biofilm structure [1,7, 8], many of these processes seem to be specific to particular wild strains. An exception is the Flo11p cell wall adhesin, a key protein involved in several developmental processes including cell adhesion [9] and the formation of colony biofilms [10], flor biofilms [11], and mats [12]. Deletion of FLO11 results in the formation of smooth colonies in various non-isogenic wild strains isolated from different habitats [13] as well as in Σ1278-derived strains [10,14]. FLO11 mRNA levels are elevated in colony biofilms and lowered after phenotypic switching called domestication, during which cells are reprogrammed to form smooth colonies similar to colonies of laboratory strains, in which key features of biofilm-life style are switched off [13,15]. The FLO11 promoter extends to 3 kb and contains at least four upstream activation sequences and nine elements involved in repression [16]. Thus, FLO11 gene expression integrates signals from diverse signaling cascades, including the Ras-cyclic AMP-dependent kinase, mitogen-activated protein kinase (which controls filamentous growth) and the main glucose repression pathways. These pathways positively or negatively regulate FLO11 expression in accordance with growth stage and nutritional conditions [1,16–18]. The expression of FLO11 is also controlled by epigenetic mechanisms, including histone deacetylation, chromatin remodeling, non-coding RNAs and prion formation [1,19–23]. The Cyc8p (Ssn6p) -Tup1p complex is mostly known to function as a transcriptional co-repressor that is conserved in eukaryotic organisms including mammals [24]. Four molecules of Tup1p in concert with one molecule of Cyc8p form this complex [25] with a tendency to oligomerize [26], which regulates hundreds of S. cerevisiae genes involved in diverse pathways, such as glucose, starch and oxygen utilization, the response to osmotic stress, DNA repair, mating, sporulation, meiosis and flocculation [24,27,28]. Cyc8p-Tup1p does not bind directly to DNA but is brought to promoters via interactions with sequence-specific regulatory binding proteins, which coordinate the expression of specific subsets of genes [24]. Some data indicate that Cyc8p may play a more direct role in the repression [29]. Cyc8p-Tup1p can interact with Mig1p and Nrg1p, which bind to the promoters of glucose-repressed genes, such as FLO11, in the presence of glucose [17,30]. Cyc8p-Tup1p can also act as a transcriptional co-activator of various genes such as HAP1 [31,32], FRE2 [33], ARG1 and ARG4 in cooperation with Gcn4p [34], TAT1 and TAT2 [35], and genes induced by Hog1p in cooperation with Sko1p [36]. Genome-wide profiling of changes in nucleosome organization and gene expression that occur following the loss of CYC8 or TUP1 in S. cerevisiae laboratory strains show significant overlap, but additional changes result from the absence of either TUP1 or CYC8 [37]. Thus, the major function of Cyc8p and Tup1p in S. cerevisiae, identified so far, is the repression of pleiotropic gene targets mostly in the form of the Cyc8p-Tup1p co-repressor complex. In addition, several mutually independent repressor functions of Tup1p and Ssn6p (a functional homologue of S. cerevisiae Cyc8p) have been reported in Candida albicans, in which filamentous growth and hypha-specific genes are repressed by Tup1p independently of Ssn6p, whereas Ssn6p may act as a repressor of phenotypic switching independently of Tup1p [38]. Ssn6p was recently identified as a negative regulator of the opaque cell transcription program in white C. albicans cells and of the white cell transcription program in opaque cells [39]. Tup1p was reported to be a repressor of the opaque state and, together with its negative regulator Wor1p, has been proposed to control the opaque switch under different circumstances [40]. In addition to its interaction with Tup1p, Ssn6p interaction with histone deacetylase Rpd31p has been reported in C. albicans [41]. In this study, Ssn6p appeared to be a repressor of filamentation as well as of wrinkled colony morphology under particular conditions, independently of Tup1p, and some of these repressive effects were enhanced by deletion of RPD31. MoTUP1 was recently identified in Magnaporthe oryzae (a rice pathogen), and its deletion causes decreased pathogenicity of the fungus [42]. These studies suggest that Tup1p and Cyc8p play important roles in the pathogenicity of different fungi and that, in addition, these factors could have independent roles. In this study, we provide clear evidence of the functions of the Tup1p and Cyc8p regulators in biofilm colony formation. We present evidence that Cyc8p itself is a repressor of FLO11 gene expression and of the formation of the structured architecture of colony biofilms, whereas Tup1p counteracts Cyc8p, being a positive regulator of FLO11 expression and colony complexity. Furthermore, we show that Tup1p regulates Flo11p accumulation at two different levels—gene expression and Flo11p stability. In addition to Flo11p, other features that are important for colony biofilm formation, such as cell invasiveness, adhesion to solid surfaces and presence of fibers connecting the cells, are also antagonistically regulated by Cyc8p and Tup1p. Conversely, features that are related to other types of multicellularity, such as cell flocculation, are co-repressed by both regulators. A series of strains was constructed producing different levels of Cyc8p and Tup1p regulators (Table 1) derived from the parental BR-F strain (wt strain; [43]), which forms structured colony biofilms [6]. The tup1 strain (tup1/tup1) was prepared by deleting both alleles of TUP1, but we did not succeed in preparing a cyc8 strain (cyc8/cyc8). As the CYC8 gene is essential in the Σ1278 strain-background [44], resembling in several aspects wild yeast strains, this gene may also be essential in the BR-F strain. Therefore, we constructed strain pGAL-CYC8 (cyc8/pGAL-CYC8), in which one CYC8 allele is deleted and the second placed under the control of the GAL1-inducible promoter (pGAL), which provides only very low (basal) level of CYC8 expression in the absence of galactose. The decreased level of CYC8 mRNA and level of Cyc8p in this pGAL-CYC8 strain (grown without galactose), compared with the BR-F strain, was confirmed by northern blot (S1A Fig) and LC-MS/MS (see below), respectively. We also prepared strain pTEF-CYC8 (CYC8/pTEF-CYC8) constitutively over-expressing CYC8 from the TEF1 promoter (pTEF). Unexpectedly, deletion of TUP1 and CYC8 over-expression resulted in a similar, very prominent change in colony architecture indicating opposing roles of Tup1p and Cyc8p in biofilm formation (Fig 1A). In both cases, the strains formed smooth colonies. Conversely, although reduced CYC8 expression slowed the growth of the pGAL-CYC8 strain, this strain formed structured colony biofilms that gradually developed morphology similar to that of wt strain biofilms (S1B Fig). Hence, 3-day-old pGAL-CYC8 colonies exhibited an architecture (Fig 1B) with features typical of younger (40-h-old) structured biofilms formed by the wt strain and 5-day-old pGAL-CYC8 colony biofilms resemble 3-day-old biofilms of the wt strain (S1B Fig and [6]). Flo11p is essential for colony biofilm formation. Therefore, we investigated the potential role of Tup1p and Cyc8p in Flo11p expression. We prepared strains pGAL-CYC8-Flo11p-GFP (cyc8/pGAL-CYC8-Flo11p-GFP) and pGAL-TUP1-Flo11p-GFP (tup1/pGAL-TUP1-Flo11p-GFP) (derived from the BR-F-Flo11p-GFP strain; [15]), in which CYC8 and TUP1 expression is inducible by galactose, to monitor Flo11p-GFP levels in the context of colony biofilm morphology. Presence of galactose in GMA partially affects the colony appearance, slightly reducing the structured morphology, as has similarly been shown for glucose YEPD medium [45]. pGAL-TUP1-Flo11p-GFP and pGAL-CYC8-Flo11p-GFP colonies were, therefore, first grown on GMA plates without galactose for 3 days and then expression of pGAL-controlled genes was induced for ~18 h by adding galactose to wells in the agar. Fig 2A shows that in areas of higher galactose concentration (near the wells), colony morphologies changed due to the induction of TUP1 (smooth → structured) or CYC8 (structured → smooth), whereas colonies located far from the galactose source retained their original morphologies. Western blots showed that Flo11p-GFP is produced in high levels in pGAL-TUP1-Flo11p-GFP colonies induced by galactose (Fig 2B, lane 4), whereas Flo11p-GFP production was totally abolished when CYC8 over-expression was induced by galactose in pGAL-CYC8-Flo11p-GFP colonies (lane 8). In accordance, Flo11p-GFP was undetectable in pTEF-CYC8-Flo11p-GFP (CYC8/pTEF-CYC8-Flo11p-GFP) colonies constitutively overexpressing CYC8 (lane 5), whereas Flo11p-GFP level in pTEF-TUP1-Flo11p-GFP (TUP1/pTEF-TUP1-Flo11p-GFP) (lane 1) colonies was similar to that of wt colonies (lane 2). Two photon excitation confocal microscopy (2PE-CM) showed that in 3-day-old wt colonies, Flo11p-GFP is present at higher levels in cells at the aerial surface of wt colonies and in cells forming the tips of “roots” invading the agar (Fig 2C). A similar pattern of Flo11p-GFP was observed in structured pGAL-TUP1-Flo11p-GFP colonies near the galactose source and in structured pGAL-CYC8-Flo11p-GFP colonies that were localized far from the galactose source and were thus not induced (Fig 2D). However, Flo11p-GFP was hardly detectable in smooth colonies of both strains. To further clarify regulatory functions of Tup1p and Cyc8p, we compared amounts of TUP1 and CYC8 mRNAs and proteins in wt colonies and colonies of above described strains with differently manipulated levels of Cyc8p or Tup1p (Fig 3A, left part, and 3B). Colonies were grown for 3 days on GMA and then induced by galactose (or treated with distilled water as a control) for 4 hours. This induction greatly increased TUP1 and CYC8 mRNA levels, respectively, in pGAL-TUP1 and pGAL-CYC8 strains (Fig 3A, lanes 4 and 6, respectively). Conversely, amounts of both CYC8 and TUP1 mRNAs were only slightly increased, as compared with mRNA levels in wt colonies, when expression was controlled by the moderate, constitutive pTEF promoter (lanes 7 and 8). Labeling of both Tup1p and Cyc8p proteins with GFP or 6HA tags resulted in dysfunctional proteins and commercial anti-Tup1p and anti-Cyc8p primary antibodies generated high unspecific background. Therefore, we quantified Tup1p and Cyc8p approximate concentrations in cells from 3-day-old colonies induced/non-induced by galactose for 4 hours by label free LC-MS/MS (Fig 3B). Contrary to TUP1 and CYC8 mRNA levels, which both were highly elevated when expression was induced by galactose (Fig 3A, lanes 4 and 6), differing enhancement of Cyc8p and Tup1p protein concentration was observed in pGAL-CYC8 and pGAL-TUP1 3-day-old colonies. Whereas Cyc8p level increased only by 40% (~1. 4 times), Tup1p level increased more than 5 times as compared with wt colonies (Fig 3B). In accordance with mRNA analysis (Fig 3A, lines 5 and 3), neither Cyc8p nor Tup1p were detected without galactose induction in pGAL-CYC8 and pGAL-TUP1 colonies, respectively (Fig 3B). Analyses of FLO11 mRNA levels (Fig 3A) showed that Cyc8p and Tup1p affect FLO11 gene expression in opposite ways and with differing efficiencies. CYC8 constitutive overexpression in pTEF-CYC8 colonies resulted in absence of FLO11 mRNA (Fig 3A, lane 7) and of Flo11p-GFP protein (Fig 2B, lane 5), thus confirming Cyc8p as a FLO11 gene repressor. TUP1 deletion in the presence of functional Cyc8p (tup1 strain) resulted in the absence of Flo11p (Fig 2B, lane 9), but a small amount of FLO11 mRNA was still detectable (Fig 3A, lane 2). This result confirmed that FLO11 transcription is enhanced by Tup1p, but when the TUP1 gene was deleted, some transcription of FLO11 still occurred. In accordance, the level of FLO11 mRNA was significantly reduced after 4 h of galactose induction of pGAL-CYC8 colonies and the level of FLO11 mRNA was significantly increased after 4 h of induction of pGAL-TUP1 colonies (Fig 3A, lane 6 and 4, respectively). 18 h after galactose induction, Flo11p protein levels increased from non-detectable to a level comparable with the wt strain in pGAL-TUP1 colonies (Fig 2B, compare lanes 3 and 4; Fig 2D) and decreased from a wt-like to non-detectable level in pGAL-CYC8 colonies (Fig 2B, compare lanes 7 and 8; Fig 2D). To further examine mutual effects of both regulators, we constructed an additional set of strains derived from the BR-F and BR-F-Flo11p-GFP strains, in which amounts of both regulators were adjustable by the inducing compound (galactose or copper) (Table 1, last four strains). We then evaluated levels of TUP1, CYC8 and FLO11 gene expression (mRNA) and levels of respective proteins. Results of CYC8 and TUP1 mRNA analysis after 4 h of galactose and/or copper induction of colonies of strains pGAL-CYC8/pCUP-TUP1 (cyc8/pGAL-CYC8/tup1/pCUP-TUP1) and pGAL-TUP1/pCUP-CYC8 (tup1/pGAL-TUP1/cyc8/pCUP-CYC8) (Fig 3A, lanes 9–16) were consistent with results of induction experiments performed with strains, in which expression of only one of the regulators was adjustable and the second was controlled by its native promoter (Fig 3A, lanes 3–6). Only the level of pGAL-regulated mRNA (of both TUP1 and CYC8) was partially diminished when copper was also present during galactose induction (Fig 3A; compare lanes 14 and 16 for CYC8 and lanes 10 and 12 for TUP1, decreased level of mRNA is marked by asterisk). Since pGAL-regulated expression of CYC8 and TUP1 was also diminished by copper in pGAL-CYC8 and pGAL-TUP1 colonies, respectively (S2 Fig), copper seem to partially reduce transcription from the pGAL promoter. Consistently with pGAL-CYC8 and pGAL-TUP1 induction experiments, increased level of Cyc8p caused a decrease in FLO11 mRNA (Fig 3A, lanes 11 and 14) and in Flo11p concentrations (Fig 3C, lanes 4 and 7) in pGAL-CYC8/pCUP-TUP1 and pGAL-TUP1/pCUP-CYC8 colonies. However, the basal FLO11 mRNA level when neither Cyc8p nor Tup1p was induced (Fig 3A, lanes 9 and 13) was higher than under conditions where a wt-level of Cyc8p was present (tup1 or pGAL-TUP1 colonies without galactose, Fig 3A, lanes 2 and 3). 4 h-induction of Tup1p by either inducing compound did not significantly increase the FLO11 mRNA level (Fig 3A, lanes 10 and 15) above the basal level identified in the absence of both inducing compounds (lanes 9 and 13). In fact this basal level was lower in the pCUP-CYC8/pGAL-TUP1 strain than in pGAL-CYC8/pCUP-TUP1 strain (Fig 3A, compare lanes 9 and 13), possibly because of traces of copper in the medium which can slightly increase CYC8 expression and thus the amount of Cyc8p repressor from the onset of colony growth. Altogether, these data further confirmed that Cyc8p is the main repressor of expression of the FLO11 gene and indicated that Tup1p modulates the level of Cyc8p repressor, potentially via formation of Tup1p-Cyc8p complex (Fig 4). Analysis of Flo11p-GFP protein levels however suggested an additional function of Tup1p. Flo11p-GFP full length protein was almost undetectable in the absence of both regulators, whereas a high level of free GFP was present in these samples indicating that Flo11p-GFP synthesis was relatively high (in accordance with high basal level of FLO11 mRNA, Fig 3A, lane 9 and 13), but that the protein was efficiently degraded (Fig 3C, lanes 2 and 6, arrows mark free GFP). These data indicate dual roles of Tup1p in regulation of Flo11p concentration in colonies and thus in regulation of colony biofilm complexity: counteracting CYC8 repression of FLO11 gene expression and preventing Flo11p degradation, possibly by repressing expression of a specific protease. Flo11p is associated with the cell wall and it is partially shed from cells into the extracellular space [46]. We therefore examined further whether extracellular Flo11p-GFP is degraded and whether presence of Tup1p influences such degradation. As expected, neither Flo11p-GFP nor free GFP was detected in extracellular fluid from colonies of pTEF-CYC8 and tup1 strains (Fig 3D, lanes 4 and 5), in which FLO11 expression is repressed by Cyc8p. In extracellular fluid from biofilm colonies of wt strain and pGAL-CYC8 strain without galactose, both partially degraded Flo11p-GFP and high level of free GFP were detected (Fig 3D, lanes 1 and 6), indicating degradation of a fraction of Flo11p-GFP, perhaps during its shedding. In colonies of both pGAL-TUP1/pCUP-CYC8 and pCUP-TUP1/pGAL-CYC8 strains with high basal levels of FLO11 gene expression and protein production in the absence of both inducing compounds, free GFP only was present in extracellular fluid (Fig 3D, lanes 2 and 3). Consistently, colonies of these strains are smooth. These data indicate that Tup1p prevents degradation of extracellular Flo11p-GFP possibly via repression of expression of a cell wall associated or extracellularly localized protease. Differences in Flo11p processing (at several positions within the protein) were found in a strain defective in the kexin Kex2p [46], serine protease which cleaves precursors of secreted proteins in the trans-Golgi network. However, Flo11p was not identified in the screen of possible Kex2p substrates and does not contain prominent Kex2p cleavage sites (Lys-Arg at P1 and P2 position) [47]. Hence, Flo11p is probably not a direct target of Kex2p, but it could be cleaved by another secreted protease, the secretion and/or processing of which requires Kex2p. Next, we examined Cyc8p and Tup1p roles in regulation of other processes that are specific to colony biofilms, such as cell-substrate adhesion and agar penetration and cell-cell interaction via cell wall fibers. Long fibers forming Velcro-like structures in contact sites between the cells were identified in colony biofilms, but not in smooth colonies of the BR-F-flo11 strain, by transmission electron microscopy (TEM) of chemically fixed cells [6]. Here we used high-pressure freezing and freeze substitution TEM that improves identification of these structures and revealed some less abundant, extracellular fibrillar material even on the surface of cells within BR-F-flo11 colonies. Fig 5A thus shows that cells in both structured and smooth colonies are covered on their surface by extracellular fibrillar material, which connects adjacent cells. However, the fibers in this material were significantly (20–30%) longer in the structured colony biofilms of the BR-F and non-induced pGAL-CYC8 strains than in the smooth colonies of pTEF-CYC8, tup1 and flo11 strains (Fig 5B), in which shorter fibers are occasionally visible despite the material appearing to be less structured. The differences are evident also in cell-cell contact sites, where Velcro-like connections were visible among cells in colony biofilms, whereas less structured material was present at contact sites in smooth colonies (Fig 5C). Velcro-like connections may be caused by interaction of N-terminal Flo11A domains of Flo11p as reported in [48] (Fig 5C, indicated by red mark), although direct proof of the presence of Flo11p in these fibers is still lacking. Adhesion to solid surfaces and invasive growth are typical features of fungal biofilms [12] as well as of colony biofilms [13], which are evident particularly in the area of the colony roots [6]. Cell adhesion and agar invasion are absent in S. cerevisiae flo11 colonies [10,13,49]. Figs 1 and 6A show that the pTEF-CYC8 and tup1 strains exhibited defects in invasive growth and adhered poorly to the agar. However, similar to the wt strain, cells of the non-induced pGAL-CYC8 strain adhered to the agar even with robust washing. These data show that both organization of extracellular fibrillar material involved in cell-cell contact and cell adhesion to surfaces are antagonistically regulated by Cyc8p and Tup1p. Adhesion to, and invasiveness into agar did not correlate with cell morphology. BR-F colony biofilms were formed by both oval and elongated cells in the aerial part and by pseudohyphae consisting of elongated cells in the subsurface part (Figs 1B and 6B). In contrast, there was a failure to form elongated cells by, not only smooth colony-forming strains with decreased level of Tup1p (tup1) and increased level of Cyc8p (pTEF-CYC8), but also a colony-biofilm forming strain with reduced level of Cyc8p (pGAL-CYC8). Thus, although the pGAL-CYC8 strain formed (in the absence of galactose) a structured colony biofilm, its root part was formed by chains of rounded cells that divided by monopolar budding and invaded the agar (Fig 1B). Consistently, some wild S. cerevisiae strains form structured colony biofilms despite being unable to form typical pseudohyphae composed of elongated cells [13]. Tup1p and Ssn6p (Cyc8p) are repressors of invasive/filamentous growth in C. albicans [38,41,50]. Our data show that in S. cerevisiae, an imbalance in the Cyc8p and Tup1p levels, rather than the presence or absence of an individual regulator, diminishes cell elongation. This defect, however, does not influence the colony biofilm morphology. The Cyc8p-Tup1p complex has been implicated in the repression of genes involved in cell flocculation, such as FLO1 [17,51]. Consistent with the literature [52–54], either deletion of TUP1 (tup1 strain) or substantial reduction of CYC8 expression (pGAL-CYC8 without galactose) or reduction of TUP1 and CYC8 expression (pCUP-CYC8/pGAL-TUP1 or pCUP-TUP1/pGAL-CYC8 strain without inducing compound) resulted in the formation of macroscopic flocs (clusters of cells) that sedimented efficiently (Fig 6C and 6D), indicating de-repression of the flocculation genes. In striking contrast, the wt strain BR-F and the CYC8- or TUP1- over-expressing strains (pTEF-CYC8 or pTEF-TUP1 strains) did not form cell clusters. These data show that in contrast to the antagonistic functions of Cyc8p and Tup1p in processes involved in colony biofilm formation, Tup1p and Cyc8p in concert repress other flocculation genes such as FLO1 in the BR-F strain. These results are in agreement with findings showing that i) in contrast to FLO11, the expression of other flocculation genes (FLO1, FLO9 and FLO10) is equivalent in colony biofilms and in smooth domesticated colonies and ii) citrate buffer treatment and the presence of mannose in the medium (both of which eliminate the flocculation caused by Flo1p but not that of Flo11p) affect BR-F cell flocculation in liquid culture but not the adhesiveness of cells from BR-F colonies [43] and iii) FLO1 and FLO5 play roles in cell aggregation and flocculation, whereas FLO11 expression promotes invasive growth and biofilm formation [55]. Our findings highlight a previously unknown antagonistic function of Tup1p and Cyc8p in the regulation of complexity of yeast colony biofilms. While Tup1p is essential for the formation of colony biofilms, increased levels of Cyc8p prevent formation of colony biofilms leading to formation of smooth colonies similar to those of laboratory strains. The antagonistic functions of Tup1p and Cyc8p are specific to features typical of yeast biofilm life-style, such as cell invasiveness, adhesion to semisolid surfaces and cell-cell adhesion by cell-wall fibers. Properties important for other types of multicellularity, such as cell flocculation [51], are regulated differently, being repressed by both regulators. In accordance, deletion of genes NRG1, MIG1 or SFL1 for repressors recruiting the Cyc8p-Tup1p co-repressor complex to promoters [56–58], did not prevent Cyc8p-mediated repression of colony biofilm formation in pTEF-CYC8 strain (S3 Fig). Flo11p adhesin is key protein in colony biofilm formation affecting most of the above mentioned biofilm-specific processes [10,13,14]. We therefore tested a hypothesis that Cyc8p and Tup1p regulate biofilm-specific processes via regulation of Flo11p. Indications exist in previous research of a possible relationship between Cyc8p/Tup1p and FLO11 expression, but findings were not consistent. Both positive [59–61] and negative [59] effects of TUP1 deletion on FLO11 mRNA levels have been reported and deletion of the CYC8 gene has been shown to increase FLO11 mRNA levels [56]. Our in depth analyses revealed that Tup1p and Cyc8p regulate the level of Flo11p adhesin in the opposite manner and at different steps in its expression (Fig 4). Firstly, Cyc8p itself represses FLO11 gene transcription, whereas Tup1p counteracts Cyc8p function, thus contributing positively to FLO11 expression. Efficiency of Cyc8p-based FLO11 gene repression depends on the comparative levels of Cyc8p and Tup1p proteins and we hypothesize that Tup1p can balance the level of free Cyc8p by forming a Cyc8p-Tup1p complex, which apparently does not regulate biofilm specific processes, but can regulate other cellular properties such as expression of flocculins. Four molecules of Tup1p interact with one molecule of Cyc8p [26], which means that, in accordance with our data, a smaller change in Cyc8p than in Tup1p levels has a stronger effect on FLO11 expression. Secondly, Tup1p also positively regulates the level of Flo11p protein in colony biofilms by preventing its degradation. Flo11p is targeted to the cell wall via the secretory pathway and is partially shed into the extracellular space [46]. The mechanism of its degradation and involvement of specific protease (s) are currently unknown. Our data showed that Tup1p prevents degradation of extracellular Flo11p-GFP. Tup1p thus may repress expression of a gene coding for a cell wall protease that is involved in Flo11p degradation. Interestingly, Tup1p represses a set of secreted aspartyl proteinases (SAPs) in C. albicans [62] and derepression of genes coding for extracellular proteases was observed in an Aspergilus nidulans strain deleted in the TUPA gene (an ortholog of TUP1) [63]. In summary, our study identifies Cyc8p and Tup1p as important regulators of Flo11p gene expression and protein stability, both affecting the final Flo11p amount in cells and the extracellular space of yeast multicellular structures. According to Flo11p concentration, the structures then acquire different levels of complexity ranging from smooth colonies to colony biofilms. Fine tuning of the amounts and mutual effects of Cyc8p and Tup1p regulators in colony/biofilm cell subpopulations could also provide a mechanism for balancing Flo11p levels and related cellular properties at different positions within the structure and, potentially, at different times during its development. In this respect, further studies are required to uncover potential role of Cyc8p and Tup1p in regulating the amount of Flo11p in different parts of colony biofilms, such as in the internal colony parts, where Flo11p seem not to be present. Orthologs of both Tup1p and Cyc8p are present in different yeast and other fungal species and their role in filamentation, phenotypic switching and virulence of yeast/fungal pathogens has been recently suggested [39–41,50,64]. Identification of Tup1p as an important positive regulator of the formation of colony biofilms brings practical advantages, making TUP1 orthologs in pathogenic yeasts prospective gene targets for new antifungal treatment strategies. All strains prepared in this study (Table 1) were derived from the wild yeast strain BR-F from a collection at the Institute of Chemistry (Slovak Academy of Sciences) and its derivative BR-F-Flo11p-GFP [15]. Colonies were grown on GMA (3% glycerol, 1% yeast extract, and 2% agar) at 28°C unless otherwise indicated, at densities ranging from 103 to 6 x 103 cells per plate. For the flocculation tests, the strains were grown in liquid GM (GMA without agar). For the strain constructions, G418 and nurseothricin concentrations in GMA were 200 and 100 μg/ml. In galactose/Cu2+ induction experiments, colonies were grown 3 days on GMA. Then, the agar was supplemented by galactose and/or Cu2+ to a final concentrations of either 2% galactose and/or 3 mM CuSO4 for colony incubation for 4 h (for Northern blot and LC-MS/MS) or 0. 1% galactose and/or 0. 25 mM CuSO4 for longer 18 h incubations used for morphology experiments and determination of Flo11p-GFP levels by Western blots (lower concentrations of both galactose and Cu2+ were needed to avoid artificially affecting colony morphology in longer incubations). Colony images were captured in incident and/or transmitted light. A ProgRes CT3 CMOS camera with a Navitar objective and NIS Elements software (Laboratory Imaging, s. r. o, Prague, CZ) were used. Strains with gene deletions and with genes under the control of an artificial promoter (pCUP, pGAL and pTEF) were prepared according to [65–67]; primers and plasmids are listed in S1 Table. Yeast cells were transformed as described in [68]. Correct genomic integration of cassettes was verified by PCR using specific primers and by sequencing. Cre-lox system was used to remove antibiotic resistance genes in strains subjected to multiple manipulations [66]. The internal architecture of the microcolonies was visualized by two photon excitation confocal microscopy (2PE-CM) according to [6,69]. In brief, colonies were embedded in agarose and cut vertically down the middle. The cut surface was placed on a coverslip, and colony side views were obtained by 2PE-CM. When required, the cross-sections were stained with 1 μg/ml Calcofluor white. Excitation wavelengths of 920 nm and 790 nm and the emission bandwidths of 480–595 nm and 400–550 nm were used for GFP and Calcofluor white. An overview of the morphology of colonies was obtained simultaneously with green GFP fluorescence as autofluorescence in the 600-740-nm wavelength range. Images of the whole colonies (Figs 1A, 1B, 2C and 2D) and the central parts of the colonies (Fig 1B) were obtained by combining two or three images from neighboring fields of view. The detection of GFP tagged Flo11p (in cell lysates or the extracellular fluid) by western blots was performed as described [70]. In brief, cells harvested from colonies were broken by glass beads in the presence of protease inhibitors, and proteins (25 μg/lane) of cell lysates were subjected to SDS-PAGE. GFP was detected by mouse monoclonal horseradish peroxidase (HRP) -conjugated anti-GFP antibody (Santa Cruz). Membranes stained by Coomassie blue were used as loading controls (S4 Fig). Extracellular proteins were extracted by phosphate-saline buffer from 3-day-old colonies. After centrifugation, proteins of the supernatant were precipitated by methanol/chloroform treatment [71]. Extracellular proteins extracted from 50 mg of wet biomass were loaded to each slot. For nanoLC-MS-MS analysis, the cells were disrupted in 100 mM triethylammonium bicarbonate buffer using glass beads. Protein aliquots (30 μg; determined by the bicinchoninic acid assay, Sigma) were solubilized using sodium deoxycholate (1% (w/v) final conc.), reduced with tris (2-carboxyethyl) phosphine, alkylated with S-methyl methanethiosulfonate, digested sequentially with trypsin and extracted with ethylacetate saturated with water [72]. Samples were desalted using C18 sorbent (Supelco pn: 66883-U) and eluents were dried and resuspended in 20 μl of 1% trifluoroacetic acid. Peptide (2 μg) from each sample were separated on 50-cm C18 column using 2. 5 h elution gradient and analyzed in a DDA mode on Orbitrap Fusion Tribrid (Thermo Scientific) mass spectrometer. Three biological replicates were run for each strain and condition. Resulting raw files were processed in MaxQuant (v. 1. 5. 8. 3) [73]. Searches were performed against latest version of S. cerevisiae Uniprot database and common contaminant database. Further analysis was performed in Perseus (v. 1. 5. 5. 3) [74]. The cell-cell adherence (flocculation) assay was performed according to [75]. In brief: 2-day-old cell cultures grown in GM medium were harvested, flocculation disrupted by EDTA (pH 8,50 mM final concentration) and OD600 of the cell suspension determined (reading A). Then, cells were washed twice by dH2O and suspended in 30 mM CaCl2. After 60 s, OD600 at upper layers of the cell suspension was measured (reading B). Flocculation (%) was calculated according to the formula: 100* (A-B) /A. Average of 4 independent measurements +/- SD is shown. The flocs or free cells were photographed using transmission light microscopy (Microscope DMR, Leica, Germany). In the invasive growth assay [76] cells were streaked onto standard GMA plates and grown at 28°C for 3 days. Plates were vigorously washed with water and photographed. Samples were prepared according to [77] with some modifications. Briefly, 3-day- and 5-day-old colonies were frozen in an EM PACT2 high-pressure freezer (Leica, Germany). The samples were freeze-substituted in an automatic FS machine (Leica, Germany) in 100% acetone containing 2% osmium tetroxide as follows: -90°C for 96 h, 5°C increase per hour for 14 h, -20°C for 24 h, 3°C increase per hour for 8 h, and 4°C for 18 h. The substituted samples were embedded in pure Epon. Ultrathin sections were cut using a Reichert-Jung Ultracut E ultramicrotome and stained using uranyl acetate and lead citrate. The sections were examined using a JEM-1011 transmission electron microscope (JEOL, Japan) operating at 80 kV. Fine structure measurements were performed using a Veleta camera and iTEM 5. 1 software (Olympus Soft Imaging Solution GmbH). Colonies were suspended in TES buffer (10 mM Tris, pH 7. 5,10 mM EDTA, 0. 5% SDS) and total RNA isolated using the hot phenol method [78]. Fifteen micrograms of total RNA was denatured in loading buffer with formamide, separated in 1. 5% agarose gel and transferred to a positively charged nylon membrane (Amersham Hybond-XL, GE Healthcare Ltd). The membranes were hybridized with specific DNA probes prepared using a random primer labeling kit (Takara). The rRNA content was visualized by ethidium bromide staining of gels and used as a loading control (S2 Fig).

Title: Cyc8p and Tup1p transcription regulators antagonistically regulate Flo11p expression and complexity of yeast colony biofilms Summary: Yeast biofilms have become an increasingly important clinical problem over the years. Biofilms are often associated with infections resistant to antifungals, which are particularly prevalent in immunosuppressed patients. High resistance is mediated by cell reprogramming leading to the specific organization of internal biofilm structure and development of numerous protective mechanisms including extracellular matrix formation. Colony biofilms, with architecture and protective mechanisms similar to natural biofilms, represent an ideal model for studying molecular mechanisms and regulations behind biofilm development and organization. Here, we describe a new mechanism of antagonistic regulation of biofilm-specific processes and formation of complex colony biofilm structure by Cyc8p and Tup1p transcriptional regulators. Both these regulators are widespread and conserved among yeasts forming clinically important biofilms, including pathogenic yeasts of Candida spp. The identification of Tup1p as a positive regulator of biofilm formation makes Tup1p-orthologs in yeast pathogens potential targets for the design of new strategies of treatment of biofilm infections.

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Summarize: Background Consumers may access location-based services through smartphones or from in-car location-based services. Four types of companies are primarily responsible for smartphone products and services in the United States: mobile carriers, such as AT&T and Verizon; developers of operating systems, such as Apple’s iPhone iOS and Google’s Android; manufacturers, such as HTC and Samsung; and developers of applications such as games like Angry Birds, social networking applications like Facebook, or navigation tools like Google Maps. We refer to these companies as mobile industry companies. In-car location- based services are delivered by in-car communications systems known as “telematics” systems, portable navigation devices, and map and navigation applications for mobile devices. Companies can obtain location data in various ways. Mobile devices and in-car navigation devices determine location information through methods such as cell tower signal-based technologies, Wi-Fi Internet access point technology, crowd-sourced positioning, and GPS technology. Assisted- GPS (A-GPS), a hybrid technology that uses more than one data collection methodology, is also widely used. For example, companies such as Google and Apple use customer data to compile large databases of cell tower and Wi-Fi access points. Non-carriers use these crowd- sourced location maps to determine location by analyzing which cell tower and Wi-Fi signals are received by a device. Consumers’ location data are transmitted over the cellular network or Wi-Fi access points to companies providing the services. These location data may then be shared with third parties for various uses. For example, companies may choose to partner with third parties to provide a specific location-based service, such as real-time traffic information. Several agencies have responsibility to address consumers’ privacy and create related guidance. The Federal Trade Commission (FTC) has authority to enforce action against unfair or deceptive acts or practices of companies; the Federal Communications Commission (FCC) has regulatory and enforcement authority over mobile carriers; and the Department of Commerce’s (Commerce) National Telecommunications and Information Administration (NTIA) advises the President on telecommunications and information policy issues. Additionally, the Department of Justice disseminates guidance on how law enforcement might request electronic evidence, such as a person’s current or historical location data. Companies Primarily Collect and Share Location Data to Provide and Improve Consumer Services Representatives from mobile industry companies we spoke to for the September 2012 report and in-car navigation service companies we spoke to for the December 2013 report told us they primarily collect and share location data to provide location services and to improve those services. Mobile carriers and application developers offer a diverse array of services that use location information, such as services providing navigation and social networking services that are linked to consumers’ locations. To provide these services, carriers and developers need to quickly and accurately determine location. Location data can also be used to enhance the functionality of other services that do not need to know the consumer’s location to operate. Search engines, for example, can use location data as a frame of reference to return results that might be more relevant. For instance, if a consumer were to search for a pizza restaurant using a location-aware search engine, the top result may be a map of nearby pizza restaurants instead of the homepage of a national chain. In- car location services use location data to provide services such as turn- by-turn directions or roadside assistance. Representatives from both mobile industry companies and in-car navigation services companies told us they also use location data to improve the accuracy of their services. Representatives from some in-car navigation service companies said they share aggregated location data associated with traffic flows with third parties to augment and improve the accuracy of real-time traffic services provided to consumers. Additionally, as we reported in 2012, mobile industry companies can use and sell location data to target the advertising that consumers receive through mobile devices. Doing so may make an advertisement more relevant to a consumer than a non-targeted advertisement, boosting advertising revenue. Advertising is particularly important to application developers, as many developers give their products away and rely on advertising to consumers through free applications for revenue. Companies may also aggregate and store individual consumer data to create consumer profiles. Profiles can be used to tailor marketing or service performance to an individual’s preferences. Mobile industry companies and providers of in-car location services must also share consumer location data if a court finds that the information is warranted for law enforcement purposes. Because consumers generally carry their mobile devices with them, law enforcement can use device location data to determine the consumer’s location. Because of this correlation, location data are valuable to law enforcement for tracking the movements of criminal suspects. Mobile carriers must comply with court orders directing the disclosure of historical location data (i.e., where the device was in the past) and in certain circumstances, real-time location data (i.e., where the device is now). Companies’ Collection and Sharing of Location Data May Put Consumers’ Privacy at Risk Although consumers can benefit from location-based services designed to make their lives easier, consumers also expose themselves to privacy risks when they allow companies to access their location data. In some cases, consumers of location-based services may be unaware that companies share their location data for purposes other than providing those services. As we stated in our September 2012 and December 2013 reports, these privacy risks include, but are not limited to the following: Disclosure to Unknown Third Parties for Unspecified Uses: According to privacy advocates, when a consumer agrees to use a service that accesses location data, the consumer is unlikely to know how his or her location data may be used in ways beyond enabling the service itself. For example, location data may be shared with third parties unknown to the consumer. Because consumers do not know who these entities are or how they are using consumers’ data, consumers may be unable to judge whether they are disclosing their data to trustworthy entities. Third parties that receive shared location information may vary in the levels of security protection they provide. If any of these entities has weak system protections, there is an increased likelihood that the information may be compromised. Tracking Consumer Behavior: When location data are collected and shared, these data could be used in ways consumers did not intend, such as to track their travel patterns or to target consumers for unwanted marketing solicitations. Since consumers often carry their mobile devices with them and can use them for various purposes, location data along with data collected on the device may be used to form a comprehensive record upon which an individual’s activities may be inferred. Amassing such data over time allows companies to create a richly detailed profile of individual behavior, including habits, preferences, and routines—private information that could be exploited. Consumers may believe that using these personal profiles for purposes other than providing a location-based service constitutes an invasion of privacy, particularly if the data are used contrary to consumers’ expectations and results in unwanted solicitations or other nuisances. Identity Theft: Criminals can use location data to steal identities when location data are disclosed, particularly when they are combined with other personal information. The risk of identity theft grows whenever entities begin to collect data profiles, especially if the information is not maintained securely. By illicitly gaining access to these profiles, criminals acquire information such as a consumer’s name, address, interests, and friends’ and co-workers’ names. In addition, a combination of data elements—even elements that do not by themselves identify anyone, such as individual points of location data—could potentially be used in aggregate to identify or infer a consumer’s behavior or patterns. Such information could be used to discern the identity of an individual. Furthermore, keeping data long-term, particularly if it is in an identifiable profile, increases the likelihood of identity theft. Personal Security: Location data may be used to form a comprehensive record of an individual’s movements and activities. If disclosed or posted, location data may be used by criminals to identify an individual’s present or probable future location, particularly if the data also contain other personally identifiable information. This knowledge may then be used to harm the individual or his property through, for instance, stalking or theft. Access to location information also raises child safety concerns as more children access mobile devices and location-based services. According to the American Civil Liberties Union (ACLU), location updates that consumers provide through social media have been linked to robberies, and GPS technology has been involved in stalking cases. Surveillance: Law enforcement agencies can obtain location data through various methods, such as a court order, and such data can be used as evidence. However, according to a report by the ACLU, law enforcement agents could potentially track innocent people, such as those who happened to be in the vicinity of a crime or disturbance. Consumers generally do not know when law enforcement agencies access their location data. In addition to information related to a crime, the location data collected by law enforcement may reveal potentially sensitive destinations, such as medical clinics, religious institutions, courts, political rallies, or union meetings. Selected Companies Have Not Consistently Implemented Practices to Protect Consumers’ Location Privacy; Federal Agencies Have Taken Actions but Federal Privacy Law Is Not Comprehensive Private Sector Entities Have Implemented Some Recommended Practices to Protect Consumers’ Location Privacy, but Not Consistently Industry and privacy advocacy groups have recommended practices for companies to follow in order to better protect consumers’ privacy while using their personal information. These recommended practices include: (1) providing disclosures to consumers about data collection, use, and sharing; (2) obtaining consent and providing controls over location data; (3) having data retention practices and safeguards; and (4) providing accountability for protecting consumers’ data. For the September 2012 report, we examined 14 mobile industry companies, and the for December 2013 report, we examined 10 in-car navigation services companies. These companies have taken steps that are consistent with some, but not all, of the recommended practices: Disclosures: All of the companies we examined for both reports have privacy policies, terms-of-service agreements, or other practices—such as on-screen notifications—to notify consumers that they collect location data and other personal information. However, some companies have not consistently or clearly disclosed to consumers what they are doing with these data or which third parties they may share them with. For example, most of the in-car navigation service companies we examined for the 2013 report provide broadly worded reasons for collecting location data that potentially allow for unlimited data collection and use. One of those company’s terms of service states that the provided reasons for location data collection were not exhaustive. Furthermore, about half of the in-car navigation service companies’ disclosures allow for sharing for location data when they are de-identified, but most of these companies’ disclosures did not describe the purposes for sharing such data. Consent and Controls: All of the companies we examined for both reports indicated they obtain consumer consent to collect location data and obtain this consent in various ways, some of which are more explicit than others. Companies also reported providing methods for consumers to control collection and use of location data, but the methods and amount of control varied. For example, most of the 14 mobile industry companies we examined for the 2012 report indicated that consumers could control smartphones’ use of their location data from the phone; however, the ability to control this varied by operating system, with some providing more options. For example, the iPhone iOS operating system displays a pop-up window the first time a consumer activates a new application that includes location-based services. The pop-up states that the application seeks to use the consumer’s location and allows the consumer to accept or decline at that time. Similarly, Android smartphones notify consumers that an application will use location data at the time a consumer downloads a new application and seeks consumer consent through this process. Some in-car navigation systems we examined for the 2013 report use similar methods to notify consumers that they will collect location data to provide services. In contrast, other in- car navigation services obtain consent when a consumer purchases a vehicle. According to one privacy group we met with, if consent is obtained in this manner, consumers may not be as likely to review a company’s stated privacy practices because they may be a part of a larger set of documentation about the vehicle. Additionally, none of the 10 in-car navigation service companies we examined allow consumers to delete the location data that are, or have been, collected. Retention and Safeguards: Officials from most of the companies we interviewed for the 2012 and 2013 reports said they kept location data only as long as needed for a specific purpose; however, in some cases, this could mean keeping location data indefinitely. Most of the privacy policies of the 14 mobile services companies we examined did not state how long companies keep location data, and there was wide variation in how long in-car navigation services companies retain vehicle-specific or personally identifiable location data when a customer requests services, ranging from not at all to up to 7 years. All the mobile industry companies we examined reported ways they safeguard consumers’ personal information. However, in some cases, it was not clear whether these protections covered location data, since some privacy policies did not state whether location was considered a form of personal information. Thus it was unclear whether stated safeguards for personal information applied to location data. As we reported in 2013, companies may safeguard location data that they use or share, in part, by de-identifying them, but companies we examined used different de-identification methods. De-identified data are stripped of personally identifiable information. The de-identification method a company uses affects the extent to which consumers may be re-identified and exposed to privacy risks. Location data that are collected along with a consumer’s name or other identifying information are, by definition, personally identifiable data and present the greatest privacy risks to consumers because a consumer’s identity is known. Privacy risks decrease when companies de-identify location data, but the level of risk falls on a spectrum depending on how easy it is to re-identify consumers. For example, de-identifying location data with unique identification numbers prevents the direct association of location data with a specific vehicle or individual. However, if the same identification number is re- used for the same consumer on multiple trips, then the consumer’s history or patterns can potentially be discerned. Conversely, consumers face little to no privacy risks when location data are stripped of any identification numbers and aggregated with other consumers’ data because the data are anonymous, meaning that the data cannot be linked to an individual at all (see fig. 1). All of the in-car navigation service companies we examined stated in their disclosures, or in interviews with us, that they use or share de-identified location data. Accountability: We reported in 2012 and 2013 that companies’ accountability practices varied. For example, all 10 of the in-car navigation services companies we examined for the 2013 report stated in their disclosures or in interviews with us that they take steps to protect location data that they share with third parties. Additionally, some mobile carriers we examined for the 2012 report said they use their contracts with third parties they share consumers’ personal data with to require those third parties to adhere to industry recommended practices for location data. In the 2013 report, we found that while not disclosed to consumers, representatives of in-car navigation services companies said their employees must follow the companies’ internal polices to protect data, including location data, and some of the representatives further explained that employees who violate such policies are subject to disciplinary action and possibly termination. Separately, representatives from one of the in-car navigation service companies told us that it had conducted an independent audit of its practices to provide reasonable assurance that it was in line with company privacy policies. Additionally, three of the mobile industry companies we examined for the 2012 report had their privacy practices certified by TRUSTe, a company that helps companies address privacy issues by certifying businesses’ privacy programs. Lacking clear information about how companies use and share consumers’ location data, consumers deciding whether to allow companies to collect, use, and share data on their location would be unable to effectively judge whether their privacy might be violated. Federal Agencies Have Taken Actions to Protect Consumer Privacy In our September 2012 report on mobile device location data, we reported that federal agencies that have responsibility for consumer data privacy protection have taken steps to promote awareness of privacy issues, such as providing educational outreach and recommending actions aimed at improving consumer privacy. For example, in February 2012, NTIA prepared a report for the White House on protecting privacy and promoting innovation in the global digital economy. The report offered a framework and expectations for companies that use personal data. The framework includes a consumer privacy bill of rights, a multistakeholder process to specify how the principles in the bill of rights apply in particular business contexts, and effective enforcement. In February 2012, FTC issued a report on privacy disclosures for mobile applications aimed at children. This report highlighted the lack of information available to parents prior to downloading mobile applications for their children and called on the mobile industry to provide greater transparency about their data practices. FTC also issued a consumer privacy report in March 2012 with recommendations for companies that collect and use consumer data, including location data. Finally, the Department of Justice has developed guidance on how law enforcement may obtain mobile location data. In our 2012 report, we concluded that NTIA and FTC could take additional actions to further protect consumers. For example, we found that NTIA had not defined specific goals, milestones, or performance measures for its proposed multistakeholder process, which consists of different groups involved with consumer privacy coming together to discuss relevant issues with the goal of developing codes of conduct for consumer privacy. Therefore, it was unclear whether the process would address location privacy. Consequently, we recommended that NTIA, in consultation with stakeholders in the multistakeholder process, develop specific goals, time frames, and performance measures for the multistakeholder process to create industry codes of conduct. In a December 2012 response to our report, the Department of Commerce (NTIA is an agency of Commerce) said it disagreed with this recommendation, stating that it is the role of the stakeholders, not the agency, to develop goals, time frames, and performance measures for the multistakeholder process. Additionally, the letter stated that stakeholders had made progress to develop their own goals, time frames, and performance measures for their efforts to create a code of conduct for mobile application transparency. We will continue to monitor NTIA’s efforts in this area. Additionally, we found that FTC had not issued comprehensive guidance to mobile industry companies with regard to actions companies should take to protect mobile location data privacy. Doing so could inform companies of FTC’s views on the appropriate actions companies should take to protect consumers’ mobile location privacy. We recommended that FTC consider issuing industry guidance establishing FTC’s views of the appropriate actions mobile industry companies could take to protect mobile location data privacy. In February 2013, FTC issued a staff report on mobile privacy disclosures; the report provided guidance for mobile industry companies to consider when disclosing their information collection and use practices. In particular, the report suggested best practices for operating systems, application developers, advertising networks and other third parties, and trade associations and other experts and researchers. For example, FTC said that operating systems should provide disclosures at the point in time when consumers access location- based services and obtain their affirmative express consent before allowing applications to access sensitive content like location data. Federal Law Addressing Location Data Privacy Is Not Comprehensive Currently, no comprehensive federal privacy law governs the collection, use, and sale of personal information by private-sector companies; rather, various federal laws pertain to the privacy of consumers’ data: The Federal Trade Commission Act prohibits unfair or deceptive acts or practices in or affecting commerce and authorizes FTC enforcement action. This authority allows FTC to take remedial action against a company that engages in a practice that FTC has found is unfair or deceives customers. For example, FTC could take action against a company if it found the company was not adhering to the practices to protect a consumer’s personal information that the company claimed to abide by in its privacy policy. The Electronic Communications Privacy Act of 1986 (ECPA), as amended, sets out requirements under which the government and providers of electronic communications can access and share the content of a consumer’s electronic communications. ECPA also prohibits providers of electronic communications from voluntarily disclosing customer records to government entities, with certain exceptions, but companies may disclose such records to a person other than government entities. The act does not specifically address whether location data are considered content or part of consumers’ records. Some privacy groups have stated that ECPA should specifically address the protection of location data. The act also provides legal procedures for obtaining court orders to acquire information relevant to a law enforcement inquiry. The Communications Act of 1934 (Communications Act), as amended, imposes a duty on telecommunications carriers to secure information and imposes particular requirements for protecting information identified as customer proprietary network information (CPNI), including the location of customers when they make calls.The Communications Act requires that companies obtain express authorization from consumers before they access or disclose call location information, subject to certain exceptions. Carriers must also comply with FCC rules implementing the E911 requirements of the Wireless Communications and Public Safety Act of 1999, including providing location information to emergency responders when mobile phone consumers dial 911. We have previously concluded that the current privacy framework warrants reconsideration in relation to a number of issues. In our 2013 report on consumer data collected and shared by information resellers, we found that changes in technology and the marketplace have vastly increased the amount and nature of personal information, including location data that are collected, used, and shared. We reported that while some stakeholders’ views differed, the current statutory framework does not fully address these changes. Moreover, we reported that while current laws protect privacy interests in specific sectors and for specific uses, consumers have little control over how their information is collected, used, and shared with third parties. This includes consumers’ ability to access, correct, and control their personal information used for marketing, such as location data, and privacy controls related to the use of new technologies and applications, such as mobile and in-car navigation devices. In 2012, FTC and NTIA called on Congress to pass data privacy legislation that would provide a minimum level of protection for consumer data, including location data. Some Members of Congress have introduced legislative proposals that address the privacy of consumers’ location data. Chairman Franken, Ranking Member Flake, and Members of the Subcommittee, this concludes my prepared remarks. I am happy to respond to any questions that you or other Members of the Subcommittee may have at this time. GAO Contact and Staff Acknowledgement For questions about this statement, please contact Mark L. Goldstein, Director, Physical Infrastructure Issues, at (202) 512-2834 or goldsteinm@gao.gov. In addition, contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this statement. Individuals who made key contributions to this statement include Andrew Von Ah (Assistant Director), Michael Clements, Roshni Davé, Colin Fallon, Andrew Huddleston, Lori Rectanus, and Crystal Wesco. 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: Smartphones and in-car navigation systems give consumers access to useful location-based services, such as mapping services. However, questions about privacy can arise if companies use or share consumers' location data without their knowledge. Several agencies have responsibility to address consumers' privacy issues, including FTC, which has authority to take enforcement actions against unfair or deceptive acts or practices, and NTIA, which advises the President on telecommunications and information policy issues. This testimony addresses (1) companies' use and sharing of consumers' location data, (2) consumers' location privacy risks, and (3) actions taken by selected companies and federal agencies to protect consumers' location privacy. This testimony is based on GAO's September 2012 and December 2013 reports on mobile device location data and in-car location-based services and December 2012 and May 2013 updates from FTC and NTIA on their actions to respond to the 2012 report's recommendations. Fourteen mobile industry companies and 10 in-car navigation providers that GAO examined in its 2012 and 2013 reports-including mobile carriers and auto manufacturers with the largest market share and popular application developers-collect location data and use or share them to provide consumers with location-based services and improve consumer services. For example, mobile carriers and application developers use location data to provide social networking services that are linked to consumers' locations. In-car navigation services use location data to provide services such as turn-by-turn directions or roadside assistance. Location data can also be used and shared to enhance the functionality of other services, such as search engines, to make search results more relevant by, for example, returning results of nearby businesses. While consumers can benefit from location-based services, their privacy may be at risk when companies collect and share location data. For example, in both reports, GAO found that when consumers are unaware their location data are shared and for what purpose data might be shared, they may be unable to judge whether location data are shared with trustworthy third parties. Furthermore, when location data are amassed over time, they can create a detailed profile of individual behavior, including habits, preferences, and routes traveled-private information that could be exploited. Additionally, consumers could be at higher risk of identity theft or threats to personal safety when companies retain location data for long periods or in a way that links the data to individual consumers. Companies can anonymize location data that they use or share, in part, by removing personally identifying information; however, in its 2013 report, GAO found that in-car navigation providers that GAO examined use different de-identification methods that may lead to varying levels of protection for consumers. Companies GAO examined in both reports have not consistently implemented practices to protect consumers' location privacy. The companies have taken some steps that align with recommended practices for better protecting consumers' privacy. For example, all of the companies examined in both reports used privacy policies or other disclosures to inform consumers about the collection of location data and other information. However, companies did not consistently or clearly disclose to consumers what the companies do with these data or the third parties with which they might share the data, leaving consumers unable to effectively judge whether such uses of their location data might violate their privacy. In its 2012 report, GAO found that federal agencies have taken steps to address location data privacy through educational outreach events, reports with recommendations to protect consumer privacy, and guidance for industry. For example, the Department of Commerce's National Telecommunications and Information Administration (NTIA) brought stakeholders together to develop codes of conduct for industry, but GAO found this effort lacked specific goals, milestones, and performance measures, making it unclear whether the effort would address location privacy. Additionally, in response to a recommendation in GAO's 2012 report, the Federal Trade Commission (FTC) issued guidance in 2013 to inform companies of the Commission's views on the appropriate actions mobile industry companies should take to disclose their privacy practices and obtain consumers' consent.

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