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Da den fynske optiker Finn Juncker tog til Senegal for at være med til at uddele brugte briller til befolkningen, var han opfyldt af idealisme og entusiasme. Fem år senere måtte han sande, hvor vanskeligt det kan være at hjælpe andre. Finn Juncker har et freelance-optikerfirma. Med udgangspunkt i Fåborg arbejder han også i Albertslund, i Malaga og i Grønland, og i perioder har han arbejdet i Senegal. Det sidste for egen regning. Det afrikanske eventyr begyndte, da han så en annonce i bladet Optikeren, hvor man søgte optikere til et projekt om brugte briller til befolkningen i Senegal. Projektet gik ud på, at indsamle brugte briller i Danmark. Brillerne blev renoveret og repareret af tidligere alkoholikere og narkomaner på et behandlingshjem i Nykøbing Falster og herefter sendt til Senegal. Bag projektet stod en dansk sygeplejerske. Hun søgte to optikere, en sygeplejerske mere og en medhjælper. I 1998 rejste disse fire, inklusive Finn Juncker, til Senegal. De skulle selv betale rejsen og opholdet, og de måtte bruge deres sommerferie på det. - Vi boede i lerhytter hos de lokale og sov på gulvet, og vores tre daglige måltider bestod af ris, som vi selv skulle betale for. Vi havde også udgifter til chauffør og benzin. Vi var der knap tre uger, og vi foretog 200 synsprøver og udleverede 150 par briller hver dag. Folk var kommet gående flere dagsmarcher for at få briller. Desværre kom der også mange blinde og folk med grå stær, som håbede, at vi kunne kurere dem. Det var hårdt at være vidne til. Men de mange, vi kunne hjælpe, var hele rejsen værd, fortæller Finn Juncker. Kort efter projektets start søgte den ledende sygeplejerske om midler hos Danida. Omkring en halv million kr. blev bevilget til de tre år, projektet skulle løbe. Det var nu et Danida-projekt. Nogle af pengene gik til at få senegalesere til Danmark, hvor Finn Juncker oplærte dem, så de kunne fungere som optikere hjemme og således overtage danskernes arbejde. Der gik tre år på denne måde. Finn Juncker brugte to-tre uger hvert år af sin sommerferie på at arbejde som optiker i Senegal, og om vinteren oplærte han senegalesere i Danmark. Men økonomien knirkede. - Der var et eller andet helt galt. Vi betalte alt for meget for den mad, vi spiste, dvs. ris, og alt for meget for benzin og til chaufførens løn. Målt med lokale alen. F.eks. var det den danske stats kilometertakst, der blev anvendt, når vi skulle køre ud og betjene befolkningen. Men brillerne blev leveret, og behovet var stort. Så vi betalte, beretter Finn Juncker. De var jævnligt ude for en del ubehagelige hold-ups ved forskellige checkpoints af krigere med maskingeværer. Området var plaget af uro, og det var ikke ufarligt at bevæge sig omkring dernede. Da projektet udløb efter tre år, opfordrede Finn Juncker tre optikere, han kendte som folk med hjertet på rette sted, til at tage med ham til Senegal i sommerferien og videreføre det påbegyndte arbejde. Han kontaktede folkene i Senegal og fortalte, at et hold på fire danske optikere med et læs briller var klar til at tage af sted på egen hånd. Det vil sige uden Danida-midler. Optikerne ville ikke kunne betale de samme summer til senegaleserne som hidtil for mad og transport. Da kølnedes interessen mærkbart hos senegaleserne, og de fire optikere kom aldrig af sted. Men Finn Juncker er gjort af stædigt stof. Han havde lært en gambianer at kende, som var indehaver af en brillebutik i Gambia, men som ikke var uddannet optiker. De to indgik en forretningsaftale om kompagniskab. Finn Juncker skulle lære ham op og hjælpe ham i gang som partner i foretagenet. Det gik også fint. Finn Juncker uddannede gambianeren i hans butik og havde medbragt en hel del kostbare måleinstrumenter til synsprøver. Meningen var, at gambianeren skulle købe måleinstrumenterne for de penge, han solgte briller for, men han ville hellere købe biler. Da Finn Juncker forklarede ham, at han gerne ville have sine måleinstrumenter tilbage, tilkaldte gambianeren politiet og bildte betjentene ind, at Finn Juncker havde foræret ham instrumenterne. Finn Juncker måtte opgive at få sine instrumenter igen. En ekstra bitter krølle på det eventyr var, at Finn Juncker i Danmark forgæves kæmpede med skattevæsnet, der ikke ville anerkende hans rejse som forretning og udgifterne i den forbindelse som fradragsberettiget. - Jeg indså, at det var slut. Min indsats og mine penge var tabt. De senegalesere, vi havde lært op som optikere her i Danmark, har aldrig fået lov at praktisere i Senegal. Jeg må sige, at jeg har fået et temmelig desillusioneret syn på ulandsstøtte. Pengene går i de forkerte lommer. Og vores projekt er desværre ikke enestående. Det er kendetegnende for en række afrikanske lande, at nogle få magtfulde høvdinge sidder på alt, og de er villige til at gå langt for at få del i støttemidlerne. De bruger pengene til deres egen familie, til at sende deres børn på dyre skoler, til rejser og luksus. Det var situationen omkring brilleprojektet, og det var grunden til, at vi skulle betale så meget for mad og kørsel. Tilbage står tusindvis af fattige og syge, som kunne have været hjulpet ofte for et forholdsvis beskedent beløb, siger Finn Juncker. Han har ikke fortrudt sin indsats i Afrika, men han er skuffet og ærgerlig. Sin trang til at gøre nytte og hjælpe andre får han så udløb for i Grønland.

Writing my last blog post really got me thinking about that guy and all the crazy things we done. The more I thought about it, the more memories came flooding back and I decided that this blog has been a little too sad/awkward so far and anyone reading must think I’ve had a whole bunch of horrible experiences. It’s about time I chronicled the peaks of my journey so far and this guy is definitely a peak. In fact, I almost peak myself just thinking about him. We met in a field. I was there “camping” with friends. There were no tents. Our trip basically involved trekking into a little patch of woodland on a farm outside of town and getting really drunk/high and having sex round a badly made campfire. It was surprisingly fun. I hadn’t been for a few months because I had actually fallen pregnant there, made it nearly three months into the pregnancy and miscarried. The dad wanted nothing to do with me and I hadn’t told my mum. So instead of facing up to the tragedy and going to hospital, I decided that going back and getting high in conception field was a better idea. I was clearly a child genius. I hated the entire night. I hadn’t been around for months, I didn’t feel like I belonged anymore and I secretly resented everyone around the campfire for not being a little more sensitive to my situation. I was just a little ball of anger in the corner, curled up in a giant “goth” hoodie and unable to even look at the bush in which I had regretfully created life in a drunken fumble. I was starting to see this entire thing for what it really was. Irresponsible and moronic. That’s when he showed up. He looked like an angel in the firelight. He was ballsy and arrogant yet he noticed right away that I wasn’t right. He was the only person that night to ask me if I was ok and he was the only one I hadn’t met before. Don’t get me wrong, he was hardly playing the role of my saviour but there was something about him, beyond the air of arrogance, that just drew me in. I could tell that he also hated the whole “drinking in the woods” scene. At one point he asked me to go home with him but I said no, having just suffered the consequences of losing the only good thing to come out of my last drunken encounter. Everyone got drunk and stripped to their pants except me. There was a lot of jumping over the fire and running around nearly naked. It was fun to watch actually. He played guitar by the fire as we got high until the sun came up and it was just us two left. The night ended with him spooning me on the muddy floor and in the morning, he left with my number and my story. He said I needed reminding of what life is really about and that I was going about it the wrong way in those muddy woods. If I’m honest I expected it to end there, I was broken and he was very aware of how hot he was. I didn’t see why he’d bother with a girl so obviously damaged. But he did. A week later, he calls me and invites me round for pizza and beer at his flat. I don’t know why I went but I did. There was something about his attitude towards life that appealed to me at that time in my life. I’d just miscarried my drunken regret, I was just about to sit important exams, my friends were horrible people and I was self-harming badly. I walked into that guy’s flat a broken mess and by the time we were over, I’d turned my life around and discovered who I really was. One day, I’ll thank him for that. Anyways, back on track with the sex part of “sex blog”. He was a serial charmer and holy god, he was good at it. He made me dinner, we got high and he listened to me as I told him more about how I’d gotten to be where I was in life. Not to mention that he was unbelievably sexy. Before long, he was helping me out of my oversized slipknot hoodie and I had forgotten entirely about my new found chastity. He kissed away all of my resistance with a style of kissing I have since adopted myself, passionate with a lot of lip but no tongue. I was mesmerised and more turned on than I’d ever been before. For once, I wasn’t having sex for attention, for reassurance, for control. I was driven entirely by lust and I loved it. Now, when we were at the campsite the previous week he’d told me that he’d always been insecure about his small penis. He genuinely seemed so down about it which really didn’t match his personality at all as he oozed confidence. At the time, I was flattered that he’d confided in me about it and hadn’t thought much of it. However, as he knelt between my legs about to unveil his tiny penis I was suddenly filled with dread. I was terrified that if I didn’t at least pretend to have the time of my life, his complex would be worsened and I didn’t wanna do that to him. So as he was about to pull off his (very tight and very sexy) boxers, I braced myself for a shrivelled little thing to greet me. Now, the room was dimly lit and I’d been wearing jeans so I had no idea what was about to pop out. It turns out he’d lied about his penis being tiny purely because he liked the look on girls’ faces when they came face to face with what I can only describe as the most perfect penis I’ve ever seen after expecting something insignificant. How deviously twisted of him. I found it hilarious, personally. It definitely tickled me pink. What followed was literally life-changing sex. His tongue gave me tingles in places I didn’t even know could tingle and he done things with his penis that I have never been able to replicate since. It was raw, carnal and rough. I’d been with dominant guys before but I’d always felt like ultimately, I was in control. Not this time. He truly owned every part of me that night and I loved it. He held me down and teased me until I begged and then kept teasing. A few hours in, he’d turned me from a timid and sad girl into a ferocious sex toy and I had never felt so alive. I spent that night acting purely upon selfish impulses and the desire for new sensations rather than thought and logic like usual. He was exactly what I needed to wake me up from the pit of teenage angst I’d fallen into. I went home aching everywhere and grinning ear to ear. I was hooked. I was back the next day and he knew I would be. This time I thought I was ready for him, that I knew what was coming but I’d soon learn that would never be the case with this one. He loved pushing boundaries and was inherently unpredictable. This time we had shower sex and he found it hilarious that I was unable to stay standing when I orgasmed. The next time he tied me up with duct tape and teased me for hours. I just kept coming back for more. Soon I started skipping school to spend time with him. We had high sex, drunk sex,, rough sex, morning sex, slow sex, half asleep sex, angry sex, and ultimately sad sex. It didn’t take me long to convince my immature self that I loved this boy. He’d woken me up from my sadness and I soon confused this new found happiness and emotion with love. I wanted him to myself and even then it was apparent that he was untameable. One time I showed up at his place with fresh cuts on my legs. He was so disappointed. He’d spent weeks building my confidence, teaching me to stop wearing my heart on my sleeve and now I’d relapsed. This was when he really turned my life around. He took me shopping, made me try on girly clothes instead of the usual baggy black clothes I usually wore. He bought me my first lingerie corset-suspender set and my first vibrator (Hamish). He took me to his flat, dressed me up in the lingerie and went over every inch of my body telling me what he found sexy about it. He cuddled me after sex and told me about his view of life, about how I was way too young to be carrying so much bitterness and sadness around on my sleeve. How I’d regret missing out on being young when I’m older. Then he played his guitar and sung his own songs naked (honestly). Since that night I have never cut myself on purpose again. We continued our whirlwind romance. He sneaked me into pubs despite both of us being underage, his charm got him served easily. I tried my first shot with him. He took me to his favourite shop – a little vintage place outside of the city centre. He taught me how to roll a joint and we’d fall asleep during high sex, him still inside me. He encouraged me to help myself along during sex and I had the most powerful orgasms I’d ever had doing that. I starred in my very first sex video with him. I tried anal for he first time after he tied me up with my stockings, gagged me and used my vibrator on me. I found out all of my limits with him and loved every second and every time he pushed me just a little bit further. He pushed me to look for who I really was but despite all of the sweet and sexy things he’d do for me I knew deep down he wasn’t just mine. He refused to commit, he saw other girls and didn’t keep in regular contact. I hated it and pushed for commitment. Then, he found out he was gonna be a dad. He’d gotten some other girl pregnant. I was upset but in my stupid teenage infatuation, I stayed with him. Then I walked in on him and that other girl and that’s where my last post starts. It wasn’t how I’d hoped it would end but by that stage I’d grown in confidence so much that I’d realised that he had given me all I needed to get by on my own. He was a wild one and had no plans for the future and had a baby on the way. I had just aced all of my exams and my future was whatever I wanted it to be. Limitless. The girl went on to have a baby boy. He now has a 4 year old son and last I heard was selling drugs and in a band. He texted me later on and asked if I wanted to have another threesome with him and the baby’s mum or wanted any drugs. I said no and that’s the last I ever heard of the wild boy who taught me how to push boundaries and to be proud of my own body and what I can do with it.

Slapp straff, men mister lappen Ungdommene som ble tatt i hasjaksjonene i Fana la alle kortene på bordet. Cannabisbruken de innrømmet i avhør, brukes nå til å ta fra dem lappen. Publisert Publisert 16. august 2014 LA KORTENE PÅ BORDET: Dette er noen av bildene politiet fant på mobiltelefonene til dem som ble pågrepet i cannabisaksjonene. Bildet i midten er fra et beslag politiet gjorde i forkant av aksjonene. Foto: POLITIET Denne artikkelen er over seks år gammel Flere av ungdommene som ble tatt i hasjaksjonene i Bergen sør i februar i år har mistet retten til å føre motorvogn. Det skjer etter at det i midten av juni i år dumpet et brev ned i postkassen til flere av dem. — I avhør skal De ha opplyst at De har røyket marihuana, omtrent annenhver helg, i forbindelse med fest. (...) Det at De har brukt marihuana over en lengre periode, fra 2013 til 2014, vitner om at deres bruk ikke er forenlig med å inneha førerretten til motorvogn. På denne bakgrunn har politiet skjellig grunn til å tro at De har et rusproblem og således ikke fyller kravene til helse som kreves for å inneha retten til å føre førerkortpliktig motorvogn, står det i ett av brevene fra Fellesforvaltningsenheten til Hordaland politidistrikt. Det skal være et titall ungdommer som nå har mistet sertifikatet. Flere av dem har hyret inn advokat for å bestride saken. - Ingen heksejakt Det er også flere som har ønsket å kjøre opp til lappen, som ikke får lov av førerkortkontoret. Også det er en følge av cannabisbruken de har innrømmet i avhør. Ifølge Morten Ørn, stasjonssjef ved Bergen Sør politistasjon, er det ikke snakk om noen koordinert aksjon for å frata dem som ble pågrepet førerkortet. — Det er en administrativ sak som går parallelt med Tidlig ute-prosjektet, sier han. De fleste av ungdommene som ble pågrepet, fikk påtaleunnlatelse mot at de gjennomførte det forebyggende programmet. Tre ganger på to år Jurist Kirsti Fürstenberg ved Forvaltningsavdelingen sier de tar utgangspunkt i håndhevingsinstruksen fra Politidirektoratet og en dom fra Borgarting lagmannsrett når de avgjør om en person skal miste førerretten som følge av en bekymringsmelding. — Så gjøres det en skjønnsmessig helhetsvurdering i hvert tilfelle. Vi går konkret inn i hvert tilfelle og ser gjennom dokumentene vi har. I dommen fra lagmannsretten, som legges til grunn for tilbakekallelsen av førerretten, står det at én overtredelse i året ikke er tilstrekkelig for å tibakekallelse. «To kan være det men formuleringen flere tyder på at det må være minst tre overtredelser i løpet av siste par år», står det i dommernes tolkning av Vegtrafikkloven. Tar ikke hensyn til lovlighet Før ungdommene mistet lappen, fikk de et forhåndsvarsel fra Forvaltningsenheten. Deretter fikk de 14 dager på seg med å komme med informasjon de mener er relevant for saken. — Det er mange som kommer med uttalelser. Det inngår som en del av sakens dokumenter, og blir en del av helhetsvurderingen, sier Fürstenberg, som igjen understreker at det gjøres en skjønnsmessig vurdering i hvert tilfelle, og at hun uttaler seg på generelt grunnlag. Forvaltningsenheten tar ikke hensyn til lovligheten av rusmiddelet som benyttes, når det gjelder vurderingen av edruelighet. — Vi ser på en konkret helhetsvurdering av ruseffekten, sier Fürstenberg. Alkohol = narkotika I prinsippet kan dermed alkoholbruk og narkotikabruk dermed behandles likt med tanke på om man får beholde førerkortet. — Det vi må vurdere er om bruken tilsier en økt risiko i trafikken. For å få sertifikatet tilbake, må ungdommene vise til dokumentert rusfrihet i ett år. — Det blir en konkret vurdering i hvert tilfelle, men de må sannsynliggjøre edruelighet. Det er opp til personen selv og skaffe bevis for dette, gjerne fra lege, sier hun. Økning i førerkortbeslag Fra 1. januar til 5. august i år, har politiet i Hordaland fattet 644 vedtak om tilbakekallelse av førerretten. 550 av disse vedtakene kommer som følge av vedtak fattet av fylkesmannen, mens 94 av dem er på bakgrunn av saker politiet selv har initiert. Samme periode i fjor, var det totalt 578 vedtak, og bare 34 av dem kom på bakgrunn av saker politiet selv initierte. — Vi ser at antallet egeninitierte saker går opp etter at vi innførte den nye metodikken. Det er et mer strømlinjeformet system, som vi er glade for, sier Ane Kvaal, politiinspektør og leder for forvaltningsenheten i Hordaland-politiet. Nytt prosjekt Det som har skjedd i mellomtiden, er nemlig at politiet har implementert prosjektet Forebygging gjennom forvaltning. Det er i praksis en ny struktur for å fange opp meldinger fra politiets egne ansatte om personer hvor det er grunn til å tro at vedkommende ikke lenger fyller vilkårene for å inneha en tillatelse fra politiet. Kvaal er forsiktig med å tilskrive det nye prosjektet all æren for at antallet vedtak som følge av egeninitierte saker går opp. — Økningen kan jo skyldes at politiet har blitt flinkere til på melde fra, eller at det finnes flere personer som ikke burde ha førerkort. Men det er vanskelig med sikkerhet å si hva sammenhengen er. Vet du noe om saken? Send MMS/SMS til 2211, eller skriv e-post Publisert Publisert: 16. august 2014 16:51 Oppdatert: 16. august 2014 18:15

/*********************************************************************** !!!!!! DO NOT MODIFY !!!!!! GacGen.exe Resource.xml This file is generated by Workflow compiler https://github.com/vczh-libraries ***********************************************************************/ #ifndef VCZH_WORKFLOW_COMPILER_GENERATED_DEMOREFLECTION #define VCZH_WORKFLOW_COMPILER_GENERATED_DEMOREFLECTION #include "Demo.h" #ifndef VCZH_DEBUG_NO_REFLECTION #include "GacUIReflection.h" #endif #if defined( _MSC_VER) #pragma warning(push) #pragma warning(disable:4250) #elif defined(__GNUC__) #pragma GCC diagnostic push #pragma GCC diagnostic ignored "-Wparentheses-equality" #elif defined(__clang__) #pragma clang diagnostic push #pragma clang diagnostic ignored "-Wparentheses-equality" #endif /*********************************************************************** Reflection ***********************************************************************/ namespace vl { namespace reflection { namespace description { #ifndef VCZH_DEBUG_NO_REFLECTION DECL_TYPE_INFO(::demo::MainWindow) DECL_TYPE_INFO(::demo::MainWindowConstructor) #endif extern bool LoadDemoTypes(); } } } #if defined( _MSC_VER) #pragma warning(pop) #elif defined(__GNUC__) #pragma GCC diagnostic pop #elif defined(__clang__) #pragma clang diagnostic pop #endif #endif

Q: ¿Porqué en este loop de JavaScript la impresión de la variable es desde counter y no desde counter-1? en mi búsqueda por aprender programación por mis propios medios, me he topado con el tema de recursividad y este simple código... mi pregunta ya que la variable counter comienza desde 10 y dentro del loop While el contador resta 1, porqué en la "impresión" aparece desde el 10. Sé que si quisiera empezar desde 10 colocaría el contador en 11... pero obviamente tengo la curiosidad y no entiendo. var counter = 10; while(counter > 0) { console.log(counter--); } resultado: 10 9 8 7 6 5 4 3 2 1 A: La razón es simple, en recursividad lo que haces es pasar una variable o arreglo en la mayor parte de los caso para modificarlos o simplemente imprimirlos, en tu caso quieres restar un numero por cada iteracion dentro de tu ciclo while pero aqui lo que tu quieres conseguir es que primero te imprima el 9 por la lógica que encuentras en tu programa y aunque no es del todo errónea eso no sucederá jamas por la siguiente razón. En tu codigo lo que tienes es la impresion de tu variable e imprimes lo que es counter-- y a pesar de que si te resta -1 en esa misma iteracion sucede que primero te imprimira la variable antes de hacer dicha operacion ya que es lo que primero lee javascript, es como si tu codigo estuviera dividido en dos partes. EJEMPLO var counter = 10; while(counter > 0) { console.log(counter); // Lee antes el valor variable counter--; // Después realiza operación } Esto sucede asi porque es como funciona internamente lo que realizas con javascript ya que a pesar de que parece un metodo simple de resta internamente esta compuesto de dos partes. Para cuando javascript hace la operacion tu valor ya esta en pantalla. EJEMPLO VISUAL Primera iteración: counter = 10 | counter-- | counter = 9 counter = 9 | counter-- | counter = 8 counter = 8 | counter-- | counter = 7 ... counter = 1 | counter-- | counter = 0 counter = 0 | counter-- | counter = -1 -> En este caso ya no cumples con la condición por lo cual nunca se imprime. Para realizar el proceso que quieres en el caso de que primero quieras que se imprima el 9 entonces deberas de hacer lo siguiente: var counter = 10; while(counter > 0) { counter--; console.log(counter); } .as-console-wrapper { max-height: 100% !important; top: 0; }

Dorsomedial hypothalamic lesions alter intake of an imbalanced amino acid diet in rats. Within 3 h of ingesting an imbalanced amino acid diet (IAAD), rats show attenuated intake. The associated conditioned taste aversion can be ameliorated by giving the serotonin3 receptor blocker, tropisetron (TROP). A recent c-fos study indicated that the dorsomedial hypothalamic nucleus (DMN) may be activated 2-3 h after ingestion of IAAD. In Experiment 1, DMN-lesioned rats (DMNL) or sham-operated (SHAM) rats were injected with saline (SAL) or TROP just before introduction of IAAD. By 3 h, SAL-DMNL rats consumed more (P < 0.01) of the IAAD than did the SAL-SHAM rats. Thereafter, over the next 21 h, the intake of the SAL-DMNL group returned to control levels. TROP treatment enhanced the intake of the treated groups; the TROP and the lesion effect were additive (P < 0.01). By d 4 of receiving the IAAD, the DMNL groups were eating less than SHAM rats (P < 0.05). The data suggest that the DMN may be involved in the early detection of the amino acid deficiency induced by IAAD, is not involved in the TROP effect and is necessary for proper long-term adaptation to an IAAD.

For the Democratic Party, a pliant and friendly news media is both a blessing and a curse. On the one hand, the corporate press's eagerness to rehabilitate Democratic officials after even the most shameful and despicable acts has made it possible for some of the party’s worst actors to maintain political power with relative ease. Then again, the news media’s reflexively and reliably soft coverage of left-wing lawmakers has made the chief benefactors of this cozy relationship both soft and complacent. Democratic officials and candidates have been lulled into a false sense of security, ignoring or forgetting the benefits that come with being tempered by a thorough, brutal vetting. The downside to the corporate media’s mostly hands-off approach to covering Democratically aligned lawmakers was never clearer than it was during the Feb. 19 primary debate in Las Vegas, Nevada. Former New York City Mayor Michael Bloomberg, who spent much of the evening having his entrails ripped out by his 2020 opponents and paraded around the stage, managed to get in one good blow against Sen. Bernie Sanders, going after the Vermont lawmaker for his conspicuous wealth. “What a wonderful country we have,” said the former mayor. “The best-known socialist in the country happens to be a millionaire with three houses. What did I miss here?” “Well,” an irate Sanders said, “You'll miss that I work in Washington, house one. ” Bloomberg interjected, “That's the first problem.” “Live in Burlington, house two,” the senator continued. The former mayor interrupted again, “That's good.” “And like thousands of other Vermonters, I do have a summer camp. Forgive me for that. Where is your home? Which tax haven do you have your home?” Sanders demanded, hopelessly losing the exchange. “New York City, thank you very much,” Bloomberg said to applause, “and I pay all my taxes.” The most remarkable thing about the exchange is not that Sanders was so easily baited by Bloomberg into making a sloppy, blindly angry defense but that it marked the first time in the 2020 campaign season that anyone, Democrat, reporter, or debate moderator, pressed the 2020 Democratic primary front-runner to reconcile his own considerable fortune with his core message of confiscating wealth from business owners. How did Sanders make it through nine debates without a fellow Democrat or debate moderator asking him to respond to the most obvious criticism he will face should he go up against Trump? The 2016 election was a “takers versus makers” election. It is astonishing, then, that it took for a 2020 Democratic primary candidate who is not even on the ballot in Nevada or South Carolina to ask the millionaire socialist senator about his three houses. I guess that is what happens when you schedule all your primary debates with only friendly news networks. This gets to the precise problem of the too-friendly relationship between the Democratic Party and the corporate media. It is a double-edged sword that allows terrible men to weather what should be career-ending scandals and missteps but also leads to their entire party getting lazy and soft. Not having to worry about a tough, combative press is how you get incidents such as the 2020 Democratic front-runner being totally unprepared to answer the most obvious criticism of his campaign. If Sanders's disastrous handling of Bloomberg's no-brainer broadside is a portent of things to come, the general election meat grinder will be a straight-up bloodbath for Democrats.

MANILA (Reuters) - Philippine security forces on Saturday killed a foreign national and his female companion who were suspected of being connected to a militant group supporting Islamic State, police officials said, two days after the group’s leader was also killed. The foreigner, believed to be Pakistani and identified as Abu Naila, resisted arrest and attempted to throw a grenade while a police and military team was conducting a manhunt in Sarangani province, Chief Superintendent Cedrick Train, a police regional director, said. They were conducting an operation against members of the militant Ansar Al-Khilafah Philippines (AKP), one of a handful of small groups that have pledged allegiance to Islamic State and blamed for years of unrest in the Philippine south. On Thursday, police chief Ronald dela Rosa said security forces had effectively broken the backbone of AKP with the killing of its leader, Mohammad Jaafar Maguid, and the arrest of his three AKP colleagues. He has warned of “retaliation” by other AKP members and said security forces were on full alert as Filipino Catholics are set to celebrate the feast of the Black Nazarene, with millions of devotees expected to join processions on Monday in several parts of the country, including Manila. Authorities have linked Maguid’s group to several crimes ranging from arson and murder to bombings. Regional police spokesman Romeo Galgo said they were still verifying the nationality of the foreigner killed on Saturday. “Officers were forced to fire at the suspects when the grenade was lobbed at them,” Train said. President Rodrigo Duterte has warned against Islamic State taking root in the southeast Asian country, saying it needed to avoid “contamination”.

Facebook has hired the Patriot Act's co-author as a general counsel - Jerry2 https://boingboing.net/2019/04/22/mass-surveillance-r-us.html ====== javagram “Jennifer Newstead helped craft the Patriot Act, a cowardly work of treasonous legislation foisted on the American people in the wake of the 9/11 attacks;” Source seems a little biased. Treasonous? That’s gotta require a lot of cortortion around the definition of treason. Patriot Act provisions have been repeatedly reauthorized by the democratically elected legislature since it was originally passed. This isn’t a case of foisting anything upon the people, the people are perfectly happy to vote in supporters of the Patriot Act. [https://en.wikipedia.org/wiki/Patriot_Act#Reauthorizations](https://en.wikipedia.org/wiki/Patriot_Act#Reauthorizations) ~~~ thundergolfer It's well known that many members of congress passed through the act _without having read it_. Given the enormity of the act's effects on the country, this is quite a problematic thing. I don't it was democracy that saw that bill through. It was crisis politics. Democracy requires a well-informed public, and capable representatives. With the USA PATRIOT act there was neither. ~~~ foxyv With the current state of campaign finance, congress is essentially two corporations with congressmen/women as employees. If you don't vote the party line or you don't secure funding for the party you get defunded on your next election. Surprising they don't bother to read the bills they are told to pass. ------ canada_dry A perfect fit really. This guy figures it's ok to allow personal records like telephone, e-mail, financial, and business records to be surreptitiously captured without full due process/transparency. Facebook would love to push the (no-)privacy envelope much further: a complete data free-for-all for their commercial gain. ------ Jerry2 It's unfortunate that mods decided sink this story. Any explanation as to why? ------ tuxxy What exactly... do they think is going to happen when news outlets hear this? ~~~ joshmn The 30 minute news cycle we've had for the last 3 years of course. ~~~ isoskeles Yeah unlike when the Patriot Act passed, and the news media spoke truth to power or whatever, and saved us all from that treasonous law. Apologies for the snark but it’s been like this for more than 20 years. ~~~ thundergolfer To add to your comment. _Manufacturing Consent_ came out in 1988, 31 years ago. That book manfully built the case that this stuff has been going on for well over a century, but that it really kicked up in the post WW2 era with the erosion of labour-class news media. Today 6 US media companies control 90% of US media, and any hope one has of the internet disarming them dims more than a little at the sight of a P.A.T.R.I.O.T act author crossing over into the arms of a tech giant.

Q: bootstrap.min.css sets transparency where not wanted I have a small chatbox at the bottom of my page which seems to be inheriting CSS style from bootstrap.min.css and that chatbox is transparent which is a nuisance because the underlying text on the page shows through and what is worse, is that hyperlinks on the page are over-riding clickable areas in the chatbox for opening, closing and submitting messages. I have tried adding CSS style to the chatbox for opacity and rgba. Even tried adding a background image but to no effect. I have since modified the chatbox to display an iFrame from a different site that does not use bootstrap.min.css. But even the iFrame page is affected by transparency. I can remove the transparency setting in bootstrap.min.css but that will not solve my bigger problem... I am intending to use this chatbox on several sites and may not have control of the site's CSS. So I need a way to override the parent site's CSS just for the chatbox. If that is impossible, then I can weed out the transparency from bootstrap.min.css that is used on my own sites. However I do wonder what is the point of such transparency when it is useless here... A: It's a z-index problem which is common when integrating iframes, apply z-index: 2000; (or whatever number as long as it comes on top) on your chatbox div so your chatbox will still stay upfront.

Allele-specific wild-type blocker quantitative PCR for highly sensitive detection of rare JAK2 p.V617F point mutation in primary myelofibrosis as an appropriate tool for the monitoring of molecular remission following therapy. Screening of JAK2 V617F point mutation becomes more and more important in monitoring of JAK2 positive MPN following stem cell transplantation. In an attempt to achieve the required high sensitivity (1:10(5)), specifity and robustness we created an approach applicable on bone marrow biopsies where we adapted the principle of wild-type blocker PCR with allele-specific Q-PCR. The significance of the assay was demonstrated on a retrospective series of sequential bone marrow biopsies as diagnosis of molecular relapse now preceded the diagnosis of clinical relapse by far. This method offers the urgently needed tool for a systematic molecular analysis of sequential biopsies in the course of stem cell transplantation to develop guidelines for the management of these patients.

Benjamin Lok describes how the robot butt sensors work 2:26 Prostate exams are potentially-life saving. But the process of getting one can be nerve-racking — both for the doctor and the patient. A group of scientists from Drexel University and the Universities of Wisconsin and Florida are hoping to assist with that. They've designed a robot to help medical students give better prostate exams. The robot's name is "Patrick" and he's an interactive butt. Professor Benjamin Lok "Patrick is part of a simulation where students get to practice prostate exams," lead researcher and University of Florida professor Benjamin Lok explains to As It Happens guest host Tom Harrington. "The simulator itself is a piece of plastic and around the anus area is a foam, rubbery material. It's anatomically correct, and inside, they have placed a prostate so that they can actually feel what a prostate would feel like." But the physical examination is actually the last part of the training process. Initially, students start using the simulator to work on their beside manner. "Students have to talk to Patrick for about five to eight minutes," the University of Florida professor says. "They work on their social skills. They try to obtain a patient history, but also have to work through anxiety that Patrick's having." A student interacting with "Patrick" (Courtesy of Andrew Robb, University of Florida) As the interview progresses — and the student realizes that Patrick needs a prostate exam — Patrick becomes quite hesitant to proceed. Lok says the robot will say, "Do you really have to do this? I don't understand why we have to do this right now." Once Patrick is convinced, the student begins the examination. "We can show you in real-time, as you're doing the exam... whether you're pressing all the regions and if you're pressing with enough pressure," Lok continues. The sensor displays traffic light-style signals of green, yellow or red depending on the appropriate pressure applied. "That helps educate the user and what a good prostate exam should feel like," he says. But why not have Patrick yell or respond in a more human-like way? Lok says they thought about doing this, but for freshmen medical students, that would increase the level of anxiety. "We want the system to provide positive experiences, where they can get good feedback but also help reduce some of the anxiety before they first practice on what are often called standardized patients, which are called actors." Many medical schools pay professional actors who are specially trained to receive numerous prostate exams by students. However, as you might imagine, there are a limited number of actors willing to do this. Postate exam program, "Patrick" (Courtesy of Andrew Robb, University of Florida) Like a pilot who practices in a simulator before actually flying a plane, Lok hopes Patrick will serve the same purpose for medical students. "You can make mistakes with Patrick and start over, that's one of the advantages of a simulator." Patrick is currently being used by medical students at the University of Florida and Drexel University. Lok hopes the technology will be used in more medical schools across the United States.

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It was a Pink Burn-out YO!PS - anyone else see the creator pot-hole on lane 2 there...You can fall in there and they'll never find you!

Autosomal dominant polycystic kidney disease (ADPKD) is a common monoallelic disorder associated with progressive cyst development and resulting in end stage renal failure (ESRD) in 50% of patients by 60y. However, there is considerable phenotypic variability, extending from in utero onset to patients with adequate renal function into old age. Autosomal dominant polycystic liver disease (ADPLD), as traditionally defined, results in PLD with minimal renal cysts. Classically there have been considered two ADPKD genes, PKD1 and PKD2, encoding PC1 and PC2, and two ADPLD genes, PRKCSH and SEC63, but in the past few years greater genetic heterogeneity has been described, with nine genes now implicated overall. Recent data also indicates an overlap in etiology and pathogenesis associated with ADPKD and ADPLD, with the efficient biogenesis and localization of the PC-complex central to both disorders. During the last funding period we identified a novel gene, GANAB, which is associated with both disorders, where the encoded protein, GII?? is involved in the maturation and trafficking of PC1. In this proposal we will take advantage of advances in next generation sequencing (NGS) methodologies, and large populations of ADPKD and ADPLD patients that have been assembled and screened for the classic genes, to hunt for novel genes for these disorders (Aim 1). The phenotype associated with these genes will be characterized (Aim 3) along with their mechanism of action (Aim 2). NGS methods will be perfected to screen the segmentally duplicated locus, PKD1, and to identify missed mutations at the known loci, including those present in just some cells due to mosaicism (Aim 1). The significance of many PKD1 nontruncating variants has been difficult to evaluate (classed as variants of unknown significance; VUS), but recently evidence that some are incompletely penetrant alleles partially explains phenotypic variability in PKD1 populations. In Aim 2 improved in silico predictions, in combination with machine learning, will improve the understanding of the pathogenicity and penetrance of VUS. A cellular assay of the biogenesis and trafficking of this PC-complex will also be employed to quantify the penetrance of VUS. The mechanism of pathogenesis will be explored in animal models with ultralow penetrant (ULP) Pkd1 or Pkd2 alleles. Employing the large clinically, imaging, and genetically well-defined populations phenotypic groupings of patients will be defined that will then be compared to the genic and PKD1 allelic groups (Aim 3). This iterative process will allow the Variant Score (VS) associated with each PKD1 VUS to be refined. In a separate population the revised VS, alone and in combination with clinical, functional, and imaging data, will be employed to generate a comprehensive, predictive algorithm for ADPKD (Aim 3). Disease modifiers to severe disease, via biallelic ADPKD, and due to alleles at other loci will also be identified and characterized in the cellular assay and in vivo in combination with the Pkd1 hypomorphic, RC model. The final aim will exploit the newly identified information that some PKD1 and PKD2 VUS are rescuable, folding mutations that in a maturation-fostering environment can traffic and function appropriately. A screening scheme based on the level of cell surface PC1 will be improved and new chaperone drugs specific for the PC complex will be sought in collaboration with Sanford Burnham Prebys. A second mutation group that will be explored therapeutically are nonsense mutations. A cellular assay for readthrough efficiency is being developed and will be used for screening. Identified chaperone or readthrough drugs will be tested in available mouse models. Overall this proposal will better explain the etiology and the genetic causes of phenotypic variability in ADPKD/ADPLD, develop better prognostic tools for individual selection of patients for treatment that are now becoming available, and explore allele based treatments for ADPKD.

Working Women, Special Provision and the Debate on Equality There has been considerable coverage in the media recently about the possibility of offering women in employment paid leave from work during their menstrual period. This has generated a broad range of responses relating to long-standing discussions about ‘equality’ and ‘difference’: is women’s equality best achieved by treating them the same as men or by making provisions that recognise their differences in terms of physiological constitution and biological functions? If the UK introduces such an initiative, it would not be the first country in the contemporary world to do so. Many countries in Asia already make the provision and Russia debated introducing a law in 2013. The policy also has a significant historical precedent. A whole chapter of my book Women Workers in the Soviet Interwar Economy: From ‘Protection’ to ‘Equality’ (Macmillan, 1999), based on extensive research conducted for my PhD, is devoted to ‘Provision for “Menstrual Leave”’. In the 1920s, scientific researchers and labour hygiene specialists in the Soviet Union conducted extensive investigations into the impact of menstruation on women’s capacity to work in manual and industrial jobs requiring a significant degree of physical labour. Their recommendations led to two decrees being issued that targeted specific categories of women workers: Decree ‘On the release from work during menstruation of machinists and iron press workers working on cutting machines without mechanised gears in the garment industry’, 11 January 1922 Decree ‘On the working conditions of women tractor and lorry drivers’, 9 May 1931 These decrees arose from research that suggested, amongst other things, that inadequate seating at machines and on tractors resulted in congestion and tension in the abdomen that was exacerbated during menstruation. In practice, the decrees did not provide for regular absence from work. Women seeking to benefit from the provision had to provide a doctor’s note, similar to the usual requirements for sick leave. The official research into the impact of menstruation on women’s capacity to work and the application of the decrees in practice raised a number of issues on both sides of the argument. I offer only a summary of the contemporary research findings and observer commentary here: For the provision: • employers have a responsibility to protect the health of their workers and unhealthy, poor and inadequate working environments can have a detrimental impact on women’s reproductive health • women’s labour productivity and output would rise as a result • it is essential to protect the professionalism of certain categories of workers: the debates here centred on performance artists and female theatrical employees engaged in highly physical and intensely emotional work • heavy physical labour and strenuous exercise can lead to disruptions of the menstrual cycle • women’s physical and intellectual capacities are reduced during menstruation; women lose muscular strength and powers of concentration • women’s biological constitution and reproductive functions require specific recognition in law Against the provision: • employers are less likely to appoint women if they are guaranteed paid time off work during menstruation • (often from male workers, who viewed the employment of women as competition) women should not be employed in jobs for which they lack the physical strength and mental capacity • if necessary, women could be transferred to different tasks involving easier work during menstruation • the provision would be open to uneven application and abuse • women cannot expect to be considered equal with men if they are given special treatment in the law It is worth noting also that the various research projects often revealed that the vast majority of women reported no regular problems or abnormalities with menstruation, and that men commonly reported higher levels of sickness than their female colleagues. Many of the problems experienced by women in the workplace could be mitigated by the introduction of improvements to their physical working conditions (not sitting down or standing up in the same position for long periods of time) or by the simple introduction of very short breaks that would allow women to walk around and get some exercise. Debates in the UK, on the TV and in the press, are unlikely to reach a consensus on this issue. What do you think?

At our best, we motivate ourselves every day to get dressed and go to work or school. Although there are larger incentives at work, it's our own volition that powers us through our innumerable daily tasks. If we could learn to control the motivational centers of our brains that drive volition, would it lead us toward healthier, more productive lives? Using a new brain imaging strategy, Duke University scientists have now taken a first step in understanding how to manipulate specific neural circuits using thoughts and imagery. The technique, which is described in the March 16 issue of the journal Neuron, is part of a larger approach called 'neurofeedback,' which gives participants a dynamic readout of brain activity, in this case from a brain area critical for motivation. "These methods show a direct route for manipulating brain networks centrally involved in healthy brain function and daily behavior," said the study's senior investigator R. Alison Adcock, an assistant professor of psychiatry and behavioral sciences and associate director of the Center for Cognitive Neuroscience in the Duke University Institute for Brain Sciences. Neurofeedback is a specialized form of biofeedback, a technique that allows people to monitor aspects of their own physiology, such as heart rate and skin temperature. It can help generate strategies to overcome anxiety and stress or to cope with other medical conditions. Neurofeedback has historically relied on electroencephalography or EEG, in which patterns of electrical activity are monitored noninvasively by electrodes attached to the scalp. But these measures provide only rough estimates of where activity occurs in the brain. advertisement In contrast, the new study employed functional magnetic resonance imaging (fMRI), which measures changes in blood oxygen levels, allowing more precisely localized measurements of brain activity. Adcock's team has been working on ways to use thoughts and behavior to tune brain function for the past eight years. In this time, they've developed tools allowing them to analyze complex brain imaging data in real time and to display it to participants as neurofeedback while they are in the fMRI scanner. This study focused on the ventral tegmental area (VTA), a small area deep within the brain that is a major source of dopamine, a neurochemical well known for its role in motivation, experiencing rewards, learning, and memory. According to Adcock's previous research, when people are given incentives to remember specific images, an increase in VTA activation before the image appears predicts whether the participants are going to successfully remember the image. External incentives like money work well to stimulate the VTA, but it was unclear whether people could exercise this area on their own, said co-author Jeff MacInnes, a postdoctoral researcher in Adcock's lab. advertisement In the new study, the team encouraged participants in the scanner to generate feelings of motivation -- using their own personal strategies -- during 20-second intervals. They weren't able to raise their VTA activity consistently on their own. But when the scientists provided participants with neurofeedback from the VTA, presented in the form of a fluctuating thermometer, participants were able to learn which strategies worked, and ultimately adopt more effective strategies. Compared to control groups, the neurofeedback-trained participants successfully elevated their VTA activity. Participants reported using a variety of different motivational strategies, from imagining parents or coaches encouraging them, to playing out hypothetical scenarios in which their efforts were rewarded, said co-author Kathryn Dickerson, a postdoctoral researcher in Adcock's group. The self-generated boost in VTA activation worked even after the thermometer display was removed. Only the participants who had received accurate neurofeedback were able to consistently raise their VTA levels. "Because this is the first demonstration of its kind, there is much still to be understood," Adcock added. "But these tools could offer benefits for everyone, particularly those with depression or attention problems." The neurofeedback training also activated other regions involved in learning and experiencing rewards, confirming that, at least in the short term, the brain changes its activity more broadly as a result of neurofeedback, Dickerson said. Adcock said one caveat of the study is that the team has not tested whether the neurofeedback drove changes in behavior. The group is working on those studies now and also plans to conduct the same study in participants with depression and attention deficit hyperactivity disorder (ADHD). This research was supported by the National Institute of Mental Health (MH9743, MH100764), the Alfred P. Sloan Foundation, the Esther A. & Joseph Klingenstein Fund, and the Dana Foundation.

Q: Como passar objetos entre controllers no MVC utilizando POO Basicamente, eu preciso que ser o login for bem sucedido salvar o nome de usuário em uma variável e utilizar-lá em outro controller. Model.php: public function login($email, $password) { session_start(); $sql = "SELECT * FROM users WHERE email = :email AND password= :password;"; $query = $this->db->prepare($sql); $parameters = array(':email' => $email, ':password' => $password); $query->execute($parameters); $rows = $query->fetch(PDO::FETCH_NUM); if($rows > 0) { header ("Location: " . URL . "home"); } else { exit ('Email or password incorrect'); } } Controller.php public function login() { if (isset($_POST['login_submit']) AND isset($_POST['email']) AND isset($_POST['password'])) { $this->model->login($_POST['email'], $_POST['password']); } } A: Não foi explicito mas parece que você quer que seja mandado por session. Sendo assim você pode simplesmente setar na sessão e pegar de volta no outro controle. <?php // declaração da classe Pessoa class Pessoa { public $nome; } // No Controller que envia os parametros session_start(); $joao = new Pessoa(); $joao->nome = "João"; $_SESSION['pessoa'] = $joao; // No Controller que recebe os dados session_start(); $joao = $_SESSION['pessoa']; print_r($joao); Ou se quiser padronizar isso e jogar no paradigma de orientação a objetos <?php // controller que envia $joao = new Pessoa(); $joao->nome = "João"; SessionUtils::setPropriedade('pessoa', $joao); // controller que recebe $joao = SessionUtils::getPropriedadeLimpar('pessoa'); print_r($joao); // declaração da classe Pessoa class Pessoa { public $nome; } // classe util para a sessão class SessionUtils { private static $BASE_PROPRIEDADES = "props"; /** * Pega uma propriedade da sessão * @return a propriedade ou null se não existir */ public static function getPropriedade($nome){ self::configurarSessao(); $sessao = self::getSessao(); return @$sessao[$nome]; } /** * Pega uma propriedade da sessão e depois a exclui da mesma * @return a propriedade ou null se não existir */ public static function getPropriedadeLimpar($nome){ self::configurarSessao(); $sessao = self::getSessao(); $valor = @$sessao[$nome]; self::setPropriedade($nome, null); return $valor; } /** * Seta uma propriedade na sessão */ public static function setPropriedade($nome, $valor){ self::configurarSessao(); $_SESSION[self::$BASE_PROPRIEDADES][$nome] = $valor; } /** * Configura a sessão para guardar os itens */ private static function configurarSessao(){ if(!isset($_SESSION)){ session_start(); } if(!self::getSessao() || !is_array(self::getSessao())){ self::setSessao(array()); } } private static function getSessao(){ return $_SESSION[self::$BASE_PROPRIEDADES]; } private static function setSessao($valor){ $_SESSION[self::$BASE_PROPRIEDADES] = $valor; } }

Introduction {#sec1-1} ============ Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFX have been reported \[[@ref9]-[@ref11]\]. Recently, a genome-wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with *FcγRΙΙΙA* -158V/V genotype had a better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\]. The aim of this study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD. Patients - Methods {#sec1-2} ================== Patients {#sec2-1} -------- We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX. IFX was administered intravenously at a dose of 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table 2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. ###### Grading of endoscopic mucosal lesions \[[@ref21],[@ref22]\]  ###### Classification of the study population due to response to infliximab therapy  Genotyping {#sec2-2} ---------- Genomic DNA from whole blood containing EDTA was extracted using standard techniques (NucleoSpin Blood kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG TG-3′ and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect the SNP in the −308 gene allele and MspI to detect the polymorphism of the −238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers-van de Straat et al \[[@ref25]\] using the primers 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC ACT CAA AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch to ensure that reagents had not been contaminated. Statistical Analysis {#sec2-3} -------------------- Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, San Diego, CA). The p values are all two-sided. Correction for multiple testing was not applied in this study. *P* values of \< 0.05 were considered to be significant. Results {#sec1-3} ======= Patient demographic and clinical characteristics are given in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders, 25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non-responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantly lower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly. ###### Demographic, clinical and biological characteristics of the study population  The -238 G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/F polymorphism in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions. ###### Genotype frequency in complete responders, partial responders and non responders  In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serum CRP levels was greatest in VV homozygotes (78.15 ± 33.68%) and lowest in FF homozygotes (69.84 ± 28.7%) but this difference was not significant. Due to the small number of cases we did not stratify the genotype frequencies according to age. Discussion {#sec1-4} ========== The mechanism of IFX action in IBD seems to be multifactorial and the response to IFX is a complex phenomenon influenced by several parameters \[[@ref1]\]. Interestingly, a certain proportion of patients do not respond to IFX at all whereas a significant proportion will lose response over time \[[@ref3]-[@ref7]\]. This is the first Greek study aiming at identifying any significant associationbetween the -238 G/A, -308 G/A, and -857 C/T polymorphisms in the promoter region of the TNF gene and the -158V/F polymorphism in *FcγRΙΙΙA* gene and response to IFX in a cohort of adult and paediatric patients with CD and it was negative. Efficacy of IFX was assessed by clinical, serological and endoscopic parameters. Clinical response to IFX was evaluated using the HBI, which has been used in many clinical trials, is simple to use and has shown good correlation with the Crohn's Disease Activity Index (CDAI) \[[@ref26]\]. Serological evaluation of response to IFX was based on changes in serum levels of CRP, which has shown a good correlation with clinical activity and to a certain degree with endoscopic activity of CD \[[@ref27]\]. Finally, endoscopic activity of disease was assessed before and after IFX therapy using a simple description of healing of ulcerative and non ulcerative lesions \[[Table 1](#T1){ref-type="table"}\] as has been previously described \[[@ref21],[@ref22]\]. Endoscopic healing was assessed after 12-20 weeks of IFX treatment. It is conceivable that 12 weeks may be early to assess mucosal healing induced by biologic therapies \[[@ref27]\] but the vast majority of patients underwent endoscopy at least 16 weeks after initiation of IFX therapy (average time 17.6 weeks) and therefore it is unlikely that we have not obtained an objective view of the intestinal mucosal at follow up ileocolonoscopy. Regarding the *TNF* genotypes, our results are in agreement with Louis et al \[[@ref11]\] who did not find any significant difference between response groups when genotyped CD patients for the TNF -308G/A polymorphism and compared response rates after IFX treatment. The same results were reported by Mascheretti et al \[[@ref10]\] and Dideberg et al \[[@ref13]\]. Moreover, our results are in agreement with Tomita et al \[[@ref28]\] who reported no significant difference on *TNFa*, *FcgammaRIIA* and *FcgammaRIIIA* between responders and non responders 8 weeks after IFX treatment as well as with results of ACCENT I study where the relative decrease in serum CRP levels after IFX treatment was greatest in -158 VV homozygotes and lowest in FF homozygotes \[[@ref18]\]. In contrast, Louis et al \[[@ref17]\] observed a significant association between the -158V/F polymorphism in *FcγRΙΙΙA* and both the proportion of patients who had a drop in serum CRP levels after IFX treatment and the magnitude in decrease of serum CRP levels. This may account for the relatively small population of patients in our study, genetic differences in the studied populations and/or methodological differences between studies. Although it would be useful to genetically differentiate 'responders' from 'non-responders', there are not enough data on TNF polymorphisms in IBD and often only selected polymorphisms are genotyped. Small studies have shown possible associations between poor response to IFX and increasing mucosal levels of activated NF-kappaB, homozygosity for the polymorphism in exon 6 of TNFR2 (genotype Arg196Arg), positivity for perinuclear antineutrophil cytoplasmic antibodies and with the presence of increased numbers of activated lamina propia mononuclear cells producing interferon-gamma and TNFa \[[@ref29]\]. In conclusion, our study did not detect any associations between three TNFα gene polymorphisms or the -158 V/F polymorphism in the *FcγRΙΙΙA* gene and response to IFX in CD. However, in view of discrepant results in the literature large-scale pharmacogenetic studies in different populations, with similar baseline disease phenotypes and treatment protocols are needed to adequately estimate associations between genetic polymorphisms and treatment outcomes. Conflict of interest: None ^a^Evangelismos Hospital, ^b^Laboratory of Biology, School of Medicine, ^c^1^st^ Department of Pediatrics, School of Medicine, University of Athens, Greece

Over every mountain there is a path, although it may not be seen from the valley I'm now taking commissions, if you're interested in some high quality terrain PM me. WorldPainter (for creating the map) Chunky (for rendering the map) Thanks for stopping by, if you enjoyed this submission please consider leaving a diamond! The sweeping sunset of the Roughhew Rocks is deceiving. The mountain becomes beautiful, the forests become picturesque and the rugged rocks look tamed. Even the birds sing a loud song heard far and wide over the coastal rocks. Don't be fooled, you're not safe. The beauty is the danger, one feels so safe, so alive, so free in Roughhew. But the same cannot be said for the human. Only two people have escaped Roughhew alive, and the thousands that are enticed here each season are slain. Some say it is nature that kills them, others say it's haunted, some claim the beauty was too much for them. While nobody knows the cause of death of these people there is one thing that cannot be disputed - this region has the highest rate of death in all the land.I like this map but I found it incredibly difficult to get nice renders of it. As such it marks the first map I've made where the cover photo is from the ground rather than a top down isometric view. This map I changed normal smooth stone for rock, a combination of stone and cobblestone, to create a rougher and more rugged terrain and I'm pleased with how it turned out. I also used gravel in the valleys to show heavy erosion and an unstable land, despite the towering mountain looming over it.I used the following tools to create this map:

POV: Henry vs Martin + a poll I won’t make claims as to their gifts and charms, but H & M do resemble me in various ways :) I usually like to write stories from a single point of view. It’s obviously a limited perspective, but I enjoy the constraints. As far as I’m concerned, there’s no such thing as a reliable narrator. Characters misinterpret things, miss things, draw the wrong conclusions, and it can be tricky and fun to work the “truth” into a story alongside the character’s perceptions. For instance, I think it’s obvious to the reader that Martin is DTF from the get-go, but Henry, equipped with the same amount of information, simply doesn’t get it. When I started writing the Ganymede Quartet books, it seemed obvious to me that the story needed to be told from the master’s point of view. Whether or not he’s actually prepared to take responsibility, the fact remains that Henry’s the one in charge and he sets the tone. It’s Martin’s job to adapt and respond and accommodate and serve. Obviously, Martin is better-equipped to steer this particular ship, but, unfortunately for Henry, the roles in this relationship weren’t assigned based on fitness or merit. If you’ve read A Most Personal Property (GQ Book 1), you know that when the opportunity finally arises for Martin to take charge, he does so with great effect, but he does wait for Henry to create the opportunity. He’s very well-trained. I think it’s apparent that Martin is miserable for most of AMPP, and writing weeks of self-doubt and misery even greater than Henry’s, from the perspective of a character who has even less power to effect change…I don’t think anyone wants to read that book, actually. Henry also needed to be the POV character for the main books because Henry is the one who has the most growing to do. They’re both young, both immature, but Martin is less immature, his sense of self is more solid and, well, he’s a lot smarter. Henry learns a lot over the course of the series, which is not to say that Martin doesn’t, but as the one nominally in charge, Henry’s growth has a greater impact on both of them. It was possibly something of a risk, but I left out or delayed certain trains of thought because Henry isn’t necessarily considering all aspects and implications of the master/slave dynamic from early on in their relationship. He’s very loving, but he’s not the most insightful person, and it takes him awhile to consider things that a savvier fellow might have questioned from the beginning. It really does take Henry a long time to wonder how Martin’s position and training impact the way Martin responds to him. I anticipate going a little deeper into Martin’s background, in a way, for the story that will accompany Book 3. I also have a pretty good idea which aspect of Book 4 I’ll present from Martin’s perspective. So far, the Martin stories have been really fun to write, and I definitely look forward to doing them. I think they’re so easy and enjoyable to work on because they revisit territory that I’ve already covered from Henry’s perspective to some extent, and when I’m writing Henry, I’m always considering how Martin might view a given situation, as well. Offering Martin’s POV at all was actually a pretty late development. It occurred to me shortly before publishing A Most Personal Property that the stories I was busy telling myself about Martin’s past would probably be of interest to anyone who was interested in AMPP, and so I quickly wrote A Superior Slave. I hoped that people who enjoyed reading ASS (ugh, that acronym!) for free might be interested in paying for AMPP, and I think that did happen to some small extent. I’ve gotten the impression (whether it’s true or not) that Martin might be the reader favorite by a small margin, so it just seems like a nice idea to continue offering Martin POV stories alongside the main books. While I think a person can enjoy the main books and Henry’s POV without side stories, I like to think Martin’s perspective is a valuable addition. I plan on adding additional points of view from other characters in the universe. I’ve got stories written about a couple of Henry’s friends to show how slave ownership works in private for other people. I’ve got at least two stories I want to write about Henry’s cousin Jesse. I think Tom gets his own novella :D With A Proper Lover (GQ Book 2) and A Master’s Fidelity (GQ Book 2.5) released, I’m just going immediately into editing Book 3 and fleshing out the notes I have for the Martin story. I’d had vague ideas about taking a break, but I honestly don’t know what that would mean at this point. I don’t know what I’d be doing during a break! Right now, the idea of downtime just makes me cranky. Knowing that there are people eager for the next books makes me want to work on getting them out. Besides, working on Martin’s POV is a treat :)

Q: React typescript ref return null in conditional rendering I want to use React refs, it works fine in static rendering, e.g: <footer ref="ftr"></footer> But, not in conditional rendering, e.g: {footer ? <footer ref="ftr"></footer> : null} When I called ReactDOM.findDOMNode(this.refs.ftr);, the first way returned the element (fine) but the second returned me undefined. How to do the right way in conditional rendering? Any answer would be appreciated. A: You should not use string refs as written in the docs: We advise against it because string refs have some issues, are considered legacy, and are likely to be removed in one of the future releases You can do this: let footerElement: HTMLElement | null = null; ... {footer ? <footer ref={ el => footerElement = el }></footer> : null} ... if (footerElement != null) { ... }

The two classes `KinesisRecorder` and `KinesisFirehoseRecorder` allow you to interface with Amazon Kinesis Data Streams and Amazon Kinesis Data Firehose to stream analytics data for real-time processing. ## What is Amazon Kinesis Data Streams? [Amazon Kinesis Data Streams](http://aws.amazon.com/kinesis/) is a fully managed service for real-time processing of streaming data at massive scale. Amazon Kinesis can collect and process hundreds of terabytes of data per hour from hundreds of thousands of sources, so you can write applications that process information in real-time. With Amazon Kinesis applications, you can build real-time dashboards, capture exceptions and generate alerts, drive recommendations, and make other real-time business or operational decisions. You can also easily send data to other services such as Amazon Simple Storage Service, Amazon DynamoDB, and Amazon Redshift. The Kinesis Data Streams `KinesisRecorder` client lets you store your Kinesis requests on disk and then send them all at once using the [PutRecords](https://docs.aws.amazon.com/kinesis/latest/APIReference/API_PutRecords.html) API call of Kinesis. This is useful because many mobile applications that use Kinesis Data Streams will create multiple requests per second. Sending an individual request under `PutRecord` action could adversely impact battery life. Moreover, the requests could be lost if the device goes offline. Thus, using the high-level Kinesis Data Streams client for batching can preserve both battery life and data. ## What is Amazon Kinesis Data Firehose? [Amazon Kinesis Data Firehose](http://aws.amazon.com/kinesis/firehose/) is a fully managed service for delivering real-time streaming data to destinations such as Amazon Simple Storage Service (Amazon S3) and Amazon Redshift. With Kinesis Data Firehose, you do not need to write any applications or manage any resources. You configure your data producers to send data to Firehose and it automatically delivers the data to the destination that you specified. The Amazon Kinesis Data Firehose `KinesisFirehoseRecorder` client lets you store your Kinesis Data Firehose requests on disk and then send them using the [PutRecordBatch](https://docs.aws.amazon.com/firehose/latest/APIReference/API_PutRecordBatch.html) API call of Kinesis Data Firehose. For more information about Amazon Kinesis Data Firehose, see [Amazon Kinesis Data Firehose](http://docs.aws.amazon.com/firehose/latest/dev/what-is-this-service.html). ## Integrating Amazon Kinesis Set up AWS Mobile SDK components by including the following libraries in your `app/build.gradle` dependencies list. ```groovy dependencies { implementation 'com.amazonaws:aws-android-sdk-kinesis:2.15.+' implementation ('com.amazonaws:aws-android-sdk-mobile-client:2.15.+@aar') { transitive = true } } ``` * `aws-android-sdk-kinesis` library enables sending analytics to Amazon Kinesis. * `aws-android-sdk-mobile-client` library gives access to the AWS credentials provider and configurations. Add the following imports to the main activity of your app. ```java import com.amazonaws.mobileconnectors.kinesis.kinesisrecorder.*; import com.amazonaws.mobile.client.AWSMobileClient; import com.amazonaws.regions.Regions; ``` To use Kinesis Data Streams in an application, you must set the correct permissions. The following IAM policy allows the user to submit records to a specific data stream, which is identified by [ARN](http://docs.aws.amazon.com/general/latest/gr/aws-arns-and-namespaces.html). ```json { "Statement": [{ "Effect": "Allow", "Action": "kinesis:PutRecords", "Resource": "arn:aws:kinesis:us-west-2:111122223333:stream/mystream" }] } ``` The following IAM policy allows the user to submit records to a specific Kinesis Data Firehose delivery stream. ```json { "Statement": [{ "Effect": "Allow", "Action": "firehose:PutRecordBatch", "Resource": "arn:aws:firehose:us-west-2:111122223333:deliverystream/mystream" }] } ``` This policy should be applied to roles assigned to the Amazon Cognito identity pool, but you need to replace the `Resource` value with the correct ARN for your Amazon Kinesis or Amazon Kinesis Data Firehose stream. You can apply policies at the [IAM console](https://console.aws.amazon.com/iam/). To learn more about IAM policies, see [Using IAM](http://docs.aws.amazon.com/IAM/latest/UserGuide/IAM_Introduction.html). To learn more about Amazon Kinesis Data Streams policies, see [Controlling Access to Amazon Kinesis Data Streams Resources with IAM](http://docs.aws.amazon.com/kinesis/latest/dev/kinesis-using-iam.html). To learn more about Amazon Kinesis Data Firehose policies, see [Controlling Access with Amazon Kinesis Data Firehose](http://docs.aws.amazon.com/firehose/latest/dev/controlling-access.html). ## Working with the API You can use `AWSMobileClient` to setup the Cognito credentials that are required to authenticate your requests with Amazon Kinesis. ```java AWSMobileClient.getInstance().initialize(getApplicationContext(), new Callback<UserStateDetails>() { @Override public void onResult(UserStateDetails userStateDetails) { Log.i("INIT", userStateDetails.getUserState().toString()); } @Override public void onError(Exception e) { Log.e("INIT", "Initialization error.", e); } } ); ``` Once you have credentials, you can use `KinesisRecorder` with Amazon Kinesis. The following snippet creates a directory and instantiates the `KinesisRecorder` client: ```java String kinesisDirectory = "YOUR_UNIQUE_DIRECTORY"; KinesisRecorder recorder = new KinesisRecorder( myActivity.getDir(kinesisDirectory, 0), Regions.<YOUR-AWS-REGION>, AWSMobileClient.getInstance() ); // KinesisRecorder uses synchronous calls, so you shouldn't call KinesisRecorder methods on the main thread. ``` To use `KinesisFirehoseRecorder`, you need to pass the object in a directory where streaming data is saved. We recommend you use an app private directory because the data is not encrypted. ```java KinesisFirehoseRecorder firehoseRecorder = new KinesisFirehoseRecorder( context.getCachedDir(), Regions.<YOUR-AWS-REGION>, AWSMobileClient.getInstance()); ``` Configure Kinesis: You can configure `KinesisRecorder` or `KinesisFirehoseRecorder` through their properties: You can configure the maximum allowed storage via the `withMaxStorageSize()` method of `KinesisRecorderConfig`. You can retrieve the same information by getting the `KinesisRecorderConfig` object for the recorder and calling `getMaxStorageSize():` ```java KinesisRecorderConfig kinesisRecorderConfig = recorder.getKinesisRecorderConfig(); Long maxStorageSize = kinesisRecorderConfig.getMaxStorageSize(); // Do something with maxStorageSize ``` To check the number of bytes currently stored in the directory passed in to the `KinesisRecorder` constructor, call `getDiskBytesUsed()`: ```java Long bytesUsed = recorder.getDiskBytesUsed(); // Do something with bytesUsed ``` To see how much space the `KinesisRecorder` client is allowed to use, you can call `getDiskByteLimit()`. ```java Long byteLimit = recorder.getDiskByteLimit(); // Do something with byteLimit ``` With `KinesisRecorder` created and configured, you can use `saveRecord()` to save records and then send them in a batch. ```java recorder.saveRecord( "MyData".getBytes(), "MyStreamName"); recorder.submitAllRecords(); ``` For the `saveRecord()` request above to work, you would have to have created a stream named `MyStreamName`. You can create new streams in the [Amazon Kinesis console](https://console.aws.amazon.com/kinesis). If `submitAllRecords()` is called while the app is online, requests will be sent and removed from the disk. If `submitAllRecords()` is called while the app is offline, requests will be kept on disk until `submitAllRecords()` is called while online. This applies even if you lose your internet connection midway through a submit. So if you save ten requests, call `submitAllRecords()`, send five, and then lose the Internet connection, you have five requests left on disk. These remaining five will be sent the next time `submitAllRecords()` is invoked online. Here is a similar snippet for Amazon Kinesis Data Firehose: ```java // Start to save data, either a String or a byte array firehoseRecorder.saveRecord("Hello world!\n"); firehoseRecorder.saveRecord("Streaming data to Amazon S3 via Amazon Kinesis Data Firehose is easy.\n"); // Send previously saved data to Amazon Kinesis Data Firehose // Note: submitAllRecords() makes network calls, so wrap it in an AsyncTask. new AsyncTask<Void, Void, Void>() { @Override protected Void doInBackground(Void... v) { try { firehoseRecorder.submitAllRecords(); } catch (AmazonClientException ace) { // handle error } } }.execute(); ``` To learn more about working with Kinesis Data Streams, see the [Amazon Kinesis Data Streams resources](http://aws.amazon.com/kinesis/developer-resources/). To learn more about the Kinesis Data Streams classes, see the [class reference for KinesisRecorder](https://aws-amplify.github.io/aws-sdk-android/docs/reference/com/amazonaws/mobileconnectors/kinesis/kinesisrecorder/KinesisRecorder.html). To learn more about the Kinesis Data Firehose classes, see the [class reference for KinesisFirehoseRecorder](https://aws-amplify.github.io/aws-sdk-android/docs/reference/com/amazonaws/mobileconnectors/kinesis/kinesisrecorder/KinesisFirehoseRecorder.html).

Michele Orecchia Michele Orecchia (26 December 1903 – 11 December 1981) was an Italian professional road bicycle racer, who won one stage in the 1932 Tour de France. He also competed in the individual and team road race events at the 1928 Summer Olympics. Major results 1927 Giro del Sestriere 1929 Giro d'Italia: 9th place overall classification 1932 Tour de France: Winner stage 8 References External links Official Tour de France results for Michele Orecchia Category:1903 births Category:1981 deaths Category:Italian male cyclists Category:Italian Tour de France stage winners Category:Sportspeople from Marseille Category:Olympic cyclists of Italy Category:Cyclists at the 1928 Summer Olympics Category:Tour de France cyclists Category:French male cyclists

INTRODUCTION {#s1} ============ Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the *Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions but are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different from that of virions ([@B15][@B16][@B17]), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]). In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], [@B7]). Only the NCs with relatively mature viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]). Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in culture supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients. RESULTS {#s2} ======= Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt/wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detected only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractions containing virions (fractions 9 to 11), naked capsids (fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), whereas only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids ranged from the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid-associated and extracellular HBV RNA (not shown). Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown). {#F1} To confirm the above-described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from 1.23 to 1.25 g/cm^3^. In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"}, HBV virions contained only the mature viral DNA (RC or DSL DNA), while naked capsids contained viral DNA replicative intermediates that ranged from the nascent minus-strand DNA to mature viral DNA ([Fig. 2B](#F2){ref-type="fig"} and [C](#F2){ref-type="fig"}). The lengths of viral minus- and plus-strand DNA in naked capsids and virions were determined by alkaline agarose gel electrophoresis analysis, a condition where denatured single-stranded DNA molecules migrate according to their lengths. In contrast to the complete minus- and mostly complete plus-strand DNA (closed to 3.2 knt) in virions, in naked capsids the minus-strand DNA and the plus-strand DNA can be both complete and incomplete (shorter than 3.2 knt) ([Fig. 2D](#F2){ref-type="fig"} and [E](#F2){ref-type="fig"}). Moreover, the length of HBV RNAs within naked capsids still ranged from 3.5 knt of pgRNA to shorter than the 0.7 knt of HBx mRNA. Full-length pgRNA accounted for only 10% of total RNA signal detected by Northern blotting (quantified from gray density of bands shown in [Fig. 2F](#F2){ref-type="fig"}). In contrast, HBV RNA species in virions are relatively shorter and barely detectable. In addition, we also determined viral DNA and RNA copy numbers in pooled naked capsids (fractions 3 to 7) and virions (fractions 10 to 21) by quantitative PCR. Quantification results showed that viral DNA in naked capsids and in virions accounted for about 60% and 40%, respectively, of total viral DNA signal in the HepAD38 cell culture supernatant ([Fig. 2G](#F2){ref-type="fig"}). More importantly, 84% of the HBV RNA was associated with naked capsids, while merely 16% was detected within virions ([Fig. 2G](#F2){ref-type="fig"}). Additionally, the DNA/RNA ratio was 11 in virions and 3 in naked capsids ([Fig. 2H](#F2){ref-type="fig"}), suggesting that more HBV RNA is present in naked capsids. {ref-type="fig"}).](zjv0241840640002){#F2} Extracellular HBV RNAs and immature viral DNA are detected in sera from CHB patients. {#s2.2} ------------------------------------------------------------------------------------- Employing the HepAD38 cell culture system, we demonstrated the presence of extracellular HBV RNAs and immature and mature viral DNA packaged in both the naked capsids and virions. Interestingly, Southern blot analyses showed that SS DNA could also be observed in serum samples from some CHB patients. We speculated that SS DNA in circulation would be carried by capsid particles that were released by HBV-infected hepatocytes into patients' bloodstreams. However, we reasoned that due to strong immunogenicity of naked capsids ([@B32], [@B33]), it would be difficult to detect them as free particles; rather, they would form complexes with specific anti-HBcAg antibodies and therefore circulate as antigen-antibody complexes ([@B25], [@B32][@B33][@B34]). To entertain this possibility, we then used protein A/G agarose beads to pull down the immune complexes. Forty-five serum samples obtained from CHB patients, with HBV DNA titers higher than 10^7^ IU per ml, were examined for the presence of particles containing SS DNA by a combination of protein A/G agarose bead pulldown assay and Southern blot analysis ([Fig. 3A](#F3){ref-type="fig"} and [B](#F3){ref-type="fig"}). SS DNA was detected, albeit to a different extent, in 34 serum samples ([Fig. 3A](#F3){ref-type="fig"} and [B](#F3){ref-type="fig"}, upper). The particles containing SS DNA were pulled down by protein A/G agarose beads from 11 out of the 34 samples ([Fig. 3A](#F3){ref-type="fig"} and [B](#F3){ref-type="fig"}, lower). Patient sera negative for SS DNA (patients 37, 38, 14, and 35) or positive for SS DNA (patients 17, 21, 42, and 44), as determined by the protein A/G agarose bead pulldown experiments, were selected for further studies ([Fig. 3C](#F3){ref-type="fig"}). {#F3} Northern blot analyses showed that HBV RNA was only detected in serum samples from patients 17, 21, and 42 ([Fig. 3D](#F3){ref-type="fig"}). Moreover, total viral DNA was analyzed by Southern blotting, and SS DNA was readily observed in serum samples from patients 17, 21, and 42 ([Fig. 3E](#F3){ref-type="fig"}). We also analyzed the lengths of DNA minus and plus strands in patients' sera. Despite the finding that most minus-strand DNA was complete, a small amount of viral DNA (that of patients 38, 35, 17, 21, and 42) was shorter than 3.2 knt ([Fig. 3F](#F3){ref-type="fig"}). Compared with viral minus-strand DNA, the length of plus-strand DNA, particularly in sera from patients 17, 21, and 42, was more variable, ranging from shorter than 2 knt to ∼3.2 knt ([Fig. 3G](#F3){ref-type="fig"}). Naked capsids form CACs with anti-HBcAg antibody in blood of CHB patients. {#s2.3} -------------------------------------------------------------------------- We showed that particles containing SS DNA were present in CHB patients' sera. To further examine these particles, we used CsCl density gradient centrifugation to fractionate a serum mixture from patients 37, 38, 14, and 35. In agreement with our earlier results ([Fig. 2A](#F2){ref-type="fig"}, lower, fractions 13 to 15, and B) and previous reports, HBV virions, with the characteristic mature viral DNA (RC or DSL DNA), were detected in fractions 12 to 14 with densities between 1.26 and 1.29 g/cm^3^ ([Fig. 4A](#F4){ref-type="fig"}) ([@B2]). Careful inspection of the blots revealed that SS DNA could be detected, albeit at very low level, in fractions 8 and 9, with densities from 1.33 to 1.34 g/cm^3^, and in fractions 18 to 21, with densities from 1.20 to 1.23 g/cm^3^ ([Fig. 4A](#F4){ref-type="fig"}). In contrast, CsCl density gradient separation of viral particles from serum of patient 17 showed a mixture of mature and immature viral DNA species. As SS DNA was detected at densities ranging from 1.37 to 1.20 g/cm^3^ ([Fig. 4B](#F4){ref-type="fig"}), no distinct viral DNA (mature RC or DSL DNA) specific to virions could be identified at densities between 1.27 and 1.29 g/cm^3^. Similar results were obtained using CsCl density gradient fractionation of sera from patient 21 (not shown) and patient 46 ([Fig. 4E](#F4){ref-type="fig"}). {#F4} We hypothesized that naked capsids could be released into blood circulation of CHB patients but were bound to specific antibodies. As SS DNA was detected in both high- and lower-density regions in CsCl gradient ([Fig. 4B](#F4){ref-type="fig"} and [E](#F4){ref-type="fig"}), we envisaged that the binding with specific antibodies led to a change of capsids' buoyant density. To test this, anti-HBcAg antibody was mixed with HepAD38 cell culture supernatant to mimic the postulated CACs in serum samples. The results demonstrated that in contrast to SS DNA from naked capsids, distributed to three fractions at densities between 1.33 and 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}, lower, and B), the mixture of naked capsids and CACs (SS DNA) was distributed more widely and could be detected in the lower density region (1.25 to 1.32 g/cm^3^) ([Fig. 4C](#F4){ref-type="fig"}, fractions 11 to 16). Similarly, intracellular capsids from HepAD38 cells were incubated with anti-HBcAg antibody, and a density shift of CACs to a lower-density region was also observed (not shown). To further confirm the lower density of CACs, NCs in virions secreted to HepAD38 cell culture supernatant were treated with NP-40 and mixed with anti-HBcAg antibody. CsCl fractionation showed that naked capsids and virion-derived NCs have become a homogenous mixture banding at densities from 1.37 to 1.27 g/cm^3^ ([Fig. 4D](#F4){ref-type="fig"}). Likewise, virion-derived NCs, obtained by treatment of serum sample from patient 46 with NP-40 bound with antibody, further formed new homogeneous CACs that settled at densities between 1.23 and 1.27 g/cm^3^ ([Fig. 4E](#F4){ref-type="fig"} versus F). However, NP-40 treatment alone did not produce a homogeneous mixture of naked capsids and virion-derived NCs, as these two particles still settled at distinct density regions with their characteristic viral DNA content ([Fig. 4G](#F4){ref-type="fig"}). On the other hand, DNA molecules in the two types of capsids still banded at densities between 1.38 and 1.31 g/cm^3^, further confirming that CACs have relatively lighter density ([Fig. 4G](#F4){ref-type="fig"}). Alternatively, the appearance of a homogenous mixture of virion-derived NCs and naked capsids ([Fig. 4D](#F4){ref-type="fig"} and [F](#F4){ref-type="fig"}) suggests the formation of higher-order antibody-mediated complexes of capsids. For instance, the complexes might not represent individual antibody-coated capsid particles but rather big CACs consisting of several capsid particles interconnected by antibodies. To verify whether intercapsid immune complexes exist, anti-HBcAg antibody was added to the purified HBV capsids expressed by Escherichia coli, and this mixture was examined by an electron microscope. E. coli-derived capsids were scattered as separate, distinct particles ([Fig. 5A](#F5){ref-type="fig"}). However, addition of antibody caused capsids to aggregate into clusters, making them too thick to be properly stained ([Fig. 5B](#F5){ref-type="fig"}). Despite this, a few capsids, which might not have been bound by antibodies or might have been associated with antibodies but did not form intercapsid antibody complexes, could be observed by electron microscopy (EM) ([Fig. 5B](#F5){ref-type="fig"}). {#F5} We then examined CACs in serum samples from CHB patients by EM. Sera from patients 11, 17, 21, 22, 23, 27, 28, 30, and 41, positive for SS DNA, were combined. Serum mixtures, with diminished HBsAg particles by centrifugation through a 20% and 45% (wt/wt) sucrose cushion, were examined by EM. The 27-nm capsid particles or CACs were visible ([Fig. 5C](#F5){ref-type="fig"}, arrow) along with the 42-nm HBV virions ([Fig. 5C](#F5){ref-type="fig"}, arrowheads) and the 22-nm spheres and rods of residual HBsAg particles (not indicated). However, the picture was not clear enough for us to conclusively determine if capsids were connected by or bound with antibodies, as described for unrelated virus in *in vitro* experiments ([@B35]). In addition, it is possible that some of the CACs are not visible by EM, as the complexes maybe too thick to gain clear contrast between lightly and heavily stained areas ([Fig. 5B](#F5){ref-type="fig"}). Lastly, CACs might be heterogeneous, having different molecular sizes and isoelectric points (pI) in hepatitis B patients' blood circulation. *In vitro* binding of naked capsids derived from HepAD38 cell culture supernatant with anti-HBcAg antibody changed their electrophoretic behavior and made them unable to enter the TAE-agarose gel ([Fig. 6A](#F6){ref-type="fig"}). Moreover, viral particles from sera of patients 0, 37, 38, 14, 35, 17, 21, 42, and 44 could not enter agarose gels prepared in TAE buffer. However, in buffer with higher pH value (10 mM NaCHO~3~, 3 mM Na~2~CO~3~, pH 9.4), they appeared as smearing bands on blots ([Fig. 6B](#F6){ref-type="fig"} and [C](#F6){ref-type="fig"}). Hence, the irregular electrophoretic behavior of these viral particles may result from changes in molecular size and/or pI value of capsid particles (pI  4.4) following their association with specific immunoglobulin G (or other types of antibodies) having different pI values (pI of human IgG may range from 6.5 to 9.5) ([@B36][@B37][@B39]). {#F6} Circulating HBV RNAs are of heterogeneous lengths and associated with CACs and virions in hepatitis B patient's plasma. {#s2.4} ----------------------------------------------------------------------------------------------------------------------- To characterize HBV RNAs circulating in CHB patients' sera, a plasma sample from patient 0 was studied. Similar to results obtained for patients 17, 21, and 46 ([Fig. 4B](#F4){ref-type="fig"} and [E](#F4){ref-type="fig"} and not shown), viral DNA in the plasma sample of patient 0 was detected in a broad density range in CsCl gradient and no distinct bands specific to HBV virions or naked capsids could be identified, indicating the presence of a mixture of virions and CACs ([Fig. 7A](#F7){ref-type="fig"}). {#F7} Furthermore, viral particles were pelleted through a 20% sucrose cushion and separated in a sucrose gradient. HBsAg was detected in fractions 5 to 14, peaking at fraction 11. The PreS1 antigen was found in fractions 5 to 12 with the peak at fractions 7 and 10, indicating its presence in HBsAg particles and HBV virions ([Fig. 7B](#F7){ref-type="fig"}, upper). Viral DNA, representing a combination of both mature and immature viral DNA, was detected in fractions 4 to 9 ([Fig. 7B](#F7){ref-type="fig"}, middle), suggesting the localization of CACs and virions in these fractions. HBV RNA was detected between fractions 5 and 7 and appeared in the same peak as viral DNA ([Fig. 7B](#F7){ref-type="fig"}, lower), indicating that HBV RNA is incorporated in the same viral particles as viral DNA. Therefore, circulating HBV RNA may be localized within CACs and/or virions. To better characterize HBV RNA in CACs and virions, plasma sample from patient 0 was centrifuged through a 20% sucrose cushion and pellets were fractionated in a homogenous CsCl solution (1.18 g/cm^3^) as previously described ([@B8]). However, possibly due to a tendency of capsid particles to aggregate and stick to the wall of the centrifugation tube and the low density of the initial CsCl solution ([@B8], [@B40]), only mature DNA species from virions were detected in densities ranging from 1.22 to 1.24 g/cm^3^ ([Fig. 7C](#F7){ref-type="fig"}, upper). Northern blot analyses demonstrated that the lengths of virion-associated HBV RNAs were approximately several hundred nucleotides ([Fig. 7C](#F7){ref-type="fig"}, lower). Virion-associated RNAs were unlikely to be contaminated by CAC-associated HBV RNAs, since the immature SS DNA could not be observed even after a long exposure of X ray film. Moreover, RNA molecules would have been longer if there were CAC contamination ([Fig. 7D](#F7){ref-type="fig"}, lower). Viral nucleic acids in pellets recovered from the centrifugation tube sidewalls could be readily detected on Northern ([Fig. 7C](#F7){ref-type="fig"}, lower, lane P) or Southern ([Fig. 7C](#F7){ref-type="fig"}, upper, lane P) blots using plus-strand-specific rather than minus-strand-specific riboprobe. To analyze viral nucleic acids in CACs, concentrated plasma sample was separated in a higher CsCl density gradient (1.18 g/cm^3^ and 1.25 g/cm^3^). Both mature and immature viral DNA species were only detected in fractions with densities from 1.21 to 1.26 g/cm^3^ ([Fig. 7D](#F7){ref-type="fig"}, upper), indicating the presence of a mixture of HBV virions and CACs. Viral RNAs were detected and ranged in length from a little shorter than the full-length pgRNA to a few hundred nucleotides ([Fig. 7D](#F7){ref-type="fig"}, lower). Compared to virion-associated RNAs ([Fig. 7C](#F7){ref-type="fig"}, lower), HBV RNA species detected in the mixture of CACs and virions were longer, with the longer RNA molecules possibly being associated with CACs. Extracellular HBV RNAs could serve as templates for synthesis of viral DNA. {#s2.5} --------------------------------------------------------------------------- Intracellular NCs are known to contain viral nucleic acids in all steps of DHBV DNA synthesis, including pgRNA, nascent minus-strand DNA, SS DNA, and RC DNA or DSL DNA ([@B5]). Our results showed that naked capsids contained almost the same DNA replicative intermediates as intracellular NCs ([Fig. 1B](#F1){ref-type="fig"} and [2B](#F2){ref-type="fig"}) ([@B7], [@B11]). We also demonstrated that extracellular HBV RNAs within the naked capsids, CACs, and virions were heterogeneous in length ([Fig. 1B](#F1){ref-type="fig"}, lower, [2F](#F2){ref-type="fig"}, and [7C](#F7){ref-type="fig"} and [D](#F7){ref-type="fig"}). In the presence of deoxynucleoside triphosphates (dNTPs), viral RNA could be degraded and reverse transcribed into minus-strand DNA by the endogenous polymerase *in vitro* ([@B5], [@B41], [@B42]). Also, incomplete plus-strand DNA with a gap of about 600 to 2,100 bases could be extended by endogenous polymerase ([@B43], [@B44]). Based on these results, we wished to examine whether extracellular HBV RNAs could serve as RNA templates for viral DNA synthesis and be degraded by polymerase in the process. As shown in [Fig. 8](#F8){ref-type="fig"}, endogenous polymerase assay (EPA) treatment of extracellular viral particles from either culture supernatant of HepAD38 cells or plasma sample from patients led to DNA minus ([Fig. 8A](#F8){ref-type="fig"} and [C](#F8){ref-type="fig"})- and plus ([Fig. 8B](#F8){ref-type="fig"} and [D](#F8){ref-type="fig"})-strand extension and, more importantly, HBV RNA signal reduction ([Fig. 8E](#F8){ref-type="fig"}, lane 4 versus 6 and lane 8 versus 10). The apparent low efficiency of EPA reaction might have been due to our hybridization method, which detected both extended and unextended DNA strands rather than detecting only newly extended DNA. {#F8} In the process of HBV DNA replication, prior to minus-strand DNA synthesis, capsid-associated RNA is the full-length pgRNA. Upon transfer of viral polymerase-DNA primer to the 3′ DR1 region of pgRNA and cleavage of the 3′ epsilon loop RNA (a 3.2-knt pgRNA fragment remained), minus-strand DNA synthesis initiates and the pgRNA template is continuously cleaved from 3′ to 5′ by RNase H activity of viral polymerase. Consequently, from the initiation to the completion of minus-strand DNA synthesis, there will be a series of pgRNA fragments with receding 3′ ends ranging from 3.2 knt to 18 nt of the 5′ cap RNA primer ([@B2], [@B21][@B22][@B24]), representing the RNA templates that have not yet been reverse transcribed into minus-strand DNA. In addition to pgRNA with receding 3′ ends, there are also short RNA fragments arising from intermittent nicks by the RNase H domain of polymerase. Therefore, we used RNA probes spanning the HBV genome to map whether these RNA molecules are present in extracellular naked capsids and virions. Five probes that spanned the HBV genome, except for the overlapping region between the 5′ end of pgRNA and the RNA cleavage site (nt 1818 to 1930), were prepared to map the extracellular HBV RNAs from HepAD38 cell culture supernatant ([Fig. 9A](#F9){ref-type="fig"}). Intracellular nucleocapsid-associated HBV RNA from HepAD38 cells was used as a reference. As the probes moved from the 5′ end to 3′ end of pgRNA, especially for probes 1 to 4, RNA bands shifted from a wider range, including both short and long RNA species, to a narrower range, close to full-length pgRNA, with fewer RNA species detected ([Fig. 9A](#F9){ref-type="fig"}, upper, lanes 2, 5, 8, 11, 14, and 17). Similarly, with the probes moving from the 5′ end to the 3′ end of pgRNA, a stronger intensity band representing extracellular HBV RNAs detected by each probe, especially for probes 1 to 4, was also shifting toward a longer RNA migration region ([Fig. 9A](#F9){ref-type="fig"}, upper, lanes 3, 6, 9, 12, 15, and 18). It should be noted that the shifting pattern was more apparent when RNAs were detected with probes 1 to 4 but not with probe 5. It is possible that the reverse transcription speed is relatively quicker in the initial step (from the 3′ end of pgRNA, which overlaps the probe 5 sequence), and as a result, fewer pgRNA fragments will harbor RNA sequence for probe 5. Also, a short RNA species from either intracellular nucelocapsids or naked capsids and virions migrated faster than 0.7 knt and could be detected by all probes ([Fig. 9A](#F9){ref-type="fig"}, upper, lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18). These RNA molecules likely represent the pgRNA fragments that have been hydrolyzed by the RNase H domain of viral polymerase (including the 3′ epsilon loop RNA cleaved by polymerase in the reverse transcription step) ([@B24]). Collectively, as predicted, longer extracellular HBV RNA species that migrated slower and closer to the position of pgRNA had longer 3′ ends, the shorter viral RNA molecules that migrated faster had relatively shorter 3′ ends, and the RNA species detected by all probes may represent products of pgRNA hydrolysis. ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.](zjv0241840640009){#F9} These results were further confirmed by employing a 3′ rapid amplification of cDNA ends (RACE) method. Various 3′ ends spanning the HBV genome were identified ([Fig. 9B](#F9){ref-type="fig"}), validating the presence of 3′ receding RNA and the heterogeneous nature of extracellular HBV RNAs. EPA treatment clearly demonstrated that extracellular HBV RNAs could be used as templates for DNA synthesis, and the presence of 3′ receding-end pgRNA fragments further confirmed not only the existence but also the use of such molecules as templates for viral DNA synthesis. Therefore, just like the viral RNA counterpart within intracellular NCs, extracellular HBV RNA molecules represent the RNA molecules generated in the process of viral DNA replication. ETV reduces viral DNA level but increases extracellular HBV RNA level in naked capsids and virions *in vitro*. {#s2.6} -------------------------------------------------------------------------------------------------------------- Entecavir (ETV), widely used in anti-HBV therapy, is a deoxyguanosine analog that blocks the reverse transcription and plus-strand DNA synthesis steps in the HBV DNA replication process ([@B45][@B46][@B47]). Treatment of CHB patients with nucleos(t)ide analogs (NAs), including entecavir, efficiently reduces the level of serum viral DNA but at the same time increases circulating HBV RNA levels ([@B28], [@B48][@B49][@B52]). We examined the effect of entecavir on the levels of both intracellular and extracellular viral nucleic acids in HepAD38 cell culture. Total viral RNA level remained unchanged or marginally increased upon entecavir treatment ([Fig. 10A](#F10){ref-type="fig"}), and the intracellular capsid-associated viral RNA level was increased ([Fig. 10B](#F10){ref-type="fig"}, upper). In contrast and as expected, the intracellular capsid-associated viral DNA level was decreased ([Fig. 10B](#F10){ref-type="fig"}, lower). Similarly, extracellular viral DNA synthesis was significantly inhibited, while viral RNA was increased ([Fig. 10C](#F10){ref-type="fig"} and [D](#F10){ref-type="fig"}). Quantitative results showed that entecavir suppressed extracellular viral DNA to about one-tenth but at the same time increased viral RNA by about twofold the level for the untreated group ([Fig. 10E](#F10){ref-type="fig"}). {ref-type="fig"}. Viral particles in each fraction were separated by native agarose gel electrophoresis, followed by immunoblotting with anti-HBcAg antibody. Viral DNA and RNA were extracted and subjected to Southern or Northern blot analyses.](zjv0241840640010){#F10} Since viral DNA and RNA were enclosed in both naked capsids and virions, CsCl density gradient was used to separate these particles and to further study the antiviral effect of entecavir. As shown in [Fig. 10](#F10){ref-type="fig"}, DNA-containing naked capsids were detected in fractions 6 to 11 and virions in fractions 15 to 24 ([Fig. 10F](#F10){ref-type="fig"}). Entecavir effectively reduced viral DNA ([Fig. 10G](#F10){ref-type="fig"}, fractions 6 to 10 and 15 to 17; this was also seen in a longer exposure of [Fig. 10G](#F10){ref-type="fig"} \[not shown\]) but increased viral RNA content mainly in naked capsids ([Fig. 10H](#F10){ref-type="fig"}, fractions 6 to 9). Moreover, the increase in RNA content within naked capsids led to an increased density of naked capsids ([Fig. 10F](#F10){ref-type="fig"}, fractions 6 and 11, lower, versus fractions 6 and 11, upper). Interestingly, entecavir seemed to reduce HBcAg signal within virions (i.e., empty virions) ([Fig. 10F](#F10){ref-type="fig"}, fractions 15 to 21, upper, versus fractions 15 to 21, lower) while increasing the egress of naked capsids from HepAD38 cells (data not shown). DISCUSSION {#s3} ========== The RNA molecules in either intracellular NCs or extracellular virions were reported more than three decades ago ([@B5], [@B41], [@B42]), and naked capsids were shown to carry pgRNA *in vitro* ([@B9], [@B11]). Recently, it was suggested that the extracellular or circulating HBV RNA could serve as a surrogate marker to evaluate the endpoint of hepatitis B treatment ([@B27], [@B30], [@B48][@B49][@B53]). With this in mind and to facilitate its application as a novel biomarker for viral persistence, we studied the origin and characteristics of extracellular HBV RNA. In the present study, we extensively characterized extracellular HBV RNAs and demonstrated that extracellular HBV RNAs were mainly enclosed in naked capsids rather than complete virions in supernatant of HepAD38 cells ([Fig. 1B](#F1){ref-type="fig"} and [2F](#F2){ref-type="fig"}). These RNAs were of heterogeneous lengths, ranging from full-length pgRNA (3.5 knt) to a few hundred nucleotides. Furthermore, circulating HBV RNAs, also heterogeneous in length, were detected in blood of hepatitis B patients ([Fig. 3D](#F3){ref-type="fig"} and [7C](#F7){ref-type="fig"} and [D](#F7){ref-type="fig"}). Interestingly, the detection of HBV RNAs coincided with the presence of immature HBV DNA ([Fig. 3D](#F3){ref-type="fig"} and [E](#F3){ref-type="fig"}). Isopycnic CsCl gradient ultracentrifugation of RNA positive serum samples exhibited a broad range of distribution of immature HBV DNA, which contrasted with the results obtained in HepAD38 cells ([Fig. 2B](#F2){ref-type="fig"} versus [@B4]B and E, [@B7]A). For the first time, we provided convincing evidence that unenveloped capsids containing the full spectrum of HBV replication intermediates and RNA species that are heterogeneous in length could be detected in the circulation of chronic hepatitis B patients. In view of our results and literature reports ([@B2], [@B21][@B22][@B24]), the presence of extracellular HBV RNAs could easily be interpreted in the context of the HBV DNA replication model ([Fig. 11A](#F11){ref-type="fig"}). Since naked capsids contain viral DNA at all maturation levels, they will also carry HBV RNA molecules originating from pgRNA, including full-length pgRNA prior to minus-strand DNA synthesis, pgRNA with 3′ receding ends, and the pgRNA hydrolysis fragments. On the other hand, virions that contain only mature forms of viral DNA species would likely bear only the hydrolyzed short RNA fragments remaining in the nucleocapsid ([@B43]). Likewise, the HBV RNA species found in CACs are longer than those in virions in sera of hepatitis B patients ([Fig. 7D](#F7){ref-type="fig"}, lower, versus C, lower). In line with this reasoning, treatment of HepAD38 cells with entecavir reduced viral DNA in naked capsids and virions ([Fig. 10C](#F10){ref-type="fig"}, [E](#F10){ref-type="fig"}, and [G](#F10){ref-type="fig"}) but at the same time increased HBV RNA content within naked capsids ([Fig. 10H](#F10){ref-type="fig"}). This may be a result of the stalled activity of viral RT with concomitant shutdown of RNA hydrolysis ([@B46], [@B54]). {#F11} Contrary to a recent report claiming that the pgRNA-containing NCs can be enveloped and secreted as virions ([@B27]), we clearly demonstrated that secreted naked capsids carry the majority of HBV RNAs ([Fig. 1B](#F1){ref-type="fig"} and [2F](#F2){ref-type="fig"}) and that virion-associated RNAs are approximately several hundred nucleotides long ([Fig. 1B](#F1){ref-type="fig"} and [7C](#F7){ref-type="fig"}). Our results are consistent with earlier reports demonstrating that only mature nucleocapsids with RC/DSL DNA are enveloped and secreted as virions ([@B6][@B7][@B8], [@B11]), and under this condition, virions carry only short RNase H-cleaved pgRNA ([Fig. 11A](#F11){ref-type="fig"}, step 3). In this research, we were unable to separate hydrolyzed pgRNA fragments from the pgRNA and pgRNA with 3′ receding ends. Thus, the length of these RNA molecules could not be determined. The existence of hydrolyzed RNA products during reverse transcription is not without precedent. In some retroviruses, DNA polymerization speed of RT is greater than the RNA hydrolysis speed of RNase H, thus hydrolysis of RNA template is often incomplete ([@B55], [@B56]). For example, RT of avian myeloblastosis virus (AMV) hydrolyzed RNA template once for every 100 to 200 nt, while cleavage frequency of RTs of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) appeared to be around 100 to 120 nt ([@B57]). Moreover, RNA secondary structures, such as hairpins, may stall the RT activity promoting RNase H cleavage, producing shorter RNA fragments ([@B55], [@B56]). Furthermore, the cleaved RNA fragments may not disassociate but anneal to the nascent minus-strand DNA forming the DNA-RNA hybrids until they are displaced by plus-strand DNA synthesis ([@B55], [@B56]). Although similar studies on HBV replication were hampered by lack of fully functional viral polymerase *in vitro* ([@B58][@B59][@B61]), the reported presence of DNA-RNA hybrid molecules clearly indicated the existence of degraded pgRNA fragments that still annealed to the minus-strand DNA ([@B5], [@B41], [@B42], [@B62]). Consistent with a previous study, our results also showed that at least part of the SS DNA is associated with RNA molecules as the DNA-RNA hybrid molecules, as detected by either RNase H digestion or the cesium sulfate density gradient separation method ([@B5] and data not shown). Given the fact that HBV RNA and immature HBV DNA are packaged in naked capsids ([Fig. 1B](#F1){ref-type="fig"} and [2B](#F2){ref-type="fig"} and [F](#F2){ref-type="fig"}) ([@B11]), we postulated that, in CHB patients, unenveloped capsids are released into circulation, where they rapidly form CACs with anti-HBcAg antibodies ([Fig. 11B](#F11){ref-type="fig"}) ([@B25], [@B33], [@B34]). In support of this notion, we showed that protein A/G agarose beads could specifically pull down particles with mature and immature HBV DNA from sera of CHB patients, implying the involvement of antibody. Addition of anti-HBcAg antibody to HepAD38 cell culture supernatant led to a shift of naked capsids' buoyant density to lower-density regions ([Fig. 4C](#F4){ref-type="fig"} and [D](#F4){ref-type="fig"}), a pattern similar to that obtained in HBV RNA-positive serum samples ([Fig. 4B](#F4){ref-type="fig"} and [E](#F4){ref-type="fig"}, and [7A](#F7){ref-type="fig"}). These particles exhibited heterogeneous electrophoretic behavior that differed from that of particles in HepAD38 culture supernatant, suggesting that they are not individual naked capsid particles but are associated with antibodies and have nonuniform compositions ([Fig. 6](#F6){ref-type="fig"} and [11B](#F11){ref-type="fig"}) ([@B36][@B37][@B38]). In CHB patients, the high titers of anti-HBcAg antibodies, which exceed 10,000 IU/ml, preclude circulation of antibody-unbound naked capsids ([@B63]). Indeed, the excessive amounts of anti-HBcAg antibodies present in the plasma sample of patient 0 were able to pull down naked capsids from the culture supernatant of HepAD38 cells (not shown). We have demonstrated the presence of circulating CACs as the new form of naked capsids in CHB patients. It is known that naked capsid particles can be secreted either by the natural endosomal sorting complex required for transport (ESCRT) pathway ([@B15][@B16][@B17]) or possibly by cell lysis consequent to liver inflammation. Our preliminary clinical data (not shown) are in agreement with a recent study showing an association of circulating HBV RNA with serum ALT level ([@B64]). However, this connection can be interpreted in a different manner, as the capsid-antibody complexes might constitute a danger signal triggering inflammation. Interestingly, the release of naked capsids seems to be an intrinsic property of hepadnaviruses preserved through evolution. Recent studies by Lauber et al. provided evidence as to the ancient origin of HBV descending from nonenveloped progenitors in fish, with their envelope protein gene emerging *de novo* much later ([@B65]). Thus, it is reasonable to propose that the active release of HBV capsid particles should be deemed a natural course of viral egress. Apart from HBV particles, it was also reported that exosomes could serve as HBV DNA or RNA carriers ([@B29], [@B66], [@B67]). However, HBV DNA and RNA was detected in naked capsids or CACs and virion fractions rather than in lower-density regions where membrane vesicles like HBsAg particles (density of 1.18 g/cm^3^) and exosomes (density of 1.10 to 1.18 g/cm^3^) would likely settle ([@B2], [@B27], [@B48], [@B68], [@B69]) ([Fig. 1](#F1){ref-type="fig"} and [7B](#F7){ref-type="fig"}). As a result, it is not likely that exosomes serve as the main vehicles carrying HBV DNA or RNA molecules. Numerous pieces of data showed that HBV spliced RNAs also represent a species of extracellular HBV RNAs ([@B28], [@B70], [@B71]). However, in HepAD38 cells, as most of the RNAs are transcribed from the integrated HBV sequence other than the cccDNA template, pgRNA packaged into nucleocapsids is the predominant RNA molecule ([Fig. 9A](#F9){ref-type="fig"} and [10D](#F10){ref-type="fig"}), and viral DNA derived from pgRNA is the dominant DNA form ([Fig. 2D](#F2){ref-type="fig"} and [E](#F2){ref-type="fig"} and data not shown). For the same reason, it would be difficult for us to estimate the amount of spliced HBV RNAs in clinical samples. Although we could not completely rule out the possibility that HBV RNAs are released into blood circulation by association with other vehicles or other pathways, it is possible that the spliced HBV RNAs also egress out of cells in naked capsids and virions like the pgRNA. In summary, we demonstrated that extracellular HBV RNA molecules are pgRNA and degraded pgRNA fragments generated in the HBV replication process *in vitro*. Moreover, we provided evidence that HBV RNAs exist in the form of CACs in hepatitis B patients' blood circulation. More importantly, the association of circulating HBV RNAs with CACs or virions in hepatitis B patients suggests their pgRNA origin. Hence, our results here suggest the circulating HBV RNAs within CACs or virions in hepatitis B patients could serve as novel biomarkers to assess efficacy of treatment. MATERIALS AND METHODS {#s4} ===================== Cell culture. {#s4.1} ------------- HepAD38 cells that replicate HBV in a tetracycline-repressible manner were maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum, and doxycycline was withdrawn to allow virus replication ([@B31]). Patients and samples. {#s4.2} --------------------- Serum samples from 45 chronic hepatitis B patients with HBV DNA titer higher than 10^7^ IU per ml were randomly selected. Detailed medical records of these patients are included in [Table 1](#T1){ref-type="table"}. ###### Medical records of hepatitis B patients used in this research[^*a*^](#T1F1){ref-type="table-fn"} Patient no. Sex Age (yr) HBV DNA titer (IU/ml) HBeAg (IU/ml) HBsAg (IU/ml) ALT (IU/liter) SS DNA result ------------- ----- ---------- ----------------------- --------------- --------------- ---------------- --------------- 0 NA NA 2.67E + 06 4,932 396 \+ 1 M 54 1.24E + 07 25 \>250 69 \+ 2 F 32 1.20E + 07 1,067 69,384 38 \+ 3 F 21 1.36E + 07 1,712 200 149 \+ 4 M 33 \>5.00E + 07 4,812 113,933 133 \+ 5 NA NA 1.25E + 07 3,423 33 − 6 M 26 1.17E + 07 545 2,759 22 − 7 M 36 1.77E + 07 4,332 19,541 136 **+** 8 M 35 \>5.00E + 07 1,199 \>250 104 **+** 9 M 26 2.20E + 07 \>250 143 − 10 M 30 \>5.00E + 07 2 4,265 123 − 11 F 23 \>5.00E + 07 20 5,757 120 **+** 12 M 37 2.07E + 07 2,315 16,128 177 **+** 13 M 28 \>5.00E + 07 3,495 60,676 58 NA 14 F 28 \>5.00E + 07 16,515 89,575 78 \+ 15 M 37 1.62E + 07 574 +, ND 112 \+ 16 M NA \>5.00E + 07 1,601 \>250 22 NA 17 M 15 2.28E + 07 2,038 32,739 180 \+ 18 M 41 2.71E + 07 694 \>250 313 \+ 19 M 34 2.35E + 07 80 32,514 148 \+ 20 F 44 \>5.00E + 07 1,596 4,306 172 − 21 M NA 3.48E + 07 107 \>250 103 \+ 22 NA NA \>5.00E + 07 2024 45,873 147 \+ 23 M 20 1.32E + 07 13,411 12,387 344 \+ 24 M 48 \>5.00E + 07 5,511 76,914 33 − 25 M NA 3.15E + 07 15,984 366 − 26 M 31 4.16E + 07 10,251 50,469 442 \+ 27 M 60 1.35E + 07 749 \>250 105 \+ 28 F 41 \>5.00E + 07 4,173 \>52,000 194 \+ 29 NA NA \>5.00E + 07 4,233 49,125 39 \+ 30 M 29 1.42E + 07 25 5,800 940 \+ 31 M 27 2.34E + 07 1,117 22,412 129 \+ 32 M 37 2.65E + 07 70 109 NA 33 NA NA 2.03E + 07 4,902 111 \+ 34 M 32 \>5.00E + 07 993 43,582 249 \+ 35 NA NA 2.94E + 07 4,641 93,336 12 \+ 36 NA NA \>5.00E + 07 10,956 2,496 108 \+ 37 F 43 \>5.00E + 07 1,021 \>250 74 \+ 38 F 28 \>5.00E + 07 215 446 26 \+ 39 M 31 \>5.00E + 07 +, ND 38,165 194 \+ 40 NA NA \>5.00E + 07 25 \>250 69 \+ 41 M 26 1.52E + 07 +, ND +, ND 95 \+ 42 M 25 \>5.00E + 07 6,300 43,151 373 \+ 43 M 22 \>5.00E + 07 3,844 23,620 329 \+ 44 M 27 1.36E + 07 1,185 11,106 149 \+ 45 M 44 1.28E + 07 663 23,330 425 − 46 F 29 \>5.00E + 07 +, ND +, ND 667 \+ NA, not available; ND, not determined; M, male; F, female; sera from patients 0 and 46 were not included with sera from other patients for SS DNA screening. Plasma sample was the plasma exchange product obtained from an HBeAg-negative hepatitis B patient (patient 0) (HBV genotype B with A1762T, G1764A, and G1869A mutation) who died of fulminant hepatitis as a consequence of reactivation of hepatitis B ([Table 1](#T1){ref-type="table"}). Ethics statement. {#s4.3} ----------------- All samples from HBV-infected patients used in this study were from an already-existing collection supported by the National Science and Technology Major Project of China (grant no. 2012ZX10002007-001). Written informed consent was received from participants prior to collection of clinical samples ([@B72]). Samples used in this study were anonymized before analysis. This study was conducted in compliance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of the Shanghai Public Health Clinical Center. Preparation of viral particles. {#s4.4} ------------------------------- HepAD38 cell culture supernatant was mixed with polyethylene glycol 8000 (PEG 8000) to a final concentration of 10% (wt/vol) and incubated on ice for at least 1 h, followed by centrifugation at 925 × *g* for 20 min. Pellets were suspended in TNE buffer (10 mM Tris-Cl \[pH 7.5\], 100 mM NaCl, and 1 mM EDTA) containing 0.05% β-mercaptoethanol to 1/150 of the original volume, followed by a brief sonication ([@B73], [@B74]). Alternatively, viral particles in HepAD38 cell culture supernatant were concentrated 50- to 100-fold by ultrafiltration using a filter unit (Amicon Ultra-15, 100 kDa). Plasma samples from patient 0 were centrifuged through a 20% (wt/vol) sucrose cushion at 26,000 rpm for 16 h in an SW 32 Ti rotor (Beckman), and pellets were resuspended in 1/200 the original volume of TNE buffer and sonicated briefly ([@B75]). Samples prepared using methods described above were either used immediately or aliquoted and stored at −80°C for later use. Sucrose density gradient centrifugation. {#s4.5} ---------------------------------------- HepAD38 cells culture supernatant concentrated by PEG 8000 was centrifugation at 500 × *g* for 5 min to remove aggregates. Ten percent, 20%, 30%, 40%, 50%, and 60% (wt/wt) sucrose gradients were prepared by underlayering and incubated for 4 h in a water bath at room temperature to allow gradient to become continuous. Five hundred microliters of concentrated sample was layered over the gradient and centrifuged at 34,100 rpm for 14 h at 4°C in a Beckman SW 41 Ti rotor. Fractions were collected from top to bottom, and the density of each fraction was determined by refractometry ([@B10]). Fractions containing viral particles were subjected to native agarose gel analysis, and HBsAg level was determined by enzyme-linked immunosorbent assay (ELISA) (Shanghai Kehua). Cesium chloride density gradient centrifugation. {#s4.6} ------------------------------------------------ HepAD38 cell culture supernatant (1.5 ml), concentrated by ultrafiltration, or serum samples from chronic hepatitis patients diluted with TNE buffer to 1.5 ml were mixed with equal volumes of 37% (wt/wt) CsCl-TNE buffer (1.377 g/cm^3^) and underlayered with 1.9 ml 34% (wt/wt) CsCl-TNE buffer (1.336 g/cm^3^), followed by centrifugation at 90,000 rpm at 4°C for 12 h (Beckman VTi 90 rotor) ([@B8]). The tube was punctured from the bottom, and every six to seven drops were collected as one fraction. Densities of separated fractions were determined by weighing. Each fraction was then desalted against TNE buffer by ultrafiltration, followed by native agarose gel separation or nucleic acid extraction. All of the CsCl density gradient centrifugation experiments were carried out at 90,000 rpm at 4°C for 12 h in a Beckman VTi 90 rotor. Native agarose gel analysis of viral particles and capsid-associated DNA. {#s4.7} ------------------------------------------------------------------------- Viral particles were resolved by native agarose gel (0.8% agarose gel prepared in Tris-acetate-EDTA \[TAE\] buffer) electrophoresis and transferred in TNE buffer to either a nitrocellulose membrane (0.45 μM) for detection of viral antigens with specific antibodies or a nylon membrane for Southern blot analysis of viral DNA. For viral antigens detection, the membrane was first fixed as previously described ([@B74]), and HBV core antigen was detected by anti-HBcAg antibody (Dako) (1:5,000). The same membrane then was soaked in stripping buffer (200 mM glycine, 0.1% SDS, 1% Tween 20, pH 2.2) and reprobed with anti-HBsAg antibody (Shanghai Kehua) (1:5,000). For Southern blot analysis of viral DNA, the membrane was dipped in denaturing buffer (0.5 N NaOH, 1.5 M NaCl) for 10 s and immediately neutralized in 1 M Tris-Cl (pH 7.0)--1.5 M NaCl for 1 min, followed by hybridization with minus-strand-specific riboprobe ([@B76]). Viral nucleic acid extraction, separation, and detection. {#s4.8} --------------------------------------------------------- **(I) Nucleic acid extraction.** To extract total viral nucleic acids (DNA and RNA), the SDS-proteinase K method was used ([@B77]). Samples were digested in solution containing 1% SDS, 15 mM EDTA, and 0.5 mg/ml proteinase K at 37°C for 15 min. The digestion mixture was extracted twice with phenol and once with chloroform. Aqueous supernatant were added with 1/9 volume of 3 M sodium acetate (pH 5.2) and 40 μg of glycogen and precipitated with 2.5 volumes of ethanol. In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer's instructions (Thermo Fisher Scientific). To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl \[pH 7.8\], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl~2~ (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase. Viral nucleic acids were released by addition of SDS to a final concentration of 1% and extracted as described above. **II. Separation. (i) TAE agarose gel.** Viral DNA was resolved by electrophoresis through a 1.5% agarose gel in 1× TAE buffer, followed by denaturation in 0.5 M NaOH--1.5 M NaCl for 30 min and neutralization with 1 M Tris-Cl (pH 7.0)--1.5 M NaCl for 30 min. **(ii) Alkaline agarose gel.** Viral DNA was denatured with a 0.1 volume of solution containing 0.5 M NaOH and 10 mM EDTA and resolved overnight at 1.5 V/cm in a 1.5% agarose gel with 50 mM NaOH and 1 mM EDTA. After electrophoresis, the gel was neutralized with 1 M Tris-Cl (pH 7.0)--1.5 M NaCl for 45 min ([@B78]). **(iii) Formaldehyde-MOPS agarose gel.** Viral RNA was obtained by treatment of total nucleic acids extracted using the above-described SDS-proteinase K method with RNase free DNase I (Roche) for 15 min at 37°C. The reaction was stopped by addition of equal amounts of 2× RNA loading buffer (95% formamide, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol FF, and 1 mM EDTA) supplemented with extra EDTA (20 mM), followed by denaturing at 65°C for 10 min. Viral RNA extracted by TRIzol LS reagent was mixed with 2× RNA loading buffer and denatured. Denatured mixtures were separated by electrophoresis through a 1.5% agarose gel containing 2% (vol/vol) formaldehyde solution (37%) and 1× MOPS (3-\[N-morpholino\]propanesulfonic acid) buffer. The gels described above were balanced in 20× SSC solution (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 20 min, and viral nucleic acids were transferred onto nylon membranes overnight with 20× SSC buffer. III. Detection. {#s4.10} --------------- Digoxigenin-labeled riboprobes used for detection of HBV DNA and RNA were prepared by *in vitro* transcription of a pcDNA3 plasmid that harbors 3,215 bp of HBV DNA (nt 1814 to 1813) by following the vendor's suggestions (12039672910; Roche). Riboprobes used for HBV RNA mapping were transcribed from DNA templates generated by PCR by incorporating T7 promoter into the 5′ end of reversed primers ([Fig. 9A](#F9){ref-type="fig"}). Hybridization was carried out at 50°C overnight, followed by two 5-min washes in 2× SSC--0.1% SDS at room temperature and two additional 15-min washes in 0.1× SSC--0.1% SDS at 50°C. The membrane was sequentially incubated with blocking buffer and anti-digoxigenin-AP Fab fragment (Roche) at 20°C for 30 min. Subsequently, the membrane was washed twice with washing buffer (100 mM maleic acid, 150 mM NaCl, and 0.3% Tween 20, pH 7.5) for 15 min, followed by detection with diluted CDP-Star substrate (ABI) and exposure to X-ray film. Protein A/G agarose bead pulldown of antibody-antigen complexes. {#s4.11} ---------------------------------------------------------------- Two hundred microliters of serum sample was first mixed with 300 μl of TNE buffer, and then 15 μl of protein A/G agarose bead slurry (Santa Cruz) was added to the mixture, followed by incubation overnight at 4°C in a sample mixer. Subsequently, protein A/G agarose beads were washed three times with TNE buffer, and viral DNA in input serum samples (40 μl) and agarose bead pulldown mixtures were extracted and subjected to Southern blot analysis. EM. {#s4.12} --- Serum samples from patients 11, 17, 21 22, 23, 27, 28, 30, and 41 were pooled (200 μl each) and mixed with 200 μl of 20% (wt/wt) sucrose. Serum mixtures were centrifuged through 2 ml of 20% (wt/wt) and 2 ml of 45% (wt/wt) (1.203 g/cm^3^) sucrose cushions at 34,100 rpm for 8 h at 4°C in an SW 41 Ti rotor (Beckman) to remove HBsAg particles. Supernatants were decanted and the centrifugation tube was placed upside down for 20 s, and residue sucrose was wiped out. One milliliter of phosphate buffer (10 mM Na~2~HPO~4~, 1.8 mM KH~2~PO~4~, and no NaCl) (pH 7.4) was added, and the bottom of the tube was gently washed without disturbing the pellet. A volume of 11.5 ml of phosphate buffer then was added into the tube and centrifuged again at 34,100 rpm for 3 h at 4°C. The pellet was resuspended in a drop of distilled water and dropped onto a carbon-coated copper grid, followed by staining with 2% phosphotungstic acid (pH 6.1) and examining in an electron microscope (Philip CM120) ([@B13], [@B79]). Viral DNA and RNA quantification. {#s4.13} --------------------------------- Viral DNA used for quantification was extracted using the SDS-proteinase K method as described above. Viral RNAs were extracted by TRIzol LS reagent, and DNase I was used to remove the remaining DNA, followed by phenol and chloroform extraction and ethanol precipitation. Reverse transcription was carried out using Maxima H minus reverse transcriptase (Thermo Fisher Scientific) with a specific primer (AGATCTTCKGCGACGCGG \[nt 2428 to 2411\]) according to the manufacturer's guidelines, except the 65°C incubation step was skipped to avoid RNA degradation. To ensure removal of viral DNA signal (below 1,000 copies per reaction), a mock reverse transcription, without addition of reverse transcriptase, was carried out. Quantitative real-time PCR (qPCR) was carried out using Thunderbird SYBR qPCR mix (Toyobo) in a StepOnePlus real-time PCR system (ABI). Primer pairs (F, GGRGTGTGGATTCGCAC \[nt 2267 to 2283\]; R, AGATCTTCKGCGACGCGG \[nt 2428 to 2411\]) conserved among all HBV genotypes and close to the 5′ end but not in the overlap region between the start codon and the poly(A) cleavage site of pgRNA were chosen. The cycling conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 5 s, 57°C for 20 s, and 72°C for 30 s. DNA fragment containing 3,215 bp of full-length HBV DNA was released from plasmid by restriction enzymes, and DNA standards were prepared according to a formula in which 1 pg of DNA equals 3 × 10^5^ copies of viral DNA. EPA. {#s4.14} ---- HepAD38 cell culture supernatant or plasma from patient 0 were concentrated as described above and mixed with equal volumes of 2× EPA buffer (100 mM Tris-Cl, pH 7.5, 80 mM NH~4~Cl, 40 mM MgCl~2~, 2% NP-40, and 0.6% β-mercaptoethanol) with or without dNTPs (dATP, dCTP, dGTP, and dTTP, each at a final concentration of 100 μM) ([@B80]). The reaction mixtures were incubated at 37°C for 2 h and stopped by addition of EDTA to a final concentration of 15 mM. 3′ RACE. {#s4.15} -------- Concentrated HepAD38 cell culture supernatant (by ultrafiltration) was digested with MNase in the presence of NP-40 (final concentration, 1%) for 30 min at 37°C. EDTA (final concentration, 15 mM) and proteinase K (final concentration, 0.5 mg/ml) were then added and incubated for another 30 min at 37°C. Viral nucleic acids were extracted with TRIzol LS reagent followed by DNase I treatment to remove residue viral DNA. Poly(A) tails were added to the 3′ end of HBV RNA by E. coli poly(A) polymerase (NEB). The preincubation step at 65°C for 5 min was omitted to reduce potential RNA degradation, and reverse transcription was carried out with Maxima H minus reverse transcriptase (Thermo Scientific) using an oligo-dT(29)-SfiI(A)-adaptor primer (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′) in reverse transcription buffer \[1× RT buffer, RNase inhibitor, 1 M betanine, 0.5 mM each dNTP, and 5 μM of oligo-dT(29)-SfiI(A)-adaptor primer\] at 50°C for 90 min, followed by heating at 85°C for 5 min and treatment with RNase H at 37°C for 15 min. PCR amplification of cDNA fragments was then performed with 5′ HBV-specific primers \[the same sequences of forward primers used for riboprobe preparation ([Fig. 9A](#F9){ref-type="fig"}), except each primer containing a flanking sequence plus a SfiI(B) site (5′-AGTGATGGCCGAGGCGGCC-3′)\] and 3′ adaptor primer (5′-AAGCAGTGGTATCAACGCAGAGTG-3′). The reaction was carried out with PrimeSTAR HS DNA polymerase (TaKaRa) at 95°C for 5 min, followed by 5 cycles of 98°C for 5 s, 50°C for 10 s, and 72°C for 210 s, 35 cycles of 98°C for 5 s, 55°C for 10 s, and 72°C for 210 s, and a final extension step at 72°C for 10 min. PCR amplicons were digested with SfiI enzyme and cloned into pV1-Blasticidin vector (kind gift from Zhigang Yi, Shanghai Medical College, Fudan University). Positive clones were identified by sequencing, and only clones with 3′ poly(dA) sequence were considered authentic viral RNA 3′ ends. We thank Zhuying Chen and Xiurong Peng for handling serum samples and compiling the clinical data used in this research. This research was supported by the National Natural Science Foundation of China (NSFC) (81671998, 91542207), National Key Research and Development Program (2016YFC0100604), National Science and Technology Major Project of China (2017ZX10302201001005), Shanghai Science and Technology Commission (16411960100), and Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-07-E00057). [^1]: **Citation** Bai L, Zhang X, Kozlowski M, Li W, Wu M, Liu J, Chen L, Zhang J, Huang Y, Yuan Z. 2018. Extracellular hepatitis B virus RNAs are heterogeneous in length and circulate as capsid-antibody complexes in addition to virions in chronic hepatitis B patients. J Virol 92:e00798-18. <https://doi.org/10.1128/JVI.00798-18>.

A&E’s hit reality TV show was ensnared in controversy after its star’s controversial comments about gays and African-Americans A record-setting reality show appears to be paddling in the wrong direction as its fifth season gets underway. Citing data from Nielsen, Variety reports that just 6.65 million viewers watched Wednesday night’s episode of Duck Dynasty on A&E. That’s down from the 8.5 million who tuned into last week’s season premiere. The ratings are the lowest for an original episode of the show since December 2012. Duck was ensnared in controversy late last year when star Phil Robertson made derogatory remarks about gays and African Americans. The show focuses on a family that made a fortune on duck-hunting accessories. Its fourth-season premiere was the most-watched cable reality TV episode in history, attracting 11.77 million viewers. Last year, it was the second most watched cable TV program overall, after AMC’s Walking Dead. [Variety]

Q: Using M-Test to show you can differentiate term by term. I have the series $\sum_{n=1}^\infty \frac{\lambda^{n-1}n}{n!}=\sum_{n=1}^\infty \frac{d}{d\lambda}\big(\frac{\lambda^n}{n!} \big)$ and I would like it to be $\frac{d}{d\lambda}\big(\sum_{n=1}^\infty \frac{\lambda^n}{n!})$. I'm trying to show that this sequence of functions converges uniformly on $(0,\infty)$ and so I'm trying the M-Test. So I need to find bounds $M_n$ for $\big|\frac{\lambda^n}{n!}\big|$, such that $\sum M_n$ converges. Thanks. This is in order to show that I can actually do the differentiation term by term. A: You deal with a power series with radius of convergence $R=+\infty$ so you can differentiate term by term.

During my pregnancy, I tried to gather as much information on how painful labor might actually be. I would often hear “mine was horrible, but everyone’s pregnancy is different” or “it was the worst pain I’ve ever felt in my life!” I heard many horror stories which often ended with, “well, don’t worry. You’ll forget about the pain as soon as your child is born.” Not the most reassuring for a first-time mother, but something I definitely kept in mind the entire time. I had feared the unknown, but on the other hand, I knew there was no turning back and that my baby was coming one way or another! Two weeks before my due date, I noticed some blood. My water didn’t break and I saw no mucous plug, but it seemed that something was happening earlier than expected. Soon after, at 1 a.m. I woke up from a notably different type of cramping. It began to occur every 5 minutes. It wasn’t that painful (yet), but uncomfortable. I felt as if I had to go diarrhea every five minutes. If this is labor, I could handle it for sure I thought, but I knew this was only the beginning. My husband nervously drove us to the hospital as if the baby would pop out any second. I had to remind him to not worry. Things usually didn’t happen that fast for first-time moms (or at least I hoped it wouldn’t). I had to go by instinct although in the back of my mind, I wasn’t sure what would happen next. We finally got to the birthing center after an hour of driving and the nurses confirmed I wasn’t even dilated. I couldn’t believe it. We were turned away and had to find a hotel because returning home wasn’t an option. It would take two hours just to return again! The diarrhea-like cramps were painful and uncomfortable; I couldn’t sleep. I was bleeding slightly and started to actually have these cramps and stomach aches over a 10 hour period. I started googling my symptoms (never a good thing!) and discovered there are people who have this uncomfortable feeling for days and weeks! “Fake labor” would not be in my cards, I had hoped. Fortunately, I had an appointment with a midwife in the afternoon and was checked again for any cervical changes. I had finally dilated 3 cm and was 90% effaced. What a relief I thought! I welcomed the pain because I wanted things to progress. I couldn’t imagine having diarrhea cramps for weeks. However, 3 cm isn’t enough to be admitted, we were told, so back to the hotel we went. “When your cramps become more regular, every 3 minutes a part, and you become more snippy, check in again” the midwife suggested. In the mean time, I tried to walk around, pausing multiple times to catch my breath. A couple hours later, I was FINALLY admitted. My husband kept asking me questions non-stop about what I wanted, needed, and more. All I could say was “if I need something, I’ll let you know. Thanks.” I literally couldn’t talk. I felt like vomiting and had heart burn for the first time in my life. As my labor progressed, I felt the urge to push before I was even 10 cm dilated. I would have a cramp, then a couple of minutes later, one that made me yell out in pain as it forced my body to push. A gush of blood would come out as this happened and I felt extremely uncomfortable because the pain was in my back and butt! It would take my breath away. However, the pain was still tolerable, believe it or not. I had a volunteer doula come in that night who helped me breathe, rubbed my back, and encouraged me. She helped me be aware of my voice and how I could use it to save energy and get through the pain. Unfortunately, she couldn’t stay the whole night, but the time she spent with me truly made a difference. Even though labor was hard work and painful, the right breathing technique and support helped ease the pain. This is probably the number one thing that helped me get through labor! As I started heading towards my second night of labor, I wondered how much longer I could go on … I questioned if it was even worth it to continue without an epidural? I went into labor without a plan. I wanted to go with the flow and make decisions as they came. I didn’t want to be tied to a bed or deliver on my back or disappointed if my perfect labor didn’t come true, so I left any expectations open. But after my second sleepless night, I started to inquire about pain medications (although deep down inside I knew I could handle more because the pain was still manageable). I was exhausted and sleep would have been nice especially if I didn’t have to feel any pain with an epidural. There were no walking epidurals available though and I didn’t want to take narcotics (which could make me dizzy), so I continued along, breathing away. A bath was an option too and this I requested and wanted. I was so uncomfortable as things progressed. I couldn’t get in the shower to relax my muscles, but somehow a bath sounded soothing and worth the effort. As soon as the bath was ready, however, I suddenly felt a pop down below as if major pressure had been released from my insides. Immediately, there was a shift. The back and butt pressure/pain I felt was no longer there. It was time to push! I knew as soon as I felt it. As the baby descended, I felt the burning sensation of the babies head crowning – a temporary stretching sting. The cramps were still there and I had no control over my own pushing. I let my body do its own work and took the breaks my body provided in between each wave of labor. I was standing up giving birth because I couldn’t get onto the bed as I would have liked and was given a stool to put my right foot onto in order to widen my pelvis. Gravity certainly assisted me. However, I never expected to be standing for 50 minutes! My legs were becoming tired and shaky, but I couldn’t move. My energy was sapped and I regretted not exercising more. Standing up was the most comfortable thing to do though and I listened to my body’s cues. I started to go along with my body’s signals to push, but after a while I felt as if the baby would never come out because things weren’t progressing fast enough. After his head came out, I thought it was all over until I heard my husband say “push, his body is stuck!” I ended up pushing as hard as I could and a gush of fluids came spewing onto the floor. It was the best sense of relief. The midwives held my baby from under me and told me to grab him. He was screaming, kicking, and punching his way into this life. He was so slippery, I was terrified to grab him. I had never held a baby before. He would be my first. I held my son and put him on my chest. I couldn’t stop looking at him in awe. He was so beautiful to me and I felt overwhelmed with love and joy. When the umbilical cord finally stopped pulsating, which happened surprisingly quick, my husband carefully snipped it. At this point, I’m glad my husband didn’t pass out. I always joked that he would get queasy and faint, but my husband did amazing! While holding my son, I had to deliver my placenta which did not hurt at all. In fact, I couldn’t even feel much down below because of the adrenaline pumping throughout my veins. Looking into my son’s eyes and holding him for the first time was the most incredible thing in the world. The pain that I felt earlier in labor vanished and I felt ecstatic to have made it through. It’s true what they say … After your baby is born, you forget the pain of labor and birth. At least most of it. Hello! Hello! It's nice to meet you! I'm Mary. Thank you for stopping by Stirring Up Delight. I hope you'll find some useful tips, recipes, and reviews or maybe a story or two that you might enjoy. Read More… Follow me Subscribe to Blog via Email Enter your email address to subscribe to this blog and receive notifications of new posts by email. Email Address Stirringupdelight.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to amazon.com For those of you who were told you might need an endometrial biopsy, here’s my experience, so you can sleep a little better at night. Although I do not know what yours will be like, I can tell you that not all of them turn out horrific like you might’ve read online. Why You Might […] Kalua pig is a dish from Hawaii that may be intimidating to make if it’s done traditionally, but modern technology has its benefits. You don’t have to roast a pig underground, but instead you can use your slower cooker to make it. How easy is it? Buy some pork butt at the store and toss […] I’m so excited to be planning my niece’s 1st birthday party this fall! For anyone who really knows me, I absolutely love planning. It is one of my obsessions. After making numerous planning mistakes, however, I would like to share with you some tips I’ve learned along the way. If you are planning your child’s […] One of my favorite drinks when I return home is plantation iced tea. Last year, when I spent a week back home in Hawaii, I ended up drinking it as often as I found it on the menu. Now that the heat of summer is here, I’m dreaming of returning home to visit again. I really […]

The Harper government is looking for security experts to help harden federal buildings in the Ottawa area against threats, including possible terror-related attacks. Public Works posted a tender Wednesday asking for professionals to conduct so-called threat risk assessments “to identify employees and assets that need protection, analyze threats against them, assess the current risk, and recommend safeguards.” The review will cover all federally owned or leased buildings in the Ottawa area and may include bridges, dams and other structures, says the document. The posting follows the Oct. 22 shootings at Parliament Hill, which exposed numerous security gaps, as well as the government’s recent claims that Canada is a target for ISIS and related groups based in the Middle East. Grade every deficiency The list of potential risks cited in the material includes terrorism, demonstrations, environmental threats and others, and the tender requires inspections of all elements of buildings, including the siting of washrooms and how the exteriors of buildings are illuminated. The consultants are to grade every deficiency up to the highest level, described as a “condition of vulnerability which exists which may be exploited by opportunistic person(s) or organizations with hostile objectives.” A lone gunman managed to penetrate deep into the Centre Block of the House of Commons on Oct. 22 before being gunned down in a hallway metres from the prime minister and dozens of other politicians. RCMP called in the Ontario Provincial Police to review the security failings exposed by the incident, and their report is expected to be handed down shortly.

Q: Not populating tableview with structure array I need to populate my tableView with an array of a structure. The first property of the structure is the name. This is what I tried... var menuArray:[Restaurant] = [Restaurant]() override func viewDidLoad() { super.viewDidLoad() let shake = Item(name: "Shake", carbs: 20) let fries = Item(name: "Fries", carbs: 30) let beverages = Category(name: "Beverages", items: [shake]) let chips_fries = Category(name: "Chips & Fries", items: [fries]) let desserts = Category(name: "Desserts", items: []) let other = Category(name: "Other Menu Items", items: []) let sandwiches_burgers = Category(name: "Sandwiches & Burgers", items: []) let sides = Category(name: "Sides", items: []) a_w = Restaurant(name: "A&W", categories: [beverages, chips_fries, desserts, other, sandwiches_burgers, sides]) let menuArray = [a_w] } override func tableView(_ tableView: UITableView, cellForRowAt indexPath: IndexPath) -> UITableViewCell { let currentCell = tableView.dequeueReusableCell(withIdentifier: "cell") let currentRestaurant = menuArray[indexPath.row] currentCell?.textLabel!.text = currentRestaurant.name return currentCell! } override func tableView(_ tableView: UITableView, numberOfRowsInSection section: Int) -> Int { return menuArray.count } Why won't it populate my tableView Here is my class also... import Foundation struct Item { let name: String let carbs: Int } struct Category { let name: String let items: [Item] } struct Restaurant { let name: String let categories: [Category] } A: In this line let menuArray = [a_w] you are creating a local variable menuArray which is different from the property with the same name representing the data source array. Omit let menuArray = [a_w] PS: Please use more descriptive variable names than a_w.