Give it to Akshay Kumar to put in a lesson or two in nationalism even in a film about a Sikh regiment fighting on behalf of the British regime. Kesari, his new movie, delves into the 1897 Battle of Saragarhi, a less explored lesson in history. Believe it or not, there were three movies in production at a time about it. Akshay Kumar and his co-producer Karan Johar beat everyone in the race to release Kesari first. But has the race to reach the theatres ensure that Kesari gives this epic moment in history the tribute it deserves? Will Akshay Kumar and Parineeti Chopra's Kesari Score Big at the Box Office? Find Out!
You should know the story by now, since the makers have been screaming from the rooftops about it. Kesari begins in 1897 with Havaldar Ishar Singh (Akshay Kumar), a sepoy leader stationed at Gulistan fort near the Indo-Afghan border. A skirmish with some Afghanis, while trying to save a woman, earns him the ire of his British superiors who transfer Ishar to the nearby fort of Saragarhi.
There, he is given the charge of the 36 Sikh Regiment, that consists of only 20 soldiers apart from him. Before we get to know them really well, the Afghanis led by a scheming maulana, attack their fort. Deciding that they are not going to retreat for the sake of their honour, the 21 Sikhs led by Ishar Singh valiantly fight against the 10,000 odd marauders, and lay their lives down with their heads high.
There is no doubt that the Battle of Saragarhi is a story that deserves to be told. Even thinking about how 21 Sikhs stood against thousands of invaders and manage to stop them from invading is enough to give you goosebumps. The presence of Akshay Kumar helps to draw in the crowds, not to mention the geniality of Karan Johar among the trade pundits. All the makers need to do was make a decent movie.
Here's where I have issues with this retelling of history. Director Anurag Singh, who had made the underrated Raqeeb before shifting to Punjabi cinema, indulges all sorts of melodrama, mush and unwanted distractions to make a very average war drama that deserves better treatment.
The first half, especially, is a downer. Right from the start when Ishar takes on a large group of Afghanis to save a girl's life, you know that there is no place for subtlety here. Kesari then moves on to some very meandering scenes involving Ishar hallucinating about his very alive wife (Parineeti Chopra), and him clashing with his new regiment over discipline and principles. There is nothing new in those sequences that you really need to spend so much time in, with some terrible character development for the supporting cast. Even the scenes between the soldiers feel like a mishmash of a couple of Akshay films, especially Gold, Singh is King and Singh is Bliing.
It is only towards the interval where Kesari manages to spruce up your interest. The second half is somewhat arresting because Kesari finally delves into what you have come for - the fateful battle. There are a few moments of combat that get your attention, particularly the fight near the door and Ishar taking on hordes on invaders all alone. And yet, Kesari doesn't manage to get you emotionally high, because of how less we spend time with the dying heroes.
Save for Ishar, that is. Kesari puts too much focus on its star lead that it is even difficult to distinguish between the rest of his comrades.
There are plenty of influences of other war movies in Kesari, notably Border (another movie that deals with real-life war story with a similar predicament), Tango Charlie and in one weird moment, even World War Z. The action choreography is pretty decent, even with some unnecessary slow-motion shots. But the VFX is unbelievably okayish, and considering that Kesari involves big production houses, this is unforgivable. I was particularly annoyed by a scene near the end, shown gloriously in the teaser of the film, that was ruined by some bad CGI in the movie. Even the shot of stabbings, blood and explosions feel as real as Akshay's distractingly fake beard and moustache. Kesari Star Akshay Kumar Wants To See a Chapter on Battle of Saragarhi in History Textbooks.
The fallings of Kesari deserve to go to the average direction and some really weak writing. The movie seems to be have made with the sensibilities of Punjabi viewers in mind, every nearly every second dialogue talking about the pride of their large pagdi. There are also a couple of dialogues that feels like WhatsApp propaganda (Ishar talks to his friend about how they have always been slaves to Mughals and British). The depiction of the villains, belonging to a particular community, as barbarians with little care for honour and moral values may not be very conducive in the current political environment. The music is okay, but the final song "Teri Mitti" is lovely.
Akshay Kumar's commanding screen presence is what makes us bear through some of the film's weakest scenes. The actor is charismatic and effective, but this is not exactly a role that demands too much from him. Akshay was impressive, though, in the scene that shows Ishar's last stand against the enemies.
Parineeti Chopra's role is more of a cameo and the actress feels perfunctory in the role. The rest of the cast, especially the actors playing the 20 Sikhs in Ishar's regiment, are good.
Kesari works for you as a viewer if you are a fan of Akshay Kumar's brand of nationalism and don't mind a chest-thumping affair. If viewed as a historical war drama, Kesari barely manages to impress, mostly due to some very faulty editing and execution. Watch Kesari for the action-packed decent second half that teaches us a lesson in valour. But only if, you can bear through a very mediocre first half and writing!


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Talking with Al Madrigal about 'The Daily Show' and His New Standup Special
By Erik Voss
Al Madrigal is a correspondent on The Daily Show, a longtime stand-up comedian, and the co-creator of the comedy podcast network All Things Comedy with Bill Burr. As the Daily Show's "Latino correspondent," Madrigal has covered issues like Arizona's anti-immigrant legislation and Puerto Rican statehood, and he explores the subject matter of raising a family in a Mexican neighborhood in his new stand-up special Why Is The Rabbit Crying? – which will air this Friday on Comedy Central. We had a chance to catch up with Madrigal about his special, as well as what it's like to work on The Daily Show during a time of such sobering headlines.
Recently, Jon Stewart was praised for his remarks on the bombings in Boston. Jon's always the first one to remind people that TDS is a comedy show first and foremost. And you're obviously a comedian first and foremost. But with so many people turning to The Daily Show for actual relief and information, have you ever felt so outraged by a topic that overwhelmed you as a comedian and made you feel a sense of duty to expose it?
As the Latino correspondent there's been a couple of stuff I've been able to get in that I've found pretty outrageous. There was a Mexican-American studies ban in Arizona — that actually recently went in front of a higher court and did not get overturned, so it's still in effect — which I feel is crazy. A bunch of Tea Party guys found that that would be a good Tea Party MO to infiltrate low-level politics, so they took over a school board and were instrumental in banning Mexican-American studies. I found that to be one of the most offensive things I've ever come across. And being able to have access to this forum, I've been aggressively pushing as many Latino DREAM-er stories as I possibly can.
I think people at this point are tuning in for Jon's perspective on a lot of that stuff, even though a lot of the material we cover is not laughable. It's pretty upsetting. It's hard to hear day-in, day-out, and not get too discouraged. You know we comb the Internet for articles, and I was reading this scroll of articles, where it was "five dead," or "ten dead" — every single article was people being shot and killed and dying and mayhem.
I feel like it's increasingly the case where Jon has to open the show with some kind of statement like, "I know we do comedy here, but I have to take a moment to address this."
Yeah. It puts everyone in a weird spot, because there are certain times you just don't want to laugh. I got irritated the other day when the Boston bombing happened. I was in the airport at LAX, flying back to do the show — I commute back and forth because my family's in LA. And it's a bloody mess, people are rushing in, 20 minutes after it happened. And I'm at the airport bar, and there's very little conversation going on. Everyone's just staring up at the television, saying "Holy shit, what happened?" And the guy to my left turns to the chick next to him and says, "So, you're traveling light, huh?" And I was just thinking, "Shut the fuck up! How are you even talking right now?"
The same thing happened when I was working a gig in Vegas. I'm not sure if you've talked to anybody about these horrible casino gigs. When you work Harrah's, you get to eat in the cafeteria. So you're sitting there with all of the employees. It was right at the moment when Katrina happened. Guy on rooftop with dog, desperate to be saved. People are going into the Superdome. Disaster's happening. And the security guards to my left — I guess Harrah's had just taken over Caesar's — and one of them says, "So, do you think they're gonna change the color of our uniforms?" Who gives a fuck about your uniforms? People are dying!
I mean, obviously people need to move on, and despite this series of – it feels like monthly – events, people still need to go on stage and do stand up, and do The Daily Show. And it's hard, you know? I find it hard not to get discouraged.
As the show's Latino correspondent, you mentioned that you're uniquely positioned to cover some issues facing Latinos, like the crazy laws in Arizona. On the surface it seems like that's just an extension of the show's general parody of cable news, where ethnic reporters get assigned to stories about people from that ethnic group, but do you feel like you fill a genuine niche on the show, to dig deeper into some of those topics?
Yes, for sure. I'm glad that I have the opportunity to do so. And you know, just the other day, MSNBC had something where they were talking about Latinos in politics and their prevalence in society, and they had three black pundits on there. I mean, you couldn't scrounge up one Latino to talk about this? Yeah, I think it's a cool opportunity to get some of these stories out there.
Which segments have you done that stand out the most to you?
I love the field pieces more than anything. I really enjoy going out and doing the interviews. Some of those I'm really proud of, like the Mexican-American studies one, and the Detroit bridge piece. The show has allowed me to drive tanks, to drive a Chrysler 300 through the streets of Detroit while listening to Eminem. I've been able to do a lot of fun stuff.
You have a lot of experience acting and working in the industry, appearing in shows like Free Agents and Welcome to the Captain. How has working on The Daily Show been different?
It's just not a job like any other. You're doing your acting, you're doing standup, you're a reporter reading a teleprompter. It's a combination of many different skills. It's not like anything else. In the field pieces, you're an interviewer, so you're sort of this journalist with jokes. Oftentimes you'll watch a straight interview that's being done — you know, it's Bryant Gumbel's Real Sports or anything on 60 Minutes — and you think, oh, that's relatively easy. Without the jokes, it doesn't seem as difficult.
You have dual functions as an interviewer: that mock newsman persona, as well as having to actually use some journalistic skills, like following up on details that might come up during the interviews. How did you go about developing those skills and that persona?
The only way you can do it is on the job, to just get out there and do those interviews. Sitting across from someone and having fun with them can pretty difficult, especially when they aren't aware of what's funny about the situation. So it's a difficult gig. There's nothing like it, and the only way to get good at it is to go out there and do it. John Oliver has been really helpful with me, coaching me along.
And if you look at those field pieces, they're way different from when Carell was on, or when Colbert was on. It's evolved into, you know, "What are we saying here?" There's much more meaning involved as we move forward.
What do you think about John Oliver guest hosting the show while Stewart is gone over the summer?
I think it's awesome. It's an incredible opportunity for him. He's a writer and a correspondent, and he works so hard. He really understands our department and the field department. He's great. We're lucky to have people like Jason Jones, Samantha Bee, Aasif Mandvi, John Oliver — they've been here for so long, and they're great for advice on my end. But yeah, I think the show is in great hands, and it probably means the correspondents are going to see a little more action.
You co-host the podcast Minivan Men. It's interesting to hear comedians using podcasts to connect with fans on a more personal level. There's less pressure to hit with a joke every few seconds, and you have more time to explore topics.
Minivan Men — that one's been tough. I started a network with Bill Burr called All Things Comedy, so I'm familiar with several podcasts, and I talk with the Earwolf people all the time, so I'm familiar with the landscape. The podcast that we do is a little more difficult because of the schedules of everybody involved. Maz Jobrani leads this double life where he's this Persian Seinfeld, an international superstar, and then you'll see him in LA at the Laugh Factory doing 15-minute sets. He can sell out five nights at a 2000-seater in Sweden. And then we also do it with Chris Spencer, who created the show The Real Husbands of Hollywood with Kevin Hart, which is a huge success. And I'm so busy, going back and forth to LA, and doing The Daily Show, and doing stand-up shows. The fact that everyone is so busy is the biggest challenge there.
Since we're three comedians with kids, we talk about that, and the challenges there, and we thought that would be interesting. Other than just, you know, comic interviews, we could talk about parenting and spouse challenges that come up. And it's been interesting to do. It's not for everyone. And been a challenge for me, you know, to be the best father possible considering my schedule. Fitting in the family time is something a lot of parents struggle with, so we talk about all of that shit.
I noticed a lot of your stand-up focuses on your relationship with your family as well. Your special is airing Friday on Comedy Central — Why Is The Rabbit Crying? — which I assume is a reference to your "Cholo soccer dad" joke.
It's talking about everything that happens in my life, and every bit is grounded in reality, so there's not a lot of fiction. And I find myself, you know, a half-Mexican living in a Mexican neighborhood in Los Angeles, surrounded by these characters, raising a family in this weird, multi-ethnic neighborhood. It's a lot of that. There's strip malls, and the neighborhood is kind of boldly blue-tarp, so it's very fruitful for material to live around a bunch of weirdos.
You're constantly doing standup. When you do a show, do you view it as an opportunity to get back to what you truly love, in a "back to the basics" way, or is it something where you're working on other projects like The Daily Show, and you'll get new ideas that you'll be inspired to work back into your act?
I'm not doing as good a job at that as I should. There's a writer here who's a standup, and we were talking about opportunities for material as we're going through it on the show. And again, I feel like I'm so busy, it's just creative mayhem over here — I'm working on All Things Comedy, I'm working on The Daily Show, and my stand-up and acting — there's so much going on, and there's zero method to any of this. I have a great time on stage, and I love interacting with the crowd. My style also is such that I've become this six-minute bit storyteller with a lot of tangents and detail, and those don't come too easy. So in terms of writing, I need to actually sit down to piece together my bits. They start on stage, but I need to make a conscious effort to work everything out. Sometimes it's a little overwhelming. This special, Why Is The Rabbit Crying?, is a combination of a lot of those stories that have been crafted in clubs over a long period of time.
A couple years ago you did an interview with Paul F. Tompkins and Maria Bamford on KCRW's The Business in which you talked about comedians like Paul Reiser, who become TV stars and never do standup again, which was notion you were grappling with. Do you think that era is over? Can comedians afford to think that way nowadays?
I think there will always be people who get a little too busy. I was talking to another comic who had just taped an hour, and he was like, "I'm excited! Here comes the new hour. I get to write a new hour." And that whole thing, with all the shit I have going on right now, it just seems a little bit daunting. So I can totally see how people want to take a break, and how it's difficult to jump back into it.
But at the same time, I would never want to take that much time off. Meeting John Oliver has been really good for me — just watching how he goes about doing standup and balancing out family time. At this point, with me being away from my family, it's very difficult for me to fly back to LA and then say, "All right, see you guys later, I'm going to the Comedy Store." So I did my club time, which used to be constant, easily 10 sets a week at the Improv, the Laugh Factory, and the Comedy Store. It's been tapered way down. I think as people get busier, yeah, it's harder to stay in the clubs, to stay fresh. But there is a bit of a balance that I'm watching John Oliver employ, going out to clubs constantly and working on material as much as you can. But I can totally see how people like Mike Binder and Paul Reiser and standups over time sort of dropped out.
I get it. I get both sides. I think I may have mentioned this on the show with Maria and Paul, that standup will always be there for me. You know, I got into this to do standup, and everything that has come from it has been great, but I really do enjoy being on stage. I've never been as polished as I've wanted to be, and I think that's what I really enjoy — just messing around with material and never doing the same bit twice.
Erik Voss is a writer and performer living in Los Angeles. He hosts the Evil Blond Kid podcast and performs improv on the Harold team The Cartel at the iO West Theater.
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The European Physical Journal E
January 2001 , Volume 4, Issue 2, pp 183–192 | Cite as
Surface and flexoelectric polarization in a nematic liquid crystal 5CB
L.M. Blinov
M.I. Barnik
H. Ohoka
M. Ozaki
N.M. Shtykov
K. Yoshino
The temperature dependence of the surface polarization has been measured for both the planar and homeotropic orientation of a nematic liquid crystal at a solid substrate. A conventional liquid crystal 5CB, pure and doped with an azo-dye, was used in cells with controlled asymmetry for light absorption. The measurements have been made by a pyroelectric technique using short pulses of a YAG laser to create a temperature increment. The latter, in turn, was measured independently by a novel time-resolved "optical thermometer" technique monitoring temperature-dependent birefringence by a He- Ne laser beam. In accordance with the symmetry of the order parameter, the surface polarization has different sign for the two orientations, its magnitude ranges from -4 to +2pC/m. The same technique has been used for the measurement of the flexoelectric polarization in hybrid cells. The sum of the flexoelectric coefficients is e1 + e3 = - 13pC/m at 25°C.
PACS. 61.30.-v Liquid crystals – 77.70.+a Pyroelectric and electrocaloric effects – 77.84.Nh Liquids, emulsions, and suspensions; liquid crystals
Received 28 February 2000 and Received in final form 5 September 2000
© EDP Sciences, Springer-Verlag, Società Italiana di Fisica 2001
1.Institute of Crystallography, Russian Academy of Sciences, 117333, Leninsky prosp. 59, Moscow, RussiaRU
2.Department of Electronic Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, JapanJP
Blinov, L., Barnik, M., Ohoka, H. et al. Eur. Phys. J. E (2001) 4: 183. https://doi.org/10.1007/s101890170127
DOI https://doi.org/10.1007/s101890170127
Publisher Name EDP Sciences, Springer-Verlag, Società Italiana di Fisica


The Association for Children's and Youth Literature is an umbrella organization founded in 1955. Currently, it has 53 organizations from the field of libraries, research, education and publishing as members, as well as 240 individual members. It is supported financially by the German Federal Ministry for Families, Senior Citizens, Women and Youth. As a nationwide lobby it promotes children's literature and the literary education of young people.
The business office is located in Munich and provides support to the honorary board members who are elected every three years by the member assembly. Since its founding, the Association for Children's and Youth Literature has been the German Section of the International Board on Books for Young People (IBBY).
The Association for Children's and Youth Literature is responsible for awarding the German Children's Literature Award (Deutscher Jugendliteraturpreis), which is a state-sponsored prize for works of fiction. It has been awarded annually since 1956 by a jury of literature specialists and critics in four categories: picture book, children's book, young adult book and non-fiction. In addition, since 2003 an independent young adult jury gives its own award. Each category carries a value of 10,000 Euro. Furthermore, two special awards are presented alternately to German authors, illustrators or translators. This special award for lifetime achievement is set at 12,000 Euro. The newly created special award for new talents carries a value of 10,000 Euro. All winners are announced in a festive ceremony which takes place at the Frankfurt Book Fair in October.


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Farmed Vs Wild
Fish Lookup
SEAFOOD RISK ASSESSMENT
Sustainable seafood is increasing in popularity around the world, yet much of the seafood popular in Asia has not been assessed in terms of its sustainability.
Over time, the Seafood Risk Assessment site aims to gradually fill that gap, by providing sustainability assessments of seafood for the Hong Kong market. Thereby ensuring that consumers have access to the sustainable seafood of their choice.
Wild Captured Assessed Fish (14) Farmed Captured Assessed Fish (9)
The oceans of Asia are home to some of the richest and most diverse fisheries in the world. The region also dominates global aquaculture production. Yet, lack of management of Asia's marine resources, is causing overfishing and habitat destruction from northern China south to Indonesia and beyond.
Overfishing is widespread and a threat to both businesses and seafood-dependent communities.
The Seafood Risk Assessment (SRA) is an open source and affordable approach for Hong-Kong based companies sourcing seafood globally to understand key risk areas through quick risk assessments.
Members of the seafood industry can use these risk assessments to guide sourcing decisions and begin the journey of initiating and supporting improvements with suppliers, source fisheries and farms.
The fish risk assessments are derived from a risk assessment method developed by fisheries and aquaculture experts, specifically for the SE Asia region. Key source locations were assessed based on advice from members of the Hong Kong Sustainable Seafood Coalition.
There is a risk assessment method for both wild fish and farmed fish as the types of risk differ between the two.
The risk assessments are undertaken at a country (or regional) level for a given species, and scored as high, medium, or low risk.
If a source country is flagged as high / medium risk you should exercise extra due diligence when sourcing, meaning asking questions of your supplier. These Supplier Questions are found in each of the risk assessment profiles.
You can use the search feature to find the species of interest or the Fish Assessment Lookup tab in the website page menu.
For some farmed species, there is insufficient publicly available information to undertake an informed risk assessment at a country level. In such cases a general narrative has been provided based on limited information. Going forward, it is envisaged that these information gaps will be addressed in consultation with in-country experts.
If you are a HKSSC member these risk assessments are aligned with the Sourcing Code of Conduct. See the Code of Conduct in English and Traditional Chinese and the Guidance Document in English and Traditional Chinese.
Per capita, Hong Kong ranks second in terms of seafood consumption in Asia, and is the world's eighth-largest seafood consumer. Hong Kong residents consume 71.8 kg of seafood on average per year, which is more than three times higher than the global average.
Source: AFCD, 2019
In 2019, the fishing industry of Hong Kong produced an estimated 123 000 tonnes of fisheries produce valued at about US$360 million, contributing to the steady supply of marine fish to local consumers. The industry consists of some 5 030 fishing vessels and about 10 100 local fishermen.
Source: FoodExport.org
Ninety-five percent of the seafood consumed in Hong Kong is imported (around US$3 billion) and much of this is consumed in Hong Kong's vast foodservice industry, consisting of more than 200 hotels and 15,000 restaurants.
Among consumers purchasing seafood for at-home consumption, 47% will make purchases at a wet market, 30% will make purchases at fish shops and 23% at supermarkets.
About 30% of Hong Kong's imported seafood products are re-exported. The largest re-export market is China followed by the U.S., Macau and Vietnam.
THE HONG KONG SUSTAINABLE SEAFOOD COALITION (HKSSC)
The HKSSC was established in 2018 to advance the sustainable seafood market in Hong Kong, by promoting responsible purchasing and consumption. If you are working in the seafood industry why not consider joining, and work in partnership with other leading Hong Kong seafood businesses to improve the sustainability of marine, fresh water and farmed fish resources in the region?
Join HKSSC
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Escape from the hustle of New York City and head north to Niagara Falls, USA. Visit iconic Niagara Falls on the American side and take a ride to the edge of the falls on the famous Maid of the Mist boat. Enjoy time for sightseeing, shopping, dining and touring around the greater Niagara Falls region. In the evening, take in a spectacular fireworks display as it lights up the sky above the falls. Round-trip airfare, ground transportation, accommodation and tickets for the Maid of the Mist are included.
Niagara Falls Day Trip by Air including Maid of the Mist and Cave of the Winds
Traveling to Niagara Falls from New York City by air is the quickest and most efficient way to visit the iconic waterfalls in just one day. All transportation is included as you make your way to top Niagara attractions, like the Maid of the Mist (seasonal), Cave of the Winds, or Niagara Falls Observation Tower. A flexible itinerary lets you decide what you most want to see.
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Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy.
Funding: This work was supported by Cooperative Agreement no. DAMD17-93-V-3004 between the U.S. Army Medical Research and Material Command; the Henry M. Jackson Foundation for the Advancement of Military Medicine, working with the Division of AIDS, National Institute of Allergy and Infectious Diseases National Institutes of Health; the Bill & Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD), grant number 38619. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The HIV-1 envelope (Env) glycoproteins are produced as a 160 kDa polyprotein that is subsequently processed to yield virion-associated, trimeric complexes of non-covalently associated gp120-gp41 heterodimers [1,2]. The surface subunit, gp120, is responsible for the specific binding of virions to target cells; gp41, the transmembrane subunit, mediates fusion of viral and cellular membranes . Neutralizing antibodies (NAbs) can block virus entry by binding Env and inhibiting attachment or conformational changes required for fusion [4–7]. Env structural studies have primarily focused on gp120 and the extracellular domain of gp41 (i.e. ectodomain), where the small panel of known broadly NAbs bind ; the cytoplasmic tail (CT) of gp41 (endodomain) is considered to be entirely contained inside the virion [9,10] and consequently is thought not to be targeted by the host immune response. However, studies have suggested a more complex role of the gp41 endodomain [11,12], showing neutralization of HIV-1 by Abs directed to an epitope in CT of gp41 [13–15]. Mutations in the CT have been shown to affect the conformation of gp120 ectodomain [16–18], and more recently, Durham et al suggested that the CT regulates the conformation of Env at the cell surface and control epitope exposure through T cell virological synapses . These results emphasize the importance of the gp41 endodomain and the rationale to express and study the complete gp160 derived from primary isolates.
These issues intersect practically in the viral reagents that are commonly used in HIV-1 neutralization assays, which form an important component in the evaluation of candidate HIV vaccines. In support of HIV vaccine development, intensive collaborative efforts have now yielded reference panels of HIV-1 Envs representative of the worldwide viral genetic diversity and have standardized neutralization assay systems [20–24]. Most of these data measured neutralization of Env-pseudotyped viruses in TZM-bl cells [21–24]. More recently, a new HIV-1 Env expression vector has been developed, allowing for multiple rounds of replication and easy read-out in HIV-1 natural target cells [25–27]. However both assays relied solely on T-cell line-adapted B strains to express HIV-1 Env and generate functional HIV-1 viral stocks [28,29]. As execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), new HIV-1 reagents representing the full genetic diversity of non-B HIV-1 subtypes are needed to support vaccine development targeting non-B HIV strains.
To bridge this gap, we have developed new IMC.LucR HIV-1 constructs derived from native subtype C and CRF01_AE strains. We further modified these vectors to express subtype C and CRF01_AE full-length gp160 Env in subtype-matched and -mismatched HIV-1 backbones. Replication of these constructs was tested in different cell types. Monoclonal antibody (mAb) and plasma-mediated neutralization sensitivity of various HIV-1 Envs were compared when expressed in these two non-subtype B HIV-1 backbones. We observed variation in neutralization sensitivity when Env is expressed in subtype-matched vs –mismatched HIV backbone. These findings indicate that non-env genes may play an important role in subtype-specific neutralization sensitivity. Our results suggest that a library of HIV-1 backbones representative of viral diversity should be generated and their usage for analysis of immune responses carefully weighed.
For clarity, all the primers used in this study are listed in Table S1.
To insert the cassette LucR.T2A from pNL-LucR.T2A  into AE.CM235.2  and C.ETH2220.11B , we chose unique enzyme restriction sites encompassing the entire env and nef gene sequences; AarI cuts at position 5062 in pol/vif, and BglI, which cuts in CM235.2 at position 9628, and in ETH2220.11B at the position 9630 (Figure 1A). AarI is present in 93% of the HIV-1 sequences that we screened in the Los Alamos database (www.hiv.lanl.gov), making it an ideal molecular tool for the cloning of LucR.T2A into all HIV-1 subtypes.
Schematic representation of the construction of (A) primary isolate-derived IMC.LucR and (B) chimeric IMC.LucR-Env.
We first amplified three overlapping fragments, obtained using the following cycles: 1 cycle at 94C for 2 min, 27 cycles at 94C for 15s, 55C for 30s, 68C for 5 min, and a final extension step at 68C for 15 min (Platinum Taq, Invitrogen). Env of CM235.2 and ETH2220.11B were amplified using the forward primer polk3 and the reverse primer LucNheIRV, the cassette LucR.T2A in NL-LucR.T2A  was amplified with LucNheIFW and LucXbaIRV, and finally nef was amplified with LucXbaIFW and pblueKS-r8. PCR products obtained were gel purified with S.N.A.P. UV-free gel purification kit (Invitrogen) and finally quantified on 0.8% EtBr-agarose gel using Gene Ruler DNA ladder mix (Fermentas).
The three resulting fragments were then subjected to the double-step DNA amplification approach described in Shevchuck et al. . The first step was without primers (1 cycle at 94C for 60s, and 12 cycles at 94C for 15s, 68C for 10 min), and the second step was with the primers AarIENVFW and BglIENVRV (1 cycle at 94C for 60s, and 30 cycles at 94C for 15s, 68C for 11min with 5s increment and a final cycle of 72C for 15 min) (Advantage GC, Clontech).
In order to identify the enzyme restriction sites to generate chimeric IMCs for heterologous Env expression, HIV-1 sequences from all subtypes retrieved from Los Alamos database were examined (www.hiv.lanl.gov). We found two enzyme restriction sites BsiWI and MluI, which are mostly absent in HIV-1; respectively, 99.1% and 98.7% of the HIV-1 sequences do not display these restriction sites. Hence, the BsiWI site was inserted at position 6202 in HXB2, in a conserved sequence (90% conserved in the CRF01_AE and subtype C vpu sequences) of the cytoplasmic region of vpu, by replacing a Glutamate with a Valine. MluI site was added after the env stop codon (Figure 1B). Briefly, using QuickChange II site-directed Mutagenesis kit (Agilent Technologies), CM235.LucR was amplified first with the complementary primer pair A/E_BsiWIvpuFW and A/E_BsiWIvpuRV, and ETH2220.LucR DNA with C-BsiWIvpuRV and C_BsiWIvpuFW using the following cycles: 1 cycle at 95C for 30s and 18 cycles at 95C for 30s, 55C for 60s, 68C for 15min. The MluI restriction site was then introduced following the same protocol into BsiWI clones using the following complementary primer pairs: for CM235.LucR, MluI_EFW and MluI_ERV and for ETH2220.LucR, MluI_CFW and MluI_CRV.
The insertion of the restriction site BsiWI in the vpu gene generated an amino acid change (position 6202 in HXB2). To confirm the functionality of our chimeric IMC.LucR with the modified-vpu, we titered transfection-derived viruses with and without the amino acid change in vpu in TZM-bl cells, and observed no differences (Figure S1).
Purified inserts and backbones CM235.2 and ETH2220.11B were digested with the required restriction enzymes; AarI, BglI, MluI, and BsiWI (Fermentas/Thermoscientific). The clones with the right insert were determined by visualizing on 0.8% EtBr agarose gel and further confirmed by sequencing using two primers situated in the envelope to differentiate between parental plasmids and clones carrying the right insert. The clones obtained were then screened for infectivity in TZM-bl cells. The infectious ones were selected and purified via PureYield Plasmid Maxiprep System (Promega), and sequenced to confirm that the whole env gene was fully inserted.
To produce IMC stocks, a 75 cm2 flask was seeded at a density of 5x106 293T/17 cells one-day prior to transfection. 16 g proviral DNA and 72 l of FuGENE 6 transfection reagent (Roche) were used according to the manufacturer's protocol. Viral supernatants were harvested 48-60 hrs post-transfection, filtered through 0.2 m pore size filters, aliquoted into 0.5 ml or 1 ml and stored at -150C. The main reason for the extensive usage of some subtype B IMCs such as pNL4-3 or pSG3 as HIV-1 backbone is their robust infectivity due to laboratory adaptation. The two non-B IMCs that we are describing here were isolated with limited "ex vivo" cell culture and exhibit modest infectivity in some cases, but we believe these are more physiologically relevant reagents. In order to overcome this issue, when needed, virus stocks were concentrated after the filtration step using Amicon Ultra-15 Centrifugal Filter Units with Ultracel-100 membrane (Millipore) according to the manufacturer's protocol. An aliquot before centrifugation was always kept to measure the titer increase obtained with this procedure.
Pseudovirus stocks were produced similarly through co-transfecting 8 g Env expression plasmid and 8 g Env-deficient HIV-1 backbone vector (pSG3env) [7,30]. Primary isolates were produced by propagation in 5.0x106 PBMC. Culture supernatants were harvested 7 days post-infection and stored as 1ml-aliquots at -150C.
Relative viral infectivity was assessed for each virus stock by viral titration (endpoint dilution assay) to determine TCID50 (50% tissue culture infectious dose) for each virus in the different cell types, removing the need to standardize viral input according to p24 content. We use a cut-off value of 3-times over background (uninfected cells) relative light units (RLU) for Firefly Luciferase (FF) as described elsewhere. For Renilla Luciferase (LucR), we determine the cut-off as being 3x[(average of negative wells)+3SD]. In the TZM-bl reporter cell line , 96-well black plates were seeded at a density of 2x104 cells per well and infected using duplicate or triplicate wells with 4-fold virus dilutions, in the presence of DEAE-Dextran (20 g/ml, Sigma) . After a 48-hour incubation, FF activity was measured using the Bright-Glo Luciferase Assay System for pseudoviruses and IMC, and Dual-Glo Luciferase Assay System for IMC.LucR (Promega); FF and LucR activity can be both measured simultaneously in the same infected cells using the Dual-Glo Luciferase Assay System (Promega).
IMC.LucR infectivity was also tested on A3R5.7 cells and PBMC. 96-well black plates were seeded at a cell density of 1x105 with (A3R5.7 cells) and without (PBMC) the addition of of 25 g/ml DEAE-Dextran. The same virus dilutions were used on all cell types and LucR activity was quantified 94 hours post-infection (Renilla-Glo Luciferase Assay System). RLU were collected on a Victor X light Luminescence counter (Perkin-Elmer) by using an exposure time of 0.1 s/well.
Inhibition of HIV-1 infection with different mAbs and sera was analyzed in TZM-bl cells and PBMC. TZM-bl neutralization assays were performed as previously described . Neutralization was measured as a reduction in FF RLU in the presence of serial antibody/serum dilutions. PBMC neutralization assays were performed essentially as previously described [25,26]. Neutralization was measured as the reduction in LucR RLU in the presence of serial antibody/serum dilutions. For both assay platforms, viral input used was normalized to a dilution for each virus expected to produce a level of RLU at least 10-times above cut off value (as described above). The reciprocal titers were derived through averaging values from two independent assays for which the IC50 values were within 3-fold range. The following reagents were tested: 4E10, 2G12, B12, 2F5 (Polymun), sCD4 (Progenics Pharmaceuticals), subtypes B, CRF01_AE and C pooled sera/plasmas (previously described ), AIP3441 and AIP3254 (individual CRF01_AE HIV+ plasma samples from Thailand, Dr. Nicos Karasavvas), 1026, 1027 and 1028 (individual subtype B HIV+ plasma samples from the US collected under the RV149 IRB approved protocol).
HIV-1 negative PBMC were collected by leukapheresis from a single donor under the Walter Reed Army Institute of Research IRB approved clinical study RV229C (WRAIR#1386) protocol. Plasma samples 1026, 1027 and 1028 were collected under the Walter Reed Army Institute of Research IRB approved clinical study RV149 (WRAIR#1011) protocol. Written consent was obtained under a procedure approved by WRAIR institutional review board (IRB).
The two native non-subtype B IMC.LucR were generated as described in Figure 1A; the cassette LucR.T2A expressed in the proviral vector NL-LucR.T2A  was successfully inserted into the CRF01_AE IMC, CM235.2  and the subtype C IMC, ETH2220.11B  generating the CRF01_AE and subtype C IMC.LucR viruses, CM235.LucR and ETH2220.LucR, respectively. Next, we engineered unique restrictions sites into these constructs to allow for exchange of different heterologous full-length HIV-1 env into either CRF01_AE or subtype C genetic backgrounds (Figure 1B). For this study, we focused our attention on the cloning of subtypes C and CRF01_AE envs, from both acute and chronic stages of HIV-1 infection (as described in Table 1); however, this cloning strategy can be used to express Envs from any subtype.
Table 1. List of subtype C and CRF01_AE chimeric IMC.LucR.
The functionality of our various IMC.LucR constructs was first tested in TZM-bl cells. This cell line had been engineered to contain a tat-inducible firefly (FF) luciferase reporter gene to measure HIV-1 infection . In the same well that contains infected-cells, we could simultaneously monitor both cell-encoded FF and virus-encoded LucR luciferase activities as shown in Figure 2A. For both CM235.LucR and ETH2220.LucR, the plots of FF and LucR RLUs over virus input dilutions had nearly identical linear range and slope (Figure 2A) and gave an equivalent titer (104.2 TCID50 for CM235.LucR and ETH2220.LucR). We evaluated the titer of these two newly constructed IMC.LucR along with the four different viral forms expressing CM235.2 and ETH2220.11B Envs. The uncloned primary isolates, pseudoviruses, IMC, and IMC.LucR were tittered on TZM-bl cells. In general, both CM235.LucR and ETH2220.LucR transfection-derived viral stocks were infectious with somewhat lower titers than the other viral forms (Figure 2B and 2C). To compare infectivity, we first determined the p24 concentration of each virus stock, then titered the stocks using a standardized p24 input (Figure S2). All HIV molecular clone-based stocks generated by 293T/17 cell transient transfection showed similar results with no infection observed at a p24 concentration ≤ 100 pg/ml. Inversely, both PBMC-derived biological isolates were still infectious at a p24 concentration of 2 pg/ml. Louder et al. previously reported that molecularly cloned viral stocks exhibit far fewer env trimers per virion when produced by transfection of 293T/17 cells rather than infection of PBMCs . A difference in the number of Env molecules expressed at the surface of the virions may partially explain the higher infectivity obtained with biological isolates.
Figure 2. Viral titrations in TZM-bl cells.
Titrations of IMC.LucR only, CM235.LucR and ETH2220.LucR (A), of CM235.2 Env-based viruses (B) and of ETH2220.11B Env-based viruses (C). All titrations were performed in duplicate, in a 4-fold dilution format. Firefly luciferase acitivity (RLU FF, bold lines) was measured 48 hours later. For IMC.LucR only (A), Renilla luciferase activity (RLU LucR, dashed lines) was also measured simultaneously in the same wells. The different viral forms expressing Env of CM235.2 (B) and ETH2220.11B (C) were compared: virus stocks of pseudovirus (diamond), biological isolate (inverted triangle), parental IMC (triangle) and IMC.LucR (circle). Horizontal solid and dashed lines indicate the cut-off values for FF and LucR, respectively.
The chimeric IMC.LucR-Env constructs (Table 1) were titered on TZM-bl cells and ranged in titer from 102.2 to 105 TCID50. In order to clone the entire env DNA sequences (including signal peptide and entire gp41 endodomain) we have generated unique chimeric Tat and Rev proteins that could have an impact on viral replication. We significantly improved low-titer virus stocks through concentration by filtration; as an example, Figures 3A and 3B show the titers of chimeric CM235.LucR-620345 and CM235.LucR-644039 on TZM-bl cells, before and after concentration. For CM235.LucR-620345 (Figure 3A), the titer increased from 102.5 to 103.4 TCID50, and for CM235.LucR-644039 (Figure 3B), from 104 to 104.8 TCID50. Similar titer values were determined using FF and LucR readouts.
Figure 3. Titrations in TZMbl cells of chimeric IMC.LucR before and after concentration.
We measured CM235.LucR-620345 (A) and CM235.LucR-644039 (B) concentrated (open symbols) and original (plain symbols) virus stocks. Titrations were performed in duplicate, in a 4-fold dilution format, in TZM-bl cells. Firefly luciferase acitivity (RLU FF, bold lines) and Renilla luciferase activity (RLU LucR, dashed lines) were measured simultaneously in the same wells, 48 hours later. Horizontal solid and dashed lines indicate the cut-off values for FF and LucR, respectively.
The ability of CRF01_AE and subtype C IMC.LucR viruses to infect different target cells was assessed using the LucR output (as measured by RLU). We compared infectivity of CM235.LucR (Figure 4A) and ETH2220.LucR (Figure 4B) using TZM-bl cells, a CD4+T-cell line (A3R5) , and PBMC; both viral constructs infected all target cell types and exhibited relatively comparable titers in the different target cells. However, ETH2220.LucR showed lower infectivity in CD4+T cells (A3R5 and PBMC) as compared with epithelial cell line TZMbl (Figure 4B). This difference may be explained in part by the difference in CCR5 and/or CD4 level of expression on the surface of the engineered TZM-bl cells and by Env specificity and the resulting receptor-Env interactions. The high level of CCR5 expressed in the TZM-bl cell line  when compared to the two other cell types, may not reflect accurately ETH2220 entry efficiency. We confirmed productive infection and de novo replication of our constructs in infected PBMC, using Zidovudine (AZT), which inhibits the reverse transcriptase and prevents HIV-1 replication, (data not shown).
Figure 4. Titrations of CM235.LucR and ETH2220.LucR in different target cells.
We also assessed infectivity of CM235.LucR (A) and ETH2220.LucR (B), in the cell lines TZMbl (two independent experiments) and A3R5 (two independent experiments), and in PBMC (three donors). RLU LucR was measured 72 hours after infection (dashed lines). Horizontal lines represent RLU cut off for LucR (dashed) activity.
We then confirmed that the insertion of the cassette LucR did not modify the neutralization sensitivity of our parental AE- and C-IMC constructs when compared to the other infectious viral forms (IMC-LucR, biological isolate and pseudovirus) expressing CM235.2 (Figure 5A) and ETH2220.11B (Figure 5B) Envs. Virus stocks were compared using the TZM-bl neutralization assay (with firefly luciferase readout) and a panel of polyclonal sera. We found that the NAb titers of CM235.LucR and ETH2220.LucR were similar to parental IMCs and pseudoviruses with the range in ID50 values not exceeding 3-fold, (average of 2.5-fold and 2.6-fold, respectively for CM235.LucR and 1.7-fold and 1.7-fold, respectively for ETH2220.LucR). CM235 and ETH2220 primary virus isolates were poorly neutralized by most sera.
Figure 5. Neutralization profiles CM235.2 and ETH2220.11B Env-based viruses.
Inhibition of infection using HIV-1+ sera and multiple viral forms (pseudovirus, biological isolate, original IMC and IMC.LucR) of CM235.2 (A) and ETH2220.11B (B) in the TZM-bl assay, using the cell-line encoded firefly luciferase reporter endpoint. Inhibition of infection using HIV-1+ sera and CM235.LucR (C) and ETH2220.LucR (D) in the TZM-bl and PBMC assay with three different donor PBMC as assay targets, using the IMC-encoded Renilla luciferase reporter endpoint. Values are the reciprocal sera dilution at which RLU was reduced by 50% compared to the level in virus control wells. Horizontal lines represent the threshold of detection (1:40 sera dilution); values at or under the line indicate 50% inhibition was not reached.
To investigate if the HIV backbone can influence neutralization sensitivity, we first looked at the neutralization profiles of CRF01_AE CM235.2 and subtype C ETH2220.11B Envs when expressed in their native backbones, CM235.LucR and ETH2220.LucR, or in subtype-mismatched backbones, CM235.LucR-ETH2220 and ETH2220.LucR-CM235 (Table 2). Differences in neutralization sensitivity were observed between the original IMC.LucR and chimeric, subtype-mismatched IMC.LucR-Env, with mAbs as well as with individual HIV+ plasma, with six data pairs showing over 3-fold differences, but only one data pair over a 5-fold difference (using plasma 3254). However, for these chronic Envs, the subtype specificity of certain polyclonal pools could be observed no matter which backbone was used, eg. the AE-pool neutralized the AE-Env more potently than the C-Env, and the B pool neutralized the C Env, while showing no cross-neutralization of the AE Env, confirming previous observations of subtype specificities with these pools .
Table 2. Neutralization profile of parental AE- & C-IMC.LucR and subtype mismatched chimeric IMC.LucR-Env in the TZM-bl assay.
1 Mean inhibitory concentration IC50: no neutralization IC50>25 (μg/ml), poor >15, low to medium IC50 5-15 (μg/ml), and good IC50 0-5 (μg/ml).
To further investigate this phenomenon, we evaluated additional Env/backbone pairs that were subtype-matched or mismatched, by expressing three subtype C and six CRF01_AE Envs into both CRF01_AE and subtype C IMC.LucR backbones (Table 1). Most of these Envs were isolated very early from patients in Fiebeig stage I/II, and all Envs were thus heterologous to their backbones CM235.LucR and ETH2220.LucR.
Neutralization profiles of all IMC.LucR-Env were then determined in TZM-bl cells using 4E10, sCD4 and two individual HIV-positive plasma samples with either low-titer (1026) or high-titer (1028) neutralizing activity (Figure 6A, B, C, and D). Differences in neutralization sensitivity were observed; analysis of this larger data set allowed the effect of backbone on neutralization sensitivity to be visualized. Interestingly, the backbone used to express different Envs seemed to impact sensitivity to inhibition by sCD4 and by the 4E10 NAb. The chimeric IMCs having CM235.LucR or ETH2220.LucR backbones with subtype-mismatched gp160 Envs were more sensitive to inhibition by 4E10 and sCD4. The three subtype C Envs showed greater sensitivity to 4E10 when expressed in the mismatched CRF01_AE backbone (Figure 6A), but the sample size was too small to generate meaningful statistics. The CRF01_AE Envs that were resistant to sCD4-mediated inhibition in CM235.LucR subtype-matched backbone showed a trend toward increased neutralization sensitivity when expressed in the ETH2220.LucR subtype-mismatched backbone (p=0.065).
Figure 6. Neutralization sensitivity of Env expressed in subtype-matched vs mismatched backbones.
Envs from three subtype C and six CRF01_AE isolates were exchanged into both the subtype C ETH2220.LucR and the CRF01_AE CM235.LucR backbones. Most of the Envs we tested were acute, except for 6838v7 and R2184. Neutralization titers for each stock were generated in TZM-bl cells using (A) 4E10 and (B) sCD4 and polyclonal plasma (C) 1026 and (D) 1028. The Y-axis represents the IC50 (A & B) or ID50 (C & D) values for each Env in both backbones. For statistical analysis, we compare, within Env subtype, neutralization sensitivity by reagent between different backbone constructs (Wilcoxon non-parametric test); p-values for C-envs are written in grey and for AE-envs in black. Horizontal lines represent the cut-off: for (A) and (B), values under the cut-off lines (25 ug/ml) represent neutralization sensitivity, when for (C) and (D) values under the cut-off (dilution 1:40) show resistance to neutralization.
Neutralization by more potent plasma (1028) was always greater when the CRF01_AE backbone was used, regardless of Env; this difference was found to be significant for the acute CRF01_AE Envs (p=0.005). However, when using the plasma with low-titer neutralizing activity (1026), greater neutralization was detected when using subtype-matched backbones for both subtype C and CRF01_AE Envs. The NAbs, IgG1b12 and 2F5 were also tested against these viral constructs, but the IMCs were generally resistant to neutralization by these NAbs (as are the matched pseudoviruses), (data not shown).
Our data show that HIV-1 backbones may impact the neutralization sensitivity of IMC expressing various complete Env-exchanged isogenic recombinants used for neutralization assays. This observation is of importance to HIV vaccine development, as vaccine developers currently rely on measures of breadth and potency of NAb titers in human sera, against genetically diverse panels of reference HIV-1 Envs  that have been thus far expressed solely in a subtype B backbone [21–25]. Indeed, such panels have been generated for the pseudovirion TZM-bl assay [21–23], as well as for the newly developed NL-LucR.T2A-Env.ecto approach which can be used in both TZM-bl, non-reporter cell-lines and PBMC-based assays [24,25]. In the NL-LucR.Env.ecto reporter virus approach, the proviral backbone is derived from the laboratory-adapted HIV-1 subtype B strain (NL4-3), generating constructs with all non-env genes and the gp41 endodomain coding sequence of subtype B origin. It has been reported that modifications in the C-terminal tail of gp41, affecting length or amino acid composition, can have marked affects on global Env structure and function, including envelope incorporation, fusogenicity and antibody reactivity [12,16,17]. For example, the mutation of two arginine residues to glutamate in the gp41 endodomain (LLP2) of an HIV-1 provirus substantially reduced Env fusogenicity and altered antibody reactivity and neutralization sensitivity of epitopes located in gp120 and the gp41 ectodomain . Moreover, recent data suggested that CT of gp41 regulates the conformation of Env and epitope exposure at the cell surface , supporting the use of full-length gp160 in neutralization assays.
Positing that the effect of strain or subtype of the backbone should be carefully considered, we generated non-B IMC (IMC.LucR) with Thai CRF01_AE (CM235.2) and Ethiopian subtype C (ETH2220.11B) strains as HIV-1 backbones into which full-length gp160 can be exchanged. We found that the neutralizing titers for these two IMC.LucR were comparable to titers obtained when using other (non-reporter) HIV-1 backbones to express the cognate Env, with the exception that primary isolate titers were typically lower. These differences may be due to the diversity of the HIV-1 genomes present in uncloned viral stocks, or to the sources of virus stock production (293T/17 cell transfection versus PBMC propagation), which are beyond the scope of this study. To create the IMC –LucR constructs for env exchange described here, we generated viral genomes with chimeric tat and rev genes (Figure 1). It is important to note that this type of chimerization occurs naturally in HIV-1 recombinants found within infected patients. An analysis of unique and circulating recombinants within the Los Alamos database revealed that over 40% of recombinant isolates sequenced from patients in both chronic and acute infection contained recombinant tat and rev genes (http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). Thus, the chimeras produced here do not represent a complete deviation from the genetic recombinants that occur in nature.
To explore the impact of non-env genes on Env-mediated neutralization, we exchanged acute subtype C and CRF01_AE gp160 Envs into both parental backbones, CM235.LucR and ETH2220.LucR, and analyzed neutralization profiles of these chimeras in the TZM-bl assay. CRF01_AE Envs, which were resistant to sCD4-mediated inhibition in the CRF01_AE backbone, (CM235.LucR) became sensitive when exchanged into the subtype C backbone, ETH2220.LucR (p=0.065). A similar trend towards increased sensitivity in a subtype mismatched-backbone was observed with 4E10 for both AE and C Envs. Differences observed in 4E10 and sCD4 titers may indicate structural changes in Env imposed by the backbone genes on the membrane proximal external region (MPER) and CD4-binding site of gp120, respectively. Substantial backbone-related differences were also observed in plasma titers (p=0.005 with plasma 1028 for AE Envs) highlighting the importance of the IMC backbone in the analysis of HIV-1 polyclonal sera and thus, vaccinee samples, when using IMC constucts. While our results demonstrate an effect of the HIV-1 backbone on neutralization sensitivity, a greater number of IMCs should be assayed to determine the impact of strain- and subtype- specificity of the backbone on virus immunoreactivity.
Current HIV-1 vaccines include several viral targets, including Gag, Env and Pol, as both DNA and protein immunogens [40–43]. Antibodies directed against non-Env proteins, such as p17 Gag, have previously been shown to inhibit HIV-1 infection by binding directly to HIV-1 or to HIV-infected cells [24,44–46]. The PBMC assay, which includes both HIV-1 target cells (CD4+ T cells) and anti-HIV-1 effector cells, measures several types of antibody mediated viral inhibition such as ADCC and ADCVI, and may therefore detect non-Env-specific antibody activity more sensitively than cell-line based assays . In the search for relevant vaccine-induced antibody responses, constructs containing the entire genome of genetically relevant HIV-1 strains will need to be compared to strains that are chimeric in backbone/envelope in case-control correlates testing in order to determine which vector system tracks immune responses that correlate with protection from acquisition or disease .
Mutation in vpu does not affect infectivity. Infection of TZM-bl cells with (A) CM235.LucR (square) and (B) ETH2220.LucR (circle) that contain (empty symbols) or do not contain (plain symbols) the BsiWI restriction site in the vpu gene. Both viruses were titrated in duplicate, in a 4-fold dilution format and the firefly luciferase activity was measured 48 hours later (RLU FF).
The horizontal bold line represents RLU FF cut off.
p24-standardized titration in TZM-bl cells of CM235.2 Env-based viruses and of ETH2220.11B Env-based viruses. The different viral forms expressing Env of CM235.2 (A) and ETH2220.11B (B) were compared using a standardized p24 virus input: virus stocks of pseudovirus, biological isolate, parental IMC, and IMC.LucR were assessed for p24 concentration (C and D). Starting with a dose of 25 ng/ml of each virus stock, a 4-fold serial dilution was used to infect TZMbl cells in duplicate. Firefly luciferase activity was measured 48 hours later. The horizontal bold line represents RLU FF cut off.
List of primers. The sequences of the forward and reverse primers used to generate the different constructs are listed.
We thank Jenica J. Lee and William Murtaugh for their assistance with cloning, Brittani Barrows, Nickiana Lora and Nichelle Gibbs for their assistance with the neutralization assays, Dr. Gustavo Kijak for helpful discussions, and Michelle Liu for statistical support.
The views and opinions expressed herein are the private opinions of the authors and are not to be considered as official or reflecting the views of the U.S. Army of the U.S. Department of Defense. Use of trade names is for identification only and does not imply endorsement by the U.S. government.
Conceived and designed the experiments: ALC LW ESB FEMcC VRP ST. Performed the experiments: ALC LW MW TT DMP SM RMcL. Analyzed the data: ALC LW ESB VRP. Contributed reagents/materials/analysis tools: TE CO JCK. Wrote the manuscript: ALC LW IH ROC DCM CO JHK VRP ST.
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16. Edwards TG, Wyss S, Reeves JD, Zolla-Pazner S, Hoxie JA et al. (2002) Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein. J Virol 76: 2683-2691. doi:https://doi.org/10.1128/JVI.76.6.2683-2691.2002. PubMed: 11861835.
17. Kalia V, Sarkar S, Gupta P, Montelaro RC (2005) Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41. J Virol 79: 2097-2107. doi:https://doi.org/10.1128/JVI.79.4.2097-2107.2005. PubMed: 15681412.
18. Spies CP, Ritter GD Jr., Mulligan MJ, Compans RW (1994) Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain. J Virol 68: 585-591. PubMed: 8289362.
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21. Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR et al. (2005) Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol 79: 10108-10125. doi:https://doi.org/10.1128/JVI.79.16.10108-10125.2005. PubMed: 16051804.
22. Li M, Salazar-Gonzalez JF, Derdeyn CA, Morris L, Williamson C et al. (2006) Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa. J Virol 80: 11776-11790. doi:https://doi.org/10.1128/JVI.01730-06. PubMed: 16971434.
23. Seaman MS, Janes H, Hawkins N, Grandpre LE, Devoy C et al. (2010) Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J Virol 84: 1439-1452. doi:https://doi.org/10.1128/JVI.02108-09. PubMed: 19939925.
24. Montefiori DC, Karnasuta C, Huang Y, Ahmed H, Gilbert P et al. (2012) Magnitude and Breadth of the Neutralizing Antibody Response in the RV144 and Vax003 HIV-1 Vaccine Efficacy Trials. J Infect Dis 206: 431-441. doi:https://doi.org/10.1093/infdis/jis367. PubMed: 22634875.
25. Edmonds TG, Ding H, Yuan X, Wei Q, Smith KS et al. (2010) Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virology 408: 1-13. doi:https://doi.org/10.1016/j.virol.2010.08.028. PubMed: 20863545.
26. Matyas GR, Wieczorek L, Beck Z, Ochsenbauer-Jambor C, Kappes JC et al. (2009) Neutralizing antibodies induced by liposomal HIV-1 glycoprotein 41 peptide simultaneously bind to both the 2F5 or 4E10 epitope and lipid epitopes. AIDS 23: 2069-2077. doi:https://doi.org/10.1097/QAD.0b013e32832faea5. PubMed: 19710597.
27. Brown BK, Wieczorek L, Kijak G, Lombardi K, Currier J et al. (2012) The role of natural killer (NK) cells and NK cell receptor polymorphisms in the assessment of HIV-1 neutralization. PLOS ONE 7: e29454. doi:https://doi.org/10.1371/journal.pone.0029454. PubMed: 22509241.
28. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R et al. (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59: 284-291. PubMed: 3016298.
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30. Wei X, Decker JM, Liu H, Zhang Z, Arani RB et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46: 1896-1905. doi:https://doi.org/10.1128/AAC.46.6.1896-1905.2002. PubMed: 12019106.
31. Salminen MO, Ehrenberg PK, Mascola JR, Dayhoff DE, Merling R et al. (2000) Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRF01_AE) generated by the polymerase chain reaction. Virology 278: 103-110. doi:https://doi.org/10.1006/viro.2000.0640. PubMed: 11112486.
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33. Shevchuk NA, Bryksin AV, Nusinovich YA, Cabello FC, Sutherland M et al. (2004) Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res 32: e19. doi:https://doi.org/10.1093/nar/gnh014. PubMed: 14739232.
34. Peachman KK, Wieczorek L, Polonis VR, Alving CR, Rao M (2010) The effect of sCD4 on the binding and accessibility of HIV-1 gp41 MPER epitopes to human monoclonal antibodies. Virology 408: 213-223. doi:https://doi.org/10.1016/j.virol.2010.09.029. PubMed: 20961591.
35. Brown BK, Wieczorek L, Sanders-Buell E, Rosa Borges A, Robb ML et al. (2008) Cross-clade neutralization patterns among HIV-1 strains from the six major clades of the pandemic evaluated and compared in two different models. Virology 375: 529-538. doi:https://doi.org/10.1016/j.virol.2008.02.022. PubMed: 18433824.
36. Louder MK, Sambor A, Chertova E, Hunte T, Barrett S et al. (2005) HIV-1 envelope pseudotyped viral vectors and infectious molecular clones expressing the same envelope glycoprotein have a similar neutralization phenotype, but culture in peripheral blood mononuclear cells is associated with decreased neutralization sensitivity. Virology 339: 226-238. doi:https://doi.org/10.1016/j.virol.2005.06.003. PubMed: 16005039.
37. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D (1998) Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol 72: 2855-2864. PubMed: 9525605.
38. Mann AM, Rusert P, Berlinger L, Kuster H, Günthard HF et al. (2009) HIV sensitivity to neutralization is determined by target and virus producer cell properties. AIDS 23: 1659-1667. doi:https://doi.org/10.1097/QAD.0b013e32832e9408. PubMed: 19581791.
39. Rusert P, Mann A, Huber M, von Wyl V, Gunthard HF et al. (2009) Divergent effects of cell environment on HIV entry inhibitor activity. AIDS 23: 1319-1327. doi:https://doi.org/10.1097/QAD.0b013e32832d92c2. PubMed: 19579289.
40. Burgers WA, Shephard E, Monroe JE, Greenhalgh T, Binder A et al. (2008) Construction, characterization, and immunogenicity of a multigene modified vaccinia Ankara (MVA) vaccine based on HIV type 1 subtype C. AIDS Res Hum Retrovir 24: 195-206. doi:https://doi.org/10.1089/aid.2007.0205. PubMed: 18240957.
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And then the whole thing took a huge turn.
"We really don't mention God, or Atheism, or anything like that. We make it as welcoming as possible for all people. We don't talk about faith, because that ends up alienating people, but we also don't talk about not believing in it," said Sanderson Jones, the group's co-founder. Asked what they sing, the other co-founder, Pippa Evans, answered, "The songs that as you wake up in the morning, you'd be happily singing along to…." And the examples they gave were utterly content free – the lyrics didn't seem to matter.
That's when it hit me that there is a significant difference between Unitarian Universalism and the Sunday Assemblies. The movement they presented is based on a model of never challenging anyone. The guiding principal seems to be that no one who comes should ever be made to feel uncomfortable – and to me that just isn't enough. Authentic community is sometimes uncomfortable, and people need to be able to deal with hard questions of meaning. We don't have to believe in God – I don't, at least not by conventional definitions – but we should be able to be challenged, from time to time, in our assumptions.
That seems to be the fundamental difference between the Sunday Assemblies and Unitarian Universalism. Unitarian Universalists embrace diversity, talk about the many things we believe, sometimes challenging each others' assumptions just by doing that, because we recognize that being with people who see things differently than we do helps us grow and deepen as human beings. In the Sunday Assemblies – at least based on the interview – diversity of viewpoints is something that might exist but isn't talked about, assumptions go unchallenged, and everything is kept very, very safe.
Ironically, this is what Unitarian Universalists are often falsely accused of being.
I hope I'm wrong about this, because I would celebrate the emergence of a powerful, culturally relevant new Humanist movement. They certainly are doing good work in the world, and they are as yet very young. But I need a community that will help me make meaning through the tough times of life, that will challenge me to think as well as feel, and that will help me grow as a person. That's why I'm a Unitarian Universalist.
I prefer Unitarian Universalism too, for that sense of challenge. But I can understand that some people might really need a place where faith isn't discussed, just doing good and being part of a caring community. Perhaps they experienced spiritual abuse, or just didn't like the coercion or manipulation of their "home" faith, and so ANY talk of faith is threatening. (There might be UUs who feel that way too.) I'm always glad for any religious movement that inspires people and encourages life-affirming beliefs. It's not a threat to UU. In fact, it's the opposite. If this movement actually becomes very popular, surely the two groups will start to see how much they have in common, and there will be opportunities to share and grow.
One of the conversations I've been having elsewhere brought out the point that the Sunday Assemblies are very young as a movement. There's every possibility – and I hold out hope – that they will deepen over time. It will require getting past the "we never talk about anything that might make someone uncomfortable" stage, though. The world has a great deal to gain if they do.
on January 2, 2014 at 12:59 pm | Reply C.C.
I am late discovering this post, but I am glad that I have, because it offers an analysis of the Sunday Assembly movement that I had not considered. I, too, have been watching them closely, and trying to figure out where the diversion was from UUism. I am among those UU's who is not entirely comfortable with terms like "faith" and "ministry" and even "religion", and "God", and so the SA idea was looking very appealing to me… I just couldn't put my finger on what was missing. I think you've hit the nail on the head, though: I enjoy being a part of a compassionate community which, among its other functions, inspires me to question my ideas and explore my spirituality. I don't know quite what Sunday Assembly would have to offer me, other than not having to deal with those traditionally "religious" words that I dislike so much.
Its like you read my mind! You appear to know so much about this, like you wrote the book on it or something. I think that you could do with a few pics to drive the message home a bit, but other than that, this is an excellent blog. A great read. I'll definitely be back.
I am checking-out The Sunday Asswmbly. UU is deeper? Maybe TSA works for non-elitists – whom I've heard in UU and non-UUs, to be fair.
I've also seen elistists and non-elistists in UUism, but I've also seen both just about everywhere else. Part of what I love so much about the UU congregation I service (The BuxMont Unitarian Universalist Fellowship in Warrington, Pennsylvania) is that we have an honest diversity of theologies and spiritualities – Christians, Humanists, Theists, Atheists, Pagans, Buddhists and far more – and everyone talks to each other, respects the differences, and learns from them.


Care to Care LLC has appointed Leonard A. Farber, MD Senior Medical Director and Section Chief of Radiation Oncology, Care to Care.
Dr. Faber will lead the Radiation Oncology Services Program; with a focus on innovations that foster new ways to approach the complexities of treatment through the use of Care to Care's proprietary evidence based criteria. He will assist Dr. Julian Safir, Care to Care's Chief Medical Officer with health outcomes research as well as clinical integration into other programs including Medical Oncology.
"Dr. Faber is a proven leader and brings a wealth of experience and capabilities in many significant areas of medical management," said Dr. Safir. "By looking at the whole health care picture Care to Care will continue to be a leader in delivering high quality clinical programs that benefit both the payor and the consumer". I am confident Dr. Faber will contribute significantly to our continued accomplishments in the health services arena."
A Board Certified physician in Radiation Oncology Dr. Farber specializes in adult radiation oncology, with particular focus on cancers of the prostate, breast, lung, skin, and gastrointestinal and gynecologic malignancies. He has special interest in High Dose Rate (HDR) brachytherapy for various types of cancers, including breast, skin, and gynecologic malignancies, and intracranial and stereotactic body radiation therapy (SBRT), all in an outpatient setting. He and his center were the first and only facility in Manhattan to introduce a 5-day hypofractionated SBRT program for prostate cancer treatment. He has implemented volumetric arc therapy (VMAT) in the treatment of benign parotid enlargement/hypertrophy, a disfiguring condition affecting a variety of patients, especially within the HIV+ community.
After receiving his B.S. and B.A. at the State University of New York at Binghamton, Dr. Farber pursued his M.D. at the Mt. Sinai School of Medicine in New York. Following an internship in internal medicine at Beth Israel Medical Center in New York, he completed a residency in Radiation Oncology at the Hospital of the University of Pennsylvania in Philadelphia, where he served as Chief Resident. Dr. Farber is a member of the American Society for Therapeutic Radiology and Oncology, the American College of Radiation Oncology, the NY Roentgen Society, the American Brachytherapy Society, the International Society of Gastrointestinal Oncology, the New York Cancer Society, the New York Metropolitan Breast Cancer Group, the New York Head & Neck Society, and several others.
Dr. Farber's has served as principal investigator on many clinical trials and has been on the forefront of the HDR treatment for various types of malignant and benign conditions and SBRT for various malignancies and sites.
Care to Care headquartered in New York has been in operation since 2007. It has been accredited by the Utilization Review Accreditation Committee (URAC) since 2009, having achieved 100% score during both reaccreditations cycles. Care to Care was founded on a commitment to use evidence based criteria sets that were specifically designed for a prior authorization program aimed at achieving effectiveness in reducing waste while requiring the least amount of effort on the part of the requesting providers. No information is required beyond that which will allow us to approve appropriate use. Because the system is entirely our own, we are able to keep it current, and to customize it to each client's particular needs and policies. Care to Care provides utilization and network management to government, commercial and self-funded programs. .www.caretocare.com.


It seems that the bigger you are the more incentive you have to spy on people for fun and profit. I just recently read a news article on how HP sneakily slipped in a spyware program onto recent HP computers under the guise of an update.
However, it wasn't an update at all but a new program altogether and it sent information back to HP on a daily basis. People have given reports that it slowed their computers down as well.
Fortunately, this program can apparently be removed easily but that is not the principle of the thing. I would have thought that HP would have taken a lesson from Sony's debacle but apparently, they thought they could get away with it.
Not that I am surprised. This has been happening with certain operating systems since Windows XP was released. Which incidentally, happened to be the time I decided to no longer use Windows if I could help it. It is also the reason I do not use Gmail or for that matter, any mail service at all. Instead, I run my own mail server. At least then I know that nobody is trawling my email communications for advertising or other reasons.
You might think I am paranoid and may even think that if you are not doing anything wrong then you have nothing to hide. The principle here is not whether I am doing anything wrong or whether I have something to hide or not. The principle here is my basic privacy rights.
In this day and age, any of your actions, any of your movements and anything you do on the internet gives interested people more information to build up a profile on what sort of person you are.
From that profile, they can then target you for advertising, or even know what you are going to do before you even do it. Personally, I do not want people I do not even know, knowing what my likes and dislikes are, what my routines are or my shopping habits.
What are your thoughts on this matter? Tell us and big brother in the comments below.


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Jones Bros' apprentice awarded 'golden ticket' in national awards nomination13th Oct 2016
A young trainee at leading civil engineering firm Jones Bros is in the running for a national apprenticeship award after being presented with a nomination in the form of a 'golden ticket.'
Robert Geraint Fôn Williams, known as Geraint, received the special ticket by the Construction Industry Training Board (CITB) to inform him he had reached the finals of its Welsh Apprentice of the Year Awards 2016.
It comes just months after Geraint, an apprentice plant operator, was named 'Most Promising Apprentice' in the CECA (Constructing Excellence) Wales awards.
The 21-year-old will travel to London later this month for the awards ceremony, which celebrates the achievements of CITB apprentices.
Geraint, 22, who is in the second year of a two-year apprenticeship with Jones Bros, joined the company as an apprentice plant operator in March 2015. He had completed A Levels at his local college and decided to opt for an apprenticeship rather than university.
He applied for the Jones Bros plant operator apprenticeship because he wanted to learn on the job and gain formal qualifications. "Combining a great company with my interest in working on plant showed the potential to be the perfect apprenticeship for me and it is great to be honoured with an award," said Geraint.
Managing director of Jones Bros Civil Engineering UK John Dielhof said: "We are very proud of Geraint's achievements, which are extremely well-deserved.
"It's terrific to be recognised by the CITB as Jones Bros has always placed a lot of emphasis on apprenticeships and training, as well as on-going nurturing and development of our staff."
The awards ceremony will take place on October 27.
Founded in the 1950s and employing almost 350 people, Ruthin-based Jones Bros has grown significantly in the last decade. It is currently working on contracts in various sectors including the construction of waste management facilities, highways, flood and marine defence and renewable energy projects around the UK.


NatWest and RBS will be closing a total of 259 branches all around the country - find out if your branch is affected.
It blamed the decision on the increasing shift to online and mobile banking. Here is a full list of the branches which will close.


Perfectly located at Australia's most easterly point, in an absolute dress circle position, coveted by many but reserved for only a select few, at spectacular world renowned Wategos beach Byron Bay.
53 Brownell Drive, Byron Bay NSW 2481 was last sold in 1998 and 29 other 4 bedroom house in Byron Bay have recently been sold. There are currently 17 properties for sale in Byron Bay.
In this street, the most recent sale of another house with 4 beds, 3 baths, 2 parking spaces was at number 59, which sold almost 5 years ago.


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Home / News / Celebrity
Helen Mirren says it was 'flattering' to be mistaken for Keanu Reeves' new flame Alexandra Grant
Reeves sparked dating rumors after hitting the red carpet at the LACMA Art + Film Gala hand-in-hand with artist Alexandra Grant over the weekend.
By Kunal Dey
Updated On : 01:01 PST, Feb 24, 2020
Tags :Keanu Reeves
Helen Mirren, Keanu Reeves (Source : Getty Images)
While it is now clear that Helen Mirren is not dating Keanu Reeves, it seems she doesn't have a problem with the confusion.
Reeves sparked dating rumors after hitting the red carpet at the LACMA Art + Film Gala hand-in-hand with artist Alexandra Grant over the weekend, Entertainment Tonight reports.
Grant, 46, passingly resembled Mirren after she wore her natural, silver hair tied back in a bun with her bangs down. Following her sensational appearance with Reeves, the internet went wild with the misunderstanding.
Alexandra Grant and Keanu Reeves attend the 2019 LACMA Art + Film Gala Presented By Gucci at LACMA on November 02, 2019 in Los Angeles, California. (Getty Images)
Mirren was attending the premiere of her new movie, 'The Good Liar', in New York City on Wednesday when she spoke to ET and addressed the apparent mixup.
"I saw that," Mirren said, referring to the social media furor that erupted over Reeves' dating rumors. "That was very flattering on me, you know, because she's obviously lovely."
That said, Mirren has been married to director Taylor Hackford for 22 years. While speaking with the outlet, the Oscar winner said she had nothing but love for the Matrix star.
"I do know Keanu very well. He did a film with my husband and he is just the most adorable, lovely person," she said, referring to the 1997 thriller The Devil's Advocate. "So she's a lucky girl and I'm sure that he's a lucky boy."
(L-R) Producer Greg Yolen, Sir Ian McKellen, Dame Helen Mirren, and director Bill Condon attend "The Good Liar" photocall at The Corinthia Hotel on October 30, 2019, in London, England. The Good Liar releases in UK cinemas on 8th November. (Getty Images)
Meanwhile, Reeves and Grant share a long history together. He worked with her on his 2011 adult picture book, 'Ode to Happiness', which features illustrations by the LA-based artist.
The pair collaborated again in 2016 for yet another book, 'Shadows', and started a publishing company together the same year.
Having said that, 'The Good Liar' will be the first film to feature Mirren and McKellen working together on screen. McKellen credited the film's Oscar-winning director, Bill Condon, for bringing them together.
"When Bill Condon asks you to be in the movie you'll do it, even if Helen Mirren isn't in it. But then it turned out she was," McKellen said. "It was a double whammy for me."


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AACD?
Mechanisms of
Cellular Decline
of AACD
The Hallmarks of Aging
Lopez-Otin C, et al. The Hallmarks of Aging. Cell. 2013;153(6):1194-217.
Link to Original Publication: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836174/
myAACD Summary:
Aging is known to be the main factor decreasing the integrity of bodily processes and function, and increasing the threat of disease onset and poor health in older adults. For example, diabetes, heart disease, and cognitive conditions such as Alzheimer's disease manifest from accelerated age-related declines in physiological processes and cellular and metabolic mechanisms.
The nine hallmarks of aging are: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. The study of these mechanisms and how they are interlinked serves to identify promising targets for future interventions to improve health during aging.
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Stamina, sau androceul, este organul bǎrbătesc de reproducere al plantelor fanerogame. Stamina este formată din filament și anteră în care se formează grăuncioarele de polen și diferă dupa formă, mărime și suprafață.

Etimologie
Din latină stāmen, -inis.

Vezi și

 floare
 fruct
 plantă
 polen
 albină

Anatomie vegetală
Morfologia plantelor
Polenizare

redpajama_set_name: RedPajamaWikipedia