Earlier this month, while the nation was asleep, a 15-year-old girl was rewriting record books in a T20 game in St Lucia (West Indies). Rohtak girl Shafali Verma broke a record Sachin Tendulkar set 30 years ago and thereby became the youngest Indian cricketer to score an international half century. Tendulkar achieved the feat when he was 16 years and 214 days old; Shafali was 15 years and 285 days when she scored a blazing 73 off 49 balls.
Her record feat included four sixes and four fours.
India has finally found a suitable partner to open with Smriti Mandhana – the panacea India has so desperately been seeking ahead of the World T20 in Australia early next year.
Shafali, an attacking opener, followed up on her maiden 50 with an unbeaten 69 runs off 35 balls in the next game.
She was eight years old when she began playing cricket. “My brother, Sahil, is a leg-spinner. He and my father would take me to a local ground for nets,” she told HT in an interview in September. “Both would bowl for long hours and I would hit the ball hard. That’s where I learnt this rule – if the ball is there to be hit then it should be hit hard.”
Tendulkar is a big influence in Shafali’s life. Her father, Sanjeev Verma, is a Tendulkar fan but couldn’t follow cricket as a profession because of family pressure. He would often watch vidoes of Tendulkar’s innings with Sahil and Shafali.
Former Ranji Trophy cricketer Ashwini Kumar, another Tendulkar admirer, trained them at Ram Narain Cricket Academy, Rohtak. “Shafali joined my academy when she was 11-and-a-half years old. Compared to other girls of that age and even older ones, she had a strong build. Because of her confidence and her game, I allowed her to practise with the U-16 boys group. She is known to hit sixes in my 75-yard cricket ground,” said Kumar.
Shafali used to write Tendulkar’s initials ‘S T’ on the back cover of her notebooks.
“For Shafali, Sachin Tendulkar is like God. Initially, I didn’t realise that she broke his record. I confirmed it from Shafali the next day over the phone. She was excited and kept talking about the knock,” said the proud father who runs a jewellery shop in Rohtak.
When Tendulkar played his last Ranji Trophy match near Rohtak in 2013, Shafali was there with her father to catch a glimpse of her icon. Seeing the hysteria around Tendulkar in the stadium, her resolve to become a cricketer intensified even more.
When Shafali was nine, she disguised herself as a boy to play cricket. Sahil was supposed to take part in an U-12 cricket tournament but he fell sick. The fact that both the siblings looked alike and had similar hairstyles made Shafali fill in his shoes and she played the entire tournament as Sahil. She played amazing knocks in the 10-overs-a-side matches and even won the Player of the Tournament award.
She started playing for Haryana in 2013 and has represented the state in U-19, U-23 and the senior category. In February, Shafali scored 128 off 56 balls, the third highest score in women’s T20 for Haryana against Nagaland. During the T20 Women’s Challenge in Jaipur held in May, Shafali impressed everyone with her stroke play while batting for the Mithali Raj-led Velocity team. She was included in the Board President’s XI team for a game against South Africa at Surat and also in the Indian T20 team for a series against the Proteas.
By the time the T20 World Cup starts in February 2020, Shafali will be 16 and would have played more cricket. With ace players such as Smriti Mandhana, Jemimah Rodrigues, Shafali Verma and Harmanpreet Kaur in the ranks, India’s chances at the mega- event look bright.


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Scots speedster James Fleming gunning for glory in Las Vegas
The best seven sevens players in Wellington consisted of one Canadian, three Fijians, two South Africans, including the lightning quick Seabelo Senatla, and Scotland’s very own Fleming, who has travelled a long way since making his initial mark at Perthshire Rugby Club.
Fleming filled his boots in the tournament, helping himself to five tries including one at the death to beat Canada to the bronze medal. As he skipped past a Welsh defender in inches of space the commentator purred, “he absolutely owned the defensive line” but it was his efforts at the opposite end of the field that caught the eye. A track-back tackle on England’s Philip Burgess helped prevent a certain score, Fleming covering acres of ground before dislodging the ball from the Englishman’s grasp over the try line.
And what happened next put a smile on many a Scottish face. The video was doctored and re-posted with Fleming charging down a grassy slope to a background of bagpipes... a nice touch from World Rugby, of all people.
The Scotland squad are currently in Las Vegas ahead of this weekend’s tournament in the middle of the Mojave desert but we spoke to Fleming before he left and the winger confirmed that he is not the first Scot to have earned a Dream Team place.
“It actually happened in Wellington two years ago when Damien Hoyland got in the Dream Team,” Fleming recalls, “but that was my first time in the team of the tourney. It doesn’t happen too often so it was quite a privilege to get there, so that was pretty pleasing.
“It happened overnight, the whole social media thing, I didn’t notice the video, I just came across the guys laughing during breakfast. It’s a good effort from World Rugby and my Facebook page erupted. You can score as many tries as you like but fans love a good tackle!”
Fleming has been around for a while now, long enough to remember the old days when Scotland were so ordinary that the public had effectively washed their hands of them. The change in the current squad could not be more pronounced if they took to the field sporting kilts.
Like the 15-man game, Scotland Sevens are currently ranked a barely believable fifth in the world. Scotland not only won the last tournament of last season, at Twickenham of all places if you are looking for an omen ahead of next weekend, but they followed that up with a cracking start to the current campaign, finishing fifth, fourth and then third in Wellington before blotting their copybook with 15th spot in Sydney, although there were mitigating circumstances.
Since the squad’s very existence was threatened two years ago, the core of sevens specialists has shrunk to just eight players, with the remainder coming from the pro-teams, the BT Premiership or, in the case of Ally Miller in the next two rounds, from London Scottish. Five of those eight players were injured in Sydney and, even then, the only roasting they got came from the home side Australia; everyone else including New Zealand beat them by no more than one converted try.
Something has changed and, like the 15-man game, a new coach has been at the centre of Scotland’s renaissance.
“There is no denying that the coaches have made a massive difference,” says Fleming. “Kitty [coach Callum MacRae] has come in and brought new aspects to the game, individual players have improved, I feel like I have really benefitted from him being my coach and all round he has brought the squad on.
“There is a level of consistency in the squad as well. It’s a really good squad with a really good culture. All of a sudden we are getting players who are wanting to come away on the sevens, guys like Dougie Fife who is just enjoying playing.
“And we have definitely benefited from a surge in self belief. There were times when I have sat down and watched finals and I have wondered what it would be like to contest one and having been in one and having won one, that was awesome. Having come away from the first two tournaments as fourth in the world and coming away after the next two still in fifth in the world after finishing third in Wellington, our second best result ever, is amazing.”
Scotland have had to dig deep into the barrel to make up for some of the injuries for the next two rounds in Vegas and Vancouver. Edinburgh duo Tom Brown, pictured, and Sean Kennedy join Miller in making their sevens bow. Scotland XVs flanker Hugh Blake is recovered from injury and Fijian-born Joe Nayacavou is back in harness after turning out in army colours.
The Scots have a tough looking pool, alongside Fiji, Australia and Japan, and are unlikely to start as favourites in the city synonymous with gambling.
“We used to stay at Planet Hollywood and now in the Monte Carlo but the lobby of every hotel is a casino,” says Fleming. “It’s quite surreal because you are walking through a casino with your rugby boots to go training and there are people up to all sorts because this is Vegas!”

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Broadway in Bozeman 2013-14 Season
Tuesday Jul. 23rd, 2013
Montana State University and Innovation Arts & Entertainment are thrilled to announce the return of AMERICAN BANK BROADWAY IN BOZEMAN for the all-new 2013-2014 season! Direct from New York, the best of Broadway is returning to Theatre at the Brick.
Now in its ninth season, the 2013-2014 AMERICAN BANK BROADWAY IN BOZEMAN will once again showcase a variety of A-list touring Broadway productions. This year’s series will feature four dazzling titles:Stomp, Hello Dolly starring Sally Struthers, West Side Story and The Midtown Men
Season tickets range from $135 to $225. Become a subscriber today and see the exciting new titles: Hello Dolly starring Sally Struthers, West Side Story and The Midtown Men in our Gold Package. Or include the international sensation Stomp in our full Platinum Package. That’s the best of Broadway for a savings of over 30% versus single tickets.
Season subscriptions are now on sale to the general public. To order a new subscription, patrons may call the Bobcat Ticket Office at 406-994-2287 or visit www.broadwayinbozeman.
Tickets to any single show are not available at this time.
Why subscribe? Subscribers are able to reserve the best seats before the general public, get the same seats at every show and enjoy the first right to renew those seats or request a change each year. In addition, subscribers can also enjoy lost ticket insurance, special offers and over 30% in savings versus single ticket prices.
DIGITAL PHOTOS OF EACH SHOW ARE AVAILABLE
Tuesday, October 8, 2013 at 7:30PM
STOMP is explosive, provocative, sophisticated, sexy, utterly unique and appeals to audiences of all ages. The return of the percussive hit also brings some new surprises, with some sections of the show now updated and restructured and the addition of two new full-scale routines, utilizing props like tractor tire inner tubes and paint cans. STOMP. See what all the noise is about.
Hello Dolly starring Sally Struthers
Tuesday, February 4, 2014 at 7:30PM
Winner of ten Tony Awards including Best Musical, HELLO, DOLLY! is one of the most enduring Broadway classics. Emmy- award winning Sally Struthers (All In the Family, Gilmore Girls) stars as the strong-willed matchmaker Dolly, as she travels to Yonkers, NY to find a match for the ornery “well-known unmarried half-a-millionaire” Horace Vandergelder. Featuring an irresistible story and an unforgettable score including the title song, “Put on Your Sunday Clothes,” “It Only Takes A Moment,” and the show-stopping “Before the Parade Passes By,” HELLO, DOLLY! has been charming audiences around the world for nearly 50 years.
West Side Story
Tuesday, March 4, 2014 at 7:30PM
More than fifty years ago one musical changed theater forever. Now it’s back and mesmerizing audiences once again. From the first note to the final breath, WEST SIDE STORY soars as the greatest love story of all time. This revival, based on Tony Award-winning librettist Arthur Laurents’ Broadway direction, remains as powerful, poignant and timely as ever. The Bernstein and Sondheim score is considered to be one of Broadway’s finest and features such classics of the American musical theatre as “Something’s Coming,” “Tonight,” “America,” “I Feel Pretty” and “Somewhere.” As the Associated Press says, “WEST SIDE STORY remains Broadway’s best dance-driven musical. Five decades have not dimmed the extraordinary choreography or the score that pulsates throughout.”
The Midtown Men
Wednesday, April 16, 2014 at 7:30PM
THE MIDTOWN MEN – 4 Stars From The Original Broadway Cast of Jersey Boys
This sensational production reunites Four Stars from the Original Cast of Broadway’s Jersey Boys. Tony Award winner Christian Hoff, Michael Longoria, Daniel Reichard and Tony Award nominee J. Robert Spencer, star in this one-of-a-kind concert experience celebrating the music that defined the ’60s.
These talented entertainers shared the Broadway stage for over a thousand performances in the mega-hit Jersey Boys. Now, as THE MIDTOWN MEN, they have thrilled crowds on concert stages and symphony halls across the continent, and have delighted television audiences with performances on “Good Morning America,” “Access Hollywood,” “Katie,” and “The Chew.” Additionally, the Men collaborated with rock and roll icon Stevie Van Zandt on their first radio single, “All Alone On Christmas,” featuring Van Zandt and members of Bruce Springsteen’s E Street Band. The group had the distinct honor of opening the National Tree Lighting Ceremony at the White House.
Don’t miss this electrifying and soulful new production with its top-shelf choreography, incredible vocal performances and legendary onstage chemistry.


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HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.
HIV-infected individuals must receive lifelong antiviral therapy because treatment discontinuation generally results in rapid viral rebound. The field has identified a state of latency at the level of transcription of the integrated provirus as the major mechanism of persistence. A number of drugs are now tested that aim at inducing viral transcription as a step to purge the reservoir. The assessment of viral production in cells from HIV-infected individuals with optimal viral suppression revealed the failure of SAHA/vorinostat to efficiently generate viral particle production. To further investigate and characterize the process of latency at the transcriptome level, and the response to SAHA as well as various reactivating agents, we use a model of primary CD4+ lymphocytes. The main observation from this study is that viral transcripts persist during latency, and that the accumulation of viral transcripts does not result in efficient viral protein expression upon reactivation with agents such as SAHA. Our data suggest that post-transcriptional blocks also contribute to latency, and that additional strategies need to be explored to efficiently purge the viral reservoir.
Citation: Mohammadi P, di Iulio J, Muñoz M, Martinez R, Bartha I, Cavassini M, et al. (2014) Dynamics of HIV Latency and Reactivation in a Primary CD4+ T Cell Model. PLoS Pathog 10(5): e1004156. https://doi.org/10.1371/journal.ppat.1004156
Editor: Michael Emerman, Fred Hutchinson Cancer Research Center, United States of America
Received: January 21, 2014; Accepted: April 18, 2014; Published: May 29, 2014
Copyright: © 2014 Mohammadi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Swiss National Science Foundation (31003A_146579), the FP7-HEALTH 2012-INNOVATION-1 (305762) and by a Grand Challenges Explorations award from the Bill and Melinda Gates Foundation. JdI is recipient of a fellowship of the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Successful antiretroviral therapy reduces HIV plasma viremia to undetectable levels; however, the virus is not eradicated . Current and non-mutually exclusive hypotheses to explain this phenomenon include (i) the presence of anatomic viral reservoirs that are inaccessible to drugs, (ii) ongoing viral replication and cell-to-cell spread, and (iii) the existence of a long-lived cellular reservoir that is infected but does not produce viral particles and can be reactivated. With the last hypothesis, it is proposed that reactivating viral transcription in latently infected cells may help purging the HIV reservoir and contribute to HIV eradication . Therefore, a better understanding of the mechanisms involved in viral transcriptional silencing and in the reactivation from latency is essential. Less attention has been paid to post-transcriptional blocks which prevent completion of the viral replication cycle, thus limiting expression of viral proteins, de novo production of viral particles and cytopathogenesis.
Currently, there are two competing models on how HIV establishes latency in resting CD4+ T cells , . The first model suggests that activated cells are infected by HIV, and that most of them are productively infected and die within a few days. However, a minority of cells revert to a resting memory state, following the natural biology of CD4+ T cells. The second model proposes that HIV is able to directly infect resting CD4+ T cells, even if this process is poorly efficient. To study HIV latency experimentally, cell lines have been used extensively to investigate the molecular mechanisms underlying impairment of viral gene expression. Cell lines highlighted viral transcriptional silencing as a major determinant of HIV latency (reviewed in –). There is however increasing interest for work in more representative primary cell settings. In recent years, multiple systems using primary cells from various CD4+ T cell subpopulations have been developed. These systems yield sufficient numbers of cells allowing investigations that may recapitulate more closely in vivo processes (reviewed in , .
Primary cell latency models have been used for the screening and assessment of molecules that promote viral transcription . Multiple compounds  reactivate viral transcription from latently infected cells in vitro, reflecting the various mechanisms involved in viral transcriptional control, including epigenetic regulation (such as histone modifications and DNA methylation) and immune modulation (such as T cell receptor engagement and protein kinase C signaling) , , . The efficiency of these agents varies according to the HIV latency model used , , underscoring that the mechanisms leading to repression and reactivation of viral transcription may be cell-specific. Some of the agents are moving forward to clinical trials , , –.
We have previously completed a detailed analysis of the cellular response to HIV infection in a T cell line and derived a model of cell reprogramming upon viral invasion . Here, we use an extension of this experimental approach to investigate viral latency and reactivation in a model of primary CD4+ T cells. We hypothesized that a comprehensive assessment of the paired transcriptomes of the host and the virus by RNA sequencing, coupled to the analysis of viral protein expression, would allow for addressing the extent of viral transcriptional and post-transcriptional silencing, the structure of the viral transcriptome, and the nature of the cellular transcriptional program induced by pharmacological and immunological reactivating compounds. Our in vitro and ex vivo data reveal defects in viral translation and particle production that reflect a state of post-transcriptional cellular latency which may not be reversed by agents merely enhancing viral transcription.
Viral production from latently infected cells ex vivo
We compared the efficacy of T-cell receptor (TCR) stimulation and SAHA treatment to stimulate viral particle production ex vivo. For this, we tested virus release from latently infected cells isolated from HIV-infected individuals under successful treatment (Table S1). We isolated resting CD4+ T cells and cultured them for 1, 2, and 4 days in the presence of IL-2 supplemented with DMSO (control), SAHA or TCR stimulation. TCR stimulation resulted in successful viral particle production in 7 of 11 samples, with viral RNA copies ranging from 42 to 2940 copies/ml of supernatant. Viral RNA copies in controls (mock-treated cells) were at or below the limit of detection of the assay for 6 samples, and values ranging from 27 to 287 copies/ml for 5 samples (Fig. 1). SAHA was undistinguishable from control, suggesting a failure to stimulate particle production from latently infected cells ex vivo, confirming previous studies . We observed effective inhibition of histone deacetylases confirming that primary cells were exposed to appropriate concentrations of SAHA (Fig. 1).
Panel A. HIV RNA copies/ml measured in the supernatant of resting CD4+ T cells isolated from treated HIV+ individuals (S: cells from single individuals, P: pooled cells from 2-3 individuals) and stimulated ex vivo with DMSO (control, black), SAHA (blue) or TCR (green) for 1, 2 or 4 days. Panel B. Box plot showing HIV RNA copies/ml measured in the supernatant of resting CD4+ T cells isolated from treated HIV+ individuals and stimulated ex vivo with DMSO (control, black), SAHA (blue) or TCR (green) for 1, 2 or 4 days. Each circle represents a different cellular sample. Bars: min to max. Solid line: median. Panel C. Immunoblot of histones (1 µg) extracted from stimulated ex vivo cells isolated from treated HIV+ individuals. H3K27Ac: anti-histone 3, acetylated Lysine 27; H3K9Ac: anti-histone 3, acetylated Lysine 9; H3: anti-histone 3 (total).
Stability of the cellular model of latency
To further investigate the mechanisms underlying SAHA inefficiency to reactivate latently infected CD4+ T cells ex vivo and generate viral particles, we used the cellular model described by the laboratories of Cloyd  and Karn  that preferentially produces latently infected CD4+ T cells with a central memory phenotype. These cells constitute the major cellular reservoir of HIV in blood . The viral vector, previously used in latency studies, encodes Tat, Rev, and a destabilized GFP protein with an estimated half-life of 6 hours . Starting with the transduction of two million cells from a healthy donor, we sorted a population of 200,000 successfully infected cells and amplified them to obtain 50 million cells. The infected cell population was then co-cultured on a feeder layer of the H80 human brain tumor cell line over 10 weeks (Fig. S1). We obtained a population of latently infected cells carrying a stable number of viral integration events, measured at 1.06 proviruses per cell. This measure of quality control, as well as the quasi-universal reactivation of the cell population upon T cell receptor (TCR) stimulation with anti-CD3/anti-CD28 and IL-2 (see below) excluded outgrowth by non-infected cells. The expression from the env open reading frame of a destabilized viral-encoded GFP decreased significantly following infection (entry phase), and reached its nadir upon co-culture on feeder cells (week 0). However, low level GFP expression remained stable at around 3-fold above background (mock uninfected cells) through 10 weeks of co-culture (latency phase) (Fig. S2). Upon reactivation by TCR stimulation (reactivation phase), we observed up to 9-fold increase of GFP expression; with 92% of cells expressing the viral-encoded GFP, Figure 2. The activation marker IL2Rα (CD25) remained undetectable during the latency phase. Thus, the model recapitulates the process of latency, although the basal degree of GFP expression suggests incomplete silencing of HIV.
Viral-encoded GFP expression (green) and activation marker IL2Rα (CD25) expression (black) during the steps of entry into latency (week -3 to week 2), maintenance of latency (week 4 to 10), and reactivation upon TCR stimulation (8, 24, and 72 hours after week 10). Shown are the geometric mean fluorescence of intensity (geoMFI) ratios of HIV-based vector infected cells and mock non-infected control cells.
Incomplete transcriptional silencing of HIV
To understand the extent of viral silencing during latency, we assessed three parameters of viral transcriptional activity using RNA sequencing data. First, we estimated the proportion of viral transcripts among the total number of detected transcripts. From week 0 (W0) through week 10 (W10), this proportion was stable with a mean of 1.23% (95%CI; 1.15, 1.32). Second, we evaluated the distribution of viral reads along the viral vector genome. We identified a conserved pattern of coverage of the viral vector genome through the latency phase (Fig. 3A). Third, we assessed the pattern of viral splicing. All main splice variants were well represented and in conserved proportions  (Fig. 3B, Fig. S3, Fig. S4). Upon TCR stimulation, there was a proportional shift in viral splice forms. Indeed, cell activation resulted in a profound modification of the transcriptome and doubling of the RNA content of the blasts.
Panel A. Distribution of HIV reads along the vector genome. On the top is depicted the viral vector genome used (NL4-3-Δ6-drEGFP). Red crosses indicate the genes that are disrupted by stop codon insertion, frameshift or deletion. TSS: transcription start site; D: splice donor; A: splice acceptor. Reads mapping to the LTR are equally assigned to 5′ and 3′ ends, explaining the presence of viral reads upstream the TSS. Panel B. Pattern of splicing for the main viral RNA forms: genomic unspliced full-length viral RNA (US, blue), singly spliced RNAs without the Gag-Pol major intron (SS, green; spliced in D1 but not in D4), and multiply spliced subgenomic mRNAs (MS, red; spliced in D1 and in D4).
The RNA sequencing approach captures polyadenylated viral RNA (i.e. fully elongated). On these RNA species we did not observe major deficits in viral genome coverage, or splicing during latency. Thus, these viral transcripts could contribute to the residual viral-encoded GFP expression. However, these analyses do not discriminate between continuous translation from a stable pool of viral transcripts from the original infection and de novo transcription from leakiness of the latency model.
Dynamics of entry, latency, and reactivation
The host RNA profile during the dynamic process of latency was studied in time-course transcriptome analyses by grouping host genes according to their transcriptional response to HIV and to TCR stimulation with anti-CD3/CD28. Whereas the virus only minimally perturbed the transcriptional state of the cell, TCR stimulation resulted in massive cell reprogramming, impacting 66% of all detected genes. Numbers of genes differentially expressed under the various conditions are detailed in the legend of Figure 4.
Panel A. Illustrated are 9729 genes with statistically significant association to at least one of the two experimental conditions, HIV infection and TCR stimulation, as evaluated by 2-way analysis of variance of 14513 expressed genes (FDR<0.05). Data are presented in three non-exclusive groups: those regulated in concordance with viral presence (345 genes, top panel), those regulated in concordance with TCR-stimulation 9647 genes, (middle panel), and those showing non-additive synergetic effects by viral presence and TCR stimulation (59 genes, bottom panel). The bold lines represent the group average, and color intensities are proportional to statistical significance of the effects within each group. Panel B. Principal component analysis of host whole-transcriptome data supports a three-step model of entry-latency-reactivation. There is a transcriptionally coherent latency phase between week 4 and week 10, and a distinct reactivation phase at 8, 24, and 72 hours after TCR stimulation. All data, including differentially expressed genes and enrichment analysis, are available at http://litchi.labtelenti.org.
Principal component analysis revealed two transcriptionally distinct phases: entry (W0–W2) and latency (W4–W10) (Fig. 4B, Fig. S5). The tight cluster of samples during the 6 weeks of latency includes both infected and uninfected samples, indicating a stable process that is only minimally perturbed by the virus. To assess the differences between infected and uninfected cells, we compared gene expression in paired samples to pinpoint potential markers of HIV latency, consistently differentially expressed during the 6 weeks of maintenance of latency (W4–W10): we identified 103 cellular genes as upregulated and 124 as downregulated in HIV-infected samples during this phase. The differentially expressed genes exhibit moderate enrichment for pathways including chemokine and cytokine receptors and immune response (Fig. S6). All differentially expressed genes and analyses can be queried and downloaded from the open access interactive web resource http://litchi.labtelenti.org.
The RNA sequence data also allowed for estimating the diversity of integration sites during the various time points and upon reactivation. Out of these, we identified 15886 integration events with reads at a junction between host and HIV transcripts. Importantly, the data reflects host-virus splicing events that, while globally identifying the general location of an integration event, do not allow precise mapping. From this we identified 12868 integration events in the host genome across conditions; ranging from 3718 at the earliest time point (i.e. 14 days before W0) to 2216 at the latest time point (TCR 24h); 92.5% represented unique integration sites. Overall, these findings indicate minimal clonal bias in the model.
In summary, the stability of the cellular transcriptome profile between weeks 4 and 10 is consistent with maintenance of a resting state of primary CD4+ T cells and limited contribution from viral infection. TCR stimulation creates a cellular environment highly supportive of HIV expression.
Reactivation with pharmacological and immunological agents
After ten weeks of co-culture on H80, cells were collected and incubated in the presence of various pharmacological (vorinostat [SAHA], disulfiram [DSF], 5′-azacytidine [AZA]), or immunological (interleukin 7 [IL-7]) stimuli, as well as TCR stimulation. We collected RNA for sequencing and cells for FACS analysis at 8 and 24 hours after treatment start. The removal of cells from the feeder cell layer induced a 2-fold reduction of the proportion of HIV to all cellular transcripts (from 1.26% to 0.64% at 8 hours), possibly due to loss of a trophic environment provided by H80 cells (Fig. 5A). In contrast, exposure to SAHA resulted in an increase in the proportion of viral transcripts to 1.77% at 8 hours and 2.14% at 24 hours, three times that of DMSO control (Fig. 5A). The effect of TCR stimulation on the proportion of HIV transcripts was modest, but cell activation was accompanied by blast morphology and a 2-fold increase in total cellular RNA at 24 hours. Disulfiram had a transient effect, with an increase in the proportion of HIV transcripts to 1.63% at 8 hours. IL-7 had a slower effect with 1.21% at 24 hours. AZA had no detectable impact on viral transcription. The use of the various agents did not result in profound changes in viral vector genome coverage or splicing. However, the use of SAHA was associated with a relative increase in the proportion of unspliced transcripts (Fig. S3).
Panel A. Viral transcription (proportion of HIV transcripts to total cellular transcripts). Panel B. Viral expression (GFP) profiles after reactivation with the various compounds. Panel C. HIV GFP protein expression corrected by the proportion of HIV transcript reads in the cell, normalized by DMSO 8 h. The reference lines represent the W0 of latency, and DMSO 8 hour values.
Viral transcription induced by activating agents such as SAHA and disulfiram was not proportionally reflected at the protein level as expression of the viral-encoded GFP remained low (Fig. 5B). As previously reported, AZA - alone, or IL-7  had no discernible or minimal effects on viral protein expression. This observation was in contrast to the strong increase in GFP expression induced by TCR stimulation. The divergent response was maximal when estimating GFP translation per transcript (Fig. 5C). Ratios were also assessed by considering the production of GFP relative to the amount of single-spliced HIV forms, which have the capability of being translated into GFP. The general profile of transcription, translation and ratios was not modified in this analysis.
To understand the differential effect of reactivating agents on viral transcription, we compared the transcriptional signatures induced by these agents (Fig. 6, and http://litchi.labtelenti.org). TCR stimulation showed a very strong effect involving 3664 upregulated and 3220 downregulated genes, with a strong enrichment signal for upregulation of the cellular machinery (signal transduction, generic gene expression, protein synthesis, and metabolism), and upregulation of genes previously proposed to play a role in HIV biology . In contrast, the various other agents exerted limited influence on the host transcriptome (Fig. S7). SAHA induced upregulation of 730 genes and downregulation of 559 genes. Genes involved in immune response and T-cell activation processes were preferentially downregulated by SAHA. Disulfiram showed weak effects on cellular gene expression, with only 132 upregulated genes enriched for HIV cofactors, innate immunity, and apoptosis, and 57 downregulated genes enriched in defense response genes. Very little transcriptomic effects were observed in response to the other reactivating compounds (Fig. 6 and http://litchi.labtelenti.org).
Each panel summarizes over-represented pathways among the differentially expressed genes induced by the indicated treatment. Organized under ten major categories, each individual circle represents one enriched pathway in Reactome (see methods). The size is proportional to the adjusted p-value (q-value), and the y-axis corresponds to the average effect of the differentially expressed genes within the reported pathway. Only TCR stimulation using anti-CD3/CD28 and IL-2 leads to upregulation of numerous cellular pathways, as well as upregulation of reported HIV cofactors and HIV related pathways (shown in red). AZA is not shown as no enrichment was observed.
To confirm the main findings, we repeated the latency and reactivation experiment in the same and on an independent donor. These analyses also included spike-in internal controls for RNA sequencing and used culture supernatant only from H80 feeder cells (without direct cell-cell contact). The data confirmed the reproducibility of the model, and the inability of SAHA to induce viral protein expression in proportion to the increase in viral transcripts (Fig. S8). Taken together, these results suggest that, in this model of latency, agents successfully inducing HIV transcription, such as SAHA and disulfiram, do not lead to proportional effects on HIV translation. These agents may not generate a cellular environment that effectively sustains HIV protein expression. In contrast, cell activation upon TCR stimulation creates a permissive environment by systematic activation of the cellular machineries required for viral replication. These results underscore the need to explore strategies that facilitate successful completion of the viral cycle in combination with agents that act primarily on viral transcription.
A number of agents have been identified as latency reverting drugs. SAHA has been extensively studied and brought forward to clinical trials , , . However, data from Blazkova et al. , our data, and recent reports at the time this work was under consideration - indicate that SAHA fails to effectively increase particle production ex vivo. We tested the ability of SAHA to increase viral production from latently infected CD4+ T cells from virologically suppressed HIV-infected individuals. While SAHA effectively inhibited histone deacetylation, it failed to stimulate particle production as compared to TCR stimulation. Data from the Siliciano laboratory also indicates that reactivation induced by SAHA is not sufficient to trigger a cytopathic effect and cell death . We explored this paradox by using the primary CD4+ T cell model described by Cloyd  and Karn .
Use of a primary cell model allowed the detailed analysis of the dynamic process of HIV latency and persistence. We examined cellular transcriptional dynamics over 10 weeks and jointly defined cellular and viral expression patterns using RNA sequencing and FACS analysis. In this cellular model, latency appears to be a stable process. However, viral transcripts are continuously present accompanied by residual expression of viral-encoded GFP. We reviewed printed material and figures from various primary models and observed comparable levels of residual GFP or p24 expression that are generally interpreted as background , , , ,  – indeed, the original paper by Tyagi et al. identified up to 28.77% of cells expressing low levels of GFP during latency . Upon TCR stimulation, there is near universal induction of GFP expression, generally validating the model. The use of SAHA and disulfiram successfully increased viral transcription; however the reactivated cells failed to produce proportional amounts of viral protein. Overall, these observations underscore the importance of the cellular environment to allow any effect on viral transcription to translate into an efficient purging strategy .
We observed a remarkable stability of the cellular transcriptional profile after 4 weeks of co-culture on H80 feeder cells despite the presence of HIV transcripts in the cells. Few cellular genes were differentially expressed between non-infected and infected cells. These genes may represent biomarkers of HIV latency, or markers of the original subset of cells that were successfully transduced. Moreover, the pool of viral transcripts might persist from the original infection or reflect ongoing transcription. Cell-associated viral RNA can also be detected in latently infected resting memory CD4+ T cells in individuals under effective antiretroviral therapy (, -, and Hu et al., abstract 405, and Fromentin et al., abstract 412, CROI 2014), and in animal models (, , and Okoye et al., abstract 136LB, CROI 2014). The short half-life of the viral vector-encoded destabilized GFP  suggests that some residual translation takes place. Leakiness of the system could be induced by the trophic environment of the experimental system, including secreted factors from the feeder cells, or by the small concentration of IL-2 used in the model. Some level of residual activation has been observed in other primary cell models . Here, however, we only detected negligible levels of cellular activation as assessed by expression of the activation marker IL2Rα (CD25). To better understand the nature of the persisting viral transcripts, we examined the amount and distribution of HIV reads along the viral vector genome, and the pattern of splicing. The proportion of HIV transcripts remained stable at about 1.26% of all cellular transcripts during 10 weeks, without apparent defects in structure. However, the RNA sequencing approach captures polyadenylated RNA; therefore it would not represent coverage of other species of viral transcripts, including non-polyadenylated paused viral transcripts that could be relevant and contribute to the process of latency or reactivation. The leaky system bears some similarities with that of infection of cells belonging to the monocyte/macrophage lineage, which, as discussed by Van Lint et al. , may not reach complete silencing but maintain a low level of viral replication. Therefore, the model used here may recapitulate both latency and persistence of viral transcription as already observed in vivo . However, the experimental system used here has shortcomings. In our hands, the amount of materials generated is limiting, and the use of a complete viral clone is too toxic, which led us to use an attenuated viral vector that only expresses GFP (from env ORF), Rev and Tat. The use of such a truncated HIV vector may affect the level of basal HIV transcription and thus the process of latency . Although we are aware of the possibility that, in other types of vectors, specific transcripts and viral proteins other than GFP could be selectively expressed (e.g. Gag) , the detailed splice analysis militates against major differences in protein expression across the viral genome.
We tested three pharmacological agents to induce viral transcription. SAHA was an efficient inducer, resulting in over 3-fold increase in the proportion of viral transcripts in the cell, consistent with previous reports , , , , reviewed in –. The effect of disulfiram was short-lived, with a 2.5-fold induction of viral transcription at 8 hours; also consistent with previous reports . Despite the observed effects on viral transcription, these compounds contributed only minimally to viral protein expression. AZA did not affect viral transcription or translation, consistent with the mode of action of this drug which needs to be incorporated into DNA to be active and prevent methylation. This finding suggests that the cells are not dividing, which again supports the accuracy of our model. In contrast, in cell lines, which are dividing, a longer exposure allows observation of biological effects . In addition, the cellular transcriptional environment was hardly affected by the various drugs, emphasizing that the cells retain a resting state , , . Use of IL-7 contributed minimally to enhanced viral transcription or translation, despite exerting more pronounced effects on cellular transcription, consistent with recent reports on the use of this cytokine in vivo . In contrast, the effect on the transcriptome and the cellular environment created by TCR stimulation strongly favored translation.
Our data raise the possibility of post-transcriptional block as one of the mechanisms of HIV latency. This is consistent with studies from the Fauci laboratory that indicated that resting CD4+ T cells from aviremic patients did not produce quantifiable cell-free virions despite the presence of cell-associated HIV-1 RNA , . Proposed mechanisms underlying post-transcriptional blocks include splicing defects , inhibition of nuclear RNA export , inhibition of expression of viral proteins in a codon-usage-dependent manner , and inhibition of HIV translation by microRNAs , . Our current work, and results from Iglesias-Ussel et al.  suggest that latently infected cells may have multiple biochemical and metabolic blocks that are not completely released by some of the reactivating agents currently being evaluated.
Cell line models and primary CD4+ T cell models have advanced the understanding of basic mechanisms of HIV transcriptional latency and served to screen for latency reversing agents. In particular, cell lines highlighted the role of multiple cis- and trans-acting factors in the regulation of basal viral transcription , . Primary cell models provided a broader view on the complexity of latency and variability in response to reactivating agents , . Our work on the dynamics of latency and reactivation using a primary cell model underscores the existence of barriers beyond transcriptional silencing – thus helping explain clinical trials and ex vivo data that identify persistent viral transcription but impaired viral protein expression and particle production. Overcoming post-transcriptional blocks may thus require additional interventions.
Materials and Methods
Viral production from latently infected cells ex vivo
We collected 25 ml of total blood from HIV-infected individuals participating in the Swiss HIV Cohort Study (http://www.shcs.ch) with controlled viremia (Table S1). Resting CD4+ T cells were purified by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell enrichment kit supplemented with anti-HLA-DR, anti-CD25 and anti-CD69 (Stem Cell Technologies). Cells were resuspended in Opti/FCS/IL-2, split in three wells (approximately 1 million/ml/well) containing DMSO, 0.5 µM SAHA, or TCR stimulation as described below. After 1, 2 or 4 days of incubation, cell supernatant was collected and assessed for viral presence in a COBAS AMPLICOR analyzer (Roche), with a limit of detection of 20 unspliced RNA copies/ml.
Resting CD4+ T cells from HIV-infected individuals were isolated and treated for 24 h with DMSO, SAHA or TCR stimulation as indicated above. Histone extraction was performed according to Abcam's protocol. Briefly, cells were washed twice with ice-cold PBS, resuspended in Triton Extraction Buffer (PBS–0.5% Triton X100–protease inhibitors–0.02% NaN3) at 107 cells/ml and incubated 10′ on ice to allow cell lysis. Cells were centrifuged at 380 g for 10′ at 4°C and washed once with Triton Extraction Buffer. After centrifugation, the pellet was resuspended at 4×107 cells/ml in 0.2N HCl and incubated over night at 4°C to allow for histone extraction. Samples were centrifuged at 380 g for 10′ at 4°C and the histone-containing supernatant was collected and protein concentration was measured using Qubit Protein Assay (Life Technologies) following manufacturer's protocol. Histone extracts (1 µg) were separated on an 8–16% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and processed for immunoblotting. Briefly the membranes were washed with PBS, blocked for 2 h at RT in PBST-milk (PBS/0.2% Tween-5% non fat dry milk), incubated over night with anti-histone antibodies (1∶1000) at 4°C, washed three times for 10′ in PBST, incubated with swine anti-rabbit-HRP (Dako P-0217, 1∶2000) for 2 h at RT, washed again and revealed using Luminata Crescendo Western HRP substrate (Millipore). Primary antibodies were rabbit polyclonal from Abcam: anti-H3K27Ac (ab4729), anti-H3K9Ac (ab10812), anti-H3 (ab1791).
Primary cell model
Primary CD4+ T cells were purified by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell Isolation kit II (Miltenyi Biotec) (Fig. S1). Cells were activated using CD3/CD28 co-stimulation in presence of IL-2 (mimicking TCR stimulation) as described previously . Briefly, anti-CD3 antibodies (10 µg) were plated in 1 ml PBS per well of a 6-well plate and incubated for 1–2 h at 37°C. Wells were then washed once with 3 ml of PBS and filled with 106 cells/ml of primary CD4+ T cells supplemented with 1 µg/ml anti-CD28 antibodies in Optimizer CTS T-Cell expansion SFM culture medium containing 5% heat-inactivated fetal calf serum (FCS) and 100 U/ml human recombinant IL-2 (Opti/FCS/IL-2 culture medium). Three days post-stimulation, cells were collected, washed and resuspended at 106 cells/ml in Opti/FCS/IL-2 for infection with an HIV-based vector (NL4-3-Δ6-drEGFP/CXCR4; a kind gift from R.F. Siliciano). This HIV vector uses a CXCR4 tropic HIV envelope for entry, contains functional tat and rev genes, as well as a gfp reporter gene containing a PEST sequence (yielding a protein half time of ∼6 hours); this vector contains mutations (stop codons, frameshift or deletion) in gag, vif, vpr, vpu, env and nef and is thus less cytotoxic for the infected cell, thereby promoting latency in a higher proportion of infected cells . HIV particles were produced by transfection of HEK293T cells as previously described . Infection was carried out by spinoculation for 3 hours at 25°C and at 1500 g using 50 µg p24 equivalent HIV vector/106 cells in presence of 5 µg/ml polybrene. After spinoculation, cells were washed and resuspended at 106 cells/ml in Opti/FCS/IL-2. Forty-eight hours post-infection, cells were resuspended in PBS/FCS (to eliminate the interference of phenol red) and sorted by FACS according to GFP expression, yielding a typical purity of >98%. FACS sorting was performed with a Mo-Flo Astrios instrument. Upon sorting, successfully infected GFP+ cells were further processed to investigate HIV latency.
Establishment and maintenance of HIV latency was mostly carried out as previously described ,  (Fig. S1). Infected GFP+ cells stimulated using anti-CD3 and anti-CD8 antibodies in presence of IL-2 as described above were expanded in Opti/FCS/IL-2 for ∼3 weeks, typically allowing ∼2 log of cell multiplication. At this time (week 0), cells were washed, resuspended in R-10/IL-2 (RPMI-1640 culture medium supplemented with 10% heat-inactivated FCS and 40 U/ml IL-2) and added to an adherent H80 feeder cell layer. The H80 human brain tumor cell line, known to promote ex vivo survival of primary CD4+ T cells, were prepared by plating 106 cells/25 cm2 flask (T25) or 3×106 cells/75 cm2 flask (T75) the day before starting co-culture with primary CD4+ T cells. CD4+ T cells were resuspended in R-10/IL-2 and added on top of the feeder cell layer, in 16 ml R-10/IL-2 in T75 if 30-100×106 primary CD4+ T cells or in 5 ml R-10/IL-2 if >30×106 cells. Culture medium was changed three times a week by renewing half of it. Every two weeks, CD4+ T cells were collected and transferred onto a new feeder cell layer flask. During this transfer, cell samples were collected for analyses (see below). The co-culture of primary CD4+ T cells with H80 feeder cells was carried over a period of 10 weeks to allow CD4+ T cells to revert to a resting phenotype and to ensure entry into and maintenance of latency. Collected samples were used for: (i) assessing the expression of virally-encoded GFP and of the marker of activation IL2Rα (CD25) by FACS analysis, and (ii) assessing dynamic changes of the cellular and viral transcriptomes using mRNA-Seq. This analysis allowed for (a) following the cellular dynamic process from activated to resting state at the transcriptional level in non-infected as well as infected cells, (b) highlighting genes differentially expressed in HIV-infected cells as compared to mock cells, and (c) measuring concomitant viral transcriptional activity in resting CD4+ T cells. The latency process was also repeated using H80 supernatant as alternative to cell-to-cell contact conditions.
We included pharmacological and immunological agents that affect HIV transcriptional activity by different mechanisms of action and that are currently under clinical consideration. Specifically, we used a histone deacetylase inhibitor (vorinostat or SAHA, 0.5 µM), a DNA methylation inhibitor (5′-azacytidine, 1 µM), an alcohol dehydrogenase inhibitor (disulfiram, 0.5 µM). All drugs were suspended in dimethyl sulfoxyde (DMSO), thus DMSO was used as a negative control (of note, final DMSO concentration was 0.0033% corresponding to 1∶30,000 dilution). We also used immunological stimulation by interleukin-7 cytokine (100 ng/ml), and a full TCR stimulation using anti-CD3/anti-CD28 antibodies in presence of interleukin-2. At 8 h and 24 h after treatment, cells were analyzed for GFP expression by FACS and for transcriptome composition as described below.
Cell samples were stored in RNALater at the time of collection. Once all samples, from week 0 to week 10, were collected, total RNA extraction was performed using Illustra RNAspin mini isolation kit (GE Healthcare) and further processed for mRNA-Seq library preparation (TruSeq RNA sample prep kit, Illumina – that starts with capture of polyA-containing transcripts), followed by cluster generation (TruSeq single-end cluster generation kit, Illumina) and high-throughput sequencing on Illumina HiSeq2000 at the Genomics Technology Facility, University of Lausanne. Addition of RNA spike-ins were also added according to manufacturer's instructions (ERCC Exfold RNA Spike-in Mixes, Life Technologies and Loven et al. ). We obtained about 100 mio single end reads of 100 nucleotides for each sample. The sequencing reads were cleaned before alignment in order to improve the accuracy of downstream analyses. The cleaning steps included the removal of (i) Illumina's adapter (if present at the 3' end of the read) with cutadapt v0.9.5 , (ii) low quality (PHRED score<6) nucleotides at the 3′ or 5′ end of the reads, (iii) reads with mean PHRED score lower than 20 and (iv) polyA tails with prinseq v0.17 . Only reads of 30 nucleotides or longer after trimming were kept for further analyses. The cleaned reads were aligned to the reference genome with TopHat v2.0.6  using the ensembl gene GRCh37 release 68 annotation file. The reference genome was built by concatenating the human genome (hg19) and the HIV vector sequence. The number of reads per gene was extracted with a modified version of HTSeq-count v0.5.3 (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html) using the same annotation file and considering the whole HIV vector as a single gene. The modifications allowed multiply aligned reads to be weighted accordingly. Ambiguous reads were randomly attributed to one of the genomic regions they aligned to. To identify integration sites we used Tophat fusion with bowtie1 and default parameters.
Two-way analysis of variance and principle component analysis (PCA) were used for quantitative assessment of the transcriptome data presented in Fig. 4. In order to fulfill the underlying assumptions of these models, we conservatively discarded lowly expressed genes to minimize heteroscedasticity due to shot noise. Retaining genes expressing at least 100 reads in more than half of the samples yielded 14,513 genes. The resulting dataset was smoothed by adding pseudocounts (10 extra reads per gene), and log-transformed for variance stabilization.
Differential expression tests
Differential expression tests were performed assuming negative-binomially distributed read counts using the Bioconductor package DESeq . Paired-sample differential expression tests were performed using generalized linear models and dispersions were estimated using the Cox-Reid-adjusted maximum likelihood estimator. Lowly expressed genes were discarded after estimating dispersion. All p-values were corrected using a false discovery rate of 5% (Benjamini-Hochberg procedure ).
Pathway enrichment analysis and visualization
Enrichment analyses were performed using Fisher's exact test to detect overrepresentations of functional classes, regulatory motifs (among miRNA and transcription factor targets), physical locations on chromosomes, and HIV-related pathways, proteins and co-factors as described previously . HIV co-factors include genes in HIV related pathways from the Reactome pathways database Ver.40, genes identified in previous siRNA studies –, and genes encoding protein interaction partners for each viral protein ( and http://mint.bio.uniroma2.it/virusmint). All p-values were corrected using a false discovery rate of 5%.
For visualization in Fig. 6, we only considered enrichment hits from Reactome pathways and HIV co-factors databases. The major pathway categories were chosen from maximal pathways, (Pi), among all the reported unique enrichment hits pooled over all reactivating agents. In order to choose the most representative pathway categories, we defined the asymmetric coverage score of the ith pathway, Pi, over the jth pathway, Pj, as mi,j = |Pi ∩ Pj|/|Pj| and the total coverage of the ith pathway as Si = ∑j mi,j. The set of representative categories was built by those with the highest total coverage score Si. We used the nine top categories plus a pseudo-pathway called “miscellaneous” for the visualization. Each pathway was assigned to the major category that holds the highest coverage score over it. Subsequently, the pathways that are not covered more than 95% in any of the chosen major categories are assigned to the miscellaneous category.
Data and materials availability
All data, including differentially expressed genes and enrichment analyses, are freely available in interactive mode or for downloading at http://litchi.labtelenti.org.
Bioinformatics of viral transcriptome
Two splice junction aligners were used to identify HIV splicing events: Tophat v2.0.6 and STAR v2.3.0 . All acceptor-donor pairs were retrieved and their proportion calculated. Both splice junction aligners gave similar and consistent results. The HIV transcriptome profile was assessed using the Python package HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html). Coverage was normalized by the sample size factor estimated using DESeq. To calculate the percentage of HIV transcripts among all transcripts, we compared the gene length normalized counts using the length of the longest annotated transcript (isoforms with intron retention were not considered).
The main viral RNA forms were estimated as follows. The number of reads overlapping the D1 junction corresponds to unspliced reads (US, non-spliced in D1). The number of reads aligning to the left of D1 and broken at D1 (up to a different HIV position) corresponds to reads spliced in D1 and thus belongs to singly spliced or multiply spliced HIV RNA (MS+SS). The number of reads overlapping the D4 junction corresponds to singly spliced or unspliced HIV transcripts (US+SS). The number of reads aligning to the left of D4 but broken at D4 (up to different HIV position) corresponds to reads spliced in D4 and thus belong to multiply spliced transcripts (MS). Finally, the total proportion of reads (100%) is US+MS+SS, and SS can be obtained as SS = 100%–US–MS.
Experimental design. Cellular model.
FACS analysis of HIV-encoded GFP expression of representative cell samples. Panel A. CD4+ T cells were analyzed by FACS to assess the geometric mean fluorescence intensity of GFP expression (FL1-H channel). The histogram plot shows uniform populations (single peaks) for uninfected controls (W10+24 h DMSO, dotted grey line), latently infected cells (W10, red solid line), latently infected cells upon 24h reactivation with DMSO (black solid line), SAHA (blue solid line) or TCR stimulation (green solid line). Data revealed a shift in intensity of TCR-stimulated cells but not SAHA-treated cells as compared to the corresponding DMSO control. Panel B. Dot plots of FSC/SSC or FL1/FL2 of CD4+ T cells, either mock or HIV-infected, at week 10 post co-culture on H80 (W10) and 24 h post TCR stimulation (TCR). FSC/SSC dot plots shows differences in cell size and complexity between W10 and TCR, likely representative of resting CD4+ T cells and activated CD4+ T cells respectively. FL1/FL2 dot plots shows highly expressing GFP cells in R7 region upon TCR stimulation.
Features of HIV transcription under several reactivation agents. Panel A. Distribution of HIV reads along the vector genome; each panel compares one agent against DMSO as control. On the top is depicted the viral vector genome used. TSS: transcription start site; D: splice donor; A: splice acceptor. Reads mapping to the LTR are equally assigned to 5′ and 3′ ends. Panel B. Pattern of splicing for the main viral RNA forms: genomic unspliced full-length viral RNA (US, blue), singly spliced RNAs without the Gag-Pol major intron (SS, green; spliced in D1 but not in D4), and multiply spliced subgenomic mRNAs (MS, red; spliced in D1 and in D4).
Features of HIV transcription under several reactivation agents. Detailed assessment of donor-acceptor splice junction usage and graphical representation. D: splice donor; A: splice acceptor.
Principal component analysis of latency and TCR stimulation compared to H80 feeder cells. The transcriptome of H80 feeder cells (two replicates) is distinct. There is no evidence for contamination of primary CD4+ T cells during the process of latency. Upon TCR stimulation, the CD4+ T cells are removed from the H80 feeder cells.
Pathway enrichments for the differentially expressed genes during HIV latency. Each panel summarizes over-represented pathways among the differentially expressed genes induced by viral presence. Organized under nine major categories, each individual circle represents one enriched pathway in Reactome (see methods). The size is proportional to the adjusted p-value (q-value), and the y-axis corresponds to the average effect of the differentially expressed genes within the reported pathway.
Principal component analysis of modifications in the transcriptome upon exposure to the various reactivating agents. The transcriptome of CD4+ T cells exposed to the various reactivating agents cluster with that of mock and of latently infected cells (W4 to W10), suggesting a minimal impact of those compounds on the cell. Panel A shows the transcriptome data in the context of latency phase and full cell activation by TCR stimulation. Panel B shows a PCA analysis on the large cluster of cells exposed to reactivating agents. The PCA of the cluster reveals small compound- and HIV-specific transcriptome differences compared to W10 infected and uninfected CD4+ T cells.
Validation and reproducibility of the model. Panel A. Viral transcription (viral-encoded GFP transcripts normalized by internal control and by baseline DMSO values) upon SAHA or TCR stimulation on two donors. Panel B. Viral expression (GFP MFI) profile after reactivation with SAHA or TCR on two donors. Panel C. Principal component analysis of modifications in the transcriptome upon exposure to the various reactivating agents. An additional experiment was performed to include cellular samples prior co-culture with H80 cell supernatant (mD14, mD12 and mD9 corresponding to cells collected 14, 12 and 9 days before W0 respectively). This additional set of CD4+ T cell transcriptomes recapitulates the entry, latency and TCR reactivation that were already observed. Exposure to H80 did not require cell-to-cell contact as the experiment used only filtered H80 cell culture supernatant. These additional cellular samples also included RNA spike-in controls (spike) to control for RNA content differences; normalization data using library size or RNA spike-in were similar.
The plasmids used to produce the HIV-based vector, i.e. pNL4-3-Δ6-drEGFP, pC-Help and pCXCR4, were obtained from Robert Siliciano. The H80 feeder cells were obtained from Miles Cloyd. We thank Simon Quenneville and Monsef Benkirane for critical reading of the manuscript, Nitisha Pyndiah and Cyril André for technical assistance, and Vital-IT (http://www.vital-it.ch) for computing infrastructure.
Conceived and designed the experiments: AT AC NB JF. Performed the experiments: MM CT AC RM. Analyzed the data: PM JdI. Wrote the paper: AT AC PM JdI NB JF. Clinical work: MC. Designed the web-based interactive analysis tool: IB.
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domain: biology


Several U.S. senators who are negotiating the final details of immigration reform legislation toured the U.S.-Mexico border on Wednesday to take a close look at some of the border security concerns.
Arizona Republican Sens. John McCain and Jeff Flake were joined by Democratic Sens. Chuck Schumer of New York and Michael Bennet of Colorado during Wednesday’s trip to the border. All four of them are members of the ‘Gang of Eight,’ a bipartisan group of senators who have spent the last few months crafting an immigration reform bill.
McCain: Border security is key to immigration reform
Their trip comes during Congress’ two-week recess. The senators had hoped to introduce their immigration reform proposal when they returned from their two-week break, but it seems that several unresolved issues might push back their deadline.
During a town hall meeting on Monday, McCain said he hopes the trip will give the senators a firsthand look at the border and help finalize the immigration-reform negotiations.
“I wish every member of the United States Senate and Congress could see the border,” McCain told reporters after Monday’s event. “Only when you can see the expanse, the difficulties and the challenges of the border, can you really appreciate the need for our border security.”
He added, “It’s not just people who are coming across our border illegally to live here. It is the drug cartels that are smuggling billions of dollars of drugs into this country.”
Senators will find a secure border
Ali Noorani, executive director of the National Immigration Forum, noted Wednesday that the U.S. has already met or surpassed most border security benchmarks that the Senate came up with during immigration reform debates in 2007.
He had said that during their trip, the senators would find a border with increased Border Patrol staffing, better infrastructure and fewer people attempting to cross.
“We have made tremendous progress on border security, and it’s time to stop moving the goalposts,” Noorani said. “Our border will be stronger and even more secure once our immigration system is reformed.”
He added that he hopes the senators and their colleagues in Congress will come back from their two-week break “ready to push reform across the finish line.”
Senators should get a broad view of the border
Arizona Gov. Jan Brewer took a similar tour of the southern border in February. After meeting with Border Patrol agents and ranchers living in border towns, she concluded that the border is not secure and called for additional resources to improve border security.
Immigrant rights groups criticized Brewer for meeting only with people, including ranchers and officers of the National Border Patrol Council, who share her views about border security. They argued that there are many border residents who feel secure because of the border enforcement that has been added in recent years.
Arizona Daily Star columnist Tim Steller indicated Wednesday he hopes the senators won’t repeat what Brewer did during her tour of the border and instead meet with townspeople and rural residents in Southern Arizona who have different views of the border.
“It’s especially important to get broad points of view this year, when proposals for immigration reform are hinging on measures of border security,” he wrote in a column.


Share to Twitter Share to Linkedin After 18 years as CEO in Kaiser Permanente , I set my sights on improving the heatlh of the nation, hoping to find a way to achieve the same quality, technology and affordability our medical group delivered to 5 million patients on both coasts. That quest launched the Fixing Healthcare podcast in 2018, and it inspired interviews with dozens of leaders, thinkers and doers, both in and around medicine. These experts shared innovative ideas and proven solutions for achieving (a) superior quality, (b) improved patient access, (c) lower overall costs, and (d) greater patient and clinician satisfaction. This month, after 150 combined episodes , three questions emerged: Which of the hundreds of ideas presented remain most promising? Why, after five years and so many excellent solutions, has our nation experienced such limited improvements in healthcare? And finally, how will these great ideas become reality? … [Read more...] about 15 Innovative Ideas For Fixing Healthcare From 15 Brilliant Minds
Vallibel finance fixed deposit interest rates
Switzerland’s secretive Credit Suisse rescue rocks global finance
Federal Councillor and chief of the finance federal department Karin Keller-Sutter, Swiss Federal Council (Bundesrat) President and chief of the interior federal department, Alain Berset, Chairman of the Swiss National Bank Thomas Jordan and Chair of the Board of Swiss Financial Market Supervisory Authority (FINMA), Marlene Amstad attend a news conference on Credit Suisse after UBS takeover offer, in Bern, Switzerland, March 19, 2023. REUTERS/Denis Balibouse/File photo ZURICH – Days before a hastily convened press conference late on Sunday that would make the world’s front pages, Switzerland’s political elite were secretly preparing a move that would jolt the globe. While the nation’s central bank and financial regulator publicly declared that Credit Suisse was sound, behind closed doors the race was on to rescue the nation’s second-biggest bank. The chain of events, led to the erasure of one of Switzerland’s flagships, a merger backed by 260 billion Swiss francs ($280 billion) … [Read more...] about Switzerland’s secretive Credit Suisse rescue rocks global finance
Are The Superrich More Burdened And Paying The Highest Tax Rates?
Share to Twitter Share to Linkedin The 27,000 Americans with more than $10 million of income have lower average federal income tax rates than the 580,000 Americans with income between $1 million and $10 million. And though the share of federal income tax revenue from taxpayers with income in the top percentile rose from 33.2 percent in 2001 to 42.3 percent in 2020, that doesn’t mean the affluent are more oppressed or less well off. That’s because over the two decades spanning 2001 through 2020, their share of total before-tax income has risen, and their average effective income tax rate has declined. Those observations, and others discussed below, add important context to — and counter the thrust of — assertions by the editorial board of The Wall Street Journal . First, about the income tax code, the board noted: “There is no denying that it is steeply progressive.” And second, it said, “The trend over the past two decades is that the income tax burden has … [Read more...] about Are The Superrich More Burdened And Paying The Highest Tax Rates?
BSP tipped to raise key rate to 6.25%
Photo courtesy of Bangko Sentral ng Pilipinas Facebook Page MANILA – The Bangko Sentral ng Pilipinas (BSP) is expected to raise its policy rate by 25 basis points or 0.25 percentage point to 6.25 percent on March 23 amid lingering concerns about the health of the global financial system. This will represent a slower tightening that, according to ING Bank, might pave the way for a likely pause of hikes in May. Before the latest policy meeting scheduled on Thursday, the Monetary Board (MB) went for hikes of 0.5 ppt on two previous occasions—February and December 2022. This, in turn, followed a 0.75-ppt hike in November last year. Since starting the tightening cycle last May, the BSP’s key rate has risen by 4 ppt from a pandemic-prompted historic low of 2 percent. Commitment Nicholas Mapa, senior economist at ING Bank, said a smaller rate hike this week will serve as an “insurance” considering that inflation remained well-above the government’s target range at 8.6 percent in … [Read more...] about BSP tipped to raise key rate to 6.25%
Prepaid College Savings Plan Under Fire In Maryland: Should You Be Worried?
Share to Twitter Share to Linkedin Prepaid college savings plans have long been an alternative to a 529 plans when it comes to saving for college. What is a prepaid college tuition plan ? Generally speaking, these plans let parents lock in college tuition at today's prices with the goal of funding school ahead of time. The idea behind these plans is letting families purchase college units or credits with a lump sump payment, or through regular payments, at which point the plan administrator invests the money on their behalf. With the money stashed away and invested, the goal of these accounts is growing the underlying funds to keep up with rising tuition costs until the child reaches college age. At that point, the funds are transferred to the school to cover tuition costs and other eligible higher education expenses. While that all sounds good and well, something is going wrong in the prepaid college tuition industry — at least in the state of … [Read more...] about Prepaid College Savings Plan Under Fire In Maryland: Should You Be Worried?
Five Ways To Get The Most From Your 2023 Financial Check-Up
Share to Twitter Share to Linkedin If you’re working with a financial advisor, you’re probably accustomed to meeting with them in-person or via video conference at least once per year. This financial check-up is similar to your annual physical, in that it helps you assess where you are now, how much progress you’ve made over the past 12 months, and the direction you’re headed in. Here are 5 tips for getting the most from your next annual financial check-up. 1. Paint a vivid, detailed description of your financial goals. Communication is key, so express your financial and life goals concretely and as vividly as possible. Clearly state your vision for the future. If you say, “Here’s exactly what I want my life to look like from a financial standpoint in 7 years,” who could accuse you of being vague or “wishy-washy,” as I like to call it. Broad statements like “I want to leave money to my kids” just cry out for elaboration. In this case, be specific … [Read more...] about Five Ways To Get The Most From Your 2023 Financial Check-Up
Student Loan Forgiveness Timeline And Deadlines Change Again In New Updates To One-Time Adjustment
Share to Twitter Share to Linkedin The Biden administration has again updated key deadlines and the overall timeline for borrowers hoping to receive student loan forgiveness under a one-time IDR account adjustment . No formal announcement accompanied the quiet change to current Education Department guidance , which occurred this week. Here’s what borrowers should know. IDR Account Adjustment Can Provide Student Loan Forgiveness Benefits Last year, the Biden administration announced the IDR Account Adjustment, a sweeping one-time fix to address ongoing issues with federal Income Driven Repayment (IDR) plans. IDR, which describes a collection of individual plans, allows borrowers to make payments on their federal student loans according to a formula based on their income and family size. IDR plans can also result in student loan forgiveness for any remaining unpaid balance after 20 or 25 years, depending on the specific plan. Historically, … [Read more...] about Student Loan Forgiveness Timeline And Deadlines Change Again In New Updates To One-Time Adjustment
Why 186 Other Banks Could Go The Way Of Silicon Valley Bank
Share to Twitter Share to Linkedin There were no new bank failures over the weekend. But the global banking system is not in the pink of health. There are two reasons for continued concern: A March 13 study by four economists reveals that 186 banks are at risk of “impairment” of $300 billion worth of insured deposits A March 19 New York Times report reveals that the Federal Reserve began warning Silicon Valley Bank (SVB) of financial risks in 2021 — over a year before it cratered — yet SVB management did nothing. What’s more Moody’s warned SVB’s management of an imminent downgrade about a month before it announced its emergency capital raise. Shares of First Republic Bank FRC are trading well below where they were after it received $30 billion in deposits from other banks on March 16. It seems only a matter of time before more rot in the banking system comes to light. (I have no financial interest in the securities mentioned in this … [Read more...] about Why 186 Other Banks Could Go The Way Of Silicon Valley Bank
After Collecting Millions From Wall Street, Janet Yellen Now Helps Decide Which Banks Get Bailouts
After raking in over $7 million in speaking fees from financial institutions in the two years before being confirmed, Treasury Secretary Janet Yellen now plays a major role in determining which banks are systemically important, according to her testimony at a Senate Finance Committee hearing Thursday. Yellen had received more than $7 million in speaking fees from dozens of companies including Goldman Sachs, Bank of America, Citi, Credit Suisse, UBS, Charles Schwab and many more, according to Yellen’s public financial disclosure report. Though the Federal Deposit Insurance Corporation insures deposits up to $250,000, Yellen told the senators the government may insure additional deposits, as it did at the recently failed Silicon Valley Bank (SVB) and Signature Bank, if that bank’s failure would present a “systemic risk” to the economy. WASHINGTON, DC – MARCH 16: U.S. Treasury Secretary Janet Yellen testifies about the Biden Administration’s FY2024 federal budget proposal before … [Read more...] about After Collecting Millions From Wall Street, Janet Yellen Now Helps Decide Which Banks Get Bailouts
Wall St ends higher as bank contagion fears ease, Fed eyed
NEW YORK – U.S. stocks jumped on Monday after a deal to rescue Credit Suisse and central bank efforts to bolster confidence in the financial system relieved investors, while participants also weighed the likelihood of a pause in rate hikes from the Federal Reserve this week. UBS late on Sunday agreed to buy rival Credit Suisse for $3.23 billion, in a merger engineered by Swiss authorities to avoid more turmoil in the banking group. Also, major central banks moved on Sunday to bolster the flow of cash around the world. Global central banks open daily dollar taps to help banks The S&P Banking index was up 0.6 percent and the KBW Regional Banking index was up 1.5 percent following sharp losses last week. The collapse of Silicon Valley Bank and Signature Bank shook markets earlier this month, leading to a rout in banking stocks and worries that central bank monetary tightening would create a recession. While some bank shares were still lower on Monday, the … [Read more...] about Wall St ends higher as bank contagion fears ease, Fed eyed


Music from MIDNITE DA DREAD
Hy·brid [ˈhīˌbrid] NOUN a thing made by combining two different elements;
Today's technology has made music easier to create, therefore there are more artists expressing their feelings, and thoughts to the world. Listeners complain that artists sound alike, and are not able to release albums that you can enjoy from beginning, to end. There is relief within an earshot as we bring you Midnite Da Dread with his latest project: The Hybrid.
The architect of The Hybrid, Midnite Da Dread was born in St. Thomas V.I. and was raised in the Miami Fl. area. Growing up in South Florida, he was exposed to many different cultures which has helped to shape his unique cadence, and sound. He took time to harness his ability, and skill, while performing on many stages, and venues throughout the southeast. Midnite Da Dread continued his musical journey out west recording, performing, and residing in the Los Angeles, and Phoenix Areas. It took Midnite Da Dread over two years to put this project together. “I could have easily put as many as three albums out within the same time span, but I wanted to release something that was musically, and lyrically strong, and could stand the test of time,” explained by Midnite Da Dread.
The Hybrid style, and sound is a gritty mash up of dancehall reggae, and hip hop. The collection of songs on this project are strong. The rhythms provide anthems for the club, something to drive to, or something to vibe to. The track Hollywood Hillz has a nice melodic feel that provides the background of life, pain, and a vision for tomorrow. Bezerk is the First single off of the album, guaranteed to catch your ear.
The Hybrid is a must listen musical experience that is engaging from the songs to the skits. Midnite Da dread has style from the islands, attitude from the streets and has created a soon to be classic album. We encourage you to smoke, sip, enjoy -Repeat..!!!
The Hybrid from Midnite Da Dread
For Booking info:email@example.com
For MP3s and drops:firstname.lastname@example.org
Midnite Promo Vid.


text:
Date: September 24, 2020
Time: 10:00 AM Central
The National Academy of Medicine (NAM) Leadership Consortium will host a webinar on Thursday, September 24, 11 a.m. – 1:45 p.m. EDT. This HRSA-supported webinar will explore approaches and sector-by-sector opportunities to strengthen, transform, and reimagine how health care is delivered across the nation in the wake of COVID-19. U.S. Surgeon General VADM Jerome M. Adams will kick off the webinar with a keynote address. A webinar recording will be posted on the NAM website.


text:
Premier Dalton McGuinty has announced a provincial government commitment to protect an area one and a half times the size of the Maritimes in Ontario’s far North.
Fifty per cent or 22.5 million acres North of the 51st parallel will be protected from industrial development
“Although the Northern Boreal region has remained virtually undisturbed since the retreat of the glaciers, change is inevitably coming to these lands,” said Premier McGuinty in the announcement. “We need to prepare for this development and plan for it. It’s our responsibility as global citizens to get this right and to act now.”
“We need to find a balance,” reflected Minister of Northern Development and Mines, Michael Gravelle. “We have tremendous opportunities many of which First Nations communities are eager to move forward on but we need to preserve our north. Can we find a way to do both? I think we can.”
The region is home to 24,000 people living in 36 communities, the majority of which are First Nations north of Ontario’s grid of roads and power lines. Those communities will be able to develop their own land use plans within the context of the moratorium on development.
The boreal forests of the world store more carbon in trees, soil and peat than any other ecosystem.
Gillian McEachran, the senior boreal campaigner of the environmentalist organization ForestEthics, applauded protection of one of the largest remaining intact forest patches in the world in what she called “the largest commitment to conservation in Canada”.
“The premier has really stepped up and shown leadership, raising the bar for the whole planet.”
She points out the northern boreal forest stores 97 billion tones of carbon and that its maintenance will be essential for enabling species to react as the earth’s climate warms.
Scott Jackson, the policy manager of the Ontario Forest Industries Association, found the announcement to be short on details but stressed the 24 million cubic meters promised to the industry will not be compromised for the sake of forestry industry shareholders and sector workers.
“What we can say is that we hope the announcement won’t result in additional business uncertainty in this province. It’s something that the province can’t afford right now.”
The association cited a report from the Ministry of Natural Resources indicating the carbon stored in products of the forestry industry is four to five times greater than that stored in standing forests. Scott Jackson and association president Jamie Lim held up the “platinum standards” of the province’s forestry practices, reiterating their commitment to combating climate change.
“If you’re a country practicing deforestation, that’s not the case, but here in Ontario, because we reforest, that’s huge,” Jamie Lim stated.
“Remember when we’re taking about the boreal forest,” said Minister of Natural Resources Donna Cansfield. “The area of undertaking, we’re talking about the far North.”
Minister Cansfield anticipated that as the roadmap begins to take shape, it will be implemented hand in glove with the standards of the new Endangered Species Act, but that there is little to no logging currently occurring in most of the area under consideration.
As the provincial export-led mining industry generates $5-7 billion annually, the stakes are also high.
The province has been under intensifying pressure to change the 1873 Mining Act since the spring incarceration of Bob Lovelace of Ardoch Algonquin First Nation and six community leaders from Kitchenuhmaykoosib Inninwug. A parallel, united lobby of 30 environmental and human rights organizations has been vocal on the need for change.
“That was an example of how things can go wrong,” said Gravelle of the so-called KI-6 imprisonment. “That’s why we are formally bringing the consultation on the Mining Act forward in August.”
Promising to work with First Nations, industry, the Ministry of Natural Resources and the Ministry of Aboriginal Affairs, Minister Gravelle committed to have revisions on the act completed by Christmas.
Peter McBride, the manager for communications at the Ontario Mining Association, pointed out the figures in Monday’s announcement include vast tracts of land that will be nowhere near communities and that it marked the instigation of the process, not a full plan.
“The reality in Northern Ontario is outside of the confines of Thunder Bay, Kenora, Sioux Lookout, Red Lake is that you would need two decades to gather enough information to develop a viable land use plan. The government is saying that they’re going to have a land use plan in 10 to 15 years? We’ll be at the table, but it’s a huge task.”
Mr. McBride complimented Minister Gravelle’s offices on narrowing the scope of the review to First Nations consultation and claim staking, stabilizing the confidence of his organization’s membership and stockholders.
…this post forwarded by Windsor Humanist, Matt Achine, after a July 15, 2008 article by Jon Thompson in The Kenora Miner & News

domain: geography