Effect of transcription inhibition and generation of suppressive viral non-coding RNAs.

BACKGROUND: HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo. RESULTS: Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state. CONCLUSIONS: Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of "RNA Machines" that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.

Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles.

Background: Ebola virus (EBOV) mainly targets myeloid cells; however, extensive death of T cells is often observed in lethal infections. We have previously shown that EBOV VP40 in exosomes causes recipient immune cell death. Methods: Using VP40-producing clones, we analyzed donor cell cycle, extracellular vesicle (EV) biogenesis, and recipient immune cell death. Transcription of cyclin D1 and nuclear localization of VP40 were examined via kinase and chromatin immunoprecipitation assays. Extracellular vesicle contents were characterized by mass spectrometry, cytokine array, and western blot. Biosafety level-4 facilities were used for wild-type Ebola virus infection studies. Results: VP40 EVs induced apoptosis in recipient T cells and monocytes. VP40 clones were accelerated in growth due to cyclin D1 upregulation, and nuclear VP40 was found bound to the cyclin D1 promoter. Accelerated cell cycling was related to EV biogenesis, resulting in fewer but larger EVs. VP40 EV contents were enriched in ribonucleic acid-binding proteins and cytokines (interleukin-15, transforming growth factor-ß1, and interferon-γ). Finally, EBOV-infected cell and animal EVs contained VP40, nucleoprotein, and glycoprotein. Conclusions: Nuclear VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Packaging of cytokines and EBOV proteins into EVs from infected cells may be responsible for the decimation of immune cells during EBOV pathogenesis.

Exosomes from uninfected cells activate transcription of latent HIV-1.

HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1-infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR-gag, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo, especially in the presence of combination antiretroviral therapy.

Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/µl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-ß, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

The diet of Myotis lucifugus across Canada: assessing foraging quality and diet variability.

Variation in prey resources influences the diet and behaviour of predators. When prey become limiting, predators may travel farther to find preferred food or adjust to existing local resources. When predators are habitat limited, local resource abundance impacts foraging success. We analysed the diet of Myotis lucifugus (little brown bats) from Nova Scotia (eastern Canada) to the Northwest Territories (north-western Canada). This distribution includes extremes of season length and temperature and encompasses colonies on rural monoculture farms, and in urban and unmodified areas. We recognized nearly 600 distinct species of prey, of which ≈30% could be identified using reference sequence libraries. We found a higher than expected use of lepidopterans, which comprised a range of dietary richness from ≈35% early in the summer to ≈55% by late summer. Diptera were the second largest prey group consumed, representing ≈45% of dietary diversity early in the summer. We observed extreme local dietary variability and variation among seasons and years. Based on the species of insects that were consumed, we observed that two locations support prey species with extremely low pollution and acidification tolerances, suggesting that these are areas without environmental contamination. We conclude that there is significant local population variability in little brown bat diet that is likely driven by seasonal and geographical changes in insect diversity, and that this prey may be a good indicator of environment quality.

Comparison of Nanotrap® Microbiome A Particles, membrane filtration, and skim milk workflows for SARS-CoV-2 concentration in wastewater.

Introduction: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for Coronavirus Disease 2019 (COVID-19) surveillance. Grab (quantitative) and passive samples (qualitative) are two distinct wastewater sampling methods. Although many viral concentration methods such as the usage of membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. Methods: The objectives of this study were to compare two workflows (Nanotrap® Microbiome A Particles coupled with MagMax kit and membrane filtration workflows coupled with RNeasy kit) for SARS-CoV-2 recovery in grab samples and two workflows (Nanotrap® Microbiome A Particles and skim milk workflows coupled with MagMax kit) for SARS-CoV-2 recovery in Moore swab samples. The Nanotrap particle workflow was initially evaluated with and without the addition of the enhancement reagent 1 (ER1) in 10 mL wastewater. RT-qPCR targeting the nucleocapsid protein was used for detecting SARS-CoV-2 RNA. Results: Adding ER1 to wastewater prior to viral concentration significantly improved viral concentration results (P < 0.0001) in 10 mL grab and swab samples processed by automated or manual Nanotrap workflows. SARS-CoV-2 concentrations in 10 mL grab and Moore swab samples with ER1 processed by the automated workflow as a whole showed significantly higher (P < 0.001) results than 150 mL grab samples using the membrane filtration workflow and 250 mL swab samples using the skim milk workflow, respectively. Spiking known genome copies (GC) of inactivated SARS-CoV-2 into 10 mL wastewater indicated that the limit of detection of the automated Nanotrap workflow was ~11.5 GC/mL using the RT-qPCR and 115 GC/mL using the digital PCR methods. Discussion: These results suggest that Nanotrap workflows could substitute the traditional membrane filtration and skim milk workflows for viral concentration without compromising the assay sensitivity. The manual workflow can be used in resource-limited areas, and the automated workflow is appropriate for large-scale COVID-19 wastewater-based surveillance.

Diet and the evolution of ADH7 across seven orders of mammals.

Dietary variation within and across species drives the eco-evolutionary responsiveness of genes necessary to metabolize nutrients and other components. Recent evidence from humans and other mammals suggests that sugar-rich diets of floral nectar and ripe fruit have favoured mutations in, and functional preservation of, the ADH7 gene, which encodes the ADH class 4 enzyme responsible for metabolizing ethanol. Here we interrogate a large, comparative dataset of ADH7 gene sequence variation, including that underlying the amino acid residue located at the key site (294) that regulates the affinity of ADH7 for ethanol. Our analyses span 171 mammal species, including 59 newly sequenced. We report extensive variation, especially among frugivorous and nectarivorous bats, with potential for functional impact. We also report widespread variation in the retention and probable pseudogenization of ADH7. However, we find little statistical evidence of an overarching impact of dietary behaviour on putative ADH7 function or presence of derived alleles at site 294 across mammals, which suggests that the evolution of ADH7 is shaped by complex factors. Our study reports extensive new diversity in a gene of longstanding ecological interest, offers new sources of variation to be explored in functional assays in future study, and advances our understanding of the processes of molecular evolution.

Living on the EDGE: Canadian Experience With EUS-directed Transgastric ERCP (EDGE) in Patients With Roux-en-Y Gastric Bypass Anatomy.

Introduction: Roux-en-Y gastric bypass (RYGB) surgery imposes anatomic barriers to endoscopic retrograde cholangiopancreatography (ERCP). Potential options for biliary access in these patients include laparoscopic-assisted ERCP or balloon enteroscopy. However, these approaches require specialized equipment and/or operating room personnel and are associated with high rates of failure and adverse events compared to conventional ERCP. A recently described technique, EDGE, is an endoscopic approach which involves accessing the excluded stomach to facilitate ERCP. Objective: The objective of this study is to describe the results of EDGE procedures performed in Canada. Methods: Data were collected from patient cases who had undergone an EDGE procedure across centers in Canada. All patients had a history of RYGB bariatric surgery. In each procedure, a 20-mm diameter lumen-apposing metal stent (LAMS) was deployed under EUS guidance to allow access from the gastric remnant/proximal jejunum to the excluded stomach. Subsequently, during a separate procedure, a duodenoscope was passed through the LAMS to perform ERCP. Following ERCP, the LAMS was replaced with a pigtail stent or APC was used to facilitate closure of the gastro-jejunal/gastro-gastric fistula. Results: The indication for EDGE in the seven included cases was for the treatment of choledocholithiasis (six) or gallstone pancreatitis (one). The technical success rate of the EDGE procedure in these cases was 100%. Clinical success, defined by normalization of bilirubin and symptomatic relief, was observed in all cases. There were no adverse events reported. Conclusion: The results of this series support EDGE as a safe and minimally invasive approach to biliary access and therapy in patients with previous RYGB surgery.

Improved Detection of Herpesviruses from Diluted Vitreous Specimens Using Hydrogel Particles.

Infectious uveitis is a sight-threatening infection commonly caused by herpesviruses. Vitreous humor is often collected for molecular confirmation of the causative agent during vitrectomy and mixed in large volumes of buffered saline, diluting the pathogen load. Here, we explore affinity-capture hydrogel particles (Nanotrap®) to concentrate low abundant herpesviruses from diluted vitreous. Simulated samples were prepared using porcine vitreous spiked with HSV-1, HSV-2, VZV and CMV at 105 copies/mL. Pure undiluted samples were used to test capturing capability of three custom Nanotrap particles (red, white and blue) in a vitreous matrix. We found that all particles demonstrated affinity to the herpesviruses, with the Red Particles having both good capture capability and ease of handling for all herpesviruses. To mimic diluted vitrectomy specimens, simulated-infected vitreous were then serially diluted in 7 mL TE buffer. Diluted samples were subjected to an enrichment protocol using the Nanotrap Red particles. Sensitivity of pathogen detection by qPCR in diluted vitreous increased anywhere between 2.3 to 26.5 times compared to non-enriched specimens. This resulted in a 10-fold increase in the limit of detection for HSV-1, HSV-2 and VZV. These data demonstrated that Nanotrap particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix.

Underwater endoscopic mucosal resection without submucosal injection (UEMR) for large colorectal polyps: A community-based series.

BACKGROUND: Underwater endoscopic mucosal resection without submucosal injection (UEMR) is an appealing therapy for large colorectal polyps. However, this technique is not practiced widely and there are limited data evaluating UEMR in community settings. METHODS: The study comprised patients undergoing UEMR of large (≥20 mm) sessile colorectal lesions at a community-based center. Residual neoplasia was assessed via follow-up colonoscopy. RESULTS: Among 264 lesions (diameter 38 ± 18 mm; range 20-110 mm) 99% were successfully resected with UEMR. Two lesions involving the cecum/IC valve required multiple sessions. There were no cases of perforation or post-polypectomy syndrome. Delayed bleeding occurred in 1.6%, all managed conservatively. Residual neoplasia was present in 5.7% and was amenable to UEMR. CONCLUSION: This large community-based series demonstrated high efficacy and safety of UEMR for large sessile colorectal lesions. The results support UEMR as first-line therapy for these lesions. SUMMARY: Underwater endoscopic mucosal resection without submucosal injection (UEMR) is a recently developed method that has advantages over conventional EMR for treatment of large colorectal lesions. However, UEMR is not practiced widely and there are limited data evaluating this technique in everyday practice. This large community-based series demonstrated high efficacy and safety of UEMR for large sessile colorectal lesions.

Extracellular Vesicle Activation of Latent HIV-1 Is Driven by EV-Associated c-Src and Cellular SRC-1 via the PI3K/AKT/mTOR Pathway.

HIV-1 is a global health crisis that has infected more than 37 million people. Latent reservoirs throughout the body are a major hurdle when it comes to eradicating the virus. In our previous study, we found that exosomes, a type of extracellular vesicle (EV), from uninfected cells activate the transcription of HIV-1 in latent infected cells, regardless of combination antiretroviral therapy (cART). In this study, we investigated the specific mechanism behind the EV activation of latent HIV-1. We found that phosphorylated c-Src is present in EVs of various cell lines and has the ability to activate downstream proteins such as EGFR, initiating a signal cascade. EGFR is then able to activate the PI3K/AKT/mTOR pathway, resulting in the activation of STAT3 and SRC-1, culminating in the reversal of HIV-1 latency. This was verified by examining levels of HIV-1 TAR, genomic RNA and HIV-1 Gag p24 protein in cell lines and primary cells. We found that EVs containing c-Src rescued HIV-1 despite the presence of inhibitors, validating the importance of EV-associated c-Src in latent HIV-1 activation. Lastly, we discovered an increased recruitment of p300 and NF-κB in the nucleus of EV-treated infected cells. Collectively, our data suggest that EV-associated c-Src is able to activate latent HIV-1 via the PI3K/AKT/mTOR pathway and SRC-1/p300-driven chromatin remodeling. These findings could aid in designing new strategies to prevent the reactivation of latent HIV-1 in patients under cART.

Low-Level Ionizing Radiation Induces Selective Killing of HIV-1-Infected Cells with Reversal of Cytokine Induction Using mTOR Inhibitors.

HIV-1 infects 39.5 million people worldwide, and cART is effective in preventing viral spread by reducing HIV-1 plasma viral loads to undetectable levels. However, viral reservoirs persist by mechanisms, including the inhibition of autophagy by HIV-1 proteins (i.e., Nef and Tat). HIV-1 reservoirs can be targeted by the "shock and kill" strategy, which utilizes latency-reversing agents (LRAs) to activate latent proviruses and immunotarget the virus-producing cells. Yet, limitations include reduced LRA permeability across anatomical barriers and immune hyper-activation. Ionizing radiation (IR) induces effective viral activation across anatomical barriers. Like other LRAs, IR may cause inflammation and modulate the secretion of extracellular vesicles (EVs). We and others have shown that cells may secrete cytokines and viral proteins in EVs and, therefore, LRAs may contribute to inflammatory EVs. In the present study, we mitigated the effects of IR-induced inflammatory EVs (i.e., TNF-α), through the use of mTOR inhibitors (mTORi; Rapamycin and INK128). Further, mTORi were found to enhance the selective killing of HIV-1-infected myeloid and T-cell reservoirs at the exclusion of uninfected cells, potentially via inhibition of viral transcription/translation and induction of autophagy. Collectively, the proposed regimen using cART, IR, and mTORi presents a novel approach allowing for the targeting of viral reservoirs, prevention of immune hyper-activation, and selectively killing latently infected HIV-1 cells.

Stem Cell Extracellular Vesicles and their Potential to Contribute to the Repair of Damaged CNS Cells.

Neurological diseases and disorders are leading causes of death and disability worldwide. Many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. As the global burden of these pathologies continues to rise there is a significant need for the development of novel therapeutics. Due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. It is now believed that paracrine factors, such as extracellular vesicles (EVs), play a critical role in the functionality associated with stem cells. The diverse biological cargo contained within EVs are proposed to mediate these effects and, to date, the reparative and regenerative effects of stem cell EVs have been demonstrated in a wide range of cell types. While a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the CNS. Here, we report the isolation, characterization, and functional assessment of EVs from two sources of human stem cells, mesenchymal stem cells and induced pluripotent stem cells. We demonstrate the ability of these EVs to enhance the processes of cellular migration and angiogenesis, which are critical for both normal cellular development as well as cellular repair. Furthermore, we investigate their reparative effects on damaged cells, specifically those with relevance to the central nervous system. Collectively, our data highlight the similarities and differences among these EV populations and support the view that stem cells EV can be used to repair or partially reverse cellular damage. Graphical Abstract Stem cell-derived Extracellular Vesicles (EVs) for repair of damaged cells. EVs isolated from human induced pluripotent stem cells and mesenchymal stem cells contribute to the partial reversal of phenotypes induced by different sources of cellular damage.

Colonoscopic withdrawal times and adenoma detection during screening colonoscopy.

BACKGROUND: Colonoscopy is commonly used to screen for neoplasia. To assess the performance of screening colonoscopy in everyday practice, we conducted a study of the rates of detection of adenomas and the amount of time taken to withdraw the colonoscope among endoscopists in a large community-based practice. METHODS: During a 15-month period, 12 experienced gastroenterologists performed 7882 colonoscopies, of which 2053 were screening examinations in subjects who had not previously undergone colonoscopy. We recorded the numbers, sizes, and histologic features of the neoplastic lesions detected during screening, as well as the duration of insertion and of withdrawal of the colonoscope during the procedure. We compared rates of detection of neoplastic lesions among gastroenterologists who had mean colonoscopic withdrawal times of less than 6 minutes with the rates of those who had mean withdrawal times of 6 minutes or more. According to experts, 6 minutes is the minimum length of time to allow adequate inspection during instrument withdrawal. RESULTS: Neoplastic lesions (mostly adenomatous polyps) were detected in 23.5% of screened subjects. There were large differences among gastroenterologists in the rates of detection of adenomas (range of the mean number of lesions per subject screened, 0.10 to 1.05; range of the percentage of subjects with adenomas, 9.4 to 32.7%) and in their times of withdrawal of the colonoscope from the cecum to the anus (range, 3.1 to 16.8 minutes for procedures during which no polyps were removed). As compared with colonoscopists with mean withdrawal times of less than 6 minutes, those with mean withdrawal times of 6 minutes or more had higher rates of detection of any neoplasia (28.3% vs. 11.8%, P<0.001) and of advanced neoplasia (6.4% vs. 2.6%, P=0.005). CONCLUSIONS: In this large community-based gastroenterology practice, we observed greater rates of detection of adenomas among endoscopists who had longer mean times for withdrawal of the colonoscope. The effect of variation in withdrawal times on lesion detection and the prevention of colorectal cancer in the context of widespread colonoscopic screening is not known. Ours was a preliminary study, so the generalizability and implications for clinical practice need to be determined by future studies.

Purification of High Yield Extracellular Vesicle Preparations Away from Virus.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

An Omics Approach to Extracellular Vesicles from HIV-1 Infected Cells.

Human Immunodeficiency Virus-1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infecting nearly 37 million people worldwide. Currently, there is no definitive cure, mainly due to HIV-1's ability to enact latency. Our previous work has shown that exosomes, a small extracellular vesicle, from uninfected cells can activate HIV-1 in latent cells, leading to increased mostly short and some long HIV-1 RNA transcripts. This is consistent with the notion that none of the FDA-approved antiretroviral drugs used today in the clinic are transcription inhibitors. Furthermore, these HIV-1 transcripts can be packaged into exosomes and released from the infected cell. Here, we examined the differences in protein and nucleic acid content between exosomes from uninfected and HIV-1-infected cells. We found increased cyclin-dependent kinases, among other kinases, in exosomes from infected T-cells while other kinases were present in exosomes from infected monocytes. Additionally, we found a series of short antisense HIV-1 RNA from the 3' LTR that appears heavily mutated in exosomes from HIV-1-infected cells along with the presence of cellular noncoding RNAs and cellular miRNAs. Both physical and functional validations were performed on some of the key findings. Collectively, our data indicate distinct differences in protein and RNA content between exosomes from uninfected and HIV-1-infected cells, which can lead to different functional outcomes in recipient cells.

Presence of Tat and transactivation response element in spinal fluid despite antiretroviral therapy.

OBJECTIVE: The aim of this study was to measure the protein concentration and biological activity of HIV-1 Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART). DESIGN: CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years. METHODS: Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay. RESULTS: Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains. CONCLUSION: Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.

Effect of a time-dependent colonoscopic withdrawal protocol on adenoma detection during screening colonoscopy.

BACKGROUND & AIMS: Screening colonoscopy can prevent cancer by removal of adenomatous polyps. Recent evidence suggests that insufficient time for inspection during overly rapid colonoscope withdrawal may compromise adenoma detection. We conducted a study of the effect of a minimum prespecified time for instrument withdrawal and careful inspection on adenoma detection rates during screening colonoscopy. METHODS: Baseline data consisted of neoplasia detection rates during 2053 screening colonoscopies performed without a specified withdrawal protocol. During a subsequent 13-month period we performed 2325 screening colonoscopies using dedicated inspection techniques and a minimum 8-minute withdrawal time. With colonoscopists comprising the study population, we compared overall and individual rates of neoplasia detection in postintervention procedures with those in baseline examinations. RESULTS: As compared with baseline subjects, postintervention subjects had higher rates of any neoplasia (34.7% vs 23.5%, P < .0001) and of advanced neoplastic lesions per patient screened (0.080 +/- 0.358 vs 0.055 +/- 0.241, P < .01). Twenty-five percent of advanced neoplastic lesions detected in postintervention examinations were 9 mm or less in diameter, versus 10% in baseline examinations (P < .001). Endoscopists with mean withdrawal times of 8 minutes or longer had higher rates of detection of any neoplasia (37.8% vs 23.3%, P < .0001) and of advanced neoplasia (6.6% vs 4.5%, P = .13) compared with those with mean withdrawal times of less than 8 minutes. CONCLUSIONS: After implementing a protocol of careful inspection during a minimum of 8 minutes to withdraw the colonoscope, we observed significantly greater rates of overall and advanced neoplasia detection during screening colonoscopy.