POLE3 is a repressor of unintegrated HIV-1 DNA required for efficient virus integration and escape from innate immune sensing.

Unintegrated retroviral DNA is transcriptionally silenced by host chromatin silencing factors. Here, we used the proteomics of isolated chromatin segments method to reveal viral and host factors associated with unintegrated HIV-1DNA involved in its silencing. By gene silencing using siRNAs, 46 factors were identified as potential repressors of unintegrated HIV-1DNA. Knockdown and knockout experiments revealed POLE3 as a transcriptional repressor of unintegrated HIV-1DNA. POLE3 maintains unintegrated HIV-1DNA in a repressive chromatin state, preventing RNAPII recruitment to the viral promoter. POLE3 and the recently identified host factors mediating unintegrated HIV-1 DNA silencing, CAF1 and SMC5/SMC6/SLF2, show specificity toward different forms of unintegrated HIV-1DNA. Loss of POLE3 impaired HIV-1 replication, suggesting that repression of unintegrated HIV-1DNA is important for optimal viral replication. POLE3 depletion reduces the integration efficiency of HIV-1. POLE3, by maintaining a repressive chromatin structure of unintegrated HIV-1DNA, ensures HIV-1 escape from innate immune sensing in primary CD4+ T cells.

Understanding latent HIV-1 reservoirs through host genomics approaches.

Genetically intact HIV-1 proviruses are a major concern with regard to curing infection because they cause viral rebound after the cessation of antiretroviral therapy. However, intact proviruses are not prevalent in HIV-1 reservoirs. As such, it is essential to precisely determine the position of these proviruses before putting forward a better antiretroviral cure. Recently, a revised HIV-1 deeply latent reservoir concept has been proposed, stating that the progress of the establishment of HIV-1 reservoirs is influenced by immune-mediated selection during the course of infection. This selection force leads to the persistence of genetically intact proviruses as those with the best fit to avoid clearance. This hypothesis refreshes our understanding of HIV-1 latent reservoirs. For this reason, we reviewed current studies relevant to this theme and provide our perspectives to reinforce the overall understanding of HIV-1 latency in the context of the host genome.

A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level.

The outbreak of SARS-CoV-2 has made us more alert to the importance of viral diagnostics at a population level to rapidly control the spread of the disease. The critical question would be how to scale up testing capacity and perform a diagnostic test in a high-throughput manner with robust results and affordable costs. Here, the latest 26 articles using barcoding technology for COVID-19 diagnostics and biologically-relevant studies are reviewed. Barcodes are molecular tags, that allow proceeding an array of samples at once. To date, barcoding technology followed by high-throughput sequencing has been made for molecular diagnostics for SARS-CoV-2 infections because it can synchronously analyze up to tens of thousands of clinical samples within a short diagnostic time. Essentially, this technology can also be used together with different biotechnologies, allowing for investigation with resolution of single molecules. In this Mini-Review, I first explain the general principle of the barcoding strategy and then put forward recent studies using this technology to accomplish COVID-19 diagnostics and basic research. In the meantime, I provide the viewpoint to improve the current COVID-19 diagnostic strategy with potential solutions. Finally, and importantly, two practical ideas about how barcodes can be further applied in studying SARS-CoV-2 to accelerate our understanding of this virus are proposed.

The Dynamic Linkage between Provirus Integration Sites and the Host Functional Genome Property Alongside HIV-1 Infections Associated with Antiretroviral Therapy.

(1) Background: The HIV-1 latent reservoir harboring replication-competent proviruses is the major barrier in the quest for an HIV-1 infection cure. HIV-1 infection at all stages of disease progression is associated with immune activation and dysfunctional production of proinflammatory soluble factors (cytokines and chemokines), and it is expected that during HIV-1 infection, different immune components and immune cells, in turn, participate in immune responses, subsequently activating downstream biological pathways. However, the functional interaction between HIV-1 integration and the activation of host biological pathways is presently not fully understood. (2) Methods: In this work, I used genes targeted by proviruses from published datasets to seek enriched immunologic signatures and host biological pathways alongside HIV-1 infections based on MSigDb and KEGG over-representation analysis. (3) Results: I observed that different combinations of immunologic signatures of immune cell types and proinflammatory soluble factors appeared alongside HIV-1 infections associated with antiretroviral therapy. Moreover, enriched KEGG pathways were often related to "cancer specific types", "immune system", "infectious disease viral", and "signal transduction". (4) Conclusions: The observations in this work suggest that the gene sets harboring provirus integration sites may define specific immune cells and proinflammatory soluble factors during HIV-1 infections associated with antiretroviral therapy.

The chromatin landscape at the HIV-1 provirus integration site determines viral expression.

HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.

Exploring histone loading on HIV DNA reveals a dynamic nucleosome positioning between unintegrated and integrated viral genome.

The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. We found that histones are loaded on HIV-1 DNA after its nuclear import and before its integration in the host genome. Nucleosome positioning analysis along the unintegrated and integrated viral genomes revealed major differences in nucleosome density and position. Indeed, in addition to the well-known nucleosomes Nuc0, Nuc1, and Nuc2 loaded on integrated HIV-1 DNA, we also found NucDHS, a nucleosome that covers the DNase hypersensitive site, in unintegrated viral DNA. In addition, unintegrated viral DNA-associated Nuc0 and Nuc2 were positioned slightly more to the 5' end relative to their position in integrated DNA. The presence of NucDHS in the proximal region of the long terminal repeat (LTR) promoter was associated with the absence of RNAPII and of the active histone marks H3K4me3 and H3ac at the LTR. Conversely, analysis of integrated HIV-1 DNA showed a loss of NucDHS, loading of RNAPII, and enrichment in active histone marks within the LTR. We propose that unintegrated HIV-1 DNA adopts a repressive chromatin structure that competes with the transcription machinery, leading to its silencing.

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4+ T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.

Using Barcoded HIV Ensembles (B-HIVE) for Single Provirus Transcriptomics.

The latent HIV reservoir is the main barrier to curing AIDS, because infected cells escape the immune system and antiretroviral therapies. Developing new treatment strategies requires technologies to trace latent proviruses. Here, we describe a genome-wide technique called Barcoded HIV Ensembles (B-HIVE) to measure HIV expression at the single provirus level. The principle of B-HIVE is to tag the genome of HIV with DNA barcodes to trace viral transcripts produced by single proviruses in an infected cell population. This in turn reveals which proviruses are active and which are latent or expressed at low level. B-HIVE is a high-throughput method to identify and quantify thousands of individual viral transcripts per round of infection. It can be applied in different conditions, characterizing the response of single proviruses to different treatments. Overall, B-HIVE gives unprecedented insight into the expression of single proviruses in populations of HIV-infected cells. © 2018 by John Wiley & Sons, Inc.

A New Quinoline BRD4 Inhibitor Targets a Distinct Latent HIV-1 Reservoir for Reactivation from Other "Shock" Drugs.

Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or "shock" drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection.IMPORTANCE The suggested "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

Position effects influence HIV latency reversal.

The main obstacle to curing HIV is the presence of latent proviruses in the bodies of infected patients. The partial success of reactivation therapies suggests that the genomic context of integrated proviruses can interfere with treatment. Here we developed a method called Barcoded HIV ensembles (B-HIVE) to map the chromosomal locations of thousands of individual proviruses while tracking their transcriptional activities in an infected cell population. B-HIVE revealed that, in Jurkat cells, the expression of HIV is strongest close to endogenous enhancers. The insertion site also affects the response to latency-reversing agents, because we found that phytohemagglutinin and vorinostat reactivated proviruses inserted at distinct genomic locations. From these results, we propose that combinations of drugs targeting all areas of the genome will be most effective. Overall, our data suggest that the insertion context of HIV is a critical determinant of the viral response to reactivation therapies.

Machine Learning: How Much Does It Tell about Protein Folding Rates?

The prediction of protein folding rates is a necessary step towards understanding the principles of protein folding. Due to the increasing amount of experimental data, numerous protein folding models and predictors of protein folding rates have been developed in the last decade. The problem has also attracted the attention of scientists from computational fields, which led to the publication of several machine learning-based models to predict the rate of protein folding. Some of them claim to predict the logarithm of protein folding rate with an accuracy greater than 90%. However, there are reasons to believe that such claims are exaggerated due to large fluctuations and overfitting of the estimates. When we confronted three selected published models with new data, we found a much lower predictive power than reported in the original publications. Overly optimistic predictive powers appear from violations of the basic principles of machine-learning. We highlight common misconceptions in the studies claiming excessive predictive power and propose to use learning curves as a safeguard against those mistakes. As an example, we show that the current amount of experimental data is insufficient to build a linear predictor of logarithms of folding rates based on protein amino acid composition.

Eccentric mastectomy and zigzag periareolar incision for gynecomastia.

BACKGROUND: Gynecomastia is enlargement of the male breast caused by gland proliferation. Surgery is performed for symptom relief or for cosmetic reasons. The authors used a modified operative procedure, then evaluated the results and safety. METHODS: Between 2001 and 2005, 22 men (median age, 26 years; range, 13-63 years) with gynecomastia underwent surgery. The operative procedure included a zigzag periareolar skin incision, eccentric subcutaneous mastectomy, and liposuction, with postoperative compression. RESULTS: All the patients were satisfied with the results of the surgery, which produced a chest contour resembling a normal male chest rather than simply a smaller breast. The only complication was a hematoma. One patient was found to have breast cancer. CONCLUSIONS: The normal male chest contour can be restored by the described method of eccentric subcutaneous mastectomy.

The two-component system ScnRK of Streptococcus mutans affects hydrogen peroxide resistance and murine macrophage killing.

To survive macrophage killing is critical in the pathogenesis of viridians streptococci-induced infective endocarditis (IE). Streptococcus mutans, an opportunistic IE pathogen, generally does not survive well phagocytic killing in murine macrophage RAW 264.7 cells. A putative two-component system (TCS), ScnR/ScnK from S. mutans, was investigated to elucidate the mechanisms underlying bacteria-cellular interaction in this study. Both the wild-type and mutant strains were phagocytosed by RAW 264.7 cells at a comparable rate and an increased intracellular susceptibility during a 5 h incubation period was observed with the scnRK-null mutants. The amount of reactive oxygen species (ROS) in activated macrophages was reduced significantly after ingesting wild-type, but not scnRK-null mutant strains, suggesting that increased macrophage killing of these mutants is due to the impaired ability of S. mutans to counteract ROS. Additionally, both scnR- or scnRK-null mutants were more susceptible to hydrogen peroxide. Interestingly, scnRK expression was unaffected by hydrogen peroxide. These experimental results indicate that scnRK is important in counteracting oxidative stress in S. mutans, and decreased susceptibility to phagocytic killing is at least partly attributable to inhibition of intracellular ROS formation.

Epidemiology of hospitalized burns patients in Taiwan.

Previous studies based on either single hospital data or sampling of specific groups of hospitalized burns victims in Taiwan have provided only minimal epidemiological information. The study is designed to provide additional data on the epidemiology of hospitalized burns patients in Taiwan. Data were obtained from the Burn Injury Information System (BIIS), which brings together information supplied by 34 contracted hospitals. The study time course spanned a 2-year period from July 1997 to June 1999. Patient characteristics (age, sex, education level, etc.), causes and severity of injuries, and medical care measures were explored. A total of 4741 patients were registered with BIIS over the study period. The majority of hospitalized patients (67%) were male. The age distribution of burns patients showed peaks occurring at the age groups of 0-5 and 35-44 years. Over the time course of a day, burn injuries occurred more frequently from 10:00 to 12:00 h and 16:00 to 18:00 h. Injuries suspected as the result of suicide, homicide or child abuse accounted for 4.8% of hospitalized cases. More than 48% of the burns occurred in the home. The leading type of burn injury was scalding, followed by naked flame, explosion, electrical burns, and chemical burns due to caustic or corrosive substances. The mean percent total body surface area (%TBSA) for adults was 19%, and for young children was 12%. The average length of hospital stay was 18 days. In conclusion, children under 5 years and adults between 35 and 44 years of age are two high-risk groups for burn injuries. Corresponding to meal preparation time, hot substances such as boiling water, hot soup, etc. are the most common agents responsible for scalds. Prevention programs for reducing the risk of burn injuries during cooking and eating are required, especially for parents with young children.