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A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1⁺ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/10⁶ CD4⁺ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/10⁶ CD4⁺ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.

BACKGROUNDThe relative stabilities of the intact and defective HIV genomes over time during effective antiretroviral therapy (ART) have not been fully characterized.METHODSWe used the intact proviral DNA assay (IPDA) to estimate the rate of change of intact and defective proviruses in HIV-infected adults on ART. We used linear spline models with a knot at seven years and a random intercept and slope up to the knot. We estimated the influence of covariates on rates of change.RESULTSWe studied 81 individuals for a median of 7.3 (IQR 5.9-9.6) years. Intact genomes declined more rapidly from initial suppression through seven years (15.7% per year decline; 95% CI -22.8%, -8.0%) and more slowly after seven years (3.6% per year; 95% CI -8.1%, +1.1%). The estimated half-life of the reservoir was 4.0 years (95% CI 2.7-8.3) until year seven and 18.7 years (95% CI 8.2-infinite) thereafter. There was substantial variability between individuals in the rate of decline until year seven. Intact provirus declined more rapidly than defective provirus (P < 0.001) and showed a faster decline in individuals with higher CD4+ T cell nadirs.CONCLUSIONThe biology of the replication-competent (intact) reservoir differs from that of the replication-incompetent (non-intact) pool of proviruses. The IPDA will likely be informative when investigating the impact of interventions targeting the reservoir.FUNDINGDelaney AIDS Research Enterprise, UCSF/Gladstone Institute of Virology & Immunology CFAR, CFAR Network of Integrated Systems, amfAR Institute for HIV Cure Research, I4C and Beat-HIV Collaboratories, Howard Hughes Medical Institute, Gilead Sciences, Bill and Melinda Gates Foundation.

The persistence of latent HIV-1 proviruses in CD4+ T cells is a major obstacle to curing HIV. The “shock and kill” strategy involves reversing latency with latency-reversing agents (LRAs) and selectively inducing cell death in infected cells. However, current LRAs have shown limited efficacy in eliminating the ex vivo HIV reservoir and thus failed in clinical study. In this study, we repurposed PZ703b, a pro-apoptotic protein degrader initially developed for anti-leukemia therapy, to target HIV eradication. PZ703b induced the degradation of Bcl-2 and Bcl-xL, activating the non-canonical NF-kB pathway and caspases cascade, resulting in latency reversal and the selective apoptosis of infected cells. The treatment of ex vivo CD4+ T cells from ART-suppressed HIV-1 patients led to approximately a 50% reduction in the replication-competent reservoir. While this result does not reach the threshold required for a complete cure, it demonstrates the potential of a dual degrader of Bcl-2/Bcl-xL in reversing HIV latency and inducing selective cell death. Our study provides a proof-of-concept for using dual degraders of Bcl-2/Bcl-xL as a novel category of LRAs in therapeutic strategies aimed at reducing HIV reservoirs. This approach may pave the way for the further exploration of targeted interventions to eliminate the HIV-inducible reservoir.

Despite antiretroviral therapy (ART), HIV persists in latently-infected cells (the HIV reservoir) which decay slowly over time. Here, leveraging >500 longitudinal samples from 67 people living with HIV (PLWH) treated during acute infection, we developed a mathematical model to predict reservoir decay from peripheral CD4 + T cells. Nonlinear generalized additive models demonstrated rapid biphasic decay of intact DNA (week 0-5: t 1/2  ~ 2.83 weeks; week 5-24: t 1/2  ~ 15.4 weeks) that extended out to 1 year. These estimates were ~5-fold faster than prior decay estimates among chronic treated PLWH. Defective DNA had a similar biphasic pattern, but data were more variable. Predicted intact and defective decay rates were faster for PLWH with earlier timing of ART initiation, higher initial CD4 + T cell count, and lower pre-ART viral load. In this study, we advanced our limited understanding of HIV reservoir decay at the time of ART initiation, informing future curative strategies targeting this critical time. HIV reservoir decay is less well studied in acute infection. Here, the authors show that reservoir decay rates are biphasic and 5x faster in people initiating antiretroviral therapy during acute HIV than prior estimates for chronic HIV. Higher initial CD4+ counts and lower viral loads predicted faster decay.

The persistence of latent HIV-1 proviruses in CD4+ T cells is a major obstacle to curing HIV. The “shock and kill” strategy involves reversing latency with latency-reversing agents (LRAs) and selectively inducing cell death in infected cells. However, current LRAs have shown limited efficacy in eliminating the ex vivo HIV reservoir. We repurposed PZ703b, a pro-apoptotic protein degrader initially developed for anti-leukemia therapy, to target HIV eradication. PZ703b induced degradation of Bcl-2 and Bcl-xL, activating the non-canonical NF-kB pathway and caspases cascade, resulting in latency reversal and selective apoptosis of infected cells. Treatment of ex vivo CD4+ T cells from ART-suppressed HIV-1 patients achieved a ∼50% reduction in the replication-competent reservoir. Our study provides proof-of-concept for using protein degraders to reverse HIV latency and induce cell death, highlighting PZ703b’s potential in HIV cure strategies. This approach may pave the way for novel therapeutic interventions aimed at eliminating the HIV-inducible reservoir.

Jan. 2--Bordentown Township police are looking for the driver of a car that struck and killed a 15-year-old Fieldsboro girl on Saturday night. Ashlee Mohacsi was unconscious about 7 p.m. when police arrived after receiving a telephone call about a pedestrian struck on Burlington Road near the I-295 overpass. Mohacsi, a Bordentown Regional High School student, was taken to Robert Wood Johnson Hospital in Hamilton, N.J., where she died around 10:30 p.m. She was walking in the road's eastbound lane when she was struck from behind and thrown onto the shoulder, police said. The driver did not stop. Mohacsi's brother Steve said she was hit while talking on her cell phone and walking with a friend to Burlington City, where the two had New Year's Eve plans.

We use a perceptual control model of identity to examine the relationship between stigmatized appraisals (from self and other) and well-being among individuals with serious mental illness. We also examine the role of stigma resistance strategies in the identity process. Using in-depth interviews with active clients of a community mental health center (N = 156), we find that deflection, or distancing oneself from mental illness, is associated with greater self-esteem and fewer depressive symptoms. Challenging others through education is associated with higher self-esteem, and challenging stigma through activism is associated with fewer depressive symptoms. Activism also moderates the relationship between identity discrepancy (the difference between appraisals from self and other) and well-being; however, the extent to which activism is helpful or harmful depends on whether appraisals from others are more or less stigmatizing than self-views. We discuss the implications of these findings for identity and stigma research.

Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) and the nuclear receptor co-repressor1 (NCoR1) are paralogs and regulate nuclear receptor (NR) function through the recruitment of a multiprotein complex that includes histone deacetylase activity. Previous genetic strategies which deleted SMRT in a specific tissue or which altered the interaction between SMRT and NRs have suggested that it may regulate adiposity and insulin sensitivity. However, the full role of SMRT in adult mice has been difficult to establish because its complete deletion during embryogenesis is lethal. To elucidate the specific roles of SMRT in mouse target tissues especially in the context of thyroid hormone (TH) signaling, we used a tamoxifen-inducible post-natal disruption strategy. We found that global SMRT deletion causes dramatic obesity even though mice were fed a standard chow diet and exhibited normal food intake. This weight gain was associated with a decrease in energy expenditure. Interestingly, the deletion of SMRT had no effect on TH action in any tissue but did regulate retinoic acid receptor (RAR) function in the liver. We also demonstrate that the deletion of SMRT leads to profound hepatic steatosis in the setting of obesity. This is unlike NCoR1 deletion, which results in hepatic steatosis due to the upregulation of lipogenic gene expression. Taken together, our data demonstrate that SMRT plays a unique and CoR specific role in the regulation of body weight and has no role in TH action. This raises the possibility that additional role of CoRs besides NCoR1 and SMRT may exist to regulate TH action.

BackgroundA neurodevelopmental syndrome was recently reported in four patients with SOX4 heterozygous missense variants in the high-mobility-group (HMG) DNA-binding domain. The present study aimed to consolidate clinical and genetic knowledge of this syndrome.MethodsWe newly identified 17 patients with SOX4 variants, predicted variant pathogenicity using in silico tests and in vitro functional assays and analysed the patients’ phenotypes.ResultsAll variants were novel, distinct and heterozygous. Seven HMG-domain missense and five stop-gain variants were classified as pathogenic or likely pathogenic variant (L/PV) as they precluded SOX4 transcriptional activity in vitro. Five HMG-domain and non-HMG-domain missense variants were classified as of uncertain significance (VUS) due to negative results from functional tests. When known, inheritance was de novo or from a mosaic unaffected or non-mosaic affected parent for patients with L/PV, and from a non-mosaic asymptomatic or affected parent for patients with VUS. All patients had neurodevelopmental, neurological and dysmorphic features, and at least one cardiovascular, ophthalmological, musculoskeletal or other somatic anomaly. Patients with L/PV were overall more affected than patients with VUS. They resembled patients with other neurodevelopmental diseases, including the SOX11-related and Coffin-Siris (CSS) syndromes, but lacked the most specific features of CSS.ConclusionThese findings consolidate evidence of a fairly non-specific neurodevelopmental syndrome due to SOX4 haploinsufficiency in neurogenesis and multiple other developmental processes.