Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability.

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

Mosaic and mixed HIV-1 glycoprotein nanoparticles elicit antibody responses to broadly neutralizing epitopes.

An effective human immunodeficiency virus 1 (HIV-1) vaccine will most likely have to elicit broadly neutralizing antibodies (bNAbs) to overcome the sequence diversity of the envelope glycoprotein (Env). So far, stabilized versions of Env, such as SOSIP trimers, have been able to induce neutralizing antibody (NAb) responses, but those responses are mainly strain-specific. Here we attempted to broaden NAb responses by using a multivalent vaccine and applying a number of design improvements. First, we used highly stabilized SOSIP.v9 trimers. Second, we removed any holes in the glycan shields and optimized glycan occupancy to avoid strain-specific glycan hole responses. Third, we selected five sequences from the same clade (B), as we observed previously that combining Env trimers from clade A, B and C did not improve cross-reactive responses, as they might have been too diverse. Fourth, to improve antibody (Ab) responses, the Env trimers were displayed on two-component I53-50 nanoparticles (NPs). Fifth, to favor activation of cross-reactive B cells, the five Env trimers were co-displayed on mosaic NPs. Sixth, we immunized rabbits four times with long intervals between vaccinations. These efforts led to the induction of cross-reactive B cells and cross-reactive binding Ab responses, but we only sporadically detected cross-neutralizing responses. We conclude that stabilized HIV-1 Env trimers that are not modified specifically for priming naive B cells are unable to elicit strong bNAb responses, and infer that sequential immunization regimens, most likely starting with specific germline-targeting immunogens, will be necessary to overcome Env's defenses against the induction of NAbs. The antigens described here could be excellent boosting immunogens in a sequential immunization regimen, as responses to bNAb epitopes were induced.

Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions.

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

Sociodemographic variation in the utilization of minimally invasive surgical approaches for pancreatic cancer.

BACKGROUND: Minimally invasive pancreatic surgery (MIPS), when selectively utilized, has been shown to hasten recovery with outcomes comparable to open approaches, but access may not be equitable. This study explored variation in utilization of MIPS for pancreatic cancer. METHODS: The National Cancer Database was queried to identify patients diagnosed with a primary pancreatic neoplasm from 2010 to 2020. Study participants had diagnoses of clinical or pathologic stage 1-3 disease and received curative-intent surgery. Multivariable analyses assessed the association between surgical approach and patient and disease factors. RESULTS: Inclusion criteria identified 73,137 patients: 51,408 underwent open surgery and 21,729 received MIPS. In our multivariable analysis, Black race was associated with reduced odds of MIPS (AOR 0.88; p = 0.02), while older age (AOR 1.17; p = 0.01), later year of diagnosis (AOR 1.57; p < 0.001), and private insurance coverage (AOR 1.30; p = 0.05) were associated with increased odds. When patients with adenocarcinoma were analyzed in isolation, disparities in MIPS utilization persisted even when controlling for disease stage. CONCLUSION: Sociodemographic factors like age, race, and insurance coverage appear to vary in the utilization of MIPS technologies for the treatment of pancreatic malignancy. Addressing variation with robust mixed methods approaches in the future is proposed to incorporate prospective interventions with highly annotated outcomes for additional study.

Inhibition of human cytomegalovirus replication by interferon alpha can involve multiple anti-viral factors.

The shortcomings of current direct-acting anti-viral therapy against human cytomegalovirus (HCMV) has led to interest in host-directed therapy. Here we re-examine the use of interferon proteins to inhibit HCMV replication utilizing both high and low passage strains of HCMV. Pre-treatment of cells with interferon alpha (IFNα) was required for robust and prolonged inhibition of both low and high passage HCMV strains, with no obvious toxicity, and was associated with an increased anti-viral state in HCMV-infected cells. Pre-treatment of cells with IFNα led to poor expression of HCMV immediate-early proteins from both high and low passage strains, which was associated with the presence of the anti-viral factor SUMO-PML. Inhibition of HCMV replication in the presence of IFNα involving ZAP proteins was HCMV strain-dependent, wherein a high passage HCMV strain was obviously restricted by ZAP and a low passage strain was not. This suggested that strain-specific combinations of anti-viral factors were involved in inhibition of HCMV replication in the presence of IFNα. Overall, this work further supports the development of strategies involving IFNα that may be useful to inhibit HCMV replication and highlights the complexity of the anti-viral response to HCMV in the presence of IFNα.

Scalable workflow for characterization of cell-cell communication in COVID-19 patients.

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable and scalable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use this to examine eight public single-cell RNA-seq datasets (six from peripheral blood mononuclear cells, one from bronchoalveolar lavage and one from nasopharyngeal), with a total of 211 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients. Overall, this workflow can be generalized and scaled to combine multiple scRNA-seq datasets to uncover cell-cell interactions.

Opto-chemo-mechanical transduction in photoresponsive gels elicits switchable self-trapped beams with remote interactions.

Next-generation photonics envisions circuitry-free, rapidly reconfigurable systems powered by solitonic beams of self-trapped light and their particlelike interactions. Progress, however, has been limited by the need for reversibly responsive materials that host such nonlinear optical waves. We find that repeatedly switchable self-trapped visible laser beams, which exhibit strong pairwise interactions, can be generated in a photoresponsive hydrogel. Through comprehensive experiments and simulations, we show that the unique nonlinear conditions arise when photoisomerization of spiropyran substituents in pH-responsive poly(acrylamide-co-acrylic acid) hydrogel transduces optical energy into mechanical deformation of the 3D cross-linked hydrogel matrix. A Gaussian beam self-traps when localized isomerization-induced contraction of the hydrogel and expulsion of water generates a transient waveguide, which entraps the optical field and suppresses divergence. The waveguide is erased and reformed within seconds when the optical field is sequentially removed and reintroduced, allowing the self-trapped beam to be rapidly and repeatedly switched on and off at remarkably low powers in the milliwatt regime. Furthermore, this opto-chemo-mechanical transduction of energy mediated by the 3D cross-linked hydrogel network facilitates pairwise interactions between self-trapped beams both in the short range where there is significant overlap of their optical fields, and even in the long range--over separation distances of up to 10 times the beam width--where such overlap is negligible.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

Interventions Including Smart Technology Compared With Face-to-face Physical Activity Interventions in Older Adults: Systematic Review and Meta-analysis.

BACKGROUND: This is a systematic review of randomized controlled trials and a meta-analysis comparing smart technology with face-to-face physical activity (PA) interventions in community-dwelling older adults (mean age 60 years). OBJECTIVE: This study aims to determine the effect of interventions including smart technology components compared with face-to-face PA interventions on PA and physical function in older adults. The secondary outcomes are depression, anxiety, and health-related quality of life. METHODS: We searched MEDLINE, Embase, CINAHL, and AMED electronic databases from inception to February 2021. Two independent reviewers screened titles, abstracts, and full texts and performed data extraction and risk of bias assessments using the Cochrane risk of bias tool. The Grading of Recommendations Assessment, Development and Evaluation was used to evaluate the quality of the evidence. We provided a narrative synthesis on all included studies and, where possible, performed meta-analyses for similar outcomes. RESULTS: This review included 19 studies with a total of 3455 participants. Random effects meta-analyses showed that interventions with smart technology components resulted in improved step count (mean difference 1440 steps, 95% CI 500-2390) and total PA (standardized mean difference 0.17, 95% CI 0.02-0.32) compared with face-to-face alone. There was no difference between groups in terms of the measures of physical function. Smart technology alone did not show significant differences between groups in any outcome. The quality of the evidence was very low based on the Grading of Recommendations Assessment, Development and Evaluation criteria. CONCLUSIONS: Interventions that include smart technology may improve daily step counts by an average of 1440 steps in community-dwelling older adults; however, the quality of the evidence was very low. Future studies are needed to improve the certainty of these results. TRIAL REGISTRATION: PROSPERO CRD42020135232; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=135232.