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Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multidrug-resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate, because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall-acting (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could serve as effective antimicrobials for the treatment of E. faecium infections.

Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2(KLHDC10) E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.

Objective: The goal of this study was to determine if a 6-week, peer-led intervention improves health literacy and numeracy among people living with HIV (PLWH). Methods: We used a randomized controlled trial with repeated measurements, which included six, 90 minute, group-based training sessions. We recruited PLWH participants (n = 359) from safety-net practices in the New York City Metropolitan area and Rochester, NY. Participants were randomly assigned (1:1) to an intervention group (n = 180) or a control group (n = 179). Outcome measures were collected at baseline, eight weeks post-baseline, and at six months using the Brief Estimate of Health Knowledge and Action-HIV (BEHKA-HIV), the Electronic Health Literacy Scale (eHEALS), the Rapid Estimate of Adult Literacy (REALM), and the Newest Vital Sign (NVS). Results: The intervention group had statistically significant improvements in eHealth literacy and BEHKAHIV compared to the control group. There were no statistically significant changes in general health literacy or numeracy in either group. The intervention had the greatest impact on participants with the lowest levels of eHealth literacy at baseline. Conclusion: The intervention had a positive impact on participants & rsquo; HIV health literacy and eHealth literacy. Practice implications: Our findings have implications for broadening the function of peer-workers in the health care continuum. (c) 2020 Elsevier B.V. All rights reserved.

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III ( EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC(50)s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3 ' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3 ' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

Fc glycosylation profoundly impacts the effector functions of antibodies and often dictates an antibody's pro- or anti-inflammatory activities. It is well established that core fucosylation of the Fc domain N-glycans of an antibody significantly reduces its affinity for Fc gamma RIIIa receptors and antibody-dependent cellular cytotoxicity (ADCC). Previous structural studies have suggested that the presence of a core fucose remarkably decreases the unique and favorable carbohydrate-carbohydrate interactions between the Fc and the receptor N-glycans, leading to reduced affinity. We report here that in contrast to natural core fucose, special site-specific modification on the core fucose could dramatically enhance the affinity of an antibody for Fc gamma RIIIa. The site-selective modification was achieved through an enzymatic transfucosylation with a novel fucosidase mutant, which was shown to be able to use modified alpha-fucosyl fluoride as the donor substrate. We found that replacement of the core L-fucose with 6-azide- or 6-hydroxy-L-fucose (L-galactose) significantly enhanced the antibody's affinity for Fc gamma RIIIa receptors and substantially increased the ADCC activity. To understand the mechanism of the modified fucose-mediated affinity enhancement, we performed molecular dynamics simulations. Our data revealed that the number of glycan contacts between the Fc and the Fc receptor was increased by the selective core-fucose modifications, showing the importance of unique carbohydrate-carbohydrate interactions in achieving high Fc gamma RIIIa affinity and ADCC activity of antibodies. Thus, the direct site-selective modification turns the adverse effect of the core fucose into a favorable force to promote the carbohydrate-carbohydrate interactions.

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.

cerebellum is thought to have a variety of functions because it developed with the evolution of the cerebrum and connects with different areas in the frontoparietal cortices. Like neurons in the cerebral cortex, those in the cerebellum also exhibit strong activity during planning in addition to the execution of movements. However, their specific roles remain elusive. In this article, we review recent findings focusing on preparatory activities found in the primate deep cerebellar nuclei during tasks requiring deliberate motor control and temporal prediction. Neurons in the cerebellum are active during anti-saccade preparation and their inactivation impairs proactive inhibitory control for saccades. Experiments using a self-timing task show that there are mechanisms for tracking elapsed time and regulating trial-by-trial variation in timing, and that the cerebellum is involved in the latter. When predicting the timing of periodic events, the cerebellum provides more accurate temporal information than the striatum. During a recently developed synchronized eye movement task, cerebellar nuclear neurons exhibited periodic preparatory activity for predictive synchronization. In all cases, the cerebellum generated preparatory activity lasting for several hundred milliseconds. These signals may regulate neuronal activity in the cerebral cortex that adjusts movement timing and predicts the timing of rhythmic events. This article is part of a Special Issue entitled: In Memoriam: Masao Ito?A Visionary Neuroscientist with a Passion for the Cerebellum. ? 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

Background. Limited information is available on pneumococcal colonization among adults. We studied pneumococcal carriage dynamics in healthy adults using high-sensitivity approaches. Methods. Eighty-seven adults (25-50 years old) were followed for 6 months in Portugal. Nasopharyngeal, oropharyngeal, and saliva samples were obtained monthly; pneumococcal carriers were also sampled weekly. Carriage was investigated by quantitative polymerase chain reaction (targeting lytA and piaB) and culture. Positive samples were serotyped. Results. Approximately 20% of the adults were intermittent carriers; 10% were persistent carriers (>4 months). Pneumococcal acquisition and clearance rates were 16.5 (95% confidence interval [Cl], 11.2-24.2) and 95.9 (95% CI, 62.3-145.0) cases/1000 person-weeks, respectively. Living with children increased pneumococcal acquisition (hazard ratio, 9.7 [95% Cl, 2.6-20.5]; P < .001). Median duration of carriage was 7 weeks and did not depend on regular contact with children. Conclusions. The pneumococcal carrier state in healthy adults is more dynamic than generally assumed: Acquisition is frequent and duration of carriage is often long. 'Ibis suggests that some adults may act as reservoirs of pneumococci and hence, depending on the social structure of a community, the magnitude of herd effects potentially attainable through children vaccination may vary. These findings are important when designing strategies to prevent pneumococcal disease in adults.