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The kinetochore proteins CENP-N and CENP-C recognize the histone H3 variant CENP-A in the centromeric nucleosome. This ensures proper kinetochore assembly and accurate segregation of chromosomes. Chittori et al. describe the cryo-electron microscopy structure of the human CENP-A nucleosome-CENP-N complex. The interaction of CENP-N with CENP-A and the nucleosomal DNA together ensure specific and stable centromeric nucleosome recognition. Mutational analyses using both human and Xenopus CENP-A and CENP-N proteins suggest that the proteins have coevolved to preserve the interacting surfaces. Science , this issue p. 339 Cryo–electron microscopy reveals mechanisms of centromeric nucleosome recognition and initial assembly steps of the kinetochore complex. Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by the kinetochore protein CENP-N. We report cryo–electron microscopy (cryo-EM), biophysical, biochemical, and cell biological studies of the interaction between the CENP-A nucleosome and CENP-N. We show that human CENP-N confers binding specificity through interactions with the L1 loop of CENP-A, stabilized by electrostatic interactions with the nucleosomal DNA. Mutational analyses demonstrate analogous interactions in Xenopus , which are further supported by residue-swapping experiments involving the L1 loop of CENP-A. Our results are consistent with the coevolution of CENP-N and CENP-A and establish the structural basis for recognition of the CENP-A nucleosome to enable kinetochore assembly and centromeric chromatin organization.

Members of the bacterial T 6SS a midase e ffector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

The essential architectural protein HMGB1 increases accessibility of nucleosomal DNA and counteracts the effects of linker histone H1. However, HMGB1 is less abundant than H1 and binds nucleosomes more weakly raising the question of how HMGB1 effectively competes with H1. Here, we show that HMGB1 rescues H1's inhibition of nucleosomal DNA accessibility without displacing H1. HMGB1 also increases the dynamics of condensed, H1-bound chromatin. Cryo-EM shows that HMGB1 binds at internal locations on a nucleosome and locally distorts the DNA. These sites, which are away from the binding site of H1, explain how HMGB1 and H1 co-occupy a nucleosome. Our findings lead to a model where HMGB1 counteracts the activity of H1 by distorting nucleosomal DNA and by contacting the H1 C-terminal tail. Compared to direct competition, nucleosome co-occupancy by HMGB1 and H1 allows a greater diversity of dynamic chromatin states and may be generalizable to other chromatin regulators.

The nucleosome is a highly dynamic macromolecular complex that is at the center of regulating access to genetic information in eukaryotes. The structural dynamics of nucleosomes are an ensemble of the dynamics of its component parts: the globular histone octamer core, the wrapped DNA, and the flexible histone tails. The coordination of these dynamics presents modes of regulation of nucleosome function. Although nucleosomes have long been considered exceptionally static and stable complexes, it has become clear that this is not the case. This thesis builds upon past work highlighting the central importance of understanding both the nature of nucleosome dynamics and the ways in which they are regulated. This thesis first addresses how nucleosome conformational dynamics are regulated by a class of nuclear proteins termed architectural proteins. Nuclear architectural proteins globally alter nucleosome structure and dynamics to induce effects on chromatin genome wide. The two most abundant nuclear architectural proteins, the linker histone H1 and HMGB1 (high mobility group box 1), compete with one another in this respect. We present a molecular model for how HMGB1 and H1 compete at many scales. We find that HMGB1 and H1 co-occupy nucleosomes and chromatin and modulate one another’s effect on DNA accessibility and mesoscale chromatin dynamics. This leads to a model wherein the dynamics of nucleosomes and chromatin can be precisely tuned by influencing the competition between these two proteins. This model also highlights the effect of altering atomic-scale nucleosome conformational dynamics on the mesoscale function of chromatin and uncovers a global role for nucleosome conformational dynamics in chromatin regulation. In addition to the work on nuclear architectural proteins, we design a proteomics-based screening platform to identify novel regulators of nucleosome conformational dynamics. Initial results from this platform implicate a number of interesting chromatin proteins and complexes as potentially having an effect on the conformational dynamics of nucleosomes. These results also imply that nucleosome conformational dynamics can be a point of regulation in a wide variety of chromatin processes. Finally, this screening platform is highly adaptable. Future iterations of this screen could target specific subsets of chromatin, focus on the activities of chromatin modifying enzymes, and shed light on potential allosteric pathways of the nucleosome itself. Overall, this thesis explores the nature and the role of nucleosome conformational dynamics in regulating a wide variety of chromatin processes.

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions. Competing Interest Statement The authors have declared no competing interest.

Objective The aim was to conduct a systematic review and meta-analysis analyzing the impact of up to 24 h of prolonged sitting on postprandial glucose, insulin and triglyceride responses, blood pressure and vascular function, in comparison to sitting interrupted with light- to moderate-intensity physical activity. Methods To be included, studies had to examine the impact of prolonged sitting lasting < 24 h in apparently healthy males or females of any age. Studies were identified from searches of the MEDLINE, CINAHL and SportDISCUS databases on July 6, 2016. Study quality was assessed using the Downs and Black Checklist; publication bias was assessed via funnel plot. Results Forty-four studies met the inclusion criteria for the systematic review; of these, 20 were included in the meta-analysis, which compared prolonged sitting to the effects of interrupting sitting with regular activity breaks on postprandial glucose, insulin and triglycerides. When compared to prolonged sitting, regular activity breaks lowered postprandial glucose ( d  = − 0.36, 95% confidence interval [CI] − 0.50 to − 0.21) and insulin ( d  = − 0.37, 95% CI − 0.53 to − 0.20), but not triglyceride responses ( d  = 0.06, 95% CI − 0.15 to 0.26). Subgroup analyses indicated reductions in postprandial triglyceride responses only occurred 12–16 h after the intervention. The magnitude of the reductions in glucose, insulin or triglyceride response was not modified by the intensity of the activity breaks, the macronutrient composition of the test meal, or the age or body mass index of participants. Conclusion Prolonged sitting results in moderate elevations in postprandial glucose and insulin responses when compared to sitting interrupted with activity breaks. PROSPERO ID CRD42015020907.

THE white smoke rose from the chimney and the church bells rang as the crowds cheered.

The antiviral drug molnupiravir was licensed for treating at-risk patients with COVID-19 on the basis of data from unvaccinated adults. We aimed to evaluate the safety and virological efficacy of molnupiravir in vaccinated and unvaccinated individuals with COVID-19. This randomised, placebo-controlled, double-blind, phase 2 trial (AGILE CST-2) was done at five National Institute for Health and Care Research sites in the UK. Eligible participants were adult (aged ≥18 years) outpatients with PCR-confirmed, mild-to-moderate SARS-CoV-2 infection who were within 5 days of symptom onset. Using permuted blocks (block size 2 or 4) and stratifying by site, participants were randomly assigned (1:1) to receive either molnupiravir (orally; 800 mg twice daily for 5 days) plus standard of care or matching placebo plus standard of care. The primary outcome was the time from randomisation to SARS-CoV-2 PCR negativity on nasopharyngeal swabs and was analysed by use of a Bayesian Cox proportional hazards model for estimating the probability of a superior virological response (hazard ratio [HR]>1) for molnupiravir versus placebo. Our primary model used a two-point prior based on equal prior probabilities (50%) that the HR was 1·0 or 1·5. We defined a priori that if the probability of a HR of more than 1 was more than 80% molnupiravir would be recommended for further testing. The primary outcome was analysed in the intention-to-treat population and safety was analysed in the safety population, comprising participants who had received at least one dose of allocated treatment. This trial is registered in ClinicalTrials.gov, NCT04746183, and the ISRCTN registry, ISRCTN27106947, and is ongoing. Between Nov 18, 2020, and March 16, 2022, 1723 patients were assessed for eligibility, of whom 180 were randomly assigned to receive either molnupiravir (n=90) or placebo (n=90) and were included in the intention-to-treat analysis. 103 (57%) of 180 participants were female and 77 (43%) were male and 90 (50%) participants had received at least one dose of a COVID-19 vaccine. SARS-CoV-2 infections with the delta (B.1.617.2; 72 [40%] of 180), alpha (B.1.1.7; 37 [21%]), omicron (B.1.1.529; 38 [21%]), and EU1 (B.1.177; 28 [16%]) variants were represented. All 180 participants received at least one dose of treatment and four participants discontinued the study (one in the molnupiravir group and three in the placebo group). Participants in the molnupiravir group had a faster median time from randomisation to negative PCR (8 days [95% CI 8–9]) than participants in the placebo group (11 days [10–11]; HR 1·30, 95% credible interval 0·92–1·71; log-rank p=0·074). The probability of molnupiravir being superior to placebo (HR>1) was 75·4%, which was less than our threshold of 80%. 73 (81%) of 90 participants in the molnupiravir group and 68 (76%) of 90 participants in the placebo group had at least one adverse event by day 29. One participant in the molnupiravir group and three participants in the placebo group had an adverse event of a Common Terminology Criteria for Adverse Events grade 3 or higher severity. No participants died (due to any cause) during the trial. We found molnupiravir to be well tolerated and, although our predefined threshold was not reached, we observed some evidence that molnupiravir has antiviral activity in vaccinated and unvaccinated individuals infected with a broad range of SARS-CoV-2 variants, although this evidence is not conclusive. Ridgeback Biotherapeutics, the UK National Institute for Health and Care Research, the Medical Research Council, and the Wellcome Trust.

The COVID‐19 pandemic has had a world‐wide impact on all areas of individuals’ health, including physical, psychological, financial, familial, social, and vocational. In the United States, the unemployment rate rose from 3.5% (5.8 million) to 13.3% (21 million) in May 2020 before dropping to 7.9% in October 2020. Cognitive information processing (CIP)is one career theory that addresses career needs of clients and society. In this article, we examine the impact of COVID‐19 on mental health and wellness, highlight differences for marginalized groups, and demonstrate how CIP theoretical elements may have been impacted by COVID‐19, and provide strategies enhancing client growth in these domains during a time when largescale social and physical distancing is recommended. The CIP‐based differentiated service delivery model is also described as a means for extending and providing access to career services.