Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide1. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output2. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.

AlphaFold: Research accelerator and hypothesis generator.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.

Evaluation of an implementation support package to increase community mental health clinicians' routine delivery of preventive care for multiple health behaviours: a non-randomised controlled trial.

BACKGROUND: People with a mental health condition are more likely to engage in risk behaviours compared to people without. Delivery of preventive care to improve such behaviours is recommended for community mental health services, but inadequately implemented. This study assessed the effectiveness of an implementation support package on clinicians' delivery of preventive care (assessment, advice, referral) for four risk behaviours (tobacco smoking, harmful alcohol consumption, physical inactivity, inadequate fruit and vegetable intake) compared to no implementation support. The participatory approach to developing the support package, and fidelity of the implementation strategies, are also described. METHODS: A non-randomised controlled trial was undertaken in 2019-2020 with two community mental health services (control and target) in one health district in New South Wales, Australia. A 4-month support package consisting of multiple implementation strategies was delivered to one site following a two-phase participatory design process. Five implementation strategies were proposed to service managers by researchers. After consultation with managers and clinicians, the final implementation support package included four strategies: training and education materials, enabling resources and prompts, client activation material, and audit and feedback. Client-reported receipt of the three elements of preventive care for the four risk behaviours was collected from a cross-sectional sample of clients who had recently attended the service at baseline (6 months) and follow-up (5 months). Logistic regression models examined change in receipt of preventive care to assess effectiveness. RESULTS: A total of 860 client surveys were completed (control baseline n = 168; target baseline n = 261; control follow-up n = 164; and target follow-up n = 267). Analyses revealed no significant differential changes in preventive care receipt between the target and control sites from baseline to follow-up, including across the four primary outcomes: assessed for all behaviours (OR = 1.19; 95% CI 0.55, 2.57; p = 0.65); advised for all relevant risk behaviours (OR = 1.18; 95% CI 0.39, 3.61; p = 0.77); referred for any relevant risk behaviour (OR = 0.80; 95% CI 0.40, 1.63; p = 0.55); and complete care (OR = 3.11; 95% CI 0.62, 15.63; p = 0.17). Fidelity of the implementation strategies was limited as one of the four strategies (audit and feedback) was not delivered, components of two strategies (enabling resources and prompts, and client activation material) were not delivered as intended, and one strategy (education and training) was delivered as intended although some components were offered late in the implementation period. CONCLUSIONS: The implementation support package was ineffective at increasing preventive care delivery. Further investigation is required to determine optimal participatory design methods to develop effective implementation strategies, including those that support delivery of care in community mental health settings within the ongoing context of uncertain environmental challenges. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry ACTRN12619001379101.

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN domain.

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5' cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here, we determine high-resolution cryoelectron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain.

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5'-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

Backward masking implicates cortico-cortical recurrent processes in convex figure context effects and cortico-thalamic recurrent processes in resolving figure-ground ambiguity.

Introduction: Previous experiments purportedly showed that image-based factors like convexity were sufficient for figure assignment. Recently, however, we found that the probability of perceiving a figure on the convex side of a central border was only slightly higher than chance for two-region displays and increased with the number of display regions; this increase was observed only when the concave regions were homogeneously colored. These convex figure context effects (CEs) revealed that figure assignment in these classic displays entails more than a response to local convexity. A Bayesian observer replicated the convex figure CEs using both a convexity object prior and a new, homogeneous background prior and made the novel prediction that the classic displays in which both the convex and concave regions were homogeneous were ambiguous during perceptual organization. Methods: Here, we report three experiments investigating the proposed ambiguity and examining how the convex figure CEs unfold over time with an emphasis on whether they entail recurrent processing. Displays were shown for 100 ms followed by pattern masks after ISIs of 0, 50, or 100 ms. The masking conditions were designed to add noise to recurrent processing and therefore to delay the outcome of processes in which they play a role. In Exp. 1, participants viewed two- and eight-region displays with homogeneous convex regions (homo-convex displays; the putatively ambiguous displays). In Exp. 2, participants viewed putatively unambiguous hetero-convex displays. In Exp. 3, displays and masks were presented to different eyes, thereby delaying mask interference in the thalamus for up to 100 ms. Results and discussion: The results of Exps. 1 and 2 are consistent with the interpretation that recurrent processing is involved in generating the convex figure CEs and resolving the ambiguity of homo-convex displays. The results of Exp. 3 suggested that corticofugal recurrent processing is involved in resolving the ambiguity of homo-convex displays and that cortico-cortical recurrent processes play a role in generating convex figure CEs and these two types of recurrent processes operate in parallel. Our results add to evidence that perceptual organization evolves dynamically and reveal that stimuli that seem unambiguous can be ambiguous during perceptual organization.

Semantic clustering on common list-learning tasks: a systematic review of the state of the literature and recommendations for future directions.

INTRODUCTION: On some list-learning tasks, such as the California Verbal Learning Test (CVLT) or Hopkins Verbal Learning Test (HVLT), examinees have the opportunity to group words based on semantically related categories (i.e., semantic clustering). Semantic clustering (SC) is often considered the most efficient organizational strategy and adopting SC is presumed to improve learning and memory. In addition, SC is conceptualized as reflecting higher-order executive functioning skills. Although SC measures have intuitive appeal, to date, there are no comprehensive reviews of the SC literature base that summarize its psychometric utility. In this systematic review, we synthesize the literature to judge the validity of SC scores. METHOD: We conducted a systematic literature search for empirical articles reporting SC from the CVLT and HVLT. We qualitatively described the relationship of SC with other list-learning and cognitive test scores and clinical diagnoses, contrasting SC with serial clustering and total learning scores when possible. RESULTS: SC was inversely correlated with serial clustering. Higher SC was strongly associated with better learning and memory performances. When compared with cognitive tests, SC tended to have the strongest relationships with other memory measures and modest relationships with tests of executive functioning. SC had negligible to small relationships with most other cognitive domains. Traditional memory scores yielded stronger relationships to cognitive test performances than did SC. SC across clinical groups varied widely, but clinical groups tended to use SC less often than healthy comparison groups. CONCLUSION: Our comprehensive review of the literature revealed that SC is strongly related to measures of learning and memory on the CVLT and HVLT and is correlated with a wide range of cognitive functions. SC has been understudied in relevant populations and additional research is needed to test the degree to which it adds incremental validity beyond traditional measures of learning and memory.

Co-development of implementation strategies to assist staff of a mental health community managed organisation provide preventive care for health behaviours.

ISSUE ADDRESSED: People with a mental health condition are at risk of developing chronic physical disease due to smoking tobacco, inadequate nutrition, high alcohol consumption, low physical activity and poor sleep (SNAPS). Community managed organisations (CMOs) represent an opportune setting to support mental health consumers to improve their health behaviours through providing preventive care. Reporting of methods used to co-develop implementation strategies to assist CMO staff to deliver preventive care for SNAPS are scarce yet warranted. OBJECTIVES: This study aims to: (1) describe a co-development workshop involving CMO staff and researchers to identify preferred implementation support strategies to help staff routinely provide preventive care; (2) describe the strategies that emerged from the workshop; and (3) report staff ratings of the workshop on four co-development principles. METHODS: A three-hour co-development workshop was conducted on two occasions with staff of one CMO in New South Wales, Australia. Twenty staff participated in the workshops. RESULTS: Participants generated and ranked a total of seven discrete implementation strategies within five categories (training, point of care prompts, guidelines, continuous quality improvement and consumer activation). Training for staff to have difficult conversations about behaviour change was ranked highest in both workshops. Participants rated the workshops positively across four co-development principles. CONCLUSIONS: The co-development workshop enabled implementation strategies to be developed within the context in which they were to be delivered and tested, potentially increasing their feasibility, acceptability, appropriateness and impact. SO WHAT?: Implementation strategies selected from the workshops will inform a pilot implementation support trial to assist CMO staff to provide preventive care to people with mental health conditions.

Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

Intravenous aviptadil and remdesivir for treatment of COVID-19-associated hypoxaemic respiratory failure in the USA (TESICO): a randomised, placebo-controlled trial.

BACKGROUND: There is a clinical need for therapeutics for COVID-19 patients with acute hypoxemic respiratory failure whose 60-day mortality remains at 30-50%. Aviptadil, a lung-protective neuropeptide, and remdesivir, a nucleotide prodrug of an adenosine analog, were compared with placebo among patients with COVID-19 acute hypoxaemic respiratory failure. METHODS: TESICO was a randomised trial of aviptadil and remdesivir versus placebo at 28 sites in the USA. Hospitalised adult patients were eligible for the study if they had acute hypoxaemic respiratory failure due to confirmed SARS-CoV-2 infection and were within 4 days of the onset of respiratory failure. Participants could be randomly assigned to both study treatments in a 2 × 2 factorial design or to just one of the agents. Participants were randomly assigned with a web-based application. For each site, randomisation was stratified by disease severity (high-flow nasal oxygen or non-invasive ventilation vs invasive mechanical ventilation or extracorporeal membrane oxygenation [ECMO]), and four strata were defined by remdesivir and aviptadil eligibility, as follows: (1) eligible for randomisation to aviptadil and remdesivir in the 2 × 2 factorial design; participants were equally randomly assigned (1:1:1:1) to intravenous aviptadil plus remdesivir, aviptadil plus remdesivir matched placebo, aviptadil matched placebo plus remdesvir, or aviptadil placebo plus remdesivir placebo; (2) eligible for randomisation to aviptadil only because remdesivir was started before randomisation; (3) eligible for randomisation to aviptadil only because remdesivir was contraindicated; and (4) eligible for randomisation to remdesivir only because aviptadil was contraindicated. For participants in strata 2-4, randomisation was 1:1 to the active agent or matched placebo. Aviptadil was administered as a daily 12-h infusion for 3 days, targeting 600 pmol/kg on infusion day 1, 1200 pmol/kg on day 2, and 1800 pmol/kg on day 3. Remdesivir was administered as a 200 mg loading dose, followed by 100 mg daily maintenance doses for up to a 10-day total course. For participants assigned to placebo for either agent, matched saline placebo was administered in identical volumes. For both treatment comparisons, the primary outcome, assessed at day 90, was a six-category ordinal outcome: (1) at home (defined as the type of residence before hospitalisation) and off oxygen (recovered) for at least 77 days, (2) at home and off oxygen for 49-76 days, (3) at home and off oxygen for 1-48 days, (4) not hospitalised but either on supplemental oxygen or not at home, (5) hospitalised or in hospice care, or (6) dead. Mortality up to day 90 was a key secondary outcome. The independent data and safety monitoring board recommended stopping the aviptadil trial on May 25, 2022, for futility. On June 9, 2022, the sponsor stopped the trial of remdesivir due to slow enrolment. The trial is registered with ClinicalTrials.gov, NCT04843761. FINDINGS: Between April 21, 2021, and May 24, 2022, we enrolled 473 participants in the study. For the aviptadil comparison, 471 participants were randomly assigned to aviptadil or matched placebo. The modified intention-to-treat population comprised 461 participants who received at least a partial infusion of aviptadil (231 participants) or aviptadil matched placebo (230 participants). For the remdesivir comparison, 87 participants were randomly assigned to remdesivir or matched placebo and all received some infusion of remdesivir (44 participants) or remdesivir matched placebo (43 participants). 85 participants were included in the modified intention-to-treat analyses for both agents (ie, those enrolled in the 2 x 2 factorial). For the aviptadil versus placebo comparison, the median age was 57 years (IQR 46-66), 178 (39%) of 461 participants were female, and 246 (53%) were Black, Hispanic, Asian or other (vs 215 [47%] White participants). 431 (94%) of 461 participants were in an intensive care unit at baseline, with 271 (59%) receiving high-flow nasal oxygen or non-invasive ventiliation, 185 (40%) receiving invasive mechanical ventilation, and five (1%) receiving ECMO. The odds ratio (OR) for being in a better category of the primary efficacy endpoint for aviptadil versus placebo at day 90, from a model stratified by baseline disease severity, was 1·11 (95% CI 0·80-1·55; p=0·54). Up to day 90, 86 participants in the aviptadil group and 83 in the placebo group died. The cumulative percentage who died up to day 90 was 38% in the aviptadil group and 36% in the placebo group (hazard ratio 1·04, 95% CI 0·77-1·41; p=0·78). The primary safety outcome of death, serious adverse events, organ failure, serious infection, or grade 3 or 4 adverse events up to day 5 occurred in 146 (63%) of 231 patients in the aviptadil group compared with 129 (56%) of 230 participants in the placebo group (OR 1·40, 95% CI 0·94-2·08; p=0·10). INTERPRETATION: Among patients with COVID-19-associated acute hypoxaemic respiratory failure, aviptadil did not significantly improve clinical outcomes up to day 90 when compared with placebo. The smaller than planned sample size for the remdesivir trial did not permit definitive conclusions regarding safety or efficacy. FUNDING: National Institutes of Health.

A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance.

Coronaviruses (CoVs) encode nonstructural proteins 1-16 (nsps 1-16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7-16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase.

When narcissists exemplify ethics: Contingent consequences of ethical leadership.

Organizations increasingly encourage, recognize, and reward ethical leadership to preempt the economic and reputational risks associated with ethical failures. At the same time, organizational leadership positions are disproportionately occupied by individuals higher in narcissism. We highlight how the combination of these two phenomena carries important organizational implications by examining how ethical leadership behaviors differentially impact leaders based on their level of narcissism. Building upon self-concordance theory, we introduce a model of contingent consequences of ethical leadership. Our model identifies motivational (i.e., self-efficacy of the leader) and social (i.e., admiration of the leader) mechanisms that explain why ethical leadership positively predicts leadership effectiveness for some leaders, but not for others. We test our model using a field study and two experiments. Findings from these three studies point to a problematic leadership paradox: When leaders higher in narcissism behave more ethically, they incur higher motivational costs and reap fewer social benefits compared to their peers who are lower in narcissism. Results reveal risks to leadership effectiveness for narcissistic leaders who attempt to lead more ethically. We discuss implications for ethical leadership research and practice. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis.

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.

Above the law? How motivated moral reasoning shapes evaluations of high performer unethicality.

Recent revelations have brought to light the misconduct of high performers across various fields and occupations who were promoted up the organizational ladder rather than punished for their unethical behavior. Drawing on principles of motivated moral reasoning, we investigate how employee performance biases supervisors' moral judgment of employee unethical behavior and how supervisors' performance-focus shapes how they account for moral judgments in promotion recommendations. We test our model in three studies: a field study of 587 employees and their 124 supervisors at a Fortune 500 telecom company, an experiment with two samples of working adults, and an experiment that directly varied explanatory mechanisms. Evidence revealed a moral double standard such that supervisors rendered less punitive judgment of the unethical acts of higher performing employees. In turn, supervisors' bottom-line mentality (i.e., fixation on achieving results) influenced the degree to which they incorporated their punitive judgments into promotability considerations. By revealing the moral leniency afforded to higher performers and the uneven consequences meted out by supervisors, our results carry implications for behavioral ethics research and for organizations seeking to retain and promote their higher performers while also maintaining ethical standards that are applied fairly across employees. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

A general mechanism for transcription bubble nucleation in bacteria.

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.

Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB.

Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis. The WhiA/B regulons and binding sites have been elucidated in Streptomyces venezuelae (Sven), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of Sven transcriptional regulatory complexes comprising RNA polymerase (RNAP) σA-holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter sepX. These structures reveal that WhiB binds to domain 4 of σA (σA4) of the σA-holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the -35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σA4 housekeeping σ-factors and the -35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein-DNA interactions reduces or abolishes developmental cell division in Sven, confirming their significance. Finally, we compare the architecture of the WhiA/B σA-holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis, an infectious disease that inflicts major health and economic costs around the world 1 . Mtb encounters a diversity of environments during its lifecycle, and responds to these changing environments by reprogramming its transcriptional output 2 . However, the transcriptomic features of Mtb remain poorly characterized. In this work, we comprehensively profile the Mtb transcriptome using the SEnd-seq method that simultaneously captures the 5' and 3' ends of RNA 3 . Surprisingly, we find that the RNA coverage for most of the Mtb transcription units display a gradual drop-off within a 200-500 nucleotide window downstream of the transcription start site, yielding a massive number of incomplete transcripts with heterogeneous 3' ends. We further show that the accumulation of these short RNAs is mainly due to the intrinsically low processivity of the Mtb transcription machinery rather than trans-acting factors such as Rho. Finally, we demonstrate that transcription-translation coupling plays a critical role in generating full-length protein-coding transcripts in Mtb. In sum, our results depict a mycobacterial transcriptome that is dominated by incomplete RNA products, suggesting a distinctive set of transcriptional regulatory mechanisms that could be exploited for new therapeutics.

Perceived Hospital Stress, Severe Acute Respiratory Syndrome Coronavirus 2 Activity, and Care Process Temporal Variance During the COVID-19 Pandemic.

OBJECTIVES: The COVID-19 pandemic threatened standard hospital operations. We sought to understand how this stress was perceived and manifested within individual hospitals and in relation to local viral activity. DESIGN: Prospective weekly hospital stress survey, November 2020-June 2022. SETTING: Society of Critical Care Medicine's Discovery Severe Acute Respiratory Infection-Preparedness multicenter cohort study. SUBJECTS: Thirteen hospitals across seven U.S. health systems. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We analyzed 839 hospital-weeks of data over 85 pandemic weeks and five viral surges. Perceived overall hospital, ICU, and emergency department (ED) stress due to severe acute respiratory infection patients during the pandemic were reported by a mean of 43% ( sd , 36%), 32% (30%), and 14% (22%) of hospitals per week, respectively, and perceived care deviations in a mean of 36% (33%). Overall hospital stress was highly correlated with ICU stress (ρ = 0.82; p < 0.0001) but only moderately correlated with ED stress (ρ = 0.52; p < 0.0001). A county increase in 10 severe acute respiratory syndrome coronavirus 2 cases per 100,000 residents was associated with an increase in the odds of overall hospital, ICU, and ED stress by 9% (95% CI, 5-12%), 7% (3-10%), and 4% (2-6%), respectively. During the Delta variant surge, overall hospital stress persisted for a median of 11.5 weeks (interquartile range, 9-14 wk) after local case peak. ICU stress had a similar pattern of resolution (median 11 wk [6-14 wk] after local case peak; p = 0.59) while the resolution of ED stress (median 6 wk [5-6 wk] after local case peak; p = 0.003) was earlier. There was a similar but attenuated pattern during the Omicron BA.1 subvariant surge. CONCLUSIONS: During the COVID-19 pandemic, perceived care deviations were common and potentially avoidable patient harm was rare. Perceived hospital stress persisted for weeks after surges peaked.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.