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One aspirational goal of computational chemistry is to predict potent and drug-like binders for any protein, such that only those that bind are synthesized. In this Roadmap, we describe the launch of Critical Assessment of Computational Hit-finding Experiments (CACHE), a public benchmarking project to compare and improve small molecule hit-finding algorithms through cycles of prediction and experimental testing. Participants will predict small molecule binders for new and biologically relevant protein targets representing different prediction scenarios. Predicted compounds will be tested rigorously in an experimental hub, and all predicted binders as well as all experimental screening data, including the chemical structures of experimentally tested compounds, will be made publicly available, and not subject to any intellectual property restrictions. The ability of a range of computational approaches to find novel binders will be evaluated, compared, and openly published. CACHE will launch 3 new benchmarking exercises every year. The outcomes will be better prediction methods, new small molecule binders for target proteins of importance for fundamental biology or drug discovery, and a major technological step towards achieving the goal of Target 2035, a global initiative to identify pharmacological probes for all human proteins.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Nipah virus causes highly lethal disease, with case-fatality rates ranging from 40% to 100% in recognised outbreaks. No treatments or licensed vaccines are currently available for the prevention and control of Nipah virus infection. In 2019, WHO published an advanced draft of a research and development roadmap for accelerating development of medical countermeasures, including diagnostics, therapeutics, and vaccines, to enable effective and timely emergency response to Nipah virus outbreaks. This Personal View provides an update to the WHO roadmap by defining current research priorities for development of Nipah virus medical countermeasures, based primarily on literature published in the last 5 years and consensus opinion of 15 subject matter experts with broad experience in development of medical countermeasures for Nipah virus or experience in the epidemiology, ecology, or public health control of outbreaks of Nipah virus. The research priorities are organised into four main sections: cross-cutting issues (for those that apply to more than one category of medical countermeasures), diagnostics, therapeutics, and vaccines. The strategic goals and milestones identified in each section focus on key achievements that are needed over the next 6 years to ensure that the necessary tools are available for rapid response to future outbreaks of Nipah virus or related henipaviruses., Competing Interests: Declaration of interests CCB declares grant U19AI142764 from the National Institute of Allergy and Infectious Diseases (NIAID), the National Institutes of Health (NIH). CCB received royalties from the licensing of Hendra sG to Zoetis (Equivac HeV), Auro Vaccines (subsidiary of Aurobindo Pharma USA), and monoclonal antibodies to Henipaviruses, Absolute Antibody, UK, created and operating under the laws of the UK. CCB declares patents planned, issued, or pending for “Soluble forms of Hendra and Nipah virus G glycoprotein” (Australian patent number 2005327194; US patent numbers 8865171, 9045532, 9056902, 9533038, and 10053495); “Soluble forms of Hendra and Nipah virus F glycoprotein and uses thereof” (Australian patent numbers 2013276968; US patent numbers 10040825 and 10590172); “Human monoclonal antibodies against Hendra and Nipah viruses” (US patent numbers 7988971, 8313746, and 8858938); “Antibodies against F glycoprotein of Hendra and Nipah viruses” (US patent numbers 9982038 and 10738104); and “Cedar virus and methods of use” (US patent number 10227664). EdW received financial support from the Intramural Research Program, NIAID, NIH; and royalties related to the vesicular stomatitis virus Nipah vaccine mentioned in this manuscript. ESG received funding (through a grant to ESG's institution) from Stanford University (Coalition for Epidemic Preparedness Innovations [CEPI] was the primary donor) to work on two studies related to development of Nipah vaccines, including development of consent documents for future trials in Bangladesh. ESG is also the principal investigator of a U01 (cooperative agreement) from NIAID, NIH for the study of Nipah virus spillovers in Bangladesh. ESG also received funding (grant to institution) from the Defense Advanced Research Projects Agency to study the dynamics of Nipah virus transmission in bats in Bangladesh. ESG received a consultancy contract from Montana State University to provide technical guidance to a study collecting biological samples from bats in Bangladesh; and served as an unpaid elected board member for the American Society for Tropical Medicine and Hygiene. LTM received financial support as a science consultant at FIND; and received consulting fees as a science consultant at FIND. SPL received research funding from CEPI to support the development of two vaccine candidates against Nipah virus. SP received research funding from Public Health Vaccines through a subaward from Stanford University. CFS declares patents planned, issued, or pending for “Nipah Henipavirus virus replicon particles and their use” (US patent number WO2023/133077 A1). KAM, AJM, NMM, JTO, MTO, and AKU received financial support for the completion of this Personal View from the Wellcome Trust (grant number 226538/Z/22/Z; payments were made to the University of Minnesota). Wellcome supported travel to attend the workshop identified in this Personal View for CCB, EdW, ANF, ESG, KH, SPL, LTM, KAM, JTO, M-PP, MTO, SP, and MR. PCF, PJH, and JPG worked at the Wellcome Trust during the time that this Personal View was completed. No authors were paid by a pharmaceutical company or any other agency to write this Personal View. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

In 2016, WHO designated Lassa fever a priority disease for epidemic preparedness as part of the WHO Blueprint for Action to Prevent Epidemics. One aspect of preparedness is to promote development of effective medical countermeasures (ie, diagnostics, therapeutics, and vaccines) against Lassa fever. Diagnostic testing for Lassa fever has important limitations and key advancements are needed to ensure rapid and accurate diagnosis. Additionally, the only treatment available for Lassa fever is ribavirin, but controversy exists regarding its effectiveness. Finally, no licensed vaccines are available for the prevention and control of Lassa fever. Ongoing epidemiological and behavioural studies are also crucial in providing actionable information for medical countermeasure development, use, and effectiveness in preventing and treating Lassa fever. This Personal View provides current research priorities for development of Lassa fever medical countermeasures based on literature published primarily in the last 5 years and consensus opinion of 20 subject matter experts with broad experience in public health or the development of diagnostics, therapeutics, and vaccines for Lassa fever. These priorities provide an important framework to ensure that Lassa fever medical countermeasures are developed and readily available for use in endemic and at-risk areas by the end of the decade., Competing Interests: Declaration of interests KAM, AJM, NMM, JTO, MTO, and AKU declare receiving financial support for the completion of this Personal View from Wellcome (grant number 226538/Z/22/Z), payments were made to the University of Minnesota. ADTB is a member of the Vaccine Research, Development and Manufacturing Committee of the Coalition for Epidemic Preparedness Innovations (CEPI). DME is employed by FIND. RFG declares grants U01AI151812, U19AI135995, R01AI132223, R01AI132244, and U19AI142790, all awarded from the National Institute of Allergy and Infectious Diseases, LEAP4WA awarded by the European & Developing Countries Clinical Trials Partnership, and ESEP1904 and INTU1901, both awarded by CEPI; stocks in and royalties or licenses from Zalgen Labs; ownership of a monoclonal antibody patent (WO2018106712A1); and travel support from Wellcome to attend the workshop identified in this Personal View. SG declares financial support for this Personal View from WHO and Wellcome; the receipt of drugs for clinical trials from Toyama–Fujifilm; grants (all payments were made to SG's institutions) from the German Research Foundation (grants GU 883/4-2, GU 883/5-1, and GU 883/7-1), the European Commission (project 101103204—INTEGRATE), the German Government, Ministry of Health (projects ZMI1-2521WHO002 and ZMII2-2523GHP006), CEPI (framework agreement for implementing partner services); the Kirmser Foundation (donation of equipment); speaker honoraria for continuing medical education on viral haemorrhagic fevers; and support for attending meetings from WHO, Wellcome, and CEPI, including travel support from Wellcome to attend the workshop identified in this Personal View. LTM declares being a science consultant at FIND, for which she receives grants, contracts, and consulting fees; and travel support from Wellcome to attend the workshop identified in this Personal View. SAO declares a consulting contract with CEPI (payments made to SAO); is a member of the INTEGRATE consortium with grants from the European & Developing Countries Clinical Trials Partnership (payments made to SAO's institution); and support from CEPI and EDCTP funds (payments made to travel agencies and through SAO's institution). CR declares participation on a Data Safety Monitoring Board or Advisory Board (this advisory board was designated after CR's participation in the work leading to this Personal View). WKA is Executive Director of the African Vaccine Manufacturing Initiative, and Cheif Executive of National Vaccine Institute, Ghana. DAA, IMA, WKA, ADTB, DGB, EF-C, IdC, DSG, SBG, MJ, KAM, JTO, MTO, AKU, and CSS declare Wellcome supported for travel to attend the workshop identified in this Personal View. PCF, PJH, and JPG worked at Wellcome during the time that this Personal View was completed. All other authors declare no competing interests. No authors were paid by a pharmaceutical company or any other agency to write this Personal View. The authors alone are responsible for the views expressed in this Personal View and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. Editorial note: The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

We assessed human immunodeficiency virus (HIV) load in plasma and semen during primary HIV infection using serial samples of semen and plasma during the first 24 weeks after diagnosis in untreated participants and those who started antiretroviral therapy (ART) immediately at diagnosis. In the absence of treatment, semen viral load was >1000 copies/mL in almost all specimens (83%) collected 2-10 weeks after the estimated date of HIV acquisition and remained >1000 copies/mL in 35% of untreated participants at the last observed time point. Thus, in the absence of ART, semen viral load remained at a level consistent with transmissibility throughout primary infection., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Statistical analysis to evaluate mechanistic pathways can be limited by non-causal associations as well as co-linearity of high-dimensional data. Here, we present a protocol evaluating statistical associations between multiple exposure variables (sociodemographic and behavioral), immune biomarkers, and HIV acquisition. We describe steps for study setup, combining Least Absolute Shrinkage and Selective Operator with the standard regression approach, and building nested models. This approach can determine to what extent associations between risks for exposure contributes to HIV acquisition with or without associated changes in immune activation. For complete details on the use and execution of this protocol, please refer to Bender Ignacio et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: We categorized levels of self-reported stress, anxiety, worry, and sleep among US college and university students during the COVID-19 pandemic., Methods: We conducted an anonymous online survey between May 7 and June 21, 2020., Results: Nearly all participants reported worry about the pandemic. Nearly half (95% CI: 43.3-51.3) reported moderate-to-severe anxiety, and 42.0% (95% CI: 38.0-45.9) reported experiencing poor sleep quality. Those with moderate-to-severe anxiety were more likely (OR: 3.3; 95% CI: 2.4-4.7) to report poor sleep quality than those with less anxiety. Moderate or extreme worry about the pandemic was associated with poor sleep quality (OR: 1.5; 95% CI: 1.1-2.1)., Conclusions: Our survey found high levels of stress, worry, anxiety, and poor sleep among US college and university students during the early months of the pandemic. Universities should prioritize access to resources for healthy coping to help students manage anxiety and improve sleep quality as the pandemic continues.

Background: Sex and gender are believed to influence vaccine response. Yet, the relationship between sex and gender and COVID-19 vaccine efficacy is poorly understood and remains under-investigated., Methods: We conducted a systematic review to determine whether and to what extent post-approval COVID-19 vaccine effectiveness (VE) studies report sex-disaggregated VE data. We searched four publication and pre-publication databases and additional grey literature sources for relevant published/preprint studies released between 1 January 2020 and 1 October 2021 (i.e., pre-Omicron era). We included observational studies providing VE estimates for one or more licensed/approved COVID-19 vaccines and including both males and females. Two reviewers independently assessed study eligibility, extracted data, and assessed risk-of-bias through a modified version of Cochrane's ROBINS-I tool. A qualitative data synthesis was performed., Results: Here we show that, among 240 eligible publications, 68 (28.3%) do not report the sex distribution among participants. Only 21/240 (8.8%) studies provide sex-disaggregated VE estimates, and high between-study heterogeneity regarding design, target population, outcomes, and vaccine type/timing prevent the assessment of sex in determining COVID-19 VE across studies., Conclusions: Our findings indicate that few COVID-19 vaccine research publications account for sex. Improved adherence to recommended reporting guidelines will ensure that the evidence generated can be used to better understand the relationship between sex and gender and VE., (© 2023. The Author(s).)

COVID-19 continues to be a public health concern in the United States. Although safe and effective vaccines have been developed, a significant proportion of the US population has not received a COVID-19 vaccine. This cross-sectional study aimed to describe the demographics and behaviors of Minnesota adults who have not received the primary series of the COVID-19 vaccine, or the booster shot using data from the Minnesota COVID-19 Antibody Study (MCAS) collected through a population-based sample between September and December 2021. Data were collected using a web-based survey sent to individuals that responded to a similar survey in 2020 and their adult household members. The sample was 51% female and 86% White/Non-Hispanic. A total of 9% of vaccine-eligible participants had not received the primary series and 23% of those eligible to receive a booster had not received it. Older age, higher education, better self-reported health, $75,000 to $100,000 annual household income, mask-wearing, and social distancing were associated with lower odds of hesitancy. Gender, race, and previous COVID-19 infection were not associated with hesitancy. The most frequently reported reason for not receiving a COVID-19 vaccination was safety concerns. Mask-wearing and being age 65 or older were the only strong predictors of lower odds of vaccine hesitancy for both the primary series and booster analyses.

Broadly protective coronavirus vaccines are an important tool for protecting against future SARS-CoV-2 variants and could play a critical role in mitigating the impact of future outbreaks or pandemics caused by novel coronaviruses. The Coronavirus Vaccines Research and Development (R&D) Roadmap (CVR) is aimed at promoting the development of such vaccines. The CVR, funded by the Bill & Melinda Gates Foundation and The Rockefeller Foundation, was generated through a collaborative and iterative process, which was led by the Center for Infectious Disease Research and Policy (CIDRAP) at the University of Minnesota and involved 50 international subject matter experts and recognized leaders in the field. This report summarizes the major issues and areas of research outlined in the CVR and identifies high-priority milestones. The CVR covers a 6-year timeframe and is organized into five topic areas: virology, immunology, vaccinology, animal and human infection models, and policy and finance. Included in each topic area are key barriers, gaps, strategic goals, milestones, and additional R&D priorities. The roadmap includes 20 goals and 86 R&D milestones, 26 of which are ranked as high priority. By identifying key issues, and milestones for addressing them, the CVR provides a framework to guide funding and research campaigns that promote the development of broadly protective coronavirus vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Galit Alter: Employed by Moderna. Dan Barouch: Co-inventor on vaccine patents that have been licensed to Janssen. Ralph Baric: Holds IP on sarbecovirus universal vaccine design, on the SAB for VacArt, and has collaborated with Adiago and NIH on Moderna mRNA vaccines. Norman Baylor: Provides regulatory advice and strategy to the regulated biopharma industry. Luciana Borio: In addition to holding a position at the Council on Foreign Relations, is a venture partner at Arch Venture Partners, an early-stage life sciences and biotechnology venture firm. Rachel Chikwamba: Has a grant with the Bill & Melinda Gates Foundation on localizing biologics manufacturing in South Africa. Cheryl Cohen: Has received grant support from Sanofi Pasteur, the US Centers for Disease Control and Prevention (CDC), Wellcome Trust, the Programme for Applied Technologies in Health (PATH), the Bill & Melinda Gates Foundation, and the South African Medical Research Council (SA-MRC). Bruce Gellin: Works for The Rockefeller Foundation, which is one of the funders of this work. Jonathan Heeney: Funded by CEPI and the Bill & Melinda Gates Foundation/Flu Lab to develop pre-pandemic vaccines for coronaviruses and influenza, respectively. Florian Krammer: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays (US Provisional Application Numbers: 62/994,252, 63/018,457, 63/020,503 and 63/024,436) and NDV-based SARS-CoV-2 vaccines (US Provisional Application Number: 63/251,020) which list FK as co-inventor. Patent applications were submitted by the Icahn School of Medicine at Mount Sinai. Mount Sinai is seeking to commercialize a mucosal NDV-based SARS-CoV-2 vaccine; therefore, the institution and its faculty inventors could benefit financially. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. FK has consulted for Merck, Seqirus, Curevac, and Pfizer, and is currently consulting for Pfizer, Third Rock Ventures, Merck, and Avimex. The FK laboratory is also collaborating with Pfizer on animal models for SARS-CoV-2. Teresa Lambe: Vaccine Taskforce via NIHR Support: Grant to support the running of the trial paid to University of Oxford. AstraZeneca Support: Support for medical writing. Vaccitech Consultant Fees for an unrelated project. Seqirus honoraria: Meeting relating to influenza meeting—unrelated work. Named as an inventor on a patent application for a vaccine against SARS CoV-2. Jason McLellan: Is an inventor on patents and patent applications regarding coronavirus vaccines and antibodies. Angela Mehr: Holds small amount of shares in AstraZeneca and Moderna. Kayvon Modjarrad: Current affiliation is as an employee of Pfizer, Inc. Peter Openshaw: Has participated in scientific advisory boards for GSK, Moderna, Janssen, Seqirus, and Pfizer. Peter Paradiso: Consultant to Pfizer, Member of Board of Directors at Dynavax. Stanley Plotkin: Consultant to Moderna, Sanofi, Merck, Janssen, Inovio, NTx Bio, Codagenix, Vaxinnity, Valneva, Meissa, and Rational. Gregory Poland: Offers consultative advice on COVID-19 vaccine development to AstraZeneca, Pfizer, Medicago, Johnson&Johnson/Janssen, Novavax, and Moderna. GAP has received grant funding from ICW Ventures for preclinical studies on a peptide-based COVID-19 vaccine for which he holds a patent. These activities have been reviewed by the Mayo Clinic Conflict of Interest Review Board and are conducted in compliance with Mayo Clinic Conflict of Interest policies. Andrew Pollard: Oxford University has an agreement with AstraZeneca for development of a COVID-19 vaccine. AJP led the clinical development of the Oxford-AstraZeneca vaccine. He is chair of the UK Government's Joint Committee on Vaccination and Immunisation, but does not participate in the COVID-19 committee. Melanie Saville: Has shares with Sanofi, a vaccine company; is an employee of CEPI, an organization that funds SARS CoV 2 and broadly protective coronavirus vaccine development. Lin-fa Wang: Co-inventor of patents on test, vaccine, and monoclonal antibodies for SARS-related coronaviruses. Daniela Weiskopf: The La Jolla Institute for Immunology has filed for patent protection for various aspects of T-cell epitope and vaccine design work. E. John Wherry: Is a member of the Parker Institute for Cancer Immunotherapy. EJW is an advisor for Danger Bio, Janssen, Merck, Marengo, New Limit, Pluto Immunotherapeutics, Related Sciences, Santa Ana Bio, Synthekine, and Surface Oncology. EJW is a founder of and holds stock in Surface Oncology, Danger Bio, and Arsenal Biosciences. Michael Worobey: Has received consulting fees on SARS-CoV-2 and the COVID-19 pandemic.], (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Seasonal influenza vaccination rates remain low, and contribute to preventable influenza cases, hospitalizations, and deaths in the US. While numerous interventions have been implemented to increase vaccine uptake, there is a need to determine which interventions contribute most to vaccine willingness, particularly among age groups with vaccination rates that have plateaued at suboptimal levels. This study aimed to quantify the relative effect of multiple interventions on vaccine willingness to receive influenza vaccine in three age groups using a series of hypothetical situations with different behavioral interventions. We assessed the relative impact of four categories of interventions: source of vaccine messages, type of vaccination messages, vaccination incentives, and ease of vaccine access using a discrete choice experiment. Within each category, we investigated the role of four different attributes to measure their relative contribution to willingness to be vaccinated by removing one option from each of the intervention categories. Among the 1,763 Minnesota residents who volunteered for our study, participants expressed vaccine willingness in over 80% of the scenarios presented. Easy access to drop-in vaccination sites had the greatest impact on vaccine willingness in all age groups. Among the younger age group, small financial incentives also contributed to high vaccine willingness. Our results suggest that public health programs and vaccination campaigns may improve their chances of successfully increasing vaccine willingness if they offer interventions preferred by adults, including facilitating convenient access to vaccination and offering small monetary incentives, particularly for young adults., Competing Interests: The authors declare that the funders had no role in the role in the design of the study or in the analysis and interpretation of data., (© 2023 The Authors.)

Background: The majority of healthcare workers (HCW) in the US report being fully vaccinated against COVID-19, yet little is known about vaccine decision-making for their household members, including children., Methods: Cross-sectional survey July-August 2021 of HCW and their household members in Minnesota., Results: 94 % of eligible participants were vaccinated with the most common reasons being wanting to protect oneself, family and loved ones. Safety concerns were the most commonly reported reasons for not being vaccinated; a significantly higher proportion of unvaccinated compared to vaccinated HCW (58 % vs 12 %, p = 0.0035) and household adults (25 % vs 5 %, p = 0.03) reported prior SARS-CoV-2 infection. Nearly half of unvaccinated adults and two-thirds of unvaccinated children would be vaccinated if a vaccine mandate were in place., Conclusions: Despite high COVID-19 vaccine acceptance among HCWs, more research is required to identify and address the needs and concerns of healthcare workers who decline COVID-19 vaccination despite availability., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors reports financial support was provided by Minnesota Department of Health. All authors reports financial support was provided by Centers for Disease Control and Prevention., (Copyright © 2022. Published by Elsevier Ltd.)

Objective: To assess the frequency of preventative COVID-19 behaviors and vaccination willingness among United States (US) college and university students during the COVID-19 pandemic. Participants: Participants ( N  = 653) were ≥18 years old and students at institutions for higher education in the US in March 2020. Methods: Students self-reported preventative behaviors, willingness to be vaccinated, and social contact patterns during four waves of online surveys from May-August 2020. Results: Student engagement in preventative behaviors was generally high. The majority of students intended to be vaccinated (81.5%). Overall, there were no significant differences in the proportion adopting preventative behaviors or in willingness to be vaccinated by sex or geographic location. The most common reason for willingness to get vaccinated was wanting to contribute to ending COVID-19 outbreaks (44.7%). Conclusions: Early in the pandemic, college students primarily reported willingness to vaccinate and adherence to preventative behaviors. Outreach strategies are needed to continue this momentum.

Importance: The measurement of laboratory biomarkers plays a critical role in managing patients with COVID-19. However, to date most systematic reviews examining the association between laboratory biomarkers and mortality in hospitalized patients early in the pandemic focused on small sets of biomarkers, did not account for multiple studies including patients within the same institutions during overlapping timeframes, and did not include a significant number of studies conducted in countries other than China., Objective: To provide a comprehensive summary and an evidence map examining the relationship between a wide range of laboratory biomarkers and mortality among patients hospitalized with COVID-19 during the early phase of the pandemic in multiple countries., Evidence Review: MEDLINE, EMBASE, and Web of Science were searched from Dec 2019 to March 9, 2021. A total of 14,049 studies were identified and screened independently by two raters; data was extracted by a single rater and verified by a second. Quality was assessed using the Joanna Briggs Institute (JBI) Case Series Critical Appraisal tool. To allow comparison across biomarkers, standardized mean differences (SMD) were used to quantify the relationship between laboratory biomarkers and hospital mortality. Meta-regression was conducted to account for clustering within institutions and countries., Results: Our systematic review included 94 case-series studies from 30 countries. Across all biomarkers, the largest and most precise SMDs were observed for cardiac (troponin (1.03 (95% CI 0.86 to 1.21)), and BNP/NT-proBNP (0.93 (0.52 to 1.34)), inflammatory (IL-6 (0.97 (0.67 to 1.28) and Neutrophil-to-lymphocyte ratio (0.94 (0.59 to 1.29)), and renal biomarkers (blood urea nitrogen (1.01 (0.79 to 1.23)) and estimated glomerular filtration rate (-0.96 (-1.42 to -0.50)). There was heterogeneity for most biomarkers across countries with studies conducted in China generally having larger effect sizes., Conclusions and Relevance: The results of this study provide an early pandemic summary of the relationship between biomarkers and mortality in hospitalized patients. We found our estimated ESs were generally attenuated compared to previous systematic reviews which predominantly included studies conducted in China. Despite using sophisticated methodology to examine studies across countries, heterogeneity in reporting of case-series studies early in the pandemic limits clinical interpretability., Competing Interests: The authors have declared that no competing interests exist.

The relationship between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dose, infection, and coronavirus disease 2019 (COVID-19) outcomes remains poorly understood. This review summarizes the existing literature regarding this issue, identifies gaps in current knowledge, and suggests opportunities for future research. In humans, host characteristics, including age, sex, comorbidities, smoking, and pregnancy, are associated with severe COVID-19. Similarly, in animals, host factors are strong determinants of disease severity, although most animal infection models manifest clinically with mild to moderate respiratory disease. The influence of variants of concern as it relates to infectious dose, consequence of overall pathogenicity, and disease outcome in dose-response remains unknown. Epidemiologic data suggest a dose-response relationship for infection contrasting with limited and inconsistent surrogate-based evidence between dose and disease severity. Recommendations include the design of future infection studies in animal models to investigate inoculating dose on outcomes and the use of better proxies for dose in human epidemiology studies., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Sabes , a treatment-as-prevention intervention among men who have sex with men and transgender women in Lima, Peru, was developed to identify HIV during early primary infection (<3 months from acquisition) through monthly serologic assays and HIV RNA tests. Newly diagnosed individuals were rapidly linked to care and offered to initiate ART. In this study we sought to study the cost-effectiveness of Sabes compared to the standard of care (SOC) for HIV testing and initiation of treatment., Methods: We adapted a compartmental model of HIV transmission to evaluate the cost-effectiveness of the Sabes approach compared to the SOC using a government health care perspective, 20-year time horizon, and 3% annual discounting. We estimated the proportion of cases of HIV detected during early primary infection, reduction in HIV incidence and prevalence, incremental cost-effectiveness ratio (ICER), and net monetary benefit. We analyzed costs using data from the Sabes study, the Peruvian Ministry of Health, published literature, and expert consultation., Findings: The Sabes intervention is projected to identify 9294 early primary HIV infections in Lima, Peru over 20 years. The intervention costs $6,896 per early primary infection diagnosed and by 2038 is expected to decrease the fraction of early infections among prevalent infections by 62%. Sabes is expected to improve health, resulting in greater total discounted QALYs per person than the SOC (16·7 vs 16·4, respectively). Sabes had an ICER of $1431 (22% per capita GDP in Peru) per QALY compared to SOC., Interpretation: Our analysis suggests that in Lima, Peru the Sabes intervention could be a cost-effective approach to reduce the burden of HIV even under stringent cost-effectiveness criteria. This finding suggests that programs that use frequent HIV testing, rapid linkage to care and initiation of ART should be considered as part of a comprehensive HIV prevention strategy., Funding: National Institutes of Health., Competing Interests: BA is an employee of Flatiron Health, an independent subsidiary of Roche. DW all work was completed prior to joining Amazon. For the remaining authors none were declared., (© 2022 Published by Elsevier Ltd.)

Background: There is a critical need to identify the drivers of willingness to receive new vaccines against emerging and epidemic diseases. A discrete choice experiment is the ideal approach to evaluating how individuals weigh multiple attributes simultaneously. We assessed the degree to which six attributes were associated with willingness to be vaccinated among university students in Uganda., Methods: We conducted a single-profile discrete choice experiment at Makerere University in 2019. Participants were asked whether or not they would be vaccinated in 8 unique scenarios where attributes varied by disease risk, disease severity, advice for or against vaccination from trusted individuals, recommendations from influential figures, whether the vaccine induced indirect protection, and side effects. We calculated predicted probabilities of vaccination willingness using mixed logistic regression models, comparing health professional students with all other disciplines., Findings: Of the 1576 participants, 783 (49.8%) were health professional students and 685 (43.5%) were female. Vaccination willingness was high (78%), and higher among health students than other students. We observed the highest vaccination willingness for the most severe disease outcomes and the greatest exposure risks, along with the Minister of Health's recommendation or a vaccine that extended secondary protection to others. Mild side effects and recommendations against vaccination diminished vaccination willingness., Interpretation: Our results can be used to develop evidence-based messaging to encourage uptake for new vaccines. Future vaccination campaigns, such as for COVID-19 vaccines in development, should consider acknowledging individual risk of exposure and disease severity and incorporate recommendations from key health leaders., Competing Interests: The authors have declared that no competing interests exist.

Transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is possible among symptom-free individuals. Patients are avoiding medically necessary healthcare visits for fear of becoming infected in the healthcare setting. We screened 489 symptom-free healthcare workers for SARS-CoV-2 and found no positive results, strongly suggesting that the prevalence of SARS-CoV-2 was <1%.

Background: Monitoring COVID-19 infection risk among health care workers (HCWs) is a public health priority. We examined the seroprevalence of SARS-CoV-2 among HCWs following the fall infection surge in Minnesota, and before and after COVID-19 vaccination. Additionally, we assessed demographic and occupational risk factors for SARS-CoV-2 infection., Methods: We conducted two rounds of seroprevalence testing among a cohort of HCWs: samples in round 1 were collected from 11/22/20-02/21/21 and in round 2 from 12/18/20-02/15/21. Demographic and occupational exposures assessed with logistic regression were age, sex, healthcare role and setting, and number of children in the household. The primary outcome was SARS-CoV-2 IgG seropositivity. A secondary outcome, SARS-CoV-2 infection, included both seropositivity and self-reported SARS-CoV-2 test positivity., Results: In total, 459 HCWs were tested. 43/454 (9.47%) had a seropositive sample 1 and 75/423 (17.7%) had a seropositive sample 2. By time of sample 2 collection, 54% of participants had received at least one vaccine dose and seroprevalence was 13% among unvaccinated individuals. Relative to physicians, the odds of SARS-CoV-2 infection in other roles were increased (Nurse Practitioner: OR[95%CI] 1.93[0.57,6.53], Physician's Assistant: 1.69[0.38,7.52], Nurse: 2.33[0.94,5.78], Paramedic/EMTs: 3.86[0.78,19.0], other: 1.68[0.58,4.85]). The workplace setting was associated with SARS-CoV-2 infection (p = 0.04). SARS-CoV-2 seroprevalence among HCWs reporting duties in the ICU vs. those working in an ambulatory clinic was elevated: OR[95%CI] 2.17[1.01,4.68]., Conclusions: SARS-CoV-2 seroprevalence in HCW increased during our study period which was consistent with community infection rates. HCW role and setting-particularly working in the ICU-is associated with higher risk for SARS-CoV-2 infection., Competing Interests: Dr. Baumgartner is employed by Quansys Biosciences (Utah, USA), the manufacturer and testing site of the assays used. There are no patents, products in development, or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Most SARS-CoV-2 antibody assays cannot distinguish between antibodies that developed after natural infection and those that developed after vaccination. We assessed the accuracy of a nucleocapsid-containing assay in identifying natural infection among vaccinated individuals. A longitudinal cohort composed of health care workers in the Minneapolis/St. Paul area was enrolled. Two rounds of seroprevalence studies separated by 1 month were conducted from November 2020 to January 2021 among 81 participants. Capillary blood from rounds 1 and 2 was tested for IgG antibodies against spike proteins by enzyme-linked immunosorbent assay (spike-only assay). During round 2, IgGs reactive to SARS-CoV-2 nucleocapsid protein (nucleocapsid-containing assay) were assessed. Vaccination status at round 2 was determined by self-report. Area under the curve was computed to determine the discriminatory ability of the nucleocapsid-containing assay for identification of recent infection. Participants had a mean age of 40 years (range, 23 to 66 years); 83% were female. Round 1 seroprevalence was 9.5%. Before round 2 testing, 46% reported vaccination. Among those not recently infected, in comparing vaccinated vs unvaccinated individuals, elevated levels of spike 1 (P<.001) and spike 2 (P=.01) were observed, whereas nucleocapsid levels were not statistically significantly different (P=.90). Among all participants, nucleocapsid response predicted recent infection with an area under the curve of 0.93 (95% CI, 0.88 to 0.99). Among individuals vaccinated more than 10 days before antibody testing, the specificity of the nucleocapsid-containing assay was 92%, whereas the specificity of the spike-only assay was 0%. An IgG assay identifying reactivity to nucleocapsid protein is an accurate predictor of natural infection among a partially vaccinated population, whereas a spike-only assay performed poorly., (Copyright © 2022 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

After an outbreak of meningococcal B (MenB) disease at a university, we surveyed students regarding their vaccination status 2 months and 20 months after campus-led vaccination campaigns and compared students' self-report to vaccination records. Nearly all participants accurately reported the number of vaccine doses at both visits. Among those who received two doses of the vaccine, accurate recall of the timing of MenB vaccination was 85.7% (95% CI: 82.7-88.6) in the short term and 62.9% (95% CI: 56.0-69.8) in the long term. After the outbreak, only one-third reported feeling 'very confident' in their MenB disease and vaccine knowledge. Our findings suggest that the validity of self-reported vaccination status among university students in an outbreak setting is high, but that if the duration of protection is unknown and additional doses of vaccine may be needed, documented vaccination records may be preferred over self-report to assess timing of vaccine receipt.

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: Sexual dysfunction affects at least one half of patients after a stroke. Problems related to sexual function are rarely assessed or addressed by physicians., Objective: To determine the frequency and factors associated with sexual dysfunction among stroke survivors and self-reported barriers of physicians to discuss sexual dysfunction during regular consultation., Methods: We administered a questionnaire to a cross-sectional sample of stroke survivors to assess the frequency and factors associated with sexual dysfunction and the aspects of sexuality most commonly affected by stroke in a reference hospital in Peru. A qualitative approach was used to determine the willingness to address sexual issues and related barriers among neurology physicians participating in the study., Results: Among 150 patients, sexual dysfunction was identified in 89 (59%). Only 10% self-reported their sexual function as optimal. Markedly decreased frequency of sexual encounters (49%) and markedly decreased sexual desire (33%) were the aspects of sexual function most commonly reported by patients after a stroke. Fear of having a new stroke [OR:3.2, 95% CI (1.5-6.3)], depression [OR:2.1, 95% CI (1.0-4.3)], and self-perception of having impaired motor function [OR:2.5, 95% CI (1.2-5.0)] were significantly associated with sexual dysfunction. In the qualitative assessment of physicians (N = 15), when asked how often they addressed sexual aspects during regular consultation with a stroke survivor, none answered "very often", and only 8 (51%) answered "sometimes". At the end of the study, 10 (66%) physicians verbalized the perception that addressing this issue encouraged their patents to be more open to personal concerns and prompted a stronger doctor-patient relationship., Conclusion: Sexual dysfunction affected more than a half of stroke survivors, and was significantly associated with depression, fear of having a new stroke, and with the self-perception of impaired motor function. Addressing sexual issues during the regular consult by physicians was infrequent. Barriers reported by physicians included limited time during regular consultation and the belief that this issue should be addressed under the scope of other specialties., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is unclear how broadly aware parents are of the concept of herd immunity and whether parents consider community benefits of vaccination when making decisions about their child's vaccinations. We aimed to determine whether educating parents about community-level benefits of measles, mumps, and rubella (MMR) vaccination and local vaccination rates would impact concern about their child's risk of measles and risk of a measles outbreak. We conducted an electronic survey among Minnesota parents of children aged 6-18 years in August 2016. We assessed baseline knowledge of herd immunity, asked participants to estimate MMR vaccination coverage in their county, and asked participants to estimate the minimum coverage needed to prevent measles outbreaks. We then delivered a short, educational intervention via the survey to inform participants about the benefits of herd immunity, the actual MMR vaccination coverage in their county, and that at least 95% MMR vaccination coverage is needed to prevent measles outbreaks. Pre- and post-intervention, participants were asked to report how concerned they were that their child might get measles. We used logistic regression models to assess factors associated with awareness of herd immunity, change in concern about one's child's measles risk, and overall concern for a measles outbreak. Among 493 participants, 67.8% were aware of herd immunity at baseline. Post-intervention, 40.2% (n = 198) of parents learned that MMR vaccination rates in their county were higher than they expected. All participants found out that their county MMR rates were lower than the measles herd immunity threshold of 95%. Overall, 27.0% (n = 133) of participants reported an increase in concern that their child might get measles after learning about local vaccination coverage and the coverage needed to achieve herd immunity. We found that our short, educational intervention aimed to increase awareness about herd immunity and local vaccination led to an increase in concern about disease risk among less than a third of parents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Little is known about adherence to American Heart Association/American Stroke Association (AHA/ASA) stroke performance measures in developing countries like Peru., Aims: We assessed adherence and determined factors associated with adherence to the AHA/ASA stroke performance measures at a reference center for neurological diseases in Lima, Peru., Methods: We conducted a retrospective chart review of 150 stroke patients admitted to the Neurological Institute of Neurological Science from 2014 to 2016 to ascertain adherence to 15 different AHA/ASA stroke performance measures. Adherence was measured as a simple proportion, with both single and composite measures. Associations were analyzed with nonparametric statistics and multivariate logistic regression., Results: Mean adherence to AHA/ASA stroke performance measures was 47%. We observed a statistically significant relationship between adherence to ischemic stroke performance measures and being married (OR = 3.78, 95% CI: 1.05-13.55), as well as an inverse relationship with an onset of symptoms of greater than 4.5 h prior to arrival at the hospital compared to those with ≤ 4.5 h (OR = 0.14, 95% CI: 0.02-0.97). Compared to patients with a lower National Institutes of Health Stroke Scale (NIHSS) score (<13), those with a score of ≥13 were less likely to have good adherence (OR = 0.11, 95% CI: 0.04-0.31)., Conclusions: The mean composite measure of adherence to internationally recognized standards of stroke management in our Peruvian institution was below the level needed for an achievement award by AHA/ASA. An intervention targeted toward stroke prevention and training could lead to improved outcomes of stroke patients in Peru., Competing Interests: Declaration of Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The availability of intravenous tissue plasminogen activator (IV-tPA) remains limited worldwide, especially in low-income countries, where the burden of disability due to ischemic stroke is the highest., Aims: To evaluate outcomes and safety of IV-tPA at the only Peruvian reference institute for neurologic diseases., Methods: We conducted a prospective, observational study of stroke patients who received IV-tPA between 2009 and 2016. We assessed characteristics associated with good outcome (modified Rankine scale 0-2) at 3 months using a multivariate regression model; and factors correlated with clinical improvement (delta National Institute of Health Stroke Scale (NIHSS)) using linear regression., Results: Only 1.98% (39/1,1962) of patients presenting with ischemic stroke received IV-tPA. Nearly half (41%) were younger than 60 years, 56.4 % were men, and most strokes were cardioembolic (46.2%). The majority (64.1%) were treated within 3-4.5 hours. The median NIHSS on admission and discharge was 9 and 4, respectively; 42.1% of patients had an mRS of 0-1 at 3 months. Three patients (7.7%) developed hemorrhagic conversion, and 1 patient died (2.6%). Patients with good outcomes had lower pretreatment systolic blood pressure (138.9 versus 158.1 mm Hg, P < .007), fewer complications during hospitalization (5 versus 9 events, P < .001), shorter hospital stay (14 versus 21 days, P < .03) and, paradoxically, longer last known well -to-door times (148.3 versus 105 minutes, P < .0022). Clinical improvement was associated with shorter door-to-tPA times and obesity., Conclusions: Our findings indicate that IV-tPA has similar safety and outcomes compared to developed countries. All internal metrics (door-to-tPA, door-to-CT, and CT-to-tPA time) improved over time, highlighting areas for future implementation science studies to further expedite the administration of IV-tPA., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: In Peru, there is an ongoing high-incidence HIV epidemic among men who have sex with men (MSM) and transgender women (TW). Sexual concurrency, or having sex with a partner in between two acts of sex with another partner, may be a key factor in onward HIV transmission. In this study, we quantify concurrency, evaluate factors associated with concurrency, and assess condom use with concurrent partners among MSM and TW in Peru., Methods: We conducted a secondary analysis of data from the 2011 Peruvian Biobehavioral Survey. Pearson's Chi-squared test was used to identify individual-level characteristics associated with concurrency. We estimated the association between participant characteristics, concurrent partnerships, partnership type (stable vs. non-stable), and CLAI within the context of concurrent partnerships using multivariate and repeated-measure Poisson regression., Results: 3-month cumulative prevalence of concurrency was higher among TW compared to MSM (30.7% vs 25.2%, p = 0.014). Among those with concurrent stable and non-stable partners, 45% used condoms with both partners (95% CI: 40%-50%) and 30% preferentially had CLAI with the stable partner only (95%CI: 26%-35%). Factors associated with CLAI within the context of concurrent partnerships varied between MSM and TW., Conclusions: Although concurrency is common among TW and MSM in Peru, patterns of concurrency and differential condom use may vary between TW and MSM. Future research may explore differential condom use with stable and non-stable partners to better understand behavioral factors that may alter vulnerability to HIV in TW compared to MSM., Competing Interests: The authors have declared that no competing interests exist.

Background: Pre-mRNA splicing involves the stepwise assembly of a pre-catalytic spliceosome, followed by its catalytic activation, splicing catalysis and disassembly. Formation of the pre-catalytic spliceosomal B complex involves the incorporation of the U4/U6.U5 tri-snRNP and of a group of non-snRNP B-specific proteins. While in Saccharomyces cerevisiae the Prp38 and Snu23 proteins are recruited as components of the tri-snRNP, metazoan orthologs of Prp38 and Snu23 associate independently of the tri-snRNP as members of the B-specific proteins. The human spliceosome contains about 80 proteins that lack obvious orthologs in yeast, including most of the B-specific proteins apart from Prp38 and Snu23. Conversely, the tri-snRNP protein Spp381 is one of only five S. cerevisiae splicing factors without a known human ortholog., Results: Using InParanoid, a state-of-the-art method for ortholog inference between pairs of species, and systematic BLAST searches we identified the human B-specific protein MFAP1 as a putative ortholog of the S. cerevisiae tri-snRNP protein Spp381. Bioinformatics revealed that MFAP1 and Spp381 share characteristic structural features, including intrinsic disorder, an elongated shape, solvent exposure of most residues and a trend to adopt α-helical structures. In vitro binding studies showed that human MFAP1 and yeast Spp381 bind their respective Prp38 proteins via equivalent interfaces and that they cross-interact with the Prp38 proteins of the respective other species. Furthermore, MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, suggesting that they are parts of equivalent spliceosomal sub-complexes. Finally, similar to yeast Spp381, human MFAP1 partially rescued a growth defect of the temperature-sensitive mutant yeast strain prp38-1., Conclusions: Human B-specific protein MFAP1 structurally and functionally resembles the yeast tri-snRNP-specific protein Spp381 and thus qualifies as its so far missing ortholog. Our study indicates that the yeast Snu23-Prp38-Spp381 triple complex was evolutionarily reprogrammed from a tri-snRNP-specific module in yeast to the B-specific Snu23-Prp38-MFAP1 module in metazoa, affording higher flexibility in spliceosome assembly and thus, presumably, in splicing regulation.

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing., (© 2016 Schütze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: Few previous investigations on correlates of HPV vaccine uptake have included Hispanics, a group known to have a disproportionately high risk of cervical cancer., Methods: Rural Hispanic mothers of daughters aged 9-17 (n=78) were recruited at local community events to participate in a standardized Spanish-language survey that examined factors related to vaccine uptake., Results: Approximately 35% of the mothers reported that their daughter had received at least one dose of the vaccine. Mothers who had heard of the HPV vaccine were more likely to have a vaccinated daughter (p<.01). Mothers who thought their daughter's father would approve were more likely to have a vaccinated daughter (p=.004). Contrary to expectation, parents who believed that only one injection is necessary were more likely to have a vaccinated daughter (p=.009)., Conclusions: HPV vaccine education programs that target both parents are needed to ensure that Hispanic parents receive the complete HPV vaccine regimen.

The fidelity of codon reading was examined in amino acid starved Escherichia coli. In one case the level of misincorporation of methionine was measured at an isoleucine residue encoded by either the commonly used AUU codon or the rarely used AUA codon. In this situation we found the frequency of methionine misincorporation to be very low and to be unaffected by the identity of the isoleucine codon. In other experiments histidine misincorporation for glutamine was measured in glutamine starved cells with normal levels of histidine-specific tRNA and cells overproducing this tRNA. Cells overproducing the tRNA had higher levels of misincorporation.

Hybridization analysis of total genomic DNA indicated that Escherichia coli K12 contains a single copy of the gene encoding the histidine-accepting tRNA. This gene was subcloned onto an inducible expression vector under the control of the tac promoter. Strains carrying the resulting plasmid showed five- to six-fold increased histidine-accepting activity after induction. This overproduction of tRNAHis did not effect the growth rate of the strain or lead to derepression of the histidine biosynthetic enzymes. Neither did it have an effect on mistranslation elicited by histidine starvation. However, in cells starved for histidine by the addition of alpha-methyl histidine, the overproduction of tRNAHis interfered with the ability of the cells to recover from starvation.

We have previously studied two mutants of Escherichia coli altered in the regulation of membrane lipid composition by temperature. One class (represented by the fabFl allele) fails to regulate upon temperature shift and is defective in cis-vaccenic acid synthesis owing to the lack of the fatty acid elongation enzyme beta-ketoacyl-acyl carrier protein synthase II(EC 2.3.1.41). A second class of mutant, given the phenotypic designation Vtr, overproduces cis-vaccenic acid at all temperatures and hence is altered in temperature regulation. In this paper we report evidence for the following conclusions. (i) The Vtr and fabFl mutations show very tight genetic linkage. (ii) The Vtr lesion is allelic to the fabFl mutation since the presence of the fabFl mutation in merodiploid strains carrying the Vtr or fabF(+) alleles results in fatty acid compositions intermediate between those of the two monoploid strains. Merodiploids carrying both the fabF(+) and Vtr alleles likewise show an intermediate composition. These results indicate intra-allelic complementation. (iii) The two E. coli proteins recently discovered by Rock (J. Bacteriol. 152:1298-1300, 1982) that form mixed disulfide cross-links to acyl carrier protein are directly demonstrated to be beta-ketoacyl-acyl carrier protein synthases I and II. (iv) The fabFl strains produce a synthase II band of altered electrophoretic mobility, indicating that the fabF locus is the structural gene for synthase II. (v) The synthase II of Vtr strains is abnormally sensitive to cerulenin, an antibiotic that specifically inhibits synthases I and II. This increased sensitivity is readily demonstrated in vivo, but in vitro we failed to detect an increased sensitivity of the Vtr synthase II to cerulenin, nor have we detected any other kinetic or structural alteration in the enzyme. We interpret these results in terms of specific interactions of synthase II with other cellular components which occur in vivo but are not duplicated in vitro.

It has previously been shown that the phenylalanine codon UUC encoding residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase, is misread as leucine at a high frequency during phenylalanine starvation. However, no misreading of the UUU encoding residue 3 was observed under these conditions. Using oligonucleotide-directed, site-specific mutagenesis, we have constructed mutants where these codons have been changed. Using these mutant argI genes we see a high level of mistranslation at position 8 during phenylalanine starvation whether the codon is UUU or UUC. With either codon at position 3 we see no leucine substitution. We also constructed a gene with a leucine codon at position 3. The product of this latter mutated gene is stable and active, indicating that preferential turnover of mistranslated protein is not obscuring an otherwise high rate of misreading. This would seem to indicate that it is the context rather than the particular phenylalanine codon which is important in determining these misreading levels.