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During cross-presentation, exogenous antigens (i.e. intracellular pathogens or tumor cells) are internalized and processed within the endocytic system and also by the proteasome in the cytosol. Then, antigenic peptides are associated with Major Histocompatibility Complex (MHC) class I molecules and these complexes transit to the plasma membrane in order to trigger cytotoxic immune responses through the activation of CD8+ T lymphocytes. Dendritic cells (DCs) are particularly adapted to achieve efficient antigen cross-presentation and their endocytic network displays important roles during this process, including a sophisticated MHC-I transport dependent on recycling compartments. In this study, we show that C. trachomatis, an obligate intracellular pathogen that exhibits multiple strategies to evade the immune system, is able to induce productive infections in the murine DC line JAWS-II. Our results show that when C. trachomatis infects these cells, the bacteria-containing vacuole strongly recruits host cell recycling vesicles, but no other endosomal compartments. Furthermore, we found that chlamydial infection causes significant alterations of MHC-I trafficking in JAWS-II DCs: reduced levels of MHC-I expression at the cell surface, disruption of the perinuclear MHC-I intracellular pool, and impairment of MHC-I endocytic recycling to the plasma membrane. We observed that all these modifications lead to a hampered cross-presentation ability of soluble and particulate antigens by JAWS-II DCs and primary bone marrow-derived DCs. In summary, our findings provide substantial evidence that C. trachomatis hijacks the DC endocytic recycling system, causing detrimental changes on MHC-I intracellular transport, which are relevant for competent antigen cross-presentation.

by Emma Peel, Yuanyuan Cheng, Julianne T. Djordjevic, Denis O’Meally, Mark Thomas, Michael Kuhn, Tania C. Sorrell, Wilhelmina M. Huston, Katherine Belov Devastating fires in Australia over 2019–20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.

Molecular simulations have played an instrumental role in uncovering the structural dynamics and physical properties of virus capsids. In this work, we move beyond equilibrium physicochemical characterization of a virus system to study a stage of the infection process that is required for viral proliferation. Despite many biochemical and functional studies, the molecular mechanism of host cell entry by non-enveloped viruses remains largely unresolved. Flock House virus (FHV) is a model system for non-enveloped viruses and is the subject of the current study. FHV infects through the acid-dependent endocytic pathway, where low pH triggers externalization of membrane-disrupting () peptides from the capsid interior. Using all-atom equilibrium and enhanced sampling simulations, the mechanism and energetics of peptide liberation and the effect of pH on this process are investigated. Our computations agree with experimental findings and reveal nanoscopic details regarding the pH control mechanism, which are not readily accessible in experiments.

The impact of seasonal coronaviruses on immune responses to SARS-CoV-2 is an active area of research. Here, Lineburg et al identify CD8+ T cells specific for a conserved and immunodominant SARS-CoV-2 epitope in HLA-B7+ individuals. Furthermore, SARS-CoV-2 epitope-specific CD8+ T cells display cross-reactivity to beta but not alpha coronaviruses due to distinct peptide–HLA conformations.

Objectives: No standard therapy, including anticoagulation regimens, is currently recommended for coronavirus disease 2019. Aim of this study was to evaluate the efficacy of anticoagulation in coronavirus disease 2019 hospitalized patients and its impact on survival. Design: Multicenter international prospective registry (Health Outcome Predictive Evaluation for Corona Virus Disease 2019). Setting: Hospitalized patients with coronavirus disease 2019. Patients: Five thousand eight hundred thirty-eight consecutive coronavirus disease 2019 patients. Interventions: Anticoagulation therapy, including prophylactic and therapeutic regimens, was obtained for each patient. Measurements and Main Results: Five thousand four hundred eighty patients (94%) did not receive any anticoagulation before hospitalization. Two-thousand six-hundred one patients (44%) during hospitalization received anticoagulation therapy and it was not associated with better survival rate (81% vs 81%; p = 0.94) but with higher risk of bleeding (2.7% vs 1.8%; p = 0.03). Among patients admitted with respiratory failure (49%, n = 2,859, including 391 and 583 patients requiring invasive and noninvasive ventilation, respectively), anticoagulation started during hospitalization was associated with lower mortality rates (32% vs 42%; p < 0.01) and nonsignificant higher risk of bleeding (3.4% vs 2.7%; p = 0.3). Anticoagulation therapy was associated with lower mortality rates in patients treated with invasive ventilation (53% vs 64%; p = 0.05) without increased rates of bleeding (9% vs 8%; p = 0.88) but not in those with noninvasive ventilation (35% vs 38%; p = 0.40). At multivariate Cox’ analysis mortality relative risk with anticoagulation was 0.58 (95% CI, 0.49–0.67) in patients admitted with respiratory failure, 0.50 (95% CI, 0.49–0.67) in those requiring invasive ventilation, 0.72 (95% CI, 0.51–1.01) in noninvasive ventilation. Conclusions: Anticoagulation therapy in general population with coronavirus disease 2019 was not associated with better survival rates but with higher bleeding risk. Better results were observed in patients admitted with respiratory failure and requiring invasive ventilation. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ccmjournal). Drs. Santoro and Núñez-Gil contributed equally and should be both considered as first authors. Drs. Romero and García Aguado received support for article research from the National Institutes of Health. Dr. Moreno Munguia received support for article research from Instituto de Investigación Sanitaria del Hospital Clínico San Carlos. The remaining authors have disclosed that they do not have any potential conflicts of interest. For information regarding this article, E-mail: natale.brunetti@unifg.it Copyright © by 2021 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo crosslinking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286-298 on the Tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286-289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

Introduction In low-income and middle-income countries, pneumonia remains the leading cause of illness and death in children<5 years. The recommended tool for diagnosing paediatric pneumonia is the interpretation of chest X-ray images, which is difficult to standardise and requires trained clinicians/radiologists. Current automated computational tools have primarily focused on assessing adult pneumonia and were trained on images evaluated by a single specialist. We aim to provide a computational tool using a deep-learning approach to diagnose paediatric pneumonia using X-ray images assessed by multiple specialists trained by the WHO expert X-ray image reading panel. Methods and analysis Approximately 10 000 paediatric chest X-ray images are currently being collected from an ongoing WHO-supported surveillance study in Bangladesh. Each image will be read by two trained clinicians/radiologists for the presence or absence of primary endpoint pneumonia (PEP) in each lung, as defined by the WHO. Images whose PEP labels are discordant in either lung will be reviewed by a third specialist and the final assignment will be made using a majority vote. Convolutional neural networks will be used for lung segmentation to align and scale the images to a reference, and for interpretation of the images for the presence of PEP. The model will be evaluated against an independently collected and labelled set of images from the WHO. The study outcome will be an automated method for the interpretation of chest radiographs for diagnosing paediatric pneumonia. Ethics and dissemination All study protocols were approved by the Ethical Review Committees of the Bangladesh Institute of Child Health, Bangladesh. The study sponsor deemed it unnecessary to attain ethical approval from the Academic and Clinical Central Office for Research and Development of University of Edinburgh, UK. The study uses existing X-ray images from an ongoing WHO-coordinated surveillance. All findings will be published in an open-access journal. All X-ray labels and statistical code will be made openly available. The model and images will be made available on request.

AbstractBackgroundVaricella zoster virus (VZV) vasculopathy is characterized by persistent arterial inflammation leading to stroke. Studies show that VZV induces amyloid formation that may aggravate vasculitis. Thus, we determined if VZV central nervous system infection produces amyloid.MethodsAβ peptides, amylin, and amyloid were measured in cerebrospinal fluid (CSF) from 16 VZV vasculopathy subjects and 36 stroke controls. To determine if infection induced amyloid deposition, mock- and VZV-infected quiescent primary human perineurial cells (qHPNCs), present in vasculature, were analyzed for intracellular amyloidogenic transcripts/proteins and amyloid. Supernatants were assayed for amyloidogenic peptides and ability to induce amyloid formation. To determine amylin’s function during infection, amylin was knocked down with small interfering RNA and viral complementary DNA (cDNA) was quantitated.ResultsCompared to controls, VZV vasculopathy CSF had increased amyloid that positively correlated with amylin and anti-VZV antibody levels; Aβ40 was reduced and Aβ42 unchanged. Intracellular amylin, Aβ42, and amyloid were seen only in VZV-infected qHPNCs. VZV-infected supernatant formed amyloid fibrils following addition of amyloidogenic peptides. Amylin knockdown decreased viral cDNA.ConclusionsVZV infection increased levels of amyloidogenic peptides and amyloid in CSF and qHPNCs, indicating that VZV-induced amyloid deposition may contribute to persistent arterial inflammation in VZV vasculopathy. In addition, we identified a novel proviral function of amylin.

by Shana Kushner Gadarian, Sara Wallace Goodman, Thomas B. Pepinsky Objective To study the U.S. public’s health behaviors, attitudes, and policy opinions about COVID-19 in the earliest weeks of the national health crisis (March 20–23, 2020). Method We designed and fielded an original representative survey of 3,000 American adults between March 20–23, 2020 to collect data on a battery of 38 health-related behaviors, government policy preferences on COVID-19 response and worries about the pandemic. We test for partisan differences COVID-19 related policy attitudes and behaviors, measured in three different ways: party affiliation, intended 2020 Presidential vote, and self-placed ideological positioning. Our multivariate approach adjusts for a wide range of individual demographic and geographic characteristics that might confound the relationship between partisanship and health behaviors, attitudes, and preferences. Results We find that partisanship—measured as party identification, support for President Trump, or left-right ideological positioning—explains differences in Americans across a wide range of health behaviors and policy preferences. We find no consistent evidence that controlling for individual news consumption, the local policy environment, and local pandemic-related deaths erases the observed partisan differences in health behaviors, beliefs, and attitudes. In further analyses, we use a LASSO regression approach to select predictors, and find that a partisanship indicator is the most commonly selected predictor across the 38 dependent variables that we study. Conclusion Our analysis of individual self-reported behavior, attitudes, and policy preferences in response to COVID-19 reveals that partisanship played a central role in shaping individual responses in the earliest months of the COVID-19 pandemic. These results indicate that partisan differences in responding to a national public health emergency were entrenched from the earliest days of the pandemic.

Antimicrobial peptides (AMPs) offer a potential solution to the antibiotic resistance crisis. Recent studies have revealed important evolutionary constraints on the evolution and horizontal spread of AMP resistance in bacteria. Here, we summarize these advances and highlight their importance for therapeutic development of AMPs.

Objective: Abnormal deposition of the antimicrobial peptide amyloid beta (Aβ) is a characteristic of Alzheimer's Disease (AD). The objective of this study was to elucidate risk factors for brain Aβ in a cohort enriched for human immunodeficiency virus (HIV) and other neurotropic pathogens. Design: Cross-sectional cohort study. Methods: We examined autopsy brains of 257 donors with a mean age of 52.8 years; 62% were male; and 194 were HIV+ and 63 HIV-. Hyperphosphorylated tau (p-tau) and Aβ were identified in frontal and temporal regions by immunohistochemistry. APOE genotyping was performed. Clinical and neuropathological predictors for Aβ were identified in univariate analyses, and then tested in multivariate regressions. Results: Cortical Aβ was identified in 32% of the sample, and active brain infection in 27%. Increased odds of Aβ were seen with increasing age and having an APOE ε4 allele; for the overall sample, HIV+ status was protective and brain infection was not a predictor. Within the HIV+ population, predictors for Aβ were duration of HIV disease and APOE alleles, but not age. When HIV disease duration and other HIV parameters were introduced into models for the entire sample, HIV disease duration was equivalent to age as a predictor of Aβ. Conclusion: We hypothesize that dual aspects of immune suppression and stimulation in HIV, and beneficial survivor effects in older HIV+ individuals, account for HIV+ status decreasing, and HIV duration increasing, odds of Aβ. Importantly, with HIV, disease duration replaces age as an independent risk for Aβ, suggesting HIV-associated accelerated brain senescence. Correspondence to Susan Morgello, MD, Department of Neurology, Box 1137, Mount Sinai Medical Center, NY NY 10029. Tel: +1 212 241 9118; fax: +1 212 241 2972; e-mail: susan.morgello@mssm.edu Received 3 February, 2021 Revised 4 March, 2021 Accepted 17 March, 2021 Copyright © 2021 Wolters Kluwer Health, Inc.

by Shengyang He, Wenlong Liu, Mingyan Jiang, Peng Huang, Zhi Xiang, Dingding Deng, Ping Chen, Lihua Xie Objective To understand the clinical characteristics of COVID-19 patients with clinically diagnosed bacterial co-infection (CDBC), and therefore contributing to their early identification and prognosis estimation. Method 905 COVID-19 patients from 7 different centers were enrolled. The demography data, clinical manifestations, laboratory results, and treatments were collected accordingly for further analyses. Results Around 9.5% of the enrolled COVID-19 patients were diagnosed with CDBC. Older patients or patients with cardiovascular comorbidities have increased CDBC probability. Increased body temperature, longer fever duration, anhelation, gastrointestinal symptoms, illness severity, intensive care unit attending, ventilation treatment, glucocorticoid therapy, longer hospitalization time are correlated to CDBC. Among laboratory results, increased white blood cell counting (mainly neutrophil), lymphocytopenia, increased procalcitonin, erythrocyte sedimentation rate, C-reaction protein, D-dimer, blood urea nitrogen, lactate dehydrogenase, brain natriuretic peptide, myoglobin, blood sugar and decreased albumin are also observed, indicating multiple system functional damage. Radiology results suggested ground glass opacity mixed with high density effusion opacities and even pleural effusion. Conclusion The aged COVID-19 patients with increased inflammatory indicators, worse lymphopenia and cardiovascular comorbidities are more likely to have clinically diagnosed bacterial co-infection. Moreover, they tend to have severer clinical manifestations and increased probability of multiple system functional damage.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.