A High-throughput screening system for SARS-CoV-2 entry inhibition, syncytia formation and cell toxicity (preprint)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to host cell is mediated through the binding of the SARS-CoV-2 Spike protein via receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds inhibiting Spike-ACE2 binding would be a promising, safe antiviral approach against COVID-19.  Methods: In the present study, we have used BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) replaced glycoprotein with spike protein of SARS-CoV-2 expressing eGFP reporter system (VSV-eGFP-SARS-CoV2) in a permissive cells harboring cytotoxicity marker. The high-throughput compatible SARS-CoV-2 permissive reporter system that encompasses cells stably expressing hACE2 tagged cerulean and nuclear H2B tagged with mCherry, as a marker of nuclear condensation that also enabled imaging of fused cells among infected EGFP positive cells and could give real-time information of syncytia formation.  Results: A limited high-throughput screening identified six natural products with marked VSV-eGFP-SARS-CoV2 inhibition at non cytotoxic dose. Molecular simulation studies with positive hits in complex with wild-type spike reaffirm their potential to impede viral entry. Real-time syncytia formation assay of the molecules revealed inhibition of syncytia with Didemnin B, and delayed inhibition with other natural products such as Scillaren A, Proscillaridin, Acetoxycycloheximide indicating that the assay is a reliable platform for any image based drug screening.  Conclusion: BSL-2 compatible assay system equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effect on syncytia. Studies using clinical isolates of SARS-CoV-2 is warranted to confirm the antiviral potency of the leads and the utility of the screening system.

Who is pregnant? defining real-world data-based pregnancy episodes in the National COVID Cohort Collaborative (N3C) (preprint)

Objective To define pregnancy episodes and estimate gestational aging within electronic health record (EHR) data from the National COVID Cohort Collaborative (N3C). Materials and Methods We developed a comprehensive approach, named Hierarchy and rule-based pregnancy episode Inference integrated with Pregnancy Progression Signatures (HIPPS) and applied it to EHR data in the N3C from 1 January 2018 to 7 April 2022. HIPPS combines: 1) an extension of a previously published pregnancy episode algorithm, 2) a novel algorithm to detect gestational aging-specific signatures of a progressing pregnancy for further episode support, and 3) pregnancy start date inference. Clinicians performed validation of HIPPS on a subset of episodes. We then generated three types of pregnancy cohorts based on the level of precision for gestational aging and pregnancy outcomes for comparison of COVID-19 and other characteristics. Results We identified 628,165 pregnant persons with 816,471 pregnancy episodes, of which 52.3% were live births, 24.4% were other outcomes (stillbirth, ectopic pregnancy, spontaneous abortions), and 23.3% had unknown outcomes. We were able to estimate start dates within one week of precision for 431,173 (52.8%) episodes. 66,019 (8.1%) episodes had incident COVID-19 during pregnancy. Across varying COVID-19 cohorts, patient characteristics were generally similar though pregnancy outcomes differed. Discussion HIPPS provides support for pregnancy-related variables based on EHR data for researchers to define pregnancy cohorts. Our approach performed well based on clinician validation. Conclusion We have developed a novel and robust approach for inferring pregnancy episodes and gestational aging that addresses data inconsistency and missingness in EHR data.

Developing pre-registration nurses’ resilience to mass casualty situations through the pedagogy of simulation

Simulation has become a core component of nursing curricula worldwide. Within a three-year, pre-registration degree, typically students would not be exposed to disaster-type situations and it was believed that a well-coordinated simulation exercise could replicate this. It was hoped that the simulation would require students to think quickly on their feet and transfer acquired skills. Worldwide disasters, including the current novel coronavirus, have heightened the need for well-prepared, resilient health professionals capable of responding to many different types of emergencies, including mass casualty situations. The simulated event involved 80 adult field student nurses, 19 probationer police officers, 6 photojournalism students, two Welsh Ambulance paramedics, five staff from 203 Field Hospital, two St John Cymru Wales officers, one community first responder and six Fire and Rescue personnel. All these individuals came together to undertake a simulated emergency response to a mass casualty incident. Behaviours and clinical skills were observed throughout the event, along with interprofessional interactions.

A Rapid Bead-Based Assay For Screening Of SARS-CoV-2 Neutralising Antibodies (preprint)

Quantitative determination of neutralizing antibodies against SARS CoV2 is of paramount importance in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Here, we describe a bead based screening assay for S1 neutralization using recombinant fluorescent proteins of hACE2 and SARS CoV2 S1, immobilized on solid beads employing nanobodies /metal-affinity tags. Nanobody-mediated capture of SARS CoV2 Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single color fluorescent imaging of ACE2 GFPspark binding to His tagged S1 Receptor Binding Domain (RBD His) immobilized beads. The second approach is dual color imaging of soluble ACE2 GFPSpark to S1 Orange Fluorescent Protein (S1 OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life time imaging of the ACE2 GFPSpark confirmed the interaction of ACE2 and S1 OFPSpark on beads. The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells.

Resistance to Racial Equity in U.S. Federalism and Its Impact on Fragmented Regions

In this commentary, we provide our ground-level observations of how the novel coronavirus disease (COVID-19 or COVID) has exposed weaknesses in our federal system to respond to local communities, particularly Black and Latina/os who live and work in the St. Louis region. Our perspectives come from a virtual town hall hosted by the Community Innovation and Action Center (CIAC) at the University of Missouri, St. Louis on April 18, 2020. Based on these initial public discussions, we use St. Louis as a lens for arguing that government?s attenuated impact is not due to a natural disaster itself, but the inevitable result of race-based policies that had worked against Black peoples over generations. The real failure involves our federalist system?s lack of a commitment to racial equity?when race no longer is used to predict life outcomes, and outcomes for all groups are improved?when designing the federal plan to respond to COVID-19 in local communities.