Increases in expression of carbohydrate sulfotransferases CHST11 and CHST15 and decline in N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB) require phospho-p38-MAPK following exposure to SARS-CoV-2 spike protein receptor binding domain in human a

Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Establishing an Intensive Care follow up clinic for patients in Belfast, Northern Ireland

Introduction: National guidance states that follow up should be offered to all patients who have spent more than four days in intensive care1 with specific guidance for the follow up of COVID patients released in May 2020.2 Prior to the pandemic, in the Belfast trust, there was no follow-up service provided for intensive care patients. The pandemic presented many new challenges to intensive care, with a high number of patients requiring follow up after discharge. It also presented a unique problem in that follow up clinics could not be delivered in the traditional face to face manner. Objective(s): To set up a follow up service that assessed patient recovery from COVID 19 and offered rehabilitation, in a manner that could be delivered safely during a national lockdown. Method(s): A database was collated of all the patients who had been treated in intensive care, during the first wave of the pandemic with a confirmed positive COVID-19 sample. A follow up pathway (Figure 1) was designed for the clinic based on the BTS and FICM guidelines.2-3 The multidisciplinary team used Microsoft Teams to complete clinic proformas for each patient, share files and perform virtual appointments. Patient questionnaires were collated using the forms app within MS Teams. Patients filled in various objective health questionnaires at both their 6 and 12 week appointments to allow the team to assess their rehabilitation. Once the appointments were completed the proformas were entered into their permanent medical record on the Northern Ireland Electronic Care Record (NIECR). Result(s): There were 42 patients treated in the pandemic's first wave, 40 were reviewed at 6 weeks and 39 at 12 weeks post hospital discharge. Anonymous feedback was gathered electronically from patients about their experience of the clinic. The feedback from the patients was overwhelmingly positive. To date the clinic has offered follow up to nearly 300 patients and is still in use. It has grown in size and has received input from the Belfast trust for further staffing and resources. The project recently received joint first prize in the innovation and transformation in care category for the Health and Social Care Quality Improvement (HSCQI) awards in the trust. Conclusion(s): This project highlights the essential requirement for follow-up after an intensive care admission with significant ongoing morbidity demonstrated in this patient cohort. It is currently still the only service with this breadth of MDT input in Northern Ireland. The initial use of MS Teams has allowed this service to run safely during a pandemic but it has since been adapted as the pandemic has evolved and is now offered to all Intensive care patients. Its collaborative platform allows for immediate communication throughout the whole team, and the ability for the team to be flexible. In essence, we have set up a unique and robust system that can be easily used to offer excellent follow up to Intensive care patients within the Belfast trust.

Convalescent Plasma Treatment of Severe Covid-19 Cases in the Bronx, Ny

Junior Physician Investigator Award Recipient Purpose of study Severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2) is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Convalescent plasma obtained from recovered persons was used for previous respiratory pandemics. Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) was proposed as an option that may hold promise as treatment for COVID-19. Our aim was to retrospectively evaluate the efficacy of CCP treatment of patients with severe to life-threatening COVID-19 hospitalized at Montefiore Medical Center (MMC) in the Bronx, NY between April 13 to May 4, 2020. Methods used We administered CCP as part of the Mayo Clinic expanded access investigational new drug (IND) program for hospitalized patients. We compared the mortality and clinical outcome of 73 patients with COVID-19 who received 200 mL of CCP with a Spike protein IgG titer >=1:2,430 (median 1:47,385) within 72 hours of admission to 1:1 propensity score-matched controls. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use (figure 1). We additionally measured Spike protein IgG and neutralizing antibody titer in CCP and pre- and post-transfusion Spike protein IgG, IgM and IgA titer in CCP recipients. The primary outcome was all-cause mortality at day 28 post-CCP. The secondary outcomes were improvement in oxygenation status or mortality at day 28 post-CCP. Exploratory outcomes were associations between pre-CCP SARS-CoV-2 antibody titers and mortality at day 28. Summary of results There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients < 65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28 (figure 2, 3). There was no association between CCP IgG or neutralizing antibody titer and clinical outcome. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses but not in multivariable analyses. Pre-transfusion Spike protein IgG titer was significantly correlated with Ddimer and detected viral load measured by cycle threshold (Ct) value of nasopharyngeal SARS-CoV-2 reverse-transcriptase- polymerase-chain-reaction (figure 4). No adverse effects of CCP were observed. Conclusions We report that CCP administration within 72 hours of hospitalization demonstrated a possible signal of reduced mortality in patients < 65 years. Pre-transfusion IgG titer may be a proxy for disease severity that may be useful in identifying those who are more likely to respond to CCP. Data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy. (Figure Presented).

The Role of SARS-CoV Non-structural protein 1 in Regulating RNA stability

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected more than 600 million people across 219 countries during the past three years. SARS-CoV-2 consists of a positive-strand RNA genome that encodes structural and nonstructural proteins and shares a 79% sequence homology with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). Nonstructural proteins are necessary for viral replication and suppression of the host cell immune response. Nonstructural protein 1 (nsp1), a small protein conserved among most beta-coronaviruses, inhibits host messenger RNA (mRNA) translation by binding to ribosomal mRNA channels. Nsp1 also triggers degradation of host mRNA while viral RNA remains intact. We have previously shown that nsp1 localizes within stress granules (SGs), non-membranous vesicles of stalled mRNA that form in response to viral infection. We also found that upon induction of stress, SGs disperses within 60- 120 minutes in the presence of nsp1. Since SGs are known to store and protect translationally stalled mRNAs that are target of nsp1, we sought to analyze the level of mRNAs accumulation in SGs in the presence of nsp1. The goal of this project is to identify the impact of nsp1 on stress granule formation during SARS-CoV infection. We used human embryonic kidney cells (HEK293) and transfected them with DNA expressing SARSCoV- 1 nsp1 or a control plasmid. Cells were then incubated at 37degreeC under 5% CO2 concentration for 16 hours. Following incubation, cells were subjected to 30 min of oxidative stress using sodium arenite. Cells were collected and lysed using lysis buffer, then centrifuge at 18 000xg to collect SG pellets used for RNA isolation. Isolated mRNAs were quantified using quantitative RT-PCR. We specifically targeted mRNAs that tend to show a preferential accumulation in SGs without any viral infection. When nsp1 was expressed, we found majority of mRNAs have shown a 2-fold decrease in accumulation in SGs. These results suggest there is a direct effect of nsp1 in dispersing of RNA from SGs. We are currently investigating the effect of viral leader sequence in their accumulation in SGs in the presence of nsp1. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis and evaluation of imidazole-bearing chalcones as a potential treatment for pulmonary aspergillosis

Pulmonary aspergillosis (PA) is a category of respiratory illnesses that significantly impacts the lives of immunocompromised individuals. However, new classifications of secondary infections like influenza associated aspergillosis (IAA) and COVID-19 associated pulmonary aspergillosis (CAPA) only exacerbate matters by expanding the demographic beyond the immunocompromised. Meanwhile anti-fungal resistant strains of Aspergillus are causing current treatments to act less effectively. Symptoms can range from mild (difficulty breathing, and expectoration of blood) to severe (multi organ failure, and neurological disease). Millions are affected yearly, and mortality rates range from 20-90% making it imperative to develop novel medicines to curtail this evolving group of diseases. Chalcones and imidazoles are current antifungal pharmacophores used to treat PA. Chalcones are a group of plant-derived flavonoids that have a variety of pharmacological effects, such as, antibacterial, anticancer, antimicrobial, and anti-inflammatory activities. Imidazoles are another class of drug that possess antibacterial, antiprotozoal, and anthelmintic activities. The increase in antifungal resistant Aspergillus and Candida species make it imperative for us to synthesize novel pharmacophores for therapeutic use. Our objective was to synthesize a chalcone and imidazole into a single pharmacophore and to evaluate its effectiveness against three different fungi from the Aspergillus or Candida species. The chalcones were synthesized via the Claisen-Schmidt aldol condensation of 4-(1H-Imizadol-1-yl) benzaldehyde with various substituted acetophenones using aqueous sodium hydroxide in methanol. The anti-fungal activity of the synthesized chalcones were evaluated via a welldiffusion assay against Aspergillus fumigatus, Aspergillus niger, and Candida albicans. The data obtained suggests that chalcone derivatives with electron-withdrawing substituents are moderately effective against Aspergillus and has the potential for further optimization as a treatment for pulmonary aspergillosis. This project was supported by grants from the National Institutes of Health (NIH), National Institute of General Medicine Sciences (NIGMS), IDeA Networks of Biomedical Research Excellence (INBRE), Award number: P20GM103466. The content is solely the responsibility of the authors and do not necessarily represent the official views of the NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Determining the Effect of KmtR Mutants, E101Q, H102Q, and H111Q on the Metal Binding Affinities

Mycobacterium tuberculosis is the second leading infectious killer after COVID-19. The bacteria utilizes several metal transport systems to help it survive in the host.With an increase in the number of multiresistant, extensively resistant and totally drug-resistant strains, the development of new therapeutic strategies that target other essential pathways in the bacteria is critical. The bacteria contain several metal transport systems which are necessary for its survival. Additionally, the bacteria has two metalloregulators that are associated with nickel and cobalt export, NmtR and KmtR. The focus of this research is on KmtR, which represses the expression of the genes, cdf (which encodes the export protein) and kmtR. The goal of our research is to identify the residues that are responsible for binding the cognate metals, nickel and cobalt, as well as the noncognate metal, zinc, to KmtR. Mutagenesis studies coupled with metal binding experiments will be used to determine how KmtR binds these metals. The E101Q, H102Q, and H111Q mutants, among others, have been made, expressed, and purified in our lab. Data obtained from Isothermal Titration Calorimetry determined that all three mutant proteins bind cobalt with nanomolar affinities and the H111Q mutant KmtR proteins binds cobalt an order of magnitude weaker than the other two mutant proteins. Research reported as supported fully by the RI Institutional Development Award (IDeA) Network for Biomedical Research Excellence (RI-INBRE) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #P20GM103430.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of CRISPR-mediated immunity and applications beyond editing

Mammals, bacteria, and archaea have domesticated transposases (e.g., RAG1 and Cas1) to form adaptive immune systems. Bacteria and archaea acquire resistance to viruses and plasmids by preferentially integrating fragments of foreign DNA at one end of a CRISPR locus. DNA motifs upstream of the CRISPR (i.e., leader) facilitate integration at the first CRISPR repeat. But how do these upstream DNA motifs act over large distances of 130 bp, or roughly 440 A, to regulate integration allosterically? Here, we determine the structure of a 560 KDa integration complex that explains how the CRISPR leader DNA recruits Cas (i.e., Cas1-2/3) and non-Cas proteins (i.e., IHF). Cas1-2/3 and IHF cooperate to fold the genome into a successive U-shaped bend and a loop. The genomic U-bend traps foreign DNA against the integrase, whereas the genomic loop positions the leader-repeat junction at the Cas1 active site. The foreign DNA and the CRISPR repeat wrap around opposite faces of Cas2, poised for a Cas1-catalyzed strand-transfer reaction. The post-integration structure suggests that strand-transfer releases tension in the DNA loop. Therefore Cas1-2/3 may harness protein-induced DNA tension to favor the completion of the isoenergetic integration reaction. Cas1-2/3 interacts extensively with the leader and repeat without making sequence-specific contacts, and we demonstrate that protein-mediated folding of DNA drives integration into diverse sequences. These results reveal Cas1-2/3 and IHF strain DNA to enhance integration allosterically and suggest a mechanism for the de novo generation of new CRISPRs. Further, to address an urgent need for inexpensive and rapid detection of viruses, we recently repurposed a CRISPR immune signaling pathway to detect SARS-CoV-2 in patient samples. A.S-F. is a postdoctoral fellow of the Life Science Research Foundation, supported by the Simons Foundation. A.S-F. is supported by the PDEP award from the Burroughs Wellcome Fund, and by the National Institutes of Health, United States grant 1K99GM147842. This work was also supported by NSF (1828765), NIH (U24 GM129539, R35GM134867).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Certificate of Excellence Completion Rate of the Cc-Erus Fellowship and Reasons for Non-Completion

INTRODUCTION AND OBJECTIVE: The Certified Curriculum of ERUS (CC-ERUS) fellowship on robot-assisted radical prostatectomy (RARP) is almost 10 years old. To complete the CCERUS outcome-based fellowship, a video of a full RARP performed by the fellow must be assessed by an expert. The aim of the current study was to 1) understand and report the completion rate of the fellowship (i.e. achievement of the Certificate of Excellence award) and 2) identify reasons for non-completion. METHOD(S): The CC-ERUS is a 6 months structured training program that includes an eLearning part, followed by one-week robotic skills course;then, trainees have 6 months of modular training at a host center. At the end of the fellowship, trainees are requested to submit a video of a full RARP performed by themselves. The video is objectively assessed by experts and, in case of positive assessment, the fellowship is completed and the fellow can receive the Certificate of Excellence. We analysed our prospectively collected data on all CC-ERUS fellows. We then conducted a telephone survey on 2018-2021 CC-ERUS fellows to investigate the reasons for noncompletion. Standardized interview format questions were used to conduct the survey. RESULT(S): Data on 87 subjects enrolled in the fellowship between were collated. While all subjects successfully completed the 1-wk robotic skills course, only 26 (30%) fellows achieved the certificate of excellence. The completion rate by year was 20% in 2018, 29% in 2019, 36.4% in 2020, and 31.4% in 2021. Therefore, the COVID-19 pandemic had only a modest impact on completion rate. The response rate to the telephone interview survey was 77%. The following reasons for non-completion emerged: insufficient console exposure (49%), insufficient fellowship duration (20%), COVID-19 pandemic (11%), logistic difficulties in submitting the video (20%). CONCLUSION(S): The CC-ERUS for RARP was the first validated robotic curriculum in the world, and still one of the very few outcome-based fellowships. Nonetheless, we observed a low completion rate that needs to be addressed with appropriate actions. To increase the fellowship completion rate, three solutions should be considered by the ERUS board: 1. Review of the Host Centers, to exclude those which do not meet the certification criteria (e.g. insufficient console time for fellows) 2. Periodical Train-The-Trainers courses for the mentors at host centers 3. Follow-up procedural diary: the fellows will be requested to submit videos of each phase while progressing in their modular training and self-assess their performance using validated RARP metrics.

Visualization and functional annotation of coronavirus spike protein glycoshields

Spike proteins of coronaviruses are highly glycosylated and responsible for host recognition and viral entry. The glycans provide a camouflaging shield to help coronaviruses evade host immunity and, in some cases, modulate functional domain structures and dynamics pertinent to host recognition. However, the glycans are chemically and conformationally heterogeneous, making it challenging to determine the chemical compositions and conformations quantitatively. Combining cryo-electron microscopy, mass spectrometry, and molecular modeling, we systematically characterize a panel of spike protein variants of human and animal coronaviruses, including those of the variants of concern of SARS-CoV-2. We have established a robust workflow to quantify the heterogeneity of individual N-glycans by mass spectrometry. We also demonstrated the ability to visualize long glycan structures directly in regions where the dynamics are restricted. In places where the N-glycans are too dynamic, their structural information is generally lost after extended cryo-EM data processing that aims to achieve high resolution. To address this issue, we developed a computational tool called GlycoSHIELD to generate ensembles of glycan conformers to recapitulate the fuzzy structures that are in quantitative agreement with the experimental cryo-EM data. The ability to generate fully glycosylated spike protein models enables the prediction of hitherto unknown receptor and antibody binding sites. This work was supported by Academia Sinica intramural fund, an Academia Sinica Career Development Award, Academia Sinica to STDH (AS-CDA-109- L08), an Infectious Disease Research Supporting Grant to STDH (AS-IDR- 110-08), and the Ministry of Science and Technology (MOST), Taiwan (MOST 109-3114-Y-001-001, MOST 110-2113-M-001- 050-MY3 and MOST 110-2311-B-001-013-MY3) to STDH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Interaction Between the Viral RNA Leader Sequence and nsp1 in SARS Coronavirus

Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS-CoV), inhibits host translation thorough cleaving host mRNA and blocking the translation initiation site on the 40S ribosome. Stem-Loop-1 (SL-1) of the viral RNA leader sequence has been identified to bind to nsp1, allowing viral RNA to escape translation repression. However, the specific residues on nsp1 and the specific sequences on SL-1 important to binding have not been experimentally verified. To investigate this binding, we used gel-shift assay and RNA pull-down to verify binding between nsp1 and SL-1. By mutating SL- 1, we seek to identify the nucleotides of SL-1 that bind to nsp1. Based on recent literature, we hypothesized that disrupting the stem region of SL-1 will decrease binding between nsp1 and SL-1. Moreover, we seek to identify the residues important to binding to SL-1 by mutating specific amino acids of nsp1. Interestingly, nsp1 is a small protein (180 amino acids) with intrinsically unstructured regions at both C- and N-terminal ends of the protein. Based on recent literature we hypothesize that disrupting the R124 and K125 residues will decrease binding to SL-1. The results of this study will increase the knowledge of how viral RNA is able to escape suppression of host gene expression. To investigate the binding of nsp1 to SL1, we used nsp1 purified from bacterial lysate using glutathione beads followed by precision protease cleavage of GST-nsp1, and biotinylated RNA. LightShift Chemiluminescence RNA EMSA Kit (Promega) was used to detect the RNA in complex with nsp1 using a gel shift assay. Contrary to our hypothesis, we found an increase in nsp1 binding to the RNA carrying stem mutation, and a decrease in nsp1 binding to the RNA with the loop mutation. Moreover, we observed two distinct bands in the stem mutant indicating two possible binding sites on SL-1. Using an electrophoretic mobility shift assay, the loop region of SL-1 has been determined to be vital for binding to nsp1 in vitro. We hypothesize when the stem was mutated, we created a new binding site for nsp1. Currently we are further investigating several mutations in SL-1 to identify the actual binding site. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Ultra High-Throughput Screening Assays for Inhibitors of TMPRSS2 and SPIKE Interaction

SARS-CoV-2, the coronavirus that causes the disease COVID- 19, was identified over three years ago, yet current small molecule therapies have limited usefulness and resistance to therapies and vaccines is inevitable. Ultra high-throughput screening (uHTS) assays for novel and repurposed inhibitors of a protein-protein interaction in the viral life cycle could be used to screen a vast number of compounds with a specific target of action. In particular, the interaction between viral SPIKE protein and human TMPRSS2 is an understudied, yet critical step in viral entry. Thus, we aim to create uHTS assays to rapidly and affordably identify inhibitors of the TMPRSS2 and SPIKE interaction for further biochemical studies and therapeutic development for SARS-CoV-2.We first sought to create a Time Resolved-Forster/Fluorescence Energy Transfer (TR-FRET) assay which uses lysates of cells with overexpressed SPIKE and TMPRSS2 and fluorescently labeled antibodies to detect interactions between these proteins. Initially, we developed and optimized this TR-FRET assay in a 384-well plate then miniaturized to a 1536-well plate. We conducted a pilot screen of compounds with known biological activity to test this assay's screening capabilities. To further narrow the hits from this TR-FRET screen, we developed an orthogonal uHTS Nanoluciferase Binary Technology (NanoBiT) assay to detect the interaction between tagged SPIKE and TMPRSS2 in live cells.With these two assays in hand, we expanded our TR-FRET screen to over 100 000 compounds and identified several that were also positive in the orthogonal NanoBiT assay. Four of these compounds were found to potentially interact with either SPIKE or TMPRSS2 from thermal shift experiments, providing support for their action as SPIKE and TMPRSS2 interaction inhibitors. Thus, we have developed TR-FRET and NanoBiT orthogonal uHTS assays which have allowed for the discovery of several possible repurposed and novel inhibitors of the SPIKE/ TMPRSS2 interaction. These uHTS assays can be employed as a model for future drug discovery efforts and the compounds identified may be used as exciting starting points for development of inhibitors of SARS-CoV-2. This research was supported in part by The Emory School of Medicine COVID Catalyst-I3 award, the NCI Emory Lung Cancer SPORE (SR, HF;P50CA217691) Career Enhancement Program (AI, P50CA217691), Emory initiative on Biological Discovery through Chemical Innovation (AI) and R01AI167356 (SS).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Exploring the Effects of Psychosocial Factors on STEM Persistence at an HBCU through a SSTEM Scholarship Program

Lane College, founded in 1882 by former slave Bishop Isaac Lane, has strong history of educating underserved, minority students through student-center approaches. The Lane College S-STEM program aims to increase the number of low-income students entering STEM fields by providing scholarships, co-curriculum and high impact program activities to support Lane College students majoring in biology, chemistry, computer science, mathematics, or physics. These activities include development and implementation of new first-year STEM courses, including CHE 110 Scientific Literacy and Critical Problem Solving and PHY 111 Galileo and the Church. We have successfully recruited 3 cohorts of S-STEM scholars who have participated in several project activities including bi-weekly cohort meetings, undergraduate research projects, community outreach, internships, mentoring, and professional development. For the research project, we are exploring the development of non-cognitive factors known to impact student persistence in STEM (Grit, Self-efficacy, Growth Mindset, etc.) as S-STEM scholars matriculate through the program. Our initial measures of Grit and Growth Mindset for cohort 1 do not show a significant difference between S-STEM scholars and the control group of academically talented Power of Potential scholars. However, we have experienced low student participation in surveys during the period of remote instruction in response to the COVID-19 pandemic. For cohort 2, we measured self-efficacy for students in cohort 2 using the self-efficacy formative questionnaire developed by Gaumer Erickson and colleagues. Our data for the five students cohort 2 who completed the survey show an average score of 80.8% for focus, 60.8% for steps, and overall 72.5%. These initial data suggest that the students will benefit from targeted instruction aimed at developing growth mindset and self-efficacy. This research is supported by National Science Foundation Award DUE # 1833960.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Certificate of excellence completion rate of the CC-ERUS Fellowship and reasons for non-completion

Introduction & Objectives: The Certified Curriculum of ERUS Fellowship (CC-ERUS) on robot-assisted radical prostatectomy (RARP) is almost 10 years old. To complete the ERUS outcome-based fellowship, a video of a full RARP performed by the fellow must be assessed by an expert. The aim of the current study was to 1) understand and report the completion rate of the fellowship (i.e., achievement of the Certificate of Excellence award) 2) identify reasons for non-completion. Material(s) and Method(s): The CC-ERUS consists of a structured training program that includes an eLearning part, followed by one-week robotic skills course, and then 6 months of modular training at a host center. At the end of the fellowship, trainees are requested to submit a video of a full RARP performed by themselves. After the video is objectively assessed by experts, the fellowship is completed and the fellow can receive the Certificate of Excellence (after positive assessment). We analysed our database which includes prospectively collected data on all CC-ERUS fellows. We then conducted a telephone survey on 2018-2021 CC-ERUS Fellows to investigate the reasons for non-completion. Standardized interview format questions were used to conduct the survey. Result(s): Data on 87 subjects who were enrolled in the fellowship between January 2018 and December 2021 were collated. All subjects successfully completed the CC-ERUS training in the lab but only 26 (29.9%) fellows achieved the certificate of excellence, while 61 (70.1%) did not. The completion rate by year was 20% in 2018, 29% in 2019, 36.4% in 2020, and 31.4% among the 2021 fellows. Therefore, the COVID-19 pandemic had only a modest impact on the completion rate. The response rate to the telephone interview survey was 77%. The following reasons for non-completion emerged: insufficient console exposure (49%), insufficient fellowship duration (20%), COVID-19 pandemic (11%), logistic difficulties in submitting the video (20%). Conclusion(s): The CC-ERUS for RARP was the first validated robotic curriculum in the world, and still one of the best and the very few outcome-based fellowships. Nonetheless, we observed a low fellowship completion rate that needs to be addressed with appropriate actions. To increase the fellowship completion rate, three solutions should be considered by the ERUS board: 1. Review of the Host Centers, to exclude those which do not meet the certification criteria (amongst whom insufficient console time for the fellow) 2. Periodical Train-The-Trainers courses for the mentors in the Host Center 3. Follow-up procedural diary: the fellows will be requested to submit videos of each phase while progressing in their modular training and self-assess their performance using validated RARP metrics.Copyright © 2023 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Teleconsultations in paediatric dermatology: a UK-wide clinician survey

The COVID-19 pandemic catapulted dermatology services into a digital era, with the rapid introduction of teleconsultations. Potential benefits include widening access to healthcare and environmental sustainability. Barriers to successful teleconsultations include reduced diagnostic accuracy and technical issues. National Health Service operational planning guidance recommends that >= 25% of consultations are delivered remotely (https://www.england.nhs.uk/wp-content/uploads/ 2021/03/B0468-implementation-guidance-21-22-prioritiesand- operational-planning-guidance.pdf). Yet there is a lack of data regarding the acceptability and effectiveness of paediatric dermatology teleconsultations. We conducted a survey to explore clinicians' experience of teleconsultations in paediatric dermatology, focusing on paediatric eczema, to inform a future clinical trial. The survey was created using an online platform (Microsoft Forms) and piloted by paediatric dermatologists. It was circulated via email to members of the British Society for Paediatric Dermatology, the British Association of Dermatologists and the UK Dermatology Clinical Trials Network (DCTN). It remained open for 7 weeks from July to September 2021. Descriptive analysis was undertaken using Microsoft Excel. There were 120 responses, the majority from consultant dermatologists (59%). Prior to COVID-19, the most commonly provided teleconsultation service was advice and guidance (A+G) to general practitioners (GPs;55% responses). The majority of responders (63%) conducted no teleconsultations. Teleconsultations accounted for < 25% of all consultations in 98% responses. Since the pandemic there has been a marked shift in practice. Forty-nine per cent of responders now conduct > 25% of consultations as teleconsultations. Only 8% provide no teleconsultations. Teleconsultations are now being offered for new consultations [n = 62 (80%)], followup consultations [n = 101 (84%)] and A+G for GPs [n = 96 (80%)]. The most common format is telephone consultations with photographs (72%). For follow-up of paediatric eczema, the most effective format was felt to be telephone consultations with photographs [n = 52 (43%)], followed by video consultations with photographs [n = 32 (27%)]. Over half of responders (54%) felt that teleconsultations were less effective than face-to-face appointments for follow-up of paediatric eczema. Seventeen (21%) responders felt they could accurately assess eczema severity using a video vs. 27 (34%) using photographs. Reported barriers to teleconsultations included poorquality images, technical problems, reduced diagnostic accuracy and impaired communication. Importantly, the issue of safeguarding children was a concern. The majority of clinicians felt that teleconsultations were beneficial for reducing footfall in hospitals and minimizing time missed from school. Our results demonstrate variation in clinician opinion on the effectiveness of teleconsultations and the optimal format for paediatric eczema follow-up appointments. As part of a UK DCTN-themed research call award, we plan to conduct a patient survey, qualitative patient interviews and a health economics analysis of teleconsultations for paediatric eczema. This feasibility work will help to inform a future clinical trial.

Modeling a public-private grant initiative to address breast cancer care disparities at the community level

Background: Breast cancer disparities between Black and White women have persisted in the US, with breast cancer death rates 40% higher in Black women compared to White women (American Cancer Society Cancer Facts & Figures for African American/Black People 2022-2024). Education and interventions at the community level can potentially reduce racial gaps, particularly in curbing late-stage diagnoses that disproportionately affect Black women with breast cancer. Together, the American Cancer Society (ACS) and Pfizer Global Medical Grants (Pfizer) developed a collaborative model to support health systems in engaging communities to reduce breast cancer disparities between Black and White women. This collaboration aimed to identify novel interventions and provide foundational support for these communities to advance their work in bridging the gap in breast cancer disparities. Method(s): This collaborative grant program divided project responsibilities, in which Pfizer provided funding and ACS provided project oversight and technical support. An advisory committee provided input on the areas of most need, impact and project direction. Funding applicants were required to partner with local organizations to implement evidence-based initiatives for education and/or quality improvement within the respected community. The grant award selection committee comprised of experts in the field, including breast cancer survivors and individuals from racial/ethnic minority groups. In response to a Request for Proposals, over 100 applications were systematically reviewed based on the National Cancer Institute grant selection process. The committee selected 9 grantees with innovative proposals addressing breast cancer disparities for Black women along the cancer-care continuum. Bi-annual progress reports were used to measure progress, with a final report to mark projects' impact and reach. The COVID-19 pandemic presented numerous obstacles during the project period and the ability to convene with partners virtually through web-based sessions helped to foster opportunities for collaboration and knowledge sharing among leaders in cancer disparities research. Result(s): The projects occurred from January 2020 to June 2022, with no-cost extensions given to accommodate COVID-19 pandemic delays. During this period grantees successfully completed project goals in one of three areas: screening, identifying areas of need and education. Approximately 10,000 patients and 200 healthcare professions were impacted among three projects focused on increasing mammography efforts in Black women during the project period. Three projects incorporated surveys and focus groups to identify novel areas for intervention/need and interviewed over 350 patients and over 60 health care professionals. The remaining three grantee projects that focused on education successfully implemented advertisement campaigns and lecture series to target patients and healthcare professionals. The projects selected under this model independently completed their goals within the project period while also laying a foundation to continue work in reducing disparities along the cancer care continuum with their enhanced community partner relations. Additionally, the project period also provided opportunities for external collaborations and discussion among all grantees through 8 ACS-coordinated online sessions and 3 summits. Conclusion(s): Projects selected by the public-private grant initiative model can enhance community relationships and provide infrastructure to continue work along the cancer care continuum. We believe this collaborative competitive grant program can be used for future efforts to address breast cancer and other health disparities at the community level. Similar collaborative funding projects related to prostate and pan-tumor disparities have been launched and are currently ongoing.

Optimizing the Interdisciplinary Design of Adaptive Platform Trials

Adaptive platform trials (APTs) are often complex clinical trials that, ideally, are well suited to answer the motivating clinical questions effectively and efficiently, with the motivating clinical questions and associated treatment arms expected to evolve over time as evidence accumulates. Recently, APTs have played a pivotal role in informing public health policy by efficiently generating compelling evidence regarding the effectiveness of therapies for COVID-19. For APTs to be maximally effective in informing future public health policy, they must be carefully tailored to address the right clinical questions, with the right balance of size, scope, rigor, and flexibility. The design process requires input from clinical and statistical domain experts and often includes input from trial implementation personnel, ethicists, and patient representatives. The design process is inherently iterative, with proposed designs evaluated through trial simulation, the identification of strengths and weaknesses of the proposed design, and revision by the team to address weaknesses. This iterative design process requires effective communication and collaboration between the statistical and clinical domain experts. This session is intended to present a current best practice in facilitating and enhancing the collaborative design process for APTs, including how best to present simulation-based trial performance to the design team and ensure effective interdisciplinary communication. The speakers have extensive experience in leading the design of APTs across multiple therapeutic areas, in both academic and industry settings. The session will begin with a brief presentation by Dr. Lewis on the basic structure of an APT and the tasks and challenges associated with the multidisciplinary design process. The subsequent discussion will be organized by the following themes: (1) considerations in the selection of the study population and primary outcome metric;(2) selecting treatment domains and factors to be compared;(3) trial simulation and communication of performance metrics to both statistical and non-statistical team members;and (4) defining and calibrating interim decision rules. Each of the 4 panel members will outline a recommended approach to facilitating 1 of the 4 design tasks, with examples drawn from their experience. The remaining time (15 min) will be available for a panel question-and-answer period. At the end of the session, the audience will have an understanding of the general organization of, and a process for facilitating, the design process for an adaptive platform trial. Panel Members Roger J Lewis, MD, PhD, is a Senior Physician in the Los Angeles County Department of Health Services, Professor of Emergency Medicine at the David Geffen School of Medicine at UCLA, and the Senior Medical Scientist at Berry Consultants, LLC, a group that specializes in innovative clinical trial design. He is also the former Chair of the Department of Emergency Medicine at Harbor-UCLA Medical Center. Dr. Lewis' expertise centers on adaptive and Bayesian clinical trials, including platform trials;translational, clinical, health services and outcomes research methodology;data and safety monitoring boards, and the oversight of clinical trials. Dr. Lewis was elected to membership in the National Academy of Medicine in 2009. He has authored or coauthored over 270 original research publications, reviews, editorials, and chapters. Dr. Lewis is a Past President of the Society for Academic Emergency Medicine (SAEM) and served on the Board of Directors for the Society for Clinical Trials. He is a fellow of the American College of Emergency Physicians, the American Statistical Association, and the Society for Clinical Trials. Juliana Tolles, MD, MHS, is an Assistant Professor of Emergency Medicine at the Harbor-UCLA Medical Center and the David Geffen School of Medicine at UCLA, and a Medical and Statistical Scientist at Berry Consultants, LLC. Her academic research interests include emergency medical services, resuscitation medicine, and trau a care. She has authored several reviews for Journal of the American Medical Association (JAMA) on statistical methodology and has lectured nationally on research methodology for the Society for Academic Emergency Medicine Advanced Research Methodology Evaluation and Design (ARMED) course. She is also a co-investigator for the Strategies to Innovate Emergency Clinical Care Trials (SIREN) network Southern California site. Kert Viele, PhD, is a Director and Senior Statistical Scientist with Berry Consultants, where he leads Berry Consultants' research enterprise. He is a leader in clinical trial implementation of Bayesian hierarchical modeling, with expertise in platform and basket trials as well as clinical trials incorporating the use of historical information. Prior to joining Berry Consultants in 2010, he was a faculty member at the University of Kentucky, where he received the Provost's Award for Outstanding Teaching and was an investigator for NSF and NIH-funded research. He has developed over 100 custom Bayesian adaptive clinical trials for clients in industry, government, and academia, and currently serves on several data safety monitoring boards for randomized clinical trials. A former editor of the journal Bayesian Analysis, Dr. Viele is also an author of FACTS (Fixed and Adaptive Clinical Trial Simulator), clinical trial simulation software currently licensed to multiple pharmaceutical, academic, and government organizations. William Meurer, MD, MS, is an Associate Professor of Emergency Medicine and Neurology at the University of Michigan Health System. In addition, he serves as a Medical and Statistical Scientist for Berry Consultants, LLC. He works to improve the care of patients with acute neurological disease both through his work on the acute stroke team and as a researcher. His work in the field focuses on the design of clinical trials with adaptive and flexible components. In addition, he is a principal investigator of the National Institutes of Neurological Disorders and Stroke (NINDS) Clinical Trials Methodology Course (http:// neurotrials.training) and a co-investigator in the clinical coordinating center of the Strategies to Innovate Emergency Care Clinical Trials (SIREN) network- also funded by NIH). He was a co-investigator on the Adaptive Designs Accelerating Promising Treatments into Trials (ADAPT-IT) project, as part of the FDA Advancing Regulatory Science initiative with NIH.

Presence of SARS-CoV-2 neutralizing and vaccine spike antibodies in amniotic fluid during the second trimester of pregnancy, detected by amniocentesis (Winner of the SIMP Award, in memory of Professor Giorgio Pardi)

Objective. Thanks to the SARS-CoV-2 vaccination, pregnant women are protected from the complications of COVID-19 infection, but the benefits of this vaccination in preventing morbidity and mortality in the fetus are not yet clear: it is not well understood if and how these antibodies cross the placenta. Indeed antibodies made after a pregnant person has received an mRNA COVID-19 vaccine have been found in amniotic fluid and umbilical cord blood at term and represent a safer method of enhancing neonatal antibody levels than administration of immunoglobulin preparation to the infant. The aim of the study is to test the presence of neutralizing SARS-CoV-2 antibodies and spike antibodies in the amniotic fluid in the second trimester of pregnancy, and then to compare the antibodies level in maternal serum and amniotic fluid to evaluate their correlation. Materials and Methods. This cohort study took place at the Department of Obstetrics and Gynecology of Messina at the AOU Policlinico G. Martino from September 2021 to February 2022;the study consisted of 22 pregnant women who had amniocentesis in the gestational period between 15 weeks plus 6 days and 18 weeks: we analyzed serum and amniotic fluid samples of women who contracted the SARS-CoV-2 infection, or who were vaccinated against the same virus, within one year, or never infected by SARS-CoV-2 or vaccinated against it. During the amniocentesis, all patients underwent a single sample of maternal serum and of amniotic fluid to evaluate SARS-CoV-2 neutralizing antibody and S1 receptor binding domain IgG antibody levels. Inclusion criteria were pregnant women with the need to undergo amniocentesis. Results. 22 pregnant women were enrolled in the study:10 of them were vaccinated with a mRNA COVID-19 vaccine;12 women were not vaccinated, 4 of them had developed COVID-19 infection within one year before the collection and 2 of them developed the infection during pregnancy;the other 6 never developed the infection and have not been vaccinated, enrolled as comparators. Mann-Whitney test showed that vaccinated patients had significantly higher S1 receptor binding domain antibody levels both in amniotic fluid (p < 0.006) and maternal blood (p < 0.005) than not vaccinated women;also SARS-CoV-2 neutralizing antibody levels were higher in pregnant women who developed COVID-19 infection both in amniotic fluid (p < 0.007) and maternal blood (p < 0.004) than not vaccinated women. There was a significantly high correlation between the concentrations of spikes antibody levels in vaccinated pregnant women's serum and amniotic fluid (p = 0.000), and of neutralizing antibody levels in serum and amniotic fluid of women who developed COVID-19 infection (p = 0.000). Conclusions. To the best of our knowledge, the analysis of amniotic fluid and serum showed for the first time that all the vaccinated pregnant women samples had SARS-CoV-2 spikes immunoglobulins both in maternal blood and amniotic fluid. There is a very high correlation between maternal blood and amniotic fluid S1 receptor binding domain antibody levels in vaccinated women: this demonstrates that there is an early transplacental antibody transfer. Also neutralizing antibodies were found in the amniotic fluid of infected pregnant women, with high correlation between concentrations.