Molecular Crowding Modulates SARS-CoV-2 Aptamer Affinity

SARS-CoV-2 aptamer is a favorable candidate for the recognition and detection of SARS-CoV-2, owing to its small size and easy synthesis. However, the issue of compromised binding affinities in real samples and targeting mutant SARS-CoV-2 hinder wide applications of the aptamer. In this study, it is discovered that molecular crowding could increase binding affinity of CoV2-6C3 aptamer against RBD (Receptor Binding Domain) of SARS-CoV-2 via increasing the absolute value of the enthalpy change. The values of the equilibrium dissociation constant in molecular crowding decrease by 70% and 150%, respectively, against wild-type and mutant RBD compared with those in buffer without crowding. Moreover, the detection limit of SARS-CoV-2 pseudovirus is up to 5 times lower under molecular crowding compared to dilute conditions. The discovery deepens the understanding of aptamer-target interaction mechanisms in crowding conditions and provides an effective way to apply SARS-CoV-2 aptamer for virus recognition and detection.

Modifications in SARS-CoV-2 N Linker Region Regulate RNA Interactions and Phase Separation

The N protein of the SARS-CoV-2 virion is critical for viral genome packaging via RNA binding and regulation of viral transcription at the replication-transcription complex (RTC). The N protein can be divided into five main domains, and the central region is the linker, which is predicted to be primarily disordered and has not been heavily studied. The linker is Serine-Arginine Rich, which is phosphorylated at multiple sites by host kinases during infection, thereby promoting the N protein's role in viral transcription. Phosphorylation is a critical process for the regulation of many cellular processes and can provide recognition sites for binding complexes. In a study that examined the recognition of the SARS-CoV-2 N protein by the human 14-3-3 protein, the linker was found to contain critical phosphosites for 14-3-3 binding. The goals of this project are to determine the structure, dynamics, and RNA interactions of the Serine-Arginine Rich linker region. To accomplish this, we performed Nuclear Magnetic Resonance spectroscopy (NMR) experiments to analyze the structure of the linker region of the N protein and its ability to bind viral RNA. NMR confirms predictions that the linker is not entirely unstructured and it is able to bind RNA. The linker region of the N protein with phosphoserine incorporated at S188 was also examined via an NMR titration experiment with 1-1000 RNA. Compared to wild type, the incorporation of phosphorylation decreases binding. Other biophysical techniques such as Analytical Ultracentrifugation (AUC) and Multi-Angle Light Scattering (MALS) are used to identify the association state of the linker and the size of the resulting protein-RNA complex. We are currently working to biophysically characterize the structure, dynamics, and viral RNA binding ability of a mutation found in the Delta and Omicron variants: the R203M linker, which have been shown to enhance viral infectivity. This work was supported by the NSF EAGER grant NSF/ MCB 2034446 and URSA Engage. Support to facilities includes the Oregon State University NMR Facility funded in part by NIH, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Hairy Cell Leukemia in a Filipino Male during the COVID-19 Pandemic - Report of a Rare Case

Hairy cell leukemia (HCL) is a rare, chronic, mature B-cell lymphoproliferative disorder accounting for 2% of all leukemias. In this paper, we would like to present our experience in the management of HCL in a financially limited setting where other diagnostic tests and chemotherapy are unavailable. The case report aims to emphasize the recognition of the distinctive morphology of hairy cells in the peripheral blood in the consideration of the initial diagnosis. A 60-year-old Filipino male was incidentally found to have anemia, thrombocytopenia and an absolute neutrophilic count below 1,000 in a pre-operative clearance for elective herniorrhaphy. Blood smear revealed atypical lymphocytes with hair like cytoplasmic projections. CT-scan of the abdomen showed splenomegaly and prominent paraaortic nodes. Flow cytometry of the bone marrow aspirate was consistent with an involvement of a Mature B cell neoplasm markers CD19, CD20, CD22 and surface immunoglobulin lambda and hairy cell leukemia markers CD11c, CD103 and CD25. He responded to six-weekly sessions of Cladribine with remission of the bone marrow and hematologic parameters. HCL is a rare type of a mature B cell neoplasm characterized by pancytopenia, splenomegaly, bone marrow fibrosis and the presence of atypical lymphoid cells with hairy projections in blood, bone marrow and spleen. Immunophenotyping express CD11c, CD103, CD123, and CD25. BRAF V600E mutation is the disease defining genetic event. Cladribine and Pentostatin are the first line of treatment. Cases of leukemia can be easily overlooked because of the mild derangement in the complete blood count. A meticulous differential review of the atypical lymphocyte, is the first step in the diagnosis of this rare disease.Copyright © 2022, Philippine College of Physicians. All rights reserved.

Restoration of innate immunity receptors under the action of muramyl peptides reduces depression and anxiety at the stage of rehabilitation after COVID-19

Background: Effective rehabilitation of patients who have had a SARS CoV-2 infection is essential to prevent re-infections and will improve the quality of life of people and reduce the burden on the healthcare system. Muramylpeptides are used in the prevention of seasonal diseases in children and adults in order to correct immunodeficiency states and prevent infectious complications. The aim of this study was to study the dynamic changes in the state of cellular factors of innate immunity and the levels of anxiety and depression in patients treated with glucosaminyl muramyl dipeptide (GMDP). Method(s): Patients who underwent mild to moderate COVID-19 (N = 60, mean age 54 +/- 11.7 years) were randomized to the study group (30 people, 15 men and 15 women) who received 2 courses of licopid 1 mg twice per day and a comparison group (30 people, 15 men and 15 women). Analysis of the phenotypic and functional characteristics of the cellular factors of the innate immune response was carried out before the start of immunomodulatory therapy, immediately after the end of the course, and also after 6 months. observations. To assess the quality of life of all patients, the SF-36 Health Status Survey and HADS questionnaires were used before the use of licopid, at the end of the course and after 6 months of follow-up. Result(s): In the course of assessing the effect of immunomodulatory therapy on the parameters of innate immunity of patients at the stage of rehabilitation after suffering COVID-19, an increase in the protective cytolytic activity of CD16+, CD8+Gr+ cells, as well as a persistent increase in the expression of TLR2, TLR4 and TLR9 was found, which indicates the restoration of antigenic recognition and presentations at the level of the monocytic link of the immune system. The use of GMDP as an immunomodulatory agent resulted in an 8-fold decrease in the frequency and severity of respiratory infections due to an increase in the total monocyte count, which persisted for 6 months from the start of therapy, while the use of systemic antibiotic therapy was not required, while in the comparison group -7 people were forced to resort to this therapy due to the severity of acute respiratory infections. When analyzing the assessment of the quality of life of patients against the background of the therapy, patients showed positive dynamics in role functioning, general assessment of their health status, and an increase in physical and mental well-being during 6 months of observation. In the comparison group, there was no improvement in the psycho-emotional state of patients. Conclusion(s): In this study, for the first time, it was found that the correction of immunological parameters when exposed to GMDP after a previous illness contributed not only to a decrease in the frequency and severity of respiratory infections, but also to an improvement in the psycho-emotional state of patients, and a decrease in anxiety and depression.

Insilico study of COVID-19 ORF1ab protein allergenicity

Introduction: Vaccination has become a main tool in combat against coronavirus disease 2019 (COVID-19). ORF1ab (open reading frame1ab) is biggest ORF of severe acute respiratory disease coronavirus 2 (SARS-CoV-2) genome. Moreover ORF1ab protein is early translated in infected cells. Besides ORF1ab is genetically stable and could be a valuable source of conserved epitopes appropriate to prepare effective protein vaccines for control of many SARS-CoV-2 variants. Hypersensitivity responses to SARS-CoV-2 vaccines have been reported by numerous studies. Objective(s): In this study SARS-CoV-2 ORF1ab protein allergenicity was predicted by bioinformatic. Method(s): The amino acid sequences of SARS-CoV-2 ORF1ab protein were obtained in NCBI database at www.ncbi.nlm.nih.gov in FASTA format. Next, allergenicity of the SARS-CoV-2 ORF1ab protein was evaluated with Allergen FP server V.1. Result(s): ORF1ab protein of SARS-CoV-2 was found to be an allergen as was confirmed by allergen FP server V.1. Conclusion(s): According to our data, ORF1ab protein of COVID-19 was potentially allergenic. Hypersensitivity reactions to some SARS-CoV-2 vaccines reported by various studies may be partly due to ORF1ab protein allergenicity. Meanwhile ORF1ab protein being a valuable source of conserved epitopes is suitable to make efficient protein vaccines for control of many SARS-CoV-2 variants. For preparation of safe and effective anti- SARS-CoV-2 vaccines from ORF1ab protein, recognition and elimination of its allergenic epitope (s) is necessary. Altogether immunization with an allergenically engineered ORF1ab protein might have potential implication in fighting the virus.

Bridging the gap with Pando

The Pando app is UK based and part of the NHS Clinical Communication Procurement Framework, which is designed to provide continuity of care with virtual patient management (https://www.bad.org.uk/healthcare-professionals/covid-19/re mote-dermatology-guidance), and drive tech-enabled connectivity across the National Health Service (NHS). This has also been used in the British Army to help defence medical staff connect with and seek advice from their colleagues in the UK while in the field (www.hellopando.com). Lack of on-site medical illustration, the COVID-19 pandemic and plastic surgeons operating in a NHS-funded private setting with no access to Picture Archiving and Communication System (PACS) in our Trust prompted use of the Pando app to capture prebiopsy pictures, avoid wrong-site surgery and improve interdepartmental communication. We present our multidisciplinary quality improvement project, involving dermatology and plastic surgery, evaluating the use of the Pando app from September to December 2020, mostly from 2-week-wait skin cancer clinics. All dermatology and plastic surgery colleagues downloaded the Pando app to their mobile phones and created a group entitled 'Dermatology/Plastics' to share their patient photos with identity labels. Patient photos could also be emailed to the clinicians' NHS email addresses - all done with patient consent. We evaluated our project with pre-and post-Pando feedback questionnaires. In the pre-Pando questionnaires, the majority of 14 colleagues involved were concerned with the varying quality of photos emailed by patients, the time lag in photos being uploaded to PACS and any likelihood of compromising patient safety. With post-Pando questionnaires, the majority found the app to be user-friendly, that the photographs taken were of superior quality, that there were no reported concerns with patient consent and they preferred using the app to the previous pathway. Comments suggested the Pando app to be invaluable for site recognition in patients with cognitive impairment, multiple lesions, difficult-to-see areas, medicolegal, educational and audit purposes, and local cancer multidisciplinary discussions. The drawbacks were the lack of seamless connection between the app and PACS, the inability to search for pictures in the app with patient identification and lack of access to previously shared pictures for new users. Despite some limitations, the Pando app has immensely improved patient safety and proved to be invaluable for our joint dermatology and plastic surgery interactions. However, there is an unmet need for a system with the ability to instantly transfer pictures to PACS and patient electronic records, to improve things further.

Lipid Lowering Natural Products Targeting hPCSK9 May Prevent the Severity of COVID-19 Infection

Background: The deadly outbreak of COVID-19 disease caused by novel SARS CoV2 has created an unprecedented global health crisis affecting every sectors of human life and enor-mous damage to world's economy. With >16.1 million infections and >650,000 deaths worldwide as of July 27, 2020, there is no treatment for this disease neither is there any available vaccine. Seri-ous research efforts are ongoing on all fronts including treatment, prevention and diagnosis to combat the spread of this infection. A number of targets that include both viral and host proteins have been identified and became part of intense investigation. In this respect the viral surface spike (S) glycoprotein caught the attention most. It is cleaved by multiple host proteases to allow recognition by host receptor human Angiotensin Converting Enzyme2 (hACE2) leading to fusion and viral re-plication. Natural products, small compounds, antioxidants, peptides, proteins, oligonucleotides, antibodies and other compounds are under investigation for development of antiviral agents against COVID-19. Objective(s): Recently cholesterol lowering phytocompounds Quercetin, Swertiamarin and Berberine which promote human Low Density Lipoprotein Receptor (hLDLR) via inhibition of human Pro-protein Convertase Subtilisin Kexin9 (hPCSK9) have been shown to block viral infections caused by ebola, influenza, Respiratory Syncytial Virus (RSV), Hepatitis C virus (HCV) and other RNA type viruses. Since SARS CoV2 is a RNA virus with similar genetic structure and infection machin-ery, it is hypothesised that these phytocompounds may also exhibit antiviral property against COVID-19. Method(s): Our above concept is based on recently published studies as well as our herein presented in silico modeling and computational data which suggested strong interactions of hPCSK9 with above phytocompounds and most importantly with hACE2 following its complexation with receptor binding domain (RBD) of SARS CoV2 S protein. Result(s): These results and a proposed schematic model showing association of hPCSK9 with SARS CoV2 infection are presented in this manuscript. It is proposed that hPCSK9 plays the role of a co-receptor in binding with hACE2:RBD complex and thereby facilitates viral fusion. Conclusion(s): Our studies suggest that PCSK9 inhibitors may provide beneficial effect against COVID-19 infection by retarding viral fusion through displacement of bound hPCSK9 from its complex with ACE2:RBD of SARS CoV2 S protein.Copyright © 2021 Bentham Science Publishers.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band-gap near infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence-dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant noncanonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20-and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van't Hoff transition enthalpy of hybridization on SWCNT CP was -11.9 kJ mol-1, much lower than that of hybridization in solution (-707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent-exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with a (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNTs with an analyte at 37 degrees C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that the target sequence choice is important as secondary structure formation in the target is negatively correlated with hybridization. Best performing CPs showed detection limits of 11 and 13 nM for DNA and RNA targets, respectively. Finally, we simulated sensing conditions using the saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence-specific hybridization on ssDNA-SWCNT CPs.

Spike antibody seroconversion and breadth following SARS-CoV-2 vaccination in Australian people with multiple sclerosis

Background: COVID-19 vaccination induces protective Spike antibodies. Some responses are attenuated in people with multiple sclerosis (MS) on high efficacy disease-modifying therapies (DMT).Whether antibodies afford immunity against emerging SARS-CoV-2 Variants of Concern (VoC) such as Delta and Omicron is unknown. Aim(s): To assess the longevity and breadth of Spike antibody in MS patients after COVID-19 vaccination. Objective(s): To determine seroconversion and antibody binding toVoC Spike. Method(s): Spike antibodies to Clade A SARS-CoV-2 were assessed in 535 MS sera at baseline (n=292), 1 (n=141) and 6 month (n=67) post-second dose, and 1 month post-third dose (n=35), and 489 health worker controls. When known, COVID- 19 vaccines were BNT162b2 (n= 489 controls, n=108 MS patients) and ChAdOx1-S (n=37).Spike antibody binding to VoC Delta and Omicron BA1 was assessed in 68 sera 1 month post-second dose. Demographic and DMT information was available in 269 patients. Result(s): 123/141 sera at 1 month post-second dose, 66/67 at 6 months post-second dose, and 26/35 at 1 month post-third dose were positive for Spike antibodies.Patients who did not seroconvert at 1 and 6 month post-second and 1 month post-third dose (n=28) were treated with ocrelizumab (n=22), cladribine (n=1), fingolimod (n=4), and siponimod (n=1). At 1 month post-second dose, the median and IQR Spike antibody levels were 67,224+/- 101,251 in the seroconverted MS group compared to 145,510+/- 99,669 in controls (n=489). When patient sera were assessed for binding to Clade A Spike, and VoC Delta and Omicron BA1 Spikes, most sera were able to bind the three different Spike antigens (n=61). However, Spike antibody immunoreactivity was decreased by 70% against Delta Spike and 90% for Omicron BA1 Spike compared to the original clade A Spike.As observed for Clade A Spike antibody, DMTs, such as ocrelizumab, fingolimod, and ofatumumab, decreased the antibody binding to Delta and Omicron Spike. Still, the pattern of antibody recognition was similar between the three Spikes and all DMTs analysed, i.e. alemtuzumab, natalizumab, teriflunomide, and interferons. Our data suggest that, irrespectively of DMTs, antibodies generated after vaccination did not bind Spike from recent VoCs to the same extent as the original Spike used in COVID-19 vaccines. Conclusion(s): Some DMTs reduce Spike antibody titres or prevent seroconversion. The sequence of Spike used in the first generation of vaccines may need to be updated for emerging VoC.

COVID-19-ASSOCIATED PULMONARY MUCORMYCOSIS PRESENTING AS SPONTANEOUS PNEUMOTHORAX: A RARE PRESENTATION

SESSION TITLE: COVID-19 Case Report Posters 1 SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Mucormycosis is an angio-invasive fungal infection with substantial morbidity and mortality. While diabetes and immune suppression remain well-known risk factors for mucormycosis, COVID-19 is now emerging as its independent predictor. CASE PRESENTATION: A 43-year-old male, with a history of hyperlipidemia and alcoholism, presented to the hospital with complaints of progressive dyspnea on exertion, productive cough, intermittent fever, anorexia, and chest pain over the course of 2 weeks. About 5 weeks prior to the current presentation, he was tested positive for COVID-19 by a polymerase chain reaction (PCR) based test and remained in quarantine at home. He was not vaccinated against COVID-19. He had no known immunosuppressive disease. On initial examination, he was ill-appearing and had a temperature of 101 F, blood pressure 138/83 mmHg, respiratory rate 22/minute, pulse 102/minute, and saturation of 91% on 2 L nasal cannula oxygen. A computerized tomography (CT) scan of the chest revealed small bilateral pneumothorax (2 cm and 5mm) along with extensive ground-glass opacifications in all lobes. In the next 24 hours, the right-sided pneumothorax progressed to tension pneumothorax requiring pigtail pleural drainage catheter placement. The drained pleural fluid had more than 100,000/uL total nucleated cells (91% neutrophils, 2% lymphocytes, and 1% eosinophils) and ultimately cultures grew Rhizopus spp. He was started on intravenous liposomal amphotericin-B infusion (5 mg/kg daily). On hospital discharge, he was switched to oral posaconazole (started with loading 300 mg delayed-release tablet twice a day, followed by 300 mg dosing of delayed-release posaconazole tablets daily) to complete the long term treatment course. DISCUSSION: Most of the reported cases of mucormycosis in COVID-19 were in patients with either diabetes or receiving steroids. This is a rare presentation of COVID-19–associated pulmonary mucormycosis (CAPM) as spontaneous pneumothorax, in the absence of known immunosuppression history. COVID-19 results in a considerable increase in cytokines, particularly interleukin-6 (IL-6), which increase free iron by increasing ferritin levels due to increased synthesis and decreased iron transport. Also, concomitant acidosis increases free iron by reducing the ability of transferrin to chelate iron and this available iron becomes a considerable resource for mucormycosis. [1] Also, Mucorales adheres to and invades endothelial cells by specific recognition of the host receptor glucose-regulator protein 78 (GRP-78). Acidosis associated with severe COVID-19 triggers GRP-78 and fungal ligand spore coating homolog (CotH) protein expression on endothelial cells, both contributing to angioinvasion, hematogenous dissemination, and tissue necrosis. [2] CONCLUSIONS: Mucormycosis can present as spontaneous pneumothorax after recent COVID-19 and clinicians should be aware of rare clinical presentation. Reference #1: Singh AK, Singh R, Joshi SR, et al. Mucormycosis in COVID-19: A systematic review of cases reported worldwide and in India. Diabetes Metab Syndr Clin Res Rev 2021;15:102146. doi:10.1016/j.dsx.2021.05.019 Reference #2: Baldin C, Ibrahim AS. Molecular mechanisms of mucormycosis—The bitter and the sweet. PLOS Pathog 2017;13:e1006408. doi:10.1371/journal.ppat.1006408 DISCLOSURES: No relevant relationships by Faran Ahmad No relevant relationships by AYESHA BATOOL No relevant relationships by Zachary DePew No relevant relationships by Neil Mendoza

A Quality Improvement Initiative Using EPIC Smartsets to Improve Adherence to AAP Guidelines for Obesity Management at a Primary Care Pediatrics Clinic

Purpose/Objectives: Effective management of childhood obesity is critical to prevent long-term medical and psychosocial sequelae. In 2015, the AAP issued guidelines on monitoring body mass index (BMI) and providing comprehensive obesity care based on risk factors. However, literature demonstrates that physician adherence to these guidelines is often poor. Electronic health record (EHR) clinical decision support tools can be effective in standardizing weight management. Utilizing EPIC SmartSets to improve physician adherence to AAP obesity management guidelines, we aimed to increase by 30% in 6 months the following: formal diagnosis of elevated BMI, frequency of weight follow-up visits, adherence to recommended lab screening, and subspecialty referrals. Design/Methods: Pre- and post-intervention surveys were distributed to residents/faculty at an academic primary care clinic to identify variability in practice and barriers to guideline adherence, which informed intervention designs. Cycle 1: SmartSets were implemented in July 2020 with diagnosis codes, note templates, readiness to change surveys, recommended lab and referral orders, patient handouts/questionnaires, and follow-up visit suggestions. Education was completed for providers. Cycle 2: Based on end-user input, SmartSets were integrated into preexisting well-visit templates rather than requiring separate workflow. Analysis metrics included the percentage of: well-visits with an appropriate diagnosis of elevated BMI, acute visits designated as weight follow-ups, and weight or well-visits in which labs were ordered or subspecialty referrals placed. All patients with BMI 85-94.9%ile (overweight) and BMI ≥95%ile (obese) ages 2-17.9 years old seen from 7/1/2019 to 3/31/2021 were included. Data was plotted on run/control charts to assess trends after implementation and revision. Results: A total of 748 overweight patients and 669 patients with obesity were seen during this timeframe. There was a sustained increase in appropriate diagnosis of elevated BMI from an average of 49% pre-intervention to 71% postintervention (Fig. 1), surpassing our aim. There were no significant trends in the percentage of weight visits, labs, or referrals. Appropriate utilization of the implemented EHR tools for well-visits improved after second cycle revisions (39% to 88%). Provider-perceived barriers to AAP guideline adherence included lack of family willingness to participate in management, lack of visit time, and socioeconomic factors out of the provider's control (Fig. 2). Conclusion/Discussion: The first step to instigate practice changes is through problem identification. By utilizing end-user feedback and preserving clinical workflows, the incorporation of AAP guidelines into EPIC SmartSets improved the diagnosis of elevated BMI during well-visits. However, due to COVID-19, it is unclear whether lab orders, referrals, or weight follow-ups improved. Additional EPIC modifications, such as auto-populated lab results, could minimize the need to chart review and thus improve these behaviors. While we demonstrated improved physician recognition, more studies are warranted to address the complex challenges primary care providers and families face regarding weight management. - Control Chart for BMI Diagnoses Made at Applicable Well Child Checks (WCC) by Month Percent of patients with elevated BMI seen at a well-visit from July 2019 through March 2021 who were formally given the diagnosis of elevated BMI. Goal to increase appropriate diagnoses by 30%. -Pareto Chart of Perceived Barriers to Adherence to AAP Guidelines for Weight Management Based upon surveys of residents and faculty at the academic pediatrics clinic studied.

Towards diagnostic and therapeutic avenues against SARS-CoV-2 by combining CryoEM and molecular simulations

Molecular simulations have been instrumental in identifying the structure-function relationships of biomolecules in the atomic level as well as providing a means for structure-based drug discovery, thereby explaining and guiding experimental findings. The increase in computational power, the new physics and machine-learning-based algorithms is significantly driving the boost in the field and gives access to addressing biomolecular phenomena of increasing length and timescales. In this talk I will discuss examples where using state-of-the-art integrative structural biology methods that inject Cryo-EM experimental data into the simulation, we can reveal accurate protein-functional dynamics of the SARS-CoV-2 spike protein in an atomistic level. In this way we can a) reveal virus vulnerabilities by identifying cryptic binding sites exposed during the S protein conformational transition related to the recognition to the host cell and b) provide with the molecular motion and energetics of protein-antibody complexes which enables to suggest mutations that increase the spike-antibody affinity. These predictions are validated in further CryoEM experiments.

Practical tapered optical fiber system for in-situ label-free sensing of various antigens

A practical tapered optical fiber (TOF) biosensing system was developed for label-free detection using antigen-antibody pairs with repeatable results and a very high degree of sensitivity. This was done by attaching molecular recognition agents to a tapered fiber surface for augmenting sensitivity and specificity of analyte. The entire system included three main parts: a tunable laser, a tapered fiber, and an optical detector. Light from an unpolarized tunable fiber laser was introduced into the tapered fiber from one end, and the transmitted intensity was detected by a photodetector. In the tapered fiber area, the evanescent electromagnetic field, which extends outside the fiber, was able to detect minute changes in the refractive index caused by antigen-antibody pairs. Recorded data was analyzed using an innovative Fourier analysis method to find phase changes, which are directly related to the biomolecular concentration coated on fiber, from which antibody-antigen concentrations are obtained. Two experiments were performed to confirm the concept using two very different agents. The first was the protein Interleukin-8 (IL-8). Repeatable results with a sensitivity of 10 pg/mL were achieved. The second was human coronavirus OC43 (HCoV-OC43), a surrogate viral particle for SARS-CoV-2, with a sensitivity of 50 viruses/mL. Critical sources of error were identified and addressed for the purpose of using the device for real clinical diagnosis in various real-life environments, where viruses can reside in water, phosphate-buffer solution, or saliva, the most popular three environments in real clinical diagnosis. Our device was designed according to the principle that only one specific kind of antibody and antigen can be combined together. The device demonstrated good accuracy to chosen analyte(s) tailored to specific applications and offered the potential to develop a point-of-care device used in clinics, as well as for detecting a variety of viruses and biocontaminants. The reproducibility of TOFs was confirmed through multiple fabrications and consistent results. [ FROM AUTHOR] Copyright of Optical Engineering is the property of SPIE - International Society of Optical Engineering and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

PUTTING RESEARCH INTO PRACTICE: KNOWLEDGE TRANSLATION AND IMPLEMENTATION FOR ACTION ON NUTRITION

The transfer of research evidence into practice has been historically slow, and requires an integration of many elements, including quality evidence, supportive physical and intellectual environments, and facilitation, as discussed at the NNEdPro Sixth International Summit on Nutrition and Health. Examples of applying clinical research into practice focused on the use of group consultations (also known as group clinics or shared medical appointments) to support behaviour change, the role of dietary micronutrients during the COVID-19 pandemic and the potential of Precision Nutrition. An emerging area from early implementation evidence includes group consultations, also known as shared medical appointments, as discussed by Dr Fallows. Group consultations have been shown to improve clinical outcomes for some patient groups (e.g., HbA1c, lipids, BMI), as well as improve self-care and health education, and patient and clinician satisfaction. These groups have been piloted throughout the UK both face-to-face and virtually, with initial findings suggesting they are feasible and acceptable to patients and clinicians. Further work is needed to assess whether these could be cost-effective when scaled-up in National Health Service UK primary care. During the COVID- 19 pandemic, there has been increasing emphasis on the central role of nutrition in health, including the role of dietary micronutrients, as discussed by Dr Van Dael and Shane McAuliffe. Nutrition plays an important role in immunity, yet the nutritional status of the most vulnerable population groups is likely to deteriorate further due to the health and socio-economic impacts of the novel coronavirus. Thus, implementation of this evidence into health care practice is key. Precision Nutrition, defined as an 'approach that uses information on individual characteristics to develop targeted nutrition advice, products or services', offers an exciting opportunity to further individualise dietary advice for behaviour change, as discussed by Dr Kohlmeier and Dr Hernandez. Precision nutrition is underpinned by the recognition that individuals differ in many important ways due to identifiable molecular traits and can be utilised to determine personalised weight loss interventions based on genetic variants. Use of implementation science is in line with one of the six cross-cutting pillars of the Nutrition Decade: Aligned health systems for universal coverage of nutrition actions. Dr Bell, an Advanced Accredited Practising Dietitian in Australia, provided an overview of key implementation science models and frameworks. Implementation frameworks such as the Action Research Framework, the Knowledge to Action Cycle, and the Spread and Sustain Framework, are underpinned by knowledge creation, effective education, and culture change. Dr Bell then highlighted how theoretical frameworks have provided guidance for the implementation of real world, complex nutrition interventions, including the Systematised Interdisciplinary Program for Implementation and Evaluation (SIMPLE) in Australia, and the More-2-Eat program in Canada.

Differences between Omicron SARS-CoV-2 RBD and other variants in their ability to interact with cell receptors and monoclonal antibodies.

SARS-CoV-2 remains a health threat with the continuous emergence of new variants. This work aims to expand the knowledge about the SARS-CoV-2 receptor-binding domain (RBD) interactions with cell receptors and monoclonal antibodies (mAbs). By using constant-pH Monte Carlo simulations, the free energy of interactions between the RBD from different variants and several partners (Angiotensin-Converting Enzyme-2 (ACE2) polymorphisms and various mAbs) were predicted. Computed RBD-ACE2-binding affinities were higher for two ACE2 polymorphisms (rs142984500 and rs4646116) typically found in Europeans which indicates a genetic susceptibility. This is amplified for Omicron (BA.1) and its sublineages BA.2 and BA.3. The antibody landscape was computationally investigated with the largest set of mAbs so far in the literature. From the 32 studied binders, groups of mAbs were identified from weak to strong binding affinities (e.g. S2K146). These mAbs with strong binding capacity and especially their combination are amenable to experimentation and clinical trials because of their high predicted binding affinities and possible neutralization potential for current known virus mutations and a universal coronavirus.Communicated by Ramaswamy H. Sarma.

Characterization of Aurintricarboxylic Acid (ATA) Interactions with Plasma Transporter Protein and SARS-CoV-2 Viral Targets: Correlation of Functional Activity and Binding Energetics.

In an effort to identify functional-energetic correlations leading to the development of efficient anti-SARS-CoV-2 therapeutic agents, we have designed synthetic analogs of aurintricarboxylic acid (ATA), a heterogeneous polymeric mixture of structurally related linear homologs known to exhibit a host of biological properties, including antiviral activity. These derivatives are evaluated for their ability to interact with a plasma transporter protein (human serum albumin), eukaryotic (yeast) ribosomes, and a SARS-CoV-2 target, the RNA-dependent RNA polymerase (RdRp). The resultant data are critical for characterizing drug distribution, bioavailability, and effective inhibition of host and viral targets. Promising lead compounds are selected on the basis of their binding energetics which have been characterized and correlated with functional activities as assessed by inhibition of RNA replication and protein synthesis. Our results reveal that the activity of heterogeneous ATA is mimicked by linear compounds of defined molecular weight, with a dichlorohexamer salicylic-acid derivative exhibiting the highest potency. These findings are instrumental for optimizing the design of structurally defined ATA analogs that fulfill the requirements of an antiviral drug with respect to bioavailability, homogeneity, and potency, thereby expanding the arsenal of therapeutic regimens that are currently available to address the urgent need for effective SARS-CoV-2 treatment strategies.

SARS-CoV-2 SPIKE EXPRESSION at the SURFACE of INFECTED HUMAN AIRWAY EPITHELIAL CELLS

Background: Emerging evidence points out to potential benefits from Fc-mediated effector functions in SARS-CoV-2 infection. Some Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) require recognition of the antigen at the surface of infected cells. Methods: To evaluate the expression levels of SARS-CoV-2 Spike at the surface of infected airway epithelial cells, we developed an intracellular staining against SARS-CoV-2 nucleocapsid (N). This assay allows the distinction between infected versus uninfected cells. Human primary airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. Infected cells were identified with an anti-N antibody and cell surface expression of Spike measured with the conformational-independent anti-S2 CV3-25 antibody. Results: We found robust SARS-CoV-2 Spike expression at the cell surface of pAECs. Infected cells were readily recognized with plasma from convalescent and vaccinated individuals. Importantly, recognition of SARS-CoV-2 infected cells strongly correlated with Fc-mediated effector functions measured in a cohort of vaccinated naïve and previously-infected individuals. Conclusion: Altogether, our findings further support the importance of measuring Fc-mediated effector function in infection and vaccination settings for SARS-CoV-2.

Promising Therapeutic Approach for SARS-CoV-2 Infections by Using a Rutin-Based Combination Therapy.

The development of new therapeutic approaches for SARS-CoV-2 infections is of particular current importance. The combination therapy proposed here is based on already proven, safe and inexpensive compounds. The natural compound rutin, one of the six therapy components, has the potential to inhibit both viral and host cell targets. In addition, this therapy involves the use of acetylsalicylic acid, vitamin C and vitamin D3 , which should be administered together with calcium and magnesium. The importance of each component is briefly described in this article. Due to multiple anti-infective properties of rutin, it provides a basis for combating a SARS-CoV-2 infection as well as various viral and bacterial co-infections. There are strong indications for a good effect of this simple combination therapy, especially in the early stages of infection. It has the potential to be of interest both prophylactically and therapeutically, and offers the possibility of protection against severe disease progression.

IMPLEMENTING CHANGE IN TREATMENT DELIVERY FOR CHILDREN AND YOUNG PEOPLE DURING A PANDEMIC

Background/Aims The Paediatric Rheumatology Team at the Great North Children's Hospital (GNCH) provides care to children and young people (CYP) across the northern region. On average 70 new CYP are diagnosed annually with rheumatic conditions. Approximately 1/3 will require long-term treatments and long-term rheumatology follow-up. The team support over 300 patients receiving treatments for at home, with 75 CYP requiring regular treatments administered in hospital. Tocilizumab targets IL-6 and is used to treat rheumatic conditions. Tocilizumab, approved for use in children with certain subtypes of JIA in 2011, is normally administered via an intravenous infusion on the Paediatric Day Unit, at GNCH. The procedure takes approximately 3 hours and involves a cannula for administration. Part of Continuity and Emergency Planning for the COVID- 19 pandemic the Medicines & Healthcare Products Regulatory Agency (MHRA) compiled a list in April 2020 of medications including tocilizumab, not to be exported from the UK or hoarded. Tocilizumab was a proposed treatment for hyper-inflammatory response for COVID-19 and concern that supplies would be affected. NHS hospitals were advised to reduce routine activity and footfall through the hospital. The team agreed the need to consider alternative treatment delivery. Methods A patient cohort were identified attending GNCH for intravenous infusions. 38/65 (58%) patients were receiving tocilizumab. The pharmacist agreed to switch CYP from intravenous to subcutaneous tocilizumab. A total of 34 eligible patients were identified (32/38 current patients and two new patients) Using a multi-disciplinary approach, the pharmacist established agreement, arranged supply and delivery of medication to the families. The CNS contacted all families via nurseled telephone clinic and invited eligible CYP to a face-to-face clinic, to received counselling and training to administer. The rheumatology consultants supported the change with prescription management. Over a 6-week period all eligible CYP transferred to subcutaneous form of tocilizumab administered at home. Medication was transported to families via home care delivery company. Results 34 patients switched, 9/34(26%) reported localised injection-site reactions, varying in severity but could lead to discontinuation of treatment. An inconsistent approach to managing these reactions was recognised by the pharmacist and CNS. Collaboration with the paediatric allergy teams, devised a standardised approach to manage injection-site reactions using an algorithm. Conclusion • Reduce pressure on precious hospital resources • More convenient for families - minimise long distance travel for treatment and time off school/ work • True collaboration and recognition of multidisciplinary team working swiftly to find a solution to a problem impacting upon patients, their family and the Trust • Algorithm devised by the paediatric pharmacist using easily accessible oral and topical treatments for families to use at home to manage site reactions. • Tocilizumab has proven to be useful in treatment of critically unwell patients infected with COVID-19.

Structure and function of SARS-CoV-2 spike protein and its receptor

Corona virus disease 2019 is an acute infectious disease caused by SARS-CoV-2 infection and has entered the state of global pandemic. Spike protein ( S protein) , a key protein that mediates SARS-CoV-2 to infect host cells, has the characteristics of specific receptor binding and membrane fusion, playing an important role in host tropism and virulence. The spontaneous closed and open conformation of S protein trimer is crucial for receptor binding and initiation of conformational changes in membrane fusion, and its unique furin recognition site may be a crucial factor leading to high infectivity. Therefore, to study the structure and function of SARS-CoV-2 S protein and its receptor has important implications for invasion mechanisms of SARS- CoV-2 and the development of relevant targeted drugs.