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The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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The spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is directly responsible for the binding to ACE2 receptors in host cells. While the spike protein overall is known to form trimers, the oligomerization state of the transmembrane domain of the spike protein in SARS-CoV-2 is unknown. It is believed to be essential for the function of this protein. Since the transmembrane domain of the spike protein is highly conserved in SARS-CoV-2 it is important to investigate its character and determine its relationship to the function of the protein as awhole. The goal of this project was to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the spike protein in SARS-CoV-2. The most practical method to synthesize the TM domain of the S protein is through solid phase peptide synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will then need to be purified using high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography- mass spectrometry (LCMS) to confirm the identity of the synthesized peptides as well as any potential impurities. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS-CoV-2. The function of the S protein is believed to only present when the transmembrane domain forms a trimer. Therefore, the analysis of their oligomerization states will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and tetramethylrhodamine (TAMRA) in a fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Rapid diagnosis of coronavirus disease 2019 (COVID-19)-infected patients is urgent in making decisions on public health measures. There are different types of diagnostic tests, such as quantitative PCR assay, antibody, and antigen-based and CRISPR-based tests, which detect genetic materials, viral proteins, or human antibodies in clinical samples. However, the proper test should be highly sensitive, quick, and affordable to address this life-threatening situation. This review article highlights the advantages and disadvantages of each test and compares its different features, such as sensitivity, specificity, and limit of detection to reach a reliable conclusion. Moreover, the FDA- authorized kits and studies' approaches toward these have been compared to provide a better perspective to the researchers.Copyright © 2022 Lippincott Williams and Wilkins. All rights reserved.
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Partitioning and effect of antiviral GC376, a potential SARS-CoV-2 inhibitor, on model lipid membranes was studied using dynamic light scattering (DLS), UV–VIS spectrometry, Excimer fluorescence, Differential scanning calorimetry (DSC) and Small- and Wide-angle X-ray scattering (SAXS/WAXS). Partition coefficient of GC376 between lipid and water phase was found to be low, reaching KP = 46.8 ± 18.2. Results suggest that GC376 partitions into lipid bilayers at the level of lipid head-groups, close to the polar/hydrophobic interface. Changes in structural and thermodynamic properties strongly depend on the GC376/lipid mole ratio. Already at lowest mole ratios GC376 induces increase of lateral pressures, mainly in the interfacial region of the bilayer. Hereby, the pre- and main-transition temperature of the lipid system increases, what is attributed to tighter packing of acyl chains induced by GC376. At GC376/DPPC ≥ 0.03 mol/mol we detected formation of domains with different GC376 content resulting in the lateral phase separation and changes in both, main transition temperature and enthalpy. The observed changes are attributed to the response of the system on the increased lateral stresses induced by partitioning of GC376. Obtained results are discussed in context of liposome-based drug delivery systems for GC376 and in context of indirect mechanism of virus replication inhibition.
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Background: Protecting vulnerable groups from severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is of great importance, as they suffer more often from severe or fatal courses. The immune response after a third vaccination is insufficiently studied in patients after hematopoietic stem cell transplantation (HSCT). Method(s): We analyzed the immune response before and after a third vaccination against SARS-CoV-2 in HSCT patients and healthy controls. This is the first report on neutralizing antibodies against 11 variants of SARSCoV- 2, analyzed by competitive fluorescence assay. Humoral immunity was also measured by semi-/quantitative ELISA and neutralization tests. Cellular immunity was assessed using interferon-gamma (IFN-gamma) and inter-leukin-2 (IL-2) ELISpots. Result(s): After the third vaccination, the cellular immune responses were 2.4-fold higher for IFN-gamma in healthy controls vs. HSCT patients, and 3.4- fold higher for IL-2. On average, healthy controls vs. HSCT patients had 1.5-fold higher concentrations of neutralizing antibodies against the variants and 1.6-fold higher antibody ratios and 4.4-fold higher antibody concentrations against wild-type SARS-CoV-2. Conclusion(s): We show that immune responses of HSCT patients are significantly lower vs. healthy controls, but HSCT patients also exhibit neutralizing antibodies to variants of SARS-CoV-2. Our findings may contribute to the adaptation of vaccination strategies and further protective measures for immunocompromised individuals.
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Introduction: Mononucleosis is an infectious disease caused by Epstein-Barr virus (EBV, human herpes virus type 4, HHV-4) and is characterized by asthenia, fever pharyngitis, and lymphadenopathy. In our laboratory diagnosis is made by rapid test and Epstein-Barr virus antibody assay. The presence of Epstein-Barr virus (VCA) specific IgM antibodies indicates primary infection. A marked lymphocytosis with inversion of the formula can be seen on the blood count. The smear shows numerous activated lymphocytic elements By examining the complete scattergram of patients with confirmed primary infection we noticed a peculiar arch arrangement of the lymphocytes in the FL1 x ALL specific leukocyte scatters. Method(s): In this study Vircell's Virapid mono M&G is used, an immunotest for the qualitative determination of 4 serological markers of EBV: two IgM, VCA and heterophile, and two IgG, VCA and EBNA. The presence of anti-VCA IgM antibodies and the absence of anti-EBNA antibodies are indicative of primary infection. CBC was performed on Abbott Alinity hq, which uses a combination of photometry optical counting and fluorescence analysis in order to enumerate cells and cellular constituens. The instrument utilizes eight light scatter detectors which include ALL (axial light loss), IAS (intermediate angles of light scatter), PSS (polarized side scatter), DSS (depolarized side scatter) and FL1 ( fluorescent channel). Result(s): The sixteen cases examined, all of which resulted positive for primary infection on the rapid test, showed a peculiar FL1 x ALL scattergram (see Fig.1). In the lymphocytes' scattergram cloud, we observed an archshaped trend going upwards and rightwards, thus highlighting cells with greater fluorescence and size Often these lymphocytes are identified as monocytes In cases of lymphocytosis from other causes (CLL lymphomas) we can see how the lymphocytes' scattergram cloud is totally different. In such a case the cloud seems like a short bar due to lymphocytes with increased fluorescence signal though with small size (see Fig.2). Conclusion(s): In the 16 cases of primary EBV infection examined, the blood count shows a peculiar FL1 x ALL scattergram, which compared with the scattergram of other causes of lymphocytosis highlights a substantial difference that could support the laboratory technician in the diagnostic differentiation of a lymphocytosis: lymphocytic response to viral infection (EBV, SARS-Cov19, ecc) or monoclonal lymphocyte proliferation.
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A novel fluorometric strategy for the simultaneous identification of SARS-CoV-2 and SARS-CoV was successfully established based on a hybridization-induced signal on-off-on mechanism. Here, one part of the probe (P1) of SARS-CoV-2 (P = P1/P2) is partially related to SARS-CoV, while the other part (P2) is completely irrelevant to SARS-CoV. They as smart gatekeepers were anchored on NH2-MIL-88(Fe) (MOF@P1/P2) to turn off its catalytic performance. Only the specific SARS-CoV-2 genetic target can strongly restore the peroxidase-like activity of MOF@P1/P2. In the presence of o-phenylenediamine, SARS-CoV-2 can be efficiently detected with high sensitivity, accuracy, and reliability. This strategy demonstrated excellent analytical characteristics with a linear range (10-9 M â¼ 10-6 M) under the limit of detection of 0.11 nM not only in buffer but also in 10 % serum, which partly shows its practicability. Most importantly, with the help of the auxiliary test of MOF@P1 and MOF@P2, SARS-CoV-2 and SARS-CoV can be efficiently quantified and distinguished. This novel strategy has provided a breakthrough in the development of such identification. In the whole process, only a simple one-step experiment was involved. This circumvents the trouble of pretreatment experiments in traditional methods, including complex enzymatic mixtures, specialized experimental equipment, many primers optimization as well as reverse transcriptase. Additionally, this novel strategy is rapid, low-cost, and easy-to-use tools.
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The proceedings contain 456 papers. The topics discussed include: DSLK and Kg are antithetical antigens in the RHAG blood group system;identification of a new low prevalence antigen in the RHAG glycoprotein;characterization of a novel high-prevalence antigen in the KEL blood group system;structural locations of single-nucleotide missense variants in the Kidd blood group on human urea transporter B;two new JK silencing alleles identified by a single molecule sequencer with 20 kb-long reads;nanopore sequencing to resolve Kidd blood group discrepancies;removal of HLA class-I antigens from platelets: increasing platelet availability for refractory patients;human neutrophil antigens and their clinical significance;a novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1 antibodies;blood supply chain management what has been learnt from the pandemic and others?;building towards organizational resilience: a case study of the organizational impacts and changes in Sanquin during COVID-19;and contingency planning in blood transfusion services African COVID-19 experience.
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Introduction and Objectives Novel SARS-CoV-2 virus has been implicated in prompting a bold immune response that leads to severe Coronavirus disease 2019 (COVID-19). Recent studies have shown that SARSCoV-2-infected monocytes and macrophages are stimulated to produce an overabundance of pro-inflammatory cytokines and chemokines to generate a cytokine storm. Cytokines in excess can contribute to local tissue inflammation and the pathogenesis of COVID-19. However, the mechanism by which SARS-CoV-2 signal macrophage-derived inflammatory response remains unclear. In the present study, we used RAW 264.7 cells, a wellcharacterized macrophage model, to study the in vitro effects of SARS-CoV-2 on reactive oxygen species (ROS) production and its potential role in the signal transduction of cytokine production. Methods The effect of SARS-CoV-2 on ROS and cytokine generation in macrophages was assessed by treating RAW 264.7 cells with SARS-CoV-2 heat inactivated virus (0-20 million viral particles) or recombinant proteins for 24 hours. 2',7'-Dichlorodihydrofluorescein (2',7'-DCF) fluorescence analysis was utilized to quantify ROS generation within the RAW 264.7 macrophage cell line. Cell culture medium was sampled to quantify the levels of tumor necrosis factor (TNF) using enzyme-linked immunosorbent assay (ELISA). To assess the effects of SARS-CoV-2 on mitochondrial function, cells were treated with SARS-CoV-2 heat inactivated virus (0-20 million viral particles) for 24 hrs. Mitochondria-derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator. Results Treatment of RAW 264.7 cells with inactivated SARS-CoV-2 viral particles or recombinant proteins stimulated ROS production. Mitochondria-derived superoxide and hydrogen peroxide production were increased in response to inactivated SARS-CoV-2 viral particles and recombinant protein exposure. The increased ROS generation is linked to macrophage activation induced by SARS-CoV-2 exposures. Along with the ROS generation, increased TNF production was observed. Conclusions The results of this study suggest that both SARS-CoV-2 viral proteins and heat-inactivated viral particle exposures cause significant generation of ROS and cytokines by RAW 264.7 cells. ROS generation and the subsequent cytokine release apparently play a significant role in the pathogenesis associated with the SARS-CoV-2 viral infection. The imbalanced cellular defense system against oxidative stress commonly associated with aging could explain the increased occurrence of more severe SARS-CoV-2 illness in seniors and in patients with underlying health conditions. Based on the results from this study, we propose that antioxidants such as N-acetyl-L-cysteine, resveratrol, or Vitamin E in combination with antiinflammatory drug could be used to control excess ROS and cytokines in patients with severe COVID-19.
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Introduction: During Coronavirus Disease-2019 (COVID-19) pandemic, Healthcare Workers (HCWs) are the frontline personals who are engaged in different facilities of the health system. So they always remain at a greater risk of exposure and acquiring the disease. They may also become a potential source of infection to the other patients as well to the community. Aim: To estimate the seroprevalence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2) specific Immunoglobulin G (IgG) antibodies among asymptomatic, COVID-19 negative HCWs of a tertiary care hospital. Materials and Methods: This hospital-based cross-sectional study was conducted in the Department of Microbiology in a tertiary care hospital of North-East India. A total of 215 HCWs were recruited from 15th October to 14th December 2020 after taking written and informed consent. Inclusion criteria were: A) >18 years of age and both genders, b) asymptomatic, negative for COVID-19 either by Rapid Antigen Test (RAT) or Reverse Transcription Polymerase Chain Reaction (RT-PCR), c) working in the hospital for atleast last four months. Predesigned questionnaire was used for data collection. Serum samples were tested for SARS-CoV-2 IgG antibodies by Enzyme Linked Fluorescence Assay (ELFA) using VIDAS (VITEK ImmunoDiagnostic Assay System) platform. Chi-square test was used (Epi Info version 7 software) for data analysis. Results: The prevalence of SARS-COV-2 IgG was 54 (25.12%) out of 215, which was highest in ≤30 years age group, 27 (32.14 %) out of 84 (p=0.0261). Significant seropositivity was found among cleaners 22 (61.11%) out of 36 (p<0.01) and participants who reported having COVID-19-related symptoms in the previous months (p<0.013). However gender, daily patient contact, close contact with COVID-19 cases and working in COVID-19 units showed no significance. Conclusion: The study highlighted a high burden of asymptomatic SARS-CoV-2 infection among HCWs. A proper surveillance system is needed for estimating the burden of COVID-19 among HCWs as well as in the community for better understanding of the dynamics of the infection.
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Background:SARS-COV-2 is a new Beta Corona virus discovered in China (December 2019).It is responsible for an international outbreak of respiratory illness termed COVID -19 ranging from self-limiting Respiratory Tract Illness to Severe Progressive Pneumonia, Multi Organ Failure and Death. Methods:This prospective study was done to analyze the clinical features, seroconversion and lab abnormalities in Post Covid cases from June 2020- Dec 2020.Whole blood samples were collected from Post COVID -19 cases (cases who were tested SARS COV- 2 Posi- tive with RT PCR in Government Testing Centers and were quarantined for 1 month from the day of testing) who presented to OPD with fatigue, breathing difficulty, muscle ache etc. They were tested for IgM and IgG using Biomerieux VIDAS SARS COV - 2 IgM and IgG Assay kits respectively. (Enzyme Linked Fluorescence Assay- ELFA). Clinically relevant lab investigations were also done and noted. [Formula presented] Results:Out of 40 cases tested, 37cases (92.5%) have mounted a specific anti SARS COV-2 IgM and/or IgG immune response. IgM was positive in 90% of (30-50days) Post COVID group.Ddimer levels were raised (62%) in cases with breathing difficulty and severe fatigue. Inflammatory markers like HsCRP, ESR were also raised and correlat- ed with the severity of post COVID symptoms.The test values of IgM and IgG obtained on testing singly on VIDAS instrument seem to correlate with the severity of COVID disease history given by the cases. Conclusions:Majority of post COVID cases mount a specific anti SARS COV2 response. The immune response / Seroconversion is Signifi- cant in COVID survivors who suffered from severe disease. Severe fatigue, altered taste, breathing difficulty are the common symp- toms in post COVID cases.