Seroprevalence of anti-severe acute respiratory syndrome coronavirus 2 antibody among healthy blood donors in a hospital-based blood center in Eastern India during the COVID-19 pandemic.

BACKGROUND: Few international studies have reported the prevalence of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in healthy, asymptomatic blood donors. These findings have definitely raised queries regarding blood safety and transfusion-transmitted coronavirus disease (COVID)-19. We conducted this first anti-SARS-CoV-2 seroprevalence survey among the healthy blood donors in Eastern India. MATERIALS AND METHODS: The study included 611 healthy blood donors who donated whole blood (WB) in our blood center. For detailed analysis, social and demographic details of all donors like gender, age, weight, occupation, and place of residence were included. Donor eligibility criteria for WB donation were followed as per existing national guidelines. Residual serum samples leftover after screening the mandatory infectious markers were tested for the presence of anti-SARS-CoV-2 IgG directed against domain S1 and S2 of the SARS-CoV-2 spike protein using automated enhanced chemiluminescence technology following the manufacturer's instructions. RESULTS: The mean overall seroprevalence of anti-SARS-CoV-2 antibody in blood donors was observed to be 4.4% (95% confidence interval 3.8-4.9) with a monthly increasing trend. Seroprevalence adjusted for sensitivity and specificity of the assay was 4.1%. The mean S/Co values of reactive donor samples were observed to be 2.99 and 3.42 in June and July 2020, respectively (P = 0.013). No significant variation in seroprevalence rate was observed among donor variables like donor age, gender, profession, and educational qualification. A higher significant prevalence of antibody was observed among voluntary donors and donors residing in suburban areas (P < 0.05). Among the ABO blood groups, no statistical significance of seroprevalences was observed among the various ABO blood groups. CONCLUSION: We conclude that despite many limitations in the current study, we found 4.4% seroprevalence of anti-SARS-CoV-2 antibody in the asymptomatic, healthy, epidemiologically, and medically screened blood donors. These data are definitely the tip of an iceberg and signify much higher seroprevalence in the normal population and indicate that protective measures like masking and social distancing should remain implemented for a long term.

The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.

The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective.

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TEASER: We revise Remdesivir's mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP.

Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates.

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aß (TMR-Aß) to characterize its stability and heterogeneity. Disaggregation of TMR-Aß amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aß amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.

Single-Molecule FRET Studies of the Hybridization Mechanism during Noncovalent Adsorption and Desorption of DNA on Graphene Oxide.

Remarkable observations on the adsorption and desorption mechanisms of single-stranded oligonucleotides and the hybridization of double-stranded DNA (ds-DNA) on a graphene oxide (GO) surface have been made using ensemble and single-molecule fluorescence methods. Probe and target DNAs labeled individually with fluorescence resonance energy transfer (FRET) pairs and having similar adsorption affinities toward the GO surface are used to provide detailed insights into the hybridization mechanism. Single-molecule FRET results reveal an "in situ" DNA hybridization mechanism, i.e., hybridization between the probe and target DNAs to form a ds-DNA, and simultaneous desorption from the GO surface thereafter. These results also demonstrate that the electrostatic interaction between DNA and GO is of little importance to the overall theory of interaction and the largest effects are from solvation forces, specifically the hydrophobic effect. This investigation improves the fundamental understanding of the DNA hybridization dynamics on the GO surface, opening new windows in the field of biophysics as well as in sensing and therapeutic applications.

Monitoring the Competence of a New Keto-tetrahydrocarbazole Based Fluorosensor Under Homogeneous, Micro-Heterogeneous and Serum Albumin Environments.

We present here a detailed photophysical study of a recently synthesised fluorophore 8-methyl-8,9-dihydro-5H-[1,3]dioxolo[4,5-b]carbazol-6(7H)-one. This is a synthetic precursor of bio-active carbazole skeleton Clausenalene. Spectroscopic investigation of the fluorophore has been carried out in different protic and aprotic solvents, as well as in binary solvent mixtures, using absorption, steady-state and time-resolved fluorescence techniques. This fluorophore is particularly responsive to the hydrogen bonding nature as well as polarity of the solvent molecules. When considered in micelles and ß-cyclodextrin, this behaves as a reporter of its immediate microenvironment. Steady state and time resolved fluorometric and circular dichroism techniques have been used to explore the binding interaction of the fluorophore with transport proteins, bovine serum albumin and human serum albumin. The probable binding sites of the fluorophore in the proteinous environments have been evaluated from fluorescence resonance energy transfer study. Laser flash photolysis experiments also have been performed to observe the triplet excited state interaction between the fluorophore and albumin proteins.