Biology subjects

Joshi, D. M.

Publications and source records attributed to Joshi, D. M..

3 recordsLinked to original sources

bioRxiv2021

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (Heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is high globally than the production due to increased incidences of various heart conditions. The main source of streptokinase is from various strains of Streptococcus. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD inhibitor gene for production of streptokinase that needs to be knocked out in order to increase it expression. However, FasX is a small RNA (sRNA) present in group A Streptococcus species which is responsible for post-transcriptional regulation of streptokinase (ska) gene by binding at the 5 end of ska mRNA. S. equisimilis is a {beta}-hemolysin producing streptococcus bacterium (group C) containing the orthologue of FasX and natively expresses a clinically important thrombolytic streptokinase. In order to improve the stability of mRNA and increasing the expression of streptokinase which is inhibited by SagD. We used CRISPR-Cas9 to successfully knock-out of SagD gene and observed a 13.58-fold relative quantification of streptokinase expression in the mutant strain as compared to wild type. We have also demonstrated the successful target gene knockout using CRISPR-Cas9 in S. equisimilis that engineered strain can be used further for overexpression of streptokinase for therapeutic applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/465095v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8e3ebeorg.highwire.dtl.DTLVardef@b92f4eorg.highwire.dtl.DTLVardef@1d0dd8borg.highwire.dtl.DTLVardef@180ee54_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Defective ORF8 dimerization in delta variant of SARS CoV2 leads to abrogation of ORF8 MHC-I interaction and overcome suppression of adaptive immune response

In India, the breakthrough infections during second wave of COVID-19 pandemic was due to SARS-COV-2 delta variant (B.1.617.2). It was reported that majority of the infections were caused by the delta variant and only 9.8% percent cases required hospitalization whereas, only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections was observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Down regulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant results in structural instability of ORF8 dimer caused by disruption of hydrogen bonding and salt bridges as revealed by structural analysis and MD simulation studies of ORF8 dimer. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 may not hindering the MHC-I expression thereby resulting in better immune response against SARS-CoV-2 delta variant, which partly explains the sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/457457v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@751eeaorg.highwire.dtl.DTLVardef@140b5b5org.highwire.dtl.DTLVardef@159a3a5org.highwire.dtl.DTLVardef@6c206_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

E156/G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.167.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape

Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/447321v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b92cborg.highwire.dtl.DTLVardef@1d261f7org.highwire.dtl.DTLVardef@11da73eorg.highwire.dtl.DTLVardef@1cef6ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗