Biology subjects

Raj, V. S.

Publications and source records attributed to Raj, V. S..

4 recordsLinked to original sources

bioRxiv2025

A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

Gene cloning, site-directed mutagenesis (SDM), and short gene synthesis are essential tools in molecular biology, yet existing approaches remain constrained by efficiency, flexibility, and cost. Here, we developed a versatile, simple, and rapid (VSR) 3-in-1 method that integrates seamless cloning, mutagenesis, and short gene assembly in a single platform. This approach works on the principles of overlap extension polymerase chain reaction (OPCR), uses high-fidelity DNA polymerase and short overlapping primers. Target genes can be directly amplified from genomic DNA or complementary DNA (cDNA) templates and cloned into vectors without intermediate purification, restriction enzyme digestion, or ligation. VSR supports the insertion of DNA fragments ranging from 80 bp to 33 kb at any plasmid locus while enabling the introduction of single or multiple nucleotide substitutions at one or more sites in a single reaction. Using this method, we cloned 35 genes of diverse lengths and two complete viral genomes with efficiencies ranging from 60-100% and introduced up to 17 substitutions in a single mutagenesis reaction. We further demonstrate efficient de novo synthesis of short genes from overlapping oligonucleotides. The VSR method is a reliable, rapid, and cost-effective approach with a wide range of applications for both routine and high-throughput workflows. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/682745v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1295a5aorg.highwire.dtl.DTLVardef@1eacb4eorg.highwire.dtl.DTLVardef@1c98fb1org.highwire.dtl.DTLVardef@1f8bcbe_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

A novel fluorobenzothiazole RBx 10080758 as a dual inhibitor against bacterial GyraseB (GyrB) and Topoisomerase IV (parE) of Gram-positive pathogens causing skin and respiratory infections

Development of a novel inhibitor targeting Gyrase B and Topoisomerase IV offers a potential opportunity to combat the drug resistance. In the present study, we extensively investigated the efficacy of RBx 10080758, a novel fluorobenzothiazole, against skin and respiratory infections caused by Staphylococci and Streptococci in in vitro and in vivo models. RBx 10080758 showed a potent IC50 of 0.06 M against Gyrase and Topoisomerase IV and also exhibited a strong whole cell in vitro activity with MIC ranges of 0.015-0.06, 0.015-0.03, 0.008-0.03, 0.008-0.03, 0.008-0.0.06, 0.015-0.06 g/ml against Staphylococcus. aureus, Streptococcus pneumoniae, Coagulase negative Staphylococci, Streptococcus viridans, Streptococcus pyogenes and Enterococcus, respectively. As expected from the novel class of molecule, it retains potent even against linezolid and vancomycin resistant strains. Interesting, in mouse systemic infection 10 mg/kg, IV dose protected 100% mice from lethal infections. In rat thigh infection model with MRSA WCUH29 at 45 mg/kg exhibited >3-log10 cfu reduction in thigh muscles. RBx 10080758 displayed potent in vitro and in vivo activity against a panel of MDR Gram-positive bacteria. As a novel chemical class, the fluorobenzothiazoles have the potential to become clinically viable antibiotics, to address the drug resistance problem by its unique dual targeting mechanism of action.

microbiology↗

bioRxiv2020

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100{degrees}C when lyophilized, upto 70{degrees}C in solution and stable for over four weeks at 37{degrees}C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

biophysics↗

bioRxiv2020

Green tea and Spirulina extracts inhibit SARS, MERS, and SARS-2 spike pseudotyped virus entry in vitro

Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike (S) protein to bind to a host cell surface receptor. The rapid outbreak of emerging novel coronavirus, SARS-CoV 2 in China inculcates the need for the development of hasty and effective intervention strategies. Medicinal plants and natural compounds have been traditionally used to treat viral infections. Here, we generated VSV based pseudotyped viruses (pvs) of SARS-, MERS-, and SARS-2 CoVs to screen entry inhibitors from natural products. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind on their receptors and enter into the cells. SARS and MERS polyclonal antibodies neutralize SARSpv and SARS-2pv, and MERSpv respectively. Incubation of soluble ACE2 inhibited entry of SARS and SARS-2 pvs but not MERSpv. In addition, expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARSpv, SARS-2pv, and MERSpv respectively. Next, we showed the antiviral properties of known enveloped virus entry inhibitors, Spirulina and Green tea extracts against CoVpvs. SARSpv, MERSpv, and SARS-2pv entry were blocked with higher efficiency when preincubated with either green tea or spirulina extracts. Green tea provided a better inhibitory effect than the spirulina extracts by binding to the S1 domain of spike and blocking the interaction of spike with its receptor. Further studies are required to understand the exact mechanism of viral inhibition. In summary, we demonstrate that pseudotyped virus is an ideal tool for screening viral entry inhibitors. Moreover, spirulina and green tea could be promising antiviral agents against emerging viruses.

microbiology↗