Biology subjects

Bhat, M.

Publications and source records attributed to Bhat, M..

5 recordsLinked to original sources

bioRxiv2023

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 {degrees}C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

biophysics↗

bioRxiv2022

Machine learning and hypothesis driven optimization of bull semen cryopreservation media

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 {+/-} 6.9% to 68.3 {+/-} 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

bioengineering↗

bioRxiv2022

Prolonged exposure to insulin causes epigenetic alteration leading to insulin resistance

Glucose homeostasis is maintained by insulin. It has been observed that hyperinsulinemia precedes insulin resistance and Type 2 diabetes. Insulin resistance is caused by multiple factors including genetic and diet. The molecular mechanism underlying insulin resistance (IR) is not completely understood. Using Glut4 and insulin receptor-expressing CHO cells we had previously shown that prolonged exposure of these cells to insulin in the absence of high levels of glucose led to insulin resistance in the cells. In the present study, we have shown that the underlying cause for the impaired GLUT4 trafficking is the defective PI3K/AKT pathway. This insulin resistance is likely due to epigenetic alterations as it is stable and can be maintained for several generations even when insulin is not provided, and epigenetic modifiers can reverse the insulin resistance. We extended these studies to liver cell line (BRL-3A) and show that these cells also develop impaired insulin signaling upon exposure to insulin in the absence of high levels of glucose. Transcriptomic analysis of the insulin-sensitive and -resistance cells uncover altered signaling networks involved in chromatin remodelling, Rho GTPases, and ubiquitination. Pathway analysis reveals the role of demethylase Kdm5b and lysine methyltransferase (Kmt2a and Kmt2e) in the development of insulin resistance. It is also observed that trimethylation of histone H3 at lysine 4 (H3K4me3) is increased in insulin resistance cellular models. We further showed that mice injected with low doses of insulin when fasting develop insulin resistance with impaired glucose tolerance and increased HOMA-IR index. Altogether, these findings suggest dysregulated synthesis of insulin in the absence of glucose stimulus could lead to epigenetic alterations that may lead to insulin resistance. Summary StatementInsulin stimulation in the absence of glucose leads to insulin resistance. We have developed a cell and mouse model of insulin resistance in this study to characterise the molecular signalling involved in insulin resistance and early onset of type 2 diabetes. The transcriptomic analysis provides new insights on epi-transcriptomic regulation in insulin resistance.

molecular biology↗

bioRxiv2021

A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation

Cells acquire fatty acids from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (Alk-4) that functions as a reporter of FASN-dependent protein acylation. In a FASN-dependent manner, Alk-4 selectively labeled the cellular protein interferon-induced transmembrane protein 3 (IFITM3) at its palmitoylation sites, and the HIV-1 matrix protein at its myristoylation site. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

microbiology↗

bioRxiv2021

A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of [~]80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 {degrees}C and were stable to long term storage of over 4 weeks at 37 {degrees}C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were [~] three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

microbiology↗