Biology subjects

Dwivedi, P.

Publications and source records attributed to Dwivedi, P..

5 recordsLinked to original sources

bioRxiv2026

Analytical evaluation of eukaryotic cell-free translation (CFT) systems to assess mRNA translatability

mRNA vaccines constitute a promising new platform for infectious disease prevention, having demonstrated their efficacy in response to the COVID-19 pandemic. While mRNA attributes such as purity, integrity, 5'-capping, polyA tail length, and nucleobase lipidation are critical to vaccine efficacy, another key attribute of any vaccine mRNA is its translatability - that is, how much antigen protein is produced by ribosomal translation. To assess mRNA translatability, cell-free translation (CFT) can be employed. Advantages of CFT include its rapid readout (~5 hours), minimal sample consumption, and measurement of antigen translation directly from mRNA, thereby eliminating any variability associated with cell transfection efficiency. This is in contrast to cell-based methods, which require larger amounts of mRNA material and formulation in lipid nanoparticles or lipofectamine to facilitate cell transfection. When developing a CFT method to assay mRNA translatability, one key consideration is which CFT system to use as there are several commercially available systems from a variety of different organisms and cell types. Presented here is the evaluation of three eukaryotic CFT systems - wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL) - for the purpose of developing an analytical method to assay the translatability of mRNA. The dynamic range, linear range, sensitivity to thermal stress, and sensitivity to potential impurities (e.g., EDTA, double-stranded RNA) were determined for each system. Although HCL translated less protein than either WGE or RRL systems, it showed several advantages, notably a larger linear range and greater sensitivity to mRNA thermal stress and dsRNA impurities. Therefore, while it is still worthwhile to screen different CFT systems, the results presented here suggest that HCL should be strongly considered when developing an analytical method to assay translatability for mRNA vaccines.

biochemistry↗

bioRxiv2025

Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

Cancer dissemination by perineural invasion (PNI) is associated with poor outcomes in epithelial malignancies, yet the cellular interactions critical to this process are not fully defined. Single-cell transcriptomic analysis of cutaneous squamous cell carcinoma (cSCC) with PNI highlighted KITLG-KIT signaling activation between keratinocytes and mast cells. Additionally, we observed increased expression of HRH1 on tumor cells, which encodes the H1 histamine receptor. We confirmed these changes in other cancers, including PNI-positive hepatobiliary and oral mucosal tumors. Histamine enhanced tumor invasion into nerve in organoids that recapitulate key features of PNI, an effect rescued by H1-antihistamines. Mechanistically, broad upregulation of matrix metalloproteinases (MMPs) through P38 activation underscored histamine-mediated PNI by enabling collagen degradation in the nerve sheath. Examination of multiple cancer types with PNI showed co-expression of HRH1 and MMP2 on tumor cells as well as type IV collagen loss in involved nerves. Retrospective analysis of >15,000 patient health records in two independent databases demonstrated that H1-antihistamine use improved overall survival and immunotherapy response in cancers that spread by PNI. Together, these data reveal a critical role for mast cells in PNI and present a new therapeutic strategy for targeting this process in epithelial cancers.

cancer biology↗

bioRxiv2024

pH dependent direct sulfhydrylation pathway is required for pathogenesis of Mycobacterium tuberculosis

Methionine is essential for the survival of Mycobacterium tuberculosis (M. tuberculosis) inside the host. However, the transsulfuration pathway, a major contributor of methionine, is dispensable for the growth of M. tuberculosis suggesting redundancy in the methionine biosynthesis pathway. Orthologues of MetZTB in other bacterial species are known to operate a redundant single-step methionine biosynthesis pathway called direct sulfhydrylation. In this study, we demonstrate that genetic disruption of the metZ-mediated direct sulfhydrylation pathway in M. tuberculosis hinders growth at low pH, an effect mitigated by methionine supplementation. Computational analyses, including in-silico molecular docking and molecular dynamics (MD) simulations, reveal enhanced binding of the MetZ substrate, O-succinyl homoserine (OSH), to the active site of MetZ at acidic pH. Intriguingly, despite increased intracellular ATP levels, a relative decrease in the frequency of Bedaquiline (BDQ)-induced persisters is observed in metZ-deficient strain, suggesting a role of direct sulfhydrylation pathway in modulating BDQ sensitivity. Finally, we demonstrated that the absence of metZ impedes the ability of M. tuberculosis to grow inside the host.

microbiology↗

bioRxiv2024

Phosphoglucomutase A mediated regulation of carbon flux is essential for antibiotic and disease persistence in Mycobacterium tuberculosis

The long-term survival of Mtb mandates judicious utilization of the available resources inside the host. Uninterrupted access to host-derived nutrients holds the key to the success of Mtb. Phosphoglucomutase enzyme besides synthesizing glycogen, which serves as a nutrient reservoir, also helps modulate the carbon flux in different pathogens. Studies on the role of glycogen metabolism in disease progression, reactivation, and drug susceptibility in tuberculosis are severely lacking. To investigate this, we generated an Mtb strain ({Delta}pgmA) devoid of the gene that encodes for the enzyme phosphoglucomutase A (pgmA). The absence of pgmA impedes the ability of the pathogen to survive under nutrient-limiting and reactivation conditions. In the current study, we demonstrate that the absence of cell membrane-associated glycolipids in {Delta}pgmA compromised the cell wall integrity and increased the susceptibility of {Delta}pgmA to various stresses. Interestingly, in comparison to the wild type, low cAMP levels in {Delta}pgmA imparted an enhanced growth phenotype on cholesterol. Differential gene expression and carbon flux analysis suggest that stored carbon in the form of glycogen is essential for the survival of Mtb under nutrient-limiting conditions. Finally, we demonstrate that the pgmA gene of Mtb is essential for the growth of Mtb inside the host. Overall, this study unveils the significance of pgmA-mediated regulation of membrane glycolipids and its implication on antibiotic and disease persistence in tuberculosis. Additionally, information derived from this study will help design anti-TB strategies that are novel, short, and more efficient.

microbiology↗

bioRxiv2021

Assessment of immunogenicity and protective efficacy of ZyCoV-D DNA vaccine candidates in Rhesus macaques against SARS-CoV-2 infection

Vaccines remain the key protective measure to achieve herd immunity to control the disease burden and stop COVID-19 pandemic. We have developed and assessed the immunogenicity and protective efficacy of two formulations (1mg and 2mg) of ZyCoV-D (a plasmid DNA based vaccine candidates) administered through Needle Free Injection System (NFIS) and syringe-needle (intradermal) in rhesus macaques with three dose vaccine regimens. The vaccine candidate 2mg dose administered using Needle Free Injection System (NFIS) elicited a significant immune response with development of SARS-CoV-2 S1 spike region specific IgG and neutralizing antibody (NAb) titers during the immunization phase and significant enhancement in the levels after the virus challenge. In 2 mg NFIS group the IgG and NAb titers were maintained and showed gradual rise during the immunization period (15 weeks) and till 2 weeks after the virus challenge. It also conferred better protection to macaques evident by the viral clearance from nasal swab, throat swab and bronchoalveolar lavage fluid specimens in comparison with macaques from other immunized groups. In contrast, the animals from placebo group developed high levels of viremia and lung disease following the virus challenge. Besides this, the vaccine candidate also induced increase lymphocyte proliferation and cytokines response (IL-6, IL-5).The administration of the vaccine candidate with NFIS generated a better immunogenicity response in comparison to syringe-needle (intradermal route). The study demonstrated immunogenicity and protective efficacy of the vaccine candidate, ZyCoV-D in rhesus macaques.

microbiology↗