Biology subjects

Ramu, S.

Publications and source records attributed to Ramu, S..

5 recordsLinked to original sources

bioRxiv2026

Optimisation of Solid-Phase Antimicrobial Susceptibility Testing for Hydrophobic Antibiotics Using an Agarose-Based MIC Assay

The minimum inhibitory concentration (MIC) assay is the gold standard for evaluating antimicrobial activity1. However, conventional agar-based MIC methods often underestimate the potency of physicochemically complex compounds[1, 2]. Hydrophobic and adhesive molecules, such as lipoglycopeptide antibiotics, exhibit poor diffusion and non-specific binding to agar, leading to artificially elevated MIC values compared to broth-based methods[3]. This issue complicates accurate potency assessment and is particularly an issue when attempting resistance frequency (FOR) studies, which must be conducted on solid media. Here, we developed a modified miniaturised agar MIC assay using 1% agarose, 0.002% Tween 80-supplemented tryptic soy broth (TSB), and a 24-well plate format[4]. These modifications improved compound dispersion, reduced matrix interactions, and lowered compound requirements. The optimised assay was validated with vancomycin, oritavancin, and dalbavancin against Staphylococcus aureus ATCC 43300 (MRSA) and Streptococcus pneumoniae ATCC 700677. This efficient, cost-effective, high-throughput platform overcomes the limitations of traditional agar methods, enhancing reliability in evaluating challenging antimicrobials and supporting next-generation antibiotic development.

microbiology↗

bioRxiv2025

ONCOchannelome: A computational framework to investigate altered ion channels across tumor types

Conventional approaches for analyzing ion channels in cancer primarily focus on the detection of membrane potential to record the polarization states of the cellular membrane. Although these approaches provide meaningful insights during validation, they face challenges in discovery, especially in identifying the ion channels that govern the membrane potential. Moreover, ion channels are known to exhibit context-dependent activity that often depends on the cancer type and metabolic state of the tumor. Here, we developed a computational framework, ONCOchannelome, that allows the identification of potential ion channels via transcriptomic datasets. We collected publicly available RNA-Seq datasets corresponding to 2421 normal, 5442 primary tumor and 588 metastatic samples of 15 tumor types. Using the data, we designed a computational strategy to merge the datasets, identifying differentially expressed ion channels in the tumor and metastatic states and aiding in the determination of ion channels in different tumor states. We subsequently developed strategies to determine the correlation of altered ion channels with epithelial-to-mesenchymal transition and to identify differentially coexpressed ion channel networks along with their possible transcription factors. The ONCOchannelome allows visualization of altered ion channels in the tumor and metastatic states in addition to revealing changes resulting in changes in biological processes and molecular functions as the tumor progresses. The ONCOchannelome enhances our understanding of the dependence of ion channels in different tumors in addition to revealing their observed alterations in progressing from the primary tumor state to the metastatic state.

bioinformatics↗

bioRxiv2024

Characterisation of in vitro resistance selection against second-/last-line antibiotics in methicillin-resistant Staphylococcus aureus

SYNOPSISO_ST_ABSBackgroundC_ST_ABSThe increasing occurrence of MRSA clinical isolates harbouring reduced susceptibility to mainstay antibiotics has escalated the use of second and last line antibiotics. Hence, it is critical to evaluate the likelihood of MRSA developing clinical resistance to these antibiotics. ObjectivesOur study sought to identify the rate in which MRSA develop resistance to vancomycin, daptomycin and linezolid in vitro and further determine the mechanisms underpinning resistance. MethodsMRSA was exposed to increasing concentrations of vancomycin, daptomycin, and linezolid for 20 days, with eight replicates for each antibiotic conducted in parallel. The resulting day 20 (D20) isolates were subjected to antimicrobial susceptibility testing, whole genome sequencing, autolysis assays, and growth curves to determine bacterial fitness. ResultsExposure to vancomycin or linezolid for 20 days resulted in a subtle two-fold increase in the MIC, whereas daptomycin exposure yielded daptomycin-nonsusceptible isolates with up to 16-fold MIC increase. The MIC increase was accompanied by variable changes in relative fitness and reduced resistance to autolysis in some isolates. D20 isolates harboured mutations in genes commonly associated with resistance to the respective antibiotics (e.g. walK for vancomycin, mprF and rpoB for daptomycin, rplC for linezolid), along with several previously unreported variants. Introduction of key mutations to these identified genes in the parental strain via allelic exchange confirmed their role in the development of resistance. ConclusionsIn vitro selection against vancomycin, daptomycin, or linezolid resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.

microbiology↗

bioRxiv2023

Multi-modal transcriptomic analysis unravels enrichment of hybrid epithelial/mesenchymal state and enhanced phenotypic heterogeneity in basal breast cancer

Intra-tumoral phenotypic heterogeneity promotes tumor relapse and therapeutic resistance and remains an unsolved clinical challenge. It manifests along multiple phenotypic axes and decoding the interconnections among these different axes is crucial to understand its molecular origins and to develop novel therapeutic strategies to control it. Here, we use multi-modal transcriptomic data analysis - bulk, single-cell and spatial transcriptomics - from breast cancer cell lines and primary tumor samples, to identify associations between epithelial-mesenchymal transition (EMT) and luminal-basal plasticity - two key processes that enable heterogeneity. We show that luminal breast cancer strongly associates with an epithelial cell state, but basal breast cancer is associated with hybrid epithelial/mesenchymal phenotype(s) and higher phenotypic heterogeneity. These patterns were inherent in methylation profiles, suggesting an epigenetic crosstalk between EMT and lineage plasticity in breast cancer. Mathematical modelling of core underlying gene regulatory networks representative of the crosstalk between the luminal-basal and epithelial-mesenchymal axes recapitulate and thus elucidate mechanistic underpinnings of the observed associations from transcriptomic data. Our systems-based approach integrating multi-modal data analysis with mechanism-based modeling offers a predictive framework to characterize intra-tumor heterogeneity and to identify possible interventions to restrict it.

systems biology↗

bioRxiv2018

Octapeptin C4 Induces Less Resistance and Novel Mutations in an Epidemic Carbapenemase-producing Klebsiella pneumoniae ST258 Clinical Isolate Compared to Polymyxins

Polymyxin B and E (colistin) have been pivotal in the treatment of extensively drug-resistant (XDR) Gram-negative bacterial infections, with increasing use over the past decade. Unfortunately, resistance to these antibiotics is rapidly emerging. The structurally-related octapeptin C4 (OctC4) has shown significant potency against XDR bacteria, including against polymyxin-resistant (Pmx-R) strains, but its mode of action remains undefined. We sought to compare and contrast the acquisition of XDR Klebsiella pneumoniae (ST258) resistance in vitro with all three lipopeptides to help elucidate the mode of action of the drugs and potential mechanisms of resistance evolution. Strikingly, 20 days of exposure to the polymyxins resulted in a dramatic (1000-fold) increase in the minimum inhibitory concentration (MIC) for the polymyxins, reflecting the evolution of resistance seen in clinical isolates, whereas for OctC4 only a 4-fold increase was witnessed. There was no cross-resistance observed between the polymyxin - and octapeptin-induced resistant strains. Sequencing revealed previously known gene alterations for polymyxin resistance, including crrB, mgrB, pmrB, phoPQ and yciM, and novel mutations in qseC. In contrast, mutations in mlaDF and pqiB, 1genes related to phospholipid transport, were found in octapeptin-resistant isolates. Mutation effects were validated via complementation assays. These genetic variations were reflected in phenotypic changes to lipid A. Pmx-R isolates increased 4-amino-4-deoxy-arabinose fortification to phosphate groups of lipid A, whereas OctC4 induced strains harbored a higher abundance of hydroxymyristate and palmitoylate. The results reveal a differing mode of action compared to polymyxins which provides hope for future therapeutics to combat the increasingly threat of XDR bacteria.

microbiology↗