Biology subjects

Gunasekaran, K.

Publications and source records attributed to Gunasekaran, K..

9 recordsLinked to original sources

bioRxiv2026

Three Plasmid Strategies, One Intermediate Convergence State: Lineage-Specific Resistance, Virulence Architecture in Dominant Indian Carbapenem-Resistant Klebsiella pneumoniae Clones

Carbapenem-resistant Klebsiella pneumoniae (CRKp) is a critical global healthcare threat driven by high-risk multidrug-resistant (MDR) clones that acquire hypervirulence genes. Although resistance-virulence co-occurrence is extensively documented, the plasmid-level mechanisms facilitating this convergence remain unclear. In this study, we utilized hybrid short- and long-read whole-genome sequencing of 376 clinical CRKp strains to define the evolutionary trajectories and structural plasmid dynamics of three predominant high-risk clones: ST147 (n=157), ST231 (n=108), and ST2096 (n=111). Carbapenemase genes were present in 90% of isolates, predominantly blaOXA-48-like and blaNDM-5 co-harbored with blaCTX-M-15. Virulence profiling indicated high aerobactin (iuc) prevalence (62.7%), while salmochelin and colibactin were undetected. Hypermucoviscosity occurred infrequently (6.6%) and was independent of rmpA/rmpA2, confirming a clear genotype-phenotype discordance. Comparative plasmid mapping revealed three distinct, lineage-specific plasmid configurations underlying this intermediate convergent pathotype: ST147 exhibited dynamic, mosaic hybrid IncFIB-IncHI1B plasmids; ST2096 showed structurally stabilized hybrids; and ST231 retained virulence and resistance determinants on separate, segregated plasmids. These findings show that convergence is regulated by multiple, clone-specific evolutionary routes rather than a single path, highlighting the critical need for more in-depth genomic surveillance capable of identifying convergent plasmids along with high-risk lineages

microbiology↗

bioRxiv2025

Shiny-Calorie: A context-aware application for indirect calorimetry data analysis and visualization using R

Indirect calorimetry is a cornerstone technique for metabolic phenotyping of animal models in preclinical research, with well-established experimental protocols and platforms. However, a flexible, extensible, and user-friendly software suite that enables standardized integration of data and metadata from diverse metabolic phenotyping platforms--followed by unified statistical analysis and visualization--remains absent. We present Shiny-Calorie, an open-source interactive web application for transparent data and metadata integration, comprehensive statistical data analysis, and visualization of indirect calorimetry datasets. Shiny-Calorie is compatible with data formats from widely used commercial metabolic phenotyping platforms, such as TSE and Sable Systems, and includes functionality for exporting processed data in these formats. Built using GNU R and a Shiny-based reactive interface, Shiny-Calorie enables intuitive exploration of complex, multi-modal longitudinal datasets comprising categorical, continuous, ordinal, and count variables. The platform incorporates state-of-the-art statistical methods for robust hypothesis testing, thereby facilitating biologically meaningful interpretation of energy metabolism phenotypes, including resting metabolic rate and energy expenditure. Overall, Shiny-Calorie streamlines routine analysis workflows and enhances reproducibility and transparency in metabolic phenotyping studies.

bioinformatics↗

bioRxiv2025

Preliminary genomic data on five Orientia tsutsugamushi strains isolated in Vellore, India

IntroductionOrientia tsutsugamushi, the causative agent of scrub typhus, can be isolated in Vero or L929 cells and has a small genome (2-2.5 Mb). However, genome assembly is challenging due to the presence of host DNA contamination and a high proportion of repeat regions (up to 51%). Current global data includes 11 fully annotated genomes, with none from India. Here, we present the first whole-genome sequences of O. tsutsugamushi from India. MethodsFive O. tsutsugamushi strains were cultured in Vero cells and confirmed by 47kDa real-time PCR. Genomic DNA was extracted after removal of host DNA and sequencing libraries were prepared. Whole-genome sequencing was performed using the PacBio Sequel II system in CCS/HiFi mode. The raw reads were assembled using Flye, and genome completeness was assessed with QUAST and BUSCO. Annotation was performed using the NCBI PGAP pipeline and comparative genome analysis by Roary. Phylogenetic analysis was based on the full-length 56kDa gene, which contains four variable domains. ResultsWe report five complete genomes of O. tsutsugamushi, four of which are circular and one linear. Genome sizes range from 2.1 to 2.4 Mb. The total number of predicted genes falls between 2,379 and 2,715, with an average of 1,824 coding genes and 613 pseudogenes. Repeat regions constitute 53-59% of the genome, a higher proportion than previously reported. All five genomes have been submitted to NCBI GenBank (Accession Numbers: CP166954-58). Phylogenetic analysis based on the full-length 56kDa gene revealed that two strains belong to the Karp genogroup, two to Kato, and one to TA763. ConclusionThis study presents the first whole-genome sequencing data of O. tsutsugamushi from India. Notably, the repetitive regions in these genomes are more extensive than previously reported. Further analyses with additional isolates are necessary to validate this observation. Comprehensive phylogenomic studies, particularly to elucidate evolutionary dynamics and potential recombination events will provide further information.

microbiology↗

bioRxiv2024

EEG data quality in large scale field studies in India and Tanzania

There is a growing imperative to understand the neurophysiological impact of our rapidly changing and diverse technological, social, chemical, and physical environments. To untangle the multidimensional and interacting effects requires data at scale across diverse populations, taking measurement out of a controlled lab environment and into the field. Electroencephalography (EEG), which has correlates with various environmental factors as well as cognitive and mental health outcomes, has the advantage of both portability and cost-effectiveness for this purpose. However, with numerous field researchers spread across diverse locations, data quality issues and researcher idle time due to insufficient participants can quickly become unmanageable and expensive problems. In programs we have established in India and Tanzania, we demonstrate that with appropriate training, structured teams, and daily automated analysis and feedback on data quality, non-specialists can reliably collect EEG data alongside various survey and assessments with consistently high throughput and quality. Over a 30-week period, research teams were able to maintain an average of 25.6 subjects per week, collecting data from a diverse sample of 7,933 participants ranging from Hadzabe hunter-gatherers to office workers. Furthermore, data quality, computed on the first 2,400 records using two common methods, PREP and FASTER, was comparable to benchmark datasets from controlled lab conditions. Altogether this resulted in a cost per subject of under $50, a fraction of the cost typical of such data collection, opening up the possibility for large-scale programs particularly in low- and middle-income countries. Significance StatementWith wide human diversity, a rapidly changing environment and growing rates of neurological and mental health disorders, there is an imperative for large scale neuroimaging studies across diverse populations that can deliver high quality data and be affordably sustained. Here we demonstrate, across two large-scale field data acquisition programs operating in India and Tanzania, that with appropriate systems it is possible to generate high throughput EEG data of quality comparable to controlled lab settings. With effective costs of under $50 per subject, this opens new possibilities for low- and middle-income countries to implement large-scale programs, and to do so at scales that previously could not be considered.

neuroscience↗

bioRxiv2023

First isolate of Orientia tsutsugamushi from Vellore, South India

BackgroundScrub typhus a common cause of acute febrile illness in India caused by Orientia tsutsugamushi an obligate intracellular bacterium requiring cell culture for isolation. Cell lines like Vero and L929 are most suitable for isolating and maintaining this organism. This study was undertaken to isolate and characterize of Orientia tsutsugamushi from whole blood samples at a tertiary care centre in Southern India. MethodsThe PBMCs (peripheral blood mononuclear cells) collected from scrub typhus positive (47kDa qPCR positive) patients were inoculated into Vero and L929 cell line at 80% confluence for primary isolation. The inoculated flasks were incubated at 37{degrees}C with 5% CO2 for 30 days and examined for presence of Orientia tsutsugamushi on the day 10, 15, 20 post-inoculation and everyday thereafter for a maximum of 30 days post inoculation. The scrapings were subjected to Giemsa staining, IFA, 47kDa qPCR and transmission electron microscopy (TEM). The isolates were passaged 3-4 times to ensure viability and then stored in DMEM with 10% FBS (-80{degrees}C). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene. ResultsAmongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR at 24th day of inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology with Orientia tsutsugamushi MW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype. ConclusionsWe have successfully isolated and characterised the Orientia tsutsugamushi for the first time at our centre from PBMCs. Based on the partial TSA56 gene sequence our isolates belongs to TA763 and Gilliam genotype. More number of samples are being processed for identifying further isolates followed by genomic analysis.

microbiology↗

bioRxiv2023

Genotyping of Orientia tsutsugamushi circulating in and around Vellore (South India) using TSA 56 gene

The immunodominant TSA 56 gene of Orientia tsutsugamushi, (scrub typhus agent) has four variable regions (VD-I to VD-IV) making it useful for genotyping. As of date the genotyping data from India is based on partial 56kDa gene sequence analysis. The complete TSA 56 gene sequence is important for knowing the circulating strains and for designing region specific diagnostics and vaccines. This study was undertaken to determine Orientia tsutsugamushi genotypes circulating in and around Vellore using complete and partial TSA 56 gene. Of the 379 whole blood samples from suspected scrub typhus patients, 162 were positive by 47 kDa qPCR. Long protocol to amplify the complete TSA 56 gene ({approx}1605 bp) was performed on 21 samples. On the same 21 samples the partial gene sequence was also amplified using the Horinouchi ({approx}650bp) and the Furuya ({approx}480 bp) protocol. Using a combination of Sanger and Nanopore technology complete sequence was obtained for 9 and near complete (1551 to 1596 bp) for 4 respectively. As Furuya protocol gave multiple bands we obtained 480 bp sequences from the 13 complete gene sequences by in silico analysis. In contrast, 650bp sequences were obtained for 11 samples while for the remaining two we derived the 650 bp sequences from the complete gene sequences (Long protocol). Phylogenetic analysis of the complete gene (Long protocol) which includes VD-I to VD-IV region and partial gene (Horinouchi) which amplifies the VD-I to VD-III regions showed identical genotypes. Twelve belonged to TA763 genotype and one belongs to Karp genotype. The Furuya sequence (in silico) correctly identified the Karp genotype and 10 of the TA763 genotypes. Two TA763 genotypes (identified by complete and 650 bp partial gene analysis) were misidentified by Furuya sequence analysis as Karp genotype. The limited analysis showed the commonest Orientia tsutsugamushi genotypes circulating in and around Vellore is TA763 and that the 650 bp (Sanger) sequencing could be a cost effective method for identifying the scrub typhus genotypes. However, these results need to be validated by larger prospective multi-centric studies.

microbiology↗

bioRxiv2021

Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases. SummaryBrain delivery, biodistribution and pharmacodynamics of a lysosomal enzyme fused to a moderate-affinity transferrin receptor-directed blood-brain barrier enzyme transport vehicle are superior to a traditional high-affinity anti-TfR monoclonal antibody fusion.

neuroscience↗

bioRxiv2021

Colistin resistance in Acinetobacter baumannii is driven by multiple genomic traits: Evaluating the role of ISAba1-driven eptA overexpression among Indian isolates

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrAB two component system and overexpression of eptA due to upstream insertion of ISAba1 play a major role.To characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 224 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrAB genes were characterized by Whole Genome Shotgun sequencing. Twenty eight complete genomes were further characterized for insertional inactivation of lpx genes and the association of ISAba1-eptA using hybrid assembly approach. Non-sysnonymous mutations like M12I in pmrA, A138T and A444V in pmrB and E117K in lpxD were identified. Four of the five colistin resistant A.baumannii isolates had insertion of ISAba1 upstream eptA. No mcr genes were identified.Overall, the present study highlights the diversity of colistin resistance mechanisms in A. baumannii. ISAba1-driven eptA overexpression could be responsible for colistin resistance among Indian isolates of colistin resistant A. baumannii.

microbiology↗

bioRxiv2020

Evolutionary tracking of SARS-CoV-2 genetic variants highlights intricate balance of stabilizing and destabilizing mutations

The currently ongoing COVID-19 pandemic caused by SARS-CoV-2 has accounted for millions of infections and deaths across the globe. Genome sequences of SARS-CoV-2 are being published daily in public databases and the availability of this genome datasets has allowed unprecedented access into the mutational patterns of SARS-CoV-2 evolution. We made use of the same genomic information for conducting phylogenetic analysis and identifying lineage-specific mutations. The catalogued lineage defining mutations were analysed for their stabilizing or destabilizing impact on viral proteins. We recorded persistence of D614G, S477N, A222V V1176F variants and a global expansion of the PANGOLIN variant B.1. In addition, a retention of Q57H (B.1.X), R203K/G204R (B.1.1.X), T85I (B.1.2-B.1.3), G15S+T428I (C.X) and I120F (D.X) variations was observed. Overall, we recorded a striking balance between stabilizing and destabilizing mutations, therefore well-maintained protein structures. With selection pressures in the form of newly developed vaccines and therapeutics to mount soon in coming months, the task of mapping of viral mutations and recording of their impact on key viral proteins would be crucial to pre-emptively catch any escape mechanism that SARS-CoV-2 may evolve for. STUDY IMPORTANCEAs large numbers of the SARS CoV-2 genome sequences are shared in publicly accessible repositories, it enables scientists a detailed evolutionary analysis since its initial isolation in Wuhan, China. We investigated the evolutionarily associated mutational diversity overlaid on the major phylogenetic lineages circulating globally, using 513 representative genomes. We detailed phylogenetic persistence of key variants facilitating global expansion of the PANGOLIN variant B.1, including the recent, fast expanding, B.1.1.7 lineage. The stabilizing or destabilizing impact of the catalogued lineage defining mutations on viral proteins indicates their possible involvement in balancing the protein function and structure. A clear understanding of this mutational profile is of high clinical significance to catch any vaccine escape mechanism, as the same proteins make crucial components of vaccines recently approved and in development. In this direction, our study provides an imperative framework and baseline data upon which further analysis could be built as newer variants of SARS-CoV-2 continue to appear.

genomics↗