Biology subjects

Shanmugam, S.

Publications and source records attributed to Shanmugam, S..

10 recordsLinked to original sources

bioRxiv2026

Sustainable Technology for the Fabrication of Liposomal Phases

Liposomes are self-assembled lipid vesicles capable of encapsulating both hydrophilic and hydrophobic therapeutics, making them versatile platforms in drug delivery and biomedical technology. In this study, the limitations of the classical thin-film hydration method were critically evaluated, and a sustainable, systematically optimized strategy was established for generating defined liposomal lamellar phases. Hydration conditions were optimized, and 4 mL of buffer per 10 mg of lipid was determined to be optimal for effective rehydration and improved statistical reliability of vesicle measurements. A refined probe-sonication protocol (20% amplitude, 5 s ON/55 s OFF pulse) enabled controlled transformation of multivesicular vesicles into stable multilamellar and unilamellar vesicles at net ON-times of 90 s and 185 s, respectively, without overheating or contamination. In addition, a Python-based machine-learning tool was developed for vesicle size characterization. Collectively, these optimizations provided a reproducible and sustainable framework for preparing liposomes across different lamellar phases.

biophysics↗

bioRxiv2026

EFFECTS OF TOLL-LIKE RECEPTOR 3 - DEPENDENT IMMUNE ACTIVATION IN MICE ARE SEX- AND TISSUE - SPECIFIC: IMPLICATIONS FOR ALCOHOL USE DISORDER

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.

neuroscience↗

bioRxiv2025

Dissecting the effect of single- and co- infection of TB and COVID-19 pathogens on the sputum microbiome

Tuberculosis (TB) and COVID-19 are both respiratory diseases and understanding their interaction is important for effective co-infection management. Although some studies have investigated TB and COVID-19 co-infection in terms of immune responses, microbial dysbiosis in such cases remains unexplored. In this study, we understand the interface between TB and COVID-19 by systematically inspecting the microbial composition of sputum samples collected from four groups of individuals: TB only, COVID-19 only, and both TB and COVID-19 (TBCOVID) infected patients, and uninfected group (Controls). Besides metagenomic analysis of the microbiome of these sputum samples, we also performed whole genome sequencing analysis of a subset of TB-positive samples. Different bioinformatic analyses ensured data quality and revealed significant differences in the microbial composition between Control vs. disease groups. To understand the effect of COVID-19 on TB, we compared TBCOVID vs. TB samples and observed (i) higher read counts of TB-causing bacteria in the TBCOVID group, and (ii) differential abundance of several taxa such as Capnocytophaga gingivalis, Escherichia coli, Prevotella melaninogenica and Veillonella parvula. Functional profiling with PICRUSt2 revealed significantly elevated pathways in the TBCOVID group relative to TB group, some of which are related to metabolism of pulmonary surfactant lipids (e.g., Cytidine diphosphate diacylglycerol (CDP-DAG) biosynthesis pathway with fold change of 7.46). Further clustering of these significantly elevated pathways revealed a sub-cluster of individuals with adverse treatment outcomes. Two individuals in this sub-cluster showed prevalence of respiratory pathogens like Stenotrophomonas maltophilia - knowing such information can help personalize the antibiotic treatment to match the pathogen profile of the individual. Overall, our study reveals the effect of COVID-19 in the airway microbiome of TB patients, and encourages the use of co-microbial/co-pathogen profiling to personalize TB treatment. Author summaryThe community of microbes in an individuals airway tract can play a complex role in respiratory diseases like TB and COVID-19. Although changes in microbial composition in TB and COVID-19 patients have been studied separately, we present a first-of-its-kind investigation of the airway tract microbiome of individuals simultaneously infected with TB and COVID-19 pathogens. Our results highlight that co-infection with COVID-19 in TB patients alters the abundance of certain bacterial species and their related pathways. For instance, Capnocytophaga gingivalis is abundant in co-infected patients, but not present in the TB-only patient group. This species and other differentially abundant species that we identified in the co-morbid condition, if replicated in independent cohorts, can help explain how COVID-19 could exacerbate the severity of lung infection in TB patients. Our study also stimulates future longitudinal studies using expanded datasets to understand the role of concomitant pathogens, and assess whether adjusting the antibiotic regimen accordingly can improve TB treatment outcomes.

genomics↗

bioRxiv2025

Effects of innate immune activation by Toll-like receptor agonists on ethanol consumption and preference in FVB/NJ x C57BL/6J hybrid mice.

Excessive alcohol (ethanol) consumption is a hallmark of alcohol use disorder (AUD). Activation of innate immune system and proinflammatory signaling in the brain may play a key role in promoting alcohol consumption and development of AUD in humans. Innate immune activation by toll-like receptor (TLR) agonists in rodents is associated with release of proinflammatory cytokines and changes in alcohol consumption, and these effects are genotype- and sex-dependent. For example, C57BL/6J male, but not female mice increase alcohol intake after TLR3 activation. In order to better understand the interactions between neuroimmune signaling, genotype, and sex and their effects on ethanol drinking, males and females of more genotypes need to be tested. The goal of this study was to test the effects of innate immune activation on ethanol consumption and neuroimmune molecular profiles of F1 hybrid mice from reciprocal crosses between C57BL/6J (B6) and FVB/NJ (FVB) mouse strains, which are animals with high levels of ethanol intake. Animals were randomly assigned to receive intraperitoneal injections of either saline, Poly(I:C) (PIC, 2 or 10 mg/kg), a TLR3 agonist, or lipopolysaccharide, (LPS, 0.1 mg/kg), a TLR4 agonist, administered every 4 days for a total of 10 injections and subjected to a 2-bottle choice every-other-day ethanol drinking paradigm for a total of 18 dinking sessions, which generated high levels of voluntary ethanol consumption. Six and 24 hours after the last injection, brains were removed, frontal cortex dissected, and levels of 3 proinflammatory cytokines (Tnfa, Il1b, Ccl5), as well as Tlr3, and Tlr4 were measured using qPCR. Immune activation by PIC produced escalation of ethanol drinking, while LPS resulted in a reduction of ethanol consumption or a trend to reduce drinking in males but not females of both FVB/B6 and B6/FVB crosses. Furthermore, activation of TLR3 by PIC produced sex-specific time course responses of pro-inflammatory cytokines, which may, at least in part, explain behavioral differences. Taken together, these results validate previous findings that the effects of immune activation on ethanol consumption depend on genotype, sex, and mode of activation (TLR3 vs TLR4) and suggest that FVB/B6J and B6J/FVB F1 males are a suitable model to study TLR3-dependent escalation of alcohol drinking. HighlightsO_LIImmune activation by Toll-like receptor 3 (TLR3) agonist, Poly(I:C), produced an escalation of ethanol drinking, while immune activation by TLR4 agonist, LPS, reduced ethanol intake in male but not female FVB/NJ x C57BL/6J hybrid mice. C_LIO_LIPoly(I:C)-induced escalation of alcohol consumption in males was reproducible and consistent across different Poly(I:C) doses. C_LIO_LIActivation of TLR3 by Poly(I:C) produced sex-specific time course responses of pro-inflammatory cytokines, which may, at least in part, explain sex differences in alcohol consumption. C_LIO_LIOur data suggest that FVB/NJ x C57BL/6J hybrid male mice are a suitable model to study TLR3-dependent escalation of alcohol drinking. C_LI

animal behavior and cognition↗

bioRxiv2025

Machine learning-assisted enzyme engineering through ultra-high throughput sorting and large-scale sequence-function data generation

Machine learning (ML) shows great promise in protein engineering but has yet to be integrated with ultra-high throughput sorting (ultra-HTS) and NGS for large-scale sequence-function data generation to harness its capability to explore wider search space and more complex mutation events. Here, we introduce PUSDA, a framework that rapidly sorts mutant libraries into multiple performance groups with good accuracy and generates large-scale sequence-function data to power ML-driven protein design. As a demonstration, PUSDA generated over five million sequence-function data of an enzyme, with data processing revealing over 1.3 million unique enzyme mutants being sorted in a single day. With a trained ML model that achieved 93.52% accuracy, we further analysed combinatorial mutation events and applied a ratio-based selection approach to design novel enzyme sequences. Validation experiment demonstrated a 16.67-fold improvement in efficiency of identifying high-performance enzymes using PUSDA compared to using ultra-HTS alone. The designed novel enzyme achieved 8.23-fold increase in productivity compared to wild type. PUSDA lays a foundation to integrate ultra-HTS, NGS, and ML for future predictive enzyme engineering, offering a data-driven tool for accelerating breakthroughs in biotechnology.

synthetic biology↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2022

Absence of the bile acid enzyme CYP8B1 increases brain chenodeoxycholic acid and reduces neuronal excitotoxicity in mice

BackgroundBile acids (BAs), which act in the liver-brain axis, are liver-derived signaling molecules found in the brain. However, how they modulate neurological function remains largely unknown. MethodsTo assess the role of BAs in the brain, we generated mice with absent 12-hydroxylase (Cyp8b1), a BA synthesis enzyme, and determined if brain BA levels were altered in these mice, and if and how this may modulate neuronal function. ResultsThe absence of CYP8B1 increased brain levels of the primary BA chenodeoxycholic acid (CDCA), and decreased ischemic stroke infarct area. Furthermore, CDCA administration reduced ischemic stroke lesion area in wild-type mice. Excitotoxicity due to elevated extra-cellular glutamate contributes to neuronal death in ischemic stroke. Neurons from Cyp8b1-/- mice showed reduced susceptibility to glutamate-induced toxicity, and exogenous CDCA reduced glutamate-induced toxicity in neurons from wild-type mice. These data suggest that CDCA-mediated decreases in excitotoxic neuronal death contributes to the reduced stroke lesion area in Cyp8b1-/- mice. Aberrant N-methyl-D-aspartate receptor (NMDAR) over-activation contributes to excitotoxicity. CDCA decreased NMDAR-mediated excitatory post-synaptic currents (EPSCs) in wild-type brain slices, by reducing over-activation of the NMDAR subunit GluN2B. In line with this, synaptic NMDAR activity was also decreased in Cyp8b1-/- brain slices. Expression level and synaptic distribution of GluN2B were unaltered in Cyp8b1-/- mice, suggesting that CDCA may directly antagonize GluN2B-containing NMDARs. ConclusionsOur data suggests that CDCA acts in the liver-brain axis and decreases the aberrant over-activation of neuronal GluN2B-containing NMDARs, contributing to neuroprotection.

physiology↗

bioRxiv2022

Differential effects of carbon nanotube and graphene on the tomato rhizosphere microbiome

Application of carbonaceous nanomaterials (CNMs) to the soil-plant system can affect plant physiology, with positive results ranging from enhanced seed germination and root system development to improved stress tolerance. The underlying mechanisms are not fully understood. Plant rhizosphere microbiomes at the soil-root interface are strongly influenced by the host plant and play a key role in the plant hosts development and health. Yet few studies have characterized changes in plant rhizosphere microbiomes following applications of CNMs to the soil-plant system. Here we investigated the effects of multi-walled carbon nanotube (CNT) and graphene on microbial communities in the ectorhizosphere of tomato plants versus surrounding bulk soil. Pot experiments were conducted where tomato plants were exposed to CNT or graphene at 200 mg/kg soil for four weeks. Ectorhizosphere and bulk soils were then collected and analyzed for physicochemical properties and microbiome structure and function. While graphene had a limited impact on the tomato rhizosphere microbiome, CNT significantly increased microbial alpha diversity, induced greater divergence of beta diversity, enhanced microbial interactions, and potentially impacted community functions such as aromatic compound degradation, antioxidant synthesis, and redox cofactor biosynthesis. Furthermore, CNT induced stronger and/or unique microbiome alterations in the tomato rhizosphere compared to bulk soil. Our findings reveal the differential modulating effects of two widely-used CNMs on plant rhizosphere microbiomes and highlight an imminent need to understand complex plant root-microbe interplays in the CNM-impacted rhizosphere. These results have implication for realizing the full potential of phytoapplication of CNMs toward improved and sustainable plant production.

microbiology↗

bioRxiv2021

Deciphering the role of mucosal immune responses and cervicovaginal microbiome in resistance to HIV infection in HIV-exposed seronegative (HESN) women

The female genital tract (FGT) is an essential site of HIV infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg) and levels of natural killer (NK) cells, CXCR5+ CD8+T cells, T follicular helper cells (Tfh) and T regulatory cells (T regs) in the vaginal mucosa, as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells and Tfh cells in HESN females as compared to HIV unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp in the mucosa of HESN women. The findings suggest the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators and critical T cells subsets that protect against HIV infection.

immunology↗

bioRxiv2020

Whole genome sequencing based differentiation between re-infection and relapse in Indian patients with tuberculosis recurrence, with and without HIV co-infection

Differentiation between relapse and reinfection in cases with tuberculosis (TB) recurrence has important implications for public health, especially in patients with human immunodeficiency virus (HIV) co-infection. Forty-one paired M. tuberculosis isolates collected from 20 HIV-positive and 21 HIV-negative patients, who experienced TB recurrence after previous successful treatment, were subjected to whole genome sequencing (WGS) in addition to spoligotyping and mycobacterial interspersed repeat unit (MIRU) typing. Comparison of M. tuberculosis genomes indicated that 95% of TB recurrences in the HIV-negative cohort were due to relapse, while the majority of TB recurrences (75%) in the HIV-positive cohort was due to re-infection (P=0.0001). Drug resistance conferring mutations were documented in four pairs (9%) of isolates associated with relapse. The high contribution of re-infection to TB among HIV patients warrants further study to explore risk factors for TB exposure in the community.

genomics↗