Biology subjects

Subramanian, M.

Publications and source records attributed to Subramanian, M..

At least 19 recordsLinked to original sources

bioRxiv2025

3D human skeletal muscle organoids reveal distinct effects of high-dose dihydronicotinamide riboside on muscle development

The evaluation of NAD+-boosting compounds in human skeletal muscle is hindered by limitations of traditional 2D cultures and animal models. Human-relevant, three-dimensional (3D) engineered skeletal muscle organoids offer a promising platform to assess the biological effects of metabolic modulation. Here we engineered 3D human skeletal muscle organoids to investigate the impact of dihydronicotinamide riboside (NRH), a potent NAD+ precursor. Sustained exposure to high NRH concentrations (500 {micro}M) enhanced early differentiation markers, including increased myotube fusion and fast-twitch fiber area, but concurrently induced structural defects such as disrupted sarcomeric organization, enlarged acetylcholine receptor clusters, and impaired acetylcholine-stimulated calcium signaling. These results reveal that excessive and sustained NAD+ elevation can uncouple rapid differentiation from proper maturation in muscle tissue. Our results highlight the importance of dose and duration optimization for NAD+-boosting compounds and establish 3D engineered muscle organoids as a valuable non-animal platform for mechanistic toxicology and preclinical safety assessment.

bioengineering↗

bioRxiv2025

Targeting a novel chloroquine derivative to lysosomes induces massive and irreversible damage to lysosomes and suppresses autophagosomes and lysosomes assembly in cancer

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound therapy resistance driven by lysosome dependent nutrient recycling, metabolic adaptation, and stress tolerance. Current lysosome-targeting agents such as chloroquine (CQ) and hydroxychloroquine (HCQ) show limited efficacy due to transient activity and dose-limiting toxicities. To overcome these limitations, we developed Lysostilbenes, a new class of hybrid small molecules combining the CQ pharmacophore with lysosome-disrupting trans-4,4'-dihydroxystilbene. Lysostilbene-4 emerged as the lead candidate, demonstrating [~]30-40-fold greater cytotoxicity against PDAC cells than parental compounds, while sparing non-malignant cells. At nanomolar concentrations, Lysostilbene-4 induced rapid, irreversible lysosomal membrane permeabilization (LMP), initiating a lysosome mitochondria apoptotic cascade via cathepsin-B release, BID cleavage, BAX activation, and caspase-mediated apoptosis. In parallel, it abrogated lysosomal recovery by impairing repair, lysophagy, autophagosome maturation, and uncoupling TFEB-driven transcriptional programs from effective lysosome biogenesis. TFEB knockout further sensitized PDAC cells, underscoring TFEB as a key determinant of lysosomal resilience and a potential predictive biomarker. Importantly, Lysostilbene-4 was well tolerated in preclinical mouse models at supra-therapeutic doses without systemic toxicity. These findings position Lysostilbene-4 as a first-in-class lysosome-targeting therapeutic that enforces sustained lysosomal collapse while disabling adaptive recovery mechanisms, providing a mechanistically precise and safe strategy against PDAC.

cancer biology↗

bioRxiv2025

Lipid-laden endothelial cells exhibit a transcriptomic signature linked to blood-brain barrier dysfunction, metabolic reprogramming and increased inflammation in the aging brain

Dysregulation in lipid metabolism is increasingly recognized as a key contributor to age-related diseases, including neurodegeneration and cerebrovascular dysfunction. While prior studies have largely focused on glial cells, the impact of lipid dysregulation on brain endothelial aging remains poorly understood. In this study, we conducted a secondary analysis of single-cell transcriptomic data from young and aged mouse brains, with a specific focus on endothelial cells (ECs). Our analyses revealed that aging promotes lipid droplet accumulation in brain ECs. These lipid-laden brain ECs exhibit a transcriptomic signature indicative of impaired blood-brain barrier function, increased cellular senescence, and inflammation in aging. Furthermore, lipid accumulation is associated with an altered metabolic phenotype characterized by increased fatty acid oxidation and decreased glycolysis, and impaired mitochondrial electron transport chain activity in the ECs of the aging brain. We have also validated lipid accumulation in aged ECs in vivo. Collectively, our findings indicate that lipid accumulation drives structural, functional, and metabolic impairments in the brain ECs, likely contributing to cerebrovascular aging. Understanding the mechanisms underlying lipid accumulation-induced endothelial dysfunction may offer novel therapeutic strategies for mitigating microvascular dysfunction and cognitive decline in aging.

neuroscience↗

bioRxiv2025

Taurine Inhibits Apolipoprotein E4 Aggregation

Apolipoprotein E4 (ApoE4) is a major genetic risk factor in many neurodegenerative diseases, yet effective therapeutic strategies targeting its associated pathologies remain unresolved. The aggregation of ApoE4, a key pathological feature, can be attenuated by tramiprosate and its metabolite 3-sulfopropanoic acid. In this study, we investigated the potential of taurine, a close chemical analogue of tramiprosate, to modulate ApoE4-mediated pathological processes. Using an integrated approach--including molecular dynamics simulations, static light scattering, mass spectrometry, and cerebral organoid models--we investigated taurines effects on ApoE4 aggregation. We found that taurine effectively inhibits ApoE4 aggregation. Notably, taurine significantly ameliorates the pathophysiological characteristics of ApoE4, bringing its phenotype closer to the more benign ApoE3 variant. By leveraging its neuroprotective properties, taurine may offer effects comparable to tramiprosate and 3-sulfopropanoic acid, positioning it as an accessible and promising candidate for mitigating neurodegeneration, particularly in individuals with the high-risk ApoE4/E4 genotype.

biochemistry↗

bioRxiv2025

A large global soil carbon sink informed by repeated soil samplings

Partitioning the terrestrial carbon sink between vegetation and soil is crucial for predicting future climate change, but the role of soils remains poorly quantified. Here, we compiled 3,099 soil organic carbon time series spanning five decades. We found a global soil organic carbon sink of 1.83 {+/-} 0.9 (mean {+/-} SE) petagrams per year from 1992 to 2020, driven by extratropical young forests, boreal old forests, and grasslands, while trends in tropical ecosystems remain uncertain. Our findings suggest the net land sink resides almost exclusively belowground as soil carbon, emphasizing the global opportunity of soil conservation and restoration for climate mitigation.

ecology↗

bioRxiv2025

Impaired Macrophage DNase Secretion Promotes Neutrophil Extracellular Trap Mediated Defective Efferocytosis in Atherosclerosis

Neutrophil extracellular traps (NETs) drive atherosclerosis progression and are associated with adverse clinical outcomes like myocardial infarction and stroke. While the triggers of NETosis in atherosclerotic plaque are well-characterized, the mechanisms underlying NET degradation and clearance remain unclear. Moreover, the impact of impaired NET clearance on atherosclerosis progression have not been elucidated. Here we show that macrophages are critical for the release of DNases, which degrade NETs. We identified endoplasmic reticulum (ER) stress mediated activation of PERK-ATF4 pathway as a key driver of impaired macrophage DNase secretion, leading to delayed NET clearance and their persistence. Elevated NET levels trigger cleavage of the efferocytosis receptor Mertk leading to defective macrophage efferocytosis and exacerbation of plaque necrosis. Human atherosclerotic plaques and Ldlr-/- mice treated with ISRIB, a PERK inhibitor, show enhanced DNase secretion and clearance of NETs. Together, the identification of key mechanisms of NET clearance in atherosclerosis offers new therapeutic strategies to stabilize plaques.

pathology↗

bioRxiv2024

CHK1-mediated regulation of TOP1 catalytic activity suppresses replication and transcription-associated genomic instability

The Topoisomerase 1 (TOP1) catalytic cycle involves a TOP1-DNA-covalent-complex (TOP1cc), which, if stabilized, can induce rapid accumulation of potentially lethal DNA double strand breaks (DSBs). Although TOP1cc are critically associated with genome instability, it is not yet precisely known how cells regulate TOP1cc level, under unperturbed physiological-condition, to prevent its accumulation and lethal consequences. We discovered a key role of CHK1 in phosphorylating TOP1 at Serine-320 and stimulating TOP1-catalytic cycle to minimise genome wide accumulation of TOP1cc in cancers. Pharmacological or genetic ablation of CHK1-mediated TOP1-phosphorylation leads to stalled replication forks and generates copious amounts of replication/transcription-associated DSBs, R-loops and transcription-replication collisions, eventually leading to chromosomal instability. Further, TOP1ccs stabilized due to CHK1 inhibition are not efficiently targeted by cellular TOP1cc-removal machineries. Since multiple patient clinical trials are ongoing with TOP1- and CHK1-targeting drugs, current finding of CHK1-mediated regulation of TOP1cc may help in better understanding the therapeutic outcomes.

cancer biology↗

bioRxiv2024

Chronic β3 adrenergic agonist treatment improves brain microvascular endothelial function and cognition in aged mice

Microvascular endothelial dysfunction, characterized by impaired neurovascular coupling, reduced glucose uptake, blood-brain barrier disruption, and microvascular rarefaction, plays a critical role in the pathogenesis of age-related vascular cognitive impairment (VCI). Emerging evidence points to non-cell autonomous mechanisms mediated by adverse circulating milieu (an increased ratio of pro-geronic to anti-geronic circulating factors) in the pathogenesis of endothelial dysfunction leading to impaired cerebral blood flow and cognitive decline in the aging population. In particular, age-related adipose dysfunction contributes, at least in part, to an unfavorable systemic milieu characterized by chronic hyperglycemia, hyperinsulinemia, dyslipidemia, and altered adipokine profile, which together contribute to microvascular endothelial dysfunction. Hence, in the present study, we aimed to test whether thermogenic stimulation, an intervention known to improve adipose and systemic metabolism by increasing cellular energy expenditure, could mitigate brain endothelial dysfunction and improve cognition in the aging population. Eighteen-month-old old C57BL/6J mice were treated with saline or CL ({beta}3-adrenergic agonist) for 6 weeks followed by functional analysis to assess endothelial function and cognition. CL treatment improved neurovascular coupling responses and rescued brain glucose uptake in aged animals. In addition, CL treatment also attenuated blood-brain barrier leakage and associated neuroinflammation in the cortex of aged animals. More importantly, these beneficial changes in microvascular function translated to improved cognitive performance in radial arm water maze and Y-maze tests. Our results suggest that {beta}3-adrenergic agonist treatment improves multiple aspects of brain microvascular endothelial function and can be potentially repurposed for treating age-associated cognitive decline.

neuroscience↗

bioRxiv2024

The metabolic benefits of thermogenic stimulation are preserved in aging

Adipose thermogenesis has been actively investigated as a therapeutic target for improving metabolic dysfunction in obesity. However, its applicability to middle-aged and older populations, which bear the highest obesity prevalence in the US (approximately 40%), remains uncertain due to age-related decline in thermogenic responses. In this study, we investigated the effects of chronic thermogenic stimulation using the {beta}3-adrenergic (AR) agonist CL316,243 (CL) on systemic metabolism and adipose function in aged (18-month-old) C57BL/6JN mice. Sustained {beta}3-AR treatment resulted in reduced fat mass, increased energy expenditure, increased fatty acid oxidation and mitochondrial activity in adipose depots, improved glucose homeostasis, and a favorable adipokine profile. At the cellular level, CL treatment increased uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT). However, in white adipose tissue (WAT) depots, CL treatment increased glycerol and lipid de novo lipogenesis (DNL) and turnover suggesting the activation of the futile substrate cycle of lipolysis and reesterification in a UCP1-independent manner. Increased lipid turnover was also associated with the simultaneous upregulation of proteins involved in glycerol metabolism, fatty acid oxidation, and reesterification in WAT. Further, a dose-dependent impact of CL treatment on inflammation was observed, particularly in subcutaneous WAT, suggesting a potential mismatch between fatty acid supply and oxidation. These findings indicate that chronic {beta}3-AR stimulation activates distinct cellular mechanisms that increase energy expenditure in BAT and WAT to improve systemic metabolism in aged mice. Our study provides foundational evidence for targeting adipose thermogenesis to improve age-related metabolic dysfunction.

pharmacology and toxicology↗

bioRxiv2024

Prediction of Aggregation Prone Regions in Proteins Using Deep Neural Networks and Their Suppression by Computational Design

Identification of aggregation-prone regions in proteins and their suppression through mutations is a powerful strategy to enhance protein solubility and yield, significantly expanding their application potential. Here, we developed a deep neural network-based predictor AggreProt, that generates a residue-level aggregation profile for protein sequences. The model outperformed or matched current state-of-the-art algorithms, as validated on two independent datasets comprising hexapeptides and full-length proteins with annotated aggregation-prone regions. We further validated the model experimentally using a set of 34 hexapeptides identified in the model protein haloalkane dehalogenase LinB, along with seven proteins from the AmyPro database. Experimental results agreed with our predictions in 79% of cases and also revealed inaccuracies in some database annotations. Finally, the algorithms utility was demonstrated by identifying aggregation-prone regions in the LinB enzyme and designing mutations to suppress aggregation in its exposed regions. The resulting variants exhibited reduced aggregation propensity, improved solubility, and up to a 100% increase in yield compared to the wild type. AggreProt is freely available to the scientific community via a user- friendly web server: https://loschmidt.chemi.muni.cz/aggreprot.

bioinformatics↗

bioRxiv2022

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein AAV transgene expression is silenced using the clinically validated modality of chemically modified short interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

genomics↗

bioRxiv2022

Interleukin-1α and Leukemia Inhibitory Factor Promote Extramedullary Hematopoiesis

Extramedullary hematopoiesis (EMH) expands hematopoietic capacity outside of the bone marrow in response to inflammatory conditions, including infections and cancer. Because of its inducible nature, EMH offers a unique opportunity to study the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niche. In cancer patients, the spleen frequently serves as an EMH organ and provides myeloid cells that may worsen pathology. Here, we examined the relationship between HSPCs and their splenic niche in EMH secondary to solid tumor. We identify an inflammatory gene signature characterized by TNF expression in HSPCs. We show a role for IL-1 in producing this gene signature and TNF expression in HSPCs in activating splenic niche activity. We also demonstrate that tumor-derived Leukemia Inhibitory Factor (LIF) induces proliferation of splenic niche cells. IL-1 and LIF display cooperative effects in activating EMH and are both upregulated in some human cancers. Together, these data expand avenues for developing niche-directed therapies and further exploring EMH accompanying inflammatory pathologies like cancer.

immunology↗

bioRxiv2022

Robust and durable prophylactic protection conferred by RNA interference in preclinical models of SARS-CoV-2

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral genome. We screened lipophilic small-interfering RNA (siRNA) conjugates targeting highly conserved regions of the SARS-CoV-2 genome and identified leads targeting outside of the spike-encoding region capable of achieving [≥]3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single-siRNA approach. A two-siRNA combination delivered intranasally protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach to limit SARS-CoV-2 infection that may help combat emergent variants, complement existing interventions, or protect populations where vaccines are less effective. Most importantly, this strategy has implications for developing medicines that may be valuable in protecting against future coronavirus pandemics.

pharmacology and toxicology↗

bioRxiv2021

Conserved angio-immune subtypes of the cancer microenvironment predict response to immune checkpoint blockade therapy

Tumor microenvironment (TME) shapes the tumor progression and therapy outcome. Particularly, tumor angiogenesis and immunity impact the effect of immune checkpoint blockade (ICB) therapy. Here, we analyzed the transcriptome from 11,069 patients from The Cancer Genome Atlas (TCGA) to assess 91 functional gene sets corresponding to endothelial and T-cell activity. Intriguingly, TME across 30 non-hematological tumors can be classified into three distinct conserved angio-immune subtypes: high angiogenesis with low immune activity, low angiogenesis with high immune activity, and the one in-between. Remarkably, patients displaying TME with poor angiogenic activity with corresponding high immune activity show the most significant responses to ICB therapy in many cancer types. Notably, re-evaluation of the Javelin Renal 101, renal cell carcinoma clinical trial, provided compelling evidence that the baseline angiogenic state is critical in determining responses to checkpoint blockade. This study offers a clear rationale for incorporating baseline angiogenic state for ICB treatment decision-making.

cancer biology↗

bioRxiv2021

Microenvironmental correlates of immune checkpoint inhibitor response in human melanoma brain metastases revealed by T cell receptor and single-cell RNA sequencing

Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

cancer biology↗

bioRxiv2021

Eosinophils restrict diesel exhaust particles induced cell proliferation of lung epithelial A549 cells, vial interleukin-13 mediated mechanisms: implications for tissue remodelling and fibrosis

Diesel exhaust particulates (DEPs) affect lung physiology and cause serious damage to the lungs. A number of studies have demonstrated that eosinophils play a very important role in the development of lung tissue remodelling and fibrosis. However, the exact mechanism of its pathogenesis is not known. We for the first time demonstrate that, Interleukin-13 plays a very important role in the development of tissue remodelling and fibrosis. We demonstrate that, Diesel exhaust particle significantly induce eosinophils cell proliferation and interleukin-13 release in invitro culture conditions. Supernatant collected from DEP induced eosinophils cells significantly restrict cell proliferation of epithelial cells due to exposure of diesel exhast particles. Furthermore, purified interleukin-13 decreases the proliferation of A549 cells. Notably, Etoricoxib (selective COX-2 inhibitor) did not inhibit DEP-triggered release of interleukin-13, suggesting another cell signalling pathway. In, vivo exposer of DEP to the mice lung, resulted in the high level of eosinophils degranulation as depicted by the EPX-1 immunostaining and altered level of mRNA expressions of inflammatory genes. We also found that, a-SMA, fibroblast specific protein (FSP-1) has been changed in response to DEP in the mice lungs along with the mediators of inflammation. Altogether, we elucidated the mechanistic role of eosinophils in the DEP triggered proliferation of lungs cells thus providing an inside in the pathophysiology of tissue remodelling and fibrosis of lungs.

cell biology↗

bioRxiv2020

Hypercholesterolemia Impairs Clearance of Extracellular DNA and Promotes Inflammation and Atherosclerotic Plaque Progression

Defects in clearance of extracellular DNA due to sub-optimal activity of DNase results in exacerbated inflammation and contributes to the pathophysiology of atherosclerosis and other inflammatory diseases. However, the physiological mechanisms that regulate systemic DNase levels and the basis of its functional impairment during disease are poorly understood. Using a mouse model of experimental increase in systemic extracellular DNA levels, we identify the existence of a physiologic DNA-induced DNase response. Importantly, hypercholesterolemia in mice impairs this critical DNA-induced DNase response through an endoplasmic reticulum stress-mediated mechanism with consequences in advanced atherosclerotic plaque progression including increased extracellular DNA accumulation, exacerbated inflammation, and development of pathological features of necrotic rupture-prone vulnerable plaques. From a translational standpoint in humans, we demonstrate that individuals with hypercholesterolemia have elevated systemic extracellular DNA levels and decreased plasma DNase activity. These data suggest that the restoration of DNA-induced DNase response could be a potential therapeutic strategy to promote inflammation resolution during hypercholesterolemia.

immunology↗

bioRxiv2018

Pervasive adaptation of hepatitis C virus to interferon lambda polymorphism across multiple genotypes

Genetic polymorphism in the interferon lambda (IFN-{lambda}) region is associated with spontaneous clearance of hepatitis C virus (HCV) infection and with response to interferon-based antiviral treatment. Here, we evaluate the associations between IFN- {lambda} polymorphism and HCV variation through a genome-to-genome analysis in 8,729 patients from diverse ancestral backgrounds infected with various HCV genotypes. We searched for associations between rs12979860 genotype, a tag for IFN-{lambda} haplotypes, and amino acid variants in the NS3, NS4A, NS5A and NS5B HCV proteins. We report multiple associations between host and pathogen variants in the full cohort as well as in subgroups defined by viral genotype and human ancestry. We also assess the combined impact of human and HCV variation on pre-treatment viral load. By demonstrating that IFN-{lambda} genetic variation leaves a large footprint in the viral genome, this study provides strong evidence of pervasive viral adaptation to host innate immune pressure during chronic HCV infection.

genomics↗