Biology subjects

Chaudhary, S.

Publications and source records attributed to Chaudhary, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Homothallic or heterothallic? A genomic investigation into the sexual capabilities of the ascomycete fungus Clonostachys rosea

Modes of reproduction and sexual strategies strongly influence the genetic diversity and evolutionary potential of a species. The ascomycete fungus Clonostachys rosea is reported to be homothallic (sexually self-fertile), although a rapid decay of genome-wide linkage disequilibrium is also reported, something that is not in line with an obligate homothallic mode of reproduction. To investigate this phenomenon, we identified the mating-type (MAT1) locus in 66 genome-sequenced C. rosea strains under the hypothesis that each strain contains genes from both MAT1 idiomorphs. Eleven strains indeed contained both MAT1-1 and MAT1-2 genes, suggesting homothallism. However, most strains harboured either MAT1-1 or MAT1-2 genes and co-existed in North America, Europe and China, suggesting heterothallism. The MAT1 locus of heterothallic strains was highly conserved, and the linkage disequilibrium half decay distance was 1050 bp, suggesting sexual outcrossing. The presence of conserved MAT1-1 or MAT1-2 idiomorphs in strains of other Clonostachys species shows that heterothallism is likely the ancestral state. A phylogenetic analysis of 2800 single-copy orthologous genes revealed that homothallic and heterothallic strains separated in two well-supported clades, indicating a single lineage of homothallic C. rosea, likely originating in South America, followed by intercontinental dispersal. Homothallic C. rosea strains displayed higher nucleotide diversity than heterothallic strains, indicating a lack of outcrossing. This unique case of both homothallic and heterothallic lineages within the same species provides an opportunity to study the genomic consequences of selfing in very closely related strains.

evolutionary biology↗

bioRxiv2026

The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

BackgroundLung cancer, including Non-Small Cell Lung Carcinoma (NSCLC), is the leading cause of cancer mortality worldwide. Platinum salts are the gold standard chemotherapy for NSCLC but many patients develop resistance leading to disease progression. Identifying new therapeutic strategies to counteract resistance is crucial. Pharmacological compounds targeting core components of the spliceosome machinery have emerged as promising anti-cancer agents. However, their mechanisms of action remain to be elucidated in NSCLC. MethodsVarious NSCLC cell lines were used in 2D and 3D cultures or clonogenic assays. NSCLC Patient-Derived Xenografts were also used. SF3B1 was silenced by siRNA. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. Western-blot, immunofluorescence, SIRF analysis and DNA repair assays were done to assess globally the DNA damage response. RNA-Seq, RT-qPCR and RT-PCR studies were performed to identify gene and splicing events impacted by SF3B1 inhibition. Publicly available transcriptomic and proteomic data were analyzed. ResultsSF3B1 is a core component of the spliceosome machinery. We show that NSCLC cells with acquired resistance to platinum salts are vulnerable to pladienolide B, a SF3B1 inhibitor, or SF3B1 knock-down. Importantly, pladienolide B also slows down tumor growth of NSCLC Patient-Derived Xenografts (PDXs) poorly responsive to platinum salts. Mechanistically, we show that pladienolide B leads to genomic instability and apoptosis, that correlate with early transcription-dependent replication stress and DNA-PKcs activation, followed by the shutdown of ATR/DNA-PKcs-dependent signaling. In addition, pladienolide B profoundly regulates the expression and/or splicing, particularly exon skipping, of numerous genes involved in DNA repair, leading to decreased repair capacities of DNA double strand breaks. Although exon skipping events are mostly transient, skipping of exon 8 of MLH3, a gene involved in mismatch DNA repair, persisted along time. Finally, we show that pladienolide B counteracts resistance to platinum salts in NSCLC cells as well as PDXs, which correlates with enhanced MLH3 exon 8 skipping and decrease of ATR, DNA-PKCs and MLH3 protein levels. ConclusionsAs a whole, our data highlight the targeting of SF3B1 as a potential therapeutic strategy, alone or in combination, in NSCLC patients who escape platinum salts-based chemotherapy.

cancer biology↗

bioRxiv2025

Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells

Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells. SIGNIFICANCE STATEMENTCRISPR technologies are typically engineered in bacteria, even though they must function in the far more complex environment of human cells. This gap has limited the discovery of variants with improved DNA recognition or with resistance to inhibitors that operate differently in mammalian systems. Here we establish CRISPR-MACE, a continuous evolution platform that leverages pressure from anti-CRISPR proteins to select Cas9 variants directly in human cells that have novel functions. Evolved variants show improvements in DNA binding strength and residence time, as well as striking escape from the potent Cas9 inhibitor AcrIIA4. Many anti-CRISPR proteins use distinct mechanisms, so our strategy can drive future continuous evolution campaigns in mammalian cells that expand the functional properties of genome-targeting agents.

synthetic biology↗

bioRxiv2025

The subclonal footprint of pervasive early dissemination in pancreatic cancer

Early is too late describes a central problem in pancreatic cancer referring to its relentless drive to disseminate and highlights the need to understand how this disease spreads so rapidly. In this study, we profiled 1,013 samples from 277 donors, including tissue whole genome and RNA sequencing combined with plasma whole genome sequencing at [~]25x. Strikingly, local primary tumours, including some reaching 7cm, were found to shed little to no circulating tumour DNA (ctDNA). Instead, metastatic burden in the liver but not extrahepatic sites, was a main physiologic determinant of ctDNA levels. Whole-genome duplication (WGD), high cell cycle activity, and non-glandular differentiation emerged as tumour-intrinsic features related to increased ctDNA shedding. By contrast, decreased shedding was related to extrinsic features including a reactive microenvironment and unexpectedly, humoral immunity. Analysis of tumour clonal architecture showed that in patients with low ctDNA levels, the signal disproportionately originated from subclones and this signal persisted even when the primary tumour was removed. Disseminated subclones were a significant source of ctDNA in early-stage patients. Longitudinal analysis of patients revealed that subclones seeded metastases and shed ctDNA in the blood years before detection. This first report of paired tissue and plasma whole genomes in pancreatic cancer is a unique resource and has broad implications for disease surveillance, treatment monitoring, and early detection in this disease.

cancer biology↗

bioRxiv2025

Lake size shapes the relationship between body mass and gut microbiota in threespine stickleback (Gasterosteus aculeatus)

Host-microbe interactions are shaped by both host and environmental factors. However, little is known about how host-microbe interactions vary across populations within a species. Here, we characterized the gut microbiota of 191 wild threespine stickleback fish (Gasterosteus aculeatus) from six populations from Alaskan lakes spanning a gradient of surface area. We tested how environmental context (lake size and ecotype) and host traits (sex, body mass, gravidity, Schistocephalus solidus (S. solidus) infection, and fibrosis) influence stickleback gut microbial composition using 16S rRNA gene sequencing. We found that the lake surface area strongly predicted fish gut microbial alpha diversity. Fish from intermediate-sized lakes harbored significantly more diverse microbiota than those from small and large lakes, independent of ecotype. Body mass was associated with gut microbial diversity. Model-predicted marginal effects from the mass and lake surface area interaction analysis showed that the association between fish mass and microbial alpha diversity was strongly negative in the smallest lakes, weakest in intermediate-sized lakes, and strongly positive in the largest lakes. In addition, sex and S. solidus infection were significantly associated with gut microbiota alpha and beta diversity, whereas fibrosis and gravidity showed minimal effects. Differential abundance analysis revealed lake size-dependent associations between body mass and individual taxa. Together, these results demonstrate that both habitat context and host variation interactively shape stickleback gut microbial communities in the wild. Integrating lake-level and individual-level analyses reveals how ecological setting modulates host-microbe associations, offering insights into the role of the gut microbiota in host adaptation and population divergence.

microbiology↗

bioRxiv2025

Deep-Pose-Tracker: a unified model for behavioural studies of Caenorhabditis elegans

Tracking and analyzing animal behaviour is a crucial step in fields such as neuroscience and developmental biology. Behavioural studies in the nematode C. elegans, for example, help in understanding how organisms respond to external cues and how the specific physiological responses link to either instantaneous or learned behaviours. Although tracking behaviour through locomotion patterns and postural dynamics is routine, it becomes laborious and time-consuming when performed manually. Automation of this process is therefore crucial for accurate and fast detection and analysis. To this end, we report Deep-Pose-Tracker (DPT), a YOLO (You Only Look Once)-based model for automated pose detection of C. elegans from videos and images. The module is further utilized for several downstream analysis algorithms to quantify essential behavioural features, including locomotion speed, orientation, forward or reverse locomotion, and complex body bends such as omega turns. In addition, it includes eigenworms decomposition to represent complex posture dynamics in a low-dimensional space. The model shows reliable performance on the validation and test datasets, with high inference speed, while being user-friendly. DPT, therefore, can be a valuable toolkit for automated behavioural quantification of C. elegans under varying experimental stimuli.

animal behavior and cognition↗

bioRxiv2025

Alpha diversity analysis of hepatic transcriptome reveals novel pathways inalcohol-related hepatitis

Next generation sequencing can identify novel gene expression patterns in disease. Beyond differentially expressed genes analysis, we investigated the ability of within-population diversity (-diversity) of the transcriptome to reveal new biological information in alcohol-related liver disease (ALD), comparing Differential Shannon diversity (DSD) to transcriptome heterogeneity changes. RNA sequencing data from normal livers and patients with early silent ALD and severe AH were analyzed. -diversity indices and Percent Shannon Diversity of a gene, which refers to this genes contribution to total Shannon entropy were calculated. Ingenuity pathway analysis identified canonical pathways determined by differentially expressed genes (DEG) and DSD approaches. ALD significantly decreased hepatic transcriptome -diversity correlating with increased relative contribution of select genes. These changes were driven by lower abundance gene expression loss. DEG and DSD analyses showed overlapping genes and canonical pathways, but DSD also identified novel genes and pathways not highlighted by DEG. Importantly, DSD more effectively identified differences between preclinical ALD and AH severity stages. ALD decreases hepatic transcriptome heterogeneity, favoring pathways associated with organ damage or damage response. Preclinical and clinical ALD led to differential heterogeneity patterns that may provide new disease insights. DSD analysis identified enriched pathways missed by standard DEG analyses, potentially yielding novel insight into disease mechanisms and biomarkers. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=53 SRC="FIGDIR/small/681263v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@d2d3a3org.highwire.dtl.DTLVardef@14496a8org.highwire.dtl.DTLVardef@1ff0dfeorg.highwire.dtl.DTLVardef@1efa8f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

Calpain-4 Knockdown Modulates Cholesterol Metabolism and LXRα Nuclear Localization in Alcohol-Related Liver Disease

Background & AimsEthanol affects lipid metabolism through multiple pathways, leading to fatty liver development in most ALD patients. Recent studies have highlighted the role of calpain, a calcium-dependent protease, in liver inflammation and fibrosis. Calpain activity is regulated by its essential subunit, Capns1, (calpain-4, Capn4), which stabilizes and modulates the activity of its catalytic isoforms, calpain-1 and calpain-2. This study investigated calpains impact on lipid metabolism in ALD. Approach & ResultsSix-week-old C57Bl6/J mice were injected with rAAV8 vectors encoding Capn4 shRNA or control vectors. After four weeks, mice underwent a 10-day period of ad libitum ethanol consumption, followed by a single gavaged ethanol administration on day 11. Following Capn4 knockdown, microvesicular steatosis was attenuated. While triglycerides and free fatty acids levels showed no significant changes, cholesterol levels were significantly reduced in the ethanol (EtOH) group with Capn4 knockdown. Cpt1a expression increased significantly in the EtOH group with Capn4 knockdown. Western blot analysis revealed increased Cleaved-HMGCR to Pro-HMGCR ratio in Capn4 knockdown mice, suggesting reduced HMGCR activity and suppressed cholesterol biosynthesis. LXR expression was mainly increased in the cytoplasm in the EtOH group, and following Capn4 knockdown, it was relocalized to the nucleus via its activation. In addition, RNA sequencing analysis suggests that Capn4 knockdown contributes to the reprogramming of ethanol-induced disruptions in metabolic and homeostatic pathways, primarily those involving cholesterol metabolism. ConclusionsFurther investigation into the relationship between Capn4 and cholesterol biosynthesis proteins may provide insights into using calpain inhibitors as a therapeutic approach for alcohol-related hepatitis.

molecular biology↗

bioRxiv2025

Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection

BackgroundKlebsiella pneumoniae is a major opportunistic pathogen with rising multidrug resistance and biofilm-related infections. Molecular and phage characterization is crucial to understand resistance mechanisms and explore alternative therapies such as phage therapy. MethodsWe performed whole-genome sequencing and antibiotic susceptibility testing of hospital-isolated Klebsiella pneumoniae (KP6697). MLST, plasmid replicon analysis, and resistance gene identification were conducted using bioinformatics. Phage isolation, electron microscopy-based morphological and biofilm analysis, and evaluation of lytic activity, stability, and host range were performed. Phage genome sequencing and annotation identified functional genes. ResultsThe host strain Klebsiella pneumoniae (KP6697) was multidrug-resistant, exhibiting resistance to 18 of 22 tested antibiotics, and genome analysis identified ST16 with eight plasmid replicons and 23 resistance genes, including blaCTX-M-15, blaNDM-5, and blaOXA-181. Functional annotations revealed extensive metabolic versatility and a rich repertoire of genes for biofilm formation, quorum sensing, secretion systems, and stress response. A lytic phage, Phage_KP6697_Omshanti, was isolated and classified as a Caudoviricetes member with a 45.3kb genome encoding lysis, replication, and structural genes. It demonstrated short latency, high burst size, thermal and pH stability, and broad host range against CRKP and other MDR strains. Importantly, microscopy confirmed its ability to inhibit and degrade biofilms at multiple stages, highlighting strong therapeutic potential. ConclusionComprehensive analysis of carbapenem-resistant K. pneumoniae (KP6697) revealed multidrug resistance and strong biofilm formation. The lytic phage Phage_KP6697_Omshanti, with depolymerase and endolysin activity, disrupted biofilms, and its stability, high burst size, and genomic traits suggest potential as an anti-CRKP agent, especially with antibiotics IMPORTANCEKlebsiella pneumoniae is increasing multidrug resistance and robust biofilm formation pose severe clinical challenges, limiting treatment options. Understanding the molecular basis of its resistance and exploiting bacteriophages with strong biofilm-disrupting properties provide promising alternative therapeutic strategies. This study highlights the isolation and genomic characterization of a lytic phage with potent anti-biofilm activity against carbapenem-resistant K. pneumoniae, underscoring its potential in combating resistant infections.

microbiology↗

bioRxiv2025

Engineering Escherichia coli Nissle as safe chassis for delivery of therapeutic peptides

Synthetic biology enables the integration of sophisticated genetic programs into microorganisms, transforming them into potent vehicles for therapeutic applications. Engineering strategies for microorganisms are rapidly evolving, offering promising solutions for cancer therapy, microbiome modulation, digestive health support, and beyond. Developing novel tools to engineer safe, nonpathogenic microbial platforms is essential for advancing clinical therapies. In this work, we present an innovative engineering approach for the probiotic Escherichia coli Nissle (EcN), aimed at creating a safe and efficient chassis for the bioproduction of therapeutics. The EcN endogenous pM1 and pM2 plasmids were cured and re-engineered to introduce a CRISPR-Cas12 chromosome shredding device and a therapeutic-producing genetic circuit, thereby generating a nonproliferative therapeutic-delivery system. Next, we build an AI-based bioinformatic pipeline to predict Anticancer-Cell-Penetrating Peptides (ACCPP) candidates. As a proof-of-concept, a selected ACCPP was produced in the engineered EcN chromosome-shredded (CS) chassis. This strategy yields a robust and controllable platform for the safe production and delivery of therapeutics, paving the way for the future development of microbial therapies and their clinical applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/673081v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@2430d4org.highwire.dtl.DTLVardef@1ce2borg.highwire.dtl.DTLVardef@8686cdorg.highwire.dtl.DTLVardef@1fc23fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Integrated spatial proteomics of human PDAC uncovers an expanded tumour-immune-stroma spectrum with genomic associations

Distinctively, pancreatic ductal adenocarcinoma (PDAC) consists of sparse tumour lesions intertwined with extensive desmoplastic stroma. The complexity of tumour-microenvironment interactions within this desmoplasia poses a challenge for accurate tumour profiling and patient stratification, and characterizes a profoundly chemoresistant tumour. Here we mapped the spatial relationships between tumour, stroma, and immune cell compartments delineating tumour and microenvironment types that expand the classical to basal spectrum of human PDAC. We used imaging mass cytometry to profile the in situ multi-cellular organization of 81 cell types in resected cases with paired whole genome sequencing. Cell types, functions, and pathway activation were distributed as highly reproducible environments in discrete locations throughout these tumours, which we deep-profiled using laser-capture mass spectrometry. We show that the connections between tumour phenotypes, vascularization, immune response, and stromal biophysical state are reinforced by genomic aberrations, altered by treatment, and associated with patient outcome. Predictive machine-learning models showed that spatial single cell data outperformed genomic or clinical features but integrated multi-omics models provide the best prediction of patient survival with compressed models requiring only 10 non-redundant robust molecular measures associated with the phenotypic spectrum of PDAC. Together, these findings define a phenotypic and molecular framework of PDAC that captures tumour-microenvironment co-dependencies and offers a refined basis for patient stratification and therapeutic targeting.

cancer biology↗

bioRxiv2025

Functional Inputs to the Subgenual Cingulate Cortex Distinguish Neurotypical Individuals with Severe and Mild Depressive Problems: Granger Causality and Clustering Analyses

Extant research has implicated functional connectivity of the subgenual anterior cingulate cortex (sgACC) in major depressive disorders or depressive traits in neurotypical populations. However, prior studies have not distinguished the inputs and outputs of the sgACC, and the "diagnostic" accuracy of these connectivity metrics remains elusive. Here, we analyzed data of 890 subjects (459 women, age 22 to 35) from the Human Connectome Project using Granger causality analyses (GCA) with the sgACC as the seed and 268 regions of interest from the Shens atlas as targets. Individual connectivities were assessed with an F test and group results were evaluated with a binomial test, both at a corrected threshold. We identified brain regions with significant input to and output from the sgACC. Clustering analyses of Granger causality input, but not Granger causality output or resting state connectivity features revealed distinct subject clusters, effectively distinguishing individuals with severe and mild depressive symptoms and those with comorbidities. Specifically, weaker projections from the fronto-parietal and orbitofrontal cortices, anterior insula, temporal cortices, and cerebellum to the sgACC characterized five clusters with low to high scores of depression as well as comorbid internalizing and externalizing problems. Machine learning using a logistic classifier with the significant "GCA-in" features and 5-fold cross-validation achieved 87% accuracy in distinguishing subject clusters, including those with high vs. low depression. These new findings specify the functional inputs and outputs of the sgACC and highlight an outsized role of sgACC inputs in distinguishing individuals with depressive and comorbid problems. Significance StatementDepression affects millions worldwide. Understanding the neural mechanisms would facilitate treatment of depression. In this study, we investigated how different parts of the brain send signals to and/or receive signals from a key brain region - subgenual cingulate cortex - implicated in the pathophysiology of depression. Using brain scans from nearly 900 adults, we found that weaker signals coming into this area, especially from regions involved in thinking and emotion, were linked to higher levels of depression and other behavioral problems such as anxiety and aggression. These findings help us better understand how regional communications in the brain relate to emotional well-being and may support new ways to identify and treat people with a depressive disorder.

neuroscience↗

bioRxiv2025

Mechanism of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol antioxidant, in rescuing oxidized low-density lipoprotein-induced cytotoxicity in human retinal pigment epithelial cells

Geographic atrophy or late stage dry age-related macular degeneration (AMD) is characterized by drusen deposition and progressive retinal pigment epithelium (RPE) degeneration, leading to irreversible vision loss. The formation of drusen leads to dyshomeostasis, oxidative stress and irreversible damage to RPE. In this study, we used an in vitro model of oxidized-low density lipoproteins (ox-LDL) induced human RPE damage/death model to investigate the mechanism whereby a sterically hindered phenol antioxidant compound, PMC (2,2,5,7,8-pentamethyl-6-chromanol) protects RPE against ox-LDL-induced damage. We show that PMC exerts its protective effect by preventing the upregulation of stress-responsive heme oxygenase-1 (HMOX1/HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) at mRNA and protein levels. This effect was due to PMCs blockade of ROS generation, which in turn blocked nuclear translocation of the Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, ultimately preventing the upregulation of antioxidant response elements (ARE), including HMOX1 and NQO1. A key role for HO-1 was demonstrated when the protective effect of PMC was inhibited by the knockdown of HMOX1. Additionally, treatment of PMC under different experimental conditions and time points revealed that the continuous presence of PMC is required for optimal protection against ox-LDL-induced cytotoxicity, defining the cellular pharmacokinetics of the molecule. Our data demonstrate the involvement of a key antioxidant pathway through which PMC mitigates oxidative stress induced by ox-LDL and provides a potential therapeutic strategy to suppress RPE degeneration/damage during AMD progression.

molecular biology↗

bioRxiv2025

M6A reader protein YTHDF3 regulates cardiomyocyte death and atrophy by modulating the alternative splicing program

BackgroundThe functional impact of m6A modifications on RNA is governed by reader proteins that read the m6A marks and process the RNA accordingly. Recent studies have highlighted the importance of m6A reader proteins in the heart. However, the function of a reader protein Ythdf3 in the heart remains completely unknown. ObjectiveWe aim to uncover the function of Ythdf3 in the hearts. Methods& ResultsHere, we show that Ythdf3 is indispensable for the heart, and its knockdown leads to cardiac cachexia evident by cardiomyocyte atrophy, death, and less dense myofibrils visualized by electron microscopy. We also found downregulation of Ythdf3 in response to doxorubicin, and its overexpression rescues doxorubicin-induced cardiomyocyte atrophy and death. Contrary to primarily cytoplasmic localization, we found that Ythdf3 localizes in the cardiomyocyte nucleus. Furthermore, using co-immunoprecipitation coupled with LC-MS/MS, we show that Ythdf3 interacts with splicing proteins like DDX5 and HNRNPU and regulates alternative splicing in the heart. Mechanistically, we found that Ythdf3 regulates the splicing of CaMKII{delta}, and its knockdown leads to an increase in CaMKII{delta}A and CaMKII{delta}C isoforms while a decrease in CaMKII{delta}9 isoform. ConclusionCollectively, Ythdf3 knockdown in the adult heart leads to cardiac cachexia due to the alteration of the splicing program.

cell biology↗

bioRxiv2025

Macrophages of diabetes and tuberculosis co-morbid conditions show perturbed differentiation and metabolic signatures

Diabetes negatively impacts the host immune system, leading to allergies, recurring infections, and lameness. Its impact on the distribution and molecular phenotypes of the bone marrow-derived macrophages (BMDMs) needs attention and, more importantly, in co-morbid conditions like diabetes and tuberculosis (DM-TB). In this study, total bone marrow cells were harvested at 3 w.p.i., from the low aerosol dose (100-120 CFU) of Mycobacterium tuberculosis H37Rv infected control and NA-STZ induced diabetic C57BL/6 mice. The bone marrow cells were differentiated into BMDMs, and CD11c, CD11b and F4/80 marker expressions were monitored using flow cytometry. BMDMs stimulated with Mtb H37Rv lysate were subjected to global transcriptome profiling to identify the perturbed metabolic and molecular landscape. The BMDMs of the DM-TB comorbid mice showed low CD11c, CD11b and higher F4/80 expression. The macrophages of the DM group showed a skewed pro-inflammatory response, characterized by perturbed complement response, NF-kB pathway and fatty acid metabolism, whereas macrophages of the DM-TB group showed deregulated ceramide and amino acid metabolism. Macrophages from the DM and DM-TB group stimulated with Mtb H37Rv lysate, showed a deregulated transcript signature that impacts humoral response. The dampened immune response observed in the DM-TB group macrophages highlights the importance of identifying novel targets for developing tailored drugs for comorbid conditions.

immunology↗

bioRxiv2025

Diabetes mellitus and tuberculosis comorbidity induce unique microbial gut dysbiosis and associated metabolome.

BackgroundPatients with diabetes and tuberculosis (DMTB) often exhibit atypical disease progression and poor treatment outcomes. Alterations in gut microbiota may contribute to the impaired immune response in diabetes. Understanding the microbiota composition and associated metabolite profiles in DMTB could provide insights for developing targeted adjunctive therapies to improve patient prognosis. MethodNicotinamide (NA) and streptozotocin (STZ) induced diabetic C57BL/6 mice were aerosol infected with 100-120 CFU of Mycobacterium tuberculosis H37Rv. At different time points (2 days pre-infection and 3- and 8 weeks post-infection; wpi), faecal samples were collected for microbiome and metabolome profiling using 16S rRNA sequencing and gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), respectively. ResultsDiabetic mice showed gut dysbiosis, with 16 bacterial genera significantly altered between DMTB and TB groups at 3 wpi, which reduced to 11 at 8 wpi. Among the 93 identified faecal metabolites, 11 and 10 metabolites were significantly deregulated (log2FC>{+/-}1.0, p<0.05) in the DMTB group compared to TB group at 3 and 8 wpi, respectively. A negative correlation between faecal valeric acid levels in DMTB with the abundances of Coriobacteriaceae, Granulicatella, Veillonella, Achromobacter, Erysipelatrichaceae, Atopobiaceae, Citrobacter, and Lactiplantibacillus were observed. Stronger correlations between microbiota and metabolite were observed in DMTB comorbidity compared to TB. Additionally, correlation analysis also revealed a phylogenetic distance-dependent synchrony in amino acid metabolism across five clades, each containing three or more bacterial genera. ConclusionThis study reveals significant differences in the gut microbiome and metabolome between DMTB and TB groups. The identified microbes and metabolic products may serve as potential prebiotic or probiotic candidates for improving recovery in DMTB comorbid conditions.

microbiology↗

bioRxiv2025

Convergent evolution of a fungal effector enabling phagosome membrane penetration

The ability of pathogens to evade phagosomal killing is critical for their pathogenicity. Previously, we had identified the HscA effector protein in the clinically important fungal pathogen Aspergillus fumigatus, which redirects conidia-containing phagosomes from the degradative to the non-degradative pathway. Here, we discovered a pathogenic form of this surface protein, determined by a single tyrosine residue (Y) at position 596, which is lacking in most fungi analyzed, that have a leucine (L) instead. Y596 enables HscA to penetrate the phagosomal membrane. In line, the introduction of a single L- to-Y exchange in the orthologous Ssb protein of Saccharomyces cerevisiae enabled the protein to penetrate phagosomal membranes that was reduced by deletion of one of the two Y-encoding SSB genes in the pathogenic fungus Candida glabrata. These data suggest a convergent evolution of HscA/Ssb proteins among human-pathogenic fungi and that a single amino acid exchange determines a virulence factor. HighlightsO_LIHscA with Y596 penetrates host cell membranes and causes phagosomal damage C_LIO_LIMost fungi (96%) have a leucine at the respective position C_LIO_LIAn L-to-Y mutation in Ssb enables S. cerevisiae to damage phagosomes C_LIO_LIRecruitment of ESCRT complex to the phagosomal membrane requires human p11 protein C_LI

microbiology↗

bioRxiv2025

Distinct region-specific neutralization profiles of contemporary HIV-1 clade C against best-in-class broadly neutralizing antibodies

Broadly neutralizing antibodies (bnAb) have been clinically proven to be an excellent choice for HIV-1 prevention. However, the relative effectiveness of best-in-class bnAbs against regionally relevant circulating HIV-1 forms is not clear. In the present study, we compared the degree of neutralization sensitivity of contemporary HIV-1 Indian clade C with that of South African origin. Phylogenetic analysis revealed that these clade C viruses continue to evolve distinctly from one another. Env-pseudotyped viruses prepared using contemporary HIV-1 clade C env genes (N=115) obtained from nine geographically distinct sites in India (between 2020-2023) were found to be most sensitive to V3-directed bnAbs 10-1074 and BG18, and second generation CD4 binding site (CD4bs) directed bnAbs (VRC07, N6 and 1-18), however they were found to be significantly resistant to V1/V2 apex directed bnAbs. Moreover, we observed that the degree of sensitivity varied between contemporary Indian and South African clade C viruses. Differences in degree of neutralization susceptibility were associated with differences observed in key residues that form bnAb contact sites, gp120 loop lengths and the number of N-linked glycans in the V4 hypervariable region. Interestingly, the second generation CD4bs bnAbs (VRC07, N6, 1-18) showed neutralization of VRC01 and 3BNC117 resistant viruses but with 2-7-fold reduced potency compared to the VRC01 sensitive counterparts, likely due to the enrichment of resistance associated residues observed in loop D. Predictive analysis indicated that combination of BG18, N6 and PGDM1400 can provide over 95% neutralization coverage at 1g/mL of contemporary India clade C, an observation found to be distinct to that reported for the Africa clade C viruses. Taken together, we found distinct neutralization patterns and env signatures associated with resistance to key bnAbs. Our study highlights that towards achieving clinical effectiveness, both the complementarity of bnAb classes and the regionally relevant HIV forms need to be considered. Author summaryWhile the development of vaccines to prevent HIV infection remains a global priority, their potential effectiveness is limited by the extraordinarily diversified circulating forms of HIV-1. The prospect of best-in-class bnAbs as potential prevention option has been demonstrated in several studies including the Phase II Antibody Mediated Prevention (AMP) trial; however, to be broadly applicable, bnAbs will need to overcome the substantial variability of HIV env. The present study highlights that the contemporary HIV-1 clade C viruses are evolving to be less sensitive to the best-in-class bnAbs and HIV-1 clade C that predominates in India and South Africa vary in their degree of susceptibility to best-in-class clinically relevant bnAbs. This indicates differences in the antigenic properties between globally circulating HIV-1 clade C at a population level. Overall, the outcome of this study highlights the need for periodic assessment of sequence and neutralization profiles of the circulating regionally relevant HIV-1 forms towards prioritizing the bnAb combination suitable for effective intervention.

immunology↗