Biology subjects

Kumar, A.

Publications and source records attributed to Kumar, A..

At least 181 records · Page 10Linked to original sources

bioRxiv2025

Structural Validation of the Intermediate Leptomeningeal Layer in the Human Central Nervous System

Traditionally, the human central nervous system (CNS) is described as having three meningeal layers, from outer to inner: dura mater, arachnoid mater, and pia mater. The arachnoid and pia mater are called the leptomeninges, and the space between them is filled with cerebrospinal fluid (CSF). Using gross dissection, light microscopy, and ultrastructural analysis of fresh postmortem and cadaveric CNS specimens spanning fetal to adult ages (N=61), we demonstrate a fibrocellular intermediate leptomeningeal layer (ILL) from the cortex to the caudal end of the spinal cord. The ILL divides the subarachnoid space (SAS) into two distinct structural compartments, through which vessels and nerves pass. The ILL shows unique structural features, such as dips into the brains sulci and fissures, as a double-fold membrane that bears intra-layer trabeculae, carries vessels, and forms the perivascular sheath. Moreover, throughout the CNS, it appears to be a non-sieved barrier, characterized by the presence of tight and adherens junctions. ILL, predominantly in the spinal cord, contains macrophage-like cells, indicating its layer-specific immune properties. The ILL warrants recognition as a distinct human meningeal layer with potential barrier and immune functions. SignificanceO_ST_ABSAn Intermediate Leptomeningeal Layer encloses the Central Nervous System in HumansC_ST_ABSThe integrated analysis of our macroscopic, microscopic, and ultrastructural study provides robust support for an intermediate leptomeningeal layer (ILL) in the subarachnoid space (SAS) of the human central nervous system (CNS) along the entire neural axis. The ILL is a fibrocellular macroscopic structure, with restricted permeability, that divides the cerebrospinal fluid (CSF)-filled SAS into two distinct structural compartments. Uniquely, ILL revealed the presence of cells with macrophage-like properties, suggesting a possible role in immune surveillance. The ILL may redefine the established concept of protective coverings of CNS, CSF circulation dynamics, and the role of leptomeninges in health and disease, including drug delivery.

neuroscience↗

bioRxiv2025

A T-cell Intrinsic Role for APOL1 Risk Alleles in Allograft Rejection

African Americans have an increased risk of kidney disease due to exonic variants in Apolipoprotein-L1 (G1 and G2). These prevalent variants have also been linked with kidney rejection, but outside of association with African ancestry, underpinning causal mechanisms are unknown. We investigated T-cell function using transgenic mice with physiologic expression of wild type (G0-), G1-, or G2-APOL1. Mice with variant APOL1 showed greater CD8+T-cell activation with expansion of a central memory (TCM) subset. Stimulated G1-CD8+T-cells showed enhanced proliferation and cytokine production, which reversed with APOL1 inhibition. In MHC-mismatched cardiac transplants, G1-mice demonstrated greater CD8+T-cell infiltration and reduced survival. Bulk transcriptome of G1-CD8+T-cells, and single-cell transcriptome of graft infiltrating TCMs, showed enrichment of canonical T-cell receptor (TCR) pathways including Ca2+-signaling. G1-CD8+T-cells demonstrated baseline ER-Ca2+ depletion followed by sustained increases in cytosolic-Ca2+ upon TCR stimulation. G1-CD8+T-cells were more sensitive to Ca2+ chelation, or store-operated Ca2+ entry inhibition, and relatively resistant to calcineurin antagonism vs. G0-CD8+T-cells. Analogously, in a kidney transplant cohort, APOL1-variant recipients developed rejection when they had elevated peripheral TCMs before transplantation and despite significantly higher tacrolimus levels vs G0/G0-AAs with rejection. In summary, we unravel an excitatory T-cell intrinsic mechanism for APOL1 exonic variants, causally linking them with kidney rejection.

immunology↗

bioRxiv2025

Thermodynamics and unbinding kinetics of A22 at multiple actin binding sites revealed by enhanced sampling simulations

The cytoskeletal protein plays a major role in various cellular processes. Understanding the interactions of small molecules with cytoskeletal protein therefore might help in the development of therapeutics. Employing combined molecular docking, all-atom molecular dynamics (MD) and infrequent well-tempered metadynamics (iWT-MetaD) we studied bacterial inhibitor A22 and actin protein interaction. Five probable A22 binding sites (S1-S5) were observed in actin and unbiased MD simulations of 100 ns - 500 ns showed structural stability at these sites. Interaction analyses showed A22 to mostly forms transient interactions except at sites S4 and S5 where long-lives interactions were present. Enhanced sampling simulations quantitatively estimated ligand dissociation free energy ({Delta}G) [~] - 2 kcal/mol to - 3.5 kcal/mol for sites S1-S4 with residence times [~] {micro}s to ms. For site S5, we observed the highest binding affinity ({Delta}G [~] - 6.05 {+/-} 0.48 kcal/mol) and longest residence time [~] 0.75 sec. Analyses of the dissociation trajectories predict multiple dissociation pathways for A22 and found key gatekeeper residues at specific-sites facilitating ligand dissociation. Comparison of A22 with other known inhibitors suggest that A22 binds actin with relatively lower affinity, however, exhibits site-specific unbinding kinetics. Thus, our study provides a detailed mechanistic overview of A22-actin interaction, its various binding modes, and unbinding kinetics. It also shows the importance and usage of infrequent metadynamics in exploring rare events like ligand-protein interaction. Deeper insights gained from this study expands our understanding of cytoskeletal ligand dynamics. These knowledges will be paramount in designing drug targeting cytoskeletal protein actin. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/663866v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@dea318org.highwire.dtl.DTLVardef@a592a4org.highwire.dtl.DTLVardef@3e8df1org.highwire.dtl.DTLVardef@d8dddb_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biophysics↗

bioRxiv2025

Convolvulus pluricaulis mediates its pharmacological effects via sod1, rdl, glut1, GABA-B-R1 and CG6293 orthologs in Drosophila melanogaster

Convolvulus pluricaulis, commonly known as Shankhpushpi, has been extensively used in the management of disorders of the nervous system, including depression and anxiety. The plant extract has also been demonstrated to function as an antioxidant. However, the molecular effectors underlying the beneficial effects of Shankpushpi are yet to be identified. In this study, we have utilized the fruit fly Drosophila melanogaster to identify the metabolic and molecular targets that mediate the beneficial effects of dietary Shankpushpi intake. Metabolomic analysis revealed changes in Ascorbic acid, glucose, and Adenosine monophosphate in the head tissue of fruit flies that were fed Shankpushpi for 20 days. Subsequent gene expression analysis revealed significant changes in expression of glut 1 (Glucose transporter 1), CG6293 (Ascorbate transporter), rdl (Resistant to dieldrin), GABA-B-R1 (GABA-B receptor subtype 1) and sod 1 (Superoxide dismutase 1) in the head tissue of adult flies that were exposed to different doses of Shankpushpi. Consistent with the expression data, knockdown of sod 1, glut1, GABA-B-R1, and CG6293 but not sod 2 abolished the Shankpushpi-mediated resistance to paraquat-induced oxidative stress. To uncover downstream effectors that are responsible for the antidepressant and anxiolytic effects of Shankpushpi, we examined the effects of dietary intake of Shankpushpi in a stress-induced model of depression. Administration of Shankpushpi during development alleviated depression-like behavior in Drosophila. Wild-type adult flies that were fed Shankpushpi accumulated high levels of Ascorbate in the head tissue, and knockdown of Ascorbate transporter abolished the antidepressant activity of Shankpushpi in the stress-induced model. Thus, indicating that Shankpushpi-induced antidepressant effects are associated with increased Ascorbate transport. Taken together, our analysis using Drosophila melanogaster as a model has uncovered five conserved downstream effectors responsible for the antioxidant activity and one conserved effector responsible for the antidepressant activity of Convolvulus pluricaulis.

molecular biology↗

bioRxiv2025

Low and high frequency signatures of impaired consciousness in temporal lobe seizures

Impaired consciousness is a debilitating and unpredictable outcome of mesial temporal lobe seizures whose mechanisms to date remain unclear. Moreover, questions about the relationship between impaired consciousness and lateralization, hemispheric spread and electrophysiological characteristics of seizures are yet to be answered. To address these gaps, we conducted in-depth investigation of behavioral and intracranial EEG data from 186 mesial temporal lobe seizures of 51 patients with intractable mesial temporal lobe epilepsy. We found that bilateral mesial temporal spread of seizures is not a necessary condition for impaired consciousness, although seizures with bilateral mesial temporal involvement were significantly more likely to have impaired consciousness than unilateral seizures. Contrary to prior belief, we found no relationship between the onset side (left vs. right temporal lobe or language dominant vs. non-dominant lobe) of seizures and the probability of impaired consciousness. Lastly, we established that widespread increases in slow-wave activity (delta band) in extratemporal cortical areas, as well as increases in fast activity (beta band) in the temporal lobes were both robust markers of seizures with impaired consciousness and could predict ictal impairment with up to 86% accuracy. Our findings shed new light on networks that underlie impaired consciousness in temporal lobe epilepsy and may help guide deep brain stimulation of such systems (e.g. via thalamic nuclei) as a potential intervention to improve consciousness during seizures.

neuroscience↗

bioRxiv2025

Molecular characterization of unique multi-domain harbouring fungal rhodopsin for establishing their novel opto-synthetic biological usages

Organisms employ light as an external stimulus for regulating cellular functions. The light-sensitive photoreceptors detect light at varying wavelengths, activating signaling cascades and triggering a range of physiological responses. Rhodopsin is a transmembrane heptahelical protein that functions as an ion channel, or a pump, and sensory receptor, respectively. It consists of a light-sensing chromophore, a retinal that upon absorbing light, initiates a series of signaling pathways of sensory perception, growth and survival. Modular rhodopsin (Different from Rhodopsin-Cyclase Module) has been reported in lower eukaryotes, its identification, characterisation and functional significance in the Fungal Kingdom largely unknown. Here, we report the identification of novel modular rhodopsins in fungi, which highlights their potential usages towards the unexplored opto-biotechnological applications (e.g., biomanufacturing of terpenoids, cytoskeleton regulation, DNA metabolism, light-controlled acetyltransferase, etc.) simply by illumination. Furthermore, identification of novel modular rhodopsins augments the expansion of the new optogenetic tools for a wide range of relevant applications. The structural and homology analysis of these identified domains sheds light on their evolutionary lineage and relatedness with the well-characterised bacteriorhodopsin, sensory and channelrhodopsin. The interactome analysis effector domain coupled with the microbial rhodopsin (Rh) reveals RPEL-mediated gene expression and metabolite regulation, which further modulates the retinol synthesis pathway. The role of the fungal Rh-RPEL effector domain in modulating the terpenoid and sphingolipid metabolism in response to light was successfully elucidated via protein-protein interaction and Biosynthesis Gene Cluster (BGC) analysis. This highlights the potential of these novel opto-synthetic biological usages that can induce the light-dependent production of commercially relevant fungal bioactive(s).

bioinformatics↗

bioRxiv2025

Single-cell RNA sequencing identifies accumulation of Fcgr2b+ virtual memory like CD8 T cells with cytotoxic and inflammatory potential in aged mouse white adipose tissue

Aging and obesity are associated with pro-inflammatory changes in adipose tissue. Overlapping mechanisms, such as the infiltration of inflammatory macrophages and T cells into visceral adipose tissue, have been implicated in contributing inflammation. However, a comparative analysis from both states is needed to identify distinct regulatory targets. Here, we performed single-cell RNA sequencing of stromal vascular fractions (SVF) isolated from gonadal white adipose tissue (gWAT) of young mice fed either a normal or a high-fat diet, and aged mice fed a normal diet. Our analysis revealed that physiological aging, compared to high-fat diet induced obesity, was associated with accumulation of phenotypically distinct CD8 T cells resembling virtual memory (VM) CD8 T cells. These cells expressed high levels of Cd44, Sell, Il7r, Il2rb, lacked Itga4, and exhibited elevated Fcgr2b expression which was associated with pseudotime differentiation trajectories. Flow cytometry confirmed an age-associated increase in Fcgr2b+CD49d- VM like CD8 T cells in gWAT. Notably, these Fcgr2b-expressing cells exhibited a cytotoxic profile, and expressed granzyme M. Functional analysis using recombinant granzyme M revealed its potential in inducing inflammation in mouse fibroblasts and macrophages. Together, our study has identified Fcgr2b+CD49d- VM-like CD8 T cells in the adipose tissue of aged mice with regulatory, cytotoxic and inflammatory potential.

immunology↗

bioRxiv2025

REACTIVATION PROTECTS HUMAN MOTOR MEMORIES AGAINST INTERFERENCE FROM COMPETING INPUTS

Memories are often thought to become increasingly stable and resistant to change over time. The reconsolidation framework proposes that reactivating a stable memory transiently increases its susceptibility to modification by subsequent experience, but evidence for such reactivation-induced destabilization remains mixed. Here we tested whether reactivation alters the susceptibility of a visuomotor memory to interference from competing learning. Across a series of motor adaptation experiments, we briefly reactivated a previously acquired memory before introducing an opposing perturbation. Contrary to reconsolidation predictions, reactivation did not increase interference during later retrieval. Instead, reactivation reduced interference when the competing memory was not allowed to stabilize after acquisition. Our findings challenge the view that reactivation universally destabilizes motor memories and instead suggest that it shapes which competing motor memory is expressed at retrieval.

neuroscience↗

bioRxiv2025

CRISPR/Cas9 editing of the wheat iron sensor TaHRZ1 confirms its conserved role in iron homeostasis and allocation in grains

Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum (wheat) HRZ homoeologs belonging to TaHRZ1 and TaHRZ2, were identified by BLAST search using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain in all the TaHRZ1 homoeologs, coupled with GRF4-GIF1 chimeric protein, achieved 6.4-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved iron partitioning compared to the wild type. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.

plant biology↗

bioRxiv2025

Peroxisomal import is circadian in glia and regulates sleep and lipid metabolism

Peroxisomes are critical organelles that detoxify cellular waste while also catabolizing and anabolizing lipids. How peroxisomes coordinate protein import and support metabolic functions across complex tissues and timescales remains poorly understood in vivo. Using the Drosophila brain, we discover a striking enrichment of peroxisomes in the neuronal soma and the cortex glia that enwrap them. Unexpectedly, import of peroxisomal proteins into cortex glia, but not neurons, oscillated across time and peaked in the early morning. Rhythmic peroxisomal import in cortex glia autonomously required the circadian clock and Peroxin 5 (Pex5; peroxisomal biogenesis factor 5 homolog), with import persistently elevated in clock mutants. Notably, reducing Pex5 in cortex glia, but not neurons, caused hyperactivity and reduced total sleep. Moreover, brain lipid metabolism was dramatically altered upon Pex5 knockdown, with glia impacting sphingolipids and triacylglycerols, and neurons impacting phospholipids. The cell-type specificity of these Pex5 phenotypes highlights unique roles for peroxisomal import in both sleep and lipid metabolism in the brain.

neuroscience↗

bioRxiv2025

An iPSC-based iMSC Model for Osteogenesis Imperfecta: A Platform for Disease Modeling and Drug Screening

IntroductionOsteogenesis Imperfecta (OI) is a rare genetic disorder of connective tissue, primarily caused by mutations in the COL1A1 or COL1A2 genes. Research is hampered by the limitations of primary patient-derived cells, which are obtained through invasive methods and have finite proliferative capacity and high variability. While induced pluripotent stem cell (iPSC) models exist, direct differentiation to osteoblasts is often inefficient and may not fully replicate disease characteristics. Our study aimed to develop and characterize a robust iPSC-derived mesenchymal stem cell (iMSC) model of OI using a simple two-step differentiation protocol to serve as a platform for disease modeling and drug screening. MethodsBone marrow MSCs (BMMSCs) were isolated from two OI patients with heterozygous missense mutations, in COL1A1 (c.2299G >A) and COL1A2 (c.982G>A). The patient BMMSCs were reprogrammed into iPSCs using an integration-free Sendai virus. The resulting OI-iPSCs were characterized for pluripotency via immunofluorescence and RT-PCR, trilineage differentiation potential, and karyotype analysis. A two-step protocol was used to differentiate the OI-iPSCs into OI-iMSCs via mesodermal lineage. The OI-iMSCs were then characterized for morphology, immunophenotype by flow cytometry, and trilineage differentiation capacity. Functional osteogenic differentiation was assessed by Alizarin Red S staining and RT-PCR analysis of key osteogenic genes. Sanger sequencing confirmed the retention of patient-specific mutations in the OI-iMSCs. ResultsPatient-derived BMMSCs were successfully differentiated into adipocytes and chondrocytes but showed impaired osteogenic potential. The reprogramming process successfully generated stable OI-iPSC lines that expressed key pluripotency markers, demonstrated trilineage potential, and maintained a normal karyotype. Critically, while direct osteogenic differentiation of these iPSCs failed to recapitulate the primary cell phenotype, the two-step differentiation protocol successfully produced a homogeneous population of iMSCs. These OI-iMSCs displayed characteristics of MSC surface markers (CD73+, CD90+, CD105+) and recapitulated the disease phenotype seen in the original patient BMMSCs. Specifically, COL1A1-iMSCs exhibited cellular rolling and detachment, while COL1A2-iMSCs showed poor mineralization during osteogenic differentiation. Both OI-iMSC lines showed significantly decreased calcium deposition and downregulation of key osteogenic genes (RUNX2, ALP, COL1) compared to controls. ConclusionOur study successfully established an iMSC-based cellular model of OI that recapitulates patient-specific disease phenotypes, including impaired osteogenic differentiation. The two-stage differentiation of iPSCs to iMSCs proved more reliable than direct osteogenic differentiation for modeling the disease. iMSC model circumvents the limitations of primary cells by providing a scalable and homogeneous source of patient-specific cells. Our platform offers a valuable and robust tool for investigating OI pathophysiology and for high-throughput screening of potential therapeutic molecules, advancing efforts toward personalized therapies

genetics↗

bioRxiv2025

High-resolution spatial mapping of cell state and lineage dynamics in vivo with PEtracer

Charting the spatiotemporal dynamics of cell fate determination in development and disease is a long-standing objective in biology. Here we present the design, development, and extensive validation of PEtracer, a prime editing-based, evolving lineage tracing technology compatible with both single-cell sequencing and multimodal imaging methodologies to jointly profile cell state and lineage in dissociated cells or while preserving cellular context in tissues with high spatial resolution. Using PEtracer coupled with MERFISH spatial transcriptomic profiling in a syngeneic mouse model of tumor metastasis, we reconstruct the growth of individually-seeded tumors in vivo and uncover distinct modules of cell-intrinsic and cell-extrinsic factors that coordinate tumor growth. More generally, PEtracer enables systematic characterization of cell state and lineage relationships in intact tissues over biologically-relevant temporal and spatial scales.

molecular biology↗

bioRxiv2025

Chenodeoxycholic Acid Modulation via Bacteroides intestinalis AM1 underscores a Novel Approach in Acute Liver Failure

BackgroundAcute liver failure (ALF) is associated with rapid and progressive hepatocellular injury, and severe metabolic-microbial derangements. We investigated early metabolic markers of non-survival, and a potential microbial intervention using Bacteroides intestinalis-AM1, to improve outcomes in ALF. MethodPlasma metabolomics and meta-proteomics were performed in 40 ALF patients and 5 healthy controls (training cohort). A non-survival marker panel was identified and validated in 270 ALF patients (test cohort) using high resolution mass spectrometry and machine learning. It was functionally validated in acetaminophen-induced ALF mouse model. B. intestinalis-AM1 was used to study alteration of gut bacteria and amelioration of liver injury. ResultsALF non-survivors showed a distinct metabolomic signature with elevated primary bile acids {chenodeoxycholic acid (CDCA), cholic acid (CA)}, tryptophan, tyrosine, and enrichment of pathways linked to inflammation, cell death, and stress response (p<0.01, FDR<0.01, FC>1.5). Non-survivors had higher alpha/beta diversity (p<0.05) with increase in Proteobacteria, Firmicutes, Actinobacteria (p<0.05); functionally associated with energy, amino acid and xenobiotic metabolism (p<0.05). A gut microbiota derangement in converting primary to secondary bile acids was evident as CDCA and cytotoxic metabolites (4-(2-Amino phenyl)-2,4-dioxobutanoate, L-Tyrosine) were higher. Elevated CDCA (logFC>10) levels correlated with mortality in ALF patients as well as in mouse model. In the later, administration of B. intestinalis-AM1 bacteria, (10^9) reduced CDCA and CA levels by enhancing FXR, FGF15, SLC10A1 gene expression, attenuating inflammation (IL-1beta, TLR4-signalling), necroptosis, and modulating glutathione(oxidative-repair), tryptophan(inflammation), and histidine (tissue repair) metabolism. ConclusionHigh levels of chenodeoxycholic acid (CDCA) represent a poor prognostic indicator in ALF patients. B. intestinalis-AM1, a primary-to-secondary bile acid converter, effectively reduce CDCA levels, activated FXR, reduced inflammation and protected hepatocytes, highlighting its therapeutic potential in ALF.

microbiology↗

bioRxiv2025

Engineered Fluorescent Carbon Dots for Selective Cellular Bioeffects: A Comparative Study of Cancer and epithelial Cells

Cancer remains one of the most critical global health challenges, with early detection being essential for effective treatment and improved survival rates. However, conventional diagnostic tools often fail to detect cancer at early stages due to limitations such as low sensitivity, high cost, and dependence on large tumour presence. Commercial dyes used for imaging are frequently hindered by poor water solubility, toxicity, instability, and high expense. In recent years, CDs have emerged as promising fluorescent probes due to their nanoscale size, tenable surface properties, strong fluorescence, and excellent biocompatibility. make them applicable to various biological applications such as bioimaging, drug delivery, and tissue engineering. In this study, CDs were synthesized using citric acid and ascorbic acid as carbon sources via a reflux method at 130 {degrees}C for 12 hours in a water-ethanol medium. The resulting CDs exhibited high water solubility, strong photostability, and low toxicity. Notably, they effectively distinguished cancerous cells from normal cells. And showing higher uptake in cancer cells due to increased membrane permeability and metabolic activity. Higher uptake means more accumulation within cells that leads to an increment in fluorescence intensity, based on the fluorescent intensity distinguishing the cancer cells and normal cells. These findings highlight the potential of CDs as cost-effective, biocompatible imaging agents for cancer diagnosis and cellular studies.

bioengineering↗

bioRxiv2025

Deep-Learning on condition specific expression profiles reveal critical cytosines in gene regulation

Compared to other nucleotides, the cytosines stand as the most expressive one for gene regulation in plants due to its status as methylation-based epigenetic switch. Methylation of some of these cytosines may have higher impact on downstream genes, making them critical ones. To this date not much has been done to decipher the criticality of such cytosines. This is first such pioneering study in decoding the critical Cs where a large volume of bisulfite and RNA-seq data, including 232 WGBS and 260 corresponding RNA-seq datasets from A. thaliana and rice, have been utilized. Using a deep learning system, strong relationship was established between methylation states of cytosines in contextual manner with respect to the downstream genes expression levels. Using the same system, all the 2kb upstream regions for Arabidopsis and rice genes have been annotated for critical cytosines. Experimental validation demonstrated specific methylation changes at critical cytosines under heat stress, affecting gene expression. The universal method developed may be applied to annotate other plant genomes for critical cytosine identification. GC% similarity, rather than homology, explains gene regulatory behavior under methylation control. The resource, CritiCal-C portal, is publicly accessible, paving the way for revolutionary strategies in gene regulation and expression with minimal interventions.

bioinformatics↗

bioRxiv2025

Structure of the energy converting methyltransferase (Mtr) of Methanosarcina mazei in complex with an oxygen-stress responsive small protein

Methanogenic archaea contribute 1-2 Gt of methane annually, impacting both global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr complex), which catalyzes the methyl transfer between two methanogen specific cofactors. This exergonic methyl transfer step is coupled with a vectorial sodium ion transport from the cytoplasm to the cell exterior and is the only energy conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 [A] single-particle cryo-EM structure of the full Mtr complex from Methanosarcina mazei. Our structural model encompasses the entire complex, reveals the arrangement of archaeal phospholipids, the architecture of the sodium ion binding site, and the structure and interactions of all catalytic subunits. Most strikingly, we discover and characterize MtrI, a previously unannotated small open reading frame (small ORF), encoded protein (<100 aa) conserved across the order of Methanosarcinales. MtrI binds to the cytoplasmic domain of MtrA in response to oxygen exposure, suggesting a role in oxygen stress response and protection. By binding on top of the sodium channel and anchoring to the cobamide cofactor in MtrAs cytoplasmic domain, MtrI might prevent sodium leakage and inhibit MtrA-CoM turnover. These findings offer new insights into methanogen energy conservation and uncover a potential adaptive response to oxygen exposure, expanding our understanding of methanogen survival strategies under oxidative stress.

biochemistry↗

bioRxiv2025

Episymbiotic Saccharibacteria suppresses epithelial immunoactivation through Type IV pili and TLR2 dependent endocytosis

Saccharibacteria are episymbionts that require host-bacteria to grow. They are positively associated with inflammatory diseases within the human microbiome, yet their mechanisms for interacting with the human host and contributing to diseases remain unknown. This study investigated interactions between a Saccharibacterium (Nanosynbacter lyticus), its host-bacteria (Schaalia odontolytica), and oral epithelial cells. The host-bacteria induced proinflammatory cytokines in epithelial cells, while Saccharibacteria were immune silent. Remarkably, Saccharibacteria dampened cytokine responses to host-bacteria during coinfection. This effect was driven by Saccharibacteria-induced clustering of TLR2 receptors, a process likely facilitated by type IV, ultimately leading to reduced TLR2-mediated cytokine signalling. High resolution imaging showed that Saccharibacteria were endocytosed by oral epithelial cells, and colocalized with endosome markers, eventually trafficking to lysosomes. Moreover, a subset of the Saccharibacteria survive endocytosis long-term, and retains their capability to reinfect host-bacteria, highlighting a mechanism for persistence in the oral microbiome and a vital role in mammalian immune system modulation.

immunology↗

bioRxiv2025

Functionally significant, novel variants of BMP4 are associated with isolated congenital heart disease

AbstractBone morphogenetic proteins (BMPs) are multipotent cytokines of TGF{beta} super family, involved in wide range of biological processes including embryonic development, tissue differentiation, cell proliferation, migration and organogenesis. BMP signaling also plays a pivotal role during different phases of cardiogenesis. Although genetic variations in several components of BMP signaling have been linked to congenital heart disease (CHD), many of these findings lack thorough functional validation. To assess the role of BMPs in CHD, Sanger sequencing of BMP4 gene was conducted in 285 CHD cases along with 400 healthy controls. Four missense novel pathogenic variants were detected in four unrelated CHD probands with heterogeneous phenotypes. All the four variants (p.R113G, p.E151V, p.T197I and p.R226W) were located in BMP4 pro-peptide domain. Western blotting analysis revealed a significant increase in the phosphorylation of SMAD1/5 caused by all the four variants. Further, all the four variants enhance the transactivation of BMP-responsive promoters, Id1-luc and Id3-luc in luciferase reporter assay. Moreover, qRT-PCR analysis validating the enhanced endogenous expression of downstream targets namely Smad1, Smad5, Id1, Id3, and Irx4 which further confirm the augmented activity due to all the four variants. Besides, our computational modeling of RNA structures and its features, modifications in secondary and tertiary structures and various physiochemical properties are speculated to enhanced the binding of BMP4 muteins with its respective partner and thereby boosting the SMAD-dependent BMP signaling. Altogether, both in vitro and in silico observations unveiling the gain-of-function activity of mutants which potentially perturbing the normal BMP signaling/ dynamics, consequently inducing CHD.

genetics↗