Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

Cryo-EM structures of Mycobacterium tuberculosis pyruvate carboxylase reveal allosteric activation and domain dynamics during catalysis

Pyruvate carboxylase (PC) is a biotin-dependent enzyme essential for anaplerotic flux into the tricarboxylic acid cycle and gluconeogenesis. Despite longstanding biochemical knowledge, the structural mechanisms governing its allosteric regulation and domain coordination remain poorly defined. Here, we present the cryo-EM structures of Mycobacterium tuberculosis PC (MtPC) captured in both apo and substrate-bound forms. In the absence of acetyl-CoA, MtPC forms a flexible and unstable tetramer that readily dissociates into subcomplexes. Upon binding of acetyl-CoA and the substrates, the enzyme transitions into a compact, ordered assembly, with acetyl-CoA acting as a molecular clamp that stabilizes inter-subunit interactions. Remarkably, we captured the mobile biotinylated domain simultaneously engaging each catalytic center within monomers, providing direct structural evidence for the two steps of the catalytic cycle. 3D variability analysis further revealed how this mobile domain translocates between the active sites during catalysis. Together, these findings demonstrate how acetyl-CoA stabilizes MtPC by reinforcing inter-subunit contacts and elucidate the conformational dynamics underlying domain coordination during catalysis. These findings provide new structural insights into the allosteric regulation of this essential metabolic enzyme.

biophysics↗

bioRxiv2025

AI-assisted Image-Based Phenotyping Reveals Genetic Architecture of Pod Traits in Mungbean (Vigna radiata L.)

Mungbean (Vigna radiata (L.) R. Wilczek) is a vital source of digestible proteins and is well-suited for the plant-based protein industry. In this study, we analyzed pod morphological traits in the Iowa Mungbean Diversity (IMD) panel with 372 genotypes (2022-23) with AI-assisted image phenotyping using 2,418 pod images. Pod morphological traits were extracted using deep learning image analysis, achieving excellent agreement with manual measurements (r>0.96 for pod length and seed per pod). Four complementary GWAS models identified 45 significant SNPs associated with pod curvature, length, width, and seed per pod traits. Notably, a significant SNP (5_35265704) on chromosome 1 was linked to pod dimensional traits, length, width, and curvature. A candidate gene, Vradi01g00001116, was located within the linkage disequilibrium (LD) region of this SNP, is part of the GH3 gene family, and has an Arabidopsis ortholog (AT4G27260) known for influencing organ elongation, pod, and seed development. Another SNP, 5_210437 on chromosome 2, has been found to be significantly associated with both pod length and seed per pod. A candidate gene, Vradi02g00003971, located in the LD region of this SNP, belongs to the potassium transporter family and shares homology with the HAK5 gene family (AT4G13420) in Arabidopsis, which influences pod and seed growth. Image-based measurements achieved genomic prediction accuracies ranging from 0.61 to 0.85 across various traits, exhibiting an improvement of 12-22% over manual methods. These results demonstrate the potential of AI-assisted phenomics integrated with genomic tools to accelerate selection for improved pod architecture in mungbean breeding programs across the Midwestern United States and globally.

genomics↗

bioRxiv2025

Programmable DNA-Silk fibroin hydrogel scaffold with temporal mechanical reinforcement for mesenchymal stem cells differentiation and accelerated wound healing in a full-thickness skin defect mice model

DNA-based hydrogels have attracted significant attention in biomedical applications due to their programmability, biocompatibility, and tunable functionalities. However, their clinical translation is hindered by high synthesis costs, limited mechanical strength, poor stability, and complex synthesis protocols. In this work, we present a novel DNA-silk fibroin hybrid hydrogel that overcomes these limitations by combining the unique biofunctionality of DNA with the superior mechanical and chemical properties of silk fibroin. Salmon-derived DNA, an abundant and low-cost source, was incorporated with silk fibroin to form hybrid networks via a simple, scalable process. Silk fibroin served as a robust scaffold, enhancing mechanical strength, controlling degradability, and improving structural stability, while the entangled DNA-silk network reduced rapid DNA degradation. The resulting hybrid hydrogel demonstrated temporal mechanical reinforcement and prolonged stability, with in vitro studies demonstrating that the developed hybrid hydrogel enhanced the chondrogenic and osteogenic differentiation of Infrapatellar Fat Pad Mesenchymal Stem Cells (IFP-MSCs). Additionally, hemostatic and in vivo studies demonstrated the potential of DNA-silk fibroin hydrogel as a promising biomaterial for rapid hemostasis and accelerated wound healing. Overall, the developed DNA-silk fibroin hybrid hydrogel offers strong potential for mesenchymal tissue engineering, hemostatic adjuvant and wound healing.

bioengineering↗

bioRxiv2025

VadK, a Non-Canonical Kinase that Regulates the Methylcitrate Cycle and is Essential for Mycobacterium tuberculosis Virulence

The evolution of new enzymatic functions is constrained and guided by the architecture of an organisms metabolic and regulatory networks as well as by environmental constraints. Here, we identify a previously uncharacterized kinase that has evolved from pyruvate phosphate dikinase (PPDK). Through biochemical and systems-level analyses, we show that this enzyme, encoded by Rv1127c in Mycobacterium tuberculosis (Mtb), has diverged from its ancestral role in central carbon metabolism to function as a histidine kinase in pathogenic mycobacteria and related species. We designate this enzyme Virulence Associated DiKinase (VadK), reflecting its ability to autophosphorylate and role in coordinating metabolism and virulence. VadK is essential for the utilization of exogenous carbon sources critical for survival within the host and is required for Mtb pathogenicity in murine models of tuberculosis. Furthermore, VadK interacts with key enzymes of the methylcitrate cycle, and 13C-metabolic flux analysis indicates that it fine-tunes flux through this pathway, with elevated flux proving growth limiting. Together, these findings identify VadK as a previously unrecognized regulatory kinase that integrates metabolic control with virulence in Mtb, revealing a new facet of metabolic regulation in bacterial pathogenesis and a potential target for therapeutic intervention.

microbiology↗

bioRxiv2025

Ventral tegmental area dopamine controls timing variability

Precision defines successful behavior, yet the brain mechanisms promoting precision are unclear. Here, we dissect dopaminergic circuits controlling precision along a single behavioral dimension - the timing of action. We use an interval timing paradigm that requires participants to indicate their estimate of an interval of several seconds with a motor response. We find that humans with Parkinsons disease (PD) had increased timing variability that predicted PD-related cognitive deficits and executive dysfunction. Surprisingly, lesioning ventral tegmental area (VTA) dopamine neurons increased temporal variability. Further, GCaMP6s fiber photometry demonstrated that VTA dopamine neuron activity is strongly modulated at the start of temporal intervals, and that this trial start-related activity predicted temporal variability. Finally, we found that stimulation of VTA dopamine neurons improved timing by decreasing temporal variability in both intact and dopamine-depleted animals. Our data establish a model of cognitive symptoms of human PD and provide insight into the neuronal control of temporal variability, which impacts a wide range of executive functions.

neuroscience↗

bioRxiv2025

Intrinsically Disordered Protein Coating for Oral Delivery of Peptide Drugs

AbstractAdvancing oral delivery of peptide therapeutics requires innovative materials that overcome gastrointestinal barriers. We introduce the first engineered synthetic intrinsically disordered protein (SynIDP) that self-assembles into an enteric coating, encapsulating peptide drugs to enhance gastric acid resistance and intestinal targeting. This SynIDP recapitulates the molecular design principles and phase transitions of native IDPs to exhibit temperature-controlled condensation and pH-controlled solidification--both transitions being reversible and precisely tuned to intestinal cues. Through detailed analysis of the kinetics of the liquid-to-solid phase transition, we achieve control over the nano-to-microscale morphology of the protein coating, optimizing drug encapsulation and protection. The coating protects peptide-based weight loss drugs for over 60 minutes in simulated gastric conditions, then dissolves to release the active compound. Oral delivery to obese mice results in more consistent weight loss compared to the unencapsulated drug. This modular protein-based coating is a promising platform technology for enhancing oral peptide drug delivery and improving patient compliance.

bioengineering↗

bioRxiv2025

Caffeine ingestion restores morning neuromuscular performance to evening levels in healthy females: A randomised crossover study

PurposeTo investigate whether caffeine ingestion offsets the circadian-related decline in morning neuromuscular performance in females. MethodsThirteen healthy females completed three experimental trials: i) evening placebo (PMPLAC), ii) morning placebo (AMPLAC), and iii) morning caffeine ingestion (6 mg{middle dot}kg-1; AMCAFF). A within-subject, repeated-measures, crossover, single-blind placebo design was used, with all subjects serving as their own controls. Each trial consisted of a maximal voluntary isometric contraction (MVIC) of the knee extensors, followed by intermittent isometric contractions (6 s contraction, 4 s rest) until task failure, and a post-fatigue MVIC. Time to exhaustion, torque and peak force, surface electromyography (RMS amplitude, MDF frequency), rate of perceived exertion, and tympanic temperature were all recorded across conditions. ResultsDuring PMPLAC, peak force and time to exhaustion were significantly greater (p < 0.05) than for AMPLAC. AMCAFF increased (p < 0.01) peak force in both pre-(+33 %) and post-(+45 %) fatigue MVIC trials compared to PMPLAC. Similarly, RMS was increased (p < 0.01) during both AMCAFF and PMPLAC compared to AMPLAC, whereas MDF did not change (p > 0.05) across trials. AMCAFF also improved (p < 0.01) time to exhaustion by 43 % compared to AMPLAC, and significantly reduced (p < 0.01) rate of perceived exertion, but without any change (p > 0.05) in temperature. ConclusionsMorning ingestion of 6 mg{middle dot}kg-1 of caffeine effectively reverses diurnal reductions in neuromuscular performance of females by restoring peak force and time to exhaustion to evening levels. No change in MDF, but an increased RMS suggests a central rather than peripheral mechanism for caffeines ergogenic effects.

physiology↗

bioRxiv2025

SIRT2 attenuates stress-induced skeletal muscle atrophy by inhibiting glucocorticoid receptor signaling

Skeletal muscle atrophy occurs in several diseases and is associated with chronic stress. Studies indicate that glucocorticoid receptor signalling is the major signalling pathway that mediates stress-induced muscle degeneration. Although the glucocorticoid signalling pathway is relatively well characterized, there is a need to identify modulators of this pathway that may be useful for drug targeting to ameliorate muscle atrophy. SIRT2 is a mammalian Sirtuin isoform known to mediate the longevity benefits of calorie restriction and exercise. Currently, the role of SIRT2 in regulating stress-induced skeletal muscle atrophy is unclear. Our study found that SIRT2 is a critical regulator of muscle homeostasis and is required to protect against stress-induced muscle atrophy. Interestingly, SIRT2 levels are reduced during glucocorticoid-induced muscle atrophy in mice. SIRT2 depletion exacerbates glucocorticoid-induced reduction in myotube diameter and atrophy gene expression. In contrast, SIRT2 overexpression ameliorates myotube atrophy in primary myotubes. Our findings indicate that SIRT2 knockout mice are susceptible to glucocorticoid-induced muscle atrophy, while muscle-specific SIRT2-transgenic mice exhibit improved muscle function and are protected from glucocorticoid-induced atrophy. Mechanistically, SIRT2 binds to the glucocorticoid receptor to negatively regulate its activity, possibly via deacetylation of critical residues in its DNA-binding domain. Our findings suggest that SIRT2 activation may protect against glucocorticoid-induced skeletal muscle atrophy and serve as a potential therapeutic target for treating muscle atrophy.

pathology↗

bioRxiv2025

Germline-targeting HIV immunogen induces cross-neutralizing antibodies in outbred macaques

Germline-targeting-(GT) is a promising strategy to activate rare broadly neutralizing antibody (bnAb)-producing B cells against HIV, but induction of such responses in outbred animals has not been achieved. Using antibody-guided structure-based design, we engineered a germline-targeting trimer immunogen Q23-APEX-GT2 that primes diverse V2-apex bnAb precursors. Q23-APEX-GT2 efficiently activated V2-apex-specific B cells in humanized knock-in mice and consistently elicited immunofocused antibody responses in rhesus macaques, priming multiple long CDRH3-loop bnAb-B cell lineages. Monoclonal antibodies from immunized macaques exhibited broad heterologous HIV trimer binding and cross-neutralization. Atomic-level structural studies confirmed precise epitope targeting and revealed CDRH3-paratope configurations that mirrored those of human V2-apex bnAbs. This study provides proof-of-principle for successful priming and maturation of authentic V2-apex bnAb precursors in outbred macaques, underscoring the potential of V2-apex-targeted vaccines. HIGHLIGHTSO_LIEngineered Q23-APEX-GT2 trimer to stimulate diverse V2-apex bnAb B cell precursors C_LIO_LIQ23-APEX-GT2 primed rare V2-apex bnAb B cells in mice and outbred rhesus macaques C_LIO_LIQ23-APEX-GT2 elicited immunofocused antibody responses and diverse V2-apex B cell lineages with desirable long-CDRH3 paratope properties C_LIO_LIQ23-APEX-GT2 alone induced V2-apex antibodies with broad HIV trimer binding and modest neutralization breadth C_LIO_LIStructural analysis confirmed bnAb site targeting, mirroring human and rhesus V2-apex bnAbs C_LI

immunology↗

bioRxiv2025

REST elevation-dependent chromatin remodeling and alternative Grk6 transcript synthesis hyperactivates Cxcr4-Sdf1 signaling in cerebellar granule cell progenitors

RE1 Silencing Transcription Factor (REST) is a repressor of transcriptional initiation of genes involved in neurogenesis. Here, we show that conditional REST elevation in cerebellar granule cell progenitors (CGNPs) of RESTTG mice perturbed foliation, increased cell migration, and sustained C-X-C motif receptor 4 (Cxcr4) signaling, a pathway key to postnatal CGNP migration. Mechanistic studies uncovered a novel role for REST in controlling transcript diversity and exon skipping in CGNPs. Alternative transcript expression was detected in known Cxcr4 signaling regulator, G-protein-coupled receptor kinase-6 (Grk6). Further analysis of Grk6s isoform expression revealed an upregulation of a transcript lacking exon 10a (Grk6-207) in RESTTG CGNPs. Grk6-207 expression in wildtype CGNPs hyperactivated Cxcr4 signaling and increased chemotaxis. Structural modeling of Grk6-207 predicted changes in active site conformation and interactions with Cxcr4 and {beta}-Arrestin-1, supporting impairment of Cxcr4 signaling desensitization. Interestingly, REST elevation promoted increased chromatin accessibility at the exon10a-10b junction and exon 10a exclusion. Integrated multiomic analyses identified the enhancer of zeste (Ezh2) as a potential mediator of alternative transcript generation which demonstrated increased occupancy at the exon10a-10b locus in RESTTG CGNPs. Pharmacological inhibition of Ezh2 downregulated Grk6-207, confirming a role for Ezh2 in Grk6 exon10a exclusion and the increased migration in RESTTG CGNPs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/682654v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@166db32org.highwire.dtl.DTLVardef@1c0c5e0org.highwire.dtl.DTLVardef@ff2d6borg.highwire.dtl.DTLVardef@fed170_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Title - ATG7 vs. ATG5: Distinct Autophagy Pathways Shaping TGF-β Signaling and Endothelial Function

Autophagy and transforming growth factor-beta (TGF-{beta}) signalling are critical cellular processes that maintain homeostasis and regulate various physiological functions, including endothelial cell function. This study explores the distinct roles of two essential autophagy regulators, autophagy-related gene 5 (ATG5) and ATG7, in modulating TGF-{beta} signalling and endothelial function. To this end, ATG5 and ATG7 were selectively silenced in endothelial cells, with knockdown efficiency confirmed via RT-qPCR and Western blot analyses. Gene expression profiling revealed differential regulation of TGF-{beta} signalling components following ATG5 or ATG7 silencing. Functional assays demonstrated that ATG5 knockdown enhanced endothelial cell proliferation and migration, whereas ATG7 silencing produced distinct, less pronounced effects. Furthermore, downstream effectors of the TGF-{beta} pathway exhibited gene-specific modulation, underscoring divergent roles of ATG5 and ATG7 in this signalling cascade. Collectively, these findings highlight the non-redundant functions of ATG5 and ATG7 in coordinating TGF-{beta} signalling pathways, offering new insights into their contribution to endothelial physiology and potential as therapeutic targets in vascular pathologies.

cell biology↗

bioRxiv2025

Accelerated osteocytic citrate production in chronic kidney disease is associated with protection of the kidney

Patients with chronic kidney disease (CKD) face elevated fracture incidences, but mechanisms underlying CKD-related bone loss remain unclear. Using the adenine-induced chronic kidney injury (AdKI) murine model, we identified that AdKI induces dysregulated glucose metabolism in bones and kidneys via in vivo and ex vivo metabolic tracing. Ex vivo 13C-metabolic tracing of osteocyte-enriched femora revealed accelerated citrate production from [1,2-13C]-glucose and [U-13C]-glutamine in AdKI mice. These metabolic changes were observed together with increased circulating citrate and Slc13a5 overexpression in bones from AdKI mice. Thus, to explore the role of citrate in AdKI, we utilized mice harboring a loss of function mutation in the citrate importer SLC13A5 (Slc13a5R337*/R337*). Mutant mice displayed elevated osteocytic citrate production, and elevated circulating citrate, without significantly worsened AdKI-related bone loss. Coincident with this, Slc13a5R337*/R337* mutant mice were significantly protected from loss of kidney function with attenuated AdKI-induced nephrolithiasis. We also confirmed that Slc13a5 is highly expressed in cortical bone compared to the kidney, suggesting the effect of the mutation is mediated by SLC13A5s function outside the kidney. Altogether, this study finds that accelerated osteocytic citrate production in CKD is associated with protection of kidney function, and modulation of citrate handling may be a site for therapeutic intervention in CKD.

physiology↗

bioRxiv2025

Genome-wide screening reveals producer-cell modifications that improve virus-like particle production and delivery potency

Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of gene editing agents. While extensive particle engineering has yielded efficient eVLPs, it remains underexplored whether engineering the cells used to produce eVLPs could further improve eVLP properties. We developed a genome-wide screening approach to systematically investigate how genetic perturbations in producer cells influence eVLP production. This approach generates eVLPs loaded with guide RNAs that identify the genetic perturbation in the cell that produced a particular particle; the abundance of each guide RNA in eVLPs therefore reflects how the corresponding genetic perturbation influences eVLP production or cargo loading. We applied this approach to identify several genes that regulate eVLP cargo expression and loading into particles during the production process. Leveraging these insights, we engineered producer cells that support increased eVLP cargo packaging and a 2- to 9-fold increase in eVLP delivery potency across several cargo, particle, and target-cell types in cultured cells and in mice. Our findings suggest the potential of producer-cell engineering as a useful strategy for improving the utility of eVLPs and related delivery methods.

synthetic biology↗

bioRxiv2025

CellMAPS: A no-code model-based customisable multiplex image analysis workflow

Although multiplex imaging allows simultaneous mapping of complex tissue architectures and cellular phenotypes, the dearth of user-friendly robust image analysis workflows remains a significant limitation to its widespread use, restricting multiplex imaging to laboratories with image analysis expertise. Here, we introduce a no-code model-based customisable suite, CellMAPS, which allows both image processing and spatial analyses by non-specialists. We also developed new tools for tissue de-arraying, image normalisation and cell segmentation to increase usability and reduce analysis subjectivity. We have also introduce new spatial analysis tools for the partition of tissue architectures and cellular microenvironments. We demonstrate the capabilities of CellMAPS in various diseases captured using different multiplex imaging platforms. Overall, CellMAPS provides a platform for inexperienced laboratories to implement multiplex imaging and complex spatial analyses, which ultimately should lead to the broader adoption of these technologies in the biomedical field.

bioinformatics↗

bioRxiv2025

Spatial genomics of the cardiac sarcomere

Structure-function mapping of proteins has improved our understanding of disease mechanisms and protein domains while human population genetics has provided a window into variational tolerance that can be modeled at a structural level. Here, to reveal novel insight into the cardiac sarcomere, the motor unit of the heart, we develop a novel analytical framework to integrate data from over 17,000 patients with hypertrophic cardiomyopathy (HCM) and combine it with two population-scale genomic databases that incorporate data from more than 800,000 individuals. We integrate in silico genetic predictions of gene variant pathogenicity with 3 dimensional integrative spatial scanning across multiple structural models of cardiac motor proteins. Results reveal both recognized and novel regions of structural variant intolerance across the critical genes of the cardiac sarcomere including novel insights into protein function. We discuss the structural relevance of variant enrichment in the context of sarcomere organization and destabilization of sequestered myosin leading to hypercontractility seen in HCM, by incorporating the recently defined high-resolution structure of human cardiac myosin filament. We extend and validate these findings through pathogenic variant class enrichment and reveal novel associations with the earlier onset of disease in a large clinical cohort. In summary, our study provides a multi-dimensional framework for integrating structural, genomic, and modeling data to reveal novel insight into the cardiac sarcomere.

genomics↗

bioRxiv2025

Consistent Induction of Broadly Neutralizing HIV Antibodies by a Novel Two-Step Mechanism Informs Immunogen Design

A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a novel two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for Envs with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

immunology↗

bioRxiv2025

Photoacoustic imaging assessment of acute kidney injury associated with experimental necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is a devastating gastrointestinal condition increasingly recognized to cause systemic complications, including acute kidney injury (AKI). However, the underlying mechanisms and variations in renal impairment remain largely unknown, and there is a lack of non-invasive imaging methods for detecting early kidney injury. MethodsWe evaluated kidney injury through a multimodal approach, using a neonatal rat model of experimental NEC. Plasma and renal tissue were analyzed for pro-inflammatory cytokines (TNF-, IL-6, IL-1{beta}) by ELISA. Histological analysis and immunofluorescence staining were used to identify renal abnormalities and expressions of KIM-1 and NGAL. Photoacoustic imaging (PAI) was used to assess renal oxygenation and total hemoglobin as functional imaging biomarkers of kidney injury. ResultsNEC pups showed significant elevations of systemic and renal cytokines, as well as increased expression of KIM-1, NGAL and CD31 in proximal tubules of the kidney. Histological examination further confirms renal injury in the NEC cohort. PAI demonstrated reduction in renal oxygen saturation, offering non-invasive assessment of physiological kidney damage. ConclusionThis study characterizes AKI associated with NEC as an inflammatory and hypoxic response in the neonatal rat NEC model and introduces PAI as a promising imaging modality for non-invasive evaluation of renal injury in NEC.

bioengineering↗

bioRxiv2025

CytokineFindeR: an R-package for benchmarking methods and databases for identifying cytokines

Cytokines play a central role in disease but are hard to study due to their short half-life and low abundance. Computational approaches have utilized gene-expression for pinpointing cytokine drivers of diseases. We benchmark various cytokine identification methods and found little congruency between gene sets for the same ligand, and variable performance in identifying cytokines based on curated ligand-receptor interactions. CytoSig, a model-based approach, was generally better at identifying the correct disease cytokine but failed for certain cytokines such as IL-13. We developed CytokinefindeR, an R package that enables comparative analysis of cytokine detection across 10 databases and four computational approaches.

bioinformatics↗