Biology subjects

Bharadwaj, A.

Publications and source records attributed to Bharadwaj, A..

17 recordsLinked to original sources

bioRxiv2026

Joining Two Perspectives of the Proteome with the Trypsin+HTA Protease Workflow

Proteolysis can be completed in a single-step, rapid (5-30 minutes) reaction using hyperthermoacidic archaeal (HTA) proteases. Protocol simplicity and stability of HTA proteases in ambient storage conditions removes many logistical barriers to widespread deployment of proteomic analysis. HTA protease cleavage selectivity is complicated but quantitatively reproducible and generates peptides that negligibly overlap with trypsin. Tryptic and HTA protease peptides emphasize different proteins and provide complementary coverage of amino acid sequence and post-translational modification (PTM) sites. Digestion of a sample's separate aliquot with HTA protease is a minor addition to trypsin-based workflows, but LC-MS analysis of both sets of peptides is not practical for large cohorts. This manuscript evaluates the identifications and reproducibility of LC-MS analysis of tryptic and HTA protease peptide mixtures. Adding HTA protease peptides to 20% increased total protein identifications by 24% on average in neat plasma and identified 216 additional non-artifact modifications compared to conventional trypsin-only bottom-up proteomic analysis. This approach is reproducible and does not increase sample preparation or analysis time. Peptides from each protease present independent opportunities to quantify overlapping proteins and a quantification model was developed in which the protease with the most complete data is selected for each protein while the second protease is used to infer missing values. This cross-protease inference is more accurate than the alternative minimum, mean, or nearest neighbor imputations. Finally, biomarkers discovered through HTA protease peptides are amenable to rapid HTA protease only proteomic workflows thus streamlining translation from discovery to fieldable applications.

biochemistry↗

bioRxiv2026

Colonial legacies in eponymous species names: a global network perspective

Eponymous etymologies (i.e., organisms named after people) are prevalent in global taxonomy worldwide, across scientific and common names. The persistent usage of eponyms has been intensely debated in recent times, across various sociopolitical, applied and philosophical perspectives. However, a rigorous statistical analysis of eponyms and their enduring colonial legacies remains hitherto unexplored. Here, we use a global and spatially-explicit network analysis to examine 2416 contemporary bird eponyms (21% of all bird species on earth) and their relationships with historical European colonialism. Additionally, we develop and utilise a Colonial Exposure Index (CEI) that summarizes the geopolitical influence of various colonial empires on modern-day countries. Our results show that an overwhelming majority of bird eponyms worldwide are named after people belonging to countries where the bird species does not occur. A majority of these eponyms honor scientists, aristocrats and army officers from erstwhile colonial empires, with eponymous species descriptions peaking during colonial expansion. Importantly, our analysis highlights a positive relationship between the Colonial Exposure Index (a metric factoring the duration and area of a country colonized by a specific empire) and the number of eponymous bird species honoring persons from that specific empire. Our temporal analysis, however, shows that the use of eponyms has recently shifted towards honoring native persons and distinguished conservation champions. Overall, this study presents an important statistical examination of eponyms and their colonial legacies, thereby providing valuable context for ongoing debates on the reform or retention of eponyms worldwide.

ecology↗

bioRxiv2026

Targeting AATF reprograms the tumor microenvironment and suppresses hepatocellular carcinoma via MIR100HG TGFβ signaling

Hepatocellular carcinoma (HCC), a leading cause of cancer death, has a dynamic and heterogeneous tumor microenvironment (TME) that drives progression and therapeutic resistance. We previously elucidated that apoptosis antagonizing transcription factor (AATF) drives angiogenesis in HCC. However, its role in TME remains unexplored. We employed an orthotopic xenograft mouse model, implanting human HCC cells into the liver, and achieved liver-specific silencing via tail vein injection of AAV8 carrying mouse-specific siAATF or siControl. Histological, biochemical, and molecular analyses, combined with whole-genome transcriptomics mapped to mouse and human genomes, were used to study TME and tumor compartments separately. Silencing of AATF in the TME significantly reduced tumor growth compared with controls. Furthermore, AATF loss disrupted key processes in TME, including inflammation, immune response, angiogenesis, and extracellular matrix remodeling. Mechanistically, TGF-{beta} signaling was significantly suppressed in the TME, thereby affecting tumor cell cycle and metabolic activity, ultimately leading to tumor regression. The long noncoding RNA (lncRNA) analysis identified MIR100HG as a key downstream regulator of AATF in the TGF-{beta} signaling pathway. These findings expand the oncogenic role of AATF to include regulation of the TME via the AATF-MIR100HG-TGF-{beta} axis, highlighting its potential as a therapeutic target in HCC.

cancer biology↗

bioRxiv2026

Interactive segmentation of membrane and membrane mimic densities in cryo-EM maps

Contextual density arising from biological membranes or membrane mimics such as detergent micelles or lipid nanodiscs, is ubiquitous in cryo-EM maps of integral and peripheral membrane proteins, or membrane-associated complexes, from single-particle analysis and subtomogram averaging. Here, we describe SURFER, a lightweight, GPU-accelerated extension for UCSF ChimeraX that enables rapid semantic differentiation of membrane or membrane mimic density from macromolecular signal. SURFER operates on unfiltered maps to automatically generate segmentation masks that can be flexibly applied to any aligned target map, including reference volumes for local refinement or post-processed maps optimised for visualisation and interpretation. By allowing contextual density to be selectively toggled or subtracted, SURFER enables direct interactive comparison between representations with and without membrane-related features to facilitate consistent analysis and reporting of membrane context in cryo-EM structures. An interactive graphical interface within ChimeraX supports convenient threshold adjustment, connectivity filtering, and immediate visual evaluation of the resulting segmentation.

biophysics↗

bioRxiv2026

Differential Assembly of Native ENaC Complexes Across Mouse Epithelial Tissues

The epithelial sodium channel (ENaC) governs sodium and fluid absorption in the lung, kidney, and colon, but the organization of native ENaC complexes has remained difficult to define because of their low abundance and biochemical instability. To enable direct analysis of native assemblies, we generated a knock-in mouse in which the endogenous {gamma} subunit is fused at its C terminus to mVenus, a 3C protease cleavage site, and a 3xFLAG epitope (ENaC{gamma}-VF). The tag preserves physiological ENaC function, as ENaC{gamma}-VF mice display normal electrolyte handling, benzamil affinity, and amiloride-sensitive Na:K responses indistinguishable from wild-type animals. Using fluorescence-detection size-exclusion chromatography and single-molecule pull-down, we directly monitor intact native ENaC complexes from lung, kidney, and colon and uncover marked tissue-to-tissue differences in channel abundance and apparent complex size. Dual-color analysis with a fluorescent Fab against ENaC marks fully assembled {beta}{gamma} channels, while {gamma}-based fluorescence reports the broader population of {gamma}-containing assemblies. In combination, the ENaC{gamma}-VF line provides a biochemical anchor for identifying regulatory and trafficking proteins that co-purify with native ENaC complexes. These data show that ENaC architecture in vivo is heterogeneous, and establish ENaC{gamma}-VF mice as a platform for dissecting how epithelial environments shape ENaC assembly, composition, and regulation.

biochemistry↗

bioRxiv2026

A single-cell transcriptomic atlas of the periventricular proliferative zone in the late gestation fetal brain in the pigtail macaque

BackgroundThe fetal brain undergoes rapid changes in late gestation, when waves of neurogenesis and gliogenesis shape cortical circuitry. The periventricular proliferative region and adjacent white matter are enriched in neuroprogenitor cells, newborn neurons, and interneurons, which is challenging to study in the late gestation human fetal brain. The nonhuman primate (NHP) provides a powerful translational model to overcome this limitation, given its close similarity to human neurodevelopmental trajectories. The study objective was to construct a single-cell RNA-Seq (scRNA-Seq) atlas of the late-gestation fetal brain of the pigtail macaque (Macaca nemestrina), focused on the periventricular proliferative zone. MethodsA sample of the lateral ventricular wall, subventricular zone, and overlying white/gray matter was dissociated into single cells and processed through the 10X Genomics pipeline, followed by SoupX removal of ambient RNA, and Seurats pipeline to aggregate, cluster and annotate single-cell populations. Monocle3 was used to determine pseudotime and map lineage progression. ResultsThis analysis captured diverse populations of neuroprogenitors, newborn neurons, developing lineages of excitatory and inhibitory neurons, oligodendrocyte and astrocyte lineages, and resident immune and endothelial cells. ConclusionsSingle-cell populations from the third-trimester nonhuman primate fetal brain are highly similar to those in the human fetus. This late-gestation single-cell atlas of the periventricular proliferative zone provides a unique reference for progenitor, neuronal, glial, vascular, and immune cell states during a critical window of primate neurodevelopment, enabling mechanistic interrogation of how inflammatory, infectious, or hypoxic insults disrupt vulnerable neurogenic niches.

genomics↗

bioRxiv2025

Confidence-guided cryo-EM map optimisation with LocScale-2.0

Cryogenic sample electron microscopy (cryo-EM) maps often display uneven quality, with high-resolution features coexisting alongside weak or poorly ordered regions. Such variation complicates structural interpretation, especially for heterogeneous macromolecular assemblies. Here, we present LocScale-2.0, a context-aware map optimisation framework that operates without prior knowledge of molecular structure or composition. By leveraging general expectations of electron scattering by biological macromolecules, it enhances local detail and connectivity while preserving weak but biologically relevant structural context. We further introduce LocScale-FEM, a Bayesian-approximate deep-learning approach that emulates this optimisation to generate feature-enhanced maps. LocScale-FEM provides voxel-wise confidence scores that give a statistically grounded measure of reliability, are sensitive to local phase error in the feature-enhanced map, and highlight regions where interpretation warrants caution. Selected examples illustrate how such maps can aid biological interpretation and how confidence-guided map optimisation can increase objectivity in cryo-EM density analysis.

biophysics↗

bioRxiv2024

Vocal species are more central in Eastern Himalayan Mixed-Species bird flocks

Mixed-species flocks (MSF) represent an important form of social organisation in bird communities worldwide. Despite its likely importance in flock formation and cohesion, the role of vocal communication in the formation and maintenance of MSF in birds is hitherto understudied. In this study, we examine if a species centrality within a mixed-species flock is influenced by its vocal behaviour during the dawn chorus, i.e., the time of MSF formation. Using acoustic sampling and field observations, we studied the bird species found in MSF in the Eastern Himalayas. Our results show differential vocal activity patterns among MSF-forming bird species and suggest a positive correlation between calling rates and closeness centrality (species importance in a flock) in understory MSFs. We also found a more synchronised vocalisation pattern in the understory MSFs, with a consistent peak in vocal activity in the early morning hours, whereas no consistent vocal pattern was found for canopy flocks. Overall, our results suggest a potential mechanism that drives MSF formation wherein the vocal activity of central species precedes and likely attracts participation from other attendant species.

ecology↗

bioRxiv2024

eQTL in diseased colon tissue identifies novel target genes associated with IBD

Genome-wide association studies (GWAS) have identified over 300 loci associated with the inflammatory bowel diseases (IBD), but putative causal genes for most are unknown. We conducted the largest disease-focused expression quantitative trait loci (eQTL) analysis using colon tissue from 252 IBD patients to determine genetic effects on gene expression and potential contribution to IBD. Combined with two non-IBD colon eQTL studies, we identified 194 potential target genes for 108 GWAS loci. eQTL in IBD tissue were enriched for IBD GWAS loci colocalizations, provided novel evidence for IBD-associated genes such as ABO and TNFRSF14, and identified additional target genes compared to non-IBD tissue eQTL. IBD-associated eQTL unique to diseased tissue had distinct regulatory and functional characteristics with increased effect sizes. Together, these highlight the importance of eQTL studies in diseased tissue for understanding functional consequences of genetic variants, and elucidating molecular mechanisms and regulation of key genes involved in IBD.

genomics↗

bioRxiv2023

Understanding inter-individual social networks in mixed-species bird flocks

Mixed-species flocks (MSFs) are an important form of social organisation in forest bird communities worldwide. MSFs provide participants with the benefits of reduced predation risk and/or enhanced foraging efficiency. Recent work has shown that participation in MSFs confers long-term survival benefits in the face of anthropogenic change. However, our understanding of MSFs mainly comes from studies that examine species-level networks, where each node is a unique species and the edges or connections between nodes are associations/interactions between species. While valuable, such approaches might not allow us to understand and investigate the mechanisms that drive MSF formation and structure because social interactions and their effects occur at the individual-level. Empirical studies on multi-species, individual-level MSF social networks have seldom been undertaken due to the various complexities and logistical challenges involved. In this study, we use mist-netting and colour-ringing followed by a standardised observation protocol to construct individual-level social networks in MSFs at 2000m ASL in Eaglenest Wildlife Sanctuary, Arunachal Pradesh, Eastern Himalaya, India. First, we found two separate flocktypes at our study site, comprising two distinct sets of understorey species. The mechanisms contributing to individual-level co-occurrences are likely to differ between these flocktypes, and with MSFs in the Neotropics. The Rusty-fronted Barwing (the nuclear species of one flocktype) shows spatially disjunct territories for each flock while the Yellow-throated Fulvetta (nuclear species of the other flocktype) shows large spatial overlap in its MSF networks, which is likely driven by non-individual-specific benefits such as predation risk dilution. Further, the addition of associating individuals to the social networks has opposite impacts on the two networks. The addition of Coral-billed Scimitar Babblers to the barwing networks greatly reduces network modularity because the associating individuals bridge two modules of barwings (both spatially and in the social network). On the contrary, territorial Rufous-capped Babblers and Grey-cheeked Warblers increase the modularity of the spatially overlapping fulvetta network. Our study provides novel insights into flock formation mechanisms in the Eastern Himalaya, likely applicable to other multi-species flock systems in the Old World.

ecology↗

bioRxiv2023

THE RIBOREGULATION MECHANISM OF HUMAN SERINE HYDROXYMETHYLTRANSFERASE IS ROOTED IN AN ALLOSTERIC SWITCH

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzymes reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

biochemistry↗

bioRxiv2022

Long-term bird abundance in a threatened grassland ecosystem: an assessment using daily bird lists

Open Natural Ecosystems (ONEs), such as tropical grasslands, are among the most threatened habitats on Earth today. The long-term monitoring of ONEs is an important research domain that is essential for understanding anthropogenic impacts and facilitating conservation action. Using a simple day-listing method over a 13-year period, we studied species trends in a central Indian grassland-agriculture mosaic experiencing several land-use changes. Our results indicate that some grassland species (such as the Great Indian Bustard Ardeotis nigriceps) show steep declines during the study period, while other generalist species (such as the Indian Peafowl Pavo cristatus) show an increasing trend. Daily listing also reveals distinct seasonal patterns, and we discuss the Great Indian Bustard and Western Marsh Harrier Circus aeruginosus as examples. Our study highlights the utility of consistent checklist surveys to monitor population trends of bird communities within a changing landscape.

ecology↗

bioRxiv2022

Abiotic niche predictors of long-term trends in body mass and survival of Eastern Himalayan birds

The synergistic impacts of climate change and habitat degradation threaten tropical species worldwide. However, how species abiotic niches affect their demographic vital rates and phenotypic changes under anthropogenic change remains poorly understood. Using an 11-year mark-recapture dataset from primary and selectively logged forest in the Eastern Himalayas, we investigated how temperature-humidity niche characteristics predicted body mass and survival trends in understorey insectivorous birds over time in each habitat. Our results show that logged forest is hotter and drier than primary forest, and the arthropod community shows dramatic shifts in composition upon selective logging. In understorey insectivores, the degree of dissimilarity between species-specific primary and logged forest niches was strongly and negatively correlated with survival and body mass trends in logged forest. Here, we show that temperature-humidity niche shifts in response to anthropogenic habitat modification can impact demographic vital rates and body condition crucial for population persistence. This work has the potential to inform prompt, targeted conservation efforts toward species that are the most threatened in a warmer and more degraded world.

ecology↗

bioRxiv2022

Single-cell Spatial Proteomic Imaging for Human Neuropathology

Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimers disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield, in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves as a baseline for mechanistic and interventional understanding in human neurodegeneration.

systems biology↗

bioRxiv2020

Multiplexed Single-cell Metabolic Profiles Organize the Spectrum of Human Cytotoxic T Cells

Cellular metabolism regulates immune cell activation, differentiation and effector functions to the extent that its perturbation can augment immune responses. However, the analytical technologies available to study cellular metabolism lack single-cell resolution, obscuring metabolic heterogeneity and its connection to immune phenotype and function. To that end, we utilized high-dimensional, antibody-based technologies to simultaneously quantify the single-cell metabolic regulome in combination with phenotypic identity. Mass cytometry (CyTOF)-based application of this approach to early human T cell activation enabled the comprehensive reconstruction of the coordinated metabolic remodeling of naive CD8+ T cells and aligned with conventional bulk assays for glycolysis and oxidative phosphorylation. Extending this analysis to a variety of tissue-resident immune cells revealed tissue-restricted metabolic states of human cytotoxic T cells, including metabolically repressed subsets that expressed CD39 and PD1 and that were enriched in colorectal carcinoma versus healthy adjacent tissue. Finally, combining this approach with multiplexed ion beam imaging by time-of-flight (MIBI-TOF) demonstrated the existence of spatially enriched metabolic neighborhoods, independent of cell identity and additionally revealed exclusion of metabolically repressed cytotoxic T cell states from the tumor-immune boundary in human colorectal carcinoma. Overall, we provide an approach that permits the robust approximation of metabolic states in individual cells along with multimodal analysis of cell identity and functional characteristics that can be applied to human clinical samples to study cellular metabolism how it may be perturbed to affect immunological outcomes.

immunology↗

bioRxiv2019

Single-cell transcriptional diversity is a hallmark of developmental potential

Single-cell RNA-sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation without prior knowledge has remained challenging. Here we describe a simple yet robust determinant of developmental potential--the number of detectably expressed genes per cell-- and leverage this measure of transcriptional diversity to develop a new framework for predicting ordered differentiation states from scRNA-seq data. When evaluated on ~150,000 single-cell transcriptomes spanning 53 lineages and five species, our approach, called CytoTRACE, outperformed previous methods and ~19,000 molecular signatures for resolving experimentally-confirmed developmental trajectories. In addition, it enabled unbiased identification of tissue-resident stem cells, including cells with long-term regenerative potential. When used to analyze human breast tumors, we discovered candidate genes associated with less-differentiated luminal progenitor cells and validated GULP1 as a novel gene involved in tumorigenesis. Our study establishes a key RNA-based correlate of developmental potential and provides a new platform for robust delineation of cellular hierarchies (https://cytotrace.stanford.edu).

genomics↗

bioRxiv2019

Benchmarking algorithms for gene regulatory network inference from single-cell transcriptomic data

We present a comprehensive evaluation of state-of-the-art algorithms for inferring gene regulatory networks (GRNs) from single-cell gene expression data. We develop a systematic framework called BEELINE for this purpose. We use synthetic networks with predictable cellular trajectories as well as curated Boolean models to serve as the ground truth for evaluating the accuracy of GRN inference algorithms. We develop a strategy to simulate single-cell gene expression data from these two types of networks that avoids the pitfalls of previously-used methods. We selected 12 representative GRN inference algorithms. We found that the accuracy of these methods (measured in terms of AUROC and AUPRC) was moderate, by and large, although the methods were better in recovering interactions in the synthetic networks than the Boolean models. Techniques that did not require pseudotime-ordered cells were more accurate, in general. The observation that the endpoints of many false positive edges were connected by paths of length two in the Boolean models suggested that indirect effects may be predominant in the outputs of the algorithms we tested. The predicted networks were considerably inconsistent with each other, indicating that combining GRN inference algorithms using ensembles is likely to be challenging. Based on the results, we present some recommendations to users of GRN inference algorithms, including suggestions on how to create simulated gene expression datasets for testing them. BEELINE, which is available at http://github.com/murali-group/BEELINE under an open-source license, will aid in the future development of GRN inference algorithms for single-cell transcriptomic data.

bioinformatics↗