Biology subjects

Selvaraj, S.

Publications and source records attributed to Selvaraj, S..

17 recordsLinked to original sources

bioRxiv2026

Ancient Somatosensory Circuit Architectures Employ Flexible Molecular Strategies

The extent to which conserved neural circuit architectures depend on shared molecular specification programs remains unclear. Here, we address this question by examining the somatosensory system of the little skate, Leucoraja erinacea, an early-diverging vertebrate that retains ancestral features of both finned and limb-based body plans. We show that core features of somatosensory circuit organization, including laminar organization of the spinal cord and dorsally restricted targeting of sensory afferents, are deeply conserved. Unexpectedly, the molecular programs specifying dorsal root ganglion (DRG) sensory subtypes diverge extensively from those of mammals. Although DRG neuron subtype specification and spinal connectivity rely on target-derived cues, skates employ distinct neurotrophin receptor and transcription factor identity codes. These findings support a model in which conserved spinal circuit architectures provide a stable scaffold that leverages flexible sensory neuron specification programs, enabling the evolutionary diversification of vertebrate somatosensory systems.

neuroscience↗

bioRxiv2026

TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

Precise gene editing through homology directed repair (HDR) is one of the most versatile genome editing approaches with broad applications. Achieving high HDR gene editing efficiency is critical to realizing the full potential of this approach. Although many strategies have been explored to enhance HDR editing efficiency, inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of the non-homologous end joining (NHEJ) pathway remains one of the most effective. Here we describe a novel, highly potent DNA-PKcs small molecule inhibitor, TFU72 which enhances HDR gene editing efficiency remarkably by up to 30-fold in cell lines and human primary cells. We assessed the previously reported genotoxic outcomes associated with DNA-PKcs inhibition such as off-target mutations, chromosomal translocations and large deletions and describe approaches to mitigate these outcomes to safely enhance HDR gene editing efficiency with TFU72. This optimized approach enables broad application of TFU72 for HDR-based precise gene editing applications in both therapeutic and research settings.

molecular biology↗

bioRxiv2025

Clonal Heterogeneity in Human Pancreatic Ductal Adenocarcinoma and Its Impact on Tumor Progression

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by profound desmoplasia and cellular heterogeneity, which cannot be fully resolved using traditional bulk sequencing approaches. To understand the contribution of this heterogeneity to PDAC biology, we analyzed a large cohort of primary human PDAC samples (n = 62), profiling 443,451 single cells and 53,236 spatial transcriptomic spots using a combined single-cell RNA sequencing and spatial transcriptomics approach. Our analysis revealed significant intratumoral heterogeneity, with multiple genetically distinct neoplastic clones co-existing within individual tumors. These clones exhibited diverse transcriptional states and subtype profiles, challenging the traditional binary classification of PDAC into basal and classical subtypes; instead, our findings support a transcriptional continuum influenced by clonal evolution and spatial organization. Additionally, these clones each interacted uniquely with surrounding cell types in the tumor microenvironment. Phylogenetic analysis uncovered a rare but consistent classical-to-basal clonal transition associated with MYC amplification and immune response depletion, which were validated experimentally, suggesting a mechanism driving the emergence of a more aggressive basal clonal phenotype. Spatial analyses further revealed dispersed clones enriched for epithelial-to-mesenchymal transition (EMT) activity and immune suppression, correlating with metastatic potential and colonization of lymph node niches. These dispersed clones tended to transition toward a basal phenotype, contributing to disease progression. Our findings highlight the critical role of clonal diversity, transcriptional plasticity, and TME interactions in shaping human PDAC biology. This work provides new insights into the molecular and spatial heterogeneity of PDAC and offers potential avenues for therapeutic intervention targeting clonal evolution and the mechanisms driving metastasis.

cancer biology↗

bioRxiv2024

Bulk and Single-cell Transcriptomic Brain Data Identify Overlapping Processes and Cell-types with Human AUD and Mammalian Models of Alcohol Use

This study explores the neurobiological underpinnings of alcohol use disorder (AUD) by integrating bulk and single-cell transcriptomic data from humans, primates, and mice across three brain regions associated with addiction (i.e., prefrontal cortex (PFC), nucleus accumbens (NAc), and central amygdala (CeA)). We compared AUD RNA expression and cell-type abundance from 92 human brain to data from 53 primates and 90 mice engaged in diverse alcohol use paradigms. The findings revealed significant and reproducible correlations between human AUD and mammalian models of alcohol use that vary by tissue, species, and behavioral paradigm. The strongest correlations occurred between primate and mouse models of binge drinking (i.e., high drinking in the dark). Certain primate models demonstrated that the brain RNA correlations with human alcohol use disorder (AUD) were approximately 40% as strong as the correlations observed within human samples themselves. By integrating single-cell transcriptomic data, this study observed decreased oligodendrocyte proportions in the PFC and NAc of human AUD with similar trends in animal models. Gene co-expression network analyses revealed conserved systems associated with human AUD and animal models of heavy/binge alcohol consumption. Gene co-expression networks were enriched for pathways related to inflammation, myelination, and synaptic plasticity and the genes within them accounted for [~]20% of the heritability in human alcohol consumption. Identified hub genes were associated with relevant traits (e.g., impulsivity, motivation) in humans and mice. This study sheds light on conserved biological entities underlying AUD and chronic alcohol use, providing insights into the cellular, genetic, and neuromolecular basis across species.

genetics↗

bioRxiv2024

Predicted mechanistic impacts of human protein missense variants

Genome sequencing efforts have led to the discovery of tens of millions of protein missense variants found in the human population with the majority of these having no annotated role and some likely contributing to trait variation and disease. Sequence-based artificial intelligence approaches have become highly accurate at predicting variants that are detrimental to the function of proteins but they do not inform on mechanisms of disruption. Here we combined sequence and structure-based methods to perform proteome-wide prediction of deleterious variants with information on their impact on protein stability, protein-protein interactions and small-molecule binding pockets. AlphaFold2 structures were used to predict approximately 100,000 small-molecule binding pockets and stability changes for over 200 million variants. To inform on protein-protein interfaces we used AlphaFold2 to predict structures for nearly 500,000 protein complexes. We illustrate the value of mechanism-aware variant effect predictions to study the relation between protein stability and abundance and the structural properties of interfaces underlying trans protein quantitative trait loci (pQTLs). We characterised the distribution of mechanistic impacts of protein variants found in patients and experimentally studied example disease linked variants in FGFR1.

bioinformatics↗

bioRxiv2024

Human striatal progenitor cells that contain inducible safeguards and overexpress BDNF rescue Huntington's disease phenotypes in R6/2 mice

Huntingtons disease (HD) is an autosomal-dominant neurodegenerative disorder characterized by striatal atrophy. Reduced trophic support due to decreased striatal levels of neurotrophins (NTs), mainly brain-derived neurotrophic factor (BDNF), contributes importantly to HD pathogenesis; restoring NTs has significant therapeutic potential. Human pluripotent stem cells (hPSC) offer a scalable platform for NT delivery but has potential safety risks including teratoma formation. We engineered hPSCs to constitutively produce BDNF and contain inducible safeguards to eliminate these cells if safety concerns arise. This study examined the efficacy of intrastriatally transplanted striatal progenitor cells (STRpcs) derived from these hPSCs against HD phenotypes in R6/2 mice. Engrafted STRpcs overexpressing BDNF alleviated motor and cognitive deficits and reduced mutant huntingtin aggregates. Activating the inducible safety switch with rapamycin safely eliminated the engrafted cells. These results demonstrate that BDNF delivery via a novel hPSC-based platform incorporating safety switches could be a safe and effective HD therapeutic.

neuroscience↗

bioRxiv2024

Geranylgeranylated-SCFFBXO10 Regulates Selective Outer Mitochondrial Membrane Proteostasis and Function

E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide does not contain a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and the interaction with two cytosolic factors, PDE6{delta} (a prenyl group-binding protein), and HSP90 (a mitochondrial chaperone) orchestrate specific OMM targeting of prenyl-FBXO10 across diverse membrane compartments. The geranylgeranylation-deficient FBXO10(C953S) mutant redistributes away from the OMM, leading to impaired mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial fragmentation. Phosphoglycerate mutase 5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative mass spectrometry analyses of enriched mitochondria (LFQ-MS/MS), leveraging the redistribution of FBXO10(C953S). FBXO10, but not FBXO10(C953S), promoted polyubiquitylation and degradation of PGAM5. Examination of the role of this pathway in a physiological context revealed that the loss of FBXO10 or expression of prenylation-deficient-FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human iPSCs and murine myoblasts. Our studies identify a mechanism for selective E3-ligase mediated regulation of mitochondrial membrane proteostasis and metabolic health, potentially amenable to therapeutic intervention.

cell biology↗

bioRxiv2024

Engineering inducible signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible EPO receptors (iEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of iEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media--EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how synthetic biology and genome editing may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated cell engineering strategies.

synthetic biology↗

bioRxiv2023

Human assembloids reveal the consequences of CACNA1G gene variants in the thalamocortical pathway

Abnormalities in crosstalk between the thalamus and the cerebral cortex are thought to lead to severe neuropsychiatric disorders, such as epilepsy and psychotic disorders. Pathogenic variants in the CACNA1G gene, which encodes the 1G subunit of the thalamus-enriched T-type voltage-gated calcium channel CaV3.1, are associated with absence seizures, intellectual disability, and schizophrenia, but the cellular and circuit level consequences of these genetic variants in humans remain unknown. Here, we developed an in vitro human assembloid model of the thalamocortical pathway to systematically dissect the contribution of genetic variants in T-type calcium channels. We discovered that a CACNA1G variant (M1531V) associated with seizures led to changes in T-type currents in human thalamic neurons, as well as correlated hyperactivity of thalamic and cortical neurons in thalamo-cortical assembloids. In contrast, CACNA1G loss, which has been associated with risk of schizophrenia, resulted in abnormal thalamocortical connectivity that was related to both increased spontaneous thalamic activity and aberrant thalamic axonal projections. Taken together, these results illustrate the utility of organoid and assembloid systems for interrogating human genetic disease risk variants at both cellular and circuit level.

neuroscience↗

bioRxiv2022

MicroRNA-mRNA networks are dysregulated in opioid use disorder postmortem brain: further evidence for opioid-induced neurovascular alterations

To understand mechanisms and identify potential targets for intervention in the current crisis of opioid use disorder (OUD), postmortem brains represent an under-utilized resource. To refine previously reported gene signatures of neurobiological alterations in OUD from the dorsolateral prefrontal cortex (Brodmann Area 9, BA9), we explored the role of microRNAs (miRNA) as powerful epigenetic regulators of gene function. Building on the growing appreciation that miRNAs can cross the blood-brain barrier, we carried out miRNA profiling in same-subject postmortem samples from BA9 and blood tissues. miRNA-mRNA network analysis showed that even though miRNAs identified in BA9 and blood were fairly distinct, their target genes and corresponding enriched pathways were highly overlapping, with tube development and morphogenesis, and pathways related to endothelial cell function and vascular organization, among the dominant enriched biological processes. These findings point to robust, redundant, and systemic opioid-induced miRNA dysregulation with potential functional impact on transcriptomic changes. Further, using correlation network analysis we identified cell-type specific miRNA targets, specifically in astrocytes, neurons, and endothelial cells, associated with OUD transcriptomic dysregulation. Our refined miRNA-mRNA networks enabled identification of novel pharmaco-chemical interventions for OUD, particularly targeting the TGF beta-p38MAPK signaling pathway. Finally, leveraging a collection of control brain transcriptomes from the Genotype-Tissue Expression (GTEx) project, we identified correlation of OUD miRNA targets with TGF beta, hypoxia, angiogenesis, coagulation, immune system and inflammatory pathways. These findings support previous reports of neurovascular and immune system alterations as a consequence of opioid abuse.

bioinformatics↗

bioRxiv2022

Alcohol, Opioid, and Combined Alcohol and Opioid Use Disorders Affect Shared and Unique Pathways: A Proteomic Analysis of Postmortem Brains

Substance misuse is a major burden for not only patients but the healthcare system. Understanding how substance misuse alters a patients genome, proteome, and neurocircuitry is an important step toward identifying targets for treatments. In this exploratory study, we performed a proteomic analysis of postmortem brain from individuals with substance misuse disorder (n=29), including alcohol (n=11) and opioid misuse disorder (n=12), or comorbidity with both disorders (n=6), compared to controls (n=12). The results demonstrate that these substances affect both common and different biological pathways compared to controls. Alcohol misuse affects primarily protein translation, rRNA processing, and energy metabolism pathways, while opioid influences G-protein signaling, protein translation, rRNA processing, energy metabolism, and angiogenesis pathways. Alcohol and opioid misuse combined appears to have a synergistic effect on protein expression of the TCA cycle, respiratory transport, mitochondrial function, LGI-ADAM interactions, interleukin signaling, neuronal system changes, and actin polymerization. These findings offer new insights into how substance misuse leads to protein alterations and how combined substance misuse leads to different protein changes compared to single substance misuse.

neuroscience↗

bioRxiv2020

Deconstructing the mouse olfactory percept through an olfactory ethological atlas

Odor perception in non-humans is poorly understood. Here, we generated the most comprehensive murine olfactory ethological atlas to date, consisting of behavioral responses to a diverse panel of 73 odorants, including 12 at multiple concentrations. These data revealed that the mouse behavior is incredibly diverse, and changes in response to odor identity and intensity. Using only behavioral responses, ~30% of the 73 odorants could be identified with high accuracy (>96%) by a trained classifier. Mouse behavior occupied a low-dimensional space, consistent with analyses of human olfactory perception. While mouse olfactory behavior is difficult to predict from the corresponding human olfactory percept, three fundamental properties are shared: odor valence is the primary axis of olfactory perception; the physicochemical properties of odorants can predict the olfactory percept; and odorant concentration quantitatively and qualitatively impacts olfactory perception. These results provide a template for future comparative studies of olfactory percepts among species.

neuroscience↗

bioRxiv2020

In silico tools for accurate HLA and KIR inference from clinical sequencing data empower immunogenetics on individual-patient and population scales

Immunogenetic variation in humans is important in research, clinical diagnosis and increasingly a target for therapeutic intervention. Two highly polymorphic loci play critical roles, namely the human leukocyte antigen (HLA) system, which is the human version of the major histocompatibility complex (MHC), and the Killer-cell immunoglobulin-like receptors (KIR) that are relevant for responses of Natural Killer (NK) cells and some subsets of T cells. Their accurate classification has typically required the use of dedicated biological specimens and a combination of in vitro and in silico efforts. Increased availability of next generation sequencing data has led to the development of ancillary computational solutions. Here, we report an evaluation of recently published algorithms to computationally infer complex immunogenetic variation in the form of HLA alleles and KIR haplotypes from whole-genome or whole-exome sequencing data. For both HLA allele and KIR gene typing, we identified tools that yielded >97% overall accuracy for 4-digit HLA types, and >99% overall accuracy for KIR gene presence, suggesting the readiness of in silico solutions for use in clinical and high-throughput research settings.

bioinformatics↗

bioRxiv2019

Telomere-to-telomere assembly of a complete human X chromosome

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the [~]2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

bioinformatics↗

bioRxiv2019

Spatial co-fragmentation pattern of cell-free DNA recapitulates in vivo chromatin organization and identifies tissues-of-origin

Three-dimensional chromatin organization varies across cell types and is essential for gene regulation. However, current technologies are unable to assess in vivo genome-wide chromatin organization non-invasively. Here we show that distant correlations in the fragment length of cell-free DNA (cfDNA) recapitulate three-dimensional chromatin organization. The inferred organization is highly concordant with that measured by Hi-C in white blood cells from healthy donors, and is not explained by technical bias or sequence composition. Furthermore, the inferred organization reflects different genomic organization in the various cell types contributing to cfDNA, allowing identification and quantification of tissues of origin. This approach is concordant with previous methods, but with more complete representations of cfDNA. Our results, demonstrated in cfDNA from healthy individuals and cancer patients, may enable noninvasive monitoring of in vivo genome organization and accurate quantification of cell death in different clinical conditions.

genomics↗

bioRxiv2018

Integration of phased Hi-C and molecular phenotype data to study genetic and epigenetic effects on chromatin looping

While genetic variation at chromatin loops is relevant for human disease, the relationships between loop strength, genetics, gene expression, and epigenetics are unclear. Here, we quantitatively interrogate this relationship using Hi-C and molecular phenotype data across cell types and haplotypes. We find that chromatin loops consistently form across multiple cell types and quantitatively vary in strength, instead of exclusively forming within only one cell type. We show that large haplotype loop imbalance is primarily associated with imprinting and copy number variation, rather than genetically driven traits such as allele-specific expression. Finally, across cell types and haplotypes, we show that subtle changes in chromatin loop strength are associated with large differences in other molecular phenotypes, with a 2-fold change in looping corresponding to a 100-fold change in gene expression. Our study suggests that regulatory genetic variation could mediate its effects on gene expression through subtle modification of chromatin loop strength.

genomics↗

bioRxiv2018

Integrating Hi-C links with assembly graphs for chromosome-scale assembly

Long-read sequencing and novel long-range assays have revolutionized de novo genome assembly by automating the reconstruction of reference-quality genomes. In particular, Hi-C sequencing is becoming an economical method for generating chromosome-scale scaffolds. Despite its increasing popularity, there are limited open-source tools available. Errors, particularly inversions and fusions across chromosomes, remain higher than alternate scaffolding technologies. We present a novel open-source Hi-C scaffolder that does not require an a priori estimate of chromosome number and minimizes errors by scaffolding with the assistance of an assembly graph. We demonstrate higher accuracy than the state-of-the-art methods across a variety of Hi-C library preparations and input assembly sizes. The Python and C++ code for our method is openly available at https://github.com/machinegun/SALSA\n\nAuthor summaryHi-C technology was originally proposed to study the 3D organization of a genome. Recently, it has also been applied to assemble large eukaryotic genomes into chromosome-scale scaffolds. Despite this, there are few open source methods to generate these assemblies. Existing methods are also prone to small inversion errors due to noise in the Hi-C data. In this work, we address these challenges and develop a method, named SALSA2. SALSA2 uses sequence overlap information from an assembly graph to correct inversion errors and provide accurate chromosome-scale assemblies.

bioinformatics↗