Biology subjects

Khan, Z.

Publications and source records attributed to Khan, Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Chronic lower limb pain is not associated with a loss of inhibitory neurons in the human lumbar spinal dorsal horn

The spinal dorsal horn is the primary processing site of nociceptive sensory input from the periphery. Excitatory spinal interneurons releasing glutamate can amplify this information before it is sent to the brain, whereas inhibitory neurons releasing GABA and/or glycine can suppress the outflow of nociceptive signals. An imbalance favoring excitation is thought to underlie certain aspects of chronic pain. Although rodent studies have identified spinal mechanisms underlying hyperalgesia and allodynia, little is known about the anatomical changes associated with chronic pain in the human spinal cord, a gap in knowledge we sought to address in this study. Using immunohistochemistry and in situ hybridization on lumbar spinal cord tissue recovered from organ donors, we characterized neuronal size and density across the human dorsal horn and confirmed the presence of the human equivalent of the lateral spinal nucleus in many individuals. Chronic lower limb pain was not associated with changes in neuronal density in the dorsal horn. Likewise, the ratio of excitatory (SLC17A6+) to inhibitory (PAX2+) neurons remained consistent across laminae for age, sex and chronic pain state, providing no evidence for selective loss of inhibitory neurons with chronic pain in humans. We found no differences in the size or density of the postsynaptic markers Homer1 and gephyrin between groups, suggesting glutamatergic and GABAergic postsynaptic sites remain structurally stable. These findings provide a thorough evaluation of cellular anatomy of the human dorsal horn and form a foundation for future studies investigating neuronal changes that may contribute to chronic pain in humans.

neuroscience↗

bioRxiv2026

VDAC1 regulates stress-associated matrix localization of DJ-1 to support mitochondrial homeostasis and neuronal survival

DJ-1 is a redox-sensitive protein implicated in early-onset Parkinson's disease, and its mitochondrial localization protects against oxidative stress, but the mechanisms regulating its submitochondrial targeting and functional impact on mitochondrial integrity remain poorly understood. We identify voltage-dependent anion channel 1 (VDAC1) as a regulator of the submitochondrial distribution of DJ-1 during stress. Endogenous DJ-1 interacted with VDAC1, and loss of VDAC1 reduced stress-induced DJ-1 accumulation within the mitochondrial matrix. VDAC1-deficient neurons exhibited mitochondrial fragmentation, impaired oxidative phosphorylation, reduced ATP levels, altered reactive oxygen species (ROS) responses, and increased sensitivity to MPP+;. Matrix-targeted, but not outer-membrane-targeted, DJ-1 rescued basal, ATP-linked, and maximal respiration, improved mitochondrial morphology, and enhanced neuronal survival. ATP synthase inhibition also rapidly increased mitochondrial DJ-1, suggesting bioenergetic stress promotes its mitochondrial accumulation. Our findings identify compartment-specific localization as a key determinant of DJ-1 function and establish VDAC1-dependent matrix targeting as a critical mechanism supporting mitochondrial integrity during stress.

neuroscience↗

bioRxiv2026

MYC pathway reprogramming through a TIP60 coactivator switch in neuroendocrine lineage transition in prostate cancer

Prostate adenocarcinomas (PRAD) can acquire resistance to androgen receptor signaling inhibitors through lineage transition to a cell state known as neuroendocrine prostate cancer (NEPC). Using a panel of isogenic PRAD and NEPC mouse tumoroids, we show that NEPC cells acquire new transcription factor (TF) dependencies that function in a previously undefined network. Through selective perturbation of each TF, we identify ASCL1 as a key regulator of NE lineage fate whereas MYCL functions downstream to drive NEPC growth/survival by recruitment of the TIP60/KAT5 acetyltransferase. Interestingly, while dependencies on specific TF family paralogs can vary across NEPC models, all show markedly enhanced dependency on TIP60. Moreover, the H2A.Z-acetyltransferase activity of the TIP60 complex (TIP60-C) is required for NEPC as well as the acetyl-reader BRD8, which is newly incorporated as a TIP60-C subunit with the NEPC transition. Targeted degradation studies in isogenic tumoroids reveal increased dependence on MYCL in NEPC relative to its paralog MYC in PRAD. In addition to a paralog switch (MYC to MYCL), the MYC pathway-addicted NE state is accompanied by a chaperone switch (from TIP60-C to SRCAP) for H2A.Z histone exchange and a coactivator switch (to TIP60) for MYC target gene expression. The NE-specific coupling of MYCL with TIP60 reveals a previously unappreciated opportunity to target MYC-driven NE diseases through pharmacological inhibition of TIP60.

cancer biology↗

bioRxiv2026

Blood-to-tissue translation in autoimmune disease: paired single-cell evidence from systemic sclerosis

AO_SCPLOWBSTRACTC_SCPLOWThe biology that governs progression and therapeutic response in autoimmune disease is organized in affected tissue, but direct molecular readout of that biology requires invasive biopsy and is rarely repeated during clinical trials or routine care. Using paired blood-skin single-cell RNA-sequencing from a systemic sclerosis (SSc) cohort of 74 individuals (57 patients and 17 matched controls, 192,809 cells across 53 annotated cell states), we show that peripheral blood carries a recoverable projection of tissue-resident molecular state. Across 63 pathways scored in both compartments, 43 same-pathway blood-skin associations reach FDR < 0.05; at cell-type resolution, 212 cross-compartment associations survive residualization for disease status and sex. Per-patient classifiers recover tissue-defined molecular states out of fold with AUCs between 0.62 and 0.79, with the strongest recoveries on fibroblast subtype programs that have no direct circulating analog: fibroblast COMP at 0.79, COCH at 0.75, MYOC2 at 0.74, POSTN at 0.74. Tissue programs route through different blood compartments at different representational levels: fibroblast programs resolve through T-cell, Treg, monocyte and B-cell axes at compositional and distributional levels, while interferon resolves through expression state across multiple cell types. Within SSc alone, a cross-validated partial least squares model learns a shared blood-skin latent axis at r = 0.486 (permutation p = 0.006); the induced patient ranking recovers tissue-interferon-high patients at 86% precision at the top-20% screening threshold against a 50% base rate. A paired multiview autoencoder, trained on module-level dependency structure under contrastive alignment, paired reconstruction, neighborhood preservation and tissue-target supervision, learns a shared latent geometry in which blood-only projections land in the same tissue-state region as their matched tissue samples and supports recovery of held-out tissue targets above simpler baselines and above two permutation null families. These results map the empirical geometry of cross-compartment inference in autoimmune disease and position peripheral blood as a substrate for tissue-state inference at trial and clinical scale.

immunology↗

bioRxiv2026

Macrophage Antigen Presentation Is Unleashed by Pan-RAS Inhibition to Promote Antitumor Immunity

Pan-RAS inhibitors have emerged as potent targeted therapies designed to suppress oncogenic signaling in RAS-mutant tumors. However, whether these inhibitors possess therapeutic utility beyond tumors with intrinsic RAS dependency remains unclear. Here we show that the clinical-stage pan-RAS(ON) inhibitor RMC-6236 (Daraxonrasib) induces robust regression of NRAS-wild-type B16 tumors in immunocompetent mice, a response mediated by a mechanism independent of direct tumor-cell targeting. Instead, RMC-6236 acts by remodeling the tumor microenvironment, specifically enhancing the antigen-presenting capacity of tumor-associated macrophages through upregulated MHC-I expression. This anti-tumor efficacy is abrogated in T cell-deficient hosts, identifying a macrophage-dependent, T cell-mediated mechanism of control. Mechanistically, RMC-6236 does not directly block canonical RAS signaling in macrophages. Rather, it suppresses a Myc-Hdac2 epigenetic program, leading to enhanced histone H4 acetylation at the Nlrc5 locus and subsequent activation of the MHC-I master regulator NLRC5. Together, our findings reveal a macrophage-centered, epigenetically wired immune mechanism for pan-RAS inhibition that broadens the therapeutic scope of targeting the "undruggable" RAS pathway beyond tumor genotype towards systemic immune modulation.

immunology↗

bioRxiv2025

Exploratory Multi-Omics Profiling of Rheumatoid Arthritis Heterogeneity

Rheumatoid arthritis (RA) is a clinically heterogeneous disease with highly variable treatment responses despite widespread use of conventional and biologic disease-modifying therapies. Existing clinical and serological markers only partially explain this variability and are not routinely used to personalise drug choice. We hypothesised that integrated plasma proteomic- metabolomic profiling could capture dimensions of RA molecular heterogeneity that are invisible to standard phenotyping and highlight characteristic patterns of metabolic dysregulation. We profiled 384 proteins and 1,063 metabolites in 80 RA patients and 40 healthy controls. Unsupervised consensus clustering of the integrated omics matrix identified two stable molecular subtypes within the RA cohort. Anti-citrullinated protein antibody (ACPA) status, which in this cohort was evenly split overall (40 ACPA-positive, 40 ACPA-negative), was similarly distributed across the two subtypes, suggesting that the molecular groupings reflect heterogeneity orthogonal to conventional serology. Protein-metabolite association network analysis revealed a substantial reduction in association edges in RA versus controls, with pathway-specific effects: pronounced connectivity loss in amino-acid metabolism but paradoxical gains in inflammatory mediators including glutamate and sphingolipids. Preserved correlation strength among surviving edges is consistent with selective pathway disconnection rather than uniformly weakened coordination. These hypothesis-generating findings, limited by cross-sectional design, medication heterogeneity, and single-centre recruitment, suggest that RA comprises molecularly distinct subtypes not captured by standard serological categories and is associated with extensive metabolic network reorganisation. This multi-omics framework illustrates how network-level signatures could support future precision stratification efforts and motivates further investigation of metabolic network restoration as a potential therapeutic avenue.

immunology↗

bioRxiv2025

The free fatty acid 2 receptor regulates the activation potency of C5a and the sensitivity to the antagonist avacopan

The complement derived neutrophil chemoattractant C5a, is a potent activator of the neutrophil superoxide anion generating NADPH oxidase. An allosteric modulator specific for the free fatty acid 2 receptor increases the activating potency but not the efficacy of C5a. The allosteric modulator also decreases the inhibitory effect of the C5a receptor antagonist avacopan, suggesting that the NADPH oxidase is activated by two different signaling pathways downstream of the receptor for C5a. While the allosteric modulator affected the C5a-mediated activation of the NADPH oxidase, the C5a-induced rise in the intracellular concentration of free calcium ions was unaffected. The C5a receptor and the free fatty acid receptor belong to the family of G protein-coupled receptors family. Our results show that the activated C5a receptors generate signals that directly activate the NADPH oxidase and allosterically modulated free fatty acid receptors which secondarily generate signals that elicit NADPH oxidase activity. This is in line with an earlier described receptor transactivation model, by which the fatty acid receptor is activated by receptor downstream signals generated by several different neutrophil receptors to which we now add the receptor for C5a. In addition, the fatty acid receptor was higher ranked than the receptor for C5a, in the neutrophil receptor hierarchy. The dual receptor trans-regulatory effects, by which the receptor for C5a activates the fatty acid receptor and by which this receptor reduces the C5a response, represent new regulatory mechanisms of importance for the NADPH oxidase activity in neutrophils.

cell biology↗

bioRxiv2025

Mitochondrial DNA damage in substantia nigra parscompacta astrocytes exacerbates dopaminergic neuron lossin a 6-hydroxydopamine mouse model of parkinsonism

Parkinsons disease (PD) is the fastest growing neurological disorder with no known cure. Our ability to develop disease-modifying treatments that slow down the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is hindered by a dearth of knowledge on roles for non-neuronal elements such as astrocytes during PD pathogenesis. More specifically, the extent to which mitochondrial DNA (mtDNA) damage in SNc astrocytes contributes to SNc DA neuron loss during PD remains unknown. To address this knowledge gap, we utilized an adeno-associated virus (AAV) called Mito-PstI that expresses the restriction enzyme PstI as an approach to damage mtDNA in SNc astrocytes and assess the effect of astrocytic mtDNA damage on SNc DA neuron function and viability in mice. Mito-PstI-induced mtDNA damage in SNc astrocytes disrupted mitochondrial morphology, caused increased wrapping of SNc astrocytic processes around SNc DA neurons, and abnormally increased dopamine release by SNc DA neuron axonal terminals within the dorsolateral striatum (DLS). In addition, mice injected with Mito-PstI in the SNc showed increased spontaneous and apomorphine-induced rotations contralateral to the side with SNc Mito-PstI injections. In further experiments, we used a parkinsonian mouse model with low dose 6-hydroxydopamine (6-OHDA) injection into the DLS to show that Mito-PstI expression in SNc astrocytes caused a worsening of 6-OHDA-induced spontaneous contralateral rotational behavior, and exacerbated SNc DA neuron loss. These results suggest that mitochondria in SNc astrocytes are not only critical for the function of SNc DA neurons, but are also a new target for developing disease-modifying strategies against PD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/673287v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b4a98aorg.highwire.dtl.DTLVardef@e6e2fcorg.highwire.dtl.DTLVardef@402b93org.highwire.dtl.DTLVardef@ecdfd2_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsO_LIMito-PstI expression in mouse SNc astrocytes increases dopamine release C_LIO_LIMito-PstI expression in SNc astrocytes worsens contralateral rotational behavior in mice C_LIO_LIMito-PstI expression in SNc astrocytes increases DA neuron loss in 6-OHDA mice C_LI

neuroscience↗

bioRxiv2025

Characterization of membrane structures regulating primary ciliogenesis by quantitative isotropic ultrastructure imaging

The trafficking, docking, and fusion of membrane vesicles at the mother centriole (MC) are required to construct the primary cilium. Here, we determined the three-dimensional (3D) membrane ultrastructures, and associated proteins, involved in primary cilium assembly upstream of axoneme growth. Our work reveals that the enlargement of small vesicles docked to the MC is a key trigger for ciliogenesis progression, a process requiring the MC distal appendage protein CEP164. We show these vesicles subsequently fuse to form tubular C-shaped and an unprecedented toroidal membrane intermediates, which ultimately organize into the ciliary vesicle covering the MC distal end. The formation of these previously uncharacterized tubular membrane ciliogenesis intermediates is orchestrated by the membrane trafficking regulators EHD1 and RAB8, and requires the IFT-B complex protein IFT88. Remarkably, we show that EHD1, through its membrane tubulation function, regulates ciliogenesis progression by directly promoting CP110/CEP97 removal from the MC cap. The establishment of these tubular membrane structures is also associated with the recruitment of the ciliary gate transition zone proteins. This study changes the architectural framework for understanding ciliogenesis mechanisms and highlights the application of isotropic ultrastructure imaging and three-dimensional quantitative analysis in understanding membrane trafficking and organelle biogenesis mechanisms.

cell biology↗

bioRxiv2025

Pre-Sequencing Assessment of RNA-Seq Library Quality Using Real-Time qPCR

RNA sequencing (RNA-Seq) is an essential sequencing assay for studying transcriptome profiling. Ribosomal RNA (rRNA) comprises more than 80 - 90% of total cellular RNA, efficient rRNA removal is essential for accurately capturing the transcriptome, particularly to sequence low-abundance mRNAs. Inefficient rRNA removal during library preparation can result from variations in sample quality, preparation methods and handling. Estimating rRNA content in RNA-Seq libraries pre-sequencing is therefore challenging due to the absence of a reliable and cost-effective assessment method. This study addresses the issue by introducing a scalable qPCR-based assay targeting 18S rRNA to evaluate rRNA depletion efficiency pre-sequencing. qPCR efficiency was optimized using serial dilutions of Universal Human Reference (UHR) control and Ct thresholds were established using pilot data from 644 libraries. Following this optimization, analysis of 1,748 Total RNA-Seq libraries and 445 Poly A+ two widely used RNA-Seq library methods, demonstrated a strong correlation between 18S rRNA qPCR results and post-sequencing rRNA rates. This assay was also used to evaluate the performance of Oligo dT beads from four different vendors to enrich mRNA. This 18S rRNA qPCR assay is a cost-effective, scalable approach for reliably predicting rRNA read percentage in RNA-Seq libraries pre-sequencing. Method SummaryThe 18S rRNA-specific qPCR assay is a cost-effective method for evaluating rRNA depletion efficiency in RNA-Seq libraries prior to sequencing by targeting the 18S ribosomal RNA. qPCR efficiency was optimized, and Ct thresholds were set from pilot data and validated across 1,748 Total RNA and 445 Poly A+ RNA-Seq libraries. A Ct threshold of [≥]16 for Total RNA and Ct 13-14 or greater for Poly A+ libraries demonstrates a strong correlation between qPCR results and post-sequencing rRNA read percentages. The assay enables early identification of poorly depleted samples, reducing unnecessary sequencing costs. Additionally, the method was employed to benchmark oligo-dT beads from four vendors, showing its utility in evaluating RNA-seq library preparation kits. This scalable approach supports quality control in both research and high-throughput sequencing environments.

molecular biology↗

bioRxiv2025

Transdiagnostic Profiles of BOLD Signal Variability in Autism and Schizophrenia Spectrum Disorders: Associations with Cognition and Functioning

BackgroundAutism spectrum disorder (autism) and schizophrenia spectrum disorders (schizophrenia) exhibit overlapping social and neurocognitive impairment and considerable neurobiological heterogeneity. Blood-oxygen-level-dependent (BOLD) signal variability captures the brains moment-to-moment fluctuations, offering a dynamic marker of neural flexibility that is sensitive to cognitive capacity. This study aimed to examine intra-regional BOLD signal variability during rest and task across schizophrenia, autism, and typically developing controls (TDC) to explore transdiagnostic patterns of brain signal variability and their relationship with cognitive and functional outcomes. MethodsIntra-regional BOLD variability, measured by mean squared successive difference (MSSD), was obtained from resting-state and Empathic Accuracy task fMRI in 176 SSD, 89 autism, and 149 TDC participants. ANCOVAs, controlling for age, sex, and motion, assessed group differences in regional and network-level BOLD variability and dimensional associations with social cognition, neurocognition, social functioning, and symptom severity. ResultsBoth autism and schizophrenia exhibited lower BOLD signal variability than TDC across rest and task, with reduced variability observed in somatomotor, visual, and auditory networks (pFDR < 0.01). Greater network variability was positively associated with better social cognitive, neurocognitive, and functional scores across the sample. Resting-state variability showed stronger group-based differences and cognitive associations than task-based variability. ConclusionsBOLD signal variability is positively associated with social cognition, neurocognition, and social functioning across groups, suggesting that variability impacts cognitive efficiency and behaviour. Reduced variability in autism and schizophrenia may indicate similar patterns of neural rigidity among these related conditions, positioning BOLD variability as a potential biomarker for neural flexibility and a valuable target for future transdiagnostic clinical interventions.

neuroscience↗

bioRxiv2025

The SHR/Akr Y chromosome reveals repeated turnover of the rat pseudoautosomal region

Crossing-over between Chr X and Chr Y was first observed 90 years ago, in the brown rat, Rattus norvegicus. However, the sequence of the rat pseudoautosomal region (PAR) has remained a mystery. We produced a near-complete sequence of Chr Y from the SHR strain, along with nearly a megabase of sequence from both telomeres of Chr X. Both telomeric ends of Chr Y display extensive homology to Chr X, but no homology to the ancestral PAR of placental mammals. Using rat Y BACs probes for FISH on meiotic cells, we show that pairing almost always occurs between the tips of Yq and Xp, which are virtually identical in nucleotide sequence, but contain no protein-coding genes. Homology at the other ends of Chr X and Chr Y is likely the result of a recent transposition of five genes from Xq to Yp. These sequences are only 99.5% identical and pair infrequently, but show signs that it may have been pseudoautosomal in the ancestor of the rat genus. The rat Chr Y sequence presents opportunities for experimental studies of meiotic phenomena in a tractable model organism. The short PAR, with a correspondingly high recombination rate, represents a unique substrate for molecular studies of crossing-over. Likewise, the absence of extensively co-amplified testis gene families on the rat X and Y suggests they might serve as control for the intense competition between selfish elements that completely remodeled the mouse sex chromosomes.

genomics↗

bioRxiv2025

BRCA2 reversion mutation-independent resistance to PARP inhibition in prostate cancer through loss of function perturbations in the DNA pre-replication complex

Recent approvals of PARP inhibitors (PARPi) for BRCA-mutant metastatic castration resistant prostate cancer (mCRPC) necessitate an understanding of the factors that shape sensitivity and resistance. Reversion mutations that restore homologous recombination (HR) repair are detected in [~]50-80% of BRCA-mutant patients who respond but subsequently relapse, but there is currently little insight into why only [~]50% of BRCA-mutant patients display upfront resistance. To address this question, we performed a genome-wide CRISPR screen to identify genomic determinants of PARPi resistance in murine Brca2{Delta}/{Delta} prostate organoids genetically engineered in a manner that precludes the development of reversion mutations. Remarkably, we recovered multiple independent sgRNAs targeting three different members (Cdt1, Cdc6, Dbf4) of the DNA pre-replication complex (pre-RC), each of which independently conferred resistance to olaparib and the next generation PARP-1 selective inhibitor AZD5305. Moreover, sensitivity to PARP inhibition was restored in Brca2{Delta}/{Delta}, Cdc6-depleted prostate cells by knockdown of geminin, a negative regulator of Cdt1, further implicating the critical role of a functional pre-RC complex in PARPi sensitivity. Furthermore, [~]50% of CRPC tumors have copy number loss of pre-RC complex genes, particularly CDT1. Mechanistically, prostate cells with impaired pre-RC activity displayed rapid resolution of olaparib-induced DNA damage as well as protection from replication fork degradation caused by Brca2 loss, providing insight into how Brca2-mutant cancer cells can escape cell death from replication stress induced by PARP inhibition in the absence of HR repair. Of note, a pharmacologic inhibitor that targets the CDT1/geminin complex (AF615) restored sensitivity to AZD5305, providing a potential translational avenue to enhance sensitivity to PARP inhibition in BRCA-mutant cancers. SignificanceHere, we address a major limitation in the effectiveness of PARP inhibitors in BRCA-mutant prostate cancer treatment: only [~]50% of patients respond despite clear genomic evidence of defective homologous recombination. Prior efforts to study PARP inhibitor resistance in prostate cancer have been plagued by the lack of suitable cell lines. We overcame this challenge using primary prostate organoids coupled with genome-wide CRISPR screening. The key finding is that loss of function mutations in the DNA pre-replication complex confer PARP inhibitor resistance. These genes map to chromosomal regions frequently lost in prostate cancer and could therefore serve as potential biomarkers of treatment response. Pharmacologic inhibition of geminin, a negative regulator of the pre-replication complex, can restore PARP inhibitor sensitivity.

cancer biology↗

bioRxiv2025

Hyper-responsiveness of cancer stem cells to microenvironmental cues controls metastasis and therapy response through YAP/TAZ/TEAD

Cancer stem cells (CSCs) are key drivers of metastasis and therapy resistance but have been challenging to visualize and study in situ. Using a fluorescent CSC reporter, we observed very different population dynamics for CSCs and nonCSCs during metastatic lung colonization in breast cancer models. CSC expansive self-renewal drives early lesion formation before switching to a maintenance mode of balanced self-renewal and differentiation, whereupon nonCSC proliferation takes over as the main driver of metastatic expansion. Mechanistic analyses showed that CSCs are hyper-responsive to microenvironmental cues such as cell crowding and nutrient availability, suggesting a novel role for CSCs as sensors and early responders to fluctuating local conditions in the tumor. Incoming signals converge on YAP/TAZ/TEAD, with heightened CSC sensitivity and response supported by elevated receptor expression and increased chromatin accessibility around enhancers with TEAD binding sites. Targeting inputs to the YAP/TAZ/TEAD node reversed chemotherapy-induced enrichment of CSCs in lung metastases. HighlightsO_LIDifferent population dynamics for breast cancer stem cells (CSCs) and their differentiated progeny in early metastatic colonization C_LIO_LICSCs are hyper-responsive to microenvironmental cues and serve as sensors of local conditions for the tumor C_LIO_LIMany microenvironmental inputs converge on YAP/TAZ to regulate self-renewal vs differentiation fate decisions in the CSC C_LIO_LITargeting YAP/TAZ input pathways blocks chemotherapy-induced enrichment of CSCs C_LI

cancer biology↗

bioRxiv2024

The Alzheimers Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

Genetic variations in MS4A4A and MS4A6A are linked to the regulation of cerebrospinal fluid soluble TREM2 (sTREM2) levels and are associated with Alzheimers disease (AD) risk and progression. Using CRISPR knockout and MS4A4A-degrading antibodies in primary human microglia, non-human primates (NHP), and a xenotransplantation model of amyloid pathology, we provide evidence that MS4A4A and MS4A6A are negative regulators of both the transmembrane and soluble TREM2 proteins. They also negatively regulate microglia proliferation, survival, metabolism, lysosomal function, energetics, phagocytosis, and disease-fighting states. Mechanistically, we find that MS4A4A exerts negative regulation by interacting with MS4A6A and protecting it from degradation. MS4A6A in turn forms a complex with and blocks the co-receptor DAP12, which is required for the stability, cell surface localization, and signaling of TREM2 and other receptors. Taken together, the data indicate that MS4A4A and MS4A6A are cooperating, post-transcriptional negative regulators of TREM2 and microglial function, and potential drug targets for AD.

neuroscience↗

bioRxiv2024

Enhancing Gene Set Overrepresentation Analysis with Large Language Models

Gene set overrepresentation analysis is widely used to interpret high-throughput transcriptomics and proteomics data, but traditional methods rely on static, human-curated gene set databases that lack flexibility. We introduce llm2geneset, a framework that leverages large language models (LLMs) to dynamically generate gene set databases tailored to input query genes, such as differentially expressed genes (DEGs) and a biological context specified in natural language. These databases integrate with methods, such as overrepresentation analysis (ORA), to assign biological functions to input genes. Benchmarking against human-curated gene sets demonstrates that LLMs generate gene sets comparable in quality to those curated by humans. llm2geneset also identifies biological processes represented by input gene sets, outperforming traditional ORA and direct LLM prompting. Applying the framework to RNA-seq data from iPSC-derived microglia treated with a TREM2 agonist highlights its potential for flexible, context-aware gene set generation and improved interpretation of high-throughput biological data. llm2geneset is available as open source at https://github.com/Alector-BIO/llm2geneset and via a web interface at https://llm2geneset.streamlit.app.

bioinformatics↗

bioRxiv2024

Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples--a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants. IMPORTANCERespiratory syncytial virus (RSV) and human noroviruses (HuNoV) are NIAID category C and category B priority pathogens, respectively, that inflict significant health consequences on children, adults, immunocompromised patients, and the elderly. Due to the high strain diversity of RSV and HuNoV genomes, obtaining complete genomes to monitor viral evolution and pathogenesis is challenging. In this paper, we present the design, optimization, and benchmarking of a comprehensive oligonucleotide target capture method for these pathogens. All 85 RSV samples and 49/55 HuNoV samples were patient-derived with six human intestinal enteroids. The methodology described here results has a higher success rate in obtaining full-length RSV and HuNoV genomes, enhancing the efficiency of studying these viruses and mutations directly from patient-derived samples.

molecular biology↗

bioRxiv2024

Coordinated Tbx3 / Tbx5 transcriptional control of the adult ventricular conduction system

The cardiac conduction system (CCS) orchestrates the electrical impulses that enable coordinated contraction of the cardiac chambers. The T-box transcription factors TBX3 and TBX5 are required for cardiac conduction system development and associated with overlapping and distinct human cardiac conduction system diseases. We evaluated the coordinated role of Tbx3 and Tbx5 in the murine ventricular conduction system (VCS). We engineered a compound Tbx3:Tbx5 conditional knockout allele for both genes located in cis on mouse chromosome 5. Conditional deletion of both T-box transcriptional factors in the ventricular conduction system, using the VCS-specific MinK:Cre, caused loss of VCS function and molecular identity. Combined Tbx3 and Tbx5 deficiency in the adult VCS led to conduction defects, including prolonged PR and QRS intervals and elevated susceptibility to ventricular tachycardia. These electrophysiological defects occurred prior to detectable alterations in cardiac contractility or histologic morphology, indicative of a primary conduction system defect. Tbx3:Tbx5 double knockout VCS cardiomyocytes revealed a transcriptional shift towards non-CCS-specialized working myocardium, indicating a change to their cellular identity. Furthermore, optical mapping revealed a loss of VCS-specific conduction system propagation. Collectively, these findings indicate that Tbx3 and Tbx5 coordinate to control VCS molecular fate and function, with implications for understanding cardiac conduction disorders in humans.

developmental biology↗