Biology subjects

Moore, C.

Publications and source records attributed to Moore, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Computer-Assisted Systematic Chemical-Space Mapping of a First-in-Class Peripherally Restricted α2AAR Agonist through Scaffold-Seeded Enumeration

CC10137 is a first-in-class peripherally restricted 2A-adrenergic receptor (2AAR) agonist with broad-spectrum analgesic efficacy and a favorable safety profile. Systematic exploration of the chemical space surrounding first-in-class leads is important for defining series boundaries and guiding continued optimization, but conventional analogue-by-analogue medicinal chemistry samples only a small fraction of the accessible structural space. Here, we used a scaffold-seeded enumeration strategy to expand the chemical space surrounding CC10137 from four SAR-informed seed compounds comprising CC10137 and three closely related structural variants. Application of predefined medicinal chemistry transformation rules in StarDrop generated a virtual library of 16,601,163 unique structures. Morgan fingerprint-based principal component analysis indicated that the library occupied a highly multidimensional structural space involving variation in scaffold substitution, peripheral functional groups, and side-chain composition. A retrospective comparison set of 43 compounds independently designed and experimentally characterized in the earlier CC10137 program represented only approximately 0.00026% of the 16.6-million-member library, yet all 43 were recovered as exact structural matches. Three compounds selected directly from the virtual library retained 2AAR binding affinity and agonist potency below 25 nM. Five representative compounds further showed significant anti-allodynic effects in the in vivo spared nerve injury model, with inhibition rates ranging from 39.3% to 55.7%. These findings support scaffold-seeded computational enumeration as a practical strategy for systematic chemical-space mapping around a first-in-class lead and for identifying additional pharmacologically active structural regions for further optimization.

pharmacology and toxicology↗

bioRxiv2026

A quantitative coordinate system for developmental dynamics

Quantitative comparison of morphogenesis across individuals remains a fundamental challenge, as developing embryos vary in shape, orientation and developmental tempo. Moreover, real-time three-dimensional imaging generates large, heterogeneous four-dimensional datasets that are difficult to directly align. As a result, developmental variability is typically described qualitatively rather than measured. Here we introduce STERN, a quantitative framework that learns continuous spatiotemporal representations of morphogenesis directly from in vivo 4D imaging data. By embedding embryos into a shared spatiotemporal space, STERN defines a quantitative developmental coordinate system that enables direct comparison of developmental trajectories across individuals without requiring explicit registration or staging. Applied to mouse embryogenesis, STERN reveals that embryos follow conserved developmental trajectories while progressing at distinct temporal rates, providing a quantitative measure of developmental heterochrony. Extending this framework to zebrafish neural crest light-sheet timelapse imaging, we further show that developmental order is preserved across distinct imaging views even with altered anatomical coverage, supporting the generality of the learned representation across vertebrate imaging contexts. Finally, in developing mouse hearts, where morphogenesis proceeds through subtle and continuously evolving structural changes, STERN resolves fine-scale developmental dynamics at minute-scale temporal resolution that are difficult to localize reproducibly using human experts or general-purpose multimodal AI. Together, these results establish a shared quantitative coordinate system for morphogenesis, in which developmental trajectories become directly comparable across individuals and developmental variability becomes a measurable property.

bioinformatics↗

bioRxiv2026

An ancient polymorphism in myosin I a/b determines the left-right asymmetry of Japanese snails

Left-right (LR) asymmetry is a fundamental feature of animal development, yet progress in understanding its establishment has been limited by the fact that almost all animals exhibit invariant chirality. Snails are the exception because some wild-living species show abundant chiral variation, yet the existing knowledge is from snails in which the chirality mutation is rare and causes pathology. Here, we show that a chiral polymorphism in Japanese Euhadra snails is due to functional variation in an unconventional myosin I a/b, an isoform not previously implicated in LR specification in any animal. Both the sinistral and dextral gene variants are ancient, impart minimal transcriptional differences in the single-cell embryo and are not pathological. Phylogenetic and structural modelling suggests that dominant-acting amino acid substitutions in the myosin actin-binding domain and/or motor-level junction were enabled by relaxed selection. These results broaden the known molecular repertoire underlying LR asymmetry, suggest key mutations and positions that should be explored in other model animals, and highlight snails as a powerful model for understanding the origins of animal LR asymmetry.

evolutionary biology↗

bioRxiv2026

Precise measurement of rodent drinking using CLiQR (Capacitive Lick Quantification in Rodents)

Accurate quantification of rodent licking behavior is essential for studies of fluid intake, including investigations of alcohol use disorder and obesity. Lickometry systems vary widely in sensing modality, cost, scalability, and data resolution. Also, many available systems require specialized housing or store only binary lick/no lick data based on thresholding. Here, we present CLiQR (Capacitive Lick Quantification in Rodents), an open-source capacitive lickometry system designed for high-throughput recording of licking behavior in home-cage environments while preserving the full capacitance time series. The system uses MPR121 capacitive sensors connected to custom metal-tipped serological pipette sippers and a centralized desktop computer to record data from up to 24 mice concurrently, with capacity for two-bottle choice experiments. Validation experiments demonstrated that the capacitive signals reliably distinguish licking from non-licking interactions. Total lick counts showed a positive correlation with measured fluid consumption (R2 = 0.849, p < 0.0001), confirming that detected events provide a meaningful proxy for intake. All information necessary to reproduce the system is shared openly in this manuscript and online. By combining scalability, full-trace data acquisition, and low cost, CLiQR provides a flexible and extensible platform for high-throughput behavioral neuroscience experiments and enables retrospective improvement of lick-detection algorithms.

animal behavior and cognition↗

bioRxiv2026

Preferential formation of NUP98-KDM5A condensates at specific H3K4me3-rich loci drives leukemogenic gene expression

Chromosomal translocations involving NUP98 generate fusion proteins that alter gene expression programs, yet the fundamental principles governing their gene targeting and condensate behavior remain poorly understood. Using NUP98-KDM5A as a model, we integrate cellular imaging, in vitro reconstitution, and genomic analyses to dissect how chromatin engagement shapes condensate formation. We find that NUP98-KDM5A forms sub-diffraction-limited, gel-like condensates whose assembly is potentiated by binding to H3K4me3. This interaction creates a quantitative targeting mechanism in which, at the native expression level, condensates preferentially form at genomic loci with high local H3K4me3 density. Such local density-dependent recruitment explains selective enrichment at the leukemogenic HOX gene clusters, despite widespread presence of H3K4me3 across the genome. Analysis of single-cell sequencing data from patients further supports a correlation between local H3K4me3 density and transcriptional activation in NUP98-KDM5A-driven leukemia. Together, our findings reveal how activating chromatin marks and condensate-forming proteins synergize to generate specificity within euchromatin, offering a generalizable framework for understanding how chromatin-associated condensates interpret epigenetic landscapes.

cell biology↗

bioRxiv2026

Targeting CPSF73, the mRNA 3' End Processing Endonuclease, Moves Cancer Cells Away from the Mesenchymal State

BackgroundMetastasis significantly contributes to cancer-related mortality and therapeutic failure. Cancer cells acquire metastatic potential by losing epithelial characteristics and gaining mesenchymal properties through the epithelial-mesenchymal transition (EMT). Differential poly(A) site (PAS) usage, known as alternative polyadenylation (APA), generates mRNA isoforms differing in coding sequence, subcellular localization, stability, or translation efficiency. In cancer, 3'UTR shortening increases expression of proto-oncogenes by escaping miRNA-mediated repression. High expression of CPSF73, which cleaves mRNA precursors at PASs, is associated with unfavorable prognoses in cancer patients. However, the role of APA in regulating EMT remains poorly understood. MethodsIn this study, to investigate the role of APA in EMT, we employed JTE-607, a small-molecule inhibitor of CPSF73 activity, to examine the impact of catalytic inhibition of CPSF73 on proliferation and EMT in MDA-MB-231, MCF7, A549, and HepG2 cancer cells. To identify differential usage of PASs, global profiling of APA changes, and differential gene expression analysis were performed in MDA-MB-231 cells. Additionally, antisense oligonucleotides were used to block the use of a specific PAS whose APA change may be a driver of EMT reversal. ResultsOur findings showed that catalytic inhibition of CPSF73 not only attenuates cancer cell proliferation but also moves the cells away from the mesenchymal state across all four cell lines tested. Global profiling of APA changes following CPSF73 inhibition revealed widespread 3'UTR lengthening and suppression of intronic PASs in MDA-MB-231 cells. APA shifts were observed in key EMT-related genes, accompanied by decreased expression of corresponding proteins across all four cell lines. We used antisense morpholino oligonucleotides to block the proximal PAS of AKT2, shifting the balance of AKT2 mRNA isoforms toward the long isoform. This shift caused EMT reversal, marked by reduced AKT2 protein expression, changes in EMT-related markers, and impaired invasion by MDA-MB-231 cells. ConclusionTogether, these findings identify APA-mediated 3UTR lengthening, with functional consequences in EMT-related genes, as a coordinated mechanism leading to an attenuated EMT phenotype, highlighting a significant connection between APA and the EMT process. Interfering with these APA changes may offer a promising therapeutic strategy to suppress metastasis, with potential efficacy across multiple pathways. Statement of SignificanceOur findings highlight APA-mediated 3 UTR lengthening as a coordinated mechanism that promotes EMT reversal and support CPSF73 inhibition or APA-targeting strategies as potential therapeutic approaches to suppress metastasis across multiple pathways.

cancer biology↗

bioRxiv2026

The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

Macrophage antimicrobial programs are regulated not only by transcriptional networks but also by RNA processing mechanisms affecting signal transduction and effector responses. One such mechanism, alternative polyadenylation (APA), determines mRNA fate by changing the length of the 3' UTR. However, our understanding of the impact of APA on antibacterial functions and how we can manipulate it to influence infection outcomes remains limited. In this study, we identify the APA regulator CFIm25 (NUDT21) as a promoter of macrophage defense against Salmonella enterica serovar Typhimurium (STM). STM infection is known to drive macrophages toward an M2-like immunosuppressive state conducive to bacterial survival. Concurrent with this transition, the CFIm25 level is reduced, and the 3 UTRs of CFIm25 targets encoding key immune proteins, such as TAB2 and TBL1XR1, are lengthened, suggesting a role for APA changes in the response to STM. Overexpression of CFIm25 in infected macrophages blocks these Salmonella-induced 3 UTR changes, leading to greater mRNA and protein expression. Significantly, the increase in CFIm25 suppresses infection, thereby creating a more antimicrobial intracellular environment, improving macrophage survival, and reducing M2 properties that support bacterial replication. Specifically, CFIm25 enhances production of the antibacterial peptide LL-37, increases reactive oxygen species and nitric oxide levels, suppresses arginase activity and lactate production, and stimulates release of pro-inflammatory cytokines while inhibiting anti-inflammatory cytokines. Depletion studies show TAB2 mediates CFIm25s antibacterial effects by activating both MAPK and NF-kB pathways. Our findings highlight APA regulation as a potential target for boosting immune defenses and developing treatments for chronic bacterial infections.

immunology↗

bioRxiv2025

Oncogenic RAS activity is linked to immune priming and adenosine-driven immune evasion in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is a leading cause of cancer death worldwide, with RAS signalling as a key oncogenic driver. Although KRAS mutations have been linked to immune evasion in preclinical models, the relationship between RAS activity and tumour immunity or response to immunotherapy in patients remains unclear. Here, we applied our previously validated RAS84 transcriptional signature to stratify LUAD patient cohorts and dissect the immune landscape associated with RAS signalling. We report that tumours with elevated RAS activity exhibited features of immune priming, including increased immune infiltration, interferon response, and immune checkpoint gene expression, and showed improved progression-free survival in an independent cohort of patients treated with anti-PD-1. Yet, in both LUAD tumours and cell lines, RAS activity also correlated with elevated immunosuppressive interstitial adenosine mediated by transcriptional regulation of several components of the adenosinergic pathway. In orthotopic pre-clinical models of high-RAS activity lung tumours, blocking adenosine signalling delayed tumour growth and improved response to anti-PD-1 and KRAS inhibition, with a significant effect on innate immunity. This study reveals a dual role for RAS signalling in tumour progression, fostering a pro-immunogenic environment whilst simultaneously dampening anti-tumoural immunity via mechanisms including extracellular adenosine accumulation. Stratifying patients based on RAS transcriptional activity, rather than genetic alterations alone, could inform immunotherapy strategies and improve clinical outcomes.

cancer biology↗

bioRxiv2025

Leveraging death of drug-sensitive cancer cells to promote immune-mediated bystander killing of subclones of drug-resistant tumor cells

The response of lung cancer patients to drugs targeting the G12C mutant form of KRAS is limited by the development of resistance through multiple mechanisms. In order to achieve lasting benefit with these therapies, effective strategies for tackling the evolution of drug resistance are required. We have developed a preclinical model system to mimic the development of resistance to KRAS G12Ci inhibitors (G12Ci) such as adagrasib and RMC-4998. Treatment of tumours containing a minor subpopulation of resistant cancer cells with G12Ci leads to their rapid outgrowth to replace the drug-sensitive cells within a few weeks. However, when combined with therapies that, at least in part, target the immune response, such as SHP2 inhibitors or PD-1 blockade, drug-resistant cells can be eliminated, even by drug combinations that do not impact their growth in the absence of drug-sensitive cells. This bystander killing of drug-resistant cells when drug-sensitive cells are targeted is dependent on an intact adaptive immune system. Mechanistically, these combination therapies lead to profound remodelling of the tumour immune microenvironment, with influx of T cells recognising a tumour associated antigen shared between drug-resistant and drug-sensitive cancer cells. Promotion of immune-mediated bystander killing of drug-resistant cells may provide a paradigm for tackling the problem of drug resistance in cancer more broadly.

cancer biology↗

bioRxiv2025

Reducing Reactive Lipids Improves Cardiac Metabolic and Diastolic Function in Pulmonary Hypertension Models

BackgroundReactive oxygen species are increased across most pulmonary hypertension (PH) etiologies, resulting in increased reactive lipid dicarbonyls, which form protein adducts and impair mitochondrial function. We hypothesized that reducing reactive lipids would reduce right ventricular systolic pressure (RVSP) and improve cardiac function by eliminating protein-lipid damage feedback loops. MethodsWe used 2-hydroxybenzylamine (2-HOBA) to scavenge reactive lipids in three complimentary mouse models of PH: AKR-high fat diet (HFD, metabolic stress), LNAME-HFD (cardiometabolic syndrome), and pulmonary artery banding (PAB, load stress). Cardiac function was measured by echocardiography and catheterization. RV energy metabolism was determined by oxygraphy. Mass spectrometry analyzed lipids and ceramides; O-link and RNA-Seq evaluated proteomic and gene expression in lungs, RV, and LV. ResultsReducing reactive lipids with 2-HOBA resulted in a [~]10% reduction in RVSP, reduced diastolic dysfunction, reduced plasma lipids and ceramides, and normalized RV fatty acid oxidation that was severely impaired in the AKR-HFD and PAB models. Proteomic and RNA changes in the lungs, RV, and LV suggested reduced oxidative damage and inflammatory signaling and altered developmental and actin organization signaling; these changes are plausibly associated with the improved adaptation. Some changes were sex specific, including a 4x higher cardiac fatty acid content in males than females. ConclusionsReactive lipid scavenging improves cardiac metabolic and diastolic function and pulmonary vascular resistance through restoration of mitochondrial function and reduced oxidative protein damage. The magnitude of hemodynamic improvement combined with substantial diastolic function improvement suggests clinical potential, particularly for PH patients with metabolic comorbidities.

molecular biology↗

bioRxiv2025

Divergent Cell-Type Specific Hypoxia Responses in Human Stem Cell-Derived and Primary Islets

BackgroundThe success of stem cell-derived islet (SC-islet) therapy for type 1 diabetes is limited by poor graft survival in the hypoxic post-transplantation microenvironment. While the response of SC-islets to chronic hypoxia has been studied, a direct comparison to primary human islets during the acute hypoxic phase has not been performed. Here, we conduct a comparative single-cell transcriptomic and functional analysis of human SC-islets and primary islets exposed to acute hypoxia (1% O2) over 48 hours. ResultsOur analysis reveals two divergent response patterns. Primary islets exhibit an energy-conserving response, characterized by a {beta}-cell-specific suppression of identity genes (PDX1, MAFA) and pro-apoptotic factors like DDIT3, alongside a shift toward metabolic quiescence. In contrast, the SC-islet response is characterized by lineage instability, a significant metabolic shift toward glycolysis, and the activation of pro-apoptotic pathways. Functionally, these transcriptomic differences result in a loss of glucose-stimulated insulin secretion in both islet types, but through different mechanisms: a suppression of secretion in primary islets versus dysregulated, glucose-unresponsive insulin release in SC-islets. ConclusionThese findings demonstrate that SC-islets are particularly vulnerable under hypoxic stress, exhibiting an unstable, plastic phenotype. This comparative dataset provides a resource for developing source-specific therapeutic interventions to overcome the hypoxic barrier and improve the efficacy of cell replacement therapies.

cell biology↗

bioRxiv2025

CFIm25-Dependent Alternative Polyadenylation in AKT2 mRNA Programs Macrophage Polarization

Macrophage polarization is essential for immune responses, tissue homeostasis, and progression of many diseases. It is a tightly regulated process involving an intricate network of signaling pathways and control mechanisms at the level of transcription, alternative mRNA splicing, translation and mRNA stability. However, regulation through alternative mRNA polyadenylation (APA), remains poorly understood. This study explores the function of CFIm25, a key APA regulator, in macrophage polarization. Our findings show that CFIm25 overexpression drives M1 polarization, as evident from increased nitric oxide synthase activity, CD80 expression, and pro-inflammatory cytokine secretion, but dampens the M2 phenotype. Conversely, CFIm25 knockdown suppresses M1 traits and promotes M2 characteristics. Functionally, CFIm25 enhances phagocytosis, migration, and cancer cell inhibition. Mechanistically, CFIm25 favors proximal polyadenylation site usage of AKT2 mRNA, increasing Akt2 protein levels to support M1 polarization. Blocking this site with an antisense oligonucleotide reduces Akt2 expression and M1 traits. These findings establish CFIm25 as a crucial regulator of macrophage identity, offering insights into RNA-based immune regulation and potential therapeutic targets. In briefCFIm25 drives macrophage polarization toward the M1 phenotype through specific regulatory pathways. The presence of CFIm25 profoundly shifts surface marker expression, enhancing M1 markers while suppressing M2 signatures. This extends to biochemical properties, where CFIm25 boosts nitric oxide and pro-inflammatory cytokine production while reducing anti-inflammatory mediators. Functionally, CFIm25 enhances phagocytosis, inflammatory migration, and cancer cell killing. Mechanistically, this orchestration of the M1 polarization program involves CFIm25s regulation of AKT2 mRNA alternative polyadenylation which increases Akt2 protein expression and amplifies NF-{kappa}B pathway activation, a central driver of M1 polarization. HighlightsO_LICFIm25 overexpression enhances M1 surface markers and biochemical properties including nitric oxide production and pro-inflammatory cytokine secretion. C_LIO_LICFIm25 promotes M1 functional activities including phagocytosis, migration, and cancer-cytotoxic effects. C_LIO_LIBlocking the AKT2 proximal polyadenylation site usage causes a decrease in Akt2 protein, suppressing M1 polarization phenotypes including NOS activity and cytokine profiles. C_LIO_LIThe CFIm25-Akt2-NF-{kappa}B axis represents a novel target for macrophage polarization reprogramming. C_LI O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/660629v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@15e28f6org.highwire.dtl.DTLVardef@d35314org.highwire.dtl.DTLVardef@1f382feorg.highwire.dtl.DTLVardef@1d083d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Alternative Polyadenylation Releases PCBP1-Mediated Suppression of CFIm25 During Macrophage Differentiation

CFIm25, a key component of the CFIm complex needed for mRNA 3 end processing, shows increased protein expression during monocyte-to-macrophage differentiation despite stable mRNA levels. We demonstrate that PCBP1 suppresses CFIm25 translation in monocytes by binding to its long 3UTR. During differentiation, alternative polyadenylation generates a shorter CFIm25 3UTR lacking PCBP1 binding sites. RNA immunoprecipitation confirms PCBP1 binding to the long 3UTR, while ribosome association analysis shows enhanced translation without this interaction. PCBP1 knockdown increases CFIm25 protein specifically in undifferentiated cells and induces macrophage differentiation markers without stimulation. These findings reveal how alternative polyadenylation controls CFIm25 expression during immune cell differentiation by modulating RNA-binding protein interactions and provide insight into post-transcriptional regulation of RNA processing factors.

molecular biology↗

bioRxiv2025

Multi-cohort, multi-sequence harmonisation for cerebrovascular brain age

IntroductionHigher brain-predicted age gaps (BAG), based on anatomical brain scans, have been associated with cognitive decline among elderly participants. Adding a cerebrovascular component, in the form of arterial spin labelling (ASL) perfusion MRI, can improve the BAG predictions and potentially increase sensitivity to cardiovascular health, a contributor to brain ageing and cognitive decline. ASL acquisition differences are likely to influence brain age estimations, and data harmonisation becomes indispensable for multi-cohort brain age studies including ASL. In this multi-cohort, multi-sequence study, we investigate harmonisation methods to improve the generalisability of cerebrovascular brain age. MethodsA multi-study dataset of 2608 participants was used, comprising structural T1-weighted (T1w), FLAIR, and ASL 3T MRI data. The single scanner training dataset consisted of 806 healthy participants, age 50{+/-}17, 18-95 years. The testing datasets comprised four cohorts (n=1802, age 67{+/-}8, 37-90 years). Image features included grey and white matter (GM/WM) volumes (T1w), WM hyperintensity volumes and counts (FLAIR), and ASL cerebral blood flow (CBF) and its spatial coefficient of variation (sCoV). Feature harmonisation was performed using NeuroComBat, CovBat, NeuroHarmonize, OPNested ComBat, AutoComBat, and RELIEF. ASL-only and T1w+FLAIR+ASL brain age models were trained using ExtraTrees. Model performance was assessed through the mean absolute error (MAE) and mean BAG. ResultsASL feature differences between cohorts decreased after harmonisation for all methods (p<0.05), mostly for RELIEF. Negative associations between age and GM CBF (b=-0.37, R2=0.13, unharmonised) increased after harmonisation for all methods (b<-0.42, R2>0.12) but weakened for RELIEF (b=-0.28, R2=0.14). In the ASL-only model, MAE improved for all harmonisation methods from 11.1{+/-}7.5 years to less than 8.8{+/-}6.2 years (p<0.001), while BAGs changed from 0.6{+/-}13.4 years to less than -1.03{+/-}7.92 years (p<0.001). For T1w+FLAIR+ASL, MAE (5.9{+/-}4.6 years, unharmonised) increased for all harmonisation methods non-significantly to above 6.0{+/-}4.9 years (p>0.42) and significantly for RELIEF (6.4{+/-}5.2 years, p=0.02), while BAGs non-significantly differed from -1.6{+/-}7.3 years to between -1.3{+/-}4.7 and -2.0{+/-}8.0 years (p>0.82). In general, the ASL-specific parameter harmonisation method AutoComBat performed nominally best. DiscussionHarmonisation of ASL features improves feature consistency between studies and also improves brain age estimations when only ASL features are used. ASL-specific parameter harmonisation methods perform nominally better than basic mean and scale adjustment or latent-factor approaches, suggesting that ASL acquisition parameters should be considered when harmonising ASL data. Although multi-modal brain age estimations were improved less by ASL-only harmonisation, possibly due to weaker associations between age and ASL features compared to T1w features feature importance, studies investigating pathological ASL-feature distributions might still benefit from harmonisation. These findings advocate for ASL-parameter specific harmonisation to explore associations between cardiovascular risk factors, brain ageing, and cognitive decline using multi-cohort ASL and cerebrovascular brain age studies.

neuroscience↗

bioRxiv2025

Single Cell Transcriptome Signatures of Sarcoidosis in Lung Immune Cell Populations

RationaleTo identify cell specific molecular changes associated with sarcoidosis risk and progression, we aimed to characterize the cellular composition, gene expression patterns, and cell-cell interactions in BAL cells from patients with sarcoidosis (both progressive and non-progressive) and healthy controls. MethodsSingle cell RNA-seq data were collected on 12 sarcoidosis and 4 control participants. We combined scRNA-seq data from these participants with our previously collected data on 4 sarcoidosis and 10 control participants for a final sample size of 16 sarcoidosis cases (8 progressive and 8 non-progressive) and 14 controls. Following initial preprocessing in CellRanger, data were quality controlled, combined, and clustered in Seurat. We tested differences in cell proportions by disease group using F-tests on cell proportions and differences in gene expression using pseudobulk analysis. Cell to cell communication and pathway analysis were performed using CellChat. ResultsWe identified five macrophage populations: resident, high metallothionein (MT) resident, recruited, profibrotic recruited, and proliferating macrophages. Each subpopulation displayed unique gene expression profiles, with notable differential expression of genes and pathways linked to sarcoidosis in resident macrophages, recruited macrophages, and proliferating macrophages. We also observed changes in gene expression associated with disease progression in resident and recruited macrophages. In non-macrophages cells, we observed a significant reduction in the number of B cells in sarcoidosis patients compared to controls. Among T cell populations, we identified specific transcriptional alterations at gene and pathway level. Additionally, we observed distinct differences in cell-to-cell interactions of macrophages and T cells between sarcoidosis patients and healthy controls. ConclusionsThese findings underscore the complexity of immune cell involvement in sarcoidosis and highlight potential cellular and molecular targets for further investigation.

genomics↗

bioRxiv2025

Transcriptional responses to prolonged oxidative stress require cholinergic activation of G-protein-coupled receptor signaling

Organisms have evolved protective strategies that are geared toward limiting cellular damage and enhancing organismal survival in the face of environmental stresses, but how these protective mechanisms are coordinated remains unclear. Here, we define a requirement for neural activity in mobilizing the antioxidant defenses of the nematode Caenorhabditis elegans both during chronic oxidative stress and prior to its onset. We show that acetylcholine-deficient mutants are particularly vulnerable to chronic oxidative stress. We find that extended oxidative stress mobilizes a broad transcriptional response which is strongly dependent on both cholinergic signaling and activation of the muscarinic G-protein acetylcholine coupled receptor (mAChR) GAR-3. Gene enrichment analysis revealed a lack of upregulation of proteasomal proteolysis machinery in both cholinergic-deficient and gar-3 mAChR mutants, suggesting that muscarinic activation is critical for stress-responsive upregulation of protein degradation pathways. Further, we find that GAR-3 overexpression in cholinergic motor neurons prolongs survival during chronic oxidative stress. Our studies demonstrate neuronal modulation of antioxidant defenses through cholinergic activation of G protein-coupled receptor signaling pathways, defining new potential links between cholinergic signaling, oxidative damage, and neurodegenerative disease.

neuroscience↗

bioRxiv2024

Use of Antibody Structural Information in Disease Prediction Models Reveals Antigen Specific B Cell Receptor Sequences in Bulk Repertoire Data

Convergent antibodies are highly similar antibodies elicited in multiple individuals in response to the same antigen. Convergent antibodies provide insight into shared immunological responses and show great promise as diagnostic biomarkers. They have typically been identified using methods that consider the amino acid sequence of the third complementarity-determining region (CDR3) of immunoglobulin heavy chain (IgH). In this study, we extend the definition of convergent antibodies to use structural information about the three IgH CDR regions (CDR1-3). We benchmark the performance of both definitions of convergence by their ability to predict disease status from bulk IgH sequencing data for two different diseases (HIV infection and food sensitization). We show that using predicted structural information outperforms prior approaches for the prediction of food sensitization status and performs on par for HIV infection status. Additionally, the structurally convergent antibody groups driving HIV prediction are from known HIV binders. Thus, the use of structural information allows for the identification of antigen specific antibody groups from bulk IgH sequencing data.

immunology↗

bioRxiv2024

Alternative mRNA Splicing and Polyadenylation Events are Synchronous with Podocyte Injury and Slit Diaphragm Dysregulation in Glomerular Disease Models

BackgroundGlomerular disease, characterized by podocyte injury and proteinuria, can lead to chronic kidney disease and end stage kidney disease. We hypothesized that the glomerular pathophysiology is associated with mRNA alternative splicing and polyadenylation of glomerular genes and of critical podocyte and slit diaphragm components that regulate the filtration barrier. MethodsGlomerular damage, accompanied by proteinuria, was induced by puromycin-aminonucleoside or adriamycin to mimic human minimal change disease or focal segmental glomerulosclerosis (FSGS), respectively, and RNA-seq analyses was performed. Alternatively spliced and alternatively polyadenylated events through differential exon and poly(A) site usage were queried in JunctionSeq and APATrap pipelines. These events were further mapped on podocyte and glomerular landscape, analyzed and modulated for slit diaphragm components, and cis- and trans-regulatory elements were identified. ResultsAltered glomerular mRNA processing by alternative splicing/polyadenylation was identified in 136/71 and 1875/746 genes in minimal change disease and FSGS models, respectively. Transcript annotation and prioritization of significant alternative splicing and polyadenylation identified key events in several podocyte and slit diaphragm genes with novel and established roles. Alternative splicing of critical slit diaphragm components, the junction protein TJP1/ZO1 and microtubule associating protein ITM2B was further characterized. Alternative polyadenylation of core members of the slit diaphragm, NPHS1, NPHS2 and NEPH1 was analyzed with potential alteration of microRNA binding sites between the proximal vs distal poly (A) site usage in their mRNAs. Concomitantly, dysregulation of trans-regulatory elements (polyadenylation and splicing factors), was discovered in these models of nephropathies. Additionally, beneficial proteinuria-reducing treatments, pioglitazone and GQ16 reversed many alternatively spliced and polyadenylated events. Moreover, GWAS SNPs as potential cis-regulatory elements were identified in several genes from the human nephrotic syndrome database. Finally, we demonstrated proof-of-concept principle of chemically modified splice switching oligonucleotides in modulating TJP1 alternative splicing in podocytes. ConclusionFindings from our studies identified that glomerular pathophysiology and disruption of the filtration barrier is associated with alternative splicing and alternative polyadenylation of glomerular genes, many of which are crucial determinants of podocyte structure and function and the slit diaphragm complex. Key PointsO_LIAlternative mRNA processing adds an important layer of transcriptome complexity in podocyte and slit diaphragm components during glomerular injury. C_LIO_LITrans- and cis-elements may offer potential mechanisms to regulate alternative splicing and polyadenylation during podocyte injury. C_LIO_LIAntisense oligonucleotides can regulate mRNA processing events, thus offering a potential RNA-based therapeutic strategy to treat glomerular disease. C_LI

molecular biology↗