Biology subjects

Huang, A.

Publications and source records attributed to Huang, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Robust and accessible in situ sequencing with an open-source automated sequencer

Targeted in situ sequencing underlies many spatial transcriptomic, barcoded connectomic, and multiplexed perturbation techniques. Each experiment requires many cycles of chemical reactions and imaging that are time-consuming and error-prone to perform manually, and few commercial platforms can sequence highly diverse RNA barcodes in situ at cellular resolution across large tissue sections. Here we present Seq-o-Matic, an open-source automated in situ sequencer built entirely from commercially available components, which integrates fluidics, heated incubation, and imaging of up to three continuous 18 mm x 50 mm areas on standard microscope slides. Relative to manual sequencing, Seq-o-Matic reduced hands-on time 6- to 17.5-fold and total run time about 2.6-fold. Across three runs spanning two mouse strains and a neonatal marmoset, sequencing quality, measured by signal retention, channel specificity, and phasing, was similar to that of four manually sequenced mouse brains. In four mouse hemispheres sequenced in separate runs (two C57BL/6J, one NOD, and one WSB), consensus cell types were present in every brain and corresponded to those of a manually sequenced C57BL/6J brain. Within cortical excitatory neurons, the two C57BL/6J brains were more similar to each other than were brains of different strains, suggesting that technical variation between runs was small relative to strain differences. Seq-o-Matic thus makes consistent, high-throughput in situ sequencing accessible to laboratories without specialized in situ sequencing expertise.

genomics↗

bioRxiv2026

UnibindMD: An Integrated AI and Molecular Dynamics Platform for De Novo Peptide Binder Design

UnibindMD is an integrated computational platform that turns a protein target into validated de novo peptide-binder leads by chaining five orthogonal stages: deep-learning binder design (BindCraft), independent structure prediction and confidence assessment (AlphaFold3-class / Boltz-2), binding-affinity prediction (PRODIGY), developability profiling (ADMET-AI), and all-atom molecular-dynamics validation (GROMACS/CHARMM36m). The platform targets a persistent weakness of single-method design, namely that confidence metrics such as ipTM and pLDDT are self-consistent but not independent, so a design predicted to be confident may still be physically unstable. We demonstrate UnibindMD on three Andes virus (ANDV) intervention points, a setting of urgent need given a case fatality rate approaching 40%, a 2026 MV Hondius cruise-ship outbreak (13 cases, 3 deaths), and no approved therapeutic: the Gn-Gc glycoprotein complex (viral entry), the L-protein endonuclease domain (cap-snatching; PDB 6Q99), and the host VEGFR2 kinase (vascular permeability; PDB 2XIR). Across nine BindCraft campaigns (4,475 trajectories) the platform produced 464 accepted binders (acceptance 2.3-67.6%), with top candidates reaching ipTM = 0.94 and pLDDT = 0.94. The three selected leads were predicted by PRODIGY to bind with G = -9.1 to -11.1 kcal mol-1 (Kd 8 nM-0.2 M), and all three remained stably bound over 100 ns of unrestrained molecular dynamics (receptor CRMSD [≤] 2.6 [A], retained peptide fold, [≥] 99.5% of frames preserving more than half of the initial interface contacts, and 75-82% helical content). ADMET-AI flagged the expected poor oral bioavailability of large polar peptides and no PAINS alerts. By converting single-prediction design into a cross-validated, dynamics-tested workflow, UnibindMD provides a rapid, scalable, and generalizable path from any protein target to validated peptide-binder candidates.

bioinformatics↗

bioRxiv2026

A Human Genome-wide CRISPR-Cas9 Screen Reveals Host Factors That Support Epstein-Barr Virus B cell infection

Epstein-Barr virus (EBV) infects 90% of the population worldwide and is associated with multiple malignancies, including Burkitt, Hodgkin and post-transplant lymphomas, nasopharyngeal and gastric carcinoma. EBV also triggers autoimmune diseases, including multiple sclerosis and systemic lupus erythematosus. While host factors important for EBV B-cell entry have been defined, including CD21/CR2, MHC class II and R9AP, much remains to be learned about host factors that support post-entry steps of EBV infection. To gain insight, we conducted a human genome-wide CRISPR-Cas9 screen. In addition to known EBV B cell receptors/co-receptors, we identified multiple new factors and pathways essential for EBV B cell infection. These included multiple B cell signaling pathways, actin cytoskeleton and nuclear import factors. Knockout of CD19, its tetraspanin chaperone CD81 or karyopherin subunit alpha 1 (KPNA1, also called importin-5) significantly impaired establishment of EBV infection. EBV and CD19 co-localized at early stages of EBV infection, and the CD19 cytoplasmic tail was important for EBV infection, suggesting CD19 signaling supports EBV uptake. Depletion of KPNA1 significantly impaired establishment of EBV infection, but did not impair production of infectious EBV upon reactivation of latently infected B cells. Together, these results provide insights into pathways that support EBV B cell infection and identify potential therapeutic targets.

microbiology↗

bioRxiv2026

Extrachromosomal Circular DNA Function as Mobile Elements to Alter the Mammalian Germline Genome

The viability of any species including the human requires that the germline genome is kept stable as it is transmitted across generations by the germ cells. Failure to safeguard the genome integrity and stability would lead to inherited diseases and infertility. Thus, a better understanding of the mechanisms that alter the germline genome is crucial to ensure human health and our continuation as a species. Here, we show that the extrachromosomal circular DNA (eccDNA) in the mouse and human male germline represents a new mechanism in altering the mammalian germline genome. To enable the tracking of germline eccDNA in vivo, we established a novel mouse model that allows the generation of a reporter eccDNA in a cell type-specific manner. Using this mouse model, we showed that eccDNA formed in the developing male germ cells can integrate into the germline genome. Using eccDNA-containing sperm for in vitro fertilization led to the eccDNA sequence being inherited by the embryos. By analyzing a large cohort of long-read whole genome sequencing data, we showed that eccDNA-mediated germline genome insertions represent an important source of human genome structural variations. Finally, by leveraging human sperm samples, we found that diabetes induces an increase in sperm eccDNA quantity, which is mediated at least in part through poly (ADP-ribose) polymerases. Together, our results provide new insights into how the mammalian germline genome can be altered, with important implications for human health and genome evolution.

developmental biology↗

bioRxiv2026

PromptBio: An Agentic Platform for End-to-End Computational Biomedical Research

Modern biomedical research increasingly depends on complex computational analyses, yet translating a scientific question into a reliable workflow still requires substantial technical expertise and manual coordination. PromptBio is a multi-agent AI platform available through a web portal at https://promptbio.ai that addresses this challenge. Through natural-language interaction, its agent harness, PromptGenie, translates a research objective into an inspectable plan, executes the required research and analysis, and adapts subsequent steps as evidence and results emerge. PromptBio integrates reasoning with managed execution while preserving human oversight and a traceable record of the research process. It can apply validated methods, construct custom analyses, and incorporate external workflows, enabling researchers to move beyond fixed pipelines while supporting reproducibility. We evaluate the platform through benchmarks of end-to-end bioinformatics analysis and biomedical deep research, validation of representative omics and machine-learning skills, and a hypothesis-driven regulatory-genomics case study. PromptGenie achieved higher analytical accuracy and stronger evidence retrieval and synthesis than the comparison agents, while the evaluated domain skills produced results consistent with established methods. The case study further demonstrates how PromptBio can integrate human-specific genomic and epigenomic features to investigate cortical development. Together, these findings show that PromptBio can coordinate complex biomedical analyses under researcher oversight, offering an accessible approach for accelerating scientific iteration and transforming research questions into transparent, reusable computational workflows.

bioinformatics↗

bioRxiv2026

Time-Resolved Phenotypic and Transcriptomic Responses of Primary Canine Dermal Fibroblasts to Prolonged Hypothermic Stress

Prolonged low-temperature exposure may extend the preservation window of mammalian cells but can also disrupt cellular homeostasis and ultimately compromise cell viability. This study investigated the time-dependent phenotypic and transcriptomic responses of primary canine dermal fibroblasts to sustained hypothermic stress. Passage-three fibroblasts were continuously maintained at 15 for up to 15 days, with samples collected on Days 0, 3, 6, 9, 12, and 15. Cellular morphology, metabolic activity and viability, and apoptosis were evaluated using bright-field microscopy, Cell Counting Kit-8 assays, and Annexin V-FITC/propidium iodide flow cytometry, respectively. RNA sequencing was performed to characterize dynamic transcriptional changes throughout the exposure period. Early low-temperature exposure was associated with relatively preserved cellular morphology and viability, suggesting a transient adaptive response. With increasing exposure duration, fibroblasts exhibited progressive morphological deterioration, reduced metabolic activity, loss of adhesion, and increased apoptosis. Time-series transcriptomic analysis further revealed temporally coordinated and stage-dependent gene-expression programs associated with metabolic regulation, cellular stress responses, structural homeostasis, and cell survival. Integration of phenotypic and transcriptomic data demonstrated that the response of primary canine dermal fibroblasts to 15 was dynamic rather than linear, progressing from early adaptation to cumulative dysfunction during prolonged exposure. These findings provide a framework for defining the low-temperature tolerance of primary canine dermal fibroblasts and may inform the optimization of protocols for their short- to medium-term preservation and transportation.

cell biology↗

bioRxiv2026

Dietary fiber intake associates with improved survival and microbiomecomposition in allogeneic hematopoietic cell transplantation

Diet is linked to changes in gut microbiota and metabolite production with clinical relevance in several disease settings, although these effects remain poorly defined. We performed prospective, real-time diet monitoring (37,929 food items, 3,837 patient days) and longitudinal microbiome and metabolite profiling (1,230 fecal samples) in a clinical cohort of 173 patients undergoing allogeneic hematopoietic cell transplantation. Patients with pre-transplant fiber intake above the cohort average had significantly improved overall survival (p=0.014) and reduced incidence of grades 2-4 acute graft-versus-host disease (GVHD) (p=0.032) post-transplant. Those consuming insoluble fiber had increased microbial diversity, enriched butyrate-producing taxa, and depleted Enterococcus. Those who developed lower gastrointestinal GVHD had reduced fecal butyrate levels. In a GVHD preclinical model, we confirmed that a fiber-enriched diet increased survival, cecal butyrate, and regulatory-to-conventional T cell ratio. Thus, we demonstrated that dietary fiber has clinical significance as a modifiable factor with microbiome-mediated effects.

cancer biology↗

bioRxiv2026

Type I Interferon-Driven Monocyte Dysregulation and MAS-associated CD8+ T cells During Macrophage Activation Syndrome

Macrophage activation syndrome (MAS) is driven by a hyperinflammatory response characterized by aberrant activation of lymphocytes and phagocytes. While monocytes and macrophages are thought to be important in MAS pathogenesis, their role remains poorly understood. We used bulk and single-cell RNA sequencing (RNA-Seq) on sorted monocytes from children with MAS and healthy controls to identify transcriptional changes during MAS. We defined a MAS signature in classical monocytes that correlated with ferritin and was elevated in monocytes from systemic lupus erythematosus and COVID-19 patients. We also identified a subset of classical monocytes with high levels of interferon-stimulated genes (ISGs) that expanded during MAS. Surprisingly, the transcriptional signature of these cells was driven by type I IFNs, rather than IFN{gamma}. Consistent with this finding, we detected increased levels of circulating IFN{beta} during MAS, suggesting that IFN{beta} plays an unrecognized role in driving MAS monocyte responses. We also identified a MAS-associated CD8+ T cell population with a distinctive transcriptional signature. We used cell-cell communication algorithms to predict increased immunoregulatory interactions between monocytes and T cells during MAS. Together, these results provide new evidence for a role for type I IFN during MAS and identify a unique CD8+ T cell population that may contribute to MAS pathophysiology.

immunology↗

bioRxiv2026

TKI-Tolerant Persisters Emerge from a PKCα-Dependent and Highly Plastic Subpopulation of Stem-Like Cells in NSCLC

Reversible drug-tolerant persister states are emerging as key drivers of limited therapeutic durability, offering a complementary non-genetic perspective distinct from traditional models of acquired resistance. This is of particular interest in lung adenocarcinoma where EGFR tyrosine kinase inhibitors (TKIs) elicit dramatic responses, yet residual surviving cells persist and ultimately seed relapse. To define mechanisms that enable survival during this earliest residual-disease phase, we focused on the drug-tolerant persister population that remains after EGFR TKI exposure and can later give rise to outgrowth. Initial observations of elevated transcript levels of PRKCA, which encodes PKC, in established TKI-resistant models, together with markedly delayed tumor relapse following PKC suppression in vivo, nominated PKC as a candidate regulator of the persister-to-relapse transition. Genetic ablation of PRKCA or its inhibition with enzastaurin reduced residual survival and outgrowth after TKI exposure, indicating that PKC functions as an early dependency of drug-tolerant persisters rather than as a general mediator of acquired resistance. Mechanistically, PKC was required for persister-associated EMT, migratory capacity, and robust induction of ALDH1A1, the latter constraining oxidative stress and enhancing persister survival. Functionally, PKC was specifically necessary for survival of a rare, pre-existing CD44High stem-like subpopulation that exhibited marked plasticity and ultimately seeded persistence. Together, these data identify a PKC-dependent EMT/stemness/ROS pathway as a critical survival program in EGFR TKI-tolerant persister cells and support therapeutic strategies aimed at eliminating residual disease to prolong clinical responses.

cancer biology↗

bioRxiv2026

MyD88 deficiency modestly attenuates disease in a Leigh syndrome mouse model while enrofloxacin accelerates disease

Primary genetic mitochondrial diseases (GMDs) are a clinically and genetically diverse group of diseases estimated to impact over 1 in 4,000 individuals. Leigh syndrome (LS) is the most common pediatric presentation of GMD. LS typically presents within the first years of life and is a severe progressive multi-system disorder. Symmetric progressive inflammatory brain lesions are a defining feature of the disease. Patients can also present with seizures, metabolic dysfunction, muscle weakness, and other symptoms. No effective clinical treatments currently exist. Recent data from the Ndufs4(-/-) mouse model shows that peripheral macrophages contribute to brain lesions in LS, that disease is causally driven by innate immune populations, and that depletion of innate immune cells prevents LS disease. However, the precise mechanisms underlying immune activation remain unknown. Certain mitochondrial macromolecules retain bacterial signatures and can act as potent agonists for innate immune pathways. For example, cytoplasmic mitochondrial RNA and mitochondrial DNA are detected by Toll-like receptors (TLRs) 7 and 9, respectively, at the endosome. Accordingly, these are considered strong candidates for mediating innate immune activation in LS. Here, we generated TLR signaling deficient Ndufs4(-/-)/MyD88(-/-) animals to assess whether TLR signaling plays a role in disease onset or progression in LS. Loss of MyD88 in Ndufs4(-/-) animals statistically significantly increased survival and delayed the onset of some symptoms, but the benefits were modest compared to CSF1R inhibition from prior work. We conclude that Myd88-mediated immune signaling is not a primary driver of LS. Notably, prophylactic enrofloxacin treatment, which was necessary for production of innate immune deficient MyD88(-/-) animals, modestly decreased survival and accelerated disease. The impact of enrofloxacin and similar drugs in the context of mitochondrial disease warrants further investigation.

neuroscience↗

bioRxiv2026

Autoimmune-like CD8⁺ T Cell Responses Drive Atherosclerotic Plaque Instability and Predict Cardiovascular Events

Background and aimsAtherosclerotic plaque rupture is a major cause of myocardial infarction and stroke. However, the precise drivers of plaque destabilisation remain elusive. We hypothesised that antigen-driven, autoimmune-like T cell responses are central to the destabilisation and rupture of atherosclerotic plaques. MethodsTo dissect T cell responses specifically in unstable compared to stable plaques, we leveraged near-infrared autofluorescence (NIRAF) imaging-guided dissection of human carotid plaques. We also used our tandem stenosis model reflecting plaque instability as seen in patients to differentiate between unstable and stable plaques in mice. To explore T cell involvement, we studied T cell differentiation states and T cell receptor (TCR) repertoires by single-cell multi-omics. Then, testing if antigen-driven CD8+ T cell responses drive plaque instability in mice, we applied a combination of AAV8-PCSK9-induced atherosclerosis, tandem stenosis and TCR transgenic mice. Finally, we leveraged data from the AtheroExpress Biobank Study to link T cell immunity to histology-defined instability and cardiovascular outcomes. ResultsT cell responses in unstable versus stable atherosclerosis were distinct. Unstable human plaques contained highly expanded, autoimmune-like CD8 T cells with markedly increased cytotoxic signatures, reduced exhaustion and distinct clonal repertoires compared to stable regions. Most plaque CD8 T cells exhibited a pronounced tissue-resident transcriptional program. Moreover, the transcriptional signature of these plaque resident T cells was distinct from multiple other human tissues. Autoimmune-like cytotoxic and tissue-resident CD8+ T cell responses were also evident in murine atherosclerosis, where restricting the activation of antigen-driven CD8+ T cells prevented plaque destabilisation. Importantly, analysis of carotid endarterectomy samples from >1000 patients identified that intraplaque cytotoxic CD8 T cell gene signatures strongly correlated with histological instability and predicted future strokes. ConclusionsIntegrated human, murine and clinical analyses demonstrate that autoimmune-like, cytotoxic CD8 T cell responses are central drivers of plaque instability and major cardiovascular events. Targeting pathogenic CD8 T cell responses may thus offer a compelling immunomodulatory strategy to stabilise plaques and reduce the risks of stroke and myocardial infarction. Graphical AbstractO_ST_ABSKey QuestionC_ST_ABSRupture of unstable atherosclerotic plaques is a typical cause of myocardial infarction and stroke. To understand the underlying cause and to prevent plaque rupture, we addressed the central hypothesis that autoimmune-like T cell responses drive plaque destabilisation and rupture. Key FindingsCD8+ T cells are clonally expanded with increased cytotoxic signatures in unstable versus stable plaques (mice and humans) and require antigen recognition to drive plaque instability. Cytotoxic CD8+ T cell signatures in excised plaques correlate with increased future cardiovascular events. Take Home MessageAutoimmune-like adaptive immune reactions, dominated by CD8+ T cells, are a major driver of plaque instability/rupture. Therefore, targeting pathogenic CD8 T cell responses offers a compelling immunomodulatory strategy to stabilise plaques and reduce the risk of myocardial infarction and stroke. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/720043v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@bd83dcorg.highwire.dtl.DTLVardef@1bee3borg.highwire.dtl.DTLVardef@1b56bc0org.highwire.dtl.DTLVardef@1b5176f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Passive neuromodulation: an energy-driven mechanism for closed-loop suppression of epileptic seizure

For decades, electrical neuromodulation has been used as a therapeutic mechanism to disrupt and desynchronize pathological neural activity in various neurological disorders. Despite notable progress, however, patient outcomes remain highly variable, particularly in medically intractable epilepsy where surgery still provides the greatest chance of seizure freedom. Here we propose passive neuromodulation (PNM) as a radical alternative to conventional neurostimulation, whereby analogue feedback is used to drain energy from an epileptic circuit and thus suppress the initiation or spread of electrographic seizures. We provide pilot evidence on the efficacy and robustness of PNM using two computational models of epileptic dynamics: a detailed biophysical network model of dentate gyrus, and the Epileptor neural mass model of seizure dynamics. Despite the vast differences between these models, our results show the robust ability of PNM to suppress seizures in both models. We further demonstrate the efficacy and robustness of responsive PNM, whereby brief (50ms) windows of PNM are triggered by a simultaneously-running seizure detection algorithm, as well as the safe and tunable nature of PNM, where more robust seizure suppression can be achieved by parametrically titrating the amount of power drained from the tissue, without inducing any seizures even if applied interictally. Overall, our results provide strong evidence on the promise of PNM for the closed-loop control of epileptic seizures and other neurological disorders where damping pathological network activity can restore healthy dynamics.

neuroscience↗

bioRxiv2026

Identification of a lineage-agnostic splicing signature caused by PRMT5 inhibition

PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein WDR77 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including apoptosis, DNA damage response and RNA processing. Some of the best characterized PRMT5 substrates are the small nuclear ribonucleoproteins SNRPB, SNRPD1 and SNRPD3, which are critical for spliceosome assembly and RNA splicing fidelity. MTAP-deleted cancers exhibit increased sensitivity to PRMT5 inhibition due to elevated levels of methylthioadenosine (MTA), a natural inhibitor of PRMT5. This vulnerability is exploited by MTA-cooperative PRMT5 inhibitors, exemplified by TNG908 and TNG462 which selectively target PRMT5 in MTAP-deleted cells while sparing MTAP-wildtype (WT) cells. Consistent with this mechanism, treatment with TNG908 in preclinical studies induces widespread splicing alterations in MTAP-deleted cancer models, with minimal effects in MTAP-WT cells. These splicing changes are consistent across diverse MTAP-deleted tumor types, including glioblastoma, pancreatic, and non-small cell lung cancer, indicating a histology-agnostic response to PRMT5 inhibition. Moreover, treatment of MTAP-WT cells with exogenous MTA mimics the splicing alterations observed with PRMT5 inhibition, as does pharmacologic inhibition of MTAP further supporting a mechanistic link between MTA accumulation, PRMT5 modulation, and aberrant splicing. Given that MTAP deletions occur in approximately 10-15% of human cancers, the identification of a robust RNA splicing signature offers a valuable pharmacodynamic biomarker for monitoring the activity of PRMT5 inhibitors. This splicing-based readout may also serve as a predictive biomarker of therapeutic response, offering greater specificity than global SDMA levels. Collectively these data suggest that a PRMT5-dependent RNA splicing signature can monitor the pharmacodynamic activity of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells.

cancer biology↗

bioRxiv2026

Brain-wide mapping and synaptic localization of C1QL3 using a novel epitope-tagged knock-in mouse

Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity; many of which are implicated in neuropsychiatric disorders. Members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql32HA), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql32HA mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and post-synaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a powerful tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.

neuroscience↗

bioRxiv2026

Morphoelectric Diversity and Specialization of Neuronal Cell Types in the Primate Striatum

The basal ganglia are evolutionary ancient subcortical nuclei that form interconnected loops with the neocortex and limbic system to regulate movement, learning, habit formation, emotion, and motivation. Their dysfunction contributes to major neurological and psychiatric disorders, yet most cellular-level insights derive from rodent studies, leaving knowledge gaps in humans and translationally relevant primate species. To address this, we generated multi-modal Patch-seq data linking transcriptomic identity with morphological and electrophysiological properties in macaque striatum, the input nucleus of the basal ganglia. We found underappreciated diversity among medium spiny neurons, including non-canonical types, and variation aligned with functional gradients. Interneurons also exhibited spatial variation and even greater morphoelectric diversity, highlighting their functional modularity. Despite broad evolutionary conservation, we identified primate-specific features and key differences from rodent striatal neurons. By integrating molecular classification with cellular properties that shape network function, our findings provide insights into the functional organization of the primate striatum.

neuroscience↗

bioRxiv2026

A Cross-Species Enhancer-AAV Toolkit for Cell Type-Specific Targeting Across the Basal Ganglia

The mammalian basal ganglia (BG) orchestrate motor, cognitive, and affective functions, yet cell type-specific genetic access remains limited, especially beyond rodents. Key structures implicated in movement and psychiatric disorders, including pallidum, subthalamic nucleus, and dopaminergic midbrain, lack scalable tools for cross-species targeting. Here, we present a comprehensive enhancer-AAV library enabling selective labeling and manipulation of major BG neuronal populations: striatal projection neuron subtypes, pallidal and subthalamic neurons, and midbrain dopaminergic and GABAergic populations. Using an evolutionarily informed discovery pipeline, we identified enhancers targeting canonical, non-canonical, and disease-relevant cell types, with validation demonstrating robust cross-species conservation of specificity between mouse and macaque. Computational modeling revealed sequence features predictive of in vivo performance, including motif grammar, chromatin accessibility, and evolutionary conservation, and identified distinct regulatory architectures across glial, projection, and interneuron lineages. This work establishes a comprehensive cross-species viral toolkit for the BG, unlocking previously inaccessible cell types for circuit dissection.

neuroscience↗

bioRxiv2026

MEA-LINK identifies the CCL4-CCR5 axis in neuronal hyperactivity control by human microglia

Microglia, the resident immune cells of the brain, act along a spectrum to maintain CNS homeostasis, respond to perturbations, and control neuronal activity. Disentangling the molecular mechanisms of human microglia-neuron crosstalk remains challenging due to the context-dependent, dynamic nature of their interaction. We introduce MEA-LINK, a systems-approach leveraging natural variation to screen for immune modulators of neuronal activity. This multi-modal platform integrates human induced pluripotent stem cell (hiPSC) technology with micro-electrode array (MEA) recordings and proteomic analyses of secreted immune factors, allowing for longitudinal samples and correlations across modalities. We applied MEA-LINK to explore microglia-neuron interactions during development and hyperactivity challenges. We show that human microglia accelerate neuronal network development and rescue hyperactive network phenotypes. Linking the secretome adaptations to neuronal network activity variations, we identified CCL4 as a top candidate in microglia-mediated hyperactivity control. Then, we functionally validated the context-dependent role of microglial CCL4 to neuronal CCR5 signaling in human neuronal networks. Our findings support a neuron-specific function of chemokines and their receptors in the brain and provide a new perspective for immune signaling in neuronal hyperactivity control. The MEA-LINK platform thus offers a foundation for comprehensive, systematic studies of human microglia-neuron interactions. HighlightsO_LIMEA-LINK integrates micro-electrode array recordings with proteomics of longitudinal samples to identify immune modulators of neuronal activity. C_LIO_LIHuman microglia rescue neuronal hyperactivity induced by pharmacological and genetic challenges. C_LIO_LIMicroglial CCL4 dampens neuronal activity via CCR5 signaling in a context-dependent manner. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/703799v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@18e44f7org.highwire.dtl.DTLVardef@151c450org.highwire.dtl.DTLVardef@12f7b89org.highwire.dtl.DTLVardef@57651b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

HistoSweep enables cellular-resolution tissue quality control for gigapixel images in digital pathology and spatial omics

High-resolution histology images are indispensable for pathology and increasingly serve as the structural backbone for spatial omics. Yet whole-slide images (WSIs) frequently contain artifacts, acellular voids, and background regions that, when included in computational workflows, introduce noise, degrade model accuracy, and compromise biological interpretation. Existing tools provide only coarse foreground-background separation, leaving a gap in fine-grained quality control (QC). Here we present HistoSweep, a scalable framework that generates morphology-aware tissue masks at cellular resolution. By integrating density filtering, texture descriptors, and adaptive thresholding, HistoSweep systematically removes non-informative tissue regions while preserving biologically meaningful microstructures. It processes billion-pixel WSIs in minutes on standard CPUs, requiring no GPU acceleration, and is deployable across research and clinical settings. Across 25 WSIs spanning distinct tissues, disease states, and spatial omics platforms, HistoSweep consistently outperformed existing methods. It enhanced visualization and segmentation, improved virtual cell type predictions, and safeguarded spatial transcriptomics integrity by detecting transcript leakage and transcript-histology misalignment. By enabling fine-grained, scalable QC, HistoSweep provides a foundational preprocessing step for reliable and reproducible digital pathology and spatial omics analyses.

bioinformatics↗