Biology subjects

Huang, Q.

Publications and source records attributed to Huang, Q..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Dual-Logistic Analysis of Time- and Concentration-Dependent Phenotypic Efficacy Evaluation Integrating Drug Targets Information

Tumor heterogeneity highlights the necessity of precision cancer medicine, making the evaluation and screening of anticancer drugs a core challenge in cancer therapy. However, current cell-based efficacy assessment methods struggle to quantify the holistic impact of drugs on cellular behavior through specific target engagement. Here, we proposed a novel approach (DL-TCP-FRET) that integrates phenotypic and target-related evaluations: the logistic fitting analysis is performed on time- and concentration-dependent cellular phenotypic characteristics to construct a phenotypic score (P), while a target score (T) is established based on the FRET efficiency between target proteins. These two scores were then further combined to generate a unified drug efficacy score (PT). Validation in A549 cells demonstrated that our method can reliably distinguish EGFR-TKIs from non-targeted drugs. DL-TCP-FRET simplifies the experimental workflow of drug efficacy evaluation and improves the accuracy of targeted drug identification, providing a novel strategy for advancing precision cancer therapy.

cell biology↗

bioRxiv2026

Cellular transcriptomics reveals evolutionary adaptation and rumination of vertebrate stomachs

Stomach, an essential digestive organ in vertebrates, has undergone significant changes in its morphology, chamber numbers, and physiology, in response to dietary diversity. However, its cellular and molecular basis underlying evolutionary adaptation remains largely uncharacterized. Here we report a single-cell and spatial transcriptomic atlas of stomachs from 23 vertebrate species with diverse feeding habits (e.g., omnivores, carnivores, and herbivores) and gastric architectures, including monogastric (e.g., primates), two- (e.g., aves), three- (e.g., camel) and four-chambered stomachs (e.g., sheep). We reveal conservation and divergence in cell-type composition, developmental trajectory, evolutionary origins and forces, spatial distribution, gene regulatory network and effect, metabolic signature and disease susceptibility associated with the feeding habits and stomach chamber. The diversification of plant based diets, particularly the expansion of coarse fibrous plants that required enhanced mechanical processing, driving the rapid evolution of specific cells and associated genes. Ruminants have evolved chamber-specific cell types with enhanced expressions of relevant functional genes (e.g., KRT6A in spinous cells of forestomach, LUC7L in SMCs of abomasum, and TSPYL4 in abomasal enteroendocrine cells), which were validated by fluorescence in situ hybridization and spatial transcriptomic profiling. The three cell-specific expressed genes showed significant effects on cell proliferation and migration by RNA interference to knock down their expressions. In particular, the knockdown of LUC7L in SMCs promotes a transition from a contractile to a synthetic phenotype, and Luc7l knock-out in mice delays gastric emptying and impairs gastric motility, demonstrating that this gene is essential for the coordinated multi-chambered gastric motility that underlies rumination in ruminants. These findings elucidate the cellular and molecular adaptations underlying the evolution of stomach coupled with the emergence of diet land plants. Our results provide potential cellular and gene targets for engineering monogastric animals to acquire functional rumination digestive capabilities as well as for medication of gastric mobility.

evolutionary biology↗

bioRxiv2026

Early clonal dominance at priming sets the trajectory for broad HIV serum neutralization

Inducing broadly neutralizing antibodies (bnAbs) remains a central challenge in HIV vaccine development 1-3. Germline-targeting immunogens are designed to activate rare bnAb precursor B cell lineages 4-12, yet the relationships between priming efficiency, clonal dominance, and downstream serum neutralization remain poorly defined. We recently demonstrated that vaccination with an engineered V2-apex germline-targeting trimer Q23-APEX-GT2 successfully recruits and activates rare long-CDRH3 B cell precursors in outbred macaques 13. Here, we dissect the immunological mechanisms governing bnAb precursor priming and early B cell expansion and define clonal features that drive progression to serum neutralization breadth. Our antigen-specific B cell analyses showed that Q23-APEX-GT2 consistently engaged long-CDRH3 precursors, although priming efficiency varied across animals. Longitudinal deep lineage tracing across lymph node and blood compartments revealed that early recruitment of multiple diverse long-CDRH3 lineages, followed by preferential expansion and dominance of one or two clones, strongly predicted serum neutralization potency. Subsequent CAP256.SU SHIV infection efficiently recalled vaccine-seeded clones, accelerated affinity maturation, and drove broad heterologous neutralization in most animals. Notably, one macaque with diverse and expanded V2-apex lineages rapidly achieved [~]70% serum neutralization breadth. Importantly, longitudinal tracing revealed that bona fide bnAbs can emerge from vaccine-primed precursors, while also uncovering "born-wrong" bnAb-like lineages that expand yet remain non-neutralizing, despite structurally validated recognition of the V2-apex bnAb site. Together, these findings establish priming efficiency coupled with early clonal dominance as key determinants of serum bnAb induction and provide a mechanistic framework to guide rational HIV vaccine design.

immunology↗

bioRxiv2026

Adaptive sequential eye-movement sampling and replay under different task demands

Human cognition is capacity-limited, requiring strategies to actively structure information. Eye movements offer a natural mechanism for sequential sampling, but whether such sequences organize mnemonic representations is unknown. We developed a working-memory task where color-frequency pairings created a consistent latent ordinal structure to optimally reduce memory load. Across two experiments, gaze patterns spontaneously aligned with this structure. Participants sampled items following this sequence during encoding and covertly replayed them during maintenance. Critically, the expression of this structure depended on cognitive demand. In a 3-item task, high performers showed robust sequential sampling during encoding, whereas lower performers compensated with replay-like revisitation during maintenance. Under higher demand (4 items), encoding-based organization was disrupted, and structured replay emerged primarily during maintenance to support memory. These findings show that eye movements do more than reflect memory; they actively organize it, revealing a flexible, behavioral analogue to neural replay when encoding resources are strained.

neuroscience↗

bioRxiv2026

Mid-infrared light produces anti-ageing effects through resonant absorption by living organisms

Aging is commonly viewed as a consequence of cumulative molecular damage and declining stress resilience. However, this perspective overlooks the possibility that aging may primarily arise from an efficiency loss of integrated cellular operation. Here, we show that aging can be systemically prevented by enhancing cellular efficiency using frequency-specific mid-infrared (MIR) light, as revealed by organismal-, cellular- and molecular-level analyses of Caenorhabditis elegans. Remarkably, super-weak 34-THz MIR light ([~]1 W mm-{superscript 2}) prolonged the median lifespan of worms by 60%, delaying aging onset and preventing abrupt "cliff-edge" mortality, without detectable thermal effects. At the cellular level, MIR exposure enhanced global gene transcription in parallel with the establishment of a mitochondrial state of high-efficiency energy metabolism, together preserving youthful cellular homoeostasis during aging. These cellular effects were associated with vibrational modes of phosphate groups in nucleic acids and mitochondrial phospholipids within the 33-35 THz range, providing a frequency-matched molecular context for MIR modulation. Together, our results support an efficiency-first, systemic anti-aging model in which frequency-specific MIR light enhances the integrated kinetic efficiency of cellular systems, spanning gene transcriptional dynamics and mitochondrial bioenergetic flux, rather than the activation of damage-repair or stress-response pathways. These findings advance our understanding of light-matter interactions in living systems and establish a non-biochemical, physical strategy for modulating aging.

biophysics↗

bioRxiv2026

Forward genetics reveals microsporidian drug targets and recombination-based resistance

Microsporidia are a diverse group of intracellular fungal parasites that infect humans and many agriculturally important animals. Several microsporidia inhibitors have been characterized, but the targets of these inhibitors in microsporidia have not been confirmed, and the mechanisms by which microsporidia evolve resistance are unknown. Using the model organism Caenorhabditis elegans and its naturally occurring microsporidian parasite Nematocida parisii, we developed a technique to continually passage infected animals for multiple generations under increasing inhibitor concentrations. We obtained multiple independent N. parisii strains that are specifically resistant to the microsporidia inhibitors albendazole or dexrazoxane. The albendazole-resistant strains all contained either heterozygous or homozygous mutations in beta-tubulin, with several strains containing beta-tubulin variants that have been observed in other albendazole-resistant organisms. Strains containing homozygous variants are resistant to several albendazole analogs, whereas the heterozygous variant-containing strains are only resistant to albendazole. Several of the albendazole-resistant strains also contain mutations in alpha-tubulin. The dexrazoxane-resistant strains all contain heterozygous mutations in topoisomerase II and several of these mutations occur in the binding site of this inhibitor. By mapping heterozygosity of the resistant strains across the N. parisii genome, we observed loss of heterozygosity spanning the beta-tubulin locus in the albendazole-resistant strains containing homozygous beta-tubulin variants. Our study demonstrates the utility of forward genetics for identifying microsporidian drug targets. We also find that drug resistance arises through de novo heterozygous mutations that can subsequently become homozygous, likely via mitotic recombination.

microbiology↗

bioRxiv2026

Mechanism study on lactic acid promoting intervertebral disc degeneration by regulating senescence and DNA damage of cartilage endplate stem cells

Intervertebral disc degeneration, a leading cause of low back pain with incompletely elucidated molecular mechanisms, was studied via integrated in vivo/vitro approaches. This study first reveals that lactic acid accelerates intervertebral disc degeneration by inducing cartilage endplate stem cells senescence and DNA damage, thereby activating the P16/P21/P53-centered senescence pathway. In a rat tail vertebra puncture-induced intervertebral disc degeneration model, degenerated discs exhibited increased lactic acid levels, narrowed intervertebral spaces, and disrupted nucleus pulposus structure (P<0.05). In vitro, 0/2/6/10 mM lactic acid dose-dependently suppressed cartilage endplate stem cells viability (10 mM group: 15.7% of the control), elevated intracellular reactive oxygen species (ROS, 2.8-fold relative to the control), induced G0 cell cycle arrest (10 mM group: 85.63%), reduced EdU-positive cells (8.62%), and increased {beta}-galactosidase-positive cells (10 mM group: 33.06%) and {gamma}-H2AX foci (all P<0.01).Molecularly, lactic acid significantly upregulated P16 (2.1-fold), P21 (3.1-fold), P53 (2.4-fold), and {gamma}-H2AX (1.8-fold). In vivo intervertebral disc injection confirmed a positive correlation between lactic acid concentration and intervertebral disc degeneration severity. This study clarifies lactic acids role in intervertebral disc degeneration via the "oxidative stress-cell cycle arrest-cellular senescence" axis, advancing understanding of intervertebral disc degeneration pathogenesis and providing a basis for targeted therapies against lactic acid metabolism.

cell biology↗

bioRxiv2026

Gn1a rice promotes symbiotic fish growth via reprogramming soil microbiome

Rice-fish symbiotic ecosystems significantly contribute to world food supply. Mutation in rice (Oryza sativa) quantitative trait locus Gn1a, encoding a cytokinin oxidase/dehydrogenase (OsCKX2), leads to cytokinin accumulation in plant, which promotes tillering and panicle branching to achieved higher yield and also shows a selective effect on soil microbial composition. Given that the growth of aquatic animals can be shaped by their surrounding microbial communities, we asked whether adjusting rice cytokinin levels might synergistically boosts both rice yield and fish growth. Herein, we co-cultured common carp (Cyprinus carpio) with ckx2 rice plants to determine the effect of cytokinins on fish. Surprisingly, compared to wild-type plants, the ckx2 plants enhanced nearly 10% fish growth and improved fish antibacterial capacity against Aeromonas hydrophila and Edwardsiella tarda. In fact, the growth-promoting effect on fish requires mediation by the soil microbiota. Mechanistically, ckx2 plants secreted more cytokinins into soil to reprogram the microbial composition, which subsequently translocated to fish gut and modulated the keystone microbes in gut to facilitate short-chain fatty acid production and carbohydrate degradation. These findings provide the first evidence that rice-derived cytokinins exert a cross-kingdom growth-promoting effect on fish, providing a boosting dual-production strategy for advancing rice-fish symbiotic ecosystem.

plant biology↗

bioRxiv2025

SF3A2 deacetylation at lysin 10 mitigates myocardial pathological remodeling by reprogramming cardiomyocyte metabolism

BackgroundDysregulation of splicing factor-mediated pre-mRNA alternative splicing (AS) underlies the progression of complex diseases, but the specific involvement of AS in myocardial pathological remodeling remains unclear. This study aimed to elucidate the role of splicing factor 3A, subunit 2 (SF3A2) in the development of myocardial pathological remodeling by regulating AS and its effect on metabolic reprogramming. MethodsWe evaluated the function and mechanism of SF3A2 in myocardial pathological remodeling using murine models induced by myocardial infarction (MI) and isoproterenol. The gain- and loss-of-function models (AAV9-cTNT delivery-mediated knock-in and lentivirus-mediated knockdown) combined with acetylomics, metabolomics, lipidomics, 13C-glucose tracing and immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (IP-MS) analyses were used to demonstrate the mechanism of SIRT7-mediated SF3A2 deacetylation at lysin 10 (K10). Unbiased RNA sequencing and AS analysis were conducted to identify the downstream effector of SF3A2. ResultsSF3A2 acetylation at lysine 10 was upregulated in cardiomyocytes in response to myocardial pathological remodeling. Functionally, the knock-in of a deacetylation-mimetic SF3A2 (K10R) mutant attenuated myocardial hypertrophy, fibrosis, and improved heart function. These phenotypes were accompanied by a redirection of glucose flux from anaerobic glycolysis to the TCA cycle, along with a promotion of fatty acid {beta}-oxidation by inhibiting CD36 translocation and decreasing ACLY activity. Mechanistically, SF3A2 mitigated metabolic reprogramming and mitochondrial dysfunction by regulating Nnt AS, a process involving the SF3A2-interacting protein Ddx1. IP-MS further identified SIRT7 as the deacetylase for SF3A2, which was found to be activated by ginsenoside Rg1 in a high-throughput screen, leading to improved heart function. ConclusionsOverall, our findings establish the SIRT7-SF3A2-Nnt axis as a critical regulator in metabolic reprogramming to delay pathological myocardial remodeling. Promoting SF3A2 deacetylation, mediated by AAV9 delivery or pharmacological activation of SIRT7, improved heart function, suggesting its potential as a therapeutic target for pathological myocardial remodeling.

molecular biology↗

bioRxiv2025

Flu Virus Infection in Juvenile Mice Leads to Lifelong Multi-Organ Damage and Parkinsonian Pathological Changes in Aging

The widespread prevalence of Long COVID underscores the potential for viral infections to exert prolonged effects on hosts long after initial recovery. Despite this recognition, the long-term and even lifelong consequences of viral infections remain poorly understood. In this study, we infected juvenile mice with influenza viruses and systematically examined the lifelong effects (from 4 to 450 days post-infection) of these infections. Pathological analysis revealed persistent lifelong damage in multiple organs, including chronic lung inflammation and fibrosis, along with significant cardiac and renal pathology. Viral infection led to substantial neuronal loss in key brain regions of mice (hippocampal CA1, CA3, and striatum) during middle and old age. Strikingly, we identified two hallmark pathological features of Parkinsons disease - dopaminergic neuron degeneration and Lewy body-like -synuclein inclusions - in middle and aged infected mice. Transcriptomic profiling demonstrated sustained upregulation of inflammation-related genes in both lung and blood tissues, correlating with observed pathological changes. Lung-derived secreted proteins, encoded by differentially expressed genes, may mediate cross-organ communication affecting cardiac, renal, and neurological function. Brain transcriptome analysis at three months post-infection revealed downregulation of neurodevelopmental genes, potentially contributing to subsequent neuronal loss and Parkinsonian pathology. These findings collectively suggest that pulmonary influenza infection can induce systemic multi-organ effects through both secretory pathways and chronic inflammatory responses. The study offers crucial insights for developing more effective strategies to prevent and manage infectious diseases and their long-term sequelae.

microbiology↗

bioRxiv2025

COPS5 is Essential for Sertoli Cell Function and Male Fertility in Mice

The COP9 signalosome subunit 5 (COPS5) is a multifunctional protein that regulates ubiquitin-dependent processes. Global knockout of Cops5 is embryonically lethal, and while it is known to be vital in germ cells and testicular smooth muscle, its function in Sertoli cells, the key somatic supporters of spermatogenesis, remains entirely unknown. This study investigates the critical role of COPS5 within Sertoli cells. Using Sertoli cell-specific Cops5 knockout mouse models, we demonstrate that COPS5 is essential for maintaining male fertility. Sertoli-specific Cops5 ablation resulted in age-dependent male infertility, despite normal initial development. Mutants exhibited progressive testicular atrophy, oligoasthenospermia, and significantly reduced testis weight. Histology showed vacuolated, disorganized tubules devoid of germ cells and sperm. Crucially, COPS5 loss disrupted Sertoli cell polarity, evidenced by aberrant cytoplasmic mislocalization of the nuclear marker WT1 and detachment from the basement membrane. Integrity of the blood-testis barrier (BTB) was severely compromised, with discontinuous and punctate expression of the tight junction adaptor ZO-1. Intercellular communication was also impaired, shown by a stark reduction in Connexin 43 gap junction signals. Our findings establish that COPS5 is an indispensable intrinsic regulator of Sertoli cell function. Its loss disrupts cell polarity, BTB architecture, and gap junction communication, leading to failed support of spermatogenesis and consequently infertility. This work defines a novel and critical somatic function for COPS5 in male reproduction. We conclude that COPS5 is intrinsically required in Sertoli cells to maintain their polarity and BTB function, which are foundational for supporting germ cell development and ensuring male fertility. This identifies COPS5 as a novel, essential regulator within the testicular somatic compartment.

genetics↗

bioRxiv2025

PARP1 Writes N3-Cytidine ADP-Ribosylation in DNA

Recent evidence indicates that mono - and poly-ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1-deoxyadenosine (N1-dA). Here, we report that PARP1 catalyzes N3-specific ADP-ribosylation of deoxycytidine (N3-dC) in single-stranded DNA. Analogous to N1-dA PARylation, which is prone to spontaneous adenine-to-inosine deamination, N3-dC PARylation promotes cytosine deamination, yielding N3-PARylated-deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC-MS/MS, namely N1-ribosyl-deoxyinosine (N1-r-dI) and N3-ribosyl-deoxyuridine (N3-r-dU). We synthesized both N1-r-dI and N3-r-dU as diagnostic standards and established absolute quantification of base ADP-ribosylations by LC-MS/MS. Quantitative analysis of PARylated dA and dC in ssDNA reveals pronounced sequence preferences of PARP1. Removal of these base modifications differs markedly, since ADP-ribose glycohydrolase TARG1 removes PAR from both dA and dC, whereas PARG acts exclusively on dA. Our results establish cytidine ADP-ribosylation as a novel DNA modification, with potential roles in DNA metabolism, epigenetic regulation, or genome stability.

biochemistry↗

bioRxiv2025

An Lrs14 family protein functions as a nucleoid-associated protein regulating cell cycle progression in Sulfolobales

Archaea of the order Sulfolobales possess eukaryotic-like cell cycle. The chromatin organization in these archaea is supposed to rely on nucleoid-associated proteins (NAPs). Genomes of Sulfolobales species encode multiple members of the Lrs14 protein family, which have been postulated to function as NAPs. Here, we show that one of the Lrs14 family protein, named Sul14a, is a potential key player in dynamic chromatin organization in Saccharolobus islandicus. Sul14a is transcribed in a cell cycle-dependent manner and appears to be essential for cell viability. The protein exhibits DNA-binding activity preferring AT-rich sequences and can bridge DNA in vitro. ChIP-seq analysis revealed that Sul14a is enriched at AT-rich regions of the chromatin. Overexpression of Sul14a leads to cell cycle arrest at G2 phase. High-throughput chromosome conformation capture (Hi-C) and transcriptomic analyses indicate that Sul14a overexpression results in chromatin contact reduction and global transcriptional changes. In addition, we observed that the chromatin contact pattern changes markedly between 1h and 5h after release from G2 synchronization, corresponding to the lowest and highest levels of sul14a transcription, respectively, during the cell cycle. Our results establish that Sul14a is an NAP intimately associated with chromatin dynamics and global transcriptional changes during the cell cycle in Sulfolobales.

microbiology↗

bioRxiv2025

Efficient mixed representation of active and passive motion in the mouse visual thalamus during natural behaviour

Most retinal motion is generated by the observer's movements. These are often self-generated (active) but can also arise from external forces such as gravity (passive). Active movements modulate spontaneous and visually evoked activity throughout the visual system, providing neural circuits with information about behavioural state. By contrast, much less is known about how visual circuits represent passive movements. Critically, it remains unclear whether the retina-recipient thalamus tracks passive motion and, if so, whether this representation is separable from active motion. To address these questions, we developed a paradigm that dissociates active and passive motion in freely moving mice and used it to examine their effects on neural activity. Mice actively explored an arena while intermittent floor tilts induced passive movement. By combining 3D reconstructions of animal pose and arena motion, we quantified active and passive linear and angular motion independently, even when they occurred simultaneously. We found that neurons in the dorsal lateral geniculate nucleus (dLGN) and surrounding regions tracked combinations of active and passive motion signals both in the dark and during visual stimulation. Individual neurons differentially weighted active and passive motion signals. This diversity enabled active and passive motion signals to be linearly decoded and partially separated from population activity, while recurrent dynamics retained passive motion information over timescales of several seconds. These findings identify a coding strategy through which early visual circuits integrate active and passive motion into an estimate of self-motion while preserving information about whether visual inputs arise from self-generated or externally imposed movements.

neuroscience↗

bioRxiv2025

Three transcription factors coordinate the archaeal cell cycle progression through a regulatory braking point mechanism

Archaea of the order Sulfolobales execute a well-structured cell cycle program similar to that of eukaryotic cells. However, unlike eukaryotes, archaea lack cyclins and cyclin-dependent kinases and thus the mechanism of cell cycle regulation remained enigmatic. Here, we show that three essential ribbon-helix-helix domain transcription factors, namely, aCcr1, aCcr2, and aCcr3, play pivotal roles in controlling the cell cycle progression in the thermoacidophilic archaeon Saccharolobus islandicus. Coordinated expression of aCcr1 during M-G1 phase, aCcr3 during G1-S phase, and aCcr2 throughout the cell cycle ensures the timely transcription of the key genes that define the cell cycle phases. These include genes involved in chromosome dimer resolution, chromosome segregation, cell division, chromatin organization, DNA replication and repair, protein phosphorylation and degradation and metabolisms of amino acids, tRNA and carbohydrates. The synergy between aCcr1, aCcr2, and aCcr3 is achieved through their differencial affinities for the promoters and the levels of protein expression. We propose that the global regulation of the Sulfolobales cell cycle may be achieved not through transcriptional activation, but rather by repression of the key genes during strategic moments of the cell cycle. We propose a braking point model for the cell cycle control in Sulfolobales, which may represent a simple evolutionary intermediate on the way to the more complex cell cycle regulation in eukaryotes.

microbiology↗

bioRxiv2025

Programmable Aptamer-Embedded Circular RNAs for Targeted Antigen-Presenting Cells Immunotherapy

The targeted delivery of mRNA to specific cell types in vivo remains a major unmet need in mRNA-based therapeutics. Conventional strategies rely on nanocarriers engineered with various modifications. However, these exogenous materials could cause adverse reactions, and their complex manufacturing processes often hinder clinical application. By leveraging the innate programmability of RNA sequences, we herein report a carrier-free, scalable, and programmable aptamer-embedded circular RNAs (Apt-circRNAs) design that confers targeting capability through the strategic incorporation of different aptamers at varying densities, while retaining circular RNA stability and protein production capability. Experimental results show that antigen-loaded Apt-circRNAs enable targeted delivery to antigen-presenting cells (APCs), drive endogenous antigen expression, activate antigen-specific T cells, and elicit potent immune responses that mediate clearance of both early-and late-stage tumors in mouse models. In humanized models of colorectal cancer, the combination of Apt-circRNA and PD-1 blockade produced synergistic antitumor effects and significantly reshaped the tumor microenvironment. In a first-in-human (FIH) clinical trial, Apt-circRNA vaccine encoding KRAS G12D/G12V neoantigens exhibited a favorable safety profile and induced robust immune activation, T cell mobilization, and neoantigen-specific immune responses. With this study, we have established a new paradigm in carrier-free, APC-targeted RNA vaccine design, offering a safe and effective platform for next-generation cancer immunotherapy.

bioengineering↗

bioRxiv2025

SPACE: spatially resolved multiomic analysis for high-throughput CRISPR screening in 3D models

Current spatial CRISPR screening technologies are limited by targeted readouts and high costs, restricting the scope of biological discovery. Here we present SPAtial Cell Exploration (SPACE), a spatial CRISPR screening platform that integrates whole-transcriptome profiling ([~]18,000 genes), multiplexed protein detection ([~]68 markers), and CRISPR perturbation mapping at subcellular resolution. SPACE significantly reduces whole-transcriptome profiling costs compared to sequencing methods while preserving spatial context. We demonstrate SPACE by screening 43 CRISPR knockouts (KOs) across [~]100,000 cells in hundreds of cancer-associated fibroblast (CAF)-tumor spheroids, obtaining whole-transcriptome and multiplexed protein readout from the same exact cells. SPACE revealed previously unknown regulatory mechanisms on tumor extracellular matrix (ECM) remodeling, and identified spatially-resolved ligand-receptor interactions and perturbation-specific spatial gene signatures that are not detectable with dissociation-based methods. This scalable, cost-effective platform provides a transformative framework for high-throughput spatial perturbation studies in complex tissue models.

cell biology↗

bioRxiv2025

Identification of a novel biological role of arenavirus NP exonuclease (exoN) activity in a safe Pichinde virus model

Arenaviruses such as Lassa virus (LASV) and Junin virus (JUNV) can cause lethal hemorrhagic fever diseases with no FDA-approved vaccines or effective therapeutics. The arenaviral nucleoprotein (NP) contains an exoribonuclease (exoN) domain that suppresses type I interferon (IFN-I) production by degrading pathogen-associated molecular-pattern (PAMP) RNAs. The biological roles of NP exoN during viral infection have not been well characterized for highly pathogenic arenaviruses, largely due to biosafety constraints and challenges in generating viable NP exoN mutants. We previously developed a recombinant tri-segmented viral vector (rP18tri) based on non-pathogenic Pichinde virus (PICV), which allows functional studies of transgenes during arenavirus infection in a BSL-2 setting. In this study, we introduced point mutations at individual catalytic residues to abolish PICV NP exoN activity and generated exoN-deficient rP18tri(NPm) reporter viruses. These mutants failed to replicate in IFN-competent A549 cells, accompanied by strong IFN-I induction. Expression of wild-type (WT) JUNV NP, but not its exoN-deficient mutant, in the rP18tri(NPm)-G backbone restored single-cycle infectivity and suppressed IFN-I induction. Furthermore, exoN-deficient viruses produced disproportionately high levels of defective virion particles relative to virion RNA from infected A549 cells, a defect that was rescued by expression of WT JUNV NP but not the exoN-deficient NP as a transgene. Together, these data demonstrate an essential role of NP exoN in IFN-I suppression and identify a novel biological function of NP exoN in promoting the production of infectious virion particles in IFN-competent cells. ImportanceArenaviruses, such as Junin virus (JUNV), can cause severe viral hemorrhagic fever in humans, with no FDA-approved vaccines or effective therapeutics. Arenaviral nucleoprotein (NP) contains an exoribonuclease domain (exoN), which helps to evade the host immune system by blocking the production of type I interferons (IFNs). However, the biological role of NP exoN in arenavirus infection is not well understood. In this study, we used a recombinant, tri-segmented non-pathogenic Pichinde virus (PICV) with NP exoN mutations as a safe and infectious arenavirus vector to express JUNV NP as a transgene. We found that JUNV NP exoN plays a key role in suppressing IFN-I responses and discovered a previously unrecognized function of exoN in producing infectious virion particles in IFN-competent cells. These findings improve our understanding of arenavirus infection and identify NP exoN as a promising target for developing new antiviral therapies.

microbiology↗