Biology subjects

Wang, J.

Publications and source records attributed to Wang, J..

At least 199 records · Page 11Linked to original sources

bioRxiv2026

Speech and music exploit distinct intrinsic timescales of the sensorimotor system

Humans produce a remarkable diversity of vocal behaviors throughout life, from innate sounds such as cry, laughter, and babble to learned, sophisticated sequences such as speech and song. Whether these diverse vocalizations share basic principles in their temporal organization remains unknown. Here, we show that, despite their distinct functions and extraordinary cultural variation, these vocalizations consistently occupy two characteristic rhythmic regimes supporting distinct functionalities. Corpus analyses revealed that, by approximately three years of age, the acoustic rhythms of speech and song settle into two distinct frequency ranges: one between 4 and 8Hz, the other below 2Hz. Critically, these two temporal regimes are already evident in infant cry, laughter, and babble, prior to the emergence of language and song. Sensorimotor synchronization experiments, where participants vocalized or tapped to sound sequences presented at different rates, revealed that individual vocal production exhibited maximal resonance between 4 and 8Hz, whereas synchronization across individuals was strongest below 2Hz. Together, these findings identify two functionally distinct sensorimotor timescales that organize diverse human vocal behavior: a faster timescale supporting stable individual vocal production and a slower timescale facilitating interpersonal synchronization.

animal behavior and cognition↗

bioRxiv2026

Cerebellar microRNA-206 tunes Purkinje neuron firing dynamics to control sensorimotor gating

MicroRNAs are potent regulators of gene expression in the brain, yet the cellular mechanisms through which they shape neuronal activity and behavior remain poorly understood. MicroRNA-206 (miR-206) has been genetically and transcriptionally linked to schizophrenia and other neuropsychiatric disorders, but its functions in the nervous system are largely unknown. Here we show that miR-206 expression in the brain is restricted to postnatal cerebellar Purkinje cells (PCs). miR-206 was dispensable for PC cell fate specification, dendritic morphogenesis, and cerebellar-regulated motor coordination. Transcriptional profiling with single-nucleus and spatial resolution, integrated with Ago2-associated miRNA-target repression mapping (HITS-CLIP) and ribosome-associated RNA profiling (TRAP-seq), showed that miR-206 regulates translational programs in PCs controlling neuronal excitability. Accordingly, miR-206 deficiency increased the tonic firing of PCs, whereas elevating miR-206 expression shifted PCs from tonic to high-frequency burst firing. Constitutive or PC-specific deletion of miR-206 impaired prepulse inhibition (PPI) of the acoustic startle response, a conserved form of sensorimotor gating disrupted in schizophrenia and related disorders. Restoration of miR-206 expression in PCs rescued PPI deficits in miR-206-deficient mice, while elevating miR-206 expression in PCs impaired PPI in wild-type animals. Together, these findings reveal that a schizophrenia-linked microRNA tunes Purkinje neuron firing dynamics to control sensorimotor gating.

neuroscience↗

bioRxiv2026

Species-Specific Roles of RIPK1 and TRADD in TNF-Induced Cell Death Reveal a Translational Gap Between Mouse Models and Human Biology

Mouse models have historically been central to studies of TNF-induced cell death and guided pharmaceutical translation into clinic, based on the assumption that TNF signaling is conserved between human and mouse. Here, our work uncovers critical species-specific differences between the two. By systematically dissecting the roles of RIPK1, TRADD, and sensitivity to RIPK1 inhibitors in TNF signaling--including RIPK1 kinase-dependent and-independent apoptosis--we found that both apoptosis modalities diverge between human and mouse cells. In mouse cells, RIPK1 suppresses TRADD-mediated kinase-independent apoptosis, whereas in human cells, RIPK1 and TRADD act redundantly. Moreover, RIPK1 inhibitors block kinase-dependent apoptosis in mouse but not human cells, despite effectively inhibiting RIPK1 S166 phosphorylation. Cross-species complementation revealed that these discrepancies stem not from RIPK1 itself but from cell-context differences. These findings echo the clinical failures of RIPK1 inhibitors despite efficacy in mouse models and underscore the need for humanized models and therapeutics that more faithfully predict clinical outcomes.

Cell Biology↗

bioRxiv2026

A brain-tumour axis links chronic stress to gastric cancer progression through NPY signalling

Chronic stress promotes gastric tumour growth, but the central neural pathways remain largely enigmatic. Here, we identify a brain-tumour axis, implicating the central amygdala (CeA) activity in promoting gastric cancer growth through sympathetic neuropeptide Y (NPY) signalling occurring within the tumour. Using clinical datasets, patient samples, pathway tracing, electrophysiology and chemogenetic manipulation, we demonstrate that stress selectively increases NPY Y1 receptor (NPY1R) synthesis in the patients gastric cancer tissue; moreover, the NPY1R density correlates with advanced cancer stage and poor prognosis. In mice, daily constraint chronic stress enhances CeA excitability and promotes sympathetic neurotransmitter norepinephrine release within the tumour microenvironment. Norepinephrine, in turn, increases tumour endogenous NPY/NPY1R production and the activation of downstream MAPK/Erk1/2 signalling pathway, which drives cancer cell proliferation and invasion. Manipulating CeA neuronal activity alone can regulate tumour growth, whereas selectively blocking tumour NPY1R prevents stress-induced tumour progression in vivo and cancer cell migration in vitro. This study identifies a central-to-peripheral circuit linking chronic stress to gastric cancer growth, and highlights NPY1R as a potential therapeutic target.

Cancer Biology↗

bioRxiv2026

mTORC1 Directs TNF-Induced Life-or-Death Decisions via Complex I Destabilization

The transition from tumor necrosis factor (TNF)-induced plasma membrane-bound complex I to cytosolic death-inducing complex II switches cells from survival to death. However, the precise regulation of this fatal decision is incompletely understood. Here, we show that mTORC1 promotes this transition by destabilizing later-stage complex I without affecting its initial assembly. Inhibition of mTORC1 unleashes ATG9A and FIP200 activity, thereby promoting the accumulation of CHUK (IKK) in complex I. CHUK scaffolds the kinase-active IKK{beta} to stabilize complex I and prevent complex II formation. Activation of this ATG9A/FIP200-CHUK/IKK{beta} axis protects against TNF-induced fulminant hepatitis while compromises antibacterial defense against Staphylococcus aureus. This mTORC1-governed life-or-death transition provides therapeutic insight into TNF-related pathologies--including cancer, metabolic tissue injury, and microbial infections--where mTORC1 activity is frequently suppressed.

Cell Biology↗

bioRxiv2026

Kaposi's Sarcoma-associated herpesvirus uses a novel protein fold to hijack RNA Polymerase II for viral late gene transcription

Herpesviruses, including the oncogenic human pathogen Kaposis Sarcoma-associated herpesvirus (KSHV), rely on cellular RNA Polymerase II (RNAPII) for expression of their protein-coding genes. Late in the lytic cycle, transcription initiation on a subset of KSHV genes depends on ORF24, a viral transcriptional activator that binds to viral late promoters through structural mimicry of a host transcription factor. Here, we present a structure of the N-terminal domain of ORF24, and show that it adopts a novel fold that facilitates direct binding of the RNAPII C-terminal domain (CTD). We further show that ORF24 is highly sensitive to the phosphorylation state of the RNAPII CTD, allowing for selective binding of the hypophosphorylated form to viral preinitiation complexes. By combining promoter binding and polymerase recruitment into a single protein, KSHV has streamlined essential steps in transcription complex assembly. Our work lays the groundwork for mechanistic dissection of a viral preinitiation complex.

microbiology↗

bioRxiv2026

Mechanism of tandem-repeat DNA synthesis by an antiviral reverse transcriptase

Defense-associated reverse transcriptases (DRTs) employ DNA synthesis to protect bacteria against phage infection1,2. We previously showed that DRT10, a tripartite system comprising an RT, a noncoding RNA (ncRNA), and a SLATT effector protein, catalyzes protein-primed, tandem-repeat DNA synthesis in a mechanism strikingly analogous to eukaryotic telomerase3. However, the structural basis by which the RT-ncRNA complex directs repeat addition processivity and controls repeat length remains unknown. Here we present cryo-EM reconstructions of two evolutionarily diverse DRT10 RT-ncRNA systems that reveal an unanticipated 2:1 architecture, wherein two RT monomers bind opposite sides of a single, pseudo-symmetric ncRNA. Biochemical experiments demonstrate that each RT monomer reverse transcribes the template encoded on its respective side of the ncRNA, but only one generates the long repetitive product, with the template sequence defined by the distance between two flanking stem-loop anchors. Together with earlier studies of DRT2, DRT3, and DRT94-6, our findings identify a conserved mechanistic logic underlying ncRNA-templated tandem-repeat synthesis across Class 2 UG antiviral systems, despite vastly different architectural solutions.

microbiology↗

bioRxiv2026

Depleting prion protein using splice-switching small molecules

Prion diseases are fatal neurodegenerative disorders driven by prion protein (PrP) misfolding, and lowering cellular PrP represents a promising therapeutic strategy. Here we report a small-molecule approach that reduces PrP by modulating pre-mRNA splicing of the PRNP gene. Through chemical modification of the clinically approved splicing modulator risdiplam, we generated CP3, the first class of compounds that selectively activate a cryptic exon in PRNP and routes its mRNA product for degradation, reducing PrP by [~]70% in cells. We demonstrate that CP3 activity critically depends on alternative splicing factor Luc7L, revealing a novel requirement for alternative splicing factors in small molecule splicing modulation. Strikingly, co-administration of CP3 with a Luc7L activator PTC258 significantly lowers PrP levels in the brains of transgenic mice. These results establish splicing modulation as a powerful strategy for PrP reduction and highlight the potential of small-molecule cooperativity for therapeutic RNA targeting.

biochemistry↗

bioRxiv2026

Extended Amygdala CRF Projections to Striatal Striosomes Potentiate Dopamine Suppression During Fentanyl Withdrawal

Opioid withdrawal is a powerful negative affective state that promotes continued drug use and relapse, and is often associated with hypodopaminergic states. Although corticotropin-releasing factor (CRF) signaling in the extended amygdala has been strongly implicated in stress and negative affect, how withdrawal-recruited CRF systems suppress dopamine release remains poorly understood. Here, we identify an extended amygdala-striosome-dopamine pathway engaged during fentanyl withdrawal. Naloxone-precipitated fentanyl withdrawal selectively recruited CRF neurons in the central amygdala and bed nucleus of the stria terminalis. These CRF neurons provided monosynaptic input to dorsostriatal medium spiny neurons (MSNs). Because striosomal MSNs are positioned to regulate dopamine release, we next found that CRF enhanced glutamatergic synaptic transmission onto striosomal MSNs, with CRF receptor 1 (CRFR1) signaling required for the postsynaptic strengthening of excitatory transmission. In vivo dopamine photometry further revealed that naloxone-precipitated withdrawal amplified striosome-mediated suppression of dopamine release, and this amplification was prevented by CRFR1 antagonism. Together, these findings reveal a mechanism by which withdrawal-recruited CRF signaling potentiates striosomal control of dopamine release. This extended amygdala-striosome-dopamine pathway may contribute to the hypodopaminergic negative affective state that drives opioid withdrawal and relapse vulnerability. HighlightsO_LIFentanyl withdrawal selectively recruits CeA and BNST CRF neurons C_LIO_LICeA and BNST CRF neurons provide monosynaptic input to striatal MSNs C_LIO_LICRF potentiates glutamatergic transmission onto striosomal MSNs via CRFR1 C_LIO_LICRFR1 signaling amplifies striosome-mediated dopamine suppression during withdrawal C_LI

neuroscience↗

bioRxiv2026

PlantGeneAnn: a strand-specific genome foundation model for ab initio gene structure annotation of plant genomes

High-quality plant genome assemblies are rapidly increasing, but accurate structural annotation remains reliant on transcript and homology evidence, limiting applications in newly sequenced and non-model species. Here, we present PlantGeneAnn, a plant-optimized, strand-specific genome foundation model for ab initio gene structure annotation. Fine-tuned on only nine high-quality model plant annotations, PlantGeneAnn outperformed a multi-species model trained on 42 species, showing that annotation quality is more important than token volume. On a stringent 13-species benchmark covering rosids, asterids, and monocots, PlantGeneAnn surpassed four state-of-the-art baselines across five evaluation levels, from base-level classification to complete transcript recovery. It achieved higher intron precision and better captured complex gene structures. In zero-shot variant effect prediction, PlantGeneAnn identified cryptic splice donors and premature stop codons in maize and rice, with saturation mutagenesis confirming single-nucleotide, context-dependent sensitivity. It also retained generalizability for epigenomic track prediction, highlighting its value for pan-genomics, crop improvement, and non-model plant research.

bioinformatics↗

bioRxiv2026

ESCRT-0 regulates AMPA receptor currents and Ca2+- dependent signaling

Membrane protein trafficking is essential for synaptic growth, maintenance, function, and plasticity, and involves the regulated exocytosis and endocytosis of proteins to and from the pre-and post-synaptic membranes. Defects in the clearance of membrane proteins can lead to the accumulation of ubiquitinated membrane proteins and contribute to neurodegenerative disease. The ESCRT (endosomal sorting complexes required for transport) machinery binds and sorts ubiquitinated membrane proteins into lysosomes for degradation, yet the presence and function of ESCRTs in sorting ubiquitinated AMPA and other receptors at the post-synapse remain unclear. Here we show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and levels are modulated by neuronal activity, increasing and decreasing with higher and lower neuronal activity, respectively. Phosphoproteomic profiling of Hrs-depleted post-synaptic membranes revealed a role for Hrs in glutamatergic synaptic transmission, including long-term potentiation. In addition, Hrs-depleted neurons showed faster AMPAR current kinetics and reduced amplitude in whole-cell patch-clamp recordings. Genetic deletion of neuronal Hgs in mice led to reductions in phosphorylated CaMKII- and -{beta} (T286/T287) and structural proteins, PSD-95 and gephyrin, suggestive of LTD (long-term depression)-like synaptic depression. In contrast, Hrs overexpression led to increases in Ca2+-dependent signaling, including protein kinase C (PKC) and PKC substrate, AMPAR subunit GluA1-S831, a site which increases conductance. Together, these findings identify a dynamic, bidirectional role for Hrs at the post-synapse as it both senses and is modulated by neuronal activity, ultimately impacting excitatory synaptic strength. Significance StatementSynaptic plasticity relies on dynamic trafficking and turnover of membrane proteins, including AMPA-type glutamate receptors (AMPARs), yet how receptor trafficking intersects with ubiquitin-mediated sorting pathways at synapses remains unclear. We show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and its abundance is bidirectionally regulated by neuronal activity. Genetic depletion of Hrs in mice reduces CaMKII phosphorylation and impacts AMPAR channel surface localization. In contrast, neuronal-specific Hrs overexpression led to enhanced GluA1 and protein kinase C substrate phosphorylation, suggesting altered AMPAR trafficking, subunit composition, and/or function. Thus, Hrs emerges as a modulator of glutamatergic signaling, coupling ubiquitin-mediated receptor sorting to the fine-tuning of synaptic transmission, with direct implications for learning and memory in health and disease.

neuroscience↗

bioRxiv2026

Intravenous midazolam alters short-interval paired-pulse TMS responses differently in younger and older adults

ObjectiveAging is associated with changes in cortical excitability and altered responsiveness to benzodiazepines, but the effects of benzodiazepine challenge on motor cortical paired-pulse physiology in older adults remain incompletely understood. We examined whether intravenous midazolam differentially modulates corticospinal excitability and short-interval paired-pulse transcranial magnetic stimulation (TMS) responses in younger and older adults. MethodsFifteen younger adults (18-35 years) and fifteen older adults (50-69 years) underwent single-pulse and paired-pulse TMS of the left primary motor cortex at baseline and during intravenous midazolam administration. Single-pulse motor evoked potential (MEP) amplitude was used to assess corticospinal excitability. Short-interval paired-pulse responses were quantified as the ratio of conditioned to unconditioned MEP amplitude. ResultsAt baseline, younger adults showed greater corticospinal excitability than older adults, reflected by larger single-pulse MEP amplitudes (adjusted p = 0.04). Younger adults demonstrated paired-pulse inhibition at baseline, reflected by a conditioned/unconditioned MEP ratio below 1.0 (ratio = 0.73; adjusted p < 0.01), whereas older adults did not show inhibition and instead had a mean ratio above 1.0 (ratio = 1.25). Midazolam reduced single-pulse MEP amplitudes in both groups. During midazolam administration, paired-pulse inhibition was no longer observed in younger adults, and older adults continued to show no evidence of inhibition. ConclusionsYounger and older adults differed in baseline corticospinal excitability and in short-interval paired-pulse TMS responses. Intravenous midazolam reduced corticospinal excitability and altered paired-pulse response patterns, eliminating baseline paired-pulse inhibition in younger adults while producing little measurable change in older adults. These findings suggest that aging may modify the net motor cortical response to benzodiazepine challenge. The results should be interpreted in relation to the paired-pulse stimulation parameters used and support further studies using complementary approaches to characterize age-related differences in inhibitory and facilitatory motor cortical circuits.

neuroscience↗

bioRxiv2026

Engineering of CAR-less lentiviral vectors via ER retention-mediated CAR blockade

Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of various tumor types by redirecting and activating T cells against tumor cells. However, CAR-T cell manufacturing approaches remain challenging and limit their widespread use in clinical settings. In vivo CAR-T therapy bypasses ex vivo cell manufacturing and patient preconditioning limitations; however, it faces a significant safety concern as CAR proteins on viral packaging cells are incorporated into budding virions, leading to off-target transduction of tumor cells. Here, we address this risk by developing the CAR-Less ER-Anchor Vector (CLEAN-V) system. By exploiting endoplasmic reticulum (ER) retention, CLEAN-V prevents the CAR protein from trafficking to the cell surface during viral packaging, thereby blocking its incorporation into the viral envelope. CLEAN-V particles exhibit near-complete loss of CAR-mediated tumor cell transduction. Furthermore, CLEAN-V integrates seamlessly into existing third-generation LVV workflows in four- or five-plasmid formats and generates CAR-T cells with preserved phenotypic and functional integrity. These results establish CLEAN-V as a robust platform for developing safe, targeted lentiviral vectors for in vivo CAR-T therapy.

bioengineering↗

bioRxiv2026

Field–Flow–Front mapping of breast cancer evolutionary dynamics in situ

Tumour progression reflects not only which clones arise but where, when and in what context they expand--dimensions genotype-centred reconstructions leave unresolved. Here we reconstruct breast cancer evolution in situ across 34 Visium HD, 16 Xenium, 32 MIBI-TOF and 10 CODEX samples, serial-section 3D reconstruction, single-cell and bulk transcriptomes, and genome-wide CRISPR dependency profiles. We partition tumours into 296 cancer microzones--stroma-bounded units within which expansion is reconstructed--and define in each a Cancer Progression Metric(CPM) coupling a transcriptomic clock to expansion geometry. Projected onto tissue, CPM yields a Field-Flow-Front model rendering progression as a continuous physical process and resolving subclonal architecture into spatially coherent domains rather than predefined branches. Unexpectedly, the most advanced fronts were not the most proliferative but low-dependency, slow-cycling populations with directional expansion, driven by an extracellular matrix programme whose evolutionary force exceeded inflammation by nearly an order of magnitude yet whose genes were the least cell-autonomous. Under chemotherapy it persisted while its clonal carriers reshuffled, marking a transferable, stroma-coupled front process--not a fixed clone--as the unit of advance. Distilled into an evolutionary advantage load, this front process predicted recurrence and survival across independent cohorts and improved on conventional staging, establishing a tissue-embedded paradigm for mapping tumour evolutionary dynamics, from local fronts to patient outcome.

Cancer Biology↗

bioRxiv2026

A human intracranial map of consciousness returning from anesthesia

How consciousness arises from the brain remains a central yet unsolved question. Although substantial effort has been devoted to characterizing the neural correlates of conscious perception during wakefulness, how consciousness re-emerges from unconscious states remains poorly understood. Here, we took advantage of the rare opportunity to record human intracranial local field potentials from propofol-induced general anesthesia through the transition to behavioral engagement and subsequent full wakefulness, capturing both the stable states of unconsciousness and consciousness and the reconstituting neural dynamics that bridge them. Anesthesia cannot be defined by any single electrophysiological signature; rather, it is an organized low-frequency regime characterized by a constellation of coordinated neural phenomena, including aperiodic slow waves, alpha/beta periodicity, global alpha synchronization, and slow-wave-alpha/beta phase-amplitude coupling. Following anesthetic cessation, this regime progressively dissolved as neural excitability and dynamical complexity climbed. The appearance of conscious behavior coincided with a fast and dramatic transformation in high-gamma activity, from stochastic and unpredictable bursts to structured responses that were distributed, task-selective, and event-locked. These findings suggest that the recovery of consciousness is a multiscale reorganization in which distinct neural underpinnings rise and fall. Together, this work charts an electrophysiological map of how conscious cognition is extinguished, reconfigured, and restored in the human brain.

neuroscience↗

bioRxiv2026

An Integrated muti-omics cell atlas of the human trabecular meshwork and ciliary body

The trabecular meshwork (TM) and ciliary body (CB) regulate aqueous humor dynamics and intraocular pressure (IOP), and TM/Schlemms canal (SC) dysfunction underlies glaucoma. Here, we present a spatially resolved multi-omics atlas of human TM and CB, integrating snRNA-seq, scRNA-seq, and snATAC-seq from over one million cells and nuclei across 112 donors with Xenium spatial transcriptomics. We identified 9 major cell classes and 21 cell types, revealing heterogeneity, including undercharacterized fibroblast and epithelial subpopulations. Spatial mapping supported TM fibroblast zonation and CB epithelial organization. Regulatory analyses identified cell type-specific programs, including OTX/PAX networks in CB epithelium and SMAD3/TGF-{beta} signaling in fibroblasts. Integration with glaucoma loci showed enrichment of non-coding variants in regulatory elements associated with POAG and PACG. Age- and ancestry-associated remodeling revealed divergent fibroblast aging with increased PIEZO1, suggesting impaired outflow and elevated IOP. Together, this high-resolution atlas links cellular, regulatory, and genetic variation to anterior segment function and glaucoma susceptibility.

genomics↗

bioRxiv2026

Human cardiomyocytes with trisomy 21 exhibit heightened susceptibility and immunological response to SARS-CoV-2 infection

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is associated with significant cardiovascular complications, including myocardial injury and long-term cardiac dysfunction. Individuals with Down syndrome (trisomy 21) exhibit increased susceptibility to severe COVID-19 outcomes, yet the cardiomyocyte-intrinsic mechanisms underlying this vulnerability remain poorly understood. To investigate genotype-specific responses to SARS-CoV-2 infection, we generated induced pluripotent stem cell-derived cardiomyocytes from individuals with trisomy 21 and their euploid, sex-matched biological relatives. Cardiomyocytes were inoculated with SARS-CoV-2, and viral susceptibility was assessed by immunofluorescence. Bulk RNA sequencing was performed under baseline and infected conditions to define transcriptional programs associated with viral response. Trisomy 21 iPSC-CMs exhibited increased susceptibility to SARS-CoV-2 infection, with greater viral protein expression and a higher proportion of infected cardiomyocytes compared to controls. Baseline transcriptomic analysis revealed no significant differences in canonical viral entry factors including ACE2 and TMPRSS2, suggesting that differential susceptibility is not driven by entry receptor availability. Following infection, both trisomy 21 and euploid control groups activated conserved antiviral pathways; however, trisomy 21 cardiomyocytes displayed a markedly amplified transcriptional response, with substantially greater numbers of differentially expressed genes. Upregulated pathways included interferon signaling, NF-{kappa}B activation, cytokine and chemokine signaling, and innate immune responses, while downregulated pathways were enriched for cardiomyocyte structural integrity, calcium handling, and metabolic processes. Notably, inflammatory and cytokine-related transcripts were significantly more elevated in trisomy 21 cells, consistent with an exaggerated immune response. These findings provide mechanistic insight into the increased cardiovascular risk observed in individuals with Down syndrome and highlight dysregulated immune signaling as a potential therapeutic target in this high-risk population.

Cell Biology↗

bioRxiv2026

Label-free Pathogen Identification with Microscopy Imaging and Deep Learning

Rapid and accurate pathogen identification is crucial for the clinical management of infectious diseases, particularly sepsis and severe respiratory infections, yet standard clinical workflows remain slow and resource-intensive. Here, we developed an automated, high-throughput imaging platform built on standard, clinically accessible bright-field microscopy, and generated a large dataset comprising 24.9 million label-free bacterial cells across six focal pathogens. Leveraging this resource, we trained a neural network (ESKAPe-ResNet) to identify ESKAPe species at the single-bacterium level. The model achieved >92% accuracy in species-level classification and >82% accuracy in quantifying ESKAPe abundance in mock mixtures, with high specificity against non-ESKAPe bacteria. In clinical validation using sputum, bronchoalveolar lavage fluid and blood samples from patients with respiratory infections and sepsis, the approach correctly identified the dominant ESKAPe pathogen in >78% of samples after minimum broth culture enrichment. The imaging-to-identification pipeline was completed in under 10 minutes, and coupled with brief cultivation, the median time to accurate identification was reduced to 5-6 hours, compared with days for conventional blood culture-based workflows. This work establishes the proof-of-principle for label-free, hardware-minimal rapid pathogen identification, providing a clinically deployable workflow to expedite diagnosis and reduce mortality in severe bacterial infections.

microbiology↗