Biology subjects

Zhou, T.

Publications and source records attributed to Zhou, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Rapid and efficient oligodendrocyte differentiation from human pluripotent stem cells via dual inhibition of BMP and Notch signaling

The protracted timing required for oligodendrocyte differentiation from human pluripotent stem cells (hPSCs) has limited their use in disease modeling, drug screening, and cell therapy. In particular, the signals that drive oligodendrocyte specification and maturation after neural induction and ventral patterning remain poorly understood. Here, we present a protocol to derive human oligodendrocytes from hPSCs that is based solely on extrinsic cues, and we identify dual inhibition of BMP and Notch signaling as critical drivers of oligodendrocyte commitment and maturation. By day 42 of differentiation, up to 70% of the cells are positive for the oligodendrocyte marker O4, with minimal astrocyte contamination, and show robust expression of mature myelin markers including MBP, MOG, and MAG. These hPSC-derived oligodendrocytes closely match the molecular identity of primary fetal human oligodendrocytes as assessed by single-cell RNA sequencing and are functional as shown by in vitro myelination assays. In addition to the rapid generation of myelinating oligodendrocytes, the new protocol can be modularly adapted for the efficient production of PDGFR+ oligodendrocyte precursors or mixed glial populations containing AQP4+ astrocytes, thereby providing a cellular toolbox for the study of human glial lineages in translational applications.

developmental biology↗

bioRxiv2026

A human developmental and adult brain atlas benchmarks dopaminergic stem cell models and cell therapy candidates

Parkinsons disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons1. Stem cell-derived mDA neurons hold promise for disease modelling2,3 and are currently in clinical trials for cell replacement therapy4-6. However, systematic benchmarking has been limited by the lack of a unified high-resolution reference and methods that quantify incomplete or mixed lineage specification in vitro7. We establish a single-cell and spatial atlas of the human developing diencephalon-midbrain-hindbrain axis resolving 93 cell subtypes, including 39 lacking prior single-cell characterization and 4 entirely novel populations. Using this atlas as a reference, we integrate 19 hPSC-derived mDA datasets, both published2,8-25 and unpublished, to build the Human Dopaminergic Neural Atlas (HDNA) spanning 2D, 3D, and graft models, including those used in clinical trials. To classify cells and quantify lineage fidelity, we develop CapybaraBrain, a marker-driven non-negative decomposition framework that assigns each cell continuous identity scores across all 93 developmental programs, enabling systematic discrimination of discrete, transitioning, and cross-lineage hybrid states26. We uncover a pervasive landscape of off-target populations reflecting relaxed transcriptional boundaries in vitro, including a previously unrecognized TH-PITX2 midbrain neuronal population, and we validate atlas-predicted latent lineage plasticity through inducible genetic fate mapping in mouse models. We further define maturation-associated transcriptional programs by harmonizing adult mDA subtype atlases, revealing that dopaminergic identity and maturation are partially decoupled across protocols. Finally, projecting PD patient-derived tri-cultures onto the HDNA uncovers genotype- and cell-type-specific transcriptional dysregulation. Together, these integrated atlases and computational framework establish a unified standard for benchmarking differentiation fidelity, exposing off-target states, and guiding next-generation PD models and cell therapies.

Developmental 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↗

bioRxiv2026

Cobamide-Dependent Dichloromethane Fermentation by Dehalobacter Reveals a Hidden Acetogenic Route for Organohalide Biotransformation

Halogenated one-carbon (C1) compounds, such as dichloromethane (DCM), drive critical fluxes in global carbon and halogen cycles. While the genus Dehalobacter is canonically defined by obligate organohalide respiration, its physiological and ecological roles in anaerobic C1 metabolism have remained fundamentally ambiguous. Here, we document a paradigm-shifting metabolic capacity within a sediment-derived microbial consortium: the autonomous fermentation of DCM by a novel population, Candidatus Dehalobacter formatiformans strain J1. Over successive transfers, strain J1 outcompeted co-existing Dehalobacterium formicoaceticum to become the overwhelmingly dominant population (>80% relative abundance), converting DCM stoichiometrically to acetate and formate (4:1) without auxiliary substrates. Genome-resolved metagenomics revealed that strain J1 couples a distinct mec gene cassette--mediating methyl-transfer reactions during DCM activation--to a complete Wood-Ljungdahl pathway for efficient C1 assimilation. Crucially, strain J1 lacks the complete genetic repertoire for de novo cobamide biosynthesis. Physiological validation confirmed that this fermentative pathway is strictly dependent on exogenous cobamides, exposing a profound reliance on community cross-feeding. These findings reveal an unexpected acetogenic lifestyle within Dehalobacter, a lineage historically viewed as comprising obligate organohalide-respiring bacteria. More broadly, this work identifies cobamide-dependent methyl-transfer metabolism as an ecological control on anaerobic DCM fermentation and expands the known roles of Dehalobacter in carbon-halogen cycling in anoxic environments.

microbiology↗

bioRxiv2026

The in vivo inhibitory function of the MHC-I alpha3 domain/CD8alpha interaction

The interaction between the Major Histocompatibility Complex Class I (MHC-I) 3 domain and CD8 has classically been viewed as a positive coreceptor interaction that stabilizes TCR signaling during antigen recognition. However, its physiological function in mature peripheral CD8+ T cells in vivo remains incompletely understood. Here, we identify the MHC-I 3 domain-CD8 interaction as a previously unrecognized inhibitory pathway that tonically restrains peripheral CD8+ T-cell activation and maintains T-cell tolerance in vivo. Antibody-mediated disruption of the MHC-I 3 domain-CD8 interaction induced spontaneous activation of peripheral CD8+ T cells without impairing their survival, lowered the threshold for antigen-induced activation, and enhanced responsiveness to cognate peptide stimulation. In peptide-induced OT-I T-cell anergy models, blockade of either H-2Db or H-2Kb 3 domain interactions with CD8 prevented the induction of anergy and restored responsiveness of previously anergic T cells. Notably, blockade of the H-2Kb 3 domain enhanced OT-I responses despite simultaneously disrupting the classical positive coreceptor interaction within the TCR-peptide-MHC complex, indicating that tonic inhibitory signaling mediated by the MHC-I 3 domain predominates under these conditions. Together, these findings redefine the classical MHC-I-CD8 interaction as a bidirectional pathway that not only supports antigen recognition but also imposes tonic inhibitory control over peripheral CD8+ T cells. These results identify the MHC-I 3 domain-CD8 axis as a potential target for reversing T-cell tolerance and enhancing antitumor or antiviral immunity.

immunology↗

bioRxiv2026

Twitchin kinase, a mechanoreceptor in the muscle sarcomere, is a catalytically-primed moonlighting kinase

To explore conserved mechanisms and functions across mechanosensory kinases associated with the skeletal architectures of the cell, we investigated in vitro and in vivo the substrate targeting of twitchin kinase (TwcK), a mechanoreceptor from the muscle sarcomere. Specifically, we elucidated the crystal structure of TwcK in complex with substrates, used real-time 31P-NMR spectroscopy and luminescence-based assays to identify the phosphorylation site on a model peptide substrate, mined the C. elegans proteome to reveal the myosin regulatory protein MLC-4 as a substrate candidate and used CRISPR/Cas9 genome-edited and transgenic C. elegans strains to query the relation of twitchin and MLC-4 in muscle. Contrary to expectations, we find that TwcK undergoes activating conformational changes that are regulated by an N-terminal tail sequence that blocks hinge dynamics in the kinase fold. This distinct mechanism is conserved across sarcomeric, but not cytoskeletal, kinases. Functionally, cytoskeletal and sarcomeric kinases share an evolutionarily conserved phosphorylation targeting of myosin light chain (MLC) proteins. Yet, we find TwcK and its MLC4 substrate to segregate in vivo and not to constitute a functional kinase/substrate pair. Thus, canonical substrate targeting cannot be delivered by TwcK in its cellular context, where it has adopted a moonlighting role. We deduce this result to apply to other intrasarcomeric kinases. Our findings highlight how the cell context confers functional individuality to non-diffusible, otherwise conserved skeletal kinases.

biophysics↗

bioRxiv2026

Structural Basis of Ligand-Specific Orthosteric-Allosteric Coupling and Sensory Tuning in the Human Bitter Taste Receptor TAS2R14

The bitter taste receptor TAS2R14 recognizes hundreds of structurally diverse ligands, yet the mechanisms governing its broad promiscuity, graded agonist efficacy, and multi-site regulation remain unclear. Here, we report cryo-EM structures of TAS2R14-Gi1 complexes bound to 3,5-diiodosalicylic acid (DA), flufenamic acid (FA) and aristolochic acid (AA) at resolutions of 2.44 [A], 2.39 [A], and 2.69 [A], with a clear efficacy hierarchy of AA > FA > DA. Transmembrane helix 6 (TM6) acts as a core regulatory hub, with agonist binding triggering 12 [A] lever-like TM6 rearrangements to reshape receptor interfaces and modulate G-protein coupling strength. We also identify cholesterol hemisuccinate (CHS) as a new TAS2R14 orthosteric agonist, selectively potentiating DA-mediated signaling potency by [~]5-fold via allosteric-orthosteric coupling without affecting FA or AA. Our findings show TM6 dynamics unify TAS2R14s key functional features, advancing insights into bitter receptor activation and sterol regulation.

molecular biology↗

bioRxiv2026

Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

Post-traumatic stress disorder (PTSD) involves complex neuroimmune-synaptic crosstalk in the hippocampus. Here we show that interleukin-18 (IL-18), an extensively studied pro-inflammatory cytokine, serves a protective role against traumatic fear memory through a microglia-to-neuron signaling axis. Traumatic stress induces sustained upregulation of IL-18 in the hippocampus. Exogenous IL-18 administration attenuates fear memory, whereas blockade of IL-18 signaling exacerbates it. Mechanistically, microglial-derived IL-18 acts on neuronal IL-18R1 to restore stress-impaired synaptic plasticity and reduce perineuronal net density, thereby facilitating structural synaptic remodeling. In addition, IL-18 modulates the synaptic organization of fear memory-encoding engram cells within hippocampal ensembles. Together, these findings redefine IL-18 as a homeostatic regulator of post-trauma hippocampal synaptic function and identify the hippocampal IL-18 pathway as a potential therapeutic target for PTSD.

neuroscience↗

bioRxiv2026

Coronavirus envelope protein drives iron sensing disorder by hijacking the TAp73-FDXR axis

Iron overload is increasingly recognized as a critical contributor to coronavirus pathogenesis1, yet the underlying induction mechanisms remain unclear. Here, we uncover a fundamental pathway by which coronavirus drives IRP1 RNA-binding activity to induce iron accumulation2 via targeting the TAp73-FDXR axis. Specifically, coronavirus infection represses transcription of FDXR (encoding the key rate-limiting enzyme in host iron-sulfur cluster synthesis3), thereby impairing host iron-sulfur cluster generation to trigger the functional conversion of the cytosolic aconitase 1 (ACO1) into iron-regulatory protein 1 (IRP1)4, ultimately leading to the hosts persistently false perception of iron deficiency. We identify TAp73 as the primary transcription factor governing FDXR expression, and demonstrate that the coronavirus envelope protein (CoV-E) orchestrates TAp73 nuclear export. Subsequently, CoV-E binds TAp73 through a critical valine residue within its C-terminal PBM domain, inducing the K48-linked ubiquitination and proteasomal degradation of TAp73. Furthermore, we developed a CoV-E-targeting molecule, DPTP-FC, which blocks CoV-E-TAp73 interaction via forming steric hindrance and effectively alleviates iron accumulation and tissue damage caused by PEDV, PDCoV, and SARS-CoV-2 infection. Our study reveals the central role of the TAp73-FDXR axis in CoV-induced iron accumulation, highlighting CoV-E as an attractive antiviral target and DPTP-FC as a promising therapeutic candidate.

microbiology↗

bioRxiv2026

Paternal behavior is controlled by preoptic Trpc5 neurons

Male parenting behavior is highly conserved and activated only after offspring are born. Here we show that the behavioral switch from infanticide in virgin male mice to caregiving in wild-type sires is associated with enhanced expression and activation of Transient receptor potential channel 5 (Trpc5) in estrogen receptor (Esr1)-expressing neurons in the hypothalamic medial preoptic area (MPOA). While selective deletion of Trpc5 from Esr1MPOA neurons diminishes paternal behavior in sires, overexpression of Trpc5 in these neurons converts the otherwise infanticidal virgin males to exhibit care to pups. Mechanistically, Trpc5-dependent changes in neuronal excitability underlie fatherhood-associated Esr1MPOA neuron plasticity. Notably, Trpc5 overexpression also enhances escape behavior and elicits exploratory diving behavior, suggesting that these neurons control a broader adaptive parenting response in males. These findings establish a Trpc5-dependent MPOA neural signal as a critical regulator of paternal behavior.

neuroscience↗

bioRxiv2026

Inosine misincorporation into mRNA triggers the integrated stress response and activates an innate immune gene expression signature

The inosine triphosphate pyrophosphatase (ITPase) enzyme restricts levels of the non-canonical nucleotides (deoxy)inosine triphosphate (dITP/ITP) and prevents their aberrant misincorporation into nucleic acids. ITPase deficiency is associated with dilated cardiomyopathy and epileptic encephalopathy in humans and is usually fatal in infancy. It leads to pronounced inosine misincorporation into RNA but the cellular consequences of this misincorporation are not well understood and the pathogenic basis of ITPase deficiency remains unknown. Here we show that cellular transfection of mRNA with inosine misincorporation activates the integrated stress response (ISR) with an innate immune gene expression signature. This stress response triggers stress granule formation and is modulated by the double stranded RNA sensor protein Kinase R (PKR). Inosine nucleoside treatment of ITPase-deficient cells leads to endogenous inosine misincorporation into mRNA and activation of the ISR. Further, differentiation of human ITPase-deficient induced pluripotent stem cells into neurons results in a low-level stress response. Thus, our study establishes inosine misincorporation into mRNA as an unappreciated form of cellular stress. This is normally prevented by the ITPase enzyme, with implications for the pathogenesis of ITPase deficiency.

molecular biology↗

bioRxiv2026

THIK-1 channel mediates microglial glucose sensing and modulates AgRP neurons

Microglia play essential roles in maintaining energy homeostasis, and their dysfunction contributes to metabolic disease. Although high-fat diet (HFD) exposure induces microglial activation, the underlying mechanisms remain poorly defined. Here, we identified a previously unrecognized role for THIK-1 channel in mediating glucose sensing of microglia in arcuate nucleus of the hypothalamus (ARH), during HFD-induced obesity. Pharmacological inhibition of THIK-1 channel with tetrapentylammonium (TPA) suppresses feeding and attenuates body-weight gain in diet induced obese mice. Mechanistically, inhibition of agouti-related peptide (AgRP) neurons is indispensable for TPA-induced hypophagia. Moreover, THIK-1 inhibition promotes microglial phagocytosis of perineuronal nets (PNNs), leading to reduced AgRP neuronal activity and feeding suppression. Together, these findings establish THIK-1 as a critical glucose sensor in hypothalamic microglia and uncover a microglia-dependent pathway through which overnutrition modulates AgRP neuronal activity via PNN remodeling to regulate energy balance, highlighting THIK-1 as a potential therapeutic target for treatment of diet-induced obesity.

neuroscience↗

bioRxiv2026

MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

ATG2A transfers glycerophospholipids from the endoplasmic reticulum (ER) to support expansion of the isolation membrane at ER membrane contact sites. While a role for ATG18/WIPI4 in targeting ATG2A to the IM is well established, how ATG2A is recruited to the ER remains unclear. In this study, we found that MOSPD3, an atypical VAP family protein, acts an adaptor that recruits ATG2A to the ER. MOSPD3 colocalizes with ATG2A and is specifically enriched at ER sites juxtaposed with the IM during autophagosome formation. Recruitment is mediated by direct interactions between the FFNT (two phenylalanines in a neutral tract) motif in the N-terminal region (NT) of ATG2A and the major sperm protein (MSP) domain of MOSPD3. Co-expression of MOSPD3 with ATG2A-NT, but not a MOSPD3-binding-defective mutant ATG2A-NT-T362A, markedly rescued the autophagic defect caused by ATG2A/B double knockout (DKO). MOSPD3 depletion abolishes ATG2A recruitment to the ER and impedes autophagic flux. Together, this study demonstrates that MOSPD3 functions as an ER adaptor for ATG2A in autophagy.

cell biology↗

bioRxiv2025

MAGNET: an all-in-one foundation model for cross-modal and cross-dimensional microscopic image restoration

The application of emerging Foundation models in current microscopy has remained limited due to task-specific model designs, strong modality dependence and insufficient robustness under complex degradations. Here, we introduce MAGNET (Microscopic All-in-one General fouNdation model for imagE resToration), the first end-to-end All-in-One foundation model designed for universal microscopic image restoration. MAGNET integrates multi-task, cross-modal and cross-dimensional (2D + 3D) image restoration capabilities within a single unified architecture, and it is specifically designed to handle composite degradations in practical imaging systems. The framework comprises three components: (i) a task-aware, prompt-guided feature enhancement module for adaptive learning across diverse degradations; (ii) an LIIF-based reconstruction module enabling continuous, resolution-adaptive restoration; and (iii) a dimension-compatible triple-plane projection module supporting both 2D and 3D data. Trained on large-scale datasets spanning 5 major microscopy modalities (structured illumination, confocal, light-sheet, wide-field and two-photon) and 8 representative restoration tasks, MAGNET achieves state-of-the-art performance across super-resolution, denoising, deblurring, background suppression, isotropic reconstruction, aberration correction, de-scattering and virtual staining. Beyond supervised training, MAGNET supports direct inference on unseen systems, efficient few-shot fine-tuning and self-supervised test-time optimization, enabling robust performance across diverse data and imaging conditions. MAGNET serves as a unified, generalizable, and scalable computational framework for microscopy, enabling more efficient and reliable downstream analyses in cell biology, neuroscience, and pathology.

cell biology↗

bioRxiv2025

A stem cell knockout village reveals lineage rewiring and a non-canonical islet cell fate in monogenic diabetes

Genetics studies have identified a core set of regulators essential for pancreatic {beta} cell development, many of which are mutated in monogenic diabetes. However, how these mutations alter developmental trajectories to produce pathological cell states remains elusive. Here we introduce a knockout village framework that enables longitudinal scRNA-seq profiling of 79 human pluripotent stem cell mutant lines targeting 30 developmental regulators, including 15 diabetes genes, across five islet differentiation stages. We show that loss of lineage regulators impairs {beta} cell formation in a stage-specific manner and rewires developmental trajectories towards competing lineages. Notably, several monogenic diabetes gene mutations drive a shift from {beta} cells to enterochromaffin (EC)-like cells, a recently recognized non-canonical islet cell fate. These EC-like cells exhibit incomplete activation of hormone regulation programs, along with elevated neuron signatures. Leveraging the diversity of cell fate outcomes across mutants, we predicted and experimentally validated ISL1 as a key downstream effector of PDX1 and PAX6 that safeguards {beta} cell identity against an EC-like fate. Together, our findings reveal cell fate rewiring as a widespread, previously underappreciated pathological mechanism in monogenic diabetes and establish a scalable platform for uncovering developmental vulnerabilities in human genetic disorders.

developmental biology↗

bioRxiv2025

Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography

{beta}-Lactamases catalyze {beta}-lactam antibiotic hydrolysis and are important contributors to bacterial antimicrobial resistance; {beta}-lactamase inhibitors are widely used to overcome {beta}-lactamase-mediated antibiotic resistance. Nucleophilic serine {beta}-lactamases (SBLs) react with their substrates and clinically available inhibitors via a covalent reaction to give complexes which can undergo further reaction. Using room temperature drop on fixed target serial crystallography, where ligands are rapidly mixed with microcrystals, and classical single-crystal crystallography at cryogenic temperatures, we investigate the reversible covalent reaction of the SBL CTX-M-15 with the diazobicyclooctane inhibitor avibactam. We observe avibactam covalently reacted (ring-opened) with the nucleophilic Ser70, at timepoints from 80 ms to minutes (room temperature) and hours (100 K). These crystallographic data reveal time-dependent movement of the avibactam carbamoyl complex, from 1.3 s onwards, that has implications for the 5-exo-trig recyclization mechanism that determines inhibitor reformation. Combined with molecular dynamics simulations and quantum mechanics calculations at the density functional theory level, the results show that in the first seconds of the reaction the avibactam N-sulfate nitrogen is poorly positioned for recyclization. This subsequently equilibrates after 10 s to a stable endpoint that is in a conformation potentially primed to initiate recyclization through attack of the N-sulfate nitrogen on the carbamoyl carbon. These results further demonstrate the capacity of room-temperature serial crystallography to capture time-resolved changes in ligand conformation at an enzyme active site, complementing discrete classical cryo-crystallography. These data inform on ligand dynamics and the stereoelectronics of diazobicyclooctane inhibition, aiding drug discovery efforts to develop inhibitors of nucleophilic enzymes.

biochemistry↗

bioRxiv2025

Drop on fixed target reaction initiation approach for serial and time resolved crystallography

We describe the design and implementation of a drop on fixed target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume (40 - 90 pL) aqueous droplets, containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals immobilised on a solid support. The system was tested with various enzyme systems, including lysozyme and two -lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free electron laser facilities was evaluated. This drop on fixed target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

biophysics↗

bioRxiv2025

Single-cell transcriptomic dynamics exposes hidden survival trajectory under antibiotic treatment

Heterogeneity plays a major role in bacterial resistance to antibiotic treatment while the mechanism at single-cell level is largely unknown. Here, we employed a robust integration of bulk and bacterial single-cell RNA-seq (scRNA-seq) to uncover how individual cells of Acinetobacter baumannii reorganise the heterogenous transcriptome in response to antibiotic. Using polymyxin as a representative, bulk RNA-seq showed canonical envelope- and efflux-centred responses but obscured underlying heterogeneity. Single-cell profiling resolved these averages into discrete subpopulations whose abundances shifted with concentration and time. Specifically, in early time an envelope-stress programme predominated survival at the low concentration, whereas an outer-membrane repair/efflux programme dominated survival at the high concentration. These patterns revealed structured and time-resolved heterogeneity, highlighting an ingenious bacterial stress responsive strategy. Through trajectory inference, we further revealed a concentration-dependent bifurcation of cell fates: low-concentration treated cells detoured through a transient stress state and rejoined growth, whereas high-concentration treated survivors diverted into a slow-growing, tolerant branch. Perturbing marker genes from these programmes altered fitness eventually and rapidly increased permeability, depolarisation and reactive-oxygen burden, linking state to survival capability. Collectively, we map a dynamic, concentration-structured landscape of antibiotic responses at the single-cell level, revealing diverse survival trajectories that are obscured in conventional population-averaged analyses. Our developed single-cell based framework revealed tolerant bacterial cells with rewiring of the transcriptional landscape, causing emergence of antibiotic resistance. Importantly, these findings urge precision antimicrobial therapy in patients to minimise emergence of antibiotic resistance.

microbiology↗