Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 415 records · Page 23Linked to original sources

bioRxiv2025

Comprehensive annotation of olfactory and gustatory receptor genes and transposable elements revealed their evolutionary dynamics in aphids

Understanding the molecular evolution of genes involved in parasite adaptation and the role of transposable elements (TEs) in driving their diversification is key to unraveling how populations adapt to their environments. In phytophagous insects like aphids, olfactory (OR) and gustatory receptor (GR) genes are crucial for host recognition, yet their post-duplication evolution remains insufficiently explored. Here, we analyzed 521 OR and 399 GR genes, alongside TEs, across 12 aphid genomes with varying host ranges. Aphid lineages with broader host ranges exhibited higher evolutionary rates, driven by gene family expansions linked to host interaction, including lipid metabolism, immune function, and transposase activity. The evolution of OR and GR genes post-duplication was shaped by diversifying selection, with bursts of positive selection followed by long periods of purifying selection, consistent with adaptation to new hosts. OR and GR genes originated from proximal and tandem duplications, with younger TE activity enriched near these genes compared to other genomic regions, suggesting a role for TEs in catalyzing tandem duplications and fueling diversification. The star-like topology of the OR phylogenetic tree, low synteny, and recent TE activity around OR genes support a faster evolutionary rate for ORs than GRs - a trend observed in other insect taxa. This study provides insights into molecular mechanisms underlying host adaptation in aphids and presents the first high-quality genome assembly of Dysaphis plantaginea, a major apple pest, with a comprehensive annotation of chemosensory genes and TEs. These resources offer a foundation for research on aphid genome evolution, insect-plant interactions.

molecular biology↗

bioRxiv2025

The H3K27me3 reader UAD-2 recruits a TAF-12-containing transcription condensates to initiate piRNA expression within heterochromatic clusters

Metazoans utilize the small RNA pathway to regulate gene expression and maintain genome integrity. This pathway directs histone H3 lysine 9 tri-methylation (H3K9me3) or histone H3 lysine 27 tri-methylation (H3K27me3) at target loci to induce transcriptional gene silencing. Interestingly, some small RNAs are generated from genomic loci enriched in H3K9me3 or H3K27me3. However, the transcription mechanism of small RNA precursors from these heterochromatic regions remains unclear. In C. elegans, piRNAs originate from two genomic clusters enriched with H3K27me3 marks, which recruit the H3K27me3 reader UAD-2 and the upstream sequence transcription complex (USTC). Here, we demonstrate that piRNA transcription in C. elegans relies on TAF-12, a subunit of the basal transcription factor IID (TFIID). Depletion of TAF-12 reduces the production of both piRNA precursors and mature piRNAs. TAF-12 interacts with UAD-2 and facilitates piRNA focus formation in germ cell nuclei. We further show that TAF-12 triggers piRNA transcription by recruiting the RNA polymerase II subunit RPB-5, the Mediator complex subunit MDT-8, and the general transcription factors GTF-2F2 and GTF-2H2C. Thus, piRNA transcription within heterochromatic regions depends on the collaboration between histone modification readers, piRNA-specific transcription factors, and core transcription machinery.

genetics↗

bioRxiv2025

Tissue-wide profiling of human lungs reveals spatial sequestration of macrophages in tuberculosis

The immune response to human tuberculosis (TB), particularly in the context of complex lung pathology, remains incompletely understood. Here, we employed whole-slide spatial proteomics to map immune cell organization in TB-affected human lung tissues. Our analysis revealed pronounced spatial segregation of major immune cell populations in non-necrotizing TB lesions. At the tissue level, macrophages and lymphocytes formed distinct cellular communities associated with specific pathological features. At the lesion level, macrophages and B cells showed an inverse relationship in both abundance and spatial distribution. Proinflammatory T cells preferentially accumulated in macrophage-rich lesions but remained largely separated from macrophages. Interestingly, lesions exhibiting clear segregation between T cells and macrophages were more common in subclinical TB than in active disease. These findings suggest that spatial isolation of macrophages from effector lymphocytes may help temper inflammation and potentially prevent lesion progression to necrosis, while also enabling immune evasion by Mycobacterium tuberculosis. One Sentence SummaryXiao et al. reveal spatial segregation of immune cells in TB-lung tissue and link the microenvironmental dynamics to disease states of tuberculosis.

immunology↗

bioRxiv2025

Modular Engineering of Escherichia coli for Enhanced Nickel Uptake, Survival, Biomineralization, and Hydrogen-Supported Bioremediation

Nickel contamination poses increasing environmental and industrial challenges, demanding effective and sustainable remediation strategies. Here, we present a modular synthetic biology approach to engineer Escherichia coli for efficient nickel enrichment, resilience in toxic environments, intracellular biomineralization, and hydrogen-based co-culture stability. Our system comprises four functional modules: (1) a nickel enrichment module, in which the fusion protein NixA-F1v outperformed existing uptake systems, and a mutant repressor RcnRC35L enhanced retention by suppressing nickel efflux; (2) a survival module where heterologous expression of Helicobacter pylori Hpn conferred tolerance to high nickel concentrations, and YejM increased phage resistance; (3) a nickel microparticle module enabling intracellular bioconversion of nickel into detectable microparticles even without engineered uptake proteins; and (4) a hydrogen supply module supporting co-culture stability through E. coli-cyanobacteria adhesion for sustained hydrogen production. These modules were experimentally validated through comparative uptake assays, survival profiling, and microscopy-based particle detection. Our findings demonstrate a customizable and integrative microbial platform for metal bioremediation and biosensing, with potential applications in environmental engineering and industrial waste treatment.

synthetic biology↗

bioRxiv2025

Protective Effects of Minocycline and Acetazolamide on Visual Function in Simulated Microgravity Rats

This study established a tail-suspension rat model to investigate the effects of simulated microgravity on intraocular biomechanical homeostasis and visual function, while evaluating the protective roles of acetazolamide (AZE) and minocycline (MINO). Forty-eight rats were divided into ground control (CTRL, n=12) and simulated microgravity groups (n=36), with the latter further categorized by treatment (PBS, AZE, or MINO) and exposure duration (2 or 4 weeks; n=6/group). Assessments included intraocular pressure (IOP), intracranial pressure (ICP), translaminar cribrosa pressure difference (TLCPD), electroretinography (ERG), optomotor response (OMR), and optical coherence tomography (OCT), supplemented by immunofluorescence staining and Sholl analysis. Simulated microgravity significantly elevated IOP and ICP, altered TLCPD, and induced neuroimmune activation, leading to ERG and OMR impairments. AZE effectively reduced IOP and ICP, mitigating mechanical stress, whereas MINO suppressed microglial and astrocytic activation, attenuating retinal neurodegeneration. ERG and OMR demonstrated that MINO restored photopic negative response (PhNR) amplitudes near control levels by 4 weeks. OCT revealed that both AZE and MINO inhibited retinal thinning, particularly in the outer nuclear layer (ONL). Immunofluorescence confirmed MINOs suppression of microglial activation and morphological changes. These findings suggest that AZE preserves visual function by modulating intraocular pressure dynamics, while MINO exerts neuroprotection by mitigating neuroinflammation, offering potential dual therapeutic strategies for spaceflight-associated visual impairment.

molecular biology↗

bioRxiv2025

The piRNA PETPIR Regulates Trophoblast Function by Modulating YTHDF2-Mediated m6A Methylation of NDRG1 in Preeclampsia

Preeclampsia is marked by abnormal placental development and impaired trophoblast function. We identified Preeclampsia-associated trophoblast piRNA (PETPIR) as an important regulator of trophoblast function and m6A modifications. Placental samples from preeclamptic and healthy pregnancies were analyzed using piRNA microarrays and m6A epitranscriptomic profiling. Functional assays in trophoblast cells, transcriptomic analysis, and an in vivo PETPIR-overexpressing mouse model were employed to investigate PETPIRs mechanistic roles. We found that PETPIR was significantly upregulated in preeclamptic placentas. PETPIR inhibited trophoblast cell proliferation, migration, and invasion by inducing cell cycle arrest and apoptosis. Transcriptome analysis demonstrated that PETPIR activated hypoxia-responsive pathways. Additionally, PETPIR enhanced global m6A methylation and stabilized NDRG1 transcripts by interacting with the m6A reader YTHDF2. In vivo, PETPIR overexpression in pregnant mice recapitulated key features of preeclampsia, including maternal hypertension and fetal growth restriction. These results suggest PETPIR is a key player in the pathogenesis of preeclampsia, functioning through the PETPIR/YTHDF2/NDRG1 axis to drive hypoxia and m6A epitranscriptomic dysregulation. We provide novel insights into preeclampsias molecular mechanisms, highlighting piRNA as a promising biomarker and therapeutic target.

molecular biology↗

bioRxiv2025

Capturing Natural Evolution in Function-guided RNA Design via Genomic Foundation Models

RNAs play critical roles in gene expression regulation, catalysis, genetic information transmission, and immune response. However, current RNA engineering approaches face limitations in designing RNA for efficient in vivo folding and functional execution. Here, we present a novel, zero-shot strategy that integrates genomic large language models (LLMs) with inverse folding models (IFMs) to generate RNA sequences that mirror natural evolutionary patterns while preserving the structural integrity of critical functional regions. Benchmarking 9 state-of-the-art genomic foundation models, we show that their unsupervised log-likelihood scores correlate strongly with RNA fitness, enabling zero-shot sequence design. Applied to fluorescent RNA aptamers, our approach yielded 20 Broccoli variants, over half of which achieved up to 155% in fluorescence and a twofold increase in binding affinity, with robust performance both in vitro and in vivo. For Pepper, two rounds of optimization produced 40 variants, 16 of which demonstrated up to 2.6 fold in fluorescence and a threefold boost in binding affinity, including five with superior in vivo brightness.This approach offers a scalable, efficient, and resource-light method for RNA design, with significant implications for RNA-based therapeutics and diagnostics. By leveraging general foundation models to facilitate RNA evolution in a zero-shot fashion, we believe our work represents a major advancement in the field of RNA engineering.

evolutionary biology↗

bioRxiv2025

SLC44A1 deficiency impedes myelin development in the central nervous system

Axon-wrapping myelin sheath is essential for the proper functioning of the central nervous system (CNS). Defects in myelination occur in various neurodevelopmental disorders. Human deficiency of the solute carrier 44A1 (SLC44A1) causes a new type of childhood-onset neurodegeneration with leukoencephalopathy. Currently, there is no effective treatment for this devastating disease, and little is known about its pathological mechanisms. In this study we identified that SLC44A1 is enriched in oligodendrocytes and required for myelin development in the CNS of zebrafish and rodents, which is crucial for normal motor coordination and cognitive function. Defective oligodendroglial maturation and myelinogenesis can be visualized by in vivo time-lapse imaging in the spinal cord of zebrafish with Slc44a1b deficiency. Mechanistically, SLC44A1 deficiency disrupts the expression of genes involved in phosphatidylcholine production pathway, and subsequently inhibiting phospholipid biosynthesis and disturbing the lipid composition of myelin sheaths. More importantly, supplementation with citicoline, a natural choline metabolite, restores developmental myelination in SLC44A1-deficient animals. These findings demonstrate that SLC44A1 is essential for CNS myelination and citicoline supplementation represents a potential therapeutic strategy to address the developmental hypomyelination.

neuroscience↗

bioRxiv2025

The GSA Family in 2025: A Broadened Sharing Platform for Multi-Omics and Multimodal Data

The Genome Sequence Archive Family (GSA family) provides a suite of database resources for archiving, retrieving, and sharing multi-omics data for the global academic and industrial communities. It currently comprises four distinct database members: the Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/), the Genome Sequence Archive for Human (GSA-Human, https://ngdc.cncb.ac.cn/gsa-human/), the open Archive for Miscellaneous Data (OMIX, https://ngdc.cncb.ac.cn/omix/), and the Open Biomedical Imaging Archive (OBIA, https://ngdc.cncb.ac.cn/obia). Compared to the 2021 version, the GSA family has significantly expanded by introducing a new database member, the OBIA, and through comprehensive upgrades to the existing ones. Notable enhancements to the existing members include broadening the range of accepted data types, enhancing the quality control systems, fine-tuning the data retrieval system, and refining the data-sharing management mechanisms.

genomics↗

bioRxiv2025

PrPC-induced signaling in human neurons activates phospholipase Cγ1 and an Arc/Arg3.1 response

Synaptic dysfunction and loss correlate with cognitive decline in neurodegenerative diseases, including Alzheimers disease (AD) and prion disease. Neuronal hyperexcitability occurs in the early stages of AD and experimental prion disease, prior to the onset of dementia, yet the underlying drivers are unclear. Here we identify an increase in the immediate early gene, Arc/Arg3.1, in the human prion disease-affected frontal cortex, suggestive of neuronal hyperactivity. To investigate early signaling events initiated by prion aggregates (PrPSc) in human neurons, we stimulated PrPC in human iPSC-derived excitatory neurons (iNs) with a known PrPSc-mimetic antibody (POM1), which recapitulated the Arc/Arg3.1 response within two hours. Proteomics, RNAseq, and a phosphokinase array in iNs revealed alterations in the EGF receptor and increased phosphorylated phospholipase C (PLC)-{gamma}1 (Y783), which was also observed in the cerebral cortex of prion-infected mice. Thus, PrPC ligands can induce a PLC-{gamma}1 intracellular signaling cascade together with an Arc response, suggestive of a neuronal activity response.

neuroscience↗

bioRxiv2025

Boosting Antitumour Efficacy and Immunity by Boron Neutron Capture Therapy with Size-Controlled Nanoparticles

Boron neutron capture therapy (BNCT) is emerging cancer radiotherapy requiring 10B sensitizer. Although boronophenylalanine (BPA)-BNCT is approved clinically in Japan, the low tumour selectivity and retentivity result in the long-time infusion of high doses. Based on our finding of the size-controllable mechanochemical synthesis of boron-10 carbide nanoparticles (10B4C NPs), the 50 nm size NPs grafted with poly(glycerol), {superscript 1}BC(50)-PG, is found to show superior tumour selectivity and retentivity to enhance the eradication efficacy at much lower dosage (5 mg [10B] / kg (mouse)). The dosage is further reduced by twice neutron irradiation or combination with an immune checkpoint inhibitor (ICI). Antitumour immunity is found to be boosted by {superscript 1}BC(50)-PG-BNCT to induce abscopal effect to treat remote or metastatic tumours and long-term memory to prevent cancer recurrence. Additionally, minimal side effects and gradual NP excretion are observed for one year. The 10B4C(50)-PG is concluded to be a promising 10B carrier for clinical application of BNCT due to the prominent antitumour efficacy and immune-activation with minimal toxicity.

cancer biology↗

bioRxiv2025

Allo-PED: Leveraging protein language models and structure features for allosteric site prediction

Allosteric regulation plays a pivotal role in modulating protein function and allosteric sites represent a promising target for drug discovery. However, identifying allosteric sites remains challenging due to their structural and evolutionary diversity. Here, we present AlloPED, a novel framework that combines protein language models and machine learning to predict allosteric sites with high accuracy. AlloPED consists of two modules: AlloPED-pocket, an ensemble model leveraging physicochemical features to predict allosteric pockets; and AlloPED-site, a dilated convolutional neural network (DCNN) augmented with a comprehensive attention mechanism for residue-level prediction. AlloPED-pocket achieves state-of-the-art performance on benchmark datasets, yielding an MCC of 0.544 and an AUC of 0.920, outperforming existing methods such as AllositePro and PARS. AlloPED-site further refines predictions using high-dimensional sequence embeddings from the ProtT5 protein language model, achieving a precision of 0.601, a recall of 0.422, and a specificity of 0.661. These results highlight the effectiveness of integrating ensemble learning and deep learning for allosteric site prediction. AlloPED also identifies critical determinants of allosteric sites, including residue clustering coefficients, van der Waals volume, and hydrophobic microenvironments. In summary, this framework provides a robust tool for advancing our understanding of allosteric regulation and facilitating structure-based drug design.

bioinformatics↗

bioRxiv2025

MouseGPT: A Large-scale Vision-Language Model for Mouse Behavior Analysis

Analyzing animal behavior is crucial in advancing neuroscience, yet quantifying and deciphering its intricate dynamics remains a significant challenge. Traditional machine vision approaches, despite their ability to detect spontaneous behaviors, fall short due to limited interpretability and reliance on manual labeling, which restricts the exploration of the full behavioral spectrum. Here, we introduce MouseGPT, a Vision-Language Model (VLM) that integrates visual cues with natural language to revolutionize mouse behavior analysis. Built upon our first-of-its-kind dataset--incorporating pose dynamics and open-vocabulary behavioral annotations across over 42 million frames of diverse psychiatric conditions--MouseGPT provides a novel, context-rich method for comprehensive behavior interpretation. Our holistic analysis framework enables detailed behavior profiling, clustering, and novel behavior discovery, offering deep insights without the need for labor-intensive manual annotation. Evaluations reveal that MouseGPT surpasses existing models in precision, adaptability, and descriptive richness, positioning it as a transformative tool for ethology and for unraveling complex behavioral dynamics in animal models.

animal behavior and cognition↗

bioRxiv2025

A GPCR-G protein-β-arrestin megacomplex induced by an allosteric modulator

GPCRs signal through both G protein pathways and {beta}-arrestin pathways. Previous work suggests that {beta}-arrestins bind to GPCRs through different modes, including core engagement and tail engagement. Core engagement competes with G proteins and terminates G protein signaling, while tail engagement can coexist with G proteins, mediating sustained intracellular activation of the receptor - a process dependent on the high affinity between {beta}-arrestin and the phosphorylated C-terminus of the receptor. In this study, we determined the structure of a GPCR - G protein - {beta}-arrestin-1 complex stabilized by an allosteric modulator. The compound, atazanavir, acts like molecular glue to anchor {beta}-arrestin-1 to the receptors TM6 and TM7 regions. This pendulum binding mode is structurally compatible with simultaneous G protein binding. We further demonstrate that the atazanavir-mediated {beta}-arrestin recruitment does not require the receptors C-terminal region. This work illustrates a novel paradigm of GPCR-G protein-{beta}-arrestin1 megacomplex assembly and opens up new avenues for modulating GPCR function.

biochemistry↗

bioRxiv2025

Identifying Key Cells for Fibrosis by Systematically Calling Cell Type-Phenotype Associations across Massive Heterogenous Datasets

Fibrotic diseases pose a significant burden on health care, yet the key pathogenic fibroblasts involved remain unclear. We developed the fibrotic disease fibroblast atlas (FDFA), which comprises 394 single-cell and 38 spatial transcriptomic samples from 11 common fibrotic diseases. To perform a cell-type phenotype association study in large-scale heterogeneous datasets, we developed the single-cell phenotype association research kit for large-scale dataset exploration (SPARKLE). SPARKLE handles heterogeneity by incorporating confounding information into its generalized linear mixed models (GLMMs). The application of SPARKLE to FDFA revealed that matrix fibroblasts (MTFs) constitute a crucial pathogenic cell group in fibrosis. Their increased proportion correlate with the fibrotic process. MTFs also synergize with MYO-Fs, increasing their degree of fibrosis. Based on MTF, we identified 25 potential antifibrotic targets for broad-spectrum antifibrotic therapies. This study enhances our understanding of fibrosis and provides a reliable framework for large-scale cell type-phenotype association research. HighlightsO_LIA cross-tissue, multidisease fibrotic disease fibroblast atlas (FDFA) reveals diverse fibroblast subtypes in fibrosis diseases. C_LIO_LIA new toolkit, SPARKLE, is introduced for detecting robust cell type-phenotype associations in large-scale heterogeneous datasets. C_LIO_LIMultiple pathogenic fibroblasts associated with common fibrosis disease (MTF) onset and progression have been identified, suggesting potential cell-specific therapeutic strategies for fibrosis-related diseases. C_LI

bioinformatics↗

bioRxiv2025

Soluble Immune Factor Profiles in Blood and CSF Associated with LRRK2 Mutations and Parkinson's Disease

Background and ObjectivesMutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are one of the most common genetic causes of Parkinsons disease (PD) and are linked to immune dysregulation in both the central nervous system and periphery. However, peripheral and central profiles of soluble immune factors associated with LRRK2 mutations and PD have not been comprehensively characterized. Using serum and CSF samples from the LRRK2 Cohort Consortium (LCC), this study aimed to probe a broad range of soluble immune biomarkers associated with LRRK2 mutations and PD. MethodsWe investigated the levels of soluble immune regulators in the serum (n=651) and cerebrospinal fluid (CSF, n=129) of LRRK2 mutation carriers and non-carriers, both with and without PD. A total of 65 cytokines, chemokines, growth factors, and soluble receptors were assessed by Luminex immunoassay. A multivariable robust linear model was used to determine levels associated with LRRK2 mutations and PD status, adjusting for age, sex, and sample cohort. Correlations were assessed using the Spearman correlation coefficient. LRRK2 G2019S knock-in mice were used to validate the associations identified in the LCC. ResultsIn this extensive discovery cohort, we identified several elevated serum immune regulatory factors associated with LRRK2 mutations. In particular, serum stromal cell-derived factor-1 alpha (SDF-1 alpha) levels, as supported by findings in LRRK2 G2019S knock-in mice, and tumor necrosis factor receptor II (TNF-RII) were significantly increased after multiple comparison adjustment. In contrast, LRRK2 mutations were associated with reduced soluble immune markers, including BAFF, CD40-Ligand, I-TAC, MIP-3 alpha, NGF beta, and IL-27 in CSF. Those with clinically diagnosed PD, with or without LRRK2 mutations, did not show strong signals in serum but reduced inflammatory analytes in CSF, including MIF, MMP-1, CD30, Tweak, and SDF-1 alpha. In addition, we found that the serum levels of these soluble immune factors display varied correlations with their corresponding CSF levels. DiscussionThis study highlights distinct immune profiles associated with LRRK2 mutations and PD in the periphery and CNS. Serum levels of SDF-1alpha and TNF-RII were elevated in LRRK2 mutation carriers, while CSF immune markers were reduced. In PD, irrespective of LRRK2 status, reduced CSF inflammatory analytes and weak serum signals were observed. These results provide insight into immune dysregulation linked to LRRK2 mutations. If replicable in independent datasets, they offer potential avenues for biomarker and therapeutic exploration.

neuroscience↗

bioRxiv2025

Quantitative Analysis of Coupled Carbon and Energy Metabolism for Lignin Carbon Utilization in Pseudomonas putida

Soil Pseudomonas species, which can thrive on lignin-derived phenolic compounds, are widely explored for biotechnology applications. Yet, there is limited understanding of how the native metabolism coordinates phenolic carbon processing with cofactor generation. Here, we achieve quantitative understanding of this metabolic balance through a multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate as a non-aromatic reference, proteomics data reveal >140-fold increase in proteins for transport and initial catabolism of each phenolic substrate, but metabolomics profiling reveals that bottleneck nodes in initial phenolic compound catabolism maintain more favorable cellular energy state. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling via pyruvate carboxylase promotes fluxes in the tricarboxylic acid cycle to provide 50-60% NADPH yield and 60-80% NADH yield, resulting in 2-fold higher ATP yield than for succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. The quantitative blueprint elucidated here explains deficient versus sufficient cofactor rebalancing during manipulations of key metabolic nodes in lignin valorization.

microbiology↗

bioRxiv2025

Biphasic bactericidal activity of nitroxoline against Acinetobacter baumannii isolates from urinary tract infections

Acinetobacter baumannii is a critical pathogen which can cause hospital-acquired infections, particularly urinary tract infections (UTIs). The antimicrobial resistance (AMR) of A. baumannii is rising which poses a significant challenge to clinical management. Nitroxoline, an old antibiotic for treating uncomplicated UTIs, has gained renewed interest as a potential therapeutic option. Here, we investigates the bactericidal activity of nitroxoline against 34 A. baumannii (17 carbapenem-resistant and 17 carbapenem-sensitive isolates) collected from UTI patients. Nitroxoline exhibited a biphasic bactericidal effect, characterized by enhanced efficacy up to an optimal bactericidal concentration (OBC), followed by declined activity at higher nitroxoline concentrations. The OBCs, minimum bactericidal concentration (MBC), minimum inhibitory concentration (MIC), and time-killing curves were evaluated to elucidate bactericidal activity. Raman deuterium stable isotope probing (Raman-DIP) was employed to validate nitroxolines bactericidal effect and its impact on bacterial metabolic activity and survival rates. These results demonstrate that nitroxoline exhibits excellent inhibitory and bactericidal activity against A. baumannii. While nitroxoline exhibits biphasic bactericidal activity with OBC50/90 values of 4/8 mg/L. Raman spectroscopy identified a decreased C-D ratio as nitroxoline concentration increased, indicating reduced metabolic activity. Notably, an inverse correlation (r = -0.7594, p <0.0001) was observed between bacterial survival and the ccratio at concentrations above the OBC. These findings underscore the necessity of optimizing dosing regimens to enhance nitroxolines therapeutic efficacy and alleviate AMR development. Raman-DIP emerges as a robust tool for assessing nitroxolines effects on bacterial metabolism and determining MIC, offering valuable insights for future clinical applications.

microbiology↗