Biology subjects

Gao, J.

Publications and source records attributed to Gao, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Discovery of Inverted Repeats and Palindromes in Viral Genomes

An inverted repeat (IR) in DNA is a sequence of nucleotides that is followed by its complementary bases but in reverse order, occurring on the same strand (e.g., TCACCGCGGTGA). If the two complementary sequences occur one after the other without other bases between them, they are referred to as DNA palindromes. IRs could form hairpin and cruciform secondary structures, which endanger genomic stability. They are found to be prevalent in viral DNA at origins of replication, and they play a crucial role in various biological processes including gene silencing, duplication, and genomic evolution. IRs have been less explored, which stems from the scarcity of sequence analysis tools allowing accurate detection on large viral genome data. Here, using the Biological Language Modeling Toolkit (BLMT), we analyzed 14 thousand viral genomes for occurrences of IRs, resulting in the identification of over 19 million IRs longer than 20 bases, including 134 IRs that are 2000 bases long, and around 1,300 IRs per virus.

bioinformatics↗

bioRxiv2025

Anillin-dependent actin assembly at PML NBs protects genome stability

Nuclear filamentous actin (F-actin) is emerging as a key regulator of genome stability1-6. During replication stress, transient bursts of nuclear actin assembly in S-phase promote fork remodelling and repair3,4, but how these bursts are initiated, regulated, and whether nuclear F-actin also act on DNA lesions inherited across cell cycles, remains unknown. Here, we show that replication stress induces nuclear actin assembly at liquid-liquid phase-separated (LLPS) promyelocytic leukaemia nuclear bodies (PML NBs), driven by the actin-binding protein Anillin. Although best known as a cytokinesis factor, Anillin localises to PML NBs in interphase, where it concentrates monomeric actin (G-actin) and establishes a favourable environment for spontaneous actin assembly. Filament growth within these condensates remodels PML NB morphology and composition, releasing sequestered AKT into the nucleoplasm. AKT is subsequently phosphorylated and activated, and this nuclear AKT activity is required to regulate 53BP1 nuclear bodies in G1. These protective LLPS compartments shield under-replicated DNA inherited from the stressed S-phase until the following S-phase. This prevents premature engagement by aberrant repair pathways that would otherwise generate toxic intermediates and culminate in mitotic failure. Together, these findings define a condensate-to-filament pathway in which nuclear F-actin dynamically reorganises phase-separated compartments to safeguard genome stability across cell cycles.

cell biology↗

bioRxiv2025

Single-Cell Immune Profiling Reveals Non-classical Monocyte-Driven Immunodepression in Tuberculosis Treatment Non-Responders

Sputum culture conversion (SCC) at 2 months is a critical predictor of tuberculosis (TB) treatment outcomes, yet the underlying immune alterations driving treatment non-response remain unclear. We performed single-cell RNA sequencing on peripheral blood mononuclear cells from 8 TB patients, stratified by 2-month SCC status, to characterize cellular heterogeneity and intercellular communication. Treatment non-responders exhibited marked expansion and terminal differentiation of non-classical monocytes with elevated TB progression risk signatures. These cells showed reduced outgoing but enhanced incoming signaling, prominently via IL-16 and TRAIL pathways, potentially amplifying regulatory T cell (Treg)-mediated immunosuppression. CD4 Tregs in non-responders engaged in intensified GALECTIN signaling toward CD8 cytotoxic T cells and mature NK cells, contributing to effector cell exhaustion. NK cell subsets, particularly mature NK c2, displayed increased inhibitory receptor expression and terminal differentiation states. Our findings delineate a dysregulated "non-classical monocyte -Treg -cytotoxic lymphocyte" axis in TB treatment failure, highlighting candidate biomarkers and host-directed therapeutic targets.

bioinformatics↗

bioRxiv2025

JAK1/2 Inhibition Delays Cachexia and Improves Survival through Increased Food Intake

Lung cancer is the leading cause of cancer-related death and is frequently accompanied by reduced food intake and cachexia, a debilitating syndrome characterized by weight loss and skeletal muscle wasting. We sought to identify contributors to cachexia using a murine model of lung cancer that reproduces key features of this syndrome. A multiplex cytokine screening approach, integrated with western blot and transcriptomic analyses, identified tumor-derived inflammatory mediators and downstream signaling pathways associated with cachexia. Notably, IL-6 superfamily members were elevated in the tumor and plasma of mice and patients with cachexia. The JAK-STAT3 signaling was upregulated in liver and skeletal muscle, driving the acute phase response and impairing lipid metabolism. Pharmacologic inhibition of JAK1/2 with ruxolitinib improved body weight, fat mass, and overall survival without altering tumor burden. These effects were driven primarily by blunted hypothalamic leptin receptor signaling, which increased food intake early in the disease course. In the liver, JAK inhibition reduced STAT3 activity, restored fatty acid oxidation, and decreased the production of acute-phase proteins. These findings support JAK inhibition as a therapeutic strategy for lung cancer-associated cachexia. Statement of SignificanceCancer cachexia is a lethal complication of lung cancer that lacks effective treatment. We show that JAK inhibition by ruxolitinib restores weight, fat mass, and prolongs survival in murine models of lung cancer. These effects were independent of tumor burden, underscoring the relevance of addressing cachexia to improve survival in cancer patients and supporting clinical testing of JAK inhibition for cancer cachexia

cancer biology↗

bioRxiv2025

A spatially-resolved human brain proteome atlas for understanding function and disease

While the brain performs specialized functions across distinct regions, the spatial organization of the human brain proteome remains largely uncharted. Here we present a comprehensive spatially-resolved proteome atlas of the human brain, analyzing over two thousand MRI- guided locations across four individuals. Proteome analysis integrated with transcriptomics reveals extensive post-transcriptional regulation, with cortical regions showing markedly higher protein diversity than transcript. Unsupervised molecular clustering defines distinct brain territories that transcend anatomical boundaries, instead reflecting metabolic demands and functional specialization patterns. Application to epilepsy brain tissue uncovered disrupted astrocyte metabolism, protein homeostasis and therapeutic targets including the seizure-associated purinergic receptor P2RX7. This resource bridges molecular and systems neuroscience to accelerate neurological drug discovery.

neuroscience↗

bioRxiv2025

Auditory Stimulus Information Entropy Modulates Inter-Brain Synchronization: Evidence from Wireless EEG Hyperscanning

Inter-brain synchronization (IBS) - reflecting inter-individual correlated neural activity during interaction - marks shared experiences like music listening. The ability of complex auditory stimuli (e.g., music) to induce IBS links to their information dynamics, notably the uncertainty they evoke, which challenges the nervous systems predictive coding. Based on mutual prediction theory (interacting individuals simultaneously process their own behavior and predict their partners; accurate mutual predictions lead to convergent neural representations and thus IBS), this study hypothesized that higher stimulus uncertainty enhances IBS (heightened uncertainty reduces independent predictability, promoting convergent representations and stronger IBS). Using information entropy to quantify uncertainty, the study conducted hyperscanning, manipulated entropy across Resting State and 6 Hz auditory stimuli (ASSR, MMN, AHER, Dream Wedding), and measured IBS via phase-locking values (PLV). Results showed frequency specificity: 6 Hz PLV increased with entropy (DW {approx} AHER > MMN {approx} ASSR > Resting State); Alpha band had highest PLV in Resting State. Critically, PLVs differed significantly between any two conditions, and each experimental conditions PLV was also significantly different from that of the Resting State. Findings confirm a 6 Hz-specific positive association between auditory uncertainty and IBS, suggesting musical elements may facilitate social interaction by modulating entropy, with entropy-IBS relations showing frequency dependence.

neuroscience↗

bioRxiv2025

ctsGRN: Inferring cell type-specific gene regulatory networks in Arabidopsis

Gene regulatory networks (GRNs) govern life processes. And exploring GRNs is of great value for understanding biological functions and developing species resources. At present, studies on plant GRNs are limited, mostly focusing on the tissue level, which hinders the discovery of cell type-specific regulatory networks and mechanisms. To address this, we have proposed a GRN research framework named ctsGRN (CellTypeSpecGRN) for conducting cell-specific GRN research at the cell type resolution. In this framework, we employed three representative algorithms from statistics (e.g., correlation analysis), traditional machine learning (e.g., random forest) and deep learning (e.g., neural networks) to construct GRNs for different cell types in Arabidopsis roots. Among these, traditional machine learning methods showed the best performance. Experimental validation through yeast one-hybrid (Y1H) assays and ChIP-seq data confirmed the high reliability and predictive power of these inferred GRNs. By analyzing these networks, we mined novel cell type-specific regulatory relationships, identified core transcription factor (TF) AT5G08790 involved in root development, discovered 249 cell-type-specific hub TFs, and 136 key functional modules were found to be enriched in biological processes. Additionally, we revealed significant heterogeneity of TF-target gene interactions across various cell types. This comprehensive analysis offers a detailed overview of TF-mediated cell type-specific transcriptional regulation in Arabidopsis roots, providing new insights into the molecular mechanisms of root development and establishing a guideline framework for GRN research at the cell-type level in plants.

bioinformatics↗

bioRxiv2025

Automatic Change Detection of Human Attractiveness: Comparing Visual and Auditory Perception

Change detection of social cues across individuals plays an important role in human interaction. Here we investigated the automatic change detection of facial and vocal attractiveness in 19 female participants by recording ERPs. We adopted a deviant-standard-reverse oddball paradigm where high- or low-attractive items were embedded as deviants in a sequence of opposite attractive standard stimuli. Both high- and low-attractive faces and voices elicited mismatch negativities (MMNs). Furthermore, low-attractive versus high-attractive items induced larger mismatch negativities in the voice condition but larger P3 amplitudes in the face condition. These data indicate that attractiveness can be automatically detected but that differences exist between facial and vocal attractiveness processing. Generally, change detection seems to work better for unattractive than attractive information possibly in line with a negativity bias.

neuroscience↗

bioRxiv2025

Fragile nucleosomes are essential for RNA Polymerase II to transcribe in eukaryotes

Nucleosomes are barriers to RNA Polymerase II (Pol II) transcribing along gene bodies in eukaryotes. We found that a fragile "tailless nucleosome" could be generated to resolve the Pol II pausing caused by the +1 nucleosome in metazoans. We hypothesized that "pan-acetylated nucleosomes" along gene bodies are fragile and are generated to resolve the resistance of nucleosomes to the elongation of Pol II. To confirm this hypothesis, we systematically analyzed pan-acetylation and pan-phosphorylation of the entire human genome in HEK293T cells. Herein, we show that both pan-acetylation and pan-phosphorylation of histones in nucleosomes along gene bodies are coupled with the activities of Pol II. Our results show that there are four major types of fragile nucleosomes required for Pol II to transcribe along gene bodies: nucleosomes without tails, nucleosomes with dominant acetylation, nucleosomes with dominant phosphorylation, and nucleosomes with both acetylation and phosphorylation.

genetics↗

bioRxiv2025

The nuclear receptor NR4A1 serves as a neutrophil-intrinsic regulator mitigating stroke severity

Ischemic stroke is accompanied by recruitment and activation of immune cells which play an important role in the progression of the brain damage. The nuclear receptor NR4A1 emerged as a key regulator within the inflammatory response of several immune diseases by regulating immune cell activation. In this study, we investigated the role of NR4A1 in the activation and recruitment of brain resident and peripheral immune cells after cerebral ischemia. Here, we show that NR4A1 mediates an anti-inflammatory and damage-limiting effect after stroke. This effect is largely mediated by neutrophil recruitment and importantly, NR4A1 activation with its ligand Cytosporone B improves functional outcome and reduces brain damage. Modulation of NR4A1 is therefore a promising therapeutic target for the treatment of the nuclear receptor NR4A1 in the activation and recruitment of peripheral and brain resident immune cells after cerebral ischemia and its consequences for stroke outcome. We demonstrate that NR4A1 ablation augments neutrophil activation and CNS recruitment within days after stroke thereby increasing infarct size, CNS inflammation, neuronal damage and deteriorating functional outcome. This effect is mediated via modulation of cell-intrinsic neutrophil function and maturation as illustrated by neutrophil-specific NR4A1 ablation and mixed bone-marrow chimera experiments. Notably, the NR4A1 agonist Cytosporone B reduced CNS neutrophil infiltration, infarct size and functional outcome after stroke in a bicentric preclinical stroke trial, demonstrating that NR4A1-mediated control of neutrophil reactivity is amenable to pharmacological modulation. In humans, NR4A1 expressing neutrophils are present in the peripheral blood of stroke patients and neutrophil NR4A1 expression correlates with improved long-term outcome after 3 months. Furthermore, NR4A1 expression in brain parenchyma neutrophils is negatively correlated with neuronal cell loss, illustrating a role of NR4A1 in regulating neutrophil mediated neuronal cell death in human stroke. Together our data reveal the nuclear factor NR4A1 as a brake of intrinsic neutrophil activity controlling neutrophil-mediated brain inflammation and neurotoxicity in stroke which may serve as a novel therapeutic target to limit inflammation-associated augmentation of ischemic damage after stroke.

neuroscience↗

bioRxiv2025

Bidirectional Crosstalk Between Bladder Cancer Cells and Normal Fibroblasts Drives Phenotypic Reprogramming and Modulates Chemosensitivity

During early bladder cancer progression, invading tumour cells first encounter normal fibroblasts (NFs) residing in the lamina propria. Whether and how this interaction shapes tumour behaviour and treatment response, however, has not been systematically examined. While cancer-associated fibroblasts (CAFs) are established drivers of tumour progression and therapy resistance, the role of their precursor NFs in shaping bladder cancer behaviour has been largely overlooked. Here we dissect bidirectional interactions between bladder cancer cells and NFs using indirect (conditioned media) and direct co-culture systems. Fibroblast-conditioned media (FCM) significantly reduced proliferation while accelerating migration in both RT112 and T24 bladder cancer cells, consistent with a pro-migratory "go-or-grow" phenotypic shift. Immunofluorescence revealed FCM-induced cadherin switching reduced E-cadherin and elevated N-cadherin, indicative of an epithelial-to-mesenchymal transition (EMT)-like state. Reciprocally, NFs acquired CAF-like features within 48 hours of tumour cell exposure, with significant upregulation of SMA and FAP confirmed by both immunofluorescence and flow cytometry. Functionally, increasing fibroblast-to-cancer cell ratio progressively attenuated mitomycin C (MMC)-induced cytotoxicity, demonstrating a stromal-mediated, ratio-dependent protective effect against intravesical chemotherapy. Data from the Cancer Genome Atlas (TCGA) for Urothelial Bladder Carcinoma provided corroboratory evidence that fibroblast-enriched tumours exhibited a strong transcriptional correlation with EMT, elevated resistance-associated transcriptional signatures, and significantly poorer overall survival. Collectively, these findings establish tumour-NF crosstalk as a critical and bidirectional regulator of bladder cancer phenotypic plasticity and chemoresistance, and suggest that fibroblast-targeting strategies combined with intravesical chemotherapy may offer a rational approach to overcoming stromal-mediated treatment failure.

cancer biology↗

bioRxiv2025

Multi-Scale Simulations of Excited-State Energy Transfer Pathways in the C2S2-Type PSII-LHCII Supercomplex of Spinach

Photosystem II-light-harvesting complex II (PSII-LHCII) supercomplex achieves efficient excitation energy transfer (EET), yet how directional transfer emerges from its nearly flat energy landscape has remained unclear. Here, we investigate the C2S2-type spinach PSII-LHCII supercomplex containing 206 pigments by integrating microsecond-scale molecular dynamics, QM/MM site energy calculations, and generalized Forster theory. The computed site energy landscape shows overall downhill gradients from peripheral antennas to the reaction center (RC), with small variations fully explained by dynamic structural fluctuations. Pairwise transfer rates reveal a dense multi-timescale EET network, where fast intra-subunit and interface couplings provide robust channels and slower links maintain global connectivity. Key interface pigments act as energy-funneling hotspots that converge excitations toward the RC. Within the PSII core, both the D1 and D2 branches ultimately transfer energy downhill to ChlD1 and ChlD2, respectively, ensuring thermodynamic feasibility of delivery. Three major transfer pathways were identified, all falling within functional timescales (< 250 ps). Bidirectional dynamics were also observed, with rapid reverse transfers enabling excess excitation to be safely dissipated through peripheral antennas. Together, these results demonstrate that PSII maintains fully downhill energy transfer through the combined effects of structural constraints and dynamic regulation, thereby achieving both efficient energy delivery and photoprotection.

biochemistry↗

bioRxiv2025

A framework to detect positive selection using variant effect predictions reveals widespread adaptive evolution of human neurons

Detecting positive selection is essential to understanding evolution. Many methods to detect positive selection use simple classifications of genetic variants (e.g. synonymous/nonsynonymous). Here, we propose that these methods can be considered special cases of a more general framework to detect positive selection with any variant effect prediction method. Using evolutionary conservation and deep learning-based variant effect predictions, we apply this framework genome-wide in the human lineage and identify positive selection on protein sequences of genes involved in brain and heart development, chromatin accessibility in binding sites of dozens of transcription factors, and non-coding substitutions in compact genomic regions with reinforcing cell type-specific effects on cis-regulatory activity. Consistently, the dominant theme was positive selection on genes regulating neuronal connectivity, suggesting that fine-scale changes in brain wiring were essential to the evolution of human cognition. Overall, this framework represents a powerful, versatile tool to investigate adaptive evolution across the tree of life.

genomics↗

bioRxiv2025

Single-cell transcriptomics and machine learning reveal RNF144B and C5AR1 as immune-related biomarkers and therapeutic targets in myocardial infarction

BackgroundMyocardial infarction (MI) is a life-threatening cardiovascular disease characterized by high morbidity and mortality. Although advances in clinical management have improved patient outcomes, early diagnosis and effective immunomodulatory therapies remain limited. MethodsIn this study, we integrated multiple transcriptomic datasets and applied machine learning approaches, including LASSO regression, to identify a robust 13 key genes significantly associated with MI. Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were subsequently performed to explore their potential biological functions. The immunological relevance of these genes was evaluated by analyzing their correlations with inflammation-related genes and those involved in immune cell migration. In addition, transcription factor (Johnson, Law et al.) and microRNA (miRNA) regulatory networks were constructed to elucidate upstream regulatory mechanisms. The expression levels of the 13 key genes were validated in MI mouse model. Furthermore, molecular docking was performed to identify candidate small molecules targeting core genes. ResultsAmong 11 cardiac cell populations identified, myeloid cells contributed most prominently to MI pathogenesis. A robust 13-gene predictive signature was established, with RNF144B and C5AR1 showing strong associations with immune modulation and disease severity. GSEA and GSVA further revealed that RNF144B was enriched in the neutrophil degranulation pathway, while C5AR1 was associated with the complement cascade. Correlation analysis demonstrated a significant positive relationship of RNF144B and C5AR1 with immunological roles. Both genes were also positively correlated with classical MI marker genes SERPINE1 and RUNX1. TF-gene and miRNA-mRNA regulatory networks supported the post-transcriptional regulation of these genes. In the MI mouse model, expression of the 13 genes was consistent with the risk-prediction model. Molecular docking identified CCX168 as a promising small-molecule candidate targeting RNF144B and C5AR1. ConclusionThis study reveals immune-related transcriptional signatures and signaling pathways that drive MI progression. The identified 13-gene signature, particularly RNF144B and C5AR1, holds promise as a diagnostic biomarker and therapeutic target, providing new insights for immunomodulatory and precision medicine strategies in MI. Keywords: Immune microenvironment, myeloid cells, inflammation, RNF144B, MI, immune-targeted therapy HighlightsO_LIA 13-gene signature was identified to predict MI risk C_LIO_LIRNF144B and C5AR1 are key immune regulators in MI progression C_LIO_LIGSEA and GSVA reveal immune-related pathways of RNF144B and C5AR1 C_LIO_LICCX168 is identified as a potential small-molecule therapy for MI C_LIO_LIMouse model validates gene expression consistent with the prediction model C_LI

cell biology↗

bioRxiv2025

Moderate-Intensity Intermittent Training Inhibits miR-34a-5p to Reduce Prefrontal Cortex Apoptosis and Improve Cognitive Ability in Aging Rats Reduced by D-galactose

Changes in microRNA (miRNA) play a role in brain aging. They are considered potential therapeutic targets. Regular long-term exercise benefits brain health. However, its exact mechanism is not fully understood. This study explored how moderate-intensity intermittent training (MIIT) improves cognitive function and reduces apoptosis in the aging brain. This study induced aging in rats by giving them D-gal injections (150 mg/kg/day) for 6 weeks. After confirming the model, the rats underwent MIIT. They exercised 45 minutes a day, 5 days a week, for 8 weeks. The results from behavioral, morphological, and molecular tests showed that 8 weeks of MIIT significantly slowed the decline in spatial learning and memory in D-gal aging rats. The exercise also improved the structure of the prefrontal cortex (PFC) and reduced apoptosis. Aging caused an overexpression of miR-34a-5p in the prefrontal cortex. MIIT reduced this overexpression. It also up-regulated Notch1 and inhibited excessive apoptosis by regulating Bcl-2 and Bax expression. In conclusion, MIIT may improve brain health by targeting miR-34a-5p and regulating apoptosis-related pathways.

physiology↗

bioRxiv2025

OpenFISH enables integrated high-resolution spatial transcriptomics and metabolomics on a single tissue section

Spatial transcriptomics enables in situ mapping of gene expression, yet no current platform provides single-cell, same-section integration with metabolomics, limiting direct links between transcriptional programsand metabolic phenotypesin native tissue. We present OpenFISH, a rapid, imaging-based spatial transcriptomics method operable on standard microscopes, requiring no proprietary hardware, and fully compatible with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). OpenFISH resolves hundreds of transcripts at subcellular resolution within 24 h and can be performed after MALDI-MSI, preserving metabolite distributions for cell-accurate co-registration on the same section. In mouse brain, integration with MALDI-MSI resolved metabolic heterogeneity at the level of individual cells. OpenFISH also quantified cell type-specific transcriptional activation of transposable elements after systemic lipopolysaccharide (LPS) challenge and detected disrupted spatial organization of D1 striatal neurons in Reeler mutants. Benchmarking showed performance comparable to or exceeding commercial platforms at [~]0.5% of per-sample cost. By enabling same-section, near-single-cell co-mapping of transcripts and metabolites in an accessible workflow, OpenFISH provides a scalable framework for high-content spatial multi-omics across neuroscience, immunology, cancer biology, and beyond.

molecular biology↗

bioRxiv2025

Mutations in VPS18 lead to a neutrophil maturation defect associated with disturbed vesicle homeostasis

Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18, we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.

physiology↗

bioRxiv2025

SCAMP3 is essential for proper formation and function of neutrophil granules

Host defense functions of neutrophils during infection critically depend on microbicidal and proteolytic proteins stored in primary, secondary and tertiary granules that are released into the phagosome or into the extracellular space upon degranulation. Granules are generated during granulopoiesis and impaired granule production or granule protein sorting has been linked to inefficient pathogen clearance resulting in recurrent bacterial and fungal infections. Here, we studied the role of the membrane protein secretory carrier associated membrane protein 3 (SCAMP3) for neutrophil defense functions. We generated Scamp3 knockout (KO) Hoxb8 cells and found that killing of Escherichia coli by Scamp3 KO Hoxb8 cell-derived neutrophils (dHoxb8 cells) was compromised as compared to control dHoxb8 cells in vitro. Mass spectrometric and Western blot analyses revealed a significant reduction of primary, secondary, and tertiary granule proteins in the genetic absence of Scamp3, resulting in a reduced overall granularity of these cells. Accordingly, degranulation was reduced in Scamp3 KO dHoxb8 cells compared to control dHoxb8 cells. Similarly, SCAMP3 deficiency in zebrafish resulted in reduced neutrophil granularity in comparison to wild-type animals. However, neutrophil migration towards sites of E. coli infection was unaffected in scamp3 KO zebrafish larvae. In summary, SCAMP3 represents an important novel player in granule equipment and degranulation, with key functions in neutrophil defense mechanisms during host-pathogen interactions in vitro. Brief summary sentence: The membrane protein secretory carrier associated membrane protein 3 (SCAMP3) is essential for proper protein equipment of neutrophil granules and Scamp3-deficient neutrophils have an impaired bacterial killing capacity in vitro.

physiology↗