Biology subjects

Liu, H.

Publications and source records attributed to Liu, H..

At least 271 records · Page 15Linked to original sources

bioRxiv2025

Rare Subset of T Cells Form Heterotypic Clusters with Circulating Tumor Cells to Foster Cancer Metastasis

The immune ecosystem is central to maintaining effective defensive responses. However, how immune cells in the periphery blood interact with circulating tumor cells (CTCs) - seeds of metastasis - remains largely understudied. Here, our analysis of the blood specimens (N=1,529) from patients with advanced breast cancer revealed that over 75% of the CTC-positive blood specimens contained heterotypic CTC clusters with CD45+ white blood cells (WBCs). Detection of CTC-WBC clusters correlates with breast cancer subtypes (triple negative and luminal B), racial groups (Black), and decreased survival rates. Flow cytometry and ImageStream analyses revealed diverse WBC composition of heterotypic CTC-WBC clusters, including overrepresented T cells and underrepresented neutrophils. Most strikingly, a rare subset of CD4 and CD8 double positive T (DPT) cells showed an up to 140-fold enrichment in the CTC clusters versus its frequency in WBCs. DPT cells shared part of the profiles with CD4+ T cells and others with CD8+ T cells but exhibited unique features of T cell exhaustion and immune suppression with higher expression of TIM-3 and PD-1. Single-cell RNA sequencing and genetic perturbation studies further pinpointed the integrin VLA4 (4{beta}1) in DPT cells and its ligand VCAM1 in tumor cells as essential mediators of heterotypic WBC-CTC clusters. Neoadjuvant administration of anti-4 (VLA4) neutralizing antibodies markedly blocked CTC-DPT cell clustering and inhibited metastasis for extended survival in preclinical mouse models in vivo. These findings uncover a pivotal role of rare DPT cells with immune suppressive features in fostering cancer dissemination through direct interactive clustering with CTCs. It lays a foundation for developing innovative biomarkers and therapeutic strategies to prevent and target cancer metastasis, ultimately benefiting cancer care. Brief summaryOur findings uncover a fostering role of immune-suppressive T cells in contact with circulating tumor cells and identify therapeutic approaches to eliminate devastating cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/646421v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@13b7862org.highwire.dtl.DTLVardef@15c97b2org.highwire.dtl.DTLVardef@1eb4fd2org.highwire.dtl.DTLVardef@1c588e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Using rotational integration of oblique interferometric scattering (RO-iSCAT) to track axial spatiotemporal responses of membrane protrusions

Despite the crucial importance of dynamic membrane protrusions for understanding phagocytosis, cellular communication and mechanobiology, current imaging modalities struggle to quantitatively track their real-time, 3D spatiotemporal dynamics with sufficient molecular specificity and minimal perturbation. Many membrane protrusions studies still utilize confocal microscopy where its axial resolution and high phototoxicity remains a key limiting factor for live axial imaging. We discovered that multiple rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon provides a foundation to generate speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free, and minimally invasive imaging of diverse membrane protrusions within complex co-cultures. RO-iSCAT enables nanoscale-sensitive tracking of membrane protrusion dynamics along the axial direction. This allows for the construction of dynamic axial variance maps, facilitating quantitative measurements of membrane protrusion formation at tens to hundreds of nanometer displacements, without requiring 3D volumetric imaging. RO-iSCAT empowers real time quantitatively dissection of the axial spatiotemporal complexities of membrane protrusions and unlock future insights into fundamental processes like cell migration, durotaxis, and intercellular communication. Key pointsO_LIDiscovered that multiple integrated rotational oblique interference scattering (RO-iSCAT) generates speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. C_LIO_LIRemoved need for 3D volumetric imaging to quantified axial motion of membrane protrusion forming tethers, trails and bridge with within [~] tens of nanometer accuracy. C_LIO_LIEnabled classification of membrane protrusions that, despite possessing identical chemical compositions, are differentiated by their interactions, thus offering a qualitative comparison of membrane protrusions at the nanoscale in living cells. C_LI Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/644841v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@b183a4org.highwire.dtl.DTLVardef@39a944org.highwire.dtl.DTLVardef@197f019org.highwire.dtl.DTLVardef@1438b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Respiratory Airway Secretory Cells act as Immune Sentinels in Human Distal Airways

Pulmonary immunity in the human distal respiratory airways is essential for lung function but remains poorly explored, mainly due to limited physiologically relevant models. Here, we develop distal lung organoids containing respiratory airway secretory (RAS) cells, a recently identified epithelial population unique to the distal airways of humans and large mammals, using human pluripotent stem cells (hPSCs). Lineage tracing identified RAS cells as descendants of SOX9brightNKX2-1bright progenitors. Single-cell transcriptomics elucidated that RAS cells exhibited a distinct immune-competent phenotype with enriched genes associated with viral host entry and pattern recognition receptor signaling. Functionally, RAS cells infected by respiratory syncytial virus (RSV) exhibited enhanced antiviral activity marked by upregulation of interferon-stimulated genes (ISGs) and complement component 3 (C3). In contrast, bacterial flagellin or Pseudomonas aeruginosa (PAO1) triggered a TLR5-driven immune response in RAS cells, inducing complement system activation, a response suppressed by selective TLR5 inhibition. Notably, in chronic obstructive pulmonary disease (COPD), RAS cells displayed dysregulated immune activation, characterized by elevated expression of adaptive immune mediators. Together, our findings identify RAS cells as previously unrecognized sensors and effectors of mucosal immunity in the human distal airways and reveal their roles in viral infection, bacterial stimulation, and chronic inflammatory lung disease, providing potential therapeutic targets for respiratory diseases.

cell biology↗

bioRxiv2025

Human Body Single-Cell Atlas of 3D Genome Organization and DNA Methylation

Higher-order chromatin structure and DNA methylation are critical for gene regulation, but how these vary across the human body remains unclear. We performed multi-omic profiling of 3D genome structure and DNA methylation for 86,689 single nuclei across 16 human tissues, identifying 35 major and 206 cell subtypes. We revealed extensive changes in CG and non-CG methylation across almost all cell types and characterized 3D chromatin structure at an unprecedented cellular resolution. Intriguingly, extensive discrepancies exist between cell types delineated by DNA methylation and genome structure, indicating that the role of distinct epigenomic features in maintaining cell identity may vary by lineage. This study expands our understanding of the diversity of DNA methylation and chromatin structure and offers an extensive reference for exploring gene regulation in human health and disease.

genomics↗

bioRxiv2025

Single-cell-guided identification of logic-gated antigen combinations for designing effective and safe CAR therapy

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, its application in solid tumors remains limited because single targets are unlikely to suffice due to tumor antigen heterogeneity and off-tumor toxicities. To overcome these obstacles, we developed LogiCAR designer, a computational approach that utilizes single-cell transcriptomics data from patient tumors to systematically identify the cancer-specific antigen circuits with logic gates ("AND," "OR," and "NOT") that target the majority of cancer cells in a tumor while sparing normal cells and tissues as much as possible. LogiCAR designer efficiently scales to higher-order antigen combinations involving up to five genes. Applied to a large-scale dataset encompassing approximately 2 million cells (including > 620k tumor cells) from 342 clinical patient samples across all major breast cancer subtypes, LogiCAR designer identified antigen circuits with enhanced tumor-targeting efficacy and improved safety profiles compared to both previously reported circuits and single-target therapies in clinical trials. However, even these optimized shared circuits still proved insufficient for some patients. We hence systematically studied LogiCAR designers ability to identify highly effective CAR circuits that are individualized to each patient. Remarkably, such personalized CAR circuits provide estimated tumor-targeting efficacy tantamount to complete response in 76% of patients and partial response for all patients. Taken together, this analysis is the first systematic quantification of the efficacy and safety of all possible CAR circuits, showing that: (a) the quality of existing solutions leaves much to be desired; (b) the ability of shared circuits optimized across many patients is moderate, and finally, (c) individually tailored circuits offer significantly higher tumor-targeting efficacies for patients. LogiCAR designer offers a rigorous, data-driven way to facilitate the rational design of safe and effective CAR-based immunotherapies for cancer. Statement of Significance Development of a computational approach that efficiently identifies logic-gated CAR target combinations, called circuits, from single-cell transcriptomics, addressing a critical unmet clinical need. Application to the largest ensemble of breast cancer datasets to date, comprising [~]2 million cells (> 620k tumor cells) from 17 clinical cohorts, to identify CAR circuits predicted to be effective. Comprehensive safety profiling of candidate circuits spanning major tissues at both RNA and protein levels. Logic-gated CAR circuits generated by our pipeline address tumor heterogeneity and achieve efficacy and safety scores that surpass clinical trial and previously computationally identified circuits. Individualized rational CAR design offers a transformative approach to deliver precision-engineered CAR therapies with unprecedented efficacy.

bioinformatics↗

bioRxiv2025

Regulation of alternative polyadenylation events by PABPC1 affects erythroid progenitor cell expansion

Erythropoiesis is precisely regulated by multilayer networks and is crucial for maintaining steady-state hemoglobin levels and ensuring effective oxygen transport. Alternative polyadenylation (APA) is a post-transcriptional regulatory mechanism that generates multiple mRNA isoforms from a single gene, based on specific 3-untranslated region sequences. While APA plays a vital role in various cellular processes, its mechanism in erythropoiesis remains unexplored. Here, we employed an integrative approach combining bioinformatics and experimental validation to systematically analyze APAs role in erythropoiesis. We mapped the APA landscape during erythroid differentiation, discovering notable APA shifts crucial for the maturation from burst-forming unit erythroid to colony-forming unit erythroid. Notably, our findings highlighted PABPC1 as the primary APA regulator of these stages. Functional investigations revealed that knocking down PABPC1 disrupts erythroid progenitor cell proliferation and differentiation, implicating the proteins essential role in modulating cell fate through APA regulation. We further identified that decreased PABPC1 levels led to the increased usage of the proximal polyA site of TSC22D1 and gene overexpression, revealing a novel mechanism through which APA affects erythroid progenitor expansion and differentiation. These insights uncover a novel dimension of APA regulation in early erythropoiesis, providing new strategies for the treatment of diseases associated with erythropoiesis disorders.

developmental biology↗

bioRxiv2025

De Novo Negatively Charged Binders Targeting MMLVRT Nucleic Acid Binding Sites Overcome Stability-Activity Trade-offs

The limited thermostability and storage-induced inactivation of Moloney murine leukemia virus reverse transcriptase (MMLV RT) have constrained its applications. In this study, a high-stability mutant, MMLV RT-SV, was generated through a multi-site mutagenesis strategy targeting the nucleic acid-binding pocket. Through de novo protein design, three highly negatively charged binders with affinities of 45 nM, 362 nM, and 829 nM were developed to specifically target the positively charged nucleic acid-binding region on the surface of MMLV RT-SV. Experimental results demonstrated that these binders formed complexes with the enzyme via electrostatic interaction, significantly enhancing the thermostability and long-term storage stability of MMLV RT-SV while not affecting its RNA- dependent DNA polymerase function. This work pioneers the rational design of negatively charged binders targeting strongly positively charged nucleic acid-binding sites, overcoming the traditional stability-activity trade-off inherent in site-directed mutagenesis and directed evolution. The proposed strategy not only provides an innovative solution to address the thermal sensitivity and storage instability of reverse transcriptase but also establishes a novel paradigm for De Novo-based precision engineering of enzyme functions, demonstrating significant potential in molecular diagnostics, gene editing, and related fields.

bioengineering↗

bioRxiv2025

BioBatchNet: A Dual-Encoder Framework for Robust Batch Effect Correction in Imaging Mass Cytometry

MotivationImaging Mass Cytometry (IMC) is a cutting-edge technology for analysing spatially resolved protein expression at the single-cell level. However, its downstream analyses are often hindered by batch effects, which introduce systematic biases and obscure true biological variations. Existing correction methods, largely developed for scRNA-seq data, struggle to achieve precise control, leading to either over-correction by removing critical biological information, or under-correction by leaving residual batch effects. Moreover, these methods face challenges in adapting to IMC data due to differences in data characteristics. Furthermore, IMC data often feature imbalanced and overlapping cell populations, complicating clustering and downstream analysis. These challenges underscore the need for a robust and controllable batch effect correction approach tailored to IMC data. ResultsWe present BioBatchNet, a dual-encoder framework utilising adversarial training to explicitly disentangle batch-specific and biological signals. BioBatchNet enables controllable batch effect correction, effectively balancing correction with the preservation of biological variation. We evaluated BioBatchNet on three IMC datasets, where it outperformed seven benchmarking methods with robustness in both correction and biological signal conservation. Additionally, we developed a Constrained Pairwise Clustering (CPC) method, which employs constrained pairs to improve clustering performance, even in datasets with imbalanced and overlapping cell populations. To validate its generalisability, BioBatchNet was also applied to four scRNA-seq datasets, where it delivered competitive performance compared to eight typical methods. These results demonstrate BioBatchNets generalisability and robustness in correcting batch effects across diverse single-cell datasets and underscore its potential for large-scale biological analyses.

bioinformatics↗

bioRxiv2025

Progressively reduced cerebral oxygen metabolism and elevated plasma NfL levels in the zQ175DN mouse model of Huntington disease

Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by a CAG-repeat expansion in exon-1 of the huntingtin gene. Currently, no disease-modifying therapies are available, with a significant challenge in evaluating therapeutic efficacy before clinical symptoms emerge. This highlights the need for early biomarkers and intervention strategies. Therefore, it is essential to develop and characterize accurate mouse models and identify early biomarkers for preclinical therapeutic development. In this study, we characterized the pathological progression of the heterozygous zQ175 neo-deleted knock in (zQ175DN) mouse model across four age groups: 3, 6, 10, and 16 months to identify human translatable outcome measures. T2-relaxation-under-spin-tagging (TRUST) MRI was used to assess global CMRO2, while T2-weighted MRI was used to analyze brain volumes. Significant brain volume loss was detected as early as 6 months of age, worsening progressively with age in the zQ175 DN mice, resembling HD brain volumetric changes. A decline in CMRO2 was observed in 6-month-old zQ175 DN mice, with significant and progressive reductions in 10- and 16-months old HD mice. Additionally, PHP1-positive mutant huntingtin (mHTT) aggregates were present in the striatum of zQ175 DN mice at all four age groups, with intranuclear localization prior to 6 months, transitioning to both intranuclear and neuropil aggregates in older zQ175 DN mice, suggesting that the localization of mHTT aggregates may reflect the severity of HD pathogenesis. Interestingly, plasma neurofilament light chain (NfL) protein concentrations were significantly elevated at 6 months of age and older zQ175DN mice. These findings provide valuable insights for selecting outcome measures in preclinical evaluations of HD therapies using the zQ175 DN mouse model.

neuroscience↗

bioRxiv2025

Gene expression plasticity in the antibiotic environment is predominantly adaptive in the budding yeast

Phenotypic plasticity plays a key role in adaptation to fluctuating environments. However, its evolutionary significance remains debated, with conflicting views on whether it is actively maintained by natural selection or a neutral consequence of molecular constraints. In this study, we investigate the evolutionary role of gene expression plasticity in yeast populations exposed to antibiotic and osmotic stress. Using mutation accumulation (MA) lines to separate the effects of natural selection from genetic drift, we compare gene expression responses (referred to as plastic change) between 22 natural strains, 21 MA lines, and their progenitor under non-stressed and stressed conditions. Our results show that, in the antibiotic environment, gene expression plasticity is selectively maintained, as indicated by its reduction in magnititude, reversal in directionality, and loss of stress-responsive pathways in MA lines. In contrast, plasticity in the osmotic stress condition appears neutral, with random variation across MA lines. This study provides direct evidence for the adaptive role of gene expression plasticity in the antibiotic environment and sheds light on its molecular mechanisms.

evolutionary biology↗

bioRxiv2025

Benchmarking AlphaFold3-like Methods for Protein-Peptide Complex Prediction

Protein-short peptide interactions are central mechanisms regulating signal transduction, chaperone functions, and drug targeting design. However, the precise prediction of their complex structures has long been constrained by the limited accuracy of traditional methods. The breakthrough of AlphaFold2 resolved the challenge of predicting protein monomer structures. Its derivative method, AlphaFold2-multimer, surpassed traditional docking approaches in predicting protein complexes, yet its accuracy remains suboptimal. AlphaFold3 and its replication models, such as Protenix, Chai-1, and Boltz-1, have further improved prediction accuracy for protein-protein interactions. To evaluate the performance of these methods in protein-short peptide structure prediction, this study conducted predictions and comprehensive comparisons on two benchmark datasets of protein-short peptide complexes. One dataset did not intentionally exclude structures from the training set of these methods, while the other did. For the former, the results demonstrate that the new-generation structural method significantly increased the success rate under stringent criteria (Fnat[≥] 0.8) to 70-80%, compared to AlphaFold2-multimer (53%), with Protenix achieving the highest accuracy at 80.8%. For the second dataset, AlphaFold2-multimer showed consistent performance, but the success rates of the new-generation methods all dropped significantly (to 40-56%). Our analysis suggests that the main challenge for the performance decline comes from the accurate prediction of binding sites. For all datasets, A multi-method combination strategy enabled a higher-quality prediction success rate compared to the individual models, highlighting synergistic advantages among methods. Notably, inter-model consistency analysis provided an efficient metric for selecting optimal predictions.

bioinformatics↗

bioRxiv2025

Harnessing Uniform Design to Enhance AI-Driven Predictions of Physicochemical Properties of Short Peptides

Short peptides hold significant promise in drug discovery and materials science due to their biocompatibility, multifunctionality, and ease of synthesis. However, accurately predicting their physicochemical properties, a prerequisite for application development, remains a challenge. This study presents an innovative approach integrating uniform design (UD) with artificial intelligence (AI) to enhance prediction of key physicochemical properties, including aggregation propensity (AP), hydrophilicity (logP), and isoelectric point (pI). Using UD, we generate 31 distinct peptide datasets, with a consistent amino acid occupation fraction of 5% at each position, thereby creating unbiased training data for AI models. The performance of each AI model is rigorously evaluated using various testing schemes, and optimal sample sizes are determined for accurate prediction of each property. Additionally, Shapley Additive Explanations (SHAP) analysis identifies aromaticity, logP, net charge, and pI as the primary factors affecting peptide aggregation. This work provides comprehensive datasets on the physicochemical properties of all tetrapeptides, develops robust AI-based predictive models, and elucidates the relationships between key physicochemical characteristics and self-assembly behavior. By integrating experimental design, AI modeling, and peptide domain knowledge, our approach facilitates the discovery and optimization of functional peptides, offering new opportunities for peptide-based therapeutic applications.

bioinformatics↗

bioRxiv2025

Obesity Increases Atherosclerosis Susceptibility via Inter-tissue miR-30e-SLC7A11 Axis

BackgroundWith obesity as a risk factor for atherosclerotic disease, recent research suggests that adipose tissue in obese animal models and humans can generate endocrine-like molecules that affect arterial health later on. Previous studies showed that microRNA-30e (miR-30e) level is elevated in atherosclerosis and solute carrier family 7 member 11 (SLC7A11), a cystine/glutamate transporter, is involved in atherogenesis. However, whether an endocrine-like link between the adipose-derived miR-30e-5p and SLC7A11 in the vascular endothelium can lead to obesity-caused atherosclerosis is unknown. MethodsMiRNA data mining and RT-PCR validations were used to demonstrate the positive association among serum level of miR-30e-5p, obesity, and coronary arterial disease in human patients. Transcriptomics (RNA-seq and single-nucleus RNA-seq), metabolomics, and in silico analysis were used to establish a miR-30e-5p-SLC7A11 regulation of central carbon metabolism, mitochondrial and endothelial cell (EC) function. Mouse models with EC-specific Slc7a11 knockout (EC-Slc7a11-/-) and gain or loss of function of miR-30e-5p were used to elucidate the detrimental role of this endocrine-like axis in obesity-related atherosclerosis. ResultsThe level of adipocyte-derived miR-30e-5p was significantly upregulated in obese humans with coronary artery disease and obese and atherosclerotic mice. Via serum exosomes, the adipocyte-generated miR-30e-5p targeted SLC7A11 mRNA in vascular ECs. SLC7A11 deficiency due to miR-30e-5p targeting dysregulated glutamate/cystine metabolism increased glycolysis, reduced oxidative phosphorylation, and impaired mitochondrial function. The EC dysfunction could be rectified by SLC7A11 overexpression or miR-30e-5p antagonism. Exogenously delivered miR-30e-5p phenocopied the increased atherosclerosis in EC-Slc7a11-/- mice. In contrast, miR-30e-5p antagomir treatment reduced atherosclerosis in Apoe-/- and ob/ob mice. ConclusionOur multi-omics approaches demonstrates that the adipose-derived miR-30e-5p downregulated SLC7A11 mRNA in ECs via tissue crosstalk. The resulting EC dysfunction led to obesity-related atherosclerosis in mice. These findings underscore a causality between obesity and atherosclerosis in the context of cardiovascular-kidney-metabolic syndrome.

molecular biology↗

bioRxiv2025

Oxytocin receptor and HER2 interactions in breast cancer

Breast cancer affects women globally, with HER2 being one of the most aggressive subtypes. Despite advances in HER2-targeted therapies, many patients fail to respond to such treatments. In search of alternative therapeutic targets, we investigated the role of the oxytocin (OT) and oxytocin receptor (OTR) signalling system in HER2+ breast cancer. Survival analysis of HER2 subtype breast cancer patients revealed high OTR expression correlating with significantly improved relapse-free survival. OTR overexpression or knockdown in HER2+ SK-BR-3 cells decreased or increased cell viability, respectively. HER2 was downregulated in OTR-overexpressing cells, which could contribute to the decreased Pertuzumab efficacy. Importantly, HER2 was internalised upon OT treatment and interacted with OTR to form OTR-HER2 complexes. OT treatment furthermore induced a transient inhibition of HER2 phosphorylation without affecting the total HER2 protein levels. In addition, OT inhibited ERK1/2 phosphorylation but enhanced Akt phosphorylation, linked to increased cell viability. In summary, this work describes a novel HER2-OTR interaction and mechanism, expanding our understanding of HER2 cancer biology that might lead to new prognostic and therapeutic opportunities centred around the OT/OTR signalling system for patients suffering from HER2+ breast cancer.

cancer biology↗

bioRxiv2025

Mechanism and cellular actions of the potent AMPK inhibitor BAY-3827

Inhibition of AMP-activated protein kinase (AMPK) is under increasing investigation for its therapeutic potential in many diseases, including certain cancers. However, existing AMPK- inhibitors available as tool compounds are largely limited to compound C/dorsomorphin and SBI-0206965, both of which suffer from poor selectivity and off-target effects. Here we describe the structure-based molecular insights and cellular actions of a recently identified potent AMPK inhibitor, BAY-3827. Kinase selectivity profiling and sequence analyses of kinases that are highly or weakly inhibited by BAY-3827 uncovered key conserved residues involved in its inhibitory mechanism. A 2.5 [A] co-crystal structure of the AMPK kinase domain (KD)-BAY-3827 complex and comparison with known KD-inhibitor structures, revealed an overlapping site in the ATP-binding pocket and an C helix-out conformation. A distinct feature of the BAY-3827-bound state is the formation of a disulfide bridge between the D helix Cys106 and the activation loop residue Cys174. This bridge appears to stabilize the activation loop such that Asn162 repositions the DFG motif Phe158 toward the C-terminal kinase lobe, displacing His137 and disrupting the regulatory spine, thereby promoting an inactive state. In hepatocytes, 2.5-5 M BAY-3827, but not the structurally resembling inactive BAY-974, fully blocked AMPK activator (MK-8722)-mediated phosphorylation of ACC1 and corresponding inhibition of lipogenesis. Unbiased transcriptome analysis in MK- 8722-treated wild-type and AMPK-null hepatocytes revealed that 5 M BAY-3827 downregulated >30% of MK-8722-stimulated AMPK-dependent genes. Based on its greater selectivity and potency substantiated by comprehensive structural and cellular investigations, BAY-3827 is a powerful tool to delineate AMPK functions. One-sentence summaryWe provide the mechanism of action of the potent and selective AMPK inhibitor BAY-3827, which blocks AMPK-dependent cellular functions.

biochemistry↗

bioRxiv2025

Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

TAR DNA-binding protein 43 (TDP-43) pathology is associated with a spectrum of clinical dementias including limbic-predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC). Post-translational modifications (PTM) are linked to TDP-43 toxic gain-of-function and cytoplasmic aggregation1-3. Phosphorylation remains the most investigated PTM and a standard criterion for determining pathology progression and clinical subclassification in TDP-43 proteinopathies4-7. However, full spectrum of PTMs on TDP-43 structure and biology remain unknown. Utilizing mass-spectrometry analysis we identified citrullination as a novel and irreversible "bona-fide" PTM of TDP-43 protein. We recognized peptidyl arginine deiminase 2 and 4 (PAD2 and PAD4) to mediate the conversion of arginine (R) to citrulline (citR) in vitro and demonstrated increased PAD2 and PAD4 expression and TDP-43 citrullination in a human wildtype TDP-43 mouse model (Tg (Thy1-TARDBP4). Transmission electron microscopy imaging analysis revealed citrullination induced vast structural changes while ThT analysis demonstrated altered aggregation kinetics of citrullinated (citR) TDP-43 protein. We further provided mechanistic evidence on reduced electrostatic and pi-pi interactions of citR TDP-43 Low Complexity Domain (LCD) with RNA, favoring liquid-solid phase separation and condensate formation. Generation and validation of several citR TDP-43 specific antibodies against several TDP-43 epitopes revealed epitope and domain-specific effects of citrullination on TDP-43 solubility in vivo. Importantly, we found distinct reactivities of citR TDP-43 antibodies shedding light into the contribution of epitope-specific properties of human citR TDP-43 to novel pathological citR TDP-43 assemblies in human brain tissue from LATE-NC, with or without comorbid Alzheimers disease neuropathologic changes (ADNC). These findings provided a unique look into the temporal citR TDP-43 signatures, and the potential clinical relevance associated with progression of pure LATE-NC and comorbid ADNC + LATE-NC. Collectively, these data reveal the existence of irreversible TDP-43 citrullination at targeted sites via induced PAD2/PAD4 activities, presenting a critical step in TDP-43 proteinopathy.

neuroscience↗

bioRxiv2025

Validation of bronchial airway gene expression associated with bronchiectasis in nasal epithelium

ObjectivesExamination of bronchial epithelium-derived gene expression signature of bronchiectasis (BE) in nasal epithelium. MethodsWe studied 220 participants from the Detection of Early Lung Cancer Among Military Personnel study with bulk RNA-seq of nasal epithelium brushings. Gene set enrichment analysis (GSEA) was used to examine whether genes previously identified as increased or decreased in the bronchial epithelium of individuals with radiologic BE are significantly enriched among the genes most significantly altered in the nasal epithelium. GSEA and cell-type specific gene expression signatures were used to examine changes in the cellular composition of nasal epithelium associated with radiologic BE. ResultsGenes previously found to have significantly altered expression in bronchial epithelium in radiologic BE are concordantly enriched among the genes most increased or decreased in nasal epithelium in radiologic BE in this validation set (p = 3x10-7, increased; p = 6x10-8, decreased). GSEA performed using cell-type signatures demonstrates that in nasal epithelium, BE is associated with increased multiciliated and deuterosomal cell-related gene expression and decreased expression of basal cell- related gene expression, consistent with our previous findings in bronchial epithelium. ConclusionOur work validates our bronchial signature of BE in an independent dataset and demonstrates that similar gene expression changes are associated with radiologic BE in the bronchial and nasal epithelium. These findings support considering nasal epithelium brushing as a less invasive tool for screening and monitoring BE. SummaryThis study validates a previously reported bronchial signature of bronchiectasis (BE) in nasal epithelium. Changes in gene expression and cell-type composition associated with radiologic BE are similar across both bronchial and nasal epithelium.

bioinformatics↗

bioRxiv2025

Structural implications of glycosylation on the voltage-gated sodium channel β3-subunit

Voltage-gated sodium (NaV) channel -subunits are modulated by associated {beta}-subunits that affect their localization, trafficking and gating behaviour. The {beta}-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length {beta}3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric {beta}3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated {beta}3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of {beta}3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the {beta}3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length {beta}3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length {beta}3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric {beta}-subunits with corresponding -subunit sodium channels.

biochemistry↗