Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

Non-disruptive 3D profiling of combinations of epigenetic marks in single cells

Recent advancements in single-cell sequencing and spatial omics technologies have enhanced our understanding of diverse cellular identities, compositions, architectures, and functions. However, the single-cell three-dimensional (3D) organization of the epigenome is still not well understood, due to an absence of spatial single-cell methods that allow high-resolution, locus-specific detection of combinations of epigenetic marks while maintaining the 3D organization of the genome. Here, we develop Epigenetic Proximity Hybridization Reaction (Epi-PHR), a non-disruptive image-based single-cell spatial epigenetic profiling technology. Epi-PHR enables locus-specific and high-resolution in situ detection of combinations of epigenetic marks at hundreds of single gene targets within the same individual cells, while retaining the 3D organization of the genome. Dual-mark Epi-PHR in hippocampus tissue sections revealed region-specific epigenetic profiles. Phased Epi-PHR combined with chromatin tracing simultaneously detects allele-specific epigenetic states and chromatin conformations of a paternally imprinted Meg3 gene cluster in single cells, revealing associations between specific epigenetic mark enrichment and chromatin folding features for the distinct alleles from different parental origins. Surprisingly, we found that H3K9me3 abundance is positively associated with a chromatin domain boundary at the Meg3 locus among maternal copies. We expect Epi-PHR will be broadly applicable in research requiring single-cell spatial epigenetic information, and will help extend our understanding of the combinatorial epigenetic code and its relationship with chromatin organization.

genetics↗

bioRxiv2025

Molecular Mechanisms of DNMT3A-3L-Mediated de novo DNA Methylation on Chromatin

De novo DNA methylation is a fundamental epigenetic process essential for early development. It is mediated by DNA methyltransferase DNMT3A and DNMT3B, in cooperation with catalytically inactive DNMT3L. Abnormal DNA methylation contributes to genomic instability and oncogenesis, yet the mechanisms governing its establishment and regulation remain unclear. Here, we present high-resolution cryo-EM structures of the nucleosome-bound full-length DNMT3A2-3L and its oligomeric assemblies in the nucleosome-free state. Strikingly, DNMT3L C-terminal "Switching Helix" displayed a distinct conformation with a 180{degrees} rotational difference compared to its DNMT3B3 counterpart in the presence of nucleosome, preventing direct interactions with the nucleosome acidic patch. Instead, DNMT3L ADD domain promotes nucleosome binding, while DNMT3A PWWP domain inhibits it, indicating multi-layer regulation. Additionally, the novel oligomeric arrangement of DNMT3A2-3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. Furthermore, we identified the critical role of DNMT3L as a sensor of histone modifications, guiding the complexs chromatin interaction. These findings uncover a previously unknown mechanism by which DNMT3A-3L mediates de novo DNA methylation on chromatin through complex reorganization, histone tail sensing, dynamic DNA search, and nucleosome engagement, providing key insights into epigenetic regulation.

molecular biology↗

bioRxiv2025

Effects of diffusion MRI spatial resolution on human brain short-range association fiber reconstruction and structural connectivity estimation

Short-range association fibers (SAFs) are critical for cortical communications but are often underestimated in conventional resolution diffusion magnetic resonance imaging (dMRI) since they locate within a [~]1.5mm thin layer of superficial white matter. With the emergence of high-resolution diffusion imaging techniques, this study timely evaluated the effects of image spatial resolution on SAF reconstruction using simulation data and multi-resolution (2, 1.5, and 0.96 mm iso.) empirical data acquired on the same 20 healthy subjects using the gSlider sequence and 20 widely used tractography approaches. Resolution effects were qualitatively assessed through model fitting and tractography results and quantitatively evaluated using global and regional short-range connectivity strength (SCS). It is found that lower resolution systematically reduces SCS across all methods in a spatially varying manner. Moreover, tractography methods exhibit significant differences in resolution sensitivity, with diffusion tensor imaging (DTI) based single-tissue single-fiber tractography showing greater vulnerability than constrained spherical deconvolution (CSD)-based multi-tissue multi-fiber tractography. Probabilistic tracking with anatomical constraints (ACT) and filtering (SIFT) improves robustness. Finally, up-sampling data to nominally higher resolution partially mitigates resolution-induced degradation and improves SAF reconstruction accuracy, particularly for DTI tractography. Based on these findings, higher resolution and multi-shell imaging is recommended if possible. For a given dataset, data up-sampling and DTI-based probabilistic tracking with ACT is recommended for single-shell low b-value data. CSD-based probabilistic tracking with ACT and SIFT is recommended for single-shell higher b-value data and multi-shell data. In summary, this study systematically and quantitatively evaluated resolution effects on SAF reconstruction and structural connectivity estimation and provided practical guidelines for more accurate mapping of SAFs. These advances hold promises to improve the characterization of healthy and diseased human brains in a wide range of neuroscientific and clinical applications.

neuroscience↗

bioRxiv2025

Reduced inhibition, bursting, and accelerated oscillations drive early hippocampal hyperactivity in Alzheimer's disease

Early hippocampal hyperactivity is a well-documented yet poorly defined phenomenon in Alzheimers disease (AD). While reported in both patients and animal models, its functional manifestations and underlying neurophysiological mechanisms in vivo remain unclear. Here, we address this gap using in vivo high-resolution patch-clamp, high-throughput single-unit, and local field recordings in young amyloidopathy mice, at a stage when A{beta} remains largely soluble. We uncover previously unidentified cellular mechanisms in vivo, characterised by reduced inhibitory synaptic input, hypoactivity of fast-spiking interneurons, and enhanced bursting in pyramidal neurons. At the network level, we reveal accelerated hippocampal oscillations, marked by increased theta and beta power, a departure from the conventional view of oscillation slowing in AD. Mechanistically, this acceleration stems from strengthened synchrony of excitatory currents at higher frequencies and an overall reduction in oscillation-associated inhibitory currents. Our findings provide the first direct in vivo evidence linking early hippocampal hyperactivity to specific synaptic transmission and network dysfunctions, resolving a long-standing ambiguity. Moreover, we propose accelerated oscillations in the hippocampus as a functional biomarker for early AD and a potential therapeutic target for restoring network stability before cognitive decline occurs.

neuroscience↗

bioRxiv2025

Underappreciated role of environmental enrichment in alleviating depression and anxiety: Quantitative evidence synthesis of rodent models

Environmental enrichment has long been recognized as a non-pharmacological intervention to mitigate mental health issues, yet its efficacy, and heterogeneity of treatment effects across experimental contexts remain underexplored. Heterogeneity of treatment effects, which reflects variability in individual responses to interventions, is a critical factor in determining the generalizability and personalization needs of treatments. Here, we conducted a registered meta-analysis of 62 studies and 1,112 comparisons in rodent models to evaluate the impact of environmental enrichment on depressive and anxiety-like behaviours. We found that environmental enrichment reduced these behaviours of animal models by 16% on average and decreased inter-individual variability by 12%, indicating not only effectiveness but also low heterogeneity of treatment effects, which suggests consistent effects across individuals. Environmental enrichment further nullified the adverse effects of stressors, demonstrating a significant antagonistic interaction. These effects were robust across multiple sensitivity analyses, including model-based predictions, post-stratification, multi-model inference, publication bias correction, and critical appraisal of study quality. Moderator analyses highlighted the importance of exposure timing and the inclusion of social enrichment components. Taken together, our pre-clinical evidence on rodent models supports environmental enrichment as a low-cost, scalable, and biologically grounded intervention with translational relevance for developing equitable and accessible treatments for depression and anxiety. Given the importance of innovation and personalization in mental health care, the low heterogeneity of treatment effects of environmental enrichment positions it as a promising avenue for non-pharmacological therapeutic strategies that can be broadly applied without extensive tailoring.

animal behavior and cognition↗

bioRxiv2025

Joint Modeling of Longitudinal Biomarker and Survival Outcomes with the Presence of Competing Risk in Nested Case-Control Studies with Application to the TEDDY Microbiome Dataset

MotivationLarge-scale prospective cohort studies collect longitudinal biospecimens alongside time-to-event outcomes to investigate biomarker dynamics in relation to disease risk. The nested case-control (NCC) design provides a cost-effective alternative to full cohort biomarker studies while preserving statistical efficiency. Despite advances in joint modeling for longitudinal and time-to-event outcomes, few approaches address the unique challenges posed by NCC sampling, non-normally distributed biomarkers, and competing survival outcomes. ResultsMotivated by the TEDDY study, we propose "JM-NCC", a joint modeling framework designed for NCC studies with competing events. It integrates a generalized linear mixed-effects model for potentially non-normally distributed biomarkers with a cause-specific hazard model for competing risks. Two estimation methods are developed. fJM-NCC leverages NCC sub-cohort longitudinal biomarker data and full cohort survival and clinical metadata, while wJM-NCC uses only NCC sub-cohort data. Both simulation studies and an application to TEDDY microbiome dataset demonstrate the robustness and efficiency of the proposed methods.

bioinformatics↗

bioRxiv2025

Neuronal oscillations predict un-cued task switching in the Wisconsin card sorting test

Cognitive flexibility is the ability to keep or update contextual cognitive representations. The underlying neural mechanisms and how it is influenced by individual sensitivity to feedback are not known. We measured cognitive flexibility under uncertainty with the Wisconsin Card Sorting Task and brain activity with magnetoencephalography (MEG). Using a behavioral sequential learning model, we show that variability in cognitive flexibility is predicted by individual reward and punishment sensitivity to feedback and anticipation and exploration tendencies. Uncovering their brain oscillatory signatures showed that individual learning speed is predicted by suppressed alpha-beta (7-32 Hz) and increased broad-band gamma (52-96 Hz) amplitudes along with concurrent large-scale alpha (7-13 Hz) desynchronization. Importantly, these oscillatory sub-processes were explained by individual levels of distinct feedback sensitivities. These results provide a novel account on neural sub-computations underlying flexible contextual switching between stable and unstable cognitive representations that predict the individual speed of rule learning.

neuroscience↗

bioRxiv2025

Parkinson's-Linked Synaptojanin 1 Loss in Dopamine Neurons Triggers Synaptic Degeneration and Striatal TH Interneuron Compensation

Presynaptic endocytic dysfunction is an early feature of Parkinson's disease (PD), but how dopamine circuits adapt to chronic synaptic failure remains unclear. In dopamine neuron-specific Synaptojanin 1 knockout mice, loss of this PD-linked endocytic protein causes terminal dystrophy and striatal dopamine deficiency, yet motor coordination is preserved. Chronic endocytic failure induces striatal dopamine-like neurons (DALNs) with circuit-dependent dopaminergic protein accumulation. DALNs do not arise through transcriptional reprogramming; instead, they acquire dopaminergic proteins from midbrain dopamine neurons through selective long-range transfer that favors soluble cargoes and can convey disease-associated proteins, including -synuclein and tau. Functionally, DALNs support local dopamine production, partially preserve motor performance and increase resistance to acute dopaminergic lesions. This adaptation is shared across PD-linked endocytic mutants and is developmentally constrained, but is not reproduced by acute denervation. Thus, synaptic endocytic stress triggers inter-neuronal protein transfer as an adaptive circuit mechanism supporting dopamine compensation and neuroprotection in early PD.

neuroscience↗

bioRxiv2025

NKp46 Recognizes the Hyphal Form of Candida albicans and Mediates Protective Antifungal Immunity

Candida albicans is an opportunistic fungal pathogen capable of transitioning between yeast and hyphal forms, a morphological plasticity critical for its pathogenicity. Natural killer (NK) cells play a crucial role in antifungal immunity, yet the molecular basis of their interaction with C. albicans remains incompletely understood. Here, we identify NKp46 (NCR1 in mice), an activating receptor on NK cells, as a functional receptor for the hyphal--but not yeast--form of C. albicans. Using image flow cytometry and binding assays with NKp46-Ig and NCR1-Ig fusion proteins, we demonstrate that NKp46 selectively binds to hyphae. This interaction is mediated by the D2 domain of NKp46 and is sialic acid independent. Blocking NKp46 impairs NK cell degranulation and fungal killing in vitro. In vivo, mice deficient in NCR1 exhibit increased susceptibility to systemic C. albicans infection. Our findings establish NKp46 as a key sensor of invasive fungal morphology and underscore its role in early antifungal immunity.

immunology↗

bioRxiv2025

PW-GAN: Pseudo-Warping Field Guided GAN for Unsupervised Denoising of Fetal Brain MRI Images

Fetal brain magnetic resonance imaging is of great importance for prenatal neural disorder diagnosis. To improve signal-to-noise ratio and coverage, fetal brain MRI often uses thick-slice scanning to reduce motion artifacts and ensure image quality for single slices. Reconstructing 3D MR volumes from multiple motion-corrupted stacks of 2D slices has shown promise in imaging of moving subjects like fetal MRI. However, raw scans acquired with varying slice thicknesses present substantial disparities in quality when retrospectively reconstructed into isotropic high-resolution volumes (e.g. 0.8 mm slice thickness). In particular, thick-slice acquisitions (e.g. 5-6 mm) tend to yield suboptimal reconstruction results compared to thin-slice scans (e.g. 2-3 mm), often exhibiting residual noise, interpolation-induced artifacts, and inter-slice structural discontinuities. This challenge has highlighted the need to further reduce noise caused by reconstruction errors when the reconstructed volumes are used for downstream tasks. With the success of neural networks in computer vision, deep learning methods based on architectures such as convolutional neural networks (CNNs) and generative adversarial networks (GANs) have achieved outstanding performance in single-image denoising. Nevertheless, CNN-based methods still heavily rely on large-scale datasets comprising noisy-clean image pairs. Although unsupervised GAN variants such as CycleGAN have been developed to mitigate this dependency, the inherent variability of GAN-generated outputs often results in tiny anatomical distortions, significantly limiting the applicability of GAN-based methods. While several approaches have introduced regularizations to address this issue, they largely focus on denoising individual slices, overlooking inter-slice structural inconsistencies that arise from treating slices independently. In this work, we propose Pseudo-Warping Field Guided Unsupervised Generative Adversarial Network (PW-GAN), which formulates post-reconstruction optimization as an unpaired style transfer problem between low-quality and high-quality MRI domains. Moreover, by incorporating a pseudo-deformation field module based on optical flow estimation, our method significantly enhances inter-slice continuity in the reconstructed volumes while effectively suppressing residual noise and interpolation artifacts introduced during the reconstruction process. Evaluations on both simulated and in vivo data demonstrate that our method outperforms existing unsupervised models and achieves performance on par with several state-of-the-art supervised methods.

neuroscience↗

bioRxiv2025

Salmonella SteD and mammalian SUSD6 use TMEM127 to co-disinhibit the WWP2 E3 ubiquitin ligase

The NEDD4-like E3 ubiquitin ligase, WWP2, is involved in a range of host processes from cell differentiation to T cell immunity. Ligase activity is tightly regulated with WWP2 being held in an autoinhibited state. Binding of a PY motif containing adaptor, an Ndfip, via the WW domains of NEDD4-like E3 ubiquitin ligases leads to their disinhibition. Here, we show that the canonical Ndfip, NDFIP2, requires multiple PY motifs for interaction with and activation of WWP2. In contrast, the single PY-motif containing Ndfips TMEM127 and SUSD6 function as a co-disinhibitory pair. TMEM127 and the Salmonella protein SteD also function as a co-disinhibitory pair. However, SteD interacts with a different region of the WWP2, the C2 domain, and this interaction results in disinhibition of WWP2. These findings demonstrate a range of ways that Ndfips can disinhibit WWP2. To our knowledge, these are the first examples of two Ndfips functioning as co-disinhibitory pairs, and of a bacterial effector that disinhibits an E3 ubiquitin ligase.

cell biology↗

bioRxiv2025

Spatial transcriptomics AI agent charts hPSC-pancreas maturation in vivo

Spatial transcriptomics has revolutionized our understanding of tissue organization by simultaneously capturing gene expression and spatial localization within intact tissues. However, analyzing these increasingly complex datasets requires specialized expertise across computational biology, statistics, and biological context. To address this challenge, we introduce the Spatial Transcriptomics AI Agent (STAgent), an autonomous multimodal agentic AI that integrates multimodal large language models (LLMs) with specialized computational tools to transform weeks-long analysis tasks into minutes of automated processing. Unlike conventional machine learning approaches that are limited to narrow, predefined tasks, STAgent leverages the emergent capabilities of multimodal LLMs - such as flexible reasoning, contextual understanding, and cross-modal integration - which allow it to adapt to novel data, execute multi-step analyses, and generate biologically meaningful insights with minimal human input. STAgent enables autonomous deep research through integrated capabilities, including dynamic code generation for complex analytical workflows, visual reasoning for interpreting spatial patterns, real-time retrieval of relevant peer-reviewd scientific literature, and synthesis of comprehensive, actionable reports. We applied STAgent to investigate the in vivo maturation of human stem cell-derived pancreatic cells (SC-pancreas) transplanted into immunodeficient mice. We generated single-cell spatial transcriptomics data spanning multiple developmental timepoints. STAgent autonomously (1) identified the maturation of initially scattered endocrine cells into well-defined islet-like structures, with predominantly peripheral -cells surrounding {beta}-cell cores supported by an expanding mesenchymal network; (2) revealed strengthening endocrine-endocrine cell interactions over time and, through context-aware gene set analysis, uncovered spatially resolved biological processes driving maturation; (3) unlike traditional analytical approaches, STAgent offers mechanistic explanations of spatial patterns, contextualizing findings with relevant literatures and developing cohesive insights into human pancreatic development. This agentic approach establishes a new paradigm in spatial transcriptomics analysis by substantially lowering the expertise barrier and reducing analysis time, accelerating biological and biomedical discovery.

genomics↗

bioRxiv2025

Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

High-risk human papillomavirus (HPV) replication requires deregulation of host DNA damage response (DDR) and inflammatory pathways. DNA topoisomerase 2{beta} (Top2{beta}) was previously shown to promote HPV replication, while the function of its paralog Top2 in the viral life cycle remains unknown. Elevated levels of Top2 are consistently observed in cervical intraepithelial lesions and the related carcinomas, as well as in HPV-positive cell lines. Silencing Top2 with shRNA severely suppresses HPV genome maintenance and amplification, but in a DDR-independent manner. Instead, Top2 facilitates secretion of interleukin (IL)-6 and IL-8, which are necessary for HPV replication. Mechanistically, this manipulation is regulated by toll-like receptor 4 (TLR4). Top2 binds to the TLR4 promoter to transcriptionally induce TLR4 expression. Blockade of TLR4 signaling by the specific inhibitor TAK-242 significantly reduces the secreted IL-6/IL-8 levels and HPV replication. Overall, our results reveal a novel role of Top2 to shape the inflammatory microenvironment that benefits HPV replication, making it a promising therapeutic target for HPV-associated diseases. IMPORTANCEHuman papillomaviruses (HPVs) are oncogenic pathogens responsible for many HPV-related diseases such as cervical cancer. HPV replication depends on the subversion of host cellular machineries, particularly pathways governing DNA damage response (DDR) and inflammation. However, the roles of enzymes that directly introduce DNA nicks or mediate strand rejoining remain poorly characterized. Here we highlight a novel role of DNA topoisomerase 2 (Top2) in promoting HPV genome maintenance and amplification but in a DDR-independent manner. Unlike its previously studied paralog Top2{beta}, Top2 supports HPV replication by driving the secretion of inflammatory molecules interleukin (IL)-6 and IL-8, which are critical for the viral genome persistence and copy number expansion. To our knowledge, this is the first evidence that a DDR-associated protein manipulates cytokine secretion, rather than expression, to support HPV replication. Furthermore, blocking toll-like receptor 4 (TLR4) activities significantly reduced Top2-dependent IL-6/IL-8 secretion and impaired HPV replication. These findings establish Top2 as a crucial player in HPV pathogenesis, with a therapeutic potential for treating HPV-associated malignancies.

microbiology↗

bioRxiv2025

DEC1 Regulates Human β Cell Functional Maturation and Circadian Rhythm

Stem cell-derived islet (SC-islet) organoids offer hope for cell replacement therapy in diabetes, but their immature function remains a challenge. Mature islet function requires the {beta}-cell circadian clock, yet how the clock regulates maturation is unclear. Here, we show that a circadian transcription factor specific to maturing SC-{beta} cells, DEC1, regulates insulin responsiveness to glucose. SC-islet organoids form normally from DEC1-ablated human pluripotent stem cells, but their insulin release capacity and glucose threshold fail to increase during in vitro culture and upon transplant. This deficit reflects downregulation of maturity-linked effectors of glucose utilization and insulin exocytosis, blunting glycolytic and oxidative metabolism, and is rescued by increasing metabolic flux. Moreover, DEC1 is needed to boost SC-islet maturity by synchronizing circadian glucose-responsive insulin secretion rhythms and clock machinery. Thus, DEC1 links circadian rhythms to human {beta}-cell maturation, highlighting the essential role of circadian control in generating fully functional SC-islet organoids.

developmental biology↗

bioRxiv2025

Loss of Kmt2c/d promotes gastric cancer initiation and confers vulnerability to mTORC1 inhibition and anti-PD1 immunotherapy

KMT2C and KMT2D (KMT2C/D) are frequently mutated in gastric adenocarcinoma, yet their function in cancer initiation remains poorly understood. In this study, based on the observation that loss-of-function mutations of KMT2C and KMT2D are enriched and co-occur in gastric adenocarcinoma, we developed genetically engineered mouse models to selectively knock out Kmt2c and Kmt2d in gastric epithelial cells with Tmprss2-CreERT2. Through histological staining and single-cell RNA sequencing, we observed that Kmt2c/d loss led to nuclear dysplasia and expansion of cells with mixed gastric lineage markers. When combined with Pten deletion, Kmt2c/d loss drove rapid development of muscle-invasive gastric adenocarcinoma as early as 3 weeks post Cre-mediated gene deletion. The adenocarcinoma exhibited decreased expression of gastric lineage markers and increased expression of intestinal differentiation markers, phenocopying human gastric adenocarcinoma. Kmt2c/d knockout reduced protein synthesis but upregulated transcription of ribosomal proteins, rendering sensitivity to mTORC1 inhibitors. Additionally, Kmt2c/d knockout increased MHC-I molecule expression and enhanced antigen presentation. Combination of mTROC1 inhibition and anti-PD1 immunotherapy significantly suppressed tumor growth in immune-competent mice. Together, these findings reveal the role of Kmt2c/d loss in gastric cancer initiation and suggest the potential therapeutic strategies for KMT2C/D-deficient gastric cancer.

cancer biology↗

bioRxiv2025

IL-25-induced memory ILC2s mediate long-term small intestinal adaptation

The adaptation of intestinal helminths to vertebrates evolved strategies to attenuate host tissue damage to support reproductive needs of parasites necessary to disseminate offspring to the environment. Helminths initiate the IL-25-mediated tuft cell-ILC2 circuit that enhances barrier protection of the host although viable parasites can target and limit the pathway. We used IL-25 to create small intestinal adaptation marked by anatomic, cell compositional and immunologic changes that persisted months after induction. Small intestinal adaptation was associated with heightened resistance to barrier pathogens, including in the lung, and sustained by transcriptionally and epigenetically modified, tissue-resident, memory-effector ILC2s distinct from those described by innate training; epithelial stem cells remained unaltered. Despite requiring IL-25 for induction, memory ILC2s maintained an activated state in the absence of multiple alarmins and supported mucosal resilience while avoiding adverse sensitization to chronic inflammation, revealing a pathway for deploying innate immune cells to coordinate a distributed mucosal defense.

immunology↗

bioRxiv2025

Hydrogen-Induced Calcium Influx via the TRPC4-TRPC4AP Axis

BackgroundCalcium ions (Ca{superscript 2}) serve as universal intracellular messengers regulating diverse physiological processes, while dysregulated Ca{superscript 2} homeostasis triggers cytotoxicity. Molecular hydrogen (H2) exhibits protective effects against oxidative stress-related pathologies, but its mechanism of action remains incompletely understood. Transient receptor potential canonical 4 (TRPC4) channels and their associated protein TRPC4AP are critical mediators of Ca{superscript 2} influx ( [Ca{superscript 2}]i), yet their role in H2-mediated calcium signaling is unexplored. This study investigates the molecular mechanism by which H2 modulates Ca{superscript 2} dynamics through the TRPC4-TRPC4AP axis, aiming to establish its therapeutic potential for calcium-related disorders. MethodsThe study employed heterogeneous cellular models (e.g., mesenchymal stem cells, neurons, fibroblasts) and in vivo two-photon calcium imaging in C57BL/6J mice. Techniques included CRISPR-Cas9 knockout, siRNA-mediated gene silencing, molecular docking (AlphaFold 3), and protein-protein interaction analysis. Calcium flux was quantified via fluorescence imaging, while mitochondrial integrity and cytoskeletal dynamics were assessed using JC-1 staining, ATPase activity assays, and live-cell imaging. Structural validation of TRPC4-TRPC4AP binding sites utilized mutagenesis and complementation experiments. ResultsH2 selectively enhanced extracellular Ca{superscript 2} influx via TRPC4-TRPC4AP, with no cytotoxicity or mitochondrial dysfunction observed. Key arginine residues (730Arg-731Arg) in the TRPC4 CIRB domain formed hydrogen-bond networks essential for channel activation. In vivo, H2 increased neuronal Ca{superscript 2} transient frequency and amplitude in the primary motor cortex. TRPC4AP knockout abolished H2-induced Ca{superscript 2} influx, while mutagenesis of 730Arg/731Arg disrupted channel activity. H2 also promoted cytoskeletal remodeling and cell motility, dependent on TRPC4AP-mediated Ca{superscript 2} signaling. ConclusionsThis study identifies H2 as a novel calcium agonist that activates the TRPC4-TRPC4AP axis to regulate extracellular Ca{superscript 2} influx. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic basis for H2-based therapies targeting calcium dysregulation in neurodegenerative, inflammatory, and metabolic diseases, while highlighting TRPC4AP as a pivotal molecular switch for gasotransmitter signaling.

biochemistry↗

bioRxiv2025

High-resolution NMR spectroscopy of proteins in intact mitochondria

Understanding protein structure and function within mitochondria is essential for unraveling the molecular mechanisms underlying cellular energy production, stress response, and disease. Here, we present a novel approach for NMR observation of proteins within intact mitochondria by delivering proteins directly into isolated mitochondria via electroporation. Using this method, we investigate the interaction of -synuclein (-syn) with the mitochondria membrane and examine how post-translational modifications regulate this interaction. Additionally, we assessed the stability of GB1 and the dimerization of its variant within mitochondria, achieving quantitative insights into mitochondrial environmental impact on protein function. This approach offers a valuable framework for exploring mitochondrial related biomolecular events at atomic-resolution within intact mitochondria, paving the way for a more comprehensive understanding of the molecular events governing mitochondrial health and dysfunction.

biochemistry↗