Biology subjects

Liu, K.

Publications and source records attributed to Liu, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Sequential infection reprograms the immune landscape to shape future responses

Mouse models have been instrumental in defining immune mechanisms but often fail to capture the complexity of human immunity, limiting clinical translation. A major limitation is the immunological immaturity of specific pathogen-free (SPF) mice relative to pathogen-experienced adult humans. Here, we use a sequential infection (SI) model that recapitulates cumulative pathogen exposure and define its impact on immune composition and function. Beyond the previously reported expansion of memory T cells, SI induced durable, system-wide remodeling across lymphoid and non-lymphoid tissues, reshaping innate and adaptive immune populations, tissue-resident immunity, and hematopoietic output. Single-cell transcriptomic analyses revealed inflammatory imprinting of naive CD4 and CD8 T cells, whereas memory T cells acquired enhanced effector programs coupled with reduced biosynthetic activity, transcriptional states that more closely resemble those of pathogen-experienced adult humans. Functionally, SI mice recapitulated the human response to anti-CD28 super-agonist and exhibited altered magnitude and differentiation of acute and chronic antiviral T cell responses, demonstrating that cumulative pathogen exposure reshapes both existing immunity and the generation of future immune responses. Thus, cumulative pathogen exposure coordinately remodels hematopoiesis and naive and memory lymphocyte states, establishing a durable inflammation-experienced immune landscape that reshapes both immune memory and future immune responses, with broad implications for the translational fidelity of preclinical mouse models.

immunology↗

bioRxiv2026

B cell-intrinsic IRF8 transcriptionally reprograms antigen presentation to sustain CD8⁺ T cell antitumor immunity

Interferon regulatory factor 8 (IRF8) is a master transcription factor of myeloid differentiation, but whether IRF8 intrinsically controls B cell function in tumors remains unknown. Using paired single-cell transcriptomic and chromatin accessibility profiling of tumors from wild-type and Irf8-deficient mice, we identify a B cell-intrinsic IRF8 axis regulating antigen presentation and sustaining anti-tumor CD8 T cell immunity. IRF8 establishes conserved chromatin accessibility programs across myeloid cells and plasmablasts centered on antigen processing and MHC class I presentation, but engages distinct motifs by lineage: myeloid cells preferentially utilize ISRE and ETS-composite elements, whereas plasmablasts are selectively enriched for EICE elements, reflecting B lineage-specific IRF8-IRF4 cooperation. Loss of IRF8 disrupts these programs, skews B cells toward plasmablast differentiation and reduces antigen presentation machinery. B cell depletion accelerated tumor growth, while CD40 agonism activated B cells, expanded T cells, and enhanced anti-tumor immunity. B cell-specific IRF8 deletion alone accelerated tumor growth, establishing a cell-intrinsic requirement independent of myeloid IRF8 function. The IRF8-regulated B cell signature was enriched in PD-1 blockade cancer patient responders, and plasmablast abundance correlated with response in pembrolizumab-treated cancer patients. These findings establish IRF8 as a lineage-adapted regulator of antigen presentation and define the IRF8-B cell axis as a determinant of anti-tumor immunity. HighlightsIRF8 establishes a conserved chromatin accessibility across tumor-infiltrating myeloid and B cells Myeloid cells engage ISRE motifs, whereas plasmablasts rely on EICE motifs as IRF8 lineage-specific cis-regulation IRF8 regulates an antigen presentation in B cells to sustain anti-tumor T cell immunity B cell-intrinsic IRF8 transcription signature predicts patient response to PD-1 blockade immunotherapy

immunology↗

bioRxiv2026

A feedback control for restraining autoimmune γδ T cells: reprogramming into ILC1s

{gamma}{delta} T cells are promising mediators of cancer immunotherapy, yet their potential to drive autoimmunity remains incompletely understood. Here, we identify a feedback mechanism in which innate-like V{gamma}1.1V{delta}6.3 T cells are reprogrammed into ILC1-like cells, thereby restraining autoimmune pathology. We define a previously unrecognized ILC1 subset whose development depends on an intact Tcrd locus. These cells predominantly harbor productive V{gamma}1.1 and V{delta}6 rearrangements, consistent with their origin from V{gamma}1.1V{delta}6.3 T cells. Mechanistically, TCR signaling induces Id3, which suppresses E protein-dependent activation of T cell-specific genes, including that encoding V{delta}6.3. Id3 ablation drives robust expansion of V{gamma}1.1V{delta}6.3 T cells and severe autoimmunity, characterized by tissue infiltration, autoantibody production, enhanced T follicular helper cell differentiation, and accumulation of germinal center and age-associated B cells. Together with previously described exocrine dysfunction, these features resemble human Sjogrens disease. Consistent with this, we observed in the salivary glands of Sjogrens disease patients an increased frequency of CD4-CD8- T cells enriched for {gamma}{delta} T cells, including subsets functionally analogous to murine V{gamma}1.1V{delta}6.3 cells. Collectively, these findings uncover a TCR-Id3-dependent reprogramming pathway that limit the pathogenic potential of harmful {gamma}{delta} T cells.

immunology↗

bioRxiv2026

Alignment-free phylogenetic inference via hyperbolic protein language models

Conventional phylogenetic methods rely on multiple sequence alignments which are computationally intensive and often fail for highly divergent lineages. Here, we introduce LucaPhylo, an alignment-free framework that infers evolutionary relationships directly from unaligned sequences. Through a cascaded learning strategy LucaPhylo integrates protein language models with hyperbolic geometry, a representation space naturally suited to hierarchical branching, to capture deep evolutionary constraints without explicit homology matching. Using highly divergent RNA virosphere as a test case, LucaPhylo places unaligned sequences into phylogenetic trees with an accuracy comparable to leading alignment-based tree construction tools, while retaining divergent sequences that conventional pipelines frequently discard. It further enables the integration of divergent viral lineages into phylogenetic trees, thereby expanding the evolutionary landscape of RNA viruses. Together, LucaPhylo establishes an AI-driven, alignment-free paradigm for phylogenetic inference and provides a robust computational foundation for resolving deep evolutionary relationships among RNA viruses and other biological systems.

evolutionary biology↗

bioRxiv2026

CHOP promotes the transition to chronic integrated stress response signaling with suppression of hepatocyte identity

The transcription factor CHOP promotes cell death during ER stress, but it is strongly induced even by moderate stresses that do not result in appreciable cell death. Its role during less severe stresses--especially in intact tissues in vivo--is poorly understood. Here, we both deleted and restored CHOP specifically in hepatocytes and challenged animals with ER stress in vivo. We found that CHOP influenced stress-dependent hepatocyte gene expression through two previously unappreciated mechanisms. It directly suppressed the expression of transcriptional master regulators of hepatocyte identity and metabolism. And more broadly, it exacerbated ER stress through the promotion of protein synthesis, which led to persistent activation of the integrated stress response (ISR) despite dephosphorylation of eIF2. This shift to second-phase ISR signaling was phenocopied by deletion of the protective UPR sensor ATF6, suggesting that it reflects a transition from an acute stress response to a chronic one. Our findings show that CHOP augments the capacity of the ISR and UPR to continue to mount a protective response even after eIF2 phosphorylation has been suppressed. In vivo, where ISR signaling intersects with hepatocyte gene regulatory networks, this transition favors lipid dysregulation, highlighting a pathway through which CHOP impacts tissue function independent of cell death.

cell biology↗

bioRxiv2026

Engineering a pacemaker-driven human mini-heart guided by spatial multi-omics of sinoatrial node development

The human sinoatrial node (SAN) functions as the primary pacemaker of the heart and coordinates the hierarchical electrical activity that drives cardiac contraction. However, experimental systems capable of reconstructing pacemaker-driven cardiac organization in human tissues remain limited. Here we integrate spatial multi-omics of the human fetal SAN with stem-cell engineering to generate pacemaker organoids ("Sinoids") and assemble them into a pacemaker-driven human mini-heart composed of sinoatrial, atrial and ventricular cardiac modules. High-resolution spatial transcriptomics and single-nucleus multi-omic analyses of human fetal SAN tissues identify regulatory pathways guiding pacemaker lineage specification, which we leverage to engineer human pluripotent stem cell-derived SAN organoids with robust pacemaker identity and electrophysiological activity. When integrated with atrial and ventricular cardioids, Sinoids initiate and coordinate electrical activation across assembled cardiac tissues, establishing directional propagation of electrophysiological signals within structured mini-heart organoids. Combining AI-guided perturbation modeling with functional validation further identifies conserved regulatory pathways controlling pacemaker specification and regionalization, including YAP-TEAD and NRG-ERBB signaling. Together, these results establish a multi-omic-guided strategy for engineering pacemaker tissues and reconstructing cardiac conduction hierarchy in vitro. The pacemaker-driven mini-heart platform provides a modular human cardiac system for studying pacemaker biology, modeling arrhythmia mechanisms and enabling electrophysiological drug discovery.

bioinformatics↗

bioRxiv2026

Somatic-to-germline transmission of horizontally acquired extrachromosomal circular DNA in Brassica graft chimeras

Whether somatically acquired traits can contribute to heritable variation has remained an open question in plant biology for over a century. Here, using graft-induced periclinal chimeras between Brassica juncea and Brassica oleracea, we identified extrachromosomal circular DNA (eccDNA) as mobile genetic elements capable of crossing histological boundaries and entering the germline. We demonstrated that grafting could promote the horizontal transfer of eccDNA between somatic cell layers, enabling its stable maintenance in recipient tissues and permitting transmission through sexual reproduction across multiple generations. We found that transmitted eccDNA is non-randomly distributed across the genome, preferentially originating from gene-dense regions, and enriched for features associated with molecular persistence, such as inverted repeats, hairpin-forming sequences, and autonomously replicating sequence (ARS) consensus motifs. These genetic regions conferred replication competence, as evidenced by autonomous propagation in an ARS-less heterologous yeast system. Sexual progeny carrying graft-acquired eccDNA exhibited reproducible and lineage-dependent alterations in leaf morphology and drought tolerance, accompanied by coordinated transcriptional reprogramming. Notably, a subset of inherited eccDNA remained transcriptionally active in progeny, producing transcripts absent from self-grafted controls. Our findings establish eccDNA as heritable extrachromosomal elements that link graft-mediated somatic genetic transfer with stable germline transmission, thereby expanding the molecular scope of heritable variation in plants and providing a conceptual framework for graft-based trait transmission.

plant biology↗

bioRxiv2026

ProteinFlux: accurate, rapid and scalable generative prediction of protein dynamics driven by post-translational modifications

The function of proteins, the building blocks of life, in health and disease depends not only on their 3D-conformational states but most importantly on the dynamic transition between states controlled by a wide array of post-translational modifications (PTMs). Recent major advances have been made in our ability to predict static 3D structures; however, understanding and predicting the impact of PTMs on protein conformational dynamics remains a major question and challenge in the field. Molecular dynamics (MD) simulation remains the major computational approach for studying protein dynamics. However, the high computational cost, lack of integration of PTMs as conditioning inputs and inefficient generation of continuous protein dynamics largely precludes PTM-regulated conformational dynamics and the study of slow conformational processes. To address this critical bottleneck, we developed ProteinFlux, a flow-matching generative framework that links PTM-conditioned conformational dynamics to evolutionary constraints encoded by PTM sites. Evolutionary information plays a critical role in capturing conformational dynamics beyond sequence identity, and PTM sites inherently encode evolutionary constraints critical to protein functional regulation. We therefore built FluxSite, a dual-modal PTM site predictor that integrates sequence evolutionary information and 3D structural features to generate a continuous conditional signal encoding conservation and functional importance for each predicted site. FluxSite achieves robust generalization across 18 PTM types and 30 disease-associated proteomes. ProteinFlux generates phosphorylation-conditioned, all-atom conformational trajectories across diverse protein fold classes, faithfully reproducing both thermodynamic properties such as free energy landscapes and kinetic features such as conformational transition pathways. It outperforms state-of-the-art predictors while achieving inference speeds several orders of magnitude faster than traditional MD. In addition, we introduce DynaMo-phos, a benchmark dataset of phosphorylated protein MD simulations. Together, ProteinFlux, FluxSite and DynaMo-phos provide a scalable, high-throughput platform for elucidating PTM-driven conformational mechanisms, with potential applications across allosteric drug design, functional annotation of disease-associated modifications and mechanism-guided therapeutic development.

bioinformatics↗

bioRxiv2026

Striatal FOXP2 is essential for stable vocal production in non-human primates

The transcription factor FOXP2 is the most well-known language-related gene in humans, yet its role in primate vocalization remains poorly understood. Here we report that knockdown of FOXP2 in the striatum markedly disrupts vocalization stability in the marmoset monkey, a valuable non-human primate model for studying vocal behavior. FOXP2 exhibited high expression in the marmoset striatum, especially during early development. Using the CRISPR-Cas12 system, we achieved specific in vivo editing of the FOXP2 gene and effective knockdown of FOXP2 protein expression in the marmoset striatum. Two neonatal marmosets received bilateral striatal injections of the gene-editing and control virus, respectively, and were raised together in the same family. In three such marmoset pairs, analysis of vocalizations recorded during 6-15 weeks post-injection revealed that striatal FOXP2 knockdown significantly altered vocal features and increased intra-individual variability in phee syllables--the most common marmoset vocalization, often produced repetitively as multi-syllable phee calls. Notably, in FOXP2-edited marmosets, acoustic alterations were minimal in the first syllable of phee calls but became progressively more pronounced in subsequent syllables, which exhibited a marked upward shift in the frequency spectrum over time with progressively steeper slopes. These temporal dynamics in vocal features reflect a reduction in the stability of continuous vocal production. In line with the known striatal functions in motor control, our findings provide the first evidence of FOXP2 in controlling vocalization in non-human primates, thereby opening new avenues for investigating the neural mechanisms underlying FOXP2 function.

neuroscience↗

bioRxiv2026

Coordinated phase separation and phase transition underlie synaptic ribbon condensate formation and plasticity

Mesoscale molecular apparatuses with diverse functions are widespread in cells. How mesoscale cellular apparatuses are formed and regulated is poorly understood. The synaptic ribbon in sensory neurons, which can dynamically adjust its size and shape in response to light or sound, is a mesoscale molecular assembly. In this study, we demonstrate that RIBEYE, the backbone protein of synaptic ribbons, via combined ordered and disordered interactions, forms micron-sized dynamic molecular assemblies. Integrated cryo-electron microscopy and tomography studies revealed that the N-terminal SAM domain and the C-terminal B domain of RIBEYE form distinct nanoscale filaments and further assemble into ribbon-like architectures via phase transition. Unexpectedly, the N-terminal intrinsically disordered region of RIBEYE softens the solid mesoscale ribbon structures via phase separation. The mesoscale size and shape of the RIBEYE condensate are bidirectionally tuned by physiological modulators such as Piccolino and CtBP1, linking synaptic ribbon structural plasticity to functional adaptability. Thus, our study demonstrates that combined phase transition and phase separation of nanoscale protein oligomers can build mesoscale functional cellular apparatuses.

biochemistry↗

bioRxiv2026

The tongue-brain axis mediates a hidden amino acid appetite

Selecting a diet containing all essential amino acids (EAAs) is critical for health. Following EAA deprivation, animals can select a nutritiously complete food source; however, the underlying mechanisms in vertebrates remain unclear. In mice, we show that leucine deficiency activates hypothalamic agouti-related protein (AgRP) neurons, which project to the paraventricular thalamus (PVT) via {gamma}-aminobutyric acid and are required for EAA deficiency-induced leucine appetite in mice. Furthermore, the peripheral tongue amino acid sensor general control nonderepressive-2 (GCN2) mediates acute EAA appetite via AgRP neurons. Together, these findings identify a tongue-AgRP-PVT circuit underlying EAA appetite, which is important for the rapid and accurate selection of essential nutrients.

neuroscience↗

bioRxiv2026

Programming Biomolecular Interactions with All-Atom Generative Model

Biomolecular interactions lie at the core of cellular life, spanning diverse molecular modalities from small molecules to nucleic acids and proteins. Nevertheless, design strategies remain separated despite shared physicochemical principles of molecular recognition. Here we present AnewOmni, a unified generative framework trained on more than 5 million biomolecular complexes, that enables transferable molecular design across molecular scales by assembling chemically meaningful building blocks at atomic resolution. We further introduce programmable graph prompts to support user-defined chemical, topological, and geometric steering during generation, exploring hybrid and unconventional chemistries beyond canonical structures. We demonstrate that transferable learning of interaction patterns and physical constraints across molecular modalities is possible, via an atom-to-block latent space capturing both atomic details and structural priors. The framework successfully designed small molecules, peptides, and nanobodies targeting the challenging KRAS G12D switch II pocket, as well as orthosteric peptides and allosteric small-molecule inhibitors for PCSK9 in the absence of known binding site, achieving 23%-75% success with only low-throughput validation, bypassing modality-specific high-throughput screening. AnewOmni is the first to succeed in functional molecular design across all scales, from small chemical entities to large biologics, and represents a stepstone towards general molecular reasoning engines, advocating a generative foundation model for biomolecular interactions to enter regimes where data and human intuition remain limited.

bioengineering↗

bioRxiv2026

Red yeast rice-derived MKA ameliorates cardiac hypertrophy in hypertensive rats by inhibiting ERK1/2/c-Fos pathway

BackgroundCardiac hypertrophy is a key pathological process in hypertensive heart failure, yet current antihypertensive therapies do not directly target it. Red yeast rice (RYR), rich in monacolin K {beta}-hydroxy acid (MKA), is known for lipid-lowering effects, but its potential to ameliorate cardiac hypertrophy is unreported. PurposeTo investigate the effects of RYR-derived MKA on cardiac hypertrophy in spontaneously hypertensive rats (SHR) and elucidate its molecular mechanisms. MethodsSpontaneously hypertensive rats (SHR) were treated with 0.6% red yeast rice for 8 weeks to assess its effects on blood pressure, cardiac function (echocardiography), cardiac hypertrophy and fibrosis (histopathology), and multi-organ toxicity (histopathology). A multigenerational study was conducted to evaluate protective effects in offspring. Network pharmacology and transcriptomic analysis were integrated to predict molecular targets, which were subsequently validated by molecular docking and experiments. ResultsEight-week RYR treatment significantly reduced blood pressure, inhibited cardiac hypertrophy and fibrosis, and improved cardiac function without gender differences. No pulmonary, hepatic, or renal toxicity was observed. Offspring from treated parents exhibited further reduced hypertrophy upon continued treatment. Mechanistically, MKA bound ERK1/2 with high affinity, inhibiting its phosphorylation and downstream c-Fos expression, thereby downregulating hypertrophy markers. ConclusionRed yeast rice improves hypertensive cardiac hypertrophy via MKA-mediated inhibition of the ERK1/2/c-Fos pathway. Its multi-organ safety and transgenerational effects offer a novel dual-therapy strategy for hypertension and cardiac hypertrophy. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=139 HEIGHT=200 SRC="FIGDIR/small/710945v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@8225acorg.highwire.dtl.DTLVardef@775f43org.highwire.dtl.DTLVardef@10f5201org.highwire.dtl.DTLVardef@d0cd88_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Learning the All-Atom Equilibrium Distribution of Biomolecular Interactions at Scale

Biomolecular functions are governed by dynamic conformational ensembles rather than static structures. While models like AlphaFold have revolutionized static structure prediction, accurately capturing the equilibrium distribution of all-atom biomolecular interactions remains a significant challenge due to the high computational cost of molecular dynamics (MD). We present AnewSampling, a transferable generative foundation framework designed for the high-fidelity sampling of all-atom equilibrium distributions, which is the first model to faithfully reproduce MD at the all-atom level. It uses a quotient-space generative framework to ensure mathematical consistency and leverages the largest self-curated database of protein-ligand trajectories to date, with over 15 million conformations. Statistically, AnewSampling consistently outperforms all prior generative methods on the ATLAS monomer benchmark, and the all-atom capabilities of AnewSampling enable close statistical alignment with ground-truth MD for evaluating atomic biomolecular interactions in protein-ligand dynamics. Furthermore, AnewSampling successfully recovers coupled ligand and side-chain motions in CDK2 systems, overcoming a major sampling hurdle inherent to conventional MD. AnewSampling enables rapid exploration of conformational landscapes prior to intensive simulations, elucidating fundamental biophysical mechanisms and accelerating the broader design of functional biomolecules.

bioinformatics↗

bioRxiv2026

Therapeutic scheduling of WEE1 inhibition preserves T cell function and promotes immune control of HPV⁺ tumors

Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV OPC) is driven by viral E6 and E7 oncoproteins, which disrupt G1 checkpoint control and impose selective dependency on WEE1-mediated G2/M regulation. While this vulnerability confers sensitivity to WEE1 inhibition, its immunologic consequences remain poorly defined, and the challenge of eliciting antitumor immunity without compromising immune fitness has limited clinical translation. Here, we show that WEE1 inhibition elicits durable antitumor immunity in immunocompetent models of HPV OPC. Using murine and human preclinical systems, we demonstrate that the WEE1 inhibitor azenosertib (ZN-c3) mediates tumor control through both cell-autonomous cytotoxicity and immune-dependent mechanisms requiring T cells and conventional dendritic cells. Mechanistically, HPV tumor cells are deficient in STING signaling and fail to mount canonical type I interferon responses. Instead, tumor cell-intrinsic cGAS drives immune activation through STING-competent host cells within the tumor microenvironment, revealing a non-cell-autonomous relay that circumvents viral immune evasion. Intermittent WEE1 inhibition preserves T cell fitness while maintaining antitumor efficacy, and mice achieving complete responses develop immunologic memory capable of rejecting tumor rechallenge. These findings establish intermittent WEE1 inhibition as an immune-permissive therapeutic strategy that enables antigen-specific T cell responses in HPV-driven malignancies and provides a mechanistic rationale for combination with immunotherapy.

cancer biology↗

bioRxiv2026

Synthetic Immunological Niche Reveals Early Immune Dysregulationand Stratifies Therapeutic Response in Type 1 Diabetes

In Type 1 Diabetes (T1D), disease onset and response to immunotherapy vary widely among individuals, reflecting heterogeneous stage-specific immune dysregulation that remains undefined. To investigate this heterogeneity, we engineered a microporous polycaprolactone scaffold that forms a synthetic immunological niche (IN) upon subcutaneous implantation, enabling in vivo capture of systemic immune dysregulation. In non-obese diabetic (NOD) mice, longitudinal transcriptomic profiling of IN-infiltrating cells identified early-stage genes relatively enriched for myeloid cells, followed by progressive increases in T cell-associated dysregulation at later stages that distinguished T1D progressors from non-progressors. We derived an early-stage IN-based T1D gene signature capturing immune alterations. The signature stratified NOD progressors from non-progressors as early as 6 weeks of age and was conserved across human T1D datasets, distinguishing T1D from non-diabetic individuals in spleen and pancreatic lymph node samples, but not peripheral blood. Signature-based stratification further revealed enrichment of macrophage-associated TNF- pathways in NOD progressors, validated in human T1D islets. Given heterogeneous response to anti-TNF- therapy, IN profiling identified resistance-associated mechanisms and enabled derivation of a pathway score that prospectively distinguished treatment-sensitive from resistant mice prior to therapy, establishing the IN as a minimally invasive platform for detecting stage-wise immune dysregulation and stratifying immunotherapy response in T1D.

bioengineering↗

bioRxiv2026

ATG5 regulates autophagy-apoptosis-ER stress dysregulation in steroid-induced osteonecrosis of the femoral head (SONFH) pathogenesis

The aim is to investigate how ATG5 regulates autophagy, endoplasmic reticulum stress (ERS), and apoptosis in steroid-induced osteonecrosis of the femoral head (SONFH), and to evaluate ATG5-targeted inhibition as a SONFH intervention. Differentially expressed genes in SONFH were screened using GEO dataset GSE74089. Autophagy/apoptosis/ERS pathway activities were analyzed via GSEA, GO/KEGG enrichment, and GSVA. Rat bone marrow mesenchymal stem cells (BMSCs) were isolated for osteoblast modeling. Groups included: control (A), steroid-treated (B, methylprednisolone), and intervention (C, steroid + ATG5-siRNA). Autophagosome formation, apoptosis rate, and ATG5/PERK/LC3 expression were assessed by electron microscopy, flow cytometry, qPCR, and western blotting. Sixty SD rats were divided into three groups (as above). The SONFH model was established via intramuscular methylprednisolone injection, with intervention group receiving ATG5-siRNA. Bone pathology, cell death, and pathway regulation were evaluated using HE/TUNEL staining, electron microscopy, and molecular detection. Transcriptome analysis revealed synergistic activation of autophagy (ATG5/BECN1), apoptosis (CASP3/9/12), and ERS (PERK) pathways in SONFH, with ATG5 strongly correlating with all three. Steroids upregulated ATG5 to overactivate autophagy, triggering PERK-mediated ERS and ERS-specific apoptosis. ATG5-siRNA intervention inhibited autophagosome formation, reduced apoptosis, downregulated PERK, and alleviated trabecular fractures and empty lacunae. ATG5 deficiency blocked PERK signaling, suppressing both autophagic death and ERS-dependent apoptosis.ATG5 drives autophagy overactivation and ERS-apoptosis cascades via PERK pathway activation, constituting a core SONFH mechanism. Targeted ATG5 silencing effectively blocks this pathology, offering novel preventive/therapeutic strategies.

molecular biology↗

bioRxiv2026

Optical Windows for Transcranial Brain Imaging in Living Mice: Skull Thinning, Clearing, and Beyond

Longitudinal, noninvasive in vivo imaging is essential for studying brain physiology and pathological mechanisms. Advances in transcranial optical windows, including thinned-skull and optical clearing techniques, have markedly improved imaging depth and resolution when combined with multiphoton microscopy. However, their optical performance often deteriorates rapidly, and quantitative studies on long-term stability and the causes of image quality loss remain limited. In this work, we systematically investigated current transcranial window approaches using multiphoton excited fluorescence microscopy (MPEFM) and adaptive optics, examining longevity, optical aberrations, and imaging resolution. Our results reveal that progressive skull regrowth is a fundamental limitation across all window types, leading to substantial declines in signal quality and resolution for conventional MPEFM thereby reducing achievable high-resolution imaging depth. To address these challenges, we developed a localized glucocorticoid (GC) delivery strategy that significantly extends window performance for up to one month. Furthermore, we demonstrated that a GC-loaded hydrogel sealing method effectively suppressed skull regrowth while preserving optimal optical properties, offering a potential and practical route to chronic, high-fidelity transcranial imaging. These findings provide mechanistic insight into window degradation and establish a framework for sustained, long-term in vivo brain imaging.

neuroscience↗