This study employed a human data-driven, metabolism-centered approach to identify endogenous compounds with rejuvenating potential for aged HSCs. Genome-scale metabolic models and single-cell transcriptomic analyses were used to characterize metabolic changes in human and mouse HSCs. Aged mice were treated with cholesterol sulfate (CS), and HSC functionality was assessed via proliferation assays, DNA damage analysis, and bone marrow transplantation. Molecular docking and in vivo experiments further explored the role of the nuclear receptor RORA. Supplementation with CS in aged mice enhanced HSC proliferation, reduced DNA damage, and restored hematopoietic output. Metabolic modeling revealed increased CS hydrolysis as a key alteration in aged HSCs, while single-cell transcriptomics showed that CS treatment suppressed inflammatory signaling and eliminated stress-responsive HSC subpopulations. Mechanistically, CS was identified as a potent ligand that activates RORA. Functional studies suggested that RORA functions as a key mediator in this process. CS supplementation reverses aging-associated HSC decline by enhancing proliferation, reducing DNA damage, and modulating inflammatory pathways, with RORA serving as an essential mediator. These findings highlight the CS-RORA axis as a promising therapeutic target for rejuvenating aged HSCs and preserving hematopoietic function.

Inflammatory bowel disease (IBD), known for its complexity and frequent relapses, urgently demands novel therapeutics due to the limited efficacy of current treatments. Cinnamaldehyde (CMA), a bioactive compound derived from Cinnamomum cassia Presl, has exhibited therapeutic potential for IBD. However, the therapeutic mechanism of CMA remains incompletely elucidated, and clinical translation is hampered by its poor oral pharmacokinetics. Using RAW 264.7 cells stimulated with either LPS or IL-4, we evaluated the effects of CMA on macrophage polarization. Subsequently, the impact of CMA on glucose metabolism in M1 macrophages was analyzed. RNA sequencing identified the signaling pathways through which CMA inhibits M1 macrophage polarization, and this was further validated through genetic or pharmacological blockade. To overcome the pharmacokinetic challenges of CMA, macrophage membrane-biomimetic CMA-loaded nanoparticles (MM@CMANP) were designed, and their pharmacokinetics and targeting to intestinal inflammation sites were evaluated. Finally, the efficacy of MM@CMANP was assessed in DSS-induced IBD mice. CMA suppresses M1 macrophage polarization in vitro. Notably, CMA disrupted M1 macrophage glucose metabolic reprogramming, characterized by glycolysis suppression and enhanced oxidative phosphorylation. RNA sequencing demonstrated a clear association with mitophagy pathway following CMA treatment, and mechanistic studies verified that CMA promotes BCL2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)-mediated mitophagy activation. Crucially, CMA-induced inhibition of M1 macrophages was mitigated by BNIP3 knockdown or autophagy inhibitors. MM@CMANP enhanced CMA accumulation in inflamed colonic tissues. In IBD mice, MM@CMANP significantly alleviated epithelial barrier disruption and mucosal inflammation. Consistent with in vitro findings, CMA modulated macrophage polarization and autophagy in vivo. These results establish mitophagy as a central mechanism underlying anti-IBD effects of CMA and position MM@CMANP as a clinically translatable nanotherapeutic platform for IBD.

Background: Sonogenetics enables non-invasive control of cellular activity using ultrasound (US)-responsive proteins. Prestin, a motor protein from outer hair cells, can render cells responsive to US when ectopically expressed, offering potential therapeutic applications for neurodegenerative diseases and epilepsy. However, current prestin variants exhibit suboptimal oligomerization, reducing their efficacy in neuromodulation. This study aimed to enhance prestin sonogenetic performance by improving oligomerization.Methods: We screened four echolocating bat prestin orthologs and point mutants of mouse prestin variants for enhanced US responsiveness. Subcellular distribution and oligomerization of prestin variants were evaluated using live-cell imaging and Förster resonance energy transfer. Calcium imaging was used to assess functional responses in HEK293T cells and cultured neurons. In vivo functionality was tested in mice by AAV-mediated expression of prestin variants in hippocampal neurons and motor cortex, followed by transcranial US stimulation and c-Fos staining to assess neuronal activation. To evaluate cross-species efficacy, we expressed prestin variants in ray sensory neurons of Caenorhabditis elegans and quantified US-evoked behavioral responses. Tissue safety was confirmed via histological analysis in mice.Results: Echolocating bat prestins did not improve US responsiveness compared to mouse prestin. In contrast, an engineered variant, Prestin(N548S, V715G), exhibited enhanced oligomerization and punctate membrane localization. This variant triggered stronger Ca²⁺ influx in vitro. In vivo, US stimulation of the cortex expressing Prestin(N548S, V715G) induced robust neuronal activation, as indicated by increased c-Fos expression and evoked tail movement in mice. In Caenorhabditis elegans, expression of Prestin(N548S, V715G) in ray neurons conferred significant US-evoked behavioral responses. No adverse histological effects were observed in mouse tissue.Conclusion: Our findings show that enhancing prestin oligomerization, rather than relying on naturally evolved echolocating variants, significantly improves its sonogenetic efficacy. The Prestin(N548S, V715G) variant represents a potent and biosafe tool for non-invasive neuromodulation and holds promise for future therapeutic applications in neurological disorders.

Background: Biglycan (BGN), a component of the extracellular matrix, has been closely associated with tumor progression. This study aimed to investigate the endocrine and paracrine roles of BGN in papillary thyroid carcinoma (PTC) to elucidate the underlying molecular mechanisms driving PTC development. Methods: Multi-omics integration was used to assess BGN expression and its clinical relevance in PTC. Functional assays, including cell viability, colony formation, and cell cycle analysis, were used to evaluate the biological functions of BGN. RNA-seq identified key signaling pathways involved in BGN-mediated PTC progression. Immune cell infiltration and macrophage polarization were analyzed using single-cell RNA-seq and flow cytometry. CRISPR interference was employed to suppress BGN enhancer activity, while luciferase assays confirmed NR2F2's role in regulating BGN expression. The therapeutic potential of the NR2F2-specific inhibitor CIA1 was tested in PTC cell lines and mouse models. Results: BGN overexpression in malignant PTC cells evaluated by BayesPrism deconvolution was linked to poor clinicopathological features in PTC. BGN knockdown inhibited cell proliferation and induced cell cycle arrest, which was rescued by recombinant BGN. Tumor-derived BGN activated the AKT signaling pathway, promoting tumor growth. Additionally, high BGN expression facilitated M2 macrophage polarization and immune evasion through TLR4 signaling. The NR2F2-BGN axis activated BGN transcription and AKT signaling. CIA1 treatment reduced BGN expression, suppressed cell proliferation, and modulated macrophage polarization. Conclusion: Our findings highlight the NR2F2-BGN axis as a critical regulator of PTC progression. Targeting this axis offers a promising therapeutic strategy for PTC treatment and immune microenvironment modulation.

The repair of large cranial defects remains a major clinical challenge, as conventional materials primarily act as inert fillers and fail to meet the complex biological requirements of cranial bone regeneration. In particular, they lack the ability to temporally coordinate angiogenesis and osteogenesis. This study aimed to develop a temporally functional composite scaffold to dynamically modulate the regenerative microenvironment and promote sequential vascularized bone regeneration. Methods: A silk fibroin-based hydrogel system was designed, incorporating salvianolic acid B (SalB)-loaded sustained-release hydrogel and mineralized silk fibroin hydrogel microspheres (MSFM). Material characterization was performed to evaluate the structural and mechanical properties of the scaffold, as well as the drug release behavior. In vitro assays were conducted to assess endothelial cell migration, tube formation, and the expression of angiogenesis-related genes, along with the osteogenic differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs). In vivo reparative efficacy was further validated using a rat cranial defect model through morphological and histological analyses. Results: Characterization confirmed that OSFM microgels were uniformly spherical with a porous internal structure and exhibited sustained release of OGP. In vitro, OSFM showed excellent cytocompatibility with BMSCs, significantly enhancing cell proliferation, ALP activity, and mineralized nodule formation compared with SFM (p < 0.05). Tube formation and scratch assays demonstrated that OSFM-conditioned medium promoted HUVEC migration and angiogenesis. In vivo, implantation of OSFM+PCL scaffolds into rat calvarial defects resulted in markedly superior bone regeneration compared with control, PCL, and SFM+PCL groups. The bone volume fraction in the OSFM+PCL group reached 52.31 ± 4.27% at the 8th weeks, significantly higher than 23.65 ± 3.81%, 30.42 ± 3.96%, and 37.86 ± 4.12% in the other groups (p < 0.05). Histological staining confirmed more mature bone formation, abundant collagen deposition, and tight integration between new bone and scaffold. Immunohistochemistry revealed upregulated expression of RUNX2, OCN, and CD31, indicating enhanced osteogenesis and angiogenesis. Conclusions: This temporally functional composite scaffold achieved a sequential "angiogenesis first, osteogenesis later" strategy by leveraging the differential degradation kinetics of its components. The findings demonstrate a biomimetic and temporally regulated approach with strong bioactivity and translational potential for cranial bone regeneration.

Aim: Despite major advances in recanalization therapy, poor functional outcomes after ischemic stroke remain common. The central challenge is not only restoring blood flow but also overcoming the bioenergetic failure that can persist during reperfusion. This study aims to identify the missing link by defining how a metabolic-epigenetic cascade drives microvascular energetic collapse, thereby elucidating mechanisms underlying sustained cerebral edema and the no-reflow phenomenon following ischemia-reperfusion.Methods: We analyzed serum samples from patients with acute ischemic stroke to evaluate associations among lactate/pyruvate (L/P) ratios, functional outcomes, and cerebral edema. High-resolution magnetic resonance imaging (MRI) and FITC-dextran extravasation test in transient middle cerebral artery occlusion (tMCAO) model were used to determine whether glycolytic inhibition reduced edema. Single-cell RNA sequencing characterized endothelial cell subpopulations after stroke, and molecular experiments examined the effects of lactate accumulation on histone H3K18 lactylation (H3K18la) and downstream ATF4-DDIT4 signaling. Mitochondrial function, electron transport chain (ETC) activity, and necroptosis-related pathways were assessed in endothelial cells.Results: An elevated L/P ratio was strongly correlated with poor neurological outcomes and was closely linked to ischemia-reperfusion-induced cerebral edema. Reducing glycolytic flux and lactate production attenuated edema formation. Single-cell sequencing identified a post-stroke hyper-glycolytic endothelial subset characterized by mitochondrial dysfunction and necroptosis activation, with greater expansion in aged tMCAO mice. Lactate accumulation increased H3K18la in endothelial cells and activated the ATF4-DDIT4 pathway, which further impaired mitochondrial and ETC function. These changes established a self-amplifying pathological loop —glycolysis/H3K18la/ATF4-DDIT4 — that intensified bioenergetic failure while promoting RIPK3-dependent necroptosis and inflammation.Conclusions: Aberrant lactate metabolism not only serves as a prognostic biomarker but also provides a mechanistic link between metabolic insufficiency and epigenetic dysregulation through histone lactylation. Targeting the feedback loop involving H3K18la and ATF4-DDIT4 offers a promising therapeutic approach to limit cerebral edema and improve functional outcomes after ischemic stroke.

Rationale: Phospholipase A2 receptor (PLA2R) is the predominant autoantigen in primary membranous nephropathy (PMN), accounting for approximately 70% of clinical cases. However, the mechanisms by which PLA2R initiates and sustains autoimmunity in PMN remain unclear. PLA2R belongs to the mannose receptor (MR) family, members of which have been shown to undergo endocytosis and lysosomal degradation for MHCII-mediated antigen presentation. This study investigates whether antibody binding promotes PLA2R internalization and lysosomal processing to enhance MHCII-mediated antigen presentation and CD4⁺ T cell activation, thereby contributing to the perpetuation of autoimmunity in PMN. Methods: Multiple PLA2R-overexpressing cell lines were generated by lentiviral-mediated overexpression of PLA2R. Imaging and western blot were employed to assess the effects of anti-PLA2R antibodies, derived from PMN patients or produced in-house, on PLA2R internalization and degradation. To define the specific endocytic pathway involved, we used pharmacological inhibitors of endocytosis as well as PLA2R constructs lacking the endocytic domain. Finally, T cell activation was evaluated using OT-II CD4⁺ T cells co-cultured with PLA2R-ovalbumin (OVA)-expressing mouse dendritic cells treated with anti-PLA2R antibodies. Results: Binding of anti-PLA2R antibodies triggers clathrin-mediated endocytosis and lysosomal trafficking of PLA2R. Antibody-induced PLA2R degradation was effectively prevented by specific endocytosis inhibitors or by deletion of the PLA2R endocytic domain. Furthermore, PLA2R-OVA-expressing mouse dendritic cells exposed to PLA2R antibodies enhanced the activation of OVA-specific CD4⁺ T cells both in vitro and in vivo. Conclusions: This study demonstrates that anti-PLA2R antibody induces internalization and lysosomal degradation of PLA2R, a process that may enhance MHC class II-mediated antigen presentation and promote the expansion of antigen-specific CD4⁺ T cells. This mechanism could establish a self-reinforcing feedback loop that perpetuates autoimmune responses in PMN.

Tumor local ablation could facilitate the release of tumor antigens, thereby activating systemic antitumor immunity. Nevertheless, clinical observations have indicated that the systemic response induced by conventional local ablation methods is rather transient, weak, and insufficient to induce protective immunity. Multimodal tumor thermal therapy (MTT), a novel local ablation technology that involves liquid nitrogen freezing followed by radiofrequency heating, has been suggested to stimulate robust and sustained antitumor immunity. However, in patients with hepatocellular carcinoma (HCC), how MTT promotes the patients' antitumor immunity remains unknown. In this study, we enrolled four patients to receive MTT and three patients to receive radiofrequency ablation (RFA), aiming to explore the mechanism by which MTT promotes antitumor immunity. We found that MTT significantly prolonged the patients' PFS compared with RFA and identified a novel T cell subset characterized by CX3CR1 and GPR56 that specifically correlated with the efficacy of MTT. MTT elicited a significant increase in CX3CR1+GPR56+ T cells and a concomitant decrease in regulatory T cells in PBMCs compared with the samples obtained from patients following RFA. CX3CR1+GPR56+ T cells express high levels of cytotoxic molecules and share TCR sequences with tumor-reactive-like T cells in tumors, and the degree of increase in the proportion of these cells is positively correlated with the PFS of patients. Compared to RFA, MTT significantly induced release of damage-associated molecular patterns (including extracellular DNA and heat shock protein 70), more effectively promoted dendritic cell maturation, and strengthened their interaction with CX3CR1+GPR56+ effector T cells via MHC I-TCR. Meanwhile, MTT diminished the interactions between DCs and Tregs through LGALS9-CD45 axis, leading to a reduction in peripheral Tregs. These findings reveal the mechanism by which MTT promotes antitumor immunity in patients with HCC and warrant further investigation in large-scale clinical studies.

Background: Splicing factors play pivotal roles in mRNA processing and are implicated in tumor progression. The aberrant expression of splicing factors is closely associated with the invasiveness and secretion profiles of pituitary neuroendocrine tumors (PitNETs). In this study, we explored the involvement of splicing factors in PIT1-lineage PitNET progression and assessed the feasibility of targeting the splicing process as a therapeutic approach.Methods: Statistical data on PitNET subtypes were obtained from the National Brain Tumor Registry of China (NBTRC), and gene expression analysis was conducted on 40 clinical samples collected for this study. Transcriptome analysis and RNA immunoprecipitation sequencing (RIP-seq) were utilized to examine FUS-mediated alternative splicing and to identify mRNA binding sites in PitNET cells. Minigene splicing assays were employed to confirm the specific exonic and intronic regions. Additionally, Annexin V/PI assays and JC-1 staining were conducted to evaluate apoptosis.Results: The expression of the splicing factor FUS was elevated in PIT1-lineage PitNETs and was correlated with increased proliferative capacity and reduced apoptosis levels. Transcriptome sequencing revealed that the knockdown of FUS led to extensive exon skipping and activated the p53 pathway. In addition to RIP-seq analysis, these findings suggest that FUS contributes to the inclusion of exon 3 to generate full-length MDM2, a well-established negative regulator of p53. Antisense oligonucleotides (ASOs) specifically designed to target binding sequences on pre-mRNAs effectively disrupted the FUS-mediated splicing process, consequently impeding the progression of PitNETs.Conclusions: Our study elucidated the critical function of FUS as a splicing factor in PitNETs. Furthermore, we illustrated that targeting the splicing mechanism associated with MDM2 could restore p53 levels, thereby impeding the progression of PitNETs. This discovery presents a potentially novel strategy for the clinical management of PIT1-lineage PitNETs.

Biomimetic nanovaccines have recently emerged as a frontier in vaccine development. These nanovaccines are designed to structurally and morphologically mimic natural pathogens, including bacteria, viruses, and certain eukaryotic cells. Engineered to replicate pathogenic surface characteristics, these nanoplatforms improve targeted delivery to antigen-presenting cells (APCs) and prolong systemic circulation, which in turn enhances antigen presentation and promotes stronger adaptive immune activation. The preparation process for biomimetic nanovaccines is also highlighted, involving isolating and purifying source cell membrane, encapsulation of synthetic nanoparticle core, and verifying. Additionally, this study offers a thorough evaluation of various biomimetic nanovaccines, particularly those with cell membrane coatings derived from tumor cells, immune cells, or bacteria. Furthermore, we also assess their capabilities when combined with common treatment modalities, including immune checkpoint inhibitors, chemotherapy, and photothermal therapy, to achieve anti-tumor effects. The study also discusses the path to clinical translation and existing challenges in manufacturing, safety, and regulatory approval.