技术资料

Many biological tissues,such as cardiac muscle,tendons,and the cornea,exhibit highly organized microstructural alignment that is critical for mechanical and physiological functions. Disruptions in this structural organization are commonly associated with pathological conditions such as fibrosis,infarction,and cancer. However,conventional histological imaging techniques rely on immunofluorescence or histochemical staining,and they evaluate tissue alignment via non-physical 2D gradient-based calculation,which is labor-intensive,antibody-dependent,and prone to variability. Here,we demonstrate label-free mid-infrared dichroism-sensitive photoacoustic microscopy (MIR-DS-PAM),an analytical imaging system for cardiac tissue assessments. By combining molecular specificity with polarization sensitivity,this method selectively visualizes protein-rich engineered heart tissue (EHT) and quantifies the extracellular matrix (ECM) alignment without any labeling. The extracted dichroism-sensitive parameters,such as the degree of dichroism and the orientation angle,enable histostructural evaluation of tissue integrity and reveal diagnostic cues in fibrotic EHT. This technique offers a label-free analytical tool for fibrosis research and tissue engineering applications. Mid-infrared dichroism-sensitive photoacoustic microscopy enables label-free,quantitative histostructural analysis by combining spectral specificity and polarization sensitivity to visualize protein-rich components and evaluate anisotropic tissue alignment. View Publication

BACKGROUND: The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). AIM: To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells (MSCs). METHODS: Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1,iMSC3),an iMatrix-free method (iMSC2),growth factor supplementation (iMSC4),and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria,requiring expression of surface markers (CD105,CD73,CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration. RESULTS: All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001,respectively),while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01,respectively). Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001),reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours). CONCLUSION: While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications. View Publication

Background: DDB1 and CUL4‐associated factor 7 (DCAF7) is a WD‐repeat adaptor that recruits substrates to the CUL4DDB1 ubiquitinligase complex,but its pan‐cancer relevance and mechanistic contribution to tumor progression remain unclear. Methods: Multi‐omics datasets (genomic,transcriptomic,epigenomic,proteomic and single‐cell) from 33 tumor types were integrated to define DCAF7 expression,regulation,and clinical significance. Somatic alterations and copy‐number variation were analysed across cohorts,and promoter methylation and RNA modification signatures were interrogated. Immune associations were assessed by computational deconvolution and checkpoint‐gene profiling. Pathway and network analyses were performed to infer DCAF7‐linked programmes. Mechanistic and functional validation was conducted in hepatocellular carcinoma (LIHC) cell lines (HepG2,Huh7) using DCAF7 perturbation and pharmacologic Wnt inhibition. Results: DCAF7 was overexpressed in most cancers,consistent with copy‐number gain,focal promoter hypomethylation and putative m6A‐linked post‐transcriptional regulation,whereas hypermethylation at two CpG loci predicted poor prognosis in LIHC. DCAF7 alterations,predominantly amplifications,were associated with shorter overall survival in LIHC and positively correlated with DCAF7 mRNA abundance across cohorts. Immunogenomic analyses linked high DCAF7 to CD4+ T‐cell enrichment,broad upregulation of checkpoint genes (PD‐1/PD‐L1,CTLA‐4,TIGIT),and increased tumour mutational burden,microsatellite instability and neoantigen load,suggesting an immune‐evasive phenotype. Network and enrichment analyses converged on canonical Wnt/β‐catenin,Hippo and cell‐cycle programs. In vitro,DCAF7 promoted LIHC cell proliferation and migration by stabilising β‐catenin via increased inhibitory Ser9 phosphorylation of GSK‐3β,thereby inducing c‐Myc and cyclin D1; DCAF7 knockdown or the Wnt inhibitor XAV939 attenuated these effects. Drug‐response modelling further predicted increased sensitivity of DCAF7‐high tumours to 17‐AAG,docetaxel and alsterpaullone. Conclusions: DCAF7 is frequently activated by genetic and epigenetic mechanisms across cancers,associates with an immunotherapy‐relevant tumour immune milieu,and drives Wnt/β‐catenindependent malignant phenotypes in LIHC. These findings support DCAF7 as a prognostic biomarker and a candidate therapeutic target,particularly for stratified intervention in LIHC. Key points: DCAF7 is up‐regulated in various tumours and correlates with poor prognosis,particularly in LIHC. High DCAF7 expression is linked to CD4+ T cell infiltration,up‐regulation of immune checkpoint genes and increased tumour mutational burden,suggesting a role in tumour immune escape. DCAF7 stabilises β‐catenin by enhancing GSK‐3β Ser9 phosphorylation,thereby driving c‐Myc/cyclin D1 expression and contributing to proliferation and migration in LIHC. DCAF7‐high tumours demonstrate therapeutic vulnerability to 17‐AAG,docetaxel and CDK/GSK‐3 inhibitor,revealing potential targeted treatment strategies. View Publication

Palmitic acid (PA) is the most common dietary saturated fatty acid,and is abundant in palm and cottonseed oil,butter,and cheese,whereas oleic acid (OA) is a monounsaturated omega-9 fatty acid found in olive oil. The differences in the cytotoxic and pro-inflammatory effects of PA and OA across endothelial cells (ECs) isolated from different vascular beds have not been investigated in detail. Here,we incubated primary human aortic valve (HAVEC),saphenous vein (HSaVEC),internal thoracic artery (HITAEC),and microvascular (HMVEC) ECs with albumin-bound PA or OA for 24 h and found that PA induced a considerable cytotoxic response,accompanied by an elevated expression of the genes encoding cell adhesion molecules (VCAM1,ICAM1,SELE,and SELP) and pro-inflammatory cytokines (MIF,PTX3,CSF2,CSF3,IL1A,IL6,CCL2,CCL5,CCL20,CSF2,CSF3,CXCL1,CXCL2,CXCL3,CXCL5,CXCL6,CXCL8,and CXCL10),followed by an increased release of interleukin-6 and interleukin-8. HAVEC and HSaVEC were more susceptible to PA,whereas OA had mild-to-moderate cytotoxic effects on HAVEC and HMVEC but did not induce generalized EC activation. Compared with other EC types,HITAEC was the most resistant to PA and OA treatment. Collectively,these results indicate considerable heterogeneity across the ECs of distinct origin in response to PA. View Publication

CD8+ T cells are an important weapon in the therapeutic armamentarium against cancer. While CD8+CD103+ T cells with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognoses,the tumor microenvironment also contains dysfunctional exhausted T (TEX) cells that exhibit a variety of TRM-like features. Here we deconvolute TRM and TEX cells across human cancers,ascribing markers and gene signatures that distinguish these populations and enable their functional distinction. Although TRM cells have superior functionality and are associated with long-term survival post-tumor resection,they are not associated with responsiveness to immune checkpoint blockade. Tumor-associated TEX and TRM cells are clonally distinct,with the latter comprising tumor-independent bystanders and tumor-specific cells segregated from cognate antigen. Intratumoral TRM cells can be forced toward an exhausted fate when chronic antigen stimulation occurs,indicating that the presence or absence of continuous antigen exposure within the microenvironment is the key distinction between tumor-associated TEX and TRM populations. These results highlight unique functions for TRM and TEX cells in tumor control,underscoring the need for distinct strategies to harness these populations for cancer therapies. Here the authors show that tissue-resident memory and exhausted T cells in tumors are distinct populations that are shaped by relative presence or absence of TCR signals,suggesting that a tailored therapeutic strategy is needed to target each subset. View Publication

Propofol is a commonly used anesthetic for sedation during surgery. This drug is reported to exhibit nonanaesthetic immunomodulatory and anti-inflammatory effects. Herein,we investigated the impact of topical propofol delivery with the aim of mitigating psoriatic inflammation. The antipsoriatic potency of propofol was evaluated in a cell-based study in which keratinocytes,macrophages,and neutrophils were used as models. A significant reduction in the proinflammatory effectors interleukin (IL)-6,IL-8,and CXC motif chemokine ligand (CXCL)1 was found in activated keratinocytes (HaCaT) treated with propofol. This reduction could enable baseline control. Immunoblotting suggested that the antioxidant enzymes nuclear factor erythroid 2-related factor (Nrf)2 and heme oxygenase (HO)-1 were involved in the protective effect of propofol on keratinocyte stimulation. The increase in Nrf2 and HO-1 was mediated by kelch-like ECH-associated protein (KEAP)1 downregulation. Propofol presented scavenging activity and decreased 2,2-diphenyl-1-picrylhydrazyl (DPPH) by 47%. The downregulation of cytokines/chemokines in activated macrophages (differentiated THP-1) and mouse neutrophils was also found after propofol treatment. Macrophage migration triggered by the conditioned medium of activated keratinocytes could be blocked with the intervention of propofol. The absorption level of propofol (3 mM) into intact pig skin was 1.2 nmol/mg. Skin deposition was increased to 3.7 nmol/mg after SC lipid removal to mimic psoriasiform skin. In silico molecular docking demonstrated the facile interaction of propofol with ceramides in the stratum corneum (SC). The treatment of imiquimod (IMQ)-sensitized mice with topical propofol suppressed erythema,acanthosis,and macrophage/neutrophil infiltration. Propofol also dramatically decreased cytokine/chemokine levels and epidermal thickness in the lesion. In summary,propofol exhibits anti-inflammatory and antioxidant properties to treat psoriasiform lesions. Topical propofol delivery is useful as an ideal route to accomplish antipsoriatic therapy and avoid systemic effects. View Publication

Staphylococcal enterotoxins (SE) crosslink the MHC‐II on antigen‐presenting cells (APC) with the T‐cell receptor,inducing a polyclonal T‐cell response. Although APCs are the initial targets of SE and are critical in shaping subsequent T‐cell activation,the effects of SE on APC function remain poorly understood. This study investigates the immunomodulatory effects of staphylococcal enterotoxin A (SEA) on monocytes and their differentiation into monocyte‐derived dendritic cells (moDC) or macrophages (MDM). Transcriptomic analyses of human monocytes via RNA sequencing revealed SEA‐induced enrichment of gene pathways associated with inflammation,infection,and dermatitis,effects that were amplified in the presence of T cells. Phenotypic and functional characterization showed that SEA‐primed monocytes differentiated into MDM with an altered polarization,deviating from classical M1/M2 pathways. SEA‐primed MDM exhibited downregulation of key markers,including HLA‐DR,CD80,CD86,and PD‐L1. Functional assays demonstrated that SEA‐primed MDM pushed hyperinflammatory T‐cell responses,with significantly enhanced proliferation and IFN‐γ secretion. In contrast,following SEA‐priming,moDC retained robust antigen‐presenting capabilities and displayed enhanced expression of molecules involved in T‐cell interactions. These findings provide mechanistic insights into SEA‐mediated immune modulation,illustrating how SEA reprograms MDM functions and amplifies proinflammatory T‐cell responses. This advances our understanding of superantigen‐driven immune interactions,offering a foundation for developing therapeutic strategies to mitigate superantigen‐mediated immune conditions. Staphylococcal enterotoxin A (SEA) alters monocyte differentiation and function,while preserving T cell stimulatory capacity. SEA‐primed macrophages downregulate antigen‐presenting markers yet drive heightened T‐cell proliferation and IFN‐γ secretion. These findings reveal mechanisms of SEA‐mediated immune modulation and superantigen‐driven inflammation. View Publication

Graft‐versus‐host disease is mediated by donor‐derived T cells reactive against the recipient's broadly expressed minor histocompatibility antigens (mHA). Regulatory T cells (Treg) have been explored as a therapeutic approach for chronic GVHD (cGVHD). The promising results from polyclonal Treg trials in this setting have led us to develop a Treg product specific for mismatched minor antigens between patient and donor (mTreg),circumventing broad immune suppression risks. HLA‐matched siblings of opposite sexes were used to obtain the sister's CD4+CD25hiCD127low Treg for co‐culture with the respective brother's dendritic cells as a source of mismatched mHA. We have established the optimal culture conditions resulting in the highest mTreg proliferation and viability. Comprehensive phenotyping during the ex vivo selection shows PD‐1,CTLA‐4,CD39 and HLA‐DR expression. Transcriptomic analysis revealed a switch in metabolic process,and up‐regulation of functional Treg genes. Furthermore,mTreg possess specific and potent suppressive activity,in which there is a dependency on cell‐to‐cell contact and a role for HLA class II expression on mTreg. This protocol would allow the generation of Treg specific to an array of mHA from the recipient's healthy tissues,likely providing a directed and strong suppression of cGVHD. We optimised a protocol for mHA‐specific Treg (mTreg) selection in an HLA‐matched context while defining its phenotype,transcriptional state and function. mTreg were highly activated and exerted specific,HLA class II‐,contact‐dependent suppression. This protocol can be explored as a highly personalised antigen‐specific Treg‐based therapy in future clinical trials for cGVHD. View Publication

Background and Objectives: Serum myelin oligodendrocyte glycoprotein (MOG) antibodies are a hallmark of the newly defined neuroinflammatory disease entity MOG antibody–associated disease (MOGAD). Yet,the lack of patient-derived recombinant human MOG (hMOG)–reactive autoantibodies limits investigations into the molecular mechanisms by which these autoantibodies mediate CNS pathology,thereby hindering rational therapeutic approaches. To understand the origins and disease-relevant mechanisms of autoantibodies in MOGAD,we generated and characterized monoclonal anti-hMOG antibodies (MOG-mAbs) from circulating B cells of patients with MOGAD.Methods: We isolated MOG-specific B-cell receptor (BCR) sequences from unique circulating B-cell clones of 6 patients with MOGAD using an antigen selection approach. BCR sequences were expressed as immunoglobulin (Ig)G1 antibodies,and their molecular features,epitope specificity,and binding to MOG isoforms were investigated. The MOG-mAbs’ ability to mediate antibody-dependent cellular phagocytosis (ADCP),natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC),and complement-dependent cytotoxicity (CDC) toward MOG-expressing cells was assessed by live cell-based assays.Results: Of the 15 MOG-mAbs generated,4 revealed evidence of affinity maturation,whereas the remaining 11 were germline encoded. Binding capacities to hMOG varied considerably,with the most frequent putative epitope mapping to a region that includes residue P42. The efficacy of these antibodies in mediating ADCP,ADCC,and CDC of MOG-expressing cells was heterogeneous and associated with their binding characteristics to MOG and its isoforms.Discussion: Taken together,the molecular characteristics and binding patterns of these patient-derived MOG-mAbs reveal a diverse repertoire of MOG-binding autoantibodies with pathogenic capacity in vitro. Consequently,these well-characterized patient autoantibodies offer a foundation for developing in vivo models of MOGAD,serve as tools to standardize diagnostic assays,and guide development of therapeutic strategies targeting either B cells or autoantibodies and their effector functions. View Publication

SF3B1,a component of the U2 snRNP pre-mRNA splicing factor,plays a critical role in splicing and is frequently mutated in cancer,particularly hematologic malignancies. We investigated the effects of the most common SF3B1 mutation,heterozygous substitution of Lysine 700 to Glutamate (K700E),in human embryonic stem cells (hESC),using CRISPR-Cas9 to generate heterozygous SF3B1K700E clones. Interestingly,we observed the upregulation of several key transcription regulators associated with hematopoiesis and a broad range of immune genes in SF3B1K700E hESCs. Despite differences in the transcriptional and splicing profiles between hESC and myelodysplastic syndrome (MDS) cells harboring the SF3B1K700E mutation,several common immune gene programs were identified in both cell types. To elucidate the molecular mechanisms underlying dysregulated gene expression in SF3B1K700E hESCs,we mapped actively engaged RNA polymerase II (RNA Pol II) using Precision Run-On sequencing (PRO-seq). These analyses revealed that the SF3B1K700E mutation alters RNA Pol II elongation properties. Specifically,we observed a general increase in pause release in SF3B1K700E hESCs,consistent with recent work in leukemia cells suggesting that the SF3B1K700E mutation affects early transcription elongation. Taken together,our study identifies several candidate genes that could contribute to the SF3B1 mutated phenotype and clarifies the role for the U2 snRNP and pre-spliceosome assembly on transcription by RNA Pol II. Further,our data suggest that mutations of SF3B1 impact immune gene expression independent of cell type,providing new insights into the role of SF3B1K700E in hematologic malignancies. View Publication

Lens transparency relies on proper intercellular communication. Exosomes are crucial mediators of intercellular communication and play a key role in organ homeostasis and development. However,their presence and dynamics in the lens remain unclear. Here,we report endogenous exosomes in the zebrafish lens using cryaa-driven Cd63-AcGFP labeling. Live imaging revealed dynamic exosome movement within lens cells and their potential transfer to adjacent tissues. Additionally,we found that the biogenesis of Cd63+ exosomes in the lens is regulated by the Syntenin-a pathway. And Syntenin-a knockdown delayed lens development by impairing lens cell differentiation,highlighting the potential role of lens cell–derived exosomes. Furthermore,ROR1+ lens progenitor cell-derived extracellular vesicles promoted lentoid differentiation in vitro,with proteomic analysis suggesting underlying mechanisms. Overall,our study addresses the gap in direct observation of endogenous lens exosomes,providing foundational insights into lens pathophysiology and a potential strategy for modulating the lens microenvironment. Visualization of endogenous exosomes in the zebrafish lens reveals their dynamic roles in intercellular communication and development,with Syntenin-a–regulated exosome biogenesis influencing lens cell differentiation. View Publication

MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening,we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover,we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program,as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets,reduces ribosome biogenesis,and impairs protein synthesis. Mechanistically,CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci,facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore,combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo,leading to more pronounced tumor growth suppression. Overall,our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation,suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB. MYC-driven medulloblastoma is an aggressive pediatric tumor with limited treatment options. Here,the authors show that CDK8 regulates ribosome biogenesis and that combined inhibition of CDK8 and mTOR demonstrates therapeutic efficacy in mouse models of this cancer. View Publication