技术资料

Embryonic stem cell (ESC)-derived cardiomyocytes are a key resource for studying cardiac development and advancing regenerative therapies. Beclin1 (Becn1),a core regulator of autophagy and cardiac morphogenesis,has an undefined role in cardiomyocyte lineage specification. This study aims to investigate the regulatory function of Becn1 during cardiac differentiation from both mouse and human ESCs. Methods: Mouse and human ESCs were differentiated into cardiomyocytes through established embryoid body (EB) formation or monolayer differentiation protocols. Stable Becn1 knockdown was achieved using short hairpin RNA (shRNA). Cardiomyocyte differentiation efficiency was evaluated by flow cytometry,immunocytochemistry,and contraction assays. Differentiation cardiomyocyte function was evaluated by sarcomere arrangement,calcium transients,and microelectrode array (MEA). Transcriptomic profiling was conducted by bulk RNA sequencing,and pathway dynamics were analyzed using qPCR and western blotting. Results: Becn1 expression declined over the course of differentiation. Knockdown of Becn1 significantly enhanced cardiomyocyte yield and promoted earlier onset of contractile activity,accompanied by increased expression of cardiac-specific markers. Mechanistically,Becn1 deficiency elicited a biphasic Wnt signaling response,characterized by early activation during mesodermal induction followed by suppression at later stages of differentiation. This shift was accompanied by sustained BMP pathway activation. Notably,Becn1-deficient ESCs underwent efficient cardiac differentiation in the absence of exogenous VEGF or FGF,with BMP signaling compensating for their omission. These findings were recapitulated in human ESCs,where BECN1 knockdown supported Wnt modulators-independent cardiomyocyte differentiation through coordinated modulation of Wnt and BMP pathways. Conclusions: Becn1 serves as a negative regulator of cardiac lineage commitment by orchestrating stage-specific Wnt/BMP signaling dynamics. Silencing Becn1 enhances cardiomyocyte differentiation and enables growth factor-independent lineage progression. These findings offer a novel approach to improve the efficiency and scalability of stem cell-derived cardiomyocyte production for regenerative applications. View Publication

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population,causing infectious mononucleosis,susceptibility to autoimmune diseases and multiple malignancies of epithelial or B cell-origin. EBV infects epithelial cells and B cells through interaction between viral glycoproteins and different host receptors,but it has remained unknown whether a common receptor mediates infection of its two major host cell targets. Here,we establish R9AP as a crucial EBV receptor for entry into epithelial and B cells. R9AP silencing or knockout,R9AP-derived peptide and R9AP monoclonal antibody each significantly inhibit,whereas R9AP overexpression promotes,EBV uptake into both cell types. R9AP binds directly to the EBV glycoprotein gH/gL complex to initiate gH/gL-gB-mediated membrane fusion. Notably,the interaction of R9AP with gH/gL is inhibited by the highly competitive gH/gL-neutralizing antibody AMMO1,which blocks EBV epithelial and B cell entry. Moreover,R9AP mediates viral and cellular membrane fusion in cooperation with EBV gp42-human leukocyte antigen class II or gH/gL-EPHA2 complexes in B cells or epithelial cells,respectively. We propose R9AP as the crucial common receptor of B cells and epithelial cells and a potential prophylactic and vaccine target for EBV. View Publication

Cancer stem cells (CSC) drive therapeutic resistance and recurrence in head and neck squamous cell carcinoma (HNSCC). We and others have shown that treatment with cytotoxic chemotherapy agents (e.g. Cisplatin,Carboplatin) induce Bmi-1 expression and increase the fraction of highly tumorigenic CSC in HNSCC. Notably,Bmi-1 is a master regulator of stem cell self-renewal and DNA repair. The purpose of this work was to test whether therapeutic inhibition of Bmi-1 sensitizes HNSCC cancer stem cells to chemotherapy. HNSCC cells (UM-SCC-1,-22A,-22B) were treated with Cisplatin or Carboplatin and subjected to stemness analyses to evaluate the impact of Bmi-1 on chemoresistance. We observed that both,shRNA-mediated Bmi-1 silencing or pharmacologic inhibition of Bmi-1 with the small molecule inhibitor PTC596,blocked chemotherapy-induced cancer stemness (i.e. increase in the fraction of ALDHhighCD44high cells),CSC self-renewal (i.e. orosphere formation) and inhibited protective DNA damage responses in HNSCC. Further,it is known that high IL-6 serum levels correlate with poor HNSCC patient survival,and that platinum-based therapies induce IL-6 signaling. Here,we observed that Bmi-1 silencing (or PTC596 treatment) inhibited the IL-6R/STAT3 signaling pathway even in presence of platinum-based cytotoxic agents (i.e. Cisplatin,Carboplatin). In vivo,Bmi-1 inhibition with PTC596 suppressed Cisplatin-mediated increase in the fraction of ALDHhighCD44high cells (cancer stemness). Collectively,these preclinical results demonstrate that Bmi-1 is a key mediator of head and neck cancer stemness and suggest that HNSCC patients might benefit from treatment with a Bmi-1 inhibitor combined with a conventional chemotherapeutic agent. View Publication

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children,exhibiting clinical and genomic heterogeneity. Of the four major subgroups,Group 3 tumors (MYC-MB),display high levels of MYC and metastasis rates. Despite treatment with surgery,radiation,and chemotherapy,patients with Group 3 MB are more likely to develop aggressive recurrent tumors with poor survival. To examine resistance mechanisms in this study,we perform single nuclei multiome analysis of matched primary and recurrent tumors. Therapy resistant Medulloblastoma demonstrates an expanded persistent progenitor population. Additionally,distinct chromatin landscapes link to altered transcription and correspond with metabolic reprogramming. In vivo modeling of radiation resistance exhibits similar chromatin-based metabolic reprogramming focused on wild-type isocitrate dehydrogenase (IDH1) activity. IDH1 inhibition reverses resistance-mediated chromatin changes and enables radiation re-sensitization. Ultimately,these findings demonstrate the efficacy of single-cell multiome analysis in elucidating resistance mechanisms and identifying targetable pathways for MYC-driven medulloblastoma. MYC-driven medulloblastoma is highly aggressive and associated with poor survival. Here,the authors perform single nuclei multi-omic analysis of matched primary and recurrent tumours and identify potential resistance mechanisms and targetable pathways. View Publication

Mutations in the GBA gene,which reduce β-glucocerebrosidase (GCase) activity,represent the most significant genetic risk factor for Parkinson’s disease (PD). Decreased GCase activity has also been observed in sporadic PD cases,supporting a broader role for GCase in the poorly understood mechanisms underlying PD etiopathogenesis. While most studies on the relationship between GBA mutations and PD have focused on neurons,evidence suggests that PD pathology promoted by GCase deficiency involves other cell types and,in particular,interactions between neuronal and glial cells.Here,we identify microglia as primary players undergoing significant alterations at early stages of the pathological processes triggered by a GCase impairment. Using both pharmacological and genetic mouse models of GCase deficiency,we observed microglial morphological,transcriptional and metabolic changes. Interestingly,these changes were associated with a cell-specific,significant reduction of microglial ATP levels. When microglial ATP depletion was reproduced in an in vitro system of co-cultured microglial and neuronal cells,the neuroprotective properties of microglia were compromised,resulting in reduced cytoprotective and detoxifying pathways in neurons.These findings underscore the role of microglia in PD pathogenesis and point to a pathogenetic mechanism by which microglial metabolic disturbances leading to ATP depletion enhance neuronal vulnerability to injury and neurodegeneration. This mechanism could be targeted for therapeutic intervention aimed at mitigating PD risk and counteracting the development of PD pathology. View Publication

Genetic variants in the X‐chromosomal gene coding for the calcium−/calmodulin‐dependent serine protein kinase (CASK) are associated with a neurodevelopmental disorder. CASK is a member of the membrane‐associated guanylate kinase (MAGUK) family of proteins. It acts as a scaffold at presynaptic sites,as a regulator of the transport of glutamate receptors,and as a transcriptional regulator. The PDZ domain of CASK has been reported to bind to presynaptic cell adhesion molecules such as Neurexin1‐3,CNTNAP2,SynCAM and SALM1. Structural analyses of related MAGUKs indicate that the canonical SH3 and GK domains combine with the PDZ domain to form the so‐called PSG supramodule. Conserved aromatic residues (Y723 and W914) flanking the GK domain contribute to the formation of a dimeric structure of two PSG modules,which is required for high‐affinity binding to the type 2 PDZ ligand motif of,for example,Neurexin. Here we identify previously uncharacterized patient variants in the SH3 domain of CASK (I672V; P673L),which alter the intermolecular binding pocket for Y723. Both variants interfere with the binding of Neurexin‐1β,in a manner similar to the previously reported Y723C variant. Intriguingly,binding to the type 1 PDZ ligand of the cell adhesion molecule SALM1 is not altered. Using a set of highly selective patient variants,we show that the binding of SALM1 to CASK is actually not mediated by the CASK PDZ domain or the PSG supramodule,but depends on other type 1 PDZ domain‐containing proteins such as SAP97 and Veli,which associate with CASK through its L27 domains. Our data underline the relevance of an intact PSG tandem of CASK for human health. View Publication

Purpose: Noninfectious uveitis is a sight-threatening autoimmune eye disease lacking effective targeted therapies. Dipyridamole (DIP),a phosphodiesterase (PDE) inhibitor,has demonstrated anti-inflammatory properties in inflammatory diseases. However,its application in uveitis remains unexplored. Methods: We used single-cell RNA sequencing (scRNA-seq) data from experimental autoimmune uveitis (EAU) mice and uveitis patients to assess the potential association of PDE gene expression with disease development. Subsequently,EAU mice received oral DIP (300 mg/kg/day),starting at different time points (preventative,early-therapeutic,or late-therapeutic),and treatment efficacy was assessed. To explore immune components and signaling changes,we profiled cervical draining lymph nodes (CDLNs) from control,EAU,and DIP-treated mice by scRNA-seq and validated key findings with additional experiments. Mechanistically,pharmacologic interventions (an adenylyl cyclase inhibitor and the STAT3 agonist) were used in vitro. Results: Expression of several PDE genes correlated with uveitis severity in both human and mouse. Preventative DIP treatment most effectively reduced fundus inflammation in EAU and modulated the Teff/Treg ratio in the CDLNs and spleens. In vitro,DIP suppressed CD4+ T cell proliferation,and inhibited pathogenic Teff. scRNA-seq analysis revealed that DIP partially reversed EAU-induced transcriptional alterations,with notable changes in immune cell composition and pathway activity. Mechanistically,DIP downregulated STAT3 activity and PIM1 expression in Th17 cells via cAMP,suggesting the involvement of the cAMP-STAT3-PIM1 axis in modulating immune homeostasis. Conclusions: DIP ameliorated intraocular inflammation,modulated Th17/Treg balance,and reduced Th17 pathogenicity in EAU,potentially via cAMP-STAT3-PIM1 signaling. These findings highlight DIP as a promising therapeutic candidate for autoimmune uveitis. View Publication