技术资料

The scaffold protein AMBRA1,which participates in the autophagy pathway,also promotes CD4+ T cell differentiation to Tregs independent of autophagy through its interactor PP2A. Here we have investigated the role of AMBRA1 in CD8+ T cell differentiation to cytotoxic T cells (CTL). AMBRA1 depletion in CD8+ T cells was associated with impaired expression of the transcription factors RUNX3 and T-BET that drive CTL differentiation and resulted in impaired acquisition of cytotoxic potential. These effects were recapitulated by pharmacological inhibition of the AMBRA1 activator ULK1 or its interactor PP2A. Based on the ability of PP2A to activate TFEB,we hypothesized a role for TFEB in the CTL differentiation program regulated by AMBRA1. We show that TFEB modulates RUNX3 and T-BET expression and the generation of killing-competent CTLs,and that AMBRA1 depletion,or ULK1 or PP2A inhibition,suppresses TFEB activity. These data highlight a role for AMBRA1,ULK1 and PP2A in CTL generation,mediated by TFEB,which we identify as a new pioneering transcription factor in the CTL differentiation program. View Publication

IntroductionSkeletal muscle satellite cells (MuSCs or stem cells) play a crucial role in muscle development,maintenance,and regeneration,supporting both hypertrophy and regenerative myogenesis. Syndecans (SDCs) act as communication bridges within the muscle microenvironment,regulating interactions with extracellular matrix components and contributing significantly to tissue repair and inflammation. Specifically,syndecan-4 (SDC4) is involved in muscle regeneration at multiple stages.MethodsThis study delves into the emerging challenge of wooden breast (WB) myopathy and its connection with SDC4. Our hypothesis proposes that disruptions in MuSC dynamics through SDC4 contribute to the increased incidence of breast myopathies observed in growing broilers. To test our hypothesis,non-affected and affected broilers were systematically selected,and the characteristics of WB myopathy were studied both in vitro and in vivo. SDC4 overexpression in MuSCs and blocking peptides (BPs) corresponding to the SDC4 ectodomain were used for investigating the role of SDC4 in muscle development and its shedding levels.Results and discussion In vivo examination of affected muscles revealed smaller fibers and changes in metabolic pathways. In vitro studies unveiled disrupted proliferation of MuSCs in WB myopathy,accompanied by the downregulation of several muscle markers. Investigation of the potential role of SDC4 in the pathogenesis of WB myopathy revealed a decreased tendency in SDC4 gene expression and increased shedding of its ectodomain. Moreover,we showed that SDC4 overexpression is linked to reduced proliferation in MuSCs and affected myogenesis. We detected an impaired proliferation of WB-affected MuSCs,revealing critical insights into the dysfunctional state of these cells in myopathy. Additionally,by treating MuSCs with blocking peptides derived from the SDC4 ectodomain,we identified altered proliferation. Taken together,this work contributes with valuable knowledge on the molecular mechanisms underlying WB myopathy and the role of SDC4 in this chicken myopathy. View Publication

Membrane proteins,especially extracellular domains,are key therapeutic targets due to their role in cell communication and associations. Yet,their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library,we conducted a flow-cytometric screen that identified several cell surface binding events,including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9,best known for its diverse roles in cancer,is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay,identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9,identical to a CLEC2 binding domain in Podoplanin (PDPN),was found to be essential for this interaction. Like PDPN,CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin’s lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling,providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion,we identified numerous interactions with monocytes and platelets and validated one,CA9,as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation,providing new insights for CA9-based therapeutic strategies. View Publication

Induced pluripotent stem cell (iPSC)-derived neurons (iNs) have been widely used as models of neurodevelopment and neurodegenerative diseases. Coating cell culture vessels with extracellular matrixes (ECMs) gives structural support and facilitates cell communication and differentiation,ultimately enhances neuronal functions. However,the relevance of different ECMs to the natural environment and their impact on neuronal differentiation have not been fully characterized. In this study,we report the use of four commonly used extracellular matrixes,poly-D-lysine (PDL),poly-L-ornithine (PLO),Laminin and Matrigel,which we applied to compare the single-coating and double-coating conditions on iNs differentiation and maturation. Using the IncuCyte live-cell imaging system,we found that iNs cultured on single Matrigel- and Laminin-coated vessels have significantly higher density of neurite outgrowth and branch points than PLO or PDL but produce abnormal highly straight neurite outgrowth and larger cell body clumps. All the four double-coating conditions significantly reduced the clumping of neurons,in which the combination of PDL+Matrigel also enhanced neuronal purity. Double coating with PDL+Matrigel also tended to improve dendritic and axonal development and the distribution of pre and postsynaptic markers. These results demonstrate that the extracellular matrix contributes to the differentiation of cultured neurons and that double coating with PDL+Matrigel gives the best outcomes. Our study indicates that neuronal differentiation and maturation can be manipulated,to a certain extent,by adjusting the ECM recipe,and provides important technical guidance for the use of the ECM in neurological studies. View Publication

Breast cancer (BC) is most frequently recognized in women and characterized by histological and molecular heterogeneity. Among the various subtypes,triple-negative BC remains the most challenging disease owing to the lack of effective molecular targets and the high frequency of breast cancer stem cells (BCSCs),which account for both recurrence and resistance to conventional treatments. Despite the availability of hormonal therapies and targeted treatments,patients still face early and late relapses,necessitating new cytotoxic and selective treatment strategies. Our study focuses on investigating the effects of protocatechualdehyde (PCA),a potent bioactive compound derived from Artemisia princeps,on CSCs in BC cells. PCA inhibited BC growth and mammosphere formation as the concentration increased. This agent decreased the fraction of the CD44+/CD24- population,the aldehyde dehydrogenase 1A-expressing population,and the protein level of Sox2 in breast CSCs by downregulating Akt and pAkt. Moreover,PCA treatment reduced the tumor volume and weight in 4T1-challenged BALB/c mice. Collectively,our findings support the anti-tumor effect of Akt/Sox2-targeting PCA,suggesting a novel utilization of PCA in BC therapy. View Publication

FAT1 cadherin exhibits dual tumor suppressor and oncogenic roles across various cancers,but its function in breast cancer remains unclear due to conflicting reports of mutational loss and overexpression. In this study,we demonstrate that FAT1 mRNA and protein levels are reduced during mammary transformation,an effect linked to promoter methylation rather than mutational events. Subtype-specific analysis reveals that high FAT1 expression correlates with poor outcomes in basal-like/triple-negative breast cancer (TNBC),while elevated FAT1 expression in luminal A/estrogen receptor-positive breast cancers is associated with improved patient prognosis. Functional studies in TNBC models using knockdown and overexpression approaches confirm that FAT1 promotes both cell proliferation and motility. High-throughput sequencing and biochemical assessments establish strong links between FAT1 phenotypes and the activation of PI3K-AKT signaling. Additionally,FAT1 manipulation induces significant changes in matrisome-related genes,extracellular matrix components,and integrin switching. Together,these findings define an oncogenic role for FAT1 in TNBC,providing mechanistic insights into how its regulation influences AKT signaling,cell proliferation,and motility. View Publication

The scarcity of human donor corneal graft tissue worldwide available for corneal transplantation necessitates the development of alternative therapeutic strategies for treating patients with corneal blindness. Corneal stromal stem cells (CSSCs) have the potential to address this global shortage by allowing a single donor cornea to treat multiple patients. To directly deliver CSSCs to corneal defects within an engineered biomatrix,we developed a UNIversal Orthogonal Network (UNION) collagen bioink that crosslinks in situ with a bioorthogonal,covalent chemistry. This cell-gel therapy is optically transparent,stable against contraction forces exerted by CSSCs,and permissive to the efficient growth of corneal epithelial cells. Furthermore,CSSCs remain viable within the UNION collagen gel precursor solution under standard storage and transportation conditions. This approach promoted corneal transparency and re-epithelialization in a rabbit anterior lamellar keratoplasty model,indicating that the UNION collagen bioink serves effectively as an in situ-forming,suture-free therapy for delivering CSSCs to corneal wounds. View Publication

Intrauterine growth restriction (IUGR) affects 5-10% of pregnancies with placental hypoxia,playing a key role as a common pathophysiological pathway of different etiologies. Despite the high metabolic rate of the placenta and its "gatekeeper" role in protecting the fetus from hypoxia,the response of placental mitochondria to hypoxic stress is not well understood. This study tested the hypothesis that transient exposure to hypoxia leads to a loss of placental mitochondria and affects their function. Human villous trophoblastic (JEG-3) cells were cultured under normoxic and hypoxic conditions for 24 h. Mitochondrial content was determined by flow cytometry before and after hypoxic exposure and after 24 h of normoxic recovery. Parameters of oxidative phosphorylation were assessed using a respirometric analyzer before hypoxic exposure and after normoxic recovery. Mitochondrial content decreased significantly from 88.5% to 26.7% during hypoxic incubation. Although it had increased to 84.2% after 24 h of normoxic recovery,oxidative phosphorylation parameters were still significantly suppressed to 1/2 to 1/3 of the pre-incubation levels. The results underscore the ability of placental cells to adapt mitochondrial content to O2 supply. Despite rapid recovery under normoxia,respiratory function remains suppressed,which may result in persistent impairment of adenosine triphosphate (ATP)-dependent synthetic and transport functions. View Publication

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Its poor prognosis is closely related to late-stage diagnosis,which results from both nonspecific symptoms and the absence of biomarkers for early diagnosis. MicroRNAs (miRNAs) exert a regulatory role in numerous biological processes and their aberrant expression has been found in a broad spectrum of diseases,including cancer. Cancer stem cells (CSCs) represent a driving force for PDAC initiation,progression,and metastatic spread. Our previous research highlighted the interesting behavior of miR-216a-5p and miR-125a-5p related to PDAC progression and the CSC phenotype. The present study aimed to evaluate the effect of miR-216a-5p and miR-125a-5p on the acquisition or suppression of pancreatic CSC traits. BxPC-3,AsPC-1 cell lines,and their CSC-like models were transfected with miR-216a-5p and miR-125a-5p mimics and inhibitors. Following transfection,we evaluated their impact on the expression of CSC surface markers (CD44/CD24/CxCR4),ALDH1 activity,pluripotency- and EMT-related gene expression,and clonogenic potential. Our results show that miR-216a-5p enhances the expression of CD44/CD24/CxCR4 while negatively affecting the activity of ALDH1 and the expression of EMT genes. MiR-216a-5p positively influenced the clonogenic property. MiR-125a-5p promoted the expression of CD44/CD24/CxCR4 while inhibiting ALDH1 activity. It enhanced the expression of Snail,Oct-4,and Sox-2,while the clonogenic potential appeared to be affected. Comprehensively,our results provide further knowledge on the role of miRNAs in pancreatic CSCs. Moreover,they corroborate our previous findings about miR-216a-5p's potential dual role and miR-125a-5p's promotive function in PDAC. View Publication

New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry,like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals,with consequent ethical and economic issues,the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however,the characterization and application of such organoids remain limited. In this study,ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs,and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types,and distinct apical-basal polarity,while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications,with Z'-factor tests indicating robust assay performance. Dose-response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology,offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health. View Publication

Mucosal-associated invariant T (MAIT) cells are unconventional innate-like T cells abundant in human mucosal tissues and are associated with protective responses to microbial infections. MAIT cells have the capacity for rapid effector functions,including the secretion of cytokines and cytotoxic molecules. In this study,we examined the longitudinal circulating MAIT cell response to the live attenuated oral vaccine Ty21a (Ty21a) against Salmonella enterica serovar Typhi (S. Typhi). We enrolled healthy adults who received a course of oral live-attenuated S. Typhi strain Ty21a vaccine and assessed peripheral blood MAIT cell longitudinal responses pre-vaccination,and at seven days and one-month post-vaccination,using flow cytometry,cell migration,and tetramer decay assays. We showed that following vaccination,circulating MAIT cells were lower in frequency,but were more activated,and had higher levels of gut-homing marker integrin α4β7 and chemokine receptors CCR9 and CCR6,suggesting the potential of MAIT cells to migrate to mucosal sites. We found no significant differences in MAIT cell functionality,cytotoxicity and T-cell receptor avidity,except in TNF expression,which was higher post-vaccination. We show that MAIT cell immune responses are modulated post-vaccination against S. Typhi. This study contributes to our understanding of MAIT cells' potential role in oral vaccination against bacterial mucosal pathogens. View Publication

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study,we report on the properties of a novel human IgA1,constructed using a single-chain variable fragment clone (2E9),selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial ?-crystallin Ag and for the human Fc?RI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-? significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls,suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-? in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of Fc?RI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis. View Publication