技术资料

MEF2C encodes a transcription factor that is critical in nervous system development. Here,to examine disease-associated functions of MEF2C in human microglia,we profiled microglia differentiated from isogenic MEF2C-haploinsufficient and MEF2C-knockout induced pluripotent stem cell lines. Complementary transcriptomic and functional analyses revealed that loss of MEF2C led to a hyperinflammatory phenotype with broad phagocytic impairment,lipid accumulation,lysosomal dysfunction and elevated basal inflammatory cytokine secretion. Genome-wide profiling of MEF2C-bound sites coupled with the active regulatory landscape enabled inference of its transcriptional functions and potential mechanisms for MEF2C-associated cellular functions. Transcriptomic and epigenetic approaches identified substantial overlap with idiopathic autism datasets,suggesting a broader role of human microglial MEF2C dysregulation in idiopathic autism. In a mouse xenotransplantation model,loss of MEF2C led to morphological,lysosomal and lipid abnormalities in human microglia in vivo. Together,these studies reveal mechanisms by which reduced microglial MEF2C could contribute to the development of neurological diseases. Coufal and colleagues generated microglia from human iPS cells to examine mechanistic roles of the transcription factor MEF2C and how these roles might relate to the autism phenotype seen following the loss of MEF2C in human microglia. View Publication

Triple-negative breast cancer (TNBC) is an aggressive and clinically challenging subtype defined by the absence of estrogen receptor,progesterone receptor,and HER2 amplification,resulting in poor prognosis and limited therapeutic options. Targeting alternative molecular pathways is urgently needed to overcome resistance and improve patient outcomes. CD147 has emerged as surface marker associated with tumor progression and immune evasion. In this study,CD147 and MHC class I—a key inhibitory ligand for natural killer cells—were analyzed in breast cancer cell lines (MCF7,MDA-MB-453,MDA-MB-231,and HCC38) using flow cytometry. The therapeutic efficacy of a humanized anti-CD147 monoclonal antibody (HuM6-1B9) was evaluated for its capacity to potentiate antibody-dependent cellular cytotoxicity (ADCC). HuM6-1B9 demonstrated the strong binding to MDA-MB-231 (KD = 4.982 nM) and HCC38 (KD = 4.523 nM),which are representative TNBC cell lines. In 3D spheroid models,HuM6-1B9 significantly enhanced PBMC-mediated ADCC,leading to a marked reduction in TNBC spheroid viability. Co-culture of CFSE-labeled MDA-MB-231 and HCC38 cells with primary NK cells confirmed robust ADCC,achieving 50% and 70% cytotoxicity,respectively,despite high MHC class I expression. Live-cell imaging demonstrated caspase-3/7 activation consistent with apoptosis in NK-targeted tumor cells,while CD107a degranulation and IFN-γ secretion confirmed the functional contribution of HuM6-1B9 to ADCC enhancement. Importantly,HuM6-1B9 did not promote migration or invasion in MDA-MB-231 cells,supporting its safety profile regarding metastasis. Collectively,these findings establish HuM6-1B9 as a promising immunotherapeutic candidate that overcomes immune resistance and selectively eliminates TNBC cells through ADCC without enhancing metastatic potential. By integrating mechanistic assays of NK cytotoxicity,apoptosis,and 3D tumor spheroids,this study provides clinically relevant insights underscoring the translational potential of HuM6-1B9 in TNBC immunotherapy. View Publication

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study,we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single,living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique,it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research. View Publication

SARS-CoV-2,a $\beta$-coronavirus,has rapidly spread across the world,highlighting its high transmissibility,but the underlying morphogenesis and pathogenesis remain poorly understood. Here,we characterize the replication dynamics,cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison,HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion,apoptosis,destruction of epithelium integrity,cilium shrinking and beaded changes are observed in the plaque regions. Taken together,our results provide important insights into SARS-CoV-2 cell tropism,replication and morphogenesis. View Publication

Membrane potential (Vmem) is a key bioelectric property of non-excitable cells that plays important roles in regulating cell proliferation. However,the regulation of Vmem itself remains largely unexplored. We found that,under nutrient starvation,during which cell division is inhibited,MKN45 gastric cancer cells were in a hyperpolarized state associated with a high intracellular chloride concentration. AMP-activated protein kinase (AMPK) activity increased,and expression of cystic fibrosis transmembrane conductance regulator (CFTR) decreased,in nutrient-starved cells. Furthermore,the increase in intracellular chloride concentration level and Vmem hyperpolarization in nutrient-starved cells was suppressed by inhibition of AMPK activity. Intracellular chloride concentrations and hyperpolarization increased after over-activation of AMPK using the specific activator AICAR or suppression of CFTR activity using specific inhibitor GlyH-101. Under these conditions,proliferation of MKN45 cells was inhibited. These results reveal that AMPK controls the dynamic change in Vmem by regulating CFTR and influencing the intracellular chloride concentration,which in turn influences cell-cycle progression. These findings offer new insights into the mechanisms underlying cell-cycle arrest regulated by AMPK and CFTR. View Publication

Dendritic cells (DC) play an essential role in innate immunity and radiation-elicited immune responses. LGP2 is a RIG-I like receptor (RLR) involved in cytoplasmic RNA recognition and anti-viral responses. Although LGP2 has also been linked to cell survival of both tumor cells and T cells,the role of LGP2 in mediating DC function and anti-tumor immunity elicited by radiotherapy remains unclear. Here we report that tumor DC are linked to the clinical outcome of breast cancer patients who received radiotherapy (RT) and the presence of DC correlates with gene expression of LGP2 in the tumor microenvironment. In preclinical models,host LGP2 was essential for optimal anti-tumor control by ionizing radiation (IR). The absence of LGP2 in DC dampened type I interferon production and the priming capacity of DC. In the absence of LGP2,MDA5-mediated activation of type I IFN signaling was abrogated. The MDA5/LGP2 agonist high molecular weight poly I: C improved the anti-tumor effect of IR. This study reveals a previously undefined role of LGP2 in host immunity and provides a new strategy to improve the efficacy of radiotherapy. View Publication

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study,we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine],a zinc chelator,protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor,diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition,we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX),p38 mitogen-activated protein kinase (MAPK),and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX,generation of reactive oxygen species (ROS),and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX,ROS accumulation,p38 activation,and caspase-3 activation. Therefore,therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation,stroke,and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death. View Publication

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However,efficient stem cell to beta cell differentiation has proven difficult,possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals,we isolated pancreatic and gastrointestinal stromal cells,and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas,whereas stroma-specific activation of signaling via loss of Hedgehog regulators,Sufu and Spop,impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly,inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation,altogether revealing the requirement for organ-specific regulation of stromal niche signals. View Publication

Respiratory syncytial virus (RSV) is increasingly recognized for causing severe morbidity and mortality in older adults,but there are few studies on the RSV-induced immune response in this population. Information on the immunological processes at play during RSV infection in specific risk groups is essential for the rational and targeted design of novel vaccines and therapeutics. Here,we assessed the antibody and local cytokine response to RSV infection in community-dwelling older adults (≥60 years of age). During three winters,serum and nasopharyngeal swab samples were collected from study participants during acute respiratory infection and recovery. RSV IgG enzyme-linked immunosorbent assays (ELISA) and virus neutralization assays were performed on serum samples from RSV-infected individuals (n = 41) and controls (n = 563 and n = 197,respectively). Nasal RSV IgA and cytokine concentrations were determined using multiplex immunoassays in a subset of participants. An in vitro model of differentiated primary bronchial epithelial cells was used to assess RSV-induced cytokine responses over time. A statistically significant increase in serum neutralization titers and IgG concentrations was observed in RSV-infected participants compared to controls. During acute RSV infection,a statistically significant local upregulation of beta interferon (IFN-$\beta$),IFN-$\lambda$1,IFN-$\gamma$,interleukin 1$\beta$ (IL-1$\beta$),tumor necrosis factor alpha (TNF-$\alpha$),IL-6,IL-10,CXCL8,and CXCL10 was found. IFN-$\beta$,IFN-$\lambda$1,CXCL8,and CXCL10 were also upregulated in the epithelial model upon RSV infection. In conclusion,this study provides novel insights into the basic immune response to RSV infection in an important and understudied risk population,providing leads for future studies that are essential for the prevention and treatment of severe RSV disease in older adults.IMPORTANCE Respiratory syncytial virus (RSV) can cause severe morbidity and mortality in certain risk groups,especially infants and older adults. Currently no (prophylactic) treatment is available,except for a partially effective yet highly expensive monoclonal antibody. RSV therefore remains a major public health concern. To allow targeted development of novel vaccines and therapeutics,it is of great importance to understand the immunological mechanisms that underlie (protection from) severe disease in specific risk populations. Since most RSV-related studies focus on infants,there are only very limited data available concerning the response to RSV in the elderly population. Therefore,in this study,RSV-induced antibody responses and local cytokine secretion were assessed in community-dwelling older adults. These data provide novel insights that will benefit ongoing efforts to design safe and effective prevention and treatment strategies for RSV in an understudied risk group. View Publication

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm,lambda Em = 550 nm). The obtained novel fluorescent product,which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries,was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose,which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway. View Publication

Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation,which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model,and near-complete suppression of cyst growth was found by tetrazolo-CFTR(inh)-172,a tetrazolo-derived thiazolidinone,and Ph-GlyH-101,a phenyl-derived glycine hydrazide,without an effect on cell proliferation. These compounds also inhibited cyst number and growth by {\textgreater}80{\%} in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR(inh)-172 and Ph-GlyH-101 to neonatal,kidney-specific PKD1 knockout mice produced stable,therapeutic inhibitor concentrations of {\textgreater}3 microM in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD. View Publication

The mechanism of uptake of the thyroid hormones,3,5,3'-triiodo-L-thyronine (L-T3) and L-thyroxine (L-T4),was studied in rat pituitary GH4C1 cells. The major portion (approximately 65{\%}) of L-T3 transport was stereospecific and saturable. Transport of L-T3 was 8-10 times more rapid than transport of D-T3. [125I]L-T3 transport was saturable at microM concentrations; a Lineweaver-Burk plot was linear with Km = 0.4 microM and Vmax = 4 pmol/min/10(6) cells. Unlabeled analogs competed with [125I]L-T3 uptake in the order L-T3 {\textgreater} or = L-T4 {\textgreater} 3,3',5'-triiodo-L-thyronine (reverse-T3),D-T3,D-T4,and L-thyronine. L-T3 and L-T4 also both effectively inhibited [125I]L-T4 transport. Uptake of [125I]L-T3 was inhibited 40-55{\%} by large neutral amino acids and 77{\%} by 80 microM beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid,an inhibitor selective for the L system of amino acid uptake. Conversely,L-T3 inhibited the transport of [3H]leucine by pituitary cells (IC50 = 2 microM),but D-T3 and 3,5,3'-triiodothyroacetic acid (Triac) did not. L-Leucine was transported much more efficiently (Vmax = 0.65 mumol/min/10(6) cells) than L-T3 by GH4C1 cells. The results show that L-T3 and L-T4 share the same stereospecific transport pathway in pituitary cells,that the transport mechanism is saturable at supraphysiological thyroid hormone concentrations,and that the L system is partially responsible for L-T3 transport. View Publication