技术资料

The thyroid hormone,3,5,3'-Triiodo-L-thyronine (T3),is essential for growth,differentiation,and regulation of metabolic functions in multicellular organisms,although the specific mechanisms of this control are still unknown. In this study,treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype,we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion,our results suggest,for the first time,that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes. View Publication

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers,including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy,it is not clear whether they can influence early disease progression. To clarify this issue,TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases],2 patients had del(17p) only,and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment,a reliable measure of disease progression,TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes,whereas del(17p) was associated with the presence of adverse prognostic factors,including CD38 positivity,unmutated-IGHV gene status,and NOTCH1 mutations. View Publication

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts,occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice,tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically. View Publication

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome,an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established,yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here,we report a non-conventional,T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous,Rip2-independent mechanism for Nod2 in uveitis. In naive animals,Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly,CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity,and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome. View Publication

The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme,named apopain,is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3,the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro,suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death. View Publication

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here,we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further,we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2,Vaccinia virus,and Zika virus through a type I interferon (IFN)-independent mechanism. In addition,4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion,NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses,including SARS-CoV2. View Publication

The generation of insulin-producing pancreatic $\beta$ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However,insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide $\beta$ cells. Here,we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive $\beta$ cells from hPSC in vitro. These stem-cell-derived $\beta$ cells (SC-$\beta$) express markers found in mature $\beta$ cells,flux Ca(2+) in response to glucose,package insulin into secretory granules,and secrete quantities of insulin comparable to adult $\beta$ cells in response to multiple sequential glucose challenges in vitro. Furthermore,these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner,and transplantation of these cells ameliorates hyperglycemia in diabetic mice. View Publication

BACKGROUND Chronic rhinosinusitis patients (CRS) suffer from chronic inflammation of the sinus mucosa associated with chronic relapsing infections. Mucosal biofilms,associated with Staphylococcus aureus,have been implicated as a cause. We compared the effect of exoproteins secreted from clinical isolates of S aureus from CRS patients in planktonic and biofilm form on the nasal epithelial barrier. METHODS Clinical S aureus isolates from 39 CRS patients were grown in planktonic and biofilm forms and their exoproteins concentrated. These were applied to primary human nasal epithelial cells grown at the air-liquid interface. Transepithelial electrical resistance,permeability of flourescein isothiocyanate-dextrans,and cytotoxicity were measured. Structure and expression of tight junctions zona occludens-1,and claudin-1 proteins were assessed by electron microscopy and immunofluorescence. The Wilcoxon signed rank test was used for statistical analyses. RESULTS S aureus biofilm exoproteins showed dose- and time-dependent reduction of transepithelial electrical resistance,increased cell toxicity,and increased permeability (p {\textless} 0.001) compared with equal concentrations of planktonic cultures. Discontinuity in zona occludens-1 and claudin-1 immunofluorescence was confirmed as disrupted tight junctions on electron microscopy. CONCLUSION S aureus biofilm exoproteins disrupt the mucosal barrier structure in a time- and dose-dependent manner and are toxic. Damage to the mucosal barrier by S aureus biofilm exoproteins may play a major role in CRS etiopathogenesis. View Publication

In neuronal growth cones,the advancing tips of elongating axons and dendrites,specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by long-chain homologues of the antibiotic tunicamycin,a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system,indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons,under conditions in which protein palmitoylation is inhibited,produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic,even in the absence of protein synthesis,or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation,rather than its previously established effects on protein glycosylation and synthesis. (a) The abilities of different tunicamycin homologues to induce growth cone collapse very systematically with the length of the fatty acyl side-chain of tunicamycin,in a manner predicted and observed for the inhibition of protein palmitoylation. Homologues with fatty acyl moieties shorter than palmitic acid (16 hydrocarbons),including potent inhibitors of glycosylation,are poor inhibitors of growth cone function. (b) The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid,which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for dynamic protein acylation in growth cone-mediated extension of neuronal processes. View Publication

The production of insulin-producing $\beta$ cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny,we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or $\beta$-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state,a principle that could be relevant to other systems. Notably,activation of the key $\beta$-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional $\beta$ cells. View Publication

Autophagy is a cell-protective and degradative process that recycles damaged and long-lived cellular components. Cancer cells are thought to take advantage of autophagy to help them to cope with the stress of tumorigenesis; thus targeting autophagy is an attractive therapeutic approach. However,there are currently no specific inhibitors of autophagy. ULK1,a serine/threonine protein kinase,is essential for the initial stages of autophagy,and here we report that two compounds,MRT67307 and MRT68921,potently inhibit ULK1 and ULK2 in vitro and block autophagy in cells. Using a drug-resistant ULK1 mutant,we show that the autophagy-inhibiting capacity of the compounds is specifically through ULK1. ULK1 inhibition results in accumulation of stalled early autophagosomal structures,indicating a role for ULK1 in the maturation of autophagosomes as well as initiation. View Publication

Uridine,a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes,is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system,at the reproduction level,with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore,uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase),responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue. View Publication