产品说明

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density,but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model,enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common ($\Delta$F508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether,targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking,but also introduces a new tool for deconstructing the ubiquitin code in situ. View Publication

The endoplasmic reticular Ca(2+)-ATPase inhibitor,thapsigargin,was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition,confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator,bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus,we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis. View Publication

Perhexiline maleate,originally classified as a calcium antagonist,is in use as an antianginal agent. The mechanism of its protective effect is unknown,but there is speculation that it involves a modification of myocardial substrate utilization,in which glycolytic sources are used rather than fatty acids. This hypothesis was tested by employing [13C]NMR isotopomer analysis to measure substrate selection in the working rat heart. Substrate utilization was measured from a mixture of substrates present at their physiological concentration,as follows: acetoacetate,glucose,lactate and long-chain fatty acids. Control perfusions were compared with those perfused with perhexiline. It was found that perhexiline increased lactate utilization,which reduced the extent of fatty acid and endogenous substrate oxidation. There was also a significant increase in cardiac output for a small and insignificant increase in oxygen consumption,which suggested an improvement in myocardial efficiency. Thus,it was confirmed by direct measurement that this drug does modify substrate oxidation,which suggests that further investigations of the role that this agent can play in the management of ischemic heart disease would be beneficial. View Publication

Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here,we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+,a principal regulator of energy metabolism and cellular fate,in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression,increased expression of senescence markers (p16INK4a,p21Waf/Cip1,ApoJ,CTGF and $\beta$-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This,we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element. View Publication

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here,we show that,in mice with EAE,the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord,decreases integrin expression in antigen-specific CD4+ T cells,increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells,a pathogenic cell population in EAE. In mice with chronic EAE,SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis. View Publication

The recent emergence of the novel,pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here,we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally,we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. View Publication

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence,this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes,Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution,the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay,which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2{\%} vs. 9.9{\%},p {\textgreater} 0.05). However,when 100 mM Z-VAD-FMK was added to the post-thaw culture media,there was a significant enhancement in the survival rate from 9.9{\%} to 14.4{\%} (p {\textless} 0.05),which was further increased to 18.7{\%} when Z-VAD-FMK was also added to the freezing solution as well (p {\textless} 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy,and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture. View Publication

The bleomycin group antitumor antibiotics have long been of interest as a consequence of their efficacy in the treatment of certain tumors,not to mention their unique structures and properties in mediating dioxygen activation and sequence selective degradation of DNA. At a chemical level,the structure originally assigned to bleomycin was subsequently reassigned and the new structure has been confirmed by total synthesis. Through the elaboration of structurally modified bleomycin congeners and fragments,synthetic efforts have also facilitated an understanding of the contribution of individual structural domains in bleomycin to sequence selective DNA binding and cleavage,and have also provided insights into the nature of the chemical processes by which DNA degradation takes place. Within the last several years,it has also become apparent that bleomycin can mediate the oxidative degradation of all major classes of cellular RNAs; it seems entirely plausible that RNA may also represent an important locus of action for this class of antitumor agent. In parallel with ongoing synthetic and mechanistic efforts using classical methods,the study of bleomycins attached to solid supports has been shown to provide important mechanistic insights,and the actual elaboration of modified bleomycins by solid phase synthesis constitutes a logical extension of such efforts. View Publication

Deregulation of apoptosis,the physiological form of cell death,is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways,both of which may provide potential targets for novel anticancer drugs. Although several ligands stimulating death receptors have been described,the actual molecular events triggering the mitochondrial pathway are largely unknown. Here,we show initiation of apoptosis by gradual depletion of the intracellular coenzyme NAD+. We identified the first low molecular weight compound,designated FK866,which induces apoptosis by highly specific,noncompetitive inhibition of nicotinamide phosphoribosyltransferase (NAPRT),a key enzyme in the regulation of NAD+ biosynthesis from the natural precursor nicotinamide. Interference with this enzyme does not primarily intoxicate cells because the mitochondrial respiratory activity and the NAD+ -dependent redox reactions involved remain unaffected as long as NAD+ is not effectively depleted by catabolic reactions. Certain tissues,however,have a high turnover of NAD+ through its cleavage by enzymes like poly(ADP-ribose) polymerase. Such cells often rely on the more readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis after inhibition of NAPRT,whereas cells effectively using the nicotinic acid pathway for NAD+ synthesis remain unaffected. In support of this concept,FK866 effectively induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC(50) of approximately 1 nM,did not directly inhibit mitochondrial respiratory activity,but caused gradual NAD+ depletion through specific inhibition of NAPRT. This enzyme,when partially purified from K562 human leukemia cells,was noncompetitively inhibited by FK866,and the inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)'),respectively. Nicotinic acid and nicotinamide were both found to have antidote potential for the cellular effects of FK866. FK866 may be used for treatment of diseases implicating deregulated apoptosis such as cancer for immunosuppression or as a sensitizer for genotoxic agents. Furthermore,it may provide an important tool for investigation of the molecular triggers of the mitochondrial pathway leading to apoptosis through enabling temporal separation of NAD+ decrease from ATP breakdown and apoptosis by several days. View Publication

BACKGROUND Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium,especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-$\beta$1,which plays a role in recruitment and activation of inflammatory cells,extracellular matrix (ECM) production and fibrosis development,and is a potent inducer of EMT. OBJECTIVE As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells,the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. METHODS We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. RESULTS Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-$\beta$1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. CONCLUSIONS AND CLINICAL RELEVANCE As an inducer of EMT and an important source of TGF-$\beta$1,neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways. View Publication

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of creeping fat" (CrF) defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria." View Publication

View Publication