Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome activation

Coronary microembolization (CME), a common reason for periprocedural myocardial infarction (PMI), bears very important prognostic implications. However, the molecular mechanisms related to CME remain largely elusive. Statins have been shown to prevent PMI, but the underlying mechanism has not been identified. Here, we examine whether the NLRP3 inflammasome contributes to CME-induced cardiac injury and investigate the effects of statin therapy on CME. In vivo study, mice with CME were treated with 40 mg/kg/d rosuvastatin (RVS) orally or a selective NLRP3 inflammasome inhibitor MCC950 intraperitoneally (20 mg/kg/d). Mice treated with MCC950 and RVS showed improved cardiac contractile function and morphological changes, diminished fibrosis and microinfarct size, and reduced serum lactate dehydrogenase (LDH) level. Mechanistically, RVS decreased the expression of NLRP3, caspase-1, interleukin-1β, and Gasdermin D N-terminal domains. Proteomics analysis revealed that RVS restored the energy metabolism and oxidative phosphorylation in CME. Furthermore, reduced reactive oxygen species (ROS) level and alleviated mitochondrial damage were observed in RVS-treated mice. In vitro study, RVS inhibited the activation of NLRP3 inflammasome induced by tumor necrosis factor α plus hypoxia in H9c2 cells. Meanwhile, the pyroptosis was also suppressed by RVS, indicated by the increased cell viability, decreased LDH and propidium iodide uptake in H9c2 cells. RVS also reduced the level of mitochondrial ROS generation in vitro. Our results indicate the NLRP3 inflammasome-dependent cardiac pyroptosis plays an important role in CME-induced cardiac injury and its inhibitor exerts cardioprotective effect following CME. We also uncover the anti-pyroptosis role of RVS in CME, which is associated with regulating mitochondrial ROS.Subject terms: Cell death, Cardiovascular diseases     

臭柏天然居群遗传多样性及演变历史分析

为了合理有效地保育天然臭柏(Juniperus sabina L.)种质资源,追溯和阐释其分布格局的历史成因,本文对我国内蒙古自治区、陕西省、甘肃省和青海省共10个天然臭柏居群388个个体的核糖体内转录间隔区(ITS)序列片段进行测序分析。结果显示:臭柏ITS序列总长度为1089 bp,共含有25个变异位点,定义32个单倍型,其中H4和H6单倍型为共有单倍型;分子变异分析(AMOVA)显示,臭柏居群变异主要来源于居群内,遗传变异为95.04%,而居群间遗传变异仅4.96%,居群间差异水平极显著(F ST=0.0496,P<0.001);Network单倍型网络分析表明,H4和H6为古老单倍型,其他单倍型是由他们衍生而来;遗传分化系数N ST(0.072)0.05)。推测臭柏起源于第三纪中新世(Miocene)中期约12.38 Mya,在第四纪冰期可能存在多个小型避难所。沙埋产生不定根的扩繁能力和较好的有性更新环境可能是沙地居群遗传多样性高于山地居群的决定性因素。     

Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca²⁺ homeostasis in the blood. The general consensus is that extracellular Ca²⁺ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca²⁺ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca²⁺ and L-Trp binding sites and conformational changes of the ECD upon Ca²⁺/L-Trp binding. However, it remains to be understood at the structural level how Ca²⁺/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca²⁺- or L-Trp-bound states, and Ca²⁺/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca²⁺ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca²⁺ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca²⁺. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.Subject terms: Cryoelectron microscopy, Calcium signalling     

Expression of injected HPRT minigene DNA in mouse embryos and its inhibition by antisense DNA

We have used a highly sensitive biochemical microassay to monitor the expression of a cloned minigene for hypoxanthine phosphoribosyl transferase (HPRT, EC.2.4.2.8) in preimplantation mouse embryos. The mouse HPRT promoter and the mouse metallothionein promoter (MT-I) function equally well in embryos at the 2-cell stage whereas the viral SV40 promoter does not allow HPRT expression. Induced HPRT activity from the MT-I HPRT minigene construct occurs in cleavage embryos cultured in the presence of cadmium. In contrast, negation of enzyme expression from the injected minigene DNA is mediated by simultaneous injection of HPRT antisense DNA.     

Crystal structure of a staphylokinase: variant a model for reduced antigenicity.

Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-A resolution, could explain a major antigenic epitope (residues A72-F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK.     

Gene expression,X-inactivation,and methylation during spermatogenesis: The case of Zfa,Zfx, and Zfy in mice

While it has become clear that X-inactivation in the female soma is complete in mouse (in contrast to being “patchy” in man), the degree of X-inactivation in the testes has not been ascertained. We have compared autosomal and X-linked zinc finger homolog expression and X-linked and Y-linked zinc finger homolog methylation in an attempt to elucidate this question. Using RTPCR, we have extended earlier studies of Zfx and Zfa expression in developing testes and find that Zfa expression starts at the time of X-inactivation while Zfx expression is continuous. Cell separation studies did not preclude continued expression of Zfx in adult germ cells. The methylation status of four CCGG residues in the Zfx promoter was studied using PCR bridging this region before and after DNA digestion with the isoschizomers Msp I and Hpa II, the latter being methylation sensitive. Hpa II resistant Zfx promoter DNA was found in all female tissues, but not in male tissues, including the testes. Previous studies have shown that Zfy is expressed at meiosis (like Zfa and unlike Zfx). Despite its expression, the Zfy gene is adjacent to, or contains, highly methylated CCGG sites since hybridization after Msp I digestion detected multiple small fragments that were not released after DNA digestion with Hpa II. Thus, Zfx is not methylated in sperm, while Zfy is, in contrast to their apparent patterns of expression. © 1993 Wiley-Liss, Inc.     

Glycogen synthase kinase 3β inhibition synergizes with PARP inhibitors through the induction of homologous recombination deficiency in colorectal cancer

Monotherapy with poly ADP-ribose polymerase (PARP) inhibitors results in a limited objective response rate (≤60% in most cases) in patients with homologous recombination repair (HRR)-deficient cancer, which suggests a high rate of resistance in this subset of patients to PARP inhibitors (PARPi). To overcome resistance to PARPi and to broaden their clinical use, we performed high-throughput screening of 99 anticancer drugs in combination with PARPi to identify potential therapeutic combinations. Here, we found that GSK3 inhibitors (GSK3i) exhibited a strong synergistic effect with PARPi in a panel of colorectal cancer (CRC) cell lines with diverse genetic backgrounds. The combination of GSK3β and PARP inhibition causes replication stress and DNA double-strand breaks, resulting in increased anaphase bridges and abnormal spindles. Mechanistically, inhibition or genetic depletion of GSK3β was found to impair the HRR of DNA and reduce the mRNA and protein level of BRCA1. Finally, we demonstrated that inhibition or depletion of GSK3β could enhance the in vivo sensitivity to simmiparib without toxicity. Our results provide a mechanistic understanding of the combination of PARP and GSK3 inhibition, and support the clinical development of this combination therapy for CRC patients.Subject terms: Cancer therapeutic resistance, Targeted therapies