IFN regulatory Factor-1 induced macrophage pyroptosis by modulating m6A modification of circ_0029589 in patients with acute coronary syndrome

BackgroundPyroptosis is identified as a novel form of inflammatory programmed cell death and has been recently found to be closely related to atherosclerosis (AS). We found that IFN regulatory factor-1(IRF-1) effectively promotes macrophage pyroptosis in patients with acute coronary syndrome (ACS). Subsequent studies have demonstrated that circRNAs are implicated in AS. However, the underlying mechanisms of circRNAs in macrophage pyroptosis remain elusive.MethodsWe detected the RNA expression of hsa_circ_0002984, hsa_circ_0010283 and hsa_circ_0029589 in human PBMC-derived macrophages from patients with coronary artery disease (CAD). The lentiviral recombinant vector for hsa_circ_0029589 overexpression (pLC5-GFP-circ_0029589) and small interference RNAs targeting hsa_circ_0029589 and METTL3 were constructed. Then, macrophages were transfected with pLC5-GFP-circ_0029589, si-circ_0029589 or si-METTL3 after IRF-1 was overexpressed and to explore the potential mechanism of hsa_circ_0029589 involved in IRF-1 induced macrophage pyroptosis.ResultsThe relative RNA expression level of hsa_circ_0029589 in macrophages was decreased, whereas the N6-methyladenosine (m6A) level of hsa_circ_0029589 and the expression of m6A methyltransferase METTL3 were validated to be significantly elevated in macrophages in patients with ACS. Furthermore, overexpression of IRF-1 suppressed the expression of hsa_circ_0029589, but induced its m6A level along with the expression of METTL3 in macrophages. Additionally, either overexpression of hsa_circ_0029589 or inhibition of METTL3 significantly increased the expression of hsa_circ_0029589 and attenuated macrophage pyroptosis.ConclusionOur observations suggest a novel mechanism by which IRF-1 facilitates macrophage pyroptosis and inflammation in ACS and AS by inhibiting circ_0029589 through promoting its m6A modification.     

分析应用速度向量成像技术评估心内强回声光点对中孕期胎儿左心室功能的影响

目的 应用速度向量成像技术(VVI)评价中孕期检出单一心内强回声光点(ICEF)时,胎儿心脏功能是否受到影响。方法 选取2014年5月至2015年5月在解放军第105医院行产前胎儿超声心动图检查发现ICEF的胎儿173例(观察组)和未见ICEF的胎儿85 例(对照组)。采集胎儿标准四腔心切面后,脱机进入VVI工作站处理分析,获得各组胎儿左心室整体及6个节段心肌的应变(S)、应变率(SR)、运动速度(V)数据,对照分析胎儿心脏功能。结果 中孕期, ICEF胎儿的左心室心肌的应变、应变率不随孕周增大而改变(P>0.05);室壁运动速度随孕周增大而增大(P<0.05),且从基底段至心尖段逐渐减低。应变、应变率、速度与对照组比较差异均无统计学意义(P>0.05)。结论 VVI技术可评价胎儿心功能,单一ICEF对胎儿心功能无明显影响。     

Machine intelligence in peptide therapeutics: A next-generation tool for rapid disease screening

Discovery and development of biopeptides are time-consuming, laborious, and dependent on various factors. Data-driven computational methods, especially machine learning (ML) approach, can rapidly and efficiently predict the utility of therapeutic peptides. ML methods offer an array of tools that can accelerate and enhance decision making and discovery for well-defined queries with ample and sophisticated data quality. Various ML approaches, such as support vector machines, random forest, extremely randomized tree, and more recently deep learning methods, are useful in peptide-based drug discovery. These approaches leverage the peptide data sets, created via high-throughput sequencing and computational methods, and enable the prediction of functional peptides with increased levels of accuracy. The use of ML approaches in the development of peptide-based therapeutics is relatively recent; however, these techniques are already revolutionizing protein research by unraveling their novel therapeutic peptide functions. In this review, we discuss several ML-based state-of-the-art peptide-prediction tools and compare these methods in terms of their algorithms, feature encodings, prediction scores, evaluation methodologies, and software utilities. We also assessed the prediction performance of these methods using well-constructed independent data sets. In addition, we discuss the common pitfalls and challenges of using ML approaches for peptide therapeutics. Overall, we show that using ML models in peptide research can streamline the development of targeted peptide therapies.     

基因修饰间充质干细胞治疗肿瘤研究进展

间充质干细胞（MSCs）是来源于中胚层的成体干细胞,体内分布广泛,可从骨髓、脂肪、牙髓、脐带/胎盘等组织中分离获取,具有高度的可增殖和分化潜能,较低的免疫原性,同时具有向炎症损伤部位微环境的趋向性,在疾病治疗方面可作为基因药物的载体实现精准治疗。癌症被认为是永远无法愈合的伤口,其组织微环境在损伤与修复中呈无休止的动态变化,MSCs在其中扮演了重要角色。利用MSCs具有的向肿瘤组织中归巢及定位的特点,对其进行抗肿瘤药物的基因工程改造可能在癌症的治疗上是一种新的策略。概述MSCs在癌症发生发展中的作用以及基因修饰MSCs治疗癌症的研究进展,旨在为临床使用基因修饰MSCs治疗癌症提供策略和见解。     

人PD-L1真核表达载体的构建及结肠癌细胞稳定表达单克隆株的筛选和鉴定

目的:构建人程序性死亡配体1（PD-L1）重组质粒载体,筛选稳定高表达人PD-L1的结肠癌单克隆细胞株。方法:提取HCT116细胞总RNA,RT-PCR克隆PD-L1基因片段,将其重组至含有HA标签和Neo真核细胞筛选抗性的pcDNA3质粒载体上,转染HCT116细胞,倍比稀释8个细胞浓度至96孔板,G418筛选两周,挑取单细胞克隆,通过免疫印迹鉴定外源性PD-L1的表达;通过MTS、Transwell和软琼脂细胞克隆形成实验比较PD-L1低表达和高表达单克隆细胞株生长增殖、迁移和细胞克隆形成能力的差别;最后通过在两组细胞中过表达帽依赖性荧光素酶报告基因阐述造成上述表型的可能机制。结果:成功构建人PD-L1的真核表达载体,并筛选获得稳定高表达PD-L1的HCT116单克隆细胞株。功能研究初步证明PD-L1可促进细胞克隆形成能力、迁移和生长增殖。过表达PD-L1可使帽依赖性荧光素酶报告基因的表达上调约3~4倍,表明其机制部分为PD-L1可上调帽依赖性蛋白质翻译。结论:获得了PD-L1重组质粒载体及可用于后续功能研究的稳定高表达人PD-L1的HCT116单克隆细胞株。     

SREBP-1c基因过表达细胞模型的构建及对HepG2细胞脂滴含量的影响

目的：构建SREBP-1c过表达HepG2细胞模型并观察其对脂滴含量的影响,为进一步深入研究肝癌脂滴代谢异常机制提供细胞模型。方法：采用DNA重组技术,将SREBP-1c基因克隆至慢病毒表达载体pLenti 6.3/V5中,并用脂质体介导法将慢病毒包装系统和带目的基因的质粒pLenti6.3-SREBP-1c共转染293T细胞,收集上清,感染HepG2细胞。通过Real-time PCR和Western blot对细胞株进行鉴定。采用免疫荧光检测HepG2细胞中脂滴的数量、大小及细胞内甘油三酯的含量。结果：成功构建了过表达SREBP-1c基因的慢病毒载体pLenti6.3-SREBP-1c,转染后HepG2细胞中可见明显的SREBP-1c蛋白表达。Real-time PCR检测结果显示转染SREBP-1c基因的HepG2细胞中SREBP-1c mRNA水平较对照组升高了约11.4倍。Western blot结果显示SREBP-1c蛋白水平较对照组提高3.2倍。免疫荧光显示过表达SREBP-1c能够明显增加HepG2细胞内脂滴的数量和大小。细胞内甘油三酯定量结果显示甘油三酯含量较对照组明显升高（P < 0.01）。结论：成功构建了SREBP-1c基因过表达的重组慢病毒载体及其稳定转染细胞株。高表达SREBP-1c的HepG2细胞增加了细胞中脂滴的数量、大小和甘油三酯的含量,可用于从脂滴代谢方面对肝癌发病分子机制的研究。     

一种新的真核细胞表达载体pEF－BOS的应用

采用一种新的真核细胞表达载体ｐＥＦ－ＢＯＳ在ＣＯＳ细胞内表达了ｇｐ１３０和ＧＡＭ两种分子，并利用免疫沉淀和Ｗｅｓｔｅｒｎ－ｂｌｏｔ观察了表达结果。发现应用ｐＥＦ－ＢＯＳ表达载体可在ＣＯＳ细胞内获得上述两种分子的较高表达，优于一般常用的ｐＳＶ表达载体。     

丙型肝炎病毒与荧光素酶融合基因细胞模型的初步建立

Objective　To establish HCV cell culture model which is easy to measure. Methods　Controllable retroviral vector with fusion gene of hepatitis C virus (HCV) cDNA and luciferase (luc) reporter gene was constructed by molecular cloning technique, the transfection of this retroviral vector in a human hepatic carcinoma cell (HHCC) line was performed by lipofectAMINE and then luciferase activity in the cellular lysate was measured by scintillation counter. Results　 ① Fusion gene of the HCV 5′ NCR-C region and luciferase reporter gene identified by restriction endonuclease cleavage have been cloned into pBPSTR1 vector. ② The luciferase activity could maintain up to 20 days at least, and could be increased by puromycin treatment and regulated by tetracycline. Conclusion　A cell model for expression of HCV C-E1 and luciferase genes was established for gene therapy studies against HCV C-E1 sequences.     

图像分割技术在中医舌诊客观化研究中的应用

舌诊是中医四诊的主要内容,是辨证论治的主要依据。客观化研究对中医辨证规范化及中医临床、教学和科研手段的现代化具有重要意义。对舌诊客观化研究中涉及的图像预处理的重要内容——舌体分割提取和舌苔舌质同类区域划分——进行了深入研究,提出了相应算法,通过实验充分证明了算法具有很好的鲁棒性。这给进一步的自动特征提取提供了保障和重要信息。