新型冠状病毒主蛋白酶抑制剂的筛选方法研究进展

新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)席卷全球,具有较高的传染性和死亡率,但目前尚缺乏安全有效的COVID-19疫苗与治疗药物.新型冠状病毒主蛋白酶(main protease,Mpro)的进化高度保守,在调控新冠病毒RNA复制中具有重要的生物学功能,已成为新型广谱抗冠状...     

系统型硬化症尿液lncRNA-mRNA差异表达基因筛选及生物信息学分析

为探讨系统性硬化症(SSc)患者尿液样本中的长链非编码RNA(lncRNA)、信使RNA(mRNA)的表达谱和生物学功能。选取6名SSc患者和3名健康对照者(HC),采集样本为中段晨尿,应用mRNA和lncRNA微阵列检测总RNA表达变异,SSc组与HC组相比。检测尿液lncRNA和mRNA表达,Gene ontology (GO)分析Kyoto Encyclopedia of Genes and Genomes (KEGG)信号通路分析差异表达的lncRNA功能分布;STRING在线网站和Cytoscape软件网络应用分析构建蛋白质相互作用网络(PPI)并筛选出核心基因(Hub Gene)。结果发现：与HC相比,SSc患者尿液中共有645个(上调546,下调99)mRNA和1 888个(上调1 647,下调241)lncRNA差异表达(Fold Change绝对值≥2,且P≤0.05)。KEGG通路结果显示富集TGF-β信号通路、氧化磷酸化、磷酸戊糖通路。SSc的GO分析显示与转录调控、DNA去甲基化、白介素6反应等相关;PPI网络分析表明主要富集在氧化磷酸化、细胞凋亡、自噬途径通路...     

Mitochondriome and Cholangiocellular Carcinoma

Cholangiocellular carcinoma (CCA) of the liver was the target of more interest, recently, due mainly to its increased incidence and possible association to new environmental factors. Somatic mitochondrial DNA (mtDNA) mutations have been found in several cancers. Some of these malignancies contain changes of mtDNA, which are not or, very rarely, found in the mtDNA databases. In terms of evolutionary genetics and oncology, these data are extremely interesting and may be considered a sign of poor fitness, which may conduct in some way to different cellular processes, including carcinogenesis. MitoChip analysis is a strong tool for investigations in experimental oncology and was carried out on three CCA cell lines (HuCCT1, Huh-28 and OZ) with different outcome in human and a Papova-immortalized normal hepatocyte cell line (THLE-3). Real time quantitative PCR, western blot analysis, transmission electron microscopy, confocal laser microscopy, and metabolic assays including L-Lactate and NAD⁺/NADH assays were meticulously used to identify mtDNA copy number, oxidative phosphorylation (OXPHOS) content, ultrastructural morphology, mitochondrial membrane potential (ΔΨ_m), and differential composition of metabolites, respectively. Among 102 mtDNA changes observed in the CCA cell lines, 28 were non-synonymous coding region alterations resulting in an amino acid change. Thirty-eight were synonymous and 30 involved ribosomal RNA (rRNA) and transfer RNA (tRNA) regions. We found three new heteroplasmic mutations in two CCA cell lines (HuCCT1 and Huh-28). Interestingly, mtDNA copy number was decreased in all three CCA cell lines, while complexes I and III were decreased with depolarization of mitochondria. L-Lactate and NAD+/NADH assays were increased in all three CCA cell lines. MtDNA alterations seem to be a common event in CCA. This is the first study using MitoChip analysis with comprehensive metabolic studies in CCA cell lines potentially creating a platform for future studies on the interactions between normal and neoplastic cells.     

FUS Interacts with HSP60 to Promote Mitochondrial Damage

FUS-proteinopathies, a group of heterogeneous disorders including ALS-FUS and FTLD-FUS, are characterized by the formation of inclusion bodies containing the nuclear protein FUS in the affected patients. However, the underlying molecular and cellular defects remain unclear. Here we provide evidence for mitochondrial localization of FUS and its induction of mitochondrial damage. Remarkably, FTLD-FUS brain samples show increased FUS expression and mitochondrial defects. Biochemical and genetic data demonstrate that FUS interacts with a mitochondrial chaperonin, HSP60, and that FUS translocation to mitochondria is, at least in part, mediated by HSP60. Down-regulating HSP60 reduces mitochondrially localized FUS and partially rescues mitochondrial defects and neurodegenerative phenotypes caused by FUS expression in transgenic flies. This is the first report of direct mitochondrial targeting by a nuclear protein associated with neurodegeneration, suggesting that mitochondrial impairment may represent a critical event in different forms of FUS-proteinopathies and a common pathological feature for both ALS-FUS and FTLD-FUS. Our study offers a potential explanation for the highly heterogeneous nature and complex genetic presentation of different forms of FUS-proteinopathies. Our data also suggest that mitochondrial damage may be a target in future development of diagnostic and therapeutic tools for FUS-proteinopathies, a group of devastating neurodegenerative diseases.     

HSV-IgM人-鼠嵌合抗体制备及稳定细胞株构建工艺开发

为实现体外大规模制备单纯疱疹病毒HSV-IgM(HSV1,HSV2)人鼠嵌合抗体,本研究通过RNA连接酶介导的cDNA末端快速扩增(RNA ligase-mediated rapid amplification of cDNA ends,RLM-RACE)技术获取其对应杂交瘤细胞基因序列,构建嵌合抗体至真核表达载体,在CHO-S细胞中稳定表达所需目的蛋白。同时优化稳定细胞株筛选工艺,对细胞池构建阶段和单克隆筛选阶段的加压条件进行摸索与探究,最后目的抗体采用蛋白L亲和纯化法进行纯化并进行生物活性检测;最终成功制备899 kDa和909 kDa的稳定高表达重组IgM抗体(HSV1,HSV2)细胞株。结果表明,最适筛选压力为20P200M(一轮加压)和50P1000M(二轮加压);使用加压培养基进行单克隆筛选抗体表达量较高,HSV1-IgM和HSV2-IgM单克隆最终表达量分别为1620 mg/L和623 mg/L。本研究为HSV1和HSV2的IgM系列重组抗体质控品开发以及体外高表达分泌IgM亚型抗体提供理论与实践基础。     

独龙族传统代粮植物食用观音座莲的研究

食用观音座莲(Angiopteris esculenta)是独龙族(中国云南人口最少的少数民族)重要的野生代粮植物,其地下部分可用于加工蕨粉以制作食物。为深入调查和记录独龙族利用食用观音座莲的传统知识,该文采用民族植物学的方法对食用观音座莲相关的传统知识进行调查和记录,并运用食品科学的方法对蕨粉的基本成分、形态、理化性质及糊化特性进行了深入探究。结果表明：(1)食用观音座莲在独龙族的传统生计中有多种用途,定量分析表明其食用用途的相对引用频率(RFC)远大于其他用途,说明食用是独龙族利用该植物最主要的方式。(2)独龙族使用传统方法采集食用观音座莲膨大叶柄基部和根茎加工并食用蕨粉主要原因是蕨粉中富含淀粉,其总淀粉含量为86.2%,并含有多种人体必需的矿物元素。(3)蕨粉具有较好理化性质,其中冻融稳定性和凝沉稳定性较强,可用于冷冻食品和淀粉产品的生产、加工;回生值和衰减值远低于常见淀粉,说明其具有良好的热糊稳定性和冷糊稳定性且不易回生。综上表明,食用观音座莲蕨粉具有较大的开发利用潜能,通过人工栽培提高产量,有利于提高当地居民收入和促进偏远地区乡村振兴。该研究为食用观音座莲后续的加工利用提供了...     

IAA和Ca^2＋对绿豆下胚轴切段伸长的影响及其相互关系

0.01 mmol/L IAA可以明显促进绿豆下胚轴切段的伸长,≤0.1 mmol/L CaCl_2也可促进其伸长,但当浓度为0.5 mmol/L时则有抑制作用。低浓度CaCl_2尚能加强IAA对绿豆下胚轴切段伸长的促进作用。Ca~(2+)专一性螯合剂EGTA、Ca~(2+)竞争性抑制剂LaCl_3及CaM拮抗剂CPZ均能抑制IAA促进绿豆下胚轴切段伸长的作用。增加培养介质中CaCl_2浓度可以逆转LaCl_3的抑制效应。     

MiR133b-mediated inhibition of EGFR-PTK pathway promotes rAAV2 transduction by facilitating intracellular trafficking and augmenting second-strand synthesis

Recombinant adeno-associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor-protein tyrosine kinase (EGFR-PTK) negatively regulated rAAV transduction, and EGFR-positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV-miRNA133b vector, which co-expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live-cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR-positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second-strand synthesis. ss-rAAV2-miRNA133b vector specifically increased rAAV2 transduction in EGFR-positive cells or tissues, while ss-rAAV2-Fluc-miRNA133b exerted an antitumor effect. rAAV-miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR-positive cell-related diseases, such as non-small-cell lung cancer.