核酸去甲基酶AlkB在毕赤酵母中的表达纯化及在tRNA相关研究中的应用

[目的]核酸的甲基化修饰是一种常见的化学修饰形式,具有重要的生物学功能,却也在一定程度上给一些核酸研究过程带来了技术难度。tRNA上具有的大量甲基化修饰会阻碍逆转录进程,从而降低荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)和高通量测序对其的检测效率。来自大肠杆菌(Escherichia coli)的AlkB蛋白是一种多功能的脱烷基化酶,可以去除DNA和RNA上多种甲基化为代表的修饰,有望解决以上问题。[方法]针对大肠杆菌来源的AlkB,分别尝试在大肠杆菌和毕赤酵母(Pichia pastoris)表达系统中进行诱导表达和纯化,对纯化获得的AlkB进行酶学性质测定。最后以tRNA_UAU^Ile等两种tRNA为代表,研究AlkB的处理对于荧光定量PCR法检测tRNA表达水平的影响。[结果]AlkB在大肠杆菌中表达时多以包涵体形式存在,但是在毕赤酵母中可以成功分泌表达。使用镍柱分离纯化后获得了纯度高于95%的AlkB蛋白,其酶学性质参数如...     

Ruthenium Triazine Composite: A Good Match for Increasing Hydrogen Evolution Activity through Contact Electrification

The development of Pt‐free catalysts for the alkaline hydrogen evolution reaction (HER), which is widely used in industrial scale water‐alkali electrolyzers, remains a contemporary and pressing challenge. Ruthenium (Ru) has excellent water‐dissociation abilities and could be an alternative water splitting catalyst. However, its large hydrogen binding energy limits HER activity. Here, a new approach is proposed to boost the HER activity of Ru through uniform loading of Ru nanoparticles on triazine‐ring (C₃N₃)‐doped carbon (triNC). The composite (Ru/triNC) exhibits outstanding HER activity with an ultralow overpotential of ≈2 mV at 10 mA cm^?2; thereby making it the best performing electrocatalyst hitherto reported for alkaline HER. The calculated metal mass activity of Ru/triNC is >10 and 15 times higher than that of Pt/C and Pt/triNC. Both theoretical and experimental studies reveal that the triazine‐ring is a good match for Ru to weaken the hydrogen binding on Ru through interfacial charge transfer via increased contact electrification. Therefore, Ru/triNC can provide the optimal hydrogen adsorption free energy (approaching zero), while maintaining the strong water‐dissociation activity. This study provides a new avenue for designing highly efficient and stable electrocatalysts for water splitting.     

黄病毒基因组非编码区的结构与功能研究进展

黄病毒科黄病毒属是由一组含单股正链RNA基因组的囊膜病毒组成,包括登革病毒、流行性乙型脑炎病毒、寨卡病毒、西尼罗病毒、黄热病病毒等,经由虫媒传播,可引起人类和动物的严重虫媒病毒病。黄病毒基因组由1个开放阅读框、5′非编码区和3′非编码区三部分组成。5′和3′非编码区含有病毒基因组复制所必需的启动元件;高度结构化的3′非编码区负责黄病毒亚基因组RNA的产生,从而有助于病毒逃避宿主免疫反应;此外3′非编码区还可作为疫苗研究的靶标。鉴于非编码区在黄病毒的蛋白翻译、基因组复制和免疫调节中发挥的重要作用,本文就黄病毒基因组非编码区的结构与功能最新研究进展作一简要综述。     

Circulating exosomes repair endothelial cell damage by delivering miR-193a-5p

Circulating exosomes delivering microRNAs are involved in the occurrence and development of cardiovascular diseases. How are the circulating exosomes involved in the repair of endothelial injury in acute myocardial infarction (AMI) convalescence (3-7 days) was still not clear. In this study, circulating exosomes from AMI patients (AMI-Exo) and healthy controls (Normal-Exo) were extracted. In vitro and in vivo, our study showed that circulating exosomes protected endothelial cells (HUVECs) from oxidative stress damage; meanwhile, Normal-Exo showed better protective effects. Through the application of related inhibitors, we found that circulating exosomes shuttled between HUVECs via dynamin. Microarry analysis and qRT-PCR of circulating exosomes showed higher expression of miR-193a-5p in Normal-Exo. Our study showed that miR-193a-5p was the key factor on protecting endothelial cells in vitro and in vivo. Bioinformatics analyses found that activin A receptor type I (ACVR1) was the potential downstream target of miR-193a-5p, which was confirmed by ACVR1 expression and dual-luciferase report. Inhibitor of ACVR1 showed similar protective effects as miR-193a-5p. While overexpression of ACVR1 could attenuate protective effects of miR-193a-5p. To sum up, these findings suggest that circulating exosomes could shuttle between cells through dynamin and deliver miR-193a-5p to protect endothelial cells from oxidative stress damage via ACVR1.     

结核分枝杆菌硫醇乙酰基转移酶基因敲除株的构建及其生物学特性分析

为探索硫醇乙酰基转移酶(mycothiol acetyltransferase,MshD)在结核分枝杆菌中的生物学特性,本实验利用噬菌体为载体的同源重组技术,构建结核分枝杆菌mshD基因敲除株、mshD基因回补株,用实时定量聚合酶链反应(real time-quantitative polymerase chain reaction, RT-qPCR)对所构建的菌株进行验证。分别收集H37Ra野生株、mshD基因敲除株、mshD基因回补株对数生长期菌液各5 mL, 离心收集菌体并培养,以观察菌落形态、生物膜形成及生长曲线测定;用5 mmol/L H₂O₂、0.05% SDS,50 ℃热激及低氧条件下分别处理基因敲出菌株和野生菌株,将菌液进行10倍梯度稀释,培养4～6周后检测抗胁迫能力并计算存活率。结果显示, 与野生株H37Ra相比,mshD基因敲除株菌落褶皱减少且菌落偏小,生长趋势较为缓慢;生物膜形成所需时间增长且褶皱明显减少;抗逆能力下降,存活率略低于野生株和回补株。揭示了mshD基因对结核分枝杆菌的生长具有重要作用,为进一步揭示该基因的功能和作用机制奠定了基础。     

Application of next-generation sequencing to screen for pathogenic mutations in 123 unrelated Chinese patients with Marfan syndrome or a related disease

Marfan syndrome(MFS) is a systemic connective tissue disease principally affecting the ocular, skeletal and cardiovascular systems. This autosomal dominant disorder carries a prevalence of 1:3,000 to 1:5,000. This study aims to define the mutational spectrum of MFS related genes in Chinese patients and to establish genotype-phenotype correlations in MFS. Panel-based targeted next-generation sequencing was used to analyze the FBN1, TGFBR1 and TGFBR2 genes in 123 unrelated Chinese individuals with MFS or a related disease. Genotype-phenotype correlation analyses were performed in mutation-positive patients. The results showed that 97 cases/families(78.9%; 97/123) harbor at least one(likely) pathogenic mutation, most of which were in FBN1; four patients had TGFBR1/2 mutations; and one patient harbored a SMAD3 mutation. Three patients had two FBN1 mutations, and all patients showed classical MFS phenotypes. Patients with a dominant negative-FBN1 mutation had a higher prevalence of ectopia lentis(EL). Patients carrying a haploinsufficiency-FBN1 mutation tended to have aortic dissection without EL. This study extends the spectrum of genetic backgrounds of MFS and enriches our knowledge of genotype-phenotype correlations.     

A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.     

OsRLCK160 contributes to flavonoid accumulation and UV-B tolerance by regulating OsbZIP48 in rice

Science China Life Sciences - Plants produce specialized metabolites to adapt to the ever-changing environments. Flavonoids are antioxidants essential for growth, development, and breeding with...     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

河流着生藻类多样性对城镇化的响应——以深圳市为例

着生藻类是河流生态状况的重要指示生物,然而关于城镇化对着生藻类多样性影响的研究还较缺乏。本研究基于深圳市9条主要河流74个样点2020年雨、旱季的调查数据,对比了城区、郊区河流样点的着生藻类α、β多样性。两次调查共鉴定出着生藻类301个分类单元,其中硅藻门种类最多。谷皮菱形藻（Nitzschia palea）、隐头舟形藻（Navicula cryptocephala）、平庸菱形藻（Nitzschia inconspicua）等污染指示种在城区组相对丰度更高,而清洁指示种微小曲丝藻（Achnanthidium minutissimum）在郊区组相对丰度更高。城镇化对着生藻类物种丰富度、香农维纳多样性指数、Pielou均匀度指数等α多样性指数均无显著影响,但对着生藻类群落结构有显著影响。城区组样点的着生藻类β多样性（用主坐标分析质心指示）低于郊区组,城区组与郊区组藻类β多样性差异在旱季更为显著。两组样点的藻类β多样性差异主要来源于物种周转组分。与城区组着生藻类群落显著相关的环境因子包括总氮、高锰酸钾指数、电导率、pH、浊度和水深,而与郊区组着生藻类群落显著相关的是总氮、溶解性无机磷和电导率。研究发现,城市人类活动导致的总氮、溶解性无机磷、高锰酸钾指数、电导率等指标的显著增高导致着生藻类β多样性的降低。为了保护河流生物多样性,建议有效控制城区生产生活污水排放,减少人类活动对河流的干扰。