AphA蛋白促进副溶血弧菌c-di-GMP合成和生物膜形成

【目的】综合运用表型和分子生化实验研究AphA蛋白对副溶血弧菌生物膜形成的调节机制。【方法】利用菌落褶皱和结晶紫染色实验比较aphA突变株(ΔaphA)和野生株(WT)的表型差异;进而利用色谱串联质谱(HPLC-MS/MS)的方法分别检测ΔaphA和WT中c-di-GMP分子含量;提取ΔaphA和WT的总RNA,采用实时定量RT-PCR的方法研究AphA对scrABC和scrG的调控关系;分别构建克隆有scrABC和scrG上游启动子区的LacZ重组质粒,并将重组质粒转入ΔaphA和WT中,通过测定并比较两株菌中β-半乳糖苷酶活性差异来进一步研究AphA对scrABC和scrG的调控关系;PCR扩增scrABC和scrG的整个启动子区DNA序列,并纯化His-AphA蛋白,通过凝胶阻滞实验(EMSA)验证AphA对靶基因启动子区是否具有直接的相互作用。【结果】表型结果显示AphA能促进c-di-GMP的合成和生物膜形成;实时定量RT-PCR和LacZ结果表明AphA能抑制scrABC和scrG的转录表达;EMSA结果证明AphA不能结合到scrABC和scrG的启动子区DNA上。【结论】AphA间接抑制scrABC和scrG的表达是其促进副溶血弧菌c-di-GMP合成及生物膜形成的机制之一。     

群体感应系统核心调控子对副溶血弧菌mshH基因的调控研究

【目的】探究副溶血弧菌群体感应(quorum sensing,QS)系统核心调控子AphA和OpaR对mshH基因的转录调控。【方法】提取特定条件下副溶血弧菌野生株(wild-type,WT)和调控子基因突变株(ΔaphA和ΔopaR)的总RNA,采用实时定量PCR (quantitative real-time PCR,qPCR)研究AphA和OpaR对mshH基因的转录调控关系以及mshH基因的时相依赖性表达特性;将mshH启动子区DNA序列克隆入pHRP309质粒β-半乳糖苷酶基因的上游,构建LacZ重组质粒,并将其转入WT、ΔaphA和ΔopaR中,获得LacZ实验菌株,再通过LacZ报告基因融合实验研究AphA和OpaR对mshH基因的调控关系以及mshH基因的时相依赖性表达特性;PCR扩增mshH上游启动子区DNA序列,并纯化His-AphA和His-OpaR蛋白,通过凝胶阻滞实验(electrophoretic mobility shift assay,EMSA)和DNase I足迹实验,研究体外条件下His-AphA和His-OpaR对靶基因启动子区DNA片段是否具有直...     

副溶血弧菌拟核相关蛋白H-NS间接抑制VP1687-1686的启动子区转录

为了探讨副溶血性弧菌拟核相关蛋白H-NS对Ⅲ型分泌系统(T3SS) VP1687-1686基因位点的转录调控,本研究提取副溶血弧菌hns突变株(Δhns)和野生株(WT)的总RNA,采用引物延伸实验研究靶基因的转录起始位点,并根据产物的丰度判断H-NS对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和Δhns中转录丰度,以判定H-NS对靶基因的转录调控关系;将靶基因启动子区域DNA序列克隆至lacZ基因上游,将重组质粒转入WT和Δhns中,得到相应的LacZ菌株,通过LacZ报告基因融合实验研究H-NS对靶基因的调控关系;用PCR扩增靶基因的启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)研究His-H-NS是否对靶基因启动子区具有直接的结合作用。研究结果显示,T3SS的VP1687-1686只含有一个转录起始位点,位于翻译起始位点上游82 bp处,且H-NS能够抑制其转录活性,但不能直接结合到VP1687-1686区的启动子区。另外,H-NS对calR的转录无调控作用,His-H-NS也不能结合到其启动子区。本研究的结果初步说明,H-NS能够间接抑制VP1687-1686的转录,该抑制机制与CalR无关联。     

副溶血弧菌拟核相关蛋白H-NS间接抑制VP1687-1686的启动子区转录

为了探讨副溶血弧菌拟核相关蛋白H-NS对Ⅲ型分泌系统(T3SS) VP1687-1686基因位点的转录调控,本研究提取副溶血弧菌hns突变株(Δhns)和野生株(WT)的总RNA,采用引物延伸实验研究靶基因的转录起始位点,并根据产物的丰度判断H-NS对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和Δhns中转录丰度,以判定H-NS对靶基因的转录调控关系;将靶基因启动子区域DNA序列克隆至lacZ基因上游,将重组质粒转入WT和Δhns中,得到相应的LacZ菌株,通过LacZ报告基因融合实验研究H-NS对靶基因的调控关系;用PCR扩增靶基因的启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)研究His-H-NS是否对靶基因启动子区具有直接的结合作用。研究结果显示,T3SS的VP1687-1686只含有一个转录起始位点,位于翻译起始位点上游82 bp处,且H-NS能够抑制其转录活性,但不能直接结合到VP1687-1686区的启动子区。另外,H-NS对calR的转录无调控作用,His-H-NS也不能结合到其启动子区。本研究的结果初步说明,H-NS能够间接抑制VP1687-1686的转录,该抑制机制与CalR无关联。     

H-NS蛋白对副溶血弧菌hcp1的转录调控

【目的】研究调控子H-NS对副溶血弧菌T6SS1结构蛋白基因hcp1的转录调控机制。【方法】利用Western blot检测Hcp1蛋白在野生株(WT)和hns基因敲除株(Δhns)中表达水平的差异。提取WT和Δhns的总RNA,采用实时定量RT-PCR的方法验证H-NS对hcp1的转录调控关系。进而采用引物延伸实验研究hcp1的转录起始位点,并根据产物的丰度判断H-NS对hcp1的调控关系。PCR扩增hcp1的整个启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)验证His-H-NS对hcp1启动子区是否具有直接的结合作用。【结果】Western blot和实时定量RT-PCR结果显示H-NS能抑制hcp1的表达;引物延伸结果显示hcp1只有一个转录起始位点T(–62)(翻译起始位点为+1),且其转录活性是H-NS和σ54依赖性的;EMSA实验表明H-NS对hcp1的启动子区具有直接的结合作用。【结论】H-NS能直接结合到hcp1启动子区而抑制其转录表达。     

副溶血弧菌ToxR截短体蛋白的表达纯化及其DNA结合活性

【目的】利用大肠杆菌BL21λDE3表达系统,表达出有活性的副溶血弧菌(Vibrio parahaemolyticus,VP)ToxR截短体蛋白,为进一步研究ToxR的转录调控机制奠定基础。【方法】以VP基因组DNA为模板,PCR扩增ToxR蛋白DNA结合结构域(ToxR-N)的DNA片段,并将其直接克隆入pET28a中,获得重组质粒;将重组质粒导入大肠杆菌BL21λDE3中,所得菌株经IPTG诱导后能表达出His-ToxR-N蛋白。利用限制级凝血酶切除His-ToxR-N中的His-标签,进而以VP的calR和VP1687为靶基因,通过体外的凝胶阻滞实验(EMSA)验证ToxR-N蛋白的DNA结合活性。分别构建克隆有calR和VP1687上游启动子区的LacZ重组质粒,并将重组质粒转入野生株(WT)和toxR突变株(ΔtoxR)中,通过测定β-半乳糖苷酶活性来比较两株重组菌中靶基因启动子活性,以验证ToxR对calR和VP1687的调控关系。【结果】成功表达出有活性的ToxR-N蛋白,该蛋白对calR启动子区具有结合活性。LacZ结果显示ToxR对calR的转录具有激活作用,而对VP1687的转录具有抑制作用。【结论】所表达的ToxR-N可用于后续的转录调控机制研究;ToxR通过直接激活calR的转录表达,而间接抑制T3SS1相关基因的表达。     

副溶血性弧菌CalR调控vopB2的分子机制

【背景】副溶血性弧菌是一种非常重要的食源性致病菌,CalR蛋白是一种全局转录调节因子。III型分泌系统2 (Type 3 secretion systems 2 T3SS2)是副溶血性弧菌主要的毒力因子,vopB2是T3SS2中的一个关键效应蛋白。【目的】研究副溶血弧菌CalR对vopB2的转录调控机制。【方法】利用引物延伸实验鉴定vopB2及vtrA的转录起始位点,并根据产物的丰度判断CalR对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和ΔcalR中转录丰度,验证CalR对靶基因的转录调控关系,进一步利用LacZ实验通过比较β-半乳糖苷酶活性的差异来判定CalR对靶基因的调控关系;利用凝胶阻滞实验分析His-CalR对靶基因启动子区是否具有直接的结合作用。【结果】vopB2有两个转录起始位点A(-130和-28)且其活性受CalR的直接抑制;引物延伸和LacZ结果表明CalR对vtrA的转录并无调控作用。【结论】CalR直接抑制vopB2的转录,该抑制作用与vtrA无关联。     

QsvR对副溶血弧菌VI型分泌系统1相关基因的转录调控

【目的】研究调控蛋白QsvR对副溶血弧菌VI型分泌系统1 （type VI secretion system 1,T6SS1）相关基因的转录调控关系。【方法】提取野生株（wild type,WT）和qsvR突变株（ΔqsvR）的总RNA,采用实时定量PCR （quantitative real-time PCR,qPCR）研究QsvR对靶基因的调控关系;进而采用引物延伸法定位靶基因的转录起始位点和核心启动子区,并根据引物延伸产物丰度判断QsvR对靶基因的调控关系;将靶基因的调控区DNA序列克隆入pHRP309质粒中的β-半乳糖苷酶基因上游（LacZ重组质粒）,并将重组质粒转化入WT和ΔqsvR中,通过LacZ报告基因融合试验研究QsvR对靶基因的调控关系;将LacZ重组质粒分别转化入含有pBAD33或pBAD33-qsvR的大肠杆菌100lpir中,进一步采用LacZ报告基因融合试验研究在异体宿主中QsvR对靶基因的调控关系;PCR扩增靶基因调控区DNA序列,同时表达并纯化His-QsvR重组蛋白,采用凝胶阻滞试验（electrophoresis mobility shift assay,EMSA）研究His-QsvR对靶基因调控区DNA序列是否具有直接的结合作用。【结果】qPCR结果显示,与WT相比,ΔqsvR中T6SS1相关基因VP1388 （操纵子VP1388-1390首基因）和hcp1 （操纵子VP1393-1406首基因）的转录水平显著性升高,表明QsvR抑制VP1388和hcp1的转录;引物延伸结果显示VP1388和hcp1各有一个转录起始位点,分别为C （-64）和T （-62）,且它们的转录活性受QsvR的抑制;LacZ报告基因融合试验结果显示QsvR可以抑制副溶血弧菌和EC100lpir中VP1388和hcp1的启动子区转录活性;EMSA结果显示His-QsvR对VP1388和hcp1的启动子区DNA序列具有直接的结合活性。【结论】QsvR对T6SS1相关操纵子VP1388-1390和VP1393-1406的转录具有直接的抑制作用。     

鼠疫菌OxyR调控子蛋白对dps的转录调控机制

[目的]利用分子生物学实验研究鼠疫菌调控子OxyR对dps的转录调控机制.[方法]提取鼠疫菌野生株(WT)和oxyR突变株(ΔoxyR)的总RNA,采用引物延伸实验研究dps的转录起始位点,并根据产物的丰度判断OxyR对dps的调控关系.进一步采用实时定量RT-PCR的方法验证OxyR对dps的调控关系.PCR扩增dps的整个启动子区DNA序列,并纯化His-OxyR蛋白,通过凝胶阻滞实验(EMSA)验证OxyR对dps启动子区是否具有直接的相互作用.利用大肠杆菌OxyR识别基序,预测鼠疫菌OxyR对dps启动子区的结合位点,从而得出鼠疫菌OxyR对dps的转录调控机制.[结果]鼠疫菌dps有一个转录起始位点G(-40)(翻译起始位点为+1),其转录表达受OxyR的激活;体外实验及生物信息学预测结果表明OxyR能结合到dps启动子区-111到-78之间的碱基上.[结论]OxyR能直接结合到dps启动子区而激活其转录表达.     

大肠杆菌Small RNA EsrE的转录调控

【背景】大肠杆菌中Small RNA EsrE调控琥珀酸脱氢酶的表达并影响细胞生长,对其调控机制的探究有利于加深EsrE对细胞生长影响的认识。【目的】探究大肠杆菌Small RNA EsrE的转录调控机制。【方法】通过双质粒报告系统筛选转录调控因子,并通过凝胶迁移实验(electrophoretic mobilityshiftassay,EMSA)和qRT-PCR研究方法验证转录调控因子。【结果】双质粒报告系统证明RNA聚合酶亚基σ~(32) (RpoH)上调P_(esrE),β-羟酰-ACP脱水酶(FabZ)下调PesrE。EMSA结果和体内实验显示RpoH结合P_(esrE)片段,FabZ不结合P_(esrE)片段。【结论】RpoH直接结合启动子序列参与调控,FabZ以其他方式间接参与Small RNA EsrE的转录调控。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.