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。     

CalR激活副溶血弧菌Ⅵ型分泌系统1相关基因的转录

【目的】研究副溶血弧菌群体感应(quorum sensing,QS)系统核心调控子AphA和OpaR对calR基因以及CalR对VI型分泌系统1 (type Ⅵ secretion system 1,T6SS1;vp1386-1420)相关基因的转录调控关系。【方法】提取副溶血弧菌野生株(wild-type,WT)和调控子基因突变株的总RNA,采用实时定量PCR (quantitative real-time PCR,qPCR)研究调控子对靶基因的转录调控关系;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调控子对靶基因的调控关系;将靶基因的启动子区克隆入pHRP309质粒的β-半乳糖苷酶基因上游,构建LacZ重组质粒,并将该重组质粒转入WT和调控子基因突变株中,获得LacZ菌株,通过LacZ报告基因融合实验进一步研究调控子对靶基因的调控关系。PCR扩增靶基因的上游调控区DNA序列,并纯化His-重组调控子蛋白,通过凝胶阻滞实验(electrophoreticmobilityshiftassay,EMSA)研究His-重组蛋白对靶基因调控区DNA序列是否具有直接的结合作...     

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。     

AphA蛋白对副溶血弧菌vopT的转录调控研究

【目的】研究副溶血弧菌AphA对vopT的转录调控机制。【方法】提取野生株(WT)和aphA突变株(ΔaphA)的总RNA,采用引物延伸实验研究vopT的转录起始位点,并根据产物的丰度差异判断AphA对其调控关系。分别将WT和ΔaphA的总RNA逆转录成cDNA,利用实时定量RT-PCR进一步研究AphA对靶基因的调控关系。将vopT的启动子区克隆入pHRP309质粒的β-半乳糖苷酶基因上游,构建LacZ重组质粒,并将该重组质粒转入WT和ΔaphA中,通过测定并比较两株菌中β-半乳糖苷酶活性的差异来判定AphA对vopT的调控关系。PCR扩增靶基因整个启动子区DNA序列,并纯化His-AphA蛋白,利用凝胶阻滞实验(EMSA)验证His-AphA对靶基因启动子区是否具有直接的结合作用。【结果】vopT只有一个转录起始位点A (?86),且其转录活性受AphA的间接抑制。RT-PCR和EMSA结果显示AphA对vtrA的转录也具有间接的抑制作用。【结论】AphA间接抑制vopT转录,且该间接抑制作用与VtrA无关。     

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。     

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。     

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...     

基于遗传片段分析系统的转录起始位点分析技术:从预测到结果评估

【目的】以遗传片段分析仪内标法替代传统放射性标记引物延伸技术进行样本转录起始位点(TSS)分析,并弥补引物延伸技术应用于未知样本缺乏前期预测和后期评估环节,形成一套基于遗传片段分析仪内标法分析未知样品TSS的完整技术方案。【方法】以粘球菌Myxococcus DK1622来源的双拷贝Gro ELs基因为素材;首先从预测出发,利用数据库进行启动子和转录起始位点预测;其次,根据预测结果设计合成荧光标记引物进行靶标m RNA的反转录;再次,应用遗传片段分析技术内标法鉴定分析粘球菌来源的双拷贝Gro ELs基因转录起始位点(TSS)及其丰度;最后,应用正态分布理论进行鉴定结果评估。【结果】明确了转录起始位点的数量、转录丰度及最可能的TSS位点:粘球菌DK1622基因组中Gro EL1拷贝存在1个启动子,TSS位点为TSS_(286);Gro EL2拷贝存在2个启动子,TSS位点分别为TSS_(548)和TSS_(502),其中TSS_(548)转录丰度是TSS_(502)的13.8倍,Gro EL1的TSS_(286)丰度是gro EL2的TSS_(548)丰度的14.3倍。【结论】预测结果指明了实验设计的范围,遗传片段分析仪内标检测法替代传统放射性标记法使实验更加简便、安全、自动、准确,正态分布理论进一步评估了实验结果的可信度,三者接合形成了完善的转录起始位点鉴定技术方案。     

利用优化的5′-RACE实验定位副溶血弧菌基因的转录起始位点

目的:优化5′-cDNA末端快速扩增(5′-RACE)实验平台,用于定位副溶血弧菌(VP)基因的转录起始位点。方法:提取VP的总RNA,用rDNaseⅠ消化去除可能污染的基因组DNA;利用T4 RNA连接酶将已知序列的寡核苷酸片段连接至RNA的5′端,进而将其逆转录成cDNA;以cDNA为模板,采用巢式PCR技术扩增目的基因DNA片段,并将其直接克隆入T载体;最后通过测序比对的方法确定靶基因的转录起始位点。利用引物延伸实验进一步研究VPA1027的转录起始位点,以检验5′-RACE实验结果的可靠性。结果:5′-RACE实验结果表明,VPA1027、scrG、scrA、cpsA及VPA0198的转录起始位点分别为G(-103)、G(-70)、T(-205)、C(-129)和G(-238)(翻译起始位点为+1);引物延伸结果显示,VPA1027的转录起始位点也为G(-103)。结论:优化后的5′-RACE实验可以精确定位VP基因的转录起始位点。     

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。     

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.