Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.     

Rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.     

Vaccine Strategies for the Control and Prevention of Japanese Encephalitis in Mainland China, 1951–2011

Japanese encephalitis (JE) is arguably one of the most serious viral encephalitis diseases worldwide. China has a long history of high prevalence of Japanese encephalitis, with thousands of cases reported annually and incidence rates often exceeding 15/100,000. In global terms, the scale of outbreaks and high incidence of these pandemics has almost been unique, placing a heavy burden on the Chinese health authorities. However, the introduction of vaccines, developed in China, combined with an intensive vaccination program initiated during the 1970s, as well as other public health interventions, has dramatically decreased the incidence from 20.92/100,000 in 1971, to 0.12/100,000 in 2011. Moreover, in less readily accessible areas of China, changes to agricultural practices designed to reduce chances of mosquito bites as well as mosquito population densities have also been proven effective in reducing local JE incidence. This unprecedented public health achievement has saved many lives and provided valuable experience that could be directly applicable to the control of vector-borne diseases around the world. Here, we review and discuss strategies for promotion and expansion of vaccination programs to reduce the incidence of JE even further, for the benefit of health authorities throughout Asia and, potentially, worldwide.     

三维技术对安徽白鹭洲战国墓M566墓主容貌的复原

2011年安徽白鹭洲发现战国时期保存完整的楚国贵族墓葬,因墓主人头发和发簪保存完好、墓主身份显赫而备受关注。该头骨保存完整,具有亚洲蒙古人种的特征,根据骨骼的形态推测其为女性,年龄为35-39岁。为展现该地区战国贵族妇女的容貌、丰富该地区考古多样性提供研究材料。本文首先使用高分辨率CT对该个体的头骨及下颌骨进行了扫描和重建,然后采用基于偏最小二乘回归的颅面复原方法实现生前容貌基本形态的复原。最后,结合考古资料,利用三维模型处理软件对面貌复原模型及其发饰进行了三维建模和纹理贴图等处理,提高了颅面复原模型的真实感,生动形象地再现战国贵族女性面部的形态特征。     

亚成体大熊猫肠道真菌多样性

【目的】分析5只亚成体大熊猫肠道真菌的多样性。【方法】采用基于ITS基因的RFLP(Restriction Fragment Length Polymorphism)方法,对肠道真菌总DNA进行ITS-RFLP分析,构建ITS克隆文库,然后用HhaI、HaeIII进行酶切指纹图谱分析,测序并绘制系统进化树。【结果】研究表明,5只亚成体大熊猫肠道真菌主要由Ascomycota(平均占46.24%)、Basidiomycota(平均占15.79%)2个门和一些未分类(平均占29.14%)、未培养(平均占8.83%)的真菌。其中,Ascomycota主要以Saccharomycetes(平均占63.74%)和Dothideomycetes(平均占35.91%)2个纲为主;Basidiomycota主要以Tremellomycetes(平均占65.80%)和Microbotryomycetes(平均占33.15%)2个纲为主。而这4个纲分别则主要以Candida、Debaryomyces;Pleosporales、Myriangium;Cystofilobasidium、Trichosporon;Leucosporidium、Leucosporidiella八个菌属为主,各样品中所占比例不等。【结论】亚成体大熊猫肠道内存在一定比例的真菌菌群,且ITS-RFLP技术能够很好地对其多样性进行分析。真菌的发现扩大了我们对大熊猫肠道微生物结构的了解,同时也有助于我们进一步研究真菌能否帮助大熊猫消化高纤维素食物。     

An improved procedure for isolating adult mouse cardiomyocytes for epicardial activation mapping

Cardiovascular disease is a leading cause of death and disability worldwide. Although genetically modified mouse models offer great potential for robust research in vivo, in vitro studies using isolated cardiomyocytes also provide an important approach for investigating the mechanisms underlying cardiovascular disease pathogenesis and drug actions. Currently, isolation of mouse adult cardiomyocytes often relies on aortic retrograde intubation under a stereoscopic microscope, which poses considerable technical barriers and requires extensive training. Although a simplified, Langendorff-free method has been used to isolate viable cardiomyocytes from the adult mouse heart, the system requires enzymatic digestions and continuous manual technical operation. This study established an optimized approach that allows isolation of adult mouse cardiomyocytes and epicardial activation mapping of mouse hearts using a Langendorff device. We used retrograde puncture through the abdominal aorta in vivo and enzymatic digestion on the Langendorff perfusion device to isolate adult mouse cardiomyocytes without using a microscope. The yields of isolated cardiomyocytes were amenable to patch clamp techniques. Furthermore, this approach allowed epicardial activation mapping. We used a novel, simplified method to isolate viable cardiomyocytes from adult mouse hearts and to map epicardial activation. This novel approach could be beneficial in more extensive research in the cardiac field.     

Isolation and Genetic Characterization of Mangshi Virus: A Newly Discovered Seadornavirus of the Reoviridae Family Found in Yunnan Province,China

Background

Seadornavirus is a genus of viruses in the family Reoviridae, which consists of Banna virus, Kadipiro virus, and Liao ning virus. Banna virus is considered a potential pathogen for zoonotic diseases. Here, we describe a newly discovered Seadornavirus isolated from mosquitos (Culex tritaeniorhynchus) in Yunnan Province, China, which is related to Banna virus, and referred to as Mangshi virus.

Methods and Results

The Mangshi virus was isolated by cell culture in Aedes albopictus C6/36 cells, in which it replicated and caused cytopathic effects, but not in mammalian BHK-21 or Vero cells. Polyacrylamide gel analysis revealed a genome consisting of 12 segments of double-stranded RNA, with a “6–4–2” pattern in which the migrating bands were different from those of the Banna virus. Complete genome sequencing was performed by full-length amplification of cDNAs. Sequence analysis showed that seven highly conserved nucleotides and three highly conserved nucleotides were present at the ends of the 5′- and 3′-UTRs in each of 12 genome segments. The amino acid identities of Mangshi virus shared with Balaton virus varied from 27.3% (VP11) to 72.3% (VP1) with Banna virus varying from 18.0% (VP11) to 63.9% (VP1). Phylogenetic analysis based on amino acid sequences demonstrated that Mangshi virus is a member of the genus Seadornavirus and is most closely related to, but distinct from, Balaton virus and Banna virus in the genus Seadornavirus of the family Reoviridae.

Conclusion

Mangshi virus isolated from mosquitoes (C. tritaeniorhynchus) was identified as a newly discovered virus in the genus Seadornavirus and is phylogenetically close to Banna virus, suggesting that there is genetic diversity of seadornaviruses in tropical and subtropical areas of Southeast Asia.     

硫酸肝素与甲病毒感染

硫酸肝素存在于细胞膜表面、基底膜及细胞外基质,是一种高度硫酸化的、带负电荷的多糖结构。研究表明辛德毕斯病毒等甲病毒可通过与细胞表面的硫酸肝素结合进入宿主细胞,完成对细胞的感染。提示细胞表面的硫酸肝素是甲病毒感染细胞的受体或共受体。     

薄毛海绵杜鹃结实和萌发特性随环境的变化

选择青藏高原东南林线地区优势植物薄毛海绵杜鹃为对象,研究薄毛海绵杜鹃结实特性和萌发特性随海拔(4183—4673 m)、坡度、坡向等环境梯度的变化规律。对比了不同环境条件下薄毛海绵杜鹃果实的长、宽、重量、单果种子数、种子千粒重等结实特性;并测试了不同环境所产种子的萌发对温度和光照的响应。结果表明:(1)在高海拔低温胁迫环境下,薄毛海绵杜鹃的生殖投入加大,果实大且质量重,种子数量多但质量轻;(2)坡向是影响单果种子数量的主要环境因子,阴坡单果种子数显著大于阳坡,生活在阴坡的薄毛海绵杜鹃的繁衍能力更强;(3)较高和较低土壤湿度都会影响薄毛海绵杜鹃的种子质量,中等土壤湿度(28.3%—32.5%)薄毛海绵杜鹃种子萌发能力最强;(4)薄毛海绵杜鹃的种子是需光性种子,在黑暗条件下几乎不萌发;(5)在光照条件下,温度对种子萌发率和萌发速率有显著影响,高温(25/5℃)有助于提高萌发率并促使萌发进程显著提前。研究揭示了不同海拔薄毛海绵杜鹃结实和萌发特性与环境之间的关系,为薄毛海绵杜鹃的种质资源保护和气候变化背景下藏东南林线动态预测提供了基础资料。