Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species

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Design,synthesis, and structure–activity-relationship of phenyl imidazoles as potent Smoothened antagonists

Through scaffold morphing of a known Smoothened antagonist Antag691, a series of novel phenyl imidazole derivatives were developed. Structure–activity-relationship studies and lead optimization led to the discovery of potent, selective and orally bioavailable Smoothened antagonist 19 that is suitable for in vivo studies.     

Balancing Selection on a Regulatory Region Exhibiting Ancient Variation That Predates Human–Neandertal Divergence

Ancient population structure shaping contemporary genetic variation has been recently appreciated and has important implications regarding our understanding of the structure of modern human genomes. We identified a ∼36-kb DNA segment in the human genome that displays an ancient substructure. The variation at this locus exists primarily as two highly divergent haplogroups. One of these haplogroups (the NE1 haplogroup) aligns with the Neandertal haplotype and contains a 4.6-kb deletion polymorphism in perfect linkage disequilibrium with 12 single nucleotide polymorphisms (SNPs) across diverse populations. The other haplogroup, which does not contain the 4.6-kb deletion, aligns with the chimpanzee haplotype and is likely ancestral. Africans have higher overall pairwise differences with the Neandertal haplotype than Eurasians do for this NE1 locus (p<10⁻¹⁵). Moreover, the nucleotide diversity at this locus is higher in Eurasians than in Africans. These results mimic signatures of recent Neandertal admixture contributing to this locus. However, an in-depth assessment of the variation in this region across multiple populations reveals that African NE1 haplotypes, albeit rare, harbor more sequence variation than NE1 haplotypes found in Europeans, indicating an ancient African origin of this haplogroup and refuting recent Neandertal admixture. Population genetic analyses of the SNPs within each of these haplogroups, along with genome-wide comparisons revealed significant F_ST (p = 0.00003) and positive Tajima''s D (p = 0.00285) statistics, pointing to non-neutral evolution of this locus. The NE1 locus harbors no protein-coding genes, but contains transcribed sequences as well as sequences with putative regulatory function based on bioinformatic predictions and in vitro experiments. We postulate that the variation observed at this locus predates Human–Neandertal divergence and is evolving under balancing selection, especially among European populations.     

A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (～1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.     

Structure of the Fusion Core and Inhibition of Fusion by a Heptad Repeat Peptide Derived from the S Protein of Middle East Respiratory Syndrome Coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical α-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design.     

Development of a cataluminescence sensor for detecting benzene based on magnesium silicate hollow spheres

A novel and sensitive gas sensor was developed for the determination of benzene based on its cataluminescence (CTL) by oxidation in air on the surface of hollow magnesium silicate spheres. Luminescence characteristics and optimum conditions were investigated. Results indicated that the as‐prepared magnesium silicate hollow spheres exhibited outstanding CTL properties such as stable intensity, high signal/noise values, and short response and recovery times. Under optimized conditions, benzene exhibited a broad linear range of 1–4500 ppm, with a correlation coefficient of 0.9946 and a limit of detection (signal‐to‐noise ratio (S/N) = 3) of 0.6 ppm, which was below the standard permitted concentration. The relative standard deviation (RSD) for 100 ppm benzene was 4.3% (n = 6). Furthermore, the gas sensor system showed outstanding selectivity for benzene compared with nine other common volatile organic compounds (VOCs). The proposed gas sensor showed good characteristics of high selectivity, fast response time and long lifetime, which suggested the promising application of magnesium silicate hollow spheres as a novel highly efficient CTL sensing material. The mechanism for the improved performance was also discussed based on the experimental results. Copyright © 2014 John Wiley & Sons, Ltd.     

基于格网的南昌市生态环境脆弱性评价

科学评价生态环境的脆弱性,对合理利用土地资源和促进区域生态安全具有重要意义。以公里格网为基本评价单元,以快速城市化区域典型代表南昌市为研究区,通过SRP概念模型构建评价指标体系并选取17个评价指标,基于空间主成分分析法、全局Moran′s I指数以及LISA聚类图,量化分析了2000—2015年南昌市生态环境脆弱性的时空分布特征及驱动力。结果表明:(1)2000—2015年南昌市生态环境脆弱性呈现由轻度脆弱向中度脆弱演变的趋势;(2)2000—2015年南昌市各县(区)生态环境脆弱性差异明显,东湖区、西湖区、青山湖区、青云谱区、湾里区脆弱性程度最高,其他县(区)脆弱性增幅较大;(3)2000和2015年南昌市生态环境脆弱性具有明显的空间自相关性,且为正相关,高高聚集区主要分布在极度脆弱区,低低聚集区与微度和轻度脆弱区相关;(4)研究时段内,南昌市生态环境脆弱性主要驱动力为人口密度、人均GDP、人均耕地、坡度、地形起伏度、年均气温、极端最高温、极端最低温、年均降水量。     

广州地区GⅡ.17型诺如病毒GZ-L343毒株病毒样颗粒的构建与免疫特征的鉴定

【背景】人源诺如病毒是急性胃肠炎暴发的主要原因,GII.4是过去几十年的主要流行基因型。2014/2015年出现的GII.17型变异株是中国首例导致大规模暴发的非GII.4流行株。通过对来自华南地区的诺如病毒GII.17型毒株的完整基因组序列进行分析,证实了该GII.17型突变株与先前确定的GII型变异株不同。【目的】制备广州地区GII.17型诺如病毒GZ-L343的病毒样颗粒,并系统表征其免疫原性及功能特性。【方法】借助杆状病毒表达系统制备GII.17-GZ-L343的病毒样颗粒,并通过氯化铯梯度超速离心对其进行纯化,制备抗血清并对其免疫功能进行评价。【结果】聚丙烯酰胺凝胶电泳和蛋白质免疫印迹结果表明所得蛋白分子量大小约为58kDa;透射电镜结果表明病毒样颗粒直径约为30nm;酶联免疫吸附测定结果显示该病毒样颗粒具有较好的免疫原性;唾液组织血型抗原的体外受体结合测定表明,该病毒样颗粒与部分A型、B型、O型及AB型分泌及非分泌血型样本存在阳性结合;效价测定结果表明免疫所得血清效价在104以上;交叉反应结果表明该抗血清与异型病毒样颗粒不存在交叉反应。此外,体外阻断结果表明,该抗血清仅能阻...     

新型鸭细小病毒样颗粒的制备及鉴定

【背景】鸭短喙侏儒综合征(beak atrophy and dwarfism syndrome, BADS)是由新型鸭细小病毒(novel duck Parvovirus, NDPV)感染导致雏鸭生长发育迟缓、上下喙萎缩的疾病。BADS的暴发给我国养鸭业造成了巨大的经济损失。【目的】利用大肠杆菌表达系统制备NDPV病毒样颗粒(virus-like particles, VLPs),为研制NDPV相关疫苗奠定基础。【方法】对NDPV VP2序列全长进行密码子优化、合成,连接至pColdTF表达载体,获得pColdTF-NDPV-VP2重组质粒,酶切、测序鉴定正确后将重组质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)对蛋白表达进行可溶性分析;使用凝血酶(thrombin)切除trigger factor (TF)标签,利用镍柱(Ni-NTA)亲和层析方法纯化重组蛋白;利用Western blotting对纯化后的VP2蛋白进行反应原性分析;利用透射电镜、动态光散射观察重组蛋白形态以及能否形成VLPs。【结果】构建了pColdTF-NDPV-VP2重组质粒,在大肠杆菌中主要以可溶性形式表达,融合蛋白TF-VP2大小约为115 kDa,去除TF标签经镍柱纯化后得到67 kDa的VP2蛋白;Western blotting试验表明VP2蛋白能与NDPV鸭阳性血清发生特异性结合;通过透射电镜可以观察到形状规则、直径约为20−25 nm的病毒样颗粒。【结论】利用大肠杆菌表达系统制备了NDPV VLPs,为下一步研发BADS相关亚单位疫苗及生物相关制品提供了基础。     

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family

Theγ-actin(ACTG1)gene is a cytoplasmic nonmuscle actin gene,which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea.Mutations in ACTG1 were found to cause autosomal dominant,progressive,sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families,respectively.In this study,a novel missense mutation (c.364A>G;p.I122V)co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls.The alteration of residue I1e122 was predicted to damage its interaction with actin-binding proteins,which may cause disruption of hair cell organization and function.These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.