An antibody-based microarray assay for small RNA detection

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 µl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs

Numerical study of inflow equivalence ratio inhomogeneity on oblique detonation formation in hydrogen-air mixtures

In this study, numerical simulations using Euler equations with detailed chemistry are performed to investigate the effect of fuel-air composition inhomogeneity on the oblique detonation wave (ODW) initiation in hydrogen-air mixtures. This study aims for a better understanding of oblique detonation wave engine performance under practical operating conditions, among those is the inhomogeneous mixing of fuel and air giving rise to a variation of the equivalence ratio (ER) in the incoming combustible flow. This work focuses primarily on how a variable equivalence ratio in the inflow mixture affects both the formation and characteristic parameters of the oblique detonation wave. In this regard, the present simulation imposes initially a lateral linear distribution of the mixture equivalence ratio within the initiation region. The variation is either from fuel-lean or fuel-rich to the uniform stoichiometric mixture condition above the oblique shock wave. The obtained numerical results illustrate that the reaction surface is distorted in the cases of low mixture equivalence ratio. The so-called “V-shaped” flame is observed but differed from previous results that it is not coupled with any compression or shock wave. Analyzing the temperature and species density evolution also shows that the fuel-lean and fuel-rich inhomogeneity have different effects on the combustion features in the initiation region behind the oblique shock wave. Two characteristic quantities, namely the initiation length and the ODW surface position, are defined to describe quantitatively the effects of mixture equivalence ratio inhomogeneity. The results show that the initiation length is mainly determined by the mixture equivalence ratio in the initiation region. Additional computations are performed by reversing ER distribution, i.e., with the linear variation above the initiation region of uniform stoichiometric condition and results also demonstrate that the ODW position is effectively determined by the ER variation before the ODW, which has in turn only negligible effect on the initiation length

Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

新型冠状病毒SARS-CoV-2在全球蔓延，给全球公共卫生带来严重威胁。快速研制疫苗、抗体和治疗药物成为科学界面临的重大挑战。由于SARS-CoV-2的高度传染性，采用病毒感染模型进行中和抗体及小分子抑制剂的药效评估需要在高等级生物安全实验室中进行，且常需要数天时间才能完成检测，限制了抗体和药物筛选的效率。发展快速、可视、不依赖于活病毒的新冠病毒入胞检测探针和细胞模型，对于加速新冠病毒抗体和药物的研究有重要意义。夏宁邵教授团队通过CHO真核表达系统高效表达制备出C端融合抗酸荧光蛋白Gamillus的重组新冠病毒spike蛋白STG。STG经SEC分子筛和冷冻电镜确认呈现与天然病毒刺突高度相似的三聚体结构，且与ACE2有很高的亲和力（18.2nM）。STG具备良好的细胞相容性和荧光性质，研究者进一步开发了可定量测定感染恢复期血清、疫苗免疫血清中和抗体（入胞阻断抗体）水平的CSBT检测方法。除了抗体检测评估方面的应用外，该研究发展的探针和模型还可用于筛选分析抑制新冠病毒入胞及胞内转运的小分子化合物。 我校博士后张雅丽，博士生王邵娟、巫洋涛，博士后侯汪衡、袁伦志和深圳市第三人民医院沈晨光博士为共同第一作者。厦门大学夏宁邵教授、袁权教授、程通教授为该论文共同通讯作者。The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system,a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed.In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.This study was supported by National Natural Science Foundation of China (81993149041 for N.X.; 81902057 for Y.Z.; 81871316 and U1905205 for Q.Y.), the National Science and Technology Major Project of Infectious Diseases (No. 2017ZX10304402‐002‐003 for T.C. and No. 2017ZX10202203‐009 for Q.Y.), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003‐005‐003 for T.C.), the Science and Technology Major Project of Fujian (2020YZ014001), the Science and Technology Major Project of Xiamen (3502Z2020YJ01), and the Guangdong Basic and Applied Basic Research Foundation (2020A1515010368 for C.S.). 该研究得到了国家自然科学基金、传染病防治国家科技重大专项、福建省应急科技攻关项目和厦门应急科技攻关项目的支持

Recruitment of MinC, an Inhibitor of Z-Ring Formation, to the Membrane in Escherichia coli: Role of MinD and MinE

In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC

Advances in critical technologies for hypersonic and high-enthalpy wind tunnel

Hypersonic and high-enthalpy wind tunnels and their measurement techniques are the cornerstone of the hypersonic flight era that is a dream for human beings to fly faster, higher and further. The great progress has been achieved during the recent years and their critical technologies are still in an urgent need for further development. There are at least four kinds of hypersonic and high-enthalpy wind tunnels that are widely applied over the world and can be classified according to their operation modes. These wind tunnels are named as air-directly-heated hypersonic wind tunnel, light-gas-heated shock tunnel, free-piston-driven shock tunnel and detonation-driven shock tunnel, respectively. The critical technologies for developing the wind tunnels are introduced in this paper, and their merits and weakness are discussed based on wind tunnel performance evaluation. Measurement techniques especially developed for high-enthalpy flows are a part of the hypersonic wind tunnel technology because the flow is a chemically reacting gas motion and its diagnosis needs specially designed instruments. Three kinds of the measurement techniques considered to be of primary importance are introduced here, including the heat flux sensor, the aerodynamic balance, and optical diagnosis techniques. The techniques are developed usually for conventional wind tunnels, but further improved for hypersonic and high-enthalpy tunnels. The hypersonic ground test facilities have provided us with most of valuable experimental data on high-enthalpy flows and will play a more important role in hypersonic research area in the future. Therefore, several prospects for developing hypersonic and high-enthalpy wind tunnels are presented from our point of view. (c) 2020 Chinese Society of Aeronautics and Astronautics. Production and hosting by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Circular RNA circPOFUT1 enhances malignant phenotypes and autophagy-associated chemoresistance via sequestrating miR-488-3p to activate the PLAG1-ATG12 axis in gastric cancer

Abstract Circular RNAs are key regulators in regulating the progression and chemoresistance of gastric cancer (GC), suggesting circular RNAs as potential therapeutic targets for GC. The roles of a novel circular RNA circPOFUT1 in GC are unknown. Here, we found that circPOFUT1 was upregulated in GC tissues and cells, and increased circPOFUT1 expression indicated poor prognosis. Overexpression of circPOFUT1 enhanced cell proliferation, migration, invasion and autophagy-associated chemoresistance in GC, which were suppressed by miR-488-3p overexpression. CircPOFUT1 reduced miR-488-3p expression via sponging miR-488-3p in GC cells. PLAG1 interacted with ATG12 and promoted its expression. MiR-488-3p bound to PLAG1 and suppressed the expression of PLAG1 and ATG12 in GC cells. Overexpression of circPOFUT1 enhanced autophagy-associated chemoresistance of GC cells in vivo, but it was inhibited by overexpression of miR-488-3p. Collectively, circPOFUT1 directly sponged miR-488-3p to activate the expression of PLAG1 and ATG12, thus enhancing malignant phenotypes and autophagy-associated chemoresistance in GC. Our findings show the potential of circPOFUT1 as biomarkers and targeting circPOFUT1 as a therapeutic strategy for GC

A theoretical and computational study of the vibration excitation on the transition criteria of shock wave reflections

In this paper, we study the vibration excitation on the reflection of shock waves in hypersonic flows by using analytical and computational approaches. First, a theoretical approach is established to solve the shock relations which are further applied to develop the shock polar analytical method for high-temperature air. Then, a comparative investigation using calorically perfect gas model and thermally perfect gas model considering vibration excitation indicates an obvious change to the overall profile of the shock polar. The post-shock pressure increases within the strong branch of the shock polar while decreases within the weak branch due to vibration excitation of air molecules. A more notable phenomenon is the increase in the maximum deflection angle of the shock polar which can significantly influence the detachment criterion of shock reflection transition in high-temperature air flows. The shock polar analysis of shock reflection shows that the vibration excitation result in an obvious increase to the detachment criterion while a slight increase to the von Neumann criterion. A series of computations are conducted to confirm the above analytical findings on the shock reflection considering the vibration excitation. A slight difference of transition criterion between the theory and computations is found to be caused by the existence of the expansion fan which is an inherent flow structure. The proposed shock polar analytical method is proved to be an effective but simple approach for the study of shock wave reflections in hypersonic flows. (C) 2019 Elsevier Masson SAS. All rights reserved