Elucidation of the stability and functional regions of the human coronavirus OC43 nucleocapsid protein

Human coronavirus OC43 (HCoV‐OC43) is one of the causes of the “common cold” in human during seasons of cold weather. The primary function of the HCoV‐OC43 nucleocapsid protein (N protein) is to recognize viral genomic RNA, which leads to ribonucleocapsid formation. Here, we characterized the stability and identified the functional regions of the recombinant HCoV‐OC43 N protein. Circular dichroism and fluorescence measurements revealed that the HCoV‐OC43 N protein is more highly ordered and stabler than the SARS‐CoV N protein previously studied. Surface plasmon resonance (SPR) experiments showed that the affinity of HCoV‐OC43 N protein for RNA was approximately fivefold higher than that of N protein for DNA. Moreover, we found that the HCoV‐OC43 N protein contains three RNA‐binding regions in its N‐terminal region (residues 1–173) and central‐linker region (residues 174–232 and 233–300). The binding affinities of the truncated N proteins and RNA follow the order: residues 1–173–residues 233–300 > residues 174–232. SPR experiments demonstrated that the C‐terminal region (residues 301–448) of HCoV‐OC43 N protein lacks RNA‐binding activity, while crosslinking and gel filtration analyses revealed that the C‐terminal region is mainly involved in the oligomerization of the HCoV‐OC43 N protein. This study may benefit the understanding of the mechanism of HCoV‐OC43 nucleocapsid formation.     

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

A Simple Solvation Model Along with A Multibody Dynamics Strategy (MBO(N)D) Produces Stable DNA Simulations that are Faster than Traditional Atomistic Methods

Abstract

We are developing solvation strategies that complement the speed advantage of MBO(N)D (a multibody simulation approach developed by Moldyn) for simulating biomolecular systems. In this report we propose to approximate the effect of bulk waters on DNA by using only a thin layer of waters proximate to the surface of DNA (which we will call the ‘thin shell approach’ or TSA). We will show that the TSA combined with substructuring (the grouping of atoms into rigid or flexible bodies) of the Dickerson dodecamer produces good comparisons with standard atomistic methods (over a nanosecond trajectory) as judged by a variety of DNA specific geometric (e.g., CURVES output) and dynamics (power spectra) properties. The MBO(N)D method, however, was faster than atomistic by a factor of six using the same solvation strategy and factor of 70 when compared to fully solvated atomistic system. The key to the speed of MBO(N)D is in its ability to use large time steps during dynamics. By keeping only a shell of molecules of water proximate to the dodecamer, we limit artifacts due to surface tension at the water-vacuum interface. These proximate waters are fairly immobile as compared to those in bulk and therefore do not severely limit the time step in the simulation. The strengths and limitations of this solvation approach, and future directions, will also be discussed.     

Salmonella enters a dormant state within human epithelial cells for persistent infection

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence.     

不同剂量地佐辛用于腹腔镜妇科手术超前镇痛的效果观察

目的：观察不同剂量地佐辛用于腹腔镜妇科手术超前镇痛的效果。方法：将90例ASAI．II级、年龄18~60岁拟行腹腔镜妇科手术的患者随机分成3组,每组30例,A组给予地佐辛0．1mg／kg开皮前15rain静脉注射;B组给予地佐辛0．15mg／kg开皮前15min静脉注射;C组给予地佐辛0．2mg／kg开皮前15min静脉注射,采用VAS评分评估术后镇痛效果并观察术后辅助镇痛药物的使用和不良反应的发生情况。结果：B、c组术后2、4、6、8h的VAS评分明显低于A组（P〈0．05）;C组术后2、4h的Ramsay评分明显高于A、B组（P〈0.05）;A组术后辅助镇痛药的使用率明显高于B组和C组（P／0．05）;3组不良反应的发生率比较均无统计学差异（P〉0．05）。结论：开皮前15rain静注地佐辛0．15mg／kg用于腹腔镜妇科手术镇痛效果好,且不增加不良反应的发生率。     

Fill Factor Losses in Cu2ZnSn(SxSe1−x)4 Solar Cells: Insights from Physical and Electrical Characterization of Devices and Exfoliated Films

Besides the open circuit voltage (V_OC) deficit, fill factor (FF) is the second most significant parameter deficit for earth‐abundant kesterite solar cell technology. Here, various pathways for FF loss are discussed, with focus on the series resistance issue and its various contributing factors. Electrical and physical characterizations of the full range of bandgap (E_g = 1.0–1.5 eV) Cu₂ZnSn(S_xSe_1?x)₄ (CZTSSe) devices, as well as bare and exfoliated films with various S/(S + Se) ratios, are performed. High intensity Suns‐V_OC measurement indicates a nonohmic junction developing in high bandgap CZTSSe. Grazing incidence X‐ray diffraction, Raman mapping, field emission scanning electron microscopy, and X‐ray photoelectron spectroscopy indicate the formation of Sn(S,Se)₂, Mo(S,Se)₂, and Zn(S,Se) at the high bandgap CZTSSe/Mo interface, contributing to the increased series resistance (R_S) and nonohmic back contact characteristics. This study offers some clues as to why the record‐CZTSSe solar cells occur within a bandgap range centered around 1.15 eV and offers some direction for further optimization.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.