Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. ¹³C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using ¹⁹F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~ 1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2–8.4 Å for L493F and L504F and 10.5 Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation.     

Revision of Corallinaceae (Corallinales,Rhodophyta): recognizing Dawsoniolithon gen. nov., Parvicellularium gen. nov. and Chamberlainoideae subfam. nov. containing Chamberlainium gen. nov. and Pneophyllum

A multi‐gene (SSU, LSU, psbA, and COI) molecular phylogeny of the family Corallinaceae (excluding the subfamilies Lithophylloideae and Corallinoideae) showed a paraphyletic grouping of six monophyletic clades. Pneophyllum and Spongites were reassessed and recircumscribed using DNA sequence data integrated with morpho‐anatomical comparisons of type material and recently collected specimens. We propose Chamberlainoideae subfam. nov., including the type genus Chamberlainium gen. nov., with C. tumidum comb. nov. as the generitype, and Pneophyllum. Chamberlainium is established to include several taxa previously ascribed to Spongites, the generitype of which currently resides in Neogoniolithoideae. Additionally we propose two new genera, Dawsoniolithon gen. nov. (Metagoniolithoideae), with D. conicum comb. nov. as the generitype and Parvicellularium gen. nov. (subfamily incertae sedis), with P. leonardi sp. nov. as the generitype. Chamberlainoideae has no diagnostic morpho‐anatomical features that enable one to assign specimens to it without DNA sequence data, and it is the first subfamily to possess both Type 1 (Chamberlainium) and Type 2 (Pneophyllum) tetra/bisporangial conceptacle roof development. Two characters distinguish Chamberlainium from Spongites: tetra/biasporangial conceptacle chamber diameter (<300 μm in Chamberlainium vs. >300 μm in Spongites) and tetra/bisporangial conceptacle roof thickness (<8 cells in Chamberlainium vs. >8 cells in Spongites). Two characters also distinguish Pneophyllum from Dawsoniolithon: tetra/bisporangial conceptacle roof thickness (<8 cells in Pneophyllum vs. >8 cells in Dawsoniolithon) and thallus construction (dimerous in Pneophyllum vs. monomerous in Dawsoniolithon).     

转录激活因子样效应物核酸酶介导的山羊β-乳球蛋白基因敲除和人乳铁蛋白基因定点整合

为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF~+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF~+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF~+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。     

持续施用缓/控释尿素条件下水田土壤NH3挥发与N2O排放特征

以持续9年施用不同缓／控释尿素的水田棕壤为试验对象,以普通大颗粒尿素为对照,研究了持续施用不同缓／控释尿素条件下水田土壤NH₃挥发与N₂O排放特征.结果表明: 与普通大颗粒尿素(U)相比,除1% 3,4－二甲基吡唑磷酸盐(DMPP)+U处理 NH₃挥发增加了25.8%外,其他缓／控释尿素肥料处理对NH₃有明显的减排效果.树脂包膜尿素(PCU)对NH₃减排效果最明显,为73.4%,硫包膜尿素(SCU)为72.2%,0.5% N-丁基硫代磷酰三胺(NBPT)+1% DMPP+U为71.9%,1% 氢醌(HQ)+3% 双氰胺(DCD)+U为46.9%,0.5% NBPT+U为43.2%,1% HQ+U为40.2%,3% DCD+U为25.5%, 1% DMPP均与施用普通大颗粒尿素差异显著;所有缓／控释尿素处理与对照相比均可显著减少N₂O排放.1% DMPP+U对N₂O减排效果最明显,为74.9%,PCU为62.1%,1% HQ+3% DCD+U为54.7%,0.5% NBPT+1% DMPP+U为42.2%,3% DCD+U为35.9%,1% HQ+U为28.9%,0.5% NBPT+U为17.7%,SCU为14.5%,均与施用普通大颗粒尿素差异显著.比较0.5% NBPT+1% DMPP+U、SCU、PCU对NH₃和N₂O减排的综合效果,3种肥料作用相近,且均明显优于其他处理,但包膜材料的成本较抑制剂高数倍.因此,同时添加脲酶和硝化抑制剂的缓释尿素是减少水田氮素损失及环境污染的首选氮肥.     

Cold stress promoting a psychrotolerant bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fragi</Emphasis> P121 producing trehaloase

A newly isolated Pseudomonas fragi P121 strain in a soil sample taken from the Arctic Circle is able to produce trehalose. The P121 strain was able to grow at temperatures ranging from 4 to 25 °C, had an optimum pH of 6.5, and an optimum salt concentration of 2 %. The P121 strain had a survival rate of 29.1 % after being repeatedly frozen and thawed five times, and a survival rate of 78.9 % when placed in physiological saline for 15 days at 20 °C after cold shock, which is far higher than the type strain Pseudomonas fragi ATCC 4973. The P121 strain could produce 2.89 g/L trehalose, which was 18.6 % of dry cell weight within 52 h in a 25 L fermention tank using the malt extract prepared from barley as medium at 15 °C, while only 11.8 % of dry cell weight at 20 °C. These results suggested that cold stress promoted the strain producing trehalose. It is the first reported cold-tolerant bacterium that produces trehalose, which may protect cells against the cold environment.     

Cell‐specific biomarkers and targeted biopharmaceuticals for breast cancer treatment

Breast cancer is the second leading cause of cancer death among women, and its related treatment has been attracting significant attention over the past decades. Among the various treatments, targeted therapy has shown great promise as a precision treatment, by binding to cancer cell‐specific biomarkers. So far, great achievements have been made in targeted therapy of breast cancer. In this review, we first discuss cell‐specific biomarkers, which are not only useful for classification of breast cancer subtyping but also can be utilized as goals for targeted therapy. Then, the innovative and generic‐targeted biopharmaceuticals for breast cancer, including monoclonal antibodies, non‐antibody proteins and small molecule drugs, are reviewed. Finally, we provide our outlook on future developments of biopharmaceuticals, and provide solutions to problems in this field.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Heavy metal contamination in surface soils of the industrial district of Wuhan,China

The Qingshan district of Wuhan City is a typical Chinese industrial area. An increase in heavy metal pollution in the region's soil, due to industrialization and urbanization, has become a serious environmental problem. Surface soil samples from 155 sites were collected and analyzed. The median concentrations of cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), and zinc (Zn) in soil were 2.3, 46.2, 24.3, 28.2, and 86.8 mg/kg, respectively. Principal component analysis coupled with hierarchical cluster analysis showed that (1) in residential and industrial areas, Pb, Cu, and Zn concentrations were mainly affected by industrial emissions and traffic emissions, whereas in agricultural areas Cu and Zn were less affected by industrial emission and traffic emission, whereas Pb was affected by agricultural activities; and (2) Cd originated from a combination of sources, including industrial activities, traffic emission, and hypergene geochemical characteristics. The integrated pollution index varied from 1.1 to 16.6 with a mean of 3.9, and 70.6% of the area is extremely contaminated, 28.1% is heavily contaminated, and the remainder is moderately contaminated.