Clumping factor B, a fibrinogen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesin of Staphylococcus aureus, also binds to the tail region of type I cytokeratin 10

The primary habitat of Staphylococcus aureus in humans is the moist squamous epithelium of the anterior nares. We showed previously that S. aureus adheres to desquamated epithelial cells and that clumping factor B (ClfB), a surface-located MSCRAMM (microbial surface components recognizing adhesive matrix molecules) known for its ability to bind to the alpha-chain of fibrinogen, is partly responsible (O'Brien, L. M., Walsh, E. J., Massey, R. C., Peacock, S. J., and Foster, T. J. (2002) Cell. Microbiol. 4, 759-770). We identified cytokeratin 10 (K10) as the ligand recognized by ClfB. Here we have shown that purified recombinant human and murine K10 immobilized on a plastic surface supports adherence of S. aureus in a ClfB-dependent manner. Furthermore, the recombinant A domain of ClfB (rClfB 45-542) bound to immobilized K10 dose-dependently and saturably. Subdomains of human and murine K10 were expressed and purified. The N-terminal head domain (residues 1-145) did not support the binding of rClfB or adherence of S. aureus ClfB+. In contrast, the C-terminal tail domains (human rHK10 452-593, mouse rMK10 454-570) promoted avid binding and adherence. Isothermal titration microcalorimetry and intrinsic tryptophan fluorescence experiments gave dissociation constants for rClfB 45-542 binding to rMK10 454-570 of 1.4 and 1.7 microM, respectively. The tail region of K10 is composed largely of quasi-repeats of Tyr-(Gly/Ser)n. A synthetic peptide corresponding to a typical glycine loop (YGGGSSGGGSSGGY; Y-Y loop peptide) inhibited the adherence of S. aureus ClfB+ to immobilized MK10 to a level of 80%, whereas control peptides had no effect. The KD of rClfB 45-542 for the Y-Y loop peptide was 5.3 microm by intrinsic tryptophan fluorescence. Thus ClfB binds to the glycine loop region of the tail domain of keratin 10 where there are probably multiple binding sites. Binding is discussed in the context of the dock-lock-latch model for MSCRAMM-ligand interactions. We provide an explanation for the molecular basis for S. aureus adherence to the squamous epithelium and suggest that nasal colonization might be prevented by reagents that inhibit this interaction.     

利用RAPD法对虹鳟鱼基因组DNA多态性研究初报

本文通过利用RAPD（随机扩增多态DNA）方法,选用20种10bp的随机引物（OPC－01－OPC—20）,用于虹鳟鱼基因组DNA多态性分析,其中发现一种引物（OPC－02）在虹鳟鱼群体中能扩增出多条带,说明该引物能反映其基因组DNA的多态性,可用于检测出不同品系间的差异。     

中国桃蚜形态学研究(英文)

对来自中国不同地区的烟草、油菜和桃树的桃蚜形态特征研究结果表明：在所研究的14个形态特征中,喙末节端部的次生毛的数目这一形态特征在所有的样本中均无显著差异。聚类分析表明采自桃树上的桃蚜的形态特征与采自其它寄生植物上的桃蚜的形态特征差异最大;其次是采自长春烟草上的桃蚜。油菜上的桃蚜在形态上比桃树上的桃蚜更接近烟草上的桃蚜。我国中南部烟草上的桃蚜又可区分为黄绿和红褐两类。在本研究中所用的14个形态特征不能明确地将我国烟草上的桃蚜与其它寄生植物上的桃蚜区分开。     

双磁珠法质粒DNA提取技术及应用

质粒是基因合成与测序领域中使用最为频繁的基因运载工具,然而传统的质粒DNA提取方法面临提取通量低、生产成本高等问题,无法满足日益增长的需求。本研究基于质粒提取原理,开发了双磁珠法(double-magnetic-bead method, DMBM)质粒提取技术,探究了磁珠投入量、质粒DNA片段大小、菌液投入量等因素对质粒提取的影响,并且对比了本技术与商业化质粒DNA提取试剂盒提取DNA质量、提取通量及提取成本。结果表明,双磁珠法质粒DNA提取技术可满足不同细胞密度、不同片段长度的质粒DNA提取。此外,该技术搭载96通道全自动核酸提取仪,提取的质粒DNA纯度更高、提取时间缩短80%、提取成本缩减57.1%,从而实现了质粒DNA提取的高通量、低成本,有效助力基因合成与测序。     

用直接注射法生产转基因鱼

本文报道了对鲤鱼、鲫鱼受精卵不加任何去膜处理,用显微操作器把外源基因直接注射到卵核附近,构建转基因鱼的方法。本法操作方便,孵化条件简单,成活率高。斑点杂交和Southern Blot杂交结果表明,外源基因的整合率与其它方法构建的转基因鱼的外源基因的整合率相近。从1988年至今,本组运用这个方法生产转基因鲤鱼、鲫鱼一万余尾。     

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast

Background  

Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.     

A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.     

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.