Development of a novel,high resolution melting analysis based genotyping method for Cryptosporidium parvum

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes.The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.     

阿霉素抗4T1乳腺癌荷瘤小鼠的免疫调控机制

为了探讨阿霉素 (Adriamycin,ADM) 对4T1乳腺癌荷瘤鼠BALB/c的免疫调节作用,采用定量蛋白质组学串联质量标签 (TMT) 标记技术检测ADM对4T1乳腺癌差异蛋白的影响,利用多重数据库对差异蛋白进行生物信息学分析。根据蛋白质组学结果,寻找差异蛋白中与免疫调节功能相关的靶点,通过酶联免疫吸附测定 (Enzyme linked immunosorbent assay,ELISA) 观察ADM对乳腺癌组织中Th1细胞 (Helper T cells 1,Th1) 和Th2细胞 (Helper T cells 2,Th2) 的影响;通过流式细胞术分析ADM对CD4+ T细胞、CD8+ T细胞和调节性T细胞 (Regulatory T cells,Tregs) 的影响;HE染色观察ADM对4T1乳腺癌荷瘤鼠胸腺的改变。ADM上调170种差异蛋白,下调58种差异蛋白。有73种差异蛋白与免疫调控过程相关,KEGG (Kyoto encyclopedia of genes and genomes,KEGG) 富集于细胞因子及受体相关的重要蛋白通路、白介素17 (Interleukin 17,IL-17) 通路和癌症的转录调控通路。与免疫功能相关的差异蛋白与CD4+ T细胞、CD8+ T细胞和Tregs细胞的功能有关,而这些细胞的分型影响乳腺癌的预后。ADM极显著升高白介素2 (Interleukin 2,IL-2),CD4+ T细胞、CD8+ T淋巴细胞含量 (P<0.01),显著降低Tregs细胞含量 (P<0.05)。ADM抗乳腺癌的免疫调节蛋白有Ighm、Igkc、S100A8、S100A9和Tmsb4x。     

Conservation of telomere protein complexes: shuffling through evolution

The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species-specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology.     

Cancer-Derived Mutations in the Fibronectin III Repeats of PTPRT/PTPρ Inhibit Cell-Cell Aggregation

Abstract

The receptor protein tyrosine phosphatase T PTPρ is the most frequently mutated tyrosine phosphatase in human cancer. PTPρ mediates homophilic cell-cell aggregation. In its extracellular region, PTPρ has cell adhesion molecule–like motifs, including a MAM domain, an immunoglobulin domain, and four fibronectin type III (FNIII) repeats. Tumor-derived mutations have been identified in all of these extracellular domains. Previously, the authors determined that tumor-derived mutations in the MAM and immunoglobulin domains of PTPρ reduce homophilic cell-cell aggregation. In this paper, the authors describe experiments in which the contribution of the FNIII repeats to PTPρ-mediated cell-cell adhesion was evaluated. The results demonstrate that deletion of the FNIII repeats of PTPρ result in defective cell-cell aggregation. Furthermore, all of the tumor-derived mutations in the FNIII repeats of PTPρ also disrupt cell-cell aggregation. These results further support the hypothesis that mutational inactivation of PTPρ may lead to cancer progression by disrupting cell-cell adhesion.     

分离自安徽土壤金龟子绿僵菌种群的遗传异质性研究

金龟子绿僵菌是昆虫种群自然控制和害虫生物防治中重要的虫生真菌,已被作为化学农药的替代品广泛应用于农林害虫的防治。其寄主范围和地理分布极广,因此种群结构也十分复杂。为明确安徽省土栖金龟子绿僵菌的种群遗传结构,采用ISSR分子标记技术对采集自安徽省不同地区土壤中的116株金龟子绿僵菌进行遗传异质性分析。分子数据显示,筛选出的8个引物共获得79个位点,其中多态位点比率为100%。不同地区种群的Nei’s基因多样性(H)为0.2796,Shannon信息指数(I)为0.4425,种群间存在一定程度的基因流(Nm=4.4282)和遗传分化(Gst=0.1015),种群内的基因多样度占总居群的89.85%,种群间占10.15%,表明安徽土栖金龟子绿僵菌的遗传变异主要来源于种群内,且种群内表现出较大的遗传变异。并采用UPGMA对所有供试材料进行聚类分析,得到7大类种群。结果表明,安徽土栖金龟子绿僵菌之间的亲缘关系与地理来源不存在相关性。     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Structural dynamics of pentapeptide repeat proteins

Pentapeptide repeat proteins (PRPs) represent a large superfamily with more than 38 000 sequences in nearly 3500 species, the majority belonging to cyanobacteria but represented among all branches of life. PRPs contain at least eight consecutive pentapeptide repeats with the consensus (A/C/S/V/T/L/I)(D/N/S/K/E/I/R)(L/F)(S/T/R/E/Q/K/V/D)(G/D/E/N/R/Q/K). PRPs fold into right-handed quadrilateral β helices, also known as repeat-five-residue (Rfr)-folds, with four consecutive pentapeptide repeats comprising a single coil, the ~90° change in polypeptide direction in square-shaped coils achieved by type I, II and IV β turns, and hydrogen bonds between coils establishing β ladders on each Rfr-fold face. PRPs are broadly categorized into group 1 and 2 involved in antibiotic resistance and group 3 currently having unknown functions. Motivated by their intriguing structures, we are investigating PRP biophysical characteristics, including Rfr-fold thermal stability, β turn and β ladder hydrogen bond amide exchange rates and backbone dynamics. Here, we present analysis of 20 ns molecular dynamics (MD) simulations and all atom normal mode analysis (aaNMA) calculations for four group 1 and group 2 and four group 3 PRPs whose structures have been determined by X-ray crystallography. The MD cross-correlation matrices and aaNMA indicated strong correlated motion between adjacent coils and weak coupled motion between coils separated by one or more intervening coils. Slow anticorrelated motions were detected between adjacent coils in aaNMA modes that we hypothesize are requisite to access exchange-competent states necessary to permit solvent exchange of amide hydrogens involved in β-ladder and β-turns hydrogen bonds, which can have lifetimes on the order of months.     

Isolation and characterization of RARE-1, a Ty1/copia-like retrotransposon domesticated in the genome of Rhizophora apiculata

Long terminal repeat (LTR) retrotransposons are predominant mobile elements that play important roles in plant genome evolution. Here, we isolated the first putative complete Ty1/copia-like retrotransposon of 6303 bp in mangrove Rhizophora apiculata, named RARE-1. RARE-1 was homologous to the soybean retroelement 1 (SORE-1) and exhibited abundant cis-regulatory motifs involved in various stress responses in its LTRs. Using the sequence-specific amplification polymorphism (S-SAP) technique, we obtained a total of 112 bands for two R. apiculata populations from Hainan, China and Ranong, Thailand. The Hainan population showed slightly higher S-SAP polymorphism but fewer unique bands than the Ranong population. Moreover, the Hainan population also had significantly more copies of RARE-1 than the Ranong population as revealed by quantitative real-time PCR (qPCR). Our results suggested that RARE-1 might have been domesticated in the R. apiculata genome, as a result of the long-term evolution of mangroves under the extreme environment.