Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer

The proteins involved in breast cancer initiation and progression are still largely elusive. To gain insights into these processes, we conducted quantitative proteomic analyses with 21T series of breast cell lines, which include a normal, primary tumor and a metastatic tumor that were isolated from a single patient. Stable isotope labeling of amino acid in cell culture followed by LC‐MS/MS analysis was performed and deregulated proteins were identified using statistical analysis. Gene ontology analysis revealed that proteins involved in metabolic processes were the most deregulated in both tumorigenesis and metastasis. Interaction network analysis indicated that ERBB2 signaling played a critical role in tumorigenesis. In addition to known markers such as ERBB2 and E‐cadherin, novel markers, including BRP44L, MTHFD2 and TIMM17A, were found to be overexpressed in 21T breast cancer cells and verified in additional breast cell lines. mRNA expression analysis as well as immunohistochemistry analysis in breast cancer tissues indicated that expression level of TIMM17A was directly correlated with tumor progression, and survival analysis suggested that TIMM17A was a powerful prognosis factor in breast cancer. More interestingly, overexpression and siRNA knockdown experiments indicated an oncogenic activity of TIMM17A in breast cancer. Our study provides a list of potential novel markers for breast cancer tumorigenesis and metastasis using a unique cell model. Further studies on TIMM17A as well as other markers on the list may reveal mechanisms that result in more effective therapeutics for cancer treatment.     

半抗原特异性抗体的筛选及亲和力成熟

噬菌体抗体库技术的出现开创了一条便捷的基因工程抗体生产路线,为小分子半抗原抗体的制备提供了一条新途径,具有极大的应用潜力,但得到的抗体片段的亲和力普遍较全分子差,需要优化筛选策略及抗体的体外亲和力成熟来解决这一问题。     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

太湖粳稻同名地方品种的遗传差异

选用45个SSR标记分析太湖流域荔枝红、老来青、太湖青和老虎稻共4组粳稻同名地方品种的遗传差异。结果表明:同名地方品种平均Nei遗传距离为0.120~0.171,遗传同一性程度较高,其中有8对同名品种难以区别,但多数品种仍然存在一定的遗传变异,且个别品种差异较大;同名品种遗传差异与种质来源、品种名称的近似程度没有关系。     

四种子囊菌甲醇提取物的抗氧化活性研究

比较了冬虫夏草Cordyceps sinensis、古尼虫草Cordyceps gunnii、蛹虫草Cordyceps militaris、黑柄炭角菌Xylaria nigripes等四种子囊菌提取物的抗氧化活性,对它们的子实体(F)和菌丝体(M)甲醇提取物清除DPPH自由基能力、还原力、金属离子鳌合能力和总酚含量进行了测定,结果显示四种子囊菌子实体的自由基清除能力、还原力和总酚含量均高于其菌丝体(P0.05),但四种子囊菌子实体的DPPH清除能力不存在显著差异(P0.05);古尼虫草和蛹虫草子实体的还原力和总酚含量显著高于冬虫夏草和黑柄炭角菌子实体(P0.05),但前两者之间不存在显著差异(P0.05)。总酚含量和DPPH自由基清除能力、还原力呈正相关。     

Antitermitic effect of the Lantana camara plant on subterranean termites (Isoptera: Rhinotermitidae)

Abstract The insecticidal effects of Lantana camara L. (flowers, leaves, stems and roots) and the soil where lantana had been growing, on foraging activity and survival of the subterranean termites Coptotermes formosanus and Reticulitermes flavipes were examined in a 3-week experiment. The soil in which lantana had been growing had no effect on termite tunneling and survival. Incorporation of chipped fresh lantana leaves and stems into soil had no effect on mortality but caused significant reduction in tunneling. The 5-cm wide barrier of soil with lantana tissue incorporated effectively repelled groups of both species from penetrating the barrier and thus prevented infestation of a piece of wood on the other side of the barrier. C. formosanus was more sensitive in avoiding the barrier than R. flavipes. Leaves, stems and flowers were more repellent than roots. These results provide preliminary evidence that fresh-cut lantana leaves, stems and flowers may have use as additives to garden mulches against termites.     

Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 μM, 100 μM, and 10 μM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.Lipopolysaccharide (LPS) is a major component of the outer membranes of Gram-negative bacteria (21). Typically, LPS molecules consist of a hydrophilic carbohydrate portion and a hydrophobic lipid A (or endotoxin). The lipid A molecule consists of a fatty acyl substituted β-d-GlcN-(1-6)-α-GlcN disaccharide unit that usually carries phosphate groups. Diphosphorylated lipid A is generally presumed to be phosphorylated at C-1 and C-4′ positions (9); however, lipid A moieties containing pyrophosphate (PP) groups have also been reported (13). The presence of phosphate groups in lipid A greatly affects the endotoxic properties of LPS (22). Deletion of either of these groups reduces an endotoxic activity of the resulting monophosphorylated LPS by approximately 100-fold (18). For example, monophosphorylated lipid A has been used as an adjuvant in a hepatitis B vaccine in Europe (1, 12).Mass spectrometry (MS) has been widely used to gain information about the heterogeneity, i.e., the number of different species of lipid A families and a distribution of fatty acids on each glucosamine residue (2, 3, 9, 16, 20, 23, 28, 29, 30, 32, 35, 36). Detailed structural information, including the phosphorylation pattern of lipid A, can be obtained by tandem mass spectrometry. Several matrices have been used for the analysis of lipid A using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and 6-aza-2-thiothymine (ATT) (8). Although DHB has been widely used for peptide analysis, it produces uneven crystals and leads to poor spot-to-spot reproducibility (3, 6, 11). Furthermore, the low solubility in the solvent compatible with lipid A and nonuniformity in a matrix layer (crystals) can lead to variations in the ionization yield across the sample resulting in formation of “hot” (or “sweet”) spots (14). On the other hand, 5-chloro-2-mercaptobenzothiazole (CMBT) was found to offer excellent spot-to-spot reproducibility because of the homogeneous crystallization of the analyte/matrix mixture over the sample spot (33). CMBT is soluble in methanol-chloroform-water (4:4:1, vol:vol:vol), a solvent compatible with lipid A molecules, especially hexaacylated species. Thus, it has been widely used for lipid A analysis (4, 9, 23, 35, 33). Interestingly, different preparation procedures for analysis of lipid A species dictate a selection of the preferred matrix system (10). For example, lipid A prepared using a TRI Reagent-based procedure with a CMBT matrix was preferable for the detection of phosphoethanolamine modifications (35). On the other hand, the analysis of lipid A prepared using an LPS extraction kit-based procedure with DHB was preferable for the detection of aminoarabinose modification (10). In addition, divalent cations, such as Ca²⁺ or Mg²⁺, can bridge the phosphorylated negatively charged groups between neighboring LPS molecules to form aggregates (24). Thus, there is a need for technologies capable of characterizing lipid A from biologically relevant samples in an accurate, rapid, and highly sensitive manner. Here we attempt to establish an optimized MALDI MS matrix system for the sensitive analysis of lipid A, especially its diphosphorylated forms, including both pyrophosphorylated and bisphosphorylated species. We also propose to incorporate a complex reagent (additive or comatrix) for reducing the interference of cations (5, 7, 15).     

Expanded Multilocus Sequence Typing and Comparative Genomic Hybridization of Campylobacter coli Isolates from Multiple Hosts

The purpose of this work was to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with certain host species. Genotyping and ClonalFrame analyses of an expanded 16-gene multilocus sequence typing (MLST) data set involving 85 isolates from 4 different hosts species tentatively supported the development of C. coli host-preferred groups and suggested that recombination has played various roles in their diversification; however, geography could not be excluded as a contributing factor underlying the history of some of the groups. Population genetic analyses of the C. coli pubMLST database by use of STRUCTURE suggested that isolates from swine form a relatively homogeneous genetic group, that chicken and human isolates show considerable genetic overlap, that isolates from ducks and wild birds have similarity with environmental water samples and that turkey isolates have a connection with human infection similar to that observed for chickens. Analysis of molecular variance (AMOVA) was performed on these same data and suggested that host species was a significant factor in explaining genetic variation and that macrogeography (North America, Europe, and the United Kingdom) was not. The microarray comparative genomic hybridization data suggested that there were combinations of genes more commonly associated with isolates derived from particular hosts and, combined with the results on evolutionary history, suggest that this is due to a combination of common ancestry in some cases and lateral gene transfer in others.Campylobacter species are a leading bacterial cause of gastroenteritis within the United States and throughout much of the rest of the developed world. According to the CDC, there are an estimated 2 million to 4 million cases of Campylobacter illness each year in the United States (37). Campylobacter jejuni is generally recognized as the predominant cause of campylobacteriosis, responsible for approximately 90% of reported cases, while the majority of the remainder are caused by the closely related sister species Campylobacter coli (27). Not surprisingly, therefore, the majority of research on Campylobacter has centered on C. jejuni, and C. coli is a less studied organism.A multilocus sequence typing (MLST) scheme of C. jejuni was first developed by Dingle et al. (13) on the basis of the genome sequence of C. jejuni NCTC 11168. There have also been a number of studies using the genome sequence data to develop microarrays for gene presence/absence determination across strains of C. jejuni and to identify the core genome components for the species (6, 15, 32, 33, 42, 43, 53, 57). Although C. coli is responsible for fewer food-borne illnesses than C. jejuni, the impact of C. coli is still substantial, and there is also evidence that C. coli may carry higher levels of resistance to some antibiotics (1). C. coli and C. jejuni also tend to differ in their relative prevalences in animal host species and various environmental sources (4, 48, 58), and there is some evidence that both taxa may include groups of host-specific putative ecotype strains (7, 36, 38, 39, 52, 56). At present, there is only a single draft genome sequence available for C. coli, and there are no microarray comparative genomic hybridization data for C. coli strains. Thus, there is no information on intraspecies variability in gene presence/absence in C. coli and how such variability might correlate with host species.The purpose of this work was to develop and apply an expanded 16-locus MLST genotyping scheme to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with isolates derived from different host species.