Molecular Biology and DNA Microarray Technology for Microbial Quality Monitoring of Water

Public concern over polluted water is a major environmental issue worldwide. Microbial contamination of water arguably represents the most significant risk to human health on a global scale. An important challenge in modern water microbial quality monitoring is the rapid, specific, and sensitive detection of microbial indicators and waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. Rapid microbiological analyses and detection of rare events in water systems are important challenges in water safety assessment since culture methods present serious limitations from both quantitative and qualitative points of view. To circumvent lengthy culture methods, newer enzymatic, immunological, and genetic methods are being developed as an alternative. DNA microarray technology is a new and promising tool that allows the detection of several hundred or even thousands DNA sequences simultaneously. Recent advances in sample processing and DNA microarray technologies provide new perspectives to assess microbial water quality.

The aims of this review are to (1) summarize what is currently known about microbial indicators, (2) describe the most important waterborne pathogens, (3) present molecular methods used to monitor the presence of pathogens in water, and (4) show the potential of DNA microarrays in water quality monitoring.     

基于DNA微阵列数据的基因聚类方法

聚类分析并不是一个新的统计问题,但是微阵列实验产生的大量复杂多元数据集对聚类的计算方法提出了新的挑战.本文对微阵列基因表达数据分析的多种聚类方法及其优缺点作了详细的介绍,包括监督聚类、非监督聚类以及基于模型的聚类.由于各种方法的有效性和适用场合不同,探索开发更为适用、聚类效果更为理想的专用于微阵列表达谱数据的聚类新方法显得尤其重要.     

The Art of Embedding Tissue for Frozen Section. Part II: Frozen Block Cryoembedding

Abstract

This article describes an original technique referred to as frozen block cryoembedding that is used for precision embedding of tissues for frozen section. Two devices are described that aid in this technique. The technique makes possible precise, on-edge embedding, even in the presence of minute samples, thin membranous or flimsy tissues, perforated and torn specimens, rubbery/curling tissues, and/or thin tubular specimens.     

Application of Recombinant Fusion Proteins for Tissue Engineering

Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Also, the development of new synthetic ECMs is very important because ECMs facilitate the localization and delivery of cells to the specific sites in the body. Therefore, the development of synthetic ECMs to replace the natural ECMs is increasingly essential and promising in tissue engineering. Recombinant genetic engineering method has enabled the synthesis of protein-based polymers with precisely controlled functionalities for the development of new synthetic ECMs. In this review, the design and construction of structure-based recombinant fusion proteins such as elastin-like polymers (ELPs) and silk-like polymers (SLPs), cell-bound growth factor-based recombinant fusion proteins such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), hybrid system composed of recombinant protein and synthetic polymer, and E-cadherin-based fusion protein by recombinant genetic engineering were explained for application of the synthetic ECMs. Modulation of mechanical properties, stimuli-sensitivity, biodegradation and cell recognition can be achieved through precise control of sequence, length, hydrophobicity and cell binding domain by recombinant genetic engineering.     

Provisional guidelines for Cell and Tumor Tissue Banking

These recommendations for cell and tissue banking were drafted by the Committee on Standardization, Collection and Distribution of Cells and Tissues of the Tissue Culture Association (TCA) jointly with the Cell and Tumor Council of the American Association of Tissue Banks (AATB).Reviewed and endorsed by the TCA Executive Board and Council (1980) and by the AATB (1980), the guidelines are reprinted here to assure their availability to the widest possible spectrum of workers involved in cell and tissue culture.Chairman, TCA Committee of Standardization, Collection and Distribution of Cells and Tissues, and AATB Cell and Tumor Council.     

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.     

Immunogenicity of DNA Vaccines Expressing Tuberculosis Proteins Fused to Tissue Plasminogen Activator Signal Sequences

Novel tuberculosis DNA vaccines encoding native ESAT-6, MPT-64, KatG, or HBHA mycobacterial proteins or the same proteins fused to tissue plasminogen activator (TPA) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. While all eight plasmids induced specific humoral responses, the constructs expressing the TPA fusions generally evoked higher antibody responses in vaccinated hosts. Although most of the DNA vaccines tested induced a substantial gamma interferon response in the spleen, the antigen-specific lung responses were 2- to 10-fold lower than the splenic responses at the time of challenge. DNA vaccines encoding the ESAT-6, MPT-64, and KatG antigens fused to TPA signal sequences evoked significant protective responses in mice aerogenically challenged with low doses of Mycobacterium tuberculosis Erdman 17 to 21 days after the final immunization. However, the protective response induced by live Mycobacterium bovis BCG vaccine was greater than the response induced by any of the DNA vaccines tested. These results suggest that the tuberculosis DNA vaccines were able to elicit substantial immune responses in suitably vaccinated mice, but further refinements to the constructs or the use of alternative immunization strategies will be needed to improve the efficacy of these vaccine candidates.     

基于网络的基因芯片数据存储分析系统

基因芯片技术是当前功能基因组研究中十分重要的工具。基于网络的基因芯片数据存储分析系统为基因芯片相关实验提供了实验室信息管理,数据存储和发布,数据分析等功能。文中介绍了该系统的基本组成,并比较了常见的基因芯片数据库和实验室信息管理系统,列举了部分基因芯片数据在线分析系统,并对系统的改进和发展进行了展望。     

Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology

Staphylococcus aureus is a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed an in silico typing scheme for the software SeqSphere⁺ (Ridom GmbH, Münster, Germany). The implemented target genes (n = 182) correspond to those queried by the Identibac S. aureus Genotyping DNA microarray (Alere Technologies, Jena, Germany). The in silico scheme was evaluated by comparing the typing results of microarray and of WGS for 154 human S. aureus isolates. A total of 96.8% (n = 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosome mec element types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.     

Progression Model Tissue Microarray (TMA) for the Study of Uterine Carcinomas

Cervical and endometrial uterine carcinomas are heterogeneous groups of cancers, which are preceded by preneoplastic lesions. More accurate tools are needed to improve the diagnosis and to define markers which may be relevant for the diagnosis, prediction of disease progression and therapeutic response.High throughput technologies for testing and validating molecular targets in cancer lesions and in their precursors are presently available. Among them, the tissue microarray (TMA) presents the advantage of a morphological control of the analyzed tissue fragment. In this article, we review the different aspects of the TMA technology with a special consideration to a uterine carcinogenesis model.     

Development of a DNA Microarray for Detection and Serotyping of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler''s diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 10⁸ CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms.Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of infectious diarrhea in the developing world, causing infantile or cholera-like disease in all age groups (2). It is among the major etiologic agents, leading to an estimated 1.5 million deaths per year worldwide (13, 14). ETEC is also a major cause of traveler''s diarrhea (3, 8, 11) and the most common pathogen among the six recognized diarrheagenic categories of E. coli, especially in the developing world (18). ETEC strains produce one or both of the following two enterotoxins: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). Two classes of STs—STa and STb—and two variants of STa—STp (initially discovered in isolates from pigs) and STh (initially discovered in isolates from humans)—have been described. The elt, estA, and estB genes encode the enterotoxins LT, STa, and STb, respectively (6, 23, 26).The O antigen comprises the outermost domain of the lipopolysaccharide molecule and is attached to the core oligosaccharide on the surfaces of Gram-negative bacteria (20). O antigens are among the most variable cellular constituents, imparting antigenic specificity. The composition of the O chain differs from strain to strain; more than 180 O-antigen structures are produced by different E. coli strains (25). The most common O serogroups reported in ETEC are O6, O8, O11, O15, O25, O27, O78, O85, O114, O115, O126, O128, O139, O148, O149, O159, O166, O167, and O173 (5, 18, 19, 31).Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST by physiological assays and immunoassays, and serotyping has depended on assays using O-serogroup-specific antisera. These traditional approaches are slow and labor-intensive, and assays using antisera can be impeded by cross-reactivity. PCR assays, which are more rapid, sensitive, and specific, have also been widely used for ETEC diagnosis (15, 24). However, molecular methods for the serotyping of ETEC have not been developed.Molecular detection and typing by PCR and microarray techniques have many advantages over traditional methods. DNA microarrays provide an efficient approach for the parallel detection and analysis of a large number of pathogenic microorganisms. This technique has been applied to the detection of pathogens from all kinds of biological samples, including water, food, and soil (4, 7, 12, 17, 21).In this study, we developed a DNA microarray for the detection and typing of ETEC. The genes encoding the enterotoxins LT and ST were used for the detection of ETEC, and the serogroup-specific genes wzx and/or wzy were used for the typing of the 19 most common ETEC O serogroups. The microarray was examined for its specificity and sensitivity, and the findings of this study indicate that it is highly sensitive and reproducible.     

GnRH受体在脑膜瘤组织芯片的表达及临床意义

目的：建立人脑膜瘤组织微阵列（组织芯片）,应用组织芯片技术研究促性腺激素释放激素受体（GnRHR）在人脑膜瘤的表达及与脑膜瘤病理分级的相关性.方法：收集手术切除的脑膜瘤蜡块标本126例,制作成直径2.0 mm 的组织芯片,以正常人脑组织为对照,采用免疫组化方法检测GnRHR在不同级别脑膜瘤中的表达.结果：①126例脑膜瘤中GnRHR的阳性表达率为69.05%,随着脑膜瘤WHO分级的增高,GnRHR表达率逐渐下降;不同级别脑膜瘤的GnRHR表达率组间有统计学意义（P=0.0015）;②不同类型脑膜瘤GnRHR阳性表达间差别无统计学意义.③正常脑组织中未见到GnRHR的表达,与脑膜瘤组比较差别有统计学意义.结论：GnRHR与人脑膜瘤发生密切相关,GnRHR在超过半数的脑膜瘤中有表达,而且对脑膜瘤的发展与演变可能起着重要的促进作用.     

Wnt Proteins in Mineralized Tissue Development and Homeostasis

The past decade has seen rapid advancement in the dissection of the molecular events and players in the development and homeostasis of mineralized tissues, that is, teeth and bones. Much of this is due to research efforts toward the regeneration of these organs and also to develop treatments for pathologies of bone, especially osteoporosis. Of late, great interest has been focused on the Wnt family of proteins and their involvement in tooth and bone development and in the regulation of postnatal bone mass. The purpose of this review is to summarize these findings and to explore new areas of Wnt research such as Wnt–bone morphogenetic protein interactions and the exciting revelation of systemic serotonin being involved in bone mass regulation.     

DNA微阵列技术在胃癌研究中的应用

多年来作为全球第二大恶性肿瘤,胃癌的研究急需一种更为整体性的研究方法。DNA微阵列技术以其高通量、自动化、平行化、快速化的优势,成为后基因组时代在转录组水平进行肿瘤基因表达谱分析的最强有力工具。本文介绍了DNA微阵列技术的应用原理和实验方法,着重回顾了这项技术在胃癌研究中的应用进展情况,并对现存的问题和应用前景进行了评价。     

冷冻组织射频比吸收率规律的研究

为了解决冷冻治疗后冰冻区域吸收RF能力显著减少这一难点,该文研究了低温条件下冷冻组织对射频能量吸收(Specific Absorption Rate,SAR)和其电导率的特点。同时采用提高冷冻组织盐离子浓度的方法来增加其对射频吸收的能力。实验结果显示,同一浓度的冰冻生物组织吸收RF的能力和电导率随温度上升而增加,且增速也随之上升。SAR和电导率随盐离子浓度的增加而近似线性增加。研究结果显示,增加冰冻组织盐离子浓度而增加其电导率的方法可以增强冷冻组织对射频的吸收能力。     

HLA-DR位点的基因芯片分型与临床应用

目的：建立一种新型的HLA－DR位点的基因分型方法，为HLA－DR的基因分型提供一个较新的思路。方法：利用基因芯片技术HLA不同基因亚型的独特序列设计探讨，制成分型芯片；待检测样品经PCR反应标记上荧光之后，与芯片进行杂交，根据杂交产生的荧光信号值分析确定样品DR位点的基因亚型，将这一方法应用于70份标准DNA和200份医院移植供受者的HLA－DR基因分型并将部分样品进行基因测序。结果：检测结果表明HLA－DR基因分型芯片可准确分辨出DR位点等位基因30大类，耗时3h 。结论：基因芯片用HLA－DR的基因分型，分辨率高，特异性强，重复性好，操作简便，对比常规的PCR－SSP和PCR－SSO方法，HLA－DR基因芯片方法更为直观，并具有集成化优势。可以在一张芯片上同时检测HLAⅠ类的A、B位点，并实现一张芯片多人份，适合于临床应用和骨髓库，脐血库的建立。     

基于支持向量聚类的肿瘤表达谱分型识别算法

对肿瘤样本进行准确的分型识别是有效治疗肿瘤的前提。首先,利用方差滤波方法选择肿瘤表达谱中具有最大方差的部分基因作为识别特征集,然后,利用支持向量聚类对肿瘤表达谱进行分型识别。针对多类型样本情况和支持向量聚类中出现的孤立点聚类问题,分别提出了有效的解决办法。对两个肿瘤表达谱数据的测试结果显示,基于支持向量聚类的方法能够准确地对肿瘤样本进行分型识别,同时能够自动发现肿瘤样本真实的亚型数量。