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Microarray Profiling of Lymphocytes in Internal Diseases With an Altered Immune Response: Potential and Methodology  [PDF]
Anatoliy Gladkevich,S. Adriaan Nelemans,Henk F. Kauffman,Jakob Korf
Mediators of Inflammation , 2005, DOI: 10.1155/mi.2005.317
Abstract: Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation.
The LO-BaFL method and ALS microarray expression analysis  [cached]
Baciu Cristina,Thompson Kevin J,Mougeot Jean-Luc,Brooks Benjamin R
BMC Bioinformatics , 2012, DOI: 10.1186/1471-2105-13-244
Abstract: Background Sporadic Amyotrophic Lateral Sclerosis (sALS) is a devastating, complex disease of unknown etiology. We studied this disease with microarray technology to capture as much biological complexity as possible. The Affymetrix-focused BaFL pipeline takes into account problems with probes that arise from physical and biological properties, so we adapted it to handle the long-oligonucleotide probes on our arrays (hence LO-BaFL). The revised method was tested against a validated array experiment and then used in a meta-analysis of peripheral white blood cells from healthy control samples in two experiments. We predicted differentially expressed (DE) genes in our sALS data, combining the results obtained using the TM4 suite of tools with those from the LO-BaFL method. Those predictions were tested using qRT-PCR assays. Results LO-BaFL filtering and DE testing accurately predicted previously validated DE genes in a published experiment on coronary artery disease (CAD). Filtering healthy control data from the sALS and CAD studies with LO-BaFL resulted in highly correlated expression levels across many genes. After bioinformatics analysis, twelve genes from the sALS DE gene list were selected for independent testing using qRT-PCR assays. High-quality RNA from six healthy Control and six sALS samples yielded the predicted differential expression for 7 genes: TARDBP, SKIV2L2, C12orf35, DYNLT1, ACTG1, B2M, and ILKAP. Four of the seven have been previously described in sALS studies, while ACTG1, B2M and ILKAP appear in the context of this disease for the first time. Supplementary material can be accessed at: http://webpages.uncc.edu/~cbaciu/LO-BaFL/supplementary_data.html. Conclusion LO-BaFL predicts DE results that are broadly similar to those of other methods. The small healthy control cohort in the sALS study is a reasonable foundation for predicting DE genes. Modifying the BaFL pipeline allowed us to remove noise and systematic errors, improving the power of this study, which had a small sample size. Each bioinformatics approach revealed DE genes not predicted by the other; subsequent PCR assays confirmed seven of twelve candidates, a relatively high success rate.
Comparative Microarray Analysis of Intestinal Lymphocytes following Eimeria acervulina, E. maxima, or E. tenella Infection in the Chicken  [PDF]
Duk Kyung Kim, Hyun Lillehoj, Wongi Min, Chul Hong Kim, Myeong Seon Park, Yeong Ho Hong, Erik P. Lillehoj
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0027712
Abstract: Relative expression levels of immune- and non-immune-related mRNAs in chicken intestinal intraepithelial lymphocytes experimentally infected with Eimeria acervulina, E. maxima, or E. tenella were measured using a 10K cDNA microarray. Based on a cutoff of >2.0-fold differential expression compared with uninfected controls, relatively equal numbers of transcripts were altered by the three Eimeria infections at 1, 2, and 3 days post-primary infection. By contrast, E. tenella elicited the greatest number of altered transcripts at 4, 5, and 6 days post-primary infection, and at all time points following secondary infection. When analyzed on the basis of up- or down-regulated transcript levels over the entire 6 day infection periods, approximately equal numbers of up-regulated transcripts were detected following E. tenella primary (1,469) and secondary (1,459) infections, with a greater number of down-regulated mRNAs following secondary (1,063) vs. primary (890) infection. On the contrary, relatively few mRNA were modulated following primary infection with E. acervulina (35 up, 160 down) or E. maxima (65 up, 148 down) compared with secondary infection (E. acervulina, 1,142 up, 1,289 down; E. maxima, 368 up, 1,349 down). With all three coccidia, biological pathway analysis identified the altered transcripts as belonging to the categories of “Disease and Disorder” and “Physiological System Development and Function”. Sixteen intracellular signaling pathways were identified from the differentially expressed transcripts following Eimeria infection, with the greatest significance observed following E. acervulina infection. Taken together, this new information will expand our understanding of host-pathogen interactions in avian coccidiosis and contribute to the development of novel disease control strategies.
Die Tissue Microarray-Technik als neues "high throughput-tool" für den Nachweis differentieller Proteinexpression  [PDF]
Merseburger AS,Hennenlotter J,Horstmann M,Kuczyk M
Journal für Urologie und Urogyn?kologie , 2003,
Abstract: Im Mittelpunkt jüngster Untersuchungen stand der Versuch, in Erg nzung zu konventionellen Tumor- bzw. Patientencharakteristika, wie beispielsweise dem Tumorstadium oder dem histologischen Differenzierungsgrad, biologische Variablen zu identifizieren. Ziel ist es, die Prognose des individuellen, an einer Tumorerkrankung leidenden Patienten durch exakte Bestimmung des im Einzelfall vorliegenden biologischen Aggressivit tspotentials zu determinieren. Auf diese Weise soll ein individualisiertes aggressiveres bzw. weniger aggressives Behandlungskonzept etabliert werden. In diesem Zusammenhang erscheint die jüngst eingeführte Tissue Microarray- (TMA) Technik als vielversprechender experimenteller Ansatz. Die TMA-Technik stellt ein "high throughput-tool" dar, das die zeit- und kostengünstige Untersuchung gro er Patientenkollektive hinsichtlich des Vorliegens von Alterationen auf DNA/RNA- bzw. Proteinebene erlaubt. Dieses initial von Kononen beschriebene Analyseverfahren erm glicht ein im Vergleich mit der konventionellen Immunhistochemie deutlich schnelleres und effizienteres Screening einer gro en Zahl von Gewebeproben, wobei im Rahmen immunhistochemischer Ans tze der Nachweis einer im Vergleich von Tumor- und Normalgewebe differentiellen Proteinexpression im Vordergrund steht. Neben der durch die gro e Probenzahl erm glichten statistischen Validierung der erhobenen Ergebnisse steht insbesondere auch die Detektion der Co-Expression solcher Proteine im Vordergrund, für die eine m gliche regulatorische Interaktion vermutet wird. Im Rahmen der vorliegenden Arbeit soll einerseits auf die durch die TMA-Technik er ffneten M glichkeiten hinsichtlich der Identifizierung prognostisch relevanter biologischer Variablen, aber auch auf aus unserer Sicht diesbezüglich erkennbare Einschr nkungen hingewiesen werden.
Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNβ-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNβ-related adverse effects in multiple sclerosis
Jun-ichi Satoh, Yusuke Nanri, Hiroko Tabunoki, Takashi Yamamura
BMC Neurology , 2006, DOI: 10.1186/1471-2377-6-18
Abstract: Total RNA of PBMC exposed to 50 ng/ml recombinant human IFNβ for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis.Among 1,258 genes on the array, IFNβ elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNβ markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.IFNβ immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNβ-related early adverse effects in MS patients in vivo.Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) white matter mediated by an autoimmune process, whose development is triggered by a complex interplay of both genetic and environmental factors [1]. Administration of interferon-gamma (IFNγ) induced acute relapses, along with activation of the systemic immune response [2], suggesting that T-lymphocytes producing proinflammatory T helper type 1 (Th1) cytokines play a pivotal role in the immunopathogenesis of MS. In contrast, interferon-beta (IFNβ) significantly reduces the frequency of clinical exacerbations and delays the progression of disability in relapsing-remitting MS (RRMS), accompanied by a reduction in the number of new brain lesions on MRI [3,4]. Furthermore, an early initiation of IFNβ delays the conversion to clinically definite MS in the patients who
Precise generation of systems biology models from KEGG pathways
Clemens Wrzodek, Finja Büchel, Manuel Ruff, Andreas Dr?ger, Andreas Zell
BMC Systems Biology , 2013, DOI: 10.1186/1752-0509-7-15
Abstract: Here, we present a precise method for processing and converting KEGG pathways into initial metabolic and signaling models encoded in the standardized community pathway formats SBML (Levels 2 and 3) and BioPAX (Levels 2 and 3). This method involves correcting invalid or incomplete KGML content, creating complete and valid stoichiometric reactions, translating relations to signaling models and augmenting the pathway content with various information, such as cross-references to Entrez Gene, OMIM, UniProt ChEBI, and many more. Finally, we compare several existing conversion tools for KEGG pathways and show that the conversion from KEGG to BioPAX does not involve a loss of information, whilst lossless translations to SBML can only be performed using SBML Level 3, including its recently proposed qualitative models and groups extension packages.Building correct BioPAX and SBML signaling models from the KEGG database is a unique characteristic of the proposed method. Further, there is no other approach that is able to appropriately construct metabolic models from KEGG pathways, including correct reactions with stoichiometry. The resulting initial models, which contain valid and comprehensive SBML or BioPAX code and a multitude of cross-references, lay the foundation to facilitate further modeling steps.
KEGG数据库在生物合成研究中的应用  [PDF]
韩增叶,田平芳
生物技术通报 , 2011,
Abstract: KEGG(KyotoEncyclopediaofGenesandGenomes)提供了一个操作平台,即以基因组信息(GENES)和化学物质信息(LIGAND)为构建模块,通过代谢网络(PATHWAY)将基因组和生物系统联系起来,然后根据功能等级进行归纳分类(BRITE)。KEGG还为各种组学研究提供相关软件,用于代谢途径重建、遗传分析和化合物比对。作为一个综合数据库,KEGG不仅指导生物燃料、药物和新材料等生物基化学品的合成,而且致力于研究日趋严重的环境问题。系统介绍了KEGG数据库的结构、功能及其相关工具的最新进展,并展望在生物合成中的应用前景。
NemaPath: online exploration of KEGG-based metabolic pathways for nematodes
Todd Wylie, John Martin, Sahar Abubucker, Yong Yin, David Messina, Zhengyuan Wang, James P McCarter, Makedonka Mitreva
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-525
Abstract: Here we present the NemaPath pathway server, a web-based pathway-level visualization tool for navigating putative metabolic pathways for over 30 nematode species, including 27 parasites. The NemaPath approach consists of two parts: 1) a backend tool to align and evaluate nematode genomic sequences (curated EST contigs) against the annotated Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database; 2) a web viewing application that displays annotated KEGG pathway maps based on desired confidence levels of primary sequence similarity as defined by a user. NemaPath also provides cross-referenced access to nematode genome information provided by other tools available on Nematode.net, including: detailed NemaGene EST cluster information; putative translations; GBrowse EST cluster views; links from nematode data to external databases for corresponding synonymous C. elegans counterparts, subject matches in KEGG's gene database, and also KEGG Ontology (KO) identification.The NemaPath server hosts metabolic pathway mappings for 30 nematode species and is available on the World Wide Web at http://nematode.net/cgi-bin/keggview.cgi webcite. The nematode source sequences used for the metabolic pathway mappings are available via FTP http://www.nematode.net/FTP/index.php webcite, as provided by the Genome Center at Washington University School of Medicine.Nematodes or roundworms are one of the most common phyla of animals, with over 20,000 different described species [1], ubiquitous in freshwater, marine, and terrestrial environments. They have remarkable life-styles both in free-living and parasitic variants, having the ability to adapt to challenging environments or to invade multiple hosts, respectively. Parasitic nematodes of humans cause sub-clinical and clinical diseases of major socio-economic importance globally as ~3 billion people are infected [2]. Financial losses caused by parasites to agriculture (domesticated animals and crops) have a major impact on farm prof
annot8r: GO, EC and KEGG annotation of EST datasets
Ralf Schmid, Mark L Blaxter
BMC Bioinformatics , 2008, DOI: 10.1186/1471-2105-9-180
Abstract: annot8r automatically downloads all files relevant for the annotation process and generates a reference database that stores UniProt entries, their associated Gene Ontology (GO), Enzyme Commission (EC) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) annotation and additional relevant data. For each of GO, EC and KEGG, annot8r extracts a specific sequence subset from the UniProt dataset based on the information stored in the reference database. These three subsets are then formatted for BLAST searches. The user provides the protein or nucleotide sequences to be annotated and annot8r runs BLAST searches against these three subsets. The BLAST results are parsed and the corresponding annotations retrieved from the reference database. The annotations are saved both as flat files and also in a relational postgreSQL results database to facilitate more advanced searches within the results. annot8r is integrated with the PartiGene suite of EST analysis tools.annot8r is a tool that assigns GO, EC and KEGG annotations for data sets resulting from EST sequencing projects both rapidly and efficiently. The benefits of an underlying relational database, flexibility and the ease of use of the program make it ideally suited for non-model species EST-sequencing projects.Protein sequences from model organisms are generally well annotated. The situation is different for non-model species where often the core of available sequence data comes from expressed sequence tags (ESTs). To date almost one thousand of the species represented in dbEST [1] have at least 100 sequences deposited. Many of these smaller EST sequencing projects are generated by laboratories that do not have the bioinformatics infrastructure available to genome sequencing centers, and there is a need for user-friendly and easy-to-use tools to assist in the functional annotation of sequences for non-model organisms on this scale. Traditionally, annotation for such projects has been based on the descriptor of the best
PathwayVoyager: pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database
Eric Altermann, Todd R Klaenhammer
BMC Genomics , 2005, DOI: 10.1186/1471-2164-6-60
Abstract: Presented here is a new software solution that utilizes the KEGG online database for pathway mapping of partial and whole prokaryotic genomes. PathwayVoyager retrieves user-defined subsets of the KEGG database and stores the data as local, blast-formatted databases. Previously selected datasets can be re-used, reducing run-time significantly. Whole or partial genomes can be automatically analyzed using NCBI's BlastP algorithm and ORFs with similarities below the user-defined threshold will be marked on pathway maps. Multiple gene hits are sorted by similarity. Since no sequence information is transmitted over the Internet, PathwayVoyager is an ideal solution for pathway mapping and reconstruction of confidential DNA sequence data.PathwayVoyager represents an alternative approach to many already existing, more complex pathway reconstructions software solutions. This software does not require any dedicated hardware or software and is flexible and straightforward to use. It is ideally suited for environments where analyses on variable datasets are desired.The ongoing sequencing of complete genomes of prokaryotes and eukaryotes reveals a tremendous amount of uncharted data. In prokaryotic genomes, roughly 25 to 30 percent of the predicted ORFeome remain functionally unknown with many Open Reading Frames (ORFs) only showing similarities to conserved hypothetical ORFs of other organisms. However, a significant number of the predicted ORFs do show similarities to functionally classified genes with defined roles in the complex network of metabolic pathways. Previously, the most common approach to determine the functionality of a gene and its gene product was to experimentally determine phenotypic changes upon inactivation or overexpression of the gene. This is the most effective approach for determining the roles of single genes, but it is unfeasible to investigate complete ORFeomes with over 2000 ORFs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) represents an ambiti
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