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  • ASSAY  (14)
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  • 1
    Keywords: EXPRESSION ; proliferation ; CELL-PROLIFERATION ; Germany ; INFORMATION ; screening ; GENE ; GENE-EXPRESSION ; GENES ; GENOME ; PROTEIN ; PROTEINS ; gene expression ; ASSAY ; DATABASE ; bioinformatics ; INTERFACE ; PROJECT ; INTEGRATION ; FEATURES ; RE ; cell proliferation ; FULL-LENGTH HUMAN ; HUMAN CDNAS ; ASSAYS ; genomic ; NORTHERN
    Abstract: LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface
    Type of Publication: Journal article published
    PubMed ID: 16381901
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  • 2
    Keywords: CANCER ; GROWTH ; INHIBITOR ; proliferation ; SURVIVAL ; tumor ; CELL-PROLIFERATION ; Germany ; KINASE ; INFORMATION ; TOOL ; DISEASE ; GENE ; GENES ; GENOME ; microarray ; PROTEIN ; PROTEINS ; transcription ; TUMORS ; RESOLUTION ; ACTIVATION ; DNA ; BIOLOGY ; cell cycle ; CELL-CYCLE ; CYCLE ; ASSOCIATION ; MOUSE ; IDENTIFICATION ; PROGRESSION ; ASSAY ; microarrays ; PROSTATE-CANCER ; STRATEGIES ; DNA-REPLICATION ; REPLICATION ; signaling ; RE ; TUMORIGENICITY ; genomics ; TRANSITION ; DNA replication ; C-ELEGANS ; cell proliferation ; PROTEIN-ANALYSIS ; development ; ASSAYS ; DIFFERENTIALLY EXPRESSED GENES ; high throughput ; HIGH-THROUGHPUT ; LONG ; PRIME ; PRINCIPLES ; REPRESSOR ; ROLES
    Abstract: Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G(1)-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies
    Type of Publication: Journal article published
    PubMed ID: 16140941
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  • 3
    Keywords: Germany ; CLASSIFICATION ; INFORMATION ; TOOL ; SITE ; CLONING ; GENOME ; PROTEIN ; PROTEINS ; TIME ; SEQUENCE ; SIGNAL ; VARIANTS ; ASSAY ; DATABASE ; LOCALIZATION ; PREDICTION ; SELECTION ; REJECTION ; SEQUENCE-ANALYSIS ; HUMAN GENES ; FUNCTIONAL GENOMICS ; CDNAS ; FEATURES ; PROGRAM ; RE ; VARIANT ; assembly ; databases ; ANNOTATION ; CPG ISLANDS ; FULL-LENGTH HUMAN ; ASSAYS ; HIGH-THROUGHPUT ; TESTS ; GENOMIC DNA ; genomic ; SIGNALS ; E ; SET ; transcriptome ; POLYADENYLATION
    Abstract: Background: The German cDNA Consortium has been cloning full length cDNAs and continued with their exploitation in protein localization experiments and cellular assays. However, the efficient use of large cDNA resources requires the development of strategies that are capable of a speedy selection of truly useful cDNAs from biological and experimental noise. To this end we have developed a new high-throughput analysis tool, CAFTAN, which simplifies these efforts and thus fills the gap between large-scale cDNA collections and their systematic annotation and application in functional genomics. Results: CAFTAN is built around the mapping of cDNAs to the genome assembly, and the subsequent analysis of their genomic context. It uses sequence features like the presence and type of PolyA signals, inner and flanking repeats, the GC-content, splice site types, etc. All these features are evaluated in individual tests and classify cDNAs according to their sequence quality and likelihood to have been generated from fully processed mRNAs. Additionally, CAFTAN compares the coordinates of mapped cDNAs with the genomic coordinates of reference sets from public available resources ( e. g., VEGA, ENSEMBL). This provides detailed information about overlapping exons and the structural classification of cDNAs with respect to the reference set of splice variants. The evaluation of CAFTAN showed that is able to correctly classify more than 85% of 5950 selected "known protein-coding" VEGA cDNAs as high quality multi-or single-exon. It identified as good 80.6% of the single exon cDNAs and 85% of the multiple exon cDNAs. The program is written in Perl and in a modular way, allowing the adoption of this strategy to other tasks like EST-annotation, or to extend it by adding new classification rules and new organism databases as they become available. We think that it is a very useful program for the annotation and research of unfinished genomes. Conclusion: CAFTAN is a high-throughput sequence analysis tool, which performs a fast and reliable quality prediction of cDNAs. Several thousands of cDNAs can be analyzed in a short time, giving the curator/scientist a first quick overview about the quality and the already existing annotation of a set of cDNAs. It supports the rejection of low quality cDNAs and helps in the selection of likely novel splice variants, and/or completely novel transcripts for new experiments
    Type of Publication: Journal article published
    PubMed ID: 17064411
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  • 4
    Keywords: CELLS ; CELL ; Germany ; PATHWAYS ; QUANTIFICATION ; SYSTEM ; SYSTEMS ; microarray ; PROTEIN ; PROTEINS ; SAMPLES ; DRUG ; BIOLOGY ; SIGNAL ; ASSAY ; microarrays ; EFFICIENT ; ELECTROPHORESIS ; BIOPSY ; PROTEOMICS ; signaling ; RECOMBINANT ; RE ; CAPACITY ; ARRAY ; GELS ; analysis ; methods ; BIOPSIES ; signaling networks ; protein arrays ; protein quantification ; reverse phase protein microarray
    Abstract: The advancement of efficient technologies to comply with the needs of systems biology and drug discovery has so far not received adequate attention. A substantial bottleneck for the time-resolved quantitative description of signaling networks is the limited throughput and the inadequate sensitivity of currently established methods. Here, we present an improved protein microarray-based approach towards the sensitive detection of proteins in the fg-range which is based on signal detection in the near-infrared range. The high sensitivity of the assay permits the specific quantification of proteins derived from as little as only 20 000 cells with an error rate of only 5%. The capacity is limited to the analysis of up to 500 different samples per microarray. Protein abundance is determined qualitatively, and quantitatively, if recombinant protein is available. This novel approach was called IPAQ (infrared-based protein arrays with quantitative readout). IPAQ offers a highly sensitive experimental approach superior to the established standard protein quantification technologies, and is suitable for quantitative proteomics. Employing the IPAQ approach, a detailed analysis of activated signaling networks in biopsy samples and of crosstalk between signaling modules as required in drug discovery strategies can easily be performed
    Type of Publication: Journal article published
    PubMed ID: 17309101
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  • 5
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; CELL ; Germany ; human ; SYSTEM ; DISEASE ; GENES ; GENOME ; PROTEIN ; PROTEINS ; ACTIVATION ; FLOW ; antibodies ; antibody ; IDENTIFICATION ; ASSAY ; CELL-DEATH ; fragmentation ; HUMAN GENOME ; FLUORESCENCE ; INHIBITORS ; CHEMISTRY ; RE ; flow cytometry ; genomics ; MEDIATED APOPTOSIS ; methods ; NUCLEAR ; USA ; function ; CANDIDATE ; microbiology ; caspase-3 ; cell-based assay ; PERMEABILITY TRANSITION PORE ; VIRUS CORE PROTEIN
    Abstract: After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers
    Type of Publication: Journal article published
    PubMed ID: 17478479
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  • 6
    Keywords: EXPRESSION ; COMBINATION ; SYSTEM ; SYSTEMS ; GENE ; GENE-EXPRESSION ; GENOME ; microarray ; COMPLEX ; MEMBERS ; EXPERIENCE ; gene expression ; MICROARRAY DATA ; ASSAY ; genetics ; DATABASE ; OUTCOMES ; heredity ; PROTEOMICS ; MANAGEMENT ; genomics ; TECHNOLOGY ; USA ; microbiology ; ENGLAND ; biotechnology ; CONSORTIUM ; UK ; DATA STANDARDS ; MINIMUM INFORMATION
    Abstract: This article summarizes the motivation for, and the proceedings of, the first ISA-TAB workshop held December 6-8, 2007, at the EBI, Cambridge, UK. This exploratory workshop, organized by members of the Microarray Gene Expression Data (MGED) Society's Reporting Structure for Biological Investigations (RSBI) working group, brought together a group of developers of a range of collaborative systems to discuss the use of a common format to address the pressing need of reporting and communicating data and metadata from biological, biomedical, and environmental studies employing combinations of genomics, transcriptomics, proteomics, and metabolomics technologies along with more conventional methodologies. The expertise of the participants comprised database development, data management, and hands-on experience in the development of data communication standards. The workshop's outcomes are set to help formalize the proposed Investigation, Study, Assay (ISA)-TAB tab-delimited format for representing and communicating experimental metadata. This article is part of the special issue of OMICS on the activities of the Genomics Standards Consortium (GSC)
    Type of Publication: Journal article published
    PubMed ID: 18447634
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  • 7
    Keywords: CELLS ; CELL ; Germany ; screening ; SYSTEM ; PROTEIN ; PROTEINS ; SAMPLE ; COMPLEX ; COMPLEXES ; TRANSPORT ; ACQUISITION ; ASSAY ; TRAFFICKING ; LOCALIZATION ; ER ; green fluorescent protein,proteomics,functional analysis,high-content screening microscopy,membrane ; MANAGEMENT
    Abstract: A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 14623100
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  • 8
    Keywords: EXPRESSION ; COMBINATION ; Germany ; KINASE ; INFORMATION ; TOOL ; CDNA ; GENE ; GENES ; PROTEIN ; PROTEINS ; DOMAIN ; TARGET ; VECTORS ; ASSAY ; REQUIRES ; DATABASE ; PREDICTION ; SELECTION ; SEQUENCE-ANALYSIS ; FUNCTIONAL GENOMICS ; HIGH-LEVEL ; PROTEOMICS ; CDNAS ; SUBCELLULAR-LOCALIZATION ; ANNOTATION ; ANTIGENIC DETERMINANTS ; STRUCTURE PREDICTION ; SUPPLEMENT ; WEB INTERFACE
    Abstract: The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins
    Type of Publication: Journal article published
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  • 9
    Keywords: CELLS ; CELL ; Germany ; human ; MICROSCOPY ; PATHWAY ; screening ; PROTEIN ; PROTEINS ; COMPLEX ; COMPLEXES ; DOMAIN ; TRANSPORT ; YEAST ; ASSAY ; MEMBRANE ; HUMAN GENOME ; GOLGI-APPARATUS ; MORPHOLOGY ; FUTURE ; ESTABLISHMENT ; REGULATOR ; REGULATORS ; DOMAINS ; ENDOPLASMIC-RETICULUM ; ER ; SUBCELLULAR-LOCALIZATION ; coiled coil ; COILED-COIL ; COPII ; MATRIX PROTEINS
    Abstract: Here we describe the establishment of microscope-based functional screening assays in intact cells that allow LIS to systematically identify new proteins involved in secretory membrane traffic, and proteins that can influence the integrity of the Golgi complex. We were able to identify 20 new proteins that affected either secretory transport, Golgi morphology, or both, when overexpressed in cells. Control experiments with human orthologs to yeast proteins with a role in membrane traffic, or already well characterized mammalian regulators of the secretory pathway, confirmed the specificity and significance of our results. Proteins localized to the Golgi complex or endoplasmic reticulum (ER) showed preferential interference in Our assays. Bioinformatic analysis of the new proteins interfering with membrane traffic and/or Golgi integrity revealed broad functional variety, but demonstrated a bias towards proteins with predicted coiled-coil domains and repeat structures. Extending our approach to a much larger set of novel proteins in the future will be an important step toward a more comprehensive understanding of the molecular basis of the secretory pathway. It will also serve as an example for similar microscope-based screens addressing different biological questions
    Type of Publication: Journal article published
    PubMed ID: 15466293
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  • 10
    Keywords: EXPRESSION ; Germany ; screening ; SYSTEM ; TOOL ; CDNA ; CLONING ; GENOME ; microarray ; PROTEIN ; PROTEINS ; INDUCTION ; BINDING ; antibodies ; antibody ; VECTORS ; ASSAY ; microarrays ; ESCHERICHIA-COLI ; VECTOR ; NUMBER ; FUSION ; FUSION PROTEINS ; HUMAN GENOME ; PURIFICATION ; STRATEGIES ; CLEAVAGE SITE ; QUANTITIES ; protein expression ; protein microarray ; PROTEIN MICROARRAYS ; FUSION PROTEIN ; BINDING PROTEIN ; BINDING-PROTEIN ; RECOMBINANT ; REQUIREMENT ; solubility ; FULL-LENGTH HUMAN ; HUMAN CDNAS ; ASSAYS ; recombinant protein ; MALDI TOF MS ; TEMPERATURE ; fusion tag (MBP,GST,NusA,hexahistidine) ; RECOMBINANT PROTEINS
    Abstract: Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility as determined at two different induction temperatures. Affinity-purified fusion, proteins were confirmed by MALDI-TOF MS
    Type of Publication: Journal article published
    PubMed ID: 16127724
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