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  • SYSTEM  (13)
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  • 1
    Keywords: RECEPTOR ; EXPRESSION ; INVASION ; CELL ; Germany ; KINASE ; PATHWAY ; PATHWAYS ; TYROSINE KINASE ; SYSTEM ; SYSTEMS ; GENE ; GENES ; GENOME ; PROTEIN ; PROTEINS ; validation ; BIOLOGY ; TARGET ; REQUIRES ; PCR ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; EPITHELIAL-CELLS ; systems biology ; TARGETS ; OVEREXPRESSION ; real-time PCR ; protein expression ; CROSS-TALK ; CYTOTOXICITY ; signaling ; SINGLE ; ARRAY ; analysis ; EVENTS ; technique ; USA ; quantitative ; SIGNALING NETWORK ; protein arrays ; combinatorial protein knockdown ; reverse-phase protein arrays
    Abstract: The elucidation of cross-talk events between intersecting signaling pathways is one main challenge in biological research. The complexity of protein networks, composed of different pathways, requires novel strategies and techniques to reveal relevant interrelations. Here, we established a combinatorial RNAi strategy for systematic single, double, and triple knockdown, and we measured the residual mRNAs and proteins quantitatively by quantitative real-time PCR and reverse-phase protein arrays, respectively, as a prerequisite for data analysis. Our results show that the parallel knockdown of at least three different genes is feasible while keeping both untargeted silencing and cytotoxicity low. The technique was validated by investigating the interplay of tyrosine kinase receptor ErbB2 and its downstream targets Akt-1 and MEK1 in cell invasion. This experimental approach combines multiple gene knockdown with a subsequent quantitative validation of reduced protein expression and is a major advancement toward the analysis of signaling pathways in systems biology
    Type of Publication: Journal article published
    PubMed ID: 17420474
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  • 2
    Keywords: RECEPTOR ; CANCER ; GROWTH-FACTOR ; CELL ; Germany ; human ; MODEL ; MODELS ; TYROSINE KINASE ; LUNG-CANCER ; SYSTEM ; SYSTEMS ; GENE ; GENOME ; PROTEIN ; FAMILY ; IMPACT ; BIOLOGY ; GROWTH-FACTOR RECEPTOR ; SEQUENCE ; breast cancer ; BREAST-CANCER ; HUMAN GENOME ; CAENORHABDITIS-ELEGANS ; NETHERLANDS ; systems biology ; QUANTITATIVE-ANALYSIS ; RECEPTORS ; PROTEOMICS ; signaling ; SINGLE ; molecular biology ; FAMILIES ; RNA INTERFERENCE ; regulation ; TECHNOLOGY ; RNAi ; CLINICAL-RESPONSE ; PROGRESS ; GROWTH-FACTOR RECEPTORS ; SIGNALING NETWORK ; QUANTITATIVE PROTEOMICS ; FEDERATION ; Vision ; PROTEIN-INTERACTION NETWORK ; DOMAIN SIGNATURES ; ERBB-signaling network ; HER2 OVEREXPRESSION ; Micro RNA
    Abstract: Substantial progress in functional genomic and proteomic technologies has opened new perspectives in biomedical research. The sequence of the human genome has been mostly determined and opened new visions on its complexity and regulation. New technologies, like RNAi and protein arrays, allow gathering knowledge beyond single gene analysis. Increasingly, biological processes are studied with systems biological approaches, where qualitative and quantitative data of the components are utilized to model the respective processes, to predict effects of perturbations, and to then refine these models after experimental testing. Here, we describe the potential of applying functional genomics and proteomics, taking the ERBB family of growth-factor receptors as an example to study the signaling network and its impact on cancer. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19303877
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  • 3
    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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  • 4
    Keywords: Germany ; human ; SYSTEM ; CDNA ; GENOME ; PROTEIN ; PROTEINS ; SAMPLE ; SEQUENCES ; DATABASE ; INTERFACE ; WEB ; TRACKING ; HUMAN GENES ; FUNCTIONAL GENOMICS ; PROTEOMICS ; CDNAS ; SUBCELLULAR-LOCALIZATION
    Abstract: We have implemented LIFEdb (http://www.dkfz.de/ LIFEdb) to link information regarding novel human full-length cDNAs generated and sequenced by the German cDNA Consortium with functional information on the encoded proteins produced in functional genomics and proteomics approaches. The database also serves as a sample-tracking system to manage the process from cDNA to experimental read-out and data interpretation. A web interface enables the scientific community to explore and visualize features of the annotated cDNAs and ORFs combined with experimental results, and thus helps to unravel new features of proteins with as yet unknown functions
    Type of Publication: Journal article published
    PubMed ID: 14681468
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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: RECEPTOR ; CANCER ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; CELL ; Germany ; INHIBITION ; KINASE ; PATHWAY ; QUANTIFICATION ; SYSTEM ; SYSTEMS ; DISTINCT ; GENOME ; microarray ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; ACTIVATION ; BIOLOGY ; MOLECULAR-BIOLOGY ; BREAST ; breast cancer ; BREAST-CANCER ; STIMULATION ; microarrays ; ARRAYS ; systems biology ; PROTEOMICS ; EPIDERMAL-GROWTH-FACTOR ; signaling ; molecular biology ; molecular ; RE ; ARRAY ; EGFR ; analysis ; methods ; HIGH-THROUGHPUT ; HER2 ; GEFITINIB ; comparison ; BREAST-CANCER-CELLS ; EGF ; ERK1/2 ; reverse phase protein array ; HER2/neu ; herceptin
    Abstract: Protein microarrays allow highly accurate comparison and quantification of numerous biological samples in parallel while requiring only little material. This qualifies protein arrays for systems biology and clinical research where only limited sample material is available, but a precise read-out is required. With the introduction of signal normalization steps to monitor the drop size of manually contact-spotted RP protein arrays, the usefulness of normalizer proteins to ensure a high-throughput but inexpensive protein analysis was demonstrated. This approach was applied for the analysis of signaling through ERBB receptor activated kinases in the breast cancer cell line MCF-7. Activation of ERK1/2 and AKT by ERBB1 (EGFR), ERRB2 (HER2/neu), and ERBB3-4 was monitored in a time-resolved manner. Analysis of pathway activation by stimulation with epidermal growth factor and heregulin, or inhibition by blocking with gefitinib or herceptin allowed a characterization of the distinct signaling properties of the different ERBB receptor subtypes
    Type of Publication: Journal article published
    PubMed ID: 18351692
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  • 7
    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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  • 8
    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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  • 9
    Keywords: RECEPTOR ; CANCER ; CELLS ; EXPRESSION ; IN-VITRO ; tumor ; SUPPORT ; SYSTEM ; GENE ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; TUMORS ; LINES ; COMPLEX ; COMPLEXES ; DOMAIN ; INDUCTION ; mechanisms ; SKIN ; MUTATION ; HETEROZYGOSITY ; MELANOMA ; CARCINOMA-CELLS ; EXCHANGE ; EPITHELIAL-CELLS ; squamous cell carcinoma ; epidermis ; TERMINAL DIFFERENTIATION ; DMBT1 ; SALIVARY AGGLUTININ ; basal cell carcinoma ; galectin-3 ; MALIGNANT BRAIN-TUMORS
    Abstract: DMBT1 and galectin-3 are potential interacting proteins with presumably complex roles in tumorigenesis. While at present a variety of mechanisms are discussed for DMBT1 and its participation in cancer, galectin-3 is commonly known to exert tumor-promoting effects. However, in vitro studies in a rodent system have suggested that DMBT1/galectin-3 interaction in the ECM triggers epithelial differentiation, which would point to tumor-suppressive properties. To improve the understanding of DMBT1/galectin-3 action in cancer, we carried out studies in skin cancer of different origins. Mutational analyses of DMBT1 identified a missense mutation in 1 of 13 melanoma cell lines. It led to an exchange of an evolutionary conserved proline residue for serine and located within the second CUB domain of DMBT1. Immunohistochemical analyses demonstrated absence of DMBT1/galectin-3 expression from melanocytes but induction of DMBT1 expression in 1 of 8 nevi and 1 of 11 melanomas and of galectin-3 expression in 3 of 8 nevi and 4 of 8 melanomas. These data suggest that DMBT1 and galectin-3 are unlikely to act as classical tumor suppressors in melanomas. DMBT1 and galectin-3 appear to be secreted to the ECM by epithelial cells within the epidermis and the hair follicle. Compared to the flanking normal epidermis, skin tumors of epithelial origin frequently displayed downregulation of DMBT1 (18 of 19 cases) and galectin-3 (12 of 12 cases). Thus, loss of DMBT1/ galectin- 3 expression may play a role in the genesis of epithelial skin cancer. This would support the view that galectin-3 can exert tumor-suppressive effects in certain scenarios, and DMBT1/galectin-3-mediated differentiation represents a candidate mechanism for this effect. (C) 2003 Wiley-Liss, Inc
    Type of Publication: Journal article published
    PubMed ID: 12673672
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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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