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  • BIOLOGY  (15)
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  • 1
    Keywords: microarray ; SAMPLE ; microarrays ; MOLECULAR-BIOLOGY ; BIOLOGY ; molecular biology
    Type of Publication: Book chapter
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  • 2
    Keywords: gene expression ; BIOLOGY ; EXPRESSION ; CELL ; GENE ; GENE-EXPRESSION ; representational difference analysis ; analysis
    Type of Publication: Book chapter
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  • 3
    Keywords: molecular biology ; MOLECULAR-BIOLOGY ; BIOLOGY ; SAMPLE
    Type of Publication: Book chapter
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  • 4
    Keywords: Germany ; human ; TOOL ; GENE ; GENES ; GENOME ; microarray ; RNA ; TIME ; DNA ; BIOLOGY ; SEQUENCE ; IN-SITU ; AMPLIFICATION ; microarrays ; ESCHERICHIA-COLI ; DATABASE ; HUMAN GENOME ; oligonucleotides ; Jun ; mutagenesis ; POLYMERASE CHAIN-REACTION ; INVITRO ; MATRIX ; databases ; LIFE ; HIGH-THROUGHPUT ; SIZE ; genomic ; PRIMERS ; array-derived oligonucleotide ; CHEMICAL SYNTHESIS ; GENE SYNTHESIS ; in situ synthesised microarrays ; matrix nucleic acid synthesiser ; synthetic biology ; synthetic gene
    Abstract: The successful completion of the Human Genome Project and other sequencing projects opened the door for another quantum jump in science advancement. The most important public sequence databases are doubling in size every 18 months. By revealing the genetic program of many organisms, these efforts endow biologists with the ability to study the basic information of life in toto as an initial step toward a comprehensive understanding of the complexity of entire organisms. We review the area of synthetic biology, defined as the making and use of biosystems founded on the chemical synthesis of the coding DNA (and potentially RNA). The recent developments discussed here introduce a rich source of oligonucleotides to the field: in situ synthesised microarrays, which in fact represent nothing else but matrix nucleic acid synthesisers. With this new way of producing the oligonucleotides used in the making of synthetic genes in a very cost-effective manner, the field of synthetic biology can be expected to change dramatically in the next decade. Synthetic genes will then be the tools of choice to obtain any sequence at any time in any laboratory. (c) 2006 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16436303
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  • 5
    Keywords: CELL ; Germany ; VOLUME ; HYBRIDIZATION ; DNA ; BIOLOGY ; SEQUENCE ; SEQUENCES ; polymorphism ; POLYMORPHISMS ; single nucleotide polymorphism ; SPECTROSCOPY ; TARGET ; IDENTIFICATION ; ASSAY ; SNP ; MUTATIONS ; specificity ; FLUORESCENCE ; PERIPHERAL-BLOOD ; real-time PCR ; IMMUNODEFICIENCY-VIRUS TYPE-1 ; SINGLE ; RESIDUES ; INCREASE ; LEADS ; SINGLE NUCLEOTIDE POLYMORPHISMS ; SNPs ; MYCOBACTERIUM-TUBERCULOSIS ; single-nucleotide ; fluorescence spectroscopy ; technique ; BREAST-CANCER PATIENTS ; ELECTRON-TRANSFER ; single-molecule spectroscopy ; RESISTANT ; CONTACT ; INCREASES ; DNA hairpin probes ; MONONUCLEOTIDE MOLECULES
    Abstract: This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17399707
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  • 6
    Keywords: CELLS ; EXPRESSION ; CELL ; GENE ; GENE-EXPRESSION ; SACCHAROMYCES-CEREVISIAE ; METABOLISM ; MECHANISM ; REDUCTION ; mechanisms ; BIOLOGY ; PHOSPHORYLATION ; SIGNAL ; ALPHA ; gene expression ; heat shock ; NUMBER ; STRESS ; MAMMALIAN-CELLS ; INCREASE ; mRNA ; LEVEL ; ENGLAND ; PROCESSING BODIES ; TRANSLATION INITIATION ; STRESS GRANULES ; P-BODIES ; TURNOVER ; AFRICAN TRYPANOSOME ; UNTRANSLATED REGION ; CYTOPLASMIC FOCI ; CELL BIOLOGY ; POSITION ; trypanosome ; Trypanosoma brucei ; BLOOD-STREAM ; eIF2 alpha ; LEISHMANIA-MAJOR ; MESSENGER-RNA DEGRADATION ; SPLICED-LEADER RNA ; stress granule
    Abstract: In trypanosomes there is an almost total reliance on post-transcriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2 alpha at the position homologous to that of serine 51 in mammalian eIF2 alpha and in the occurrence of a novel XRNA-focus
    Type of Publication: Journal article published
    PubMed ID: 18713834
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  • 7
    Keywords: CANCER ; tumor ; CELL ; Germany ; human ; SYSTEM ; SYSTEMS ; DISEASE ; liver ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; MOLECULES ; TISSUE ; TISSUES ; BIOLOGY ; MOLECULE ; IDENTIFICATION ; MEMBRANE ; INSTABILITY ; ELECTROPHORESIS ; pancreatic cancer ; SECTIONS ; molecular biology ; pancreas ; PANCREATIC-CANCER ; EXTRACTION ; SEPARATION ; USA ; protein fractionation ; CELL-CELL ; EFFECTOR MOLECULE ; ISLETS ; protein extraction
    Abstract: Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissue, one of the most challenging tissues for protein isolation because of its high protease content. Success of the different procedures varied greatly. Based on a scheme of tissue-slicing and subsequent cell isolation, we established a reliable workflow for the fractional extraction of cytosolic proteins, membrane and organelle proteins, nuclear proteins, and cytoskeletal filaments. The tissue slices also allow for a representative confirmation of individual samples' cellular status by histochemical processes, and a proper separation or mixing of cellular material from across a tumor if required
    Type of Publication: Journal article published
    PubMed ID: 19450236
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  • 8
    Keywords: CANCER ; EXPRESSION ; CELL ; Germany ; GENOME ; microarray ; PROTEIN ; PROTEINS ; PATIENT ; COMPLEX ; COMPLEXES ; QUALITY ; BIOLOGY ; antibodies ; antibody ; ASSAY ; microarrays ; DESIGN ; ARRAYS ; REPRODUCIBILITY ; CANCER-PATIENTS ; CANCER PATIENTS ; pancreatic cancer ; PROTEOMICS ; PROTEOMIC ANALYSIS ; SERUM ; FEATURES ; PANCREATIC-CANCER ; antibody microarray ; HEALTHY-SUBJECTS ; methods ; HUMAN PLASMA ; DEPLETION ; QUANTITATIVE PROTEOMICS ; proteomic ; ABUNDANCE PROTEINS ; BIOMARKER DISCOVERY
    Abstract: Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses.
    Type of Publication: Journal article published
    PubMed ID: 20164060
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  • 9
    Keywords: CANCER ; CELLS ; EXPRESSION ; tumor ; TUMOR-CELLS ; carcinoma ; CELL ; Germany ; MODEL ; INFORMATION ; SYSTEM ; SYSTEMS ; EXPOSURE ; SITE ; SITES ; GENE ; GENES ; GENOME ; transcription ; DRUG ; TUMORS ; LINES ; DNA ; BIOLOGY ; CELL-LINES ; BREAST ; PROGRESSION ; resistance ; CARCINOMA CELLS ; TUMOR PROGRESSION ; CELL-LINE ; chemotherapy ; LINE ; DNA methylation ; CANCER-CELLS ; BREAST-CARCINOMA ; FREQUENT ; drug resistance ; DRUG-RESISTANCE ; METHYLATION ; SCIENCE ; development ; PROGNOSTIC MARKER ; PROFILES ; breast carcinoma ; DRUGS ; TOPOISOMERASE-II-ALPHA ; CANCERS ; epigenetic regulation ; Type ; CONTRIBUTE ; ANTHRACYCLINES ; MCJ GENE ; MDR1 PROMOTER
    Abstract: Acquired drug resistance represents a frequent obstacle which hampers efficient chemotherapy of cancers. The contribution of aberrant DNA methylation to the development of drug resistant tumor cells has gained increasing attention over the past decades. Hence, the objective of the presented study was to characterize DNA methylation changes which arise from treatment of tumor cells with the chemotherapeutic drug doxorubicin. DNA methylation levels from CpG islands (CGIs) linked to twenty-eight genes, whose expression levels had previously been shown to contribute to resistance against DNA double strand break inducing drugs or tumor progression in different cancer types were analyzed. High-definition DNA methylation profiles which consisted of methylation levels from 800 CpG sites mapping to CGIs around the transcription start sites of the selected genes were determined. In order to investigate the influence of CGI methylation on the expression of associated genes, their mRNA levels were investigated via qRT-PCR. It was shown that the employed method is suitable for providing highly accurate methylation profiles, comparable to those obtained via clone sequencing, the gold standard for high-definition DNA methylation studies. In breast carcinoma cells with acquired resistance against the double strand break inducing drug doxorubicin, changes in methylation of specific cytosines from CGIs linked to thirteen genes were detected. Moreover, similarities between methylation profiles obtained from breast and ovarian carcinoma cell lines with acquired doxorubicin resistance were found. The expression levels of a subset of analyzed genes were shown to be linked to the methylation levels of the analyzed CGIs. Our results provide detailed DNA methylation information from two separate model systems for acquired doxorubicin resistance and suggest the occurrence of similar methylation changes in both systems upon exposure to the drug
    Type of Publication: Journal article published
    PubMed ID: 20544021
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  • 10
    Keywords: CELLS ; BLOOD ; CELL ; evaluation ; Germany ; SYSTEM ; SYSTEMS ; GENOME ; microarray ; PROTEIN ; PROTEINS ; TIME ; COMPLEX ; COMPLEXES ; T cell ; T cells ; T-CELL ; T-CELLS ; BIOLOGY ; MOLECULAR-BIOLOGY ; LINKAGE ; SIGNAL ; cytokines ; antibodies ; antibody ; PERFORMANCE ; AMPLIFICATION ; MICROARRAY DATA ; microarrays ; DESIGN ; PLASMA ; CANCER-CELLS ; PARAMETERS ; Jun ; STRATEGIES ; sensitivity ; MICROARRAY ANALYSIS ; INTERFERON-GAMMA ; IMMUNOASSAYS ; PROTEOMICS ; CYTOKINE ; molecular biology ; molecular ; CHEMISTRY ; ELISA ; monitoring ; RHEUMATOID-ARTHRITIS ; antibody microarray ; CYTOKINE PRODUCTION ; LEVEL ; analysis ; methods ; ROLLING-CIRCLE AMPLIFICATION ; EXTENT ; SPECIMENS ; MATTER ; protein profiling ; microspot kinetics ; MASS-TRANSPORT ; quantitative ; detection strategies ; INTERLEUKIN-4 PRODUCTION ; PLASMA-PROTEOME ; TRITON X-100
    Abstract: Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach
    Type of Publication: Journal article published
    PubMed ID: 17474144
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