| 1. |
- Zatloukal, Bernhard, et al.
(författare)
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Sensitivity and Specificity of In situ Proximity Ligation for Protein Interaction Analysis in a Model of Steatohepatitis with Mallory-Denk Bodies
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:5, s. e96690-
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Tidskriftsartikel (refereegranskat)abstract
- The in situ proximity ligation assay (isPLA) is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs). These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). We investigated the colocalization of all three key MDB components, namely keratin 8 (K8), keratin 18 (K18), and p62 (sequestosome 1) by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8(-/-) and Krt18(-/-) mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol.
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| 2. |
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| 3. |
- Bysani, Madhu Sudhan Reddy, et al.
(författare)
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ChIP-seq in steatohepatitis and normal liver tissue identifies candidate disease mechanisms related to progression to cancer
- 2013
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Ingår i: BMC Medical Genomics. - : Springer Science and Business Media LLC. - 1755-8794. ; 6, s. 50-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Steatohepatitis occurs in alcoholic liver disease and may progress to liver cirrhosis and hepatocellular carcinoma. Its molecular pathogenesis is to a large degree unknown. Histone modifications play a key role in transcriptional regulations as marks for silencing and activation of gene expression and as marks for functional elements. Many transcription factors (TFs) are crucial for the control of the genes involved in metabolism, and abnormality in their function may lead to disease. Methods: We performed ChIP-seq of the histone modifications H3K4me1, H3K4me3 and H3K27ac and a candidate transcription factor (USF1) in liver tissue from patients with steatohepatitis and normal livers and correlated results to mRNA-expression and genotypes. Results: We found several regions that are differentially enriched for histone modifications between disease and normal tissue, and qRT-PCR results indicated that the expression of the tested genes strongly correlated with differential enrichment of histone modifications but is independent of USF1 enrichment. By gene ontology analysis of differentially modified genes we found many disease associated genes, some of which had previously been implicated in the etiology of steatohepatitis. Importantly, the genes associated to the strongest histone peaks in the patient were over-represented in cancer specific pathways suggesting that the tissue was on a path to develop to cancer, a common complication to the disease. We also found several novel SNPs and GWAS catalogue SNPs that are candidates to be functional and therefore needs further study. Conclusion: In summary we find that analysis of chromatin features in tissue samples provides insight into disease mechanisms.
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| 4. |
- Mahajan, Vineet, et al.
(författare)
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Cross beta-Sheet Conformation of Keratin 8 Is a Specific Feature of Mallory-Denk Bodies Compared With Other Hepatocyte Inclusions
- 2011
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Ingår i: Gastroenterology. - : WB Saunders. - 0016-5085 .- 1528-0012. ; 141:3, s. 1080-U428
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND andamp; AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steato-hepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross beta-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross beta-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, alpha(1)-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross beta-sheet conformation than K18, whereas p62 never had cross beta-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly alpha-helical to cross beta-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8(-/-) mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.
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| 5. |
- Thompson, Rachel, et al.
(författare)
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RD-Connect : An Integrated Platform Connecting Databases, Registries, Biobanks and Clinical Bioinformatics for Rare Disease Research
- 2014
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Ingår i: Journal of general internal medicine. - : Springer Science and Business Media LLC. - 0884-8734 .- 1525-1497. ; 29:S3, s. S780-S787
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Forskningsöversikt (refereegranskat)abstract
- Research into rare diseases is typically fragmented by data type and disease. Individual efforts often have poor interoperability and do not systematically connect data across clinical phenotype, genomic data, biomaterial availability, and research/trial data sets. Such data must be linked at both an individual-patient and whole-cohort level to enable researchers to gain a complete view of their disease and patient population of interest. Data access and authorization procedures are required to allow researchers in multiple institutions to securely compare results and gain new insights. Funded by the European Union's Seventh Framework Programme under the International Rare Diseases Research Consortium (IRDiRC), RD-Connect is a global infrastructure project initiated in November 2012 that links genomic data with registries, biobanks, and clinical bioinformatics tools to produce a central research resource for rare diseases.
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| 6. |
- van Ommen, Gert-Jan B, et al.
(författare)
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BBMRI-ERIC as a resource for pharmaceutical and life science industries : the development of biobank-based Expert Centres
- 2015
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Ingår i: European Journal of Human Genetics. - : Springer Science and Business Media LLC. - 1018-4813 .- 1476-5438. ; 23:7, s. 893-900
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Tidskriftsartikel (refereegranskat)abstract
- Biological resources (cells, tissues, bodily fluids or biomolecules) are considered essential raw material for the advancement of health-related biotechnology, for research and development in life sciences, and for ultimately improving human health. Stored in local biobanks, access to the human biological samples and related medical data for transnational research is often limited, in particular for the international life science industry. The recently established pan-European Biobanking and BioMolecular resources Research Infrastructure-European Research Infrastructure Consortium (BBMRI-ERIC) aims to improve accessibility and interoperability between academic and industrial parties to benefit personalized medicine, disease prevention to promote development of new diagnostics, devices and medicines. BBMRI-ERIC is developing the concept of Expert Centre as public-private partnerships in the precompetitive, not-for-profit field to provide a new structure to perform research projects that would face difficulties under currently established models of academic-industry collaboration. By definition, Expert Centres are key intermediaries between public and private sectors performing the analysis of biological samples under internationally standardized conditions. This paper presents the rationale behind the Expert Centres and illustrates the novel concept with model examples.
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| 7. |
- Wichmann, H-Erich, et al.
(författare)
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Comprehensive catalog of European biobanks
- 2011
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 29:9, s. 795-797
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Tidskriftsartikel (refereegranskat)
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| 8. |
- Yuille, Martin, et al.
(författare)
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Biobanking for Europe
- 2008
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Ingår i: Briefings in Bioinformatics. - : Oxford University Press (OUP). - 1467-5463 .- 1477-4054. ; 9:1, s. 14-24
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biobanks are well-organized resources comprising biological samples and associated information that are accessible to scientific investigation. Across Europe, millions of samples with related data are held in different types of collections. While individual collections can be well organized and accessible, the resources are subject to fragmentation, insecurity of funding and incompleteness. To address these issues, a Biobanking and BioMolecular Resources Infrastructure (BBMRI) is to be developed across Europe, thereby implementing a European roadmap for research infrastructures that was developed by a forum of EU member states and that has been received by the European Commission. In this review, we describe the work involved in preparing for the construction of BBMRI in a European and global context.
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