| 1. |
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| 2. |
- Ahlin, Gustav, 1977-, et al.
(författare)
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Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs
- 2009
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 37:12, s. 2275-2283
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate the gene and protein expression profiles of important drug transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters (HeLa, HEK293) and leukaemia cell lines used to study drug resistance by ABC-transporters (HL-60, K562) were investigated, and compared with organotypic cell lines (HepG2, Saos-2, Caco-2 and Caco-2 TC7). For gene expression studies, real-time PCR was used, while monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression and nine for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1; SLC16A1 was investigated using 14C-lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression and the expression patterns were barely affected by transfection. The leukaemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, while the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. Importantly, the monocarboxylic acid transporting protein MCT1 was significantly expressed in all, and functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.
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| 3. |
- Al-Khalili Szigyarto, Cristina
(författare)
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Duchenne Muscular Dystrophy : recent advances in protein biomarkers and the clinical application
- 2020
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Ingår i: Expert Review of Proteomics. - : Informa UK Limited. - 1478-9450 .- 1744-8387. ; 17:5, s. 365-375
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Forskningsöversikt (refereegranskat)abstract
- Introduction Early biomarker discovery studies have praised the value of their emerging results, predicting an unprecedented impact on health care. Biomarkers are expected to provide tests with increased specificity and sensitivity compared to existing measures, improve the decision-making process, and accelerate the development of therapies. For rare disorders, like Duchenne Muscular Dystrophy (DMD) such biomarkers can assist the development of therapies, therefore also helping to find a cure for the disease. Area covered State-of-the-art technologies have been used to identify blood biomarkers for DMD and efforts have been coordinated to develop and promote translation of biomarkers for clinical practice. Biomarker translation to clinical practice is however, adjoined by challenges related to the complexity of the disease, involving numerous biological processes, and the limited sample resources. This review highlights the current progress on the development of biomarkers, describing the proteomics technologies used, the most promising findings and the challenges encountered. Expert opinion Strategies for effective use of samples combined with orthogonal proteomics methods for protein quantification are essential for translating biomarkers to the patient's bed side. Progress is achieved only if strong evidence is provided that the biomarker constitutes a reliable indicator of the patient's health status for a specific context of use.
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| 4. |
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| 5. |
- Al-Khalili Szigyarto, Cristina, et al.
(författare)
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The E3 Ligase Axotrophin/MARCH-7 : Protein Expression Profiling of Human Tissues Reveals Links to Adult Stem Cells
- 2010
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 58:4, s. 301-308
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Tidskriftsartikel (refereegranskat)abstract
- Axotrophin/MARCH-7 was first identified in mouse embryonic stem cells as a neural stem cell gene. Using the axotrophin/MARCH-7 null mouse, we discovered profound effects on T lymphocyte responses, including 8-fold hyperproliferation and 5-fold excess release of the stem cell cytokine leukemia inhibitory factor (LIF). Our further discovery that axotrophin/MARCH-7 is required for targeted degradation of the LIF receptor subunit gp190 implies a direct role in the regulation of LIF signaling. Bioinformatics studies revealed a highly conserved RING-CH domain in common with the MARCH family of E3-ubiquitin ligases, and accordingly, axotrophin was renamed "MARCH-7." To probe protein expression of human axotrophin/MARCH-7, we prepared antibodies against different domains of the protein. Each antibody bound its specific target epitope with high affinity, and immunohistochemistry cross-validated target specificity. Forty-eight human tissue types were screened. Epithelial cells stained strongly, with trophoblasts having the greatest staining. In certain tissues, specific cell types were selectively positive, including neurons and neuronal progenitor cells in the hippocampus and cerebellum, endothelial sinusoids of the spleen, megakaryocytes in the bone marrow, crypt stem cells of the small intestine, and alveolar macrophages in the 7 lung. Approximately 20% of central nervous system neuropils were positive. Notably, axotrophin/MARCH-7 has an expression profile that is distinct from that of other MARCH family members. This manuscript contains online supplemental material at http://www.jhc. org. Please visit this article online to view these materials. (J Histochem Cytochem 58:301-308, 2010)
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| 6. |
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| 7. |
- Ayoglu, Burcu, et al.
(författare)
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Affinity proteomics within rare diseases : a BIO-NMD study for blood biomarkers of muscular dystrophies
- 2014
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 6:7, s. 918-936
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Tidskriftsartikel (refereegranskat)abstract
- Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavo-protein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies.
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| 8. |
- Bengtsson, Sofia, et al.
(författare)
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Large-scale proteomics analysis of human ovarian cancer for biomarkers
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:4, s. 1440-1450
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Tidskriftsartikel (refereegranskat)abstract
- Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues.
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| 9. |
- Berglund, Lisa, et al.
(författare)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Forskningsöversikt (refereegranskat)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 10. |
- Berglund, Lisa, et al.
(författare)
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A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 8:14, s. 2832-2839
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Tidskriftsartikel (refereegranskat)abstract
- Here, we present an antigen selection strategy based on a whole-genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in-house generated data. The web-based bioinformatics platform has been designed for selection of multiple, non-overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain- and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome-wide analysis demonstrates that antigens for approximately 80% of the human protein-coding genes can be selected with this strategy.
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| 11. |
- Berglund, Lisa, et al.
(författare)
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Generation of validated antibodies towards the human proteome
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Here we show the results from a large effort to generate antibodies towards the human proteome. A high-throughput strategy was developed based on cloning and expression of antigens as recombitant protein epitope signature tags (PrESTs) Affinity purified polyclonal antibodies were generated, followed by validation by protein microarrays, Western blotting and microarray-based immunohistochemistry. PrESTs were selected based on sequence uniqueness relative the proteome and a bioinformatics analysis showed that unique antigens can be found for at least 85% of the proteome using this general strategy. The success rate from antigen selection to validated antibodies was 31%, and from protein to antibody 55%. Interestingly, membrane-bound and soluble proteins performed equally and PrEST lengths between 75 and 125 amino acids were found to give the highest yield of validated antibodies. Multiple antigens were selected for many genes and the results suggest that specific antibodies can be systematically generated to most human proteibs.
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| 12. |
- Cepeda, D., et al.
(författare)
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CDK-mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer
- 2013
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 5:7, s. 999-1018
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Tidskriftsartikel (refereegranskat)abstract
- SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer. FBXO28 is identified as part of a SCF complex acting as a regulator of tumor cell proliferation and an important modifier of MYC function. FBXO28 may be a new prognostic factor in breast cancer and a new potential drug target in MYC- driven tumors.
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| 13. |
- Charitou, Theodosia, et al.
(författare)
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Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRAS(G13D) mutation
- 2019
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Ingår i: British Journal of Cancer. - : NATURE PUBLISHING GROUP. - 0007-0920 .- 1532-1827. ; 121:1, s. 37-50
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFRtargeted therapies. METHODS: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRAS(G13D) allele (mtKRAS). RESULTS: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGF alpha stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGF alpha treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGF alpha-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGF alpha in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. CONCLUSIONS: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.
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| 14. |
- Danielsson, Frida, et al.
(författare)
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Transcriptome profiling of the interconnection of pathways involved in malignant transformation and response to hypoxia
- 2018
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Ingår i: Oncotarget. - : Impact Journals LLC. - 1949-2553. ; 9:28, s. 19730-19744
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Tidskriftsartikel (refereegranskat)abstract
- In tumor tissues, hypoxia is a commonly observed feature resulting from rapidly proliferating cancer cells outgrowing their surrounding vasculature network. Transformed cancer cells are known to exhibit phenotypic alterations, enabling continuous proliferation despite a limited oxygen supply. The four-step isogenic BJ cell model enables studies of defined steps of tumorigenesis: the normal, immortalized, transformed, and metastasizing stages. By transcriptome profiling under atmospheric and moderate hypoxic (3% O2) conditions, we observed that despite being highly similar, the four cell lines of the BJ model responded strikingly different to hypoxia. Besides corroborating many of the known responses to hypoxia, we demonstrate that the transcriptome adaptation to moderate hypoxia resembles the process of malignant transformation. The transformed cells displayed a distinct capability of metabolic switching, reflected in reversed gene expression patterns for several genes involved in oxidative phosphorylation and glycolytic pathways. By profiling the stage-specific responses to hypoxia, we identified ASS1 as a potential prognostic marker in hypoxic tumors. This study demonstrates the usefulness of the BJ cell model for highlighting the interconnection of pathways involved in malignant transformation and hypoxic response.
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| 15. |
- Edfors, Fredrik, et al.
(författare)
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Enhanced validation of antibodies for research applications
- 2018
-
Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
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| 16. |
- Fagerberg, Linn, et al.
(författare)
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Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics
- 2014
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:2, s. 397-406
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Tidskriftsartikel (refereegranskat)abstract
- Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody- based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to 80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.
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| 17. |
- Fagerberg, Linn, et al.
(författare)
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Contribution of antibody-based protein profiling to the human chromosome-centric proteome project (C-HPP)
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:6, s. 2439-2448
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Tidskriftsartikel (refereegranskat)abstract
- A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas (www.proteinatlas.org).
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| 18. |
- Falzarano, Maria Sofia, et al.
(författare)
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Urine-Derived Stem Cells Express 571 Neuromuscular Disorders Causing Genes, Making Them a Potential in vitro Model for Rare Genetic Diseases
- 2021
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Ingår i: Frontiers in Physiology. - : FRONTIERS MEDIA SA. - 1664-042X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Neuromuscular disorders (NMDs) are a heterogeneous group of genetic diseases, caused by mutations in genes involved in spinal cord, peripheral nerve, neuromuscular junction, and muscle functions. To advance the knowledge of the pathological mechanisms underlying NMDs and to eventually identify new potential drugs paving the way for personalized medicine, limitations regarding the availability of neuromuscular disease-related biological samples, rarely accessible from patients, are a major challenge. & nbsp; Aim: We characterized urinary stem cells (USCs) by in-depth transcriptome and protein profiling to evaluate whether this easily accessible source of patient-derived cells is suitable to study neuromuscular genetic diseases, focusing especially on those currently involved in clinical trials. & nbsp; Methods: The global transcriptomics of either native or MyoD transformed USCs obtained from control individuals was performed by RNA-seq. The expression of 610 genes belonging to 16 groups of disorders () whose mutations cause neuromuscular diseases, was investigated on the RNA-seq output. In addition, protein expression of 11 genes related to NMDs including COL6A, EMD, LMNA, SMN, UBA1, DYNC1H1, SOD1, C9orf72, DYSF, DAG1, and HTT was analyzed in native USCs by immunofluorescence and/or Western blot (WB). & nbsp; Results: RNA-seq profile of control USCs shows that 571 out of 610 genes known to be involved in NMDs, are expressed in USCs. Interestingly, the expression levels of the majority of NMD genes remain unmodified following USCs MyoD transformation. Most genes involved in the pathogenesis of all 16 groups of NMDs are well represented except for channelopathies and malignant hyperthermia related genes. All tested proteins showed high expression values, suggesting consistency between transcription and protein representation in USCs. & nbsp; Conclusion: Our data suggest that USCs are human cells, obtainable by non-invasive means, which might be used as a patient-specific cell model to study neuromuscular disease-causing genes and that they can be likely adopted for a variety of in vitro functional studies such as mutation characterization, pathway identification, and drug screening.
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| 19. |
- Fasterius, Erik, et al.
(författare)
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A novel RNA sequencing data analysis method for cell line authentication
- 2017
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 12:2
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a novel analysis method that can interrogate the authenticity of biological samples used for generation of transcriptome profiles in public data repositories. The method uses RNA sequencing information to reveal mutations in expressed transcripts and subsequently confirms the identity of analysed cells by comparison with publicly available cell-specific mutational profiles. Cell lines constitute key model systems widely used within cancer research, but their identity needs to be confirmed in order to minimise the influence of cell contaminations and genetic drift on the analysis. Using both public and novel data, we demonstrate the use of RNA-sequencing data analysis for cell line authentication by examining the validity of COLO205, DLD1, HCT15, HCT116, HKE3, HT29 and RKO colorectal cancer cell lines. We successfully authenticate the studied cell lines and validate previous reports indicating that DLD1 and HCT15 are synonymous. We also show that the analysed HKE3 cells harbour an unexpected KRAS-G13D mutation and confirm that this cell line is a genuine KRAS dosage mutant, rather than a true isogenic derivative of HCT116 expressing only the wild type KRAS. This authentication method could be used to revisit the numerous cell line based RNA sequencing experiments available in public data repositories, analyse new experiments where whole genome sequencing is not available, as well as facilitate comparisons of data from different experiments, platforms and laboratories.
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| 20. |
- Fasterius, Erik, 1987-, et al.
(författare)
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Analysis of public RNA-sequencing data reveals biological consequences of genetic heterogeneity in cell line populations
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Meta-analysis of datasets available in public repositories are used to gather and summarise experiments performed across laboratories, as well as to explore consistency of scientific findings. As data quality and biological equivalency across samples may obscure such analyses and consequently their conclusions, we investigated the comparability of 85 public RNA-seq cell line datasets. Thousands of pairwise comparisons of single nucleotide variants in 139 samples revealed variable genetic heterogeneity of the eight cell line populations analysed as well as variable data quality. The H9 and HCT116 cell lines were found to be remarkably stable across laboratories (with median concordances of 99.2% and 98.5%, respectively), in contrast to the highly variable HeLa cells (89.3%). We show that the genetic heterogeneity encountered greatly affects gene expression between same-cell comparisons, highlighting the importance of interrogating the biological equivalency of samples when comparing experimental datasets. Both the number of differentially expressed genes and the expression levels negatively correlate with the genetic heterogeneity. Finally, we demonstrate how comparing genetically heterogeneous datasets affect gene expression analyses and that high dissimilarity between same-cell datasets alters the expression of more than 300 cancer-related genes, which are often the focus of studies using cell lines.
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| 21. |
- Fasterius, Erik, 1987-
(författare)
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Exploring genetic heterogeneity in cancer using high-throughput DNA and RNA sequencing
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- High-throughput sequencing (HTS) technology has revolutionised the biomedical sciences, where it is used to analyse the genetic makeup and gene expression patterns of both primary patient tissue samples and models cultivated in vitro. This makes it especially useful for research on cancer, a disease that is characterised by its deadliness and genetic heterogeneity. This inherent genetic variation is an important aspect that warrants exploration, and the depth and breadth that HTS possesses makes it well-suited to investigate this facet of cancer.The types of analyses that may be accomplished with HTS technologies are many, but they may be divided into two groups: those that analyse the DNA of the sample in question, and those that work on the RNA. While DNA-based methods give information regarding the genetic landscape of the sample, RNA-based analyses yield data regarding gene expression patterns; both of these methods have already been used to investigate the heterogeneity present in cancer. While RNA-based methods are traditionally used exclusively for expression analyses, the data they yield may also be utilised to investigate the genetic variation present in the samples. This type of RNA-based analysis is seldom performed, however, and valuable information is thus ignored.The aim of this thesis is the development and application of DNA- and RNA- based HTS methods for analysing genetic heterogeneity within the context of cancer. The present investigation demonstrates that not only may RNA-based sequencing be used to successfully differentiate different in vitro cancer models through their genetic makeup, but that this may also be done for primary patient data. A pipeline for these types of analyses is established and evaluated, showing it to be both robust to several technical parameters as well as possess a broad scope of analytical possibilities. Genetic variation within cancer models in public databases are evaluated and demonstrated to affect gene expression in several cases. Both inter- and intra-patient genetic heterogeneity is shown using the established pipeline, in addition to demonstrating that cancerous cells are more heterogeneous than their normal neighbours. Finally, two bioinformatic open source software packages are presented.The results presented herein demonstrate that genetic analyses using RNA-based methods represent excellent complements to already existing DNA-based techniques, and further increase the already large scope of how HTS technologies may be utilised.
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| 22. |
- Fasterius, Erik, 1987-, et al.
(författare)
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SeqCAT : A bioconductor R-package for variant analysis of high throughput sequencing data [version 1; peer review: 1 approved with reservations, 1 not approved]
- 2018
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Ingår i: F1000 Research. - : F1000 Research Ltd. - 2046-1402. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- High throughput sequencing technologies are flourishing in the biological sciences, enabling unprecedented insights into e.g. genetic variation, but require extensive bioinformatic expertise for the analysis. There is thus a need for simple yet effective software that can analyse both existing and novel data, providing interpretable biological results with little bioinformatic prowess. We present seqCAT, a Bioconductor toolkit for analysing genetic variation in high throughput sequencing data. It is a highly accessible, easy-to-use and well-documented R-package that enables a wide range of researchers to analyse their own and publicly available data, providing biologically relevant conclusions and publication-ready figures. SeqCAT can provide information regarding genetic similarities between an arbitrary number of samples, validate specific variants as well as define functionally similar variant groups for further downstream analyses. Its ease of use, installation, complete data-to-conclusions functionality and the inherent flexibility of the R programming language make seqCAT a powerful tool for variant analyses compared to already existing solutions. A publicly available dataset of liver cancer-derived organoids is analysed herein using the seqCAT package, demonstrating that the organoids are genetically stable. A previously known liver cancer-related mutation is additionally shown to be present in a sample though it was not listed in the original publication. Differences between DNA- and RNA-based variant calls in this dataset are also analysed revealing a high median concordance of 97.5%.
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| 23. |
- Fasterius, Erik, 1987-, et al.
(författare)
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Single-cell RNA-seq variant analysis for exploration of genetic heterogeneity in cancer
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Inter-and intra-tumour heterogeneity is caused by genetic and non-genetic factors, leading to severe clinical implications. High-throughput sequencing technologies provide unprecedented tools to analyse DNA and RNA in single cells and explore both genetic heterogeneity and phenotypic variation between cells in tissues and tumours. Simultaneous analysis of both DNA and RNA in the same cell is, however, still in its infancy. We have thus developed a method to extract and analyse information regarding genetic heterogeneity that affects cellular biology from single-cell RNA-seq data. The method enables both comparisons and clustering of cells based on genetic variation in single nucleotide variants, revealing cellular subpopulations corroborated by gene expression-based methods. Furthermore, the results show that lymph node metastases have lower levels of genetic heterogeneity compared to their original tumours with respect to variants affecting protein function. The analysis also revealed three previously unknown variants common across cancer cells in glioblastoma patients. These results demonstrate the power and versatility of scRNA-seq variant analysis and highlight it as a useful complement to already existing methods, enabling simultaneous investigations of both gene expression and genetic variation.
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| 24. |
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| 25. |
- Forslund, Kristoffer, 1980-
(författare)
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The relationship between orthology, protein domain architecture and protein function
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lacking experimental data, protein function is often predicted from evolutionary and protein structure theory. Under the 'domain grammar' hypothesis the function of a protein follows from the domains it encodes. Under the 'orthology conjecture', orthologs, related through species formation, are expected to be more functionally similar than paralogs, which are homologs in the same or different species descended from a gene duplication event. However, these assumptions have not thus far been systematically evaluated. To test the 'domain grammar' hypothesis, we built models for predicting function from the domain combinations present in a protein, and demonstrated that multi-domain combinations imply functions that the individual domains do not. We also developed a novel gene-tree based method for reconstructing the evolutionary histories of domain architectures, to search for cases of architectures that have arisen multiple times in parallel, and found this to be more common than previously reported. To test the 'orthology conjecture', we first benchmarked methods for homology inference under the obfuscating influence of low-complexity regions, in order to improve the InParanoid orthology inference algorithm. InParanoid was then used to test the relative conservation of functionally relevant properties between orthologs and paralogs at various evolutionary distances, including intron positions, domain architectures, and Gene Ontology functional annotations. We found an increased conservation of domain architectures in orthologs relative to paralogs, in support of the 'orthology conjecture' and the 'domain grammar' hypotheses acting in tandem. However, equivalent analysis of Gene Ontology functional conservation yielded spurious results, which may be an artifact of species-specific annotation biases in functional annotation databases. I discuss possible ways of circumventing this bias so the 'orthology conjecture' can be tested more conclusively.
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| 26. |
- Johansson, Camilla, et al.
(författare)
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Identification of Gene-Therapy Responsive Blood Biomarkers for Duchenne Muscular Dystrophy
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- IntroductionAssessing muscle dystrophin expression systemically is important for understanding the effect of dystrophin-restoring therapies in Duchenne muscular dystrophy (DMD). Many potential blood biomarkers have been identified in DMD patients which are either more or less abundant in blood samples compared to healthy individuals and that have been shown to change with disease progression or respond to pharmacological treatment. In this study, it was suggested that a panel of such blood biomarker candidates could be used to monitor dystrophin rescue in microdystrophin therapies. MethodsPlasma samples from mdx mice treated with the microdystrophin therapy SGT-001 were analysed with an antibody suspension bead array consisting of 87 antibodies targeting 83 proteins previously identified as biomarker candidates for DMD. Each sample was assayed at two different plasma dilutions to cover a broader concentration range. Median fluorescent intensities (MFI) for each antibody were correlated to dystrophin expression in muscle tissue, as measured by immunohistochemistry and Western blot. 13 targets were selected and validated in a DMD and Becker muscular dystrophy (BMD) longitudinal natural history cohort using a suspension bead array. Results10 proteins were found significantly elevated in untreated mdx mice compared to C57 wild-type mice and 10 were found to correlate with dystrophin expression (Spearman’s correlation, FDR < 0.05) upon gene transfer. Abundance of TTN, ADSSL1, LONP1, OTUD5, MYL3 as well as DMD protein were associated with dystrophin expression in BMD patients. Of these, MYL3 and ADSSL1 had different abundance in DMD compared to healthy individuals, and MYL3 also displayed different age trajectories between DMD and BMD patients. DiscussionThe ten proteins identified in mouse plasma are related to muscle contraction (ADSSL1, ASAH1, CA3, MYL3, TTN), microtubule formation (TPI1), and protein degradation (PSMA2, OTUD4, LONP1). Of these, MYL3 and ADSSL1 showed the most promise as a dystrophin monitoring biomarker in patient samples.
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| 27. |
- Johansson, Camilla, et al.
(författare)
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Monitoring Biomarker Study in Becker Muscular Dystrophy using Data Independent Acquisition LC-MS/MS
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Becker muscular dystrophy (BMD) is a rare and heterogenous form of dystrophinopathy caused by reduced expression of altered dystrophin protein. Gene therapies and exon-skipping therapies for the more severe form of dystrophinopathy, Duchenne muscular dystrophy (DMD), assumes that by promoting partial dystrophin expression in DMD patients, their disease progression could be reduced. Several studies have identified potential progression biomarkers for DMD and hypothesised in their usefulness in monitoring pharmacodynamic response in gene-therapy clinical trials. However, knowledge of progression changes of blood proteome in BMD is lacking. In this study, we aimed at exploring differences in proteomic changes between DMD and BMD in a prospective longitudinal 4-year study as well as explore what proteins relate to functional performance in BMD patients. Serum from 48 BMD patients and 19 DMD patients were analysed using Data Independent Acquisition Tandem Mass Spectrometry (DIA-MS). Linear mixed effects models identified 17 proteins with altered longitudinal signatures between DMD and BMD, among these CKM, PKM and ALDOA. Furthermore, bikunin (product of AMBP gene), C3 and IGHG2 were found related to functional performance in BMD patients.
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| 28. |
- Johansson, Camilla, 1993-, et al.
(författare)
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Orthogonal proteomics methods warrant the development of Duchenne muscular dystrophy biomarkers
- 2023
-
Ingår i: Clinical Proteomics. - : Springer Nature. - 1542-6416 .- 1559-0275. ; 20:1
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundMolecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools.MethodsTwo technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS).ResultsFive, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml.ConclusionsThese results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.
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| 29. |
- Johansson, Camilla, 1993-
(författare)
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Proteomic strategies for blood biomarker development in rare dystrophinopathies
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are two rare genetic disorders of the family dystrophinopathy. They are both caused by the lack of, or reduced production of, the protein dystrophin. Due to abnormal dystrophin expression, patients experience progressive loss of muscle mass and cardio-, respiratory- and sometimes cognitive complications. DMD is the more severe form of dystrophinopathy, which manifests in young children and leads to wheelchair confinement in early teens followed by bed confinement and a shortened life expectancy. Dystrophin expression is absent or at less than 3% in DMD patients, often due to frame-shift mutations which cause protein expression to stop pre-maturely. BMD patients, on the other hand, display a higher but variable expression of dystrophin, often with large internal truncations. This partial expression results in a milder phenotype than DMD with sometimes unaffected life expectancies compared to healthy individuals. In the past decade, there has been substantial research into therapies aiming at increasing dystrophin expression in DMD patients and thereby prolonging ambulation, with the first gene-therapy gaining regulatory approval from the U.S. Food and Drug Administration (FDA) in June 2023. Current regulatory approvals for treatments of DMD patients have relied on dystrophin quantification in muscle biopsies as a biomarker and surrogate endpoint to predict a possible benefit from treatment, but these tests require repeated collection of muscle biopsies. Biomarkers are biochemical or physiological laboratory tests that measure a biological processes or condition. There is a need for monitoring biomarkers in dystrophinopathies, as well as biomarkers which can be used to predict outcome in clinical trials. As patients are often young children, it is important to develop biomarkers from less invasive and more readily available biological samples than muscle biopsies, such as blood or urine.In this thesis, we have used affinity proteomics and mass spectrometry to identify and validate biomarkers for monitoring disease progression in DMD and BMD patients from serum or plasma. The overall aim has been to identify biomarkers capable of distinguishing between patients with different levels of dystrophin expression and rates of disease progression in order to suggest gene-therapy pharmacodynamic biomarkers. In Paper I, we used suspension bead array (SBA) technology to identify biomarker candidates which reflect disease progression in DMD. Ten proteins were identified as related to disease progression. The ten biomarker candidates identified in Paper I were further analytically validated in Paper II using two orthogonal and absolute quantitative methods, parallel reaction monitoring mass spectrometry (PRM-MS) and sandwich immunoassays, which resulted in five analytically validated disease monitoring biomarkers for DMD (CA3, MYL3, LDHB, COL1A1 and FGG).Dystrophin-restoring therapies build on the hypothesis that increasing expression of internally deleted dystrophin reduces disease severity. In BMD patients, partial expression of short dystrophin molecules results in a milder phenotype than in DMD patients lacking expression of dystrophin. However, there are some differences in the nature of disease progression between DMD and BMD. In Paper III, we used Data Independent Acquisition Mass Spectrometry (DIA-MS) to study proteomic similarities and differences between DMD and BMD disease progression. This study revealed some discrepancies between disease progression biomarker candidates in the two related disorders.In Paper IV, we searched for blood biomarkers capable of reflecting changes in dystrophin expression during gene-therapy clinical trials. We identified ten proteins which correlated with dystrophin or microdystrophin expression in DMD mouse models. Out of these ten proteins, we identified that myosin light chain 3 (MYL3) declined steeper over time in dystrophinopathy patients with low or no dystrophin expression compared to patients with higher dystrophin expression. Two more biomarkers were identified in Paper IV as potentially related to dystrophin expression in muscle biopsies from both mouse models and patients. These were titin (TTN) and, interestingly, serum leakage of dystrophin. The possible presence of dystrophin in blood has not been well studied, and only one prior publication suggests that dystrophin may be a biomarker for DMD. Many DMD therapies currently in clinical trials aims at restoring dystrophin production and for those trials, the possibility of monitoring dystrophin leakage into blood could provide valuable information on therapeutic efficacy. In Paper V, we designed a proof-of-principle study to explore if dystrophin in blood can be a DMD biomarker. In conclusion, this thesis explores disease progression monitoring biomarkers and gene-therapy pharmacodynamic biomarkers for DMD and BMD. Three proteins, MYL3, TTN, and serum levels of dystrophin, are here suggested as possible gene-therapy pharmacodynamic biomarkers.
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| 30. |
- Kennedy, S. A., et al.
(författare)
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Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D
- 2020
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
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| 31. |
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| 32. |
- Klaesson, Axel
(författare)
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Development of DNA-based methods for analysis of protein interactions
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In situ proximity ligation assay (PLA) is a method for detection of protein interactions, post-translational modifications (PTMs) and individual proteins that allows information about their localization in a cell or tissue to be extracted. The method is based on oligonucleotide-conjugated antibodies (proximity probes) that upon binding of two epitopes in close proximity give rise to an amplifiable DNA circle. Rolling circle amplification (RCA) is used to create a DNA bundle of over a thousand repeats to which fluorescently labeled detection oligonucleotides are hybridized. This thesis is focused on improving the existing in situ PLA method and on developing new approaches for detection of proteins, protein-protein interactions and PTMs in situ in cells and tissues.In paper I, a new enzyme-independent method capable of in situ detection of protein-protein interactions was developed. The method combined the proximity requirement of in situ PLA and the amplification of hybridization chain reaction (HCR) creating a proximity-dependent initiation of hybridization chain reaction (proxHCR). Circumventing the need for enzymes resulted in a cost-efficient method that is less sensitive to storing conditions.Paper II addresses the problem of irregularly formed RCA products that can appear to be split into several fluorescent objects. A compaction oligonucleotide system was designed to crosslink the DNA bundle with itself and thereby reduce the size and increase the brightness of each individual RCA product.In paper III, the conventional in situ PLA was redesigned to increase the detection efficiency of protein interactions and PTMs in situ. The new set of proximity probes was designed to have circularization oligonucleotides incorporated that were unfolded through enzymatic digestion. The UnFold in situ PLA was able to generate more signals and had a higher sensitivity than the conventional in situ PLA.In paper IV, an oligonucleotide system able to generate signals for individual proteins (A or B) and their interaction (A and B) in a molecular Boolean (MolBoolean) protein analysis was designed. The MolBoolean design was able to generate signals detecting both individual proteins and their interaction in situ.
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| 33. |
- Koerkamp, M. G., et al.
(författare)
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Dissection of transient oxidative stress response in Saccharomyces cerevisiae by using DNA microarrays
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:8, s. 2783-2794
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Tidskriftsartikel (refereegranskat)abstract
- Yeast cells were grown in glucose-limited chemostat cultures and forced to switch to a new carbon source, the fatty acid oleate. Alterations in gene expression were monitored using DNA microarrays combined with bioinformatics tools, among which was included the recently developed algorithm REDUCE. Immediately after the switch to oleate, a transient and very specific stress response was observed, followed by the up-regulation of genes encoding peroxisomal enzymes required for fatty acid metabolism. The stress response included up-regulation of genes coding for enzymes to keep thioredoxin and glutathione reduced, as well as enzymes required for the detoxification of reactive oxygen species. Among the genes coding for various isoenzymes involved in these processes, only a specific subset was expressed. Not the general stress transcription factors Msn2 and Msn4, but rather the specific factor Yap1p seemed to be the main regulator of the stress response. We ascribe the initiation of the oxidative stress response to a combination of poor redox flux and fatty acid-induced uncoupling of the respiratory chain during the metabolic reprogramming phase.
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| 34. |
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| 35. |
- Lourbakos, A., et al.
(författare)
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Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients.
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| 36. |
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| 37. |
- Lu, Wenshu, et al.
(författare)
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Sun1 forms immobile macromolecular assemblies at the nuclear envelope
- 2008
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1783:12, s. 2415-2426
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Tidskriftsartikel (refereegranskat)abstract
- SUN-domain proteins form a novel and conserved family of inner nuclear membrane (INM) proteins, which establish physical connections between the nucleoplasm and the cytoskeleton. In the current study, we provide evidence that within the nuclear envelope (NE) Stint proteins form highly immobile oligomeric complexes in interphase cells. By performing inverse fluorescence recovery after photobleaching analysis, we demonstrate in vivo that both perinuclear and nucleoplasmic Sun I segments are essential for maintenance of Sun I immobility at the NE. Our data in Particular underline the self-association properties of the C-terminal coiled-coil Sun I segment, the ability of which to form dimers and tetramers is demonstrated. Furthermore, the Sun1 tertiary Structure involves interchain disulfide bonds that might contribute to higher homo-oligomer formation, although the overall dynamics of the Sun1 C-terminus remains unaffected when the cysteins involved are mutated. While a major Sun1 pool colocalizes with nuclear pore complex proteins, a large fraction of the Sun1 protein assemblies colocalize with immunoreactive foci of Sun2, another SUN-domain paralogue at the NE. We demonstrate that the Sun1 coiled-coil domain permits these heterophilic associations with Sun2. Sun1 therefore provides a non-dynamic platform for the formation of different macromolecular assemblies at the INM Our data support a model in which SUN-protein-containing multivariate complexes may provide versatile outer nuclear membrane attachment sites for cytoskeletal filaments. Crown
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| 38. |
- Nilsson, Peter, et al.
(författare)
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Towards a human proteome atlas : high-throughput generation of mono-specific antibodies for tissue profiling
- 2005
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 5:17, s. 4327-4337
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Tidskriftsartikel (refereegranskat)abstract
- A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.
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| 39. |
- Passarelli, Chiara, et al.
(författare)
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Tumor Necrosis Factor Receptor SF10A (TNFRSF10A) SNPs Correlate With Corticosteroid Response in Duchenne Muscular Dystrophy
- 2020
-
Ingår i: Frontiers in Genetics. - : Frontiers Media S.A.. - 1664-8021. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- Background Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment. Methods and Findings We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in theTNFRSF10Agene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N= 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results. Conclusion We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker.
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| 40. |
- Patrakka, Jaakko, et al.
(författare)
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Expression and subcellular distribution of novel glomerulus-associated proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik
- 2007
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Ingår i: Journal of the American Society of Nephrology. - : Ovid Technologies (Wolters Kluwer Health). - 1046-6673 .- 1533-3450. ; 18:3, s. 689-697
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Tidskriftsartikel (refereegranskat)abstract
- The glomerular capillary tuft is a highly specialized microcapillary that is dedicated to function as a sophisticated molecular sieve. The glomerulus filter has a unique molecular composition, and several essential glomerular proteins are expressed in the kidney exclusively by glomerular podocytes. A catalog of > 300 glomerulus-upregulated transcripts that were identified using expressed sequence tag profiling and microarray analysis was published recently. This study characterized the expression profile of five glomerulus-upregulated transcripts/proteins (ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik) in detail. The expression pattern of these novel glomerular transcripts in various mouse tissues was studied using reverse transcriptase-PCR, Northern blotting, and in situ hybridization. For studying the distribution of corresponding proteins, polyclonal antibodies were raised against the gene products, and Western blotting, immunofluorescence, and immunoelectron microscopic analyses were performed. Remarkably, it was discovered that all five transcripts/proteins were expressed in the kidney exclusively by glomerular cells. Ehd3 was expressed only by glomerular endothelial cells. Importantly, ehd3 is the first gene ever shown to be expressed exclusively by glomerular endothelial cells and not by other endothelial cells in the kidney. Dendrin, sh2d4a, plekhh2, and 2310066E14Rik, however, were transcribed solely by podocytes. With the use of polyclonal antibodies, dendrin, sh2d4a, and plekhh2 proteins were localized to the slit diaphragm and the foot process, whereas 2310066E14Rik protein was localized to the podocyte major processes and cell body. This study provides fresh insights into glomerular biology and uncovers new possibilities to explore the role of these novel proteins in the glomerular physiology and pathology.
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| 41. |
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| 42. |
- Previtali, Stefano C., et al.
(författare)
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Rimeporide as a first- in-class NHE-1 inhibitor : Results of a phase Ib trial in young patients with Duchenne Muscular Dystrophy
- 2020
-
Ingår i: Pharmacological Research. - : Elsevier BV. - 1043-6618 .- 1096-1186. ; 159
-
Tidskriftsartikel (refereegranskat)abstract
- Rimeporide, a first-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a promising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide's anti-fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6-11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentrations from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies.
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| 43. |
- Rossi, Rachele, et al.
(författare)
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A Proof of Principle Proteomic Study Detects Dystrophin in Human Plasma : Implications in DMD Diagnosis and Clinical Monitoring
- 2023
-
Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:6
-
Tidskriftsartikel (refereegranskat)abstract
- Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by pathogenic variations in the DMD gene. There is a need for robust DMD biomarkers for diagnostic screening and to aid therapy monitoring. Creatine kinase, to date, is the only routinely used blood biomarker for DMD, although it lacks specificity and does not correlate with disease severity. To fill this critical gap, we present here novel data about dystrophin protein fragments detected in human plasma by a suspension bead immunoassay using two validated anti-dystrophin-specific antibodies. Using both antibodies, a reduction of the dystrophin signal is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD.
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| 44. |
- Rossi, Rachele, et al.
(författare)
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Circadian Genes as Exploratory Biomarkers in DMD : Results From Both the mdx Mouse Model and Patients
- 2021
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Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients' plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.
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| 45. |
- Sander, Marie Rubin, 1988-
(författare)
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Analysis of PDGF receptor internalization and signaling using proximity ligation assays
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell signaling is mediated by signaling proteins that relay the signal in an intricate network of interactions before the signal is translated into a biological response. Short linear motifs (SLiMs) in intrinsically disordered regions of proteins serve as docking sites for protein interaction in all aspects of cell regulation including signal transduction. SLiM-mediated interactions are transient and low affinity and can be hijacked by virus. Only a small fraction of SLiMs have been described, but many more exist. Platelet derived growth factor receptor β (PDGFRβ) belongs to the family of receptor tyrosine kinases (RTKs) and controls cell growth, proliferation and migration. Dysregulation of PDGFRβ mediated signaling pathways is seen in many cancer types. To discover and characterize protein interactions, large scale high through-put methods are needed in concert with low through-put methods, that can characterize the interaction in a cellular context. The aim of this thesis has been to study protein-protein interactions in internalization and signaling of PDGFRβ and motif-mediated host-virus interactions through the use of in situ proximity ligation assay (PLA). Signaling via PDGFRβ is compartmentalized and depends on receptor internalization. In paper I we investigated the effects of dynamin inhibition for activation and signaling of PDGFRβ, and found that dynamin inhibition leads to impaired dimerization of the PDGFRβ. The results indicate that membrane localization of PDGFRβ is affected by dynamin. In paper II we developed a new method, Molboolean, for localized simultaneous detection of both free protein and protein in complex in cells. Molboolean is based on the principles from PLA with a fluorescent read out detectable with fluorescence microscopy. In paper III we mapped SLiM based host-virus interactions and explored their mechanisms. Using proteomic peptide phage display, we identified 1712 potential virus-host interactions by screening a library covering intrinsically disordered regions of the proteome for 229 RNA viruses. Clathrin mediated endocytosis was found to be a common target for viral hijacking, and viral binding of clathrin impaired PDGFRβ internalization. Some RTKs are proteolytically cleaved following ligand activation. In paper IV we characterized the Ca2+-dependent proteolytic cleavage of PDGFRβ. The cleavage resulted in two PDGFRβ fragments and was dependent on receptor internalization. Inhibition of the proteasome with bortezomib prevented internalization and cleavage and resulted in increased activation of PLCγ and STAT3. This thesis provides insight in the regulation of PDGFRβ signaling and internalization, and highlights contributions of both large-scale screenings and low through-put methods for studying protein-protein interactions.
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| 46. |
- Signorelli, Mirko, et al.
(författare)
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Longitudinal serum biomarker screening identifies malate dehydrogenase 2 as candidate prognostic biomarker for Duchenne muscular dystrophy
- 2020
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Ingår i: Journal of Cachexia, Sarcopenia and Muscle. - : Wiley. - 2190-5991 .- 2190-6009. ; 11:2, s. 505-517
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Tidskriftsartikel (refereegranskat)abstract
- AbstractBackgroundDuchenne muscular dystrophy (DMD) is a fatal disease for which no cure is available. Clinical trials have shown to be largely underpowered due to inter‐individual variability and noisy outcome measures. The availability of biomarkers able to anticipate clinical benefit is highly needed to improve clinical trial design and facilitate drug development.MethodsIn this study, we aimed to appraise the value of protein biomarkers to predict prognosis and monitor disease progression or treatment outcome in patients affected by DMD. We collected clinical data and 303 blood samples from 157 DMD patients in three clinical centres; 78 patients contributed multiple blood samples over time, with a median follow‐up time of 2 years. We employed linear mixed models to identify biomarkers that are associated with disease progression, wheelchair dependency, and treatment with corticosteroids and performed survival analysis to find biomarkers whose levels are associated with time to loss of ambulation.ResultsOur analysis led to the identification of 21 proteins whose levels significantly decrease with age and nine proteins whose levels significantly increase. Seven of these proteins are also differentially expressed in non‐ambulant patients, and three proteins are differentially expressed in patients treated with glucocorticosteroids. Treatment with corticosteroids was found to partly counteract the effect of disease progression on two biomarkers, namely, malate dehydrogenase 2 (MDH2, P = 0.0003) and ankyrin repeat domain 2 (P = 0.0005); however, patients treated with corticosteroids experienced a further reduction on collagen 1 serum levels (P = 0.0003), especially following administration of deflazacort. A time to event analysis allowed to further support the use of MDH2 as a prognostic biomarker as it was associated with an increased risk of wheelchair dependence (P = 0.0003). The obtained data support the prospective evaluation of the identified biomarkers in natural history and clinical trials as exploratory biomarkers.
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| 47. |
- Signorelli, M., et al.
(författare)
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Penalized regression calibration : A method for the prediction of survival outcomes using complex longitudinal and high-dimensional data
- 2021
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Ingår i: Statistics in Medicine. - : Wiley. - 0277-6715 .- 1097-0258. ; 40:27, s. 6178-6196
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Tidskriftsartikel (refereegranskat)abstract
- Longitudinal and high-dimensional measurements have become increasingly common in biomedical research. However, methods to predict survival outcomes using covariates that are both longitudinal and high-dimensional are currently missing. In this article, we propose penalized regression calibration (PRC), a method that can be employed to predict survival in such situations. PRC comprises three modeling steps: First, the trajectories described by the longitudinal predictors are flexibly modeled through the specification of multivariate mixed effects models. Second, subject-specific summaries of the longitudinal trajectories are derived from the fitted mixed models. Third, the time to event outcome is predicted using the subject-specific summaries as covariates in a penalized Cox model. To ensure a proper internal validation of the fitted PRC models, we furthermore develop a cluster bootstrap optimism correction procedure that allows to correct for the optimistic bias of apparent measures of predictiveness. PRC and the CBOCP are implemented in the R package pencal, available from CRAN. After studying the behavior of PRC via simulations, we conclude by illustrating an application of PRC to data from an observational study that involved patients affected by Duchenne muscular dystrophy, where the goal is predict time to loss of ambulation using longitudinal blood biomarkers.
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| 48. |
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| 49. |
- Spitali, Pietro, et al.
(författare)
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Tracking disease progression non-invasively in Duchenne and Becker muscular dystrophies
- 2018
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Ingår i: Journal of Cachexia, Sarcopenia and Muscle. - : Wiley-VCH Verlagsgesellschaft. - 2190-5991 .- 2190-6009. ; 9:4, s. 715-726
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Tidskriftsartikel (refereegranskat)abstract
- Background Analysis of muscle biopsies allowed to characterize the pathophysiological changes of Duchenne and Becker muscular dystrophies (D/BMD) leading to the clinical phenotype. Muscle tissue is often investigated during interventional dose finding studies to show in situ proof of concept and pharmacodynamics effect of the tested drug. Less invasive readouts are needed to objectively monitor patients' health status, muscle quality, and response to treatment. The identification of serum biomarkers correlating with clinical function and able to anticipate functional scales is particularly needed for personalized patient management and to support drug development programs. Methods A large-scale proteomic approach was used to identify serum biomarkers describing pathophysiological changes (e.g. loss of muscle mass), association with clinical function, prediction of disease milestones, association with in vivo(31)P magnetic resonance spectroscopy data and dystrophin levels in muscles. Cross-sectional comparisons were performed to compare DMD patients, BMD patients, and healthy controls. A group of DMD patients was followed up for a median of 4.4years to allow monitoring of individual disease trajectories based on yearly visits. Results Cross-sectional comparison enabled to identify 10 proteins discriminating between healthy controls, DMD and BMD patients. Several proteins (285) were able to separate DMD from healthy, while 121 proteins differentiated between BMD and DMD; only 13 proteins separated BMD and healthy individuals. The concentration of specific proteins in serum was significantly associated with patients' performance (e.g. BMP6 serum levels and elbow flexion) or dystrophin levels (e.g. TIMP2) in BMD patients. Analysis of longitudinal trajectories allowed to identify 427 proteins affected over time indicating loss of muscle mass, replacement of muscle by adipose tissue, and cardiac involvement. Over-representation analysis of longitudinal data allowed to highlight proteins that could be used as pharmacodynamic biomarkers for drugs currently in clinical development. Conclusions Serum proteomic analysis allowed to not only discriminate among DMD, BMD, and healthy subjects, but it enabled to detect significant associations with clinical function, dystrophin levels, and disease progression.
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| 50. |
- Stahl, A., et al.
(författare)
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Rapid degradation of the presequence of the F-1 beta precursor of the ATP synthase inside mitochondria
- 2000
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 349, s. 703-707
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the fate of the presequence of an overexpressed protein derived from the precursor of the F(1)beta subunit of ATP synthase after import and processing in mitochondria. Our studies revealed a rapid degradation of the presequence inside mitochondria catalysed by matrix-located protease(s). In contrast, the mature portion of the precursor was not degraded. This is the first experimental evidence of the rapid degradation of the F(1)beta a mitochondrial presequence in organello after in vitro import and processing.
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