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  • 1.
    Al-Amin, Abdullah
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gallant, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lööf, Sara
    Department of Oncology-Pathology, Karolinska Institutet.
    Lengqvist, Johan
    Department of Medicine, Karolinska Institutet.
    Bacanu, Smarand
    Department of Oncology-Pathology, Karolinska Institutet.
    Nordlund, Pär
    Department of Oncology-Pathology, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Sensitive Measurement of Cellular Drug-Target Engagement Using Multiplex Proximity Extension AssaysManuscript (preprint) (Other academic)
  • 2.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A cell that senses signals from its environment uses proteins for signal transduction via post translational modifications (PTMs) and protein- protein interactions (PPIs) from cell membrane into the nucleus where genes controlling cell proliferation, differentiation and apoptosis can be turned on or off, i.e. changing the phenotype or fate of the cell. Aberrations within such proteins are prone to cause diseases, such as cancer. Therefore, it is important so study aberrant signaling to be able to understand and treat diseases.

    In this thesis, signaling aberrations of PTMs and PPIs were analyzed with the use of the in situ proximity ligation assay (in situ PLA), and the thesis also contain method development of rolling circle amplification (RCA), which is the method used for signal amplification of in situ PLA reaction products.

    Paper I considers the integrity of RCA products. Here, the aim was to generate a smaller and more compact RCA product, for more accurate either visual or automated analysis. This was achieved with the use of an additional so called compaction oligonucleotide that during RCA was able to bind and pull segments of RCA products closer together. The compaction oligonucleotide served to increase the signal to noise ratio and decrease the number of false positive signals.

    The crosstalk between the Hippo and TGFβ signaling pathways were studied in paper II. Activity of the Hippo signaling pathway is regulated by cell density sensing and tissue control. We found differences in amounts and localization of interactions between the effector proteins of the two pathways depending on cell density and TGFβ stimulation.

    In paper III the NF-кB signaling pathway constitutively activated in chronic lymphocytic leukemia (CLL) was studied. A 4 base-pair frameshift deletion within the NFKBIE gene, which encodes the negative regulator IкBε, was found among 13 of a total 315 cases by the use of targeted deep sequencing. We found reduced levels of IкBε protein, decreased p65 inhibition, and increased phosphorylation, along with increased nuclear localization of p65 in NFKBIE deleted cases compared to healthy cases.

    Crosstalk between the Hippo and Wnt signaling pathway are studied within paper IV. Here, we found differences in cellular localization of TAZ/β-catenin interactions depending on colon cancer tumor stage and by further investigate Hippo/WNT crosstalk in cell line model systems we found an increase of complex formations involved in the crosstalk in sparse growing HEK293 cells compared to dense growing cells. Also, active WNT3a signaling was affected by cell density. Since cell density showed to have a big effect on Hippo/WNT crosstalk we continued to investigated the effect of E-cadherin, which has a function in cell junctions and maintenance of epithelial integrity on Hippo/WNT crosstalk. Interestingly, we found that E-cadherin is likely to regulate Hippo/WNT crosstalk.

    List of papers
    1. Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
    Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
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    2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, p. 12317:1-10, article id 12317Article in journal (Refereed) Published
    National Category
    Medical Image Processing
    Research subject
    Computerized Image Processing
    Identifiers
    urn:nbn:se:uu:diva-260286 (URN)10.1038/srep12317 (DOI)000358358900001 ()26202090 (PubMedID)
    Funder
    EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 259796Swedish Research Council
    Available from: 2015-07-23 Created: 2015-08-18 Last updated: 2018-02-27Bibliographically approved
    2. Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
    Open this publication in new window or tab >>Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
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    2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 21, p. 3407-3415Article in journal (Refereed) Published
    Abstract [en]

    The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-268434 (URN)10.1016/j.jmb.2015.04.015 (DOI)000363823200006 ()25937570 (PubMedID)
    Funder
    EU, FP7, Seventh Framework Programme, 278568Swedish Research Council
    Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2017-12-01Bibliographically approved
    3. Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia
    Open this publication in new window or tab >>Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia
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    2015 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 212, no 6, p. 833-843Article in journal (Refereed) Published
    Abstract [en]

    NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-279237 (URN)10.1084/jem.20142009 (DOI)000355569300001 ()25987724 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), b2011080Swedish Cancer SocietySwedish Research CouncilNIH (National Institute of Health), CA81554; CA081554EU, European Research Council, 259796EU, FP7, Seventh Framework Programme, 306242
    Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2018-01-10Bibliographically approved
    4. Crosstalk between WNT and Hippo signaling pathways is affected by cell density and loss of or mutated E-cadherin protein, associated to HDGC.
    Open this publication in new window or tab >>Crosstalk between WNT and Hippo signaling pathways is affected by cell density and loss of or mutated E-cadherin protein, associated to HDGC.
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-281714 (URN)
    Available from: 2016-03-29 Created: 2016-03-29 Last updated: 2016-05-12
  • 3.
    Arngården, Linda
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Crosstalk between WNT and Hippo signaling pathways changes upon colon cancer stage and is affected by cell density and loss of or mutated E-cadherin proteinManuscript (preprint) (Other academic)
  • 4. Asplund, A. C.
    et al.
    Falk, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Moens, Lotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kumar, Y.
    Bauser, C.
    Bernatowska, E.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Smith, C. I. E.
    Large-scale mutation analysis of primary immunodeficiency patients by next-generation sequencing2012In: Journal of Clinical Immunology, ISSN 0271-9142, E-ISSN 1573-2592, Vol. 32, no Suppl 1, p. 227-228Article in journal (Other academic)
  • 5.
    Bagchi, Sonchita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fuchino, Katsuya
    Department of Biology, Lund University.
    Cantlay, Stuart
    Department of Biology, Lund University.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Bergman, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kamali‐Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Flardh, Klas
    Department of Biology, Lund University.
    Ausmees, Nora
    Department of Biology, Lund University.
    Co‐operation between two coiled coil cytoskeletons in polar growth in StreptomycesManuscript (preprint) (Other academic)
  • 6. Barisic, Ivan
    et al.
    Schoenthaler, Silvia
    Ke, Rongqin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Noehammer, Christa
    Wiesinger-Mayr, Herbert
    Multiplex detection of antibiotic resistance genes using padlock probes2013In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 77, no 2, p. 118-125Article in journal (Refereed)
    Abstract [en]

    The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse beta-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the beta-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of beta-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.

  • 7.
    Beghini, A.
    et al.
    Univ Milan, Dept Hlth Sci, Milan, Italy..
    Lazzaroni, F.
    Univ Milan, Dept Hlth Sci, Milan, Italy..
    Del Giacco, L.
    Univ Milan, Dept Biosci, Milan, Italy..
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Biasci, D.
    Univ Cambridge, Cambridge Inst Med Res, Cambridge, England..
    Turrini, M.
    Valduce Hosp, Dept Internal Med, Como, Italy..
    Prosperi, L.
    Univ Milan, Dept Biosci, Milan, Italy..
    Brusamolino, R.
    Osped Niguarda Ca Granda, Dept Pathol, Milan, Italy..
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Cairoli, R.
    Osped Niguarda Ca Granda, Dept Oncol, Hematol Unit, Milan, Italy..
    Clinical Relevance Of Recurrent Allele-Specific Recombination Expressing The Wnt10Bivs1 Allele Variant In Acute Myeloid Leukemia2016In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 668-669Article in journal (Other academic)
  • 8. Beghini, Alessandro
    et al.
    Corlazzoli, Francesca
    Del Giacco, Luca
    Re, Matteo
    Lazzaroni, Francesca
    Brioschi, Matteo
    Valentini, Giorgio
    Ferrazzi, Fulvia
    Ghilardi, Anna
    Righi, Marco
    Turrini, Mauro
    Mignardi, Marco
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Cesana, Clara
    Bronte, Vincenzo
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Morra, Enrica
    Cairoli, Roberto
    Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133(bright) Acute Myeloid Leukemia Cells2012In: Neoplasia, ISSN 1522-8002, E-ISSN 1476-5586, Vol. 14, no 12, p. 1236-+Article in journal (Refereed)
    Abstract [en]

    Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/beta-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT 10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated beta-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)gamma c(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

  • 9. Beisvåg, Vidar
    et al.
    Kauffmann, Audrey
    Malone, James
    Foy, Carole
    Salit, Marc
    Schimmel, Heinz
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Parkinson, Helen
    Huber, Wolfgang
    Brazma, Alvis
    Sandvik, Arne K.
    Kuiper, Martin
    Contributions of the EMERALD project to assessing and improving microarray data quality2011In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 50, no 1, p. 27-31Article in journal (Refereed)
    Abstract [en]

    While minimum information about a microarray experiment (MIAME) standards have helped to increase the value of the microarray data deposited into public databases like ArrayExpress and Gene Expression Omnibus (GEO), limited means have been available to assess the quality of this data or to identify the procedures used to normalize and transform raw data. The EMERALD FP6 Coordination Action was designed to deliver approaches to assess and enhance the overall quality of microarray data and to disseminate these approaches to the microarray community through an extensive series of workshops, tutorials, and symposia. Tools were developed for assessing data quality and used to demonstrate how the removal of poor-quality data could improve the power of statistical analyses and facilitate analysis of multiple joint microarray data sets. These quality metrics tools have been disseminated through publications and through the software package arrayQualityMetrics. Within the framework provided by the Ontology of Biomedical Investigations, ontology was developed to describe data transformations, and software ontology was developed for gene expression analysis software. In addition, the consortium has advocated for the development and use of external reference standards in microarray hybridizations and created the Molecular Methods (MolMeth) database, which provides a central source for methods and protocols focusing on microarray-based technologies.

  • 10.
    Bhandage, Amol K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Jin, Zhe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Korol, Sergiy V.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology. Uppsala University.
    Shen, Qiujin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Pei, Yu
    Karolinska Institute, Stockholm, Sweden.
    Deng, Qiaolin
    Karolinska Institute, Stockholm, Sweden.
    Espes, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Transplantation and regenerative medicine.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Transplantation and regenerative medicine.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Birnir, Bryndis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    GABA Regulates Release of Inflammatory Cytokines From Peripheral Blood Mononuclear Cells and CD4+ T Cells and Is Immunosuppressive in Type 1 Diabetes2018In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30, p. 283-294Article in journal (Refereed)
    Abstract [en]

    The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100nM, 500nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion.

  • 11.
    Birgisson, H
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Tsimogiannis, Kostas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Freyhult, Eva
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala Univ, Sci Life Lab, Dept Immunol Genet & Pathol, Uppsala, Sweden.
    Plasma Protein Profiling Reveal Osteoprotegerin as a Marker of Prognostic Impact for Colorectal Cancer2018In: Translational Oncology, ISSN 1944-7124, E-ISSN 1936-5233, Vol. 11, no 4, p. 1034-1043Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Due to difficulties in predicting recurrences in colorectal cancer stages II and III, reliable prognostic biomarkers could be a breakthrough for individualized treatment and follow-up. OBJECTIVE: To find potential prognostic protein biomarkers in colorectal cancer, using the proximity extension assays. METHODS: A panel of 92 oncology-related proteins was analyzed with proximity extension assays, in plasma from a cohort of 261 colorectal cancer patients with stage II-IV. The survival analyses were corrected for disease stage and age, and the recurrence analyses were corrected for disease stage. The significance threshold was adjusted for multiple comparisons. RESULTS: The plasma proteins expression levels had a greater prognostic relevance in disease stage III colorectal cancer than in disease stage II, and for overall survival than for time to recurrence. Osteoprotegerin was the only biomarker candidate in the protein panel that had a statistical significant association with overall survival (P = .00029). None of the proteins were statistically significantly associated with time to recurrence. CONCLUSIONS: Of the 92 analyzed plasma proteins, osteoprotegerin showed the strongest prognostic impact in patients with colorectal cancer, and therefore osteoprotegerin is a potential predictive marker, and it also could be a target for treatments.

  • 12.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Chen, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gustafsdottir, Sigrun M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vuu, Jimmy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Ullenhag, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Kämpe, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Autoimmunity.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Hedstrand, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0154214Article in journal (Refereed)
    Abstract [en]

    Background

    The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.

    Objectives

    To investigate SOX10 as a potential biomarker for melanoma and vitiligo.

    Methods

    In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.

    Results

    The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.

    Conclusions

    We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

  • 13.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Nong, Rachel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Darmanis, S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Hertz, E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Protein biomarker validation via proximity ligation assays2014In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1844, no 5, p. 933-939Article, review/survey (Refereed)
    Abstract [en]

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.

  • 14.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany..
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Hust, Michael
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
    Schirrmann, Thomas
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany.;YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany..
    Helmsing, Saskia
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
    Grannas, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Hertz, Ellen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Morén, Anita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Chen, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Dubel, Stefan
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens2016In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 15, no 6, p. 1848-1856Article in journal (Refereed)
    Abstract [en]

    The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

  • 15. Bohmer, Sylvia-Annette
    et al.
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bohmer, Frank-D.
    Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e62871-Article in journal (Refereed)
    Abstract [en]

    Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation.

  • 16.
    Boije, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Ring, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
    Fard, Shahrzad Shirazi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
    Grundberg, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hallbook, Finn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
    Alternative Splicing of the Chromodomain Protein Morf4l1 Pre-mRNA Has Implications on Cell Differentiation in the Developing Chicken Retina2013In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, no 2, p. 615-628Article in journal (Refereed)
    Abstract [en]

    The proliferation, cell cycle exit and differentiation of progenitor cells are controlled by several different factors. The chromodomain protein mortality factor 4-like 1 (Morf4l1) has been ascribed a role in both proliferation and differentiation. Little attention has been given to the existence of alternative splice variants of the Morf4l1 mRNA, which encode two Morf41l isoforms: a short isoform (S-Morf4l1) with an intact chromodomain and a long isoform (L-Morf4l1) with an insertion in or in the vicinity of the chromodomain. The aim of this study was to investigate if this alternative splicing has a function during development. We analysed the temporal and spatial distribution of the two mRNAs and over-expressed both isoforms in the developing retina. The results showed that the S-Morf4l1 mRNA is developmentally regulated. Over-expression of S-Morf4l1 using a retrovirus vector produced a clear phenotype with an increase of early-born neurons: retinal ganglion cells, horizontal cells and cone photoreceptor cells. Over-expression of L-Morf4l1 did not produce any distinguishable phenotype. The over-expression of S-Morf4l1 but not L-Morf4l1 also increased apoptosis in the infected regions. Our results suggest that the two Morf4l1 isoforms have different functions during retinogenesis and that Morf4l1 functions are fine-tuned by developmentally regulated alternative splicing. The data also suggest that Morf4l1 contributes to the regulation of cell genesis in the retina.

  • 17.
    Botling, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Moens, Lotte N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Sorqvist, Elin Falk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Sundström, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Nilsson, M.
    Targeted Resequencing of Formalin-Fixed, Paraffin-Embedded (FFPE) Specimens for Mutation Diagnostics in Solid Tumors2013In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 15, no 6, p. 916-916Article in journal (Other academic)
  • 18.
    Bränn, Emma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Papadopoulos, Fotios
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Psychiatry, University Hospital.
    Fransson, Emma
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden.; Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden .
    White, Richard
    Norwegian Inst Publ Hlth, Oslo, Norway.
    Edvinsson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Hellgren, Charlotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Boström, Adrian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Schiöth, Helgi B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Sundström-Poromaa, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Skalkidou, Alkistis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Inflammatory markers in late pregnancy in association with postpartum depression-A nested case-control study.2017In: Psychoneuroendocrinology, ISSN 0306-4530, E-ISSN 1873-3360, Vol. 79, p. 146-159Article in journal (Refereed)
    Abstract [en]

    Recent studies indicate that the immune system adaptation during pregnancy could play a significant role in the pathophysiology of perinatal depression. The aim of this study was to investigate if inflammation markers in a late pregnancy plasma sample can predict the presence of depressive symptoms at eight weeks postpartum. Blood samples from 291 pregnant women (median and IQR for days to delivery, 13 and 7-23days respectively) comprising 63 individuals with postpartum depressive symptoms, as assessed by the Edinburgh postnatal depression scale (EPDS≥12) and/or the Mini International Neuropsychiatric Interview (M.I.N.I.) and 228 controls were analyzed with an inflammation protein panel using multiplex proximity extension assay technology, comprising of 92 inflammation-associated markers. A summary inflammation variable was also calculated. Logistic regression, LASSO and Elastic net analyses were implemented. Forty markers were lower in late pregnancy among women with depressive symptoms postpartum. The difference remained statistically significant for STAM-BP (or otherwise AMSH), AXIN-1, ADA, ST1A1 and IL-10, after Bonferroni correction. The summary inflammation variable was ranked as the second best variable, following personal history of depression, in predicting depressive symptoms postpartum. The protein-level findings for STAM-BP and ST1A1 were validated in relation to methylation status of loci in the respective genes in a different population, using openly available data. This explorative approach revealed differences in late pregnancy levels of inflammation markers between women presenting with depressive symptoms postpartum and controls, previously not described in the literature. Despite the fact that the results do not support the use of a single inflammation marker in late pregnancy for assessing risk of postpartum depression, the use of STAM-BP or the novel notion of a summary inflammation variable developed in this work might be used in combination with other biological markers in the future.

  • 19.
    Caja, Laia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Tzavlaki, Kalliopi
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Dadras, Mahsa Shahidi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tan, E-Jean
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hatem, Gad
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Maturi, Naga Prathyusha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Morén, Anita
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wik, Lotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Watanabe, Yukihide
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Univ Tsukuba, Dept Expt Pathol, Fac Med, Tsukuba, Ibaraki, Japan.
    Savary, Katia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Univ Reims, UMR CNRS MEDyC 7369, Reims, France.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Uhrbom, Lene
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Heldin, Carl-Henrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Snail regulates BMP and TGF beta pathways to control the differentiation status of glioma-initiating cells2018In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 37, no 19, p. 2515-2531Article in journal (Refereed)
    Abstract [en]

    Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor beta (TGF beta) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGF beta 1 signaling activity. Exogenous TGF beta 1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGF beta pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

  • 20.
    Cane, Gaëlle
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Leuchowius, Karl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Jarvis, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Helbring, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Pardell, Katerina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Ebai, Tonge
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Koos, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Protein Diagnostics by Proximity Ligation: Combining Multiple Recognition and DNA Amplification for Improved Protein Analyses2017In: Molecular Diagnostics (Third Edition), 2016: Academia Press, 2017, 3, p. 219-231Chapter in book (Refereed)
    Abstract [en]

    Proximity ligation assay (PLA) is a unique method in which single-stranded oligonucleotides are conjugated to affinity binders of proteins, followed by amplification of the signal by DNA polymerization and hybridization of complementary oligonucleotides labeled with fluorogenic or chromogenic readout. Here, a brief overview of the field of protein analysis describes the background and the initial development of the technique for the detection of protein–protein interactions via the proximity probes mentioned. In this context, PLA can constrain the general problem of cross-reactivity in protein detection by affinity binders, by ensuring that only cognate pairs of proximity probes result in a signal. Thereafter, this chapter deals mainly with derivatives methods and their applications, with a particular interest in improved specificity, application to various biological materials, and multiplexing. The method has been applied in situ and in solution, adapted for the detection of posttranslational modifications such as phosphorylation and interactions between proteins and specific DNA sequences, and multiplexed to a certain extent, which illustrates its versatility. A technique free from enzymatic reaction, the hybridization chain reaction, can be considered a cost-effective alternative particularly suitable to molecular diagnostics. Finally, we explore further development toward higher-level multiplexing and sensitivity. At this point it is not clear what level can be achieved by PLA, but the assay is compatible with a wide range of readout, including separate real-time amplification reactions and novel microfluidic read-out platforms.

  • 21.
    Chen, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Molecular Tools for Biomarker Detection2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The advance of biological research promotes the emerging of new methods and solutions to answer the biological questions. This thesis describes several new molecular tools and their applications for the detection of genomic and proteomic information with extremely high sensitivity and specificity or simplify such detection procedures without compromising the performance.

    In paper I, we described a general method namely super RCA, for highly specific counting of single DNA molecules. Individual products of a range of molecular detection reactions are magnified to Giga-Dalton levels that are easily detected for counting one by one, using methods such as low-magnification microscopy, flow cytometry, or using a mobile phone camera. The sRCA-flow cytometry readout presents extremely high counting precision and the assay’s coefficient of variation can be as low as 0.5%. sRCA-flow cytometry readout can be applied to detect the tumor mutations down to 1/100,000 in the circulating tumor cell-free DNA.

    In paper II, we applied the super RCA method into the in situ sequencing protocol to enhance the amplified mRNA detection tags for better signal-to-noise ratios. The sRCA products co-localize with primary RCA products generated from the gene specific padlock probes and remain as a single individual object in during the sequencing step. The enhanced sRCA products is 100% brighter than regular RCA products and the detection efficiency at least doubled with preserved specificity using sRCA compared to standard RCA.

    In paper III, we described a highly specific and efficient molecular switch mechanism namely RCA reporter. The switch will initiate the rolling circle amplification only in the presence of correct target sequences. The RCA reporter mechanism can be applied to recognize single stranded DNA sequences, mRNA sequences and sequences embedded in the RCA products.

    In paper IV, we established the solid phase Proximity Ligation Assay against the SOX10 protein using poly clonal antibodies. Using this assay, we found elevated SOX10 in serum at high frequency among vitiligo and melanoma patients. While the healthy donors below the threshold.

    List of papers
    1. A molecular approach for single molecule counting and rare mutation detection in blood plasma
    Open this publication in new window or tab >>A molecular approach for single molecule counting and rare mutation detection in blood plasma
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Problems in biology and medicine frequently require the ability to observe, evaluate, and count even extremely rare macromolecules directly in biological samples. Examples include the detection of mutant DNA or RNA molecules in plasma or distributed in tissues in tumor patients, and highly precise, digital enumeration of proteins and other molecules of interest in clinical specimens. We describe herein a general means to magnify detection signals from individual molecules to easily recorded levels via a highly specific process - super rolling circle amplification (sRCA). We demonstrate the ability of this technique to vastly enhance in situ detection, to count individual molecules by flow cytometry or using a mobile phone camera, and to enumerate tumor-specific sequence variants in plasma from patients at very high efficiency, with specificity adequate to detect single nucleotide mutant sequences among 100,000 copies of the normal sequence. 

    Keywords
    Rolling circle amplification, cfDNA, single molecule, digital counting, rare mutation detection, PoC application
    National Category
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-331737 (URN)
    Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
    2. Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products
    Open this publication in new window or tab >>Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    We have recently developed a technology for localized sequence library preparation with rolling-circle amplification (RCA) as an approach for in situ sequencing. This method involves generation of clonally amplified and specially confined substrates for next-generation sequencing within the preserved context of cells and tissues. Our approach combines padlock probing, RCA, and sequencing-by-ligation chemistry that can resolve expression profiles of sets of genes and mutations in tissues without loss of histological context. Like other fluorescence-based assays, it can be hindered by high level of background fluorescence. To achieve high signal-to-noise ratios, we now describe a method to boost the amplification generated by RCA of padlock probes in situ by super RCA (sRCA). In this technique, a second padlock probe is hybridized, ligated and amplified on the first RCA product for enhanced, localized amplification. We describe and compare different sRCA strategies where gap-fill ligation was showed to be most efficient. The sRCA products co-localize and have comparable sizes as RCA products but they display at least two fold higher signal intensity. This increase in signal to noise also proved to result in two folds increase in the number of sRCA products detected. By combining sRCA with in situ sequencing for highly multiplex detection in tissue a four-time increase was seen. In summary, we demonstrate that sRCA can significantly increase the performance of padlock-based in situ sequencing for gene expression profiling of tissue sections, enabling detection of low abundant transcripts and the analysis of also highly auto-fluorescent samples. 

    Keywords
    in situ sequencing; super RCA (sRCA); tissue gene expression profiling, gap-fill padlock probe
    National Category
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-331741 (URN)
    Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
    3. Rolling Circle Amplification (RCA) Reporters – a new generic tool for the detection of DNA, RNA and proteins
    Open this publication in new window or tab >>Rolling Circle Amplification (RCA) Reporters – a new generic tool for the detection of DNA, RNA and proteins
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Many methods to detect biomolecules with rolling circle amplification (RCA), for example padlock probes and proximity ligation assay (PLA), are tedious and includes several reaction steps combined with long incubation times. The present investigation evaluates a new tool to be included in the toolbox of RCA based detection methods that we refer to as RCA Reporter. The main idea with the RCA Reporter is to use pre-ligated circles protected from RCA initiation by almost entire hybridization to a protector molecule that unlocks through a well characterized and highly specific strand displacement process. The RCA Reporters are a generic tool that can be used to directly detect ssDNA or RNA in a sample, to detect proteins via coupling to proximity recognition using a pair of antibodies or to further amplify ongoing RCA reactions. The latter can be used to allow for efficient digital readout of single molecules via flow cytometry with the potential to develop simple and highly accurate molecular counting assays.

    National Category
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-331743 (URN)
    Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
    4. Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay
    Open this publication in new window or tab >>Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay
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    2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0154214Article in journal (Refereed) Published
    Abstract [en]

    Background

    The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.

    Objectives

    To investigate SOX10 as a potential biomarker for melanoma and vitiligo.

    Methods

    In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.

    Results

    The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.

    Conclusions

    We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

    Keywords
    sox10 proximity ligation assay
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-289194 (URN)10.1371/journal.pone.0154214 (DOI)000374970600050 ()27110718 (PubMedID)
    Funder
    EU, FP7, Seventh Framework Programme, 294409EU, FP7, Seventh Framework Programme, 316929Swedish Research Council
    Available from: 2016-04-29 Created: 2016-04-29 Last updated: 2018-01-10Bibliographically approved
  • 22.
    Clausson, Carl-Magnus
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Making Visible the Proximity Between Proteins2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.

    In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.  

    In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.

    The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.

    The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

    List of papers
    1. Increasing the dynamic range of in situ PLA
    Open this publication in new window or tab >>Increasing the dynamic range of in situ PLA
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    2011 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, p. 892-893Article in journal, Editorial material (Refereed) Published
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-159199 (URN)10.1038/nmeth.1743 (DOI)000296891800004 ()
    Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2017-12-08Bibliographically approved
    2. Compaction of rolling circle amplification products increases signal strength and integrity
    Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal strength and integrity
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-217748 (URN)
    Available from: 2014-02-04 Created: 2014-02-04 Last updated: 2018-06-08Bibliographically approved
    3. Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
    Open this publication in new window or tab >>Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
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    2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 6, p. 1563-1571Article in journal (Refereed) Published
    Abstract [en]

    Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-203549 (URN)10.1074/mcp.O112.023374 (DOI)000319865000007 ()
    Note

    De två första författarna delar förstaförfattarskapet.

    Available from: 2013-07-15 Created: 2013-07-15 Last updated: 2017-12-06Bibliographically approved
    4. Proximity Depended Initiation of Hybridization Chain Reaction
    Open this publication in new window or tab >>Proximity Depended Initiation of Hybridization Chain Reaction