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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.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab.
    Gallant, Caroline J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Lööf, Sara
    Department of Oncology-Pathology, Karolinska Institutet.
    Lengqvist, Johan
    Department of Medicine, Karolinska Institutet.
    Bacanu, Smaranda
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sensitive Measurement of Drug-Target Engagement Using Cellular Thermal Shift Assays with Multiplex Proximity Extension Assay ReadoutManuscript (preprint) (Other academic)
    Abstract [en]

    The ability to measure target engagement in cellular contexts is key for successful drug discovery and clinical care. The cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. CETSA combined with mass spectrometry (MS) readout can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis low-throughput and requires substantial amounts of sample material. Here, we combined CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of 184 proteins from minimal sample material treated with kinase inhibitors. PEA allows analyses of large numbers of specific target proteins at high sensitivity in small sample aliquots. We observed concordant results for proteins measured by MS or PEA. This highly sensitive CETSA-PEA procedure is promising for monitoring drug-target engagement in small aliquots of patient material for analysis of drug binding in drug development and in clinical settings. 

  • 3.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab.
    Johansson, Lars
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Autoimmunity. Uppsala University, Science for Life Laboratory, SciLifeLab. Department of Medicine (Solna), Karolinska University Hospital, Karolinska Institutet.
    Löf, Liza
    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.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Blokzijl, Andries
    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.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lönn, Peter
    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. Dept. Of Immunology, Genetics and Pathology,.
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    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 University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundbäck, Thomas
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    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.
    Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in SituManuscript (preprint) (Other academic)
    Abstract [en]

    It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins.  In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins. 

  • 4.
    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.

  • 5.
    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)
  • 6. 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)
  • 7.
    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)
  • 8. 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.

  • 9.
    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)
  • 10. 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.

  • 11. 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.

  • 12.
    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.

  • 13.
    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.

  • 14.
    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.

  • 15.
    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.

  • 16.
    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.

  • 17. 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.

  • 18.
    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.

  • 19.
    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)
  • 20.
    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.

  • 21.
    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.

  • 22.
    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.

  • 23.
    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.

  • 24.
    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.

  • 25.
    Clausson, Carl-Magnus
    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.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Weibrecht, Irene
    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.
    Mahmoudi, Salah
    Farnebo, Marianne
    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.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. 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.
    Increasing the dynamic range of in situ PLA2011In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, p. 892-893Article in journal (Refereed)
  • 26.
    Clausson, Carl-Magnus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Ishaq, Omer
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Klaesson, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kühnemund, Malte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Grannas, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Koos, Björn
    Qian, Xiaoyan
    Ranefall, Petter
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Krzywkowski, Tomasz
    Brismar, Hjalmar
    Nilsson, Mats
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, p. 12317:1-10, article id 12317Article in journal (Refereed)
  • 27.
    Clausson, Carl-Magnus
    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.
    Grundberg, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    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.
    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.
    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.
    Methods for analysis of the cancer microenvironment and their potential for disease prediction, monitoring and personalized treatments2012In: The EPMA journal, ISSN 1878-5085, Vol. 3, no 7Article, review/survey (Refereed)
    Abstract [en]

    A tumor does not consist of a homogenous population of cancer cells. Therefore, to understand cancer, the tumor microenvironment and the interplay between the different cell types present in the tumor has to be taken into account, and how this regulates the growth and survival of the cancer cells. To achieve a full picture of this complex interplay, analysis of tumor tissue should ideally be performed with cellular resolution, providing activity status of individual cells in this heterogeneous population of different cell-types. In addition, in situ analysis provides information on the architecture of the tissue wherein the cancer cells thrive, providing information of the identity of neighboring cells that can be used to understand cell-cell communication. Herein we describe how padlock probes and in situ PLA can be used for visualization of nucleic acids and protein activity, respectively, directly in tissue sections, and their potential future role in personalized medicine.

  • 28.
    Dahl, Markus
    et al.
    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.
    Maturi, Varun
    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.
    Lönn, Peter
    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.
    Papoutsoglou, Panagiotis
    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.
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vanlandewijck, Michael
    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.
    van der Heide, Lars P
    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.
    Watanabe, Yukihide
    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.
    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.
    Hottiger, Michael O
    Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich, Switzerland.
    Heldin, Carl-Henrik
    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.
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 8, p. e103651-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.

    METHODS:

    A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.

    RESULTS:

    TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.

    CONCLUSION:

    Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

  • 29.
    Dahlin, Joakim S.
    et al.
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden..
    Ekoff, Maria
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden..
    Grootens, Jennine
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden..
    Löf, Liza
    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.
    Amini, Rose-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Hagberg, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Ungerstedt, Johanna S.
    Karolinska Inst, Dept Med Huddinge, Stockholm, Sweden.;Karolinska Univ Hosp, Hematol Ctr, Stockholm, Sweden..
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Nilsson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology. Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden.
    KIT signaling is dispensable for human mast cell progenitor development2017In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 130, no 16, p. 1785-1794Article in journal (Refereed)
    Abstract [en]

    Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.

  • 30.
    Darmanis, Spyros
    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.
    Cui, Tao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
    Drobin, Kimi
    KTH - Royal Institute of Technology, Stockholm, Sweden.
    Li, Su-Chen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
    Öberg, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH - Royal Institute of Technology, Stockholm, Sweden.
    Schwenk, Jochen M.
    KTH - Royal Institute of Technology, Stockholm, Sweden.
    Giandomenico, Valeria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Identification of Candidate Serum Proteins for Classifying Well-Differentiated Small Intestinal Neuroendocrine Tumors2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. e81712-Article in journal (Refereed)
    Abstract [en]

    Background

    Patients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NET) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required.

    Materials and methods

    Suspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases.

    Results

    A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification.

    Conclusions

    We propose new potential protein biomarker candidates for classifying WD-SI-NET at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NET and their eventual use in diagnostics.

  • 31.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Gallant, Caroline Julie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Marinescu, Voichita Dana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Niklasson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Segerman, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Flamourakis, Georgios
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Fredriksson, Simon
    Olink Biosci, S-75237 Uppsala, Sweden..
    Assarsson, Erika
    Olink Biosci, S-75237 Uppsala, Sweden..
    Lundberg, Martin
    Olink Biosci, S-75237 Uppsala, Sweden..
    Nelander, Sven
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells2016In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, no 2, p. 380-389Article in journal (Refereed)
    Abstract [en]

    Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  • 32.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Gallant, Caroline
    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.
    PCR-Based Multiparametric Assays in Single Cells.2012In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 58, no 12, p. 1618-1619Article in journal (Refereed)
  • 33.
    Darmanis, Spyros
    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.
    Nong, Rachel Yuan
    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.
    Vänelid, Johan
    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.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Ericsson, Olle
    Halo Genomics AB, Dag Hammarskjölds väg 36B, SE-752 37 Uppsala Sweden.
    Fredriksson, Simon
    Olink Biosciences, Dag Hammarskjölds väg 52B, SE-752 37 Uppsala, Sweden.
    Bäcklin, Christofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Gut, Marta
    Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4, 08028 Barcelona, Spain.
    Heath, Simon
    Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4, 08028 Barcelona, Spain.
    Gut, Ivo Glynne
    Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4, 08028 Barcelona, Spain.
    Wallentin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, UCR-Uppsala Clinical Research Center.
    Gustafsson, Mats G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    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.
    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.
    ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e25583-Article in journal (Refereed)
    Abstract [en]

    Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. 

  • 34.
    de la Torre, Teresa Zardan Gomez
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Strömberg, Mattias
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Göransson, Jenny
    Gunnarsson, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Svedlindh, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Molecular diagnostics using magnetic nanobeads2010In: / [ed] Goll G., Lohneysen H.V., Loidl A., Pruschke T., Richter M., Schultz L., Surgers C., Wosnitza J, 2010, Vol. 200, p. 122011-Conference paper (Refereed)
    Abstract [en]

    In this paper, we investigate the volume-amplified magnetic nanobead detection assay with respect to bead size, bead concentration and bead oligonucleotide surface coverage in order to improve the understanding of the underlying microscopic mechanisms. It has been shown that: (i) the immobilization efficiency of the beads depends on the surface coverage of oligonucleotides, (ii) by using lower amounts of probe-tagged beads, detection sensitivity can be improved and (iii) using small enough beads enables both turn-off and turn-on detection. Finally, biplex detection was demonstrated.

  • 35. de Miranda, Noel F. C. C.
    et al.
    Peng, Roujun
    Georgiou, Konstantinos
    Wu, Chenglin
    Sörqvist, Elin Falk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Berglund, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Chen, Longyun
    Gao, Zhibo
    Lagerstedt, Kristina
    Lisboa, Susana
    Roos, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    van Wezel, Tom
    Teixeira, Manuel R.
    Rosenquist, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Sundström, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    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.
    Zeng, Yixin
    Kipling, David
    Pan-Hammarstrom, Qiang
    DNA repair genes are selectively mutated in diffuse large B cell lymphomas2013In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 210, no 9, p. 1729-1742Article in journal (Refereed)
    Abstract [en]

    DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.

  • 36.
    de Oliveira, Felipe
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Nils
    Department of Medicine, Solna (MedS), K2, Karolinska Institute, Stockholm, Sweden .
    Muthelo, Phathutshedzo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Åke, Lernmark
    Department of Clinical Sciences, Skåne University Hospital SUS, Lund University, Malmö, Sweden.
    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.
    Autoimmunity detection via proximity assaysManuscript (preprint) (Other academic)
    Abstract [en]

    Since autoantibodies are recognized as valuable biomarkers for clinical diagnostics and prognostics in autoimmune diseases such as Stiff Person Syndrome (SPS) and Type 1 diabetes, detection of such markers at improved sensitivity and specificity could be of significant interest. In addition, as proximity assays have been shown to offer highly sensitive and specific detection of multiple proteins, the technique could be expanded to applications for autoimmunity detection. In the present study, we have applied the newly developed proximity ligation assay with rolling circle amplification (PLARCA), and proximity extension assay (PEA) for the detection of GADA, autoantibodies specific for glutamic acid decarboxylase 65 (GAD65). Through the use of oligonucleotide conjugated autoantigen GAD65 and anti-human antibodies, as proximity probes, we were able to apply these proximity assays to detect GADA in a set of SPS patient samples. In summary, we have applied and established both PLARCA and PEA, as a proof of concept, for the use of the specific and sensitive autoimmune detection.

  • 37.
    de Oliveira, Felipe Marques Souza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Development and Application of Proximity Assays for Proteome Analysis in Medicine2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Along with proteins, a myriad of different molecular biomarkers, such as post-translational modifications and autoantibodies, could be used in an attempt to improve disease detection and progression. In this thesis, I build on several iterations of the proximity ligation assay to develop and apply new adaptable methods to facilitate detection of proteins, autoantibodies and post-translational modifications.

    In paper I, we present an adaptation of the solid-phase proximity ligation assay (SP-PLA) for the detection of post-translational modification of proteins (PTMs). The assay was adapted for the detection of two of the most commons PTMs present in proteins, glycosylation and phosphorylation, offering the encouraging prospect of using detection of PTMs in a diagnostic or prognostic capacity. 

    In paper II, we developed a variant of the proximity ligation assay using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer, termed PLARCA. With a detection limit considerably lower than ELISA, PLARCA detected femtomolar levels of these proteins in patient samples.

    In paper III, we aim to compare detection values of samples collected from earlobe capillary, venous plasma, as well as capillary plasma stored in dried plasma spots (DPS) assessed with a 92-plex inflammation panel using multiplex proximity extension assay (PEA). Despite the high variability in protein measurements between the three sample sources, we were able to conclude that earlobe capillary sampling is a suitable less invasive alternative, to venipuncture.

    In paper IV, we describe the application of PLARCA and proximity extension assay (PEA) for the detection of GAD65 autoantibodies (GADA). Thus, offering highly sensitive and specific autoimmunity detection.

  • 38.
    de Oliveira, Felipe Marques Souza
    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.
    Mereiter, Stefan
    i3S – Instituto de Investigação e Inovação em Saúde and IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Portugal.
    Lönn, Peter
    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.
    Siart, Benjamin
    Department of Anthropology, University of Vienna, Austria; Department of Behavioral Biology, University of Vienna, Austria .
    Shen, Qiujin
    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.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Raykova, Doroteya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlsson, Niclas G.
    Department of Medical Biochemistry and Cell Biology at Institute of Biomedicine, Gothenburg University, Sweden.
    Polom, Karol
    Department of Surgical Oncology, Medical University of Gdansk, Poland; General Surgery and Surgical Oncology Department, Università deli Studi di Siena, Italy..
    Roviello, Franco
    General Surgery and Surgical Oncology Department, Università deli Studi di Siena, Italy..
    Reis, Celso A.
    ) i3S – Instituto de Investigação e Inovação em Saúde and IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Portugal; Instituto de Ciências Biomédicas Abel Salazar (ICBAS), University of Porto, Portugal; Faculty of Medicine of the University of Porto, Portugal.
    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.
    Detection of post-translational modifications using solid-phase proximity ligation assay2018In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 45, p. 51-59Article in journal (Refereed)
    Abstract [en]

    Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers

  • 39.
    de Oliveira, Felipe Marques Souza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mereiter, Stefan
    Univ Porto, i3S, Oporto, Portugal.;Univ Porto, IPATIMUP Inst Mol Pathol & Immunol, Oporto, Portugal..
    Persson, Nina
    Univ Copenhagen, Dept Chem, Copenhagen, Denmark..
    Blixt, Ola
    Univ Copenhagen, Dept Chem, Copenhagen, Denmark..
    Reis, Celso A.
    Univ Porto, i3S, Oporto, Portugal.;Univ Porto, IPATIMUP Inst Mol Pathol & Immunol, Oporto, Portugal..
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Detection of post-translational modification of cancer biomarkers via proximity ligation assay2016In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 26, no 12, p. 1455-1455Article in journal (Refereed)
  • 40.
    Dieterich, Lothar C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mellberg, Sofie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Langenkamp, Elise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Zhang, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Salomäki, Henriikka
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Teichert, M.
    Huang, Hua
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Edqvist, Per-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Kraus, T.
    Augustin, H. G.
    Olofsson, Tommie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Larsson, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Molema, G.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Georgii-Hemming, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Alafuzoff, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFβ2 in vascular abnormalization2012In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 228, no 3, p. 378-390Article in journal (Refereed)
    Abstract [en]

    Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy.

  • 41.
    Djureinovic, Dijana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Pontén, Victor
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Landelius, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery.
    Al Sayegh, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Kappert, Kai
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry; Center for Cardiovascular Research (CCR), Berlin, Germany.
    Kamali-Moghaddam, Masood
    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.
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Ståhle, Elisabeth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery.
    Multiplex plasma protein profiling identifies novel markers to discriminate patients with adenocarcinoma of the lung2019In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 19, article id 741Article in journal (Refereed)
    Abstract [en]

    Background:The overall prognosis of non-small cell lung cancer (NSCLC) is poor, and currently only patients with localized disease are potentially curable. Therefore, preferably non-invasively determined biomarkers that detect NSCLC patients at early stages of the disease are of high clinical relevance. The aim of this study was to identify and validate novel protein markers in plasma using the highly sensitive DNA-assisted multiplex proximity extension assay (PEA) to discriminate NSCLC from other lung diseases. 

    Methods:Plasma samples were collected from a total of 343 patients who underwent surgical resection for different lung diseases, including 144 patients with lung adenocarcinoma (LAC),68 patients with non-malignant lung disease, 83 with lung metastasis of colorectal cancers and 48 patients with typical carcinoid. One microliter of plasma was analyzed using PEA, allowing detection and quantification of 92 established cancer related proteins. The concentrations of the plasma proteins were compared between disease groups.

    Results:The comparison between LAC and benign samples revealed significantly different plasma levels for four proteins; CXL17, CEACAM5, VEGFR2 and ERBB3 (adjusted p-value < 0.05). A multi-parameter classifier was developed to discriminate between samples from LAC patients and from patients with non-malignant lung conditions. With a bootstrap aggregated decision tree algorithm (TreeBagger) a sensitivity of 93% and specificity of 64% was achieved to detect LAC in this risk population. 

    Conclusion:By applying the highly sensitive PEA, reliable protein profiles could be determined in microliter amounts of plasma. We further identified proteins that demonstrated different plasma concentration in defined disease groups and developed a signature that holds potential to be included in a screening assay for early lung cancer detection. 

  • 42.
    Dubois, Louise
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Hultenby, Kjell
    Department of Laboratory Medicine, Karolinska Institutet, SE-141 86 Huddinge, Sweden.
    Waldenström, Anders
    Department of Public Health and Clinical Medicine, Umeå University, SE-901 85 Umeå, Sweden.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Ronquist, K. Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents2018In: Cancer Research Frontiers, ISSN 2328-5249, Vol. 4, no 1, p. 13-26Article in journal (Refereed)
    Abstract [en]

    Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs).

    Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h.

    Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions.

    Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.

  • 43.
    Ebai, Tonge
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine.  Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment.  In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection.

    In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA.

    In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA).  We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer.

    In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold.

    In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.

  • 44.
    Ebai, Tonge
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    de Oliveira, Felipe
    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.
    Wik, Lotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Schweiger, Caroline
    Charité Comprehensive Cancer Center, University of Berlin, Germany.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Keilholtz, Ulrich
    Institute of Pathology, Medical University of Graz, Austria.
    Haybaeck, Johannes
    Charité Comprehensive Cancer Center, University of Berlin, Germany.
    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.
    Sensitive protein detection in microtiter plates by proximity ligation with rolling circle amplification (PLARCA)Manuscript (preprint) (Other academic)
  • 45.
    Ebai, Tonge
    et al.
    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.
    de Oliveira, Felipe Marques Souza
    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.
    Löf, Liza
    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.
    Wik, Lotta
    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.
    Schweiger, Caroline
    Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Institute of Pathology, Medical University of Graz, Graz, Austria; .
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Keilholtz, Ulrich
    Institute of Pathology, Medical University of Graz, Graz, Austria; .
    Haybaeck, Johannes
    Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Department of Pathology, Otto von Guericke University Magdeburg, Magdeburg, Germany..
    Landegren, Ulf
    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.
    Kamali-Moghaddam, Masood
    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.
    Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification2017In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 63, no 9, p. 1497-1505Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals.

    METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition, these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader.

    RESULTS: We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor-15 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA.

    CONCLUSIONS: PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

  • 46.
    Ebai, Tonge
    et al.
    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.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Parallel protein detection by solid-phase proximity ligation assay with real-time PCR or sequencing2015In: Current Protocol in Molecular Biology, ISSN 1934-3647, Vol. 109, no 20Article in journal (Refereed)
    Abstract [en]

    Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA-modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid-phase proximity ligation assays for soluble proteins using either real-time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed.

  • 47.
    Ebai, Tonge
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Wu, Yajun
    Chinese Academy of Inspection and Quarantine, Beijing, China.
    Kamali-Moghaddam, Masood
    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 detection by sensitive magnetic bead-based proximity extension assaysManuscript (preprint) (Other academic)
  • 48.
    Elfineh, Lioudmila
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Classon, Christina
    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.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. 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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lind, Sara Bergström
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tyrosine phosphorylation profiling via in situ proximity ligation assay2014In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 14, p. 435-Article in journal (Refereed)
    Abstract [en]

    Background: Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay. Methods: K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity. Results: Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format. Conclusions: This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.

  • 49. Engström, Anna
    et al.
    Zardán Gómez de la Torre, Teresa
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Nilsson, Mats
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Herthnek, David
    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.
    Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead- Based Readout2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, p. e62015-Article in journal (Refereed)
    Abstract [en]

    Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlockprobes were designed to target the most common mutations associated with rifampicinresistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. Fordetection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identificationof the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugatedmagnetic nanobeads and detected by measuring the frequency-dependent magneticresponse of the beads using a portable AC susceptometer.

  • 50. Engström, Anna
    et al.
    Zardán Gómez de la Torre, Teresa
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    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.
    Herthnek, David
    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.
    Detection of Rifampicin Resistance in Mycobacterium tuberculosis using Padlock Probes and a Magnetic Biosensor2012In: 33rd Annual Congress of the European Society of Mycobacteriology (ESM), 01st-04th July 2012, Brasov, Romania: Scientific Program including Abstracts / [ed] PD Dr. Stefan Niemann, 2012, p. 69-69Conference paper (Refereed)
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